In Vitro Pharmacological Characterization of , Samidorphan, and Combinations Being Developed as an Adjunctive Treatment for Major Depressive Disorder Sokhom Pin,1 Jean Bidlack,2 Brian Knapp,2 Daniel Deaver,1 Margarita Plotnikava,1 Derrick Arnelle,1 Angela Quinn,1 May Toh,1 Mark Namchuk1 1Alkermes, Inc., Waltham, MA, USA; 2University of Rochester, School of Medicine and Dentistry, Rochester, NY, USA P.796

• The dissociation T1/2 values (the time required to Table 3: EC and E Values for BUP and SAM 3 3 50 max INTRODUCTION reach 50% of maximal binding) for [ H]BUP and [ H]SAM, Alone and in Combination in Stimulating the respectively, were each longer at MOR than KOR (Figure 1). Activation of Gαi1, Gαi2, and Gαi3, Mediated by • A combination of buprenorphine and samidorphan (BUP/SAM, • In saturation binding experiments, [3H]BUP had the highest the MOR and KOR formerly ALKS 5461) at a 1:1 fixed-dose ratio is being affinity for KOR (K values 0.14 ± 0.015 nM and 0.074 ± d Gαi1 Gαi2 Gα3 investigated as an adjunctive treatment for major depressive 0.011 nM in trisaminomethane hydrochloride [Tris-HCl] and EC50 (nM) Emax (%) EC50 (nM) Emax (%) EC50 (nM) Emax (%) disorder in patients having an inadequate response to GP buffers, respectively) and [3H]SAM had highest affinity for 1,2 MOR . MOR (K values 0.046 ± 0.0027 nM and 0.044 ± 0.0051 nM, d BUP 0.70 ± 0.10 42 ± 1.0 0.40 ± 0.020 12 ± 0.25 0.72 ± 0.20 57 ± 4.0 • Although BUP is in clinical use, relatively little is known about respectively). its in vitro binding and pharmacological properties at the SAM No activity No activity 0.35 ± 0.040 9.0 ± 1.0 1:3 human receptors. 3 0.80 ± 0.30 11 ± 1.0 No activity 0.98 ± 0.030 21 ± 4.0 Figure 1: Association and Dissociation of [ H]BUP and BUP:SAM • SAM, a derivative containing a 3-carboxamido [3H]SAM with MOR and KOR KOR group, has high affinity for the µ- (MOR) and [3H]BUP A. Association Dissociation BUP 1.2 ± 0.050 31 ± 5.0 1.4 ± 0.25 19 ± 3.0 1.5 ± 0.50 43 ± 3.0 60 acts as an MOR antagonist and a at κ- and 100 3 SAM 1.9 ± 0.50 60 ± 8.0 3.4 ± 0.10 55 ± 0.50 1.9 ± 0.30 58 ± 4.0 δ-opioid receptors (KOR and DOR, respectively). 40 Tris-HCl Buffer 50 GP Buffer

MOR 1:3 • In preclinical studies, titration of SAM attenuates the abuse 20 1.4 ± 0.050 37 ± 5.0 1.2 ± 0.10 22 ± 3.0 1.2 ± 0.30 40 ± 3.0 Tris-HCI Buffer mean T1/2 BUP:SAM % Control Binding 0 Specific Binding (fmol) potential of BUP, while having no effect on the 0 GP Buffer mean T 0 50 100 150 0 5001000 1500 2000 1/2 BUP, buprenorphine; EC50, 50% maximal stimulation; Emax, maximum effect; KOR, κ-opioid 4 Time (min) Time (min) effect. 15 receptor; MOR, µ-opioid receptor; SAM, samidorphan. 100 Objective 10 50 • To investigate the in vitro pharmacological properties of BUP KOR 5 % Control Binding • BUP was almost a full agonist and SAM a partial agonist when

Specific Binding (fmol) 0 0 and SAM alone and in combination at molar ratios similar to 0 50 100150 200 0 500 1000 Time (min) Time (min) MOR signaled through Gαo and Gαz proteins (Figure 3). those observed in humans following BUP/SAM administration. [3H]SAM • On activating MOR, BUP caused partial recruitment of • To characterize μ (MOR)- and κ (KOR)-opioid receptor B. Association Dissociation 6 β-arrestin. signaling for the BUP/SAM combination across a spectrum 100 4 • When BUP and SAM were combined at 1:3 molar ratio, SAM of G proteins. 50 MOR 2 blocked β-arrestin recruitment by BUP (Figure 4).

% Control Binding 0 Specific Binding (fmol) 0 • When BUP and SAM were combined at 1:3 molar ratio, the 0 20 40 60 80 100 0 200 400 600 Time (min) Time (min) 10 efficacy of the combination matched BUP alone when METHODS 100 8 signaling through Gαi and Gαo proteins (Table 3). 6 50 Radioligand Competition Binding Assays KOR 4 • There was very little β-arrestin recruitment by BUP or SAM 2 % Control Binding

3 3 Specific Binding (fmol) 0 at the KOR. • [ H]BUP and [ H]SAM binding to membranes from Chinese 0 0 50 100 150 0 100 200 300 hamster ovary (CHO) cells stably expressing human MOR, KOR, Time (min) Time (min) and DOR were reported as mean ± SEM (standard error of the Association assays were performed by incubating [3H]BUP and [3H]SAM with hMOR-CHO Figure 4: Recruitment of β-Arrestin to the MOR by BUP and hKOR-CHO cell membranes for various times in 50 mM Tris-HCl, pH 7.4, or GP buffer. mean) from 3 separate experiments performed in triplicate. Dissociation assays were performed by incubating [3H]BUP and [3H]SAM with membranes and SAM Alone and in Combination for 90 and 60 minutes, respectively, to reach equilibrium at 25°C and then, adding 10 µM BUP 35 [ S]GTPγS Binding Assays and 1 µM SAM to displace the bound [3H]BUP and [3H]SAM, respectively. BUP, buprenorphine; CHO, Chinese hamster ovary; KOR, κ-opioid receptor; MOR, µ-opioid Inactivation • G-protein activation in CHO cell membranes expressing receptor; SAM, samidorphan; Tris-HCl, trisaminomethane hydrochloride. Activation the MOR or KOR in response to BUP and SAM alone and in βarr combination (1:1, 1:5, and 1:50 molar ratios) were similarly G G [35S]GTPγS Binding Assays βarr R reported as mean ± SEM from 3 separate experiments in • BUP was a partial agonist at the MOR (50% maximal R triplicate. 100 DAMGO stimulation [EC50 (nM)] = 0.63 ± 0.4; maximal stimulation BUP SAM 1:3 BUP:SAM Bioluminescent Resonance Energy Transfer [Emax (%)] = 57 ± 5.5) and KOR (EC50 = 0.46 ± 0.14; 75 (BRET) Assays Emax = 25 ± 1.3) (Figure 2). • Coupling of MOR and KOR to specific G proteins in HEK 293 • SAM was an antagonist at the MOR and a partial agonist at the 50 cells (Domain Therapeutics, Inc, Montreal, Canada) was used KOR (EC50 = 1.9 ± 0.1; Emax = 56 ± 0.6) (Figure 2). to study BUP and SAM, alone and in combination, to activate • Combination of BUP and SAM at 1:1, 1:5, and 1:50 molar Activation MOR % 25 opioid receptors to interact with specific G proteins, and to ratios showed that increasing the relative amount of SAM recruit β-arrestin. decreased the Emax value for the combination on the MOR • These results were reported as mean values ± SEM from (Figure 2). 0 10 –11 10 –10 10 –9 10 –8 10 –7 10 –6 10 –5 6 determinations. Log, [M] BUP, buprenorphine; G, green fluorescent protein; MOR, µ-opioid receptor; R, Renilla luciferase; Figure 2: Stimulation and Inhibition of [35S]GTPγS SAM, samidorphan. Binding to the MOR and KOR by BUP and SAM Alone RESULTS and in Combination A. Mu Agonist Activity B. Kappa Agonist Activity 80 BUP 80 BUP CONCLUSIONS 1:1 BUP:SAM 1:1 BUP:SAM Radioligand Competition Binding Assays 1:5 BUP:SAM 1:5 BUP:SAM 3 3 60 1:50 BUP:SAM 60 1:50 BUP:SAM S Binding S S Binding S SAM SAM γ • [ H]BUP and [ H]SAM bound to all 3 opioid receptors with high γ • When assessed across a panel of G-protein sensors, BUP 40 40 S]GTP affinity (Table 1; Table 2). S]GTP 35 35 acted as a partial agonist of the MOR and KOR, with Emax 20 20 values ranking higher for MOR than KOR.

Table 1: Ki Values for BUP and SAM in Inhibiting 0 0 • When assessed across a panel of G-protein sensors, SAM % Stimulation of [ Binding to the MOR, KOR, and DOR % Stimulation of [ –20 –20 largely functioned as an antagonist on the MOR and partial 0.001 0.01 0.1 1 10 100 1000 10000 0.001 0.01 0.1 1 10 100 1000 10000

MOR KOR DOR Total Concentration Total Concentration agonist on the KOR.

[3H]DAMGO [3H]U69,593 [3H] A. Stimulation of [35S]GTPgS binding at the MOR by BUP and SAM alone or various molar ratios • BUP and SAM in combination at 1:3 molar ratio of the compounds together. K (nM ± SEM) i B. Stimulation of [35S]GTPgS binding at the KOR by BUP, SAM, or various molar ratios of the (approximating the steady-state molar ratio observed for BUP 0.41 ± 0.0079 0.23 ± 0.0067 2.5 ± 0.15 compounds together. BUP/SAM in humans) retained a low level of activity at the SAM 0.052 ± 0.0 0.23 ± 0.018 2.7 ± 0.36 BUP, buprenorphine; KOR, κ-opioid receptor; MOR, µ-opioid receptor; SAM, samidorphan. MOR, due to effective competition of SAM with BUP, but Data are the mean Ki value ± SEM from 3 independent experiments performed in triplicate. functioned like BUP at the KOR. BUP, buprenorphine; DOR, δ-opioid receptor; KOR, κ-opioid receptor; MOR, µ-opioid receptor; SAM, samidorphan; SEM, standard error of the mean. BRET Assays • SAM inhibited BUP’s recruitment of β-arrestin to the MOR, • BUP was a partial agonist and SAM an antagonist when MOR suggesting that attenuation of BUP’s activation of G proteins activated Gαi proteins (Figure 3). may be responsible for the inhibition of β-arrestin recruitment. Table 2: Affinities of Full and Partial Opioid Agonists and Antagonists for Binding to the MOR Under High- • When BUP and SAM were combined at 1:3 molar ratio, the and Low-Affinity Binding Conditions efficacy of BUP at Gαi proteins was reduced by the inclusion of SAM (Table 3). High Affinity Low Affinity Klow/Khigh Compound Buffer Buffer References 3 3 [ H]DAMGO [ H] Figure 3: BRET Studies Showing BUP and SAM Alone 1. Ehrich, et al. Neuropsychopharmacology. 2015;40:1448-1455. 2. Fava, et al. Am J Psychiatry. 2016;173:499-508. Full Agonists Ki (nM ± SEM) and in Combination Activating MOR and KOR Signaling 3. Wentland, et al. Bioorg Med Chem Lett. 2009;19:2289-2294. 530 0.32 ± 0.028 170 ± 7.5 Through Gαi1, Gαi2, Gαi3, Gαoa, Gαob, and Gαz 4. Smith, et al. Neuropsychopharmacology. 2017;42:S550-551. 480 1.3 ± 0.14 630 ± 35 MOR pEC MOR E Inactivation 50 max Acknowledgments DAMGO 390 0.72 ± 0.084 280 ± 8.2 GαoA GαoA GDP GTP The study was sponsored by , Inc. Medical writing and editorial support 380 0.47 ± 0.048 180 ± 7.8 Activation β was provided by PAREXEL and funded by Alkermes, Inc. α β ̀ α ̀ G G GαoB GαZ GαoB GαZ Partial Agonists x 8 10 6 60 100 4 40 80 Disclosures x R 2 20 (-) 37 2.1 ± 0.088 78 ± 5.1 SP, DD, MP, DA, AQ, MT, and MN are employees of Alkermes. 19 0.70 ± 0.023 13 ± 0.18 R JB and BK were supported by Alkermes, Inc. to perform this research. 13 0.24 ± 0.015 3.2 ± 0.054 BUP/MOR Gαi1 Gαi3 Gαi1 Gαi3 5.5 0.69 ± 0.053 3.8 ± 0.42 100 Gαi1 Gαi2 Gαi2 BUP Gαi2 SAM BUP 0.40 0.35 ± 0.064 0.14 ± 0.0071 Gαi3 KOR pEC50 BUP:SAM KOR Emax 75 G oA α GαoA GαoA Antagonists GαoB GαZ Naloxone 2.2 0.87 ± 0.12 1.9 ± 0.14 50 GαoB GαZ GαoB GαZ 8 6 60 100 Naltrexone 1.4 0.31 ± 0.012 0.42 ± 0.014 4 40 80 2 20 0.92 0.24 ± 0.022 0.22 ± 0.010 % MOR Activation 25 SAM 0.49 0.19 ± 0.020 0.094 ± 0.0073 0 Gαi1 Gαi3 Gαi1 Gαi3

Klow/Khigh is the Ki value obtained under low affinity binding conditions in GP buffer divided by the 10–11 10–10 10–9 10–8 10–7

Ki value obtained under high-affinity Tris-HCl buffer conditions. BUP, Log[M] Gαi2 Gαi2 Data are the mean K values ± SEM from 3 separate experiments performed in triplicate. i BRET, bioluminescent resonance energy transfer; BUP, buprenorphine; Copies of this poster obtained through this QR (Quick Response) BUP, buprenorphine; MOR, µ-opioid receptor; SAM, samidorphan; SEM, standard error of the FPO code are for personal use only and may not be reproduced E , maximum effect; G, green fluorescent protein; KOR, κ-opioid receptor; without permission of Alkermes. mean; Tris-HCl, trisaminomethane hydrochloride. max MOR, µ-opioid receptor; R, Renilla luciferase; SAM, samidorphan. For permission contact: [email protected]

Presented at the 31st European Congress of Neuropsychopharmacology, Barcelona, Spain, October 6-9, 2018.