Cognitive-Enhancing Effects of Acetylcholine Receptor Agonists in Group-Housed Cynomolgus Monkeys Who Drink Ethanol.

The cognitive impairments that are often observed in patients with alcohol use disorder (AUD) partially contribute to the extremely low rates of treatment initiation and adherence. Brain acetylcholine receptors (AChR) mediate and modulate cognitive and reward-related behavior, and their distribution can be altered by long-term heavy drinking. Therefore, AChRs are promising pharmacotherapeutic targets for treating the cognitive symptoms of AUD. In the present study, the procognitive efficacy of two AChR agonists, xanomeline and varenicline, were evaluated in group-housed monkeys who self-administered ethanol for more than 1 year. The muscarinic AChR antagonist scopolamine was used to disrupt performance of a serial stimulus discrimination and reversal (SDR) task designed to probe cognitive flexibility, defined as the ability to modify a previously learned behavior in response to a change in reinforcement contingencies. The ability of xanomeline and varenicline to remediate the disruptive effects of scopolamine was compared between socially dominant and subordinate monkeys, with lighter and heavier drinking histories, respectively. We hypothesized that subordinate monkeys would be more sensitive to all three drugs. Scopolamine dose-dependently impaired performance on the serial SDR task in all monkeys at doses lower than those that produced nonspecific impairments (e.g., sedation); its potency did not differ between dominant and subordinate monkeys. However, both AChR agonists were effective in remediating the scopolamine-induced deficit in subordinate monkeys but not in dominant monkeys. These findings suggest xanomeline and varenicline may be effective for enhancing cognitive flexibility in individuals with a history of heavy drinking. SIGNIFICANCE STATEMENT: Procognitive effects of two acetylcholine (ACh) receptor agonists were assessed in group-housed monkeys who had several years' experience drinking ethanol. The muscarinic ACh receptor agonist xanomeline and the nicotinic ACh receptor agonist varenicline reversed a cognitive deficit induced by the muscarinic ACh receptor antagonist scopolamine. However, this effect was observed only in lower-ranking (subordinate) monkeys and not higher-ranking (dominant monkeys). Results suggest that ACh agonists may effectively remediate alcohol-induced cognitive deficits in a subpopulation of those with alcohol use disorder.

The development of diphenyleneiodonium analogs as GPR3 agonists.

G protein-coupled receptor 3 (GPR3) is an orphan receptor potentially involved in many important physiological processes such as drug abuse, neuropathic pain, and anxiety and depression related disorders. Pharmacological studies of GPR3 have been limited due to the restricted number of known agonists and inverse agonists for this constitutively active receptor. In this medicinal chemistry study, we report the discovery of GPR3 agonists based off the diphenyleneiodonium (DPI) scaffold. The most potent full agonist was the 3-trifluoromethoxy analog (32) with an EC50 of 260 nM and 90% efficacy compared to DPI. Investigation of a homology model of GPR3 from multiple sequence alignment resulted in the finding of a binding site rich in potential π-π and π-cation interactions stabilizing DPI-scaffold agonists. MMGBSA free energy analysis showed a good correlation with trends in observed EC50s. DPI analogs retained the same high receptor selectivity for GPR3 over GPR6 and GPR12 as observed with DPI. Collectively, the DPI analog series shows that order of magnitude improvements in potency with the scaffold were attainable; however, attempts to replace the iodonium ion to make the scaffold more druggable failed.

Reinforcing and Stimulant-Like Effects of Methamphetamine Isomers in Rhesus Macaques.

Monoamine releasers such as d-methamphetamine (d-MA) can reduce cocaine use in laboratory studies and have been forwarded for the management of cocaine use disorder (CUD). However, the proven abuse liability of d-MA has limited enthusiasm for clinical use. The levorotatory isomer of MA, l-MA, appears to have lesser stimulant effects, possibly due to its preferential norepinephrine-releasing properties compared with dopamine. The present study evaluated the abuse potential of l-MA by comparing its reinforcing effects with known stimulant drugs of abuse in nonhuman primates. Adult rhesus macaques (N = 4) responded for intravenous injections of cocaine, d-MA, methcathinone (MCAT), or l-MA under a fixed-ratio (FR) schedule of reinforcement; reinforcing effectiveness was evaluated using behavioral economic demand procedures. In a separate cohort (N = 9), daily activity and food-reinforced responding were assessed during 100 days of treatment with daily dosages of l-MA (2.3 mg/kg per day, i.v.) or d-MA (0.74 mg/kg per day, i.v.) previously shown to decrease cocaine self-administration. Results show that all drugs maintained self-administration, with peak injections reaching ∼100 inj per session for cocaine, MCAT, and d-MA and ∼50 inj per session for l-MA . In demand studies, self-administration of each drug gradually decreased as FR size increased. The exponential model of demand indicated that the reinforcing effectiveness of l-MA was significantly less than the other drugs studied. Chronic l-MA treatment did not appreciably alter daily activity and only transiently suppressed food-reinforced responding. These data, coupled with previous findings that l-MA effectively reduces stimulant self-administration, suggest that l-MA, or other norepinephrine-preferring releasers, may serve as agonist medication for CUD with lesser abuse liability than common psychostimulants. SIGNIFICANCE STATEMENT: Development of pharmacotherapies for cocaine use disorder remains a formidable challenge. Agonist-based therapies show promise, but enthusiasm is tempered by the abuse liability of previously proposed medications. This study evaluated the abuse liability and chronic treatment effects of methamphetamine's levorotatory isomer (l-MA). l-MA demonstrated lower abuse liability compared with commonly abused stimulants and produced few untoward effects. In the context of recent studies demonstrating that l-MA attenuates stimulant self-administration, these findings support l-MA's potential as a pharmacotherapy for stimulant addiction.

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.

Evaluation of the Reinforcing Strength of Phendimetrazine Using a Progressive-Ratio Schedule of Reinforcement in Rhesus Monkeys.

Stimulant abuse is a persistent public health problem with no Food and Drug Administration-approved pharmacotherapy. Although monoamine-releasing drugs such as d-amphetamine can decrease cocaine self-administration in human and animal laboratory studies, their potential for abuse limits clinical utility. "Abuse-deterrent" formulations of monoamine releasers, such as prodrugs, hold greater clinical promise if their abuse potential is, as theorized, lower than that of cocaine. In these studies, we determined the reinforcing strength of phendimetrazine (PDM), a prodrug for the amphetamine-like monoamine releaser phenmetrazine; both drugs have been shown to decrease cocaine self-administration in laboratory animals. To date, no study has directly compared PDM (Schedule III) with cocaine (Schedule II) under progressive-ratio (PR) schedules of reinforcement, which are better suited than fixed-ratio schedules to directly compare reinforcing strength of drugs. Dose-response curves for cocaine (saline, 0.001-0.3 mg/kg per injection) and PDM (0.1-1.0 mg/kg per injection) were generated in six cocaine-experienced male rhesus monkeys during 4-hour sessions with a 20-minute limited hold (LH). Under these conditions, the maximum number of injections was not significantly different between cocaine and PDM. The reinforcing strength of doses situated on the peaks of the cocaine and PDM dose-effect curves were redetermined with a 60-minute LH. The mean number of injections increased for both drugs, but not for saline. Cocaine presentations resulted in significantly higher peak injections than PDM with a 60-minute LH, which is consistent with the lower scheduling of PDM. These results support PDM as Schedule III and highlight the importance of schedule parameters when comparing reinforcing strength of drugs using a PR schedule of reinforcement. SIGNIFICANCE STATEMENT: One strategy for reducing cocaine use is to identify a treatment that substitutes for cocaine but has lower abuse potential. In a rhesus monkey model of drug abuse, this study compared the reinforcing strength of cocaine and phendimetrazine, a drug that has been shown to decrease cocaine use in some studies.

In Vitro Effects of Ligand Bias on Primate Mu Opioid Receptor Downstream Signaling.

Interest has emerged in biased agonists at the mu opioid receptor (MOR) as a possible means for maintaining potent analgesis with reduced side effect profiles. While approaches measuring in vitro biased agonism are used in the development of these compounds, their therapeutic utility will ultimately be determined by in vivo functional effects. Nonhuman primates (NHPs) are the most translational model for evaluating the behavioral effects of candidate medications, but biased signaling of these drugs at NHP MOR receptors has been unstudied. The goal of the current work was to characterize MOR ligand bias in rhesus macaques, focusing on agonists that have previously been reported to show different patterns of biased agonism in rodents and humans. Downstream signaling pathways that responded to MOR activation were identified using a luciferase reporter array. Concentration-response curves for specific pathways (cAMP, NF-ĸB, MAPK/JNK) were generated using six agonists previously reported to differ in terms of signaling bias at rodent and human MORs. Using DAMGO as a reference ligand, relative cAMP, NF-ĸB and MAPK/JNK signaling by morphine, endomorphin-1, and TRV130 were found to be comparable between species. Further, the bias patterns of across ligands for NF-ĸB and MAPK/JNK were largely similar between species. There was a high degree of concordance between rhesus macaque and human MOR receptor signaling bias for all agonists tested, further demonstrating their utility for future translational behavioral studies.

Identification of Matrine as a Novel Regulator of the CXCR4 Signaling Axis in Tumor Cells.

Matrine, a quinolizidine alkaloid, is commonly employed for treating various viral and inflammatory disorders. Here, we have evaluated matrine for its activity on C-X-C chemokine receptor type 4 (CXCR4) and matrix metalloproteinases (MMP-9/2) expression, and its potential to affect tumor metastasis and invasion. The effects of matrine on CXCR4, MMP-9/2, and nuclear factor κB (NF-κB) activation in lung (A549), prostate (DU145), and pancreas (MIA PaCa-2) cells were investigated by diverse techniques. The expression level of CXCR4 and MMP-9/2 was analyzed by western blot analysis and reverse transcription polymerase chain reaction. NF-κB activation was also evaluated by western blot analysis, electrophoretic mobility shift assay as well as immunocytochemical experiments. Furthermore, we monitored cell invasion and metastasis activities by wound healing and Boyden chamber assays. We noted that matrine induced a down-regulation of CXCR4 and MMP-9/2 at both protein and mRNA levels. In addition, matrine negatively regulated human epidermal growth factor receptor 2 (HER2) and C-X-C Motif Chemokine Ligand 12 (CXCL12)-induced CXCR4 expression. Moreover, NF-κB suppression by matrine led to inhibition of metastatic potential of tumor cells. Our results suggest that matrine can block the cancer metastasis through the negative regulation of CXCR4 and MMP-9/2 and consequently it can be considered as a potential candidate for cancer therapy.

Evodiamine Mitigates Cellular Growth and Promotes Apoptosis by Targeting the c-Met Pathway in Prostate Cancer Cells.

Evodiamine (EVO) is an indoloquinazoline alkaloid that exerts its various anti-oncogenic actions by blocking phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), mitogen-activated protein kinase (MAPK), c-Met, and nuclear factor kappa B (NF-κB) signaling pathways, thus leading to apoptosis of tumor cells. We investigated the ability of EVO to affect hepatocyte growth factor (HGF)-induced c-Met/Src/STAT3 activation cascades in castration-resistant prostate cancer (CRPC). First, we noted that EVO showed cytotoxicity and anti-proliferation activities in PC-3 and DU145 cells. Next, we found that EVO markedly inhibited HGF-induced c-Met/Src/STAT3 phosphorylation and impaired the nuclear translocation of STAT3 protein. Then, we noted that EVO arrested the cell cycle, caused apoptosis, and downregulated the expression of various carcinogenic markers such as B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), cyclin D1, cyclooxygenase 2 (COX-2), survivin, vascular endothelial growth factor (VEGF), and matrix metallopeptidases 9 (MMP-9). Moreover, it was observed that in cPC-3 and DU145 cells transfected with c-Met small interfering RNA (siRNA), Src/STAT3 activation was also mitigated and led to a decrease in EVO-induced apoptotic cell death. According to our results, EVO can abrogate the activation of the c-Met/Src/STAT3 signaling axis and thus plays a role as a robust suppressor of tumor cell survival, proliferation, and angiogenesis.

Transdermal Delivery of the Free Base of 3-Fluoroamphetamine: In Vitro Skin Permeation and Irritation Potential.

This work aimed to continue our effort in establishing the feasibility of 3-fluoroamphetamine (also known as PAL-353) to be a transdermal drug candidate by studying the delivery of the base form through the human cadaver skin in lieu of the previously investigated salt form, and for the first time using an EPIDERM™-reconstructed human epidermal model to predict the skin irritation potential of PAL-353, in support of development for a matrix-type transdermal delivery system. Passive and enhanced (with chemical permeation enhancers) transdermal delivery were investigated via in vitro permeation studies that were performed on Franz diffusion cells with dermatomed human cadaver skin. After 24 h, PAL-353 free base revealed high passive permeation of 417.49 ± 30.12, 1577.68 ± 165.41, and 4295.16 ± 264.36 µg/cm2, with applied formulation concentrations of 5.5 (F1), 20 (F2), and 40 (F3) mg/mL, respectively. Oleyl alcohol produced an approximately threefold steady-state flux enhancement at 5% or 10% w/w but may not be needed as the free base alone provided therapeutically relevant permeation. Further, it was predicted that therapeutically relevant delivery would be unlikely to cause skin irritation using the EPIDERM™-reconstructed human epidermal model. In conclusion, the present study further supported the development of PAL-353 transdermal delivery systems.

Further pharmacological comparison of D-methamphetamine and L-methamphetamine in rats: abuse-related behavioral and physiological indices.

Previous preclinical research suggests that L-methamphetamine (L -MA) has potential therapeutic utility to treat psychostimulant abuse. This study examined potential abuse-related and adverse physiological effects of D -MA and L -MA alone and in combination in rats, as these effects had not been previously characterized. Potential abuse-related effects were examined in locomotor sensitization and conditioned place preference paradigms. Body temperature was monitored to assess the physiological effects of these drugs or drug combinations. In the locomotor study, D-MA induced locomotor sensitization to both D-MA and L -MA. L -MA induced locomotor sensitization only to D-MA. Responses to a combination of L-MA and D -MA were not differentially affected by L-MA or D-MA conditioning. In the conditioned place preference study, D-MA and L -MA each induced significant place preference. L -MA did not attenuate D-MA-induced place preference. In the body temperature study, D-MA induced hyperthermia and L -MA induced hypothermia. In combination, L -MA did not affect D-MA-induced hyperthermia. These data suggest that L -MA alone produces less abuse-related and adverse physiological effects than D-MA, but modulates and is modulated by concurrent and subsequent D-MA exposure, which may enhance the abuse liability of both drugs. These findings should be considered when L -MA is proposed for replacement therapy.

Conformational state interactions provide clues to the pharmacochaperone potential of serotonin transporter partial substrates.

Point mutations in SLC6 transporters cause misfolding, which can be remedied by pharmacochaperones. The serotonin transporter (SERT/SLC6A4) has a rich pharmacology including inhibitors, releasers (amphetamines, which promote the exchange mode), and more recently, discovered partial substrates. We hypothesized that partial substrates trapped the transporter in one or several states of the transport cycle. This conformational trapping may also be conducive to folding. We selected naphthylpropane-2-amines of the phenethylamine library (PAL) including the partial substrate PAL1045 and its congeners PAL287 and PAL1046. We analyzed their impact on the transport cycle of SERT by biochemical approaches and by electrophysiological recordings; substrate-induced peak currents and steady-state currents monitored the translocation of substrate and co-substrate Na+ across the lipid bilayer and the transport cycle, respectively. These experiments showed that PAL1045 and its congeners bound with different affinities (ranging from nm to µm) to various conformational intermediates of SERT during the transport cycle. Consistent with the working hypothesis, PAL1045 was the most efficacious compound in restoring surface expression and transport activity to the folding-deficient mutant SERT-601PG602-AA. These experiments provide a proof-of-principle for a rational search for pharmacochaperones, which may be useful to restore function to clinically relevant folding-deficient transporter mutants.

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.

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.

Methamphetamine-like discriminative stimulus effects of bupropion and its two hydroxy metabolites in male rhesus monkeys.

The dopamine transporter (DAT) inhibitor and nicotinic acetylcholine (nACh) receptor antagonist bupropion is being investigated as a candidate 'agonist' medication for methamphetamine addiction. In addition to its complex pharmacology, bupropion also has two distinct pharmacologically active metabolites. However, the mechanism by which bupropion produces methamphetamine-like 'agonist' effects remains unknown. The aim of the present study was to determine the role of DAT inhibition, nACh receptor antagonism, and the hydroxybupropion metabolites in the methamphetamine-like discriminative stimulus effects of bupropion in rhesus monkeys. In addition, varenicline, a partial agonist at the nACh receptor, and risperidone, a dopamine antagonist, were tested as controls. Monkeys (n=4) were trained to discriminate 0.18 mg/kg intramuscular methamphetamine from saline in a two-key food-reinforced discrimination procedure. The potency and time course of methamphetamine-like discriminative stimulus effects were determined for all compounds. Bupropion, methylphenidate, and 2S,3S-hydroxybupropion produced full, at least 90%, methamphetamine-like effects. 2R,3R-Hydroxybupropion, mecamylamine, and nicotine also produced full methamphetamine-like effects, but drug potency was more variable between monkeys. Varenicline produced partial methamphetamine-like effects, whereas risperidone did not. Overall, these results suggest DAT inhibition as the major mechanism of the methamphetamine-like 'agonist' effects of bupropion, although nACh receptor antagonism appeared, at least partially, to contribute. Furthermore, the contribution of the 2S,3S-hydroxybupropion metabolite could not be completely ruled out.

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.

Preclinical Assessment of Lisdexamfetamine as an Agonist Medication Candidate for Cocaine Addiction: Effects in Rhesus Monkeys Trained to Discriminate Cocaine or to Self-Administer Cocaine in a Cocaine Versus Food Choice Procedure.

BACKGROUND: Chronic amphetamine treatment decreases cocaine consumption in preclinical and human laboratory studies and in clinical trials. Lisdexamfetamine is an amphetamine prodrug in which L-lysine is conjugated to the terminal nitrogen of d-amphetamine. Prodrugs may be advantageous relative to their active metabolites due to slower onsets and longer durations of action; however, lisdexamfetamine treatment's efficacy in decreasing cocaine consumption is unknown. METHODS: This study compared lisdexamfetamine and d-amphetamine effects in rhesus monkeys using two behavioral procedures: (1) a cocaine discrimination procedure (training dose = 0.32mg/kg cocaine, i.m.); and (2) a cocaine-versus-food choice self-administration procedure. RESULTS: In the cocaine-discrimination procedure, lisdexamfetamine (0.32-3.2mg/kg, i.m.) substituted for cocaine with lower potency, slower onset, and longer duration of action than d-amphetamine (0.032-0.32mg/kg, i.m.). Consistent with the function of lisdexamfetamine as an inactive prodrug for amphetamine, the time course of lisdexamfetamine effects was related to d-amphetamine plasma levels by a counter-clockwise hysteresis loop. In the choice procedure, cocaine (0-0.1mg/kg/injection, i.v.) and food (1g banana-flavored pellets) were concurrently available, and cocaine maintained a dose-dependent increase in cocaine choice under baseline conditions. Treatment for 7 consecutive days with lisdexamfetamine (0.32-3.2mg/kg/day, i.m.) or d-amphetamine (0.032-0.1mg/kg/h, i.v.) produced similar dose-dependent rightward shifts in cocaine dose-effect curves and decreases in preference for 0.032mg/kg/injection cocaine. CONCLUSIONS: Lisdexamfetamine has a slower onset and longer duration of action than amphetamine but retains amphetamine's efficacy to reduce the choice of cocaine in rhesus monkeys. These results support further consideration of lisdexamfetamine as an agonist-based medication candidate for cocaine addiction.

Simple radiometric method for accurately quantitating epitope densities of hapten-protein conjugates with sulfhydryl linkages.

Control of small molecule hapten epitope densities on antigenic carrier proteins is essential for development and testing of optimal conditions for vaccines. Yet, accurate determination of epitope density can be extremely difficult to accomplish, especially with the use of small haptens, large molecular weight carrier proteins, and limited amounts of protein. Here we report a simple radiometric method that uses (14)C-labeled cystine to measure hapten epitope densities during sulfhydryl conjugation of haptens to maleimide activated carrier proteins. The method was developed using a (+)-methamphetamine (METH)-like hapten with a sulfhydryl terminus, and two prototype maleimide activated carrier proteins, bovine serum albumin (BSA) and immunocyanin monomers of keyhole limpet hemocyanin. The method was validated by immunochemical analysis of the hapten-BSA conjugates, and least-squares linear regression analysis of epitope density values determined by the new radiometric method versus values determined by matrix-assisted laser desorption/ionization mass spectrometry. Results showed that radiometric epitope density values correlated extremely well with the mass spectrometrically derived values (r(2) = 0.98, y = 0.98x + 0.91). This convenient and simple method could be useful during several stages of vaccine development including the optimization and monitoring of conditions for hapten-protein conjugations, and choosing the most effective epitope densities for conjugate vaccines.

Alpha-ethyltryptamines as dual dopamine-serotonin releasers.

The dopamine (DA), serotonin (5-HT), and norepinephrine (NE) transporter releasing activity and serotonin-2A (5-HT2A) receptor agonist activity of a series of substituted tryptamines are reported. Three compounds, 7b, (+)-7d and 7f, were found to be potent dual DA/5-HT releasers and were >10-fold less potent as NE releasers. Additionally, these compounds had different activity profiles at the 5-HT2A receptor. The unique combination of dual DA/5-HT releasing activity and 5-HT2A receptor activity suggests that these compounds could represent a new class of neurotransmitter releasers with therapeutic potential.

mGlu2 and mGlu3 receptor negative allosteric modulators attenuate the interoceptive effects of alcohol in male and female rats.

RATIONALE: The subjective effects of alcohol are associated with alcohol use disorder (AUD) vulnerability and treatment outcomes. The interoceptive effects of alcohol are part of these subjective effects and can be measured in animal models using drug discrimination procedures. The newly developed mGlu2 and mGlu3 negative allosteric modulators (NAMs) are potential therapeutics for AUD and may alter interoceptive sensitivity to alcohol. OBJECTIVES: To determine the effects of mGlu2 and mGlu3 NAMs on the interoceptive effects of alcohol in rats. METHODS: Long-Evans rats were trained to discriminate the interoceptive stimulus effects of alcohol (2.0 g/kg, i.g.) from water using both operant (males only) and Pavlovian (male and female) drug discrimination techniques. Following acquisition training, an alcohol dose-response (0, 0.5, 1.0, 2.0 g/kg) experiment was conducted to confirm stimulus control over behavior. Next, to test the involvement of mGlu2 and mGlu3, rats were pretreated with the mGlu2-NAM (VU6001966; 0, 3, 6, 12 mg/kg, i.p.) or the mGlu3-NAM (VU6010572; 0, 3, 6, 12 mg/kg, i.p.) before alcohol administration (2.0 g/kg, i.g.). RESULTS: In Pavlovian discrimination, male rats showed greater interoceptive sensitivity to 1.0 and 2.0 g/kg alcohol compared to female rats. Both mGlu2-NAM and mGlu3-NAM attenuated the interoceptive effects of alcohol in male and female rats using Pavlovian and operant discrimination. There may be a potential sex difference in response to the mGlu2-NAM at the highest dose tested. CONCLUSIONS: Male rats may be more sensitive to the interoceptive effects of the 2.0 g/kg alcohol training dose compared to female rats. Both mGlu2-and mGlu3-NAM attenuate the interoceptive effects of alcohol in male and female rats. These drugs may have potential for treatment of AUD in part by blunting the subjective effects of alcohol.

Contingent administration of typical and biased kappa opioid agonists reduces cocaine and oxycodone choice in a drug vs. food choice procedure in male rhesus monkeys.

RATIONALE: Combinations of mu and kappa-opioid receptor (KOR) agonists have been proposed as analgesic formulations with reduced abuse potential. The feasibility of this approach has been increased by the development of KOR agonists with biased signaling profiles that produce KOR-typical antinociception with fewer KOR-typical side effects. OBJECTIVE: The present study determined if the biased KOR agonists, nalfurafine and triazole 1.1, could reduce choice for oxycodone in rhesus monkeys as effectively as the typical KOR agonist, salvinorin A. METHODS: Adult male rhesus monkeys (N = 5) responded under a concurrent schedule of food delivery and intravenous cocaine injections (0.018 mg/kg/injection). Once trained, cocaine (0.018 mg/kg/injection) or oxycodone (0.0056 mg/kg/injection) was tested alone or in combination with contingent injections of salvinorin A (0.1-3.2 µg/kg/injection), nalfurafine (0.0032-0.1 µg/kg/injection), triazole 1.1 (3.2-100.0 µg/kg/injection), or vehicle. In each condition, the cocaine or oxycodone dose, as well as the food amount, was held constant across choice components, while the dose of the KOR agonist was increased across choice components. RESULTS: Cocaine and oxycodone were chosen over food on more than 80% of trials when administered alone or contingently with vehicle. When KOR agonists were administered contingently with either cocaine or oxycodone, drug choice decreased in a dose-dependent manner. Salvinorin A and triazole 1.1 decreased drug-reinforcer choice without altering total trials completed (i.e., choice allocation shifted to food), while nalfurafine dose dependently decreased total trials completed. CONCLUSIONS: These results demonstrate that salvinorin A and triazole 1.1, but not nalfurafine, selectively reduce cocaine and oxycodone self-administration independent of nonspecific effects on behavior, suggesting that G-protein bias does not appear to be a moderating factor in this outcome. Triazole 1.1 represents an important prototypical compound for developing novel KOR agonists as deterrents for prescription opioid abuse.