Psilocybin pulse regimen reduces cluster headache attack frequency in the blinded extension phase of a randomized controlled trial.

BACKGROUND: In a recent randomized, double-blind, placebo-controlled study, we observed a nonsignificant reduction of attack frequency in cluster headache after pulse administration of psilocybin (10 mg/70 kg, 3 doses, 5 days apart each). We carried out a blinded extension phase to consider the safety and efficacy of repeating the pulse regimen. METHODS: Eligible participants returned to receive a psilocybin pulse at least 6 months after their first round of study participation. Participants kept headache diaries starting two weeks before and continuing through eight weeks after the first drug session. Ten participants completed the extension phase and all ten were included in the final analysis. RESULTS: In the three weeks after the start of the pulse, cluster attack frequency was significantly reduced from baseline (18.4 [95% confidence interval 8.4 to 28.4] to 9.8 [4.3 to 15.2] attacks/week; p = 0.013, d' = 0.97). A reduction of approximately 50% was seen regardless of individual response to psilocybin in the first round. Psilocybin was well-tolerated without any unexpected or serious adverse events. DISCUSSION: This study shows a significant reduction in cluster attack frequency in a repeat round of pulse psilocybin administration and suggests that prior response may not predict the effect of repeated treatment. To gauge the full potential of psilocybin as a viable medicine in cluster headache, future work should investigate the safety and therapeutic efficacy in larger, more representative samples over a longer time period, including repeating the treatment. CLINICAL TRIALS REGISTRATION: NCT02981173.

Exploratory investigation of a patient-informed low-dose psilocybin pulse regimen in the suppression of cluster headache: Results from a randomized, double-blind, placebo-controlled trial.

OBJECTIVE: Using a patient-informed regimen, we conducted an exploratory randomized, double-blind, placebo-controlled study to systematically investigate the effects of psilocybin in cluster headache. BACKGROUND: Sustained reductions in cluster headache burden after limited quantities of psilocybin-containing mushrooms are anecdotally reported, although to date there are no controlled studies investigating these effects. METHODS: Participants were randomized to receive psilocybin (0.143 mg/kg) or placebo (microcrystalline cellulose) in a pulse of three doses, each ~5 days apart. Participants maintained headache diaries starting 2 weeks before and continuing through 8 weeks after the first drug session. A total of 16 participants were randomized to receive experimental drug and 14 were included in the final analysis. RESULTS: In the 3 weeks after the start of the pulse regimen, the change in cluster attack frequency was 0.03 (95% confidence interval [CI] -2.6 to 2.6) attacks/week with placebo (baseline 8.9 [95% CI 3.8 to 14.0]) and -3.2 (95% CI -8.3 to 1.9) attacks/week with psilocybin (baseline 9.6 [95% CI 5.6 to 13.6]; p = 0.251). Group difference in change from baseline had a moderate effect size (d = 0.69). The effect size was small in episodic participants (d = 0.35) but large in chronic participants (d = 1.25), which remained over the entire 8-week period measured (d = 0.81). Changes in cluster attack frequency were not correlated with the intensity of acute psychotropic effects during psilocybin administration. Psilocybin was well-tolerated without any unexpected or serious adverse events. CONCLUSIONS: Findings from this initial, exploratory study provide valuable information for the development of larger, more definitive studies. Efficacy outcomes were negative, owing in part to the small number of participants. The separation of acute psychotropic effects and lasting therapeutic effects underscores the need for further investigation into the mechanism(s) of action of psilocybin in headache disorders.

Exploratory study of the dose-related safety, tolerability, and efficacy of dimethyltryptamine (DMT) in healthy volunteers and major depressive disorder.

There is considerable interest in the therapeutic potential of psychedelic drugs. Dimethyltryptamine (DMT) is a potent, rapid-onset, and short-acting psychedelic drug that has not yet been independently tested for the treatment of depression. The safety, tolerability, and efficacy of intravenous DMT were investigated in treatment-resistant individuals with major depressive disorder (MDD) and healthy controls (HC) in an open-label, fixed-order, dose-escalation (0.1 mg/kg followed by 0.3 mg/kg) exploratory phase 1 study that was conducted in a typical hospital setting with strategic psychoeducation/support, but minimal psychotherapy. Tolerability, safety, cardiovascular function, abuse liability, psychedelic, and psychotomimetic effects, mood, and anxiety were assessed at each dosing session. In addition, depression was measured using the HAMD-17 in MDD participants 1 day after each dosing session. DMT was tolerated by both HC (n = 3) and MDD participants (n = 7) studied; there were no dropouts. HAMD-17 scores decreased significantly (p = 0.017) compared to baseline in MDD participants the day after receiving 0.3 mg/kg DMT (mean difference -4.5 points, 95% CI: -7.80 to -1.20, Hedge's g = 0.75). Adverse events were mostly mild with one self-limited serious event. DMT increased blood pressure, heart rate, anxiety, psychedelic effects, and psychotomimetic effects, which resolved within 20-30 min of injection. There were no dose-related differences in measures of drug reinforcement and abuse liability. In this small exploratory pilot study, intravenous DMT at doses of 0.1 and 0.3 mg/kg was mostly safe and tolerated and may have next-day (rapid) antidepressant effects in patients with treatment-resistant MDD. Further rigorous trials are warranted to replicate these findings and to determine the durability of antidepressant effects.

Psilocybin: crystal structure solutions enable phase analysis of prior art and recently patented examples.

Psilocybin {systematic name: 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl dihydrogen phosphate} is a zwitterionic tryptamine natural product found in numerous species of fungi known for their psychoactive properties. Following its structural elucidation and chemical synthesis in 1959, purified synthetic psilocybin has been evaluated in clinical trials and has shown promise in the treatment of various mental health disorders. In a recent process-scale crystallization investigation, three crystalline forms of psilocybin were repeatedly observed: Hydrate A, Polymorph A, and Polymorph B. The crystal structure for Hydrate A was solved previously by single-crystal X-ray diffraction. This article presents new crystal structure solutions for the two anhydrates, Polymorphs A and B, based on Rietveld refinement using laboratory and synchrotron X-ray diffraction data, and density functional theory (DFT) calculations. Utilizing the three solved structures, an investigation was conducted via Rietveld method (RM) based quantitative phase analysis (QPA) to estimate the contribution of the three different forms in powder X-ray diffraction (PXRD) patterns provided by different sources of bulk psilocybin produced between 1963 and 2021. Over the last 57 years, each of these samples quantitatively reflect one or more of the hydrate and anhydrate polymorphs. In addition to quantitatively evaluating the composition of each sample, this article evaluates correlations between the crystal forms present, corresponding process methods, sample age, and storage conditions. Furthermore, revision is recommended on characterizations in recently granted patents that include descriptions of crystalline psilocybin inappropriately reported as a single-phase `isostructural variant.' Rietveld refinement demonstrated that the claimed material was composed of approximately 81% Polymorph A and 19% Polymorph B, both of which have been identified in historical samples. In this article, we show conclusively that all published data can be explained in terms of three well-defined forms of psilocybin and that no additional forms are needed to explain the diffraction patterns.

N,N-Dimethyltryptamine attenuates spreading depolarization and restrains neurodegeneration by sigma-1 receptor activation in the ischemic rat brain.

Dimethyltryptamine (DMT), an endogenous ligand of sigma-1 receptors (Sig-1Rs), acts against systemic hypoxia, but whether DMT may prevent cerebral ischemic injury is unexplored. Here global forebrain ischemia was created in anesthetized rats and aggravated with the induction of spreading depolarizations (SDs) and subsequent short hypoxia before reperfusion. Drugs (DMT, the selective Sig-1R agonist PRE-084, the Sig-1R antagonist NE-100, or the serotonin receptor antagonist asenapine) were administered intravenously alone or in combination while physiological variables and local field potential from the cerebral cortex was recorded. Neuroprotection and the cellular localization of Sig-1R were evaluated with immunocytochemistry. Plasma and brain DMT content was measured by 2D-LC-HRMS/MS. The affinity of drugs for cerebral Sig-1R was evaluated with a radioligand binding assay. Both DMT and PRE-084 mitigated SDs, counteracted with NE-100. Further, DMT attenuated SD when co-administered with asenapine, compared to asenapine alone. DMT reduced the number of apoptotic and ferroptotic cells and supported astrocyte survival. The binding affinity of DMT to Sig-1R matched previously reported values. Sig-1Rs were associated with the perinuclear cytoplasm of neurons, astrocytes and microglia, and with glial processes. According to these data, DMT may be considered as adjuvant pharmacological therapy in the management of acute cerebral ischemia.

Exploratory Controlled Study of the Migraine-Suppressing Effects of Psilocybin.

While anecdotal evidence suggests that select 5-hydroxytryptamine 2A (5-HT2A) receptor ligands, including psilocybin, may have long-lasting therapeutic effects after limited dosing in headache disorders, controlled investigations are lacking. In an exploratory double-blind, placebo-controlled, cross-over study, adults with migraine received oral placebo and psilocybin (0.143 mg/kg) in 2 test sessions spaced 2 weeks apart. Subjects maintained headache diaries starting 2 weeks before the first session until 2 weeks after the second session. Physiological and psychological drug effects were monitored during sessions and several follow-up contacts with subjects were carried out to assure safety of study procedures. Ten subjects were included in the final analysis. Over the 2-week period measured after single administration, the reduction in weekly migraine days from baseline was significantly greater after psilocybin (mean, - 1.65 (95% CI: - 2.53 to - 0.77) days/week) than after placebo (- 0.15 (- 1.13 to 0.83) days/week; p = 0.003, t(9) = 4.11). Changes in migraine frequency in the 2 weeks after psilocybin were not correlated with the intensity of acute psychotropic effects during drug administration. Psilocybin was well-tolerated; there were no unexpected or serious adverse events or withdrawals due to adverse events. This exploratory study suggests there is an enduring therapeutic effect in migraine headache after a single administration of psilocybin. The separation of acute psychotropic effects and lasting therapeutic effects is an important finding, urging further investigation into the mechanism underlying the clinical effects of select 5-HT2A receptor compounds in migraine, as well as other neuropsychiatric conditions. Clinicaltrials.gov : NCT03341689.

Synthesis and characterization of high-purity N,N-dimethyltryptamine hemifumarate for human clinical trials.

Since 2006, there has been a resurgent interest in the pharmacology and therapeutics of psychedelic drugs. Psilocybin, the 4-phosphoryl ester of N,N-dimethyltryptamine (DMT), has been studied most often, but DMT itself is also appealing because of its brief but profound psychological effects and its presence as an endogenous substance in mammalian brain. Although there have been a few studies of ayahuasca, a DMT-containing water infusion, only one human study with pure DMT has been reported since the early 2000s. Newly planned clinical trials to assess the safety and efficacy of DMT in humans with major depressive disorders require high-purity water-soluble DMT for intravenous administration. Accordingly, we synthesized and characterized DMT hemifumarate for these upcoming studies. The synthetic approach of Speeter and Anthony was slightly modified to gain some efficiency in time. In particular, this is the first known report to use aluminum hydride, generated in situ from lithium aluminum hydride, to reduce the intermediate 2-(1H-indol-3-yl)-N,N-dimethyl-2-oxoacetamide to DMT. A quench protocol was developed to produce a good yield of exceptionally pure free base DMT upon workup, which was then converted to the hemifumarate salt. Analysis of the final product included differential scanning calorimetry, thermogravimetric analysis, gas chromatography-mass spectrometry (GC-MS), 1 H and 13 C nuclear magnetic resonance spectroscopy, high-performance liquid chromatography, residual solvent analysis by GC headspace sampling, X-ray powder diffraction analysis, and residual lithium analysis by inductively coupled plasma-mass spectrometry. The DMT hemifumarate was minimally 99.9% pure, with no significant impurities or residual solvents, thus meeting regulatory standards for administration to humans.

Direct Phosphorylation of Psilocin Enables Optimized cGMP Kilogram-Scale Manufacture of Psilocybin.

A second-generation kilogram-scale synthesis of the psychedelic tryptamine psilocybin has been developed. The synthesis was designed to address several challenges first encountered with the scale-up of previously described literature procedures, which were not optimized for providing consistent yield and purity of products, atom economy, or being run in pilot plant-scale reactors. These challenges were addressed and circumvented with the design of the second-generation route, which featured an optimized cGMP large-scale Speeter-Anthony tryptamine synthesis to the intermediate psilocin with improved in-process control and impurity removal over the three steps. Psilocin was subsequently phosphorylated directly with phosphorous oxychloride for the first time, avoiding a tedious and poor atom economy benzyl-protecting group strategy common to all previously described methods for producing psilocybin. In this report, the challenges encountered in a 100 g scale first-generation literature-based synthesis are highlighted, followed by a detailed description of the newly developed second-generation synthesis to provide over one kilogram of high-purity psilocybin under cGMP.

para-Trifluoromethyl-methcathinone is an allosteric modulator of the serotonin transporter.

The transporters for dopamine (DAT) and serotonin (SERT) are important targets in the treatment of psychiatric disorders including major depression, anxiety and attention-deficit hyperactivity disorder. Drugs acting at these transporters can act as inhibitors or as releasers. In addition, it has been recently appreciated that some compounds are less efficacious releasers than amphetamine. Thus, they are classified as partial releasers. Compounds can act on both SERT and DAT or display exquisite selectivity for either SERT or DAT, but the structural basis for selectivity is poorly understood. The trifluoromethyl-substitution of methcathinone in the para-position has been shown to dramatically shift the selectivity of methcathinone (MCAT) towards SERT. Here, we examined MCAT, para-trifluoromethyl-methcathinone (pCF3MCAT) and other analogues to understand (i) the determinants of selectivity and (ii) the effects of the para-CF3-substitution of MCAT on the transport cycle. We systematically tested different para-substituted MCATs by biochemical, computational and electrophysiological approaches: addition of the pCF3group, but not of other substituents with larger van der Waal's volume, lipophilicity or polarity, converted the DAT-selective MCAT into a SERT-selective partial releaser. Electrophysiological and superfusion experiments, together with kinetic modelling, showed that pCF3MCAT, but not MCAT, trapped a fraction of SERTs in an inactive state by occupying the S2-site. These findings define a new mechanism of action for partial releasers, which is distinct from the other two known binding modes underlying partial release. Our observations highlight the fact that the substrate permeation pathway of monoamine transporters supports multiple binding modes, which can be exploited for drug design. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

High dose psilocybin is associated with positive subjective effects in healthy volunteers.

AIM: The aim of the current study was to investigate the relationship between escalating higher doses of psilocybin and the potential psilocybin occasioned positive subjective effects. METHODS: Healthy participants ( n=12) were given three escalating doses of oral psilocybin (0.3 mg/kg; 0.45 mg/kg; 0.6 mg/kg) or (18.8-36.6 mg; 27.1-54.0 mg; 36.3-59.2 mg) a minimum of four weeks apart in a supervised setting. Blood and urine samples, vital signs, and electrocardiograms were obtained. Subjective effects were assessed using the Mystical Experience Questionnaire and Persisting Effects Questionnaire. RESULTS: There was a significant linear dose-related response in Mystical Experience Questionnaire total score and the transcendence of time and space subscale, but not in the rate of a complete mystical experience. There was also a significant difference between dose 3 compared to dose 1 on the transcendence of time and space subscale, while no dose-related differences were found for Mystical Experience Questionnaire total scores or rate of a mystical experience. Persisting Effects Questionnaire positive composite scores 30 days after completion of the last dose were significantly higher than negative composite scores. Persisting Effects Questionnaire results revealed a moderate increase in sense of well-being or life satisfaction on average that was associated with the maximum Mystical Experience Questionnaire total score. Pharmacokinetic measures were associated with dose but not with Mystical Experience Questionnaire total scores or rate of a mystical experience. CONCLUSIONS: High doses of psilocybin elicited subjective effects at least as strong as the lower doses and resulted in positive persisting subjective effects 30 days after, indicating that a complete mystical experience was not a prerequisite for positive outcomes.

Receptor binding profiles and behavioral pharmacology of ring-substituted N,N-diallyltryptamine analogs.

Substantial effort has been devoted toward understanding the psychopharmacological effects of tryptamine hallucinogens, which are thought to be mediated by activation of 5-HT2A and 5-HT1A receptors. Recently, several psychoactive tryptamines based on the N,N-diallyltryptamine (DALT) scaffold have been encountered as recreational drugs. Despite the apparent widespread use of DALT derivatives in humans, little is known about their pharmacological properties. We compared the binding affinities of DALT and its 2-phenyl-, 4-acetoxy-, 4-hydroxy-, 5-methoxy-, 5-methoxy-2-methyl-, 5-fluoro-, 5-fluoro-2-methyl-, 5-bromo-, and 7-ethyl-derivatives at 45 receptor and transporter binding sites. Additionally, studies in C57BL/6 J mice examined whether these substances induce the head twitch response (HTR), a 5-HT2A receptor-mediated response that is widely used as a behavioral proxy for hallucinogen effects in humans. Most of the test drugs bound to serotonin receptors, σ sites, α2-adrenoceptors, dopaminergic D3 receptors, histaminergic H1 receptors, and the serotonin transporter. DALT and several of the ring-substituted derivatives were active in the HTR assay with the following rank order of potency: 4-acetoxy-DALT > 5-fluoro-DALT > 5-methoxy-DALT > 4-hydroxy-DALT > DALT > 5-bromo-DALT. 2-Phenyl-DALT, 5-methoxy-2-methyl-DALT, 5-fluoro-2-methyl-DALT, and 7-ethyl-DALT did not induce the HTR. HTR potency was not correlated with either 5-HT1A or 5-HT2A receptor binding affinity, but a multiple regression analysis indicated that 5-HT2A and 5-HT1A receptors make positive and negative contributions, respectively, to HTR potency (R2 = 0.8729). In addition to supporting the established role of 5-HT2A receptors in the HTR, these findings are consistent with evidence that 5-HT1A activation by tryptamine hallucinogens buffers their effects on HTR. This article is part of the Special Issue entitled 'Psychedelics: New Doors, Altered Perceptions'.

Pharmacokinetics of Escalating Doses of Oral Psilocybin in Healthy Adults.

INTRODUCTION: Psilocybin is a psychedelic tryptamine that has shown promise in recent clinical trials for the treatment of depression and substance use disorders. This open-label study of the pharmacokinetics of psilocybin was performed to describe the pharmacokinetics and safety profile of psilocybin in sequential, escalating oral doses of 0.3, 0.45, and 0.6 mg/kg in 12 healthy adults. METHODS: Eligible healthy adults received 6-8 h of preparatory counseling in anticipation of the first dose of psilocybin. The escalating oral psilocybin doses were administered at approximately monthly intervals in a controlled setting and subjects were monitored for 24 h. Blood and urine samples were collected over 24 h and assayed by a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for psilocybin and psilocin, the active metabolite. The pharmacokinetics of psilocin were determined using both compartmental (NONMEM) and noncompartmental (WinNonlin) methods. RESULTS: No psilocybin was found in plasma or urine, and renal clearance of intact psilocin accounted for less than 2% of the total clearance. The pharmacokinetics of psilocin were linear within the twofold range of doses, and the elimination half-life of psilocin was 3 h (standard deviation 1.1). An extended elimination phase in some subjects suggests hydrolysis of the psilocin glucuronide metabolite. Variation in psilocin clearance was not predicted by body weight, and no serious adverse events occurred in the subjects studied. CONCLUSIONS: The small amount of psilocin renally excreted suggests that no dose reduction is needed for subjects with mild-moderate renal impairment. Simulation of fixed doses using the pharmacokinetic parameters suggest that an oral dose of 25 mg should approximate the drug exposure of a 0.3 mg/kg oral dose of psilocybin. Although doses of 0.6 mg/kg are in excess of likely therapeutic doses, no serious physical or psychological events occurred during or within 30 days of any dose. CLINICAL TRIALS IDENTIFIER: NCT02163707.

Atypical dopamine efflux caused by 3,4-methylenedioxypyrovalerone (MDPV) via the human dopamine transporter.

Synthetic cathinones are similar in chemical structure to amphetamines, and their behavioral effects are associated with enhanced dopaminergic signaling. The past ten years of research on the common constituent of bath salts, MDPV (the synthetic cathinone 3,4-methylenedioxypyrovalerone), has aided the understanding of how synthetic cathinones act at the dopamine (DA) transporter (DAT). Several groups have described the ability of MDPV to block the DAT with high-affinity. In this study, we demonstrate for the first time a new mode of action of MDPV, namely its ability to promote DAT-mediated DA efflux. Using single cell amperometric assays, we determined that low concentrations of MDPV (1nM) can cause reverse transport of DA via DAT. Notably, administration of MDPV leads to hyperlocomotion in Drosophila melanogaster. These data describe further how MDPV acts at the DAT, possibly paving the way for novel treatment strategies for individuals who abuse bath salts.

Combined Simulation and Mutation Studies to Elucidate Selectivity of Unsubstituted Amphetamine-like Cathinones at the Dopamine Transporter.

The dopamine and serotonin transporter proteins (DAT, SERT) play a vital role in behavior and mental illness. Although their substrate transport has been studied extensively, the molecular basis of their selectivity is not completely understood yet. In this study, we exploit molecular dynamics simulations combined with mutagenesis studies to shed light on the driving factors for DAT-over-SERT selectivity of a set of cathinones. Results indicate that these compounds can adopt two binding modes of which one is more favorable. In addition, free energy calculations indicated the substrate binding site (S1) as the primary recognition site for these ligands. By simulating DAT with SERT-like mutations, we hypothesize unsubstituted cathinones to bind more favorably to DAT, due to a Val152 offering more space, as compared to the bulkier Ile172 in SERT. This was supported by uptake inhibition measurements, which showed an increase in activity in SERT-I172V.

Receptor binding profiles and quantitative structure-affinity relationships of some 5-substituted-N,N-diallyltryptamines.

N,N-Diallyltryptamine (DALT) and 5-methoxy-N,N-diallyltryptamine (5-MeO-DALT) are two tryptamines synthesized and tested by Alexander Shulgin. In self-experiments, 5-MeO-DALT was reported to be psychoactive in the 12-20mg range, while the unsubstituted compound DALT had few discernible effects in the 42-80 mg range. Recently, 5-MeO-DALT has been used in nonmedical settings for its psychoactive effects, but these effects have been poorly characterized and little is known of its pharmacological properties. We extended the work of Shulgin by synthesizing additional 5-substituted-DALTs. We then compared them to DALT and 5-MeO-DALT for their binding affinities at 45 cloned receptors and transporter proteins. Based on in vitro binding affinity, we identified 27 potential receptor targets for the 5-substituted-DALT compounds. Five of the DALT compounds had affinity in the 10-80 nM range for serotonin 5-HT1A and 5-HT2B receptors, while the affinity of DALT itself at 5-HT1A receptors was slightly lower at 100 nM. Among the 5-HT2 subtypes, the weakest affinity was at 5-HT2A receptors, spanning 250-730 nM. Five of the DALT compounds had affinity in the 50-400 nM range for serotonin 5-HT1D, 5-HT6, and 5-HT7 receptors; again, it was the unsubstituted DALT that had the weakest affinity at all three subtypes. The test drugs had even weaker affinity for 5-HT1B, 5-HT1E, and 5-HT5A subtypes and little or no affinity for the 5-HT3 subtype. These compounds also had generally nanomolar affinities for adrenergic α2A, α2B, and α2C receptors, sigma receptors σ1 and σ2, histamine H1 receptors, and norepinephrine and serotonin uptake transporters. They also bound to other targets in the nanomolar-to-low micromolar range. Based on these binding results, it is likely that multiple serotonin receptors, as well as several nonserotonergic sites are important for the psychoactive effects of DALT drugs. To learn whether any quantitative structure-affinity relationships existed, we evaluated correlations among physicochemical properties of the congeneric 5-substituted-DALT compounds. The descriptors included electronic (σp), hydrophobic (π), and steric (CMR) parameters. The binding affinity at 5-HT1A, 5-HT1D, 5-HT7, and κ opioid receptors was positively correlated with the steric volume parameter CMR. At α2A, α2B, and α2C receptors, and at the histamine H1 receptor, binding affinity was correlated with the Hammett substituent parameter σp; higher affinity was associated with larger σp values. At the σ2 receptor, higher affinity was correlated with increasing π. These correlations should aid in the development of more potent and selective drugs within this family of compounds.

Reinforcing and neurochemical effects of the "bath salts" constituents 3,4-methylenedioxypyrovalerone (MDPV) and 3,4-methylenedioxy-N-methylcathinone (methylone) in male rats.

RATIONALE: 3,4-Methylenedioxypyrovalerone (MDPV) and 3,4-methylenedioxy-N-methylcathinone (methylone) are synthetic drugs found in so-called "bath salts" products. Both drugs exert their effects by interacting with monoamine transporter proteins. MDPV is a potent uptake blocker at transporters for dopamine and norepinephrine while methylone is a non-selective releaser at transporters for dopamine, norepinephrine, and serotonin (5-HT). OBJECTIVES: We hypothesized that prominent 5-HT-releasing actions of methylone would render this drug less reinforcing than MDPV. METHODS: To test this hypothesis, we compared behavioral effects of MDPV and methylone using intravenous (i.v.) self-administration on a fixed-ratio 1 schedule in male rats. Additionally, neurochemical effects of the drugs were examined using in vivo microdialysis in nucleus accumbens, in a separate cohort of rats. RESULTS: MDPV self-administration (0.03 mg/kg/inj) was acquired rapidly and reached 40 infusions per session, similar to the effects of cocaine (0.5 mg/kg/inj), by the end of training. In contrast, methylone self-administration (0.3 and 0.5 mg/kg/inj) was acquired slowly, and response rates only reached 20 infusions per session by the end of training. In dose substitution studies, MDPV and cocaine displayed typical inverted U-shaped dose-effect functions, but methylone did not. In vivo microdialysis revealed that i.v. MDPV (0.1 and 0.3 mg/kg) increased extracellular dopamine while i.v. methylone (1 and 3 mg/kg) increased extracellular dopamine and 5-HT. CONCLUSIONS: Our findings support the hypothesis that elevations in extracellular 5-HT in the brain can dampen positive reinforcing effects of cathinone-type drugs. Nevertheless, MDPV and methylone are both self-administered by rats, suggesting these drugs possess significant abuse liability in humans.

Noncompetitive inhibition of indolethylamine-N-methyltransferase by N,N-dimethyltryptamine and N,N-dimethylaminopropyltryptamine.

Indolethylamine-N-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of N,N-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and S-adenosyl-l-methionine (SAM). The biological, biochemical, and selective small molecule regulation of INMT enzyme activity remain largely unknown. Kinetic mechanisms for inhibition of rabbit lung INMT (rabINMT) by the product, DMT, and by a new novel tryptamine derivative were determined. After Michaelis-Menten and Lineweaver-Burk analyses had been applied to study inhibition, DMT was found to be a mixed competitive and noncompetitive inhibitor when measured against tryptamine. The novel tryptamine derivative, N-[2-(1H-indol-3-yl)ethyl]-N',N'-dimethylpropane-1,3-diamine (propyl dimethyl amino tryptamine or PDAT), was shown to inhibit rabINMT by a pure noncompetitive mechanism when measured against tryptamine with a Ki of 84 µM. No inhibition by PDAT was observed at 2 mM when it was tested against structurally similar Class 1 methyltransferases, such as human phenylethanolamine-N-methyltransferase (hPNMT) and human nicotinamide-N-methyltransferase (hNNMT), indicating selectivity for INMT. The demonstration of noncompetitive mechanisms for INMT inhibition implies the presence of an inhibitory allosteric site. In silico analyses using the computer modeling software Autodock and the rabINMT sequence threaded onto the human INMT (hINMT) structure (Protein Data Bank entry 2A14 ) identified an N-terminal helix-loop-helix non-active site binding region of the enzyme. The energies for binding of DMT and PDAT to this region of rabINMT, as determined by Autodock, were -6.34 and -7.58 kcal/mol, respectively. Assessment of the allosteric control of INMT may illuminate new biochemical pathway(s) underlying the biology of INMT.

Radiotracers for cardiac sympathetic innervation: transport kinetics and binding affinities for the human norepinephrine transporter.

INTRODUCTION: Most radiotracers for imaging of cardiac sympathetic innervation are substrates of the norepinephrine transporter (NET). The goal of this study was to characterize the NET transport kinetics and binding affinities of several sympathetic nerve radiotracers, including [(11)C]-(-)-meta-hydroxyephedrine, [(11)C]-(-)-epinephrine, and a series of [(11)C]-labeled phenethylguanidines under development in our laboratory. For comparison, the NET transport kinetics and binding affinities of some [(3)H]-labeled biogenic amines were also determined. METHODS: Transport kinetics studies were performed using rat C6 glioma cells stably transfected with the human norepinephrine transporter (C6-hNET cells). For each radiolabeled NET substrate, saturation transport assays with C6-hNET cells measured the Michaelis-Menten transport constants Km and Vmax for NET transport. Competitive inhibition binding assays with homogenized C6-hNET cells and [(3)H]mazindol provided estimates of binding affinities (KI) for NET. RESULTS: Km, Vmax and KI values were determined for each NET substrate with a high degree of reproducibility. Interestingly, C6-hNET transport rates for 'tracer concentrations' of substrate, given by the ratio Vmax/Km, were found to be highly correlated with neuronal transport rates measured previously in isolated rat hearts (r(2)=0.96). This suggests that the transport constants Km and Vmax measured using the C6-hNET cells accurately reflect in vivo transport kinetics. CONCLUSION: The results of these studies show how structural changes in NET substrates influence NET binding and transport constants, providing valuable insights that can be used in the design of new tracers with more optimal kinetics for quantifying regional sympathetic nerve density.

Powerful cocaine-like actions of 3,4-methylenedioxypyrovalerone (MDPV), a principal constituent of psychoactive 'bath salts' products.

The abuse of psychoactive 'bath salts' containing cathinones such as 3,4-methylenedioxypyrovalerone (MDPV) is a growing public health concern, yet little is known about their pharmacology. Here, we evaluated the effects of MDPV and related drugs using molecular, cellular, and whole-animal methods. In vitro transporter assays were performed in rat brain synaptosomes and in cells expressing human transporters, while clearance of endogenous dopamine was measured by fast-scan cyclic voltammetry in mouse striatal slices. Assessments of in vivo neurochemistry, locomotor activity, and cardiovascular parameters were carried out in rats. We found that MDPV blocks uptake of [(3)H]dopamine (IC(50)=4.1 nM) and [(3)H]norepinephrine (IC(50)=26 nM) with high potency but has weak effects on uptake of [(3)H]serotonin (IC(50)=3349 nM). In contrast to other psychoactive cathinones (eg, mephedrone), MDPV is not a transporter substrate. The clearance of endogenous dopamine is inhibited by MDPV and cocaine in a similar manner, but MDPV displays greater potency and efficacy. Consistent with in vitro findings, MDPV (0.1-0.3 mg/kg, intravenous) increases extracellular concentrations of dopamine in the nucleus accumbens. Additionally, MDPV (0.1-3.0 mg/kg, subcutaneous) is at least 10 times more potent than cocaine at producing locomotor activation, tachycardia, and hypertension in rats. Our data show that MDPV is a monoamine transporter blocker with increased potency and selectivity for catecholamines when compared with cocaine. The robust stimulation of dopamine transmission by MDPV predicts serious potential for abuse and may provide a mechanism to explain the adverse effects observed in humans taking high doses of 'bath salts' preparations.