Inhibition of Lysyl Oxidase Like-2 (LOXL2) Reduces Cardiac Interstitial Fibrosis in Mice

Inhibition of Lysyl Oxidase Like-2 (LOXL2) reduces cardiac interstitial fibrosis in mice A Buson,B LLC Cao, M MD Deodhar, dh ADFiA D Findlay, dl JSFJ S Foot, t AGA Greco, WJW Jarolimek, li k J MLPBR*Moses, L Perryman, B Rayner*, HHCS C Schilter, hilt X XT Tan, CITC I Turner, T T Yow, A Zahoor, W Zhou Drug Discovery, Pharmaxis Ltd., 20 Rodborough Road, Frenchs Forest, NSW, Australia; *Heart Research Institute, Eliza Street, Newton, NSW, Australia Introduction Lysyl oxidases are a family of enzymes Experimental protocol Cmpd A improves cardiac function responsible for the conversion of the primary EExpExperimentalperiimentltal ProtocolPProtot coll amine group of (hydroxyl-) lysine residues to the MI/Sham corresponding aldehyde. In extracellular matrix A 100 proteins, lysyl oxidases contribute to cross-linking, Monitoring survival rate thereby stabilising areas of fibrosis that occur 80 following tissue injury. Accumulation of cross- ** 60 linked extracellular matrix and the resulting excessive fibrosis can ultimately progress to organ Echocardiography at 24 hrs and 28 days post-MI 40 failure. Daily oral gavage for 28 days Heart photography 20 Yan et al (DOI: 10.1038/ncomms13710) have PBS Heart weight/body weight Ejection Fraction [%] 0 recently shown that an enzyme that crosslinks Cmpd A: 25 mg/kg, once a day Masson trichrome stain AM dA collagen—Lysyl Oxidase-Like-2 (LOXL2)—is Losartan (LOS): 15 mg/kg, once a day H LOS S +PBS mp + I C essential for interstitial fibrosis and mechanical M MI Sham group: No treatment B + dysfunction of pathologically stressed hearts, with MI other lysyl oxidase family members less This model was performed by CL Laboratory, LLC, Baltimore, MD. 80 upregulated during cardiac fibrosis. Importantly, in diseased human hearts, LOXL2 is upregulated in 60 cardiac interstitium, with levels correlating with the ** extent of collagen crosslinking and resultant 40 cardiac dysfunction. LOXL2 is also elevated in the Functional measurements serum of heart failure (HF) patients, correlating 20 with other HF biomarkers, suggesting a conserved SHAM MI + PBS MI + Cmpd A MI + LOS LOXL2-mediated mechanism of human HF. [%] Shortening Fractional 0 M S BS HA P LO Pharmaxis has developed small molecule S MI+ MI+ +CmpdA mechanism-based inhibitors that are selective for I LOXL2 over ubiquitous LOX and have drug-like M Fig. 5 A and B properties. Changes in cardiac function as measured by echocardiography at day 1 (hatched column ) and day 28 (solid column) Fig. 3 Representative photos of echocardiograms 28 days post myocardial infarction Cmpd A is aCmpdC selectivem p d A LOXL2 inhibitor 100 80 Cmpd A reduces area of fibrosis 60 Myocardial infarction 40 % inhibition 20 human fibroblast LOXL2 Amplex Red assay 20 human fibroblast LOX SHAM MI + PBS MI + Cmpd A MI + LOS ** ** * 0 15 0.001 0.01 0.1 1 10 concentration [P M] Fig. 1 10 LOX or LOXL2 secreted from human fibroblasts was pre- incubated with inhibitor for 30 min and activity was measured 5 in physiological buffer solution %areaoffibrosis 0 AM dA PBS p SH + Cm MI MI+ LOS Fig. 6 MI+ rec human LOXL2 24 Fig. 4 Area of fibrosis measured by Mason Trichrome rec mouse LOXL2 20 Representative photos of hearts at the time of sacrifice straining ) [nM] 50 human LOXL2 40 human LOX 1800 bovine LOX 1600 rec human LOXL1 1700 Inhibition (IC Summary rec human LOXL3 38 Pharmaxis has developed small molecules that selectively inhibit LOXL2 with nanomolar potency rec human LOXL4 100 LOXL2 inhibitor • improve cardiac function after myocardial infarction Table 1 Pharmacology of Cmpd A against other lysyl oxidases • and reduce the area of fibrosis. No inhibition (IC50 >30 μM) of other amine oxidases (SSAO, MAO-A and MAO-B).

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