Samidorphan Attenuates Drug-Induced Increases in Extracellular Dopamine Concentrations and Drug Self-Administration in Rats Jacobi I. Cunningham*1, Reginald L. Dean III1, Mark S. Todtenkopf1, Marc R. Azar2, George F. Koob3, Daniel R. Deaver1, David J. Eyerman1

1Alkermes, Inc., Waltham, MA, 2Behavioral Pharma, Inc., La Jolla, CA, 3The Scripps Research Institute, La Jolla, CA P.3.a.009

INTRODUCTIONINTRODUCTION RESULTS INTRODUCTION

ALKS 3831, a combination of and the Fig 1: Effects of samidorphan on ethanol-induced Fig 2: Effects of samidorphan on -induced novel receptor modulator, samidorphan increases in extracellularINTRODUCTION concentrations of DA in the NAc-sh increases in extracellularINTRODUCTION concentrations of DA in the NAc-sh e (formerly referred to as ALKS 33), is a drug 1 .0 e 4 n n i i

2 5 0 V e h ic le + E T O H l 4 0 0 V e h ic le + M O R l e e s candidate for the treatment of in 0 .8 s a a 3 B S a m id o rp h a n + E T O H B

S a m id o rp h a n + M O R

e 0 .6 e v patients with substance use disorders. v o o 2 b b A A

0 .4

A Samidorphan is an modulator A A A

D M O R D D D

1

0 .2 x * x

with potent µ-antagonist properties. Importantly, 2 0 0 a 3 0 0 a e e m E T O H m * n C n 0 .0 C 0 i opioid receptors and their endogenous ligands i l l e e s are expressed in areas of the brain, such as the s a a B mesolimbic system, that are associated with the B

1 5 0 2 0 0 t rewarding effects of drugs of abuse. In t n n e nonclinical studies, opioid receptor modulation e c c r r e alters the neurobiological responses to drugs of e P P abuse, including drug-induced increases in 1 0 0 1 0 0 extracellular concentrations of mesolimbic S a m id o r p h a n S a m id o r p h a n dopamine (DAext) and drug-seeking behavior. Therefore, the current studies were designed to -1 0 1 2 3 -1 0 1 2 3 characterize the effects of samidorphan on T im e (h o u rs ) T im e (h o u rs ) amphetamine, cocaine, ethanol and morphine- Extracellular concentrations of dopamine in the NAc-sh increased approximately 40% Extracellular concentrations of dopamine in the NAc-sh increased approximately 200% above baseline following ethanol (2.5 g/kg; PO). Subcutaneous administration of above baseline following morphine (15 mg/kg; SC). Subcutaneous administration of induced increases in DAext in the nucleus samidorphan (1 mg/kg) 30 min prior to ethanol significantly attenuated ethanol-induced samidorphan (1 mg/kg) 30 min prior to morphine significantly attenuated morphine- accumbens shell (NAc-sh), and cocaine and increases in DA (p < 0.001, drug x time interaction; p < 0.05, drug; p < 0.05, time) and induced increases in DA (p < 0.001, drug x time interaction; p < 0.001, drug; p < 0.001, maximal (Cmax) concentrations in DA (*p < 0.01) above baseline in the NAc-sh. Raw data time) and maximal (Cmax) concentrations in DA (*p < 0.01) above baseline in the NAc-sh. ethanol self-administration behavior. were converted to and reported as percent change from baseline (± SEM). n = 7 per Raw data were converted to and reported as percent change from baseline (± SEM). n = 6 group. per group. Fig 3: Effects of samidorphan on cocaine-induced Fig 4: Effects of samidorphan on amphetamine-induced METHODSINTRODUCTION increases in extracellularINTRODUCTION concentrations of DA in the NAc-sh increases in extracellularINTRODUCTION concentrations of DA in the NAc-sh

e 4 e 6 V e h ic le + A M P H n

5 0 0 V e h ic le + C O C n 5 0 0 i i l l e e s

Male Wistar rats (200-350 g) were used in all experiments. Rats s a S a m id o rp h a n + C O C a S a m id o rp h a n + A M P H 3 B B

were housed and cared for in accordance with the Guide for the 4 e e v v o Care and Use of Laboratory Animals (National Research Council o 2 b b A A A M P H A A

4 0 0 4 0 0 2011), and experiments were approved by the and A * A 2 D D D D

1

C O C x

Behavioral Pharma Institutional Animal Care and Use Committees. x e e a a * m n n m i C i C 0 0 l l e In vivo microdialysis e s Microdialysis probes were inserted into the NAc-sh using the 3 0 0 s 3 0 0 a a B following coordinates from bregma: A/P +1.70; M/L ±0.8; D/V -7.8. B

t Rats were continuously perfused with sterile artificial cerebrospinal t n fluid (aCSF) (CMA CNS Perfusion Solution, CMA-Microdialysis) via n e 2 0 0 e 2 0 0 c a syringe pump at 2.0 µL/minute. Each microdialysate fraction c r r e consisted of perfusate collected continuously over 15 minute e P intervals using a CMA 470 refrigerated autofraction collector (CMA- P Microdialysis). Four baseline fractions were collected followed by 1 0 0 1 0 0 administration of samidorphan or vehicle. Two subsequent S a m id o r p h a n S a m id o r p h a n fractions were collected and rats were administered test compounds (as noted in the figure legends) at a dose volume of 1 -1 0 1 2 3 -1 0 1 2 3 mL/kg. Microdialysis fractions were analyzed via HPLC-EC using T im e (h o u rs ) T im e (h o u rs ) an Alexys Monoamine Analyzer (Antec Leyden, Leiden, the Netherlands) using an automated sample handler. An aliquot of Extracellular concentrations of dopamine in the NAc-sh increased approximately 400% of Extracellular concentrations of dopamine in the NAc-sh increased approximately 360% each fraction (10 μL) was injected onto a 1 µm reverse-phase C18 baseline following cocaine (5 mg/kg; IP). Subcutaneous administration of samidorphan (1 above baseline following amphetamine (0.5 mg/kg; IP). Subcutaneous administration of mg/kg) 30 min prior to cocaine significantly attenuated cocaine-induced increases in DA (p samidorphan (1 mg/kg) 30 min prior to amphetamine significantly attenuated column (ALF-105, Antec Leyden) for monoamine separation. DA < 0.001, drug x time interaction; p < 0.01, drug; p < 0.001, time) and maximal (Cmax) amphetamine-induced increases in DA (p < 0.001, drug x time interaction; p = 0.13, drug; was eluted using a mobile phase (pH 6.4) consisting of 50 mM concentrations in DA (*p < 0.05) above baseline in the NAc-sh. Raw data were converted p < 0.01, time) and maximal (Cmax) concentrations in DA (*p < 0.05) above baseline in the phosphoric acid, 8 mM KCL, 0.1 mM EDTA, 10% methanol and 500 to and reported as percent change from baseline (± SEM). n = 6 (Vehicle + Cocaine) and n NAc-sh. Raw data were converted to and reported as percent change from baseline (± mg/L octane sulfonic acid. DA was detected using a Decade II = 5 (samidorphan + cocaine) per group. SEM). n = 6 per group. amperometric detector (Antec Leyden) with a glassy carbon electrode maintained at approximately 0.3 V relative to a Ag/AgCl Fig 5: Effect of samidorphan Fig 6: Effect of samidorphan on the PR cocaine self-administration ISAAC reference electrode. onINTRODUCTION FR2 ethanol self-administration INTRODUCTION

Oral ethanol self-administration A B V e h ic le + C O C Rats were trained to orally self-administer ethanol using a modified V e h ic le + C O C operant procedure. Rats were hand shaped over a one to three day 1 5 0 1 5 8 0 S a m id o rp h a n + C O C period to lever press (Fixed Ratio 1) for a 0.1% saccharine solution. S a m id o rp h a n + C O C

Rats were then started on 5% ethanol in 0.1% saccharine, and the s e

ethanol concentration gradually increased to 10%, and the s 6 0 s t s saccharine concentration was then decreased to 0.04% over the e 1 0 0

1 0 n r d i r next 20–40 sessions. Briefly, the start of the session was signaled P o

a f by the activation of the house light. A cue light above the lever was p o w

k 4 0

e r illuminated, and the rat was required to press the lever two times * a e e R

r (Fixed Ratio 2) to receive three-second access to the ethanol b 5 0 5 # B

m * cocktail from the liquid dipper. Each daily session (five days per 2 0 week) lasted 30 min. Rats that consistently drank a minimum of 0.6 u * * N g/kg/h of ethanol (approximately 60 bar presses in 30 min with a 10% ethanol in 0.04% saccharine cocktail) over a four-week period 0 0 0 were used in these studies. Approximately 60% of the rats that V e h ic le 0 .3 1 3 began training were able to meet this criterion. S a m id o rp h a n D o s e (m g /k g ) IV cocaine self-administration Lever pressing was established by the method of Hyytia et al., Rats were trained to orally administer ethanol (10% ethanol in a Rats were trained to lever press for cocaine (0.5 mg/kg/inf; IV) and tested on a PR schedule of reinforcement for six hr. (1996). Rats were catheterized and allowed three-five days to 0.04% saccharin solution) and tested on a FR2 schedule of Bars represent average (± SEM) number of cocaine infusions per session. A) Samidorphan (1 mg/kg, SC) administered 30 reinforcement. Bars represent average (± SEM) number of lever recover before self-administration sessions started. During the minutes prior to test session significantly attenuated the number of cocaine rewards (one-way ANOVA; * p < 0.01) presses per session. Samidorphan administered 30 minutes compared to vehicle control. B) Samidorphan administered 30 minutes prior to test session significantly attenuated the recovery catheter lines were flushed daily with 30 units/mL of prior to test session produced a significant, dose-dependent ® breakpoint for cocaine self-administration. (* p < 0.01). n= 9 per group. heparinized saline containing 100 mg/mL of Timentin to prevent decrease in lever presses when compared to vehicle (*p < 0.05). infection and maintain patency. Rats were tested on a six hour n=4 (vehicle) and n=5 (0.3 mg/kg, 1 mg/kg, 3 mg/kg progressive-ratio schedule, with each reward resulting in a samidorphan) per group. progressive increase in the number of lever presses required for the subsequent reward. The progression of lever presses was 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, etc., where initially the INTRODUCTION first lever press delivered a cocaine reinforcer. The subsequent CONCLUSIONS reinforcer required two presses, and the subsequent reinforcer required four lever presses for the reinforcer, etc. Breakpoint was defined as the last ratio completed with no responses for 30 •Samidorphan, a novel opioid receptor modulator, attenuated neurochemical and behavioral responses produced by minutes. After stable responding for cocaine was achieved, multiple drugs of abuse. samidorphan was administered to rats (1 mg/kg, SC) 30 minutes prior to a six hour test session. •Differences in the effectiveness of samidorphan in the neurochemical studies were noted; samidorphan was more effective in attenuating DA increases produced by ethanol, morphine and cocaine than amphetamine.

•Samidorphan is most effective when blocking NAc-sh DA increases produced by mu receptor activation (i.e., ethanol and morphine) than increases produced by drug activity at DA transporters and vesicles (i.e., cocaine and amphetamine).

•Samidorphan may contribute to the efficacy of ALKS 3831 in treating patients with schizophrenia and [email protected] disorders. All studies were funded by Alkermes, Inc. P.3.a.009 Samidorphan attenuates drug-induced increases in extracellular dopamine concentrations and drug self- administration in rats J.I. Cunningham1, R.L. Dean III1, M.S. Todtenkopf1, M.R. Azar2, G.F. Koob3, D.R. Deaver1, D.J. Eyerman1 1Alkermes Inc.a Life Sciences and Toxicologya Walthama USA 2Behavioral Pharma Inc.a La Jollaa USA 3The Scripps Research Institutea La Jollaa USA

ALKS 3831, a combination of olanzapine and the novel opioid receptor modulator, samidorphan (formerly referred to as ALKS 33) is a drug candidate for the treatment of schizophrenia in patients with substance use disorders. Samidorphan selectively binds to human mu, kappa and delta opioid receptors [1], and is characterized as a mu antagonist, and kappa/delta , as determined by a GTPγ S assay [2]. Importantly, opioid receptors and their endogenous ligands are expressed in areas of the brain, such as the mesolimbic system, that are associated with the rewarding effects of drugs of abuse [3]. In nonclinical studies, opioid receptor modulation alters the neurobiological responses to drugs of abuse, including drug-induced increases in extracellular concentrations of mesolimbic dopamine (DAext) and drug-seeking behavior. Therefore, the current studies were designed to characterize the effects of samidorphan on amphetamine, cocaine, ethanol and morphine-induced increases in DAext in the nucleus accumbens shell (NAc-sh), and cocaine and ethanol self- administration behavior.

To assess the effects of samidorphan on drug-induced increases in DA ext, in vivo microdialysis was performed in male rats with microdialysis probes stereotaxically implanted within the NAc-sh (final coordinates relative to bregma: A/P +1.70; M/L ±0.8; D/V −6.0, perpendicular to the top of the skull). For cocaine self-adm inistration, rats were trained to self-administer cocaine under a fixed ratio (FR1) and tested on a 6 hour progressive ratio (PR) schedule of reinforcement, with each cocaine reward (0.5mg/kg/inf) resulting in a progressive increase in the number of lever presses required for the subsequent reward. For ethanol self-administration, rats were trained to orally self- administer ethanol (10a ethanol in 0.04a saccharin) and tested in an FR2 schedule of reinforcement.

Within the NAc-sh, amphetamine (0.5mg/kg), cocaine (5mg/kg), ethanol (2.5g/kg) and morphine (15mg/kg) produced a 330a, 400a, 40a and 200a increase in DAext, respectively. Administration of samidorphan (1mg/kg) 30min. prior to drug administration significantly attenuated drug-induced increases in NAc-sh DAext. In behavioral studies, rats trained to self-administer cocaine under a PR schedule of reinforcement reached a breakpoint of 62 with an average of 12.4 infusions of cocaine. Samidorphan (1mg/kg) administered 30min. prior to testing significantly attenuated cocaine self-administration: rats reached a breakpoint of 18 with an average of 6.8 infusions. In rats trained to self-administer ethanol, administration of samidorphan 30min. prior to testing dose-dependently decreased ethanol self-administration behavior.

The data from these nonclinical studies highlight the importance of the endogenous opioid system in the neurobiological effects of drugs of abuse and demonstrate that a novel mixed opioid receptor modulator, samidorphan, attenuates neurochemical and behavioral effects of multiple drugs of abuse. Additionally, these data suggest that samidorphan may contribute to the efficacy of ALKS 3831 in treating patients with schizophrenia and substance use disorders.

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2. Wentland M.P., Lou R., Lu Q., Bu Y., Denhardt C., Jin J., Ganorkar R., VanAlstine M.A., Guo C., Cohen D.J., Bidlack J.M., 2009. Syntheses of novel high affinity ligands for opioid receptors. Bioorg Med Chem Lett 19(8), 2298–94.

3. Kuhar M.J., Pert C.B., Snyder S.H., 1973. Regional distribution of receptor binding in monkey and human brain. Nature 245, 447–450.

Disclosure statement: I am a full time employee of Alkermes, Inc.

Citation: Eur Neuropsychopharmacol. 2014;24aSuppl 2):S489

Keywords Schizophrenia: basic Drug dependence & abuse: basic Behavioural pharmacology