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| Targets |
Amitifadine primarily targets the human serotonin transporter (SERT), norepinephrine transporter (NET), and dopamine transporter (DAT), functioning as a “triple” reuptake inhibitor (SNDRI) with a serotonin‑preferring profile. Its inhibition potencies (IC₅₀) for the uptake of the three monoamines in HEK‑293 cells expressing human recombinant transporters are 12 nM for serotonin, 23 nM for norepinephrine, and 96 nM for dopamine. The corresponding binding affinities (Kᵢ) at the transporters are 99 nM for SERT, 262 nM for NET, and 213 nM for DAT. The in vivo pharmacological ratio for monoamine elevation is approximately 1:2:8.
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| ln Vitro |
Antidepressants like amitidine (DOV 21,947) are used. SERT, NET, and DAT have Ki values of 99 nM, 262 nM, and 213 nM, respectively. Serotonin uptake, norepinephrine uptake, and dopamine uptake had IC50 values of 12, 23, and 96 nM, respectively [1].
Amitifadine is a potent inhibitor of human SERT, NET, and DAT in vitro. Using HEK‑293 cells stably expressing the human recombinant transporters, the compound exhibits IC₅₀ values of 12 nM for inhibiting serotonin uptake, 23 nM for norepinephrine uptake, and 96 nM for dopamine uptake. The binding affinities (Kᵢ) to the three transporters are 99 nM (SERT), 262 nM (NET), and 213 nM (DAT). This profile establishes amitifadine as a serotonin‑preferring triple reuptake inhibitor, with a potency ratio for monoamine uptake inhibition of approximately 1:2:8 (5‑HT:NE:DA). The major metabolite of amitifadine, the 2‑lactam compound, only weakly inhibits monoamine uptake, suggesting that the parent compound is the primary active moiety. |
| ln Vivo |
Amitifa at 30 mg/kg significantly decreased the amount of nicotine that people self-administered. The first fifteen minutes saw a decrease in nicotine self-administration with the 5 mg/kg and 10 mg/kg doses. when taking the maximum recommended dosage of nicotine by mouth. A significant decrease in mean locomotor activity during the one-hour session and a reduction in food-induced responses were not observed with the 30 mg/kg dose of amitifa (but lower doses). At the start of the course, the 10 mg/kg dose resulted in hypoactivity, whereas the 5 mg/kg dose had no such effect. It was also established what happened after 15 rounds of continuous amitidine treatment (10 mg/kg). Amitidine dramatically decreased the amount of nicotine that was self-administered; this reduction persisted one week after the mandatory abstinence period and during the two weeks of continuous treatment. Self-administration of nicotine is greatly decreased by amitifatin. This has led to additional investigation into whether amitidin could be a useful smoking cessation treatment [2].
Amitifadine (10 mg/kg, i.p.) produces a marked and persistent increase in extracellular concentrations of serotonin, norepinephrine, and dopamine in the prefrontal cortex of freely moving rats, as measured by in vivo microdialysis. It also elevates extracellular dopamine levels in the striatum and in the nucleus accumbens, a core region of the brain’s reward circuitry. Consistent with its reuptake inhibition mechanism, amitifadine also significantly decreases the extracellular levels of monoamine metabolites (5‑HIAA, DOPAC, HVA) in these brain regions. Importantly, across a broad dose range, amitifadine does not increase locomotor activity or induce stereotypical behaviors typically associated with psychostimulants, which is an advantage for a compound that enhances dopamine transmission. A 10 mg/kg dose of amitifadine significantly reduced nicotine self‑administration in rats, an effect that persisted over two weeks of chronic treatment, suggesting potential for smoking‑cessation therapy. |
| Enzyme Assay |
The in vitro potency of amitifadine is determined using radioligand uptake inhibition assays in HEK‑293 cells expressing human recombinant SERT, NET, or DAT. Cells are plated in multi‑well plates and pre‑incubated with varying concentrations of the test compound. The uptake reaction is initiated by adding a low concentration (nanomolar range) of the respective radiolabeled neurotransmitter substrate ([³H]5‑HT for SERT, [³H]NE for NET, or [³H]DA for DAT). After a brief incubation (typically 5‑15 minutes) at 37 °C, the reaction is terminated by rapid aspiration and washing with ice‑cold buffer. The cells are then lysed, and the accumulated radioactivity in the lysate is quantified by liquid scintillation counting. Non‑specific uptake is determined in the presence of a high concentration of a specific reuptake inhibitor (e.g., fluoxetine for SERT, desipramine for NET, or GBR‑12909 for DAT). The percentage of inhibition at each compound concentration is calculated, and IC₅₀ values are derived by non‑linear regression analysis of the concentration‑inhibition curves.
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| Cell Assay |
Cell‑based assays for amitifadine utilize the same HEK‑293 cell lines stably expressing the human recombinant SERT, NET, or DAT as described in the enzyme assay section. These functional uptake assays are specifically designed to measure the compound’s ability to block the transporter‑mediated influx of radiolabeled neurotransmitters into living cells, which is the primary pharmacological action of the molecule. The protocol involves exposing the cells to the test compound and the radiolabeled substrate simultaneously. After the uptake period, the reaction is stopped by rapid washing to remove extracellular radioactivity, and the cells are lysed. The intracellular radioactivity (a direct measure of uptake) is then quantified by liquid scintillation counting. This method directly measures the functional antagonism of the transporters. No other cell‑based assays such as viability, proliferation, or toxicity studies are detailed in the referenced literature.
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| Animal Protocol |
Rat in Vivo Microdialysis: To assess the neurochemical effects of amitifadine, male Sprague‑Dawley rats are anesthetized, and guide cannulae are stereotaxically implanted into target brain regions (e.g., prefrontal cortex, striatum, or nucleus accumbens). After recovery, a microdialysis probe is inserted, and the animal is perfused with artificial cerebrospinal fluid (aCSF) at a constant flow rate (e.g., 1.5 μL/min). Following a stabilization period, baseline dialysate samples are collected every 20 minutes. Amitifadine is then administered intraperitoneally (i.p.) at a dose of 10 mg/kg, and sample collection continues for several hours. The concentrations of monoamines (5‑HT, NE, DA) and their metabolites (5‑HIAA, DOPAC, HVA) in the dialysate are analyzed by high‑performance liquid chromatography (HPLC) with electrochemical detection (ECD). Locomotor Activity Test: Sprague‑Dawley rats are placed in photocell‑equipped activity cages, and baseline locomotor activity is recorded. After a habituation period, amitifadine is administered i.p., and ambulatory counts are recorded to assess any stimulant or depressant effects on spontaneous motor activity over a broad dose range.
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| ADME/Pharmacokinetics |
In preclinical studies, amitifadine is rapidly absorbed following oral administration, with a plasma t_max of 0.7-1.2 hours. The elimination half-life (t₁/₂) is approximately 3.3-4.4 hours. Both C_max and AUC values increase in a dose-proportional manner across the dose range studied (5-150 mg). At a single dose of 10 mg, the average C_max was 78 ng/mL, and doses above this level result in plasma concentrations that exceed the in vitro IC₅₀ values for monoamine uptake inhibition. The compound shows no remarkable accumulation following 10 days of once‑daily dosing, with steady‑state being achieved within approximately one week. In humans, a single 8 mg dose of amitifadine hydrochloride produced a C_max of 2.02 ng/mL (18.4% CV) with a median T_max of 10 hours, and a mean half-life of 61.3 hours (34.2% CV).
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| Toxicity/Toxicokinetics |
In clinical trials for MDD, amitifadine has been shown to be safe and well‑tolerated. In a phase II proof‑of‑concept trial, the estimated effect size (Cohen‘s d) for amitifadine compared to placebo was −0.601 on the MADRS scale, indicating clinically meaningful antidepressant activity. Notably, amitifadine was well tolerated without causing weight gain or sexual dysfunction, which are common adverse effects limiting adherence to standard first‑line antidepressants. In a comprehensive phase IIb/IIIa safety and efficacy trial (TRIADE study) involving a total safety population of 342 patients with treatment‑resistant MDD, amitifadine demonstrated a favorable safety profile, with no significant safety signals concerning cardiovascular events, hepatotoxicity, or serious adverse events reported. The most common adverse events, if any, were likely mild and transient, consistent with its mechanism of action.
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| References | |
| Additional Infomation |
Amitifadine hydrochloride (CAS 410074-74-7; UNII 0) is the hydrochloride salt of the single enantiomer (+)-(1R,5S)-1-(3,4-dichlorophenyl)-3-azabicyclo[3.1.0]hexane. It has the molecular formula C₁₁H₁₂Cl₃N and a molecular weight of 264.58. The compound is part of the 3-azabicyclo[3.1.0]hexane class and is structurally related to the prototype triple reuptake inhibitor DOV-216,303. The synthesis of amitifadine has been described in several patents and involves a stereospecific route using (S)-epichlorohydrin to establish the cis-cyclopropane stereochemistry. The discovery that a triple reuptake inhibitor might improve outcomes in SSRI‑nonresponsive patients originated from the National Institute of Mental Health’s large‑scale STAR*D (Sequenced Treatment Alternatives to Relieve Depression) study, where combining serotonergic, noradrenergic, and dopaminergic mechanisms was associated with improved outcomes. The advanced phase IIb/IIIa TRIADE trial employed a novel clinical trial design, the Sequential Parallel Comparison Design (SPCD), to minimize the high placebo response rate that has plagued many depression trials, and also used the SAFER criteria to ensure diagnostic and treatment‑history accuracy. A previous phase II study in MDD patients indicated that the effect size for amitifadine was superior to that observed for citalopram, an active comparator, in the same trial. Beyond depression, preclinical evidence suggests amitifadine may have potential as a smoking‑cessation agent, as it significantly reduced nicotine self‑administration in an animal model.
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| Molecular Formula |
C11H12CL3N
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| Molecular Weight |
264.5787
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| Exact Mass |
263.004
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| Elemental Analysis |
C, 49.94; H, 4.57; Cl, 40.20; N, 5.29
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| CAS # |
410074-74-7
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| Related CAS # |
410074-73-6; 410074-74-7
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| PubChem CID |
11680542
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| Appearance |
White to off-white solid powder
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| LogP |
3.985
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
1
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
15
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| Complexity |
245
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| Defined Atom Stereocenter Count |
2
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| SMILES |
ClC1=C(C([H])=C([H])C(=C1[H])[C@]12C([H])([H])N([H])C([H])([H])[C@@]1([H])C2([H])[H])Cl.Cl[H]
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| InChi Key |
KAGBHVBIOJBGBD-NINOIYOQSA-N
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| InChi Code |
InChI=1S/C11H11Cl2N.ClH/c12-9-2-1-7(3-10(9)13)11-4-8(11)5-14-6-11;/h1-3,8,14H,4-6H2;1H/t8-,11+;/m1./s1
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| Chemical Name |
(1R,5S)-1-(3,4-dichlorophenyl)-3-azabicyclo[3.1.0]hexane;hydrochloride
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| Synonyms |
(-)-Amitifadine hydrochloride; DOV-21,947; EB-1010; DOV-21947; EB 1010; DOV21,947; EB1010; 8WR4Q5ZGU3; DOV-102677 hydrochloride; DOV-102,677 hydrochloride; 500733-69-7;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ≥ 100 mg/mL (~377.96 mM)
H2O : ~50 mg/mL (~188.98 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.75 mg/mL (10.39 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.75 mg/mL (10.39 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.75 mg/mL (10.39 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 10 mg/mL (37.80 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C). |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.7796 mL | 18.8979 mL | 37.7958 mL | |
| 5 mM | 0.7559 mL | 3.7796 mL | 7.5592 mL | |
| 10 mM | 0.3780 mL | 1.8898 mL | 3.7796 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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