Effects of Repeated Treatment with the Monoacylglycerol Lipase Inhibitor MJN110 on Pain-Related Depression of Nesting and Cannabinoid 1 Receptor Function in Male and Female Mice.

MJN110 inhibits the enzyme monoacylglycerol lipase (MAGL) to increase levels of the endocannabinoid 2-arachidonoylglycerol , an endogenous high-efficacy agonist of cannabinoid 1 and 2 receptors (CB1/2R). MAGL inhibitors are under consideration as candidate analgesics, and we reported previously that acute MJN110 produced partial antinociception in an assay of pain-related behavioral depression in mice. Given the need for repeated analgesic administration in many pain patients and the potential for analgesic tolerance during repeated treatment, this study examined antinociceptive effects of repeated MJN110 on pain-related behavioral depression and CB1R-mediated G-protein function. Male and female ICR mice were treated daily for 7 days in a 2 × 2 design with (a) 1.0 mg/kg/d MJN110 or its vehicle followed by (b) intraperitoneal injection of dilute lactic acid (IP acid) or its vehicle as a visceral noxious stimulus to depress nesting behavior. After behavioral testing, G-protein activity was assessed in lumbar spinal cord (LSC) and five brain regions using an assay of CP55,940-stimulated [35S]GTPÉ£S activation. As reported previously, acute MJN110 produced partial but significant relief of IP acid-induced nesting depression on day 1. After 7 days, MJN110 continued to produce significant but partial antinociception in males, while antinociceptive tolerance developed in females. Repeated MJN110 also produced modest decreases in maximum levels of CP55,940-induced [35S]GTPÉ£S binding in spinal cord and most brain regions. These results indicate that repeated treatment with a relatively low antinociceptive MJN110 dose produces only partial and sex-dependent transient antinociception associated with the emergence of CB1R desensitization in this model of IP acid-induced nesting depression. SIGNIFICANCE STATEMENT: The drug MJN110 inhibits monoacylglycerol lipase (MAGL) to increase levels of the endogenous cannabinoid 2-arachidonoylglycerol and produce potentially useful therapeutic effects including analgesia. This study used an assay of pain-related behavioral depression in mice to show that repeated MJN110 treatment produced (1) weak but sustained antinociception in male mice, (2) antinociceptive tolerance in females, and (3) modest cannabinoid-receptor desensitization that varied by region and sex. Antinociceptive tolerance may limit the utility of MJN110 for treatment of pain.

Low-Efficacy Mu Opioid Agonists as Candidate Analgesics: Effects of Novel C-9 Substituted Phenylmorphans on Pain-Depressed Behavior in Mice.

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 ~1hr. 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.

Effects of Selective and Mixed-Action Kappa and Delta Opioid Receptor Agonists on Pain-Related Behavioral Depression in Mice.

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.

Role of Efficacy as a Determinant of Locomotor Activation by Mu Opioid Receptor Ligands in Female and Male Mice.

Mu opioid receptor (MOR) agonists produce locomotor hyperactivity in mice as one sign of opioid-induced motor disruption. The goal of this study was to evaluate the degree of MOR efficacy required to produce this hyperactivity. Full dose-effect curves were determined for locomotor activation produced in male and female Institute of Cancer Research (ICR) mice by (1) eight different single-molecule opioids with high to low MOR efficacy and (2) a series of fixed-proportion fentanyl/naltrexone mixtures with high to low fentanyl proportions. Data from the mixtures were used to quantify the efficacy requirement for MOR agonist-induced hyperactivity relative to efficacy requirements determined previously for other MOR agonist effects. Specifically, efficacy requirement was quantified as the EP50 value, which is the "Effective Proportion" of fentanyl in a fentanyl/naltrexone mixture that produces a maximal effect equal to 50% of the maximal effect of fentanyl alone. Maximal hyperactivity produced by each drug and mixture in the present study correlated with previously published data for maximal stimulation of GTPÉ£S binding in MOR-expressing Chinese hamster ovary cells as an in vitro measure of relative efficacy. Additionally, the EP50 value for hyperactivity induced by fentanyl/naltrexone mixtures indicated that opioid-induced hyperactivity in mice has a relatively high efficacy requirement in comparison with some other MOR agonist effects, and in particular is higher than the efficacy requirement for thermal antinociception in mice or fentanyl discrimination in rats. Taken together, these data show that MOR agonist-induced hyperactivity in mice is efficacy dependent and requires relatively high levels of MOR agonist efficacy for its full expression. SIGNIFICANCE STATEMENT: Mu opioid receptor (MOR) agonist-induced hyperlocomotion in mice is dependent on the MOR efficacy of the agonist and requires a relatively high degree of efficacy for its full expression.

Core Outcome Measures in Preclinical Assessment of Candidate Analgesics.

All preclinical procedures for analgesic drug discovery involve two components: 1) a "pain stimulus" (the principal independent variable), which is delivered to an experimental subject with the intention of producing a pain state; and 2) a "pain behavior" (the principal dependent variable), which is measured as evidence of that pain state. Candidate analgesics are then evaluated for their effectiveness to reduce the pain behavior, and results are used to prioritize drugs for advancement to clinical testing. This review describes a taxonomy of preclinical procedures organized into an "antinociception matrix" by reference to their types of pain stimulus (noxious, inflammatory, neuropathic, disease related) and pain behavior (unconditioned, classically conditioned, operant conditioned). Particular emphasis is devoted to pain behaviors and the behavioral principals that govern their expression, pharmacological modulation, and preclinical-to-clinical translation. Strengths and weaknesses are compared and contrasted for procedures using each type of behavioral outcome measure, and the following four recommendations are offered to promote strategic use of these procedures for preclinical-to-clinical analgesic drug testing. First, attend to the degree of homology between preclinical and clinical outcome measures, and use preclinical procedures with behavioral outcome measures homologous to clinically relevant outcomes in humans. Second, use combinations of preclinical procedures with complementary strengths and weaknesses to optimize both sensitivity and selectivity of preclinical testing. Third, take advantage of failed clinical translation to identify drugs that can be back-translated preclinically as active negative controls. Finally, increase precision of procedure labels by indicating both the pain stimulus and the pain behavior in naming preclinical procedures.

Some effects of putative G-protein biased mu-opioid receptor agonists in male rhesus monkeys.

G-protein-biased mu-opioid receptor (GPB-MOR) agonists are an emerging class of compounds being evaluated as candidate analgesics and agonist medications for opioid use disorder. Most of the basic pharmacology of GPB-MOR agonists has been conducted in rodents and much less is known how the basic behavioral pharmacology of these compounds translates to nonhuman primates. The present study determined the antinociceptive potency and time course of three putative GPB-MOR agonists: (+)-oliceridine (i.e. TRV130), SR14968, and SR17018 in male rhesus monkeys (n = 3). In addition, the respiratory effects of these compounds were also indirectly determined using a pulse oximeter to measure percent peripheral oxygen saturation (%SpO2). The largest intramuscular oliceridine dose (3.2 mg/kg) produced significant antinociception at 50°C, but not 54°C, and peak effects were between 10 and 30 min. Oliceridine also decreased SpO2 below the 90% threshold that would be clinically categorized as hypoxia in two out of three monkeys. The largest intramuscular SR14968 dose (0.32 mg/kg) produced 100% MPE at 50°C, but not 54°C, in two out of three monkeys, and peak effects were between 30 and 100 min. The largest intravenous SR17018 dose (1 mg/kg) produced 100% MPE at 50°C, but not 54°C, in the same two out of three monkeys, and peak effects were between 30 and 100 min. Solubility limitations for both SR14968 and SR17018 impaired our ability to determine in-vivo potency and effectiveness on antinociceptive and %SpO2 measures for these two compounds.

Sex differences in the effectiveness of buprenorphine to decrease rates of responding in rhesus monkeys.

Sex differences in µ-opioid receptor (MOR) agonist-induced antinociception have been reported in nonhuman primates. The degree to which µ-opioid receptor agonist sex differences in nonhuman primates extend to other behavioral endpoints remains unknown. The present study compared the behavioral effects of three MOR ligands (fentanyl, buprenorphine, and naltrexone) that varied in efficacy to stimulate [S]-GTPγS binding (from highest to lowest: fentanyl, buprenorphine, and naltrexone) in male and female rhesus monkeys. Male (n=3) and female (n=3) monkeys were trained to respond under a fixed-ratio 10 schedule of food presentation during daily sessions consisting of multiple components. Once rates of responding were stable, cumulative dose-effect functions were determined for intramuscular fentanyl (0.00032-0.032 mg/kg), buprenorphine (0.001-1 mg/kg), and naltrexone (0.01-0.1 mg/kg). Fentanyl dose-dependently decreased rates of responding in both sexes and the corresponding ED50 values were not significantly different. Buprenorphine dose-dependently decreased rates of responding in females, but not males. Naltrexone did not significantly alter behavior in either females or males. Overall, these results suggest that the expression of sex differences in MOR pharmacology depends upon both the efficacy of the MOR ligand and the behavioral endpoint.

Effects of the α2/α3-subtype-selective GABAA receptor positive allosteric modulator KRM-II-81 on pain-depressed behavior in rats: comparison with ketorolac and diazepam.

This study examined effects of the α2/α3-subtype-selective GABAA receptor positive allosteric modulator KRM-II-81 in an assay of pain-related behavioral depression. Adult, male Sprague-Dawley rats responded for electrical brain stimulation in a frequency-rate intracranial self-stimulation (ICSS) procedure. Intraperitoneal injection of 1.8% lactic acid served as an acute noxious stimulus to depress ICSS. Effects of KRM-II-81 were evaluated in the absence and presence of the acid noxious stimulus. The NSAID ketorolac and the benzodiazepine diazepam were tested as comparators. Neither ketorolac nor KRM-II-81 altered ICSS in the absence of the acid noxious stimulus; however, diazepam produced facilitation consistent with its abuse liability. Ketorolac blocked acid-induced depression of ICSS, and effects of 1.0 mg/kg ketorolac lasted for at least 5 h. KRM-II-81 (1.0 mg/kg) produced significant antinociception after 30 min that dissipated by 60 min. Diazepam also attenuated acid-depressed ICSS, but only at doses that facilitated ICSS when administered alone. The lack of ketorolac or KRM-II-81 effects on ICSS in the absence of the acid noxious stimulus suggests low abuse liability for both compounds. The effectiveness of ketorolac to block acid-induced ICSS depression agrees with clinical analgesic efficacy of ketorolac. KRM-II-81 produced significant but less consistent and shorter-acting antinociception than ketorolac.

Application of Receptor Theory to the Design and Use of Fixed-Proportion Mu-Opioid Agonist and Antagonist Mixtures in Rhesus Monkeys.

Receptor theory predicts that fixed-proportion mixtures of a competitive, reversible agonist (e.g., fentanyl) and antagonist (e.g., naltrexone) at a common receptor [e.g., mu-opioid receptors (MORs)] will result in antagonist proportion-dependent decreases in apparent efficacy of the agonist/antagonist mixtures and downward shifts in mixture dose-effect functions. The present study tested this hypothesis by evaluating behavioral effects of fixed-proportion fentanyl/naltrexone mixtures in a warm-water tail-withdrawal procedure in rhesus monkeys (n = 4). Fentanyl (0.001-0.056 mg/kg) alone, naltrexone (0.032-1.0 mg/kg, i.m.) alone, and fixed-proportion mixtures of fentanyl/naltrexone (1:0.025, 1:0.074, and 1:0.22) were administered in a cumulative-dosing procedure, and the proportions were based on published fentanyl and naltrexone Kd values at MOR in monkey brain. Fentanyl alone produced dose-dependent antinociception at both 50 and 54°C thermal intensities. Up to the largest dose tested, naltrexone alone did not alter nociception. Consistent with receptor theory predictions, naltrexone produced a proportion-dependent decrease in the effectiveness of fentanyl/naltrexone mixtures to produce antinociception. The maximum effects of fentanyl, naltrexone, and each mixture were also used to generate an efficacy-effect scale for antinociception at each temperature, and this scale was evaluated for its utility in quantifying 1) efficacy requirements for antinociception at 50 and 54°C and 2) relative efficacy of six MOR agonists that vary in their efficacies to produce agonist-stimuated GTPγS binding in vitro (from lowest to highest efficacy: 17-cyclopropylmethyl-3,14ß-dihyroxy-4,5α-epoxy-6α-[(3'-isoquinolyl)acetamindo]morphine, nalbuphine, buprenorphine, oxycodone, morphine, and methadone). These results suggest that fixed-proportion agonist/antagonist mixtures may offer a useful strategy to manipulate apparent drug efficacy for basic research or therapeutic purposes.

Interactions between Cocaine and the Putative Allosteric Dopamine Transporter Ligand SRI-31142.

Drugs that inhibit the dopamine (DA) transporter (DAT) include both therapeutic agents and abused drugs. Recent studies identified a novel series of putative allosteric DAT inhibitors, but the in vivo effects of these compounds are unknown. This study examined the abuse-related behavioral and neurochemical effects produced in rats by SRI-31142 [2-(7-methylimidazo[1,2-a]pyridin-6-yl)-N-(2-phenyl-2-(pyridin-4-yl)ethyl)quinazolin-4-amine], one compound from this series. In behavioral studies, intracranial self-stimulation (ICSS) was used to compare the effects produced by SRI-31142, the abused and nonselective DAT inhibitor cocaine, and the selective DAT inhibitor GBR-12935 [1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine]. In neurochemical studies, in vivo microdialysis was used to compare the effects of SRI-31142 and cocaine on levels of DA and serotonin in nucleus accumbens (NAc). The effects of SRI-31142 in combination with cocaine were also examined in both procedures. In contrast to cocaine and GBR-12935, SRI-31142 failed to produce abuse-related increases in ICSS or NAc DA; instead, SRI-31142 only decreased ICSS and NAc DA at a dose that was also sufficient to block cocaine-induced increases in ICSS and NAc DA. Pharmacokinetic studies suggested low but adequate brain penetration of SRI-31142, in vitro binding studies failed to identify likely non-DAT targets, and in vitro functional assays failed to confirm DA uptake inhibition in an assay of DAT-mediated fluorescent signals in live cells. These results indicate that SRI-31142 does not produce cocaine-like abuse-related effects in rats. SRI-31142 may have utility to block cocaine effects and may warrant further study as a candidate pharmacotherapy; however, the role of DAT in mediating these effects is unclear, and side effects may be a limiting factor.

Lack of paclitaxel effects on intracranial self-stimulation in male and female rats: comparison to mechanical sensitivity.

Paclitaxel is a cancer chemotherapy with adverse effects that include peripheral neuropathy, neuropathic pain, and depression of behavior and mood. In rodents, hypersensitive paw-withdrawal reflexes from mechanical stimuli serve as one common measure of paclitaxel-induced pain-related behavior. This study tested the hypothesis that paclitaxel would also depress rates of positively reinforced operant responding as a measure of pain-related behavioral depression. Male and female Sprague-Dawley rats were equipped with electrodes targeting the medial forebrain bundle, trained to lever press for electrical brain stimulation in an assay of intracranial self-stimulation (ICSS), and treated with four injections of varying paclitaxel doses (0.67, 2.0, or 6.0 mg/kg/injection×4 injections on alternate days). Mechanical sensitivity, body weight, and ICSS were evaluated before, during, and for 3 weeks after paclitaxel treatment. Paclitaxel doses sufficient to produce mechanical hypersensitivity did not reliably depress ICSS in male or female rats. Moreover, the degree of behavioral suppression in individual rats did not correlate with mechanical sensitivity. Paclitaxel treatment regimens commonly used to model chemotherapy-induced neuropathic pain in rats are not sufficient to depress ICSS.

Relief of Pain-Depressed Behavior in Rats by Activation of D1-Like Dopamine Receptors.

Clinically significant pain often includes a decrease in both behavior and mesolimbic dopamine signaling. Indirect and/or direct dopamine receptor agonists may alleviate pain-related behavioral depression. To test this hypothesis, the present study compared effects of indirect and direct dopamine agonists in a preclinical assay of pain-depressed operant responding. Male Sprague-Dawley rats with chronic indwelling microelectrodes in the medial forebrain bundle were trained in an intracranial self-stimulation (ICSS) procedure to press a lever for pulses of electrical brain stimulation. Intraperitoneal injection of dilute lactic acid served as an acute noxious stimulus to depress ICSS. Intraperitoneal lactic acid-induced depression of ICSS was dose-dependently blocked by the dopamine transporter inhibitor methylphenidate and the D1-selective agonist SKF82958, but not by the D2/3-selective agonists quinpirole, pramipexole, or sumanirole. The antinociceptive effects of methylphenidate and SKF82958 were blocked by the D1-selective antagonist SCH39166. Acid-induced stimulation of a stretching response was evaluated in separate groups of rats, but all agonists decreased acid-stimulated stretching, and antagonism experiments were inconclusive due to direct effects of the antagonists when administered alone. Taken together, these results suggest that D1-receptor stimulation is both sufficient to block acid-induced depression of ICSS and necessary for methylphenidate antinociception in this procedure. Conversely, D2/3-receptor stimulation is not sufficient to relieve pain-depressed behavior. These results support the hypothesis that pain-related depression of dopamine D1 receptor signaling contributes to pain-related depression of behavior in rats. Additionally, these results support further consideration of indirect dopamine agonists and direct D1 receptor agonists as candidate treatments for pain-related behavioral depression.

Effects of Acute and Chronic Treatments with Dopamine D2 and D3 Receptor Ligands on Cocaine versus Food Choice in Rats.

Dopamine D3 receptor ligands are potential medications for psychostimulant addiction. Medication assessment may benefit from preclinical studies that evaluate chronic medication effects on choice between an abused drug and an alternative, nondrug reinforcer. This study compared acute and chronic effects of dopamine D2- and D3-preferring ligands on choice between intravenous cocaine and palatable food in rats. Under baseline conditions, cocaine maintained dose-dependent increases in cocaine choice and reciprocal decreases in food choice. Acutely, the D2 agonist R-(-)-norpropylapomorphine (NPA) and antagonist L-741,626 [3-[[4-(4-chlorophenyl)-4-hydroxypiperidin-l-yl]methyl-1H-indole] produced leftward and rightward shifts in cocaine dose-effect curves, respectively, whereas the partial agonist terguride had no effect. All three drugs dose-dependently decreased food-maintained responding. Chronically, the effects of R-(-)-norpropylapomorphine and L-741,626 on cocaine self-administration showed marked tolerance, whereas suppression of food-reinforced behavior persisted. Acute effects of the D3 ligands were less systematic and most consistent with nonselective decreases in cocaine- and food-maintained responding. Chronically, the D3 agonist PF-592,379 [5-[(2R,5S)-5-methyl-4-propylmorpholin-2-yl]pyridin-2-amine] increased cocaine choice, whereas an intermediate dose of the D3 antagonist PG01037 [N-[(E)-4-[4-(2,3-dichlorophenyl)piperazin-1-yl]but-2-enyl]-4-pyridin-2-ylbenzamide] produced a therapeutically desirable decrease in cocaine choice early in treatment; however, tolerance to this effect developed, and lower and higher doses were ineffective. D3 ligands failed to significantly modify total cocaine intake but caused persistent decreases in food intake. Thus, D2-and D3-preferring ligands showed distinct profiles, consistent with different pharmacological actions. In addition, these results highlight the role of acute versus chronic treatment as a determinant of test drug effects. With the possible exception of the D3 antagonist PG01037, no ligand was promising in terms of cocaine addiction treatment.

Oral modafinil facilitates intracranial self-stimulation in rats: comparison with methylphenidate.

Modafinil is a low-potency inhibitor of dopamine transporters (DAT) approved clinically to promote wakefulness. In most procedures used for abuse-liability assessment, modafinil produces effects similar to those of abused DAT inhibitors such as cocaine and methylphenidate, although modafinil often shows lower effectiveness. However, modafinil has failed to maintain drug self-administration or produce conditioned place preferences in rats. The low potency and poor solubility of modafinil complicate its delivery by parenteral routes of administration commonly used in rats, and this may contribute toward negative results. This study evaluated the effects of orally administered modafinil in rats using an assay of intracranial self-stimulation (ICSS) that has been used to examine the effects of other DAT inhibitors. Adult male Sprague-Dawley rats equipped with electrodes in the medial forebrain bundle responded for pulses of brain stimulation that varied across a range of frequencies (158-56 Hz) during daily behavioral sessions. Modafinil (20-600 mg/kg, orally) and methylphenidate (1.0-10 mg/kg, intraperitoneally; 3.2-32 mg/kg, orally) produced dose-dependent and time-dependent facilitation of ICSS, an effect produced by abused DAT inhibitors and other classes of abused drugs. These results are in agreement with other evidence for stimulant-like abuse liability of modafinil and show the sensitivity of ICSS to orally administered drug.

Cocaine-like discriminative stimulus effects of alpha-pyrrolidinovalerophenone, methcathinone and their 3,4-methylenedioxy or 4-methyl analogs in rhesus monkeys.

Synthetic cathinones are beta-ketone amphetamine analogs that have emerged as a heterogeneous class of abused compounds that function as either monoamine transporter substrates or inhibitors. Pre-clinical drug discrimination procedures are useful for interrogating structure-activity relationships of abuse-related drug effects; however, in vivo structure-activity relationship comparisons between synthetic cathinones with different mechanisms of action are lacking. The aim of the present study was to determine whether the cocaine-like discriminative stimulus effects of the monoamine transporter inhibitor alpha-pyrrolidinovalerophenone (alpha-PVP) and the monoamine transporter substrate methcathinone were differentially sensitive to 3,4-methylenedioxy and 4-methyl substitutions. Male rhesus monkeys (n = 4) were trained to discriminate intramuscular cocaine (0.32 mg/kg) from saline in a two-key food-reinforced discrimination procedure. Potency and timecourse of cocaine-like discriminative stimulus effects were determined for (±)-alpha-PVP, (±)-methcathinone and their 3,4-methylenedioxy or 4-methyl analogs. Alpha-PVP and methcathinone produced dose- and time-dependent cocaine-like effects. A 3,4-methylenedioxy addition to either alpha-PVP or methcathinone (methylone) did not alter the potency or efficacy to produce cocaine-like effects, but did prolong the time course. A 4-methyl addition to alpha-PVP (pyrovalerone) did not alter the potency or efficacy to produce cocaine-like effects, but did prolong the time course. In contrast, addition of a 4-methyl moiety to methcathinone (4MMC; mephedrone) significantly attenuated efficacy to produce cocaine-like effects. Overall, these results suggest different structural requirements for cocaine-like discriminative stimulus effects of monoamine transporter inhibitor and substrate synthetic cathinone analogs. Given that 4MMC is more hydrophobic than MDMC, these results suggest that hydrophobicity may be an important determinant for limiting monoamine transporter substrate abuse-related behavioral effects.

Intracranial self-stimulation to evaluate abuse potential of drugs.

Intracranial self-stimulation (ICSS) is a behavioral procedure in which operant responding is maintained by pulses of electrical brain stimulation. In research to study abuse-related drug effects, ICSS relies on electrode placements that target the medial forebrain bundle at the level of the lateral hypothalamus, and experimental sessions manipulate frequency or amplitude of stimulation to engender a wide range of baseline response rates or response probabilities. Under these conditions, drug-induced increases in low rates/probabilities of responding maintained by low frequencies/amplitudes of stimulation are interpreted as an abuse-related effect. Conversely, drug-induced decreases in high rates/probabilities of responding maintained by high frequencies/amplitudes of stimulation can be interpreted as an abuse-limiting effect. Overall abuse potential can be inferred from the relative expression of abuse-related and abuse-limiting effects. The sensitivity and selectivity of ICSS to detect abuse potential of many classes of abused drugs is similar to the sensitivity and selectivity of drug self-administration procedures. Moreover, similar to progressive-ratio drug self-administration procedures, ICSS data can be used to rank the relative abuse potential of different drugs. Strengths of ICSS in comparison with drug self-administration include 1) potential for simultaneous evaluation of both abuse-related and abuse-limiting effects, 2) flexibility for use with various routes of drug administration or drug vehicles, 3) utility for studies in drug-naive subjects as well as in subjects with controlled levels of prior drug exposure, and 4) utility for studies of drug time course. Taken together, these considerations suggest that ICSS can make significant contributions to the practice of abuse potential testing.

Abuse-Related Neurochemical Effects of Para-Substituted Methcathinone Analogs in Rats: Microdialysis Studies of Nucleus Accumbens Dopamine and Serotonin.

Methcathinone (MCAT) is a monoamine releaser and parent compound to a new class of designer drugs that includes the synthetic cathinones mephedrone and flephedrone. Using MCAT and a series of para-substituted (or 4-substituted) MCAT analogs, it has been previously shown that expression of abuse-related behavioral effects in rats correlates both with the volume of the para substituent and in vitro neurochemical selectivity to promote monoamine release via the dopamine (DA) versus serotonin (5-HT) transporters in rat brain synaptosomes. The present study used in vivo microdialysis to determine the relationship between these previous measures and the in vivo neurochemical selectivity of these compounds to alter nucleus accumbens (NAc) DA and 5-HT levels. Male Sprague-Dawley rats were implanted with bilateral guide cannulae targeting the NAc. MCAT and five para-substituted analogs (4-F, 4-Cl, 4-Br, 4-CH3, and 4-OCH3) produced dose- and time-dependent increases in NAc DA and/or 5-HT levels. Selectivity was determined as the dose required to increase peak 5-HT levels by 250% divided by the dose required to increase peak DA levels by 250%. This measure of in vivo neurochemical selectivity varied across compounds and correlated with 1) in vivo expression of abuse-related behavioral effects (r = 0.89, P = 0.02); 2) in vitro selectivity to promote monoamine release via DA and 5-HT transporters (r = 0.95, P < 0.01); and 3) molecular volume of the para substituent (r = -0.85, P = 0.03). These results support a relationship between these molecular, neurochemical, and behavioral measures and support a role for molecular structure as a determinant of abuse-related neurochemical and behavioral effects of MCAT analogs.

Preclinical Abuse Potential Assessment of Flibanserin: Effects on Intracranial Self-Stimulation in Female and Male Rats.

INTRODUCTION: Flibanserin is a serotonin receptor subtype 1A agonist and 2A antagonist that has been approved by the Food and Drug Administration for treating female sexual interest and arousal disorder. Little is known about the abuse potential of flibanserin. AIM: To examine abuse-related effects of flibanserin in rats using an intracranial self-stimulation (ICSS) procedure previously used to evaluate the abuse potential of other drugs. METHODS: Adult female and male Sprague-Dawley rats with electrodes implanted in the medial forebrain bundle were trained to press a lever for electrical brain stimulation under a "frequency-rate" ICSS procedure. In this procedure, increasing frequencies of brain stimulation maintain increasing rates of responding. Drugs of abuse typically increase (or "facilitate") ICSS rates and produce leftward and upward shifts in ICSS frequency-rate curves, whereas drugs that lack abuse potential typically do not alter or only decrease ICSS rates. Initial studies determined the potency and time course of effects on ICSS produced by acute flibanserin administration (1.0, 3.2 and 10.0 mg/kg). Subsequent studies determined the effects of flibanserin (3.2-18 mg/kg) before and after a regimen of repeated flibanserin administration (5.6 mg/kg/d for 5 days). Effects of the abused stimulant amphetamine (1.0 mg/kg) were examined as a positive control. MAIN OUTCOME MEASURES: Flibanserin effects on ICSS frequency-rate curves in female and male rats were examined and compared with the effects of amphetamine. RESULTS: Baseline ICSS frequency-rate curves were similar in female and male rats. Acute and repeated administrations of flibanserin produced only decreases in ICSS rates, and rate-decreasing effects of the highest flibanserin dose (10 mg/kg) were greater in female than in male rats. In contrast to flibanserin, amphetamine produced an abuse-related increase in ICSS rates that did not differ between female and male rats. CONCLUSION: These results suggest that flibanserin has low abuse potential. In addition, this study suggests that female rats might be more sensitive than male rats to the rate-decreasing effects of high flibanserin doses.

Pharmacological modulation of neuropathic pain-related depression of behavior: effects of morphine, ketoprofen, bupropion and [INCREMENT]9-tetrahydrocannabinol on formalin-induced depression of intracranial self-stimulation in rats.

Neuropathic pain is often associated with behavioral depression. Intraplantar formalin produces sustained, neuropathy-associated depression of intracranial self-stimulation (ICSS) in rats. This study evaluated pharmacological modulation of formalin-induced ICSS depression. Rats with intracranial electrodes targeting the medial forebrain bundle responded for electrical brain stimulation in an ICSS procedure. Bilateral intraplantar formalin administration depressed ICSS for 14 days. Morphine (0.32-3.2 mg/kg), ketoprofen (0.1-10 mg/kg), bupropion (3.2-32 mg/kg), and [INCREMENT]9-tetrahydrocannabinol (THC; 0.32-3.2 mg/kg) were evaluated for their effectiveness to reverse formalin-induced depression of ICSS. Drug effects on formalin-induced mechanical allodynia were evaluated for comparison. Morphine and bupropion reversed both formalin-induced ICSS depression and mechanical allodynia, and effects on ICSS were sustained during repeated treatment. Ketoprofen failed to reverse either formalin effect. THC blocked mechanical allodynia, but decreased ICSS in control rats and exacerbated formalin-induced depression of ICSS. The failure of ketoprofen to alter formalin effects suggests that formalin effects result from neuropathy rather than inflammation. The effectiveness of morphine and bupropion to reverse formalin effects agrees with other evidence that these drugs block pain-depressed behavior in rats and relieve neuropathic pain in humans. The effects of THC suggest general behavioral suppression and do not support the use of THC to treat neuropathic pain.

Cocaine-like discriminative stimulus effects of phendimetrazine and phenmetrazine in rats.

Phendimetrazine is a clinically available anorectic and candidate medication for the treatment of cocaine addiction. Phendimetrazine can be metabolized to the amphetamine-like monoamine releaser phenmetrazine, but it is unclear if phendimetrazine functions as an inactive prodrug or might have activity on its own. As one method to address this issue, the present study compared the potency and time course of phendimetrazine and phenmetrazine to produce cocaine-like discriminative stimulus effects in adult, male rats (N=5) trained to discriminate cocaine (5.6 mg/kg, intraperitoneally) from saline in a two-key food-reinforced discrimination procedure. We hypothesized that, if metabolism to phenmetrazine was required for phendimetrazine effects, then phendimetrazine would be less potent and have a slower onset and offset of effects than phenmetrazine. Both phendimetrazine and phenmetrazine produced dose-dependent cocaine-like discriminative stimulus effects, and phendimetrazine was 7.8-fold less potent than phenmetrazine. However, the time courses of discriminative stimulus effects produced by phendimetrazine and phenmetrazine were similar, with peak effects at 10 min and offset by 100 min. These results show the effectiveness of phendimetrazine to rapidly produce cocaine-like behavioral effects in rats and support other nonhuman primate evidence to suggest that metabolism to phenmetrazine may not be required for phendimetrazine effects.