- Nicholas Vito Cozzi, Ph.D. is a scientist and educator with background and training in pharmacology, chemistry, toxic... moreNicholas Vito Cozzi, Ph.D. is a scientist and educator with background and training in pharmacology, chemistry, toxicology, and neuroscience. He holds a Ph.D. in Pharmacology and a B.S. in Pharmacology and Toxicology, both from the University of Wisconsin-Madison School of Pharmacy.
Dr. Cozzi’s research involves the design, chemical synthesis, and pharmacological testing of substances with central nervous system activity, especially psychedelics, empathogens, antidepressants, and psychostimulants. He is interested in how these agents act in the brain to promote neuronal health, improve mood, enhance cognition, and increase awareness, and in their clinical value in treating addiction, depression, post-traumatic fear, neurological diseases, and other health ailments. Dr. Cozzi has published numerous articles focused on the chemistry and pharmacology of psychoactive drugs and is internationally recognized for his work.
As an educator, Dr. Cozzi taught pharmacology at the Brody School of Medicine at East Carolina University, the UW-Madison School of Medicine and Public Health, and the UW-Madison School of Pharmacy, and he is a frequent guest lecturer at other academic institutions around the United States. He has received several teaching and research awards, including a Distinguished Basic Science Teaching Award from the UW-Madison and a prestigious NARSAD Award from the Brain and Behavior Research Foundation for his work involving the serotonin uptake transporter.
Dr. Cozzi's research has been supported by grants from the National Institutes of Health, the UW-Madison, and private foundations. Outside acdemia, he serves as a scientific consultant for legal, pharmaceutical industry, and government clients. Dr. Cozzi is also Co-Founder, President, and Director of Pharmacology at the Alexander Shulgin Research Institute, a psychedelics discovery, development, and educational institute located in Lafayette, CA.edit
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).... more
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.
Research Interests:
A urine sample was submitted to our laboratory for drugs of abuse testing. The ordering provider mentioned that he was specifically interested in assessing the presence of 3,4-methylenedioxymethamphetamine (MDMA). Screening was performed... more
A urine sample was submitted to our laboratory for drugs of abuse testing. The ordering provider mentioned that he was specifically interested in assessing the presence of 3,4-methylenedioxymethamphetamine (MDMA). Screening was performed with an automated immunoassay (Abbott Alinity) for opiates, oxycodone/oxymorphone, benzodiazepines, ethanol, THC, cocaine, barbiturates, methadone, and amphetamine/methamphetamine. The only component that screened positive was amphetamine/ methamphetamine. The positive amphetamine result reflexed confirmatory testing. Confirmatory testing was performed using enzyme hydrolysis followed by analysis using a SciEx Triple Quad 5500 with a Kinetex, 2.6 um Biphenyl 100 Å, LC Column 50 × 3.0 mm. Confirmatory testing with LC-MS/MS revealed a possible trace amount of the MDMA qualifier ion at the expected retention time but did not meet criteria to justify a finding of MDMA (qualifier and quantifier ions for MDMA are 194.1/163.1 and 194.1/105.1, respectively). The presence of a peak corresponding to the retention time of MDMA, as well as the positive amphetamine screen result, led to further inquiry of the ordering provider as to the origins of the specimen.
Research Interests:
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... more
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, though there are no controlled studies investigating these effects.
Methods: Subjects were randomized to receive psilocybin (0.143 mg/kg) or placebo (microcrystalline cellulose) in a pulse of 3 doses approximately 5 days apart each. Subjects maintained headache diaries starting two weeks before and continuing through eight weeks after the first drug session. Fourteen subjects were included in the final analysis.
Results: In the three weeks after the start of the pulse regimen, cluster attack frequency was +0.03 (95% Confidence Interval: -2.6 to 2.6) attacks/week with placebo and -3.2 (-8.3 to 1.9) attacks/week with psilocybin (p = 0.251). Group difference in change from baseline had a moderate effect size (d = 0.69), though was not statistically significant (p = 0.251). The effect size in episodic subjects was small (d = 0.35), but large in chronic subjects (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. 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. Clinicaltrials.gov: NCT02981173
Background: Sustained reductions in cluster headache burden after limited quantities of psilocybin-containing mushrooms are anecdotally reported, though there are no controlled studies investigating these effects.
Methods: Subjects were randomized to receive psilocybin (0.143 mg/kg) or placebo (microcrystalline cellulose) in a pulse of 3 doses approximately 5 days apart each. Subjects maintained headache diaries starting two weeks before and continuing through eight weeks after the first drug session. Fourteen subjects were included in the final analysis.
Results: In the three weeks after the start of the pulse regimen, cluster attack frequency was +0.03 (95% Confidence Interval: -2.6 to 2.6) attacks/week with placebo and -3.2 (-8.3 to 1.9) attacks/week with psilocybin (p = 0.251). Group difference in change from baseline had a moderate effect size (d = 0.69), though was not statistically significant (p = 0.251). The effect size in episodic subjects was small (d = 0.35), but large in chronic subjects (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. 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. Clinicaltrials.gov: NCT02981173
Research Interests:
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... more
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.
Research Interests:
Psilocybin {systematic name: 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl dihydrogenphosphate} is a zwitterionic tryptamine natural product found in numerous species of fungi known for their psychoactive properties. Following its structural... more
Psilocybin {systematic name: 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl dihydrogenphosphate} 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.
Research Interests:
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... more
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.
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While anecdotal evidence suggests that select 5-hydroxytryptamine 2A (5-HT 2A) receptor ligands, including psilocybin, may have long-lasting therapeutic effects after limited dosing in headache disorders, controlled investigations are... more
While anecdotal evidence suggests that select 5-hydroxytryptamine 2A (5-HT 2A) 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-HT 2A receptor compounds in migraine, as well as other neuropsychiatric conditions. Clinicaltrials.gov: NCT03341689
Research Interests:
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... more
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 disorder 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, 1H and 13C nuclear magnetic resonance spectroscopy, high-performance liquid chromatography, residual solvent analysis by gas chromatography 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.
Research Interests:
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... more
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.
Research Interests:
• Para-CF3-substitution of MCAT allows to selectively target SERT over DAT. • MCAT and para-CF3-MCAT are partial releasers of SERT. • The partial release evoked by para-CF3-MCAT involves the allosteric site of SERT. • The substrate... more
• Para-CF3-substitution of MCAT allows to selectively target SERT over DAT.
• MCAT and para-CF3-MCAT are partial releasers of SERT.
• The partial release evoked by para-CF3-MCAT involves the allosteric site of SERT.
• The substrate permeation pathway of SERT affords different binding modes.
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 (p-CF3-MCAT) and other analogues to understand (i) the determinants of selectivity and (ii) the effects of the para-CF 3-substitution of MCAT on the transport cycle. We systematically tested different para-substituted MCATs by biochemical, computational and electrophysiological approaches: addition of the p-CF3-group, 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 p-CF3-MCAT, 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.
• MCAT and para-CF3-MCAT are partial releasers of SERT.
• The partial release evoked by para-CF3-MCAT involves the allosteric site of SERT.
• The substrate permeation pathway of SERT affords different binding modes.
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 (p-CF3-MCAT) and other analogues to understand (i) the determinants of selectivity and (ii) the effects of the para-CF 3-substitution of MCAT on the transport cycle. We systematically tested different para-substituted MCATs by biochemical, computational and electrophysiological approaches: addition of the p-CF3-group, 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 p-CF3-MCAT, 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.
Research Interests:
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... more
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.
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.
Research Interests:
Substantial effort has been devoted toward understanding the psychopharmacological effects of trypt-amine hallucinogens, which are thought to be mediated by activation of 5-HT 2A and 5-HT 1A receptors. Recently, several psychoactive... more
Substantial effort has been devoted toward understanding the psychopharmacological effects of trypt-amine hallucinogens, which are thought to be mediated by activation of 5-HT 2A and 5-HT 1A 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-, 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-HT 2A receptor-mediated response that is widely used as a behavioral proxy for hallucinogen effects in humans.
Research Interests:
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... more
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.
Key Points:
-Psilocybin, as its active metabolite psilocin, demonstrates linear pharmacokinetics over the 0.3–0.6 mg/kg oral dose range tested.
-Less than 2% of the psilocin in plasma is excreted in urine in that form, suggesting minimal effect of renal dysfunction in elimination of the active metabolite.
-A fixed oral dose of 25 mg is expected to approximate the area under the concentration–time curve and concentration profile of the 0.3 mg/kg oral dose used in this study.
Key Points:
-Psilocybin, as its active metabolite psilocin, demonstrates linear pharmacokinetics over the 0.3–0.6 mg/kg oral dose range tested.
-Less than 2% of the psilocin in plasma is excreted in urine in that form, suggesting minimal effect of renal dysfunction in elimination of the active metabolite.
-A fixed oral dose of 25 mg is expected to approximate the area under the concentration–time curve and concentration profile of the 0.3 mg/kg oral dose used in this study.
Research Interests:
A B S T R A C T 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... more
A B S T R A C T 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 (1 nM) 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.
Research Interests:
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... more
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.
Research Interests:
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 12-20 mg range, while... more
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 12-20 mg 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 nanomolar 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.
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Abstract 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... more
Abstract
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.) selfadministration
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, MDPVand cocaine displayed typical inverted Ushaped
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.
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.) selfadministration
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, MDPVand cocaine displayed typical inverted Ushaped
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.
Research Interests:
BACKGROUND AND PURPOSE Methcathinone (MCAT) is a potent monoamine releaser and parent compound to emerging drugs of abuse including mephedrone (4-CH3 MCAT), the para-methyl analogue of MCAT. This study examined quantitative... more
BACKGROUND AND PURPOSE
Methcathinone (MCAT) is a potent monoamine releaser and parent compound to emerging drugs of abuse including mephedrone (4-CH3 MCAT), the para-methyl analogue of MCAT. This study examined quantitative structure–activity relationships (QSAR) for MCAT and six para-substituted MCAT analogues on (a) in vitro potency to promote monoamine release via dopamine and serotonin transporters (DAT and SERT, respectively), and (b) in vivo modulation of intracranial self-stimulation (ICSS), a behavioural procedure used to evaluate abuse potential. Neurochemical and behavioural effects were correlated with steric (Es), electronic (σp) and lipophilic (πp) parameters of the para substituents.
EXPERIMENTAL APPROACH
For neurochemical studies, drug effects on monoamine release through DAT and SERT were evaluated in rat brain
synaptosomes. For behavioural studies, drug effects were tested in male Sprague-Dawley rats implanted with electrodes targeting the medial forebrain bundle and trained to lever-press for electrical brain stimulation.
KEY RESULTS
MCAT and all six para-substituted analogues increased monoamine release via DAT and SERT and dose- and time-dependently modulated ICSS. In vitro selectivity for DAT versus SERT correlated with in vivo efficacy to produce abuse-related ICSS facilitation. In addition, the Es values of the para substituents correlated with both selectivity for DAT versus SERT and magnitude of ICSS facilitation.
Methcathinone (MCAT) is a potent monoamine releaser and parent compound to emerging drugs of abuse including mephedrone (4-CH3 MCAT), the para-methyl analogue of MCAT. This study examined quantitative structure–activity relationships (QSAR) for MCAT and six para-substituted MCAT analogues on (a) in vitro potency to promote monoamine release via dopamine and serotonin transporters (DAT and SERT, respectively), and (b) in vivo modulation of intracranial self-stimulation (ICSS), a behavioural procedure used to evaluate abuse potential. Neurochemical and behavioural effects were correlated with steric (Es), electronic (σp) and lipophilic (πp) parameters of the para substituents.
EXPERIMENTAL APPROACH
For neurochemical studies, drug effects on monoamine release through DAT and SERT were evaluated in rat brain
synaptosomes. For behavioural studies, drug effects were tested in male Sprague-Dawley rats implanted with electrodes targeting the medial forebrain bundle and trained to lever-press for electrical brain stimulation.
KEY RESULTS
MCAT and all six para-substituted analogues increased monoamine release via DAT and SERT and dose- and time-dependently modulated ICSS. In vitro selectivity for DAT versus SERT correlated with in vivo efficacy to produce abuse-related ICSS facilitation. In addition, the Es values of the para substituents correlated with both selectivity for DAT versus SERT and magnitude of ICSS facilitation.
Research Interests:
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... more
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.
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.
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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... more
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 [11C]-(−)-meta-hydroxyephedrine,
[11C]-(−)-epinephrine, and a series of [11C]-labeled phenethylguanidines under development in our
laboratory. For comparison, the NET transport kinetics and binding affinities of some [3H]-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 [3H]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 (r2=
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.
norepinephrine transporter (NET). The goal of this study was to characterize the NET transport kinetics and
binding affinities of several sympathetic nerve radiotracers, including [11C]-(−)-meta-hydroxyephedrine,
[11C]-(−)-epinephrine, and a series of [11C]-labeled phenethylguanidines under development in our
laboratory. For comparison, the NET transport kinetics and binding affinities of some [3H]-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 [3H]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 (r2=
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.
Research Interests:
Cathinones are a class of drugs used to treat various medical conditions including depression, obesity, substance abuse, and muscle spasms. Some "designer" cathinones, such as methcathinone, mephedrone, and methylone, are used... more
Cathinones are a class of drugs used to treat various medical conditions including depression, obesity, substance abuse, and muscle spasms. Some "designer" cathinones, such as methcathinone, mephedrone, and methylone, are used nonclinically for their stimulant or entactogenic properties. Given the recent rise in nonmedical use of designer cathinones, we aimed to improve understanding of cathinone pharmacology by investigating analogs of methcathinone with a CF3 substituent at the 2-, 3 , or 4-position of the phenyl ring (TFMAPs). We compared the TFMAPs with methcathinone for effects on monoamine uptake transporter function in vitro and in vivo, and for effects on locomotor activity in rats. At the serotonin transporter (SERT), 3-TFMAP and 4 TFMAP were 10-fold more potent than methcathinone as uptake inhibitors and as releasing agents, but 2 TFMAP was both a weak uptake inhibitor and releaser. At the norepinephrine and dopamine transporters (NET and DAT), all TFMAP isomers were less potent than methcathinone as uptake inhibitors and releasers. In vivo, 4-TFMAP released 5-HT, but not dopamine, in rat nucleus accumbens and did not affect locomotor activity, whereas methcathinone increased both 5-HT and dopamine and produced locomotor stimulation. These experiments reveal that TFMAPs are substrates for the monoamine transporters and that phenyl ring substitution at the 3- or 4-position increases potency at SERT but decreases potency at NET and DAT, resulting in selectivity for SERT. The TFMAPs might have therapeutic value for a variety of medical and psychiatric conditions and may have lower abuse liability compared to methcathinone due to their decreased DAT activity.
Research Interests:
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... more
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 [3H]dopamine (IC50 = 4.1 nM) and
[3H]norepinephrine (IC50 = 26 nM) with high potency but has weak effects on uptake of [3H]serotonin (IC50 = 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.
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 [3H]dopamine (IC50 = 4.1 nM) and
[3H]norepinephrine (IC50 = 26 nM) with high potency but has weak effects on uptake of [3H]serotonin (IC50 = 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.
Research Interests:
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N,N-dimethyltryptamine (DMT) is a potent plant hallucinogen that has also been found in human tissues. When ingested, DMT and related N,N-dialkyltryptamines produce an intense hallucinogenic state. Behavioral effects are mediated through... more
N,N-dimethyltryptamine (DMT) is a potent plant hallucinogen that has also been found in human tissues. When ingested, DMT and related N,N-dialkyltryptamines produce an intense hallucinogenic state. Behavioral effects are mediated through various neurochemical mechanisms including activity at sigma-1 and serotonin receptors, modification of monoamine uptake and release, and competition for metabolic enzymes. To further clarify the pharmacology of hallucinogenic tryptamines, we synthesized DMT, N-methyl-N-isopropyltryptamine (MIPT), N,N-dipropyltryptamine (DPT), and N,N-diisopropyltryptamine. We then tested the abilities of these N,N-dialkyltryptamines to inhibit [3H]5-HT uptake via the plasma membrane serotonin transporter (SERT) in human platelets and via the vesicle monoamine transporter (VMAT2) in Sf9 cells expressing the rat VMAT2. The tryptamines were also tested as inhibitors of [3H]paroxetine binding to the SERT and [3H]dihydrotetrabenazine binding to VMAT2. Our results show that DMT, MIPT, DPT, and DIPT inhibit [3H]5-HT transport at the SERT with K I values of 4.00 ± 0.70, 8.88 ± 4.7, 0.594 ± 0.12, and 2.32 ± 0.46 μM, respectively. At VMAT2, the tryptamines inhibited [3H]5-HT transport with K I values of 93 ± 6.8, 20 ± 4.3, 19 ± 2.3, and 19 ± 3.1 μM, respectively. On the other hand, the tryptamines were very poor inhibitors of [3H]paroxetine binding to SERT and of [3H]dihydrotetrabenazine binding to VMAT2, resulting in high binding-to-uptake ratios. High binding-to-uptake ratios support the hypothesis that the tryptamines are transporter substrates, not uptake blockers, at both SERT and VMAT2, and also indicate that there are separate substrate and inhibitor binding sites within these transporters. The transporters may allow the accumulation of tryptamines within neurons to reach relatively high levels for sigma-1 receptor activation and to function as releasable transmitters.
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The sigma-1 receptor is widely distributed in the central nervous system and periphery. Originally mischaracterized as an opioid receptor, the sigma-1 receptor binds a vast number of synthetic compounds but does not bind opioid peptides;... more
The sigma-1 receptor is widely distributed in the central nervous system and periphery. Originally mischaracterized as an opioid receptor, the sigma-1 receptor binds a vast number of synthetic compounds but does not bind opioid peptides; it is currently considered an orphan receptor. The sigma-1 receptor pharmacophore includes an alkylamine core, also found in the endogenous compound N,N-dimethyltryptamine (DMT). DMT acts as a hallucinogen, but its receptor target has been unclear. DMT bound to sigma-1 receptors and inhibited voltage-gated sodium ion (Na+) channels in both native cardiac myocytes and heterologous cells that express sigma-1 receptors. DMT induced hypermobility in wild-type mice but not in sigma-1 receptor knockout mice. These biochemical, physiological, and behavioral experiments indicate that DMT is an endogenous agonist for the sigma-1 receptor.
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A large number of N,N-dialkylated tryptamines are known to induce psychoactive effects in humans. This has resulted in their increased attention within clinical and forensic communities. Deuterated tryptamines are ideal for use as... more
A large number of N,N-dialkylated tryptamines are known to induce psychoactive effects in humans. This has resulted in
their increased attention within clinical and forensic communities. Deuterated tryptamines are ideal for use as internal standards during MS bioanalysis or of use in biochemical NMR studies. The present study reports on a microwave-enhanced synthesis of 22 N,N-dialkylated-[a,a,b,b-d4]-tryptamines via the reduction with lithium aluminium deuteride of glyoxalylamide precursors obtained by the procedure of Speeter and Anthony. Syntheses were carried out using a single-mode system under elevated pressure conditions where anhydrous tetrahydrofuran was used as the solvent at 150 °C. Good yields were obtained within 5 min.
their increased attention within clinical and forensic communities. Deuterated tryptamines are ideal for use as internal standards during MS bioanalysis or of use in biochemical NMR studies. The present study reports on a microwave-enhanced synthesis of 22 N,N-dialkylated-[a,a,b,b-d4]-tryptamines via the reduction with lithium aluminium deuteride of glyoxalylamide precursors obtained by the procedure of Speeter and Anthony. Syntheses were carried out using a single-mode system under elevated pressure conditions where anhydrous tetrahydrofuran was used as the solvent at 150 °C. Good yields were obtained within 5 min.
Research Interests:
Three new 2-(4-fluorophenoxy)-2-phenyl-ethyl piperazines, 1-(3-chlorophenyl)-4-[2-(4-fluorophenoxy)-2-phenylethyl]-piperazine 7, 1-[2-(4-fluorophenoxy)-2-phenylethyl]-4-(2-methoxyphenyl)-piperazine 8, and... more
Three new 2-(4-fluorophenoxy)-2-phenyl-ethyl piperazines, 1-(3-chlorophenyl)-4-[2-(4-fluorophenoxy)-2-phenylethyl]-piperazine 7, 1-[2-(4-fluorophenoxy)-2-phenylethyl]-4-(2-methoxyphenyl)-piperazine 8, and 1-[2-(4-fluorophenoxy)-2-phenylethyl]-4-(3-trifluoromethylphenyl)-piperazine 9, modeled after the potent antidepressant fluoxetine and coupled with several functionalized piperazines, have been prepared by chemical synthesis as selective serotonin reuptake inhibitors (SSRIs) with a potentially improved adverse reaction profile. Typical SSRIs, although very effective in the treatment of depression, still face the troublesome side effect of sexual dysfunction. A number of pharmacological agents-notably, drugs in the piperazine class-have been used to reverse SSRI-induced sexual dysfunction, and evidence for developing an improved SSRI by coupling a fluoxetine congener with the pharmacophore of a reversal agent holds promise. Preliminary data indicates that the hydrochloride (HCl) salts 10, 11, and 12 each exhibit single-site binding at the site of the serotonin reuptake transporter (SERT). However, each of the three compounds are much less potent than typical SSRIs, showing micromolar (μM) affinity for the SERT with IC50 values of 1.45 μM, 3.27 μM, and 9.56 μM, respectively. Further biological evaluation of compounds 10, 11, and 12 is needed before definitive conclusions can be made with regard to each compound's potential for use as an SSRI-type candidate which is devoid of sexual side effects. Nevertheless, the initial findings are quite encouraging, thus lending credence to the idea of hybridizing an SSRI congener with that of the pharmacophore of an agent known to reverse or treat SSRI-induced sexual dysfunction.Graphic
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We previously reported that the psychostimulant drug methcathinone inhibits serotonin accumulation via the plasma membrane serotonin uptake transporter. By analogy to known substrates for the serotonin transporter, we hypothesized that... more
We previously reported that the psychostimulant drug methcathinone inhibits serotonin accumulation via the plasma membrane serotonin uptake transporter. By analogy to known substrates for the serotonin transporter, we hypothesized that methcathinone is also a substrate for this transporter and that inhibition of serotonin uptake by methcathinone occurs in part through competition for substrate recognition sites within the transporter. To test the hypothesis we preloaded human platelets with [3H]5-HT then superfused the platelets with either methcathinone or with the known
serotonin uptake transporter substrate para-methylthioamphetamine. Under superfusion conditions, transporter substrates will evoke an increase in released [3H]5-HT through a carrier-mediated exchange process. For direct assessment of
methcathinone transport via the serotonin uptake transporter, we tested whether [3H]methcathinone would be accumulated by cells stably expressing the cloned human serotonin uptake transporter (293SERT cells). Supporting the hypothesis,
superfusion of [3H]5-HT-containing platelets with methcathinone or with para-methylthioamphetamine produced a large increase in tritium efflux. The efflux declined when the drugs were removed. When increasing concentrations of
[3H]methcathinone were incubated with 293SERT cells under conditions used to assess serotonin transport, saturable, single-site accumulation of radiolabel was observed. The uptake of [3H]methcathinone was temperature, inhibitor, and
sodium-sensitive, and was not observed in wild-type HEK 293 cells. Non-linear regression analysis of specific [3H]methcathinone uptake produced values for KM and Vmax of 244∫51 nM and 202∫25 fmol/min./mg protein, respectively. These
data support the notion that the reported serotonergic neurotoxicity of methcathinone may arise through accumulation of the drug within serotonergic neurones.
serotonin uptake transporter substrate para-methylthioamphetamine. Under superfusion conditions, transporter substrates will evoke an increase in released [3H]5-HT through a carrier-mediated exchange process. For direct assessment of
methcathinone transport via the serotonin uptake transporter, we tested whether [3H]methcathinone would be accumulated by cells stably expressing the cloned human serotonin uptake transporter (293SERT cells). Supporting the hypothesis,
superfusion of [3H]5-HT-containing platelets with methcathinone or with para-methylthioamphetamine produced a large increase in tritium efflux. The efflux declined when the drugs were removed. When increasing concentrations of
[3H]methcathinone were incubated with 293SERT cells under conditions used to assess serotonin transport, saturable, single-site accumulation of radiolabel was observed. The uptake of [3H]methcathinone was temperature, inhibitor, and
sodium-sensitive, and was not observed in wild-type HEK 293 cells. Non-linear regression analysis of specific [3H]methcathinone uptake produced values for KM and Vmax of 244∫51 nM and 202∫25 fmol/min./mg protein, respectively. These
data support the notion that the reported serotonergic neurotoxicity of methcathinone may arise through accumulation of the drug within serotonergic neurones.
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The atypical antidepressant drug bupropion and the psychostimulant drug methcathinone are both members of a chemical class known as aminopropiophenones. Differences in the psychoactive effects of these two drugs result from small... more
The atypical antidepressant drug bupropion and the psychostimulant drug methcathinone are both members of a chemical class known as aminopropiophenones. Differences in the psychoactive effects of these two drugs result from small variations in their chemical structures, but the relationship between chemical structure and psychoactivity has not been characterized. To investigate how structural modifications to aminopropiophenones affect antidepressant or stimulant activity, we synthesized several analogs of bupropion and methcathinone and tested the new compounds for antidepressant-like or psychostimulant effects. The synthesized compounds are 2-(methylamino)-1-(3-bromophenyl)propan-1-one (3-BMAP), 2-(methylamino)-1-(4-bromophenyl)propan-1-one (4-BMAP), 2-(iso-propylamino)-1-phenylpropan-1-one (i-PAP), and -(tert-butylamino)-1-phenylpropan-1-one (t-BAP). Bupropion, methcathinone, desipramine, and the newly-synthesized aminopropiophenones were administered to rats for behavioral testing. We used the Porsolt swim test to assess antidepressant-like activity and a locomotor activity assay to test for psychostimulant effects. All of the compounds displayed antidepressant-like effects in the Porsolt swim test. Some compounds, including bupropion, increased locomotor activity at moderate-to-high doses. A halogenated analog of methcathinone, 4-BMAP, increased swim time but did not stimulate locomotor activity, even at the highest dose tested. The data indicate that phenyl ring substitution or branched alkylamines can shift the psychopharmacological profile of aminopropiophenones from stimulant activity to antidepressant-like activity. Several of the new drugs may be effective antidepressants in humans with fewer stimulant-like side effects compared to bupropion. Drug Dev. Res. 60:252–260, 2003. © 2003 Wiley-Liss, Inc.
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Meta-hydroxyephedrine (HED) comprises four stereoisomers consisting of two enantiomeric pairs related to ephedrine and pseudoephedrine. HED is transported into adrenergic neurons and radiolabeled HED has been employed in positron emission... more
Meta-hydroxyephedrine (HED) comprises four stereoisomers consisting of two enantiomeric pairs related to ephedrine and pseudoephedrine. HED is transported into adrenergic neurons and radiolabeled HED has been employed in positron emission tomography (PET) to image adrenergic neurons in vivo. To extend structure-activity analyses of binding sites within monoamine transporters and to determine which stereoisomer displayed the best selectivity for PET imaging applications, we tested the HED compounds for their abilities to inhibit [3H]neurotransmitter uptake into platelets, transfected cells, and chromaffin vesicles. We hypothesized that the HED compounds would be most potent at the norepinephrine transporter (NET) compared to the serotonin or dopamine transporters and that the 1R diastereomers would be more effective than 1S diastereomers. Supporting the hypotheses, all stereoisomers were most potent at the NET and the 1R,2S stereoisomer was the most potent inhibitor overall. However, the 1S,2R isomer may be preferred for PET applications because of better selectivity among the transporters and reduced neuronal recycling.
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N-ethylmaleimide (NEM) inhibits [3H]desipramine binding and [3H]noradrenaline uptake at the rat noradrenaline transporter (rNET) by covalently modifying cysteine residues. We report here that NEM also inhibits [3H]desipramine binding and... more
N-ethylmaleimide (NEM) inhibits [3H]desipramine binding and [3H]noradrenaline uptake at the rat noradrenaline transporter (rNET) by covalently modifying cysteine residues. We report here that NEM also inhibits [3H]desipramine binding and [3H]noradrenaline uptake at the cloned human noradrenaline transporter (hNET) stably expressed in C6 glial cells. The IC50 for NEM inhibition of [3H]noradrenaline uptake was 43.6±5.5 µM. We tested several compounds for their abilities to inhibit [3H]noradrenaline uptake via the hNET and for their abilities to protect against NEM inactivation of [3H]desipramine binding. We found that the substrate analogs bupropion, 3-bromomethcathinone, and 4-bromomethcathinone all inhibit uptake at the hNET with IC50 values of 1370±140, 158±20, and 453±30 nM, respectively. These compounds as well as methamphetamine, methcathinone, and desipramine also protected the hNET from NEM inactivation of [3H]desipramine binding. The ability of substrate analogs and desipramine to protect the [3H]desipramine binding site is consistent with the hypothesis that the desipramine binding site and the substrate binding site are mutually exclusive. It also supports the use of structure-activity relationships derived from substrate analogs in the rational design of hNET uptake inhibitors. The hNET contains 10 cysteine residues whereas the rNET contains 12 cysteine residues. Since the hNET and the rNET are both inhibited by NEM, and because the NEM inhibition is protectable by desipramine and substrate analogs, we conclude that the two additional cysteine residues (C28 and C447) present in the rNET are not likely to be involved in desipramine binding or uptake function.
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Methcathinone and methylone, the β-ketone analogues of methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA), respectively, were tested for neurotransmitter uptake inhibition in vitro. The β-ketones were threefold less potent than... more
Methcathinone and methylone, the β-ketone analogues of methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA), respectively, were tested for neurotransmitter uptake inhibition in vitro. The β-ketones were threefold less potent than the nonketo drugs at inhibiting platelet serotonin accumulation, with IC50's of 34.6±4.8 μM and 5.8±0.7 μM, respectively. Methcathinone and methylone were similar in potency to methamphetamine and MDMA at catecholamine transporters individually expressed in transfected glial cells. For dopamine uptake, IC50's were 0.36±0.06 μM and 0.82±0.17 μM, respectively; for noradrenaline uptake, IC50 values were 0.51±0.10 μM and 1.2±0.1 μM, respectively. In chromaffin granules, IC50's for serotonin accumulation were 112±8.0 μM for methcathinone and 166±12 μM for methylone, 10-fold higher than the respective values for methamphetamine and MDMA. Our results indicate that methcathinone and methylone potently inhibit plasma membrane catecholamine transporters but only weakly inhibit the vesicle transporter.
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N-Ethyl-5-trifluoromethyl-2-aminoindan (ETAI) and 5-trifluoromethyl-2-aminoindan (TAI) were synthesized to examine the effects of side-chain cyclization on the pharmacology of the anorectic drugs fenfluramine (FEN) and norfenfluramine... more
N-Ethyl-5-trifluoromethyl-2-aminoindan (ETAI) and 5-trifluoromethyl-2-aminoindan (TAI) were synthesized to examine the effects of side-chain cyclization on the pharmacology of the anorectic drugs fenfluramine (FEN) and norfenfluramine (norFEN), respectively. ETAI and TAI inhibited synaptosomal accumulation of 5-HT but were less effective at inhibiting catecholamine uptake than FEN or norFEN, respectively. In vivo, ETAI and TAI were less neurotoxic than FEN or norFEN; decreases in the number of [3H]paroxetine-labeled 5-HT uptake sites were 50% less than the decreases produced by FEN or norFEN. Rats treated with ETAI, TAI, FEN, and norFEN lost 10–15% of their pretreatment body weight over a 4-day period, while saline-treated control animals gained 8%. In two-lever drug discrimination (DD) assays in rats, TAI fully substituted for the 5-HT releaser/uptake inhibitor, (+)-MBDB [(+)-N-methyl-1-(1,3-benzodioxol-5-yl)-2-aminobutane]. ETAI produced only partial substitution in this test. Neither TAI nor ETAI mimicked (+)-amphetamine in the DD assay. These studies demonstrate that incorporation of the side-chain of phenylisopropylamines into the five-membered ring of a 2-aminoindan changes both the molecular pharmacology and the neurotoxic profile of FEN and norFEN, but does not diminish the drugs’ ability to reduce body weight.
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Several drugs selective for the serotonin 5-HT2A receptor were tested for their effects on spontaneous and K+-evoked [3H]γ-aminobutyric acid (GABA) release from slices of rat frontal cortex. Under K+ stimulation, the antagonists... more
Several drugs selective for the serotonin 5-HT2A receptor were tested for their effects on spontaneous and K+-evoked [3H]γ-aminobutyric acid (GABA) release from slices of rat frontal cortex. Under K+ stimulation, the antagonists ketanserin, spiperone, R-(+)-α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenethyl)-4-piperidinemethanol (MDL 100,907) and ritanserin inhibited GABA release by 12–31%. Rats were treated with the serotonin-depleting agent para-chlorophenylalanine and with the serotonergic neurotoxin para-chloroamphetamine. In para-chlorophenylalanine-treated animals, stimulated GABA release in the presence of ketanserin remained depressed. In animals treated with both para-chlorophenylalanine and para-chloroamphetamine, ketanserin or the hallucinogenic agonist (2,5-dimethoxy-4-iodophenyl)-2-aminoethane (2C-I) each appeared to decrease stimulated GABA release but this was not significant. However, when ketanserin and 2C-I were both present in the superfusion buffer an additive inhibitory effect was observed, and GABA release was decreased 30%. These results suggest that serotonin facilitates GABA release in cortex via 5-HT2A receptors and that the functional response of this system is resistant to serotonin depletion.
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Santo Daime Church of Oregon v. United States Department of Justice document.
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Santo Daime Church of Oregon v. United States Department of Justice document.
Santo Daime Church of Oregon v. United States Department of Justice document.
Indolethylamine-N-methyl transferase (INMT), a member of a family of small molecule methyltransferase enzymes, adds methyl groups from S-adenosyl-L-methionine (SAM) to the side chain amino group of indole amines such as tryptamine and... more
Indolethylamine-N-methyl transferase (INMT), a member of a family of small molecule methyltransferase enzymes, adds methyl groups from S-adenosyl-L-methionine (SAM) to the side chain amino group of indole amines such as tryptamine and serotonin; for example, tryptamine methylation produces N-monomethyl tryptamine (MMT) and the hallucinogen, N,N-dimethyltryptamine (DMT). In animals, the endogenous production of DMT has been proposed to be an important regulator of mood and cognition through activation of serotonin and other receptors.
In contrast to the INMT from human, rabbit, rat and chicken, INMT from mouse (referred to as thioether methyl transferase, TEMT) has been independently characterized as an enzyme that utilizes SAM to methylate sulfur, selenium and tellurium compounds to produce their respective “onium “ products (Mozier et al. JBC 263,10 4527-4531 (1988)). TEMT may thus regulate the levels of endogenous thiols and trace metal containing compounds.
To explore the functional differences and similarities between human INMT (hINMT) and mouse TEMT (mTEMT), we evaluated the efficiency of TEMT to produce MMT and DMT using the co substrate 14C-SAM. TEMT in mouse lung homogenates produced negligible amounts of N-methylated tryptamine derivatives when compared to rabbit lung homogenates. Similarly pure recombinant mTEMT was not efficient in tryptamine methylation compared to the methylation of thiol containing compounds such as cysteamine, and methyl thio ethyl amine (MTEA). The Km for methylation of MTEA by TEMT was found to be 0.7 mM.
On the other hand, pure recombinant hINMT methylated both cysteamine, N-acetyl cysteamine and MTEA as well as tryptamine. The Km for MTEA methylation at pH 7.2 was determined to be approximately 1.2 mM compared to a previously reported tryptamine Km of 2.9 mM [Thomson et al. Genomics 61, 285-297 (1999)] Furthermore, over the pH range of 6.3-8.0, we found that more acidic conditions favored methylation of MTEA, while alkaline pH favored methylation of tryptamine. At pH 6.8, which is the average intracellular pH, the methylation levels of 10 mM tryptamine and 10 mM MTEA were approximately equal.
These data suggest that hINMT regulates the metabolism of thiols and possibly similar selenium and tellurium derivatives. Thus hINMT may compete with tryptamine (and serotonin) methylation especially under conditions of intracellular pH fluctuations (e.g. increased acidification).
In contrast to the INMT from human, rabbit, rat and chicken, INMT from mouse (referred to as thioether methyl transferase, TEMT) has been independently characterized as an enzyme that utilizes SAM to methylate sulfur, selenium and tellurium compounds to produce their respective “onium “ products (Mozier et al. JBC 263,10 4527-4531 (1988)). TEMT may thus regulate the levels of endogenous thiols and trace metal containing compounds.
To explore the functional differences and similarities between human INMT (hINMT) and mouse TEMT (mTEMT), we evaluated the efficiency of TEMT to produce MMT and DMT using the co substrate 14C-SAM. TEMT in mouse lung homogenates produced negligible amounts of N-methylated tryptamine derivatives when compared to rabbit lung homogenates. Similarly pure recombinant mTEMT was not efficient in tryptamine methylation compared to the methylation of thiol containing compounds such as cysteamine, and methyl thio ethyl amine (MTEA). The Km for methylation of MTEA by TEMT was found to be 0.7 mM.
On the other hand, pure recombinant hINMT methylated both cysteamine, N-acetyl cysteamine and MTEA as well as tryptamine. The Km for MTEA methylation at pH 7.2 was determined to be approximately 1.2 mM compared to a previously reported tryptamine Km of 2.9 mM [Thomson et al. Genomics 61, 285-297 (1999)] Furthermore, over the pH range of 6.3-8.0, we found that more acidic conditions favored methylation of MTEA, while alkaline pH favored methylation of tryptamine. At pH 6.8, which is the average intracellular pH, the methylation levels of 10 mM tryptamine and 10 mM MTEA were approximately equal.
These data suggest that hINMT regulates the metabolism of thiols and possibly similar selenium and tellurium derivatives. Thus hINMT may compete with tryptamine (and serotonin) methylation especially under conditions of intracellular pH fluctuations (e.g. increased acidification).
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N,N-dimethyltryptamine (DMT) is a naturally-occurring indole hallucinogen found in plants, animals, and humans, but its biological role has not been fully characterized. DMT has been proposed to act as a neurotransmitter in humans and to... more
N,N-dimethyltryptamine (DMT) is a naturally-occurring indole hallucinogen found in plants, animals, and humans, but its biological role has not been fully characterized. DMT has been proposed to act as a neurotransmitter in humans and to be involved in psychosis, dreaming, near-death experiences, and spiritual exaltation. DMT is biosynthesized from tryptamine through the actions of the enzyme indolethylamine N-methyltransferase (INMT). Using S-adenosyl methionine as the methyl donor, INMT catalyzes the addition of methyl groups to tryptamine and analogous indole alkylamines.
Human INMT was cloned and sequenced in 1999. Assessment of human INMT expression by Northern blot analysis in 35 tissues revealed widespread INMT mRNA distribution with high levels in thyroid, adrenal gland, and lung. However, in the central nervous system, INMT mRNA was detected only in the spinal cord, but not in whole brain or in seven brain subregions. This observation suggested that INMT may not be involved in DMT biosynthesis in the brain and calls into question the role, if any, of endogenous DMT in producing exceptional mental states. To explore the possibility that INMT is expressed in nervous tissue but that in some situations, INMT mRNA is not detectable by Northern analysis (e.g. the INMT gene is inducible, INMT expression is limited to specific brain nuclei, or INMT mRNA in brain is short-lived), we probed three primate nervous system tissues with antibodies to INMT itself. The antibodies were generated against the C-terminus of human INMT during the original cloning of the human INMT gene. Rhesus macaque spinal cord, pineal gland, and retina were probed with rabbit polyclonal antibodies to human INMT and with mouse antibodies to synaptophysin and synaptotagmin. Nuclear DNA was visualized with 4',6-diamidino-2-phenylindole (DAPI). Binding of primary antibodies to the tissues was visualized with fluorescent goat anti-rabbit and goat anti-mouse secondary antibodies. All three of the nervous tissues tested were positive for INMT immunoreactivity. In agreement with earlier Northern studies in human tissue, INMT immunoreactivity was detected in spinal cord where it was localized in ventral horn motoneurons. In the pineal gland, the INMT signal was robust and punctuate but did not colocalize with synaptophysin, synaptotagmin, or DAPI. Strong INMT immunoreactivity was detected in retinal ganglion neurons and at synapses in the inner and outer plexiform layers. We conclude that INMT protein is expressed in some primate central nervous system tissues, but whether INMT expression is associated with the biosynthesis of DMT in neurons remains to be investigated.
Human INMT was cloned and sequenced in 1999. Assessment of human INMT expression by Northern blot analysis in 35 tissues revealed widespread INMT mRNA distribution with high levels in thyroid, adrenal gland, and lung. However, in the central nervous system, INMT mRNA was detected only in the spinal cord, but not in whole brain or in seven brain subregions. This observation suggested that INMT may not be involved in DMT biosynthesis in the brain and calls into question the role, if any, of endogenous DMT in producing exceptional mental states. To explore the possibility that INMT is expressed in nervous tissue but that in some situations, INMT mRNA is not detectable by Northern analysis (e.g. the INMT gene is inducible, INMT expression is limited to specific brain nuclei, or INMT mRNA in brain is short-lived), we probed three primate nervous system tissues with antibodies to INMT itself. The antibodies were generated against the C-terminus of human INMT during the original cloning of the human INMT gene. Rhesus macaque spinal cord, pineal gland, and retina were probed with rabbit polyclonal antibodies to human INMT and with mouse antibodies to synaptophysin and synaptotagmin. Nuclear DNA was visualized with 4',6-diamidino-2-phenylindole (DAPI). Binding of primary antibodies to the tissues was visualized with fluorescent goat anti-rabbit and goat anti-mouse secondary antibodies. All three of the nervous tissues tested were positive for INMT immunoreactivity. In agreement with earlier Northern studies in human tissue, INMT immunoreactivity was detected in spinal cord where it was localized in ventral horn motoneurons. In the pineal gland, the INMT signal was robust and punctuate but did not colocalize with synaptophysin, synaptotagmin, or DAPI. Strong INMT immunoreactivity was detected in retinal ganglion neurons and at synapses in the inner and outer plexiform layers. We conclude that INMT protein is expressed in some primate central nervous system tissues, but whether INMT expression is associated with the biosynthesis of DMT in neurons remains to be investigated.
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To determine whether accumulation of IP is altered by manipulations that significantly alter neurotransmitter release, inositol phospholipid metabolism was examined under experimental conditions used to study acetylcholine (ACh) and... more
To determine whether accumulation of IP is altered by manipulations that significantly alter neurotransmitter release, inositol phospholipid metabolism was examined under experimental conditions used to study acetylcholine (ACh) and dopamine (DA) release from rat neostriatal slices. Slices from Fischer 344 rats (3 months old) were labeled with myo[2-3H]-inositol, and IP accumulation was determined in the presence of LiCI (10 mM). Effects of nondepolarizing and depolarizing conditions, acetylcholinesterase (AChE) inhibition, and muscarinic agents were tested. Under nondepolarizing conditions, IP accumulation(% of total 3H-inositol incorporation) was 2.4% ± 0.1% (n = 3) during a 25-minute incubation. If the slices were depolarized an additional 5 minutes following the nondepolarizing incubation, IP accumulation increased another 78% (4.28% ± 0.28%; n = 3, p < 0.05), indicating that conditions used to stimulate neurotransmitter release also increase IP accumulation. In the presence of physostigmine (PHY), an AChE inhibitor, IP accumulation following a 25-minute nondepolarizing/5-minute depolarizing incubation was 3.3-fold greater (p < 0.05) than in its absence. Oxotremorine-M (OXO-M), a muscarinic agonist, enhanced IP accumulation fivefold (5 μM) and ninefold (100 μM) during a 25-minute nondepolarizing incubation, and 29% and 14% more during an additional 5-minute depolarization. Pirenzepine (50 μM), a muscarinic antagonist, decreased IP accumulation in the presence of OXO·M and PHY by 74% (p < 0.01) and 68% (p < 0.01), respectively. These manipulations also significantly affected ACh and DA release, indicating that IP accumulation in neostriatal slices is significantly altered during manipulations used to study neurotransmitter release.