USOO7378095B2
`
`(12) United States Patent
`Cao et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,378,095 B2
`*May 27, 2008
`
`(54)
`
`(75)
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`(73)
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`METHODS OF TREATING TYPE I
`DABETES BY BLOCKING
`VEGF-MEDIATED ACTIVITY
`
`Inventors: Jingtai Cao, Chappaqua, NY (US);
`Li-Hsien Wang, Somers, NY (US);
`Hsin Chieh Lin, Yorktown Heights, NY
`(US); Mark W. Sleeman, Mahopac,
`NY (US): Stanley J. Wiegand,
`Croton-on-Hudson, NY (US)
`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 300 days.
`
`This patent is Subject to a terminal dis
`claimer.
`
`(21)
`(22)
`(65)
`
`(60)
`
`(51)
`
`(52)
`
`(58)
`
`Appl. No.: 11/193,746
`
`Filed:
`
`Jul. 29, 2005
`
`Prior Publication Data
`US 2006/OO24309 A1
`Feb. 2, 2006
`
`Related U.S. Application Data
`Provisional application No. 60/592,628, filed on Jul.
`30, 2004.
`
`Int. C.
`(2006.01)
`A6 IK 38/18
`(2006.01)
`C07K I4/7
`(2006.01)
`CI2N 5/62
`U.S. Cl. ............................... 424/134.1; 424/192.1;
`514/2; 514/12:530/350,536/23.4
`Field of Classification Search ..................... None
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`6,524,583 B1* 2/2003 Thorpe et al. ........... 424,145.1
`6,833,349 B2 12/2004 Xia et al.
`2003/0044841 A1
`3/2003 Baker et al.
`2005/0260203 A1* 11/2005 Wiegand et al. ......... 424,145.1
`2006/0030529 A1* 2/2006 Wiegand et al. .............. 514/12
`
`FOREIGN PATENT DOCUMENTS
`
`WOOO,753.19
`WO
`WO O2/060489
`WO
`WO WO 2004/0872O6
`
`12/2000
`8, 2002
`10, 2004
`
`OTHER PUBLICATIONS
`Flyvbjerg, A., et al. (2002) Diabetes 5:3090-3094.
`Eremina, E., et al., (2003) J. Clin. Invest. 111:707-716.
`De Vriese, A., et al., (2001) J. Am. Soc. Nephrol. 12:993-1000.
`Kobayashi, T., et al. (2002) Am. J. Physiol. Heart Circ. Physiol.
`283(5): H1761-H1768.
`Chiarelli, F., et al., (2000) Diabetic Medicine 17(9):650-656.
`* cited by examiner
`
`Primary Examiner Christine J Saoud
`Assistant Examiner—Jon M Lockard
`(74) Attorney, Agent, or Firm Valeta Gregg, Esq.
`
`(57)
`
`ABSTRACT
`
`Methods of treating diabetes in mammals, particularly
`humans, by blocking or inhibiting VEGF-mediated activity.
`A preferred inhibitor of VEGF-mediated activity is a VEGF
`antagonist such as the VEGF fusion protein trap of SEQ ID
`NO:2 capable of binding and blocking VEGF. The method
`of the invention may be combined with other therapies, such
`as with insulin therapy.
`
`5 Claims, 2 Drawing Sheets
`
`Mylan Exhibit 1039
`Mylan v. Regeneron, IPR2021-00880
`Page 1
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`Joining Petitioner: Apotex
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`

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`U.S. Patent
`
`May 27, 2008
`
`Sheet 1 of 2
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`US 7,378,095 B2
`
`Before VGT
`48 hr after VGT
`
`700
`
`
`
`6OO
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`5OO
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`400
`
`2OO
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`1 OO
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`CB+saline
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`Dia--saline
`
`Dia+VGT12.5mg
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`Fig. 1
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`Mylan Exhibit 1039
`Mylan v. Regeneron, IPR2021-00880
`Page 2
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`Joining Petitioner: Apotex
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`

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`U.S. Patent
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`May 27, 2008
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`Sheet 2 of 2
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`US 7,378,095 B2
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`initiate
`Trap treatment
`
`
`
`-
`"S a
`
`raaf.
`
`O
`9.
`
`()
`O
`C2
`d
`
`--Buffer-hC
`--STL-FC
`--STZ-hVTrap
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`40
`Time (Days)
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`60
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`Fig. 2
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`Mylan Exhibit 1039
`Mylan v. Regeneron, IPR2021-00880
`Page 3
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`Joining Petitioner: Apotex
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`US 7,378,095 B2
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`1.
`METHODS OF TREATING TYPE I
`DABETES BY BLOCKING
`VEGF-MEDIATED ACTIVITY
`
`RELATED APPLICATIONS
`
`This application claims the benefit under 35 U.S.C. 119(e)
`to U.S. Ser. No. 60/592,628 filed 30 Jul. 2004, which
`application is incorporated by reference in its entirety.
`
`BACKGROUND
`
`1. Field of the Invention
`The field of the invention is generally related to methods
`of treating diabetes by administering agents capable of
`decreasing serum glucose levels. In particular, the field of
`the invention is methods of treating type I diabetes by
`administering agents capable of blocking, inhibiting, or
`ameliorating VEGF-mediated activity.
`2. Description of Related Art
`Streptozotocin (STZ)-induced diabetes is widely accepted
`as an animal model for human type I diabetes (see, for
`example, Susztak et al. (2004) Diabetes 53:784-794).
`Vascular endothelial growth factor (VEGF) has been
`recognized as a primary stimulus of angiogenesis in patho
`logical conditions. Approaches to methods of blocking
`VEGF are described, for example, in PCT WO/0075319
`which describes a VEGF-specific fusion protein which binds
`and inhibits VEGF.
`
`BRIEF SUMMARY OF THE INVENTION
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`The present invention is based, in part, on the observation
`that administration of a vascular endothelial growth factor
`(VEGF) antagonist is able to return blood glucose levels to
`normal in an animal model of human type I diabetes.
`Accordingly, in a first aspect, the invention features a
`method of treating type I diabetes in a subject, comprising
`administering to the Subject an agent capable of blocking,
`inhibiting, or ameliorating VEGF-mediated activity. In spe
`cific embodiments, the method of treatment of the invention
`results in decreased serum glucose levels, improved glucose
`tolerance, and/or improved glycemic control.
`The agent capable of blocking, inhibiting, or ameliorating
`45
`VEGF-mediated activity in specific embodiments is a VEGF
`antagonist. More specifically, the VEGF antagonist is a
`VEGF trap antagonist is a fusion protein selected from the
`group consisting of acetylated Flt-1(1-3)-Fc, Flt-1(1-3
`x
`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) (SEQ ID NOS:1-2). In a preferred
`embodiment, the VEGF antagonist is VEGFR1R2-FcAC1
`(a). In other embodiments, the VEGFantagonist is a VEGF
`specific antibody, a nucleic acid Such as an inhibitory
`55
`ri0bozyme or antisense molecule, a small molecule, an
`aptamer, a carbohydrate, peptidomimetic, or hapten.
`Administration of the agent may be by any method known
`in the art, including Subcutaneous, intramuscular, intrader
`mal, intraperitoneal, intravenous, intranasal, or oral routes of
`60
`administration. Preferred methods of administered a VEGF
`trap antagonist is by Subcutaneous or intravenous adminis
`tration.
`The subject treated is preferably a human suffering from
`type I diabetes.
`Other objects and advantages will become apparent from
`a review of the ensuing detailed description.
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`BRIEF DESCRIPTION OF THE FIGURES
`
`FIG. 1. A single injection of VEGF trap antagonist sig
`nificantly reduces blood glucose levels in diabetic rats.
`Normal control rats (CB+saline, n=4); Diabetic rats treated
`with saline (STZ--saline, n=6); Diabetic rats treated with
`VEGF trap (VGT) (12.5 mg/kg) (Dia+VGT 12.5 mg, n=6).
`FIG. 2. Changes in blood glucose levels produced by
`repeated administration of VEGF trap in diabetic mice.
`Normal control mice (CB) treated with a control protein
`(hFc, n=7, —0—); Diabetic mice (STZ-induced) treated
`with VEGF trap (hVTrap, n=9, —A ); Diabetic mice
`(STZ-induced) treated with a control protein (hFc, n=9,
`— ).
`
`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 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 have 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, the
`preferred methods and materials are now described. All
`publications mentioned herein are incorporated herein by
`reference in their entirety.
`General Description
`The invention is based in part on the finding that admin
`istration of an agent capable of blocking or inhibiting
`VEGF-mediated activity is capable of decreasing serum
`glucose and improving glucose disposal in an animal model
`of human type I diabetes. These findings represent the first
`time an agent capable of blocking or inhibiting VEGF
`mediated activity has been shown to ameliorate type I
`diabetes. Thus, the invention provides for methods of treat
`ing diabetes in a mammal by administering a VEGF antago
`nist. More specifically, the method of the invention may be
`practiced with a VEGF antagonist Such as a dimeric protein
`composed of two fusion polypeptides (“VEGF trap') (SEQ
`ID NO:2), as shown below, or a VEGF-specific antibody.
`Definitions
`By the term “therapeutically effective dose' is meant a
`dose that produces the desired effect for which it is admin
`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).
`By the term “blocker”, “inhibitor', or “antagonist' is
`meant a Substance that retards or prevents a chemical or
`physiological reaction or response. Common blockers or
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`Mylan Exhibit 1039
`Mylan v. Regeneron, IPR2021-00880
`Page 4
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`Joining Petitioner: Apotex
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`inhibitors include but are not limited to antisense molecules,
`antibodies, antagonists and their derivatives. More specifi
`cally, an example of a VEGF blocker or inhibitor is a VEGF
`receptor-based antagonist including, for example, an anti
`VEGF antibody, or a VEGF trap such as VEGFR1R2
`FcAC1(a) (SEQID NOS:1-2). For a complete description of
`VEGF-receptor based antagonists including VEGFR1R2
`FcAC1(a), see PCT publication WO/00/75319, the contents
`of which is incorporated in its entirety herein by reference.
`A “small molecule' is defined herein to have a molecular
`weight below about 500 Daltons, and may include chemical
`as well as peptide molecules.
`Nucleic Acid Constructs
`Individual components of the VEGF-specific fusion pro
`teins of the invention may be constructed by molecular
`biological methods known to the art with the instructions
`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; a multimerizing component. Such
`as an Fc.
`Specific embodiments of the VEGF-specific fusion pro
`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
`ably, the multimerizing component comprises an immuno
`globulin derived domain. Suitable multimerizing compo
`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 are inserted into an
`expression vector by methods known to the art, wherein the
`nucleic acid molecule is 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 Pichia pastoris, an insect cell,
`Such as Spodoptera frugiperda, or a mammalian cell. Such as
`a COS, CHO, 293, BHK or NS0 cell.
`Antisense Nucleic Acids
`In one aspect of the invention, VEGF-mediated activity is
`blocked or inhibited by the use of VEGF antisense nucleic
`acids. The present invention provides the therapeutic or
`prophylactic use of nucleic acids comprising at least six
`nucleotides that are antisense to a gene or cDNA encoding
`VEGF or a portion thereof. As used herein, a VEGF “anti
`sense' nucleic acid refers to a nucleic acid capable of
`hybridizing by virtue of some sequence complementarity to
`a portion of an RNA (preferably mRNA) encoding VEGF.
`The antisense nucleic acid may be complementary to a
`coding and/or noncoding region of an mRNA encoding
`VEGF. Such antisense nucleic acids have utility as com
`55
`pounds that prevent VEGF expression, and can be used in
`the treatment of diabetes. The antisense nucleic acids of the
`invention are double-stranded or single-stranded oligonucle
`otides, RNA or DNA or a modification or derivative thereof,
`and can be directly administered to a cell or produced
`intracellularly by transcription of exogenous, introduced
`Sequences.
`The VEGF antisense nucleic acids are of at least six
`nucleotides and are preferably oligonucleotides ranging
`from 6 to about 50 oligonucleotides. In specific aspects, the
`oligonucleotide is at least 10 nucleotides, at least 15 nucle
`otides, at least 100 nucleotides, or at least 200 nucleotides.
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`The oligonucleotides can be DNA or RNA or chimeric
`mixtures or derivatives or modified versions thereof and can
`be single-stranded or double-stranded. In addition, the anti
`sense molecules may be polymers that are nucleic acid
`mimics, such as PNA, morpholino oligos, and LNA. Other
`types of antisence molecules include short double-stranded
`RNAs, known as siRNAs, and short hairpin RNAs, and long
`dsRNA (>50 bp but usually 2500 bp).
`Inhibitory Ribozymes
`In another aspect of the invention, diabetes may be treated
`in a Subject Suffering from Such disease by decreasing the
`level of VEGF activity by using ribozyme molecules
`designed to catalytically cleave gene mRNA transcripts
`encoding VEGF, preventing translation of target gene
`mRNA and, therefore, expression of the gene product.
`Ribozymes are enzymatic RNA molecules capable of
`catalyzing the specific cleavage of RNA. The mechanism of
`ribozyme action involves sequence-specific hybridization of
`the ribozyme molecule to complementary target RNA, fol
`lowed by an endonucleolytic cleavage event. The composi
`tion of ribozyme molecules must include one or more
`sequences complementary to the target gene mRNA, and
`must include the well known catalytic sequence responsible
`for mRNA cleavage. For this sequence, see, e.g., U.S. Pat.
`No. 5,093,246. While ribozymes that cleave mRNA at
`site-specific recognition sequences can be used to destroy
`mRNAs encoding VEGF, the use of hammerhead ribozymes
`is preferred. Hammerhead ribozymes cleave mRNAs at
`locations dictated by flanking regions that form complemen
`tary base pairs with the target mRNA. The sole requirement
`is that the target mRNA has the following sequence of two
`bases: 5'-UG-3'. The construction and production of ham
`merhead ribozymes is well known in the art. The ribozymes
`of the present invention also include RNA endoribonu
`cleases (hereinafter “Cech-type ribozymes') such as the one
`that occurs naturally in Tetrahymena thermophila (known as
`the IVS, or L-19 IVS RNA). The Cech-type ribozymes have
`an eight base pair active site that hybridizes to a target RNA
`sequence where the cleavage of the target RNA takes place.
`The invention encompasses those Cech-type ribozymes that
`target eight base-pair active site sequences that are present
`in the gene encoding VEGF.
`Generation of Antibodies to VEGF Proteins
`In another aspect of the invention, the invention may be
`practiced with an anti-VEGF antibody or antibody fragment
`capable of binding and blocking VEGF activity. Anti-VEGF
`antibodies are disclosed, for example, in U.S. Pat. No.
`6,121,230, herein specifically incorporated by reference.
`The term “antibody” as used herein refers to a polypeptide
`comprising a framework region from an immunoglobulin
`gene or fragments thereof that specifically binds and recog
`nizes an antigen. The recognized immunoglobulin genes
`include the kappa, lambda , alpha, gamma, delta, epsilon,
`and mu constant regions, as well as the myriad immunoglo
`bulin variable region genes. Light chains are classified as
`either kappa or lambda. Heavy chains are classified as
`gamma, mu, alpha, delta, or epsilon, which in turn define the
`immunoglobulin classes, IgG, IgM, IgA, Ig|D, and IgE,
`respectively. Within each IgG class, there are different
`isotypes (eg. IgG, IgG, etc.). Typically, the antigen-bind
`ing region of an antibody will be the most critical in
`determining specificity and affinity of binding.
`Antibodies exist as intact immunoglobulins, or as a num
`ber of well-characterized fragments produced by digestion
`with various peptidases. For example, pepsin digests an
`antibody below the disulfide linkages in the hinge region to
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`Mylan Exhibit 1039
`Mylan v. Regeneron, IPR2021-00880
`Page 5
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`produce F(ab)', a dimer of Fab which itself is a light chain
`joined to V-C1 by a disulfide bond. The F(ab), may be
`reduced under mild conditions to break the disulfide linkage
`in the hinge region, thereby converting the F(ab)' dimer into
`an Fab' monomer. The Fab' monomer is essentially Fab with
`part of the hinge region. While various antibody fragments
`are defined in terms of the digestion of an intact antibody,
`one of skill will appreciate that such fragments may be
`synthesized de novo either chemically or by using recom
`binant DNA methodology. Thus, the terms antibody, as used
`herein, also includes antibody fragments either produced by
`the modification of whole antibodies, or those synthesized
`de novo using recombinant DNA methodologies (e.g., single
`chain Fv) (scFv) or those identified using phase display
`libraries (see, for example, McCafferty et al. (1990) Nature
`348:552-554).
`Methods for preparing antibodies are known to the art.
`See, for example, Kohler & Milstein (1975) Nature 256:
`495-497; Harlow & Lane (1988) Antibodies: a Laboratory
`Manual, Cold Spring Harbor Lab., Cold Spring Harbor,
`N.Y.). The genes encoding the heavy and light chains of an
`antibody of interest can be cloned from a cell, e.g., the genes
`encoding a monoclonal antibody can be cloned from a
`hybridoma and used to produce a recombinant monoclonal
`antibody. Gene libraries encoding heavy and light chains of
`monoclonal antibodies can also be made from hybridoma or
`plasma cells. Random combinations of the heavy and light
`chain gene products generate a large pool of antibodies with
`different antigenic specificity. Techniques for the production
`of single chain antibodies or recombinant antibodies (U.S.
`Pat. Nos. 4,946,778; 4.816,567) can be adapted to produce
`antibodies used in the fusion proteins and methods of the
`instant invention. Also, transgenic mice, or other organisms
`Such as other mammals, may be used to express human or
`humanized antibodies. Alternatively, phage display technol
`ogy can be used to identify antibodies and heteromeric Fab
`fragments that specifically bind to selected antigens.
`Antibody Screening and Selection. Screening and selec
`tion of preferred antibodies can be conducted by a variety of
`methods known to the art. Initial Screening for the presence
`40
`of monoclonal antibodies specific to a target antigen may be
`conducted through the use of ELISA-based methods, for
`example. A secondary Screen is preferably conducted to
`identify and select a desired monoclonal antibody for use in
`construction of the multi-specific fusion proteins of the
`invention. Secondary screening may be conducted with any
`suitable method known to the art. One preferred method,
`termed “Biosensor Modification-Assisted Profiling” (“Bi
`aMAP) is described in co-pending U.S. Ser. No. 10/699,
`361 filed 31 Oct. 2003, herein specifically incorporated by
`reference in its entirety. BiaMAP allows rapid identification
`of hybridoma clones producing monoclonal antibodies with
`desired characteristics. More specifically, monoclonal anti
`bodies are sorted into distinct epitope-related groups based
`on evaluation of antibody:antigen interactions.
`Methods of Administration
`The invention provides 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
`preferably an animal, e.g., Such as cows, pigs, horses,
`chickens, cats, dogs, etc., and is preferably a mammal, and
`most preferably human.
`Various delivery systems are known and can be used to
`administer an agent of the invention, e.g., encapsulation in
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`liposomes, microparticles, microcapsules, recombinant cells
`capable of expressing the compound, receptor-mediated
`endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem.
`262:4429-4432), construction of a nucleic acid as part of a
`retroviral or other vector, etc. Methods of introduction can
`be enteral or parenteral and include but are not limited to
`intradermal, intramuscular, intraperitoneal, intravenous,
`Subcutaneous, intranasal, and oral routes. The compounds
`may be administered by any convenient route, for example
`by infusion or bolus injection, by absorption through epi
`thelial or mucocutaneous linings (e.g., oral mucosa, rectal
`and intestinal mucosa, etc.) and may be administered
`together with other biologically active agents. Administra
`tion can be systemic or local. Administration can be acute or
`chronic (e.g. daily, weekly, monthly, etc.) or in combination
`with other agents.
`In another embodiment, the active agent can be delivered
`in a vesicle, in particular a liposome (see Langer (1990)
`Science 249:1527-1533). In yet another embodiment, the
`active agent can be delivered in a controlled release system.
`In one embodiment, a pump may be used (see Langer (1990)
`Supra). In another embodiment, polymeric materials can be
`used (see Howard et al. (1989) J. Neurosurg. 71:105). In
`another embodiment where the active agent of the invention
`is a nucleic acid encoding a protein, the nucleic acid can be
`administered in vivo to promote expression of its encoded
`protein, by constructing it as part of an appropriate nucleic
`acid expression vector and administering it so that it
`becomes intracellular, e.g., by use of a retroviral vector (see,
`for example, U.S. Pat. No. 4,980.286), or by direct injection,
`or by use of microparticle bombardment (e.g., a gene gun;
`Biolistic, Dupont), or coating with lipids or cell-surface
`receptors or transfecting agents, or by administering it in
`linkage to a homeobox-like peptide which is known to enter
`the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.
`Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid
`can be introduced intracellularly and incorporated within
`host cell DNA for expression, by homologous recombina
`tion.
`Cellular Transfection and Gene Therapy
`The present invention encompasses the use of nucleic
`acids encoding the VEGF-specific fusion proteins of the
`invention for transfection of cells in vitro and in vivo. These
`nucleic acids can be inserted into any of a number of
`well-known vectors for transfection of target cells and
`organisms. The nucleic acids are transfected into cells ex
`vivo and in vivo, through the interaction of the vector and
`the target cell. Reintroduction of transfected cells may be
`accomplished by any method known to the art, including
`re-implantation of encapsulated cells. The compositions are
`administered (e.g., by injection into a muscle) to a subject in
`an amount Sufficient to elicit a therapeutic response. An
`amount adequate to accomplish this is defined as "a thera
`peutically effective dose or amount.”
`In another aspect, the invention provides a method of
`treating diabetes in a human comprising transfecting a cell
`with a nucleic acid encoding a VEGF-specific fusion protein
`of the invention, wherein the nucleic acid comprises an
`inducible promoter operably linked to the nucleic acid
`encoding the VEGF-specific fusion protein. For gene
`therapy procedures in the treatment or prevention of human
`disease, see for example, Van Brunt (1998) Biotechnology
`6:1149-1154.
`Combination Therapies
`In some embodiments, the VEGF-specific fusion proteins
`of the present invention may be administered in combination
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`7
`with insulin or an insulin derivative. Combination therapy
`includes administration of a single pharmaceutical dosage
`formulation which contains a VEGF-specific fusion protein
`and insulin; as well as administration of a VEGF-specific
`fusion protein pharmaceutical dosage formulation and insu
`lin in its own separate pharmaceutical dosage formulation.
`For example, a VEGF-specific fusion protein of the inven
`tion and insulin can be administered to the patient together
`in a single oral dosage composition Such as a tablet or
`capsule, or each agent administered in separate oral dosage
`formulations. Where separate dosage formulations are used,
`the VEGF-specific fusion protein of the invention and insu
`lin can be administered at essentially the same time, i.e.,
`concurrently, or at separately staggered times, i.e., sequen
`tially. In specific embodiments, insulin may be delivered
`through islet cell transfer or using infusion pump.
`Pharmaceutical Compositions
`Pharmaceutical compositions useful in the practice of the
`method of the invention include a therapeutically effective
`amount of an active agent, and a pharmaceutically accept
`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
`peutic is administered. Such pharmaceutical carriers can be
`sterile liquids, Such as water and oils, including those of
`petroleum, animal, vegetable or synthetic origin, Such as
`peanut oil, soybean oil, mineral oil, Sesame oil and the like.
`Suitable pharmaceutical excipients include starch, glucose,
`lactose, Sucrose, gelatin, malt, rice, flour, chalk, silica gel.
`Sodium Stearate, glycerol monostearate, talc, Sodium chlo
`ride, dried skim milk, glycerol, propylene, glycol, water,
`ethanol and the like. The composition, if desired, can also
`contain minor amounts of wetting or emulsifying agents, or
`pH buffering agents. These compositions can take the form
`of Solutions, Suspensions, emulsion, tablets, pills, capsules,
`powders, Sustained-release formulations and the like. The
`composition can be formulated as a Suppository, with tra
`ditional binders and carriers such as triglycerides. Oral
`formulation can include standard carriers such as pharma
`ceutical grades of mannitol, lactose, starch, magnesium
`Stearate, sodium saccharine, cellulose, magnesium carbon
`ate, etc. 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 pharmaceutical
`composition adapted for intravenous, Subcutaneous, or intra
`muscular administration to human beings. Where necessary,
`the composition may also include a solubilizing agent and a
`local anesthetic Such as lidocaine to ease pain at the site of
`the injection. Where the composition is to be administered
`by infusion, it can be dispensed with an infusion bottle
`containing sterile pharmaceutical grade water or saline.
`Where the composition is administered by injection, an
`ampoule of sterile water for injection or saline can be
`provided so that the ingredients may be mixed prior to
`administration.
`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, tar
`taric acids, etc., and those formed with free carboxyl groups
`Such as those derived from Sodium, potassium, ammonium,
`
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`US 7,378,095 B2
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`calcium, ferric hydroxides, isopropylamine, triethylamine,
`2-ethylamino ethanol, histidine, procaine, etc.
`The amount of the active agent of the invention that will
`be effective in the treatment of diabetes can be determined
`by standard clinical techniques based on the present descrip
`tion. In addition, in vitro assays may optionally be employed
`to help identify optimal dosage ranges. The precise dose to
`be employed in the formulation will also depend on the route
`of administration, and the seriousness of the condition, and
`should be decided according to the judgment of the practi
`tioner and each Subject's circumstances. Effective doses
`may be extrapolated from dose-response curves derived
`from in vitro or animal model test systems.
`For systemic administration, a therapeutically effective
`dose can be estimated initially from in vitro assays. For
`example, a dose can be formulated in animal models to
`achieve a circulating concentration range that includes the
`IC, as determined in cell culture. Such information can be
`used to more accurately determine useful doses in humans.
`Initial dosages can also be estimated from in Vivo data, e.g.,
`animal models, using techniques that are well known in the
`art. One having ordinary skill in the art could readily
`optimize administration to humans based on animal data.
`Dosage amount and interval may be adjusted individually
`to provide plasma levels of the compounds that are sufficient
`to maintain therapeutic effect. One having skill in the art will
`be able to optimize therapeutically effective local dosages
`without undue experimentation.
`The amount of compound administered will, of course, be
`dependent on the Subject being treated, on the Subjects
`weight, the severity of the affliction, the manner of admin
`istration, and the judgment of the prescribing physician. The
`therapy may be repeated intermittently while symptoms are
`detectable or even when they are not detectable. The therapy
`may be provided alone or in combination with other drugs.
`Kits
`The invention also provides an article of manufacturing
`comprising packaging material and a pharmaceutical agent
`contained within the packaging material, wherein the phar
`maceutical agent comprises at least one VEGF-specific
`fusion protein of the invention and wherein the packaging
`material comprises a label or package insert which indicates
`that the VEGF-specific fusion protein can be used for
`treating diabetes.
`Other features of the invention will become apparent in
`the course of the following descriptions of exemplary
`embodiments which are given for illustration of the inven
`tion 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
`compositions 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 indicated otherwise, parts are parts by weight,
`molecular weight is average molecular weight, temperature
`is in degrees Centigrade, and pressure is at or near atmo
`spheric.
`
`Mylan Exhibit 1039
`Mylan v. Regeneron, IPR2021-00880
`Page 7
`
`Joining Petitioner: Apotex
`
`

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`9
`Example 1
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`US 7,378,095 B2
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`10
`Example 2
`
`Chronic administration of VEGF Trap to diabetic mice
`A single injection of VEGF Trap reduces blood glucose
`results in a progressive reduction in blood glucose to normal
`levels in a rat model of Type I diabetes.
`glycemic levels.
`Adult, male Sprague Dawley rats (n=16) were fasted
`Apollipoprotein E (APOE) deficient mice were rendered
`diabetic by 5 consecutive daily int