Developing safe and effective pain medications is an ongoing challenge for human health. Agonists for the µ-opioid receptor (MOR) are essential pain medications, but their high intrinsic efficacy also induces adverse side effects, including respiratory depression, constipation, tolerance, dependence, withdrawal and addiction1-7. Strategies to limit adverse effects traditionally include developing MOR agonists that have low intrinsic efficacy or that preferentially activate G-protein signalling over β-arrestin signalling8. Here we identify a novel MOR agonist with supramaximal intrinsic efficacy and a unique pharmacological profile that produced effective analgesia in rodents with minimal adverse effects. N-desethyl-fluornitrazene (DFNZ) was derived from a class of synthetic benzimidazole opioids called nitazenes. DFNZ has impaired brain penetrance, a unique spatiotemporal MOR cellular signalling profile, and diminished efficacy at the MOR-galanin 1 receptor (GAL1) heteromer. DFNZ does not induce respiratory depression, tolerance or MOR downregulation after repeated exposure. Compared with other MOR agonists, DFNZ has limited effects on dopamine neurotransmission in nucleus accumbens and weaker reinforcing effects in the drug self-administration procedure. These results provide novel insights about MOR and nitazene pharmacology, have important implications for pain and addiction treatment, and challenge the prevailing dogma that high-efficacy MOR agonists cannot constitute safe and effective therapeutic agents.

Mu opioid receptor (MOR) analgesics like morphine have high intrinsic MOR efficacy but produce side effects. Lower efficacy MOR agonists like buprenorphine can produce analgesia with improved safety. We recently described a series of novel phenylmorphan-based MOR agonists with graded MOR efficacies, including several with sub-buprenorphine efficacies, and we described their acute effectiveness in mice to produce (a) antinociception in an assay of pain-related locomotor depression, and (b) improved safety on a range of side effects. Effects of repeated treatment with these compounds have not been examined. The present objective was to compare effects of repeated treatment with four opioids that ranged from high to low MOR efficacy (morphine> buprenorphine > JL-2-39 > DC-1-76.1). Female and male ICR mice were treated once daily for 7 days with a selected dose of each drug ± intraperitoneal lactic acid (IP acid) as a noxious stimulus and evaluated for vertical and horizontal locomotor activity on Days 1 and 7. IP acid alone produced sustained, concentration-dependent, and pain-related locomotor depression. When administered as a daily pretreatment to IP acid, all four opioids produced sustained antinociception expressed as significant alleviation of IP acid-induced behavioral depression on Days 1 and 7. However, when these opioids were administered alone, effects were efficacy dependent. Morphine produced locomotor stimulation and sensitization; buprenorphine and JL-2-39 produced locomotor stimulation without sensitization; DC-1-76.1 produced neither stimulation or sensitization. These findings support continued consideration of low-efficacy MOR agonists as candidate analgesics that can produce sustained antinociception with reduced side effects during repeated treatment.

Opioid use disorder (OUD) remains a global public health concern, exerting a significant economic burden on societies worldwide. To combat the rise of substance use disorder, a complementary vaccine-based intervention was developed to support and strengthen the existing treatment modalities. Hapten-protein conjugate vaccines are employed as a promising novel strategy for neutralizing nonimmunogenic small-molecule drugs such as opioids by eliciting drug-specific antibodies that sequester opioids in circulation before reaching the brain. In this study, multiple parameters to optimize the efficacy of the TT-6-AmHap heroin conjugate vaccine were investigated in mice and rats, focusing on dose optimization, hapten density, booster timing, and cross-reactivity with other prescription opioids. Binding antibody responses were quantified, and vaccine efficacy was assessed using nociceptive assays following a drug challenge. Immunization with TT-6-AmHap vaccine in combination with adjuvants ALF43 and Alhydrogel® (ALFA), demonstrated a robust in-vivo efficacy across multiple opioid challenges including heroin, hydrocodone, and hydromorphone. Both antibody titers to 6-AmHap and protective efficacy were maintained following repeated heroin challenges up to 54 weeks. Notably, these repeat heroin challenges did not induce any measurable decline in antibody titers. Further characterization of the vaccine responses indicated a strong correlation between hapten density on the carrier protein and both binding antibody titers and overall vaccine efficacy. These findings support the continued development of hapten-based conjugate vaccines for OUD and demonstrate that an optimized vaccine can provide long-lasting protection against the antinociceptive effects of opioids in animals, laying the groundwork for future translational studies.

Environmental cues that have been associated with drug-taking can evoke drug-craving and drug-seeking and drive relapse. Using the New Response Acquisition procedure, we evaluated the extent to which activation of delta opioid receptors (DORs) changes responding for cocaine-associated stimuli. We hypothesized that activation of DORs, either directly via agonists or indirectly via protected concentrations of endogenous enkephalin peptides, would increase the conditioned reinforcing effects of cues. First, animals undergo Pavlovian conditioning during which rats received 5 infusions of cocaine (0.32 mg/kg/inf) and either paired or unpaired presentations of a stimulus (light + tone) per day for 10 days. Next, nosepokes were added to the operant chamber and rats were allowed to respond for presentations of cocaine-associated stimuli (acquisition). Consistent with previous findings, animals assigned to paired Pavlovian conditioning emitted more responses for cue presentations than animals assigned to the unpaired control. Interestingly, acute administration of SNC80 (DOR agonist; 3.2 mg/kg s.c.) on acquisition session 4 led to robust increases in responding for the cocaine-paired cues in the paired, but also increased responding for cues in rats assigned to unpaired and saline control groups. Further, the enkephalinase inhibitor RB101 (10 mg/kg intravenous), which maintains extracellular concentrations of enkephalins, increased active responding in a DOR-dependent manner. These data suggest that activation at DORs influences behaviors maintained by cues and sheds light on the neurobiology underlying the conditioned reinforcing effects of drug-associated stimuli. SIGNIFICANCE STATEMENT: We used a more rigorous test of conditioned reinforcement to show that activation of delta opioid receptors increases the reinforcing effects of cocaine-paired cues, depending on conditioning history, which implicates the delta opioid receptor system as a target to reduce relapse.

Background and PurposeThe use of 'bath salts' drug preparations has been associated with high rates of toxicity and death. Preparations often contain mixtures of drugs, including multiple synthetic cathinones or synthetic cathinones and caffeine. Little is known about the interactions of 'bath salts' constituents and adverse effects often reported by users.Experimental ApproachThis study used adult male Sprague-Dawley rats to characterise the cardiovascular effects, locomotor effects and pharmacokinetics of methylone, methylenedioxypyrovalerone (MDPV) and caffeine, administered alone and as binary mixtures. Dose-addition analyses were used to determine the effect levels of a strictly additive interaction for dose pairs.Key ResultsMethylone, MDPV and caffeine increased heart rate (HR) and locomotion, with methylone producing the largest increase in HR, MDPV producing the largest increase in locomotor activity and caffeine being the least effective in stimulating HR and locomotor activity. MDPV and caffeine increased mean arterial pressure (MAP), with caffeine being more effective than MDPV. The nature of the interactions between methylone and MDPV tended towards sub-additivity for all endpoints, whereas interactions between MDPV or methylone and caffeine tended to be additive or sub-additive for cardiovascular endpoints, and additive or supra-additive for increases in locomotion. No pharmacokinetic interactions were observed between individual constituents, but methylone appeared to display nonlinear pharmacokinetics at the largest dose evaluated.Conclusion and ImplicationsThese findings demonstrate that 'bath salts' preparations can impact both cardiovascular and locomotor effects and suggest that interactions among constituent drugs could contribute to the 'bath salts' toxidrome reported by human users.

Replacement of the phenolic hydroxy in 3-((1R,5S,9R)-2-phenethyl-9-vinyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol (DC-1-76.2), a potent efficacious MOR agonist, with an amide bioisosteric moiety provided a MOR partial agonist with morphine-like potency in the forskolin-induced cAMP accumulation assay and in the [35S]GTPγS functional assay. This amide, 5, had superior metabolic stability in comparison to its precursor in human and mouse liver microsomes. However, in an antinociception study, an assay of pain-depressed locomotion in mice, it was found to possess shorter antinociceptive activity than its precursor. The in vitro and in vivo data enabled the characterization of amide, 5, as a functionally selective, low-efficacy, and low-potency MOR agonist with a relatively short duration of action in vivo. Modification of the N-phenethyl substituent in DC-1-76.2 gave a number of highly interesting partial agonists and the unexpectedly potent antagonist, 17. The results of molecular docking and binding free energy calculations for DC-1-76.2 and 17 provided details about their receptor interactions and supported their functional roles. Several analogs synthesized were found to have sufficient potency in vitro to warrant further study.

Substance use disorder is associated with a high rate of relapse driven by the rewarding and motivational properties of drug-associated stimuli. We examined the extent to which the δ opioid receptor (DOR) mediated the conditioned reinforcing properties of drug-associated stimuli across 4 experiments and 2 operant procedures in Sprague-Dawley rats. In experiment 1, we assessed the extent to which SNC80 (DOR agonist) altered responding during extinction in a remifentanil self-administration procedure. In experiment 2, we assessed the extent to which SNC80 altered responding for a remifentanil-associated stimulus in the new response acquisition procedure. In experiment 3, we assessed the extent to which the effects of SNC80 were altered in standard versus enhanced stimulus control conditions in the new response acquisition procedure. In experiment 4, we assessed the extent to which naltrindole (DOR antagonist) was able to block SNC80-induced alterations in responding for a remifentanil-associated stimulus in the new response acquisition procedure. In experiment 5, we assessed the extent to which pretreatments of SNC80 altered Pavlovian conditioning in the new response acquisition procedure. These experiments support the role of the DOR system in mediating the rewarding properties of drug-associated stimuli, such that SNC80 dose-dependently enhanced responding for a remifentanil-associated stimulus across self-administration and new response acquisition procedures. These effects were blocked by naltrindole, demonstrating that these are DOR-mediated effects. DORs may represent a promising target to address relapse. SIGNIFICANCE STATEMENT: Conditioned reinforcing effects of an opioid-associated cue were enhanced following stimulation of δ opioid receptors (DORs), and this effect was blocked via DOR antagonism, which suggests the role of DORs in mediating drug-associated conditioned reinforcement.

Background/Objectives: Opioid use disorder (OUD) remains a severe health problem globally, resulting in substantial social and economic challenges. While existing medications for managing OUD are proven to be effective, they also present certain challenges. A vaccine offers a promising therapeutic strategy to combat OUD and potentially reduce the risk of overdose death. The TT-6-AmHap heroin conjugate vaccine has effectively reduced heroin-induced pharmacological effects in behavioral assays as well as demonstrated the induction of high titer and high affinity antibody responses in mice and rats. In this GLP study conducted in rabbits, the potential local and systemic toxicity of the TT-6-AmHap heroin vaccine in combination with or without adjuvants ALF43 and Alhydrogel® (ALFA) was investigated. Methods: Male and female New Zealand White rabbits were administered with vaccines or a saline control intramuscularly at two-week intervals over a 57-day study period. The presence, persistence or reversibility of any toxic effects of the vaccine was determined over a four-week recovery period. Results: Administration of TT-6-AmHap with or without the adjuvants induced high antibody-specific IgG in treatment groups compared to the controls. The study found no TT-6-AmHap-related effects on mortality, physical examinations, dermal Draize observations, body weights, body weight changes, food consumption, ophthalmology, clinical pathology (hematology, coagulation, clinical chemistry, and urinalysis), macroscopic pathology, or organ weights. Conclusions: Under the conditions of this study, these results demonstrate that the TT-6-AmHap vaccine with or without adjuvants was well tolerated, immunogenic, and the effects were not considered adverse in both male and female rabbits.

The word opium derives from the ancient Greek word ὄπιον (ópion) for the juice of any plant, but today means the air-dried seed capsule latex of Papaver somniferum. Alkaloid chemistry began with the isolation of morphine from crude opium by Friedrich Wilhelm Adam Sertürner in 1804. More than a century later, Hungarian pharmacist János Kabay opened new perspectives for the direct isolation of morphine from dry poppy heads and straw without the labor-intensive harvesting of opium. In 2015, Kabay's life and achievements obtained official recognition as constituting a «Hungarikum», thereby entering the national repository of matters of unique cultural value. To this day, the study of Papaver alkaloids is a focus of medicinal chemistry, the (perhaps unstated) aspiration of which is to obtain an opioid with lesser abuse potential and side effects, while retaining good analgesic properties. We begin this review with a brief account of opiate biosynthesis, followed by a detailed presentation of semisynthetic opioids, emphasizing the efforts of the Alkaloida Chemical Company, founded in 1927 by János Kabay, and the morphine alkaloid group of the University of Debrecen.

IntroductionStudies suggest that serotonin (5-HT) plays an important role in alcohol use disorder (AUD). While several receptor subtypes modulate the role of 5-HT in AUD, evidence suggests that 5-HT2A and 5-HT2C receptors may be directly involved in alcohol drinking due to their interaction with the mesolimbic dopaminergic system. The aim of the present study was to investigate the effects of 5-HT2A and 5-HT2C antagonists, alone or in combination, on the acquisition and expression (i.e., return to alcohol drinking after a period of abstinence/treatment) of voluntary alcohol drinking in male mice.MethodsAnimals had intermittent access to alcohol (10% v/v) in a two-bottle choice procedure for 30 days (acquisition), and were then submitted to alcohol re-exposure sessions after periods of abstinence. Vehicle, the 5-HT2A receptor antagonist M100907 (M100, 1 mg/kg) and/or the 5-HT2C receptor antagonist SB242084 (SB, 1 mg/kg) were administered either prior to acquisition (Experiment 1) or during the abstinence period preceding re-exposure sessions (Experiment 2). During re-exposure tests, animals were submitted to the same conditions as during acquisition, with no treatments prior to those sessions.ResultsOur findings show that combined treatment with 5-HT2A and 5-HT2C antagonists, but not treatment with the antagonists separately, reduced alcohol drinking and preference when administered immediately before acquisition (Experiment 1). Combined treatment with 5-HT2A and 5-HT2C antagonists after the establishment of voluntary alcohol drinking did not alter the expression of drinking behavior (Experiment 2). On the other hand, while post-acquisition treatment with a 5-HT2A antagonist alone decreased alcohol intake and preference during re-exposure, co-administration of a 5-HT2C antagonist blocked these effects.DiscussionOur findings suggest that 5-HT2A and 5-HT2C receptors differentially modulate the acquisition and expression of voluntary alcohol drinking in mice.

Recent data suggest that 3,4-methylenedioxypyrovalerone (MDPV) has neurotoxic effects; however, the cognitive and neurochemical consequences of MDPV self-administration remain largely unexplored. Furthermore, despite the fact that drug preparations that contain MDPV often also contain caffeine, little is known regarding the toxic effects produced by the co-use of these two stimulants. The current study investigated the degree to which self-administered MDPV or a mixture of MDPV+caffeine can produce deficits in recognition memory and alter neurochemistry relative to prototypical stimulants. Male Sprague Dawley rats were provided 90 min or 12 h access to MDPV, MDPV+caffeine, methamphetamine, cocaine, or saline for 6 weeks. Novel object recognition (NOR) memory was evaluated prior to any drug self-administration history and 3 weeks after the final self-administration session. Rats that had 12 h access to methamphetamine and those that had 90 min or 12 h access to MDPV+caffeine exhibited significant deficits in NOR, whereas no significant deficits were observed in rats that self-administered cocaine or MDPV. Striatal monoamine levels were not systematically affected. These data demonstrate synergism between MDPV and caffeine with regard to producing recognition memory deficits, highlighting the importance of recapitulating the manner in which drugs are used (e.g., in mixtures containing multiple stimulants, binge-like patterns of intake).

The synthesis of stereochemically pure oximes, amines, saturated and unsaturated cyanomethyl compounds, and methylaminomethyl compounds at the C9 position in 3-hydroxy-N-phenethyl-5-phenylmorphans provided μ-opioid receptor (MOR) agonists with varied efficacy and potency. One of the most interesting compounds, (2-((1S,5R,9R)-5-(3-hydroxyphenyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-9-yl)acetonitrile), was found to be a potent partial MOR agonist (EC50 = 2.5 nM, %Emax = 89.6%), as determined in the forskolin-induced cAMP accumulation assay. Others ranged in potency and efficacy at the MOR, from nanomolar potency with a C9 cyanomethyl compound (EC50 = 0.85 nM) to its totally inactive diastereomer, and three compounds exhibited weak MOR antagonist activity (the primary amine 3, the secondary amine 8, and the cyanomethyl compound 41). Many of the compounds were fully efficacious; their efficacy and potency were affected by both the stereochemistry of the molecule and the specific C9 substituent. Most of the MOR agonists were selective in their receptor interactions, and only a few had δ-opioid receptor (DOR) or κ-opioid receptor (KOR) agonist activity. Only one compound, a C9-methylaminomethyl-substituted phenylmorphan, was moderately potent and fully efficacious as a KOR agonist (KOR EC50 = 18 nM (% Emax = 103%)).

We recently developed a series of nalfurafine analogs (TK10, TK33, and TK35) that may serve as non-addictive candidate analgesics. These compounds are mixed-action agonists at the kappa and delta opioid receptors (KOR and DOR, respectively) and produce antinociception in a mouse warm-water tail-immersion test while failing to produce typical mu opioid receptor (MOR)-mediated side effects. The warm-water tail-immersion test is an assay of pain-stimulated behavior vulnerable to false-positive analgesic-like effects by drugs that produce motor impairment. Accordingly, this study evaluated TK10, TK33, and TK35 in a recently validated assay of pain-related behavioral depression in mice that are less vulnerable to false-positive effects. For comparison, we also evaluated the effects of the MOR agonist/analgesic hydrocodone (positive control), the neurokinin 1 receptor (NK1R) antagonist aprepitant (negative control), nalfurafine as a selective KOR agonist, SNC80 as a selective DOR agonist, and a nalfurafine/SNC80 mixture. Intraperitoneal injection of dilute lactic acid (IP lactic acid) served as a noxious stimulus to depress vertical and horizontal locomotor activity in male and female ICR mice. IP lactic acid-induced locomotor depression was alleviated by hydrocodone but not by aprepitant, nalfurafine, SNC80, the nalfurafine/SNC80 mixture, or the KOR/DOR agonists. These results suggest that caution is warranted in advancing mixed-action KOR/DOR agonists as candidate analgesics.

Abstract ID 129587Poster Board 515One contributor to relapse is the ability of environmental cues that have been associated with drug-taking to evoke drug-craving and -seeking behaviors. The conditioned reinforcing properties of drug-paired cues are often evaluated following contingent drug-self administration in reinstatement procedures and are thought to be dopamine mediated, yet other systems, such as opioid receptor systems, have also been implicated. We sought to evaluate whether activation of the delta opioid receptors (DORs) would alter responding for cocaine-paired cues in a stringent test of cocaine conditioned reinforcement (New Response Acquisition). We hypothesized that activation of DORs, either indirectly via protected concentrations of endogenous enkephalin peptides or directly via agonists, would increase the conditioned reinforcing properties of cues in the New Response Acquisition procedure. This procedure begins with Pavlovian Conditioning in which subjects receive five infusions of cocaine (320 ug/kg/inf) and either simultaneous (Paired) or separate (Unpaired) presentations of a light+tone stimulus per day for 10 days. Then, novel operant manipulanda are introduced into the chamber, and responses produce presentations of cues formerly associated with cocaine (Acquisition). During Acquisition, drug pretreatments were administered acutely before the start of the session and responding was evaluated. Consistent with previous findings, subjects in the Paired group make more active responses than inactive responses for cue presentations and more active responses than Unpaired subjects. Interestingly, administration of the enkephalinase inhibitor RB101 (10 mg/kg intravenous), which maintains extracellular concentrations of enkephalins, increased responding for cocaine-paired cues and this effect was blocked by the DOR selective antagonist naltrindole (3.2 mg/kg subcutaneous). Further, the DOR agonist SNC80 (3.2 mg/kg subcutaneous) produced robust increases in responding for the cocaine-paired cues in the Paired group when given acutely on either Acquisition 4 or 8, and these effects dissipate across days following repeated administration. Surprisingly, SNC80 also produced greater levels of responding for cues in Unpaired groups, potentially indicating that these cues acquire weak conditioning reinforcing properties. The effects of SNC80 are unlikely due to locomotor stimulation, as psychomotor stimulants have been shown previously to not alter responding for cues in this procedure. These data suggest that activation at DORs alters behaviors maintained by cocaine-paired cues. This work may provide novel insight into the underlying neurobiology mediating the conditioned reinforcing effects of drug-paired cues. Funding for this work was provided by: R01DA042092, F31DA053697-01, and T32DA007281-23.Funding for this work was provided by: R01DA042092, F31DA053697-01, and T32DA007281-23.

Low-efficacy mu opioid receptor (MOR) agonists may serve as novel candidate analgesics with improved safety relative to high-efficacy opioids. This study used a recently validated assay of pain-depressed behavior in mice to evaluate a novel series of MOR-selective C9-substituted phenylmorphan opioids with graded MOR efficacies. Intraperitoneal injection of dilute lactic acid (IP acid) served as a noxious stimulus to depress locomotor activity by mice in an activity chamber composed of two compartments connected by an obstructed door. Behavioral measures included (1) crosses between compartments (vertical activity over the obstruction) and (2) movement counts quantified as photobeam breaks summed across compartments (horizontal activity). Each drug was tested alone and as a pretreatment to IP acid. A charcoal-meal test and whole-body-plethysmography assessment of breathing in 5% CO2 were also used to assess gastrointestinal (GI) inhibition and respiratory depression, respectively. IP acid produced a concentration-dependent depression in crosses and movement that was optimally alleviated by intermediate- to low-efficacy phenylmorphans with sufficient efficacy to produce analgesia with minimal locomotor disruption. Follow-up studies with two low-efficacy phenylmorphans (JL-2-39 and DC-1-76.1) indicated that both drugs produced naltrexone-reversible antinociception with a rapid onset and a duration of ∼1 h. Potency of both drugs increased when behavior was depressed by a lower IP-acid concentration, and neither drug alleviated behavioral depression by a non-pain stimulus (IP lithium chloride). Both drugs produced weaker GI inhibition and respiratory depression than fentanyl and attenuated fentanyl-induced GI inhibition and respiratory depression. Results support further consideration of selective, low-efficacy MOR agonists as candidate analgesics. SIGNIFICANCE STATEMENT This study used a novel set of mu opioid receptor (MOR)-selective opioids with graded MOR efficacies to examine the lower boundary of MOR efficacy sufficient to relieve pain-related behavioral depression in mice. Two novel low-efficacy opioids (JL-2-39, DC-1-76.1) produced effective antinociception with improved safety relative to higher- or lower-efficacy opioids, and results support further consideration of these and other low-efficacy opioids as candidate analgesics.

Substance use disorder (SUD) is a heterogeneous disorder, where severity, symptoms, and patterns of use vary across individuals. Yet, when rats self-administer cocaine under short-access conditions, their behavior tends to be well-regulated, though individual differences can emerge with long- or intermittent-access. In contrast, significant individual differences emerge when rats self-administer 3,4-methylenedioxypyrovalerone (MDPV), even under short-access conditions, wherein ~30 % of rats exhibit high levels of drug-taking. This study assessed SUD-like phenotypes of male and female rats self-administering MDPV or cocaine by comparing level of drug intake, responding during periods of signaled drug unavailability, and sensitivity to footshock punishment to determine whether: (1) under short-access conditions, rats that self-administer MDPV will exhibit a more robust SUD-like phenotype than rats that self-administer cocaine; (2) female rats will have a more severe phenotype than male rats; and (3) compared to short-access, long- and intermittent-access to MDPV or cocaine self-administration will result in a more robust SUD-like phenotype. Compared to cocaine, rats that self-administered MDPV exhibited a more severe phenotype, even under short-access conditions. Long- and intermittent-access to cocaine and MDPV temporarily altered drug-taking patterns but did not systematically change SUD-like phenotypes. Behavioral and quantitative autoradiography studies suggest phenotypic differences are not due to expression of dopamine transporter, dopamine D2 or D3 receptors, or 5-HT1B, 5-HT2A, or 5-HT2C receptors. This study suggests individuals who use synthetic cathinones may be at greater risk for developing a SUD, and short-access MDPV self-administration may provide a useful method to study the transition to disordered substance use in humans.