International Journal of Nanomedicine
`
`Open Access Full Text Article
`
`Dovepress
`open access to scientific and medical research
`
`REvI Ew
`
`Liposomes and nanotechnology in drug
`development: focus on ocular targets
`
`Miki Honda1
`Tomohiro Asai2
`Naoto Oku2
`Yoshihiko Araki3
`Minoru Tanaka1
`Nobuyuki Ebihara1
`1Department of Ophthalmology,
`Juntendo University Urayasu
`Hospital, Chiba, Japan; 2Department
`of Medical Biochemistry, School of
`Pharmaceutical Sciences, University
`of Shizuoka, Shizuoka, Japan; 3Institute
`for Environmental and Gender-
`Specific Medicine, Juntendo University
`Graduate School of Medicine,
`Chiba, Japan
`
`Correspondence: Miki Honda
`Department of Ophthalmology,
`Juntendo University Urayasu Hospital,
`2-1-1 Tomioka, Urayasu-City,
`Chiba 279-0021, Japan
`Tel +81 47 353 3111
`Fax +81 47 355 5949
`Email m-honda@juntendo-urayasu.jp
`
`Abstract: Poor drug delivery to lesions in patients’ eyes is a major obstacle to the treatment
`of ocular diseases. The accessibility of these areas to drugs is highly restricted by the presence
`of barriers, including the corneal barrier, aqueous barrier, and the inner and outer blood–retinal
`barriers. In particular, the posterior segment is difficult to reach for drugs because of its structural
`peculiarities. This review discusses various barriers to drug delivery and provides comprehen-
`sive information for designing nanoparticle-mediated drug delivery systems for the treatment
`of ocular diseases. Nanoparticles can be designed to improve penetration, controlled release,
`and drug targeting. As highlighted in this review, the therapeutic efficacy of drugs in ocular
`diseases has been reported to be enhanced by the use of nanoparticles such as liposomes, micro/
`nanospheres, microemulsions, and dendrimers. Our recent data show that intravitreal injection
`of targeted liposomes encapsulating an angiogenesis inhibitor caused significantly greater sup-
`pression of choroidal neovascularization than did the injection of free drug. Recent progress in
`ocular drug delivery systems research has provided new insights into drug development, and
`the use of nanoparticles for drug delivery is thus a promising approach for advanced therapy
`of ocular diseases.
`Keywords: intravitreal injection, drug delivery system, age-related macular degeneration,
`APRPG-modified PEGylated liposome, DDS
`
`Introduction
`Nanotechnology is a general term used for technologies that use the properties of
`nanoscale substances to develop new functions for these substances and improve their
`properties. Many research projects are underway in this domain around the world.
`Nanotechnology has been used for various applications in the medical field, such as
`diagnosis, biosensors, and drug delivery, and has thus provided novel nanomedicines
`and nanodevices.1
`For the effects of a drug to be maximized, the molecules of the drug need to reach
`specific locations within the target tissue. Because drug molecules typically cannot
`selectively reach their site of action, there is a need for carriers that can efficiently
`deliver the required amount of the drug to its target site. The eye, particularly the pos-
`terior segment, is composed of tissues that are difficult for drugs to penetrate because
`of structural peculiarities such as the barrier function. Thus, many research studies on
`nano-sized drug carriers have been conducted in the field of ophthalmology.2,3
`This review provides an overview of recent progress in drug delivery methods for
`posterior ocular disease and the use of nanotechnology for intravitreal injection as a
`strategy for overcoming these issues.
`
`submit your manuscript | www.dovepress.com
`Dovepress
`http://dx.doi.org/10.2147/IJN.S30725
`
`495
`International Journal of Nanomedicine 2013:8 495–504
`© 2013 Honda et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article
`which permits unrestricted noncommercial use, provided the original work is properly cited.
`
`Novartis Exhibit 2022.001
`Regeneron v. Novartis, IPR2020-01318
`
`

`

`Honda et al
`
`Dovepress
`
`Eye diseases and structural
`peculiarities of the eye
`Among eye diseases, age-related macular degeneration
`(AMD) with neovascularization, pathologic myopia, central
`retinal vein occlusion/diabetic retinopathy, and cytomegalo-
`virus-associated macular edema cause severe blindness.4
`Structures that function as a barrier to limit the permeability
`of the eye to pharmacological agents include the sclerocorneal
`parenchyma, the corneal epithelium and endothelium, the inner
`and outer blood–retinal barriers, and the retinal inner limiting
`membrane (Figure 1).5 Topical administration using eye drops,
`which is the most commonly used method for administering
`medications into the eye, results in excretion of the drug through
`tear drainage and peribulbar and choroidal blood flow. As a
`result, less than 5% of the drug can cross the corneal barrier
`and gain access to the inner eye.6–8 In contrast, although sys-
`temically administered drugs can reach the choroid membrane,
`drug delivery into the retina or the vitreous body is difficult to
`achieve through conventional methods because of the presence
`of the blood–aqueous barrier and the inner and outer blood–
`retinal barriers in those structures.9 As a result, large doses
`are often required, which leads to concern over systemic side
`effects. Based on this background, intravitreal administration
`of pharmacological agents has been performed for vitreoretinal
`diseases wherein drug delivery is difficult (Figure 2).10
`Overview of the treatment
`modalities used and barriers
`encountered in the past
`Most current clinical applications of intravitreal injections
`involve the treatment of AMD. AMD causes neovascularization
`
`Blood–aqueous barrier
`(Iridal vascular endothelium)
`(Ciliary nonpigmented epithelium)
`
`Cornea
`Epithelium
`Stroma
`Endothelium
`
`Vitreous
`
`Aqueous outflow
`
`Internal limiting membrane
`
`Tear drainage
`
`Inner blood–retinal barrier
`(Retinal vascular endothelium)
`
`Outer blood–retinal barrier
`(Retinal pigment epithelium)
`
`Figure 1 Structural particularities of the eye.
`Notes: There are many barriers to drug delivery to the retina. Drugs cannot be
`easily delivered to the retina by topical administration, for example, by using eye
`drops, because of the presence of tear drainage and peribulbar and choroidal blood
`flow. In contrast, systemically administrated drugs hardly enter the retina because
`of the presence of the blood–aqueous barrier and the inner and outer blood–retinal
`barriers.
`
`Figure 2 Intravitreal injection.
`Note: Intravitreal injection of drug is recently used for treatment of several ocular
`diseases.
`
`in the choroid membrane in the macular region, which leads
`to blindness, and it has a high prevalence in developed
`countries.11,12 Vascular endothelial growth factor (VEGF)
`plays a major role in the development of choroidal neovas-
`cularization (CNV), and VEGF is expressed in choroidal
`neovascularized tissues as well as in the retinal pigment
`epithelium.13–15 Therefore, drugs capable of inhibiting VEGF
`are being developed, and currently in the United States of
`America, three such drugs have been approved by the Food and
`Drug Administration (FDA) for use in the treatment of AMD,
`namely, Macugen® (Eyetech Pharmaceuticals, Palm Beach
`Gardens, FL, USA), Lucentis® (Genentech, San Francisco, CA,
`USA), and Eylea® (Regeneron, Tarrytown, NY, USA).
`Because intravitreal injections allow the administration
`of small amounts of the drug in each injection, they enable
`an increase in intraretinal and intravitreal drug concentra-
`tions while avoiding the adverse effects caused by systemic
`administration.4,16 However, because the drug remains in the
`eye for only a short duration, it needs to be administered fre-
`quently into the vitreous body, and there are concerns about
`the development of complications such as intravitreal hemor-
`rhage, retinal detachment, cataracts, and endophthalmitis.17
`
`Development of a drug
`delivery system (DDS) by using
`nanotechnology
`The development of a DDS that can be used for the posterior
`segment of the eye and that involves nanocarriers to over-
`come the issue of frequent intravitreal administration has
`received considerable attention.
`DDSs with appropriate spatiotemporal characteris-
`tics are designed to allow drugs to affect the target tissue
`
`496
`
`submit your manuscript | www.dovepress.com
`Dovepress
`
`International Journal of Nanomedicine 2013:8
`
`Novartis Exhibit 2022.002
`Regeneron v. Novartis, IPR2020-01318
`
`

`

`Dovepress
`
`Nano drug delivery systems for ocular targets
`
`efficiently, and three classes of techniques are generally used:
`(1) absorption promotion – promotion of the passage of a drug
`through the tissue, which functions as a barrier; (2) controlled
`release – efficient time-controlled sustained release of a
`locally administered drug; and (3) drug targeting – to allow
`it to act efficiently and exclusively on the target tissue.5
`Among ophthalmic drug delivery systems (DDS)
`that use nanocarriers, liposomes and micro/nanospheres
`have been studied the most extensively; other systems
`include emulsions and dendrimers (Figure 3). In this review,
`we have provided a description of nanocarriers used in the
`field of ophthalmology, focusing on those that are delivered
`intravitreally.
`
`Liposomes
`Liposomes are closed vesicles (small lipid vesicles) com-
`posed of a phospholipid bilayer, and water-soluble drugs
`can be incorporated into their aqueous phase, whereas
`lipid-soluble drugs can be incorporated into their lipid
`phase.18 Liposomes have various advantages as drug car-
`riers: (1) Because they are noncovalent aggregates, their
`lipid composition, size, and electric charge can be easily
`controlled;19,20 (2) Their modification, with surface polymers,
`carbohydrates, and antibodies, can be easily achieved to
`facilitate targeting;21 (3) Liposomes have almost no toxicity
`
`and low antigenicity;22 (4) Liposomes can be biodegradable
`and metabolized in vivo;22 (5) Properties such as mem-
`brane permeability can be controlled to some extent;23 and
`(6) Liposomes can hold various types of solutes with dif-
`ferent properties and molecular weights, such as fat-soluble
`molecules,23 water-soluble molecules,24 and amphiphilic
`molecules.25 Because of these characteristics, studies have
`been conducted on the intravitreal injection of drug-bearing
`liposomes and have demonstrated that the release of the
`drug can be controlled, the half-life of the drug inside the
`vitreous body can be prolonged, and the toxicity of the drug
`can be reduced.26–31
`With the goal of eventually using liposome-encapsulated
`ganciclovir (GCV) for the treatment of cytomegalovirus
`retinitis in acquired immune deficiency syndrome (AIDS)
`patients, liposome-encapsulated GCV and free GCV were
`injected into the vitreous body of rabbits in a previous study.32
`The intraocular concentration of GCV was determined by
`an enzyme linked immunosorbent assay (ELISA) assay of
`the vitreous after a single intravitreal injection of different
`doses of the free drug (0.2–20 mg) or 1 mg of liposome-
`encapsulated GCV. At 72 hours after the intravitreal injection,
`only the vitreous of rabbits injected with a free GCV dose
`greater than or equal to 5 mg showed therapeutic levels of
`the drug. In addition, free GCV caused retinopathy at doses
`
`Hydrophobic
`
`A
`
`Hydrophilic
`
`B
`
`Drug
`
`C
`
`Liposome
`
`Microemulsion (nano) sphere
`
`Micro (nano) capsule
`
`D
`
`E
`
`Phospholipid
`
`Surface modification
`
`Microemulsion
`
`Dendrimer
`
`Figure 3 various formulations of injectable particles. (A) Liposomes: Liposomes are closed vesicles (small lipid vesicles) composed of a phospholipid bilayer, and water-
`soluble drugs can be incorporated into their aqueous phase, whereas lipid-soluble drugs can be incorporated into their lipid phase; (B) Micro/nanocapsule: Drugs are
`encapsulated in synthetic and natural polymers to permit sustained local release and tissue targeting; (C) Micro/nanocapsules are similar to microspheres, and both names are
`used without a clear distinction; (D) Microemulsion: A microemulsion is a type of dispersion system that is composed of two types of liquids. The diameter of the micelles
`is as low as approximately 100 nm or less. These micelles are thermodynamically stable and can be formed easily without requiring strong agitation; and (E) Dendrimer:
`Dendrimers are repetitive/single molecules with a regularly branched structure, and they are composed of a central molecule known as the “core” and side chain moieties.
`
`International Journal of Nanomedicine 2013:8
`
`submit your manuscript | www.dovepress.com
`Dovepress
`
`497
`
`Novartis Exhibit 2022.003
`Regeneron v. Novartis, IPR2020-01318
`
`

`

`Honda et al
`
`Dovepress
`
`of 15 mg or higher. In contrast, no retinopathy was found in
`the group that received 1 mg of liposome-encapsulated GCV,
`and the concentration of the drug demonstrated therapeutic
`levels up to 14 days after injection.32 Furthermore, compared
`with the effects of injection of free GCV as a control, intrav-
`itreal injection of 0.25 mL (1 mg) of liposome-encapsulated
`GCV in patients with AIDS-induced cytomegalovirus
`retinitis prevented the progression of retinal hemorrhages,
`retinal detachment, and cytomegalovirus (CMV) retinitis. In
`addition, previous reports have shown that fewer intravitreal
`injections were required for liposome-encapsulated GCV
`than for free GCV.33
`The stability of the drug inside the vitreous body can be
`improved even further by adding polyethylene glycol (PEG)
`to the surface of the liposomes, since PEG-modification of
`liposomes sterically stabilizes them by covering the lipo-
`somal surface with a fixed aqueous layer.31
`Other reports have also demonstrated the usefulness
`of liposomal formulations after intravitreal injection
`(Table 1), and the drugs delivered in these studies have
`included amikacin,34 amphotericin B,35 a model anti-
`sense oligonucleotide,31,36 bevacizumab,37 cyclosporine,38
`5-fluorouridine 5′-monophosphate,39,40 fluconazole,41 GCV,
`gentamicin,28 tacrolimus,42 tobramycin,43 vasoactive intes-
`tinal peptide,44 an angiogenesis inhibitor,45 tilisolol,46 and
`ofloxacin.47
`The toxicity of various doses of intravitreal amphotericin
`B deoxycholate, amphotericin B lipid complexes (ABLC),
`
`and liposomal amphotericin B (L-AmB) (AmBisome®;
`Astellas Pharma Inc, Tokyo, Japan) in rabbits was examined
`by Cannon et al.35 Eye examination was performed before
`and after injection, and at the designated times, the vitreous
`humor was aspirated, and amphotericin B concentrations
`were determined, followed by enucleation for histological
`studies. Vitreous band formation was significantly higher in
`the ABLC-treated eyes than in those treated with L-AmB.
`Vitreal inflammation was higher in eyes treated with
`L-AmB, amphotericin B deoxycholate, and ABLC than in
`the control eyes. Based on histological data, increased doses
`of all three agents appeared to be associated with increas-
`ing toxicity; however, based on ophthalmic data, L-AmB
`appeared to be less toxic than amphotericin B deoxycholate
`and ABLC.35
`Fishman et al28 investigated the effect of liposome encap-
`sulation on the pharmacokinetics of gentamicin, after injection
`in rabbits. The final liposomal suspension contained 10 mg/mL
`gentamicin with 95% encapsulation.28 Each rabbit received
`an intravitreal injection of 100 mg liposome-encapsulated
`gentamicin or 100 mg gentamicin in 0.1 mL of phosphate-
`buffered saline. The peak free drug concentration in the
`vitreous was significantly greater for liposome-encapsulated
`gentamicin than for gentamicin at 24, 72, 120, and 192 hours.
`The areas under the drug concentration-time curve for the total
`drug and for the free drug in the case of liposome-encapsulated
`gentamicin were twofold and 1.5-fold higher, respectively,
`than those for gentamicin.
`
`Table 1 Improvement of drug pharmacokinetics by intravitreal injection of the liposomal drug
`Drug
`Observations
`Amikacin
`The half-life of the drug in vitreous;
`The toxic intravitreal dose of liposomal amphotericin B
`The concentration after intravitreal injection of various
`doses of the drug
`The concentration in the aqueous humor and vitreous
`Retinal toxicity
`The half-life of the drug
`The half-life of the drug
`The concentration of the drug in the vitreous
`The number of intravitreal injections
`The pharmacokinetics of the drug
`Retinal toxicity
`Distribution and clearance of the drug in the vitreous
`The efficacy and safety of the drug on EAU in rats
`Retention in the vitreous; concentration of the drug
`The duration of administration of the drug at
`therapeutic concentrations
`45
`Targeting in the CNv and release
`Tyrosine kinase inhibitor
`44
`The concentration and safety of the drug on EIU in rats
`vasoactive intestinal peptide
`Abbreviations: EAU, experimental autoimmune uveoretinitis; CNv, choroidal neovascularization; vIP, vasoactive intestinal peptide; EIU, endotoxin-induced uveitis.
`
`Amphotericin B
`
`Bevacizumab
`Cyclosporine
`5-fluorouridine 5′-monophosphate
`Fluconazole
`Ganciclovir
`Ganciclovir
`Gentamicin
`Ofloxacin
`Oligonucleotide
`Tacrolimus (FK-506)
`Tilisolol
`Tobramycine
`
`Reference(s)
`34
`
`35
`
`37
`38
`39, 40
`41
`32
`33
`28
`47
`31, 36
`42
`46
`43
`
`498
`
`submit your manuscript | www.dovepress.com
`Dovepress
`
`International Journal of Nanomedicine 2013:8
`
`Novartis Exhibit 2022.004
`Regeneron v. Novartis, IPR2020-01318
`
`

`

`Dovepress
`
`Nano drug delivery systems for ocular targets
`
`Bevacizumab (Avastin®, Genentech/Roche, San Francisco,
`CA, USA), molecular weight, 149 kDa, is a synthetic mono-
`clonal antibody against VEGF and has been approved by the
`FDA for colorectal cancer treatment. Intravitreal injection of
`bevacizumab has also been recently used for the treatment
`of several ocular diseases. In one study, the liposomal beva-
`cizumab concentration in the aqueous humor and vitreous
`was investigated after injection.37 The mean concentration
`of free bevacizumab in the eyes that received liposomal
`bevacizumab was onefold and fivefold higher at days 28 and
`42, respectively, than that in the eyes injected with soluble
`bevacizumab. The results of this study showed the beneficial
`effects of liposomes in prolonging the time that bevacizumab
`is retained in the vitreous.
`The authors of this review encapsulated SU5416
`(a VEGF receptor tyrosine kinase inhibitor) into PEGylated
`liposomes that had been modified to display Ala-Pro-Arg-
`Pro-Gly (APRPG) (a peptide targeting newly formed blood
`vessels)48,49 and injected the liposomes intravitreally into a
`rat experimental model of CNV to determine whether it was
`possible for the CNV-targeted drug to be released inside the
`vitreous body (Figure 4).
`First, excessive laser irradiation of the retina of Brown
`Norway (BN) rats was performed; then, APRPG-liposomal
`SU5416 and liposomal SU5416 without APRPG (0.1 mL)
`were each injected once into the vitreous body of the rats.
`At 4 days and 2 weeks postinjection, reflux staining was
`performed using fluorescein isothiocyanate (FITC-dextran),
`after which the rats’ eyes were removed, and a flat mount
`was created. Using fluorescence microscopy, we determined
`whether CNV targeting occurred. Compared with liposomal
`SU5416 without APRPG, APRPG-liposomal SU5416 accu-
`mulated markedly in CNV, which suggests that APRPG-
`liposomal SU5416 could be used to target CNV lesions
`
`PEG-modified liposome
`
`APRPG-modified liposome
`
`APRPG peptide
`
`PEG
`
`Figure 4 APRPG-modified PEGylated liposomes for angiogenic vessel targeting.
`Notes: APRPG-PEG-Distearoylphosphatidylethanolamine was synthesized and
`incorporated into liposomes to prepare APRPG-modified PEGylated liposomes. The
`APRPG peptide was originally identified by in vivo biopanning using a phage-displayed
`peptide library. PEGylated liposomes were used as nontargeted control liposomes.
`Abbreviations: APRPG, Ala-Pro-Arg-Pro-Gly; PEG, polyethylene glycol.
`
`(Figure 5). APRPG-liposomal SU5416 was still found in
`the choroid membrane of the rats even at 2 weeks after the
`intravitreal injection.45
`In addition, the following experiments were performed to
`examine the inhibitory effect of APRPG-liposomal SU5416
`on CNV.45 APRPG-liposomal SU5416, balanced-salt solu-
`tion (BSS), APRPG-liposomes, and soluble SU5416 were
`injected once into the vitreous body of CNV model rats at
`a dose of 0.1 mL.
`At 1 week and 2 weeks after the injections, flat mounts
`were created using the method described above, and the
`inhibitory effect on CNV was examined by using a fluores-
`cence microscope to measure the area occupied by CNV.
`The inhibitory effect on CNV at 1 week after injection was
`almost the same as that in the group injected with APRPG-
`liposomal SU5416 and in the group injected with soluble
`SU5416. However, at 2 weeks after injection, CNV was only
`significantly inhibited in the group injected with APRPG-
`liposomal SU5416 (Figure 6).
`Barriers to the use of liposomes include the blurring
`of vision after injection of the suspension into the vitre-
`ous body; the limited storage conditions, depending on
`the composition of the drug and the liposomes; the poor
`maintenance of efficiency when a water-soluble substance
`is encapsulated; the predominant usage of cationic lipo-
`somes in gene delivery;50 and potential proinflammatory
`effects.
`
`Figure 5 Double labeling of liposomal SU5416 (red) and choroidal vascularization
`(green).
`Notes: Localization of APRPG-liposomal SU5416 at 4 days after intravitreal injection.
`Asterisks: optic nerve; arrows: CNv region; arrowheads: normal choroidal vessels
`Copyright © 2011. American Medical Association. All rights reserved. Honda M, Asai T,
`Umemoto T, Araki Y, Oku N, Tanaka M. Suppression of choroidal neovascularization
`by intravitreal injection of liposomal SU5416. Arch Ophthalmol. 2011;129(3):317–321.45
`Abbreviations: APRPG, Ala-Pro-Arg-Pro-Gly; CNv, choroidal neovascularization.
`
`International Journal of Nanomedicine 2013:8
`
`submit your manuscript | www.dovepress.com
`Dovepress
`
`499
`
`Novartis Exhibit 2022.005
`Regeneron v. Novartis, IPR2020-01318
`
`

`

`Honda et al
`
`Dovepress
`
`micrometers or less. In some cases, particles with a smaller
`diameter are also considered nanospheres.4 Drugs are encap-
`sulated in synthetic and natural polymers to permit sustained
`local release and tissue targeting of the drugs. Micro/nano-
`capsules are similar to microspheres, and both names are
`used without a clear distinction. However, in the strict sense
`of the terms, microspheres are multinuclear microcapsules.
`The most common substrates are poly(lactic acid) (PLA),
`polyglycolic acid (PGA), and their copolymer, poly(lactic-
`co-glycolic acid) (PLGA). These substrates are nonenzymati-
`cally hydrolyzed and degraded in vivo. Intravitreally injected
`PLA and PLGA do not exhibit electrophysiological or
`histological toxicity in the retina.51,52
`A GCV intraocular implant is the first FDA-approved
`sustained-release formulation (Vitrasert®; Bausch and Lomb,
`Rochester, NY, USA) that is nondegradable in vivo and is
`being used in the treatment of cytomegalovirus retinitis in
`AIDS patients. This device, which uses the biodegradable
`polymers ethylene vinyl acetate (EVA) and polyvinyl alcohol
`(PVA), is implanted through a 5.5-mm scleral incision gener-
`ated using a pulse planner and allows the sustained release
`of 450 mg of GCV for 6 to 8 months.53
`However, in some cases, if CMV retinitis is not alleviated
`after the sustained release of the drug, removal of the device
`followed by insertion of a new device is often considered.
`In previous reports, the sub-Tenon capsule placement of
`Vitrasert using a 8-0 nylon suture, followed by removal of
`the device after sustained release of the drug, did not result
`in any complications.54
`The in-vivo biodegradable implant Ozurdex® allows the
`sustained intravitreal release of 350 mg and/or 700 mg of
`dexamethasone. Visual acuity and retinal thickness have both
`been reported to show a marked improvement at 180 days
`after the intravitreal injection of Ozurdex for the treatment
`of macular edema associated with retinal vein occlusion.55
`The efficacy of Ozurdex against uveitis56 and diabetic
`retinopathy57 has also been confirmed.
`After intravitreal injection, microspheres are likely to be
`trafficked to retinal pigment epithelial cells.58,59 The tissue dis-
`tribution of microspheres after intravitreal injection depends
`on the diameter of the particles. When fluorescent 2000 nm,
`200 nm, and 50 nm nanospheres were injected into the vitre-
`ous body of rabbits, the 2000 nm particles were found in the
`intravitreal cavity and the trabecula, whereas the 200 nm and
`50 nm particles were found even inside the retina.60
`When devices that are non-degradable in vivo are used,
`drug release is stable for a long period, but surgical removal
`of the device after sustained drug release is generally difficult
`
`*
`
`*
`
`BSS
`
`APRPG-Lip
`
`Soluble-
`SU5416
`
`APRPG-
`Lip-SU5416
`
`*
`
`*
`
`BSS
`
`APRPG-Lip
`
`Soluble-
`SU5416
`
`APRPG-
`Lip-SU5416
`
`A
`
`30
`
`20
`
`10
`
`0
`
`B
`
`40
`
`30
`
`20
`
`10
`0
`
`Mean choroidal neovascular membrane area (x103 µm2)
`
`Figure 6 Quantitative analysis of the CNv area in each experimental group. Data
`obtained at 1 week (A) and 2 weeks (B) after intravitreal injection.
`Notes: The results are expressed as simple average CNv area ± SE (n = 6). The asterisks
`show statistically significant differences between the indicated groups (P , 0.05).
`Copyright © 2011. American Medical Association. All rights reserved. Honda M, Asai T,
`Umemoto T, Araki Y, Oku N, Tanaka M. Suppression of choroidal neovascularization
`by intravitreal injection of liposomal SU5416. Arch Ophthalmol. 2011;129(3):317–321.45
`Abbreviations: CNv, choroidal neovascularization; SE, standard error; BSS,
`balanced-salt solution; APRPG, Ala-Pro-Arg-Pro-Gly; Lip, liposome.
`
`Visudyne® (Valeant Pharmaceuticals Int, Montreal,
`Canada; Novartis AG, Basel, Switzerland) (principal agent:
`verteporfin), which is a liposomal formulation that is used
`in the treatment of AMD but not administered through intra-
`vitreal injection, has already been released on the market.
`When intravenously administered Visudyne is activated
`by irradiation of the diseased region (with a nonthermal
`laser), selective occlusion of newly formed blood vessels
`occurs, resulting in a decrease in bleeding and exudation.
`The formulation is a lyophilized product and contains 15 mg
`of the principal agent per vial; the liposomal membrane is
`composed of egg yolk phosphatidylglycerol (egg yolk PG)
`and dimyristoylphosphatidylcholine (DMPC), and other
`additives include lactose, palmitic acid, ascorbic acid, and
`dibutyl hydroxytoluene. In this case, liposomes are used to
`formulate hydrophobic verteporfin in an injectable form for
`intravenous injection.
`
`Micro/nanospheres
`Microspheres are spherical preparations in which particles
`have a diameter ranging from 1 µm to several hundreds of
`
`500
`
`submit your manuscript | www.dovepress.com
`Dovepress
`
`International Journal of Nanomedicine 2013:8
`
`Novartis Exhibit 2022.006
`Regeneron v. Novartis, IPR2020-01318
`
`

`

`Dovepress
`
`Nano drug delivery systems for ocular targets
`
`to achieve. When biodegradable implants are used, they do
`not need to be removed after sustained drug release; however,
`in comparison with implants that are non-biodegradable
`in vivo, intravitreal drug concentration is unstable, and the
`sustained-release period is shorter.61
`
`Other nanocarriers
`Microemulsions
`A microemulsion is a type of dispersion system composed of
`two types of liquids. The diameter of the micelles is as low as
`approximately 100 nm or less. These micelles are thermody-
`namically stable and can be formed easily without the need
`for strong agitation. They look transparent or semitransparent
`because of the limited dispersion of visible light arising from
`their small size.62 They comprise three components, including
`two types of immiscible substances (represented by water [W]
`and oil [O]) and a surfactant. In some cases, they may contain
`auxiliary agents. The properties of microemulsions depend on
`the nature and composition of these components.
`Microemulsions have good tissue permeability because of
`the small size of the micelles and the presence of a surfactant
`among the components; as a result, studies on DDSs have
`been conducted mainly in the field of ophthalmic drugs.63
`The instillation of dexamethasone-containing microemul-
`sions in the eyes of rabbits has been shown to result in
`enhanced intraocular permeability.64
`Because sterilization is normally performed by autoclaving,
`microemulsions are unsuitable when the drug is water-soluble
`or insoluble (does not dissolve in water or oil), thermolabile, or
`if the drug should appear transparent externally. In comparison
`with microspheres and liposomes, microemulsions are also
`unsuitable for long-term sustained drug release.
`
`Dendrimers
`Dendrimers are repetitive/single molecules with a regularly
`branched structure, and they are composed of a central
`
`mole cule known as the “core” and side chain moieties known
`as “dendrons.” Dendrimers are included in the category of
`polymer compounds, and they have a spherical structure
`and homogenous molecular weight. Hydrophobic drugs can
`be incorporated into the core. In addition, their chemical
`structure, physical properties, and size can be controlled at
`the molecular level, and attempts to use them as drug carriers
`are underway.65,66
`Lipophilic and cationic dendrimers have been used to
`mediate the delivery of a sense oligonucleotide, ODN-1, and
`to inhibit the expression of VEGF (in vitro). Furthermore,
`when fluorescence leakage from the CNV was evaluated in
`a rat laser model, using fluorescein fundus angiography after
`intravitreal injection of two types of dendrimers selected from
`in vitro experiments, fluorescence leakage from CNV was
`prominently inhibited in both cases.67
`Dendrimers can incorporate a lower amount of drugs than
`other carriers. In addition, among the dendrimers synthe-
`sized so far, few have been proven to be safe in vivo when
`compared with liposomes and microspheres. As described
`above, advantages and disadvantages of the different types
`of nanocarriers are listed in Table 2.
`
`Future prospects
`With the introduction of the intravitreal injection of VEGF
`inhibitors, an improvement in visual acuity has been achieved
`in many patients. However, discontinuation of the injection of
`anti-VEGF antibodies often results in recurrence of CNV, and
`in some cases, the treatment has to be continued for the rest
`of the patient’s life. The frequent and long-term intravitreal
`injection of drugs poses a heavy burden on the patient, not only
`in terms of complications but also financially, emotionally, and
`physically. DDSs are considered to be essential for overcom-
`ing the current limitations with regard to drug efficacy, and
`the currently used method of sustained release of anti-VEGF
`antibodies is considered to have great potential.
`
`Liposome
`
`Table 2 Advantages and disadvantages of different types of nanocarriers
`Advantages
`• Low toxicity and antigenicity
`• Biodegradable and metabolized in vivo
`• Can prolong the drug half-life in the vitreous
`• Reduces drug toxicity
`• Drug release is stable for a long time (nondegradable)
`
`Micro/nanosphere
`
`Microemulsion
`Dendrimer
`
`• Good tissue permeability
`• Physical properties and size can be
`controlled at the molecular level
`
`Disadvantages
`• Blurring of vision after intravitreous injection
`• Limited storage conditions
`
`• Need to be removed surgically (nondegradable)
`• Sustained release period is short (biodegradable)
`• Unsuitable for long-term sustained drug release
`• Conjugates low amount of drugs
`• Safety is unclear in vivo
`
`International Journal of Nanomedicine 2013:8
`
`submit your manuscript | www.dovepress.com
`Dovepress
`
`501
`
`Novartis Exhibit 2022.007
`Regeneron v. Novartis, IPR2020-01318
`
`

`

`Honda et al
`
`Dovepress
`
`Intraocular implants in the posterior segment of the
`eye, which are promising for sustained drug action, have
`already been used for clinical applications. Implants with
`the same shape as that of Vitrasert and that ensure a sus-
`tained release of fluocinolone acetonide are also used in
`the treatment of uveitis, and recently, clinical trials have
`been initiated for their use in the treatment of macular
`edema. Moreover, implants having this shape might be
`used for sustained release of drug candidates for the treat-
`ment of AMD.
`More potent drugs might be required for use in the new
`devices. In the future, there will be a growing demand for
`DDS formulations using biodegradable implants, partic