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Kratom alkaloids have mostly been evaluated for their opioid activity but less at other targets that could contribute to their physiological effects. Here, we investigated the in vitro and in vivo activity of kratom alkaloids at serotonin receptors (5-HTRs). Paynantheine and speciogynine exhibited high affinity for 5-HT(1A)Rs and 5-HT(2B)Rs, unlike the principal kratom alkaloid mitragynine. Both alkaloids produced antinociceptive properties in rats via an opioid receptor-independent mechanism, and neither activated 5-HT(2B)Rs in vitro. Paynantheine, speciogynine, and mitragynine induced lower lip retraction and antinociception in rats, effects blocked by a selective 5-HT(1A)R antagonist. In vitro functional assays revealed that the in vivo 5-HT(1A)R agonistic effects may be due to the metabolites 9-O-desmethylspeciogynine and 9-O-desmethylpaynantheine and not the parent compounds. Both metabolites did not activate 5-HT(2B)R, suggesting low inherent risk of causing valvulopathy. The 5-HT(1A)R agonism by kratom alkaloids may contribute to the mood-enhancing effects associated with kratom use.

RATIONALE: Methylphenidate and d-amphetamine, medications used for treatment of attention deficit hyperactivity disorder (ADHD), are used recreationally and self-administered by laboratory animals. Benztropine (BZT) analogs, like those medications, increase synaptic dopamine levels but are less effective in maintaining self-administration, suggesting clinical utility with less abuse liability. OBJECTIVES: The current study was designed to evaluate potential therapeutic effects of BZT analogs related to ADHD. METHODS: Rats responded under a delay-discounting procedure in which responses on one lever produced immediate delivery of a single food pellet and alternative responses produced four food pellets either immediately or with various temporal delays, with those delays arranged in ascending or random orders in different groups of rats. Selection of the smaller more immediate reinforcer has been suggested as an aspect of “impulsivity,” a trait with suggested involvement in ADHD. Other rats were studied under fixed-interval (FI) 300-s schedules to assess drug effects on behavior under temporal control. RESULTS: d-Amphetamine, methylphenidate, and the BZT analog AHN 1–055, but not AHN 2–005 or JHW 007 increased selection of the large, delayed reinforcer with either arrangement of delays. All drugs changed the temporal distribution of responses within the FI from one with responses concentrated at the end to a more uniform distribution. Changes in the temporal distribution of FI responding occurred with drugs that did not affect discounting suggesting that discounting does not arise directly from the same temporal control processes controlling FI responding. CONCLUSIONS: AHN 1–055 may be of clinical utility in the treatment of ADHD.

The α4β2* nicotinic acetylcholine receptor (nAChR) subtypes are targeted for the development of smoking cessation aids, and the use of drug discrimination in mice provides a robust screening tool for the identification of drugs acting through nAChRs. Here, we established that the α4β2* nAChR agonist epibatidine can function as a discriminative stimulus in mice. Male C57BL/6J mice discriminated epibatidine (0.0032 mg/kg, subcutaneously) and were tested with agonists varying in selectivity and efficacy for α4β2* nAChRs. The discriminative stimulus effects of epibatidine were characterized with the nonselective, noncompetitive nicotinic antagonist mecamylamine, with the selective β2-substype-containing nAChR antagonist dihydro-β-erythroidine hydrobromide (DHβE), and the α7 antagonist methyllycaconitine (MLA). Nicotine (0.32-1.0 mg/kg, subcutaneously), the partial nAChR agonist cytisine (1.0-5.6 mg/kg, subcutaneously), and the α7 nAChR agonist N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide (10-56 mg/kg, intraperitoneally) produced no more than 33% epibatidine-appropriate responding. The partial α4β2* nAChR agonists varenicline and 2'-fluoro-3'-(4-nitro-phenyl)deschloroepibatidine produced 61 and 69% epibatidine-appropriate responding, respectively. DHβE and mecamylamine, but not MLA, significantly antagonized the discriminative stimulus effects of epibatidine. These results show that epibatidine may be trained as a discriminative stimulus in mice and has utility in elucidating the in-vivo pharmacology of α4β2* nAChR ligands.

σRs are unique intracellular chaperone proteins [8] initially thought to be opioid receptor subtypes [9]. They have been classified into two subtypes based on specific radioligand binding assays using [3H](+)pentazocine for σ1Rs and [3H]1,3-di-o-tolylguanidine ([3H]DTG, in the presence of dextrallorphan to mask the σ1R) for σ2Rs in rat liver and kidney membranes [10]. Currently, the more selective σ1R ligand (+)-pentazocine has replaced dextrallorphan to mask the σ1R [7,11-14]. The σ1R has already been cloned as a 25-29 kDa chaperone protein composed of 223 amino acids [4,8,15]. It is widely distributed throughout the body [16-20]. Upon binding with agonists or under cellular stress, σ1Rs translocate from their primary endoplasmic reticulum (ER) location to different subcellular compartments where they can regulate ion channels and G-protein-coupled-receptor (GPCR) signaling [8,21-24]. In vivo functional studies on σ1Rs suggest that they play a substantial role in various cellular functions. Drugs acting at this receptor have been studied for their potential therapeutic effects in cancer, human immunodeficiency virus (HIV) infection, various psychiatric disorders, and substance abuse [1,25].

Sigma(1) receptors (σ(1)Rs) represent a structurally unique class of intracellular proteins that function as chaperones. σ(1)Rs translocate from the mitochondria-associated membrane to the cell nucleus or cell membrane, and through protein-protein interactions influence several targets, including ion channels, G-protein-coupled receptors, lipids, and other signaling proteins. Several studies have demonstrated that σR antagonists block stimulant-induced behavioral effects, including ambulatory activity, sensitization, and acute toxicities. Curiously, the effects of stimulants have been blocked by σR antagonists tested under place-conditioning but not self-administration procedures, indicating fundamental differences in the mechanisms underlying these two effects. The self administration of σR agonists has been found in subjects previously trained to self administer cocaine. The reinforcing effects of the σR agonists were blocked by σR antagonists. Additionally, σR agonists were found to increase dopamine concentrations in the nucleus accumbens shell, a brain region considered important for the reinforcing effects of abused drugs. Although the effects of the σR agonist, DTG, on dopamine were obtained at doses that approximated those that maintained self administration behavior those of another agonist, PRE-084 required higher doses. The effects of DTG were antagonized by non-selective or a preferential σ(2)R antagonist but not by a preferential σ(1)R antagonist. The effects of PRE-084 on dopamine were insensitive to σR antagonists. The data suggest that the self administration of σR agonists is independent of dopamine and the findings are discussed in light of a hypothesis that cocaine has both intracellular actions mediated by σRs, as well as extracellular actions mediated through conventionally studied mechanisms. The co-activation and potential interactions among these mechanisms, in particular those involving the intracellular chaperone σRs, may lead to the pernicious addictive effects of stimulant drugs.

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT's role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H+ antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects., Amphetamines are known to enhance extracellular dopamine levels, but the underlying mechanisms are unclear. Utilising a new pH biosensor for synaptic vesicles, the authors show that amphetamines diminish vesicle pH gradients, disrupting dopamine packaging and leading to increased neurotransmitter release.

The reuptake inhibition of dopamine (DA) through the DA transporter (DAT) is thought to be the primary mechanism underlying the reinforcing effects of cocaine [1,2]. However, there are several DA reuptake inhibitors that do not produce appreciable cocaine-like effects in vivo [2-4]. Previously, there were several relatively viable hypotheses related to the mechanisms underlying the atypical effects of such agents, but new studies have decreased this number [3,5]. The new findings may contribute to the development of a better understanding of what constitutes cocaine-like behavioral effects.

Interactions between distinct receptor proteins have been assessed in vitro and in vivo. Among these studies are G-Protein-Coupled Receptor (GPCR) heteromers. GPCR heteromers form a popular area for study in molecular pharmacology and are expected to contribute valuable information to clinical needs for development of medications discovery in various neuropsychiatric diseases and symptoms. For example, physical formation of GPCR heteromers could result in a novel signaling mechanism that is distinct from those of individual GPCR monomers or their homomer. However, significance of various studies on dopamine (DA) D1/D2 receptor heteromers was challenged by a recent report [1]. Javitch et al. [1] provided compelling evidence against D1/D2 heteromers, one of the most prominent subjects of interest in neuropsychiatry field. In the study Javitch et al. performed an “in vivo” assessment of the first demonstration for the presence of DA D1/D2 receptor heteromers in animal tissues [1]. Surprisingly, despite the successful creation of DA D1/D2 receptor heteromers “in vitro”, their immunohistochemical studies discovered the virtual absence of physical DA D1/D2 receptor heteromers in the shell region of the nucleus accumbens (NAS) from C57Bl/6J mouse brain [1]. In the NAS, DA D1 and D2 receptors seem to co-express in the same neuron for some neurons but did not localize [1]. The virtual absence of physical DA D1/D2 receptor heteromers “in vivo” was further substantiated by the discovery of lack of localization across species. For example, there was little if any signal indicative of the presence of DA D1/D2 receptor heteromers in the NAS from a rat and the ventral striatum from rhesus monkey. Since various drugs abused by humans are known to increase extracelluar DA levels in the NAS, the “in vivo” negative results against the presence of DA D1/D2 receptor heteromers in the NAS could have a substantial impact at least on research for drug abuse. In sumary, the failure to demonstrate in vivo the presence of DA D1/D2 receptor heteromers [1] undoubtly suggests reconsideration of the use of the “in vitro preclinical” models using receptor couplings. There is a clear need to reassess the validity of this approach for the development of medications discovery regarding various neuropsychiatric diseases and symptoms.

Benztropine (BZT) analogs inhibit dopamine uptake but are less effective than cocaine in producing behavioral effects predicting abuse liability. The present study compared reinforcing effects of intravenous BZT analogs with those of standard monoamine uptake inhibitors and the effects of their oral pretreatment on cocaine self-administration. Responding of rats was maintained by cocaine [0.032–1.0 mg/kg/injection (inj)] or food reinforcement under fixed-ratio five-response schedules. Maximal rates of responding were maintained by 0.32 mg/kg/inj cocaine or substituted methylphenidate, with lower rates maintained at lower and higher doses. The N-methyl BZT analog, AHN 1-055 (3α-[bis(4′-fluorophenyl)methoxy]-tropane), also maintained responding (0.1 mg/kg/inj), although maximal rates were less than those with cocaine. Responding was not maintained above vehicle levels by the N-allyl, AHN 2-005 (N-allyl-3α-[bis(4′-fluorophenyl)methoxy]-tropane), and N-butyl, JHW 007 [N-(n-butyl)-3α-[bis(4′-fluorophenyl)methoxy]-tropane], BZT analogs, and it was not maintained with nisoxetine or citalopram. Presession treatment with methylphenidate (3.2–32 mg/kg) dose-dependently shifted the cocaine self-administration dose-effect curve leftward, whereas nisoxetine and citalopram effects were not significant. An intermediate dose of AHN 1-055 (32 mg/kg) increased responding maintained by low cocaine doses and decreased responding maintained by higher doses. A higher dose of AHN 1-055 completely suppressed cocaine-maintained responding. Both AHN 2-005 and JHW 007 dose-dependently (10–32 mg/kg) decreased cocaine self-administration, shifting its dose-effect curve down. Decreases in cocaine-maintained responding occurred at doses of methylphenidate and BZT analogs that left food-maintained responding unchanged. During a component in which injections were not available, methylphenidate and AHN 1-055, but not AHN 2-005 or JHW 007, increased response rates. These findings further support the low abuse liability of BZT analogs and their potential development as medications for cocaine abuse.