I 1111111111111111 11111 1111111111 11111 111111111111111 IIIII IIIIII IIII IIII IIII
`US007303748B2
`
`c12) United States Patent
`Wiegand et al.
`
`(IO) Patent No.:
`(45) Date of Patent:
`
`US 7,303,748 B2
`Dec. 4, 2007
`
`(54) METHOD OF TREATING EYE INJURY
`WITH LOCAL ADMINISTRATION OF A
`VEGF INHIBITOR
`
`(75)
`
`Inventors: Stanley J. Wiegand,
`Croton-on-Hudson, NY (US); Jingtai
`Cao, Chappaqua, NY (US)
`
`(73) Assignee: Regeneron Pharmaceuticals, Inc.,
`Tarrytown, NY (US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 94 days.
`
`(21) Appl. No.: 11/346,009
`
`(22) Filed:
`
`Feb. 2, 2006
`
`(65)
`
`Prior Publication Data
`
`US 2006/0172944 Al
`
`Aug. 3, 2006
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/649,232, filed on Feb.
`2, 2005.
`
`(51)
`
`Int. Cl.
`A61K 38/18
`(2006.01)
`C07K 14171
`(2006.01)
`C12N 15162
`(2006.01)
`(52) U.S. Cl. ............................... 424/134.1; 424/192.1;
`514/2; 514/12; 530/350; 536/23.4
`(58) Field of Classification Search ..................... None
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5,851,999 A
`
`12/1998 Ullrich et al.
`
`6,0ll,003 A
`6,270,993 Bl
`6,897,294 B2
`
`1/2000 Charnock-Jones
`8/2001 Shibuya
`5/2005 Davis-Smyth et al.
`
`FOREIGN PATENT DOCUMENTS
`
`WO
`WO
`WO
`WO
`WO
`WO
`
`WO 97/44453
`WO 98/13071
`WO 99/13909
`WO 00/75319
`WO 03/072029
`WO 2004/106378
`
`11/1997
`4/1998
`3/1999
`12/2000
`9/2003
`12/2004
`
`OTHER PUBLICATIONS
`
`Holash, J., et al., (2002) PNAS, 99(17):ll393-ll398.
`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):lll97-ll204.
`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(ll):1519-1524.
`Cao, J., et al., (2004) Investigative Ophthalmolgy and Visual
`Science, 45 (Suppl. l):U922.
`
`Primary Examiner----Christine J. Saoud
`Assistant Examiner-Jon M Lockard
`(74) Attorney, Agent, or Firm-Valeta Gregg, Esq.
`
`(57)
`
`ABSTRACT
`
`Methods of reducing or treating angiogenesis and/or inflam(cid:173)
`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 provided. 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).
`
`10 Claims, 7 Drawing Sheets
`
`Mylan Exhibit 1148
`Mylan v. Regeneron, IPR2021-00881
`Page 1
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 1 of 7
`
`US 7,303,748 B2
`
`0
`<">
`
`0
`N
`
`-
`(%) eaJy Je1noseA
`
`0
`
`0
`
`C. ca ...
`I-
`u.
`CJ w
`> I
`Cl) ...
`:::::J -:::::J en
`
`Cl)
`CJ
`.c
`Cl) > I
`Q) ...
`:::::J -:::::J en
`
`C.
`
`ca ...
`I-
`u.
`CJ
`w
`>
`cb ...
`:::::J -:::::J en
`
`Q)
`CJ
`.c
`Cl)
`> I
`Q) ...
`:::::J -:::::J en
`
`T"""
`
`C)
`
`u:::
`
`0
`I/'\
`
`0
`ST
`
`Cil
`
`1--,
`
`0
`I/'\
`
`0
`ST
`
`0
`<">
`
`0
`N
`
`::
`
`0
`
`<(
`
`(%) eaJy Je1noseA
`
`Mylan Exhibit 1148
`Mylan v. Regeneron, IPR2021-00881
`Page 2
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 2 of 7
`
`US 7,303,748 B2
`
`C. m
`...
`I-
`LL
`CJ
`UJ
`> I
`G) ... ::,
`.... ::, en
`G) -(,) ·-.c
`a,
`> I
`G) ... ::,
`.... ::, en
`
`C\J .
`C)
`LL
`
`0
`1£)
`
`0
`N
`
`0
`
`-
`
`0
`
`Mylan Exhibit 1148
`Mylan v. Regeneron, IPR2021-00881
`Page 3
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 3 of 7
`
`US 7,303,748 B2
`
`Ol
`:::J
`0
`LI)
`
`Ol
`:::J
`......
`LI)
`N u
`.0
`:::s
`II)
`.......
`Cl.
`ra
`L.
`I-
`LL
`Ol ~
`:::J w
`0 >
`0
`T""'i
`
`C")
`
`0)
`i.L..
`
`Ol
`:::J
`IJ"l
`N
`
`Ol
`:::J
`0
`IJ"l
`
`......
`u
`(/)
`.......
`Cl.
`ra
`L.
`I-
`LL
`~ w
`Ol >
`:::J
`0
`0
`T""'i
`
`0
`L..
`.J-J
`C
`
`0 u
`
`0
`
`0
`0
`N
`
`0
`0
`CX)
`
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`~ v
`~ v
`N
`0
`.-4
`...-l
`(wn) 4:a6ua1 1assal\
`
`.-4
`
`.-4
`
`0
`0
`0
`N
`
`0
`0
`00
`.-4
`
`Mylan Exhibit 1148
`Mylan v. Regeneron, IPR2021-00881
`Page 4
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 4 of 7
`
`US 7,303,748 B2
`
`..-..
`(.)
`.c
`::::, en
`.__.
`a.
`n, ...
`I-
`LL c.,
`w
`>
`
`..-..
`0
`.c
`::::,
`en
`.__.
`
`en
`::::s
`It)
`
`Q)
`
`C ·--ns
`U) -ns
`E ...
`0 z
`
`e ..,
`
`C
`0
`0
`
`,q-
`.
`Cl
`LL
`
`0
`0
`N
`
`0
`It)
`""'-
`
`0
`It)
`
`Mylan Exhibit 1148
`Mylan v. Regeneron, IPR2021-00881
`Page 5
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 5 of 7
`
`US 7,303,748 B2
`
`-0 en -C.
`ca .. t-
`
`LL
`C>
`w
`>
`
`-0 .c
`:::I en -C.
`
`i!
`t-
`LL
`C>
`w
`>
`
`C)
`:::I
`0
`
`0 -
`
`C)
`
`:::I an
`N
`
`C)
`:::I
`0
`
`0 -
`
`C)
`
`:::I an
`N
`
`C)
`
`:::I an
`
`-0
`z~ -
`
`U) .c
`
`e
`0 .a
`
`.. :::I
`- U)
`C
`I
`0 C
`0
`0 z
`
`LO
`
`CJ)
`
`iL
`
`0
`0
`,q-
`
`0
`0
`0
`0
`It)
`It)
`N
`(Y)
`(Y)
`(wn) ssau)l~!llt leauJo::,
`
`0
`0
`N
`
`Mylan Exhibit 1148
`Mylan v. Regeneron, IPR2021-00881
`Page 6
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 6 of 7
`
`US 7,303,748 B2
`
`--u
`
`.c
`::::,
`U)
`.._,.
`a.
`cu
`L.. r-
`LL
`l.!J
`LU >
`
`.......... u
`l/)
`..........
`a.
`IU
`L.. r-
`LL
`l.!J
`LU >
`
`CJ)
`::,
`lJ")
`
`O"I
`:::I
`LI)
`N
`
`O"I
`:::I
`0
`0
`T-i
`
`CJ)
`::,
`LI)
`N
`
`O"I
`:::I
`0
`0
`T-i
`
`0
`L..
`~
`C
`0 u
`
`<O
`
`C')
`LL
`
`~ 0
`0
`IX)
`
`~ 0
`0
`I.O
`
`-:R. 0
`0
`,:;j"
`
`~ 0
`0
`M
`
`-:R. 0
`0
`N
`
`~ 0
`0
`
`Mylan Exhibit 1148
`Mylan v. Regeneron, IPR2021-00881
`Page 7
`
`

`

`U.S. Patent
`
`Dec. 4, 2007
`
`Sheet 7 of 7
`
`US 7,303,748 B2
`
`c..
`ro
`~
`LL
`l?
`UJ >
`
`,,-....
`U)
`C.
`0
`1-
`"'O
`I
`Q)
`>-Q)
`...........
`
`f'O u ·-0.
`
`0
`~
`
`QJ
`
`u ·-..c
`QJ >
`
`Q)
`1-
`:,
`-1-1 :,
`CJ)
`
`0
`1-
`-1-1
`C
`
`0 u
`
`0
`1-......,
`C
`0 u
`
`I'--
`
`C)
`LL
`
`0
`0
`00
`
`0
`0
`I',
`
`0
`0
`
`"°
`
`0 0 0 0 0 0
`0
`0
`0
`0
`0
`V
`N
`M
`Ll'l
`~
`
`Mylan Exhibit 1148
`Mylan v. Regeneron, IPR2021-00881
`Page 8
`
`

`

`US 7,303,748 B2
`
`1
`METHOD OF TREATING EYE INJURY
`WITH LOCAL ADMINISTRATION OF A
`VEGF INHIBITOR
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application claims the benefit under 35 USC § 119( e)
`of U.S. Provisional 60/649,232 filed 2 Feb. 2005, which
`application is herein specifically incorporated by reference 10
`in its entirety.
`
`BACKGROUND
`
`1. Field of the Invention
`The field of the invention is related to local administration
`ofVEGF 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).
`
`BRIEF SUMMARY OF THE INVENTION
`
`20
`
`25
`
`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 second agent, such as
`an immunosuppressive agent, for example, one or more of a
`corticosteroid, dexamethasone, or cyclosporin A.
`Local administration includes, for example, administra(cid:173)
`tion 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 inhib(cid:173)
`iting vascular endothelial growth factor (VEGF)-mediated
`15 activity to a subject in need thereof, such that the eye injury
`heals.
`In a third aspect, the invention features a method of
`reducing or ameliorating angiogenesis associated with an
`eye injury, comprising locally administering an effective
`amount of an agent capable of blocking or inhibiting vas(cid:173)
`cular endothelial growth factor (VEGF)-mediated activity to
`a subject in need thereof, such that the angiogenesis asso(cid:173)
`ciated with the eye injury is reduced or ameliorated.
`In a fourth aspect, the invention features a method of
`reducing or ameliorating inflammation associated with an
`eye injury, comprising locally administering an effective
`amount of an agent capable of blocking or inhibiting vas(cid:173)
`cular endothelial growth factor (VEGF)-mediated activity to
`a subject in need thereof, such that the inflammation asso(cid:173)
`ciated with the eye injury is reduced or ameliorated.
`In a fifth aspect, the invention features an ophthalmic
`composition comprising a VEGF antagonist, for example
`the VEGF trap VEGFR1R2-FcllCl(a), in a pharmaceuti-
`35 cally acceptable carrier. Such pharmaceutical compositions
`may be liquid, gel, ointment, salve, slow release formula(cid:173)
`tions or other formulations suitable for ophthalmic admin(cid:173)
`istration. In various embodiments, the pharmaceutical com(cid:173)
`position is for local administration comprising a VEGF trap,
`40 buffer, stabilizer, isotonizer, and a pharmaceutical carrier. In
`a preferred embodiment, the pharmaceutical composition is
`administered in the form of eye drops. In specific embodi(cid:173)
`ments, the pharmaceutically acceptable carrier comprises as
`least one ophthalmically acceptable excipient, wherein the
`ophthalmically acceptable excipient can reduce a rate of
`removal of the VEGF antagonist from the eye by lacrima(cid:173)
`tion. 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 subconjuctival administration such as subconjuctival
`injection and subconjuctival implantation.
`In a sixth aspect, the invention features a method of
`administering a VEGF antagonist for treatment of angio(cid:173)
`genesis 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
`60 a review of the ensuing detailed description.
`
`30
`
`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(cid:173)
`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 inhib(cid:173)
`iting vascular endothelial growth factor (VEGF)-mediated
`activity to a subject in need thereof, such that the eye injury
`is ameliorated or improved. Preferably, the eye injury is a
`corneal injury or conjunctiva! injury and the method of
`treatment reduces angiogenesis and inflanimation 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
`(l-3R->N)-Fc, Flt-1(1-3,v,)-Fc, Flt-1(2-3,v,)-Fc, Flt-1(2-3)(cid:173)
`Fc,
`Flt-lD2-VEGFR3D3-FcllCl(a),
`Flt-lD2-Flk-1D3-
`FcllCl (a), and VEGFR1R2-FcllCl(a). In a specific and
`is VEGFRl
`preferred embodiment,
`the VEGF
`trap
`R2-FcllCl(a) (also termed VEGF trapR 1R2 ) comprising the 50
`nucleotide sequence set forth in SEQ ID NO: 1 and the
`amino acid sequence set forth in SEQ ID 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 SEQ ID NO: 1 and/or the 55
`amino acid sequence set forth in SEQ ID NO:2.
`The method of the invention is useful to treat acute and
`sub-acute corneal injury or conjunctiva! injury. Acute cor(cid:173)
`neal injury may be treated within 24 hours of occurrence,
`and includes corneal injury or conjunctiva! 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 65
`trauma, e.g., surgical injuries, chemical burn, corneal trans(cid:173)
`plant, infectious or inflammatory diseases.
`
`45
`
`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 µg or (B)
`three 10 µg doses.
`
`Mylan Exhibit 1148
`Mylan v. Regeneron, IPR2021-00881
`Page 9
`
`

`

`US 7,303,748 B2
`
`4
`cularization; corneal vascular area and vessel length follow(cid:173)
`ing suture injury being comparable to that of a normal
`untreated (nonsutured) cornea. Treatment with VEGF trap
`eye drops following suture injury also effectively reduced
`5 neovascularization in suture-injured cornea.
`In addition to quantification of neovascularization as
`measured by an increase in either blood vessel length or
`blood vessel area, suture-injury produced a marked influx of
`leucocytes into the injury site. When VEGF trap was admin-
`10 istered 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
`15 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.
`
`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 µg 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 µg VEGF trap injections
`administered subcutaneously (SC) or subconjunctivally
`(SubC). (Control=right non-sutured eye).
`FIG. 4. Quantification of blood vessel length in sutured
`rats receiving subconjunctival normal saline, 5 µg, 25 µg, or
`100 µg VEGF trap on day 0, 3 and 6. (Control=right
`non-sutured eye).
`FIG. 5. Quantification of corneal edema as evidenced by
`corneal thickness in sutured rats receiving 25 or 100 µg
`VEGF trap SC or normal saline, 5 µg, 25 µg, or 100 µg
`VEGF
`trap SubC.
`(Control=right non-sutured eye)
`(NS=sutured eye, normal saline administered SubC).
`FIG. 6. Percent reduction of edema. Effect ofVEGF trap
`on inflammation as determined by measurement of corneal
`thickness. All animals were sutured (control=sutured+sys- 20
`temic injection (SC) of normal saline).
`FIG. 7. Blood vessel length in suture-injury. Control=no
`suture mJury.
`Suture
`control=suture+no
`treatment.
`Vehicle=suture-injury+vehicle provided as eye drops. VEGF
`trap=suture-injury+l drop three times per day (412 µg 25
`VEGF trap protein/drop).
`
`Definitions
`The phrase "therapeutically effective dose" includes a
`dose that produces the desired effect for which it is admin(cid:173)
`istered. 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 Com-
`pounding).
`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,
`antisense molecules, antibodies, antagonists and their
`derivatives. More specifically, an example of a VEGF
`blocker or inhibitor includes a VEGF receptor-based antago(cid:173)
`nist comprising, for example, an anti-VEGF antibody, or a
`VEGF trap such as VEGFR1R2-FcllCl(a) (SEQ ID 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
`transient effects such as minor irritation or a "stinging"
`sensation are common with topical ophthalmic administra(cid:173)
`tion 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. However, preferred formulations, compositions and
`ingredients are those that cause no substantial detrimental
`effect, even of a transient nature.
`
`30
`
`35
`
`40
`
`45
`
`50
`
`VEGF Antagonists
`In various embodiments, the VEGF trap is selected from
`the group consisting of acetylated Flt-1(1-3)-Fc, Flt-1(1-3R-
`55 >N)-Fc, Flt-1 (1-3 ,v,)-Fc, Flt-1 (2-3,v,)-Fc, Flt-1 (2-3)-Fc, Flt-
`1 D2-VEGFR3D3-FcllC1 ( a), Flt-1 D2-Flk-1 D3-FcllC1 ( a),
`and VEGFR1R2-FcllCl(a). For a more detailed description
`of these and other VEGF-receptor-based antagonists, includ(cid:173)
`ing pegylated receptor-based blockers, see PCT WO/00/
`60 75319, the contents of which are incorporated in their
`entirety herein by reference.
`In addition to the VEGF receptor-based antagonists dis(cid:173)
`closed in PCT WO/00/75319, which publication is herein
`specifically incorporated by reference in its entirety, variants
`65 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
`
`DETAILED DESCRIPTION
`
`Before the present methods are described, it is to be
`understood 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 limiting, 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
`understood 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 practice 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(cid:173)
`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(cid:173)
`mediated 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 neovas-
`
`Mylan Exhibit 1148
`Mylan v. Regeneron, IPR2021-00881
`Page 10
`
`

`

`US 7,303,748 B2
`
`6
`Preferably, the pharmaceutical compositions of the inven(cid:173)
`tion are administered to the area in need of treatment by
`topical administration. Topical drug delivery is the most
`common treatment for diseases or disorders of the anterior
`segment 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 angiogen(cid:173)
`esis inhibitor to the eye or cornea can be an effective
`treatment for treating neovascularization and/or inflamma(cid:173)
`tion. A preferred method of administering the pharmaceuti(cid:173)
`cal 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.
`
`5
`
`5
`one or more additions, insertions, deletions and/or substitu(cid:173)
`tions of one or more nucleotides of the sequence set forth in
`SEQ ID NO: 1. Changes to a nucleotide sequence may result
`in an amino acid change at the protein level, or not, as
`determined by the genetic code. Thus, nucleic acid accord-
`ing to the present invention may include a sequence different
`from the sequence shown in SEQ ID NO: 1, yet encode a
`polypeptide with the same amino acid sequence as SEQ ID
`NO: 2. On the other hand, the encoded polypeptide may
`comprise an amino acid sequence which differs by one or 10
`more amino acid residues from the amino acid sequence
`shown in SEQ ID NO:2. A nucleic acid encoding a polypep(cid:173)
`tide which is an amino acid sequence variant or derivative of
`the sequence shown in SEQ ID NO:2 is further provided by
`the present invention. A nucleic acid encoding such a 15
`polypeptide may show at the nucleotide sequence and/or
`encoded amino acid level greater than about 90%, 95%,
`98%, or 99% homology with the coding sequence shown in
`SEQ ID NO: 1 and/or the amino acid sequence shown in
`SEQ ID NO:2. Amino acid "homology", may be understood 20
`to be similarity (according to the established principles of
`amino acid similarity, e.g. as determined using the algorithm
`GAP (Genetics Computer Group, Madison, Wis.)) or iden(cid:173)
`tity. GAP uses the Needleman and Wunsch algorithm to
`align two complete sequences that maximizes the number of 25
`matches and minimizes the number of gaps. Generally, the
`default parameters are used, with a gap creation penalty=12
`and gap extension penalty=4.
`Individual components of the VEGF-specific fusion pro(cid:173)
`teins of the invention may be constructed by molecular 30
`biological 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, VEGFRl; a second cellular receptor protein, such
`as, for example, VEGFR2; and a multimerizing component, 35
`such as, for example, an Fe.
`Specific embodiments of the VEGF-specific fusion pro(cid:173)
`teins useful in the methods of the invention comprise a
`multimerizing component which allows the fusion proteins
`to associate, e.g., as multimers, preferably dimers. Prefer- 40
`ably, the multimerizing component comprises an immuno(cid:173)
`globulin-derived domain. Suitable multimerizing compo(cid:173)
`nents are sequences encoding an immunoglobulin heavy
`chain hinge region (Takahashi et al. 1982 Cell 29:671-679);
`immunoglobulin 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
`expression control sequence. Host-vector systems for the
`production 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.
`
`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 accept(cid:173)
`able 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
`generally 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 thera(cid:173)
`peutic is administered. Examples of suitable pharmaceutical
`carriers are described in "Remington's Pharmaceutical Sci-
`ences" by E.W. Martin. In a preferred embodiment, the
`composition is formulated in accordance with routine pro(cid:173)
`cedures as a pharmaceutical composition adapted for topical
`administration to human beings. Such pharmaceutical com(cid:173)
`positions may be liquid, gel, ointment, salve, slow release
`formulations or other formulations suitable for ophthalmic
`administration. The composition comprises an effective
`amount of VEGF antagonist and, optionally, at least one
`ophthalmically acceptable excipient, wherein the excipient
`is able to reduce a rate of removal of the composition from
`45 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,
`50 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
`55 gels.
`Preferably the liquid composition is aqueous. Alterna(cid:173)
`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
`60 aqueous solution. Such a composition can comprise a gel(cid:173)
`ling 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
`thermosetting polymers such as tetra-substituted ethylene
`65 diamine block copolymers of ethylene oxide and propylene
`oxide (e.g., poloxamine 1307); polycarbophil; and polysac(cid:173)
`charides such as gellan, carrageenan (e.g., kappa-carrag-
`
`Methods of Administration
`The invention comprises methods of treatment compris(cid:173)
`ing 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 sub(cid:173)
`stances that limit its effect or produce undesired side(cid:173)
`effects ). The subject is preferably an animal, e.g., such as
`cows, pigs, horses, chickens, cats, dogs, etc., and is prefer(cid:173)
`ably a mammal, and most preferably human.
`
`Mylan Exhibit 1148
`Mylan v. Regeneron, IPR2021-00881
`Page 11
`
`

`

`US 7,303,748 B2
`
`7
`eenan and iota-carrageenan), chitosan and alginate gums.
`The phrase "in situ gellable" includes not only liquids oflow
`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 5
`exhibit substantially increased viscosity or gel stiffness upon
`administration to the eye or area surrounding the eye.
`Aqueous compositions of the invention have ophthalmi(cid:173)
`cally compatible pH and osmolality. Preferably these com(cid:173)
`positions incorporate means to inhibit microbial growth, for 10
`example through preparation and packaging under sterile
`conditions and/or through inclusion of an antimicrobially
`effective amount of an ophthalmically acceptable preserva(cid:173)
`tive. Suitable preservatives non-restrictively include mer(cid:173)
`cury-containing substances such as phenylmercuric salts 15
`( e.g., phenylmercuric acetate, borate and nitrate) and thime(cid:173)
`rosal; stabilized chlorine dioxide; quaternary ammonium
`compounds such as benzalkonium chloride, cetyltrimethy(cid:173)
`lammonium bromide and cetylpyridinium chloride; imida(cid:173)
`zolidinyl urea; parabens such as methylparaben, ethylpara- 20
`ben, propylparaben and butylparaben, and salts thereof;
`phenoxyethanol; chlorophenoxyethanol; phenoxypropanol;
`chlorobutanol; chlorocresol; phenylethyl alcohol; disodium
`EDTA; and sorbic acid and salts thereof.
`The composition can comprise an ophthalmic depot for- 25
`mulation comprising an active agent for subconjunctival
`administration. The ophthalmic depot formulation com(cid:173)
`prises microparticles of essentially pure active agent, e.g.,
`the VEGF trap of SEQ ID NO:2. The microparticles com(cid:173)
`prising VEGF trap can be embedded in a biocompatible 30
`pharmaceutically acceptable polymer or a lipid encapsulat(cid:173)
`ing 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 35
`site of administration after release of all or substantially all
`the active agent. The depot formulation can be liquid for(cid:173)
`mulation, comprising a pharmaceutical acceptable polymer
`and a dissolved or dispersed active agent, Upon injection,
`the polymer forms a deot at the injections site, e.g. by 40
`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 conjunctiva! sac, where the article
`releases the active agent. Release from such an article is 45
`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 50
`non-bioerodible. Bioerodible polymers that can be used in
`preparation of ocular implants carrying a VEGF trap in
`accordance with the present invention include without
`restriction aliphatic polyesters such as polymers and copoly(cid:173)
`mers of poly(glycolide ), poly(lactide ), poly( E-caprolactone ), 55
`poly(hydroxybutyrate)
`and
`poly(hydroxyvalerate ),
`polyamino acids, polyorthoesters, polyanhydrides, aliphatic
`polycarbonates and polyether lactones. Illustrative of suit(cid:173)
`able non-bioerodible polymers are silicone elastomers.
`The active agents of the invention can be formulated as 60
`neutral or salt forms. Pharmaceutically acceptable salts
`include those formed with free amino groups such as those
`derived from hydrochloric, phosphoric, acetic, oxalic, tar(cid:173)
`taric acids, etc., and those formed with free carboxyl groups
`such as those derived from sodium, potassium, ammonium, 65
`calcium, ferric hydroxides, isopropylamine, triethylamine,
`2-ethylamino ethanol, histidine, procaine, etc.
`
`8
`
`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, includ(cid:173)
`ing 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
`co