In a Rat Model of Opioid Maintenance, the G Protein–Biased Mu Opioid Receptor Agonist TRV130 Decreases Relapse to Oxycodone Seeking and Taking and Prevents Oxycodone-Induced Brain Hypoxia

Journal Pre-proof In a rat model of opioid maintenance, the G-protein-biased MOR agonist TRV130 decreases relapse to oxycodone seeking and taking, and prevents oxycodoneinduced brain hypoxia Jennifer M. Bossert, Eugene Kiyatkin, Hannah Korah, Jennifer K. Hoots, Anum Afzal, David Perekopskiy, Shruthi Thomas, Ida Fredriksson, Bruce E. Blough, S. Stevens Negus, David H. Epstein, Yavin Shaham PII:

S0006-3223(20)30103-7

DOI:

https://doi.org/10.1016/j.biopsych.2020.02.014

Reference:

BPS 14134

To appear in:

Biological Psychiatry

Received Date: 11 June 2019 Revised Date:

13 February 2020

Accepted Date: 13 February 2020

Please cite this article as: Bossert J.M., Kiyatkin E., Korah H., Hoots J.K., Afzal A., Perekopskiy D., Thomas S., Fredriksson I., Blough B.E., Negus S.S., Epstein D.H. & Shaham Y., In a rat model of opioid maintenance, the G-protein-biased MOR agonist TRV130 decreases relapse to oxycodone seeking and taking, and prevents oxycodone-induced brain hypoxia, Biological Psychiatry (2020), doi: https:// doi.org/10.1016/j.biopsych.2020.02.014. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc on behalf of Society of Biological Psychiatry.

2-13-20 Biological Psychiatry (BPS-D-19-00900R2) Main text

In a rat model of opioid maintenance, the G-protein-biased MOR agonist TRV130 decreases relapse to oxycodone seeking and taking, and prevents oxycodone-induced brain hypoxia

Jennifer M. Bossert

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, Eugene Kiyatkin , Hannah Korah , Jennifer K. Hoots , Anum Afzal , David

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Perekopskiy , Shruthi Thomas , Ida Fredriksson , Bruce E. Blough , S. Stevens Negus , 4

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David H. Epstein , and Yavin Shaham 1

Behavioral Neuroscience Branch, IRP/NIDA/NIH, Baltimore, MD, U.S.A. Center for Drug Discovery, Research Triangle Institute, Research Triangle Park, NC, U.S.A. 3 Dept. of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, U.S.A. 4 Clinical Pharmacology and Therapeutics Research Branch, IRP/NIDA/NIH, Baltimore, MD, U.S.A. 2


Correspondence: Jennifer M. Bossert ([email protected]) Short title: opioid agonist maintenance therapy with TRV130 Text information Abstract: 250 words Introduction: 699 words Discussion: 1269 words Total text: 3998 words Figures: 5 References: 68 Supplementary text: 3336 words Supplementary figures: Figures S1-S4 Supplemental tables: Table S1 Key words: buprenorphine, context, drug cues, drug self-administration, extinction, G-protein-biased MOR agonists, oxycodone, reacquisition, reinstatement, relapse, sex differences, TRV130 Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper are available upon request from the corresponding author. Author’s contributions: JMB, HK, JKH, IF, AA, DP, ST, and EK carried out the experiments; JMB, HK, JKH, EK, and YS performed data analysis. JMB and YS designed the study and wrote the manuscript with DE and SSN. BEB provided TRV130 and SSN provided critical input on pharmacological aspects of the study. All authors critically reviewed the content and approved the final version before submission.

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Abstract Background: Maintenance treatment with opioid agonists (buprenorphine, methadone) is effective for opioid addiction but does not eliminate opioid use in all patients. Here, we modeled maintenance treatment in rats that self-administered the prescription opioid oxycodone. The maintenance medication was either buprenorphine or the G-protein-biased mu opioid receptor (MOR) agonist TRV130. We then tested prevention of oxycodone seeking and taking during abstinence using a modified context-inducedreinstatement procedure, a rat relapse model. Methods: We trained rats to self-administer oxycodone (6-h/d, 14-d) in context A; infusions were paired with discrete tone-light cues. We then implanted osmotic pumps containing buprenorphine or TRV130 (0, 3, 6, or 9 mg/kg/d) and performed three consecutive tests: lever pressing reinforced by oxycodone-associated discrete cues in non-drug context B (extinction responding), context-induced reinstatement of oxycodone seeking in context A, and reacquisition of oxycodone self-administration in context A. We also tested whether TRV130 maintenance would protect against acute oxycodone-induced decreases in nucleus accumbens oxygen levels. Results: In males, buprenorphine and TRV130 decreased extinction responding and reacquisition of oxycodone self-administration but had a weaker (non-significant) effect on context-induced reinstatement. In females, buprenorphine decreased responding in all three tests while TRV130 only decreased extinction responding. In both sexes, TRV130 prevented acute brain hypoxia induced by moderate doses of oxycodone. Conclusion: TRV130 decreased oxycodone seeking and taking during abstinence in a partly sexspecific manner and prevented acute oxycodone-induced brain hypoxia. We propose that G-proteinbiased MOR agonists, currently in development as analgesics, should be considered as relapse prevention maintenance treatment for opioid addiction.

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Introduction Over the two past decades, there has been a large increase in the abuse of prescription and illegal opioids; this increase coincides with increases in opioid-related deaths (1, 2). This so-called ‘opioid crisis’ persists despite the availability of FDA-approved treatments (methadone, buprenorphine, and naltrexone) (3). A critical challenge is the occurrence of lapses or relapses in treated patients, especially given that lapses carries a risk of overdose (4). Maintenance treatment with the mu opioid receptor (MOR) agonist methadone (5) or the partial MOR agonist/kappa opioid receptor (KOR) antagonist buprenorphine (6) causes cessation of other opioid use for some, but not all, patients (7, 8). For methadone, success is limited by prototypical MOR agonist side effects like respiratory suppression, sedation, and constipation (9), effects on appetite and weight, sweating, and sexual dysfunction (10, 11). These side effects, particularly respiratory suppression, can hinder maintaining patients on high enough doses to prevent lapses to use of other opioids (12, 13). Buprenorphine is less liable to induce respiratory depression on its own but can do so when taken with benzodiazepines or alcohol (14, 15). Additionally, as a partial MOR agonist, buprenorphine’s maximal efficacy is lower than that of methadone (8) and in some patients, buprenorphine can precipitate withdrawal in the presence of higher efficacy MOR agonists like heroin (16). Therefore, there is an unmet need to advance MOR medications that may overcome limitations of methadone and buprenorphine. Here, we used a rat model of opioid maintenance (17-19) combined with a modified version of our ABA renewal model of context-induced reinstatement (20, 21) to compare the efficacy of buprenorphine with that of the G-protein-biased MOR agonist TRV130 (22); Renewal refers to the resumption of learned responses in the original training context after extinction in a different context (23). The rationale for MOR biased agonists is that they can preferentially produce relatively high efficacy activation of the G-proteincoupled pathway over the β-arrestin pathway (24), producing more selective behavioral and physiological effects. In rodent studies, TRV130 and other G-protein-biased MOR agonists show less propensity toward inducing antinociceptive tolerance than traditional opioids (24). In some studies, they also produce less respiratory depression and constipation (22, 25), although these findings are less consistent (26-28). In human studies, TRV130 decreases moderate to severe acute pain and, compared to morphine, TRV130 trended towards having a higher therapeutic index (analgesia versus side effects related to

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respiratory depression and nausea/vomiting) (29-32). Overall, TRV130 may have utility as a maintenance medication because it may be safer than methadone while having higher efficacy than buprenorphine. To test TRV130 as a maintenance medication, we first established the predictive validity of our rat model using buprenorphine. Rats previously trained to self-administer the prescription opioid oxycodone in a drug-associated context A were maintained on buprenorphine with Alzet osmotic minipumps (19). Starting 3-4 days after maintenance treatment, we assessed drug seeking and taking during abstinence using three measures commonly employed in animal models of relapse: (1) extinction responding in a non-drug context B where responding produces drug-paired discrete cues but not drug, (2) contextinduced reinstatement in context A after extinction of operant responding reinforced by the drug-paired discrete cues in context B, and (3) reacquisition of drug self-administration in context A (33-36). These three measures model different aspects of human craving and relapse: (1) craving expressed as attraction to drug cues where drugs are not available (37), (2) craving and relapse induced by the drug context after successful extinction of cue reactivity in nondrug settings (38), and (3) craving and continued use induced by reexposure to the drug (39). After validation studies with buprenorphine, we tested the effect of the long-acting KOR antagonist nor-binaltorphimine (nor-BNI) (40) to determine whether buprenorphine’s effects on the three measures were attributable to antagonism at KORs (41). We then tested the effect of TRV130 maintenance. We first tested buprenorphine, nor-BNI, and TRV130 in consecutive experiments (Exp. 1A, 2A, 3A) in male rats. Next, we tested the generality of the positive (buprenorphine and TRV130) and negative (nor-BNI) results to female rats (Exp. 1B, 2B, 3C). Finally, we used oxygen sensors implanted into nucleus accumbens (NAc) to determine whether TRV130 delivery would prevent brain hypoxia induced by intravenous oxycodone (42) in both sexes (Exp. 4).

Materials and Methods Exp. 1-3: Oxycodone self-administration, extinction responding, context-induced reinstatement, and reacquisition Subjects

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We used male (n=138) and female (n=112) Sprague-Dawley rats (Charles River) weighing 250-350 g (males) or 175-225 g (females) before surgery. We maintained the rats under a reverse 12:12 h light/dark cycle (8:00 A.M lights off) with food and water freely available. We housed two rats/cage prior to surgery and individually after surgery. We performed the experiments in accordance with NIH Guide for the Care and Use of Laboratory Animals (8th edition), under protocols approved by NIDA-IRP ACUC. We excluded 7 rats due to health problems or death during minipump surgery. Surgery, Drugs, and Apparatus: see SOM. Oxycodone self-administration, extinction, context-induced reinstatement, and reacquisition The experiments include six phases: (1) oxycodone self-administration training (context A, 14 days, 6 h/day), (2) minipump surgery or nor-BNI injection (1 day), (3) 3-4 days off after surgery or nor-BNI injection, (4) extinction test (context B, 7 days, 6 h/day), (5) context-induced reinstatement of oxycodone seeking test (contexts A and B, 2 days, 6 h/day), and (6) reacquisition of oxycodone self-administration test (context A, 1 day, 6 h). We modified our procedure from a previous study (20) to perform the three tests within the minipump active period (14 days). Timelines are shown in Fig. 1-3. Exp. 1: Buprenorphine We used 55 males (Exp. 1A) and 25 females (Exp. 1B). After training, we assigned male rats (matched for infusions/day during self-administration) to either vehicle or buprenorphine (3, 6, or 9 mg/kg/day; n=11-17/dose) and female rats to either vehicle or buprenorphine (9 mg/kg/day; n=1213/dose). Exp. 2: nor-BNI We used 27 males (Exp. 2A) and 28 females (Exp. 2B). After training, we assigned rats (matched for infusions/day) to either vehicle or nor-BNI (20 mg/kg, i.p.; males: n=13-14/dose; females: n=14/dose). Exp. 3: TRV130 We used 53 males (Exp. 3A) and 55 females (Exp. 3B). After training, we assigned rats (matched for infusions/day) to either vehicle or TRV130 (3, 6, or 9 mg/kg/day; males: n=13-14/dose females: n= 1314/dose). Statistical analyses: see SOM. Exp. 4: Effect of TRV130 on oxycodone-induced changes in NAc oxygen

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We used 8 males and 8 females. We determined the effect of vehicle (n=4/sex) or TRV130 (9 mg/kg/d; n=4/sex) on acute oxycodone-induced changes in NAc oxygen; we tested the rats on days 1314 of vehicle or TRV130 delivery (minipump ON). We also determined oxycodone’s effect on changes in NAc oxygen 1, 3, and 6 days after removal of the vehicle- or TRV130-containing minipumps (minipump OFF, withdrawal). On days 13-14 of minipump delivery, we tested two oxycodone doses (0.3 and 0.6 mg/kg, i.v., 3 injections/session; 120 min inter-injection-interval). After minipump removal (minipump OFF, withdrawal), we tested oxycodone (0.6 mg/kg, i.v., 2 injections/session) effect on days 1, 3, and 6. Subjects, Surgery, Electrochemical detection; Experimental procedures; Data analysis: see SOM.

Results Oxycodone self-administration (context A) In Exp. 1-3 we trained the rats to self-administer oxycodone at 0.1 mg/kg/infusion for the first 7 days, followed by 0.05 mg/kg/infusion for the next 7 days. Rats of both sexes demonstrated reliable oxycodone self-administration as indicated by an increase in the number of oxycodone infusions and active lever presses over days, and a compensatory increase in the number of infusions earned when we halved the dose (Fig. 1-3 and Table S1 for statistics). There were no significant sex differences in oxycodone selfadministration (Table S1). Effect of buprenorphine on extinction, context-induced reinstatement, and reacquisition In Exp. 1, we used buprenorphine to establish the predictive validity of our opioid maintenance model. We trained rats to self-administer oxycodone, then implanted vehicle- or buprenorphine-containing Alzet minipumps and performed three drug-seeking and drug-taking tests described below. We first tested males using 3 doses (3, 6, and 9 mg/kg/d, Exp. 1A) and then tested the generality of buprenorphine’s effect to females using the highest dose (Exp. 1B). Extinction responding (Fig. 1B) Buprenorphine decreased active lever presses reinforced by the discrete tone+light cue in context B (extinction responding) in both sexes. For males, the ANCOVA (covariate: inactive lever) with the withinsubjects factor Session (1-7) and the between-subjects factor Buprenorphine Dose (0, 3, 6, 9 mg/kg), showed significant effects of Buprenorphine Dose (F3,50=3.1, p=0.033) and Buprenorphine Dose x

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Session (F3,50=2.8, p=0.047). For females, the ANCOVA showed significant effects of Buprenorphine Dose (F1,22=4.3 p=0.049), Session (F6,132=2.4 p=0.033), and Buprenorphine Dose x Session (F6,132=2.2 p=0.043). Context-induced reinstatement (Fig. 1C) Buprenorphine decreased context-induced reinstatement in females, but not reliably in males. For males, the ANCOVA, which included the within-subjects factor Context and the between-subjects factor Buprenorphine Dose, showed a significant effect of Context (F1,50=15.5, p<0.001), but not Buprenorphine Dose or interaction. For females, the ANCOVA showed significant effects of Context (F1,22=14.1, p=0.001) and Buprenorphine Dose (F1,22=5.4, p=0.03); the interaction approached the significance criterion (F1,22=3.9, p=0.06). Reacquisition (Fig. 1D) Buprenorphine decreased reacquisition of oxycodone self-administration in both sexes. For males, the ANOVA, which included the within-subjects factor Session Hour (1-6) and the between-subjects factor Buprenorphine Dose, showed significant effects of Buprenorphine Dose (F3,38=10.7, p<0.001), Hour (F5,190=10.2, p<0.001), and Buprenorphine Dose x Hour (F15,190=2.3, p=0.005). The interaction reflected lower responding in the buprenorphine groups than in the vehicle group during hours 2-6. For females, the ANOVA showed significant effects of Buprenorphine Dose (F1,23=37.4, p<0.001) and Hour (F5,115=2.6, p=0.031), but no interaction. The results of Exp. 1 support the predictive validity of our opioid maintenance model in both sexes, with chronic buprenorphine decreasing extinction responding and reacquisition in males, and extinction responding, context-induced reinstatement, and reacquisition in females. [See SOM for analyses that include Sex as a factor. These analyses should be interpreted with caution because the male and female experiments were performed over a year apart.] Effect of nor-BNI on extinction, context-induced reinstatement, and reacquisition The goal of Exp. 2 was to determine whether buprenorphine’s effects in Exp. 1 were due to antagonism at KORs (41). We used the long-acting irreversible KOR antagonist nor-BNI (40). Extinction responding (Fig. 2B)

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nor-BNI had no effect on extinction responding in either sex. For males, the ANCOVA, which included the within-subject factor Session and the between-subjects factor nor-BNI Dose (0, 20 mg/kg), showed a significant effect of Session (F6,144=11.5, p<0.001), but not nor-BNI Dose or interaction. For females, the ANCOVA showed a significant effect of Session (F6,150=5.3, p<0.001), but not nor-BNI Dose or interaction. Context-induced reinstatement (Fig. 2C) nor-BNI had no effect on context-induced reinstatement in either sex. For males, the ANCOVA, which included the within-subjects factor Context and the between-subjects factor nor-BNI Dose showed a significant effect of Context (F1,24=7.4, p=0.012) but not nor-BNI Dose or interaction. For females, the ANCOVA showed a significant effect of Context (F1,25=10.1, p=0.004) but no effect of nor-BNI Dose or interaction. Reacquisition (Fig. 2D) nor-BNI had no effect on reacquisition of oxycodone self-administration in either sex. For males, the ANOVA showed significant effects of Hour (F5,110=3.7, p=0.004) and Hour x nor-BNI Dose (F5,110=2.6, p=0.031), but not nor-BNI Dose. The significant interaction reflects somewhat higher responding in the nor-BNI group during hours 1-2 and somewhat lower responding during hour 6. For females, the ANCOVA showed a significant effect of Hour (F5,105=3.0, p=0.014) but not nor-BNI Dose or interaction. The results of Exp. 2 indicate that buprenorphine’s effects on drug seeking and taking in Exp. 1 are mediated by its action on MORs, but not KORs. Effect of TRV130 on extinction, context-induced reinstatement, and reacquisition Extinction responding (Fig. 3B) TRV130 decreased extinction responding in both sexes. For males, the ANCOVA, which included the within-subjects factor Session and the between-subjects factor TRV130 Dose, showed significant effects of Session (F6,288=9.5, p<0.001) and Session x TRV130 Dose (F18,288=2.3, p=0.002), but not TRV130 Dose. For females, the ANCOVA showed significant effects of TRV130 Dose (F3,50=5.5, p=0.002), Session (F6,300=13.2, p<0.001), and interaction (F18,300=2.5, p=0.001). In both analyses, the significant interactions reflect the stronger effect of the two high doses of TRV130 in some of the extinction sessions. Context-induced reinstatement (Fig. 3C)

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TRV130 modestly decreased context-induced reinstatement in males but had no effect in females. For males, the ANCOVA, which includes the within-subjects factor Context and the between-subjects factor TRV130 Dose, showed significant effects of Context (F1,48=21.1, p<0.001) and TRV130 Dose (F3,48=3.4, p=0.026), but no interaction. Subsequent post-hoc one-way ANCOVAs on active lever presses in contexts A and B showed a significant effect of TRV130 Dose for context A (F3,52=4.0, p=0.013) but not context B. For females, the ANCOVA showed a significant effect of Context (F1,50=13.5, p<0.001) but not of TRV130 Dose or interaction. Reacquisition (Fig. 3D) TRV130 decreased reacquisition of oxycodone self-administration in males but not females. For males, the ANOVA, which included the within-subjects factor Hour and the between-subjects factor TRV130 Dose, showed significant effects of TRV130 Dose (F3,45=5.2, p=0.003) and Hour (F5,225=2.8, p=0.018) but no interaction. For females, the ANOVA showed a significant effect of Hour (F5,165=5.2, p<0.001) but not TRV130 Dose or interaction. The results of Exp. 3 demonstrate that in male rats TRV130 reliably decreased extinction responding and reacquisition while its effect on context-induced reinstatement was more modest. In contrast, TRV130 was less effective in female rats, decreasing extinction responding in context B, but not contextinduced reinstatement or reacquisition in context A. Effect of TRV130 on oxycodone-induced changes in NAc oxygen In Exp. 4, we used males and females to determine whether TRV130 maintenance would prevent oxycodone-induced brain hypoxia. We focused on the NAc, a brain region critical for opioid reward and relapse (43-49). We tested acute oxycodone-induced changes in NAc oxygen in rats maintained on either vehicle or TRV130 (9 mg/kg/d, 14 days). We tested the rats on days 13-14 of vehicle or TRV130 delivery (minipump ON) and on days 1, 3, and 6 after minipump removal (minipump OFF, withdrawal). The timeline is shown in Fig. 4. Minipump ON (Fig. 4A): In vehicle-maintained rats of both sexes, acute oxycodone (0.3 and 0.6 mg/kg, i.v.) had a biphasic effect on NAc oxygen levels: an initial rapid decrease followed by a reboundlike increase. The decrease was dose-dependent and stronger in males than in females. In contrast, in TRV130-maintained rats of both sexes, oxycodone moderately increased NAc oxygen levels. The

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increases in the TRV130-treated rats were tonic, prolonged, and relatively similar for both oxycodone doses. There were significant group differences in the initial changes in NAc oxygen in both sexes and at both oxycodone doses. For males, the ANOVA, which included the between-subjects factor TRV130 Dose (0, 9 mg/kg/d) and the within-subjects factors Oxycodone Dose (0.3, 0.6 mg/kg) and Time (0, 1, 2,…10 min), showed significant effects of TRV130 Dose x Time (F10,60=17.3, p<0.001), Oxycodone Dose x Time (F10,60=6.7, p<0.001), and TRV130 Dose x Oxycodone Dose x Time (F10,60=4.3, p<0.001). For females, the ANOVA

showed significant effects of TRV130 Dose x Time (F10,60=17.3, p<0.001), Oxycodone Dose x Time (F10,60=9.3, p<0.001), and TRV130 Dose x Oxycodone Dose x Time (F10,60=3.7, p<0.001). For a full statistical report, see Table S1. Minipump OFF (withdrawal, Fig. 4B): In male and female rats previously maintained on vehicle, the initial hypoxic effect of oxycodone (0.6 mg/kg) persisted on days 1, 3, and 6 after minipump removal, and this effect became weaker over days (likely reflecting tolerance to oxycodone). The delayed rebound increase in NAc oxygen remained relatively stable over time. In contrast, in males and females previously maintained on TRV130, oxycodone did not induce NAc hypoxia for up to 6 days after minipump removal. The predominant effect continued to be long-lasting increases in NAc oxygen levels. For males, the ANOVA, which included the between-subjects factor TRV130 Dose, and the within-subjects factors Withdrawal Day (1, 3, 6) and Session Time, showed significant effects of TRV130 Dose x Time (F10,60=8.7, p<0.001), Withdrawal Day x Time (F20,120=2.0, p=0.014), and TRV130 Dose x Withdrawal Day x Time (F20,120=2.5, p<0.001). For females, the ANOVA showed significant effects of TRV130 Dose x Time (F10,60=5.8, p<0.001) but no other interactions. For full statistical reports, see Table S1. The results of Exp. 4 demonstrate that in both sexes, chronic TRV130 delivery prevented acute oxycodone-induced brain hypoxia, an effect that unexpectedly persisted for up to 6 days after withdrawal from TRV130.

Discussion We combined a rat model of opioid maintenance (17-19) with a modified version of the ABA contextinduced-reinstatement model (20, 21) to study prevention of relapse to oxycodone seeking and taking

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under chronic delivery of the G-protein biased MOR agonist TRV130. We report four main findings. First, the relapse model itself showed predictive validity when tested with the FDA-approved treatment buprenorphine (50). Second, buprenorphine’s effects in the model were not mimicked by the KOR antagonist nor-BNI, indicating that buprenorphine’s effects are not mediated by its action on KOR. Third, TRV130 decreased oxycodone seeking in the three drug-seeking and drug-taking measures in male rats, but only decreased extinction responding in context B in female rats. Fourth, TRV130 prevented brain hypoxia induced by acute oxycodone injections, and in both sexes this protection persisted for up to 6 days after treatment cessation. Together, TRV130 decreased opioid seeking and taking during abstinence in male rats while its effects were weaker and more variable in female rats. TRV130 also protected against oxycodone-induced brain hypoxia in both sexes. A rat model of opioid agonist maintenance treatment Our results with buprenorphine are consistent with those of Sorge et al. (19) who reported that buprenorphine (1.5 or 3 mg/kg/d) decreased extinction responding and drug priming-induced reinstatement in male rats previously trained to self-administer heroin and cocaine. However, our reacquisition results with oxycodone are different from those of a subsequent study by the same group (51), in which buprenorphine (3 mg/kg/d) had no effect on ongoing heroin self-administration. The reason for the difference is unknown, but it may be due to our use of extended-access training (6 h/d) and reacquisition testing versus the use of limited-access training (3 h/d) and testing during ongoing self*

administration in the Sorge et al. study . It is unlikely that the reduction in lever presses during the drug seeking and taking tests was due to performance deficits, because tolerance to MOR agonists’ sedative effects develops quickly (52-54). Also, we started testing 3-4 days after implantation, when tolerance to the rate-decreasing effects of the MOR agonists had developed (Fig. S1). Finally, the three buprenorphine doses had similar effects on the different drug seeking and taking measures. This lack of dose-response effect agrees with results from studies with minipump delivery of buprenorphine using different measures of drug seeking (19, 51). The

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Note: We did not test buprenorphine’s effect on oxycodone priming-induced reinstatement because in pilot studies, we did not observe a reliable priming effect after either IV, IP, or SC injections of different oxycodone doses.

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similar effect across dose levels may be due to buprenorphine’s partial-agonist actions at MOR and the use of relatively high doses on the plateau rather than on the ascending limb of buprenorphine doseeffect function (41, 55). For example, previous studies in rhesus monkeys found that lower maintenance doses of buprenorphine caused dose-dependent decreases in heroin self-administration (56, 57). TRV130 for opioid agonist maintenance treatment In humans, TRV130 decreases acute pain with a trend towards fewer side effects (respiratory depression and constipation) than morphine (29-31, 58). This more favorable clinical profile may (24) or may not (26, 27) be due to a bias away from activating the β-arrestin pathway. For opioid addiction treatment, TRV130 and related G-protein-biased MOR agonists with higher efficacy than buprenorphine could theoretically mimic some of the beneficial effects of methadone with a safety profile like that of buprenorphine. This possibility was the basis of our study. In our male rats, TRV130 maintenance decreased drug-seeking behaviors on all three measures. However, TRV130’s protection against reacquisition was not as pronounced as that of buprenorphine (Fig. 1D versus Fig. 3D). One possibility is that the maximal TRV130 dose we tested (9 mg/kg/d) was only as effective as the lower buprenorphine doses. Tentative support for this interpretation is that one study showed that buprenorphine at 0.2 mg/kg was equianalgesic to TRV130 at 0.9 mg/kg (22). In considering advancing TRV130 for maintenance treatment, three issues arise. First, due to pharmacokinetic factors, TRV130 in humans can only be administered intravenously (58), making it unsuitable for daily maintenance. This problem is surmountable by use of orally active analogs such as TRV734 (59), or, better still, by developing depot formulations of TRV130 (or TRV734) that could be administered monthly, as is now done with buprenorphine (60). The second issue is abuse liability: in rats, TRV130 is self-administered, produces fentanyl-like discriminative-stimulus effects, and decreases threshold for brain-stimulation reward (26, 61-63). These findings suggest that its abuse liability may not be separable from its beneficial actions. This issue could be addressed by using depot formulations or oral combination products that deter use by problematic parenteral routes of administration (e.g., buprenorphine/naloxone (64)). Third, we found that the effect of TRV130 on relapse was more robust in males than in females. Thus, to the degree that results from preclinical relapse/reinstatement studies

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generalize to human relapse (34, 65), our data suggest that TRV130 maintenance may be more effective for men. A question for future research is the mechanisms underlying the sex difference we found; it might reflect sex differences in MOR signaling (66, 67) or potentially in TRV130 pharmacokinetics and central receptor occupancy. We also do not know if the weaker effect of TRV130 in female rats was due to fluctuation in estrous cycle. In this regard, gonadal hormone fluctuations modulate morphine's effect on locomotor activity in female but not male rats (68). Effect of chronic TRV130 on oxycodone-induced brain oxygen levels We examined the effect of chronic TRV130 delivery on oxycodone-induced decreases in NAc oxygen levels because respiratory depression followed by brain hypoxia is the most dangerous side effect of opioid drugs (52). Consistent with previous studies (42, 69), we found that oxycodone-induced NAc oxygen response is biphasic, with a rapid but transient decrease that is followed by a prolonged tonic increase. Our studies with oxygen recordings from the subcutaneous space and multi-site temperature recordings indicate that the initial decrease in oxygen is caused by respiratory depression (70). The subsequent increase is caused by cerebral vasodilation due to strong peripheral vasoconstriction, which causes increased blood flow from periphery to brain (70). Importantly, the vascular effect appears at low doses of oxycodone and is masked by a rapid drop in oxygen due to respiratory depression at higher doses (69). NAc oxygen responses to oxycodone in TRV130-maintained rats were drastically different, with rapid (but small) increases in NAc oxygen levels that persisted much longer than the decreases in the vehiclemaintained rats. Thus, in both sexes, chronic TRV130 delivery protected against brain hypoxia induced by moderate doses of oxycodone (69). Unexpectedly, in both sexes, this protective effect persisted after termination of TRV130 treatment for at least 6 days. The reasons for this prolonged protective effect are unknown, but we speculate that it reflects a shift-to-the-right or tolerance to the acute hypoxic effect of oxycodone, because of prior history of chronic exposure to TRV130. Finally, our results suggest that TRV130 effect on oxycodone-induced brain hypoxia is similar across sexes. However, these results should be interpreted with caution, because in the vehicle minipump condition, the hypoxic response to oxycodone alone was weaker in female rats.

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Conclusions We used a rat model of opioid maintenance and relapse and found that TRV130 decreased oxycodone seeking and taking during abstinence in a partly sex-specific manner. We also found that chronic TRV130 delivery prevented acute oxycodone-induced brain hypoxia in both sexes. Based on these results, we propose that TRV130 and related G-protein-biased MOR agonists should be considered for opioid maintenance treatment. In this regard, a recent study showed that monthly buprenorphine depot injections for 6 months led to ~40% abstinence compared with ~5% in a placebo condition (60). Impressive as that is, it leaves room for additional treatment options for opioid users who are not willing to enroll in methadone treatments because of stringent government regulations or do not respond to either methadone or buprenorphine.

Acknowledgment and Financial Disclosure: The authors report no biomedical financial interests or potential conflicts of interest. The research was supported by the Intramural Research Program of NIDA funds to the Neurobiology of Relapse Section (PI: Yavin Shaham).

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Figure legends Figure 1. Effect of chronic delivery of buprenorphine on extinction in context B, context A-induced reinstatement, and reacquisition in context A. Timeline of experiment. Left panels: male rats; right panels: female rats (A) Self-administration training in context A: Number of oxycodone infusions and inactive and active lever presses during self-administration training (total n=55 [males] and n=25 [females]). (B) Extinction in context B. Total number of active and inactive lever presses during the seven 6-h extinction sessions in rats implanted with Alzet osmotic minipumps containing vehicle or buprenorphine (3, 6, or 9 mg/kg/d; n=11-17 per group [males] and n=12-13 per group [females]). Active lever presses led to contingent presentations of the tone-light cue previously paired with oxycodone infusions. Number of active lever presses during each extinction session. (C) Context A-induced reinstatement: Total number of active and inactive lever presses during the 6-h reinstatement tests in context B and context A. Active lever presses led to contingent presentations of the tone-light cue; n per vehicle or buprenorphine dose is the same as that for extinction in context B. Number of active lever presses in context A at each hour of testing. (D) Reacquisition in context A: Total number of oxycodone infusions (0.05 mg/kg/infusion) during reacquisition (n=10-11 per group [males], n=12-13 per group [females]). Number of oxycodone infusions per hour. All data are mean±SEM. * All doses different from vehicle, # 6 & 9 mg/kg/d different from vehicle, p<0.05

Figure 2. Effect of nor-BNI on extinction in context B, context A-induced reinstatement, and reacquisition in context A. Timeline of experiment. Left panels: male rats; right panels: female rats (A) Self-administration training in context A: Number of oxycodone infusions (left panel) and inactive and active lever presses (right panel) during self-administration training (total n=27 [males] and n=28 [females]). (B) Extinction in context B. Left panel: Total number of active and inactive lever presses during the seven 6-h extinction sessions in rats acutely injected with vehicle or nor-BNI (20 mg/kg, i.p; n=13-14 per group [males] and n=14 per group [females]). Active lever presses led to contingent presentations of the tone-light cue previously paired with oxycodone infusions. Right panel: Number of active lever presses during each extinction session. (C) Context A-induced reinstatement: Left panel: Total number of active and inactive lever presses during the 6-h reinstatement tests in context B and context A. Active

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lever presses led to contingent presentations of the tone-light cue; n per vehicle or nor-BNI dose is the same as that for extinction in context B. Right panel: Number of active lever presses in context A at each hour of testing. (D) Reacquisition in context A: Left panel: Total number of oxycodone infusions (0.05 mg/kg/infusion) during reacquisition; n=12 per group [males] and n=11-12 per group [females]). Right panel: Number of oxycodone infusions per hour. All data are mean±SEM.

Figure 3. Effect of chronic delivery of TRV130 on extinction in context B, context A-induced reinstatement, and reacquisition in context A. Timeline of experiment. Left panels: male rats; right panels: female rats (A) Self-administration training in context A: Number of oxycodone infusions (left panel) and inactive and active lever presses (right panel) during self-administration training (total n=53 [males] and n=55 [females]). (B) Extinction in context B. Left panel: Total number of active and inactive lever presses during the seven 6-h extinction sessions in rats implanted with Alzet osmotic minipumps containing vehicle or TRV130 (3, 6, or 9 mg/kg/d; n=13-14 per group [males], n=13-14 per group [females]). Active lever presses led to contingent presentations of the tone-light cue previously paired with oxycodone infusions. Right panel: Number of active lever presses during each extinction session. (C) Context Ainduced reinstatement: Left panel: Total number of active and inactive lever presses during the 6-h reinstatement tests in context B and context A. Active lever presses led to contingent presentations of the tone-light cue; n per vehicle or TRV130 dose is the same as that for extinction in context B. Right panel: Number of active lever presses in context A at each hour of testing. (D) Reacquisition in context A: Left panel: Total number of oxycodone infusions (0.05 mg/kg/infusion) during reacquisition; n=12-13 per group [males) and n=8-11 per group [females]). Right panel: Number of oxycodone infusions per hour. All data are mean±SEM. * All doses different from vehicle, # 6 & 9 mg/kg/d different from vehicle, p<0.05

Figure 4. Effect of chronic delivery of TRV130 on acute oxycodone-induced brain hypoxia. Timeline of Experiment (A) Minipump ON: Effect of acute oxycodone (0.3 and 0.6 mg/kg, i.v) on nucleus accumbens (NAc) oxygen levels in freely-moving rats implanted with minipumps containing either vehicle (n=4 males/4 females) or TRV130 (9 mg/kg/d, n=4 males/4 females) and tested on days 13-14 of minipump delivery and analyzed at 1-min intervals. (B) Minipump OFF (withdrawal): Effect of acute

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oxycodone (0.6 mg/kg, i.v) on NAc oxygen levels 1, 3, and 6 days after minipump removal. Data are mean±SEM percent change from the pre-injection baseline (100%). Data are shown separately for males and females, and asterisks (*) show time intervals during which mean changes in NAc oxygen were significantly different from vehicle (p<0.05).

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Figure 1. Minipump implantation

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20 0

0 B A Test context

0 1 2 3 4 5 6 Session hour

0 1 2 3 4 5 6 Session hour

D. Reacquisition: Context A

60 30 0

10 5

15

60 30 0

0 0 20 nor-BNI dose (mg/kg)

Infusions per hour

90 Infusions (6 h)

Infusions

Infusions (6 h)

Total infusions (6 h)

Infusions per hour 15

Infusions

Total infusions (6 h) 90

0 1 2 3 4 5 6 Session hour

10 5 0

0 20 nor-BNI dose (mg/kg/d)

0 1 2 3 4 5 6 Session hour

Figure 3.

Oxycodone self-administration Context A Days 1-7 (6 h/d) Days 8-14 (6 h/d) 0.1 mg/kg/inf 0.05 mg/kg/inf

Minipump implantation Day 17

Extinction Context B Days 20-26 (6 h/d)

Context-induced reinstatement Contexts A & B Days 27-28 (6 h/d)

MALES

Reacquisition Context A Day 29 (6 h/d) 0.05 mg/kg/inf

FEMALES A. Self-administration: Context A

30

0.05 mg/kg/ infusion

0

300 200 100

90 0.1 mg/kg/ infusion

60 30

0.05 mg/kg/ infusion

0

0 0 2 4 6 8 101214 Training session

0 2 4 6 8 101214 Training session

Lever presses Inactive lever Active lever

Infusions Lever presses (6 h)

60

0.1 mg/kg/ infusion

Lever presses Inactive lever Active lever

Infusions (6 h)

Infusions (6 h)

90

Lever presses (6 h)

Infusions

300 200 100 0

0 2 4 6 8 101214 Training session

0 2 4 6 8 101214 Training session

B. Extinction: Context B

600 300

100

Active lever

#

Vehicle 3 mg/kg/d 6 mg/kg/d 9 mg/kg/d # *

0

0 0 3 6 9 TRV130 dose (mg/kg/d)

Total lever presses 900

Inactive lever Active lever

600

.

300

*

*

Active lever

300 Lever presses

900

#

Lever presses (7 d)

Inactive lever 300 Active lever 200 * * Lever presses

Lever presses (7 d)

Total lever presses

200 * 100

#

Vehicle 3 mg/kg/d 6 mg/kg/d 9 mg/kg/d

0

0

0 1 2 3 4 5 6 7 Extinction session

#

0 3 6 9 TRV130 dose (mg/kg/d)

0 1 2 3 4 5 6 7 Extinction session

*

50

40 20 0

0 B A Test context

150

Context A: Active lever Total active lever Vehicle 6 mg/kg/d 3 mg/kg/d 9 mg/kg/d 60 Lever presses

100

Lever presses (6 h)

150

Total active lever Context A: Active lever Vehicle 6 mg/kg/d 3 mg/kg/d 9 mg/kg/d 60 Lever presses

Lever presses (6 h)

C. Context-induced reinstatement: Context A & B

100 50 0

40 20 0

B A Test context

0 1 2 3 4 5 6 Session hour

0 1 2 3 4 5 6 Session hour

D. Reacquisition: Context A

*

*

30 0

Infusions per hour * * * *

10 5

0 1 2 3 4 5 6 Session hour

Infusions per hour 15

60 30

0

0 0 3 6 9 TRV130 dose (mg/kg/d)

Total infusions (6 h) 90 Infusions

*

Infusions

Infusions (6 h)

60

15

Infusions (6 h)

Total infusions (6 h) 90

10 5 0

0 3 6 9 TRV130 dose (mg/kg/d)

0 1 2 3 4 5 6 Session hour

Figure 4. Minipump ON

Minipump OFF

O2 sensor implant

Treatment: Vehicle or TRV-130

Withdrawal

1

13

14

1

3

6

Days Oxycodone 0.3 & 0.6 mg/kg

Nucleus accumbens

Oxycodone 0.6 mg/kg

A. Minipump ON (Days 13-14)

Change in Oxygen (%)

Males

Females

Oxycodone (0.3 mg/kg)

*

Oxycodone (0.6 mg/kg)

*

Oxycodone (0.3 mg/kg)

*

*

Oxycodone (0.6 mg/kg)

125 100 Vehicle TRV130

75

Vehicle TRV130

50 0

10 20 30 40

0

Time (min)

10 20 30 40

0

0

10 20 30 40

Time (min)

10 20 30 40 Time (min)

Time (min)

Oxycodone injection

B. Minipump OFF (Days 1, 3, 6)

Change in Oxygen (%)

Males Day 3

Day 1

*

*

Oxycodone (0.6 mg/kg)

125

Day 6 *

Oxycodone (0.6 mg/kg)

Oxycodone (0.6 mg/kg)

100 Vehicle TRV130 75 0

10

20

30

40

0

10 20 30 Time (min)

40

0

10

20

30

40

Females

Change in Oxygen (%)

Day 1 *

*

Oxycodone (0.6 mg/kg)

125

Day 3 Oxycodone (0.6 mg/kg)

Day 6 Oxycodone (0.6 mg/kg)

*

100 Vehicle TRV130 75 0

10

20

30

40

0

10 20 30 Time (min)

40

0

10

20

30

40