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A Brain-Targeted Orally Available ROCK2 Inhibitor Benefits Mild and Aggressive Cavernous Angioma Disease

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Abstract

Cavernous angioma (CA) is a vascular pathology caused by loss of function in one of the 3 CA genes (CCM1, CCM2, and CCM3) that result in rho kinase (ROCK) activation. We investigated a novel ROCK2 selective inhibitor for the ability to reduce brain lesion formation, growth, and maturation. We used genetic methods to explore the use of a ROCK2-selective kinase inhibitor to reduce growth and hemorrhage of CAs. The role of ROCK2 in CA was investigated by crossing Rock1 or Rock2 hemizygous mice with Ccm1 or Ccm3 hemizygous mice, and we found reduced lesions in the Rock2 hemizygous mice. A ROCK2-selective inhibitor, BA-1049 was used to investigate efficacy in reducing CA lesions after oral administration to Ccm1+/− and Ccm3+/− mice that were bred into a mutator background. After assessing the dose range effective to target brain endothelial cells in an ischemic brain model, Ccm1+/− and Ccm3+/− transgenic mice were treated for 3 (Ccm3+/−) or 4 months (Ccm1+/−), concurrently, randomized to receive one of three doses of BA-1049 in drinking water, or placebo. Lesion volumes were assessed by micro-computed tomography. BA-1049 reduced activation of ROCK2 in Ccm3+/−Trp53−/− lesions. Ccm1+/−Msh2−/− (n=68) and Ccm3+/−Trp53−/− (n=71) mice treated with BA-1049 or placebo showed a significant dose-dependent reduction in lesion volume after treatment with BA-1049, and a reduction in hemorrhage (iron deposition) near lesions at all doses. These translational studies show that BA-1049 is a promising therapeutic agent for the treatment of CA, a disease with no current treatment except surgical removal of the brain lesions.

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References

  1. Al-Shahi Salman R, Hall JM, Horne MA, Moultrie F, Josephson CB, Bhattacharya JJ, et al. Untreated clinical course of cerebral cavernous malformations: a prospective, population-based cohort study. Lancet Neurol. 2012;11(3):217–24. https://doi.org/10.1016/S1474-4422(12)70004-2.

    Article  PubMed  Google Scholar 

  2. Riant F, Bergametti F, Ayrignac X, Boulday G, Tournier-Lasserve E. Recent insights into cerebral cavernous malformations: the molecular genetics of CCM. FEBS J. 2010;277(5):1070–5. https://doi.org/10.1111/j.1742-4658.2009.07535.x.

    Article  CAS  PubMed  Google Scholar 

  3. Leblanc GG, Golanov E, Awad IA, Young WL. Biology of vascular malformations of the brain NWC. Biology of vascular malformations of the brain. Stroke. 2009;40(12):e694–702. https://doi.org/10.1161/STROKEAHA.109.563692.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Al-Holou WN, O’Lynnger TM, Pandey AS, Gemmete JJ, Thompson BG, Muraszko KM, et al. Natural history and imaging prevalence of cavernous malformations in children and young adults. J Neurosurg Pediatr. 2012;9(2):198–205. https://doi.org/10.3171/2011.11.PEDS11390.

    Article  PubMed  Google Scholar 

  5. Plummer NW, Gallione CJ, Srinivasan S, Zawistowski JS, Louis DN, Marchuk DA. Loss of p53 sensitizes mice with a mutation in Ccm1 (KRIT1) to development of cerebral vascular malformations. Am J Pathol 2004;165(5):1509–1518. https://doi.org/10.1016/S0002-9440(10)63409-8.

  6. Horne MA, Flemming KD, Su IC, Stapf C, Jeon JP, Li D, et al. Clinical course of untreated cerebral cavernous malformations: a meta-analysis of individual patient data. Lancet Neurol. 2016;15(2):166–73. https://doi.org/10.1016/S1474-4422(15)00303-8.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Porter PJ, Willinsky RA, Harper W, Wallace MC. Cerebral cavernous malformations: natural history and prognosis after clinical deterioration with or without hemorrhage. J Neurosurg. 1997;87(2):190–7. https://doi.org/10.3171/jns.1997.87.2.0190.

    Article  CAS  PubMed  Google Scholar 

  8. Sahoo T, Johnson EW, Thomas JW, Kuehl PM, Jones TL, Dokken CG, et al. Mutations in the gene encoding KRIT1, a Krev-1/rap1a binding protein, cause cerebral cavernous malformations (CCM1). Hum Mol Genet. 1999;8(12):2325–33. https://doi.org/10.1093/hmg/8.12.2325.

    Article  CAS  PubMed  Google Scholar 

  9. Laberge-le Couteulx S, Jung HH, Labauge P, Houtteville JP, Lescoat C, Cecillon M, et al. Truncating mutations in CCM1, encoding KRIT1, cause hereditary cavernous angiomas. Nat Genet. 1999;23(2):189–93. https://doi.org/10.1038/13815.

    Article  CAS  PubMed  Google Scholar 

  10. Bergametti F, Denier C, Labauge P, Arnoult M, Boetto S, Clanet M, et al. Mutations within the programmed cell death 10 gene cause cerebral cavernous malformations. Am J Hum Genet. 2005;76(1):42–51. https://doi.org/10.1086/426952.

    Article  CAS  PubMed  Google Scholar 

  11. McDonald DA, Shi C, Shenkar R, Gallione CJ, Akers AL, Li S, et al. Lesions from patients with sporadic cerebral cavernous malformations harbor somatic mutations in the CCM genes: evidence for a common biochemical pathway for CCM pathogenesis. Hum Mol Genet. 2014;23(16):4357–70. https://doi.org/10.1093/hmg/ddu153.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Akers AL, Johnson E, Steinberg GK, Zabramski JM, Marchuk DA. Biallelic somatic and germline mutations in cerebral cavernous malformations (CCMs): evidence for a two-hit mechanism of CCM pathogenesis. Hum Mol Genet. 2009;18(5):919–30. https://doi.org/10.1093/hmg/ddn430.

    Article  CAS  PubMed  Google Scholar 

  13. Gault J, Sain S, Hu LJ, Awad IA. Spectrum of genotype and clinical manifestations in cerebral cavernous malformations. Neurosurgery. 2006;59(6):1278–84; discussion 84-5. https://doi.org/10.1227/01.NEU.0000249188.38409.03.

    Article  PubMed  Google Scholar 

  14. Yao L, Romero MJ, Toque HA, Yang G, Caldwell RB, Caldwell RW. The role of RhoA/Rho kinase pathway in endothelial dysfunction. J Cardiovasc Dis Res. 2010;1(4):165–70. https://doi.org/10.4103/0975-3583.74258.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Borikova AL, Dibble CF, Sciaky N, Welch CM, Abell AN, Bencharit S, et al. Rho kinase inhibition rescues the endothelial cell cerebral cavernous malformation phenotype. J Biol Chem. 2010;285(16):11760–4. https://doi.org/10.1074/jbc.C109.097220.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Stockton RA, Shenkar R, Awad IA, Ginsberg MH. Cerebral cavernous malformations proteins inhibit Rho kinase to stabilize vascular integrity. J Exp Med. 2010;207(4):881–96. https://doi.org/10.1084/jem.20091258.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Whitehead KJ, Chan AC, Navankasattusas S, Koh W, London NR, Ling J, et al. The cerebral cavernous malformation signaling pathway promotes vascular integrity via Rho GTPases. Nat Med. 2009;15(2):177–84. https://doi.org/10.1038/nm.1911.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Fischer A, Zalvide J, Faurobert E, Albiges-Rizo C, Tournier-Lasserve E. Cerebral cavernous malformations: from CCM genes to endothelial cell homeostasis. Trends Mol Med. 2013;19(5):302–8. https://doi.org/10.1016/j.molmed.2013.02.004.

    Article  CAS  PubMed  Google Scholar 

  19. Shenkar R, Shi C, Rebeiz T, Stockton RA, McDonald DA, Mikati AG, et al. Exceptional aggressiveness of cerebral cavernous malformation disease associated with PDCD10 mutations. Genet Med. 2015;17(3):188–96. https://doi.org/10.1038/gim.2014.97.

    Article  CAS  PubMed  Google Scholar 

  20. Beckers CM, Knezevic N, Valent ET, Tauseef M, Krishnan R, Rajendran K, et al. ROCK2 primes the endothelium for vascular hyperpermeability responses by raising baseline junctional tension. Vasc Pharmacol. 2015;70:45–54. https://doi.org/10.1016/j.vph.2015.03.017.

    Article  CAS  Google Scholar 

  21. Mueller BK, Mack H, Teusch N. Rho kinase, a promising drug target for neurological disorders. Nat Rev Drug Discov. 2005;4(5):387–98. https://doi.org/10.1038/nrd1719.

    Article  CAS  PubMed  Google Scholar 

  22. Loirand G. Rho kinases in health and disease: from basic science to translational research. Pharmacol Rev. 2015;67(4):1074–95. https://doi.org/10.1124/pr.115.010595.

    Article  CAS  PubMed  Google Scholar 

  23. Green J, Cao J, Bandarage UK, Gao H, Court J, Marhefka C, et al. Design, synthesis, and structure-activity relationships of pyridine-based rho kinase (ROCK) inhibitors. J Med Chem. 2015;58(12):5028–37. https://doi.org/10.1021/acs.jmedchem.5b00424.

    Article  CAS  PubMed  Google Scholar 

  24. Xin YL, Yu JZ, Yang XW, Liu CY, Li YH, Feng L et al. FSD-C10: A more promising novel ROCK inhibitor than Fasudil for treatment of CNS autoimmunity. Biosci Rep. 2015;35(5). https://doi.org/10.1042/BSR20150032.

  25. Rosen KM, Abbinanti MD, Ruschel J, McKerracher L, Bond L. Rho kinase inhibitor BA-1049 (R) and active metabolites thereof, United States Patent 10,106,525, October 23, 2018. . In: USPTO Patent Full-Text and Image Database 2018. patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=10106525.PN.&OS=PN/10106525&RS=PN/10106525. Accessed 29 Jul 2019.

  26. Gunel M, Awad IA, Finberg K, Anson JA, Steinberg GK, Batjer HH, et al. A founder mutation as a cause of cerebral cavernous malformation in Hispanic Americans. N Engl J Med. 1996;334(15):946–51. https://doi.org/10.1056/NEJM199604113341503.

    Article  CAS  PubMed  Google Scholar 

  27. McDonald DA, Shenkar R, Shi C, Stockton RA, Akers AL, Kucherlapati MH, et al. A novel mouse model of cerebral cavernous malformations based on the two-hit mutation hypothesis recapitulates the human disease. Hum Mol Genet. 2011;20(2):211–22. https://doi.org/10.1093/hmg/ddq433.

    Article  CAS  PubMed  Google Scholar 

  28. Jiang SX, Lertvorachon J, Hou ST, Konishi Y, Webster J, Mealing G, et al. Chlortetracycline and demeclocycline inhibit calpains and protect mouse neurons against glutamate toxicity and cerebral ischemia. J Biol Chem. 2005;280(40):33811–8. https://doi.org/10.1074/jbc.M503113200.

    Article  CAS  PubMed  Google Scholar 

  29. Zhu M, Liu PY, Kasahara DI, Williams AS, Verbout NG, Halayko AJ, et al. Role of rho kinase isoforms in murine allergic airway responses. Eur Respir J. 2011;38(4):841–50. https://doi.org/10.1183/09031936.00125010.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Shenkar R, Peiper A, Pardo H, Moore T, Lightle R, Girard R, et al. Rho kinase inhibition blunts lesion development and hemorrhage in murine models of aggressive Pdcd10/Ccm3 disease. Stroke. 2019;50(3):738–44. https://doi.org/10.1161/STROKEAHA.118.024058.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Girard R, Zeineddine HA, Orsbon C, Tan H, Moore T, Hobson N, et al. Micro-computed tomography in murine models of cerebral cavernous malformations as a paradigm for brain disease. J Neurosci Methods. 2016;271:14–24. https://doi.org/10.1016/j.jneumeth.2016.06.021.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Shenkar R, Shi C, Austin C, Moore T, Lightle R, Cao Y, et al. RhoA kinase inhibition with Fasudil versus simvastatin in murine models of cerebral cavernous malformations. Stroke. 2017;48(1):187–94. https://doi.org/10.1161/STROKEAHA.116.015013.

    Article  CAS  PubMed  Google Scholar 

  33. Shi C, Shenkar R, Zeineddine HA, Girard R, Fam MD, Austin C, et al. B-cell depletion reduces the maturation of cerebral cavernous malformations in murine models. J NeuroImmune Pharmacol. 2016;11(2):369–77. https://doi.org/10.1007/s11481-016-9670-0.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Conover WJ. Practical nonparametric statistics. New York: John Wiley and Sons; 1999.

    Google Scholar 

  35. Zhu Y, Wu Q, Fass M, Xu JF, You C, Muller O, et al. In vitro characterization of the angiogenic phenotype and genotype of the endothelia derived from sporadic cerebral cavernous malformations. Neurosurgery. 2011;69(3):722–32. https://doi.org/10.1227/NEU.0b013e318219569f.

    Article  PubMed  Google Scholar 

  36. Newell-Litwa KA, Badoual M, Asmussen H, Patel H, Whitmore L, Horwitz AR. ROCK1 and 2 differentially regulate actomyosin organization to drive cell and synaptic polarity. J Cell Biol. 2015;210(2):225–42. https://doi.org/10.1083/jcb.201504046.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kim H-H, Ayata C. Rho-associated kinases in cerebrovascular disease. In: Caplan LR, Leary MC, Thomas AJ, Zhang JH, Biller J, Lo EH et al., editors. Primer on cerebrovascular diseases (second edition). Amsterdam: Elsevier; 2017. pp. 265–268.

  38. Terry S, Nie M, Matter K, Balda MS. Rho signaling and tight junction functions. Physiology (Bethesda). 2010;25(1):16–26. https://doi.org/10.1152/physiol.00034.2009.

    Article  CAS  Google Scholar 

  39. Hoang MV, Whelan MC, Senger DR. Rho activity critically and selectively regulates endothelial cell organization during angiogenesis. Proc Natl Acad Sci U S A. 2004;101(7):1874–9. https://doi.org/10.1073/pnas.0308525100.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. McDonald DA, Shi C, Shenkar R, Stockton RA, Liu F, Ginsberg MH, et al. Fasudil decreases lesion burden in a murine model of cerebral cavernous malformation disease. Stroke. 2012;43(2):571–4. https://doi.org/10.1161/STROKEAHA.111.625467.

    Article  CAS  PubMed  Google Scholar 

  41. Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J. 2000;351(Pt 1):95–105. https://doi.org/10.1042/0264-6021:3510095.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Wang Y, Zheng XR, Riddick N, Bryden M, Baur W, Zhang X, et al. ROCK isoform regulation of myosin phosphatase and contractility in vascular smooth muscle cells. Circ Res. 2009;104(4):531–40. https://doi.org/10.1161/CIRCRESAHA.108.188524.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Hartmann S, Ridley AJ, Lutz S. The function of rho-associated kinases ROCK1 and ROCK2 in the pathogenesis of cardiovascular disease. Front Pharmacol. 2015;6:276. https://doi.org/10.3389/fphar.2015.00276.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Defert O, Boland S. Rho kinase inhibitors: a patent review (2014 - 2016). Expert Opin Ther Pat. 2017;27(4):507–15. https://doi.org/10.1080/13543776.2017.1272579.

    Article  CAS  PubMed  Google Scholar 

  45. Lisowska J, Rodel CJ, Manet S, Miroshnikova YA, Boyault C, Planus E et al. The CCM1-CCM2 complex controls complementary functions of ROCK1 and ROCK2 that are required for endothelial integrity. J Cell Sci. 2018;131(15). https://doi.org/10.1242/jcs.216093.

  46. Kurokawa M, Akino K, Kanda K. A new apparatus for studying feeding and drinking in the mouse. Physiol Behav. 2000;70(1–2):105–12. https://doi.org/10.1016/s0031-9384(00)00226-2.

    Article  CAS  PubMed  Google Scholar 

  47. Mikati AG, Khanna O, Zhang L, Girard R, Shenkar R, Guo X, et al. Vascular permeability in cerebral cavernous malformations. J Cereb Blood Flow Metab. 2015;35(10):1632–9. https://doi.org/10.1038/jcbfm.2015.98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Polster SP, Stadnik A, Akers AL, Cao Y, Christoforidis GA, Fam MD, et al. Atorvastatin treatment of cavernous angiomas with symptomatic hemorrhage exploratory proof of concept (AT CASH EPOC) trial. Neurosurgery In press. 2018. https://doi.org/10.1093/neuros/nyy539.

  49. Eisa-Beygi S, Wen XY, Macdonald RL. A call for rigorous study of statins in resolution of cerebral cavernous malformation pathology. Stroke. 2014;45(6):1859–61. https://doi.org/10.1161/STROKEAHA.114.005132.

    Article  PubMed  Google Scholar 

  50. Uchida S, Watanabe G, Shimada Y, Maeda M, Kawabe A, Mori A, et al. The suppression of small GTPase rho signal transduction pathway inhibits angiogenesis in vitro and in vivo. Biochem Biophys Res Commun. 2000;269(2):633–40. https://doi.org/10.1006/bbrc.2000.2315.

    Article  CAS  PubMed  Google Scholar 

  51. Mori K, Amano M, Takefuji M, Kato K, Morita Y, Nishioka T, et al. Rho-kinase contributes to sustained RhoA activation through phosphorylation of p190A RhoGAP. J Biol Chem. 2009;284(8):5067–76. https://doi.org/10.1074/jbc.M806853200.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Cenk Ayata (Massachusetts General Hospital), Mark Bear (Massachusetts Institute of Technology), Guy Rouleau (McGill), Dennis Choi (State University of New York), Rebecca Stockton (LABioMed), and Dongdong Zhang (University of Chicago) for helpful discussions throughout the study. We also thank Peter Pytel (University of Chicago) for help with the autopsy of mice as well as Heidy Pardo and Erin Griffin (both of Duke University) for help with transgenic mice breeding.

Funding

Supported by a National Institute of Health Small Business Innovation Research (SBIR) 1R44NS095420-01 grant to LM, DM, and IA.

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Author notes

  1. Douglas Marchuk and Issam Awad contributed equally to this work.

Authors and Affiliations

  1. BioAxone BioSciences Inc., Cambridge, MA, USA

    Lisa McKerracher, Matthew Abbinanti, Joerg Ruschel & Kenneth Rosen

  2. Department of Neurology and Neurosurgery, McGill University, Montreal, Canada

    Lisa McKerracher

  3. Neurovascular Surgery Program, Section of Neurosurgery, Department of Surgery, The University of Chicago Medicine, Chicago, IL, USA

    Robert Shenkar, Ying Cao, Rhonda Lightle, Thomas Moore, Nicholas Hobson, Janne Koskimäki, Romuald Girard & Issam A. Awad

  4. Department of Molecular Genetics and Microbiology, Duke University, Durham, NC, USA

    Amy Peiper, Carol Gallione & Douglas A. Marchuk

  5. Section of Cardiology, Department of Medicine, The University of Chicago, Chicago, IL, USA

    James K. Liao

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  1. Lisa McKerracher
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Correspondence to Issam A. Awad.

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Competing Interests

Dr. McKerracher, CEO of BioAxone, holds an ownership interest in the company and has a significant competing interest. Drs. Matthew Abbinanti, Ken Rosen, and Joerg Ruschel were employees of BioAxone and have a modest conflict of interest. Drs. Doug Marchuk and Issam Awad were recipients of sponsored research through the SBIR grant to BioAxone. The other authors declare no conflict of interest.

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All animal protocols were approved by the Tufts University Institutional Animal Care and Use Committee (IACUC) or the Duke University IACUC, and mice were bred at Duke University in compliance with the NIH Guide for the Care and Use of Laboratory Animals.

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McKerracher, L., Shenkar, R., Abbinanti, M. et al. A Brain-Targeted Orally Available ROCK2 Inhibitor Benefits Mild and Aggressive Cavernous Angioma Disease. Transl. Stroke Res. 11, 365–376 (2020). https://doi.org/10.1007/s12975-019-00725-8

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