US008377886B2 (12) United States Patent (10) Patent No.: US 8,377,886 B2 Susztak et al. (45) Date of Patent: Feb. 19, 2013 (54) USE OF GAMMA SECRETASE INHIBITORS 7,144.910 B2 12/2006 Madin et al. AND NOTCH PATHWAY INHIBITORS FOR E.R. 358 (SR." TREATMENT AND PREVENTION OF RENAL 2003/0166894 A1* 9/2003 Kapeller Libermann et al. .......... 536,231 DISEASE 2006/0264380 A1* 11/2006 Hellstrom et al. .............. 514, 19 2007/0066568 A1 3/2007 Dalton et al. (75) Inventors: Katalin Susztak, Cresskill, NJ (US); Bernhard Bielesz, Rye, NY (US); FOREIGN PATENT DOCUMENTS Thiruvur G. Niranjan, Bronx, NY (US) WO WO2004/073630 * 9/2004 (73) Assignee: Albert Einstein College of Medicine of OTHER PUBLICATIONS Yeshiva University, Bronx, NY (US) “Glomerulonephritis' (in WWW. intellihealth.com/ IH/ht?t=31198& p=~br, IHWI-st, 24479-r, WSIH (*) Notice: Subject to any disclaimer, the term of this W000-b.* liprevent).* patent is extended or adjusted under 35 Vanes et al. in Nature 435(16), pp. 959-963 (2005).* U.S.C. 154(b) by 78 days. Masuda et al. in American Journal of Pathology, 159(2) (2001).* Yum et al. in Human Pathology 15(10), 921-927 (1984).* PCT International Preliminary Report on Patentability (dated Mar. (21) Appl. No.: 12/733,339 16, 2010) in connection with PCT International Patent Application No. PCT/US2003/010362, 6 pages. (22) PCT Filed: Sep. 4, 2008 Cheng HT et al., entitled Notch2, but not Notch1, is required for proximal fate acquisition in the mammalian nephron, Development (86) PCT NO.: PCT/US2O08/O10362 134,801-811 (2007). S371 (c)(1) Cheng HTetal. entitled “The role ofNotch signaling in specification (2), (4) Date. Apr. 30, 2010 of podocyte and proximal tubules within the developing mouse kid s e a V8 ney.” Kidney Int., Nov. 2005:68(5): 1951-2, Abstract Only. Cheng HT et al., entitled “Y-Secretase activity is dispensable for (87) PCT Pub. No.: WO2009/035522 mesenchyme-to-epithelium transition but required for podocyte and PCT Pub. Date: Mar. 19, 2009 proximal tubule formation in developing mouse kidney.” Develop ment 130, 2003, 5031-5042. 65 Prior PublicationO O Data Niranjan Tet al., entitled “The Notch ppathway y in podocytesp playplays a (65) role in the development of glomerular disease.” Nature Medicine, US 2010/0222283 A1 Sep. 2, 2010 vol. 14, No. 3, Mar. 2008, 290-298, with attached supplemental figures and tables, 13 pages. O O Kretzler Metal., entitled “Notch inhibition reverses kidney failure.” Related U.S. Application Data Nature Medicine, vol. 14, No. 3, Mar. 2008, 246-247. (60) Provisional application No. 60/993,865, filed on Sep. Konishi J et al., entitled "Gamma-Secretase Inhibitor Prevents 14, 2007. Notch3 Activation and Reduces Proliferation in Human Lung Can cers.” Cancer Res 2007:67:(17), pp. 8051-8057. (51) Int. Cl. PCT Notification of Transmittal of The International Search Report and The Written Opinion of The International Searching Authority, or 3. s 3. the Declaration dated Nov. 28, 2008 by the U.S. Patent Office in ( .01) connection with PCT International Patent Application No. PCT/ A6 IK3I/225 (2006.01) US2008/10362, 8 pages. A6IPI3/2 (2006.01) (52) U.S. Cl. ...... 514/19:514/217: 514/548: 514/212.04 * cited by examiner (58) Field of Classification Search ........................ None See application file for complete search history. Primary Examiner — Dennis Heyer (74) Attorney, Agent, or Firm — Amster, Rothstein & (56) References Cited Ebestein U.S. PATENT DOCUMENTS (57) ABSTRACT 6,756,511 B2 6/2004 Pineiro et al. Methods are provided for treating and preventing renal dis 6,984,6266,890,956 B2 5,1/2006 2005 ChurcherNadin et al. et al. ease in a Subject by administering a gamma Secretase inhibiinhih 7,049,296 B2 5, 2006 Pineiro et al. tor or a Notch pathway inhibitor to the subject. 7,101,895 B2 9, 2006 Churcher et al. 7,138.400 B2 11/2006 Collins et al. 10 Claims, 12 Drawing Sheets U.S. Patent Feb. 19, 2013 Sheet 1 of 12 US 8,377,886 B2 2:7 "...a8 . a isf ..is a. f if : sil. 3. d Figure 1A-1E U.S. Patent US 8,377,886 B2 a Figure 2A-2C U.S. Patent Feb. 19, 2013 Sheet 3 of 12 US 8,377,886 B2 g f th 6000 S. 500 E 4000 3000 f 2000 1. i W NC1 A TA CN1 e f 000 tACN1 0. O. g5 : . is. is. 3. ; Figure 3A-3F U.S. Patent Feb. 19, 2013 Sheet 4 of 12 US 8,377,886 B2 a strain (podocin it? A mice) doxycyclire -a- icni (tetO-NICD mice) 33 O5 5 TACN1 Figure 4A-4D U.S. Patent Feb. 19, 2013 Sheet 5 of 12 US 8,377,886 B2 a b 1 40 Vector EIN E E EIN EN EN 120 Tam - 0.75 0.75 1.5 3 100 80 val1744N1 60 40 3-actin 20 Vector El E E EIN EN EN Cleaved Tam P s 15 0.75 15 3 PARP C : Cleaved is us dis (wi is gwo (O Cl 1. 9 D S x t C N Tam - - 3 0.75 15 3 3 3 Z-wad - - - - - - 10 - Z-LEHD - - - - - - - 10 d 35 DVEGF 30 2 TGF 25 2 30 É 20 a 25 S 15 2 | 2N 20 2 2 2N 15 i: 10 3.2N | 2N3S 10 5 23.2 2S 5O E° N E. E.- Tanvector EIN- E- 1.5E 0.75ENENEN 1.5 3 Figure 5A-5E U.S. Patent Feb. 19, 2013 Sheet 6 of 12 US 8,377,886 B2 - Relative mRNA a 0.75 0.75t 1.5M 3u M Tam an Tan EGFP an EGFP EGFP EGFP CT CN1 EGFP CN1 TICN CN1 100 109 109 427 720 1413 1.88 180 1435 26.62 1704 181 112 0.79 152 106 125 0.78 0.58 169 063 Wector EN E E EIN EN EN c Tam - 0.75 0.75 15 3 Figure 6A-6C U.S. Patent Feb. 19, 2013 Sheet 7 of 12 US 8,377,886 B2 a SB PD Y GS - - -- -- -- - - - - TGFB(min) - 5 30 60 120 240 5 30 60 120 240 60 60 60 val1744N1 is is s 3-Actin b GSI TGFB(hr) P-p53 + + - PF C GS 1 4 24 1 4 24 24 10 5 0.5 O O CTL TGFb TGFb TGFB CTL TGFb TGFb TGFs GSXX PF GS X's GSX Figure 7A-7E U.S. Patent Feb. 19, 2013 Sheet 8 of 12 US 8,377,886 B2 + + + -- -- a 5 30 60 120 240 5 30 60 120 240 val1744N1 3-Actin P TGFB val1744N1 Figure 8A-8B U.S. Patent Feb. 19, 2013 Sheet 9 of 12 US 8,377,886 B2 b CL Het DM OKO DM 100 80 CTL Het-DN KO-DM d Nephrin Fodocin C 10 Nephrin 3E 8 CTL S s5 6 4 C 2 O Het DM CTL Het DM KODM 200 e CL OOO Het DM 3. 800 o KO DM Z c coo e KO DM 400 200 TGFB VEGF Figure 9A-9E U.S. Patent Feb. 19, 2013 Sheet 10 of 12 US 8,377,886 B2 a Nephrin 18 Podocin S WEGF 14 12 0.8 OS O4 O.2 O 8:8 R pjwTWT Rbplox/WT Rbpjioxiflox b ce ce ce 5 350 3. 25 2O) SO 5 RbpjwTiwT RbptioxWT Rbpjfoxiflox Rbpiloxiflox Ce Ce Cre Cre DM Mice n= Weight Glucose WT 5 34.94 1286 KO 5 32.65.4 17123 CTL Het DM 10 23.43.4 6000 Het Dr. KO DM 10 24.315 576-28 23.4.56 KCM Combined Cellularity Mesangial Expansion Figure 10A-10D U.S. Patent Feb. 19, 2013 Sheet 11 of 12 US 8,377,886 B2 a S 4000 5 g 3000 2000 S E 1000 C < 0 CTL PAN PAN DBZ C s S 4.5 3. C 4. z 3.5 & E 3. S 25 is 2 S : i.S. E. 1 did 0.5 2 c PAN 82. f PAN 4.5 4. op53 C 33.5 pe21 O. 5 redO E 3. 9. 2.5 2 e 15 A. 0.5 CTL PAN O32 Pai Figure 11A-11F U.S. Patent Feb. 19, 2013 Sheet 12 of 12 US 8,377,886 B2 O2 3.ss CTL PAN OBZPAN Netc. Ne2 O2 3 es Hes5 ey 4. 6 35 5, 30ao Ya 2 2500 s 2. s 5 1500 m 56 k c AOR AREZ Figure 12A-12C US 8,377,886 B2 1. 2 USE OF GAMMA SECRETASE INHIBITORS it has not been known whether protection from podocyte loss AND NOTCH PATHWAY INHIBITORS FOR or dysfunction would influence glomerular disease develop TREATMENT AND PREVENTION OF RENAL ment. DISEASE The Notch signaling pathway comprises a family of trans membrane receptors, their ligands, negative and positive CROSS-REFERENCE TO RELATED modifiers, and transcription factors'''. To date, 4 mamma APPLICATIONS lian receptors (Notch1 through Notch4) and at least 5 ligands (Delta 1, 3, and 4 and Jagged 1, 2) have been identified. This application is a national stage entry under 35 U.S.C. Binding of the ligand renders the Notch receptor susceptible S371 of PCT International Patent Application No. PCT/ 10 to metalloprotease- and Y-secretase-mediated proteolytic US2008/010362, filed Sep. 4, 2008, and claims priority to cleavage, which in turn results in the release of the Notch U.S. Provisional Patent Application No. 60/993,865, filed intracellular domain (ICN) from the plasma membrane and Sep.