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6.6 Transfer Through the Vitreoretinal Border
6.6.1 The Role of the Blood–Retinal Barrier
Passive penetration of substances into the retina is restricted by the blood retinal
barrier (BRB) and this barrier can be divided into the inner and outer BRB. The
inner BRB, positioned at the inner section of the retina, is closest to the vitreous and
is formed by capillary endothelial cells, connected via tight junctions. The outer
BRB consists of the melanin-rich, RPE. Blood is supplied to the initial two-thirds of
the retina, closest to the vitreous, via the inner BRB and the rest is nourished by the
choriocapillaris via the outer BRB.
Capillary endothelial cells surrounding the vessel lie within a network of neurons, astrocytes and Műller cells and control movement between the retina and
blood supply (Gardner et al. 2000). This movement is controlled mainly via
influx and efflux transporters, which ensure the retina receives a rich energy, supply of glucose, lactate and creatine, alongside antioxidants including vitamin C
and cysteine and amino acids; leucine and taurine. Many of these transporters
have low affinity and therefore respond to increased gradients by increasing flux –
for example, lactate transport by monocarboxylate transporters (Hertz and Dienel
2004). Influx and efflux transporters are also responsible for controlling the
movement of organic anions in and out of the retina (Hosoya et al. 2009a, b).
Urtti and colleagues completed an extensive review of the role of transporters in
the eye including those involved into the anterior tissues (Mannermaa et al.
2006). In view of this, we have specifically directed our discussion with regard
to those transporters involved in movement of compounds from the vitreous into
the retina.
6.6.1.1 Amino Acid Transport
The mechanisms of amino acid transport from the vitreous to the retina could have
potential application in drug delivery and are therefore of relevance, although most
observations are limited to cell culture models such as those described by Hosoyo
et al. (2001). In the intact eye, microdialysis has been useful in elucidating the role
of the large neutral amino acid transporter system (LAT) on the retinal uptake of
and also in the presence of known LAT inhibitors. Retinal uptake of l-Phe was
shown to be inhibited following administration of the blockers, demonstrating the
role of LAT in amino acid movement from the vitreous to the retina (Atluri et al.
2008). In addition, amino acid transporter system A is important in the vitreous to
retina movement of proline. The rate of vitreous elimination of proline was shown
to differ from that of the bulk flow marker and was indicative of active transport in
the rat (Yoneyama et al. 2010).
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6.6.1.2 P-Glycoprotein
The multidrug transporter P-glycoprotein (P-gp) is a member of the ATP-binding
cassette (ABC) family of transporters and is involved in various functions including
cell signalling, ion transport, nutrient uptake and efflux of waste compounds.
A number of isoforms of P-gp have been identified including multidrug resistant
1 (MDR1) and multidrug resistant 2 (MDR2), shown to be expressed in human cell
lines (Hennessy and Spiers 2007).
Earlier ocular studies examined the role of the MDR pump on the uptake of fluorescein sodium (FS) and benzcylamine phenyl sulfonylglcine (BAPSG) into human RPE
cells. BAPSG is a selective aldosterone reductase inhibitor, considered for its potential
role in the treatment of diabetic retinopathy. Indomethacin, verapamil and probenecid
were co-administered with FS and BAPSG as known MDR inhibitors. Both indomethacin and probenecid were shown to increase FS accumulation in cells, with no significant
effect demonstrated by verapamil. BAPSG accumulation also increased in the presence
of indomethacin and probenecid but also verapamil when the concentration of inhibitor
was increased to 10
mm. The efflux of BAPSG from RPE cells was also shown to be
significantly higher when the inhibitors were not present (Aukunuru et al. 2001).
Using various RPE cell lines the penetration of rhodamine 123, a known P-gp substrate, into cells has been investigated. In one cell line, the presence of the P-gp inhibitor,
verapamil, resulted in an increased uptake of rhodamine 123 by approximately 13-fold.
Little effect was noted on the other cell lines studies, reportedly due to poor expression
of P-gp (Constable et al. 2006). The appreciation of the importance of P-gp has prompted
investigations of the inner blood–retinal-barrier cell line (TR-iBRB) transfected with
P-gp. Uptake studies of rhodamine 123 were performed in the presence and absence of
a series of test compounds; AGN 194716, AGN 195127, AGN 197075, acebutolol,
alprenolol, atenolol, brimonidine, carbamazepine epoxide (CBZ-E), metoprolol, nadolol
and sotalol, to identify potential P-gp inhibitors. Using TR-iBRB cell lines, rhodamine
123 uptake identified only AGN 197075 as an inhibitor of P-gp-mediated efflux of rhodamine 123 from compounds studied (Shen et al. 2003).
Steuer et al. described both P-gp and multidrug resistant protein (MRP) expression
in the outer BRB in the pig. Verapamil and rhodamine 123 were applied to both the
choroid and retina independently and a higher cell permeability of both compounds
into cells was demonstrated when the compounds were applied to the retinal side of the
outer BRB. Increases in permeability were 3.5-fold for verapamil and 2.6-fold for rhodamine, leading to the conclusion that P-gp expression in the outer BRB must be
greater at the choroidal side. The authors also investigated the penetration of FS from
retina to choroid in the presence and absence of the MRP inhibitor, probenecid. FS
permeability increased 11-fold following addition of the inhibitor (Steuer et
al. 2005).
6.6.1.3 Organic Cationic Transporters
Organic cationic transporters (Oct) are members of the solute carrier transporter
gene family and are involved in the transport of small organic cations and hydrophilic
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compounds (Kusuhara and Sugiyama 2004). Known substrates of Oct include
tetraethylammonium and monoamine neurotransmitters (Tsuji 2005). The Oct has
been shown to be expressed in mouse RPE (Rajan et al. 2000) and its involvement
in retinal drug transport demonstrated (Han et al. 2001). Using human RPE cell
lines, RPE/Hu and ARPE-19, the uptake of verapamil into RPE was shown to be a
saturable process, with an apparent Km equal to 7.2 mM. The rate of verapamil
uptake decreased in the presence of metabolic inhibitors, when the temperature was
reduced, and in the presence of some organic cations: including quinidine, pyrilamine, quinacrine and diphenhydramine. Cationic drugs – diltiazem, timolol and
propranolol – commonly used in the treatment of glaucoma also inhibited uptake.
However, no change in uptake rate was seen in the presence of other organic cations,
including tetraethylammonium and cimetidine, revealing the expression of a new
Oct subtype.
6.6.1.4
Organic Anion Transporters
Organic anion transporters (Oat) are also members of a family of solute carrier transporters and involved in energy-independent efflux transport (Brasnjevic et al. 2009).
Using polymerase chain reaction (PCR), rOat3 (rat organic anion transporter) was
found to be expressed in the retina and retinal endothelial cells of rats. In the intact eye,
a series of radio-labelled drug candidates were administered via intravitreal injection
and concentration changes measured by microdialysis. P-aminohippuric acid, benzylpenicillin and 6-mercaptopurine showed a biexponential elimination pattern from the
vitreous. The elimination rate of all three was reduced in the presence of rOat3 inhibitor, probenecid, demonstrating the role of rOat3 in drug efflux (Hosoya et al. 2001).
Oat involvement in transport was also shown for [3H]-estradiol 17-beta glucuronide
([3H]E17bG). The study was performed in rats using microdialysis with co-administered
[14C]d-mannitol as a marker of bulk flow movement. Removal of both compounds from
the vitreous followed a biexponential pattern. In the initial phases of drug elimination
from the vitreous, the elimination rate was similar for both compounds. However, the
second phase of decline differed, with elimination of [3H]E17bG shown to be significantly greater than that of [14C]d-mannitol, with elimination constants of 9.0 × 10−3/min
for [3H]E17bG and 5.0 × 10−3/min for [14C]d-mannitol. The author suggested that the
first phase represents the drug diffusing across the whole of the vitreous whereas the
second phase, the true rate of drug elimination out of the vitreous. In the presence of
probenecid, the rate of elimination of [3H]E17bG was reduced similar fluxes to that of
[14C]d-mannitol, leading to the conclusion that [3H]E17bG undergoes efflux transport
via a probenecid-sensitive organic anion transport process, likely to take place at the
BRB (Hosoya et al. 2003).
6.6.1.5 Other Transporters
In the investigation of fluorescein and fluorescein monoglucuronide elimination from
rabbit vitreous, an elimination rate for fluorescein was calculated to be 0.22 ± 0.03/h
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and that for fluorescein monoglucuronide 0.07 ± 0.01/h. After addition of probenecid,
the rate of fluorescein loss was reduced to 0.13/h with less effect on the rate of clearance of metabolite, fluorescein monoglucuronide. Since fluorescein is eliminated via
the posterior route, a probenecid-sensitive transport process was suggested to be
involved in fluorescein transport from vitreous to retina (Kitano and Nagataki 1986)
which could be related to Oat and Oct transporters discussed previously.
The involvement of an oligopeptide transporter system on the distribution of a
model labelled peptide [3H] glycylsarcosine has been demonstrated. At steady state,
the area under the curve (AUC) detailing the penetration rate of the peptide from
vitreous to plasma was shown to be 1.61
peptide transporter substrates, glucylproline, carnosine and captopril, the uptake of
the model peptide was inhibited. In addition, when non-peptide transporter substrates were added, no effect on uptake rate was noted (Atluri et al. 2003).
P2Y2 is a G protein coupled receptor known to have involvement in transfer of
extracellular nucleotides. The impact of this transporter system on drug clearance was
investigated using P2Y2 receptor agonists, UTP and INS542, administered by intravitreal injection to rabbits, together with fluorescein. UTP had no effect on fluorescein
levels; leading the author to conclude that it was likely that UTP was degraded in the
vitreous before exerting an effect on the receptor. INS542, on the other hand, significantly reduced fluorescein to metabolite fluorescein glucuronide ratios in the vitreous,
when compared to eyes dosed with phosphate-buffered saline instead of agonist.
Therefore, a larger proportion of the administered fluorescein was transferred out of
the vitreous into the retina after administration of the agonist. This increase in transport is likely due to P2Y2 receptor activation (Takahashi et al. 2004).
A retinal transporter has also been suggested to be important for the elimination
of anti-VEGF agent bevacizumab although the specific mechanisms involved has
not been elucidated (Heiduschka et al. 2007).
From the studies described, it is apparent that drug transporters will play a key
role in intravitreal drug delivery. Understanding the extent of the effect of specific
transporter subgroups on specific ocular treatments could markedly improve drug
targeting to the retina, improving therapeutic options and disease prognosis.
± 0.49 nmol min/mL. After the addition of
6.7 The Ageing Vitreous
6.7.1 Underlying Mechanisms of Vitreous Degeneration
With age, the vitreous humour undergoes progressive structural and biochemical
changes (Sebag 1998; Bishop 2000; Ciferri and Magnasco 2007). Neither the vitreous
humour nor the inner limiting membrane undergo renewal in later life and do not regenerate after vitrectomy. Halfter et al. describe the detachment of the vitreous body from
the inner limiting membrane as consequences of the low synthetic rate and deterioration (Halfter et al. 2005). These processes are usually associated with vitreous syneresis
(contraction) and synchisis (liquefaction), the rate of occurrence increasing with age.
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The mechanism by which vitreous degeneration happens remains uncertain,
although a photochemical reaction has been proposed to be a potential underlying
cause (Ueno et al. 1987; Akiba et al. 1994). In the studies, the authors demonstrated that visible light excites riboflavin to generate radicals and oxygen species
including the superoxide anion, hydrogen peroxide, hydroxyl radicals and singlet
anions. These components are known to account for the degradation of hyaluronan and cross-linking of calf collagen in vitro (Akiba et al. 1994; Kakeshi et al.
1994) and in vivo (Ueno et al. 1987). Riboflavin is a photosensitiser that exists
naturally in the vitreous environment; the authors proposed that light exposure
could be the mechanism underlying the occurrence of age-related vitreous degeneration. Another hypothesis is based on the work by Akiba et
showed that whole serum and a combination of serum protein (transglutaminase)
and fibronectin promotes collagen cross-linking leading to vitreous gel contraction. The leakage of plasma proteins from the intravascular space into the vitreous
body is possible due to vascular incompetence associated with ageing retinal and
ciliary body vasculature. As a result, the concentration of soluble proteins present
in the vitreous increases from approximately 0.5–0.6 mg/mL at ages 13–50 to
0.7–0.9 mg/mL at ages 50–80 and 1.0 mg/mL above 80 years (Sebag 1989).
In addition, age-related increase in proteolytic activities within the vitreous may
also be a contributing factor to vitreous liquefaction (Thomas et al. 2000). The
concentration of plasmin, a proteolytic enzyme in the vitreous, increases with age,
possibly caused by tissue degeneration such as the retina. In the vitreous, plasmin
may combine with membrane type matrix metalloproteinase-1 (MMP-1) to activate progelatinase-A (proMMP-2), which has been documented to have the capability to cleave off hybrid type of V/XI collagen and liquefying the vitreous gel
in vitro (Brown et al. 1996).
al. (1995), who
6.7.2 Physical Changes Involved in the Ageing Vitreous
Balaz and Denlinger established the progression of human vitreous liquefaction in
post-mortem biopsy tests performed on 610 human eyes aged between 5 and 90
years (Balazs and Denlinger 1982). The volume of vitreous gel and liquid phase
were measured and related to the age of the eye. It was found that the vitreous of
young human adults of around 20 years of age was 80% gel phase, which decreased
to almost 50% beyond 60 years. The decrease in gel volume was accompanied by a
parallel increase in liquid volume as illustrated in Fig. 6.8.
Sebag and Balaz illustrated the changes on maturation using dark-field microscopy
illuminated with a slit lamp (Sebag 1987, 2005). Human vitreous from donors, aged
53–88 years, were dissected from the sclera, choroid and retina with the anterior segment remained attached. The non-fixed vitreous was mounted in a transparent chamber containing isotonic saline and sucrose (3.5 g/L) and trans-illuminated. Bundles of
parallel and thick fibres coursing along the anterior–posterior direction as well as
areas of liquid pockets were seen in the vitreous of a middle-aged man (Fig. 6.9).
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Fig. 6.8 Age-related changes in the volume of the vitreous gel and liquid phase. The sustained
onset of liquefaction throughout life is evident (adapted from Balazs and Denlinger 1982)
Fig. 6.9 Vitreous structural changes with age. (a) Vitreous humour of a 6 year-old child (4×)
(adapted from Sebag 1987, with permission); (b) vitreous humour of a 59-year-old adult (8.3×);
(c) vitreous humour of a 88-year-old adult (2.7×). The lower part of the image indicates the position of the lens (adapted from Sebag
Ophthalmological Society)
These structures become more prominent in an older person (80–90 years) where
fibres were no longer parallel and linear in shape but rather tortuous and broken.
Additionally, enlarged liquid pockets in areas devoid of collagen fibres were seen at
the central and peripheral areas of the vitreous. This suggests that disruption of the
fibrous structure and advanced liquefaction leads to eventual collapse of the whole
vitreous; observations which can be explained by changes to the organisation of the
vitreous components. Chondroitin sulphate, hyaluronan and opticin, which previously
filled the space in between fibrils, are dissociated from the collagen fibrils leading to
its lateral aggregation into bundles of fibres. As a result, areas devoid of collagen
2005, republished with permission of the American
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fibrils are filled with liquid vitreous containing depolymerised hyaluronan and other
soluble substances (Bishop 2000). As opposed to a juvenile vitreous, the elderly vitre-
ous is more fibrous in appearance and smaller as a result of syneresis (Fig. 6.9).
6.7.2.1 Pre-Clinical Model of Ageing Vitreous
Despite the wide recognition of vitreous degeneration with age, pre-clinical drug
development for retinal therapeutics is generally conducted in young laboratory animals with an intact vitreous structure. Studies have demonstrated that the percentage vitreous gel content of the laboratory Dutch-belted rabbits (3 months to 2 years
old) was ~20% (Tan et al. 2011), a measurement similar to that established by Balaz
and Denlinger for young human adults (Balazs and Denlinger 1982). This suggests
that laboratory rabbit is a relevant model for younger populations but may not be
representative of the elderly eye.
Based on the fact that an elderly model will be useful for ocular disposition studies,
our group has established a rabbit model with partial vitreous liquefaction using
ovine testicular hyaluronidase. The generated degree of vitreous liquefaction was
representative to that seen in the elderly of age around 60 years. The enzyme-induced
liquefaction approach was demonstrated to be reproducible without gross ocular tissue
changes observed using fundus examination. The model was successfully utilised in
assessing intravitreal drug disposition of different molecular weight fluorescent compounds of which results will be discussed in next section (Sect. 6.7.2.2).
6.7.2.2 Effects of Vitreous Liquefaction on Intravitreal Drug Delivery
The effects of vitreous liquefaction have been evaluated on the distribution kinetics of
sodium fluorescein, fluorescein dextran (FD) 150 kDa and 1 mm fluorescent particles
(Tan et al. 2011). In the study, it was found that sodium fluorescein (MW ~ 376 Da)
and FD 150 kDa were distributed and cleared faster from the partially liquefied vitreous as compared to normal vitreous as illustrated in Figs. 6.10 and 6.11, respectively.
The faster rate of clearance in the liquefied vitreous suggested that the capability of
the vitreous in retaining small and large molecules has significantly reduced and
injected substances were expected to have a shorter intravitreal half-life. Nevertheless,
ocular fluorophotometry data revealed similar gradient pattern of fluorescent probes
along the optical axis in both normal and liquefied vitreous. This shows that although
the rate of clearance has accelerated, the elimination pathway by which molecules
were cleared was not affected by the vitreous state. In case of microparticles, distribution was found to be more dispersed in the liquefied eye and a faster rate of particle
sedimentation was observed as shown in Fig. 6.12. Findings based on these data led
us to conclude that vitreous diffusivity and convective forces were enhanced in the
partially liquefied vitreous leading to a faster rate of drug clearance.
Clinically, the increased vitreous diffusivity in the liquefied vitreous has been
illustrated by Moldow et al. using a fluorescein profile of a 52-year-old patient.
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Fig. 6.10 HRA images showing the distribution of sodium fluorescein at 2 and 5 h following
intravitreal injection. After 5 h, the fluorescent mass remained well retained in the normal vitreous
but appeared to be more diffuse in the liquefied vitreous, suggesting increased vitreous diffusivity
(adapted from Tan et al. 2011, with permission)
Fig. 6.11 HRA wide-angle images of intravitreally injected fluorescein dextran 150 kDa in the
normal and liquefied vitreous models from 1 h to day 30 after injection. The amount of FD 150 kDa
remained in the vitreous was lower in the liquefied vitreous as compared to normal from day 6
onwards, suggesting that FD 150
environment (adapted from Tan et al. 2011, with permission)
The diagnosis was retinitis pigmentosa with vitreous liquefaction or detachment
(Moldow et al. 1998). The lack of diffusional gradient across the vitreous cavity
observed 30 min after injection into a superficial arm vein could partly be attributed
to lower vitreous diffusivity. Additionally, Spielberg and Leys have reported that
older patients (mean age: 68.5 years) treated with intravitreal bevacizumab for myopic
kDa has a shorter intravitreal half-life in a more liquefied vitreous
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Fig. 6.12 HRA images showing the distribution kinetics of intravitreally injected 1 mm particle
suspension 1 h until day 30 after injection. The upper part of the image represents the superior area
of the rabbit vitreous. Microparticles remained relatively in place at the injection site (superiortemporal) in both models for at least 5 h post-injection, however, particle sedimentation occurred
at a faster rate in the liquefied vitreous model (adapted from Tan et al. 2011, with permission)
choroidal neovascularisation required more frequent dosing, with an average of
3.75 injections as compared to 1.75 injections in the younger aged patients (mean
age: 39.5 years), for a similar degree of visual improvement (Spielberg and Leys
2009). The more frequent dosing observed in the elderly could be attributed to the
faster rate of drug clearance associated with the liquefied vitreous humour. More
importantly, the clinical study has the crucial implication that treating patients of all
age groups with a standard dosing regimen is inappropriate and might result in subtherapeutic efficacy.
6.7.3 Vitrectomised Eyes
Vitrectomy is a commonly used technique for conditions such as rhegmatogeneous
retinal detachment (Lai et al. 2008; Nakin et al. 1992), macular hole (Shimada et al.
2009) and vitreoretinopathies (Park et al. 2010). The primary aim of the surgical
procedure is to relieve the tractional forces exerted by the degenerating vitreous on
the retina, before cellular remodelling occurs (Mura et al. 2009). In some cases,
post-operative endophthalmitis and hypotony may develop depending on the surgical
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techniques used, with higher risks reported with smaller gauge (25G) vitrectors
(Kunimoto et al. 2007; Shimada et al. 2008) and sutureless vitrectomy (Acar et al.
2008), respectively. When the vitreous is removed, it is replaced by intravitreal gas
(Ruby et al. 1999), silicone oil (Tognetto et al. 2005) or air (Poliner and Schoch
1987; Itakura et al. 2009), and the decision will be based on treatment modalities.
Itakura and colleagues examined the concentration of hyaluronan in the fluid
samples during fluid–air exchange from patients with macular hole and diabetic
retinopathy after vitrectomy and discovered a significant lower amount of high
molecular weight hyaluronan in the replacement fluid, which led the authors to the
conclusion that hyaluronan is no longer produced in the vitreous after surgery. This
observation is due to two possible reasons: (1) loss of hyalocytes to secrete vitreous
hyaluronan and (2) loss of vitreous collagen meshwork serving as a scaffold for the
assembly of high molecular weight hyaluronan. Therefore, the vitreous can no
longer be reformed once it is removed. This will have a considerable impact on the
overall functions of the surrounding tissues, which have been discussed in the recent
reviews by Stefánsson (
2009) and Holekamp (2010).
6.7.3.1 Intravitreal Drug Distribution and Clearance in Silicone Oil
Silicone oil used in surgical vitrectomy stays within the vitreous cavity a few months
as a tamponade to facilitate retinal reattachment using the physicochemical properties – low density and interfacial tension – to work against the subretinal fluid
(Giordano and Refojo 1998). The duration for which silicone oil is left in the eye
depends on individual patient prognosis; however, prolonged residence is not recommended as it can result in long-term ocular complications including glaucoma,
cataracts and post-operative keratopathy (Falkner et al. 2001; Tiedel et al. 1990).
Nevertheless, silicone oil is increasingly utilised as a drug vehicle during the tamponade period, mainly for antiproliferative agents to simultaneously treat underlying problems such as iris neovascularisation (Singh and Stewart
2008) and
proliferative vitreoretinopathy (Ahmadieh et al. 2008). This clinical application has
led to considerable interest in understanding drug kinetics, safety and other pharmaceutical issues such as the solubility of the agent in silicone oil.
An early clinical study in 1980s observed patients with silicone-fluid filled eyes
presenting with lower incidence of sight-threatening neovascular glaucoma. This
led to an experimental hypothesis that silicone oil behaves as a diffusional or convective barrier to oxygen transport from the anterior chamber (de Juan et al. 1986).
In order to test the hypothesis, de Juan and coworkers compared the oxygen pressure (PO
) at the anterior chamber between one eye with lensectomy-vitrectomy
2
and the contra-lateral eye that went through the same surgical procedures but had
replacement with silicone oil. The result showed significantly higher anterior chamber PO
in silicone oil treated eyes as compared to the fellow eye leading the authors
2
to confirm their earlier hypothesis which suggested that silicon oil may protect the
anterior segment from the occurrence of neovascularisation. Coincident with this
observation, McLeod reported in another study where patients with complete silicone
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