THAT INA MI CANUS009945845B2 WITH AN ALTA MONITORI (12 ) United States Patent ( 10 ) Patent No. : US 9 , 945 ,845 B2 Brändli et al. ( 45 ) Date of Patent : Apr. 17 , 2018 ( 54 ) METHODS OF SCREENING USING ( 56 ) References Cited AMPHIBIANS U . S . PATENT DOCUMENTS (71 ) Applicants :André W . Brändli, Zollikon (CH ) ; 2006 /0159676 A1 7 / 2006 Krieg Roland E . Kälin , Berlin (DE ) 2007 /0107072 A15 /2007 Carmeliet 8(72 ) Inventors: André W . Brändli , Zollikon (CH ) ; Roland E . Kälin , Berlin (DE ) FOREIGN PATENT DOCUMENTS WO 2006 /076736 A2 7 /2006 ( * ) Notice : Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U . S . C . 154 ( b ) by 0 days. OTHER PUBLICATIONS Seiffert et al. (2000 ) J . Biol. Chem . 275 : 34086 - 34091. ( 21 ) Appl. No. : 15 /803 ,662 Kälin et al. (2007 ) Developmental Biol . 305 :599 -614 . Kälin et al . (2009 ) Blood 114 : 1110 - 1122 . ( 22 ) Filed : Nov . 3 , 2017 Gu et al. ( 1999 ) Am . J . Physiol . Heart Circ . Physiol. 277 : 595 -602 . Ny et al . (2005 ) Nature Medicine 11 :998 - 1004 . (65 ) Prior Publication Data Wheeler et al . (2009 ) Developmental Dynamics 238 : 1287 - 1308 . Ny et al. ( 2008 ) Blood 112 : 1740 - 1749 . US 2018 /0059097 A1 Mar. 1 , 2018 Tomlinson et al . ( 2009 ) Molecular BioSystems 5 : 376 -384 . Tomlinson et al . ( 2005 ) Molecular BioSystems 1 : 223 - 228 . Related U . S . Application Data Feoktistov et al. ( 2002 ) Circulation Research 90 :531 - 538 . (63 ) Continuation of application No. 13 /256 , 209 , filed as application No . PCT /EP2010 /053613 on Mar. 19 , Primary Examiner — Nancy J Leith 2010 , now abandoned . (74 ) Attorney , Agent, or Firm — VLP Law Group LLP ; (60 ) Provisional application No . 61 / 161, 497 , filed on Mar. David A . Roise 19, 2009 . (51 ) Int. Ci. (57 ) ABSTRACT C120 1/ 68 ( 2018 .01 ) GOIN 33 /50 ( 2006 .01 ) High - throughput methods of screening agents for activities ( 52 ) U . S . CI. affecting renal , cardiac , blood or lymphatic vascular devel CPC . GOIN 33/ 5088 ( 2013 . 01) ; GOIN 2333 /4606 opment and functions in amphibians in multiwell plates are (2013 . 01 ) provided . Also provided are novel compounds thatmodulate ( 58 ) Field of Classification Search blood and lymphatic vascular development. None See application file for complete search history . 23 Claims, 8 Drawing Sheets atent Apr . 17 , 2018 Sheet 1 of 8 US 9 , 945 , 845 B2 Matings Embryos : . - y . .52 .. : 61 , 1 . 1. .. .. - . - . .. *: . / . / . * . : • y . - - - - - * : * . " . :. ees . Chemical librarybrary KAM hermainanto * D tre moltes somte therespectnationtoiletentertainmentwithin WAO W rite things WinXY wwwboybang . Site RES 3 poun ? Edema * A * 1???? 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SB1 .5 - { ???? , K | gf} S4312 { ? .{ } 4 Silv # KA # # # # # # # # # # # GU54 16 {] t { Tyf _ infin & 4GA wwwwwwwwwwwwwwwwwwwwwwww janmak {} 5 1 461 23 | £ 100 } % of Control * f { { } {} } atent Apr . 17 , 2018 Sheet 7 of 8 US 9 , 945 , 845 B2 HUVEC 337 0.1%DMSO Nicardipine 7-Cyclo VOX radio 2-methoxyestradiolPhenylxanthineINOX Figure* 7 atent Apr . 17 , 2018 Sheet 8 of 8 US 9 ,945 , 845 B2 VEGFA naphthyridine Control VEGFA up Lymphatic vessels Blood vessels number? mm ? area as % ROI number/ mm2 g area as % ROI wwwwwwwwwwwwwwwww * * * www * 0 .0018 *Makikit 2008 * * * * D 0 . 0021 p = 0 . 05 ?????? ?????????????? 3 *wwwwwwwwwww C THY D *Hotti 1 * t * north their * MYW.TR sob i j o WWWWWWWWWWWWWWW . o JAMES * VEGF - * # . j Naph . - * . Figure 8 US 9 ,945 ,845 B2 METHODS OF SCREENING USING tor - C (VEGFC ), tumor lymphangiogenesis and lymph node AMPHIBIANS metastasis has been found in human and in experimental tumors (Pepper et al ., 2003 ) . VEGFC promotes lymphan CROSS -REFERENCE TO RELATED giogenesis by activating VEGF receptor- 2 (VEGFR2 ) and APPLICATION 5 VEGFR3 on lymphatic endothelial cells (Makinen et al. , 2001) . VEGF - C - deficient mice fail to develop a functional This application is a continuation of U . S . patent applica lymphatic system ( Karkkainen et al. , 2004 ) , and transgenic tion Ser . No . 13 /256 ,209 , filed on Sep . 12 , 2011 , which is a expression of a soluble VEGFR - 3 results in pronounced national phase application of PCT International Application lymphedema (Makinen et al. , 2001 ) . However , blockade of the VEGF - C /VEGFR - 3 axis only partially inhibits lym the benefit of U . S . Provisional Application No. 61/ 161 ,497 , phatic metastasis , indicating that additional pathways are filed Mar. 19 , 2009 , the disclosures of which are incorpo involved in mediating the formation and growth of lym rated herein by reference in their entireties. phatic vessels . There have been previous attempts to identify lymphatic specific receptors and pathways by transcriptional FIELD OF THE INVENTION and proteomic profiling of cultured lymphatic endothelial The present invention provides for in vivo chemical cells (LEC ) (Hirakawa et al. , 2003 ; Petrova et al. , 2002 ; screening methods involving a simple phenotypic read -out Roesli et al ., 2008 ) . However , large - scale functional in vivo ( edema formation or lethality ) , optionally followed by in screens to identify molecular pathways or drug - like small situ hybridization or immunohistochemistry to screen for 20 molecule modulators of lymphatic vessel formation have agents modulating cardiac, vascular, lymphatic , or renal been missing to date . development or organ functions in amphibian embryos or In the last years , cost - efficient maintenance together with tadpoles in a multiwell format. Furthermore , active com abundant experimental techniques and molecular tools , have pounds interfering with blood vascular and lymphatic devel - made zebrafish the only vertebrate model used for large opment in Xenopus laevis are disclosed . Also provided are 25 scale in vivo drug screens (Zon and Peterson , 2005 ) . screening methods to identify pathways that mediate lym - Amphibians offer many of the same experimental advan phatic and /or vascular development in an amphibian . tages that have favored zebrafish in the past , such as rapid extra - uterine development, the transparency of developing BACKGROUND OF THE INVENTION tadpoles , and the permeability of the skin for small mol 30 ecules , but they have to date not been employed for large Lymphatic vessels play a major role in tissue pressure scale chemical library screens to gain insight into vascular homeostasis , immune responses , and the uptake of dietary development. Amphibians have a common evolutionary fat and fat - soluble vitamins , as well as in inflammation and history with mammals that is an estimated 100 million years cancer progression (Cueni and Detmar, 2006 ) . Recent stud - longer than between zebrafish and mammals (Brändli , ies indicate that both lymphatic and blood vessels are 35 2004 ) . Being both tetrapods, amphibians and mammals involved in chronic inflammatory diseases such as rheuma - share extensive synteny at the level of the genomes and have toid arthritis , inflammatory bowel disease and psoriasis many similarities in organ development, anatomy, and ( Alitalo et al. , 2005 ; Carmeliet, 2003 ; Cueni and Detmar, physiology (Christensen et al. , 2008 ; Raciti et al. , 2008 ) . 2006 ) . But the formation and activation of both types of These traits favor the use of amphibians for large- scale in endothelium have also important roles in the progression 40 vivo drug screens . In the past , embryos and tadpoles of the and metastasis of the majority of human cancers ( Alitalo et African clawed frog ( Xenopus laevis) have served as a al ., 2005 ; Carmeliet, 2003 ) . Tumors need to induce the powerful animal model to study blood vascular development growth of new blood vessels (angiogenesis ) in order to and angiogenesis (Cleaver and Krieg