Lysyl oxidase-like 2 (LOXL2) is an extracellular matrix (ECM) associated enzyme that oxidizes the terminal e-amines of lysine and hydroxylysine residues in collagen and/or elastin resulting in cross-linking of these molecules. The increase in cross-links renders the extracellular matrix (ECM) more resistant to degradation by matrix metalloproteases and also increases the stiffness of the ECM. This increase in matrix stiffness induces signaling changes within resident fibroblasts which promotes activation to a myofibroblast phenotype and initiates a pro-fibrotic feed-forward loop that can lead to scarring and destruction of the normal tissue architecture. LOXL2 is found circulating in the plasma/serum of healthy subjects at low concentrations, but is elevated in the serum of patients with idiopathic pulmonary fibrosis (IPF), HBV- and HVC-related fibrosis, non-alcoholic steatohepatitis, primary sclerosing cholangitis and systolic heart failure (SHF).

PharmAkea has developed and validated an IHC method for analysis of LOXL2 expression in fixed tissues. In normal mouse lung, LOXL2 is expressed at relatively low levels with both extracellular expression and intracellular expression in the cytoplasm of Type II alveolar epithelial cells and the nucleus of a small percentage of bronchial epithelial cells. Extracellular LOXL2 is increased dramatically in mouse lung after bleomycin instillation, particularly in fibrotic areas that show increased collagen deposition. Additionally, nuclear and cytoplasmic expression of LOXL2 increases in lung fibroblasts and/or macrophages after bleomycin instillation. The intracellular targets of LOXL2 in these pulmonary cell types are not known, however, others have shown that LOXL2 regulates epithelial-mesenchymal transition (EMT) in cancer cells through direct effects on the transcriptional regulators, SNAIL and Histone H3K4trimethyl.

PAT-1251 is a small molecule, irreversible inhibitor that potently inhibits LOXL2 activity in human blood and is selective over other amine oxidases. Importantly, PAT-1251 binds at the active site and inhibits the oxidation of biologically relevant amines (e.g. hydroxylysine, tropoelastin and fibronectin). The antibody AB0023 (mouse version of Simtuzumab) does not bind to the catalytic domain and does NOT inhibit the oxidative activity of LOXL2.

The anti-fibrotic efficacy of PharmAkea’s clinical candidate, PAT-1251, was evaluated in a mouse bleomycin model of lung fibrosis using Ashcroft score as the endpoint (Hubner et al., 2008, BioTechniques). In a prophylactic dose-response study, PAT-1251 treatment resulted in a dramatic improvement in Ashcroft score when dosed at 30 or 60 mg/kg/day (Rowbottom et al., 2017, J Med Chem). This anti-fibrotic effect was dose-related, as treatment with 10 mg/kg/day resulted in a smaller, but significant, reduction in Ashcroft score whereas treatment with 3 mg/kg/day was without effect. The improvement in anti-fibrotic scores was further supported by reductions in lung weights and measurements of inflammation and matrix deposition in BAL fluid.

In other mouse bleomycin studies, anti-fibrotic efficacy of PAT-1251 was evaluated using a variety of dosing paradigms. In a 21-day lung bleomycin study (A), PAT-1251 treatment administered therapeutically from day 7-21, reduced the lung Ashcroft score to a similar extent as prophylactic dosing. In a reversal/recovery study (B), dosing of PAT-1251 for two weeks starting at the time of peak fibrosis (Day 14) accelerated the reversal of the established fibrosis. In yet another study, the dose frequency of PAT-1251 was varied from daily dosing (QD), to every other day (Q2D) or every third day (Q3D) dosing. While maximal anti-fibrotic activity required daily dosing, partial efficacy was maintained with Q2D and Q3D dosing. Because pharmacokinetic analyses have demonstrated rapid clearance of PAT-1251,  continued efficacy with the Q2D and Q3D dosing paradigms supports the hypothesis that PAT-1251 is a mechanism-based, pseudo-irreversible inhibitor of LOXL2 and that matrix-associated LOXL2 is a stable protein with a long half-life.

Efficacy of PAT-1251 in the mouse bleomycin lung model was compared to rAB0023, a recombinantly-produced version of the LOXL2 monoclonal antibody, AB0023. Whereas prophylactic treatment with PAT-1251 resulted in a significant reduction in fibrotic score, prophylactic treatment with rAB0023 (at twice the published efficacious dose) showed only a trend for reduction in Ashcroft score. Similarly, PAT-1251 was compared head-to-head with BMS-986020 (AM152; an LPA1 receptor antagonist) and it also showed superior efficacy in the lung bleomycin model. In a model of liver fibrosis, PAT-1251 treatment significantly reduced fibrosis as assessed by the area fraction of Picrosirius red (collagen) positive staining, while rAB0023 showed only a trend for reduction. In a renal fibrosis model, treatment with PAT-1251 significantly reduced both tubulointerstitial fibrosis as well as glomerulosclerosis. These data demonstrate the robust and broad anti-fibrotic effect of the small molecule LOXL2 inhibitor, PAT-1251, and demonstrate superiority of PAT-1251 over the LOXL2 monoclonal antibody.