BU08073 a buprenorphine analogue with partial agonist activity at µ-receptors in vitro but long-lasting opioid antagonist activity in vivo in mice.

BACKGROUND AND PURPOSE: Buprenorphine is a potent analgesic with high affinity at µ, δ and κ and moderate affinity at nociceptin opioid (NOP) receptors. Nevertheless, NOP receptor activation modulates the in vivo activity of buprenorphine. Structure activity studies were conducted to design buprenorphine analogues with high affinity at each of these receptors and to characterize them in in vitro and in vivo assays. EXPERIMENTAL APPROACH: Compounds were tested for binding affinity and functional activity using [(35) S]GTPγS binding at each receptor and a whole-cell fluorescent assay at µ receptors. BU08073 was evaluated for antinociceptive agonist and antagonist activity and for its effects on anxiety in mice. KEY RESULTS: BU08073 bound with high affinity to all opioid receptors. It had virtually no efficacy at δ, κ and NOP receptors, whereas at µ receptors, BU08073 has similar efficacy as buprenorphine in both functional assays. Alone, BU08073 has anxiogenic activity and produces very little antinociception. However, BU08073 blocks morphine and U50,488-mediated antinociception. This blockade was not evident at 1 h post-treatment, but is present at 6 h and remains for up to 3-6 days. CONCLUSIONS AND IMPLICATIONS: These studies provide structural requirements for synthesis of 'universal' opioid ligands. BU08073 had high affinity for all the opioid receptors, with moderate efficacy at µ receptors and reduced efficacy at NOP receptors, a profile suggesting potential analgesic activity. However, in vivo, BU08073 had long-lasting antagonist activity, indicating that its pharmacokinetics determined both the time course of its effects and what receptor-mediated effects were observed. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Activities of mixed NOP and mu-opioid receptor ligands.

BACKGROUND AND PURPOSE: Compounds that activate both NOP and mu-opioid receptors might be useful as analgesics and drug abuse medications. Studies were carried out to better understand the biological activity of such compounds. EXPERIMENTAL APPROACH: Binding affinities were determined on membranes from cells transfected with NOP and opioid receptors. Functional activity was determined by [(35)S]GTPgammaS binding on cell membranes and using the mouse vas deferens preparation in vitro and the tail flick antinociception assay in vivo. KEY RESULTS: Compounds ranged in affinity from SR14150, 20-fold selective for NOP receptors, to buprenorphine, 50-fold selective for mu-opioid receptors. In the [(35)S]GTPgammaS assay, SR compounds ranged from full agonist to antagonist at NOP receptors and most were partial agonists at mu-opioid receptors. Buprenorphine was a low efficacy partial agonist at mu-opioid receptors, but did not stimulate [(35)S]GTPgammaS binding through NOP. In the mouse vas deferens, each compound, except for SR16430, inhibited electrically induced contractions. In each case, except for N/OFQ itself, the inhibition was due to mu-opioid receptor activation, as determined by equivalent results in NOP receptor knockout tissues. SR14150 showed antinociceptive activity in the tail flick test, which was reversed by the opioid antagonist naloxone. CONCLUSIONS AND IMPLICATIONS: Compounds that bind to both mu-opioid and NOP receptors have antinociceptive activity but the relative contribution of each receptor is unclear. These experiments help characterize compounds that bind to both receptors, to better understand the mechanism behind their biological activities, and identify new pharmacological tools to characterize NOP and opioid receptors.

Dopamine receptor DRD2 genotype and smoking cessation outcome following treatment with bupropion SR.

The A1 allele of the dopamine D2 receptor gene (DRD2) is associated with a reduced number of dopamine binding sites in the brain and with the increased likelihood of substance abuse and addictive behavior. In a study of smokers enrolled in an open-label, randomized effectiveness trial, we investigated whether variants in the DRD2 receptor gene are associated with smoking cessation outcomes following treatment with a combination of bupropion SR and behavioral counseling. Adherence to treatment and point-prevalent smoking status were assessed at 3 and 12 months, respectively, following a target quit date. Compared to women who carry both A2 alleles, women with at least one A1 allele were more likely to report having stopped taking bupropion due to medication side effects (odds ratio (OR)=1.91, 95% confidence interval (CI)=1.01-3.60; P<0.04) and at 12 months were somewhat more likely to report smoking (OR=0.76, 95% CI=0.56-1.03; P<0.076). Significant associations or trends were not observed in men. In women, individual variability in responsiveness to bupropion-based treatment may be partially due to differences in genetic variants influencing dopamine receptor function.

N-terminal modifications leading to peptide ORL1 partial agonists and antagonists.

Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino acid peptide that is the endogenous ligand for the G protein-coupled receptor (opioid receptor like 1, ORL1), a member of the opioid receptor family. Although it is clear that this receptor system is involved in a variety of physiological functions, including analgesia, the precise actions of N/OFQ remain largely uncharacterized. One reason for this has been limited high affinity ligands to ORL1, and particularly the lack of availability of useful specific antagonists. Herein we describe the pharmacological activity of a series of N-terminally modified hexapeptides with high affinity for ORL1. These compounds were tested for binding affinity using [3H]N/OFQ binding to human ORL1 in CHO cells, and functional activity by measuring stimulation of [35S]GTPgammaS binding in CHO cell membranes. The N-terminal modifications have produced compounds that maintained very high receptor affinity, but led to significant changes in intrinsic activity. One compound, pentanoyl-RYYRWR-NH2, with barely measurable agonist activity was tested in vivo. It was found to possess modest analgesic activity, but it was unable to block the morphine modulatory activity of N/OFQ.

Dopamine D1- and D2-like receptor mechanisms in relapse to cocaine-seeking behavior: effects of selective antagonists and agonists.

Dopaminergic mechanisms are thought to be critical in mediating relapse to cocaine-seeking behavior. This study examined the different roles of D1- and D2-like receptor mechanisms in the relapse process. Squirrel monkeys were given extended histories of i. v. cocaine self-administration under conditions in which responding was maintained jointly by response-contingent cocaine injections and a cocaine-paired visual stimulus (second-order schedule). Responding was then extinguished by substituting saline for cocaine injections and omitting presentations of the cocaine-paired stimulus. Subsequently, noncontingent priming injections of cocaine combined with restoration of the cocaine-paired stimulus induced dose-dependent reinstatement of drug-seeking behavior, with response rates approaching those maintained by active cocaine self-administration. The priming effects of cocaine were attenuated by several D1- and D2-like receptor antagonists and low efficacy agonists but not by the D3-preferring antagonists UH 232 and AJ-76. The priming effects of cocaine were mimicked by the D2-like receptor agonists R(-)-propylnorapomorphine hydrochloride (NPA) and quinpirole, less consistently by 7-OH-DPAT, and not by the D1-like receptor agonists SKF-81297 and SKF-82958, the D3-preferring agonist PD-128,907, or any low efficacy agonist. Cotreatment with NPA, PD-128,907, and 7-OH-DPAT did not alter reinstatement of drug-seeking behavior induced by a maximally effective priming dose of cocaine, whereas cotreatment with D1-like receptor agonists attenuated the priming effects of cocaine. The results suggest that D1- and D2-like receptors play fundamentally different roles in the relapse process. Although stimulation of D2-like, but probably not D3-like, receptors appears necessary for induction of relapse, either stimulation or blockade of D1-like receptors appears to be inhibitory with respect to relapse.

Pharmacological and environmental determinants of relapse to cocaine-seeking behavior.

Animal models have been developed that simulate relevant features of relapse to cocaine-seeking behavior in humans. These models have provided valuable information about pharmacological and environmental factors that precipitate reinstatement of extinguished cocaine-seeking in rats and monkeys, as well as new insights about potential pharmacotherapies for relapse prevention. Reinstatement of cocaine-seeking behavior in animals can be induced by cocaine priming or by cocaine-paired environmental stimuli: however, maximum reinstatement of drug-seeking appears to be induced when cocaine priming and cocaine-paired stimuli are combined. Drugs that share cocaine's indirect dopamine agonist properties or that act as direct agonists at D2-like dopamine receptors also induce reinstatement of cocaine-seeking behavior, whereas with some exceptions (e.g., caffeine, morphine) drugs from other pharmacological classes do not. D1-like receptor agonists block rather than mimic the priming effects of cocaine, suggesting different roles for D1- and D2-like receptor mechanisms in cocaine relapse. Although considerable overlap exists, drugs that exhibit cocaine-like discriminative stimulus and/ or reinforcing effects in other situations do not invariably induce cocaine-like reinstatement of drug-seeking and vice versa, implying that these effects are not simply different behavioral expressions of a unitary neurobiological process. Finally, recent findings with D1-like receptor agonists, partial agonists, and antagonists suggest that some of these drugs may be viable candidates for development as antirelapse pharmacotherapies.

Behavioral interactions produced by co-administration of 7-OH-DPAT with cocaine or apomorphine in the rat.

Previous research from our laboratory suggests that low doses (<0.1 mg/kg) of the dopamine (DA) D3-preferring agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) attenuate conditioned place preference (CPP) produced by the indirect DA agonist d-amphetamine, but enhance d-amphetamine-induced stereotypic behaviors. This study further examined the effects of 7-OH-DPAT on behaviors produced by the indirect DA agonist, cocaine, and the non-selective direct DA agonist, apomorphine. To examine whether 7-OH-DPAT would alter cocaine and apomorphine dose-response curves for motor behaviors and CPP, 0.1 mg/kg 7-OH-DPAT was co-administered with 0-30 mg/kg cocaine and 0-3 mg/kg apomorphine. To establish place conditioning, drug injections were paired with one of two distinctly different compartments, whereas saline injections were paired with the other compartment. Locomotion, sniffing, oral stereotypy, and headbobbing were measured following acute and repeated drug administration during conditioning, and place conditioning was assessed 24 h following the last conditioning day. 7-OH-DPAT enhanced cocaine- and apomorphine-induced stereotypies following repeated administration. 7-OH-DPAT also attenuated cocaine-CPP, but potentiated apomorphine-CPP. Furthermore, 7-OH-DPAT attenuated locomotion produced by high doses of apomorphine. The attenuation of cocaine-CPP by 7-OH-DPAT likely involves stimulation of D2/D3 autoreceptors in the mesolimbic pathway, whereas the potentiation of apomorphine-CPP likely involves stimulation of D2/D3 postsynaptic receptors. Furthermore, it is suggested that attenuation of apomorphine-induced locomotion by 7-OH-DPAT likely involves stimulation of postsynaptic D3 receptors in the mesolimbic pathway. Thus, if postsynaptic D3 receptors are involved in mediating CPP and locomotion, then stimulation of D3 receptors may facilitate CPP but inhibit locomotion.

Differential effects of 7-OH-DPAT on amphetamine-induced stereotypy and conditioned place preference.

Low doses of the dopamine D3-preferring agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) produce a behavioral profile that is opposite to that produced by the psychomotor stimulants cocaine and amphetamine. For example, low doses of 7-OH-DPAT produce conditioned place aversion and hypolocomotion, whereas psychomotor stimulants produce conditioned place preference (CPP) and hyperlocomotion. In experiment 1, the effects of low doses of 7-OH-DPAT (0.01-0.1 mg/kg) on d-amphetamine-induced (1 mg/kg) motor behaviors and CPP were assessed. In experiment 2, the effects of 0.1 mg/kg 7-OH-DPAT on d-amphetamine (0-10 mg/kg) dose-response curves for the same behaviors were examined. During conditioning, drug injections were paired with a distinct compartment, whereas saline injections were paired with another compartment. Locomotion and headbobbing were measured following acute and repeated drug administration during conditioning and place conditioning was assessed 24 h following the last conditioning day. In experiment 1, d-amphetamine-induced locomotion was dose-dependently decreased by 7-OH-DPAT following repeated administration, which was probably due to the emergence of headbobbing, a behavior not observed with d-amphetamine alone. d-Amphetamine-CPP was not altered by co-administration of 0-0.03 mg/kg 7-OH-DPAT, but was attenuated by co-administration of 0.1 mg/kg 7-OH-DPAT. In experiment 2, 7-OH-DPAT co-administered with low doses of d-amphetamine (0-0.5 mg/kg) produced a decrease in locomotion following acute administration. However, 7-OH-DPAT produced sensitization of locomotion at the 0.5 mg/kg dose of d-amphetamine and an increase in headbobbing at the 0.5-10 mg/kg doses of d-amphetamine following repeated administration. In contrast, d-amphetamine-CPP was attenuated by co-administration of 7-OH-DPAT. These findings suggest that 0.1 mg/kg 7-OH-DPAT attenuates the reinforcing effects of d-amphetamine despite enhancing stereotypic behaviors.

Effects of intraaccumbens administration of SCH-23390 on cocaine-induced locomotion and conditioned place preference.

The effects of systemic (0-1.0 mg/kg) or intraaccumbens (0-1.0 microg/side) administration of SCH-23390 on cocaine-induced (0 or 4.2 mg/kg, i.v.) locomotion, sniffing, and conditioned place preference (CPP) were investigated in rats. After behavioral testing was completed, animals were injected with their respective dose of SCH-23390 into the nucleus accumbens (NAc), followed by a systemic injection of the irreversible antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Receptors occupied by intraaccumbens SCH-23390, and therefore protected from EEDQ-induced inactivation, were then quantified from autoradiograms of sections labeled with 3H-SCH-23390. Systemic administration of 0.5 and 1.0 mg/kg SCH-23390 reversed cocaine-induced locomotion, sniffing, and CPP, suggesting that stimulation of D1-like receptors is necessary for these behavioral changes. Intraaccumbens administration of 1.0 microg/side SCH-23390 reversed cocaine-CPP, and this dose occupied D1-like receptors primarily in the rostral pole of the NAc. Intraaccumbens administration of 0.5 microg/side SCH-23390 reversed cocaine-induced locomotion. However, this dose occupied a similar number of D1-like receptors in the NAc as a lower and behaviorally ineffective dose of 0.1 microg/side, but occupied more receptors in the caudate-putamen relative to both the 0.1 and 1.0 microg/side doses. These findings suggest that stimulation of D1-like receptors in the NAc is necessary for cocaine-CPP, but not for cocaine-induced locomotion.

Effects of SCH-23390 on dopamine D1 receptor occupancy and locomotion produced by intraaccumbens cocaine infusion.

This study examined the effects of both systemic and intraaccumbens administration of SCH-23390 in rats on dopamine D1 receptor occupancy and on locomotor activity produced by intraaccumbens infusion of cocaine. In experiment 1, rats received SCH-23390 (0-1 mg/kg, i.p.) 15 minutes prior to intraaccumbens infusion of cocaine (0 or 100 microg/side). In experiment 2, rats received coinfusion of SCH-23390 (0-1 microg/side) and cocaine (0 or 100 microg/side) into the nucleus accumbens (NAc). After behavioral testing, receptors occupied by SCH-23390 were quantified by injecting animals with their respective dose of SCH-23390, followed by a systemic injection of the irreversible antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Receptors occupied by SCH-23390, and therefore protected from EEDQ-induced inactivation, were quantified from autoradiograms of sections labeled with 3H-SCH-23390. Systemic administration of SCH-23390 dose-dependently (0.1-1.0 mg/kg) reversed cocaine-induced locomotion and occupied 72-100% of D1-like receptors in the anterior NAc. D1 receptor occupancy following systemic administration of SCH-23390 was evident as an inverted U-shaped, dose-dependent change, with the greatest occupancy observed at the intermediate dose of 0.3 mg/kg. Intraaccumbens infusion of SCH-23390 did not alter cocaine-induced locomotor activity despite occupying 40-60% of D1-like receptors in the anterior NAc core and shell. The findings that systemic, but not intraaccumbens, administration of SCH-23390 potently reversed locomotion produced by intraaccumbens cocaine infusion suggest that stimulation of D1 receptors in regions other than the NAc is involved in locomotion produced by intraaccumbens infusion of cocaine, and that stimulation of D1 receptors in the NAc is not necessary for this behavior.

Effects of D3-preferring agonists 7-OH-PIPAT and PD-128,907 on motor behaviors and place conditioning.

Dose-dependent effects of 7-OH-PIPAT and PD-128,907 on motor behaviors and place conditioning were examined in rats. Four 2-day conditioning trials were conducted over 8 consecutive days. On one day of each trial, animals received an injection of either saline, one of six doses of 7-OH-PIPAT (0.01-10.0 mg/kg), or one of five doses of PD-128,907 (0.01-1.0 mg/kg), and were placed into a distinct compartment for 40 min. On the other day, animals received an injection of saline and were placed into a different compartment for 40 min. Locomotion, sniffing, and yawning were measured following the first and last drug injections. Place conditioning was assessed the day following the last conditioning trial. None of the doses of 7-OH-PIPAT or 0-0.3 mg/kg PD-128,907 produced place conditioning. However, 1 mg/kg PD-128,907 produced conditioned place preference (CPP). Across doses, both 7-OH-PIPAT and PD-128,907 produced a U-shaped change in sniffing and locomotion and an inverted U-shaped change in yawning. Across time, lower doses produced a decrease in sniffing and locomotion and an increase in yawning that were evident immediately, whereas higher doses produced a biphasic change in that there was an initial decrease followed by an increase in sniffing and locomotion. Behaviors produced by both low and high doses were sensitized following repeated administration. PD-128,907 produced CPP and was more potent than 7-OH-PIPAT in altering motor behaviors, possibly due to its greater selectivity for the D3 receptor.

Differential effects of intra-accumbens sulpiride on cocaine-induced locomotion and conditioned place preference.

The effects of intra-accumbens sulpiride on conditioned place preference and locomotion produced by i.v. cocaine were investigated. Every other day during conditioning, rats received infusions of sulpiride (0-0.4 microgram) into the nucleus accumbens (NAc) or caudate-putamen. Fifteen min later, they were placed into a distinct compartment and injected with saline or cocaine (4.2 mg/kg, i.v.). On the alternate days, rats received sham intracranial injections and were placed into a different compartment. Locomotion and stereotypies were assessed after the first and last injection, and conditioned place preference was assessed 24 hr after the last conditioning day. After behavioral testing, receptors occupied by sulpiride were quantified by injecting rats intracranially with their respective dose of sulpiride, followed by a systemic injection of the irreversible antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Receptors protected from EEDQ induced inactivation by sulpiride were revealed on autoradiograms of sections labeled with 3H-sulpiride. Sulpiride did not alter cocaine-conditioned place preference or cocaine-induced stereotypies. However, the two lowest doses of intra-accumbens sulpiride attenuated cocaine-induced locomotion and occupied > 42% of the sulpiride binding sites in the NAc, and the highest dose completely reversed cocaine-induced locomotion and occupied > 96% of the sulpiride binding sites in the NAc. Intracaudate sulpiride also attenuated cocaine-induced locomotion without occupying a significant number of binding sites in the NAc. These findings suggest that D2-like receptors in the NAc and anterior medial caudate-putamen are involved in cocaine-induced locomotion, but not cocaine-conditioned place preference.

Dose-dependent characterization of the rewarding and stimulant properties of cocaine following intraperitoneal and intravenous administration in rats.

Dose-dependent differences in the rewarding and stimulant properties of cocaine administered intravenously (IV) and intraperitoneally (IP) were compared. Six 2-day conditioning trials were conducted over consecutive days. Rats received cocaine and were placed into a compartment on one day of the trial, and were directly placed into a different compartment without drug on the other day. Rats were exposed to the compartments for either 20 or 40 min. The effects of cocaine on stimulant behaviors, including locomotion and stereotypies, were compared following the first and last injection. After conditioning, three tests were given with 1 rest day intervening each: (1) conditioned place preference (CPP) was measured as an increase in the amount of time animals spent in the injection compartment relative to the noninjection compartment when given access to both, (2) conditioned activity (CA) was measured as an increase in stimulant behaviors in cocaine-treated animals relative to saline controls following an injection of saline in the injection compartment and (3) context-independent sensitization was measured as an increase in stimulant behaviors following an injection of cocaine in the noninjection compartment relative to the animals' behavior following the first injection. Cocaine did not reliably produce sensitization of locomotion under any of the conditions examined. Cocaine produced sensitization of headbobbing that was more robust following IP administration than it was following IV administration. In both cases, sensitization of headbobbing involved a context-independent component. Cocaine produced CPP and CA with both routes of administration. CPP was established more readily with 40-min relative to 20-min exposures following IV administration, whereas CA was more prevalent with 20-min relative to 40-min exposures. This study provides a thorough characterization of the behavioral effects of cocaine administered IV and a new efficient method for assessing the effects of cocaine on conditioned and unconditioned behaviors following repeated administration.

Dose-dependent effects of the D3-preferring agonist 7-OH-DPAT on motor behaviors and place conditioning.

Dose-dependent effects of 7-OH-DPAT on several behaviors, including place preference, were assessed. Three 2-day conditioning trials were conducted. On 1 day, animals received an injection of one of eight doses of 7-OH-DPAT (0-5 mg/kg) and were placed into a distinct compartment for 40 min. On the other day, animals received an injection of saline and were placed into a different compartment for 40 min. Locomotion, sniffing, and yawning were measured following the first and last injection of 7-OH-DPAT. Place conditioning was assessed on the day following the last trial. 7-OH-DPAT produced a U-shaped dose-dependent change in locomotion and sniffing, and an inverted U-shaped dose-dependent change in yawning. Additionally, repeated administration of 0.1 mg/kg sensitized yawning, whereas 5 mg/kg sensitized locomotion. None of the doses of 7-OH-DPAT produced conditioned place preference, however, there was a trend for conditioned place aversion at 0.03 mg/kg. By contrast, LiCl (127 mg/kg) produced conditioned place aversion and amphetamine (1 mg/kg) produced conditioned place preference using the same conditioning parameters. A subsequent experiment in which the number of animals and conditioning trials were increased demonstrated that the 0.03 mg/kg dose of 7-OH-DPAT produced conditioned place aversion. 7-OH-DPAT has a higher affinity for D3 receptors relative to D2 receptors. Therefore, it is suggested that intermediate doses (0.01-0.1 mg/kg) that increase yawning, and decrease locomotion and sniffing, may preferentially occupy D3 receptors. Furthermore, the results suggest that these putative D3-preferring doses have weak aversive effects.