Drug Delivery to the Posterior Segment from Drops

SURVEY OF OPHTHALMOLOGY VOLUME 47 • SUPPLEMENT 1 • AUGUST 2002

Drug Delivery to the Posterior Segment from Drops David M. Maurice, PhD Department of Ophthalmology, Columbia University, New York, New York, USA Abstract. The published evidence that instilled drugs can affect the blood supply to the retina and optic nerve head in humans is examined. As a background, seven techniques that have been used to measure flow are briefly described and criticized. For timolol, the corresponding measurements, obtained by a number of investigators are evaluated. The outcome is very erratic and does not allow any conclusion as to the effect of this drug on flow. Consideration is then given to the possible mechanism whereby a drug could affect blood flow; directly, by diffusion to receptors on the vessels, or indirectly, through more anterior receptors. The question is raised whether the small changes in circulation induced by drugs would not be swamped by those resulting from natural alterations in the ambient light level. The literature was analyzed in the hope of identifying discrete entry pathways, for example, through the lens or the suprachoroidal space, that are sufficiently permeable to allow a significant quantity of drug to pass. There was an indication that a drug might diffuse through the lens cortex in sufficient quantity to cause a measurable rise in its concentration in the vitreous. In general, however, there was insufficient quantitative data to allow any meaningful predictions to be made. Stimulated by recent evidence, it is suggested that drug penetration from the tear fluid takes place by direct diffusion across the conjunctiva into the sclera and orbit when the head is supine. (Surv Ophthalmol 47 (Suppl 1):S41-S52, 2002. © 2002 by Elsevier Science Inc. All rights reserved.) Key words. drug penetration • glaucoma • pharmacokinetics • intraocular pressure ocular perfusion pressure • optic nerve • retinal blood flow • topical application

There have been frequent suggestions in recent years that therapeutic benefits, in particular the maintenance of an adequate blood supply to the retina or optic nerve head (ONH), may result from the topical application of drugs. On the other hand, conventional ocular pharmacokinetics has downplayed the possibility of any effective transfer of a drug from an eye drop to the vitreous humor or retina of a patient. It has been intuitively assumed that the distance a drug must travel from an eye drop to the fundus of the eye and the barriers it encounters in its passage will prevent the build-up of a therapeutic concentration in the posterior vitreous or at the vasomotor receptors on the capillaries that supply the retinal circulation. The purpose of this review is to examine in detail the basis of these contradictory opinions. First, the

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published evidence for drug-induced changes in blood flow will be examined. There are a number of physical techniques in use for measuring the rate of blood flow in the retina or ONH and these are specified and their limitations discussed. The experimental findings on the effect of eye drops they give rise to are compared: only in the case of timolol is there sufficient published data to make such a comparison meaningful. Little agreement exists between the findings of the various workers, so little confidence can be felt in statements that the drug raises or lowers the blood flow. Whether an appreciable quantity of drug enters the posterior segment of an eye from a drop is approached more directly by experiments on animals. Unfortunately, there are technical difficulties associated with these measurements that most investigaS41

© 2002 by Elsevier Science Inc. All rights reserved.

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tors have not been able to resolve. A few studies that are acceptable indicate that the concentration of drug in the vitreous rises only to about one-millionth of that in the drop after a single instillation. The next section discusses the fate of this small quantity of drug which enters the globe: whether it would continue its passage to interact directly with the receptors on the capillaries or whether it could exert any effect by a more indirect mechanisms. An attempt was then made to identify any route, for example, the lens, whereby a drug might pass from the tear film to the retina and to estimate where possible the build-up in concentration it might give rise to. Little success attended these efforts as far as drug transport through the globe is concerned. However, there is a strong indication that entry from the conjunctival cul-de-sac to the posterior sclera and orbit can occur and this possibility should be the object of further studies.

Evidence for Drug Penetration from an Eye Drop to the Fundus of the Globe Two approaches to establishing whether a pharmacologically active concentration can be built up will be presented. The first examines published reports of changes in the blood flow of the capillaries of the retina, ONH, and choroid that result from the instillation of a drop in humans. The second collects data from experiments in animals in which any accumulation of drug in the vitreous is measured after the instillation of a drop. CHANGES IN BLOOD FLOW AFTER A DROP IN HUMANS

Consideration is limited to experiments where a single drop was instilled because repetition leads to a complex situation not readily susceptible to pharmacokinetic analysis. However, the outcome of single dose and longer term treatment was not found to be different in many cases.28,33,36,37,72 MEASUREMENT TECHNIQUES

A variety of non-invasive techniques, based on entirely distinct physical principles of measurement, have been used in humans to identify changes in blood flow in the retinal, choroidal, and ONH systems. The specificity and accuracy of most of these techniques have been discussed previously,35,40 and there is disagreement over the significance of many of the results38,39,107 because of instrumental unreliability and of uncertainty over which blood vessels are implicated in the velocity changes observed. An understanding of the principles of the operation of these seven techniques is essential to assessing the validity of their results and it is relevant to set them out. Furthermore, some of them have only recently

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come into use and many investigators may be unfamiliar with them. Ultrasound Doppler Sonography (Color Doppler Imaging) This makes use of the changes in frequency of a beam of ultrasound scattered back from the blood flowing in a vessel.76 The beam of ultrasound is able to penetrate to invisible vessels—for example, the ophthalmic and central arteries that are deep and large. The blood flow can be displayed over a 20color range. Laser Doppler Velocimetry (LDV) This measures the frequency change of light scattered back from the blood in order to estimate its flow in the larger visible vessels as well as that in the capillaries in the vessel free regions of the ONH.78,79 Because it employs infrared light, which has a significant but limited penetration into the tissues, particularly the ONH, the depth of the capillaries being studied is often not clear.71,74 It has been combined with a confocal scanning system to create a more complex instrument, generally known as the Heidelberg Retinal Flowmeter after its manufacturer,31 that provides a two-dimensional image of the blood flow velocities, and a better depth resolution.65 Speckle Blur This recently introduced method relies on the analysis of the speckle that is observed when a laser illuminates an object. The speckle becomes measurably blurred when the object, in this case the column of red blood cells, moves. It does not provide absolute flow values but only relative values in an individual subject, but it is reported to give reproducible values that correlate with more invasive measures.93,94,98 The correlation with LDV measurements is rather disappointing, however.107 Fluorescein Angiography A bolus of fluorescein is injected intravenously and a series of retinal arteriograms is recorded. These are analyzed to determine the time taken for the front of the fluorescent blood to travel from the arterial to the venous side of the ONH or over some other defined path. Recordings are acquired photographically or with a scanning laser ophthalmoscope. Although this is a versatile method, reservations about its accuracy have been expressed.19,40,97 Intraocular Pulse It is claimed that pulsatory excursions make up the greater part of the blood flow through the choroid, hence the magnitude of the IOP pulse is its measure.47 However, the basis for this conclusion is not clear. There does not even appear to be a con-

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sensus as to whether the elastic element responsible for the transmission of the pulse corresponds to the vessel walls of the choroid20 (as I believe) or to the resilience of the sclera as in the Friedenwald model.47 Furthermore, although our ultimate concern should be the total blood perfusion, pulsatile and steady, through tissues at risk from anoxia, there is no reason to expect a simple relationship between this rate of perfusion and the raw pulse amplitude. On the contrary, in my experience8,55 the pulse amplitude may be altered many times over by postural changes, such as sitting down, that would not be expected to promote ischemia in the retina. Fundus Pulse This method uses an optical interference system to measure pulsatile movements of the macula and optic disk, which should allow the retinal and choroidal systems to be separated.85 Although it is more specific in its target than the intraocular pulse method, the relationship between the amplitude of the pulse and the continuous blood flow through the vascular system remains unclear. Entoptic Leukocyte Observation (Blue Field Simulation)

have been demonstrated to cause a significant drop in IOP. Most attention has been paid to adrenergic agents, particularly the beta-blockers. Contralateral Effects Drugs can enter the eye not only by direct penetration into the globe but also by way of the systemic circulation after absorption across the conjunctiva. This can give a false impression of the effectiveness of the local route and measurements were rejected from consideration unless the contralateral eye was untouched and the observed responses compared to those in the treated eye. Vascular responses in the contralateral eye have not been described frequently, but changes in its IOP were noted in several articles.26,63,108 This is a general effect with beta-blockers, for in more than 800 subjects, 35% contralateral eyes showed a reduction of 3 mm Hg or more, compared to a 6 mm Hg reduction in the treated eye.73 This is probably the effect of systemic delivery, although it has been suggested that it is due to some central nervous mechanism evoked by the drug.64 Timolol

This is a subjective technique in which the movement of leukocytes in the parafoveal capillaries is visualized.81 Subjects have been able to count the number of leukocytes passing through a single capillary loop over a 30-second interval and this has been used to estimate the action of timolol.77 A more convenient system matches the overall leukocyte movement for velocity and density with a variable computer-driven image.81 MEASUREMENTS ON HUMANS

These techniques have been used to estimate vasomotor changes induced by a variety of drugs that

Timolol has been studied by eight groups of investigators, who between them have used every one of the techniques listed above, and sometimes compared the outcome of two or more of them.6,26,27,31, 77,86,95,108 In brief, it was reported that the blood flowed more rapidly in 2 series of experiments, more slowly in 6, and did not change or gave equivocal results in 3 (Table 1). The various techniques often purport to measure flow in different vascular systems, but there was no consensus among the separate groups who used only one technique—LDV, for example.

TABLE 1

Effect of Timolol Drop in Humans Blood Flow Change, % Technique Laser Doppler26 Laser Doppler108 Heidelberg31 Ultrasound Doppler86 Ultrasound Doppler33 Fundus Pulsation86 Speckle Blur95 Transit Time6 Transit Time77 Blue Field77 Ocular Pulse108

Where Central retinal vein Retinal artery Macular capillaries Ophthalmic and retinal arteries Orbital vessels Disc and macula ONH capillaries ONH capillaries Central retinal vein Macular capillaries All vessels

Treated

Contralateral

13 7 15 0 0 13 0 25 9 13 30

2 18

12

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Betaxolol

Systemic Transfer

The results of studies of betaxolol were more consistent in that 5 measuring techniques used in 6 experimental series by 4 groups of investigators agreed in showing that there were no significant vascular changes evoked by this drug.31,37,86,95 On another two occasions, a significantly increased flow was determined.6,33 There are a few single studies on other adrenergic agents, for example metipranolol,106 in which the rate of flow was found to rise significantly, but most lack conviction because questions have been brought forward about the validity of the techniques employed.

Most of the drug in a conventional-sized drop is absorbed into the blood system across the conjunctiva or in the naso-lacrimal duct or digestive system, and thence it can penetrate the ocular tissues of both eyes. The penetration is controlled by the blood retinal barrier, whose properties have been summarized in several articles.15,56 The systemic route of penetration can provide a major portion of the very small amount of drug found in the vitreous or retina after topical administration, particularly in an animal with a relatively small ratio of body weight to eye size, such as the rabbit, but significant contralateral effects have also been reported in humans, as noted above. For meaningful estimates of the direct penetration, it is best, therefore, that the tests be carried out unilaterally and that the values found in the control eye are subtracted from those found in the experimental eye. In addition, it is important to ensure that the blood level remains very low throughout the experiment. Unfortunately, these simple procedures have not been universally carried out, and more than one-half of the publications examined were removed from consideration on this basis.

Dorzolamide Studies of dorzolamide showed that significant reductions in arteriovenous transit times were noted on some occasions,34,36,37 even if only in limited sectors of the retina. In other studies,52 Doppler ultrasound showed that dorzolamide increased the velocity in the retina and ONH but had no effect on the retrobulbar vessels34,36,37 or ONH.29 No significant changes in any vessels were found by LDV.72 Latanoprost LDV scanning has been used in only one study of the effects of latanoprost on humans,88 and this failed to detect any changes in the vascular system. This is in agreement with the results of a study on monkeys,91 which used the invasive microsphere method, in which only the anterior scleral vascular system showed a change. CHEMICAL ANALYSIS OF PENETRATION IN ANIMALS

There have been a number of measurements of the quantity of a drug that is found in the vitreous, retina, and other parts of the eye, in an animal (generally a rabbit) after the instillation of a drop. The interpretation of the data is attended with many problems, to be addressed in the next section. Before that, several technical difficulties must be overcome to ensure that the drug level in the sample of tissue being assayed corresponds to that resulting from direct penetration from the eye drop. Sources of Error The maximum concentration developed in the vitreous humor after an eye drop is approximately a hundred-thousandth of that in the drop itself and great care must be taken to avoid artifacts during the collection of tissues. The following are some sources of error that have been identified.

Contamination Potentially, the most serious error is from contamination of the globe that occurs while it is being enucleated and its contents collected. Excess fluid instilled into an eye tends to dry out on the lid margins and eyelashes, and it is likely that they may require more than flushing with a few milliliters of saline to remove the encrusted drug.57 The residue can be smeared over the surface of the globe while it is being enucleated and may have an opportunity of reaching the vitreous or retina to a degree dependent on the subsequent separation procedures. Because contamination is largely a matter of chance, it can often be detected by the erratic data it provides. Miscellaneous Errors A source of inaccuracy can be the sampling of the vitreous humor through a syringe needle. The drug distribution can be very uneven in the gel, particularly during the early stages of penetration, when it is concentrated near the hyaloid or retina. A needle is more likely to sample the fluid from the center of the vitreous body where the concentration can be very small. A method such as freezing that can cleanly separate the entire vitreous from the retina is desirable. The postmortem redistribution of drug may also present a problem, particularly in tissues like the ONH that are small, and are collected after a delay.

DRUG DELIVERY TO THE POSTERIOR SEGMENT FROM DROPS

Drug Penetration A detailed examination of a no-doubt incomplete selection of publications concerning the vitreous or retinal penetration from an eye drop in rabbits has been carried out. In view of the errors, noted above, which can vitiate the results, very few studies could be accepted as providing valid data. The barrier to penetration into the posterior segment may be represented as a dilution; in this case the concentration of drug in the eye drop divided by the maximum concentration reached in the vitreous. The measured dilutions are approximately 250, 000 for imirestat,13 and 1,000,000 for dexamethasone acetate.32 Although these vitreous levels are very low, they may amount to therapeutic concentrations.

Mechanism of Drug Action It is relevant to consider the fate of a drug after it has penetrated the globe: first, the factors that influence its passage to the vascular receptors; and, second, the nature of the interaction between the activated receptors and the circulation in the capillaries. These are complex issues that cannot be dealt with in depth in this review and attention will be limited to a few aspects that should be the subject of further studies.

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the binding of a drug should correspond to extending the volume of the vitreous, so that the concentration of the unbound pharmaceutically active form in it will be reduced during the entry stages of administration and prolonged when the treatment is interrupted or terminated. At later times, when a quasiequilibrium has been established between the vitreous and the pigmented tissues, the concentration of free drug should be about the level that would be found in the absence of any absorption. Some workers are reluctant to accept the vitreous level as representing the concentration of free drug throughout the posterior segment, presumably because it is so low; however, it is the appropriate value because it is in equilibrium with the high-affinity drug-loaded tissues. It should also be kept in mind that a high drug concentration in the peripheral shell of the vitreous is not necessarily an indication that it is exerting a correspondingly large biological action on the adjacent retina; it may only represent the absence of vasomotive receptors in the retinal tissues or lack of access to them. Conversely, a relatively low concentration in the peripheral vitreous during the early stages of penetration does not necessarily mean that its effect is being diminished by loss to the blood or binding by inert tissue, but it could result from a large active uptake by vasomotive receptors. Melanin Affinity

KINETICS OF DRUG IN POSTERIOR SEGMENT

When drug molecules enter the vitreous, most likely at its anterior zone, they can progress further to the fundus by diffusion through the gel when it is formed or by convection when it is liquefied. The movement can be visualized by fluorescent tracers or, potentially, by MRI,3,10 or can be computed by the engineering technique of finite element modeling, based on the experimental finding that diffusion in the gel is virtually unrestricted. Such computations in the human eye show22 that if a mass, D, is released near the hyaloid membrane, it will build up a concentration at the ONH of 0.06 D/ml for a drug to which the retina is impermeable or 10 times less for a drug with a high outward retinal permeability, such as fluorescein. The rise to the peak concentration can take a long time but levels one-tenth of this, when the concentration begins to rise rapidly, are achieved in 4–5 hours.

One well-established form of binding is the affinity of drugs to melanin.100 This has been reviewed by Salazar-Bookaman et al,83 and instances of the prolongation of action of various drugs are cited, in addition to two further studies on timolol.5,9 Detailed studies of the effect of pigment on the uptake of beta-blockers after the instillation of one drop into the rabbit eye have been published.5,23,24 In the albino animal,84 the iris concentration remains 2–3 times higher than that in the aqueous, and the concentrations fell rapidly for the 8 hours over which the experiments were carried out. In a pigmented strain, on the other hand, the iris concentration remains much greater than that in the aqueous, at its maximum,1–3 days after instillation, as much as 80 times more. The drug is lost from the tissues far more slowly than from the albino, less than onehalf disappearing over the 1–3 day interval. Similar relationships may be expected between the retina and vitreous, but the data are less reliable.

Drug Binding

Cell Membrane Solubility

Many drugs, including beta-blockers, are strongly bound to the uveal and retinal tissue. This limits the amount of free drug available to act on the vascular receptors in the early stages of drug penetration into the posterior segment. In pharmacokinetic terms,

A form of sequestration similar to binding is the solution of lipid-soluble drugs in the cell membrane of the tissues. The uptake of fluorescein by the lens will be discussed later. The retina is another potential depot for this dye, where its uptake from the

Movement through Vitreous

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blood has been observed directly,25 and where its function as a depot has been semi-quantitatively analyzed.101 The binding of timolol and betaxolol by Tenon’s capsule in patients who have been long-term users of these drugs has recently been documented.90 Although the levels found were sometimes very high, it is not evident that the release from a depot external to the sclera would be enough to provide a significant concentration within the globe. INTERACTION OF DRUG WITH RECEPTORS

Physical Mechanisms The vascular complex of the eye, including the ONH, involves the interpenetration of flows from very many component circulations, large and small, and these are affected by the IOP and the elasticity of the sclera and the blood vessels. A drug can affect the hydraulic resistance of any component of the vascular network, thus resetting the pressures and flows throughout the whole of it. In some cases where an eye drop reduces the IOP by cutting down on the ciliary blood flow, it is possible that the blood supply to the retina and ONH may be inadvertently improved as a result of a resetting of the system. It is possible to envision specific physical mechanisms whereby this might be effected. One theory that has come under consideration is that the drop in IOP caused by the drug will itself encourage an increase in the retinal blood flow, by way of increasing the ocular perfusion pressure (OPP). There is little direct evidence to support this hypothesis, although the finding by Grunwald26 that there is a strong correlation between OPP and blood velocity in the central vein of the retina argues in its favor. On the other hand, increasing the OPP by trabulectomy or by subsequent revision of the filtering bleb resulted in no change in the retinal blood flow, as estimated by the speckle blur method.96 A similar mechanism could apply in those cases where an eye drop fails to evoke any change in the ophthalmic artery flow. Many Doppler ultrasound measurements show this to be a common occurrence. In these cases a diminution of blood flow to the anterior uvea, related to the hypotensive action of the drug, would inevitably cause a rise in the flow to the retina and choroid. Neurological Pathway Apart from the physical mechanisms just mentioned, some form of amplification or increase in reliability of the pharmacological response at the retina resulting from interaction between the drug and receptors in the anterior segment is conceivable.

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Some form of neural pathway, passing for example from receptors in the root of the iris to the retinal or ONH capillary wall, might be envisaged. Finally, it may be questioned whether the degeneration that accompanies low tension glaucoma can be explained on the basis of vasomotor responses alone or whether other factors, particularly mechanical, need to be invoked.53 EFFECT OF LIGHT

Many workers believe that a principal function of the choroidal circulation is to carry off the heat that is produced by the absorption of light in the retina and the retinal pigment epithelium; it has been claimed7, 67–70 that the choroidal circulation increases by more than 70% when a bright light is shone on the retina or that of the contralateral eye. Recently, however, these conclusions have been justifiably challenged.50 It is reasonable to suppose that there should be mechanism in place to stabilize the temperature of the retina, in particular against the heating caused by bright light, and this could be provided by an increase in choroidal blood flow. Such an increase can be provoked by other means; for example, a 2–3 times rise in the flow has been shown to result from stimulation of the facial nerve in rabbits.66 In the retinal circulation, on the other hand, the situation may be reversed;18,82 laser Doppler velocimetry has been proposed to show that flow in the retinal veins was about 80% higher in the dark than in the light, in human subjects.80 This was considered to be result of the higher metabolic requirement of the photoreceptors in the dark. Very recently, however, the group involved has undergone a reversal in their views.82 It has been reported that the effect of light on both the choroidal and retinal circulations could be as much as 6 times greater than that found for the most active of the vasomotive drugs. This requires that the validity of the methods of estimating blood flow must be reconsidered. Many involve shining a bright light in the eye in order to collect the required information, so that it is to be expected that the flow rates are changing in the period when they are being measured. Only in one instance does this source of error seem to have been appreciated and accounted for.50 Similarly, a shifting baseline could result from a period of acclimatization before the measurements. Furthermore, the changes in ONH blood flow that can be attributed to a drug seem unimportant in comparison with those that would occur when a patient goes out in the sun or closes his eyes in sleep. Under these conditions, the drug-induced adjustments to the circulation would be irrelevant.

DRUG DELIVERY TO THE POSTERIOR SEGMENT FROM DROPS

Analysis of Entry Pathways I find it difficult to conceive that any biological purpose could be served by a system that directs a solute from the tear film and presents it to the retina at about one-millionth of its original concentration. It seems intrinsically more probable that the transfer would be a consequence of a small failure in the system of barriers that protect the retina from potentially toxic factors in the external environment. Where such a low (but potentially pharmacologically active) concentration of a drug is encountered in the vitreous, it may seem fruitless to analyze by what mechanism it arrived there. However, the possible clinical benefit that may be associated with such a break down of the barrier justifies an examination of its structure so that it might be understood and controlled. Therefore, possible routes of penetration will be examined in some detail. There are two general pathways whereby a drug can reach the posterior blood vessels from a drop: 1. Corneal: into the anterior chamber, and then through the lens, the pupil, or the iris or its root 2. Conjunctival: either directly across the sclera, choroid, choriocapillaris and retinal pigment epithelium to the retina, or indirectily into the retrobulbar space and thence the ONH.

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The movement of fluorescein in the lens and its exchange with the aqueous humor has been experimentally determined and analyzed in terms of a diffusion model,44 but unfortunately the study was not extended to the vitreous body and retina. Another in vitro study2 generally supports this model, but it lacks confirmation from experimentally determined concentration contours. In the absence of a complete diffusional model, the amount of drug can diffuse back to the retina may be sensed by recourse to published data collected over limited experimental periods. However, a quasi-steady state takes days to evolve in the living eye and only two studies have extended over a period of time sufficiently long to be meaningful, and they are lacking in breadth and detail. Apart from fluorescein, I could find only two substances whose lens and vitreous concentrations have been followed over a period of days, namely imirestat, 13 an aldose reductase inhibitor, and timolol. It should be possible from this type of data to estimate whether the level of drug found in the vitreous could be maintained by its rate of leakage from the depot that was originally created in the lens. A rough analysis suggests that this is not the case and that, in the long term, less than a quarter of the vitreous concentration could be derived from the lens depot. Other reservoirs need to be identified, but which are the corresponding structures and how they are originally filled is not clear.

CORNEAL ROUTE

The dynamics of drug penetration from a drop into the anterior chamber of the human and rabbit eye are well documented.56 Because the amount of drug that penetrates does not depend on the volume of the drop, as long as it is above 10 l,62,104 I prefer to express it in terms of the concentration of the drug, not its total mass. The maximum concentration in the aqueous humor occurs from 0.5 to 3 hours after instillation and, in humans, it is about a 150,000 dilution of the drop for a hydrophilic drug such as fluorescein,43 and a 1,500 dilution for a lipophilic drug such as metipranolol.45 A drug can travel back from the anterior chamber through the lens, the margin of the pupil, or the iris, or through the root of the iris by the alternative outflow pathway. Lens Drugs that are lipophilic, but, according to my unsystematic observations, not those that are lipophobic, can partition into the lens from the aqueous humor and then diffuse around the cortex and pass into the vitreous body. In the rabbit, measurable quantities of drug have been found in the lens after the instillation of a single drop.2,13,30,32,103

Pupillary Margin It is generally believed that there is no passage of drug into the posterior chamber around the pupillary margin. This is because there is a flow of aqueous humor in the opposite direction, and the iris and lens may be expected to form a valve, preventing any reflux.87 However, Sherman et al89 found a considerable quantity of fluorescein in the posterior chamber after it had been perfused through the anterior chamber of the monkey eye. Although this observation does not seem to have been confirmed by others, an explanation for the dye’s presence needs to be sought. To start with, it has been noted that the aqueous does not flow continuously into the anterior chamber from all points around the pupillary border, but occurs in bursts of a few l from one point on the perimeter at intervals of several minutes.42,59 This pulsatile flow could be developed by the formation of a seal between the iris and lens, which is opened periodically either by a build-up of pressure in the posterior chamber or by a centrally controlled release in tension of the iris sphincter. It has been suggested, moreover, that under normal conditions there is little need for the lens to be in contact with the iris or to exert a

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force upon it.4 A reflux of fluid from the anterior to the posterior chamber is conceivable while the seal is broken, but this is not readily amenable to calculation. Alternatively, it is possible that the transfer of dye from the anterior to the posterior chamber could be a post-mortem artifact encouraged by intraocular pressure gradients created within the soft eyeball during the manipulations involved in enucleation. Iris The anterior face of the iris is not covered by a cell layer, and drugs dissolved in the aqueous humor can rapidly diffuse up to the epithelium on its posterior surface.89 In one study, the rabbit iris was isolated,16 and its inward-directed permeability to fluorescein was determined to be 1.7  106 cm/ sec. This corresponds to a barrier about double that represented by the retinal pigment epithelium,46 but four times less than that of the human conjunctival epithelium51 and 500 times less than that of the corneal epithelium.43 This value for the permeability of the iris corresponds to a flux across the tissue of roughly 1.5  1010 g/hr, on the assumption that the concentration gradient of fluorescein across the iris is simply that of the concentration of dye in the anterior chamber, 3  108 g/ml at 7 days.44 The concentration of fluorescein in the vitreous body that would be created by this flux was estimated from this figure and the loss coefficient of fluorescein from the vitreous, 0.3/hr,56 and its volume 1.5 ml. This concentration, 3  1010 g/ml, is approximately two magnitudes smaller than the level found experimentally.44 Thus, even when the influence of the iridial vascular system in hindering the progress of the dye is ignored, the iris route appears to be an insignificant component of the backward-directed flux. Alternative Outflow Route It has long been established that much of the aqueous humor flows out of the anterior chamber through the root of the iris and then continues backward in the suprachoroidal space.12 This space is 30 m wide41 and is bounded on one face by the sclera and on the other by the choroid. As the aqueous flows backward, it quickly loses its lower molecular weight components across these boundaries,99 so that their presence cannot be detected much beyond the ciliary body.14 On the other hand, high molecular weight compounds do not escape readily and low levels of radioactive albumin has been shown to be present in the posterior sclera of the human eye after in vivo perfusion through the anterior chamber for several hours.11

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It does not seem likely that any appreciable quantity of a medium-sized drug can be carried back to the posterior circulation of the globe by this mechanism. There might be a small breakthrough into the vitreous near the pars plana where the drug could have less risk of being swept away by the circulation in the choroid and choriocapillaris. Uveal Vasculature Unfortunately, drug exchanges with the dense vasculature of the uvea may complicate the simplified relationships considered above. All regions of the uvea and the retina present this problem and it should be solvable by either of two methods. First, model the anatomy of the vascular system; assign values to the capillary endothelial permeability; and compute the absorption of the layer using a numerical solution. Such techniques have already been developed in engineering but they do not seem to have been applied to the eye. Second, more reliably, drug transfer can be followed in circumscribed regions of the in situ eye over short periods of time, to ensure that only a predetermined barrier is involved in the transfer (see below). CONJUNCTIVAL ROUTE

There is evidence that mechanically blocking off the corneal surface has little effect on drug penetration into the posterior tissues,1,87 which suggests that the conjunctival route is the more important for drug delivery. Sclera The general anatomy of the retinal and uveal tissue seems to be similar over the anterior and posterior segments, at least in humans. In many experiments on delivery across the sclera, drugs have been injected subconjunctivally in order to limit the area over which penetration can occur. However, relatively large volumes of 10 l or more have been injected that raise the subconjunctival pressure and force the depot to spread forward to the limbus and enter the aqueous and anterior vitreous at artefactually high levels. In the rabbit, furthermore, there is a possibility of regurgitation of the drug solution through the injection hole, which leads to a similar error.105 In a recent study,54 these dangers have been overcome by injecting volumes of only 0.2 l; in addition, the eyeballs were frozen immediately after the animal was killed at the end of the experimental period, and freeze-dried and separated into their separate components. For the very lipophobic fluorescent tracer sulforhodamine B, the amount that could be recovered from either the retina or the vitreous was approximately 1/100,000 of the mass in-

DRUG DELIVERY TO THE POSTERIOR SEGMENT FROM DROPS

jected. For the very lipophilic tracer, rhodamine B, these quantities were about 10 times greater. Pars Plana This area is mentioned separately because it has features that might favor the penetration of drugs in this region. It is bathed externally by a high concentration of drug, initially comparable to that in the drop. Furthermore, the retina is missing and illustrations of the vasculature sometimes show it as being less dense than in other areas of the uvea;41 a casual view of scanning electron micrographs of vascular casts does not support this representation,92 however. Orbit In a recent study (Araie, personal communication), monkey (Cynomolgus) eyes were frozen 15 and 60 minutes after the topical instillation of 14C-nipradilol (an autonomic hypotensive agent) and then sectioned so that an autoradiograph of the whole head could be developed that showed the distribution of activity across both eyes and their orbits. These sections allowed some unexpected phenomena to be observed: 1. Even at 15 minutes, heavy accumulations of radioactivity are visible in the orbit of the instilled eye, seemingly lining the orbital wall. 2. The retina/choroid very rapidly gains a notably high level of radioactivity which is similar on the experimental and control sides and that this uptake is roughly uniform over the tissue surface except for holes where it is penetrated by the iris and ONH. There is no smearing of linear structures, such as the retina, in the autographs and the technique used is unlikely to cause contamination of the orbital surfaces. On the other hand, it is to be expected that distortion of the more liquid structures, the vitreous and orbital contents will occur during freezing but this should not affect the main conclusions. The concentration of solutes at the wall of a compartment is an established phenomenon in some cases of relatively slow freezing. The animals were supine and the autoradiographs show the labeled drug in the tear fluid had penetrated into the conjunctival fornices as far as the equator. By probing the fornices of the human eye, Ehlers17 showed that they also extend backward beyond the equator of the globe. Thus, they form potential reservoirs for excess fluid in a drop. Drug dissolved in the fluid could transfer across the bulbar conjunctiva and enter the posterior sclera and orbit; the distance from the fornix to the globe or the orbital contents is only a few mm. and should take

S49

place rapidly. Some support for this mechanism is provided by studies5,23,24,84 that show a considerable concentration of a labeled drug in the posterior sclera shortly after its instillation as an eye drop. Moreover, penetration to the retina from the orbital space is known to occur; soon after the retrobulbar injection of fluorescein in human subjects60 the ONH became brightly stained and a little later the dye is seen to enter the vitreous over the entire retinal surface. The concentration of drug in the tear film beneath the lids after the instillation of a drop has been little studied58 and most of our information comes from the studies of Fraunfelder who used the technique of scintigraphy.21 He showed that if a very small drop is trapped beneath the lids it will generally be squeezed into the interpalpebral space, and if it is placed between the lids it will not cross their margins to enter the fornices. PENETRATION HYPOTHESIS

Although Fraunfelder may have underestimated the concentration in the very thin tear film in the fornices because of the limited sensitivity of the scintigraphy camera, it appears that the penetration of a drop into the cul-de-sac with the head held normally upright would be very restricted and thus the transfer into the posterior sclera and orbit of the drug dissolved in it could be negligible. If, on the other hand, the patient was supine and a large drop was allowed to flood the interpalpebral space, the fluid could fall under gravity and distend the cul-de-sac. In that case, the drug would have the opportunity to penetrate into the posterior sclera and orbit from a concentrated solution for as long as the patient remained in that position. Thus, the penetration from a drop to the posterior segment might be increased many orders of magnitude by the technique of instillation or from pathological conditions such as lax eyelids and give rise to therapeutic effects. It is possible to make an order of magnitude estimate of how much drug can reach the vitreous from a drop in contact with the posterior conjunctiva. Direct experimental results on the penetration of fluorophores as stated unequivocally in two publications are used. The first51 states that the conjunctival permeability to fluorescein in humans is 2.5  105 cm/min. The probably correct assumption is made that the permeability to carboxyfluorescein is the same as to fluorescein, and the more dubious one that the human and rabbit numbers are the same. The further approximation is made that the area of contact between the posterior conjunctiva and the globe is 4 cm2, and the eye drop is assigned a concentration of 0.1 g/ml. Therefore, the total mass of drug entering the globe each minute will be (2.5  105)  (4  101)  105 g.

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Surv Ophthalmol 47 (Supp 1) August 2002

This drug will be assumed to take up a subconjunctival position. The second publication101 concerns the movement of fluorophores from the subconjunctival space to the vitreous. After injecting 20 l of 25% fluorescein or carboxyfluorescein subconjunctivally, near the equator, the vitreous concentration immediately behind the lens was followed non-invasively with a fluorometer. In the case of carboxyfluorescein the vitreous concentration reached a maximum averaging 8  108 g/ml in 7 hours; for fluorescein the recorded level was 1.2  108 g/ml after 4.5 hours, but the results were very variable. Combining the results of both papers, it works out that one minute of exposure of the conjunctival fornices to a 10% drug solution should lead to a maximum vitreous concentration of 1.5  1012 g/ml, 7 hours later. For fluorescein this brings it into the range of therapeutic concentration. Clearly a 10-minute rather than a 1-minute exposure to the drop would correspondingly increase the vitreous concentration. Although these calculations are based on some unverified assumptions, I believe they provide insight into the penetration mechanism, and indicate the direction progress can be made. Similar estimates should be provided for other recently proposed systems that rely on drug transfer across the sclera. The possible central importance of direct entry of drug from the tears across the posterior conjunctiva, though obvious enough, has not been previously recognized. For example, a recent review48 makes no reference to this entry mechanism except in the context of subconjunctival injection, and rejects its possible significance. The role of the conjunctiva has been considered almost exclusively as absorbing a drug from the tears,49,75,102 not as allowing its passage to the globe.

MAURICE 2. 3. 4. 5. 6.

7. 8. 9.

10.

11. 12.

13.

14. 15. 16. 17.

Method of Literature Search The literature up to the 1960s was represented by a collection of reprints in the author’s files, supplemented in some cases by his memory. From 1966 to the present several thousand articles were searched in Medline under broad terms such as pharmacokinetics, ocular, retina, blood flow, and IOP. These assemblages were reduced to groups of manageable size by adding terms such as tears, choroid, drug-binding, and so on. An attempt was made to create sets of only about 100 articles that could more readily sifted in a preliminary examination. Articles in European languages were translated by the author; for Japanese the translator sat beside him.

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MAURICE 94.

Tamaki Y, Araie M, Kawamoto E, et al: Non-contact, twodimensional measurement of tissue circulation in choroid and optic nerve head using laser speckle phenomenon. Exp Eye Res 60:373–83, 1995 95. Tamaki Y, Araie M, Tomita K, et al: Effect of topical betablockers on tissue blood flow in the human optic nerve head. Curr Eye Res 16:1102–10, 1997 96. Tamaki Y, Araie M, Hasegawa T, Nagahara M: Optic nerve head circulation after intraocular pressure reduction achieved by trabeculectomy. Ophthalmology 108:627–32, 2001 97. Tomic L, Maepea O, Sperber GO, Alm A: Comparison of retinal transit times and retinal blood flow: a study in monkeys. Invest Ophthalmol Vis Sci 42:752–5, 2001 98. Tomidokoro A, Araie M, Tamaki Y, Tomita K: In vivo measurement of iridial circulation using laser speckle phenomenon. Invest Ophthalmol Vis Sci 39:364–71, 1998 99. Toris CB, Gregerson DS, Pederson JE: Uveoscleral outflow using different-sized fluorescent tracers in normal and inflamed eyes. Exp Eye Res 45:525–32, 1987 100. Tsuchiya M, Hayasaka S, Mizuno K: Affinity of ocular acidinsoluble melanin for drugs in vitro. Invest Ophthalmol Vis Sci 28:822–5, 1987 101. Tsukahara Y, Maurice DM: Local pressure effects on vitreous kinetics. Exp Eye Res 60:563–73, 1995 102. Urtti A: Delivery of antiglaucoma drugs: ocular vs systemic absorption. J Ocular Pharmacol 10:349–57, 1994 103. Valeri P, Palmery M, Martinelli B, Catanese B: Absorption of bendazac lysine after topical application to the rabbit eye. Pharmacol Res Commun 19:517–25, 1987 104. Whitson JT, Love R, Brown RH, et al: The effect of reduced eyedrop size and eyelid closure on the therapeutic index of phenylephrine. Am J Ophthalmol 115:357–9, 1993 105. Wine NA, Gornall AG, Basu PK: The ocular uptake of subconjunctivally injected C14 hydrocortisone. 1. Time and major route of penetration. Am J Ophthalmol 58:362–6, 1964 106. Wolf S, Werner E, Schulte K, Reim M: Acute effect of metipranolol on the retinal circulation. Br J Ophthalmol 82: 892–6, 1998 107. Yaoeda K, Shirakashi M, Funaki S, et al: Measurement of microcirculation in the optic nerve head by laser speckle flowgraphy and scanning laser Doppler flowmetry. Am J Ophthalmol 129:734–9, 2000 108. Yoshida A, Feke GT, Ogasawara H, et al: Effect of timolol on human retinal, choroidal and optic nerve head circulation. Ophthalmic Res 23:162–70, 1991

Supported by NIH EY00431 and RPB institutional grant. The author has no proprietary or commercial interest in any product or concept discussed in this article. The author is greatly indebted to Dr. T Nagasaki for assistance in all phases of the preparation of this article. Reprint address: David Maurice, PhD, Dept Ophthalmology, Columbia University, 630 West 168th Street, New York, NY 10032; Email: [email protected].