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USOO8936806B2 (12) United States Patent Kuhn (54) PERIOSTIN INDUCES PROLIFERATION OF CARDIOMYOCYTES AND PROMOTES CARDIAC REGENERATION (75) Inventor: Bernhard Kuhn, Brookline, MA (US) (73) Assignee: Children’s Medical Center Corporation, Boston, MA (US) (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 707 days. (10) Patent N0.: (45) Date of Patent: US 8,936,806 B2 Jan. 20, 2015 Litvin J, S Zhu, R Norris, and R Markwald. 2006. Perio stin Family of Proteins: Therapeutic Targets for Heart Disease. Anat Rec A Discov Mol Cell Evol Biol. 287(2): 1205-1212.* Crackower, MA. et al. Regulation of myocardial contractility and cell size by distinct PI3K-PTEN signaling pathways. Cell 110, 737 49 (2002). del Monte, F. et al. Abrogation of ventricular arrhythmias in a model ofischemia and reperfusion by targeting myocardial calcium cycling. Proc Natl Acad Sci U S A 101, 5622-7 (2004). Engel, F.B., et al. Proc. Natl. Acad. Sci. USA 103, 15546-15551 (2006). Engel, F.B. et al. p38 MAP kinase inhibition enables proliferation of adult mammalian cardiomyocytes. Genes Dev 19, 1175-87 (2005). Fazel, S. et al. Cardioprotective c-kit+ cells are from the bone marrow (21) Appl. No.: 12/524,097 (22) PCT Filed: Jan. 22, 2008 and regulate the myocardial balance of angiogenic cytokines. J Clin Invest 116, 1865-77 (2006). He, T.C. et al. A simpli?ed system for generating recombinant (86) PCT No.: PCT/US2008/051659 adenoviruses. Proc Natl Acad Sci U S A 95, 2509-14 (1998). Howard, C.V. & Reed, M. Unbiased Stereology: Three-Dimensional § 371 (0)0), (2), (4) Date: (87) Measurement in Microscopy, (BIOS Scienti?c Publishers, Oxford, Feb. 11, 2010 PCT Pub. No.: WO2008/091867 PCT Pub. Date: Jul. 31, 2008 (65) Prior Publication Data US 2010/0166827 A1 Jul. 1,2010 2005). Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 110:673-87. 2002. Katsuragi, N. et al. Periostin as a novel factor responsible for ventricular dilation. Circulation 110, 1806-1813 (2004). Litvin, J. et al. Cardiovasc. Pathol. 15, 24-32 (2006). Ma, H., Sumbilla, C.M., Farrance, I.K., Klein, M.G. & Inesi, G. Am J Physiol Cell Physiol 286, C556-64 (2004). Miranti, C.K. & Brugge, J .S. Sensing the environment: a historical perspective on integrin signal transduction. Nat Cell Biol 4, E83-90 (2002). Related US. Application Data (60) (51) Provisional application No. 60/881,938, ?led on Jan. Circulation 114,1206-1213 (2006). Pasumarthi, K.B., et al. Circ. Res. 96, 110-118 (2005). 22,2007. Pasumarthi, K.B. & Field, L.J. Cardiomyocyte cell cycle regulation. Int. Cl. A61F 2/00 Circ Res 90, 1044-54 (2002). Prunier, F. et al. Am J Physiol Heart Circ Physiol (2006). Rape, M. & Kirschner, M.W. Autonomous regulation of the (2006.01) C12N5/077 A61K38/17 (52) U.S.Cl. CPC ........ .. (2010.01) (2006.01) C12N5/0657(2013.01);A61K38/1709 (2013.01); C12N2501/585 (2013.01); CIZN 2501/998 (2013.01) USPC ........... .. 424/426; 424/93.7; 424/423; 514/12 (58) Nakajima, H., et al. Circ. Res. 94, 1606-1614 (2004); W00,Y.J. et al. Field of Classi?cation Search anaphase-promoting complex couples mitosis t0 S-phase entry. Nature 432, 588-95 (2004). Rubinson, D.A. et al. Nat. Genet. 33, 401-406 (2003. Sadoshima, J. & Izumo, S. Annu Rev Physiol 59, 551-71 (1997). Shi, Q. & King, R.W. Nature 437, 1038-42 (2005). Soonpaa, M.H., Kim, K.K., Pajak, L., Franklin, M. & Field, L.J. Cardiomyocyte DNA synthesis and binucleation during murine development. Am J Physiol 271, H2183-9 (1996). Strejan et al., J. Neuroimmunol. 7:27 (1984). (Continued) None See application ?le for complete search history. (56) References Cited U.S. PATENT DOCUMENTS 6,444,642 B1 2005/0042254 A1* 2007/0264254 A1 Primary Examiner * Carlos Azpuru Assistant Examiner * David Browe (74) Attorney, Agent, 0rFirm * Nixon Peabody LLP; David S. Resnick; Leena H. Karttunen Contarino 9/2002 Sklar et al. 2/2005 Freyman et al. ............ .. 424/426 11/2007 Zhou OTHER PUBLICATIONS Gillan, L, D Matei, DA Fishman, CS Gerbin, BY Karlan, and DD Chang. 2002. Periostin secreted by epithelial ovarian carcinoma is a ligand for alpha v-beta 3 and alpha v-beta 5 integrins and promotes cell motility. Cancer Research; 62: 5358-5364.* Visconti RP and RR Markwald. 2006. Recruitment of New Cells into the Postnatal Heart: Potential Modi?cation of Phenotype by Periostin. Ann NYAcad Sci. 1080: 19-33.* Nadal-Ginard B, J Kaj stura, A Leri, and P Anversa. 2003. Myocyte Death, Growth, and Regeneration in Cardiac Hyperttrophy and Fail ure. Circ Res. 92: 139-150.* (57) ABSTRACT The present invention provides compositions and methods for increasing proliferation, increasing cell cycle activity, and/or proliferation of postmitotic mammalian differentiated cardi omyocytes. The invention can be used to slow, reduce, or prevent the onset of cardiac damage caused by, for example, myocardial ischemia, hypoxia, stroke, or myocardial infarc tion in vivo. In addition, the methods and compositions of the invention can be used to enhance proliferation of differenti ated cardiomyocytes in vitro and/ or ex vivo, which can then be used in tissue grafting. 9 Claims, 17 Drawing Sheets US 8,936,806 B2 Page 2 (56) International Preliminary Report on Patentability mailed Feb. 2, 20 12 References Cited for Application No. PCT/US2010/042565 (7 pages). International Search Report and Written Opinion mailed Apr. 20, 2011 for Application No. PCT/US2010/042565 (13 pages). OTHER PUBLICATIONS Tai, I.T., Dai, M. & Chen, L.B. Carcinogenesis 26, 908-15 (2005). Litvin, J. et al., Cardiovascular Pathlogy, vol. 13, pp. S139-S200, Jackson-Fisher et al., Formation of Neu/ErbB2-induced mammary XP02573717 abstract. tumors is unaffected by loss of ErbB4. Oncogene. Sep. 14, Litvin, J. et al., The Anatomical Record, Part A, Discoveries in Molecular, Cellular, and Evolutionary Biology, vol. 287, pp. 1205 1212, 2005. Kim et al., International Journal ofCancer, vol. 117, pp. 51-58, 2005. 2006;25(41):5664-72. Epub May 1, 2006. Keefe DL. Trastuzumab-associated cardiotoxicity. Cancer. Oct. 1, 2002;95(7):1592-600. Ahuja et al., Cardiac myocyte cell cycle control in development, Kuhn et al., Periostin induces proliferation of differentiated disease, and regeneration. Physiol Rev. Apr. 2007;87 (2)1521-44. Bersell et al., Neuregulinl/ErbB4 signaling induces cardiomyocyte Bettencourt-Dias et al., Heterogeneous proliferative potential in cardiomyocytes and promotes cardiac repair. Nat Med. Aug. 2007;13(8):962-9. Epub Jul. 15, 2007. Lee et al., Requirement for neuregulin receptor erbB2 in neural and cardiac development. Nature. Nov. 23, 1995;378 (6555):394-8. Liebermanet al., (1980) Eds., Pharmaceutical Dosage Forms, Marcel regenerative adult newt cardiomyocytes. J Cell Sci. Oct. 1, Decker, New York, N.Y Table of Contents. 21 pages. 2003;116(Pt 19):4001-9. Epub Aug. 19, 2003. Liu et al., Neuregulin-1/erbB-activation improves cardiac function and survival in models of ischemic, dilated, and viral cardiomyopathy. J Am Coll Cardiol. Oct. 3, 2006;48(7):1438-47. proliferation and repair of heart injury. Cell. Jul. 23, 2009;138(2):257-70. Chaudhry et al., Cyclin A2 mediates cardiomyocyte mitosis in the postmitotic myocardium. J Biol Chem. Aug. 20, 2004;279(34):35858-66. Epub May 24, 2004. Epub Sep. 14, 2006. Engel et al., FGFl/p38 MAP kinase inhibitor therapy induces cardiomyocyte mitosis, reduces scarring, and rescues function after Meyer et al.,Multiple essential functions of neuregulin in develop ment. Nature. Nov. 23, 1995;378(6555):386-90. Erratum in: Nature myocardial infarction. Proc Natl Acad Sci U S A. Oct. 17, Dec. 14, 1995;378(6558):753. 2006;103(42):15546-51. Epub Oct. 10, 2006. Sawyer, Peptide Based Drug Design, ACS, Washington (1995) Table Fuller et al., ErbB receptors, their ligands, and the consequences of their activation and inhibition in the myocardium. J Mol Cell Cardiol. of Contents. 38 pages. May 2008;44(5):831-54. Epub Mar. 4, 2008. Gao et al., A Phase II, randomized, double-blind, multicenter, based on standard therapy, placebo-controlled study of the ef?cacy and safety of recombinant human in patients with chronic heart failure. J Am Coll Cardiol. May 4, 1010:55(18):1907-14. Soriano, Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. Jan. 1999;21(1):70-1. Weaver et al., Dual origin of tissue-speci?c progenitor cells in Drosophila tracheal remodeling. Science. 2008;321(5895):1496-9. Epub Jul. 31, 2008. Sep. 12, Gassmann et al., Aberrant neural and cardiac development in mice Wills et al., Regulated addition of new myocardial and epicardial cells fosters homeostatic cardiac growth and maintenance in adult lacking the ErbB4 neuregulin receptor. Nature. Nov. 23, zebra?sh. Development. Jan. 2008;135(1):183-92. Epub Nov. 28, 1995;378(6555):390-4. 2007. Guha et al., Organ renewal and cell divisions by differentiated cells in Drosophila. Proc Natl Acad Sci U S A. Aug. 5, 2008;105(31):10832 6. Epub Jul. 29, 2008. Yoshizumi et al., Disappearance of cyclin A correlates with perma nent withdrawal of cardiomyocytes from the cell cycle in human and rat hearts. J Clin Invest. May 1995;95(5):2275-80. Zhao et al., Neuregulins promote survival and growth of cardiac myocytes. Persistence of ErbB2 and ErbB4 expression in neonatal and adult ventricular myocytes. J Biol Chem. Apr. 24, Hoover, John E., Remington’s Pharmaceutical Sciences, (1975), 1998;273(17):10261-9. Golub et al., Behavioral characteristics of a nervous system-speci?c erbB4 knock-out mouse. Behav Brain Res. Aug. 12, 2004;153(1):159-70. Mack Publishing Co., Easton, Pennsylvania Table of Contents. 3 pages. * cited by examiner US. Patent Sheet10f17 Jan.20,2015 Fig. 1. a o 5E.382:.? b <B>02EuLm 15285:? av m 2 US 8,936,806 B2 US. Patent Jan.20,2015 US 8,936,806 B2 Sheet20f17 Fig. 2. Lu “a by 543210 perioslin Au US. Patent Jan. 20, 2015 Sheet 3 0f 17 cardiomyte cardiomyte(cyto%kin)es US 8,936,806 B2 (sDynN%theA)si U' A 9 no PN FGF stim. 5 Q ‘1' ' no PN FGF stim. O 1|) DNA synthesis 75 DscyntNohkieAs , (%ofmamal) 5 cytoklnesis N WW..— 0 3 5 9 12 duration of stimulation (days) (D cardlomytesDynthNeiAlw 50 [inc stim .PN P=0.0Q99 cardiomyteckinslwe Una stim. .PN P=o.goza US. Patent Jan. 20, 2015 Sheet 4 0f 17 US 8,936,806 B2 Fig. 4. a 07‘ E a q; 4 P>0.05 g L "‘ 20° .2 M1 05 G) g |___'__, S x- 2 8 £ @100 2 o 2 buffer PN 0 0 0 buffer PM O lsheart cardiomyteDsyNntAhes 7,500 cardxomyteckinslheart 5524;: 5,000 2,500 1.500- P<0.01 .—-—1 1,000 500 0 buffer PM 0 buffer PN FN PM US. Patent Jan. 20, 2015 Sheet 5 0f17 US 8,936,806 B2 FIGURE 5 a b '3 2.0 " 5515 . ° 3 R1 = 0.12 ‘ 0 m §~A 2.0 >35 g2 1.5_ 33-935 g; 2.0 " E% 1.5 - 3210K gm E z: 551-0 is“) L E Fibroblast content (%) l' ,9}? 7 Fibroblast content n‘c rd, :2 density 5’ low?ncg) US. Patent Jan. 20, 2015 Sheet 6 6f 17 FIGURE 6 sDCyarndNtiohmeAscy ofctr)neg (% US 8,936,806 B2 US. Patent Jan. 20, 2015 Sheet 7 0f 17 US 8,936,806 B2 FIGURES 7A & 7B cBiardU-opmysc?vte I] No stim. I PM P<0.05 __ I FGF [————“ P> 0.05 po(eristnf-%muiated) Carrier Q *’< (2+ sszoasso 3‘ III Nostim. I PM cBradU-ipomsytve po(eristfn-%?mulaed) P> 0.05 50 Ow. I NFlG US. Patent Jan. 20, 2015 Sheet 8 0f 17 FIGURES 7C & 7D No {rims ooéirol PT'EN caP'lsK n-Akr US 8,936,806 B2 US. Patent Jan. 20, 2015 Sheet 9 0f 17 FIGURE 7E 30,000 m[3H(u1cLp.etamik.ne) DNO stlm. I PM I PE 20,000 10,000 Days-O-S DaysS-S Days 6-9 [3H1Leucine label period US 8,936,806 B2 US. Patent Jan. 20, 2015 Sheet 10 0f 17 US 8,936,806 B2 U m 5..,\° :25; 75 3% 50 5'5 5% 25 periostin) U 0 2 4 s 810 actin). ‘ time(weeks) control c d . periosigin _ . ejectlon E (“M 50 shortening ' Ion 0/0 f(rac“tlonM 50 shortening ii 1 12 a 1 ejectlon 75 fract 12 1 time (weeks) e A 15° 12 control B) :1: E i E = W W 2 a. O 50 100 1 50 200 250 volume (uL) f 8 15° periostin :: _ _ E E. 9 I w (I! 2 D 0 50 1 time (weeks) 100 150 volume (pL) 200 250 12 US. Patent US 8,936,806 B2 Sheet110f17 Jan.20,2015 9 21 nu 150 AU P=0.0003 10 $8535“new £@562 5 5 Time after M! (weeks) GB23 : D. O. D n“ ctr septum infarct 5o PM f P=o.51 AEmv 05 he _3 4 ll 2 54321o 9i. ch O1 0oc2%;>:3“5?;? $agB5>!umo“E2-DN PM? [email protected]%»;? ArEa9g31-“52cv mw$523: : m, w. wa00.- P=D.0012 752 PN 2 US. Patent Jan. 20, 2015 Sheet 12 0f 17 US 8,936,806 B2 FIGURES 10A & 10B A' Q“ 2-5 O m“ 2-0 15-1; 1-5 _>_ 0) new. IPN P<0.01 . 8%); 0.5 1‘0 P<0.05 55% 002 I i i “1%o 01011;] I a12I O 1 Time after Ml (weeks) B. A .2 (D § w 0'3“ 3% 3 . .P>OO5 3 5‘ 0'2 - |'—-'1 o “1 > L2“ E 0.1 3 |_. a o P>0.05 I'_l 0 1 12 Time after MI (Weeks) US. Patent Jan. 20, 2015 Sheet 13 0f 17 US 8,936,806 B2 FIGURESIOC&10D C. 0-5 04 9A iii new. IPN Q3 P<0.01 Emu/g 02 P<0.05 I i .0 g 0.1 [—1 '01-: 20 a 0.010 o I 0.005 O 1 > 12 Time after MI (weeks) d A UCtr. IPN 1: . $2 53? 3-0- ref-949i 5‘5 P<0.05 9% 20-P>0.05 E I; *- u S E ' ' ' 1"“ Til 0 1 1 12 Time after MI (weeks) US. Patent Jan. 20, 2015 Sheet 14 0f 17 US 8,936,806 B2 FIGURE10E&10F E. 65" E .750- ElCtr. P< 0.0063 5 IPN ~ E% 250 p 005 g 500 "g- l 0 Fl I 0 ' II 1 12 Time after MI (weeks) F. 13- EICtr. IPN 3? P<0.05 g‘6 gE 10050 E L. gv g . “J 0 F—‘P'?s "‘1 P> 0.05 1 12 Time after MI (weeks) US. Patent Jan. 20, 2015 Sheet 15 0f 17 FIGURE 10G G. 2 El Ctr. Q A 2 P>0.05 IPN (D NE > E E 2% ~ 1 91 8 3:2 51 0 Time after MI (weeks) US 8,936,806 B2 US. Patent Jan. 20, 2015 Sheet 16 0f 17 US 8,936,806 B2 FIGURE 11 A. B or 750 P<0.0063 *1 E -~ 50 E _T».,_, II} U 31:: 0 0.5 w E- 250 2 = % i Q *4 ctr PN .2 g, 0.0 FEQJL‘ US. Patent Jan. 20, 2015 Sheet 17 0f 17 Figure 12 Periostin + lntegrins i ERK1I2, Akt + P-Rb, cyclin A + Cardiomyocyte proliferation + Myocardial regeneration + Improved myocardial function US 8,936,806 B2 US 8,936,806 B2 1 2 PERIOSTIN INDUCES PROLIFERATION OF CARDIOMYOCYTES AND PROMOTES CARDIAC REGENERATION and/or proliferation of postmitotic mammalian differentiated prevent the onset of cardiac damage caused by, for example, RELATED APPLICATIONS tion in vivo. In addition, the methods and compositions of the cardiomyocytes. The invention canbe used to slow, reduce, or myocardial ischemia, hypoxia, stroke, or myocardial infarc invention can be used to enhance proliferation of differenti ated cardiomyocytes in vitro and/ or ex vivo, which can then This application claims priority of Us. Provisional Appli cation No. 60/881,938 ?led Jan. 22, 2007, which is incorpo rated by reference in its entirety. be used in tissue grafting. The invention is based, in part, on the discovery that peri ostin, a component of the extracellular matrix, and fragments GOVERNMENT SUPPORT thereof promote cardiomyocyte proliferation and myocardial This invention was made with government support under Grant No. AHA 0425772T awarded by the American Heart with scar formation, not with proliferation, the cellular basis regeneration. The adult mammalian heart responds to injury for regeneration. The insuf?cient regeneration of mammalian hearts is explained by the contractile apparatus impinging on cardiomyocyte division. The invention demonstrates that extracellular periostin can induce cell cycle re-entry of dif ferentiated mammalian cardiomyocytes. Perio stin stimulates Association. The U.S. government has certain rights in this invention. BACKGROUND OF THE INVENTION Cardiovascular diseases are a leading cause of death result 20 mononuclear cardiomyocytes, present in the adult mamma ing in almost 40% of deaths annually in the United States. lian heart, to undergo the full mitotic cell cycle. Periostin Inadequate human myocardial regeneration poses a signi? activates 0tV[31/3/5 integrins located in the cardiomyocyte cell membrane. Periostin-induced cardiomyocyte prolifera cant public health problem. It is estimated that 13 million tion results from activation the ERK1/2 and Akt signaling Americans have coronary artery disease, and more than half a million experience a myocardial infarction every year. 25 Human cardiac tissue responds to injury, e.g. myocardial remodeling and function, reduces ?brosis and infarct size, and increases angiogenesis. These results demonstrate that infarction, with scar formation. Because the human heart is incapable of adequate muscle regeneration, survivors of a myocardial infarction typically develop heart failure, arrhyth mias, thrombosis, and other complications. 30 Adult human hearts do not regenerate after injury; instead, the defect is replaced by ?brotic tissue. Most evidence to date indicates that cardiomyocyte proliferation, the cellular basis of regeneration, is not a signi?cant component of the mam malian response to acute injury. In contrast to adult cardi periostin and the pathway it regulates is a new target for innovative strategies to treat heart failure. The invention identi?es pathways that can induce prolif eration of differentiated cardiomyocytes as a potential route to restore cardiac function. In another aspect of the invention, agents are disclosed that activate the signal transduction cas 35 omyocytes, fetal cardiomyocytes do proliferate during devel opment. cade consisting of extracellular periostin, integrins, and the ERK1/2 and the PI3-kinase/Akt pathways. Such agents include periostin, truncated biologically active constructs of periostin, functional analogs of periostin and pharmaceuti Heart disease results in the loss of cardiomyocytes. It has been a signi?cant challenge to develop effective treatments for cardiac repair because adult mammalian cardiomyocytes pathways. After myocardial infarction, recombinant perio stin induces cardiomyocyte cell cycle re-entry, improves cardiac cally acceptable derivatives of periostin or its functional ana 40 logs, and combinations thereof. are highly differentiated cells and have been believed to be In another aspect, the invention discloses methods of unable to proliferate. Mammalian cardiomyocytes withdraw stimulating proliferation of post-mitotic cells using com pounds comprising periostin, fragments or variants thereof, from the cell cycle soon after birth and have lowered levels of or pharmaceutically acceptable derivatives thereof. The post cyclin A (Yoshizumi, M., et. al. (1995). J Clin Invest 95, 2275-2280). The fact that primary cardiac tumors occur rarely supports the notion that adult cardiomyocytes are highly restricted in their ability to divide. Because of its lack of proliferative potential, the primary response of the mam malian heart to injury is scar formation, which prevents car 45 diac repair. Thus the loss of cardiomyocytes after damage caused by events such as myocardial infarction generally 50 lian cardiomyocytes. In some embodiments, proliferation comprises at least one of cell cycle reentry, increased cardi omyocyte DNA synthesis and cytokinesis. In some embodi ments, periostin, fragments or variants thereof, is delivered locally. In another aspect, the invention discloses a method of inducing division of post mitotic cells, comprising adminis results in compensatory responses that are inadequate to restore function. Unreplaced loss of cardiomyocytes leads to tering an integrin activator or a pharmaceutically acceptable derivative thereof to a subject in an amount effective to stimu heart failure, a signi?cant health problem worldwide. Current therapies are also limited in their effectiveness. In mitotic cells can be cardiomyocytes, and preferably mamma 55 late de-differentiation of post-mitotic cells. The integrin acti order to suf?ciently repair cardiac injury, it is necessary to vator activates at least one of the integrin subunits selected provide a source of new cardiomyocytes. Proliferation of from the group comprising (xV, [31, and [33, and [35 integrin differentiated endogenous cardiomyocytes can enhance the subunits. The integrin activator can be periostin or fragments regenerative capacity of mammalian hearts. thereof. In some embodiments, activation of integrin causes ERK1/2 and/ or Akt phosphorylation. In some embodiments, Accordingly, there is a need in the art for methods of 60 the integrin activator or a pharmaceutically acceptable deriva tive thereof can be delivered locally to the target cells or target area. Local and/ or targeted delivery can be administered increasing and/or promoting proliferation of adult mamma lian cardiomyocytes. SUMMARY OF THE INVENTION 65 The present invention provides compositions and methods for increasing proliferation, increasing cell cycle activity, using a slow controlled release delivery system, such as, for example, a biodegradable matrix. The invention can be used with a long-term, short-term and/or controlled release deliv ery systems.