USOO7531173B2
`
`(12) United States Patent
`Wiegand et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,531,173 B2
`*May 12, 2009
`
`(54) OPHTHALMIC COMPOSITION OF A VEGF
`ANTAGONST
`
`6,270,993 B1
`6,897,294 B2
`
`8/2001 Shibuya
`5/2005 Davis-Smyth et al.
`
`(75) Inventors: Stanley J. Wiegand, Croton-on-Hudson,
`NY (US); Jingtai Cao, Chappaqua, NY
`(US)
`
`(*) Notice:
`
`(73) Assignee: Regeneron Pharmaceuticals, Inc.,
`Tarrytown, NY (US)
`s
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`This patent is Subject to a terminal dis-
`claimer
`
`(21) Appl. No.: 11/998,746
`
`(22) Filed:
`
`(65)
`
`Nov.30, 2007
`O
`O
`Prior Publication Data
`US 2008/0085276 A1
`Apr. 10, 2008
`O
`O
`Related U.S. Application Data
`(62) Division of application No. 1 1/346,009, filed on Feb.
`2, 2006, now Pat. No. 7,303,748.
`(60) frnal application No. 60/649,232, filed on Feb.
`s
`(51) Int. Cl
`(2006.01)
`A6 IK 38/18
`(2006.01)
`C07K I4/7
`(2006.01)
`CI2N 5/62
`(52) U.S. Cl. ..................... 424/134.1; 424/192.1; 514/2:
`514/12 530/350:536/23.4
`s
`s
`(58) Field of Classification Search ............... ... None
`See application file for complete search history.
`References Cited
`
`(56)
`
`U.S. PATENT DOCUMENTS
`
`5,851,999 A 12/1998 Ullrich et al.
`6,011,003 A
`1/2000 Charnock-Jones
`
`FOREIGN PATENT DOCUMENTS
`WO97/.44453
`11, 1997
`WO
`WO98, 13071
`4f1998
`WO
`WO99/13909
`3, 1999
`WO
`WOOOf75319
`12/2000
`WO
`WOO3,O72029
`9, 2003
`WO
`WO WO 2004/106378
`12, 2004
`OTHER PUBLICATIONS
`Phillips, A.J. (2001). The challenge of gene therapy and DNA deliv
`ery. J. Pharm. Pharmacol. 53:1169-1174.*
`Palu et al. (1999). In pursuit of new developments for genetherapy.J.
`Biotechnol. 68:1-13.
`Holash, J., et al., (2002) PNAS, 99(17): 11393-11398.
`Heidaran, M.A., et al., (1990).J. Biol. Chem. 265(31): 18741-18744.
`Cunningham, S.A., et al., (1997) Biochem. Biophys. Res. Comm.
`231:596-599.
`Fuh, G., et al., (1998) J. Biol. Chem. 273(18): 11 197-11204.
`Wiesmann, C., et al., (1997) Cell, 91.695-704.
`Barleon, B., et al., (1997) J. Biol. Chem. 272(16):10382-10388.
`Davis-Smyth, T., et al., (1998) J. Biol. Chem. 273(6):3216-3222.
`Wulff, C., et al., (2002) Endocrinology 143(7):2797-2807.
`Yatoh, S., et al., (1998) Transplantation 66(11): 1519-1524.
`Cao, J., et al., (2004) Investigative Ophthalmolgy and VisualScience,
`45(Suppl. 1):U922.
`* cited by examiner
`Primary Examiner Christine J Saoud
`Assistant Examiner—Jon M Lockard
`(74) Attorney, Agent, or Firm Valeta Gregg
`
`ABSTRACT
`(57)
`Methods of reducing or treating angiogenesis and/or inflam
`mation associated with eye injury in a subject in need thereof,
`comprising administering an agent capable of blocking or
`inhibiting vascular endothelial growth factor (VEGF) are pro
`vided. The methods are useful for inhibiting or ameliorating
`eye injury, particularly acute or Subacute corneal injury and
`feature local administration (for example, Subconjunctival
`injection or eye drops).
`
`2 Claims, 7 Drawing Sheets
`
`CELLTRION - EXHIBIT 1008
`
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`U.S. Patent
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`

`1.
`OPHTHALMC COMPOSITION OF A VEGF
`ANTAGONST
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a divisional of U.S. Pat. application Ser.
`No. 1 1/346,009, filed 2 Feb. 2006, now U.S. Pat. No. 7,303,
`748, which claims the benefit under 35 USC S 119(e) of U.S.
`Provisional 60/649.232 filed 2 Feb. 2005, which applications
`are herein incorporated by reference.
`
`BACKGROUND
`
`1. Field of the Invention
`The field of the invention is related to local administration
`of VEGF antagonists to treat eye-related diseases, disorders
`and injuries.
`2. Description of Related Art
`It has previously been reported that topical application of
`an anti-VEGF neutralizing antibody Suppresses acute
`allograft rejection in a rat corneal transplant model (Yatoh et
`al. (1998) Transplantation 66(11):1519-24).
`
`10
`
`15
`
`BRIEF SUMMARY OF THE INVENTION
`
`The invention is based in part on the finding that local
`administration of an agent capable of blocking, inhibiting, or
`reducing the activity of vascular endothelial growth factor
`(VEGF) is useful in treating of angiogenesis and inflamma
`tion associated with eye injuries or infection.
`In a first aspect, the invention features a method of treating
`an eye injury, comprising locally administering an effective
`amount of an agent capable of blocking or inhibiting vascular
`endothelial growth factor (VEGF)-mediated activity to a sub
`ject in need thereof. Such that the eye injury is ameliorated or
`improved. Preferably, the eye injury is a corneal injury or
`conjunctival injury and the method of treatment reduces
`angiogenesis and inflammation associated with the eye
`injury.
`In specific embodiments, the agent capable of blocking,
`inhibiting, or ameliorating VEGF-mediated activity is a
`VEGF antagonist comprising a fusion polypeptide selected
`from the group consisting of acetylated Flt-1(1-3)-Fc, Flt-1
`(1-3 -)-Fc, Flt-1 (1-3A)-Fc., Flt-1 (2-3A)-Fc, Flt-1 (2-3)-
`Fc, Flt-1D2-VEGFR3D3-FcAC1(a), Flt-1D2-Flk-1D3
`FcAC1 (a), and VEGFR1R2-FcAC1(a). In a specific and
`preferred embodiment, the VEGF trap is VEGFR1R2-FcAC1
`(a) (also termed VEGF trap) comprising the nucleotide
`sequence set forth in SEQ ID NO: 1 and the amino acid
`sequence set forth in SEQID NO: 2. The invention comprises
`the use of a VEGF trap that is at least 90%. 95%, 98%, or at
`least 99% homologous with the nucleotide sequence set forth
`in SEQID NO: 1 and/or the amino acid sequence set forth in
`SEQID NO:2.
`The method of the invention is useful to treat acute and
`Sub-acute corneal injury or conjunctival injury. Acute corneal
`injury may be treated within 24 hours of occurrence, and
`includes corneal injury or conjunctival injury caused by a
`penetrating object, a foreign body, or a chemical or burn
`injury. A Sub-acute injury may be treated up to two weeks
`post-injury and may include the above listed injuries as well
`as infectious etiologies.
`In various embodiments, the eye injury is caused by
`trauma, e.g., Surgical injuries, chemical burn, corneal trans
`plant, infectious or inflammatory diseases.
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`US 7,531,173 B2
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`2
`Length of treatment will vary according to the injury, but
`treatment duration may be short, e.g., up to one month, and
`may include a 3-6 month observation period, during which
`retreatment may be provided.
`Administration may also include a secondagent, such as an
`immunosuppressive agent, for example, one or more of a
`corticosteroid, dexamethasone, or cyclosporin A.
`Local administration includes, for example, administration
`of the VEGF antagonist in eye drops applied to the eye, or
`Subconjunctival injection to the eye.
`In a second aspect, the invention features a method of
`healing an eye injury, comprising locally administering an
`effective amount of an agent capable of blocking or inhibiting
`vascular endothelial growth factor (VEGF)-mediated activity
`to a subject in need thereof. Such that the eye injury heals.
`In a third aspect, the invention features a method of reduc
`ing or ameliorating angiogenesis associated with an eye
`injury, comprising locally administering an effective amount
`of an agent capable of blocking or inhibiting vascular endot
`helial growth factor (VEGF)-mediated activity to a subject in
`need thereof. Such that the angiogenesis associated with the
`eye injury is reduced or ameliorated.
`In a fourth aspect, the invention features a method of reduc
`ing or ameliorating inflammation associated with an eye
`injury, comprising locally administering an effective amount
`of an agent capable of blocking or inhibiting vascular endot
`helial growth factor (VEGF)-mediated activity to a subject in
`need thereof, such that the inflammation associated with the
`eye injury is reduced or ameliorated.
`In a fifth aspect, the invention features an ophthalmic com
`position comprising a VEGF antagonist, for example the
`VEGF trap VEGFR1R2-FcAC1(a), in a pharmaceutically
`acceptable carrier. Such pharmaceutical compositions may
`be liquid, gel, ointment, salve, slow release formulations or
`other formulations suitable for ophthalmic administration. In
`various embodiments, the pharmaceutical composition is for
`local administration comprising a VEGF trap, buffer, stabi
`lizer, isotonizer, and a pharmaceutical carrier. In a preferred
`embodiment, the pharmaceutical composition is adminis
`tered in the form of eye drops. In specific embodiments, the
`pharmaceutically acceptable carrier comprises as least one
`ophthalmically acceptable excipient, wherein the ophthalmi
`cally acceptable excipient can reduce a rate of removal of the
`VEGF antagonist from the eye by lacrimation. In various
`preferred embodiments, the pharmaceutical composition has
`an effective residence time in the eye of about 2 to about 24
`hours.
`In other embodiments, the pharmaceutical composition is
`for Subconjunctival administration Such as Subconjunctival
`injection and Subconjunctival implantation.
`In a sixth aspect, the invention features a method of admin
`istering a VEGF antagonist for treatment of angiogenesis
`and/or inflammation associated with eye injury or infection,
`comprising local administration by eye drops comprising a
`VEGF trap, or subconjunctival administration by injection or
`implantation.
`Other objects and advantages will become apparent from a
`review of the ensuing detailed description.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`FIG. 1. Percent of vascularized corneal area in sutured
`mice subconjunctivally (SubC) treated with vehicle only or
`VEGF trap, at dosing regimens of (A) three 40 ug or (B) three
`10 ug doses.
`
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`3
`FIG. 2. Percent of neovascularized cornea at day 9 in
`sutured rats treated SubC with vehicle only or treated with
`VEGF trap, with a dosing regimen of 10 ug on the day of
`Suturing.
`FIG. 3. Blood vessel length in sutured rats treated at day 0,
`3 and 6 with 25, 50, or 100 ug VEGF trap injections admin
`istered subcutaneously (SC) or subconjunctivally (SubC).
`(Control-right non-sutured eye).
`FIG. 4. Quantification of blood vessellength in sutured rats
`receiving Subconjunctival normal saline, 5ug, 25ug, or 100
`ug VEGF trap on day 0, 3 and 6. (Control-right non-sutured
`eye).
`FIG. 5. Quantification of corneal edema as evidenced by
`cornealthickness in sutured rats receiving 25 or 100 ug VEGF
`trap SC or normal saline, 5ug. 25 ug, or 100 ug VEGF trap
`SubC. (Control-right non-sutured eye) (NS=sutured eye,
`normal saline administered SubC).
`FIG. 6. Percentreduction of edema. Effect of VEGF trap on
`inflammation as determined by measurement of corneal
`thickness. All animals were sutured (control Sutured--sys
`temic injection (SC) of normal saline).
`FIG. 7. Blood vessel length in suture-injury. Control—no
`Suture
`injury.
`Suture control Suture--no
`treatment.
`Vehicle-suture-injury--vehicle provided as eye drops. VEGF
`trap-suture-injury--1 drop three times per day (412 ug VEGF
`25
`trap protein/drop).
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`DETAILED DESCRIPTION
`
`4
`Suture injury being comparable to that of a normal untreated
`(nonsutured) cornea. Treatment with VEGF trap eye drops
`following Suture injury also effectively reduced neovascular
`ization in Suture-injured cornea.
`In addition to quantification of neovascularization as mea
`sured by an increase in either blood vessel length or blood
`vessel area, Suture-injury produced a marked influx of leuco
`cytes into the injury site. When VEGF trap was administered
`locally either by subconjunctival injection (SubC) or by eye
`drop, a dramatic reduction in leucocyte infiltration was
`observed (data not shown).
`In addition to the measurements reported below, serum
`levels of VEGF trap were determined in animals treated by
`subconjunctival injection or eye drops of VEGF trap. As
`evidenced by ELISA measurement for free VEGF trap in
`serum, there is little or no systemic exposure when VEGF trap
`is delivered at the effective doses by either of these local
`(SubC or eye drops) routes.
`Definitions
`The phrase “therapeutically effective dose' includes a dose
`that produces the desired effect for which it is administered.
`The exact dose will depend on the purpose of the treatment,
`and will be ascertainable by one skilled in the art using known
`techniques (see, for example, Lloyd (1999) The Art, Science
`and Technology of Pharmaceutical Compounding).
`The term “blocker”, “inhibitor', or “antagonist are used
`interchangeably to mean a substance that retards or prevents
`a chemical or physiological reaction or response. Common
`blockers or inhibitors comprise, but are not limited to, anti
`sense molecules, antibodies, antagonists and their deriva
`tives. More specifically, an example of a VEGF blocker or
`inhibitor includes a VEGF receptor-based antagonist com
`prising, for example, an anti-VEGF antibody, or a VEGF trap
`such as VEGFR1R2-FcAC1(a) (SEQID NOS:1-2).
`The phrase “ophthalmically acceptable' with respect to a
`formulation, composition or ingredient herein means having
`no persistent effect that is substantially detrimental to the
`treated eye or the functioning thereof, or on the general health
`of the subject being treated. It will be recognized that tran
`sient effects such as minor irritation or a 'stinging sensation
`are common with topical ophthalmic administration of drugs
`and the existence of such transient effects is not inconsistent
`with the formulation, composition or ingredient in question
`being “ophthalmically acceptable' as herein defined. How
`ever, preferred formulations, compositions and ingredients
`are those that cause no substantial detrimental effect, even of
`a transient nature.
`VEGF Antagonists
`In various embodiments, the VEGF trap is selected from
`the group consisting of acetylated Flt-1 (1-3)-Fc, Flt-1 (1-
`3 -)-Fc, Flt-1 (1-3A)-Fc, Flt-1 (2-3A)-Fc, Flt-1 (2-3)-Fc.
`Flt-1D2-VEGFR3D3-FcAC1(a), Flt-1D2-Flk-1D3-FcAC1
`(a), and VEGFR1R2-FcAC1(a). For a more detailed descrip
`tion of these and other VEGF-receptor-based antagonists,
`including pegylated receptor-based blockers, see PCT
`WO/00/75319, the contents of which are incorporated in their
`entirety herein by reference.
`In addition to the VEGF receptor-based antagonists dis
`closed in PCT WO/00/75319, which publication is herein
`specifically incorporated by reference in its entirety, variants
`and derivatives of such VEGF receptor-based blockers are
`also contemplated by the invention. The sequence of the
`variants or derivatives may differ by a change that can be one
`or more additions, insertions, deletions and/or Substitutions
`of one or more nucleotides of the sequence set forth in SEQ
`ID NO:1. Changes to a nucleotide sequence may result in an
`
`Before the present methods are described, it is to be under
`stood that this invention is not limited to particular methods,
`and experimental conditions described, as Such methods and
`conditions may vary. It is also to be understood that the
`terminology used herein is for the purpose of describing
`particular embodiments only, and is not intended to be limit
`ing, since the scope of the present invention will be limited
`only by the appended claims.
`As used in this specification and the appended claims, the
`singular forms “a”, “an', and “the' include plural references
`unless the context clearly dictates otherwise. Thus for
`example, a reference to “a method’ includes one or more
`methods, and/or steps of the type described herein and/or
`which will become apparent to those persons skilled in the art
`upon reading this disclosure and so forth.
`Unless defined otherwise, all technical and scientific terms
`used herein include the same meaning as commonly under
`stood by one of ordinary skill in the art to which this invention
`belongs. Although any methods and materials similar or
`equivalent to those described herein can be used in the prac
`tice or testing of the present invention, preferred methods and
`materials are now described. All publications mentioned
`herein are incorporated herein by reference in their entirety.
`General Description
`Experiments were undertaken to evaluate corneal neovas
`cularization after Surgical Suture placement in the cornea and
`to test whether corneal neovascularization following Suture
`injury can be Suppressed by local administration of an agent
`capable of blocking, inhibiting, or ameliorating VEGF-me
`diated activity. As described in the experimental section
`below, corneas of male C57BL/6 mice or Sprague-Dawley
`rats were Suture-injured. A molecular trap designed to inhibit
`VEGF-A activity was administered locally and tested for its
`ability to suppress corneal vascularization. The results
`revealed that Sutured cornea receiving Subconjunctival
`administration of VEGF trap exhibited little or no neovascu
`larization; corneal vascular area and vessel length following
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`amino acid change at the protein level, or not, as determined
`by the genetic code. Thus, nucleic acid according to the
`present invention may include a sequence different from the
`sequence shown in SEQID NO:1, yet encode a polypeptide
`with the same amino acid sequence as SEQID NO: 2. On the
`other hand, the encoded polypeptide may comprise an amino
`acid sequence which differs by one or more amino acid resi
`dues from the amino acid sequence shown in SEQID NO:2.
`A nucleic acid encoding a polypeptide which is an amino acid
`sequence variant or derivative of the sequence shown in SEQ
`10
`ID NO:2 is further provided by the present invention. A
`nucleic acid encoding Such a polypeptide may show at the
`nucleotide sequence and/or encoded amino acid level greater
`than about 90%. 95%, 98%, or 99% homology with the cod
`ing sequence shown in SEQID NO:1 and/or the amino acid
`sequence shown in SEQID NO:2. Amino acid “homology’.
`may be understood to be similarity (according to the estab
`lished principles of amino acid similarity, e.g. as determined
`using the algorithm GAP (Genetics Computer Group, Madi
`son, Wis.)) or identity. GAP uses the Needleman and Wunsch
`algorithm to align two complete sequences that maximizes
`the number of matches and minimizes the number of gaps.
`Generally, the default parameters are used, with a gap cre
`ation penalty—12 and gap extension penalty 4.
`Individual components of the VEGF-specific fusion pro
`teins of the invention may be constructed by molecular bio
`logical methods known to the art with the guidance provided
`by the instant specification. These components are selected
`from a first cellular receptor protein, Such as, for example,
`VEGFR1; a second cellular receptor protein, such as, for
`example, VEGFR2; and a multimerizing component. Such as,
`for example, an Fc.
`Specific embodiments of the VEGF-specific fusion pro
`teins useful in the methods of the invention comprise a mul
`timerizing component which allows the fusion proteins to
`associate, e.g., as multimers, preferably dimers. Preferably,
`the multimerizing component comprises an immunoglobu
`lin-derived domain. Suitable multimerizing components are
`sequences encoding an immunoglobulin heavy chain hinge
`region (Takahashi et al. 1982 Cell 29:671-679); immunoglo
`bulin gene sequences, and portions thereof.
`The nucleic acid constructs encoding the fusion proteins
`useful in the methods of the invention can be inserted into an
`expression vector by methods known to the art, wherein the
`nucleic acid molecule can be operatively linked to an expres
`sion control sequence. Host-vector Systems for the produc
`tion of proteins comprising an expression vector introduced
`into a host cell suitable for expression of the protein are
`known in the art. The suitable host cell may be a bacterial cell
`Such as E. coli, a yeast cell. Such as, for example, Pichia
`pastoris, an insect cell. Such as, for example, Spodoptera
`frugiperda, or a mammalian cell. Such as, for example, a
`COS, CHO, 293, BHK or NS0 cell.
`Methods of Administration
`The invention comprises methods of treatment comprising
`administering to a subject an effective amount of an agent of
`the invention. In a preferred aspect, the agent is substantially
`purified (e.g., Substantially free from Substances that limit its
`effect or produce undesired side-effects). The subject is pref
`erably an animal, e.g., Such as cows, pigs, horses, chickens,
`cats, dogs, etc., and is preferably a mammal, and most pref
`erably human.
`Preferably, the pharmaceutical compositions of the inven
`tion are administered to the area in need of treatment by
`65
`topical administration. Topical drug delivery is the most com
`mon treatment for diseases or disorders of the anterior seg
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`ment of the eye, including, for example, corneal diseases,
`uveitis, and glaucoma. Topical delivery can be a safer and
`more convenient delivery method for patients, and can reduce
`the risk of many side effects observed in systemic treatment
`regimens. Topical administration of an angiogenesis inhibitor
`to the eye or cornea can be an effective treatment for treating
`neovascularization and/or inflammation. A preferred method
`of administering the pharmaceutical compositions of the
`invention to the eye is by eye drops comprising a VEGF trap.
`In various preferred embodiments, the pharmaceutical
`compositions of the invention are administered to the area in
`need of treatment by subconjunctival administration. One
`preferred method of subconjunctival administration to the eye
`is by injectable formulations comprising a VEGF trap.
`Another preferred method of subconjunctival administration
`is by implantations comprising slow releasing VEGF trap.
`Pharmaceutical Compositions
`Pharmaceutical compositions useful in the practice of the
`method of the invention include a therapeutically effective
`amount of an active agent with a pharmaceutically acceptable
`carrier. The term “pharmaceutically acceptable” means
`approved by a regulatory agency of the Federal or a state
`government or listed in the U.S. Pharmacopeia or other gen
`erally recognized pharmacopeia for use in animals, and more
`particularly, in humans. The term “carrier refers to a diluent,
`adjuvant, excipient, or vehicle with which the therapeutic is
`administered. Examples of Suitable pharmaceutical carriers
`are described in “Remington’s Pharmaceutical Sciences” by
`E. W. Martin. In a preferred embodiment, the composition is
`formulated in accordance with routine procedures as a phar
`maceutical composition adapted for topical administration to
`human beings. Such pharmaceutical compositions may be
`liquid, gel, ointment, salve, slow release formulations or other
`formulations suitable for ophthalmic administration. The
`composition comprises an effective amount of VEGFantago
`nist and, optionally, at least one ophthalmically acceptable
`excipient, wherein the excipient is able to reduce a rate of
`removal of the composition from the eye by lacrimation, Such
`that the composition has an effective residence time in the eye
`of about 2 hours to about 24 hours.
`In various embodiments, compositions of the invention can
`comprise a liquid comprising an active agent in solution, in
`Suspension, or both. The term 'suspension' herein includes a
`liquid composition wherein a first portion of the active agent
`is present in Solution and a second portion of the active agent
`is present in particulate form, in Suspension in a liquid matrix.
`As used herein, liquid compositions include gels.
`Preferably the liquid composition is aqueous. Alterna
`tively, the composition can take form of an ointment. In a
`preferred embodiment, the composition is an in situ gellable
`aqueous composition, more preferably an in situ gellable
`aqueous solution. Such a composition can comprise agelling
`agent in a concentration effective to promote gelling upon
`contact with the eye or lacrimal fluid in the exterior of the eye.
`Suitable gelling agents non-restrictively include thermoset
`ting polymers such as tetra-substituted ethylene diamine
`block copolymers of ethylene oxide and propylene oxide
`(e.g., poloxamine 1307); polycarbophil; and polysaccharides
`Such as gellan, carrageenan (e.g., kappa-carrageenan and
`iota-carrageenan), chitosan and alginate gums. The phrase
`“in situ gellable' includes not only liquids of low viscosity
`that can form gels upon contact with the eye or with lacrimal
`fluid in the exterior of the eye, but also more viscous liquids
`Such as semi-fluid and thixotropic gels that exhibit Substan
`tially increased Viscosity or gel stiffness upon administration
`to the eye or area surrounding the eye.
`
`

`

`7
`Aqueous compositions of the invention have ophthalmi
`cally compatible pH and osmolality. Preferably these compo
`sitions incorporate means to inhibit microbial growth, for
`example through preparation and packaging under sterile
`conditions and/or through inclusion of an antimicrobially
`effective amount of an ophthalmically acceptable preserva
`tive. Suitable preservatives non-restrictively include mer
`cury-containing Substances such as phenylmercuric salts
`(e.g., phenylmercuric acetate, borate and nitrate) and thime
`rosal; Stabilized chlorine dioxide; quaternary ammonium
`compounds such as benzalkonium chloride, cetyltrimethy
`lammonium bromide and cetylpyridinium chloride; imidazo
`lidinyl urea; parabens Such as methylparaben, ethylparaben,
`propylparaben and butylparaben, and salts thereof; phenoxy
`ethanol; chlorophenoxyethanol; phenoxypropanol; chlo
`robutanol; chlorocresol; phenylethyl alcohol; disodium
`EDTA; and sorbic acid and salts thereof.
`The composition can comprise an ophthalmic depot for
`mulation comprising an active agent for Subconjunctival
`administration. The ophthalmic depot formulation comprises
`microparticles of essentially pure active agent, e.g., the VEGF
`trap of SEQID NO:2. The microparticles comprising VEGF
`trap can be embedded in a biocompatible pharmaceutically
`acceptable polymer or a lipid encapsulating agent. The depot
`formulations may be adapted to release all of substantially all
`the active material over an extended period of time. The
`polymer or lipid matrix, if present, may be adapted to degrade
`sufficiently to be transported from the site of administration
`after release of all or substantially all the active agent. The
`depot formulation can be liquid formulation, comprising a
`pharmaceutical acceptable polymer and a dissolved or dis
`persed active agent. Upon injection, the polymer forms a deot
`at the injections site, e.g. by gelifying or precipitating.
`The composition can comprise a solid article that can be
`inserted in a suitable location in the eye, such as between the
`eye and eyelid or in the conjunctival sac, where the article
`releases the active agent. Release from Such an article is
`preferably to the cornea, either via lacrimal fluid that bathes
`the surface of the cornea, or directly to the cornea itself, with
`which the solid article is generally in intimate contact. Solid
`articles Suitable for implantation in the eye in Such fashion
`generally comprise polymers and can be bioerodible or non
`bioerodible. Bioerodible polymers that can be used in prepa
`ration of ocular implants carrying a VEGF trap in accordance
`with the present invention include without restriction ali
`phatic polyesters such as polymers and copolymers of poly
`(glycolide), poly(lactide), poly(e-caprolactone), poly(hy
`droxybutyrate) and poly(hydroxyvalerate), polyamino acids,
`polyorthoesters, polyanhydrides, aliphatic polycarbonates
`and polyether lactones. Illustrative of suitable non-bioerod
`ible polymers are silicone elastomers.
`The active agents of the invention can be formulated as
`neutral or salt forms. Pharmaceutically acceptable salts
`include those formed with free amino groups such as those
`derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
`acids, etc., and those formed with free carboxyl groups such
`as those derived from sodium, potassium, ammonium, cal
`cium, ferric hydroxides, isopropylamine, triethylamine,
`2-ethylamino ethanol, histidine, procaine, etc.
`Combination Therapies
`In various embodiments, the VEGF antagonists of the
`present invention may be administered in combination with
`one or more additional compounds or therapies or medical
`procedures. For example, Suitable therapeutic agents for use
`in combination, either alternating or simultaneously, with the
`VEGF-binding fusion proteins of the invention, including
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`US 7,531,173 B2
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`8
`topically administered immunosuppressive agents such as
`corticosteroids, dexamethasone, cyclosporin A, FK506, or
`anti-metabolic agents, (see Barker, NH, et al., (2000) Clin
`Exp Opthal 28:357-360). Other suitable therapeutic agents
`for use in combination, either alternating or simultaneously,
`with the VEGF antagonists of the invention may include
`agents that can block the biological activity of other VEGF
`family members such as VEGF-C and VEGF-D.
`Kits
`The invention also provides an article of manufacture com
`prising packaging material and a pharmaceutical agent con
`tained within the packaging material, wherein the pharma
`ceutical agent comprises at least one VEGF-specific fusion
`protein of the invention and wherein the packaging material
`comprises a label or package insert that indicates that the
`VEGF-specific fusion protein can be used for treating eye
`injury. The kit can comprise a composition comprising a
`VEGF trap and one or more other components such as, for
`example, components to be combined prior to use either by a
`health care professional or by the subject. In one embodi
`ment, the VEGF trap is combined with one or more compo
`nents that can comprise, for example, a solution included in
`the kit to reconstitute a VEGF trap in the form of an oph
`thalmical composition Suitable for topical or Subconjunctival
`administration to a human or animal. Kit components can
`comprise, for example, normal saline Solutions and/or solu
`tions comprising one or more Suitable pharmaceutical carri
`ers, stabilizers, additives, or buffers. Preferably the kit com
`prises instructions for treatment or administration regimens
`and/or instructions for preparing or reconstituting a VEGF
`trap for use. The instructions can be in writing on paper, on
`computer media of any Suitable type, as audiovisual materials
`including, for example, CD or DVD, or any other suitable
`format.
`Other features of the invention will become apparent in the
`course of the following descriptions of exemplary embodi
`ments which are given for illustration of the invention and are
`not intended to be limiting thereof.
`
`EXAMPLES
`
`The following example is put forth so as to provide those of
`ordinary skill in the art with a complete disclosure and
`description of how to make and use the methods and compo
`sitions of the invention, and are not intended to limit the scope
`of what the inventors regard as their invention. Efforts have
`been made to ensure accuracy with respect to numbers used
`(e.g., amounts, temperature, etc.) but some experimental
`errors and deviations should be accounted for. Unless indi
`cated otherwise, parts are parts by weight, molecular weight
`is average molecular weight, temperature i