CHITINASE INHIBITORS DEVELOPED BY OAT USED AS TOOLS IN OUR MOA AND POC STUDIES ACKNOWLEDGEMENTS OATD-01 EFFICACY IN BLEOMYCIN-INDUCED PULMONARY FIBROSIS MODEL IN MICE CHRONIC HDM-INDUCED AIRWAY INFLAMMATION AND REMODELING MODEL INTRODUCTION Acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1) are enzymatically active chitinases that have been implicated in the pathology of chronic lung diseases. Significantly elevated chitinolytic activity was demonstrated in asthma, chronic obstructive pulmonary disease (COPD) and interstitial lung diseases such as idiopathic pulmonary fibrosis (IPF) and sarcoidosis. Herein, we describe our studies on targeting chitinases with small molecules as a potential therapy for pulmonary diseases. PHARMACOKINETIC PROFILE IN MICE OATD-01 has a favorable pharmacokinetics in mice suitable for once- or twice-a-day dosing regimen in mice. CONCLUSIONS ✓ OATD-01 is the first-in-class chitinase inhibitor to have entered clinical trials (currently in phase 1b). ✓ OATD-01 is a nanomolar inhibitor of AMCase and CHIT1 with optimal pharmacokinetic profile in rodents and dogs. ✓ In addition to anti-inflammatory efficacy in asthma models, OATD-01 exhibited anti-fibrotic effects in a chronic HDM- induced airway remodelling model and in a bleomycin-induced pulmonary fibrosis model in mice. These data indicate that inhibition of chitinases might represent a novel therapeutic approach for pulmonary diseases as well as several fibrotic pathologies. OATD-01, A Dual hAMCase and hCHIT Inhibitor as a Potential Therapeutic Agent for Treatment of Pulmonary Diseases. Agnieszka Bartoszewicz 1 , Szymon Kłossowski 1 , Marzena Mazur 1 , Sylwia Olejniczak 1 , Robert Koralewski 1 , Michał Kowalski 1 , Barbara Dymek 1 , Piotr Sklepkiewicz 1 , Michał Mlącki 1 , Wojciech Czestkowski 1 , Gleb Andryianau 1 , Elżbieta Pluta 1 , Piotr Niedziejko 1 , Krzysztof Matyszewski 1 , Mariusz M. Gruza 1 , Łukasz Joachimiak, Agnieszka Zagożdżon 1 , Magdalena Salamon 1 , Jakub Gołab 2 , Marcin Nowotny 3 , Agnieszka Napiórkowska 3 , Małgorzata Kwiecień 3 , Karolina Dzwonek 1 , Paweł Dobrzański 1 , Jacek Olczak 1 , Adam Golebiowski 1 1 OncoArendi Therapeutics SA, Żwirki i Wigury 101, 02-089 Warsaw, Poland; 2 Department of Immunology, Medical University of Warsaw, Nielubowicza 5, 02-097 Warsaw, Poland 3 ProBiostructures, International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland. REFERENCES **** Normal control Sarcoidosis IPF COPD 0 1 2 3 4 5 6 Chitinolytic activity [µM/µl/h] **** ** SERUM Normal control Sarcoidosis IPF COPD 0.0 0.2 0.4 0.6 1.0 1.5 2.0 2.5 3.0 Chitinolytic activity [µM/µl/h] * *** ** INDUCED SPUTUM Sarcoidosis IPF COPD 0.0 0.1 0.2 0.3 0.4 0.5 0.5 1.0 1.5 2.0 Chitinolytic activity [µM/µl/h] BALF SAR STUDY In the course of our program over 2500 compounds have been designed and synthesized, resulting in OAT-870 as our advanced lead compound. Further optimization of drug-like properties and selectivity of OAT-870 yielded a clinical candidate OATD-01. The compound bears an additional methyl group at the morpholine ring, which abrogated undesired off-target activity towards dopamine transporter (DAT). 5,6 „Preclinical research and clinical trials of a first-in-class development candidate in therapy of asthma and inflammatory bowel disease” hAMCase IC 50 = 14 nM hCHIT1 IC 50 = 232 nM mAMCase IC 50 = 19 nM mCHIT1 IC 50 = 2955 nM 17x 155x Selective inhibitor of mAMCase 2 hAMCase IC 50 = 4.7 nM hCHIT1 IC 50 = 830 nM Selective inhibitor of hAMCase 3 mCHIT1 IC 50 = 29 nM mAMCase IC 50 = 4170 nM Selective inhibitor of mCHIT 4 143x 176x Poster MEDI69 OATD-01 Pharmacokinetic Parameters Route IV PO Dose (mg/kg) 3 10 AUC 0-inf (mg·h/L) 8.6 22.2 AUC n 0 -inf (kg·h/L) 2.9 2.2 C 0 or C max (mg/L) 4.0 3.4 Tmax(h) n/a 2.0 CL (mL/min/kg) 5.8 n/a Vss (L/kg) 1.0 n/a T½ (h) 2.1 1.9 Bioavailability (F%) n/a 77 CRYSTAL STRUCTURE OF OATD-01-CHIT1 COMPLEX ✓ Treatment with OATD-01 significantly decreased chitinolytic activity in BAL indicating target engagement in the lungs in vivo. Decrease in chitinolytic activity correlated with reduction of either collagen level in the lungs or edema represented by lung weight to body weight ratio A) Stereoview of key residues (shown as green sticks) of hCHIT1 active site. Protein−ligand hydrogen bonds are represented by orange dashed lines and distances are given in Å. B) Active site pocket of hCHIT1 represented as surface and colored according to electrostatic potential. mRNA profile of chitinases in IPF lungs 0.0 0.2 0.4 0.6 0.8 Chitinolytic activity in plasma, pH 6, [µM/µl/h] Control Bleomycin (2U/kg) OATD-01 (30 mg/kg b.i.d) Pirfenidone (250 mg/kg b.i.d) **** **** Day 0 7 21 Bleomycin 2U/kg, 3x i.n. (d0-2) OATD-01 (30mg/kg; PO; b.i.d) PIRFENIDONE (250mg/kg; PO; b.i.d) 0 50 100 150 Soluble collagen [mg/lungs] ** * Control Bleomycin (2U/kg) OATD-01 (30 mg/kg b.i.d) Pirfenidone (250 mg/kg b.i.d) 0 10 20 30 Lung/Body weight ratio Control Bleomycin (2U/kg) OATD-01 (30 mg/kg b.i.d) Pirfenidone (250 mg/kg b.i.d) *** * OATD-01 reduced the goblet cell hyperplasia as assessed with periodic acid-Shiff (PAS) staining of mucins in mouse lung tissue. 1) Yang IV et al. Thorax. 2013, 68, 1114, Data extracted from the study GSE32537 (IPF n=119; Control n=50) 2) Mazur, M.; Olczak, J.; Olejniczak, S.; Koralewski, R.; Czestkowski, W.; et al. J. Med. Chem. 2018, 61, 695. 3) Mazur M. et al. US20170066743 4) Mazur M.; Bartoszewicz A.; Dymek B.; Salamon, M.; Adrianau, G.; et al. Bioorg. Med. Chem. Lett., 2018, 28, 310. 5) Mazur, M.; Dymek, B.; Koralewski, R.; Sklepkiewicz, P.; Olejniczak, S.; et. al. J. Med. Chem. 2019, ASAP 6) Koralewski R.; Dymek, B.; Mazur, M.; Sklepkiewicz, P.; Olejniczak, S.; et. al. submited Poster MEDI67 hAMCase IC 50 = 24 nM hCHIT1 IC 50 = 47 nM mAMCase IC 50 = 20 nM mCHIT1 IC 50 = 77 nM DAT 94% inhibition at 10 mM hAMCase IC 50 = 54 nM hCHIT1 IC 50 = 14 nM mAMCase IC 50 = 1 nM mCHIT1 IC 50 = 14 nM hAMCase IC 50 = 3 nM hCHIT1 IC 50 = 18 nM mAMCase IC 50 = 2 nM mCHIT1 IC 50 = 19 nM DAT 34% inhibition at 10 mM hAMCase IC 50 = 23 nM hCHIT1 IC 50 = 52 nM mAMCase IC 50 = 111 nM mCHIT1 IC 50 = 87 nM hAMCase IC 50 = 826 nM hCHIT1 IC 50 = 5100 nM mAMCase IC 50 = 1000 nM mCHIT1 IC 50 = 6400 nM hAMCase IC 50 = 9 nM hCHIT1 IC 50 = 23 nM mAMCase IC 50 = 8 nM mCHIT1 IC 50 = 28 nM DAT IA C57Bl/6 ✓ In 7-week-long HDM-induced airway inflammation model OATD-01 administered qd in a therapeutic regiment significantly reduced the total number of leukocytes and eosinophils in BALF. ✓ Anti-inflammatory activity of OATD-01 correlates with a significantly reduced chitinolytic activity in BALF and plasma. ✓ OATD-01 reduced the serum IgE concentration in a dose-dependent manner indicating reduction of allergic response. OATD-01 (30mg/kg) OATD-01 30mg/kg OATD-01 OATD-01 OATD-01 exhibited significant anti-remodeling activity: decreased airway wall thickness and reduced collagen deposition around bronchioles. 0 1 2 3 4 PAS staining mean score * Control Chronic HDM OATD-01 (3 mg/kg qd) OATD-01 (30 mg/kg qd) **** HDM Control OATD-01 30mg/kg HDM Control 0 2 4 6 Chitinolytic activity in BAL, pH 6 [µM/µl/h] (mean with SEM) ** * Control HDM OATD-01 (3 mg/kg) OATD-01 (30 mg/kg) Dexamethasone (10 mg/kg) 0 50000 100000 150000 Total number of BAL eosinophils (mean with SEM) Control HDM OATD-01 (3 mg/kg) OATD-01 (30 mg/kg) Dexamethasone (10 mg/kg) * ** 0 10000 20000 30000 40000 50000 60000 Total IgE in plasma [ng/ml] Control HDM OATD-01 (3 mg/kg) OATD-01 (30 mg/kg) Dexamethasone (10 mg/kg) 0 50000 100000 150000 200000 250000 Total BAL (CD45 + ) cell number (mean with SEM) Control HDM OATD-01 (3 mg/kg) OATD-01 (30 mg/kg) Dexamethasone (10 mg/kg) * ** Control Bleomycin (2U/kg) OATD-01 (30mg/kg, b.i.d) Pirfenidone (250mg/kg; b.i.d) ✓ OATD-01 treatment decreases pulmonary fibrosis formation in bleomycin induced fibrosis model. Co nt r ol Bl e o m y cin OAT D- 0 1 (3 0 mg/ k g bi d ) P irf e ni d one (250 mg/kg bid) 0 2 4 6 8 Ashcroft fibrosis score **** * *