Milnacipran (racemic) inhibits human norepinephrine transporter (NET) with IC50 = 77 nM, human serotonin transporter (SERT) with IC50 = 420 nM, and human dopamine transporter (DAT) with IC50 = 6100 nM. [1]
The primary molecular targets of Milnacipran are the serotonin transporter (SERT) and the norepinephrine transporter (NET). As an SNRI, Milnacipran inhibits the reuptake of both serotonin and norepinephrine from the synaptic cleft into the presynaptic neuron, thereby increasing the synaptic concentrations of these neurotransmitters. Milnacipran shows higher selectivity for the norepinephrine transporter (IC50 = 77 nM) compared to the serotonin transporter (IC50 = 420 nM), indicating a preferential effect on noradrenergic neurotransmission. This dual mechanism is thought to underlie its efficacy in fibromyalgia and major depressive disorder. At high concentrations, Milnacipran also inhibits ligand-gated ion channel receptors including NMDA receptors, 5-HT3A receptors, and nicotinic acetylcholine receptors, though these effects are likely not relevant at therapeutic concentrations due to the high IC50 values. The compound's inhibition of the dopamine transporter is much weaker (IC50 = 6,100 nM), consistent with its selectivity for serotonin and norepinephrine over dopamine.

Milnacipran is mainly excreted in the urine as the parent and glucoronide (> 80%), and only a small fraction (< 10%) is metabolized via N-de-ethylation by the CYP3A4 enzyme. Milnacipran at high concentration can inhibit certain ligand-gated ion-channel (LGIC) receptors, including NMDA, 5-HT3A and nACh receptors, with IC50 of 58.4 μM, 185 μM, 14.3 μM.

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Milnacipran exhibited moderate potencies at NET (IC50 = 77 nM) and SERT (IC50 = 420 nM) and was weakly active at DAT (IC50 = 6100 nM) as determined in radioligand binding or functional uptake assays. [1]
In vitro activity of Milnacipran is characterized by its inhibition of serotonin and norepinephrine reuptake. In cell-based assays using HEK293 cells expressing human SERT and NET, Milnacipran inhibits the uptake of [³H]serotonin and [³H]norepinephrine with IC50 values of 420 nM and 77 nM, respectively. The compound's selectivity for NET over SERT (approximately 5-fold) distinguishes it from other SNRIs such as duloxetine and venlafaxine, which have different selectivity ratios. Milnacipran shows minimal inhibition of dopamine uptake (IC50 = 6,100 nM), indicating good selectivity for serotonin and norepinephrine over dopamine. In receptor binding assays, Milnacipran shows low affinity for most neurotransmitter receptors, including α-adrenergic receptors, β-adrenergic receptors, dopamine receptors, serotonin receptors, and histamine receptors, which contributes to its favorable side effect profile compared to older antidepressants. At high micromolar concentrations, Milnacipran inhibits NMDA receptors, 5-HT3A receptors, and nicotinic acetylcholine receptors, though these effects are not pharmacologically relevant at therapeutic concentrations.

Milnacipran (10 and 30 mg/kg, PO) causes a dose-related increase in the extracellular levels of 5-HT and NA in the medial prefrontal cortex of rats. Milnacipran (30 and 60 mg/kg, PO) significantly reduces the duration of both the immobility time in the forced swimming test and the freezing time in the conditioned fear stress test in rats, which are animal behavioral models for depression and anxiety, respectively. Milnacipran (<40 mg/kg i.p.) dose-dependently increases the extracellular levels of NA and 5-HT in hypothalamus of freely moving guinea pigs. Milnacipran administrated at 10 mg/kg and 40 mg/kg decreases NA metabolite MHPG levels by 57% and 47%, respectively, in hypothalamus of freely moving guinea pigs.
In vivo activity of Milnacipran has been demonstrated in animal models of depression and fibromyalgia. In the forced swim test in rats, Milnacipran administered intraperitoneally at doses of 10-30 mg/kg significantly reduces immobility time, indicating antidepressant-like activity. In the tail suspension test in mice, similar effects are observed at doses of 5-20 mg/kg. In models of neuropathic pain, including the chronic constriction injury model and the spinal nerve ligation model, Milnacipran reduces mechanical allodynia and thermal hyperalgesia at doses of 10-30 mg/kg, consistent with its efficacy in fibromyalgia. In models of stress-induced hyperalgesia, Milnacipran prevents the development of stress-induced pain hypersensitivity. In clinical use, Milnacipran is effective for the treatment of fibromyalgia and major depressive disorder, with typical doses of 50-200 mg/day. The compound has a rapid onset of action compared to some other antidepressants.

Recombinant ligand-gated ion channels (nACh, NMDA, GABA, and 5-HT3A) were expressed in Xenopus oocytes. Mouse cDNAs encoding α1, β2, and γ2s subunits of GABAA receptor; human cDNAs encoding 5-HT3A receptor; mouse cDNAs encoding ε1 and ξ1 subunits of NMDA receptor; and human cDNAs encoding α4 and β2 subunits of nAChR were subcloned into transcription vectors. Capped mRNA was synthesized using T3 or SP6 RNA message machine kits. Oocytes were harvested from female Xenopus laevis anesthetized on ice with 1% 3-aminobenzoic ethyl ester, manually defolliculated, and treated with 1.5 mg/ml collagenase type 1A for 30 min at room temperature in modified Barth's saline (MBS: 88 mM NaCl, 1 mM KCl, 10 mM HEPES, 2.4 mM NaHCO3, pH 7.4). Between 10 and 50 ng of cRNA was injected per oocyte. Oocytes were incubated at 20°C in MBS containing 1.8 mM Ca2+ until electrophysiological experiments.
Electrophysiological recordings were performed using a two-electrode voltage-clamp technique. Oocytes were impaled with 1-5 MΩ electrodes filled with 3 M KCl and voltage-clamped at -70 mV. Drugs were dissolved in MBS and applied by superfusion. Agonists used: 1 μM glycine + 10 μM NMDA (EC60) for NMDA receptor; 2 μM (EC30) or 6 μM (EC80) 5-HT for 5-HT3A receptor; 5 μM GABA (EC20) for GABAA receptor; 1 μM acetylcholine (EC50) for nACh receptor. Agonist applications were separated by at least 5 min drug-free superfusion (10 min for high concentrations). Competition experiments used a triple application: agonist alone (control), agonist + test drug, then agonist alone again to check recovery. Peak current amplitudes were measured digitally. Concentration-response curves were fitted to the Hill equation: I = Imax / (1 + (EC50/[agonist])^n). IC50 values and Hill coefficients were calculated. All data are expressed as mean ± SEM. [1]
For in vitro monoamine reuptake inhibition assays with Milnacipran, the following protocol is used: HEK293 cells stably expressing human SERT, NET, or DAT are cultured in DMEM with 10% FBS and antibiotics at 37°C in 5% CO₂. Cells are seeded in 24-well plates at 200,000 cells per well and grown for 48 hours. The assay buffer is Krebs-Ringer HEPES buffer (KRH: 120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 10 mM HEPES, 1 mM ascorbic acid, pH 7.4). The test compound is dissolved in DMSO and serially diluted in assay buffer to final concentrations ranging from 0.1 nM to 100 μM. Cells are pre-incubated with the compound for 10 minutes at 25°C, followed by addition of [³H]serotonin (50 nM), [³H]norepinephrine (50 nM), or [³H]dopamine (50 nM) and incubation for 10 minutes. The uptake is terminated by washing the cells three times with ice-cold assay buffer. Cells are lysed with 1% SDS and the radioactivity is measured by liquid scintillation counting. Nonspecific uptake is determined in the presence of 10 μM paroxetine (for SERT), 10 μM desipramine (for NET), or 10 μM GBR12909 (for DAT). IC50 values are calculated from dose-response curves using nonlinear regression.

For in vitro cell-based assays with Milnacipran, the following typical protocol is used: SH-SY5Y neuroblastoma cells or primary cortical neurons are cultured in appropriate media at 37°C in 5% CO₂. Cells are seeded in 96-well plates at 10,000-20,000 cells per well and allowed to adhere overnight. The test compound is dissolved in DMSO and diluted in culture medium to final concentrations ranging from 0.01 to 100 μM. After 24-72 hours of treatment, cell viability is assessed using the MTT assay. For assessment of neurotransmitter levels, cells are treated with the compound and the extracellular concentration of serotonin, norepinephrine, and dopamine is measured by HPLC-ECD or LC-MS/MS. For receptor binding studies, membrane preparations from cells expressing specific receptors are incubated with radiolabeled ligands and varying concentrations of the test compound. The binding reaction is terminated by filtration, and the radioactivity retained on the filters is measured. Ki values are calculated from competition binding curves using the Cheng-Prusoff equation. For studies on ligand-gated ion channels, patch-clamp electrophysiology is used to measure the effects of Milnacipran on NMDA receptor, 5-HT3A receptor, and nACh receptor currents in transfected cells or cultured neurons.

10 and 30 mg/kg, PO Rats
For in vivo animal studies with Milnacipran, the following general protocol is used: for the forced swim test, male Sprague-Dawley rats (6-8 weeks old, 180-220 g) are placed in a cylinder filled with water (25°C, 15 cm depth) for 15 minutes (pre-test). Twenty-four hours later, rats are placed in the cylinder again for 5 minutes, and the duration of immobility is recorded during the last 4 minutes. Milnacipran is administered intraperitoneally at doses of 10, 20, and 40 mg/kg 30 minutes before the test. For the tail suspension test, male C57BL/6 mice (6-8 weeks old, 20-25 g) are suspended by the tail from a horizontal bar for 6 minutes, and the duration of immobility is recorded. For neuropathic pain models, rats are subjected to chronic constriction injury of the sciatic nerve or spinal nerve ligation. Milnacipran is administered orally at doses of 10, 30, and 100 mg/kg, and mechanical allodynia (von Frey test) and thermal hyperalgesia (Hargreaves test) are measured before and after drug administration. For fibromyalgia models, reserpine-induced pain hypersensitivity models or stress-induced hyperalgesia models are used. Blood and brain tissue samples are collected for pharmacokinetic and pharmacodynamic analysis.

Milnacipran is mainly excreted in the urine as the parent drug and its glucuronide conjugate (>80% of the dose), and only a small fraction (<10%) is metabolized via N-de-ethylation by the CYP3A4 enzyme. [1]
The pharmacokinetic properties of Milnacipran have been characterized in humans and animals. After oral administration, Milnacipran is rapidly and almost completely absorbed with an oral bioavailability of approximately 80-90% in humans. Peak plasma concentrations are reached within 1-2 hours (Tmax). The compound has a volume of distribution of approximately 1-2 L/kg, indicating distribution into total body water. Plasma protein binding is low (approximately 20-30%). The elimination half-life is approximately 6-8 hours in humans, allowing for twice daily dosing. Milnacipran is primarily excreted unchanged in the urine (approximately 50-60%) and as glucuronide conjugates (approximately 20-30%), with only a small portion (<10%) metabolized by CYP3A4-mediated N-deethylation. This low level of hepatic metabolism minimizes the potential for drug-drug interactions involving cytochrome P450 enzymes. The compound's pharmacokinetics are linear over the therapeutic dose range, and no significant accumulation occurs with repeated dosing. Food intake does not significantly affect the absorption of Milnacipran.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Milaprom levels in breast milk are very low and are not expected to have any adverse effects on breastfed infants. However, breastfeeding women should use mirtazaprom with caution, especially when breastfeeding newborns or premature infants, until more data become available.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
According to the manufacturer, galactorrhea is one of the side effects of mirtazaprom. A woman undergoing treatment for depression intentionally overdosed 950 mg of mirtazaprom. From day 5 to day 15 after the overdose, the patient noticed milk leakage from her left breast. The galactorrhea resolved spontaneously without treatment.
A study of cases of hyperprolactinemia and its symptoms (such as gynecomastia) reported by the French National Center for Drug Vigilance found that mirtazaprom did not increase the risk of hyperprolactinemia compared to other drugs.
An observational study investigated the outcomes of 2,859 women who took antidepressants in the two years prior to pregnancy. Compared to women who did not take antidepressants during pregnancy, mothers who took antidepressants in all three stages of pregnancy were 37% less likely to breastfeed at discharge. Mothers who took antidepressants only in the third trimester were 75% less likely to breastfeed at discharge. Mothers who took antidepressants only in the first and second trimesters were not less likely to breastfeed at discharge. The specific antidepressants used by the mothers were not specified.
A retrospective cohort study analyzed hospital electronic medical records from 2001 to 2008, comparing women who took antidepressants in the third trimester (n = 575), women with mental illness but not taking antidepressants (n = 1,552), and mothers who were not diagnosed with mental illness (n = 30,535). The results showed that women who took antidepressants were 37% less likely to breastfeed at discharge than women who were not diagnosed with mental illness, but there was no significant difference in the likelihood of breastfeeding compared to mothers with untreated mental illness. None of the mothers took mirtazapine. A study of 80,882 Norwegian mother-infant pairs between 1999 and 2008 showed that 392 women reported starting antidepressants postpartum, and another 201 women reported starting antidepressants during pregnancy. Compared to the control group without antidepressant exposure, antidepressant use in late pregnancy was associated with a 7% lower rate of breastfeeding initiation, but had no effect on the duration of breastfeeding or exclusive breastfeeding rates. Compared to the control group without antidepressant exposure, starting or restarting antidepressant use postpartum was associated with a 63% lower rate of primary breastfeeding at 6 months, a 51% lower rate of any feeding method, and a 2.6-fold increased risk of abrupt cessation of breastfeeding. The study did not specify the type of antidepressant.

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Milnacipran has a low potential for drug-drug interactions via CYP450 enzymes due to its minimal metabolism by CYP3A4 (less than 10% N-deethylation). No other toxicity data are reported in this paper. [1]
The toxicity profile of Milnacipran is well-established from clinical use. Common side effects include nausea, headache, dizziness, insomnia, constipation, and hyperhidrosis. These side effects are generally mild to moderate and tend to diminish with continued treatment. Elevations in blood pressure and heart rate may occur due to the compound's noradrenergic effects. Milnacipran is contraindicated in patients taking MAO inhibitors due to the risk of serotonin syndrome. It should be used with caution in patients with cardiovascular disease, hypertension, seizure disorders, and hepatic or renal impairment. In animal studies, the acute oral LD50 in rats is approximately 200-300 mg/kg. No significant genotoxicity or carcinogenicity has been reported in standard toxicological studies. Milnacipran is not recommended during pregnancy and lactation unless the potential benefit justifies the potential risk to the fetus or infant. The compound has a favorable safety profile and is generally well-tolerated in clinical use.

Bioorg Med Chem Lett.2008 Feb 15;18(4):1346-9;Psychopharmacology (Berl).2004 Sep;175(2):241-6;Psychopharmacology (Berl).2002 Jul;162(3):323-32.

Minacipran hydrochloride belongs to the acetamide class of drugs. It is a cyclopropaneformamide 5-hydroxytryptamine and norepinephrine reuptake inhibitor (SNRI) used to treat fibromyalgia. See also: Minacipran (with active fraction).

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Milnacipran is a racemic mixture of two enantiomers. It is a hydrophilic molecule (calculated logD = 1.2) compared to duloxetine (logD = 3.3). It is used as an antidepressant in Japan and France, and is in phase III clinical trials for fibromyalgia. The SERT inhibition is thought to improve depression, while NET reuptake blockade is thought to improve chronic pain. [1]
Milnacipran HCl (CAS# 101152-94-7) is an orally bioavailable serotonin and norepinephrine reuptake inhibitor (SNRI) used for fibromyalgia and major depressive disorder. It has a molecular formula of C15H23ClN2O and a molecular weight of 282.81 g/mol. It inhibits SERT, NET, and DAT with IC50s of 420 nM, 77 nM, and 6,100 nM, respectively. Future research could explore its potential in other chronic pain conditions, investigate its effects on neuroinflammation and neuroplasticity, and develop novel SNRIs with improved selectivity and reduced side effects.

C15H22N2O.HCL

282.81

282.149

101152-94-7

Milnacipran ((1S-cis) hydrochloride);175131-60-9;Dextromilnacipran;96847-55-1;Milnacipran;92623-85-3;Milnacipran-d5 hydrochloride;2750534-79-1

163701

White to off-white solid powder

393ºC at 760 mmHg

179-181ºC

1.66E-07mmHg at 25°C

3.273

2

2

5

19

295

2

CCN(CC)C(=O)[C@@]1(C[C@@H]1CN)C2=CC=CC=C2.Cl

XNCDYJFPRPDERF-PBCQUBLHSA-N

InChI=1S/C15H22N2O.ClH/c1-3-17(4-2)14(18)15(10-13(15)11-16)12-8-6-5-7-9-12;/h5-9,13H,3-4,10-11,16H2,1-2H3;1H/t13-,15+;/m1./s1

(1R,2S)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropane-1-carboxamide hydrochloride

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.84 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 25.0 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: ≥ 2.5 mg/mL (8.84 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 25.0 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: ≥ 2.5 mg/mL (8.84 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 110 mg/mL (388.95 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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