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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5636_Библиотеки_им_академика_М_И_Перельмана
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Fig. 6.3 Photomicrograph showing loss of inner limiting membrane (ILM) integrity above a vein
(v) and artery (a) (adapted from Wolter 1964, with permission)
6.3 The Vitreous As a Drug Reservoir
In ophthalmic drug delivery, the vitreous is employed as a drug reservoir for the
treatment of posterior segment diseases such as age-related macular degeneration (Yoganathan et al. 2006; Parravano et al. 2010; Konstantinidis et al. 2009),
uveitis (Antcliff et al. 2001; Young et al. 2001), cytomegalovirus retinitis (Dhillon
et al. 1998; Guembel et al. 1999) and proliferative diabetic retinopathy (Hornan et al.
2010; Modarres et al. 2009). The relatively large gelled volume and the lack of strong
morphological characteristics tend to suggest that it could be treated as a simple
reservoir, allowing radial diffusion from the point of injection to the retinal tissues.
As the skull largely encloses the posterior eye, introduction of the drug delivery system into the vitreous cavity is usually completed from the anterior aspect. It would
then be expected that drug would freely diffuse throughout the gel quickly, achieving
a uniform equilibrium at all points of retinal contact. This would make both the task
of delivery and the calculation of appropriate dose very simple.
The anterior eye is constantly undergoing fluid turnover and, being closer to the
exterior, is cooler. Thus it is reasonable to expect that convective forces might
operate in the vitreous, especially if evidence of different viscosities could be
found in central and peripheral zones or in the antral and distal regions. In addition,
as we age, the vitreous compartment undergoes liquefaction and collapses, and the
detachment of the posterior vitreous cortex from the retina provides two potential
compartments, following posterior vitreous detachment (PVD). The issue concerning the contribution of flow processes to a non-uniform distribution is therefore a
significant and inconvenient nuisance.
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6.4 Flow Processes in the Vitreous
The movement of drugs around the eye following intra-ocular administration must
occur as a function of several processes. These are grouped into three driving effects:
hydrostatic pressure, diffusional drive and convective flow (Chastain 2003; Moseley
1981). The relative importance of each in affecting clearance after administration
will be reflected in the drug and formulation, the mode of delivery, the physical state
of the vitreous, the size and shape of the eye, the relationship of the depot to the
intra-ocular structures and time. In addition, active transport mechanisms considered
later in this chapter, are important.
6.4.1 Flow Patterns
The turnover of fluid in the rabbit vitreous body was described by Duke-Elder
(1930). He proposed that the supply of liquid to the vitreous came from the ciliary
body and pars planar region, flowing posteriorly through the vitreous to exit near to
the optic nerve head. The observations by Duke-Elder led Fowlkes (1963) to investigate the vitreous flow patterns in the rabbit, using injections of Indian ink and the
highly protein-bound blue dye nitro blue tetrazolium chloride, which forms an
insoluble formazan-labelled protein in situ. The doses were administered starting in
the region of the pars planar moving radially outwards, entering the eye near to the
superior rectus and from the temporal side. The eyes were harvested and sectioned
whilst frozen. Blue formazan stained the retina immediately posterior to the injection. It was observed that when the marker was injected at a shallow depth into the
vitreous humour within 2 mm of the retina, it was swept posteriorly at a rate faster
than diffusion. Fowlkes termed this movement meridonal flow and only occurred in
live eyes. The behaviour was observed to be similar as the injection site was made
radially away from the pars planar.
In the perfused Miyake-Apple preparation, increased movement of particles can
be noted in surface zones although thermal effects may contribute to movement.
This suggests that very short needle injections into the vitreous might access the
posterior pole successfully. In mathematical simulations of flows in the eye, flow
velocities were calculated to reach a maximum around the edge of the vitreous
boundary (Missel 2002). If the viscosity is lower in the peripheral zone between the
can spread underneath the retina. This is illustrated in Fig.
Injection into the body of denser mid-vitreous within an ovine eye, as shown in
Fig. 6.4, demonstrates a more tortuous path for the particles as shown in Fig. 6.6,
probably following the cisternal margins described in previous literature (Jongebloed
and Worst 1987). The appearance of the needle track will change with the structural
and rheological properties of the vitreous humour, with pressure differences contributing to the initial disposition.
6.7.
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Fig. 6.4 Needle track of an injection of 10 mm fluorescent microparticles suspension into ovine
vitreous humour. The suspension was well retained within the central vitreous gel phase after
injection. Note the tortuous path (adapted from Laude et al. 2010, with permission)
Fig. 6.5 Injection of 10 mm fluorescent microparticles suspension into ovine vitreous humour con-
tained within a cuvette. (a) During injection, (b) immediately after injection, (c) 3 h after injection
If the vitreous humour is decanted into a cuvette, the preservation of cisternal
structure is noted, with settling of the particles injected into the humour at the top of
the cuvette occurring under the gravitational forces to outline internal boundaries as
shown in Fig. 6.5.
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Fig. 6.6 The appearance of an intravitreal depot formed in ovine vitreous, viewed in an ovine eye
in vitro
6.4.2 Injection and Hydrostatic Effects
The injection of even small volumes of liquid into an enclosed volume under pressure will cause a transient increase in hydrostatic pressure, sufficient to pose a risk
of reduced retinal blood flow. The movement of suspensions in the eye can be seen
using the Miyake-Apple technique, in which a cover slip is glued to the eye after
removing a small circle of sclera creating a window on the vitreous. Illumination
of the preparation through the lens using a high-intensity blue light emitting diode,
allows the movement of a 10 mm suspension to be followed using a camera. As can
be seen, the introduction of the needle creates a channel and a temporary, lowresistance pathway along the track of the injection path, leading to some reflux of
the material as shown in Fig. 6.6. This may reflect the reflux scenario seen clinically (Benz et al. 2006; Boon et al. 2008). Morlet and Young reported that the mean
IOP immediately following injection of six eyes in four patients with 0.1 mL of
formulation was 44.5 mmHg, a mean rise of 38 mmHg which was significantly
reduced by previous ocular decompression (Mortlet and Young 1993). Application
of pressure to the injection site upon withdrawing the needle has been demonstrated to minimise the reflux of triamcinolone acetonide through the injection
hole. Maurice has shown that injection of fluorescein made via the sclera through
extra-ocular muscle reduces the regurgitation of large injection volume of 100 mL
to 12% in rabbits; however, in this paper the loss in one animal was reported to be
32% (Maurice 1997).
In the study by Boon et al., a significant loss of fluid after injection was reported
which was associated with a restoration of IOP to below 24 mmHg. On a further
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investigation of 13 patients, the authors mixed fluorescein (1% w/v) with the dose
of bevacizumab. Ten patients showed reflux of clear liquid, not stained with fluorescein, whereas one patient had reflux of largely fluorescein-stained liquid (Boon
et al. 2008). This suggests that the non-homogeneity of vitreous humour and the
induced pressure rise will influence the amount and nature of refluxate according to
technique and operator.
Maurice described the consequences of multiple injections on the integrity of the
vitreous. He dosed both eyes with fluorescein such that the manipulations on one
eye could be compared across both eyes, using one as a control. He found that an
injection of the vitreous with a 25G needle, even without introduction of fluid,
caused a temporary change in integrity leading to increased loss of a fluorescein
marker previously injected. Multiple injections at different sites around the rabbit
eye led to even greater losses, which resolved at 48
h (Maurice 1987).
6.4.3 Diffusion
In earlier literature, flow within the vitreous humour was thought to be determined by diffusion alone (Maurice 1957; Moseley et al. 1984). Diffusion can be
defined as a “random molecular motion that leads to complete mixing” and can
be characterised using Fick’s law (Cussler 2009). Passive diffusion is the most
fundamental transport mechanism for small molecules in liquid. It requires a differential gradient to provide motive force (such as osmotic pressure and concentration) towards creation of an equilibrium state at which point, no net diffusion
occurs.
The vitreous may not be a simple, uniform gel as the structure of collagen–
hyaluronan network varies depending on the local abundance and concentration of
the macromolecules. The movement of tritiated water is slower in intact rabbit vitreous than water suggesting that structural elements constituted by the vitreous components impose a diffusional barrier to the transport of even very small molecules
(Foulds et
in the vitreous gel as compared to water (Gisladottir et al. 2009). Unlike small
molecules, which can diffuse freely across the vitreous network, the diffusion activity
of larger molecules appears to be limited by the fibrillar structure of the vitreous
meshwork.
al. 1985). Similarly, the diffusion of dexamethasone is 4–5 times slower
6.4.4 Convective Flow
Convection describes bulk movement in a fluid initiated through an applied
force, for example, due to pressure gradients or temperature differences. In the
vitreous humour, convection is thought to arise through a pressure drop between
the anterior and posterior eye from a steady permeating flow, possibly generated
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by pressure and temperature differences between the anterior chamber and the
surface of the retina.
The impact of both diffusion and convection on flow within the vitreous has
been described by several authors based on experimental data and mathematical
modelling (Fatt 1975, 1977; Xu et al. 2000; Maurice 1987). Fatt used data from a
study of Na+ flux to create a mathematical model of tracer movement within the
vitreous. Using this model, he was able to demonstrate that both diffusion and
convection played a role in the movement of the marker. Although the presence of
convection was noted, convective processes appeared to have less of an impact in
determining tracer distribution, with diffusion having an eightfold greater control
over tracer movement. Using a specially constructed diffusion cell, Fatt concluded
that hydraulic flow conductivity was greater in the bovine vitreous, when compared to the rabbit vitreous (Fatt
1977). The data was used to calculate the effec-
tive channel size through which water flows: the figure for both bovine and rabbit
vitreous was approximately 0.4 mm. More recently, the diffusion coefficient of
acid orange 8 in bovine vitreous and water were shown to differ (3.4 × 10−6 and
6.5 × 10−6, respectively). Using the data obtained, the hydraulic conductivity of
bovine vitreous was determined to be 8.4 ± 4.5 × 10−7 cm2/Pa, suggesting the convection would play a role in the movement of acid orange 8 in bovine vitreous (Xu
et al. 2000).
Dr Paul Missel has created a number of interesting finite element models to
create a 3D representation of hydraulic flow within the eye (Missel 2002), based
on data derived following intravitreal injection of different molecular weight dextrans. When the model was set up to disregard hydraulic flow, the elimination rate
of the high molecular weight dextran (157 kDa) was reduced to below the elimination rate expected for a dextran of this size. No notable effect was seen for the
lower molecular weight dextrans, leading to the conclusion that convection only
appeared to be important for larger molecular weight molecules. Stay and colleagues
reached similar conclusions using model compounds with diffusion coefficients
of 5 × 10−6 and 1 × 10−7, respectively (Stay et al. 2003). Park’s group used a high
diffusivity (1 × 10−5 cm2/s) in their simulation to represent compounds with an
approximate MW of less than 100 Da (Park et al. 2005). When convective flow
was altered by increasing vitreous outflow, little accumulation at the retina was
predicted. An increase in accumulation of only 10% for a highly diffusible small
molecule was noted, suggesting convection would have little influence on the
pharmacokinetic movement of the drug. On the other hand, when using a low diffusivity to represent larger macromolecules with a molecular weight of greater
than 40 kDa, the rate of diffusion was slow and convection appeared to have a
more obvious role, with increased vitreous outflow causing a fourfold increase in
accumulation at the retina after 50 h.
MRI data has been used in a similar manner to investigate the effect of reducing
convective flow on the pharmacokinetic movement of the low molecular weight
drug surrogate Gd-DPTA (Kim et al. 2005). Using the model, it was found that
predicted changes in Gd-DPTA concentrations (MW 590 Da) were insignificant on
switching convective flow on and off.
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6.5 Clearance Pathways from the Vitreous Compartment
It is classically accepted that drug substances administered intravitreally are cleared
either anteriorly to the aqueous chamber or posteriorly to the retina as shown in
Fig. 6.7. As has been mentioned earlier, the meridonal flow described by Fowlkes is
also important but even this is overly simplistic when considering poorly soluble
suspensions which may aggregate following an initial dispersion phase. Following
injection of suspensions of triamcinolone to the rabbit eye, aggregates of drug were
seen by fundus photography on the floor of the posterior chamber at 2–3 days and
up to at least 15 days post-injection (Scholes et al. 1985). In patients who had undergone vitrectomy and had received injections of crystalline cortisol, the material was
observed in the macular region in two patients, wherafter it disappeared at 2 months
with sequalae (Jonas et al. 2000). From these observations, it is clear that settling of
a suspension and subsequent aggregation should result in marked regional differences in distribution of drug over the inner retina.
6.5.1 Charge and Collagen Interaction
Gene delivery to the vitreous offers the prospect of a longer acting and more effective
therapy. Early attempts to utilise complexes of DNA and cationic carriers including
polyethyleneimine, poly-l-lysine and 1,2-Dioleyl-3-trimethyl ammonium-propanebased (DOTAP) liposomal vehicles revealed that vitreous humour decreased the cellular uptake of these vehicles by an retinal pigmented epithelium (RPE) cell line
D407 in cultured cells (Pitkänen et al. 2003). To a lesser extent, this behaviour was
also seen in hyaluronate solutions and it was proposed that the human vitreous would
be a diffusional barrier for cationic DNA complexes. Peeters et al. demonstrated that
intravitreal injections of polystyrene microparticles stick within the mucus, probably
by charge interaction with the collagen fibres of the vitreous (Peeters et
By formulation of the DNA lipoplexes with increasing amounts of distearoyl
al. 2005).
Fig. 6.7 (a) Diffusion towards the posterior and retina (b), forward clearance through the anterior
chamber and (c), meridonal flow as described by Fowlkes
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phosphatidylethanolamine polyethylene glycol (DSPE-PEG) to systems which are
smaller than 500 nm, no binding of the coated lipoplexes to vitreal collagen strands
was observed above a content of 16.7 mol% and a clear relationship between the
DSPE-PEG concentration and aggregation could be observed in the micrographs.
6.5.2 Aqueous Clearance
Friedrich’s group has shown that injection position and volume has significant influence on clearance kinetics of model compounds fluorescein and fluorescein
glucuronide (Friedrich et al. 1997a, b). Different injection sites (including behind
the lens, at the hyaloid membrane, central injection and injection next to the retina)
were found to influence the measured retinal permeability of fluorescein from
1.94 × 10−5 up to 3.50 × 10−5 cm/s. Thus the site of the intravitreal injection of fluorescein is predicted to influence distribution and permeability through the retina. In
addition, it was calculated that the mean concentration remaining in the vitreous at
24 h varied up to a factor of 3.8-fold dependent on initial location of the smaller
volume of 15 mL. It was also shown that increasing the volume from 15 to 100 mL
reduced the magnitude of these changes to approximately 2.5-fold at 24 h (Friedrich
et al. 1997a).
The rapid turnover of aqueous humour in the anterior chamber is the main motive
force for forward clearance. All compounds injected intravitreally can be removed
through this bulk flow system. The majority of materials can effortlessly move
across the hyaloid membrane; the central anterior position of the lens being the
main barrier to this forward movement (Xu et al. 2000; Worst and Los 1995).
Thompson and Glaser showed that the flux of 20 and 70 kDa dextran from the vitreous into the anterior chamber increased significantly after extracapsular lensectomy
with posterior capsulotomy (Thompson and Glaser 1984). In addition, according to
the study performed by Stepanova et al., the transport mechanism present at the lens
epithelium generates uni-directional flow that moves fluids towards the retina rather
than the anterior chamber (Stepanova et al. 2005). Therefore, materials tend to move
around the edge of the lens, instead of diffusing across the highly packed 20 nm
collagen meshwork (Worst and Los 1995). Substances that successfully enter the
anterior chamber are subsequently removed along with aqueous humour by the trabecular and uveoscleral outflow (Cunha-Vaz 1997).
The aqueous drainage at the anterior chamber generates a sustained “sink condition”
for intravitreally administered substances, resulting in the formation of a concentration gradient, originating from the injection pocket, that spreads across the vitreous
cavity. Maurice illustrated a clearance process parallel to the posterior capsule of
the lens with the lowest concentration located at the hyaloid membrane gradually
increasing towards the retina (Araie and Maurice 1991).
Typically, hydrophilic and larger molecules that are not able to exit through the
retina are removed via the anterior route. Atluri and Mitra investigated the vitreal
disposition of short-chain aliphatic alcohols with varying degrees of lipophilicity in
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rabbits using ocular microdialysis techniques. Their findings reveal that methanol
achieved the highest concentration at the anterior chamber, whereas the concentration of the more lipophilic 1-heptanol was undetectable (Atluri and Mitra 2003).
Araie and Maurice have also shown that, as opposed to fluorescein which is smaller
and lipophilic, larger and hydrophilic molecules such as fluorescein glucuronide
and fluorescein isothiocyanate dextran have poor retinal penetration. A steep concentration gradient from the vitreous to the posterior chamber was noted and flux
through the retina was small indicating loss through the aqueous route (Araie and
Maurice
aqueous humour, demonstrated that the anterior elimination pathway is important
for intravitreal clearance of the steroid, triamcinolone (Beer et al. 2003).
inner eye should be problematic for large molecules with low flux which would to
be removed by the aqueous system (Atluri and Mitra 2003; Urtti 2006). A relatively small biopharmaceutical, an oligonucleotide with an approximate molecular
weight of 7 kDa, exhibited rapid anterior clearance following intravitreal injection,
with less than 7% remaining after 7 days (Dvorhik and Marquis 2000). A study of
a larger sized biopharmaceutical, rituximab, in rabbits, suggested that clearance of
rituximab occurred via the aqueous route. It was suggested that Rituximab diffuses
through the vitreous, between the lens and ciliary body, into the anterior chamber
for removal (Kim et al. 2006). Similarly, removal via aqueous humour is thought
to represent the predominant clearance pathway of bevacizumb, following intravitreal administration in man (Krohne et al. 2008). Bakri et al. described the clearance kinetics of intravitreal bevacizumab in Dutch-belted rabbits using a
non-compartmental model and concluded that bevacizumab was cleared through
the anterior pathway with an estimated intravitreal half-life of 4.32 days (Bakri
et al. 2007).
1991). In addition, a small human study utilising samples taken from the
If we exclude transporter effects and phagocytosis, the retinal barrier of the
6.5.3 Retinal Clearance
The posterior elimination pathway has been proposed to be the primary route for
small and lipophilic molecules. Once removed from the retina, materials will be
subsequently transported away by the choroidal blood flow. If melanin binding is
significant, accumulation in the melanocytes of the uveal tract will occur. The RPE
is able to remove material by passive diffusion through the paracellular and/or
transcellular routes. In vitro, the retinal permeability is 8–20 times higher for lipophilic than hydrophilic molecules, suggesting a higher efficiency of the transcellular pathway (Pitkänen et al. 2005).
Inflammation of the RPE, encountered in patients with endophthalmitis, damages retinal pump function thereby decreasing the intravitreal half-life of molecules eliminated by this system (Ficker et al. 1990). The RPE therefore forms an
important component of the blood-retinal barrier and contains retinal glial cells
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6 Principles of Retinal Drug Delivery from Within the Vitreous
Forward clearance Retinal clearance
Site of activity Aqueous chamber Retina
Barrier system Blood–aqueous barrier, lens Blood–retina barrier
“Sink” condition Aqueous humour turnover Choroidal blood flow
Diffusional contour Parallel to the posterior capsule
of the lens
Parallel to the retina surface
Active transport
mechanism
Ciliary epithelium and iris Retinal pigment epithelium and
retinal capillaries
Drug molecule Hydrophilic Lipophilic
Examples Aminoglycosides (Barza et
al.
1983; Cobo and Forster 1981)
b-lactam antibiotics (Barza et
al.
1983)
Fluorescein glucuronide (Araie and
Maurice
1991)
Fluorescein (Araie and Maurice
1991)
Fluorescein dextran (Araie and
Maurice 1991)
1-Heptanol (Atluri and Mitra 2003)
Methanol (Atluri and Mitra 2003) Dexamethasone
Brimonidine
Cu
2+
ions (Bito and Baroody 1987)
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and the endothelium of retinal blood vessels (Cunha-Vaz 1997). An early study by
Mosley (1981) modelled the movement of [
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Xe] xenon from the vitreous through
the retina, using samples taken from the vortex vein and concluded that the radioisotope was cleared from the vitreous, to the retina and into the choroid circulation with a mean transit time of 27 min. A pharmacokinetic model of data from a
rabbit study suggested the antiviral used in the treatment of cytomegalovirus
retinitis, ganciclovir, is eliminated across the retinal surface (Tojo et al. 1999). In
addition, following intravitreal administration of memantine, high concentrations
were found in the choroid and RPE, suggesting posterior elimination (Koeberle
et
al. 2003).
The size of antibody fragments begins to approach nanoparticulate dimensions
and therefore data from nanoparticulate movement might be a useful predictor of
large anti-VEGF agents. Sakurai et al. showed that particles of sizes 200 nm and
below can transverse the retina but 2 mm particles were found to mainly clear
through the trabecular meshwork (Sakurai et al. 2001). Pitkänen et al. have also
reported that the permeability of carboxyfluorescein (376 Da) was 35 times higher
as compared to FITC-dextran 80 kDa (Pitkänen et al. 2005). In contrast, data
obtained by Dias and Mitra showed that FITC-dextran at a molecular weight of
38.9 kDa was predominantly removed from the vitreous through the retina, an
observation attributed to the possible presence of channel-mediated transport mechanism at the retina for large and hydrophilic molecules (Dias and Mitra 2000).
A comparison between the characteristics of forward and retinal clearance is illustrated in Table 6.1.
Table 6.1 Comparison between the forward and retinal routes of clearance
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