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(12) Patent Application Publication (10) Pub. No.: US 2010/0069301 A1 Li Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2010/0069301 A1 Li Et Al US 2010 OO69301A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0069301 A1 Li et al. (43) Pub. Date: Mar. 18, 2010 (54) COMPOSITIONS AND METHODS FOR Related U.S. Application Data TREATING PATHOLOGICANGIOGENESIS (60) Provisional application No. 60/869,526, filed on Dec. AND VASCULAR PERMEABILITY 11, 2006. Publication Classification (75) Inventors: Dean Li, Salt Lake City, UT (US); Christopher Jones, Salt Lake City, (51) Int. Cl. A638/16 (2006.01) UT (US); Nyall London, Bountiful, C07K 7/08 (2006.01) UT (US) C07K 7/06 (2006.01) C07K I4/00 (2006.01) Correspondence Address: A638/08 (2006.01) STOEL RIVES LLP - SLC A638/10 (2006.01) 201 SOUTH MAIN STREET, SUITE 1100, ONE A6IP35/00 (2006.01) UTAH CENTER (52) U.S. Cl. ........... 514/12:530/328; 530/327: 530/326; SALT LAKE CITY, UT 84111 (US) 530/325; 530/324; 514/15: 514/14: 514/13 (57) ABSTRACT (73) Assignee: UNIVERSITY OF UTAH Compounds, compositions and methods for inhibiting vascu RESEARCH FOUNDATION, Salt lar permeability and pathologic angiogenesis are described herein. Methods for producing and screening compounds and Lake City, UT (US) compositions capable of inhibiting vascular permeability and pathologic angiogenesis are also described herein. Pharma (21) Appl. No.: 12/518,730 ceutical compositions are included in the compositions described herein. The compositions described herein are use ful in, for example, methods of inhibiting vascular permeabil (22) PCT Filed: Dec. 11, 2007 ity and pathologic angiogenesis, including methods of inhib iting vascular permeability and pathologic angiogenesis (86). PCT No.: PCT/US07/25354 induced by specific angiogenic, permeability and inflamma tory factors, such as, for example VEGF, bFGF and thrombin. S371 (c)(1), Methods for treating specific diseases and conditions are also (2), (4) Date: Nov. 19, 2009 provided herein. A wild-type robo4 MO Patent Application Publication Mar. 18, 2010 Sheet 1 of 31 US 2010/0069301 A1 A-AA-A.. Patent Application Publication Mar. 18, 2010 Sheet 2 of 31 US 2010/0069301 A1 TM CCO . CC2 obo4 HA oSit2 Mock Robo4 Robo4ATail FIG. 2B Patent Application Publication Mar. 18, 2010 Sheet 3 of 31 US 2010/0069301 A1 160 Pa).<0.05 P.69se so oo: PO.47=0 OS it2 Mock Robo4 Robo4 Robo4 pCDNA3 NH2 COOH FIG. 2C IP: HA (Robo4) Blot: V5 (Hic-5) IP. HA (Robo4) Blot: HA (Robo4) Cell Lysates Blot: V5 (Hic-5) FIG. 3A Patent Application Publication Mar. 18, 2010 Sheet 4 of 31 US 2010/0069301 A1 Se-sa Blot: Hic-5 Blot: Paxillin Paxillin-V5: - -- RobO4-HA. -- pCDNA3: - -- IP: HA (Robo4) Blot: V5 (Paxillin) IP: HA (Robo4) Blot: HA (Robo4) Cell Lysates Blot: V5 (Paxillin) FIG. 3C Slit? -- w IP: HA (Robo4) Blot: Paxillin IP: HA (Robo4) Blot: HA (Robo4) is as Cell lysates Blot: Paxillin FIG. 3D Patent Application Publication Mar. 18, 2010 Sheet 5 of 31 US 2010/0069301 A1 TM CCO CC2 Amino Acids: 494 73.1 : 1039 494-1039 Gs) - 494-73 731-1039 GST-RobO4 Patent Application Publication Mar. 18, 2010 Sheet 6 of 31 US 2010/0069301 A1 Amino Acids: as a GRROCCO CC2 494-554 Gs) 555-604 SOS-674 675-73 Gs FIG. 4C GST-Robo4. M 8 s 6 s s c33. e 1. a 3° S 63 Blot: Paxillin Patent Application Publication Mar. 18, 2010 Sheet 7 of 31 US 2010/0069301 A1 GST-RobO4 Blot: Paxillin Blot: Mena Bot: Robo4 PCO.O1 s O Slit2 | Mock Robo4APM Robo4 FIG. 4F Patent Application Publication Mar. 18, 2010 Sheet 8 of 31 US 2010/0069301 A1 DSlit2 Mock Sit2: + s : -- - " Fibronectin: + + aga + - GTP-Rac Total Rac Patent Application Publication Mar. 18, 2010 Sheet 9 of 31 US 2010/0069301 A1 1.2 1.O 0.8- 0.6- 0.4- O.2 O.O L. Site: re: Fibronectin: e cDNA: Robo4. pCDNA3 FIG 5C 1OOOO a SOOO OSlit2 Mock 4000 2000 Robo4APM Robo4 FIG.SD Patent Application Publication Mar. 18, 2010 Sheet 10 of 31 US 2010/0069301 A1 ROCO4 Robo4APIM pCDNA3 Slit2: + - - - - - - - - - Fibronectin: + + - + + - + + - GTP-Rac Total Rac St. is: H se Fibronectin: s se s cDNA, ROA. Ro4AP pCONA3 FIG. 5F Patent Application Publication Mar. 18, 2010 Sheet 11 of 31 US 2010/0069301 A1 2OOO as SOO D s OOO a Sit2 Mock s SOD O Robo4. Roo4. Lacz RaCG12V FIG SG GTP-RaC Total Rac Patent Application Publication Mar. 18, 2010 Sheet 12 of 31 US 2010/0069301 A1 1.25 st 1.00 2 S. O.75 s O.50 S. O.25 s O.OO St2: s WEGF: : t FIG. 5 LD Domains Lim Domains Paxillin Paxillin-LD E. E. Paxillin-Lim PaxillinALim4 FIG. 6A Patent Application Publication Mar. 18, 2010 Sheet 13 of 31 US 2010/0069301 A1 Paxillin-LD-V5: or Paxillin-Lim-V5: - - w RobO4-HA: - + - pCDNA3: - + - + P: HA (RobO4) Blot: Paxillin , IP: HA (Robo4) Blot: HA (Robo4) Cell Lysates Blot: V5 (Paxillin) Paxillin(ALim4-V5 - s Paxillin-V5: - - - RobO4-HA: - s pCDNA3: - - H IP: HA (Robo4) Blot: V5 (Paxillin) IP: HA (Robo4) Blot: HA (Robo4) Patent Application Publication Mar. 18, 2010 Sheet 14 of 31 US 2010/0069301 A1 Ch-PaxillinMLima + A. " h Wild-Type Ch-Paxillin: - Paxillin SiRNA. -- a. -- w RObo4-HA: - a. Bot: Paxillin. | Blot: HA (Robo4) s s y OSlit2 s Mock Robo4 Robo4 Robo4 al H hPax RNA hPax RNAi s ae Ch-PaxALim4 Ch-Pax WT FIG. 6E Patent Application Publication Mar. 18, 2010 Sheet 15 of 31 US 2010/0069301 A1 100 8O SO | 40 O robo4 Morpholino: + + 3 + -- robo4 mRNA: va 4} Ax robo4ATail mRNA: - e s : s robo4API mRNA: -- (t FIG 7A integrins Motility FIG. 7B Patent Application Publication Mar. 18, 2010 Sheet 16 of 31 US 2010/0069301 A1 gG FN M. CCO CC2 RObo4 Protein (XX) robo4 Genomic mo ame Forward Rowerso Patent Application Publication Mar. 18, 2010 Sheet 17 of 31 US 2010/0069301 A1 LD Domains LIM Domains Hic5 all 234 i1-1 #3-9 i4-5 f5-4 #9-8 pGBK7 Robo4 pACT2 pACT2 pGBKT7 Robo4 Hics Hics -LT LaCZ FIG. 9B TM CCO CC2 WT Robo4 u1 Paxillin Interaction Motif: 604-PAARRFPSKLAGTSSSPWASSDSSLCSRRG LCSPRMSL-639 FIG 10 Patent Application Publication Mar. 18, 2010 Sheet 18 of 31 US 2010/0069301 A1 Sit2: Fibronectin: -- -- P H -- FIG 11 A SGAP1 s -- Robo: s - s Robo4: s " pCDNA3: ap t P: HA (Robo) Blot: Flag (sroAP1) P: HA (Robo) Blot. HA (Robo) Cell Lysates Blot: Flag (srdAP1) FIG 11B Patent Application Publication Mar. 18, 2010 Sheet 19 of 31 US 2010/0069301 A1 Wildtype Mock vs slit 5 O FIG. 12A Wildtype Mice Mock Vs Saline Mock Saline FIG. 12B Patent Application Publication Mar. 18, 2010 Sheet 20 of 31 US 2010/0069301 A1 Knockout Mock Vs Slit 20 18 16 14 12 10 ------------- 8 s 4 ------ 2 O FIG. 12C Dermal Permeability Minus Background 0.06 Netrin-1 VEGF O.05 OVEGF + Sit ZVEGF + Netrin-1 0.04 0.03 0.01 -0.01 Patent Application Publication Mar. 18, 2010 Sheet 21 of 31 US 2010/0069301 A1 Retinal Permeability O.08 Saline DVEGF OWEGF + Mock ZVEGF + Sit O O4 0.02 Dermal Permeability C, 14 IgG Vegf+lgG 0.2 O Semaphorin 3A-Fc ZVegf+ Semaphorin Ephrin-A1-Fc 0.1 BVegf+ Ephrin 0.08 0.06 0.04 Patent Application Publication Mar. 18, 2010 Sheet 22 of 31 US 2010/0069301 A1 FIG. 16 Patent Application Publication Mar. 18, 2010 Sheet 23 of 31 US 2010/0069301 A1 A B Anti-HAP Anti-HAP anti-myc Western anti-myc western cell lysates conditioned media Cells CM Rob04-HA - - + - + NRobo4-HA . Slit-myc * * * Slit-myc - - - - - Control-HEK - + - + - Contro-HEK . HEK -- - - - - HEK -- Kd 2 3 3 200 | 200 97 97 68 68 43 43 fluorescence Contro-HEK RobO4-HEK Patent Application Publication Mar. 18, 2010 Sheet 24 of 31 US 2010/0069301 A1 Detail pericytes & Overview smooth muscle Cels Detail tip cells & Inner retina Outer retinal Adult retina overview vascular plexus vascular plexus b 1. PECAM Robo Robo4 d Patent Application Publication Mar. 18, 2010 Sheet 25 of 31 US 2010/0069301 A1 Robo4/ Robo APAP Cell Migration b Tube Fortation NS d WEGF-A-induced Dermal Permeability 200 0.025 0.020 O.O15 0.010 0.05 f P-VEGFR2 (Y1175) 9 h total VEGFR2 sas Patent Application Publication Mar. 18, 2010 Sheet 26 of 31 US 2010/0069301 A1 R e Oxygen-induced Retinopathy j Choroidal Neovascularization H NS 40 100,000 sa 80,000 O Robo4" Robo4APAP 60,000 40,000 20,000 FIG. 20 Patent Application Publication Mar. 18, 2010 Sheet 27 of 31 US 2010/0069301 A1 a bFGF-induced Tube Formation b Thrombin-induced in vitro Permeability 2 200 a. 175 59 5 150 me ( ) O 125 SS SE Y 100 O CO E 75 t O 50 25 O a 0 Mock Sit2 bFGF: + + Mock: + - No Thrombin Slit2: . Thrombin (2U/ml) FIG 21 Patent Application Publication Mar. 18, 2010 Sheet 28 of 31 US 2010/0069301 A1 Cell infiltrate Protein Exudate a 750,000 250 ? 200 500,000 5 CDO 150 R() SO) 100 8 250,000 . o 50 SS O O w Saline MOCk Slit Saline MOCk Slit LPS LPS FIG. 22 Patent Application Publication Mar. 18, 2010 Sheet 29 of 31 US 2010/0069301 A1 pSecTag Å Slit2D1-03100kDa ·Slit2D140kDa Siit2D1-D4120kDa Slit2D1-D260kDa Slit2N150kDa aa19 aa19 1524Gº Migrated HMVEC cells B200 n a o oo as ns . d d d 160 180 pSecTag Na IS 2IS . V as T. to as - g 2C O aS Sc" to 2S EIVEGF(15ng/ml) –Mock+VEGF115mg/m)DNegativecontº two- or 2S gS-C aS 9 OS FIG.
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