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Safety Evaluation of Ocular Drug Delivery Formulations: Techniques and Practical Considerations
Brian G. Short
Toxicol Pathol 2008 36: 49
DOI: 10.1177/0192623307310955
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Toxicologic Pathology, 36:49-62, 2008
Copyright © 2008 by Society of Toxicologic Pathology
ISSN: 0192-6233 print / 1533-1601 online
DOI: 10.1177/0192623307310955
Safety Evaluation of Ocular Drug Delivery Formulations:
Techniques and Practical Considerations
BRIAN G. SHORT
From Allergan, Inc., Irvine, California, USA.
ABSTRACT
Development of new drug candidates and novel delivery techniques for treatment of ocular diseases has recently accelerated. Treatment of anteriorsegment diseases has witnessed advances in prodrug formulations and permeability enhancers. Intravitreal, subconjunctival, and periocular routes of
administration and sustained-release formulations of nanoparticles and microparticles, as well as nonbiodegradable and biodegradable implants to
deliver drugs to the posterior segment of the eye, are becoming popular therapeutic approaches. Without adequate regulatory guidance for ocular
drugs, such routes of administration and novel formulations can pose unique challenges to those involved in designing nonclinical programs, including
considering clinical and nonclinical factors and choosing species, strains, and ocular toxicity parameters. Toxicologic pathologists also contribute
practical experience to evaluating morphological effects of these novel formulations. Lastly, understanding species’ anatomical differences is useful
for interpreting toxicological and pathological responses to the eye and is important for human risk assessment of these important new therapies for
ocular diseases.
Keywords: Ocular drug delivery; intravitreal; subconjunctival; periocular; ocular implant.
INTRODUCTION
TABLE 1.—Leading causes of visual impairment
and ocular discomfort.
Millions of people suffer from a wide variety of ocular
diseases, many of which lead to visual impairment and ocular
blindness and cost the federal government approximately $4
billion annually (Clark and Yorio, 2003). Certain ocular diseases
are quite rare, whereas others, such as cataracts, age-related
macular degeneration (AMD), and glaucoma, are very common,
especially in the aging population (Table 1). A rapid expansion
of new technologies in ocular drug delivery and new drug candidates, including biologics, to treat these challenging diseases
in the anterior and posterior segments of the eye have recently
emerged. These approaches are necessary because the eye has
many unique barriers to drug delivery (Figure 1). Current routes
of administration include but are not limited to topical administration, systemic administration, intravitreal injections, and
intraocular implants, each of which has its own set of complications and disadvantages (Figure 2). Ocular bioavailability
after topical ocular eyedrop administration, the most common
form of ocular medication, is less than 5% and often less than
1%, and therefore, only the diseases of the anterior segment
of the eye can be treated with eyedrops. Blood-ocular barriers,
Disease
Cataract
Age-related macular
degeneration
Glaucoma
Diabetic retinopathy
Dry eye
Ocular allergy
Retinitis pigmentosa
Number of Patients
6–19% of patients older than 43 years
11–28% of patients older than 65 years
1–4% of patients older than 45 years
71–90% of diabetics older than 10 years
50–60 million (10–15% of U.S. population)
~25% of U.S. population
1 in 3000–5000
Reproduced by permission from Clark, A. F, and Yorio, T. (2003). Nat Rev Drug Discov
2, 448-59. © 2003 by Macmillan Publishers.
including tight junctional complexes between ciliary and retinal
pigmented epithelium, nonfenestrate and iridal capillaries, and
P-glycoprotein efflux pumps, are defense mechanisms to protect
the eye from circulating antigens, inflammatory mediators, and
pathogens. Unfortunately, they also act as a considerable barrier
to systemically administered drugs (Davis et al., 2004).
Ocular delivery from intravitreal, subconjunctival, and periocular sites to the posterior segment is becoming a popular
approach to support the development of new injectable and
implantable prolonged-action dosage forms (Figure 3).
Regulatory expectations for nonclinical testing of ocular
drugs are not well defined, and regional differences exist. Many
toxicologists and pathologists new to this field are responsible
for developing novel ocular drugs or novel delivery techniques
using an existing ocular or systemic drug. The objective of this
review is to briefly summarize some of the newer methods of
administering ocular drugs; to provide a spectrum of toxicological and pathological viewpoints for nonclinical development,
Address correspondence to: Brian G. Short, Allergan, Inc., 2525 Dupont
Dr. RD-2A, Irvine, CA 92612.
Abbreviations: AMD, age-related macular degeneration; CNTF, ciliary neurotrophic factor; DDS, drug delivery system; ECT, encapsulated cell technology;
ERG, electroretinogram; EVA, ethylene vinyl acetate; FIHS, first in human
studies; FDA, Food and Drug Administration; ICH, International Conference
on Harmonization; IOP, intraocular pressure; ISO, International Standards
Organization; ITV, intravitreal; NCE, new chemical entity; NZW, New Zealand
White; OECD, Organisation for European Cooperation and Development; PLA,
poly-lactic acid; PLGA, poly-lactic-glycolic acid; PVA, polyvinyl alcohol;
RTK, receptor tyrosine kinase; TA, triamcinolone acetonide; TLRs, toll-like
receptors; VEGF, vascular endothelial growth factor.
49
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TOXICOLOGIC PATHOLOGY
FIGURE 1.—Barriers to intraocular delivery. The cornea, tear drainage, episcleral blood flow, and intraocular convection limit the
influx of locally administered drugs into the posterior segment of the eye. Macromolecules as large as antibodies are unlikely to
penetrate the internal limiting membrane. A systemic approach should overcome the problem of the blood retinal barriers.
Reproduced by permission from Yasukawa et al. (2006). Expert Opin Drug Deliv 3, 261-273. © 2006 by Ashley Publications.
FIGURE 2.—Disadvantages and complications associated with ocular drug delivery. Reproduced by permission from Davis et al.
(2004). Curr Opin Mol Therap 6, 195-205. © 2004 Thomson Corporation.
including regulatory considerations, species selection, study
design, morphological evaluation, and relationship of pathology data to functional endpoints; and to ensure a comprehensive
and meaningful risk assessment for humans.
NOVEL OCULAR DRUG DELIVERY FORMULATIONS
A detailed summary of the rapidly expanding progress and
catalog of drugs used with these approaches is beyond the scope
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FIGURE 3.—Sites and methods for ocular drug delivery to the eye. Various methods for delivery of drugs to the anterior and
posterior chambers of the eye are illustrated, including sites for conventional drug delivery as well as newer routes. Reproduced
by permission from Davis et al. (2004). Curr Opin Mol Therap 6, 195-205. © 2004 Thomson Corporation.
of this review but is provided elsewhere (Davis et al., 2004;
Bourges et al., 2006; Ghate and Edelhauser, 2006; Yasukawa
et al., 2006; Hsu, 2007) and summarized briefly below with
clinical examples of therapeutic indication. Emphasis is placed
on discussion of nonconventional formulations (i.e. nontopical)
for posterior-segment diseases.
Prodrug Formulations and Permeability Enhancers
Prodrug formulations use pharmacologically inactive derivatives of drug molecules that are better able to penetrate the
cornea (e.g., they are more lipophilic) than the standard formulation of the drug (Davis et al., 2004). Within the cornea or
after corneal penetration, the prodrug is either chemically or
enzymatically metabolized to the active parent compound.
Enzyme systems identified in ocular tissues include esterases,
ketone reductase, and steroid 6β-hydroxylase. Most prodrugs
are delivered conventionally by topical application such as antiviral prodrugs ganciclovir and acyclovir, although ganciclovir
has also been delivered intravitreally by injection or as a nonbiodegradable reservoir (see below). Delivery of a drug with a
nonnatural enzyme system in the cornea has been achieved
with topical 5-flurocytosine, a prodrug of 5-fluorouracil, administered after subconjunctival transplantation of cells containing
the converting enzyme cytosine deaminase.
Increased corneal penetration into the anterior segments
can be achieved with the addition of permeability enhancers
to the drug formulation (Davis et al., 2004). Surfactants, bile
acids, chelating agents, and preservatives have all been used.
Cyclodextrins, cylindrical oligonucleotides with a hydrophilic
outer surface and a lipophilic inner surface that form complexes
with lipophilic drugs, are among the more popular permeability
enhancers. They increase chemical stability and bioavailability
and decrease local irritation, and they have been used with
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corticosteroids, choloramphenicol, diclofenac, cyclosporine, and
sulfonamide carbonic anhydrase inhibitors.
Injectable Therapies
The antisense oligonucleotide Fomivirsen (Vitravene),
which delays cytomegalovirus retinitis in patients with AIDS,
was the first biologic approved for intravitreal injection and the
first antisense oligonucleotide approved for clinical use (Perry
and Balfour, 1999). Clinical intravitreal injection of vascular
endothelial growth factor (VEGF)–specific inhibitors that
bind VEGF-A, such as the aptamer pegaptanib (Macugen; Ng
and Adamis, 2006) and the antibody fragment ranibizumab
(Lucentis; Ferrara et al., 2006), attest to the therapeutic usefulness of blocking the VEGF pathway in human AMD following
this route of delivery, either alone or in combination with
standard-of-care photocoagulation and photodynamic therapies. Bevacizumab (Avastin), a whole humanized mouse antibody that binds to VEGF and was developed for intravenous
treatment for metastatic colorectal cancer, is currently being
used intravitreally off-label for treatment of AMD, although
the intraocular safety profile remains unknown (Morris et al.,
2007).
Although treatment of the eye with antibodies, aptamers,
and oligonucleotides is relatively new, intravitreal injection is
not; this method has been used for decades to deliver a variety
of drugs including steroids for treating ocular inflammation and
AMD. Intravitreal administration of triamcinolone acetonide
(TA) has been widely used in ophthalmology for decades for the
treatment of diabetic retinopathy, uveitis, pseudophakic cystoid
macular edema, choroidal neovascularization associated with
AMD, and macular edema associated with central retinal vein
occlusion (Davis et al., 2004). Kenaog-40 (Bristol-Myers Co.)
is the most commonly used formulation for off-label intravitreal
use. Unfortunately, there have been reports of sterile endophthalmitis and vision loss, thought to be related to the preservative and/or dispersion agent. Several clinical trials sponsored
by the National Eye Institute are developing preservative-free
formulations.
Injectable therapies are also given by periocular routes
including subconjunctival, retrobulbar, peribulbar, and posterior
sub-Tenon injections (Ghate and Edelhauser, 2006). The subconjunctival route is an attempt to minimize dosing frequency
while maintaining a sustained drug delivery to the anterior and
posterior segment during a prolonged period of time. Hydrophilic
drugs, which penetrate through the sclera, are more effective
when given by the subconjunctival route, because they do not
have to penetrate the conjunctival epithelium. The angiostatic
steroid anecortave (Retaane) is injected as a depot formulation
via a specialized cannula in a posterior juxtascleral position
(sub-Tenon’s space) and is being investigated as a treatment
for AMD (Davis et al., 2004). One problem with this route of
administration is reflux of the drug from Tenon space, so further
work is currently being carried out to address this problem
(Morris et al., 2007).
TOXICOLOGIC PATHOLOGY
Particulate Drug Delivery Systems:
Nanoparticles and Microparticles
Particulate ocular drug delivery systems include nanoparticles (1 to 1,000 nm) and microparticles (1 to 1,000 µm), which
are further categorized as nanospheres and microspheres
and nanocapsules and microcapsules (Bourges et al., 2006).
Nanospheres and microspheres are a polymer–drug combination in which the drug is homogenously dispersed in the polymeric matrix. In nanocapsules and microcapsules, the drug
particles or droplets are entrapped in a polymeric membrane.
Particulate systems have the advantage of intraocular delivery
by injection, and their size and polymer composition influence
markedly their biological behavior in vivo. Poly-lactic acid
(PLA) microspheres remain in the vitreous for 1.5 months in
normal rabbit eyes and 2 weeks in vitrectomized eyes; therefore,
microparticles act like a reservoir after intravitreal injection but
may have a shorter half-life in human vitrectomized eyes (Hsu,
2007). Nanoparticles, on the other hand, diffuse rapidly and are
internalized in ocular tissues and cells of the anterior and posterior segment. Intravitreous injection of polylactides results in
transretinal movement in rats, with preferential localization
in the retinal pigment epithelium cells for 4 months after a single
injection (Bourges et al., 2003). A wide variety of ocular drugs,
including nucleic acids such as antisense oligonucleotides,
aptamers, and small interfering RNAs, are being investigated with
nanosphere and microsphere ocular drug delivery methods to
enhance their cellular penetration, protect against degradation,
and allow long-term delivery (Fattal and Bochot, 2006).
Liposomes, a type of nanoparticle or microparticle, are vesicular lipid systems of a diameter ranging between 50 nm and a
few micrometers. They allow encapsulation of a wide variety of
drug molecules such as proteins, nucleotides, and even plasmids
and can be injected under a liquid dosage form (27- to 30-gauge
needle); they provide a convenient way of obtaining slow drug
release from a relatively inert depot. Almost every class of topically or subconjunctivally applied ophthalmic drug has been
studied in liposomal form (Ghate and Edelhauser, 2006). Another
advantage of liposomes is that encapsulated drugs appear to be
less toxic, because only a limited amount of drug comes in
direct contact with ocular tissues. Similar to microparticles and
nanoparticles, however, liposomes can also impair vitreous
clarity. Furthermore, the long-term effects of liposomal injections
in the eye are unknown (Hsu, 2007).
Ocular Implants
Ocular implants have many advantages over more traditional
methods of drug administration to the eye, including delivering
constant therapeutic levels of drug directly to the site of action
and bypassing the blood–brain barrier (Davis et al., 2004; Bourges
et al., 2006; Yasukawa et al., 2006). Release rates are typically
well below toxic levels, and higher concentrations of the drug
are therefore achieved without systemic side effects. In general,
subconjunctival implantation is used for anterior-segment
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FIGURE 4.—Nonbiodegradable and biodegradable inserts. Ocusert, Prosert, Vitrasert, and Retisert are clinically available, nonbiodegradable (reservoir-type) devices. Lacrisert is a biodegradable insert. Ocusert, Prosert, and Lacrisert are inserted into the conjunctival
sac. Vitrasert and Retisert are implanted transclerally, suspended at the pars plana. Implants can be implanted into the episcleral or
intrascleral space, at the sclera, or into the vitreous cavity in various shapes, such as a disc, a sheet, a plug, a rod, and a pellet. EVA =
ethylene vinyl acetate; PVA = polyvinyl alcohol. Reproduced by permission from Yasukawa et al. (2006). Expert Opin Drug Deliv
3, 261-273. © 2006 by Ashley Publications.
diseases, whereas intravitreal and suprachoroidal methods are
typically used to treat posterior-segment diseases. Intrascleral
implants can be used for either. A significant amount of crossover
can occur, and a drug may be delivered to both segments, regardless of placement.
Subconjunctival implants are inserted through a small incision in the conjunctiva and placed in contact with the sclera.
Intrascleral devices, implanted in a small scleral pocket at one-half
the total scleral thickness, place the drug closer to its site of action
than conventional transcleral devices, making it more useful for
treatment of posterior-segment diseases with less systemic absorption of the drug than subconjunctival or peribulbar injections.
Intravitreal placement of ocular implants has the advantage
of delivering a drug directly to target tissues of the posterior
segment. The implant is inserted into the vitreous through a
sclerotomy site or injected with an applicator, which has the
advantage of no sutures with needle delivery. The site of implantation is commonly over the pars plana, which is anterior to the
insertion of the retina and posterior to the lens, and this is the
area least likely to damage those structures. Anatomical differences within this region make implantation much more difficult
in rabbits compared to larger species, discussed below.
The structures of polymeric devices for controlled, sustained
release are classified as nonbiodegradable and biodegradable
(Figure 4). Nonbiodegradable implants have the advantage of
steady, controlled release of a drug during potentially long periods
of time (years) and the disadvantage of removal and/or replacement when the drug is depleted. Biodegradable implants have
the advantage of being able to be fashioned into many shapes,
they are amenable for injection as an office procedure, they do
not require removal, and they increase the half-life of the drug
(Hsu, 2007).
Nonbiodegradable (Reservoir) Implants
Reservoir implants are typically made with a pelleted drug
core surrounded by nonreactive substances such as silicon, ethylene vinyl acetate (EVA), or polyvinyl alcohol (PVA); these
implants are nonbiodegradable and can deliver continuous
amounts of a drug for months to years (Davis et al., 2004). One of
the first reservoir implants to gain Food and Drug Administration
(FDA) approval was Vitrasert ganciclovir intraocular implant
for treatment of cytomegalovirus retinitis in patients with
acquired immunodeficiency (Figure 5). The Vitrasert implant
is fixed at the pars plana and projects into the vitreous cavity;
it releases the drug for 5 to 8 months and must be replaced.
Complications in humans include vitreous hemorrhage, retinal
detachment, and endopthalmitis.
Nonbiodegradable corticosteroid implants of dexamethasone
and fluocinolone have been investigated for their potential to
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TOXICOLOGIC PATHOLOGY
space) implant (Figure 6b) with a silicone-based matrix design
that is effective for at least 6 months.
Delivery of large-molecular-weight compounds has been
relatively unsuccessful when incorporated into reservoir implants.
One exception is encapsulated cell technology (ECT), which is
a cell-based delivery system that can be used to deliver therapeutic agents to the eye. Genetically modified cells are packaged in a hollow tube of semipermeable membrane, which
prevents immune-cell entry and allows nutrients and therapeutic
molecules to diffuse freely across the membrane. Two ends of
the polymer section are sealed, and a titanium loop is placed
on the anchoring end, which is implanted at the pars plana and
anchored to the sclera. Currently, ciliary neurotrophic factor
(CNTF, NT-501), which has been shown to protect the retina
from degeneration in many animal models, safely completed
phase 1 clinical trials as delivered by an ETC implant containing
NTC-201 cells, which are genetically engineered human retinal
pigment epithelium cells that overexpress CNTF (Neurotech;
Tao, 2006).
Biodegradable Matrix Implants
FIGURE 5.—The Vitrasert implant. (a) Front view showing drug
pellet containing Ganciclovir on the left and the schematic on
the right, with the drug pellet surrounded by two polymers, PVA
and EVA. (b) A cadaver eye from a patient with HIV infections
showing a cross-sectional view of the location of the Vitrasert
implant fixed at the pars plana. EVA = ethylene vinyl acetate;
PVA = polyvinyl alcohol. Reproduced by permission from
Davis et al. (2004). Curr Opin Mol Therap 6, 195-205. © 2004
Thomson Corporation.
treat inflammatory diseases of the eye and diseases that involve
fibrovascular proliferation. Retisert (fluocinolone acetonide
intravitreal implant) was approved by the FDA in 2005 for
chronic noninfectious uveitis and can deliver the corticosteroid
to the posterior eye tissue for up to 3 years (Yasukawa et al., 2006;
Figure 6a). Cataract development and increased intraocular (IO)
have been associated with Retisert implants in humans (Jaffe
et al., 2006).
Cyclosporine has been investigated for its use as an immunosuppressant in cases of chronic uveitis in humans and horses
(Davis et al., 2004) and keratoconjunctivitis sicca in dogs (Kim
et al., 2005) with an intravitreal or episcleral (suprachoroidal
Matrix implants are typically used to treat acute-onset diseases
that require a loading dose followed by tapering doses of the
drug during a 1-day to 6-month time period (Davis et al., 2004).
They are most commonly made from the copolymers polylactic-acid (PLA) and/or poly-lactic-glycolic acid (PLGA), which
degrade to water and carbon dioxide (Figure 7a). The rate and
extent of drug release from the implant can be decreased by
altering the relative concentrations of lactide (slow) and glycolide
(fast), altering the polymer weight ratios, adding additional coats
of polymer, or using hydrophobic, insoluble drugs. The release of
drug generally follows first-order kinetics with an initial burst
of drug release followed by a rapid decline in drug levels. The
advantage over a nonbiodegradable implant is that biodegradable implants do not require removal, as they dissolve over time
(Hsu, 2007). Biodegradable implants also allow flexibility in
dose and treatment from short duration (weeks) to longer duration
(months to a year), depending on the polymer PLA/PLGA ratio,
which is another benefit in tailoring drug delivery to disease
progression, because dose and treatment requirements may change
over time.
Effective sustained delivery has been achieved using a variety
of drugs: antiviral, antifungal, antimetabolic, immunosuppressive agents, and steroids. Biodegradable ocular inserts placed
in the lower conjunctival sac and mixed with nonbiodegradable
polymers have been used with atropine, pilocarpine, and gentamicin (Davis et al., 2004).
A biodegradable matrix implant consisting of PLGA and up
to 700 µg dexamethasone (Posurdex) drug-delivery system (DDS)
has been evaluated in patients with persistent macular edema in
phase 2 trials (Davis et al., 2004; Hsu, 2007; Kuppermann et al.,
2007; Figure 7b). The implant releases dexamethasone for up to
6 months in nonclinical studies after insertion into the vitreous
through an innovative special injector and does not disturb the
clear media (Yasukawa et al., 2006).
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FIGURE 6.—Cyclosporine intravitreal sustained-release implant and the Retisert implant. (a) Double-pellet (left) and single-pellet
(right) cyclosporine implants to deliver at different release rates. (b) Photograph of human eye with double implant in vitreous cavity.
Retisert (fluocinolone acetonide) implant showing (c) front and (d) side views. Reproduced by permission from Davis et al. (2004). Curr
Opin Mol Therap 6, 195-205. © 2004 Thomson Corporation.
Iontophoresis
FIGURE 7.—Posterior-segment drug-delivery system (PS DDS)
composed of PLA/PLGA demonstrating a rod for delivery
of dexamethasone intravitreally. PLA = poly-lactic acid;
PLGA = poly-lactic-glycolic acid. Reproduced by permission
of Kupperman et al. (2007) Arch Ophthalmol 125, 309-17. ©
2007 American Medical Association.
Iontophoresis is a noninvasive technique in which a small
electric current is applied to enhance ionized drug penetration
into tissue (Myles et al., 2005; Eljarrat-Binstock and Domb,
2006). The drug is applied with an electrode carrying the same
charge as the drug, and the ground electrode, which is of the
opposite charge, is placed elsewhere on the body to complete
the circuit. The drug serves as the conductor of the current
through the tissue. Transcorneal and transcleral iontophoresis
(Figure 8) have been studied with a variety of ophthalmic
drugs including oligonucleotides, in animals, and more limited
data are available for humans. Iontophoresis has the advantage
of being noninvasive, and therefore, it avoids the risks of
surgical implantation or intravitreal injections but does not
increase drug half-life (Hsu, 2007). Animal studies have shown
that transcleral iontophoresis can be used to deliver therapeutic
levels of bioactive proteins to the retina and the choroid, which
may be a viable and less invasive alternative for delivering antiVEGF agents. OcuPhor/pegaptanib is an example of a commercially available iontophoresis unit designed specifically for the
eye (Davis et al., 2004).
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FIGURE 8.—Illustration of drug distribution to posterior segments of the eye after transscleral iontophoresis. Reproduced and
adapted by permission from Eljarrat-Binstock and Domb. (2006). J Controlled Release 110, 479-89. © 2006 Elsevier B.V.
NONCLINICAL PROGRAMS FOR OCULAR DRUG
DELIVERY FORMULATIONS
Study Design
There is relatively little International Conference on
Harmonization (ICH) guidance of regulatory expectations for
nonclinical toxicity and pharmacokinetic studies needed for ocular drugs, including those with novel delivery approaches. Some
FDA guidance that is a decade old is published in brief form
(Avalos et al., 1997; Weir et al., 1999). Some differences exist
among or within U.S., European, and Japanese regulatory agencies. As with all nonclinical programs, design depends on the
clinical and nonclinical factors, including clinical route and indication, pharmacokinetic considerations including in vitro and in
vivo release rates, anticipated frequency and duration of treatment
in clinical trials, age range and sex of patients, exclusion and
inclusion criteria, and whether the drug product is a new chemical entity (NCE) or a reformulation of a marketed ocular and/or
systemic drug. For first-in-human studies (FIHS) of ocular drugs,
two species (usually nonrodents) by the ocular route and clinical
formulation are preferred; however, one species may be sufficient
if there is sufficient rationale (i.e., lack of biologic homology in
other species), previous nonclinical and/or clinical information
from other ocular routes with the drug, or brief (1-day) human
exposure from the ocular route (i.e., an eyedrop).
Ocular pharmacokinetics studies are usually conducted to
understand the absorption, distribution, metabolism, and excretion in various ocular compartments. Usually, one species is sufficient, and target pharmacological relevance is not required.
An in vitro melanin-binding study may be advisable for FIHS
and to provide rationale for strain selection and toxicology
parameters, although the usefulness of these data is limited (see
below).
Before conduction of pivotal repeat-dose ocular studies, an
assessment of the ocular tolerability of the test article should be
conducted. Review of physiochemical properties (e.g., pH),
structure-activity relationships, and in vitro assays to evaluate
irritancy and ocular tolerability are practiced routinely in this
laboratory with an NCE or a reformulation containing new
excipients or vehicles and are beyond the scope of this review
(Avalos et al., 1997). Ocular tolerance studies are recommended
by European regulatory authorities (CPMP/SWP/21/00) and
usually consist of a single-dose study in a small number of
rabbits (1 to 3), with a 20 to 30 µl drop size in a single dose/
concentration with hourly observation and scoring (modification of Draize scoring system), with an increase in concentration or frequency over several days and evaluation by slit-lamp
biomicroscopy. For pivotal toxicity studies, typical parameters
for ocular evaluation usually include ophthalmic examinations,
including biomicroscopy to examine the anterior segment; indirect ophthalmoscopy to examine the posterior segment; tonometry; electroretinograms (ERG) if the drug reaches the retina; and
histopathology. Systemic toxicokinetics are usually conducted,
especially if systemic tissues are evaluated. Ocular toxicokinetics are not usually conducted, because there is not enough
tissue for compartmentalization studies and microscopic examination and ocular pharmacokinetic studies suffice. Other specialized procedures, such as fluorescein angiography, ocular
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computed tomography, or vitreal pharmacokinetics may also
be conducted if considered essential.
Systemic evaluation of toxicity in at least one species by
another route (iv or po) to maximize systemic exposure is
generally expected before FIHS, as are two vitro genetic toxicity tests for mutation and clastogenicity. Full systemic evaluation can also be conducted in the ocular toxicity studies in one or
both species and/or as a separate study given by another route of
administration that maximizes systemic exposure in one species,
such as a rat. Reproductive toxicity and safety pharmacology
should be assessed according to the ICH guidelines; these studies (or carcinogenicity studies) may be needed if there is a cause
for concern in any of these areas because of pharmacological
mode of action, class effects, or sufficient systemic exposure. For
prenatal and postnatal development and carcinogenicity studies,
it is expected that one provides adequate justification for a
waiver to receive timely regulatory feedback on the nonclinical
program (Weir et al., 1999).
Species and Strain Selection Parameters
Nonrodents, such as rabbits, dogs, and/or monkeys, are usually selected as the species of choice in ocular toxicity and pharmacokinetic studies. Rabbits are the most common species used
for ocular toxicity testing, and the rabbit eye is large enough to
perform accurate ocular injections or delivery by other methods.
Some companies have used rats in intravitreal toxicity studies
based on target activity and lack of such activity in one or more
other species, although the rat eye is too small for ocular implants
designed for humans, and use of rats for these types of ocular studies may meet regulatory resistance. A nonpigmented rabbit strain,
such as the New Zealand White (NZW), is usually acceptable
when combined with a pigmented species (dog or primate). There
has been some encouragement to use pigmented rabbits, such as
New Zealand Red and Dutch-Belted (DB), if the drug binds
ocular melanin binding. DB rabbits, the most popular pigmented
strain, have some disadvantages because of lack of availability,
less ease in handling, and an increased incidence of spontaneous
corneal dystrophy and cardiomyopathy. There is some evidence
that binding of drugs to ocular melanin is not predictive of ocular
toxicity and that using nonpigmented rabbits or rodents in addition to pigmented nonrodent species is fully justified (Leblanc
et al., 1998), although regulatory requests for pigmented rabbit
strains are not uncommon, especially in Europe.
The monkey eye most closely resembles the human eye
with regard to anatomy and physiology, including presence of
a macula, and it is usually the first choice for biologics because
of higher sequence homology to humans compared to other
species, and therefore, higher relevance to pharmacological
responses in humans and less risk of antigenic response to the
test article. Drug-related inflammatory changes in the eye observed
by histopathology often need to be differentiated from incidental,
spontaneous changes. Background inflammatory cell changes
seen in control and drug-treated monkeys in these studies and
others include mononuclear cell infiltrates in the uvea, choroid,
57
and ciliary body, which are typically composed of lymphocytes
and fewer plasma cells. These changes were observed in approximately 25% to 29% of naïve, untreated cynomolgus monkeys
used in drug evaluation studies from one study (Sinha et al.,
2006). Microscopically observed mononuclear cell infiltration
in the ciliary body and choroid is not detected by ophthalmoscopic examination and is not related to any associated disease
process or functional deficit.
Dogs were cited as the most commonly used nonrodent
species in an earlier review in this forum (Heywood and
Gopinath, 1990). However, many cases of retinal toxicity in
dogs are associated with the tapetum, making these types of
findings difficult to extrapolate to humans. This fact, coupled
with the surge in research, the development directed toward
retinal diseases, and the increased use of biologics, has diminished the use of dogs and increased the use of monkeys in nonclinical safety evaluation for ocular drugs, although there are
recent examples of use of the dog as the primary nonrodent
species with Retisert, a nonbiodegradable implant (Retisert
FDA Pharmacology Review, 2004).
MORPHOLOGICAL ASSESSMENT
Methodology
Collection and Fixation
There are several good references that address this area
(Latendresse et al., 2002; Whiteley and Peiffer, 2002), and only
additional comments not provided in these sources are provided
here. In ocular toxicity studies, both treated and untreated eyes
and surrounding ocular structures, including both upper and
lower eyelids, nictitating membrane, Harderian glands, lacrimal
glands, the optic nerve, and adjacent muscles, should usually
be collected, fixed, and evaluated microscopically. Before dissection and collection, the right upper eyelid can be identified
with a suture, and a circular, palpebral skin incision is made
surrounding the eye, eyelids, and medial and lateral canthus.
The palpebral rim is reflected back to expose the orbit, the bulbar conjunctiva is grasped above the globe, the optic nerve and
remaining muscle attachments are cut, the globe is removed in
toto, and extraneous tissues are trimmed away except for 1 to
2 mm of optic nerve attached to the globe. The orbit is gently
dissected from extraocular tissue at the reflection of the bulbar
and palpebral conjuctiva in the back of the eye. For implant
studies, implant sites are marked with indelible dye before fixation (Figure 9) so that they can be sectioned and examined.
Scleral injection sites for intravitreal studies are usually too small
to be identified macroscopically and are not sectioned unless
visible and specifically requested. For microsphere studies by
subconjunctival injection, the test article is usually identifiable
macroscopically beneath the palpebral conjunctiva attached
to the globe. The globes are placed in Davidson’s fixative for
24 hours, 70% alcohol for 24 hours and processed or stored in
formalin. A pluck of extraocular tissue containing remaining
optic nerve, extraocular muscles, lacrimal glands, and Harderian
glands is fixed in 10% normal buffered formalin.
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TOXICOLOGIC PATHOLOGY
which usually are visible macroscopically without marking.
Serial or step sections are usually necessary to capture the most
optimal view of the implant or microsphere site for microscopic
evaluation. PLA/PLGA implants observed during processing
and sectioning usually dissolve during staining.
Morphometric Species Comparisons
FIGURE 9.—Ocular implant-site evaluation, rabbit eye with
cornea on the left. (a) Indelible marks are made on each side of
two injection sites for ocular implantation in the vitreous before
fixation. (b) Plane of section for trimming eye for evaluation of
injection sites.
Trimming and Sectioning
For topical or intravitreal injection studies, the nasal and
temporal edges of each eye are trimmed away perpendicular to
the posterior ciliary arteries by cutting closer to the nasal side
than the temporal side of the optic nerve and through the cornea.
The injection site is not usually processed and examined for
intravitreal injection studies, as discussed above. The trimmed
eye is placed in a deep cassette. For implant studies, the marked
implant sites are usually located in the temporal superior quadrant, and therefore, a temporal section trimmed from the globe
containing the site or sites is further trimmed to allow sectioning
across the longitudinal axis of the injection tract. A similar section
is trimmed for microsphere subconjunctival injection studies,
NZW rabbits and cynomolgus monkeys are two of the most
common strains or species used in ocular toxicity studies for
posterior-segment diseases, and anatomical difference must be
factored in for correct interpretation of test-article effects. The
rabbit eye is essentially a fisheye lens system with significantly
greater corneal area to accommodate greater peripheral vision,
with corresponding lens differences. An essential absence of
ciliary muscle in the rabbit indicates that the focal length in this
species is essentially fixed. In contrast, the monkey eye has a
rather small lens coupled to a smaller cornea that occupies a
smaller area of the globe, with a markedly greater vitreal volume
compared to the rabbit.
To quantitate these species differences, morphometric comparisons were made from representative longitudinal ocular
sections of an NZW rabbit eye and a cynomolgus monkey eye
obtained from intraocular implant studies (Holland, 2005).
Although globe size was similar between the two species, the
anterior chamber and lens of the rabbit eye were 2.3-fold and
3.9-fold larger, respectively, than similar areas of the monkey
eye. Conversely, the rabbit vitreous is one-half the size of the
monkey vitreous, and the ratio of the vitreous-to-globe area was
0.4 in the rabbit eye and 0.7 in the monkey eye. The monkey
eye is more similar than the rabbit eye to humans, which is
consistent with the ratio of vitreous-to-globe of 0.3 in rabbits and
0.6 in humans from the literature (Samuelson, 1999).
The ciliary body and pars plana region is important in
intravitreal implant studies, because it is the preferred site for
surgical penetration of the posterior segment (Koch and Kreiger,
1994). The monkey eye is distinctive in two essential ways:
One, the lens is positioned more forward and is volumetrically
smaller than in the rabbit (Figure 10). Accordingly, the monkey
lens is less likely to come in direct contact with the implant or
be damaged by the method used for placement. Two, monkey
ciliary muscle is significantly more developed than in the rabbit.
Therefore, the ciliary muscle extends proportionally much further
from the limbus toward the ora serrata. However, the monkey
pars plana area is also proportionately more extensive than in
rabbits. This is consistent with the more forward location of the
ora serrata in rabbits because of greater functional peripheral
vision in this species. In monkeys, the iris root is a better reference point for judging the point of entry into the vitreous than
is the limbus, because variable bulbar pigment creates ambiguity
in identifying this area.
There is a greatly reduced space between the lens and the
pars plana and a much reduced area occupied by the pars plana
in rabbits compared to primates (Figure 11). It is as if evolution
traded accommodation ability for peripheral visual acuity. The
experimental challenges here are to reliably detect and insert
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OCULAR DRUG DELIVERY FORMULATIONS
59
FIGURE 10.—Comparative ocular anatomy a rabbit and a primate
(cynomolgus monkey). Globe size is similar between species,
but anterior chamber and lens are larger in the rabbit than the
monkey, and the vitreous is larger in the monkey than the rabbit.
implants through a less extensive pars plana, as well as to direct
the delivery system at a more acute angle, after penetration. All
of these factors contribute to differences in outcomes following
surgical implantation in these species.
Species Differences in Vitreal Volume: Estimating
Animal/Human Safety Multiples
To enable comparison of intravitreal doses between animals
and humans, dose levels can be expressed in terms of the
initial theoretical vitreous concentration, which appropriately
normalizes the doses to the species-specific vitreous volume and
reflects size-adjusted exposure of the eye. Literature review
indicates that vitreous volume is approximately 20 µL, or 0.02
ml, in rats (Dureau et al., 2001), 1.5 ml in NZW rabbits (Leeds
et al., 1997), 1.5 to 3.2 ml in cynomolgus monkeys (Leeds et al.,
1998; Pearson et al., 1996), and 4.0 ml in humans (Friedrich,
2003). A conservative value for the vitreous volume of monkeys
of 3.2 ml should be adopted, which is consistent with the vitrealarea ratios among species presented above.
Safety Evaluation of Ocular Drug
Delivery Formulations: Case Studies
Injectable Therapies
Most injectable therapies are used intravitreally, and there is
a plethora of literature on the safety and pharmacokinetics of
these formulations by this route, mostly conducted in rabbits.
As mentioned above, Kenalog, the only commercially available
TA formulation in the United States, is toxic to the retina. Benzyl
FIGURE 11.—Lens placement relative to ciliary body. (a) Rabbit
and (b) cynomolgus monkey. Cynomolgus monkey lens (L) is
less susceptible to focal injury from intravitreal injection or
implantation because of a lens that is smaller and more anterior
than the rabbit lens. There is a larger ciliary body, because the
monkey has a longer pars plana (PP) because of a more posterior
location of the ora serrata (OS). CM = ciliary muscle; CP = ciliary
processes.
alcohol preservative has been shown to be the culprit in rabbit
studies, as injected concentrations modestly higher than what is
present in commercial Kenalog are toxic to the rabbit eye, causing
focal retinal whitening and hemorrhage with no histopathological changes at lower doses and retinal atrophy, loss of photoreceptor outer segments, retinal pigment epithelium proliferation,
and vitritis at higher doses (Morrison et al., 2006). In contrast,
preservative-free formulation at TA concentrations similar to
and higher than that used in clinical trials did not demonstrate
any retinal toxicity up to 7 months following a single intravitreal
injection in ocular toxicity studies in rabbits (McGee et al., 2005;
Kim et al., 2006).
Most intravitreal biologics produce ocular inflammation in
animals at high doses, as detected by ophthalmoscopic and/or
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microscopic examination. There is often no prediction of which
species are more sensitive to ocular inflammation induced by
high doses of intravitreal biologics; rabbits are usually more
sensitive to oligonucleotides than other species (Doug Kornbrust,
personal communication, Preclinsight, Reno, NV). Both intravitreal fomiversen and pegaptanib cause ocular inflammation
at lower doses in rabbits compared to monkeys and/or dogs
(Vitravene (fomivirsen) FDA Pharmacology Review, 1998;
Macugen FDA Pharmacology Review, 2004). This may be
because of the smaller vitreal volume, decreased retinal vascularity, and/or decreased aqueous humor outflow from the uveoscleral pathway in rabbits compared to larger species, which
increase drug half-life (Leeds et al., 1998). The mechanism of
the inflammation from these compounds has not been investigated; however, it is known that toll-like receptors (TLRs) play
a crucial role by recognizing proteins or DNA/RNA sequences
belonging to infectious agents, and activation of TLRs results
in the production of proinflammatory mediators and cytokines
and links innate adaptive responses under pathological conditions, including the various regions and diseases of the eye
(Micera et al., 2005). Therefore, it is possible that TLRs play a
role in inflammation induced by biologicals injected in the eye.
In addition, the influence of biologicals—for example, small
interfering RNAs—on inflammatory responses depends on the
contemporaneous administration of vehicles and the mode of
delivery (Aronin, 2006).
Monkeys were more sensitive to ocular inflammation than
rabbits at high intravitreal doses of ranibizumab, with no apparent correlation between the degree of ocular inflammation and
the appearance of serum antibodies to ranibizumab (Lucentis
FDA Pharmacology/Toxicology Review and Evaluation, 2006).
Drug-induced ocular inflammation in animals is reversible and
usually does not progress with repeat injections of similar or
increasing dose; it may actually decrease with repeated injections, depending on the injection interval. Drug-related ocular
inflammation is reversible, monitorable, and treatable in humans,
and therefore, it is considered an acceptable safety risk in clinical
trials for posterior-segment diseases.
Small molecules and biologics injected by periocular routes
can be associated with inflammation, and the various types, location, and investigation of the source of inflammation are important for understanding compound-related effects compared to
foreign-body reactions. For example, AG-028345, a small molecular VEGF receptor tyrosine kinase (RTK) inhibitor injected as
a single dose by sub-Tenon administration in monkeys, caused
foamy macrophages at the episcleral sub-Tenon dose site and
lymphoplasmacytic cells in the choroid or extraocular muscle
at 8 weeks posttreatment (Younis et al., 2007). The macrophage
response was consistent with foreign-body response and was
not associated with tissue injury or a fulminant inflammatory
response. On the other hand, the lymphoplasmacytic cells suggested an active immune response, because AG-028345 weakly
stimulated the proliferation of peripheral blood mononuclear
cells and IgG antibody production in in vitro culture. These
immunostimulatory effects were considered compound specific
because the reaction has not been observed with other VEGF
RTK inhibitors.
TOXICOLOGIC PATHOLOGY
Nanoparticles and Microparticles
Localized foreign-body reaction has been observed after
intravitreal injection of microspheres loaded with Ganciclovir,
PLA, or PLGA particles loaded with inert fluorochromes
(Bourges et al., 2006). Functionally, however, the intravitreal
nanoparticle injections do not affect the ERG. Intravitreally
injected liposomes of antibiotics, antivirals, antifungals, and
antimetabolic agents are less toxic than the free form because
there is less free drug in contact with tissues. Liposomes also
protect poorly stable drugs from degradation, such as phosphodiester antisense oligonucleotides and peptides.
PLA or PLGA microspheres loaded with 5-fluorouracil,
adriamycin, or retinoic acid have been used experimentally
in proliferative vitreoretinopathy. In these experiments, the
microparticles settled on the inferior quadrants of the retina and
induced a localized multinuclear giant-cell reaction (Bourges
et al., 2006). Vascular endothelia growth factor released by
PLGA microspheres was injected into the subretinal space in a
rodent model, with no observable toxicity.
The disadvantages of microparticles and nanoparticles is the
risk of injection and that intraocular injections may cause vitreous
clouding and periocular injections may cause a foreign-body
response in the case of microparticles (Herrero-Vanrell and
Refojo, 2001). For example, a whitish material in the vitreous
cavity appeared at 10 days and disappeared at 20 to 25 days
following intravitreal injection of fluorescein-loaded microspheres in rabbits, with no histological effects. In another study,
a mild, localized foreign-body reaction surrounding partially
degraded ganciclovir loaded microspheres with mononuclear
cells and multinucleated giant cells, with no involvement of the
retina or other ocular structures, was observed at 4 and 8 weeks
following intravitreal injection in rabbits, and it decreased
substantially at 12 weeks.
Ocular Implants
Insertion of implants by incision is slightly more invasive
compared to injection, and the normal wound-healing process
takes place with both procedures. Pars plana incisions and
injections have been studied in monkey eyes from 6 to 13 years
following the procedure, and the scars were found to consist of
fibrous tissue and blood vessels that extended from the epislera
into the vitreous (Koch and Kreiger, 1994). There were quantitative but no qualitative differences between the two procedures,
and there were ultrastructural features of mature scar tissue.
Nonbiodegradable Implants
The nonclinical review and evaluation of the safety of FA
(Retisert) is posted on the FDA Web site (Retisert Pharmacology
Review, 2004) and provides the most current and thorough look
at nonclinical development of an ocular implant. The nonclinical program consisted of a 1-year intravitreal implant pharmacokinetic and toxicity study in DB rabbits, a 4-week and a 1-year
intravitreal implant toxicity study in dogs, a genotoxicity battery
as recommended by ICH, an ISO-10993/OECD biocompatibility
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OCULAR DRUG DELIVERY FORMULATIONS
study of extracts of the empty implant, and a waiver for conduct
of reproductive and developmental toxicity studies. Significant
drug-related systemic toxic effects were not observed in either
rabbits or dogs. In rabbits, a mild postoperative reaction to the
implant and no FA-related reactions were observed. In dogs,
cataracts and corneal opacities were observed. Cataracts were
caused by mechanical contact between the lens and vitreous
implant. Corneal opacities were secondary to intraocular inflammation. Canine corneal opacity is often observed with immunemediated ocular inflammation or ocular infection. This finding
was considered species specific because it has not been found
in rabbits or humans.
The ocular safety of nonbiodegradable implants of ganciclovir
(Vitrasert) was studied in rabbits 80 days following implantation (Smith et al., 1992). No evidence of ocular inflammation
by indirect ophthalmoscopy was observed, but lens opacification, cataracts, and retinal-detachment ERG changes were
observed. There was no evidence of drug-related effects microscopically, although a chronic inflammatory reaction with multinucleated giant cells around the silk suture used to secure the
implant to the sclera was observed. Although Vitrasert is an
approved, marketed drug in the United States, the pharmacology
review is unavailable on the FDA Web site.
An episcleral cyclosporine implant that targeted the lacrimal
gland was studied for up to 6 months in rabbits and dogs, with
no systemic toxicity or exposure and no ocular inflammation by
ophthalmoscopy or histopathology and with a fibrous capsule
surrounding the implant with a few inflammatory cells present
(Kim et al., 2005).
Biodegradable Implants
Scleral plugs of biodegradable PLGA to deliver ganciclovir,
doxorubicin, and fluconazole to the vitreous have been evaluated for ocular toxicity up to 24 weeks following implantation
in rabbits (Yasukawa et al., 2006). Slit-lamp biomicroscopy
showed no ocular inflammatory reactions, and no substantial
changes were observed by ERG. Histology showed no abnormalities in the rabbit retinal tissue adjacent to the implant site
and the posterior pole. Inflammatory cells infiltrated the matrix
pore, and fibrous tissue closed the sclerotomy site (Yasukawa
et al., 2006). An intrascleral PLA implant of betamethasone evaluated for ocular toxicity for up to 16 weeks following implantation in rabbits showed no ERG changes and complete degradation
of the implant at 16 weeks, with replacement by loose connective
tissue and a few multinucleated giant cells with no histological
retinal changes (Okabe et al., 2003). Similarly, there was no ocular toxicity for up to 3 months following insertion of a PLGA
cyclosporine implant into the anterior chamber in rabbits (Theng
et al., 2003). Clinical safety evaluation of a PLGA rod implant
of dexamethasone DDS following intravitreal insertion by sclerotomy in a phase 2 study in patients with persistent macular
edema showed a mild increase in incidence of adverse events
in the dexamethasone DDS treatment groups compared to the
observation groups on day 8 that were expected as a result of the
surgical procedure (hyperemia, pruritis, vitreous hemorrhage,
61
and anterior chamber cells and flare; Kuppermann et al., 2007).
The rate of ocular adverse events after day 8 and up to study
termination at day 180 was similar between treatment and observation groups, including no corticosteroid-induced cataract
formation or increases in IOP.
CONCLUSIONS
Novel ocular drug-delivery formulations and methods for
localized, sustained delivery may provide the solution for treating
serious intraocular diseases. Although regulatory guidance is
sparse for ocular drugs, review of FDA Pharmacology Reviews
and selected literature can provide insights. Species and strain
differences in ocular pigmentation and anatomy, retinal vasculature, and vitreal volume are important for designing and interpreting ocular toxicity studies and human risk assessment. Novel
ocular drug formulations and sustained delivery are generally
well tolerated, and inflammatory effects appear to be more
pronounced in animals than humans. Additional investigation is
needed to determine the progression, tolerance, and pathogenesis
of inflammation in selected cases.
ACKNOWLEDGMENTS
The author gratefully acknowledges the technical assistance
of Carole Nootenboom and Maria Rivero in preparation of the
figures, Vinsa Sun for histotechnology procedures, and pathology
peer interactions with Adelekan Oyejide and Michael Holland.
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