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`US0075
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`US7,531,173 B2
`(10) Patent No.:
`a2) United States Patent
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`Wiegandetal.
`(45) Date of Patent:
`*May12, 2009
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`(54) OPHTHALMIC COMPOSITION OF A VEGF
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`ANTAGONIST
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`(75)
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`Inventors: Stanley J. Wiegand, Croton-on-Hudson,
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`NY (US); Jingtai Cao, Chappaqua, NY
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`(US)
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`(*) Notice:
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`(65)
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`(60)
`(51)
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`(56)
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`5,851,999 A
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`6,011,003 A
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`(73) Assignee: Regeneron Pharmaceuticals, Inc.,
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`Tarrytown, NY (US)
`°
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`Subject to any disclaimer, the term of this
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`patent is extended or adjusted under 35
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`U.S.C. 154(b) by 0 days.
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`This patent is subject to a terminal dis-
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`claimer
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`(21) Appl. No.: 11/998,746
`:
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`Novy.30, 2007
`(22)
`Filed:
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`ous
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`Prior Publication Data
`:
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`US 2008/0085276 Al
`Apr. 10, 2008
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`see
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`Related U.S. Application Data
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`(62) Division of application No. 11/346,009,filed on Feb.
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`2, 2006, now Pat. No. 7,303,748.
`icati
`7181
`f
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`srovisional application No. 60/649,232, filed on Feb.
`.
`,
`
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`Int. Cl
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`(2006.01)
`AGIK 38/18
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`
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`(2006.01)
`CO7K 14/71
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`_
`(2006.01)
`CI2N 15/62
`:
`Cc
`9
`.
`(9.
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`(52) US. Ch reece 424/134.1; 424/] 22.1 5914/2;
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`514/12; 530/350; 536/23.4
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`(58) Field of Classification Search ...............pee None
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`See applicationfile for complete search history.
`
`
`References Cited
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`12/1998 Ullrichet al.
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`8/2001 Shibuya
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`Phillips, A.J. (2001). The challenge of gene therapy and DNAdeliv-
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`Barleon,B.,et al., (1997)J. Biol. Chem. 272(16): 10382-10388.
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`Davis-Smyth,T., et al.. (1998) J. Biol. Chem. 273(6):3216-3222.
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`Wulff, C., et al., (2002) Endocrinology 143(7):2797-2807,
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`Yaloh, S., et al., (1998) Transplantation 66(1 1): 1519-1524.
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`Cao, J., et al., (2004) Investigative OphthalmolgyandVisual Science,
`
`
`45(Suppl. 1):U922.
`
`
`
`* cited by examiner
`
`
`
`Primary Examiner—Christine } Saoud
`Assistant Examiner—Ion M Lockard
`
`
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`(74) Attorney, Agent, or Firm—Valeta Gregg
`
`
`ABSTRACT
`67)
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`Methodsofreducing or treating angiogenesis and/or inflam-
`108)
`gs
`gs
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`mation associated with eye injury in a subject in need thereof,
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`comprising administering an agent capable of blocking or
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`inhibiting vascular endothelial growth factor (VLG) are pro-
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`vided. The methodsare useful for inhibiting or ameliorating
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`eye injury, particularly acute or subacute corneal injury and
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`feature local administration (for example, subconjunctival
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`injectionor eye drops).
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`2 Claims, 7 Drawing Sheets
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`CELLTRION- EXHIBIT 1008
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`CELLTRION - EXHIBIT 1008
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`

`

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`U.S. Patent
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`May 12, 2009
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`Sheet 1 of 7
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`US 7,531,173 B2
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`U.S. Patent
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`May 12, 2009
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`Sheet 2 of 7
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`US 7,531,173 B2
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`U.S. Patent
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`May 12, 2009
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`Sheet 3 of 7
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`U.S. Patent
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`May 12, 2009
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`Sheet 4 of 7
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`US 7,531,173 B2
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`May 12, 2009
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`May 12, 2009
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`Sheet 7 of 7
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`US 7,531,173 B2
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`Topical(eye-drops)
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`1
`OPHTHALMIC COMPOSITION OF A VEGF
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`ANTAGONIST
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`US 7,531,173 B2
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`This applicationis a divisional ofU.S. Pat. application Ser.
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`No. 11/346,009, filed 2 Feb. 2006, now U.S. Pat. No. 7,303,
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`748, which claimsthe benefit under 35 USC § 119(e) of US.
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`Provisional 60/649,232 filed 2 Feb. 2005, whichapplications
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`are herein incorporated by reference.
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`2
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`Length of treatment will vary according to the injury, but
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`treatment duration maybeshort, e.g., up to one month, and
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`may include a 3-6 month observation period, during which
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`retreatment may be provided.
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`Administration mayalso include a second agent, such as an
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`immunosuppressive agent, for example, one or more of a
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`corticosteroid, dexamethasone, or cyclosporin A.
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`Tocal administrationincludes, for example, administration
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`of the VEGFantagonist in eye drops applied to the eye, or
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`subconjunctival injection to the eye.
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`In a second aspect, the invention features a method of
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`healing an cye injury, comprising locally administering an
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`effective amountofan agent capable of blocking orinhibiting
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`vascular endothelial growth factor (VEGF)-mediated activity
`1. Yield of the Invention
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`to a subject in need thereof, such that the eye injury heals.
`The field ofthe invention is related to local administration
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`Inathird aspect, the invention features a methodof reduc-
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`of VEGFantagonists to treat eye-related diseases, disorders
`ing or ameliorating angiogenesis associated with an eye
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`and injuries.
`injury, comprising locally administering an effective amount
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`2. Description of Related Art
`of an agent capable of blocking or inhibiting vascular endot-
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`It has previously been reported that topical application of
`helial growth factor (VEGF)-mediatedactivity to a subject in
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`an anti-VEGF neutralizing antibody suppresses acute
`need thereof, such that the angiogenesis associated with the
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`allograft rejection in a rat corneal transplant model (Yatohet
`eye injury is reduced or ameliorated.
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`al. (1998) Transplantation 66(11):1519-24).
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`Ina fourth aspect, the invention features amethod ofreduc-
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`ing or ameliorating inflammation associated with an eye
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`injury, comprising locally administering an effective amount
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`of an agent capable of blocking or inhibiting vascular endot-
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`The invention is based in part on the finding that local
`helial growth factor (VEGF)-mediatedactivity to a subject in
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`administration of an agent capable of blocking,inhibiting, or
`need thereof, such that the inflammation associated with the
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`reducing the activity of vascular endothelial growth factor
`eye injury is reduced or ameliorated.
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`(VEGF)is useful in treating of angiogenesis and inflamma-
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`Ina fifth aspect, the invention features an ophthalmic com-
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`tion associated with eye injuries or infection.
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`position comprising a VEGF antagonist, for example the
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`Ina first aspect, the invention features a methodoftreating
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`VEGF trap VEGFRIR2-FcAC1 (a),
`in a pharmaceutically
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`an eye injury, comprising locally administering an effective
`acceptable carrier. Such pharmaceutical compositions may
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`amount ofan agent capable ofblocking or inhibiting vascular
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`be liquid, gel, ointment, salve, slow release formulations or
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`endothelial growth factor (VEGF)-mediatedactivity to a sub-
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`other formulations suitable for ophthalmic administration. In
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`ject in need thereof, such that the eye injury is ameliorated or
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`various embodiments, the pharmaceutical compositionis for
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`improved. Preferably, the eye injury is a corneal injury or
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`local administration comprising, a VEGF trap, buffer, stabi-
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`conjunctival
`injury and the method of treatment reduces
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`liver, isotonizer, and a pharmaceutical carrier. In a preferred
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`angiogenesis and inflammation associated with the eye
`embodiment,
`the pharmaceutical composition is adminis-
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`injury.
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`tered in the form of eye drops. In specific embodiments, the
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`In specific embodiments, the agent capable of blocking,
`pharmaceutically acceptable carrier comprises as least one
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`inhibiting, or ameliorating VEGF-mediated activity is a
`ophthalmically acceptable excipient, wherein the ophthalmi-
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`VEGFantagonist comprising a fusion polypeptide selected
`cally acceptable excipient can reducea rate of removal of the
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`from the group consisting of acetylated Flt-1(1-3)-Fe, Flt-1
`VEGF antagonist from the eye by lacrimation. In various
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`(1-3.,)-Fe, Fit-1(1-3,,,)-Fe, Flt-1(2-3,,,)-Fe, Flt-1(2-3)-
`preferred embodiments, the pharmaceutical composition has
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`Fe, Fit-1D2-VEGFR3D3-FcAC1 (a),—Fit-1D2-Flk-1D3-
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`an effective residence time in the eye of about 2 to about 24
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`hours.
`FeAC1(a), and VEGFR1R2-FeAC1(a). In a specific and
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`preferred embodiment, the VEGFtrap isVEGFR1IR2-FcAC1
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`In other embodiments, the pharmaceutical compositionis
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`(a) (also termed VEGFtrapz;,>) comprising the nucleotide ;
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`for subconjunctival administration such as subconjunctival
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`sequence set forth in SEQ ID NO:
`| and the amino acid
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`injection and subconjunctival implantation.
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`sequenceset forth in SEQ ID NO: 2. The invention comprises
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`Ina sixth aspect, the invention features a method of admin-
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`the use of a VEGFtrap that is at least 90%, 95%, 98%,or at
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`istering a VEGF antagonist for treatment of angiogenesis
`least 99% homologouswith the nucleotide sequenceset forth
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`and/or inflammation associated with eye injury or infection,
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`in SEQ ID NO:1 and/or the amino acid sequenceset forth in
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`comprising local administration by cye drops comprising a
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`SEQ ID NO:2.
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`VEGFtrap, or subconjunctival administration byinjection or
`The method of the invention is useful to treat acute and
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`implantation.
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`sub-acute comeal injury or conjunctival injury. Acute corneal
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`Other objects and advantages will become apparentfrom a
`injury may be treated within 24 hours of occurrence, and
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`review of the ensuing detailed description.
`includes corneal injury or conjunctival injury caused bya
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`penetrating object, a foreign body, or a chemical or burn
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`injury. A sub-acute injury maybe treated up to two weeks
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`post-injury and may include the abovelisted injuries as well
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`as infectious etiologies.
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`the eye injury is caused by
`In various embodiments,
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`trauma,e.g., surgical injuries, chemical burn, corneal trans-
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`plant, infectious or inflammatory diseases.
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`CROSS-REFERENCE TO RELATED
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`APPLICATIONS
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`a
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`BACKGROUND
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`BRIFF SUMMARY OF THE INVENTION
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`w °o
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`BRIEF DESCRIPTION OF THE FIGURES
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`FIG. 1. Percent of vascularized corneal area in sutured
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`mice subconjunctivally (SubC) treated with vehicle only or
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`VEGFtrap, at dosing regimensof (A) three 40 yg or (B) three
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`10 ug doses.
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`FIG. 2. Percent of neovascularized cornea at day 9 in
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`sutured rats treated SubC with vehicle onlyor treated with
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`VEGFtrap, with a dosing regimen of 10 ug on the day of
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`suturing.
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`FIG.3. Bloodvessel length in suturedrats treatedal day 0,
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`3 and 6 with 25, 50, or 100 ug VEGFtrap injections admin-
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`istered subcutaneously (SC) or subconjunctivally (SubC).
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`(Control=right non-sutured eye).
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`FIG.4. Quantification ofblood vessel length in suturedrats
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`receiving subconjunctival normalsaline, 5 ug, 25 wg, or 100
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`ug VEGFtrap on day0, 3 and 6. (Control=right non-sutured
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`FIG. 5. Quantification of corneal edema as evidenced by
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`corneal thickness in sutured rats receiving 25 or 100 ug VEGF
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`trap SC or normalsaline, 5 ug, 25 wg, or 100 ug VEGEtrap
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`SubC. (Control=right non-sutured eye) (NS=sutured eye,
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`normalsaline administered SubC).
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`FIG. 6. Percent reduction ofedema. Effect ofVEGFtrap on
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`inflammation as determined by measurement of comeal
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`thickness. All animals were sutured (control=sutured+tsys-
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`temic injection (SC) of normal saline).
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`FIG. 7. Blood vessel length in suture-injury. Control=no
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`injury.
`Suture
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`treatment.
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`Vehicle=suture-injury+vehicle provided as eye drops. VEGF
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`trap=suture-injury+1 drop three timesper day (412 pg VEGF
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`trap protein/drop).
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`DETAILED DESCRIPTION
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`Before the present methodsare described,it is to be under-
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`stood that this invention is notlimited to particular methods,
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`and experimental conditions described, as such methods and
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`conditions may vary. It is also to be understood that the
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`terminology used herein is for the purpose of describing
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`particular embodiments only, and is not intended to belimit-
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`ing, since the scope of the present invention will be limited
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`only bythe appended claims.
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`As used in this specification and the appended claims, the
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`singular forms “a’’, “an”, and “the” include plural references
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`unless the context clearly dictates otherwise. Thus for
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`example, a reference to “a method” includes one or more
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`methods, and/or steps of the type described herein and/or
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`which will become apparentto those personsskilled in theart
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`upon reading this disclosure and so forth.
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`Unless defined otherwise,all technical and scientific terms
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`used herein include the same meaning as commonly under-
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`stood by one of ordinary skill in the art to whichthis invention
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`belongs. Although any methods and materials similar or
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`equivalent to those described herein can be used in the prac-
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`lice or testing of the presentinvention, preferred methods and °
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`materials are now described. All publications mentioned
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`herein are incorporated herein byreference in theirentirety.
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`General Description
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`Experiments were undertaken to evaluate corneal neovas-
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`cularization after surgical suture placementin the cornea and
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`to test whether cornmeal neovascularization following suture
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`injury can be suppressed bylocal administration of an agent
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`capable of blocking, inhibiting, or ameliorating VEGF-me-
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`diated activity. As described in the experimental section
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`below, corneas of male C57BL/6 mice or Sprague-Dawley
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`rats were suture-injured. A molecular trap designedto inhibit
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`VEGF-A activity was administered locally and tested for its
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`ability to suppress corneal vascularization. The results
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`revealed that
`sutured cornea receiving subconjunctival
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`administration ofVEGFtrap exhibited little or no neovascu-
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`larization; corneal vascular area and vessel length following
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`US 7,531,173 B2
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`4
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`suture injury being comparable to that of a normal untreated
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`(nonsutured) cornea. Treatment with VEGF trap eye drops
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`following suture injury also effectively reduced neovascular-
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`ization in suture-injured cornea.
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`Tn addition to quantification of neovascularization as mea-
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`sured by an increase in either blood vessel length or blood
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`vessel area, suture-injury produced a marked influx of leuco-
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`cytes into the injurysite.When VEGFtrap was administered
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`locally either by subconjunctival injection (SubC) or by eye
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`drop, a dramatic reduction in leucocyte infiltration was
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`observed (data not shown).
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`In addition to the measurements reported below, serum
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`levels of VEGF trap were determined in animals treated by
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`subconjunctival injection or eye drops of VEGF trap. As
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`evidenced by ELISA measurement for free VEGF trap in
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`serum,thereis little orno systemic exposure when VEGFtrap
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`is delivered at the effective doses by either of these local
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`(SubC or eye drops)routes.
`Definitions
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`Thephrase“therapeuticallyeffective dose” includes a dose
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`that producesthe desired effect for which it is administered.
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`The exact dose will depend on the purposeofthe treatment,
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`and will be ascertainable by one skilled in the art using known
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`techniques (see, for example, T.loyd (1999) The Art, Science
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`and Technology of Pharmaceutical Compounding).
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`The term “blocker”, “inhibitor”, or “antagonist” are used
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`interchangeably to mean a substancethat retards or prevents
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`a chemical or physiological reaction or response. Common
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`blockers or inhibitors comprise, bul are not limitedto, anti-
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`sense molecules, antibodies, antagonists and their deriva-
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`tives. More specifically, an example of a VEGF blocker or
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`inhibitor includes a VEGF receptor-based antagonist com-
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`prising, for example, an anti-VEGFantibody, or a VEGFtrap
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`such as VEGFR1R2-FcAC1 (a) (SEQ ID NOs:1-2).
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`The phrase “ophthalmically acceptable” with respectto a
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`formulation, composition or ingredient herein means having
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`no persistent effect that is substantially detrimental to the
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`treated eye or the functioning thereof, or on the general health
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`of the subject being treated. It will be recognized that tran-
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`sient effects such as minorirritation or a “stinging” sensation
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`are commonwith topical ophthalmic administration of drugs
`and the existence of such transient effects is not inconsistent
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`with the formulation, composition or ingredient in question
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`being “ophthalmically acceptable” as herein defined. How-
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`ever, preferred formulations, compositions and ingredients
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`are those that cause no substantial detrimental effect, even of
`a transient nature.
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`VEGF Antagonists
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`In various embodiments, the VEGF trap is selected from
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`the group consisting of acetylated Fli-1(1-3)-Fe, Flt-1(1-
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`3xn)-Fe, Fit-1(1-3,,)-Fe, Fli-1(2-3,,)-Fe, Fll-1(2-3)-Fe,
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`Flt-1D2-VEGFR3D3-FcAC1 (a),
`Flt-1D2-Flk-1D3-FceAC1
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`(a), and VEGFR1IR2-FcAC 1 (a). For a more detailed descrip-
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`tion of these and other VEGF-receptor-based antagonists,
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`including pegylated receptor-based blockers,
`see PCT
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`WO0/00/75319, the contents ofwhich are incorporatedin their
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`entirety herein by reference.
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`In addition to the VEGFreceptor-based antagonists dis-
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`closed in PCT WO/00/75319, which publication is herein
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`specifically incorporated by referencein its entirety, variants
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`and derivatives of such VEGFreceptor-based blockers are
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`also contemplated by the invention. The sequence of the
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`variants or derivatives may differ by a changethat can be one
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`or more additions, insertions. deletions and/or substitutions
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`of one or more nucleotides of the sequenceset forth in SEQ
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`ID NO:1. Changesto a nucleotide sequence mayresult in an
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`US 7,531,173 B2
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`ment of the eye, including, for example, corneal diseases,
`amino acid changeat the proteinlevel, or not, as determined
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`uveitis, and glaucoma. Topical delivery can be a safer and
`by the genetic code. Thus, nucleic acid according to the
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`more convenientdelivery methodfor patients, and can reduce
`present invention may include a sequencedifferent from the
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`the risk of many side effects observed in systemic treatment
`sequence shownin SEQ ID NO:1, yet encode a polypeptide
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`regimens. Topical administration ofan angiogenesis inhibitor
`with the same aminoacid sequence as SEQ ID NO: 2. On the
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`to the eye or cornea canbe aneffective treatment for treating
`other hand, the encoded polypeptide may comprise an amino
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`neovascularization and/or inflammation. A preferred method
`acid, sequence which differs by one or more amino acid resi-
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`of administering the pharmaceutical compositions of the
`dues from the amino acid sequence shownin SEQ ID NO:2.
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`inventionto the eye is by eye drops comprising a VEGFtrap.
`A nucleic acid encoding, a polypeptide whichis an amino acid
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`sequence variant or derivative of the sequence shown in SEQ
`In various preferred embodiments,
`the pharmaceutical
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`ID NO:2 is further provided by the present invention. A
`compositions of the invention are administered to the area in
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`nucleic acid encoding such a polypeptide may showat the
`need of treatment by subconjunctival administration. One
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`nucleotide sequence and/or encoded aminoacidlevel greater
`preferred method of subconjunctival administrationto the eye
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`than about 90%, 95%, 98%, or 99% homology with the cod-
`is by injectable formulations comprising a VEGF trap.
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`ing sequence shown in SEQ ID NO:1 and/or the amino acid
`Anotherpreferred method of subconjunctival administration
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`sequence shown in SEQ ID NO:2. Aminoacid “homology”,
`is by implantations comprising slowreleasing VEGFtrap.
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`may be understood to be similarity (according to the estab-
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`Pharmaceutical Compositions
`lished principles of amino acid similarity, e.g. as determined
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`Pharmaceutical compositions useful in the practice ofthe
`using the algorithm GAP (Genetics Computer Group, Madi-
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`method of the invention include a therapeutically ctfective
`son, Wis.)) or identity. GAP uses the Needleman and Wunsch
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`amountof'an active agent with a pharmaceutically acceptable
`algorithm to align two complete sequences that maximizes
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`carrier. The term “pharmaceutically acceptable” means
`the number of matches and minimizes the number of gaps.
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`approved by a regulatory agency ofthe l’ederal or a state
`Generally, the default parameters are used, with a gap cre-
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`governmentorlisted in the U.S. Pharmacopeia or other gen-
`ation penalty=12 and gap extension penalty=4.
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`erally recognized pharmacopeia for use in animals, and more
`Individual components of the VEGF-specific fusion pro-
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`particularly, in humans. The term“carrier”refers to a diluent,
`teins of the invention may be constructed by molecular bio-
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`adjuvant, excipient, or vehicle with whichthe therapeutic is
`logical methods knownto the art with the guidance provided
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`administered. Fxamples of suitable pharmaceutical carriers
`by the instant specification. These components are selected
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`are described in “Remington’s Pharmaceutical Sciences” by
`fromafirst cellular receptor protcin, such as, for example,
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`F. W. Martin. In a preferred embodiment, the compositionis
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`VEGERI; a second cellular receptor protein, such as, for
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`formulated in accordance with routine procedures as a phar-
`example, VEGFR2; anda multimerizing component, such as,
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`maceutical composition adapted for topical administrationto
`for example, an Ic.
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`humanbeings. Such pharmaceutical compositions may be
`Specific embodiments of the VEGF-specific fusion pro-
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`liquid,gel, ointment, salve, slow release formulations or other
`teins useful in the methods of the invention comprise a mul-
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`formulations suitable for ophthalmic administration. The
`timerizing component which allows the fusion proteins to
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`composition comprises an effective amount ofVEGFantago-
`associate, e.g., as multimers, preferably dimers. Preferably,
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`nist and, optionally, at least one ophthalmically acceptable
`the multimerizing, component comprises an immunoglobu-
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`excipient, wherein the excipient is able to reduce a rate of
`lin-derived domain. Suitable multimerizing components are
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`removalofthe composition from the eye by lacrimation, such
`sequences encoding an immunoglobulin heavy chain hinge
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`that the compositionhasan effective residencetimein the eye
`region (Takahashi et al. 1982 Cell 29:671-679); immunoglo-
`of about 2 hours to about 24 hours.
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`bulin gene sequences, and portions thereof.
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`In various embodiments, compositions ofthe invention can
`The nucleic acid constructs encoding the fusion proteins
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`useful in the methods of the invention can be inserted into an
`comprise a liquid comprising an active agent in solution, in
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`suspension, or both. The term “suspension”herein includes a
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`expression vector by methods knownto the art, wherein the
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`liquid composition whereinafirst portion of the active agent
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`nucleic acid molecule can be operatively linked to an expres-
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`is present in solution and a secondportion ofthe active agent
`sion control sequence. Host-vector systems for the produc-
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`1s present in particulate form, in suspension in a liquid matrix.
`tion of proteins comprising an expression vector introduced
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`As used herein, liquid compositions include gels.
`into a host cell suitable for expression of the protein are
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`Preferably the liquid composition is aqueous. Alterna-
`knownin the art. The suitable host cell maybe a bacterial cell
`a 2
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`tively, the composition can take form of an ointment. In a
`such as E. coli, a yeast cell, such as, for example, Pichia °
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`preferred embodiment, the composition is an in situ gellable
`pastoris, an insect cell, such as, for cxample, Spodoptera
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`aqueous composition, more preferably an in situ gellable
`frugiperda, or a mammalian cell, such as, for example, a
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`aqueoussolution. Such a composition can comprisea gelling
`COS, CHO, 293, BHK or NSOcell.
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`agent in a concentration effective to promote gelling upon
`Methods ofAdministration
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`contact with the eye orlacrimal fluid in the exteriorofthe eye.
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`The invention comprises methodsoftreatment comprising,
`Suitable gelling agents non-restrictively include thermoset-
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`administering to a subject an effective amount of an agent of
`ting polymers such as tetra-substituted ethylene diamine
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`the invention. In a preferred aspect, the agent is substantially
`block copolymers of ethylene oxide and propylene oxide
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`purified (e.g., substantially free from substancesthat limit its
`(e.g., poloxamine 1307); polycarbophil; and polysaccharides
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`effect or produce undesired side-effects). The subject is pref-
`such as gellan, carrageenan (e.g., kappa-carrageenan and
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`erably an animal, e.g., such as cows, pigs. horses, chickens,
`iota-carrageenan), chitosan and alginate gums. The phrase
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`cats, dogs, etc., and is preferably a mammal, and most pref-
`“in situ gellable” includes not only liquids of low viscosity
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`erably human.
`that can form gels upon contact with the eye or with lacrimal
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`Preferably, the pharmaceutical compositions of the inven-
`fluid in the exterior of the eye, but also more viscous liquids
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`tion are administered to the area in need of treatment by
`such as semi-fiuid and thixotropic gels that exhibit substan-
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`topical administration. Topical drug delivery is the most com-
`tially increased viscosity or gel stiffness upon administration
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`mon treatment for diseases or disorders of the anterior seg-
`to the eye or area surrounding the eye.
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`a5S
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`7
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`Aqueous compositions of the invention have ophthalmi-
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`cally compatible