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16 Drug and Gene Therapy Mediated by Physical Methods
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Fig. 16.2 Electroporation under optimized conditions following subretinal injection. (a) In this
schematic of the eye, the injected bleb is in green and the positive electrode in red. The black electrode represents the negative electrode. This panel is based on an image from Johnson et al. (2008),
and is used with permission. (b) Actual electrodes handmade from platinum–iridium wire. The
loop diameter is approximately 1.5 mm. (c) The electroporation power generator. This is a commercial apparatus that provides square waves. Other commercial generators provide different
waveforms and square waves as well. (d) Positioning the electrodes on the mouse eye
convective movement of the plasmid across the pore into the cells. With naked
plasmid microinjected into the subretinal space, and current applied to electrodes
positioned on the sclera underlying the subretinal bleb, electron avalanche trans-
fection was highly efficient, transfecting 1,000–10,000-fold more cells compared
to standard electroporation, as demonstrated in eyes of live rabbits (Chalberg
et al. 2006).
16.3.4.2 Strengths and Weaknesses of Electroporation
Strengths – Transfection efficiency mediated by electroporation generally increases
as the number of small pores in the plasma membrane increases. This is modulated
once a point is reached where many small pores merge to become large permanent
holes that kill the cell. Reporter gene expression from naked plasmids peaks at field
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Fig. 16.3 Expression of a reporter gene in RPE cells; subretinal injection and electroporation
under optimized conditions. Red fluorescence of tdTomato expression was observed following
subretinal injection and electroporation under optimized conditions; individual RPE cells were
resolved. The cells include neighbors with and without tdTomato fluorescence (red). About 30%
of the cells in electroporated area were positive for tdTomato gene expression. This field represents
the RPE cells located directly over the anode. TdTomato-expressing cells exhibited polygonal
shapes characteristic of normal RPE cells. The electroporation conditions employed were 50
2 mm gap between electrodes, 1 ms pulse duration, and 10 pulses at 1 s intervals. (a) In this schematic of the eye, the bleb is in green and the positive electrode in red. The black electrode represents the negative electrode. (b) Presented is a wholemount of the eye after bleb formation but
without voltage applied to the electrodes. The edges of the flatmounts are outlined in white and
form a floret shape. The center of the floret corresponds to the retina while the outer half of the
“petals” correspond to the cornea. (c) Shown is a wholemount of the eye following subretinal
injection and electroporation under the optimized condition. A focused patch of red fluorescent
cells is evident near the center of the floret. Each dot represents a separate RPE cell. This region of
the retina corresponds to the bleb and the location of the anode. (d) High magnification of the fluorescent region shows about 30% of the RPE cells manifesting tdTomato fluorescence. (e) Close-up
of a cluster of tdTomato fluorescence in cells reveals a cobblestone or polygonal shape. (f) Shown
is a close-up of a single binucleate RPE cell. In (b, c), images are about 9
in (d) represents 50 m. The scale bars in (e, f) represent 25 mm. This caption is quoted from and the
figure images are reproduced with permission from Johnson et al. (2008)
V,
mm across. The scale bar
strengths of 100–200 V/cm. Also, increasing pulse number and length up to a point
increases expression. About 30–50% of cells may be transfected in vivo. In many
experiments, reporter expression increases linearly even at the maximum dose of
plasmid tested, indicating that the most effective dosage of naked plasmid was not
reached. A further increase in dose of plasmid DNA ought to enhance transfection
efficiency.
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Fig. 16.4 Subretinal injection followed by electroporation at different voltages. In all panels
1 mL 2 mg/mL pVAX-tdTomato in water were injected and three blebs were raised. Five different
voltage settings (0, 25, 40, 70, and 100 V) were used. Electroporation followed with a constant
1 ms pulse, 1 s interval, and 5 pulses on (a, c, e, g, and i, and 10 pulses with b, d, f, h, and j). In (a, b)
the samples received 0 V, in (c, d), 25 V, in (e, f), 40 V, in (g, h), 70 V, and in (i, j), 100 V. Each
panel represents a flatmount of the eye excluding the lens. The tips of the florets correspond to the
center of the cornea, and the central area from the midpoint or greatest bulge of each petal inwards
corresponds to the RPE sheet. The red fluorescence is punctate; each dot corresponds to a single
RPE cell. When no voltage was applied, there was no evidence of tdTomato expression (red). When
V was applied, there was no expression of tdTomato; however, when 40–100 V were applied,
25
there was an accumulation of tdTomato fluorescence. The scale bar represents 1 mm. This caption
is quoted from and the figure images are reproduced with permission from Johnson et al. (2008)
Weaknesses – Caution must be observed as a maximum safe voltage is reached
precipitously with further increases resulting in cell death. Loss of intracellular ATP
by leakage through electroporetic pores in the cell membrane is a concern. Some
transfection media are designed to match intracellular concentrations of ions and
ATP so that the opening of pores does not deplete the cell of essential constituents.
In some cases, these media help to reduce cell death in cell lines. Transfection efficiency seems low compared to several viral delivery systems. The size, shape, and
volume of a subretinal bleb may interfere with electrical field strength, and the
kinetics of bleb resorption have not been factored into electroporation strategies.
16.4 Outstanding Issues in Electric Fields
for the Delivery of Drugs
(1) Electrodes must be configured, designed, and placed considering the anatomy
and physiology of the region near the eye: Current must not pass along or through
the cranial nerves, optic nerve, or extraocular muscles. (2) Electrode set-up alone
may be insufficient to obtain an optimum electric field. To electroporate irregularly
shaped objects (the retina, RPE, choroid, or sclera), protection of other nearby tissues must be done. Shielding must be placed appropriately. Insulating materials
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Fig. 16.5 Expression of tdTomato in mouse RPE cells following subretinal injection and
electroporation. An adult C57BL/6J mouse was subretinally injected and electroporated as
described (Johnson et al. 2008). Three days later the eye was excised and the RPE sheet was
flatmounted. The RPE were stained for f-actin with phalloidin and imaged with a confocal microscope. The image highlights the expression and accumulation of tdTomato fluorescence in RPE
cells. The cells were established to be RPE based on several quantitative criteria. These included:
the presence of the actin ring bounding each cell, polygonality and number of neighboring cells,
size of each cell, regularity of each cell, and number of binculeate cells. Also it was apparent that
these were RPE cells based on the location and face adjacent to the removed neural retina (removed
during dissection and preparation of the flatmount), and RPE pigment that blocked show-through
of the underlying choroid. The expression pattern, including a significant fraction of RPE cells
displaying tdTomato, indicates a high transfection efficiency of the combined subretinal injection
and electroporation approach
may be placed or injected near or into adjacent sensitive tissues. Insulation placed
on electrodes may help to prevent collateral damage. (3) Electric fields may damage
plasma membrane proteins even in an electric field that does not cause thermal
denaturation. (4) Histology alone may not be sensitive enough to study subtle
changes in the tissue barriers after application of an electric field. Other special tests
are needed, c.f., ERGs. (5) Long-term safety and efficacy have not been assessed
except with a few select drugs. (6) Large animal studies have not been conducted.
(7) Electroporation likely is the basis of electro-shock therapy, and shielding the
brain from an electrical-based therapy in the eye is critical. (8) Time: These treatments take time, much longer than a simple intravitreal injection.
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16.5 Summary
In this review, we critiqued the current state of electrical fields as means of delivery of
therapeutic agents intracellularly in the posterior segment of the eye. Such physical
approaches are used in conjunction with the formulation of drugs and other physical
techniques such as the subretinal or suprachoroidal injection of agents in a location
immediately abutting the relevant target cell, for example, the RPE cell or the photoreceptor cell. Electric field–tissue interactions are complex and poorly understood,
and the application of an electric field to a tissue requires optimization for each particular ocular target. The use of electric fields should be given consideration when
other simpler delivery approaches fail or when a simple route needs augmentation.
Electric fields offer several unique advantages that other approaches lack. No other
approach aids in transiently and reversibly breaching any membrane of the cell.
Numerous examples abound in the use of electroporation in vitro. In vitro electroporation is by far the most efficient way to transfect DNA, RNA, or protein into a cell
without the use of viruses. Even viral transfection is fraught with potentially lethal
hazards. Provided that great care is taken to minimize cellular damage by the electric
field, iontophoresis and electroporation are delivery approaches worthy of further consideration in vivo.
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Chapter 17
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Protein Drug Delivery and Formulation
Development
Rinku Baid, Puneet Tyagi, Shelley A. Durazo, and Uday B. Kompella
Abstract Several therapeutic agents including low and high molecular weight
drugs intended for treating back of the eye disorders are routinely administered as
intravitreal injections. Intravitreal injection of Lucentis®, a therapeutic protein, was
approved in 2006 for treating the wet form of age-related macular degeneration.
This chapter summarizes the challenges and opportunities in delivering therapeutic
proteins to the eye. Specifically, barriers to delivery including permeability barriers,
examples of marketed therapeutic agents as well as those under development, formulation approaches for proteins, and novel delivery systems are discussed.
Wherever appropriate, other macromolecules such as aptamers that bind specific
protein targets are also discussed.
17.1 Introduction
For people between the ages of 25 and 74 in the United States, the most common
cause of blindness is diabetic retinopathy (progressive damage of retina due to
diabetes) (Congdon et al. 2004). For people aged 60 and older, cataract (impaired
vision due to the development of cloudiness or opacity in the lens), retinitis pigmentosa (RP, a retinal disease that causes progressive peripheral loss of vision leading
to central vision loss in the retina), and age-related macular degeneration (AMD,
degeneration of macula due to age, stress, poor nutrition, and other factors, leading
to loss of vision) are the major causes of blindness. Of the above disorders, diabetic
U.B. Kompella (*)
Nanomedicine and Drug Delivery Laboratory, Department of Pharmaceutical Sciences,
University of Colorado, 12850 East Montview Blvd., C238-V20, Aurora, CO 80045, USA
Department of Ophthalmology, University of Colorado, Aurora, CO, USA
e-mail: uday.kompella@ucdenver.edu
U.B. Kompella and H.F. Edelhauser (eds.), Drug Product Development for the Back of the Eye,
AAPS Advances in the Pharmaceutical Sciences Series 2, DOI 10.1007/978-1-4419-9920-7_17,
© American Association of Pharmaceutical Scientists, 2011
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retinopathy and AMD have neovascular forms of the disease. In proliferative
diabetic retinopathy (PDR) and neovascular AMD (NVAMD) or wet AMD, retinal
neovascularization and choroidal neovascularization (CNV) are evident, respectively. As discussed in this chapter, macromolecule drugs have revolutionized the
treatment of wet AMD. Currently, several other macromolecules are under development for the back of the eye. Below, prior approaches to treat wet AMD along with
examples of macromolecule therapeutics for this and other back of the eye disorders
are discussed.
At least as early as 1971, laser photocoagulation was introduced as the primary
treatment for neovascularization (new vessel growth). For laser photocoagulation,
an argon, xenon, or krypton laser is aimed at the new blood vessels in order to
destroy them using laser generated thermal energy (May et al. 1976; Group 1991).
Since thermal laser photocoagulation may lead to reduced vision due to the
destruction of photoreceptors in the targeted area of the retina, especially the fovea
(a region of the macula responsible for fine vision), it is applicable when abnormal
vessels do not occupy the foveal region. Clinical evidence shows a decrease in
the rate of severe visual loss and prevention of further contrast sensitivity loss with
laser photocoagulation. However, following laser photocoagulation, recurrence of
neovascularization occurs within 2 years of treatment (Group 1991; Yamaoka et al.
1994). Regardless of potential adverse events and poor clinical outcome after 2 years,
laser photocoagulation remains the main treatment option for PDR and investigations are underway to improve the clinical outcomes with this technique (Nagpal
et al. 2010).
Another treatment for neovascularization is photodynamic therapy (PDT)
(Kaiser 2005), which entails intravenous infusion of verteporfin, a photosensitizing
agent, which binds to low density lipoprotein (LDL) receptors that are elevated in
abnormal endothelial vessels. Subsequent application of laser energy activates
verteporfin, which then produces free radicals, ultimately causing damage to
endothelial cells and thrombus formation. Photofrin® was approved in 1995 for the
®
treatment of malignant dysphagia caused by esophageal cancer and Visudyne
was
approved in 2000 as the first pharmacotherapy for treating neovascular or wet
AMD. Compared to thermal laser photocoagulation, treatment with PDT is safer
(Schmidt-Erfurth et al. 1998). Photocoagulation and PDT are effective only during
the proliferative stage of disease. A clinical trial in 1998 found that leakage from
CNV in a majority of the patients was stabilized up to 3 months after PDT treatment, yet recurrence of CNV was observed in 50% of the eyes after 2 years of PDT
treatment (Schmidt-Erfurth et al. 1998; Wormald et al. 2007). However, with the
arrival of anti-vascular endothelial growth factor (anti-VEGF) therapy, laser photocoagulation and PDT are finding less widespread use.
Advances in anti-VEGF drug discovery introduced promising and revolutionary
macromolecule therapeutic agents for ocular diseases. Pegaptanib (Macugen®,
EyeTech), a PEGylated aptamer (oligonucleotide ligands having selective high
binding affinity for molecular targets) is effective in preventing vision loss in patients
with CNV by binding to VEGF. Ranibizumab (Lucentis®, Genentech), a monoclonal
antibody fragment, has been shown to improve visual acuity in patients with wet AMD.
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Clinical trials found that 95% of patients receiving monthly ranibizumab injections
maintained their visual acuity and 34–40% had improved vision (gaining 15 or more
letters in 12 months). Bevacizumab (Avastin®, Genentech), a full length humanized
anti-VEGF antibody, approved by the FDA to prevent regrowth of vessels at tumor
sites in patients with colon cancer, breast cancer, and nonsmall cell lung cancer, is
currently used as an off-label drug to treat wet AMD. A new VEGF analog that has
received increased attention is the VEGF trap (VEGF Trap-Eye™, Regeneron), a
modified soluble VEGF receptor analog protein that binds more tightly to VEGF
than pegaptanib (Ng et al. 2006) and ranibizumab (Stewart and Rosenfeld 2008).
With the development of anti-VEGF therapies, visual acuity of patients suffering
from wet AMD and diabetic macular edema (DME) is expected to be significantly
increased. In addition, development of therapeutics that target growth factor such as
ciliary neurotrophic factor (CNTF) are under development for treating retinal degenerative disorders using the NT-501 intravitreal implant [Neurotech, Inc. developed
an encapsulated cell technology (ECT) based implant to deliver macromolecules
directly to the site of action after cellular production], which is currently undergoing
clinical trials. The results thus far have shown that the implant is safe up to 1 year
after injection. Thus, protein and other macromolecule therapeutics are of value in
treating disorders of the eye and are currently being explored for their potential
long-term effects. The primary target for the current protein therapies of the eye are
tissues of the posterior segment of the eye. However, due to the presence of formidable biological barriers, therapeutic macromolecules such as pegaptanib and
ranibizumab as well as implants encapsulating cells are typically administered in
the vitreous humor in order to ensure that therapeutic concentrations of the drug
reach the target site in the back of the eye.
In this chapter, various routes of administration, delivery strategies, challenges
for each delivery system, macromolecule case studies, and standard protocols for
formulation development are discussed with a key focus on protein drugs. Several of
the approaches discussed might be relevant to nucleic acid therapeutics as well.
17.2 Routes of Protein Administration
Due to the unique anatomy and physiology of the eye, ocular drug delivery is
historically challenging (Lee and Robinson 1986; Kompella et al. 2010). Protein
delivery to the eye has been evaluated for various routes of administration including
topical, intracorneal, intracameral, periocular (subconjunctival, sub-Tenon, retrobulbar, and peribulbar), intravitreal, subretinal, suprachoroidal, and intravenous. The
route of administration directly influences the extent of drug delivery to various
target sites within the eye. Topical, intracorneal, intracameral, and periocular routes
typically deliver higher concentration of most therapeutic agents, especially small
molecules, to the anterior segment as compared to the posterior segment. Whereas,
intravitreal, subretinal, and suprachoroidal injections deliver higher concentration
of protein and other therapeutic agents to the posterior segment as compared to the
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anterior segment. On the other hand, systemic administration or delivery by the
intravenous route can potentially deliver proteins and other therapeutic agents,
although at low concentrations, to the anterior and/or posterior segments of the eye,
provided the drug can overcome the blood-aqueous, blood-retinal, metabolic, and
immunologic or other clearance barriers. In the following discussion, some principal routes of administration of therapeutic proteins along with some successful
examples are discussed.
17.2.1 Topical
Topical administration of drugs into the inferior fornix of the conjunctiva is typically
used for treating diseases of the anterior segment of the eye. Due to rapid clearance
from eye surface, drugs from an eye drop cannot typically reach the posterior
segment to a therapeutic level (Lee and Robinson 1986). Topically applied drugs
undergo rapid clearance and do not reside for long durations in the precorneal area,
due to mixing and dilution of drug with tears, tear turnover or tear drainage [0.7 mL/min
in rabbit and 1.0–2.0 mL/min in human (Owen et al. 2007)], and blinking of the
eye [once per 18 min for rabbits and 4–16 times per min in human (Congdon et al.
2004; Owen et al. 2007)], leading to poor drug bioavailability. In addition, the tight
junctions of the corneal and conjunctival epithelial layers further restrict the drug
from entering the eye.
Formulations such as suspensions, ointments, and gels may be used to prolong
the precorneal drug residence. In situ forming gels such as Gelrite™ were designed
to overcome the precorneal elimination problem to a certain extent (Carlfors et al.
1998). Upon instillation, these drops undergo sol-gel transition in the cul-de-sac of the
eye in the presence of mono- or di-valent cations of the lacrimal fluid. A formulation of indomethacin using Gelrite sustained drug release for 8 h in vitro and was
efficacious in treating uveitis in a rabbit model (Balasubramaniam et
Topically applied drugs may be able to reach the posterior segment of the eye to a
greater extent if the formulation has enhanced precorneal drug retention.
Interestingly, few protein drugs have been reported to permeate to the back of the
eye following topical eye drop instillation. In a recent study, tumor necrosis factor
(TNF)-a inhibitory single-chain antibody fragment (scFv; 26 kDa) (ESBA105)
when administered as topical drop at high frequency followed by persistent opening
of the eyes, showed absorption and distribution to various compartments of the eye
as opposed to an intravenous injection of an equivalent dose (Furrer et al. 2009). In
this study, rabbits were divided into three groups: two groups received ESBA105
topically as drops and one group received ESBA105 via intravenous administration.
In group one, ESBA105 was administered topically as one drop every hour for 10 h,
up to 5 mg/day for one single day (after each administration, the eyes were kept still
for 30 s). Group two was given one topical drop of ESBA105, 5 times a day, for
6 days up to 15 mg/6 days. Group three received an intravenous bolus injection of
5 mg of ESBA105, one time through the marginal ear vein. Drug concentrations
al. 2003).
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