Methamphetamine-induced Hyperlocomotion Research Articles

Pivotal role of Slitrk1 in adult striatal cholinergic neurons in mice: Implication in Tourette syndrome.

The SLIT and NTRK-like 1 (SLITRK1) gene mutation and striatal cholinergic interneurons (ChIs) loss are associated with Tourette syndrome (TS). ChIs comprise only 1-2% of total neurons but send dense arbors of fibers in the stratum, making it the most ACh-abundant brain region. Here, mice receiving Slitrk1 siRNA bilateral microinjection in the dorsal striatum, Slitrk1-knockdown (KD) mice, exhibit TS-like haloperidol-sensitive stereotypic tic behaviors and impaired prepulse inhibition, as well as delayed sensorimotor response, compared with scrambled siRNA-treated controls. These TS-like characteristics correlate with lower striatal Slitrk1 protein levels, fewer Slitrk1-containing ChIs, and fewer activated ChIs in Slitrk1-KD mice. Based on their electrophysiological properties, Slitrk1-negative ChIs are less excitable than Slitrk1-positive ChIs. Slitrk1-KD mice have lower evoked acetylcholine and dopamine levels, higher tonic dopamine levels, and downregulated dopamine transporters in the striatum, increased apomorphine-induced climbing behaviors, and impaired methamphetamine-induced hyperlocomotion compared with controls. These results suggest that Slitrk1 is pivotal in maintaining striatal ChIs activity and subsequent dopaminergic transmission for normal motor functioning. Furthermore, conditional striatal Slitrk1-KD mice may serve as a translational modality with aspects of TS phenomenology. This article is protected by copyright. All rights reserved.

Knockdown of Piccolo in the Nucleus Accumbens Suppresses Methamphetamine-Induced Hyperlocomotion and Conditioned Place Preference in Mice.

Methamphetamine (METH), the most widely distributed psychostimulant, aberrantly activates the reward system in the brain to induce addictive behaviors. The presynaptic protein "Piccolo", encoded by Pclo, was identified as a METH-responsive protein with enhanced expression in the nucleus accumbens (NAc) in mice. Although the physiological and pathological significance of Piccolo has been identified in dopaminergic signaling, its role in METH-induced behavioral abnormalities and the underlying mechanisms remain unclear. To clarify such functions, mice with Piccolo knockdown in the NAc (NAc-miPiccolo mice) by local injection of an adeno-associated virus vector carrying miRNA targeting Pclo were generated and investigated. NAc-miPiccolo mice exhibited suppressed hyperlocomotion, sensitization, and conditioned place preference behavior induced by systemic administration of METH. The excessive release of dopamine in the NAc was reduced in NAc-miPiccolo mice at baseline and in response to METH. These results suggest that Piccolo in the NAc is involved in METH-induced behavioral alterations and is a candidate therapeutic target for the treatment of drug addiction.

Apocynin-Tandospirone Derivatives Suppress Methamphetamine-Induced Hyperlocomotion in Rats with Neonatal Exposure to Dizocilpine.

Accumulating evidence implicates oxidative stress as a potential pathophysiological mechanism of schizophrenia. Accordingly, we synthesized new chemicals using apocynin and tandospirone as lead compounds (A-2, A-3 and A-4). These novel compounds decreased reactive oxygen species (ROS) concentrations in vitro and reversed decreases in glutathione levels in the medial prefrontal cortex of rats transiently exposed to MK-801, an N-methyl-d-aspartate receptor antagonist, in the neonatal period. To determine whether A-2, A-3 and A-4 show behavioral effects associated with antipsychotic properties, the effects of these compounds on methamphetamine (MAP)-induced locomotor and vertical activity were examined in the model rats. A-2 and A-3, administered for 14 days around the puberty period, ameliorated MAP-induced hyperlocomotion in MK-801-treated rats in the post-puberty period, while A-4 suppressed MAP-induced vertical activity. These findings indicate that apocynin-tandospirone derivatives present anti-dopaminergic effects and may alleviate psychotic symptoms of schizophrenia.

Effects of pretreatment with a glycogen synthase kinase-3 inhibitor on methamphetamine-induced hyperlocomotion, stereotypy and reward in mice

Effects of pretreatment with a glycogen synthase kinase-3 inhibitor on methamphetamine-induced hyperlocomotion, stereotypy and reward in mice

Metoprine, a histamine N-methyltransferase inhibitor, attenuates methamphetamine-induced hyperlocomotion via activation of histaminergic neurotransmission in mice

Metoprine increases the content of histamine in brain by inhibiting histamine N-methyltransferase (HMT), a centrally acting histamine degrading enzyme. We present data demonstrating that pretreatment with metoprine attenuates the hyperlocomotive effects of METH in mice using a multi-configuration behavior apparatus designed to monitor four behavioral outcomes [horizontal locomotion, appetitive behavior (food access), and food and water intake]. Metoprine pretreatment itself induced hyperlocomotion in mice challenged with saline during the large part of light phase. The trend was also observed during the following dark phase. This is the first report that metoprine has a long-lasting locomotor stimulating property. Similarly, in a tail suspension test, a single injection of metoprine significantly reduced total time of immobility in mice, consistent with the idea that metoprine possesses motor stimulating properties. Metoprine pretreatment did not affect other aspects of behavior. Metoprine did not affect the appetitive and drinking behavior while exerted an effect on stereotypy. No stereotyped behavior was observed in mice pretreated with vehicle followed by METH, while stereotyped sniffing was observed in mice pretreated with metoprine followed by METH. The metoprine pretreatment attenuated METH-induced hyperlocomotion during the first 2 h of light phase, suggesting that metoprine-induced locomotor stimulating property might be different from that of METH. The hypothalamic content of histamine (but not its brain metabolite) was increased after metoprine or METH administration. Both METH and metoprine reduced dopamine and histamine turnover in the striatum and the nucleus accumbens and the hypothalamus, respectively, and there is a significant metoprine pretreatment x METH challenge interaction in the histamine turnover. It is likely that metoprine may attenuate METH-induced hyperlocomotion via activation of histaminergic neurotransmission. Metoprine also might induce a long-lasting locomotor stimulating effect via a putative mechanism different from that whereby METH induces the locomotor stimulating effect.

D1R/PP2A/p-CaMKIIα signaling in the caudate putamen is involved in acute methamphetamine-induced hyperlocomotion

Drug addiction is underscored by the transition from experimental use to dependent use of addictive drugs. Acute use of methamphetamine (METH) causes a range of clinical symptoms, including hyperlocomotion. Dopamine D1 receptor (D1R)-mediated negative regulation of phosphorylated calcium/calmodulin-dependent protein kinase IIα (p-CaMKIIα, threonine [Thr] 286) is involved in the acute effects induced by single METH administration. Protein phosphatase 2A (PP2A) is a potential bridge that links D1R and p-CaMKIIα (Thr 286) after acute METH administration. However, the mechanisms underlying hyperlocomotion induced by single METH administration remain unclear. In this study, SCH23390 (a D1R inhibitor) and LB100 (a PP2A inhibitor) were administered to examine the involvement of D1R and PP2A signaling in acute METH-induced hyperlocomotion in mice. The protein levels of methylated PP2A-C (m-PP2A-C, leucine [Leu] 309), phosphorylated PP2A-C (p-PP2A-C, tyrosine [Tyr] 307), PP2A-C, p-CaMKIIα (Thr 286), and CaMKIIα in the prefrontal cortex (PFc), nucleus accumbens (NAc), and caudate putamen (CPu) were measured. Administration of 0.5 mg/kg SCH23390 reversed the acute METH-induced increase in protein levels of m-PP2A-C (Leu 309) and the decrease in protein levels of p-PP2A-C (Tyr 307) in the CPu, but not in the PFC and NAc. Moreover, prior administration of 0.1 mg/kg LB100 attenuated hyperlocomotion induced by single METH administration and reversed the decrease in protein levels of p-CaMKII (Thr 286) in the PFC, NAc, and CPu. Collectively, these results indicate that the D1R/PP2A/p-CaMKIIα signaling cascade in the CPu may be involved in hyperlocomotion after a single administration of METH.

Naltrexone attenuates methamphetamine-induced behavioral sensitization and conditioned place preference in mice

Methamphetamine addiction causes serious public health problems worldwide. However, there is no effective medication licensed for methamphetamine addiction. The endogenous opioid system is considered to be a common substrate in drug addiction due to its regulation of dopamine release. In recent clinical trials, (-)-naltrexone, an opioid receptors and Toll-like receptor 4 antagonist, has exhibited encouraging findings for treating methamphetamine addiction; however, the understanding of its pharmacological mechanisms remains insufficient. By using mice models of behavioral sensitization and conditioned place preference (CPP), the present study was performed to investigate the effects of naltrexone on the methamphetamine-associated properties of incentive salience and reward-related memory, the two crucial factors for the development of addictive process and relapse. We found that naltrexone reduced single methamphetamine-induced hyperlocomotion in mice. In the paradigm of methamphetamine-induced behavioral sensitization paired with contextual cues in mice, naltrexone suppressed the development and expression of locomotor sensitization, suggesting the decrease in incentive salience to methamphetamine and context. In the methamphetamine-induced CPP paradigm in mice, naltrexone attenuated both the expression and methamphetamine-priming reinstatement of CPP response, suggesting the impairment of either contextual cue- or drug-induced retrieval of methamphetamine-associated memory. After the establishment of methamphetamine-induced CPP in mice, naltrexone treatment during the extinction training produced conditioned place adverse response, suggesting that naltrexone facilitated negative affection-associated extinction learning. Taken together, these findings demonstrate that naltrexone could intervene in the properties of incentive salience and reward-related memory in methamphetamine addiction, which may contribute to its therapeutic effects on methamphetamine addicts in clinical studies.

In vivo evaluation of effects of histamine H3 receptor antagonists on methamphetamine-induced hyperlocomotion in mice

A single administration with METH (3 mg/kg) induced a hyperlocomotion in male ICR mice. Pretreatment of mice with pitolisant, a histamine H3 receptor antagonist (5 and 10 mg/kg), for 30 min showed a significant reduction of the hyperlocomotion induced by METH, as compared with vehicle (saline)-pretreated subjects. Pretreatment of mice with the histamine H3 receptor antagonists JNJ-10181457 (5 and 10 mg/kg) or conessine (20 mg/kg), also showed similar inhibitory effects on METH-induced hyperlocomotion, similar to pitolisant. No significant change in locomotion was observed in mice pretreated with pitolisant, JNJ-10181457, or conessine alone. The pitolisant (10 mg/kg) action on METH-induced hyperlocomotion was completely abolished by the histamine H1 receptor antagonist pyrilamine (10 mg/kg), but not by the peripherally acting histamine H1 receptor antagonist fexofenadine (20 mg/kg), the brain-penetrating histamine H2 receptor antagonist zolantidine (10 mg/kg), or the brain-penetrating histamine H4 receptor antagonist JNJ-7777120 (40 mg/kg). Pretreatment with a histamine H3 receptor agonist immepip (10 mg/kg) augmented METH--induced behavior, including hyperlocomotion and stereotyped biting, and combined pretreatment with pitolisant (10 mg/kg) significantly attenuated stereotyped biting. These observations suggest that pretreatment with histamine H3 receptor antagonists attenuate METH-induced hyperlocomotion via releasing histamine after blocking H3 receptors, which then bind to the post-synaptic histamine receptor H1 (but not H2 or H4). It is likely that activation of brain histamine systems may be a good strategy for the development of agents, which treat METH abuse and dependence.

<i>In vivo</i> evaluation of effects of various histamine H<sub>3 </sub>receptor inverse agonists on methamphetamine-induced hyperlocomotion and stereotyped behavior in mice

<i>In vivo</i> evaluation of effects of various histamine H<sub>3 </sub>receptor inverse agonists on methamphetamine-induced hyperlocomotion and stereotyped behavior in mice

Novel molecules-related drug dependence in mice

Shati/Nat8l and TMEM168 were identified from nucleus accumbens (NAcc), which received continuous methamphetamine treatments. Shati/Nat8l is a synthetic enzyme that produces N-acetylaspartate (NAA) from L-aspartate and acetyl-coenzyme NAA is converted into N-acetylaspartylglutamate (NAAG) by NAAG synthetase (NAAGS). NAAG works as a highly selective endogenous agonist for the metabotropic glutamate type 3 receptor (mGluR3). We attempted to microinjection of adeno associated virus (AAV) including Shati/Nat8l into mice NAcc. These NAcc-Shati/Nat8l mice showed attenuation of the pharmacological effects of methamphetamine. NAcc-Shati or TMEM168 mice were also produced by AAV strategy and these mice also attenuated the methamphetamine-induced hyper locomotion and place preference test. TMEM168 interacts with osteopontin in NAcc of mice and cultured cells. Further, osteopontin it self has suppressive effects of methamphetamine. TMEM168 enhances anxiety in the elevated-plus maze and light-dark box test. The anxiety is recovered by the treatment of antianxiety drug diazepam. There our serial studies demonstrate that investigation of drug dependence-related molecule could lead to new pathway for new target for psychiatric disease.

Methamphetamine-induced hyperlocomotion and cFos expression in specific brain regions involve T-type Ca<sup>2+</sup> channels in mice

Methamphetamine-induced hyperlocomotion and cFos expression in specific brain regions involve T-type Ca<sup>2+</sup> channels in mice

Cerebellar α6 -subunit-containing GABAA receptors: a novel therapeutic target for disrupted prepulse inhibition in neuropsychiatric disorders.

The pathophysiological role of α6 -subunit-containing GABAA receptors, which are mainly expressed in cerebellar granule cells, remains unclear. Recently, we demonstrated that hispidulin, a flavonoid isolated from a local herb that remitted a patient's intractable motor tics, attenuated methamphetamine-induced hyperlocomotion in mice as a positive allosteric modulator (PAM) of cerebellar α6 GABAA receptors. Here, using hispidulin and a selective α6 GABAA receptor PAM, the pyrazoloquinolinone Compound 6, we revealed an unprecedented role of cerebellar α6 GABAA receptors in disrupted prepulse inhibition of the startle response (PPI), which reflects sensorimotor gating deficits manifested in several neuropsychiatric disorders. PPI disruptions were induced by methamphetamine and NMDA receptor antagonists in mice. Effects of the tested compounds were measured in Xenopus oocytes expressing recombinant α6 β3 γ2S GABAA receptors. Hispidulin given i.p. or by bilateral intracerebellar (i.cb.) injection rescued PPI disruptions induced by methamphetamine, ketamine, MK-801 and phencyclidine. Intracerebellar effects of hispidulin were mimicked by Ro15-4513 and loreclezole (two α6 GABAA receptor PAMs), but not by diazepam (an α6 GABAA receptor-inactive benzodiazepine) and were antagonized by furosemide (i.cb.), an α6 GABAA receptor antagonist. Importantly, Compound 6 (i.p.) also rescued methamphetamine-induced PPI disruption, an effect prevented by furosemide (i.cb.). Both hispidulin and Compound 6 potentiated α6 β3 γ2S GABAA receptor-mediated GABA currents. Positive allosteric modulation of cerebellar α6 GABAA receptors rescued disrupted PPI by attenuating granule cell activity. α6 GABAA receptor-selective PAMs are potential medicines for treating sensorimotor gating deficits in neuropsychiatric disorders. A mechanistic hypothesis is based on evidence for cerebellar contributions to cognitive functioning including sensorimotor gating.

Phosphodiesterase 2A Inhibitor TAK-915 Ameliorates Cognitive Impairments and Social Withdrawal in N-Methyl-d-Aspartate Receptor Antagonist-Induced Rat Models of Schizophrenia.

The pathophysiology of schizophrenia has been associated with glutamatergic dysfunction. Modulation of the glutamatergic signaling pathway, including N-methyl-d-aspartate (NMDA) receptors, can provide a new therapeutic target for schizophrenia. Phosphodiesterase 2A (PDE2A) is highly expressed in the forebrain, and is a dual substrate enzyme that hydrolyzes both cAMP and cGMP, which play pivotal roles as intracellular second messengers downstream of NMDA receptors. Here we characterize the in vivo pharmacological profile of a selective and brain-penetrant PDE2A inhibitor, (N-{(1S)-1-[3-fluoro-4-(trifluoromethoxy)phenyl]-2-methoxyethyl}-7-methoxy-2-oxo-2,3-dihydropyrido[2,3-b]pyrazine-4(1H)-carboxamide) (TAK-915) as a novel treatment of schizophrenia. Oral administration of TAK-915 at 3 and 10 mg/kg significantly increased cGMP levels in the frontal cortex, hippocampus, and striatum of rats. TAK-915 at 10 mg/kg significantly upregulated the phosphorylation of α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptor subunit GluR1 in the rat hippocampus. TAK-915 at 3 and 10 mg/kg significantly attenuated episodic memory deficits induced by the NMDA receptor antagonist (+)-MK-801 hydrogen maleate (MK-801) in the rat passive avoidance test. TAK-915 at 10 mg/kg significantly attenuated working memory deficits induced by MK-801 in the rat radial arm maze test. Additionally, TAK-915 at 10 mg/kg prevented subchronic phencyclidine-induced social withdrawal in social interaction in rats. In contrast, TAK-915 did not produce antipsychotic-like activity; TAK-915 had little effect on MK-801- or methamphetamine-induced hyperlocomotion in rats. These results suggest that TAK-915 has a potential to ameliorate cognitive impairments and social withdrawal in schizophrenia.

Design and synthesis of 1-(1-benzothiophen-7-yl)-1H-pyrazole, a novel series of G protein-coupled receptor 52 (GPR52) agonists

G-protein-coupled receptor 52 (GPR52) is classified as an orphan Gs-coupled G-protein-coupled receptor. GPR52 cancels dopamine D2 receptor signaling and activates dopamine D1/N-methyl-d-aspartate receptors via intracellular cAMP accumulation. Therefore, GPR52 agonists are expected to alleviate symptoms of psychotic disorders. A novel series of 1-(benzothiophen-7-yl)-1H-pyrazole as GPR52 agonists was designed and synthesized based on compound 1b. Compound 1b has been reported by our group as the first orally active GPR52 agonist, but high lipophilicity and poor aqueous solubility still remained as issues for candidate selection. To resolve these issues, replacement of the benzene ring at the 7-positon of compound 1b with heterocylic rings, such as pyrazole and pyridine, was greatly expected to reduce lipophilicity to levels for which calculated logD values were lower than that of compound 1b. While evaluating the pyrazole derivatives, introduction of a methyl substituent at the 3-position of the pyrazole ring led to increased GPR52 agonistic activity. Moreover, additional methyl substituent at the 5-position of the pyrazole and further introduction of hydroxy group to lower logD led to significant improvement of solubility while maintaining the activity. As a result, we identified 3-methyl-5-hydroxymethyl-1H-pyrazole derivative 17 (GPR52 EC50 = 21 nM, Emax = 103%, logD = 2.21, Solubility at pH 6.8 = 21 μg/mL) with potent GPR52 agonistic activity and good solubility compared to compound 1b. Furthermore, this compound 17 dose-dependently suppressed methamphetamine-induced hyperlocomotion in mice.

New Scaffold for Lead Compounds to Treat Methamphetamine Use Disorders.

Despite increased methamphetamine use worldwide, pharmacotherapies are not available to treat methamphetamine use disorder. The vesicular monoamine transporter-2 (VMAT2) is an important pharmacological target for discovery of treatments for methamphetamine use disorder. VMAT2 inhibition by the natural product, lobeline, reduced methamphetamine-evoked dopamine release, methamphetamine-induced hyperlocomotion, and methamphetamine self-administration in rats. Compared to lobeline, lobelane exhibited improved affinity and selectivity for VMAT2 over nicotinic acetylcholine receptors. Lobelane inhibited neurochemical and behavioral effects of methamphetamine, but tolerance developed to its behavioral efficacy in reducing methamphetamine self-administration, preventing further development. The lobelane analog, R-N-(1,2-dihydroxypropyl)-2,6-cis-di-(4-methoxyphenethyl)piperidine hydrochloride (GZ-793A), potently and selectively inhibited VMAT2 function and reduced neurochemical and behavioral effects of methamphetamine. However, GZ-793A exhibited potential to induce ventricular arrhythmias interacting with human-ether-a-go-go (hERG) channels. Herein, a new lead, R-3-(4-methoxyphenyl)-N-(1-phenylpropan-2-yl)propan-1-amine (GZ-11610), from the novel scaffold (N-alkyl(1-methyl-2-phenylethyl)amine) was evaluated as a VMAT2 inhibitor and potential therapeutic for methamphetamine use disorder. GZ-11610 was 290-fold selective for VMAT2 over dopamine transporters, suggesting that it may lack abuse liability. GZ-11610 was 640- to 3500-fold selective for VMAT2 over serotonin transporters and nicotinic acetylcholine receptors. GZ-11610 exhibited > 1000-fold selectivity for VMAT2 over hERG, representing a robust improvement relative to our previous VMAT2 inhibitors. GZ-11610 (3-30mg/kg, s.c. or 56-300mg/kg, oral) reduced methamphetamine-induced hyperactivity in methamphetamine-sensitized rats. Thus, GZ-11610 is a potent and selective inhibitor of VMAT2, may have low abuse liability and low cardiotoxicity, and after oral administration is effective and specific in inhibiting the locomotor stimulant effects of methamphetamine, suggesting further investigation as a potential therapeutic for methamphetamine use disorder.

Involvement of the accumbal osteopontin-interacting transmembrane protein 168 in methamphetamine-induced place preference and hyperlocomotion in mice

Chronic exposure to methamphetamine causes adaptive changes in brain, which underlie dependence symptoms. We have found that the transmembrane protein 168 (TMEM168) is overexpressed in the nucleus accumbens of mice upon repeated methamphetamine administration. Here, we firstly demonstrate the inhibitory effect of TMEM168 on methamphetamine-induced behavioral changes in mice, and attempt to elucidate the mechanism of this inhibition. We overexpressed TMEM168 in the nucleus accumbens of mice by using an adeno-associated virus vector (NAc-TMEM mice). Methamphetamine-induced hyperlocomotion and conditioned place preference were attenuated in NAc-TMEM mice. Additionally, methamphetamine-induced extracellular dopamine elevation was suppressed in the nucleus accumbens of NAc-TMEM mice. Next, we identified extracellular matrix protein osteopontin as an interacting partner of TMEM168, by conducting immunoprecipitation in cultured COS-7 cells. TMEM168 overexpression in COS-7 cells induced the enhancement of extracellular and intracellular osteopontin. Similarly, osteopontin enhancement was also observed in the nucleus accumbens of NAc-TMEM mice, in in vivo studies. Furthermore, the infusion of osteopontin proteins into the nucleus accumbens of mice was found to inhibit methamphetamine-induced hyperlocomotion and conditioned place preference. Our studies suggest that the TMEM168-regulated osteopontin system is a novel target pathway for the therapy of methamphetamine dependence, via regulating the dopaminergic function in the nucleus accumbens.

The antipsychotic-like effects in rodents of YQA31 involve dopamine D3 and 5-HT1A receptor.

We previously reported that YQA31 is a dopamine D3 receptor antagonist with modest 5-HT1A receptor affinity and that it exhibits antipsychotic properties in animal models of schizophrenia. However, the contributions of D3 and 5-HT1A receptors in the anti-psychotic effects of YQA31 are not clear. The current study evaluated the role of these two receptors in the effect of YQA31 on the hyperactivity and novel object recognition deficit in mice. We used dopamine D3 receptor knockout mice and 5-HT1A receptor antagonist WAY100635 pretreatment, respectively, to investigate the involvement of these receptors in the effects of YQA31. The anti-psychotic effects were tested by inducing hyperlocomotion with methamphetamine or MK-801 and by inducing novel object recognition deficit with MK-801, which are the animal models to represent a positive symptom and a cognitive disorder. YQA31 significantly inhibited MK-801-induced hyperlocomotion and novel object recognition deficit in WT mice, which was significantly inhibited by dopamine D3 receptor knockout. The 5-HT1A receptor antagonist, WAY100635, also blocked the effect of YQA31 in MK-801-induced novel object recognition deficit but not hyperlocomotion. The effect of YQA31 on methamphetamine-induced hyperlocomotion was not reversed by either dopamine D3 receptor knockout or WAY100635 pretreatment. These results demonstrate the different roles of dopamine D3 and 5-HT1A receptors in the anti-psychotic effects of YQA31. Both dopamine D3 and 5-HT1A receptors contributed to the effects of YQA31 on the inhibition of MK-801-induced novel object recognition deficit, and the dopamine D3 receptor mediated the inhibiting effect of YQA31 on hyperlocomotion induced by MK-801.

Design, synthesis, and pharmacological evaluation of 4-azolyl-benzamide derivatives as novel GPR52 agonists

G protein-coupled receptor 52 (GPR52) agonists are expected to improve the symptoms of psychiatric disorders. During exploration for a novel class of GPR52 agonists with good pharmacokinetic profiles, we synthesized 4-(3-(3-fluoro-5-(trifluoromethyl)benzyl)-5-methyl-1H-1,2,4-triazol-1-yl)-2-methylbenzamide (4u; half maximal effective concentration (EC50)=75nM, maximal response (Emax)=122%) starting from a high-throughput screening hit 3 (EC50=470nM, Emax=56%). The structural features of a reported GPR52 agonist were applied to 3, led to design 4-azolylbenzamides as novel GPR52 agonists. A structure–activity relationship study of 4-azolylbenzamide resulted in the design of the 1,2,4-triazole derivative 4u, which demonstrated excellent bioavailability in rats (F=53.8%). Oral administration of 4u (10mg/kg) significantly suppressed methamphetamine-induced hyperlocomotion in mice. Thus, 4u is a promising lead compound for drug discovery research of GPR52 agonists.

Hispidulin alleviated methamphetamine-induced hyperlocomotion by acting at α6 subunit-containing GABAA receptors in the cerebellum.

Hispidulin is a flavonoid we isolated from Clerodendrum inerme, an herb that effectively remitted a case of intractable motor tic disorders. Hispidulin was shown to be a positive allosteric modulator (PAM) of GABAA receptors, including the α6 subunit-containing subtype (α6GABAAR) that is predominantly expressed in cerebellar granule cells and insensitive to diazepam. We explored the action mechanism(s) of hispidulin using hyperdopaminergic mouse models induced by methamphetamine and apomorphine, based on the hyperdopaminergic nature of tic disorders. Hispidulin significantly inhibited methamphetamine-induced hyperlocomotion (MIH) at i.p. doses without affecting apomorphine-induced hyperlocomotion and stereotypy behaviors or having significant benzodiazepine-like effects (BZLE), including sedation, anxiety, and motor impairment. When given by intracerebellar (i.c.b.) microinjection, hispidulin also alleviated MIH and this effect was prevented by i.c.b. coadministration of furosemide, an α6GABAAR antagonist, and mimicked by i.c.b. Ro 15-4513, an α6GABAAR PAM. Conversely, i.c.b. diazepam did not affect MIH while it reduced MIH at i.p. doses having significant BZLE. In a screening assay for 92 neurotransmitter receptors/degradation enzymes/transporters, hispidulin displayed significant (>50% inhibition of radiolabeled ligand binding at 10μM) binding affinity only at the benzodiazepine binding site of GABAARs (IC50 0.73∼1.78μM) and catecholamine-o-methyl-transferase (COMT) (IC50 1.32μM). OR-486, a more potent COMT inhibitor than hispidulin, did not affect MIH. It is suggested that hispidulin alleviates MIH via acting as a PAM of cerebellar α6GABAARs, but not through COMT inhibition or affecting dopamine receptor responsiveness. Thus, selective α6GABAAR PAMs may have the potential to be a novel treatment for hyperdopaminergic disorders.

ML314: A Biased Neurotensin Receptor Ligand for Methamphetamine Abuse.

Pharmacological treatment for methamphetamine addiction will provide important societal benefits. Neurotensin receptor NTR1 and dopamine receptor distributions coincide in brain areas regulating methamphetamine-associated reward, and neurotensin peptides produce behaviors opposing psychostimulants. Therefore, undesirable methamphetamine-associated activities should be treatable with druggable NTR1 agonists, but no such FDA-approved therapeutics exist. We address this limitation with proof-of-concept data for ML314, a small-molecule, brain penetrant, β-arrestin biased, NTR1 agonist. ML314 attenuates amphetamine-like hyperlocomotion in dopamine transporter knockout mice, and in C57BL/6J mice it attenuates methamphetamine-induced hyperlocomotion, potentiates the psychostimulant inhibitory effects of a ghrelin antagonist, and reduces methamphetamine-associated conditioned place preference. In rats, ML314 blocks methamphetamine self-administration. ML314 acts as an allosteric enhancer of endogenous neurotensin, unmasking stoichiometric numbers of hidden NTR1 binding sites in transfected-cell membranes or mouse striatal membranes, while additionally supporting NTR1 endocytosis in cells in the absence of NT peptide. These results indicate ML314 is a viable, preclinical lead for methamphetamine abuse treatment and support an allosteric model of G protein-coupled receptor signaling.