Hypocretin-Receptor Antagonists for Treatment of Methamphetamine Use Disorder

The hypocretin/orexin (HCRT) system is associated with compulsive stimulant drug use, involving both HCRT-receptor 1 (-R1) and HCRT-receptor 2 (-R2). Few studies, however, have examined the role of HCRT-R2 or combined HCRT-R1/2 on compulsive methamphetamine (METH) taking behavior. In thus study, we examine the effects of HCRT R1, R2, and R1/2 antagonists on compulsive METH self-administration, as modeled by escalated intake in rats allowed extended access to the drug. Three cohorts of male rats were allowed either short (1h; ShA; n=7-10/cohort) and long (12h; LgA; n=7-9/cohort) access to METH intravenous self-administration for 14 sessions (fixed ratio 1 schedule). Each cohort of rats was systemically administered a single- or dual-HCRT-R antagonist 30 min prior to drug-taking operant testing in a latin-square design: cohort 1, selective HCRT-R1 antagonist (RTIOX-276; RTI-R1; 0, 10, and 20 mg/kg); cohort 2, selective HCRT-R2 antagonist (JNJ-10397049; JNJ-R2; 0, 10, and 20 mg/kg); and cohort 3, dual HCRT-R1/2 antagonist (Suvorexant; SUV-R1/2; 0, 30, and 60 mg/kg). RTI-R1 and SUV-R1/2 dose-dependently reduced METH intake in LgA (p\u3c0.01, p\u3c0.05), but not ShA (p=0.14, p=0.06), rats in the first hour. Administration of JNJ-R2, however, had no effect on METH intake in the first hour in either ShA (p=0.58) or LgA (p=0.58), but had deferred effects in LgA rats reducing overall METH intake over the full 6 h session (p\u3c0.05). Beam breaks were significantly reduced following RTI-R1 (p\u3c0.05) and SUV-R1/2 (p\u3c0.05) in ShA rats only, although both JNJ-R2 (p\u3c0.05) and SUV-R1/2 (p\u3c0.05) reduced beam breaks in METH naïve rats, indicating a possible confound of generalized effects on locomotor. Combined, these results suggest HCRT-R1 neurotransmission, in particular, may contribute to compulsive METH taking associated with stimulant use disorder. Future studies will investigate the contribution of HCRT to pervasive compulsive METH use after a period of drug abstinence

Reduced methamphetamine self-administration following single or dual hypocretin-receptor blockade or viral vector hypocretin-knockdown in adult male rats

The hypocretin/orexin (HCRT) system is associated with compulsive stimulant drug use, involving both HCRT-receptor 1 (-R1) and HCRT-receptor 2 (-R2). Few studies, however, have examined the role of HCRT-R2 or combined HCRT-R1/2 on compulsive methamphetamine (METH) taking behavior. In this study, we examined the effects of HCRT-R1, -R2, and -R1/2 antagonists on compulsive METH self-administration, as modeled by escalated intake in adult male Wistar rats allowed extended access to METH. Three cohorts of rats were allowed either short (1h; ShA; n=7-10/cohort) or long (6h; LgA; n=7-9/cohort) access to METH intravenous self-administration for 14 sessions (fixed ratio 1 schedule). Each cohort was then systemically administered a single- or dual-HCRT-R antagonist 30 min prior to METH self-administration testing: cohort 1, selective HCRT-R1 antagonist (RTIOX-276; RTI-R1; 0, 10, and 20 mg/kg); cohort 2, selective HCRT-R2 antagonist (JNJ-10397049; JNJ-R2; 0, 10, and 20 mg/kg); and cohort 3, dual HCRT-R1/2 antagonist (Suvorexant; SUV-R1/2; 0, 30, and 60 mg/kg). RTI-R1 elicited a dose-dependent reduction in METH intake in LgA, but not ShA, in the first hour. Administration of JNJ-R2 had no effect on METH intake in the first hour in neither ShA nor LgA rats, but reduced METH intake during the full 6 h session at the lowest dose. SUV-R1/2 administration had no effect on METH intake in ShA rats, but showed significant attenuation of METH-taking at the highest dose in both the first hour and full 6h session for LgA rats. Locomotor activity was significantly reduced following RTI-R1 and SUV-R1/2 in ShA rats only. To further explore the role that HCRT plays in METH dependence after a period of abstinence, we used a shRNA-encoding adeno-associated viral vector (AAV) to silence Hcrt in a separate cohort of previously-escalated METH-dependent rats. Following an initial escalation phase, and prior to a 3-week period of drug abstinence, rats were injected with either a control scramble-RNA AAV (AAV-Scram; n= 4) or a Hcrt-knockdown AAV (AAV-HCRT-KD; n= 5). AAV-Scram rats showed a significant decrease in METH self-administration post-abstinence, and a subsequent increase in METH-taking following a re-escalation period. In contrast, AAV-HCRT-KD rats showed a significant attenuation of METH self-administration following the re-escalation period. Combined, these results suggest HCRT neurotransmission at both HCRT-R1 and -R2 may contribute to compulsive METH-taking behavior

Correction of sleep disturbances during abstinence following hypocretin-receptor antagonism in fentanyl-dependent rats.

Fentanyl is a potent synthetic opioid that has been shown to produce sleep disturbances, and the deterioration of sleep quality is associated with drug abuse and relapse in humans. The hypocretin/orexin neuropeptide system is a plausible pharmacological target, and dual-hypocretin antagonists such as lemborexant may mitigate sleep disturbances associated with fentanyl dependence. The current study characterizes sleep macroarchitecture (time spent asleep or awake) and microarchitecture (the number of bouts, and NREM sleep spindle characterization) prior to fentanyl vapor exposure (baseline), following one week of drug abstinence, and four weeks of drug abstinence in female and male rats. Females and males showed a reduction in the amount of time spent in rapid eye movement (REM) sleep following one week of abstinence. The pre-treatment of lemborexant the following day increased the amount of time spent in REM, compared to vehicle at both one and four weeks of abstinence. While there was no effect of fentanyl abstinence on the amount of time spent in non-rapid eye movement (NREM) sleep and wakefulness, lemborexant increased the amount time spent in NREM and decreased the amount of time spent awake. Examination of microarchitecture demonstrated a decrease in the number of NREM bouts at one week of abstinence, which lemborexant subsequently brought back to baseline levels at weeks one and four. Abstinence from fentanyl did not impact the number of NREM sleep spindles, but indicated a trend showing a decrease in intra-spindle frequency at one week of abstinence. Lemborexant, however, increased the number of spindles at weeks one and four of abstinence. Presently, findings indicate that fentanyl abstinence produces changes in sleep macroarchitecture, particularly REM sleep disruptions, which may be alleviated by lemborexant. This highlights the need for further examination of the relationship between sleep disturbances and drug abstinence, and the use of dual-hypocretin antagonists as therapeutic intervention

Insomnia Treatment Drug Lemborexant Rescues Sleep Dysfunction Associated with Methamphetamine Vapor Withdrawal

Introduction: In 2021, 2.5 million people aged 12 and older abused the addictive psychostimulant methamphetamine (MA) in the US. MA produces short-lasting euphoria, but also anxiety, erratic behavior, mood disturbance, and abnormal wakefulness. Chronic use of MA can lead to disordered sleep, particularly during withdrawal, and clinical studies have shown that sleep dysfunction is a strong predictor for drug-taking relapse. The neuropeptide hypocretin (HCRT) plays a critical role in the transition to a waking state and also modulates drug reward. Enhanced HCRT signaling in the brain underlies the sleep disorder insomnia and the HCRT-receptor antagonist lemborexant has recently been FDA-approved for treatment of insomnia in humans. Thus, in the current study we characterize sleep dysfunction associated with MA vapor withdrawal and hypothesize that HCRT signaling contributes to negative sleep outcomes. Methods: Adult male Wistar rats (N =8) were implanted with a telemetry device and electroencephalographic/electromyographic signals were recorded for 24 hours (12:12 hours, light:dark cycle). Data were analyzed prior to MA vapor exposure (baseline), and during withdrawal (after one week of MA vapor abstinence). Rats were administered lemborexant (0, and 30 mg/kg, in a counter-balanced order) during withdrawal at the beginning of the light cycle. Results: Rats showed a decrease in time spent in rapid eye movement (REM) sleep in the light cycle during withdrawal, and there was a trend for an increase in time spent in REM sleep during the dark cycle, indicating possible REM sleep rebound. There were no changes to non-REM (NREM) sleep or waking in either the light or dark cycle. The number of bouts of REM sleep decreased during the light cycle, and there was no change in average bout duration in REM sleep during withdrawal compared to baseline. The number of bouts of NREM sleep and waking increased during the dark cycle, while the average bout duration decreased during withdrawal compared to baseline, indicating periods of sleep/wake were more fragmented during the dark cycle. In addition, administration of lemborexant restored the amount of time spent in REM sleep and the number of REM sleep bouts during the light cycle. Conclusions: Overall, these findings show there is a role for HCRT neurotransmission in the observed dysregulated and fragmented sleep of male rats during MA withdrawal. Future research should look at gender differences for sleep dysfunction and MA withdrawal, as well as long-term consequences of MA use

Effects of Single and Dual Hypocretin-receptor Blockade or Knockdown of Hypocretin Amygdalar Projections on Alcohol Drinking in Dependent Male Rats

Hypocretin/Orexin (HCRT) is a neuropeptide that is associated with both stress and reward systems in humans and rodents. The different contributions of signaling at hypocretin-receptor 1 (HCRT-R1) and hypocretin-receptor 2 (HCRT-R2) to compulsive alcohol drinking are not yet fully understood. Thus, the current studies used pharmacological and viral-mediated targeting of HCRT to determine participation in compulsive alcohol drinking and measured HCRT-receptor mRNA expression in the extended amygdala of both alcohol-dependent and non-dependent male rats. Rats were made dependent through chronic intermittent exposure to alcohol vapor and were tested for the acute effect of HCRT-R1-selective (SB-408124; SB-R1), HCRT-R2-selective (NBI-80713; NB-R2), or dual HCRT-R1/2 (NBI-87571; NB-R1/2) antagonism on alcohol intake. NB-R2 and NB-R1/2 antagonists each dose-dependently decreased overall alcohol drinking in alcohol-dependent rats, whereas, SB-R1 decreased alcohol drinking in both alcohol-dependent and non-dependent rats at the highest dose (30 mg/kg). SB-R1, NB-R2, and NB-R1/2 treatment did not significantly affect water drinking in either alcohol-dependent or non-dependent rats. Additional PCR analyses revealed a significant decrease in Hcrtr1 mRNA expression within the central amygdala (CeA) of dependent rats under acute withdrawal conditions compared to nondependent rats. Lastly, a shRNA-encoding adeno-associated viral vector with retrograde function was used to knockdown HCRT in CeA-projecting neurons from the lateral hypothalamus (LH). LH-CeA HCRT knockdown significantly attenuated alcohol self-administration in alcohol-dependent rats. These observations suggest that HCRT signaling in the CeA is necessary for alcohol-seeking behavior during dependence. Together, these data highlight a role for both HCRT-R1 and -R2 in dependent alcohol-seeking behavior

Knockdown of hypocretin attenuates extended access of cocaine self-administration in rats

The hypocretin/orexin (HCRT) neuropeptide system regulates feeding, arousal state, stress responses, and reward, especially under conditions of enhanced motivational relevance. In particular, HCRT neurotransmission facilitates drug-seeking behavior in circumstances that demand increased effort and/or motivation to take the drug. The present study used a shRNA-encoding adeno-associated viral vector to knockdown Hcrt expression throughout the dorsal hypothalamus in adult rats and determine the role of HCRT in cocaine self-administration. Chronic Hcrt silencing did not impact cocaine self-administration under short-access conditions, but robustly attenuated cocaine intake under extended access conditions, a model that mimics key features of compulsive cocaine taking. In addition, Hcrt silencing decreased motivation for both cocaine and a highly palatable food reward (i.e., sweetened condensed milk; SCM) under a progressive ratio schedule of reinforcement, but did not alter responding for SCM under a fixed ratio schedule. Importantly, Hcrt silencing did not affect food or water consumption, and had no consequence for general measures of arousal and stress reactivity. At the molecular level, chronic Hcrt knockdown reduced the number of neurons expressing dynorphin (DYN), and to a smaller extent melanin-concentrating hormone (MCH), in the dorsal hypothalamus. These original findings support the hypothesis that HCRT neurotransmission promotes operant responding for both drug and non-drug rewards, preferentially under conditions requiring a high degree of motivation. Furthermore, the current study provides compelling evidence for the involvement of the HCRT system in cocaine self-administration also under low-effort conditions in rats allowed extended access, possibly via functional interactions with DYN and MCH signaling

Effects of single and dual hypocretin-receptor blockade or knockdown of hypocretin projections to the central amygdala on alcohol drinking in dependent male rats

Hypocretin/Orexin (HCRT) is a neuropeptide that is associated with both stress and reward systems in humans and rodents. The different contributions of signaling at hypocretin-receptor 1 (HCRT-R1) and hypocretin-receptor 2 (HCRT-R2) to compulsive alcohol drinking are not yet fully understood. Thus, the current studies used pharmacological and viral-mediated targeting of HCRT to determine participation in compulsive alcohol drinking and measured HCRT-receptor mRNA expression in the extended amygdala of both alcohol-dependent and non-dependent male rats. Rats were made dependent through chronic intermittent exposure to alcohol vapor and were tested for the acute effect of HCRT-R1-selective (SB-408124; SB-R1), HCRT-R2-selective (NBI-80713; NB-R2), or dual HCRT-R1/2 (NBI-87571; NB-R1/2) antagonism on alcohol intake. NB-R2 and NB-R1/2 antagonists each dose-dependently decreased overall alcohol drinking in alcohol-dependent rats, whereas, SB-R1 decreased alcohol drinking in both alcohol-dependent and non-dependent rats at the highest dose (30 mg/kg). SB-R1, NB-R2, and NB-R1/2 treatment did not significantly affect water drinking in either alcohol-dependent or non-dependent rats. Additional PCR analyses revealed a significant decrease in Hcrtr1 mRNA expression within the central amygdala (CeA) of dependent rats under acute withdrawal conditions compared to nondependent rats. Lastly, a shRNA-encoding adeno-associated viral vector with retrograde function was used to knockdown HCRT in CeA-projecting neurons from the lateral hypothalamus (LH). LH-CeA HCRT knockdown significantly attenuated alcohol self-administration in alcohol-dependent rats. These observations suggest that HCRT signaling in the CeA is necessary for alcohol-seeking behavior during dependence. Together, these data highlight a role for both HCRT-R1 and -R2 in dependent alcohol-seeking behavior

Compulsive-Like Sufentanil Vapor Self-Administration in Rats.

Opioid misuse is at historically high levels in the United States, with inhalation (ie, smoking and vaping) being one of the most common routes of consumption. We developed and validated a novel preclinical model of opioid self-administration by inhalation that does not require surgery and reliably produces somatic and motivational signs of dependence. Rats were trained to perform an operant response (nosepoke) to receive 10 s of vaporized sufentanil, a potent opioid, in 2 h daily sessions. Rats readily and concentration-dependently self-administered vaporized sufentanil. Rats exhibited a significant increase in responding for sufentanil when given the preferential μ-opioid receptor inverse agonist naloxone, suggesting the participation of μ-opioid receptors in the reinforcing properties of sufentanil vapor. Serum sufentanil concentrations significantly correlated with the number of sufentanil vapor deliveries. Rats that were given long access (LgA; 12 h/day) but not short access (ShA; 1 h/day) to vaporized sufentanil escalated their drug intake over time and exhibited both naloxone-precipitated somatic signs of opioid withdrawal and spontaneous withdrawal-induced mechanical hypersensitivity. After 6 months of forced drug abstinence, LgA rats returned to pre-escalation baseline levels of responding for sufentanil and mechanical sensitivity. Upon subsequent re-escalation (ie, after the return to extended access to sufentanil vapor), LgA rats again developed naloxone-precipitated somatic signs of withdrawal and spontaneous withdrawal-induced mechanical hypersensitivity. These findings demonstrate that the operant sufentanil vapor self-administration model has both face and construct validity and therefore will be useful for investigating the neurobiological basis of opioid addiction