Mechanisms of neurotransmitter transport and drug inhibition in human VMAT2.

Monoamine neurotransmitters such as dopamine and serotonin control important brain pathways, including movement, sleep, reward and mood1. Dysfunction of monoaminergic circuits has been implicated in various neurodegenerative and neuropsychiatric disorders2. Vesicular monoamine transporters (VMATs) pack monoamines into vesicles for synaptic release and are essential to neurotransmission3-5. VMATs are also therapeutic drug targets for a number of different conditions6-9. Despite the importance of these transporters, the mechanisms of substrate transport and drug inhibition of VMATs have remained elusive. Here we report cryo-electron microscopy structures of the human vesicular monoamine transporter VMAT2 in complex with the antichorea drug tetrabenazine, the antihypertensive drug reserpine or the substrate serotonin. Remarkably, the two drugs use completely distinct inhibition mechanisms. Tetrabenazine binds VMAT2 in a lumen-facing conformation, locking the luminal gating lid in an occluded state to arrest the transport cycle. By contrast, reserpine binds in a cytoplasm-facing conformation, expanding the vestibule and blocking substrate access. Structural analyses of VMAT2 also reveal the conformational changes following transporter isomerization that drive substrate transport into the vesicle. These findings provide a structural framework for understanding the physiology and pharmacology of neurotransmitter packaging by synaptic vesicular transporters.

Comparative study of dezocine, pentazocine and tapentadol on antinociception and physical dependence.

AIMS: Dezocine and pentazocine, widely prescribed in China for postoperative pain, were initially considered as mixed agonist/antagonist targeting µ-opioid receptors (MORs) and κ-opioid receptors (KORs). However, dezocine has been revealed to alleviate chronic neuropathic pain through MOR activation and norepinephrine reuptake inhibition (NRI). This study investigated dezocine- and pentazocine-induced antinociception and physical dependence development, compared to the typical MOR-NRI opioid tapentadol. MAIN METHODS: Calcium mobilization assay was conducted to assess the potency of the drugs while hot-plate test was performed to compare the antinociception. Physical dependence development was compared with morphine. KEY FINDINGS: Treatment with dezocine, pentazocine and tapentadol stimulated calcium mobilization in HEK293 cells stably expressed MORs but not KORs, whereas dezocine and pentazocine inhibited KOR activities. Subcutaneously injected dezocine-, tapentadol- and pentazocine-induced antinociception dose-dependently, in hot-plate test. Intrathecally injected MOR antagonist CTAP, norepinephrine depletor 6-OHDA and α2-adrenoceptor (α2-AR) antagonist yohimbine partially antagonized dezocine, pentazocine and tapentadol antinociception. Whereas specific KOR antagonist GNTI did not alter their antinociception, the putative inverse KOR agonist nor-BNI reduced dezocine and pentazocine antinociception. Moreover, combined CTAP and 6-OHDA or yohimbine blocked dezocine and tapentadol antinociception but displayed the same partial inhibition on pentazocine antinociception as CTAP alone. Furthermore, compared to morphine and pentazocine, long-term treatment with dezocine and tapentadol produced much less physical dependence-related withdrawal signs, which were restored by spinal 6-OHDA or yohimbine treatment. SIGNIFICANCE: Our findings illustrated that dezocine and tapentadol, but not pentazocine, exert remarkable antinociception in nociceptive pain with less abuse liability via dual mechanisms of MOR activation and NRI.

Tailoring Atomoxetine Release Rate from DLP 3D-Printed Tablets Using Artificial Neural Networks: Influence of Tablet Thickness and Drug Loading.

Various three-dimensional printing (3DP) technologies have been investigated so far in relation to their potential to produce customizable medicines and medical devices. The aim of this study was to examine the possibility of tailoring drug release rates from immediate to prolonged release by varying the tablet thickness and the drug loading, as well as to develop artificial neural network (ANN) predictive models for atomoxetine (ATH) release rate from DLP 3D-printed tablets. Photoreactive mixtures were comprised of poly(ethylene glycol) diacrylate (PEGDA) and poly(ethylene glycol) 400 in a constant ratio of 3:1, water, photoinitiator and ATH as a model drug whose content was varied from 5% to 20% (w/w). Designed 3D models of cylindrical shape tablets were of constant diameter, but different thickness. A series of tablets with doses ranging from 2.06 mg to 37.48 mg, exhibiting immediate- and modified-release profiles were successfully fabricated, confirming the potential of this technology in manufacturing dosage forms on demand, with the possibility to adjust the dose and release behavior by varying drug loading and dimensions of tablets. DSC (differential scanning calorimetry), XRPD (X-ray powder diffraction) and microscopic analysis showed that ATH remained in a crystalline form in tablets, while FTIR spectroscopy confirmed that no interactions occurred between ATH and polymers.

Locomotor effects of 3,4-methylenedioxymethamphetamine (MDMA) and its deuterated form in mice: psychostimulant effects, stereotypy, and sensitization.

RATIONALE: There is a renewed interest in the use of 3,4-methylenedioxymethamphetamine (MDMA) for treating psychiatric conditions. Although MDMA has entered phase II clinical trials and shows promise as an adjunct treatment, there is an extensive literature detailing the potential neurotoxicity and adverse neurobehavioral effects associated with MDMA use. Previous research indicates that the adverse effects of MDMA may be due to its metabolism into reactive catechols that can enter the brain and serve directly as neurotoxicants. One approach to mitigate MDMA's potential for adverse effects is to reduce O-demethylation by deuterating the methylenedioxy ring of MDMA. There are no studies that have evaluated the effects of deuterating MDMA on behavioral outcomes. OBJECTIVES: The purpose of the present study was to assess the motor-stimulant effects of deuterated MDMA (d2-MDMA) and compare them to MDMA in male mice. METHODS: Two experiments were performed to quantify mouse locomotor activity and to vary the drug administration regimen (single bolus administration or cumulative administration). RESULTS: The results of Experiments 1 and 2 indicate that d2-MDMA is less effective at eliciting horizontal locomotion than MDMA; however, the differences between the compounds diminish as the number of cumulative administrations increase. Both d2-MDMA and MDMA can elicit sensitized responses, and these effects cross-sensitize to the prototypical drug of abuse methamphetamine. Thus, d2-MDMA functions as a locomotor stimulant similar to MDMA, but, depending on the dosing regimen, may be less susceptible to inducing sensitization to stereotyped movements. CONCLUSIONS: These findings indicate that d2-MDMA is behaviorally active and produces locomotor effects that are similar to MDMA, which warrant additional assessments of d2-MDMA's behavioral and physiological effects to determine the conditions under which this compound may serve as a relatively safer alternative to MDMA for clinical use.

Mechanisms of the antilipolytic response of human adipocytes to tyramine, a trace amine present in food.

Tyramine is found in foodstuffs, the richest being cheeses, sausages, and wines. Tyramine has been recognized to release catecholamines from nerve endings and to trigger hypertensive reaction. Thereby, tyramine-free diet is recommended for depressed patients treated with irreversible inhibitors of monoamine oxidases (MAO) to limit the risk of hypertension. Tyramine is a substrate of amine oxidases and also an agonist at trace amine-associated receptors. Our aim was to characterize the dose-dependent effects of tyramine on human adipocyte metabolic functions. Lipolytic activity was determined in adipocytes from human subcutaneous abdominal adipose tissue. Glycerol release was increased by a fourfold factor with classical lipolytic agents (1 µM isoprenaline, 1 mM isobutylmethylxanthine) while the amine was ineffective from 0.01 to 100 µM and hardly stimulatory at 1 mM. Tyramine exhibited a partial antilipolytic effect at 100 µM and 1 mM, which was similar to that of insulin but weaker than that obtained with agonists at purinergic A1 receptors, α2-adrenoceptors, or nicotinic acid receptors. Gi-protein blockade by Pertussis toxin abolished all these antilipolytic responses save that of tyramine. Indeed, tyramine antilipolytic effect was impaired by MAO-A inhibition. Tyramine inhibited protein tyrosine phosphatase activities in a manner sensitive to ascorbic acid and amine oxidase inhibitors. Thus, millimolar tyramine restrained lipolysis via the hydrogen peroxide it generates when oxidized by MAO. Since tyramine plasma levels have been reported to reach 0.2 µM after ingestion of 200 mg tyramine in healthy individuals, the direct effects we observed in vitro on adipocytes could be nutritionally relevant only when the MAO-dependent hepato-intestinal detoxifying system is overpassed.

Deutetrabenazine: Treatment of hyperkinetic aspects of Huntington's disease, tardive dyskinesia and Tourette syndrome.

Deutetrabenazine is a derivative of tetrabenazine in which two trideuteromethoxy groups substitute two methoxy groups. The active metabolites of deutetrabenazine have a longer half-life than those of tetrabenazine, together with a greater overall absorption. However, the peak plasma concentrations are lower. Because of these pharmacokinetic differences, deutetrabenazine can be given twice daily, thus improving compliance. The lower peak concentrations may account for a lower incidence of some unwanted adverse effects. Unlike tetrabenazine, deutetrabenazine has no effect on the QT interval. Treatment with deutetrabenazine significantly improved chorea in Huntington's disease, the hyperkinetic features of tardive dyskinesia, and tics in Tourette syndrome. In all three conditions, deutetrabenazine produced an acceptable level of overall adverse effects without causing any severe adverse effects.

Mechanistic Studies of the N-formylation of Edivoxetine, a Secondary Amine-Containing Drug, in a Solid Oral Dosage Form.

Edivoxetine (LY2216684 HCl), although a chemically stable drug substance, has shown the tendency to degrade in the presence of carbohydrates that are commonly used tablet excipients, especially at high excipient:drug ratios. The major degradation product has been identified as N-formyl edivoxetine. Experimental evidence including solution and solid-state investigations, is consistent with the N-formylation degradation pathway resulting from a direct reaction of edivoxetine with (1) formic acid (generated from decomposition of microcrystalline cellulose or residual glucose) and (2) the reducing sugar ends (aldehydic carbons) of either residual glucose or the microcrystalline cellulose polymer. Results of labeling experiments indicate that the primary source of the formyl group is the C1 position from reducing sugars. Presence of water or moisture accelerates this degradation pathway. Investigations in solid and solution states support that the glucose Amadori Rearrangement Product does not appear to be a direct intermediate leading to N-formyl degradation of edivoxetine, and oxygen does not appear to play a significant role. Solution-phase studies, developed to rapidly assess propensity of amines toward Maillard reactivity and formylation, were extended to show comparative behavior with example systems. The cyclic amine systems, such as edivoxetine, showed the highest propensity toward these side reactions.

Structure-Activity Relationships of Synthetic Cathinones.

Until recently, there was rather little interest in the structure-activity relationships (SARs) of cathinone analogs because so few agents were available and because they represented a relatively minor drug abuse problem. Most of the early SAR was formulated on the basis of behavioral (e.g., locomotor and drug discrimination) studies using rodents. With the emergence on the clandestine market in the last few years of a large number of new cathinone analogs, termed "synthetic cathinones", and the realization that they likely act at dopamine, norepinephrine, and/or serotonin transporters as releasing agents (i.e., as substrates) or reuptake inhibitors (i.e., as transport blockers), it has now become possible to better examine their SAR and even their quantitative SAR (QSAR), in a more effective and systematic manner. An SAR picture is beginning to emerge, and key structural features, such as the nature of the terminal amine, the size of the α-substituent, stereochemistry, and the presence and position of aromatic substituents, are being found to impact action (i.e., as releasing agents or reuptake inhibitors) and transporter selectivity.

Synthesis of reboxetine intermediate and carnitine acetyltransferase inhibitor via NBS-induced electrophilic multicomponent reaction.

N-Bromosuccinimide-induced electrophilic multicomponent reaction has been applied to the synthesis of Reboxetine intermediate, a highly potent selective norepinephrine reuptake inhibitor. By simply changing the olefinic partner, the synthesis of a carnitine acetyltransferase inhibitor, which contains a 2,6,6-trisubstituted morpholine system, can be accomplished.

Nano-structured complexes of reserpine and quinidine drugs with chloranilic acid based on intermolecular H-bond: spectral and surface morphology studies.

The study of the drug-acceptor interaction may be useful in understanding the drug-receptor interactions and the mechanism of drug action. Here, complexes of reserpine (Res) and quinidine (Qui) drugs with chloranilic acid (CLA) have been synthesized. Then, these complexes were characterized chemically and structurally using CHN elemental analysis, infrared (IR) and electronic absorption spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM). The stoichiometry of the H-bonded complex was found to have a 1:1 ratio, so these complexes can be formulated as [(Drug)(CLA)]. IR measurements confirmed the presence of intermolecular H-bond. Application of Debye-Scherrer equation indicates that the formed complexes are in the range of nano-size. The Res complex exhibits a remarkable crystalline morphology. It was also found that the particle size of Res complex is 1.533 time higher than that of Qui complex. Interestingly, free Res molecular weight is higher than that of free Qui by the same ratio (precisely; 1.525).

Synthesis and antiproliferative action of a novel series of maprotiline analogues.

The synthesis of a diverse library of compounds structurally related to maprotiline, a norepinephrine reuptake transporter (NET) selective antidepressant which has recently been identified as a novel in vitro antiproliferative agent against Burkitt's lymphoma (BL) cell lines is reported. A series of 9,10-dihydro-9,10-ethanoanthracenes were synthesised with modifications to the bridge of the dihydroethanoanthracene structure and with alterations to the basic side chain. A number of compounds were found to reduce cell viability to a greater extent than maprotiline in BL cell lines. In addition a related series of novel 9-substituted anthracene compounds were investigated as intermediates in the synthesis of 9,10-dihydro-9,10-ethanoanthracenes. These compounds proved the most active from the screen and were found to exert a potent caspase-dependant apoptotic effect in the BL cell lines, while having minimal effect on the viability of peripheral blood mononuclear cells (PBMCs). Compounds also displayed activity in multi-drug resistant (MDR) cells.

Synthesis and biological evaluation of novel 3,4-diaryl lactam derivatives as triple reuptake inhibitors.

A series of 3,4-diarylpyrrolidin-2-one was designed, prepared and evaluated as triple reuptake inhibitors for antidepressant. Most compounds exhibited comparable in vitro efficacy as norepinephrine and dopamine transporter reuptake inhibitors. Especially, 2i showed better potency than GBR-12909 (IC50=14 nM) which was used as reference compound for dopamine transporter. In addition, 2a and 2b showed inhibition (5.17 µM-85.6 nM) for three transporters.

Future directions for serotonin and antidepressants.

Despite the widespread use of antidepressant medications that block serotonin (5-hydroxytryptamine; 5-HT) and/or norepinephrine (NE) transporters, such as SSRIs (selective serotonin reuptake inhibitors) or SNRIs (serotonin and norepinephrine reuptake inhibitors), the underlying neurobiological basis of action of these agents is poorly understood. Increases in serotonergic function are hypothesized to have beneficial effects on depressive symptoms. However, which of the 14 different neuronal receptors sensitive to 5-HT accounts for the therapeutic effects of SSRIs and SNRIs remains undetermined. The development of drugs that activate or block specific 5-HT receptors may help to circumvent the two main limitations of current antidepressants: low efficacy and delayed onset of therapeutic action. What follows is a short summary of the author's views on this matter.

Functional and structural interaction of (-)-reboxetine with the human α4ß2 nicotinic acetylcholine receptor.

The interaction of the selective norepinephrine reuptake inhibitor (-)-reboxetine with the human α4ß2 nicotinic acetylcholine receptor (nAChR) in different conformational states was studied by several functional and structural approaches. Patch-clamp and Ca(2+)-influx results indicate that (-)-reboxetine does not activate hα4ß2 nAChRs via interaction with the orthosteric sites, but inhibits agonist-induced hα4ß2 activation by a noncompetitive mechanism. Consistently, the results from the electrophysiology-based functional approach suggest that (-)-reboxetine may act via open channel block; therefore, it is capable of producing a use-dependent type of inhibition of the hα4ß2 nAChR function. We tested whether (-)-reboxetine binds to the luminal [(3)H]imipramine site. The results indicate that, although (-)-reboxetine binds with low affinity to this site, it discriminates between the resting and desensitized hα4ß2 nAChR ion channels. Patch-clamp results also indicate that (-)-reboxetine progressively inhibits the hα4ß2 nAChR with two-fold higher potency at the end of one-second application of agonist, compared with the peak current. The molecular docking studies show that (-)-reboxetine blocks the ion channel at the level of the imipramine locus, between M2 rings 6' and 14'. In addition, we found a (-)-reboxetine conformer that docks in the helix bundle of the α4 subunit, near the middle region. According to molecular dynamics simulations, (-)-reboxetine binding is stable for both sites, albeit less stable than imipramine. The interaction of these drugs with the helix bundle might alter allostericaly the functionality of the channel. In conclusion, the clinical action of (-)-reboxetine may be produced (at least partially) by its inhibitory action on hα4ß2 nAChRs.

A structural powder diffraction study of two polymorphic forms of nortriptyline hydrochloride.

Nortriptyline hydrochloride, a tricyclic antidepressant, appears in two different polymorphic forms, only one of which (hereafter, form ß) has been previously characterized by single-crystal analysis. Form ß is monoclinic, P2(1)/c, with a = 5.070(2), b = 34.088(5), c = 9.976(1) Å, and ß = 90.74(2)°. A second crystalline form (the α form) has now been characterized by structural powder diffraction methods (using both laboratory and synchrotron radiation diffraction data). Form α crystallizes in the monoclinic P2/c space group, a = 9.99126(6), b = 5.10021(3), c = 34.1636(1) Å, and ß = 98.684(6)°. The thermodynamic relationship between the two forms has been determined by differential scanning calorimetry analysis and variable-temperature thermodiffractometric experiments, revealing that the two forms are monotropically related and form α is more stable. Both phases are characterized by a sequence of hydrogen-bonded N-protonated molecules, which, in the two crystalline environments, adopt the same conformation. The difference between the two crystals can be traced back to the supramolecular arrangement characterized by one-dimensional chains, built by homochiral molecules (for conformationally driven chirality) in the α form, and by enantiomeric ones in the ß form. This observation nicely explains why, upon heating, solid-solid interconversion between the two forms does not occur.

Excretion and metabolism of milnacipran in humans after oral administration of milnacipran hydrochloride.

The pharmacokinetics, excretion, and metabolism of milnacipran were evaluated after oral administration of a 100-mg dose of [¹4C]milnacipran hydrochloride to healthy male subjects. The peak plasma concentration of unchanged milnacipran (∼240 ng/ml) was attained at 3.5 h and was lower than the peak plasma concentration of radioactivity (∼679 ng Eq of milnacipran/ml) observed at 4.3 h, indicating substantial metabolism of milnacipran upon oral administration. Milnacipran has two chiral centers and is a racemic mixture of cis isomers: d-milnacipran (1S, 2R) and l-milnacipran (1R, 2S). After oral administration, the radioactivity of almost the entire dose was excreted rapidly in urine (approximately 93% of the dose). Approximately 55% of the dose was excreted in urine as unchanged milnacipran, which contained a slightly higher proportion of d-milnacipran (∼31% of the dose). In addition to the excretion of milnacipran carbamoyl O-glucuronide metabolite in urine (∼19% of the dose), predominantly as the l-milnacipran carbamoyl O-glucuronide metabolite (∼17% of the dose), approximately 8% of the dose was excreted in urine as the N-desethyl milnacipran metabolite. No additional metabolites of significant quantity were excreted in urine. Similar plasma concentrations of milnacipran and the l-milnacipran carbamoyl O-glucuronide metabolite were observed after dosing, and the maximum plasma concentration of l-milnacipran carbamoyl O-glucuronide metabolite at 4 h after dosing was 234 ng Eq of milnacipran/ml. Lower plasma concentrations (<25 ng Eq of milnacipran/ml) of N-desethyl milnacipran and d-milnacipran carbamoyl O-glucuronide metabolites were observed.

First-in-class, dual-action, 3,5-disubstituted indole derivatives having human nitric oxide synthase (nNOS) and norepinephrine reuptake inhibitory (NERI) activity for the treatment of neuropathic pain.

A family of different 3,5-disubstituted indole derivatives having 6-membered rings were designed, synthesized, and demonstrated inhibition of human nitric oxide synthase (NOS) with norepinephrine reuptake inhibitory activity (NERI). The structure-activity relationship (SAR) within the cyclohexane ring showed the cis-isomers to be more potent for neuronal NOS and selective over endothelial NOS compared to their trans-counterparts. Compounds, such as cis-(+)-37, exhibited dual nNOS and NET inhibition (IC(50) of 0.56 and 1.0 µM, respectively) and excellent selectivity (88-fold and 12-fold) over eNOS and iNOS, respectively. The lead compound (cis-(+)-37) showed lack of any direct vasoconstriction or inhibition of ACh-mediated vasorelaxation in isolated human coronary arteries. Additionally, cis-(+)-37 was effective at reversing both allodynia and thermal hyperalgesia in a standard Chung (spinal nerve ligation) rat neuropathic pain model. Overall, the data suggest that cis-(+)-37 is a promising dual action development candidate having therapeutic potential for the treatment of neuropathic pain.