In Vitro Metabolic, CYP and Transporter Characterization of PTI-125, a Novel Small Molecule Drug Candidate for Alzheimer’s Disease Gang Luo1, Jody Wanta1, Kaitlin Bruden1, Macaulay Haller1, Courtney Lechler1, Kari Landsverk1, Daniel Albaugh1, Donald McKenzie1 and Lindsay Burns2 1Covance Laboratories Inc., Madison, WI; 2Cassava Sciences, Inc., Austin, TX

Abstract Methods Table 1. Experimental Conditions for CYP Inhibition by PTI-125 PTI-125 is an oral, small molecule drug candidate in Metabolic Stability CYP (µM) HLM (mg/mL) Incubation (min) Control for RI (µM) Control for TDI (µM) clinical development for the treatment of Alzheimer’s PTI-125 (1 µM) was incubated with mouse, rat, dog, and CYP1A2 Phenacetin (110) 0.063 10 Fluvoxamine (0.5) Furafylline (3) CYP2B6 Bupropion (120) 0.063 10 Orphenadrine (600) ThioTEPA (20) disease (AD). PTI-125 binds and reverses an altered human liver microsomes (1 mg/mL) at 37°C for 0, 15, 30 and CYP2C8 Amodiaquine (1.5) 0.013 10 (0.1) GEM (40) CYP2C9 (6) 0.1 5 Sulfaphenazole (5) Tienilic Acid (10) conformation of filamin A to suppress multiple AD 60 minutes in the presence of NADPH (1 mM). The CYP2C19 S-Mephenytoin (50) 0.063 10 Nootkatone (20) Esomeprazole (8) pathologies. In the current in vitro study, the metabolic remaining PTI-125 concentrations were quantitated by liquid CYP2D6 Dextromethorphan 0.063 10 Quinidine (0.3) Paroxetine (2) CYP3A4/5 Testosterone (65) 0.1 5 Ketoconazole (0.2) Erythromycin (100) stability of PTI-125 was characterized in mouse, rat, chromatography tandem mass spectrometry (LC-MS/MS). CYP3A4/5 Midazolam (1.5) 0.063 5 Ketoconazole (0.1) Troleandomycin (10) dog and human hepatic microsomes. Potential drug CYP Inhibition RI: Reversible inhibition; TDI: time-dependent inhibition. interactions were assessed by inhibition and induction Potential reversible and time-dependent inhibition of CYP of human hepatic CYP and by interactions enzymes by PTI-125 (eight concentrations ranging from Table 2. Experimental Conditions for CYP Induction by PTI-125 with a panel of human drug transporters. PTI-125 0.1 to 100 µM) was determined. Experimental conditions for CYP Enzyme Known Inducer (µM) Probe Substrate µM) Reaction Metabolite (1 µM) was incubated with pooled (from both male and CYP inhibitions are presented in Table 1. CYP1A2 Omeprazole Phenacetin (100) Phenacetin O-deethylation Acetaminophen female) mouse, rat, dog and human hepatic CYP2B6 Phenobarbital Bupropion (500) Bupropion hydroxylation Hydroxybupropion CYP Induction CYP3A4/4 Testosterone (250) Testosterone 6β-hydroxylation 6β-Hydroxytestosterone microsomes (1 mg/mL) in the presence of nicotinamide Potential induction of CYP1A2, CYP2B6, and CYP3A4/5 by Flumazenil (20 µM) was used as negative control. adenine dinucleotide phosphate (NADPH) for 0, 15, 30 PTI-125 (20, 60 and 100 µM) was assessed in hepatocytes and 60 minutes or in the absence of NADPH for 0 and from 3 individual donors. Both mRNA and enzyme activity 60 minutes before determining remaining Table 3. Experimental Conditions for Transporter Interactions with by PTI-125 were determined for assessment of CYP induction. concentrations of PTI-125 in the incubation samples. Transporter Test System Probe Substrate (µM) Selective Inhibitor (µM) Experimental conditions for CYP induction are presented in PTI-125 showed measurable NADPH-dependent MATE1 Transfected HEK cells 14C-Tetraethylammonium (5) Cimetidine (100) Table 2. MATE2-K Transfected HEK cells 14C-Tetraethylammonium (5) Cimetidine (100) metabolism by rat liver microsomes, but metabolism OAT1 Transfected HEK cells 14C-para-Aminohippurate (1) Probenecid (200) OAT3 Transfected HEK cells 3H-Estrone-3-sulfate (1) Probenecid (200) was not measureable by mouse, dog or human liver Transporter Interactions OCT1 Transfected HEK cells 14C-Tetraethylammonium (5) Quinidine (256) Potential interactions of PTI-125 (as a substrate and OCT2 Transfected HEK cells 14C-Metformin (1) Quinidine (256) microsomes. PTI-125 (up to 100 µM) did not show any OATP1B1 Transfected HEK cells 3H--17β-D-glucuronide (0.5) Cyclosporine A (10) reversible or time-dependent inhibition of CYP1A2, inhibitor) with a panel of human key transporters were OATP1B3 Transfected HEK cells 3H-Cholecystokinin octapeptide (1) Cyclosporine A (10) OATP2B1 Transfected HEK cells 3H-Estrone-3-sulfate (1) Rifamycin (20) CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or assessed. Probe substrates and selective inhibitors are P-gp Caco-2 cells 3H-Digoxin (1) Zosuquidar (2) 3 CYP3A4/5. Under the same experimental conditions, listed in Table 3. BCRP Caco-2 cells H-Estrone-3-sulfate (0.1) Ko143 (1) the known reversible and time-dependent inhibitors of CYP enzymes showed marked CYP inhibition. PTI-125 Results Table 4. Summary of CYP Inhibition by PTI-125 (up to 100 μM) showed no apparent cytotoxicity in a ▶ PTI-125 was slightly metabolized in rat liver microsomes CYP Enzyme Activity Assay Reversible Inhibition Time-Dependent Inhibition primary culture of human hepatocytes from a single CYP1A2 Phenacetin O-deethylase Not observed Not observed but stable in mouse, dog and human liver microsomes CYP2B6 Bupropion hydroxylase Not observed Not observed donor. PTI-125 (20 and 100 μM) was stable when (Figure 1). CYP2C8 Amodiaquine N-deethylase Not observed Not observed incubated with these single-donor human hepatocytes CYP2C9 Diclofenac 4′-hydroxylase Not observed Not observed ▶ PTI-125 showed no inhibition of CYP1A2, CYP2B6, CYP2C19 S-Mephenytoin 4′-hydroxylase Not observed Not observed for 24 hours. PTI-125 (20, 60 and 100 μM) showed no CYP2D6 Dextromethorphan O-demethylase Not observed Not observed CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A4/5 CYP3A4/5 Testosterone 6β-hydroxylase Not observed Not observed induction of CYP1A2, CYP2B6 or CYP3A4 mRNA CYP3A4/5 Midazolam 1′-hydroxylase Not observed Not observed (Table 4). levels or activities in human hepatocytes from three ▶ PTI-125 showed no induction of CYP1A2, CYP2B6 or individual donors. In the same hepatocytes, all known CYP3A4/5 (Table 5). Table 5. Summary of CYP Induction by PTI-125 inducers demonstrated marked induction of these ▶ PTI-125 was not a substrate of human transporters tested human CYP enzymes. In transporter studies, PTI-125 mRNA Enzyme Activity and showed mild inhibition of OAT1, OCT1, OCT2, Donor Fold Induction % of Positive Control Conclusion Fold Induction % of Positive Control Conclusion was not a substrate of key solute carrier (SLC) MATE1 and BCRP but not the other transporters tested CYP1A2 transporters including Organic Anion Transporter (OAT) (Table 6). 1 1.82 0.842 No 1.29 0.521 No 1, OAT3, Organic Cation Transporter (OCT) 1, OCT2, 2 1.58 3.18 No 1.42 0.962 No 3 2.15 2.37 No 1.63 5.39 No Organic Anion Transporting Polypeptide (OATP) 1B1, CYP2B6 OATP1B3, OATP2B1, Multidrug and Toxin Extrusion 1 2.00 4.74 No 2.10 1.76 No 2 2.57 6.06 No 1.34 3.57 No (MATE) 1, MATE2-K or key ATP-binding cassette 3 2.53 3.89 No 1.24 2.11 No transporters including P-glycoprotein (P-gp) and Breast CYP3A4 CYP3A4/5 Cancer Resistance Protein (BCRP). PTI-125 showed 1 2.66 2.04 No 1.69 4.28 No 2 2.14 7.79 No 1.35 2.61 No weak inhibition of OAT1, OCT1, OCT2, MATE1 and 3 3.43 6.64 No 1.32 3.88 No BCRP, but no inhibition of OAT3, OATP1B1, OATP1B3, Fold induction was calculated over vehicle control and only maximum values are presented.

OATP2B1, MATE2-K or P-gp. Although IC50 values were not accurately determined, they were estimated to Table 6. Summary of Transporter Interactions with PTI-125 be >100 µM for OAT1, OCT1, OCT2 and BCRP and Transporter Substrate Inhibitor IC50 (µM) >40 µM for MATE1. Overall, the general lack of drug MATE1 No Yes >10 but <100 metabolism (apparent only in rat), the lack of CYP MATE2-K No No NA OAT1 No Yes >100 interactions and the very weak interactions with some OAT3 No No NA transporters comprise a highly favorable profile for this OCT1 No Yes >100 OCT2 No Yes >100 novel drug candidate for AD. OATP1B1 No No NA OATP1B3 No No NA OATP2B1 No No (but activation) NA P-gp No No NA Introduction Figure 1. Metabolic stability of PTI-125 in mouse, rat, dog and BCRP No Yes >100 PTI-125 is an oral, small molecule drug candidate in human liver microsomes. clinical development for the treatment of AD. PTI-125 binds and reverses an altered conformation of filamin A Summary to suppress multiple AD pathologies. The present study PTI-125 was metabolically stable in human liver microsomes, aimed to determine metabolic stability in mouse, rat, showed no inhibition or induction of CYP enzymes, and had mild dog and human hepatic microsomes and assess inhibition of OAT1, OCT1/2 and MATE1. PTI-125 is unlikely to potential drug-drug interactions for PTI-125. have drug-drug interactions clinically.

Presented at ISSX 2019