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.
The primary targets of Amitifadine HCl are the three monoamine transporters: the serotonin transporter (hSERT), the norepinephrine transporter (hNET), and the dopamine transporter (hDAT). By inhibiting these transporters, Amitifadine blocks the reuptake of serotonin, norepinephrine, and dopamine from the synaptic cleft back into the presynaptic neuron. This leads to an increase in the concentration of these neurotransmitters in the synapse, enhancing monoaminergic neurotransmission. This triple reuptake inhibition mechanism is thought to be effective for treating depression, as it targets multiple neurotransmitter systems involved in mood regulation.

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].

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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.

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In vitro, Amitifadine HCl has been shown to be a potent inhibitor of serotonin, norepinephrine, and dopamine reuptake. Its IC50 values in HEK 293 cells are 12 nM for serotonin, 23 nM for norepinephrine, and 96 nM for dopamine. This indicates that it is most potent at the serotonin transporter, followed by the norepinephrine transporter, and then the dopamine transporter. The compound's ability to inhibit all three transporters simultaneously distinguishes it from selective serotonin reuptake inhibitors (SSRIs) or serotonin-norepinephrine reuptake inhibitors (SNRIs), which target only one or two of these transporters.

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].

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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.

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In vivo, Amitifadine HCl was evaluated in clinical trials for the treatment of depression. By increasing the levels of serotonin, norepinephrine, and dopamine in the brain, it is expected to produce antidepressant effects. The compound's triple reuptake inhibition profile may offer advantages over SSRIs or SNRIs, particularly in addressing the broader range of symptoms associated with depression, such as fatigue and anhedonia, which may be related to dopamine signaling. It was the only drug ever clinically tested in Phase 3 for treating depression as a triple reuptake inhibitor.

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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Non-cellular in vitro assays for Amitifadine HCl involve measuring its ability to inhibit the reuptake of radiolabeled monoamines. A standard protocol uses membrane preparations from cells expressing recombinant human serotonin, norepinephrine, or dopamine transporters. The membranes are incubated with a radiolabeled substrate (e.g., [³H]serotonin, [³H]norepinephrine, or [³H]dopamine) and varying concentrations of Amitifadine. The uptake is allowed to proceed for a specified time, and the reaction is terminated by rapid filtration. The radioactivity associated with the membranes is measured. The IC50 values are calculated from the inhibition curves.

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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Cellular assays for Amitifadine HCl are performed using cell lines expressing the human monoamine transporters, such as HEK293 cells. Cells are pre-incubated with Amitifadine at various concentrations for a short period. Then, a radiolabeled monoamine (e.g., [³H]serotonin) is added, and the uptake is allowed to proceed. After incubation, the cells are washed, and the intracellular radioactivity is measured. The IC50 for inhibition of uptake is determined from the concentration-response curve. This assay confirms that Amitifadine is a functional inhibitor of the transporters.

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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In vivo animal studies for Amitifadine HCl were conducted in rodent models of depression, such as the forced swim test or the tail suspension test. In these tests, mice or rats are administered Amitifadine (e.g., i.p. or p.o.) at doses such as 1-10 mg/kg, and the duration of immobility is measured. A reduction in immobility time is indicative of antidepressant-like activity. The compound's effects on neurotransmitter levels in the brain can also be measured using microdialysis to confirm its mechanism of action. However, the most significant in vivo data come from human clinical trials.

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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Amitifadine HCl is a small molecule with a molecular weight of 264.57. It is a hydrochloride salt, which enhances its aqueous solubility. Detailed pharmacokinetic parameters for Amitifadine have been reported from clinical trials. It is expected to be well-absorbed after oral administration and to cross the blood-brain barrier to reach its targets in the central nervous system. Specific PK data, such as half-life and bioavailability, are not provided in the available text, but its progression to Phase 3 trials indicates that it had acceptable pharmacokinetic properties.

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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The toxicity profile of Amitifadine HCl was evaluated in preclinical and clinical studies. As a drug that was tested in Phase 3 trials, it would have undergone extensive safety evaluations. Common side effects of triple reuptake inhibitors may include nausea, insomnia, dry mouth, and increased heart rate or blood pressure. Specific toxicity data are not provided in the available text. The compound was not approved for marketing, indicating that its benefit-risk profile may not have been favorable compared to existing treatments.

[1]. Antidepressant-like actions of DOV 21,947: a "triple" reuptake inhibitor. Eur J Pharmacol. 2003 Feb 14;461(2-3):99-104.

[2]. Amitifadine, a triple monoamine re-uptake inhibitor, reduces nicotine self-administration in female rats. Eur J Pharmacol. 2015 Jun 20;764:30-37.

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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Amitifadine HCl (DOV-21947, EB-1010) is a triple reuptake inhibitor that was investigated as a potential antidepressant. It was unique in that it was the only drug of its class to reach Phase 3 clinical trials for depression. Despite reaching this advanced stage of development, it was not approved by the FDA. The reasons for its discontinuation are not specified in the available text but may have been related to efficacy, safety, or commercial considerations. It remains a compound of interest for researchers studying the pharmacology of triple reuptake inhibitors.

C11H12CL3N

264.5787

263.004

C, 49.94; H, 4.57; Cl, 40.20; N, 5.29

410074-74-7

410074-73-6; 410074-74-7

11680542

White to off-white solid powder

3.985

2

1

1

15

245

2

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]

KAGBHVBIOJBGBD-NINOIYOQSA-N

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

(1R,5S)-1-(3,4-dichlorophenyl)-3-azabicyclo[3.1.0]hexane;hydrochloride

(-)-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;

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 100 mg/mL (~377.96 mM)
H2O : ~50 mg/mL (~188.98 mM)

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.

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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.

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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.
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 corn oil and mix evenly.

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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).

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 (Please use freshly prepared in vivo formulations for optimal results.)