Addressing Safety and Specificity with Aldose Reductase Inhibition: Development of AT-001 for Diabetic Cardiomyopathy Poster #632

Riccardo Perfetti1, Gautham Yeppuri2, Nosirudeen Quadri2, Ameen F Ghannam1, Ravichandran Ramasamy2, Shoshana Shendelman1. Applied Therapeutics1 and NYU Langone Medical Center2, New York, NY

Introduction Methods Zopolrestat (but not AT-001) Reduces Cell Zopolrestat (but not AT-001) Induces Cell Damage Diabetic cardiomyopathy (DbCM) leading to overt heart failure is a Viability of Cultured Hepatocytes in Cultured Hepatocytes as Measured by AST and Cell culture: Cryopreserved human hepatocytes cultured in standard medium. common sequalae of both type 1 and type 2 diabetes. Prior attempts to ALT Release develop treatments for DbCM via inhibition of Aldose Reductase (AR) Comparative cell hepatoxicity of AT-001 vs controls: Primary human hepatocyte demonstrated clinical efficacy. spheroids were cultured for 7 days. At Day 7, spheroid cultures were exposed to different concentrations of AT-001 and zopolrestat. Troglitazone (a known CellTiter-GloTM luminescent CellTiter-GloTM luminescent AST normalised to cell viability However, first generation AR inhibitors (ARIs) presented with liver and hepatotoxic agent) was used as a positive control. Exposure was maintained for cell viability assay cell viability assay renal safety signals. Off-target inhibition of Aldehyde Reductase, an enzyme 14 days and cell viability was assessed via ATP quantification using the CellTiter- 7.00E-06 TM necessary for cellular detoxification, was considered responsible for the Glo luminescent cell viability assay. 10000 14000 6.00E-06 unfavorable safety profile. Liver enzyme quantitation assay: Lactate dehydrogenase (LDH) release (a measure 9000 12000 In the present study, we report the in-vitro safety, selectivity and specificity of cell necrosis/injury), and liver injury markers alanine aminotransferase (ALT) 8000 and aspartate aminotransferase (AST) were measured using commercially available 5.00E-06 of AT-001, a novel small molecule ARI with optimised affinity and 7000 10000 assay kits. specificity for AR, and no off-target aldehyde reductase activity. 6000 Selectivity assessment of AT-001 vs zopolrestat: Enzymatic inhibition (both 8000 4.00E-06 on-target inhibition of AR and off-target inhibition of aldehyde reductase) was 5000 Pathogenesis of DbCM and Hyperactivation RL U RL U 6000 determined via spectrophotometric assay of substrate binding and nicotinamide 4000 3.00E-06 1,2 AST activity of Polyol Pathway adenine dinucleotide phosphate release. 3000 4000 2000 2.00E-06 2000

1000 cell viability normalised to Hexokinase Glucose-6- Glycolitic Glucose Krebs cycle 1.00E-06 phosphate pathway Results 0 0 Vehicle AT-001 ZOPOL TROGLITAZONE Vehicle AT-001 ZOPOL 5X 5X 5X (positive control) 50X 50X 50X 0.00E+00 Improved inhibitory selectivity of AT-001 [EC ] [EC ] Vehicle 50X AT-001 50X ZOPOL 50X Aldose Hyperglycaemia/ischaemia Osmotic stress 50 50 reductase (Polyol Pathway Activated) CELL DEATH for Aldose Reductase p=0.03 vs AT-001 and vehicle AT-001 ALT normalised to cell viability Sorbitol Greater AR inhibition AT-001 vs zopolrestat 6.00E-06 Redox imbalance Zopolrestat Sorbitol Dehydrogenase ROS formation Advanced glycation 5.00E-06 Fructose PKC, NF-kB* activation 100 Zopolrestat (but not AT-001) Induces Cell Damage CELL DEATH 4.00E-06 *Nf-kB is a protein complex that controls transcription of DNA, cytokine production and cell survival 80 in Cultured Hepatocytes as Measured by Lactate Dehydrogenase Release 3.00E-06

60 ALT activity 2.00E-06 LDH normalised to cell viability* First Generation Aldose Reductase 40 cell viability normalised to Inhibitor vs AT-0013 % AR Inhibition 1.00E-06 20 2.50E-05 0.00E+00 Zopolrestat Inhibition of Aldose Reductase 0 Vehicle 50X AT-001 50X ZOPOL 50X Clinical Efficacy Zopolrestat demonstrated 0.1nM 1nM 10nM 100nM 1uM 10uM 100uM Glutamate based assay comparing AT-001 and zopolrestat at 50x EC50 concentration: AST activity reported as mmol/ clinical efficacy in Phase 2 2.00E-05 Competitive inhibition studies. Off-target hepatoxicity min/mL. One unit of AST (or ALT) is the amount of enzyme that will generate 1.0 mmol of glutamate per minute at of Aldehyde Reductase pH 8.0 at 37 oC was documented. Clinical Inhibitor Concentration (AT-001 or Zopolrestat) development was terminated3 (Off-target) Summary and Conclusions Hepatotoxicity Zopolrestat (but not AT-001) Off-target AT-001 1.50E-05 Inhibits Aldehyde Reductase, an Enzyme AT-001 is a logarithmically more potent ARI than zopolrestat in inhibiting Aldose Selective inhibition of AT-001 developed through Reductase, an enzyme implicated in the development of diabetic complications Aldose Reductase rational drug design to Critical for Liver Function selectively inhibit AR without including diabetic cardiomyopathy (DbCM). Clinical Efficacy off-target inhibition of Aldehyde Reductase 1.00E-05 Cell viability assessment at high substrate concentrations demonstrated that while Aldehyde reductase inhibitory activity AT-001 zopolrestat was cytotoxic, AT-001 and vehicle were not. Zopolrestat 100 The unique structure and activity of AT-001 provide selectivity for Aldose Reductase Zopolrestat inhibits Aldehyde LDH activity (millunits/ml) cell viability normalised to n and avoids off-target inhibition of Aldehyde Reductase, which is an enzyme critical IC50 of AT-001 vs zopolrestat for Aldose Reductase Reductase activity in the Aldose 80 for normal detoxification processes in the liver. Reductase effective dose range 5.00E-06 Measurement of ALT, AST and LDH in in-vitro and ex-vivo systems demonstrated Tissue penetration (in rats) 60 that zopolrestat, but not AT-001, was toxic to hepatocytes. MTD in Compound IC 50 Systemic/ The in-vitro safety of AT-001, together with the positive safety data from the Phase 1/2 animals Nerve Retina CNS 40 Heart 0.00E+00 program, support the ongoing pivotal study in DbCM: ARISE-HF (NCT 04083339). 20 Vehicle 50X AT-001 50X ZOPOL 50X AT-001 30pM >2,000mg/kg ✓ ✓ ✓ X References 0 Zopolrestat 10nM 100mg/kg ✓ ✓ X X % Aldehyde reductase inhibitio *Cell damage in cultured hepatocytes measured by lactate dehydrogenase (LDH) release; 1. Brownlee M. Diabetes Care. 2005;54(6):1615–1625; -20 LDH is a biochemical marker for tissue injury/cellular necrosis 2. Miki T, et al. Heart Fail Rev. 2013;18(2):149–166; 0.1uM 1uM 10uM 50uM 100uM 3. Johnson, et al. Diabetes Care. 2004:448–454. Inhibitor concentration (AT-001 or zopolrestat)

DISCLOSURES: RP and SS are employees of and stockholders in Applied Therapeutics Inc. 56th Annual EASD Meeting, 21–25 September 2020 Data was first presented at ADA June 2020