The calpain inhibitor, BLD-2660, has robust anti-fibrotic activity in a rat model of non-alcoholic steatohepatitis Mozhdeh Sojoodi1, Smitha Krishnan1, Allen A. Razavi1, Michael R. Day1, Gunisha Arora1, Sarani Goshal1, Shen Li1, Derek J. Erstad1, Peter Caravan1, Kenneth K. Tanabe1, Maria E. Fuentes2, Bryan C. Fuchs1 1Massachusetts General Hospital, Harvard Medical School, Boston, United States 2BladeTherapeutics, Inc, South San Francisco, United States Background and aims Treatment of BLD-2660 decreases fibrosis. BID treatment of BLD-2660 was well-tolerated in CDAHFD rats Calpains are non-lysosomal Ca+ dependent cysteine proteases, which modulate different cellular A MC (BID) BLD-2660 (200mg/Kg, QD) BLD-2660 (60mg/Kg, QD) BLD-2660 (100mg/Kg, BID) BLD-2660 (30mg/Kg, BID) A pathways and have been involved in several diseases. Dimeric calpains (calpain 1, 2 and 9) QD L iv e r/B o d y W e ig h t B o d y W e ig h t function as obligate heterodimeric complexes between a large catalytic subunit (CAPN1, CAPN2 n s S p le e n /B o d y W e ig h t n s n s or CAPN9) and the small regulatory subunit (CAPNS1) to form the functional protease. The 5 0 0 ** 0 .0 8 0 .0 0 5 ** n s Sirius red dimeric calpains have been implicated in organ fibrosis in multiple rodent models, including 4 0 0 n s 0 .0 0 4 ** n s 0 .0 6 heart, lung and adipose tissue fibrosis. Inhibition or knock-out of dimeric calpain activity resulted 3 0 0 0 .0 0 3 r g % % 0 .0 4 in decrease fibrosis in these various models. 2 0 0 0 .0 0 2 ** 0 .0 2 Here we evaluated the effects of BLD-2660, a small molecule inhibitor of calpain 1, 2 and 9 1 0 0 0 .0 0 1 H&E Poster presented at: presented Poster provided by Blade Therapeutics Inc., in a rat model of non-alcoholic steatohepatitis (NASH). 0 0 .0 0 0 .0 0 0 C D g g C D g g C D g g K K K K K K N Q / / N Q / / N Q / / C g g C g g C g g M m m M m m M m m 0 0 0 0 0 0 0 6 0 6 0 6 2 2 2 Method BID SMA - L iv e r/B o d y W e ig h t S p le e n /B o d y W e ig h t α B o d y W e ig h t n s B * n s 5 0 0 ** 0 .0 8 Adult male Wistar rats (175-200 g) were fed either normal chow (NC) or a choline-deficient, L- * 0 .0 0 5 n s ** ** 4 0 0 n s amino acid-defined, high fat diet with 60 kcal% fat and 0.1% methionine (CDAHFD) for 12 B 0 .0 6 0 .0 0 4 3 0 0 weeks. After 5 weeks, rats were randomized to receive either vehicle control (0.5% r 0 .0 0 3 g % 0 .0 4 % QD 2 0 0 methylcellulose (MC), 200 mg/kg BLD-2660, or 60 mg/kg BLD-2660 by QD and 100 mg/kg 0 .0 0 2 0 .0 2 BLD-2660, or 30 mg/kg BLD-2660 by BID gavage (n = 8 per group). At the end of the study, 1 0 0 0 .0 0 1 liver tissue and serum were collected for further analysis. 0 0 .0 0 0 .0 0 0 C D g g C D g g g g I I C ID N /K /K N /K /K K K B B N B / / g g g g g g C C C m m m m m m M M M 0 0 0 0 0 0 0 3 0 3 0 3 1 1 1 DOI: 10.3252/pso.eu.ILC2019.2019 Results • Serum levels of Alanine transaminase (ALT), Alkaline phosphatase (ALP), Aspartate transaminase (AST) and Albumin did not change in CDAHFD rats that received BLD-2660 BID Fig4: Body weight, liver weight/body weight and spleen weight /body weight, as assessed at the time of sacrifice, remained stable in Expression of Calpain1 and 2 increased in a rat model of non-alcoholic steatohepatitis CDAHFD rats that received BLD-2660. N=8, Mean±SD, One-way ANOVA, **p < 0.01. NC = Normal Chow; 200 mg/Kg = BLD-2660 200 mg/kg one dose per day; 60 mg/Kg = BLD-2660 60 mg/kg one dose per day, MC BID = methyl cellulose twice per day; 100 mg/Kg = BLD-2660 100 mg/kg twice per day; 30 mg/Kg = BLD-2660 30 mg/kg twice per day. A Normal Chow (NC) 2 Weeks 8 Weeks 12 Weeks Conclusion In a rat model of NASH cirrhosis that more closely resembles the histology of human disease, Sirius red increase expression of calpain 1 and 2 was observed. Therapeutic treatment with 100 mg/kg BID Fig2: (A) Sirius Red, H&E and SMA staining on CDAHFD rats without drug (MC) and treated with BLD-2660. (B) BLD-2660, treated rats or 200 mg/kg QD of BLD-2660 showed reduction in fibrosis progression. No effect on steatosis have less fibrosis as measured by Collagen proportional area (CPA%), Hydroxy Proline and Smooth muscle actin (SMA) immunohistochemical staining. BLD-2660 did not change fat deposition in treated groups. N=8, Mean±SD, One-way ANOVA, *p < 0.05, was observed with the compound treatment. These results confirm the hypothesis that inhibiting **p < 0.01. Normal Chow; 200 mg/Kg = BLD-2660 200 mg/kg one dose per day; 60 mg/Kg = BLD-2660 60 mg/kg one dose per day, MC dimeric calpains represents a viable approach for an anti-fibrotic therapy. BID = methyl cellulose twice per day; 100 mg/Kg = BLD-2660 100 mg/kg twice per day; 30 mg/Kg = BLD-2660 30 mg/kg twice per day. H&E BID treatment of BLD-2660 decreased expression of pro-fibrotic genes in CDAHFD rats References B C D E S M A B C D C o l1 a 1 C a lp a in 1 C a lp a in 2 A C T G F IL -6 C A P N 2 1- McVicker, B. L. and R. G. Bennett (2017). "Novel Anti-fibrotic Therapies." Front Pharmacol 8: 318. C A P N 1 2 5 2 5 0 2 .0 8 2- Tabata, C., et al. (2010). "The calpain inhibitor calpeptin prevents bleomycin-induced pulmonary fibrosis in ** ** ns 2 0 * * 1 5 2 0 0 8 0 * * 1 5 0 4 ns mice." Clin Exp Immunol 162(3): 560-567. 1 .5 6 ** * n s 1 5 Q 1 5 0 * * n s 3- Younossi, Z., et al. (2018). "Global burden of NAFLD and NASH: trends, predictions, risk factors and Q Q Q * * * R 6 0 3 * R * R R 1 .0 4 * 1 0 1 0 1 0 0 * 1 0 0 * prevention." Nat Rev Gastroenterol Hepatol 15(1): 11-20. Q Q Q Q * R R 2 R 0 .5 2 R 4 0 5 5 0 4- Heidrich FM, Ehrlich BE (2009). Calcium, calpains, and cardiac hypertrophy: a new link. Circ Res. Jan 5 0 5 30;104(2):e19-20. 0 0 0 .0 0 2 0 1 C W W W C W W W C W W W C W W W N 2 6 2 N 2 6 2 N 2 6 2 N 2 6 2 5- uniappan L, et al. (2017). Calpain Inhibition Attenuates Adipose Tissue Inflammation and Fibrosis in Diet- 1 1 1 1 0 0 0 0 induced Obese Mice. Sci Rep. 31;7(1):14398. g g g g C D g g C D g g Cirrhosis and complications Mozhdeh Sojoodi C ID C ID I I K K K K N /K /K N /K /K N B / / N B / / B B Fig1: Histologic Sections of CDAHFD Rats. (A) H&E and Sirius Red staining indicate the progression of fat and Collagen g g g g g g g g C C C C 6- Ye T, et al. (2015). Over-expression of calpastatin inhibits calpain activation and attenuates post-infarction m m m m m m m m M M M M deposition, respectively, during 2, 6 and 12 weeks of CDAHFD. (B) Smooth muscle actin (SMA) and (C) Collagen expression 0 0 0 0 0 0 0 0 0 3 0 3 0 3 0 3 myocardial remodeling. PLoS One. 18;10(3):e0120178. increases gradually. (D, E) Calpain1 and Calpain 2 expression increases in CDAHFD rat model of NASH. N=3, Mean±SD, One-way 1 1 1 1 ANOVA, Compared to NC, *p < 0.05, **p < 0.01. NC = Normal Chow; 2W = 2 weeks after CDAHFD, 6W = 6 weeks after CDAHFD, Fig3: BLD-2660 (100 mg/Kg and 30 mg/Kg BID) decreased the expression of inflammatory markers (A-B) CTGF and IL-6. (E, F) 12W = 12 weeks after CDAHFD. Calpain 1 and 2 expression did not change after BID treatment of BLD-266D. NC N=3, BID, 100 mg/Kg and 30 mg/Kg N=8, Mean±SD, One-way ANOVA, *p < 0.05, **p < 0.01. NC = Normal Chow; MC BID = methyl cellulose twice per day; 100 mg/Kg = BLD- Any question? Please contact me [email protected] 2660 100 mg/kg twice per day; 30 mg/Kg = BLD-2660 30 mg/kg twice per day.