The invention relates to methods and compositions for the treatment or prevention of ocular angiogenesis and neovascularization associated with neovascular disease. Administration of vascular endothelial growth factor (VEGF)-A into the eye when macrophage infiltration is reduced inhibits ocular angiogenesis

Ambati, Jayakrishna

English

University of Kentucky

University of KentuckyUKnowledgeOphthalmology and Visual Science Faculty Patents Ophthalmology and Visual Science6-30-2009VEGF-A as an Inhibitor of Angiogenesis andMethods of Using SameJayakrishna AmbatiUniversity of Kentucky, jayakrishna.ambati@uky.eduClick here to let us know how access to this document benefits you.Follow this and additional works at: https://uknowledge.uky.edu/ophthalmology_patentsPart of the Ophthalmology CommonsThis Patent is brought to you for free and open access by the Ophthalmology and Visual Science at UKnowledge. It has been accepted for inclusion inOphthalmology and Visual Science Faculty Patents by an authorized administrator of UKnowledge. For more information, please contactUKnowledge@lsv.uky.edu.Recommended CitationAmbati, Jayakrishna, "VEGF-A as an Inhibitor of Angiogenesis and Methods of Using Same" (2009). Ophthalmology and Visual ScienceFaculty Patents. 19.https://uknowledge.uky.edu/ophthalmology_patents/19(12) United States Patent Ambati US007553496B2 US 7,553,496 B2 Jun. 30, 2009 (10) Patent N0.: (45) Date of Patent: (54) VEGF-A AS AN INHIBITOR 0F ANGIOGENESIS AND METHODS OF USING SAME (75) Inventor: J ayakrishna Ambati, Lexington, KY (Us) (73) Assignee: University of Kentucky Research Foundation, Lexington, KY (US) ( * ) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 247 days. (21) Appl.No.: 11/017,201 (22) Filed: Dec. 21, 2004 (65) Prior Publication Data US 2006/0135423 A1 Jun. 22, 2006 (51) Int. Cl. A61F 2/00 A61K 39/395 A61K 38/18 (2006.01) A61K 31/70 (2006.01) (52) US. Cl. ......................... .. 424/427; 514/12; 514/44; 424/ 130.1 (58) Field of Classi?cation Search ..................... .. None See application ?le for complete search history. (56) References Cited U.S. PATENT DOCUMENTS (2006.01) (2006.01) 6,177,401 Bl 6,397,849 Bl * 1/2001 Ullrich et al. 6/2002 Bowman et al. .......... .. 128/898 6,524,799 B1 * 2/2003 Walker et al. . . . . . . . . . . .. 435/6 7,335,371 B2 * 2/2008 Sackey?o et al. ......... .. 424/400 2002/0151680 A1 10/2002 Alitalo et al. 2003/0118579 A1* 6/2003 Walker et al. .......... .. 424/94.63 2004/0110757 A1* 6/2004 Arrhenius et al. 514/241 2005/0176669 A1* 8/2005 Al-Murrani ................ .. 514/44 OTHER PUBLICATIONS Kupprion et al., Journal of Biological Chemistry, 273(45):29635 29640, 1998* Shih, S. et al., Selective stimulation of VE GER-1 prevents oxygen induced retinal vascular degeneration in retinopathy of prematurity. (2003) J Clinical Invest 112, 50-57. Carmeliet, P. et al. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. (2001) Nat Med 7, 575-83. Hiratsuka, S. et al. Involvement of ?t-I tyrosine kinase (vascular endothelial growth factor receptor-1) in pathological angiogenesis. (2001) Cancer Res 61, 1207-1213. Bussolati, B. et al. Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide. (2001) Am J Pathol 159, 993-1008. Lopez, P. F., et al., T ransdijferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgi cally excised age-related macular degeneration-related choroidal neovascular membranes. (1996) Invest Ophthalmol Vis Sci 37, 855 68. Saishin, Y. et al. VE GF -TRAPR1R2 suppresses choroidal neovascularization and VEGF-induced breakdown of the blood-reti nal barrier. (2003) J Cell Physiol 195, 241-8. These data are the bases for current clinical trials of anti-VEGF-A therapy in patients With AMD. Spilsbury, K., et al., Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the devel opment of choroidal neovascularization. (2000) Am J Pathol 157, 135-44. Schwesinger, C. et al. Intrachoroidal neovascularization in transgenic mice overexpressing vascular endothelial growth factor in the retinalpigment epithelium. (2001) Am J Pathol 158, 1161-1172. Oshima, Y et al. Increased expression of VE GF in retinal pigmented epithelial cells is not su?cient to cause choroidal neovasculariza tion. J Cell Physiol Published Online: Jun. 7, 2004, DOI: 10.1002/ jcp.20110 (2004). Schmidt-Erfurth, U. et al. In?uence of photodynamic therapy on expression of vascular endothelial growth factor (VEGF), VEGF receptor 3, and pigment epithelium-derived factor. (2003) Invest. Ophthalmol. Vis. Sci. 44, 4473-4480. Grossniklaus, H. E. et al. Macrophage and retinal pigment epithelium expression of anglogenic cytokines in choroidal neovasculariZation. (2002) Mol Wu 8, 119-26. Sakurai, E. et al. Targeted disruption of the CD18 or ICAM-I gene inhibits choroidal neovascularization. (2003) Invest Ophthalmol Vis Sci 44, 2743-9. Sakurai, E., et al., Macrophage depletion inhibits experimental choroidal neovascularization. (2003) Invest Ophthalmol Vis Sci 44, 3578-85. * cited by examiner Primary ExamineriMarianne P Allen (74) Attorney, Agent, or FirmiMcDermott Will & Emery LLP (57) ABSTRACT The invention relates to methods and compositions for the treatment or prevention of ocular angiogenesis and neovas culariZation associated With neovascular disease. Adminis tration of vascular endothelial groWth factor (VEGF)-A into the eye When macrophage in?ltration is reduced inhibits ocu lar angiogenesis. 4 Claims, 20 Drawing Sheets US. Patent Jun. 30, 2009 Sheet 1 0f 20 US 7,553,496 B2 + VEGF-A + PlGF-1 + VEGF-E m .00 w a4a 5205: 2@E:3~15 g( 4| m.v El pmol - dIq0 53 21.  Fig. 1(b) lnl 5-] Oq Control‘ PBS VEGF-A PlGF-1VEGF-E Laser injury US. Patent Jun. 30, 2009 Sheet 3 0f 20 US 7,553,496 B2 Fig. 1(e) 000,000 T 000,000 - i: * 300,000 { 0 q COfltl'OI COC|2 COCIQ + H202 H2O2+ VEGF Ab VEGF Ab Fig. 10‘) 1,600,000-1 ,3 PBS I VEGFR-1 Ab (q a VEGFR-Z Ab 51,200,000 - I,’ E * 2 000,000 > ‘k 2 0 400,000_ 7 / 0 % CoCl 2 VEGF-A VEGF-E US. Patent Fig- 1(9) 1,200,000 1 CNV volume(pm3) Jun. 30, 2009 900,000 - 600,000 - 300,000 - Fig. 1(h) 1,200,000 1 CNV volume(pm3) 900,000 J 600,000 — 300,000 - Sheet 4 0f 20 US 7,553,496 B2 0 PBS I SU5 mSU1 416 498 i * VEGF-E PBS I Vegfr1 tk-l 1 VEGF-A US. Patent Jun. 30, 2009 Sheet 5 0f 20 US 7,553,496 B2 Fig. 1(i) 900,000 - ) 000,000 - CNV volume (p 3 300,000 - Week Fig. 1(j) 1,200,000 900,000 - 600,000 — CNV volume(pm3) 300,000 PBS -1 0 1 2 3 4 US. Patent Jun. 30, 2009 Sheet 6 0f 20 US 7,553,496 B2 F l9. 1 (k) 600 '1 4- Laser -I- Laser+VEGF-A164 *5 500 ‘ +~ Laser+CoC|2 a 7.2 2 E 2 LL (9 LL] Q. Fig. 1(|) 700,000 W VEGF-A154(4 ng) 600,000 — * T 500,000 - 400,000 — 300,000 A CNV volume (pm3)200,000 - 100,000 PBS 0 0.125 0.5 1.25 DBAPBA (119) US. Patent Jun. 30, 2009 Sheet 7 0f 20 US 7,553,496 B2 ("1) Fig. 1 900,000} PBS PBS gtlgG NP-l Ab rb lgG NP-2Ab Fig. 2(a) 103 102 RPE 11 VEGFR-2 fluorescence VEGFR-2 fluorescence '101 102 103 VEGFR-1 fluorescence RPE r(TC VEGFR-l fluorescence US. Patent Jun. 30, 2009 Sheet 8 0f 20 US 7,553,496 B2 Fig. 2(b) ,200 1 52: 25i.2 638mm1 Days after laser Fig. 2(0) PBS VEGF-A PlGF-1 \lEGF-E WBIpTyr Control Mr(kDa) WB: VEGFR-2 IPIVEGFR-2 US 7,553,496 B2 Injection +48 h .3 6.7t2.1 SHP-1/VEGFR-2 .9 4.9110 VEGFR-2(PIT) +BMOV sHP-H Sheet 9 0f 20 Jun. 30, 2009 Injection +30 min WB: SHP-1 PBS VEGF-A VEGF-A VEGF-A wild-type WB: pTyr WB: VEGFR-2 110 3&1 900,000T 600 000 300,000~ 3E3 @522, > 0Control US. Patent Fig. 2(d) VEGF-A Mf(kDa) _ IP: VEGi‘Fi-é +0 +0 Fig. 2(e) U.S. Patent Jun. 30, 2009 Sheet 11 0f 20 US 7,553,496 B2 Fig. 3(a) 1,250,000 1 ‘k * A1,000.000 - * T 750,000 - T 500,000 - 250,000 - 0 . . . . PBS VEGF-A PlGF-1 VEGF-E CoCl2 CNV volume(“ 3 Fig. 3(0) Control +1 +2 M,(kDa) WB: SPARC WBI GAPDH US. Patent Jun. 30, 2009 Sheet 12 0f 20 US 7,553,496 B2 Fig. 3(0) VEGF-A * ‘k I * PBS 0 ng 100 ng 300 ng 300 ng rhSPARC SPARC Ab 900,000 1 600,000 - 300,000 - 3E5 22:2 > 0Fig. 3(d) 1,200,000 1 _ 0 0 0 0 0 9 600,000 - ES: @222, > 0300,000 - VEGF-A VEGF-A+ PBS SPARC Ab US. Patent Jun. 30, 2009 Fig. 4(a) CNV volume(pm3) 800,000-} 600,000 400,000 — 200,000 - 0. Fig. 4(b) CNV volume(pm3) 1,000,0001 800,000— 600,000 - 400,000‘ 200,000 - PBS \\\\\\\\\\\\\\\\\\w A Sheet 14 0f 20 +Ccl-2 wt I PBS [3 VEGF-A Q PlGF-1 @ VEGF-E :\ llllllllllllllllllllllllllllllllllllllllllllllllllll '\ lllllllllllllllll|lllllllllllllllllllllllllllllll VEGF-A VEGF-A :\ llllll|lllllllllllllllllllllllllllllllllllllllllllllllllllllllllllIlllllllllllllllllllllll US 7,553,496 B2 * PBS I CCl-Z Cc/2-/ US. Patent Jun. 30, 2009 Fig. 4(0) 900,000} % PBMCSheet 15 0f 20 US 7,553,496 B2 Control PBS VEGF-APlGF-1VEGF-E CIOd-LIP DC I D a Day 2 (PBS) 8 Day 2 (\lEGF-A) CD14+ CD34— US. Patent Jun. 30, 2009 Sheet 16 0f 20 US 7,553,496 B2 Fig. 4(6) 2001 .w Mw260 -821-£5LwQQREO 62PBS VEGF-A VEGF-A+ Ccl-2Ab Clod-LIP ClOd L|P+ Cal-2 Cal-2 Fig. 4m 900,000 W 1 600,000 — 300,000 — Ame: Z522>76 VEGF-A PBS US. Patent Jun. 30, 2009 Sheet 17 0f 20 US 7,553,496 B2 * l . .1 -_q_ 0 O0 53 2— 522a =  9::5~603VEGF-A+ PBS VEGF-A VEGF-A+ ZnPP CuPP VEGF-A VEGF-A+ VEGF-A+ Fig. 5(b) * 1,200,0001 900,000 - 600,000 - 300,000 — 0 3&1 vwas? >76CuPP ZnPP PBS Sheet 19 0f 20 US 7,553,496 B2 US. Patent Jun. 30, 2009 VEGF-A Ab -19 -18 -17 -16 -15 -14 -13 -12 900,000 1 ) a ( 6 M F - q o 00 “Va 00 0 0 6 33E3 22:9 >2log (VEGF-A) (M) inje ntrol EGF EGF ction INo 0C0 mv lgG -A Ab (1 ng) -A Ab (2 ng) 900,000 1 3 Fig. 6(b) 1]. 0 m 0 O nu, 0 6 E1382? >20 / §\\\\\\\\\\\\\\\\\\\\\\ 7///////// . 0 0 0 O 0 3 a F Near Control 1% Vegfr1 tk-l I Control @l. ..%////////////// ea NF UQ tm - o. m0 0 0 .U; 0 ..0 o21 g63SE3 252 >20) C ( 6 F 

VEGF-A as an Inhibitor of Angiogenesis and Methods of Using Same

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VEGF-A as an Inhibitor of Angiogenesis and Methods of Using Same

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