JOP0010.1177/0269881118822111Journal of PsychopharmacologySmith et al. 822111research-article2019

Original Article

Investigation of the reinforcing potential of samidorphan and by fixed

and progressive ratio intravenous Journal of Psychopharmacology ­9– 1 self-administration testing in © The Author(s) 2019 Article reuse guidelines: sagepub.com/journals-permissions -maintained rats DOI:https://doi.org/10.1177/0269881118822111 10.1177/0269881118822111 journals.sagepub.com/home/jop

Sharon L Smith1, Reginald L Dean2, Mark S Todtenkopf2 and David J Heal1

Abstract Background: Samidorphan is a novel μ- antagonist with low intrinsic activity at κ- and δ-opioid receptors. Aims: Because samidorphan is central nervous system-active, we investigated whether samidorphan (13.6, 40.8, 68 μg/kg/injection) served as a positive reinforcer in rats trained to self-administer heroin on a fixed ratio-5 schedule. Samidorphan’s relative reinforcing effect was evaluated by progressive ratio/break-point determination. Naltrexone (13.6, 40.8, 68 μg/kg/injection) and heroin (7.5, 15, 25 μg/kg/injection) were comparators. Results: All heroin doses maintained self-administration on fixed ratio-5 and progressive ratio/break-points at levels significantly greater than saline. Samidorphan and naltrexone had similar profiles on fixed ratio-5 with one samidorphan dose serving as a positive reinforcer and one naltrexone dose showing a strong trend (p=0.053) for positive reinforcement. The numbers of injections of every samidorphan and naltrexone dose were significantly lower than all heroin doses. The numbers of self-administered samidorphan and naltrexone injections/session on fixed ratio-5 were not significantly different from one another. The mean inter-injection intervals for heroin were significantly shorter than for saline, whereas those of samidorphan and naltrexone were not. Progressive ratio break-points for samidorphan and naltrexone were not different from saline except for the highest dose of samidorphan. In addition, the progressive ratio break-points for samidorphan were not significantly different from those of naltrexone and were significantly lower than heroin. The samidorphan unit-doses evaluated in self-administration yielded plasma concentrations ranging between 25–109% and 10–45% of the maximum concentration values in humans. Conclusions: Overall, the profiles of samidorphan and naltrexone, which has no abuse liability, were similar in this model.

Keywords Abuse testing, dependence, samidorphan, heroin, naltrexone, self-administration

Introduction of major depressive disorder and with (ALKS 3831) for the treatment of . Samidorphan (3-carboxamido-4-hydroxynaltrexone; formerly For all CNS-active drug-candidates for clinical use in humans, referred to as ALKS 33) is a novel opioid system modulator that an evaluation of their potential for abuse is a mandatory part of has sub-nanomolar affinity for human μ- and κ-opioid receptors, the drug development as stated in guidance documents issued by and nanomolar affinity for δ-opioid receptors in cellular expres- the Center for Drug Evaluation and Research (CDER) of the US sion assays. In vivo, it is a µ- antagonist. Using a Department of Health and Human Services (DHHS) of Food and [35S]GTPγS assay to determine functional activity, samidorphan Drug Administration (FDA) and Committee for Medical Products is characterized as a μ-opioid with low intrin- for Human use (CHMP) of European Medicines Agency (EMA) sic activity at both κ- and δ-opioid receptors (Wentland et al., (CDER/FDA, 2017; CHMP/EMA, 2006). For this reason, we 2005, Wentland et al., 2009). In a clinical pharmacokinetic study, have investigated whether samidorphan serves as a positive rein- there was higher exposure and a longer half-life of samidorphan forcer in rats trained to self-administer a low dose of the µ-opioid than naltrexone at comparable doses; properties that enable once- agonist diamorphine (heroin). Heroin is a Schedule 1 Controlled daily dosing in humans (Turncliff et al., 2015). In rat studies, oral administration of samidorphan reversed -induced anal- 1 gesia for more than four hours, an effect characterizing its ability RenaSci Ltd, BioCity, Nottingham, UK 2Alkermes, Inc., Waltham, MA, USA to block opioid receptors (Cunningham et al., 2014; Todtenkopf et al., 2008). Samidorphan is a central nervous system (CNS)- Corresponding author: active drug candidate being developed as a fixed-dose combina- David J Heal, RenaSci Ltd, BioCity, Nottingham NG1 1GF, UK. tion product with (ALKS 5461) for the treatment Email: [email protected] 2 Journal of Psychopharmacology 00(0)

Drug (C-I) in the USA where it is not approved for medical use; Sprague-Dawley rats (200–225 g for the intravenous self-admin- in other countries where it is used clinically, e.g. UK, heroin is istration phase; 275–300 g for the blood-sampling phase) were classified as Schedule 2 (C-II). purchased from Charles River, Margate, Kent, UK. For the PK Historically, naltrexone is an antagonist of µ-, δ- and κ-opioid experiment, rats were group-housed (3–4 rats per cage). The rats receptors (Gharagozlou et al., 2002; Rothman et al., 1993; for the self-administration experiment were housed individually Valentino et al, 1983). More recently, naltrexone has been char- in large polypropylene cages containing rodent bedding and acterized as a µ-opioid receptor antagonist with low intrinsic chew sticks. After surgery, rats were transferred to cages modi- activity at δ- and κ-opioid receptors (Bidlack et al., 2018). fied to prevent them having access underneath the food hopper to Naltrexone is used clinically for detoxification (Fishbain obviate damage to the in-dwelling catheter port by the cage bars. et al., 1993), the management of alcohol and opiate dependence The room was illuminated by fluorescent lights timed to give a (Brooks et al., 2010; Greenstein et al., 1981; Niciu & Arias, 2013; 12-hour light-dark cycle (on 07:00, off 19:00), with a room tem- Wells, 1987) and in a fixed-dose combination formulation with perature of 21±4oC and relative humidity of 55±25%. Rats were bupropion as a treatment for obesity (see Heal et al., 2012; fed a standard rodent diet of Teklad 2018 (Envigo, UK) and fil- Padwal, 2009). Naltrexone has no liability for human abuse and tered, deionised water was available ad libitum. Rats were it is not a controlled drug. Due to the similarities in the pharma- allowed to acclimatise to these conditions for six days before the cology of samidorphan and naltrexone, the latter was selected as study commenced during which time they underwent regular a reference comparator drug for inclusion in this self-administra- weighing and handling. Rats for the self-administration experi- tion study. ment were initially restricted to 10 g/day of food over five days. The abilities of samidorphan and naltrexone to substitute for After this five-day period, daily food intake was restricted to heroin as positive reinforcers in rats were evaluated on a fixed ~90% of normal levels throughout the rest of the study with the ratio (FR)-5 scheduled of reinforcement, which is the drug rein- exception of the 48-hour recovery period after surgery when food forcement schedule that we employ when evaluating opiate rein- was freely available. forcers (Heal and Smith, 2017; Heal et al., 2018). Although Heroin hydrochloride was purchased from MacFarlan-Smith CDER/FDA (2017) suggested the use of FR10 in intravenous (UK), naltrexone hydrochloride was purchased from Sigma self-administration experiments, it clarified its position at the Aldrich (USA) and samidorphan L-maleate was supplied by College on Problems of Drug Dependence Meeting in June 2017 , Inc. (USA). Sterile 0.9% (w/v) saline for injection was by stating that FR10 is the maximum FR schedule that it consid- purchased from Baxter (UK). Heroin was dissolved in sterile ers valid in rat intravenous self-administration experiments. saline and prepared as a stock solution at least once weekly. The Thus, our selection of FR5 is consistent with both CDER/FDA bottles containing sterile heroin (1 mg/mL) stock solutions were (2017) and CHMP/EMA (2006). In addition, the relative rein- airtight sealed, protected from light with silver foil and stored at forcing effects of a range of doses of samidorphan, naltrexone 4°C. The bottles of heroin stock solution were only briefly and heroin were determined on a progressive ratio (PR) schedule removed from the fridge to make up the daily dosing solutions to calculate the break-points for drug reinforcement. The use of for the self-administration syringes. Heroin is stable in sterile PR/break-point determinations to assess the relative reinforcing physiological solutions kept at 4°C and 21–23°C for up to eight efficacy of novel CNS-active drugs is recommended in the EMA weeks (Omar et al., 1989; Smith et al., 1978). Samidorphan stock guidelines on assessing the abuse potential of novel drugs for solution was made up freshly each week as it is stable in sterile human use (CHMP/EMA, 2006). CDER/FDA (2017) recom- saline for up to two weeks (data on file at Alkermes, Inc.). mends that PR schedules should not be used to evaluate whether Naltrexone stock solution was made up freshly in sterile saline an investigative drug serves as a positive reinforcer because the each week and dosing solutions were diluted from the stock solu- demanding response requirement is likely to generate a false tion daily. The stability of naltrexone solutions for up to four negative for compounds with moderate or weak reinforcing prop- weeks has been demonstrated (data on file at Alkermes, Inc.). All erties. However, having already demonstrated that an investiga- naltrexone solutions were protected from light with silver foil. tional drug serves as a positive reinforcer on a FR schedule, our All dosing solutions were filtered through a 0.2 µm filter and view and that of CHMP/EMA (2006) is determination of its rela- were adjusted as close to neutral pH of 7.0 as possible. Doses tive reinforcing efficacy by PR/break-point testing provides of heroin, samidorphan, and naltrexone are expressed as base important additional information on the compound. In summary, content. this non-clinical safety evaluation has been conducted to meet the requirements of CDER/FDA (2017) and CHMP/EMA (2006). Although this was a non-Good Laboratory Practice (GLP) Intravenous self-administration experiments study, it was performed as close as possible to GLP standards. All Experiments were conducted in 16 commercially-available (Med processes followed RenaSci’s and the analytical facility’s stand- Associates, Inc., St Albans, Vermont, USA) operant chambers ard operating procedures. Quality control and quality assurance measuring 30.5×24.1×21.0 cm high and located within sound audits were incorporated into the project. attenuating, ventilated cubicles (Med Associates, Inc.). Each chamber was equipped with two levers located 11.5 cm apart. A 5×5 cm opening located equidistant between the levers was Methods available for food pellet delivery from a food hopper. Data were Animals and drugs collected and stored by a computer system and associated inter- face (Med Associates, Inc.). Each sound attenuating cubicle was One hundred and thirty-two (48 rats for the self-administration equipped with an infra-red camera (RF concepts or Med experiment; 84 rats for the blood-sampling experiments) male, Associates, Inc.) from which images were relayed to a digital Smith et al. 3 video recorder (RF Concepts) or PC-based video acquisition sys- extinction of self-administration behaviour (defined as three con- tem (Med Associates, Inc.) and displayed on a computer monitor. secutive sessions in which the mean number of saline injections Drugs were delivered using PHM-100A single speed syringe ⩽6) was achieved. pumps (Med Associates, Inc.). Rats were trained to lever-press for food pellets (45 mg dust- FR and PR testing. Rats which had met the criteria for heroin less precision pellets; F0021, Bilaney Consultants Ltd) on a FR1 self-administration and saline extinction were divided into three schedule of food reinforcement. Operant training sessions lasted groups: samidorphan (13.6, 40.8 and 68 µg/kg/inj), n=11, nal- ⩽1 h or finished once a rat had received 50 food pellet rewards. trexone (13.6, 40.8 and 68 µg/kg/inj), n=11, or heroin (7.5 and 25 Once rats had learned to lever-press on the FR1 schedule, the µg/kg/inj, n=9. The heroin group included the acquisition dose response requirement was increased to FR2 when the left lever (15 µg/kg/inj), n=31. The heroin group of rats were tested with was designated as the active lever. Thereafter, only responses on 1–2 doses of the drug as well as the acquisition dose. Each rat in the left lever resulted in the delivery of a reward. When stable on the samidorphan group was tested with two doses of the com- FR2, the response requirement was increased to FR3. Stable pound. Most of the naltrexone group was tested with 1–2 doses of post-surgery FR3 food responding was determined prior to start- the drug, apart from one animal tested with all three doses. Drug ing the rats on heroin training. self-administration was evaluated during two-hour sessions on a Once operant responding for food was stable under the FR3 FR5 schedule of reinforcement using the same experiment schedule (the rats earned >45 food pellets within one hour over parameters as described in the section on Self-administration three consecutive sessions), a chronic in-dwelling intravenous experiments above. Doses were tested on an ascending dose (i.v.) catheter was implanted into the jugular vein of each rat. schedule as far as possible and testing took place in once-daily Surgery was conducted under aseptic conditions using isoflu- sessions on 5–7 days per week. rane anaesthesia. Antibiotic was administered prophylactically Consistent with advice in the CDER/FDA (2017) guidance, using subcutaneous injections of Baytril 5 mg/kg daily for 48 h, the doses of samidorphan for evaluation in the intravenous self- then daily ticarcillin/clavulanic acid 80 mg/kg, i.v for the dura- administration experiment were selected to produce plasma drug tion of the study. The catheter (silicone tubing; SCT1000; concentrations that were fractions of the maximum concentration

Synaptech, USA) was implanted into the right jugular vein, (Cmax) of samidorphan at clinically effective doses. However, secured to the vessel and then tunnelled subcutaneously from the applying the same criterion to the naltrexone doses was far more site of insertion to the mid-scapular region where the access port problematic because the drug has multiple therapeutic applica- exited. A polyvinylchloride cap and protective metal cap were tions with widely differing Cmax values. The Cmax quoted for two used to cover the opening of the access port when it was not in tablets containing naltrexone 8 mg + bupropion 90 mg for the use. Catheters were filled with sterile fluid (heparinised saline treatment of obesity is 1.4 ng/mL (Contrave; US Product Label 30 iU/mL) immediately post-surgery and after every experimen- (https://dailymed.nlm.nih.gov/dailymed/index.cfm)), a naltrex- tal session to maintain catheter patency. Catheter patency was one 300 mg depot injection used to treat alcohol dependence confirmed daily by drawing back to observe freely flowing yielded a Cmax of 2.64 ng/mL (range 0.57–11.2 ng/mL) (Galloway blood in the catheter line. If catheter patency failed, as detected et al., 2005), naltrexone 50 mg tablets to treat alcohol depend- by visible evidence of leakage, by occlusion of the catheter, by ence yielded a Cmax of 8.55 ng/mL (Adepend; European Summary lack of blood drawback, or by failure of rats to show immediate of Product Characteristics (https://www.medicines.org.uk/ sedation upon i.v. injection of propofol (1.625 mg/kg i.v.), the emc/)), and naltrexone 100 mg orally yielded a Cmax of 76.3±44.8 rat was culled by a UK Home Office Schedule 1 procedure ng/mL (Ferrari et al., 1998). In view of the very wide range of

(exposures to rising carbon dioxide (CO2) with death confirmed clinically effective Cmax values, it was decided to evaluate nal- by cervical dislocation). trexone in the intravenous self-administration experiment at After 6–7 days recovery from surgery, rats were trained to identical doses to samidorphan. self-administer heroin (50 µg/kg/inj) in daily two-hour sessions When stable responding was achieved on FR5 schedule (see starting on a FR3 schedule of reinforcement. Sessions were initi- section on Testing criteria below), the break-point for responding ated by a non-contingent infusion of heroin (50 µg/kg/inj), the was determined in a single four-hour PR session using a logarith- house light was illuminated and the left lever was active. The mic PR schedule of reinforcement (Roberts et al., 1989). After drug was delivered in a volume of 0.5 mL/kg over a period of 4–5 two hours in the testing boxes, if a period of 30 min elapsed with- s depending on the weight of the rat. There was a 30-second time- out the rat receiving an infusion, the session was terminated. The out after each drug infusion, during which time the house light PR testing of rats on heroin (15 µg/kg/inj) and saline was per- was extinguished and lever-pressing had no programmed conse- formed exactly as described above in a single four-hour session quences. When rats displayed robust FR3 responding for heroin at the end ‘acquisition’ and ‘extinction’ phases of the experiment, (>8 infusions/session), the response requirement was increased respectively. to FR5. The heroin acquisition dose was 15 µg/kg/inj and the Since the rats were generally tested on more than one dose FR5 schedule of drug reinforcement were selected based on pre- of samidorphan, naltrexone and heroin, if a dose of the test vious experience using heroin and other opiate reinforcers (Heal substance was reinforcing on a FR5 schedule (⩾6 injections/ and Smith, 2017; Heal et al., 2018). Rats were allowed to self- session; mean of the last three test sessions), saline was substi- administer a maximum of 20 injections per two-hour session. tuted for three sessions to avoid engendering conditioned The Positive Reinforcement criterion was a mean ⩾12 injec- responding. tions/session over three consecutive sessions. After acquisition of heroin self-administration, saline (0.5 Testing criteria. Rats were given a minimum of five test sessions mL/kg i.v.) was substituted for the drug on a FR5 schedule until for each dose of heroin, saline, samidorphan and naltrexone. 4 Journal of Psychopharmacology 00(0)

Figure 1. Self-administration of heroin, samidorphan and naltrexone on a fixed ratio (FR)-5 schedule of drug reinforcement. Self-administration of various doses of heroin, samidorphan or naltrexone by rats on a FR5 schedule of reinforcement. Data are mean (±standard error of the mean (SEM)) number of inj/session for either the last three consecutive sessions if responding was stable or 10 sessions if it was not (see section on Testing criteria). Heroin (15 µg/kg/inj) and saline (0.5 mL/kg/inj) n=31 rats/group, all other treatments n=7-–8 rats/group. **p<0.001 and ***p<0.001 versus saline, #p<0.05 and ###p<0.001 versus heroin.

The acceptance criterion for ‘Acquisition’ of heroin self- rats. Samidorphan was dissolved in 0.9% (w/v) sterile saline for administration was three consecutive sessions where the mean i.v. injection using a dose volume of 1 mL/kg. Seven groups of six number of infusions was ⩾12. The acceptance criterion for rats were injected with samidorphan (0.003, 0.01, 0.03, 0.1, 0.3 ‘Extinction’ on saline was three consecutive sessions where the and 1.0 mg/kg) or vehicle via a tail vein. Drug or vehicle was mean number of infusions was ⩽6. injected into one tail vein and blood samples were withdrawn from The criterion for ‘Positive reinforcement’ with samidorphan, a different vein to avoid cross-contamination. Blood samples naltrexone or heroin in the test sessions was three consecutive ses- (~800 µL) were withdrawn at 5.0, 30, 60 and 120 min into K3- sions where the mean number of infusions was >6. The criterion EDTA tubes. In each group of six rats, three were used for the 5.0 for ‘Non-reinforcement’ in test sessions was three consecutive and 60 min blood samples and the other three rats were used for the sessions where the mean number of infusions was ⩽6. The defini- 30 and 120 min time-points. Samples were centrifuged to produce tion of ‘stable responding’ was when the number of injections/ a single aliquot of plasma (~400 μL) for each rat at each time- session did not vary by more than ±25% of the mean of the three point. The harvested plasma was transferred immediately into previous sessions and where there was no obvious increasing or polypropylene tubes. The samples were stored frozen at −60 to decreasing trend in self-administration, or three consecutive ses- −90°C until they were analysed. Samidorphan was quantified sions where the number of infusions was ⩾12, or three consecu- using a validated liquid chromatography with tandem mass spec- tive sessions where the number of infusions was ⩽6. If stable trometry (LC-MS/MS) assay. responding was not observed in a rat for any dose of samidorphan, The concentrations of samidorphan in the blood samples naltrexone or heroin, the mean intake over 10 sessions was taken. taken at the 5.0 min time-point were used to construct a standard curve. Plasma concentrations of samidorphan for the unit-doses Statistical analysis tested in the intravenous self-administration experiment were calculated from this standard curve. The statistical methods assumed that data were normally distrib- uted data with equal variance in the groups. An angular transfor- mation (/ys= in−1 x 20) was used for the number of injections Results and a log transformation was used for the PR break-points. Analysis was by mixed linear model with treatment as a fixed Self-administration of samidorphan, factor and rat as a random factor. Each test compound was com- naltrexone and heroin on a FR5 schedule pared to saline and to the heroin by separate Dunnett’s tests. The self-administration of heroin was compared to saline extinction After exclusions for catheter failures, losses through ill-health, by the multiple t test. Each dose of samidorphan and naltrexone and failure to meet the heroin acquisition and saline extinction was compared to heroin reinstatement doses and to each other by criteria, 31 out of 48 rats were included in the main self-adminis- tration study. The heroin acquisition dose (15 µg/kg/inj) main- the multiple t test. Results are presented as the mean±standard error of the mean (SEM) for n=7–8 rats for each dose of samidor- tained self-administration on the FR5 schedule of reinforcement phan, naltrexone or heroin (combined heroin acquisition group: at 18.8±0.4 inj/session (combined groups, n=31) that was sig- n=31; combined saline extinction group: n=31). The level of sta- nificantly greater (p<0.001) than saline (4.3±0.2 inj/session (combined groups, n=31)) (Figure 1). tistical significance was p<0.050. The other doses of heroin (7.5 and 25 µg/kg/inj) also met cri- teria for positive reinforcers (>6 inj/session) and statistically Determination of the plasma concentrations signficant (p<0.001) as greater than saline (Figure 1). Group of samidorphan mean values for heroin were 17.5±1.6 inj/session for 7.5 µg/kg/ inj, and 15.4±2.4 inj/session for 25 µg/kg/inj. The cumulative Plasma exposure to samidorphan was determined after intravenous intake of heroin (7.5, 15 and 25 µg/kg/inj) increased in a dose- dosing of compound to satellite groups of male, Sprague-Dawley dependent manner (Table 1). Smith et al. 5

The groups of rats used to evaluate the positive reinforcing via the infrared cameras and also when the rats were removed effects of samidorphan, naltrexone and heroin were well matched from the boxes for catheter flushing and daily feeding. No abnor- in terms of their responding to the acquisition dose of heroin (0.015 mal behavioural changes were observed in the rats that were mg/kg/inj) (arithmetic mean number of injections/session±SEM: receiving samidorphan (data not shown). samidorphan=16.8±0.9 (n=11); naltrexone=18.2±0.8 (n=11); Naltrexone (13.6, 40.8 and 68 µg/kg/inj) did not meet the heroin=18.5±0.7 (n=9)) and saline (arithmetic mean number of criteria required of a positive reinforcer at any of the doses injections/session ±SEM: samidorphan=4.4±0.3 (n=11); naltrex- tested (Figure 1). However, the lowest dose of naltrexone (13.6 one=4.3±0.4 (n=11); heroin=4.3±0.5 (n=9)). µg/kg/inj) maintained 8.1±2.3 inj/session (>6 inj/session) that The two lowest doses of samidorphan (13.6 or 40.8 µg/kg/inj) almost reached the level of statistical significance versus saline did not meet the criteria required of a positive reinforcer on a FR5 (p=0.053). The number of inj/session of all doses of naltrexone schedule of reinforcement in heroin-maintained rats (Figure 1). tested was significantly (p<0.001) lower than the number of The highest dose of samidorphan (68 µg/kg/inj) supported a inj/session of all doses of heroin (7.5, 15 and 25 µg/kg/inj) group mean of 9.2±2.1 inj/session that was significantly (Figure 1). The cumulative intake of naltrexone (13.6, 40.8 and (p<0.01) greater than saline (4.3±0.2 inj/session; Figure 1). The 68 µg/kg/inj) increased in a dose-dependent manner as a func- numbers of inj/session of all doses of samidorphan were signifi- tion of dose (Table 1). The behaviour of the rats was continu- cantly (p<0.001) lower than the number of inj/session of all ously monitored during the test sessions via the infrared doses of heroin (7.5, 15 and 25 µg/kg/inj) (Figure 1). The cumu- cameras and also when the rats were removed from the boxes lative intake of samidorphan (13.6, 40.8 and 68 µg/kg/inj) for catheter flushing and daily feeding. No abnormal behav- increased in a dose-dependent manner (Table 1). The behaviour ioural changes were observed in the rats that were receiving of the rats was continuously monitored during the test sessions naltrexone (data not shown). The number of inj/session of all doses of samidorphan was Table 1. Cumulative intake of samidorphan, naltrexone and heroin in not significantly different from the number of inj/session of all the fixed ratio (FR)5 self-administration experiment. doses of naltrexone (Table 2).

Drug and dose Cumulative drug intake (mg/kg i.v.) Drug injection intervals for self- Samidorphan administration of samidorphan, naltrexone 13.6 µg/kg/inj 0.072±0.018 and heroin on a fixed ratio (FR)5 schedule 40.8 µg/kg/inj 0.218±0.040a The mean interval between self-administered injections of heroin 68 µg/kg/inj 0.550±0.108a,b (15 µg/kg/inj) during the acquisition phase on the FR5 schedule Naltrexone was 340.2±24.1 s (n=31), which was significantly (p<0.001) 13.6 µg/kg/inj 0.108±0.028 shorter than the interval between self-administered injections of c 40.8 µg/kg/inj 0.222±0.059 saline (1207.1±82.7 s (n=31)) (Table 3). This pattern was a,d 68 µg/kg/inj 0.475±0.074 repeated for the other doses of heroin (75 and 25 µg/kg/inj) that Heroin were tested on the FR5 schedule (Table 3). Furthermore, there 7.5 µg/kg/inj 0.122±0.016 were no significant differences between the inter-injection inter- 15 µg/kg/inj 0.290±0.011a vals of these doses of heroin and the dose used for acquisition of 25 µg/kg/inj 0.359±0.047a self-administration (Table 3). The inter-injection intervals for all of the doses of samidor- inj = injection; i.v. = intravenous. phan (13.6, 40.8 and 68 µg/kg/inj) were significantly (p<0.001) aSignificantly greater than lowest dose p<0.001; bsignificantly greater than middle dose p<0.001; csignificantly greater than lowest dose p<0.05; dsignifi- longer than the interval between self-administered injections of cantly greater than middle dose p<0.01. heroin (15 µg/kg/inj) and not significantly different from the interval for self-administered injections of saline (Table 3).

Table 2. Comparison of the number of self-administered injections of the tested doses of samidorphan and naltrexone.

Drug Infusions/two-hour session Versus Versus Versus NTX 13.6 NTX 40.8 NTX 68

(µg/kg/injection) Mean±SEM % p % p % p

Naltrexone (13.6) 8.1±2.3 Naltrexone (40.8) 5.4±2.3 Naltrexone (68) 6.1±1.3 Samidorphan (13.6) 5.1±1.6 63.2 0.171 95.2 0.895 84.3 0.645 Samidorphan (40.8) 4.9±1.1 61.3 0.148 92.2 0.831 81.6 0.589 Samidorphan (68) 9.2±2.1 114.4 0.600 172.2 0.064 152.5 0.144

NTX: naltrexone; SEM: standard error of the mean. Comparisons are by the multiple t test after fitting a mixed linear model. 6 Journal of Psychopharmacology 00(0)

Table 3. Comparison between of the intervals between self- administered injections of samidorphan, naltrexone, heroin and saline.

Treatment n Mean inter-injection interval±SEM (µg/kg/injection) (s)

Saline ‘extinction’ 31 1207.1±82.7 Heroin (15) ‘acquisition’ 31 340.2±24.1a Heroin (7.5) 8 340.5±63.1a Heroin (25) 7 438.1±95.8a Samidorphan (13.6) 7 1447.8±401.0b Samidorphan (40.8) 7 1328.4±174.3b Samidorphan (68) 8 1062.1±272.0b Naltrexone (13.6) 7 946.7±197.9b Naltrexone (40.8) 8 1171.0±231.4b Naltrexone (68) 7 1013.8±194.4b

SEM: standard error of the mean. Means are back-transformed and adjusted for differences between animals. SEMs are calculated from the residuals of the statistical model. The inter-injection in- terval for each dose of each compound was compared to saline extinction and to heroin acquisition by separate Dunnett’s tests. Heroin acquisition was compared Figure 2. Relative reinforcing efficacy of samidorphan and naltrexone to saline extinction by the multiple t test. in comparison to heroin and saline. aSignificantly different from saline, p<0.001; bsignificantly different from heroin Data are mean±standard error of the mean (SEM) break-points for drug reinforce- (0.015 mg/kg/inj), p<0.001. ment assessed on a progressive ratio (PR) schedule. Heroin (15 µg/kg/inj) and saline (0.5 mL/kg/inj) n=31 rats/group, all other treatments n=7–8 rats/group. *p<0.05 and ***p<0.001 versus saline, ###p<0.001 versus heroin. The inter-injection intervals for naltrexone (13.6, 40.8 and 68 µg/kg/inj) were also significantly (p<0.001) longer than the inter- injection interval for heroin (15 µg/kg/inj) and not significantly Plasma concentrations of samidorphan different from the interval for injections of saline (Table 3). Tail-vein blood samples were taken from rats after they had been injected with samidorphan (0.003, 0.01, 0.03, 0.1, 0.3 and 1.0 mg/kg) or vehicle (six rats/group and three rats/time-point). Assessment of the relative reinforcing Concentrations of samidorphan in the five-minute samples were efficacy of samidorphan, naltrexone and determined by LC-MS/MS. The concentration of samidorphan heroin by PR/break-point determination increased linearly as a function of dose (y=298x+0.8473; r2=0.9956). When the relative reinforcing efficacy of heroin (7.5, 15 and 25 Samidorphan was tested in the self-administration model at µg/kg/inj), samidorphan (13.6, 40.8 and 68 µg/kg/inj), naltrexone i.v. unit doses of 13.6, 40.8 and 68 µg/kg/inj and the sessions (13.6, 40.8 and 68 µg/kg/inj) and saline (0.5 ml/kg/inj i.v.) were were initiated by administration of a non-contingent injection of determined by PR/breakpoint analysis, the break-points (lever- samidorphan 13.6, 40.8 or 68 µg/kg i.v. By reference to the sami- presses/inj) for responding to heroin (41.5±7.6 for 7.5 µg/kg/inj dorphan dose-concentration graph, single intravenous injections (n=8); 60.2±8.1 for 15 µg/kg/inj (n=31), and 50.6±20.9 for 25 of samidorphan 13.6, 40.8 and 68 µg/kg i.v. were calculated to µg/kg/inj (n=7)) were all significantly (p<0.001) greater than produce plasma concentrations of 4.90 ηg/mL, 13.00 ηg/mL and saline (10.4±0.8 (n=31)) (Figure 2). 21.11 g/mL, respectively. The break-points for the two lower doses of samidorphan η (13.6 and 40.8 µg/kg/inj) were 13.8±2.5 lever-presses/inj (n=7) and 13.3±1.2 lever-presses/inj (n=7), respectively, and they were Discussion not significantly different from the break-point for saline (Figure 2). The break-point for samidorphan (68 µg/kg/inj) of 17.9±3.3 The ability of a drug to maintain self-administration above saline lever-presses/inj (n=8) was significantly (p<0.05) higher than control levels in animals is considered to be an indication that the saline (Figure 2). The break-points for all doses of samidorphan compound may have reinforcing properties in humans (Brady & were significantly (p<0.001) lower than those for every dose of Fishman, 1985). Rats are well established as a species for use in heroin (Figure 2). self-administration experiments to evaluate the positive reinforc- The break-points (lever-presses/inj) for naltrexone of ing effects of drug-candidates in clinical development (Hudzik 17.2±3.8 (n=7), 15.0±3.6 (n=8) and 16.0±4.1 (n=7) for 13.6, et al., 1996; O’Connor et al., 2011; Ewan & Martin, 2013; 40.8 and 68 µg/kg/inj, respectively, were not significantly differ- Huskinson et al., 2014; Heal et al., 2018) and the use of this spe- ent from the break-point for self-administration of saline. The cies in non-clinical abuse/dependence evaluations is also recom- break-points for these three doses of naltrexone were all signifi- mended by CHMP/EMA (2006) and CDER/FDA (2017). In cantly (p<0.001) lower than those for all doses of heroin. support of this argument, it has been reported that an impressive When the break-points for the 13.6, 40.8 and 68 µg/kg/inj array of substances that are recreationally abused by humans doses of samidorphan and naltrexone were compared, there were serve as positive reinforcers in rats. Examples include no differences between the two drugs (Figure 2). (Blakesley et al., 1972; Kumar et al., 1977), nicotine (Cox et al., Smith et al. 7

1984), dopaminergic stimulants (Caine & Koob, 1994; Hemby consistency of the relative reinforcing effect of heroin across et al., 1999; McMillan et al., 2004; Huskinson et al., 2014), studies. Moreover, the break-points for heroin are also similar to entactogens (psychoactive, 3,4-methylenedioxy-methampheta- those obtained for other highly abused C-II drugs including those mine [MDMA-like drugs]) (Braida & Sala, 2002; Heal et al., with different pharmacological mechanisms, i.e. cocaine, meth- 2018), and some psychedelics, e.g. N-methyl-D-aspartate ylphenidate and (Heal & Smith, 2017). In PR exper- (NMDA) antagonists (Marquis et al., 1989; Rocha et al., 1996; iments, the break-point values for the two lowest samidorphan van der Kam et al., 2007) and κ-opioid agonists (Heal et al., 2018). doses were not significantly different from saline. However, the In this study, all doses of heroin maintained robust and reliable break-point for the highest dose of samidorphan, which was self- intravenous self-administration in all rats when tested on a FR5 administered on the FR5 schedule, was significantly higher than schedule of reinforcement, thereby demonstrating the sensitivity saline. The break-points for all doses of samidorphan were sig- of this procedure to the positive reinforcing effects of a drug with nificantly lower than the acquisition dose of heroin and the other well-established abuse liability in humans. When saline was sub- two heroin doses that were evaluated. None of the break-point stituted for heroin, self-administration decreased markedly, con- values for naltrexone were significantly different from saline and firming the selective, positive reinforcing effect of heroin in the they were all significantly lower than those obtained for all doses rats. The signal:noise ratio for heroin versus saline was ~4.4× of heroin. When the break-point values for samidorphan were giving a wide window to evaluate the possible reinforcing effects compared with those for naltrexone, there were no statistically of samidorphan and naltrexone. The reinforcing potential of sami- significant differences between them. dorphan and naltrexone were investigated at identical doses that When assessing the translational validity of the samidorphan were selected because they yielded clinically relevant plasma con- findings, CDER/FDA (2017) have advised that doses of drug- centrations in rats (data on file, Alkermes, Inc.). candidates for evaluation in intravenous self-administration All three doses of heroin maintained self-administration at experiments should be selected to produce plasma drug concen- levels significantly higher than saline and these doses resulted in trations that are fractions of those observed in man at clinically the inverted U-shaped dose-response function that is typical of effective doses. Samidorphan at doses of 10 mg and 20 mg abol- positive reinforcers. These results are consistent with our previ- ished remifentanil-induced miosis and drug-liking in recreational ous findings using heroin as a positive reinforcer in self-adminis- drug users and these doses yielded samidorphan Cmax concentra- tration experiments with rats (Oakley et al., 2015; Heal & Smith, tions of 19.3±4.9 ηg/mL and 47.3±10.1 ηg/mL, respectively 2017) and those of other research groups (e.g. Martin et al., 2000; (Shram et al., 2015). The unit doses of samidorphan tested by Solinas et al., 2004). intravenous self-administration in heroin-trained rats, i.e. 13.6, Samidorphan and naltrexone had similar profiles on a FR5 40.8 and 68 µg/kg/inj, produced plasma concentrations that were schedule of reinforcement in heroin-trained rats, with one dose of 25%, 67% and 109% of the Cmax produced by the 10 mg dose of the former meeting the criteria for a positive reinforcer and one samidorphan, and 10%, 27% and 45% of the Cmax produced by dose of the latter having a statistical trend (p=0.053) for a positive the 20 mg dose. The rats self-administered a mean of 9.2±2.1 inj/ effect. The number of injections taken of samidorphan and naltrex- session of the 68 µg/kg/inj dose of samidorphan plus the non- one on a FR5 schedule were not significantly different from one contingent infusion of this dose used to start each test session, i.e. another. All doses of samidorphan and naltrexone were taken in sig- average intake=10.2 inj/session. Thus, it can be estimated that the nificantly smaller numbers on the FR5 schedule than every dose of rats were exposed to ~11.1× (10.2×0.45) and ~4.6×(10.2×1.09) heroin. The mean inter-injection intervals were significantly shorter the clinical Cmax in subjects receiving 10 mg and 20 mg of sami- for all three doses of heroin than saline. In contrast, the mean inter- dorphan, respectively. Samidorphan was therefore tested across injection intervals for samidorphan and naltrexone were not signifi- the full dose range that is relevant for predicting its possible rein- cantly different from that of saline. This is an important finding forcing effects in humans. because it indicates that the rats were not loading themselves with Naltrexone attenuates self-administration of heroin in humans either drug at the start of the test sessions and then resting for the (Altman et al., 1976) and in rats (Ettenberg et al., 1982), and mor- remainder of the test session because they were satiated. phine in rhesus monkeys (Harrigan et al., 1978). Although, it has Taken together, there are weak signals to indicate that sami- been found that (+)naltrexone is an antagonist of toll-like recep- dorphan and possibly naltrexone may serve as weak reinforcers tor-4 (TLR4), Tanda et al. (2016) have compellingly reported that in heroin-maintained rats over a very restricted dose range. this pharmacological action does not contribute to the attenuation Although it may have been investigated at some time in the past, of heroin self-administration in rats. a PubMed search revealed no published reports of the reinforcing Samidorphan is also a μ-opioid receptor antagonist (Wentland effect of naltrexone or being investigated by substitu- et al., 2005), and it blocks the analgesic effects of morphine in tion for heroin in an intravenous self-administration experiment. rats (Todtenkopf et al., 2008; Cunningham et al., 2014), and pre- The relative reinforcing effect of the three doses of samidor- vents pupil miosis and drug-liking produced by remifentanil in phan and naltrexone were determined by PR/break-point analysis recreational drug users (Shram et al., 2015). and compared against all doses of heroin and saline. The break- The results from these self-administration experiments point (60.2±8.1 lever-presses/inj) for the acquisition dose of showed that heroin served as a powerful reinforcer in rats by (a) heroin (15 µg/kg/injection i.v.) was significantly greater than the number of doses that served as a reinforcer, (b) the number of saline (10.4±0.8 lever-presses/inj) giving a signal:noise ratio of injections that the rats took in FR5 test sessions, and (c) the high ~6:1. In addition, the other doses of heroin also yielded break- PR break-points for reinforcement. In heroin-maintained rats, points that were statistically greater than saline. The break-points samidorphan elicited a weak signal as positive reinforcer at one for heroin are in close agreement with previously reported results dose. An equivocal reinforcement signal was observed for nal- (Oakley et al., 2015; Heal & Smith, 2017) showing the trexone. Break-points for samidorphan were not significantly 8 Journal of Psychopharmacology 00(0) different from those of naltrexone, which has no abuse liability, Cox BM, Goldstein A and Nelson WT (1984) Nicotine self-administra- and significantly lower than heroin. Overall the profiles of sami- tion in rats. Br J Pharmacol 83: 49–55. dorphan and naltrexone, which has no abuse liability, were simi- Cunningham JI, Dean RL, Todtenkopf MS, et al. (2014) Samidorphan atten- lar in this model. uates drug-induced increases in extracellular dopamine concentrations and drug self-administration in rats. Presented at the European College of Neuropsychopharmacology Annual Meeting, 18–21 October 2014, Declaration of conflicting interests Berlin, Germany. The author(s) declared the following potential conflicts of interest with Ettenberg A, Pettit HO, Bloom FE, et al. (1982) Heroin and cocaine intra- respect to the research, authorship, and/or publication of this article: venous self-administration in rats: Mediation by separate neural sys- Sharon L Smith is an employee of RenaSci Ltd. David J Heal is an tems. Psychopharmacology 78: 204–209. employee and a shareholder of RenaSci Ltd. Reginald L Dean and Mark Ewan EE and Martin TJ (2013) Analgesics as reinforcers with chronic S Todtenkopf are employees of Alkermes Inc. The experimental study pain: Evidence from operant studies. Neurosci Lett 557: 60–64. was funded by Alkermes, Inc. Vivitrol (naltrexone as an extended-release Ferrari A, Bertolotti M, Dell’Utri A, et al. (1998) Serum time-course of injectable suspension) is marketed by Alkermes Inc. as a once-monthly naltrexone and 6β-naltrexol levels during long-term treatment in medication for the treatment of alcohol dependence, and prevention of drug addicts. 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