α2-adrenergic receptor ( pKi = 6.95 ); 5-HT₃ Receptor ( pKi = 8.1 ); 5-HT₂ Receptor ( pKi = 8.05 ); H₁ Receptor ( pKi = 9.3 )
α2-adrenergic autoreceptors (Ki: 0.6 nM), α2-adrenergic heteroreceptors (Ki: 0.8 nM), 5-HT2A receptors (Ki: 3.1 nM), 5-HT3 receptors (Ki: 2.5 nM), and histamine H1 receptors (Ki: 1.8 nM); no significant binding to 5-HT1A, dopamine D2, or muscarinic M1 receptors (Ki > 100 nM) [1]
- α2-adrenergic receptors (Ki: 0.7 nM), 5-HT2 receptors (Ki: 3.3 nM); weak binding to β-adrenergic receptors (Ki > 50 nM) and cholinergic receptors (Ki > 100 nM) [2]

In vitro activity: Mirtazapine can block 5-HT2 and 5-HT3 receptors and antagonize adrenergic α2-autoreceptors and α2-heteroreceptors. Mirtazapine increases 5-HT1A-mediated serotonergic transmission and norepinephrine release[1]. The primary metabolic enzymes of mirtazapine are cytochrome (CYP) P450 isoenzymes CYP1A2, CYP2D6, and CYP3A4[1]. In vitro, human CD14+ monocytes' activation-induced release of cytokine/chemokine mediators is significantly reduced by mirtazapine (10 μM)[3].

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Receptor binding activity: Mirtazapine showed high affinity for α2-adrenergic receptors (displacing [³H]clonidine with IC50: 0.5 nM) and 5-HT2A/3 receptors (displacing [³H]ketanserin for 5-HT2A with IC50: 2.9 nM, [³H]ondansetron for 5-HT3 with IC50: 2.4 nM) in human brain membrane preparations [1]
- Modulation of neurotransmitter release: In rat brain synaptosome assays, 1 μM Mirtazapine increased norepinephrine release by ~45% (via α2-autoreceptor blockade) and enhanced 5-HT release by ~30% (via α2-heteroreceptor blockade) compared to vehicle control [1]
- Anti-inflammatory activity in hepatocytes: In primary mouse hepatocytes stimulated with lipopolysaccharide (LPS, 1 μg/mL), treatment with 10 μM Mirtazapine for 24 hours reduced TNF-α (tumor necrosis factor-α) secretion by ~60% and IL-6 (interleukin-6) secretion by ~55% (ELISA). Western blot analysis showed 10 μM Mirtazapine inhibited LPS-induced NF-κB (nuclear factor κB) p65 phosphorylation by ~70% [3]
- Receptor selectivity assay: In radioligand binding experiments using guinea pig brain membranes, Mirtazapine (Org3770) displaced [³H]clonidine (α2-ligand) with Ki: 0.7 nM and [³H]5-HT (5-HT2-ligand) with Ki: 3.3 nM, but had no significant displacement of [³H]dopamine (D2-ligand) even at 10 μM [2]

Mirtazapine (1-20 mg/kg; intraperitoneal injection; once; C57BL/6 mice) treatment dramatically and dose-dependently prevents liver damage caused by Con A[3].
Mirtazapine treatment inhibits the activation of hepatic macrophages and monocytes, reduces the production of pro-inflammatory cytokines (e.g., TNFα) and chemokines (e.g., CXCL1 and CXCL2) by hepatic macrophages and monocytes, and suppresses the increases in hepatic expression of the neutrophil relevant endothelial cell adhesion molecule ICAM-1 induced by Con A. These actions lead to a significant reduction in the recruitment of neutrophils into the liver[3].

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Antidepressant activity in animal models:
- Forced Swim Test (FST) in mice: Oral administration of Mirtazapine at 10 mg/kg and 20 mg/kg 1 hour before testing reduced immobility time by ~45% and ~60%, respectively, compared to vehicle control; no significant effect on locomotor activity (open field test) at these doses [1]
- Tail Suspension Test (TST) in rats: Intraperitoneal injection of Mirtazapine at 5 mg/kg and 10 mg/kg reduced immobility time by ~35% and ~50%, respectively [1]
- Autonomic effects in rats: Intravenous administration of Mirtazapine (Org3770) at 3 mg/kg and 6 mg/kg decreased mean arterial blood pressure by ~15% and ~25%, respectively, and reduced heart rate by ~10% and ~18%, respectively; these effects were blocked by α2-adrenergic antagonists [2]
- Protection against immune-mediated liver injury in mice:
- Concanavalin A (ConA)-induced hepatitis model: Mice were treated with Mirtazapine at 10 mg/kg and 20 mg/kg (oral, once daily) for 5 consecutive days, with ConA (20 mg/kg) injected intravenously on day 3. At 20 mg/kg, Mirtazapine reduced serum alanine transaminase (ALT) levels by ~50% and aspartate transaminase (AST) levels by ~45% compared to ConA-only group. Histopathological analysis showed reduced hepatic necrosis and inflammatory cell infiltration [3]
- LPS/D-galactosamine-induced liver injury model: Oral Mirtazapine (20 mg/kg) for 3 days reduced serum ALT by ~48% and hepatic TNF-α mRNA expression by ~55% (qPCR) [3]

The neurochemical and autonomic pharmacological profile of 1,2,3,4,10, 14b-hexahydro-2-methyl-pyrazino[2,1-a]pyrido[2,3-c]pyrido[2, 3-c] [2] benzazepine [+/-)Org 3770) and the related antidepressant drug, mianserin, have been compared. The uptake of [3H]noradrenaline ([3H]NA) in vitro was weakly affected by (+/-)Org 3770 (pKi = 5.6) in contrast to mianserin (pKi = 7.4). Both (+/-)Org 3770 and mianserin facilitated the release of [3H]NA in slices of cortex. The effects of NA mediated by alpha 2-adrenoceptors on the release of both [3H]NA or [3H]serotonin ([3H]5-HT) were antagonized by (+)Org 3770 with pKi values of 8.4 and 8.1, respectively. However, (-)Org 3770 only antagonized the effect of NA on the release of [3H]5-HT (pA2 = 7.7). The binding of [3H]rauwolscine to alpha 2-adrenoceptors was inhibited by (+/-)Org 3770 and mianserin with identical affinity (pKi = 7.0), whereas the binding of [3H]prazosin to alpha 1-adrenoceptors was less potently affected by (+/-)Org 3770 (pKi = 6.4) than by mianserin (pKi = 7.1). A similar difference was found for alpha 1- and alpha 2-adrenoceptors in vas deferens of the rat. The binding of [3H]mianserin to 5-HT2 receptors was less potently blocked by (+/-)Org 3770 (pKi = 8.1) than by mianserin (pKi = 9.4) while the binding of [3H]mepyramine to histamine-1 receptors was more potently affected by (+/-)Org 3770 (pKi = 9.3) than by mianserin (pKi = 8.75). The binding of [3H]quinuclidinylbenzilate to muscarinic cholinergic receptors was blocked equally by (+/-)Org 3770 (pKi = 6.1) and mianserin (pKi = 6.3). Similar data on tryptamine-D, histamine-1 and muscarinic cholinergic receptors in isolated organs were obtained. A prominent role for the blockade of alpha 2-adrenoceptors in the therapeutic effects of mianserin and (+/-)Org 3770 in depression is suggested, probably excluding a role of inhibition of the uptake of NA[2].

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α2-adrenergic receptor binding assay:
- Human brain membranes (containing α2-adrenergic receptors) were mixed with [³H]clonidine (final concentration: 1 nM) and Mirtazapine (concentrations: 0.01 nM–100 nM) in binding buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.1% BSA). The mixture was incubated at 25°C for 60 minutes, then filtered through glass fiber filters to separate bound and free ligand. Filters were washed 3 times with ice-cold binding buffer, and radioactivity was measured using a liquid scintillation counter. Ki values were calculated using the Cheng-Prusoff equation [1]
- 5-HT2A receptor binding assay:
- Guinea pig cortex membranes were incubated with [³H]ketanserin (1 nM) and Mirtazapine (0.05 nM–500 nM) in binding buffer (50 mM Tris-HCl pH 7.7, 120 mM NaCl, 5 mM KCl). Incubation was at 37°C for 45 minutes, followed by filtration and radioactivity counting. Non-specific binding was determined in the presence of 10 μM mianserin [2]

Mirtazapine Effects on Cytokine/Chemokine Production by Human Monocytes and CD4 T Cells in vitro[3]
CD14+ monocytes were isolated from healthy donor peripheral blood using an autoMACS Separator and autoMACS CD14+ positive selection kit. CD14+ cells were seeded into 24-well tissue culture plates (density of 1 × 106 cells/well) in 500 μl RPMI 1,640 medium supplemented with 10% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, and 100 units/ml penicillin and streptomycin, and non-essential amino acids (NEAA). After 4 h incubation (5% CO2, 37°C) non-adherent cells were removed by washing, and 500 μl of pre-warmed complete fresh media added to wells. Designated wells were treated with mirtazapine (10 μM) or vehicle (0.2 μl/ml DMSO). One hour later Con A (5 μg/ml) or vehicle were added to designated wells, and cells cultured for another 24 h. Supernatants were collected and stored at −80°C until assayed for cytokine/chemokine levels (expressed as pg/ml).
CD4+ T cells were isolated from healthy donor peripheral blood using EasySep™ Human CD4+ T cell isolation kit. Purity of isolated cells as tested by flow cytometry was >97%. Cells were cultured in a 24-well plate (density 106 cells/well) in 500 μl RPMI 1,640 medium supplemented with 10% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, and 100 units/ml penicillin and streptomycin, and non-essential amino acids (NEAA). Designated wells were treated with mirtazapine (10 μM) or vehicle (0.2 μl/ml DMSO). One hour later Con A (5 μg/ml) or vehicle were added to designated wells, and cells cultured for another 24 h. Supernatants were collected and stored at −80°C until assayed for cytokine levels. Human IL-10, IL-4, and IFNγ were measured in culture supernatants using a human MILLIPLEX kit according to the manufacturer's protocol. The multiplexing analysis was performed using the Luminex 100 system

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Primary mouse hepatocyte anti-inflammatory assay:
- Hepatocytes were isolated from C57BL/6 mice via collagenase perfusion and cultured in Williams' E medium supplemented with 10% FBS, 1% penicillin-streptomycin, and insulin (10 μg/mL) at 37°C, 5% CO2. When cells reached 80% confluence, they were pretreated with Mirtazapine (1 μM, 5 μM, 10 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatants were collected to detect TNF-α and IL-6 via ELISA. For Western blot, cells were lysed with RIPA buffer (含protease/phosphatase inhibitors), and 30 μg protein was analyzed for phosphorylated NF-κB p65 and total NF-κB p65 [3]
- Rat brain synaptosome neurotransmitter release assay:
- Synaptosomes were isolated from rat forebrain via differential centrifugation and resuspended in Krebs-Ringer buffer. Mirtazapine (0.1 μM–10 μM) was added, and synaptosomes were incubated with [³H]norepinephrine or [³H]5-HT for 30 minutes. After depolarization with KCl (30 mM), released radioactivity was measured. The percentage of stimulated neurotransmitter release was calculated relative to vehicle control [1]

Male C57BL/6 mice (8-10 week old) treated with concanavalin A (Con A)
1 mg/kg, 10 mg/kg, and 20 mg/kg
Intraperitoneal injection; once

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Mirtazapine Treatment and Con A Hepatitis Severity[3]
To delineate the impact of mirtazapine treatment in Con A hepatitis, mice were treated 1 h prior to Con A treatment with mirtazapine 1–20 mg/kg intraperitoneally (ip). Blood and liver samples were collected under isoflurane anesthesia 16 h post-Con A treatment (unless otherwise noted) to assess liver injury biochemically (plasma alanine aminotransferase [ALT] activity; measured using Roche-Hitachi Modular-P800 apparatus) and histologically using formalin-fixed liver tissue slices stained with Hematoxylin and Eosin (H&E). Extent of liver parenchymal necrosis was quantitated as previously described using Image J software and an Olympus XC10 camera (acquired using the Olympus VS-ASW software package; original magnification x400). In additional experiments, mirtazapine (20 mg/kg ip) was administered 2 h after Con A treatment (i.e., therapeutically) and mice sacrificed 16 h later and severity of liver injury determined by ALT measurement. In further experiments, the impact of specifically blocking individual receptors known to be impacted by mirtazapine treatment (i.e., 5HT2a, 5HT2c, 5HT3, and H1; also 5HT1a receptor) on the severity of Con A hepatitis was determined by ALT measurement.

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Mouse Forced Swim Test (FST) protocol:
- Male ICR mice (20–25 g) were randomly divided into 3 groups (n=8/group): vehicle (0.5% methylcellulose, oral), Mirtazapine 10 mg/kg (oral), Mirtazapine 20 mg/kg (oral). Drugs were administered 1 hour before testing. Mice were placed in a cylindrical tank (25 cm diameter, 30 cm height) filled with water (25°C, 15 cm depth) for 6 minutes. Immobility time (time spent floating without active movement) was recorded during the last 4 minutes [1]
- Rat autonomic function test:
- Male Wistar rats (250–300 g) were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal). A catheter was inserted into the femoral artery to measure mean arterial blood pressure and heart rate, and a femoral vein catheter for drug administration. Mirtazapine (Org3770) was dissolved in saline and injected intravenously at 3 mg/kg and 6 mg/kg (n=6/group). Hemodynamic parameters were recorded for 60 minutes post-injection [2]
- Mouse ConA-induced hepatitis model:
- Male C57BL/6 mice (6–8 weeks old, 18–22 g) were grouped (n=6/group): vehicle (saline + 0.1% DMSO, oral), Mirtazapine 10 mg/kg (oral), Mirtazapine 20 mg/kg (oral). Drugs were administered once daily for 5 days. On day 3, mice were injected intravenously with ConA (20 mg/kg) to induce liver injury. On day 5, mice were sacrificed, serum was collected for ALT/AST measurement, and liver tissues were fixed in 4% paraformaldehyde for histopathological analysis [3]

Absorption, Distribution and Excretion
This drug is rapidly and completely absorbed. Due to first-pass metabolism in the liver and intestinal wall metabolism, its absolute bioavailability is approximately 50%. Peak plasma concentrations are reached approximately 2 hours after oral administration. Food has minimal effect on the absorption of mirtazapine; no dose adjustment is required when taken with food. Steady-state plasma concentrations are reached approximately 5 days after the first dose. The pharmacokinetics of mirtazapine vary with sex and age. Studies have shown that plasma concentrations are higher in women and older adults than in men and younger adults. This drug is primarily excreted by the kidneys. 75% is excreted in the urine and 15% in the feces. In one pharmacokinetic study, the volume of distribution after an oral steady-state dose was 107 ± 42 L. In a clinical pharmacokinetic study, the total clearance in male patients after intravenous administration was 31 L/h. Clearance in Elderly Patients: The clearance of mirtazapine is lower in older adults than in younger adults. Caution should be exercised when administering this drug to elderly patients. In a clinical trial, mirtazapine clearance was significantly lower in older men compared to younger men taking the same dose. The clearance difference between older women and younger women taking mirtazapine was smaller. Clearance in patients with hepatic or renal impairment is reduced and may require dose adjustment. Moderate renal and hepatic impairment can reduce mirtazapine clearance by approximately 30%. Severe renal impairment can reduce mirtazapine clearance by 50%.

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Metabolism/Metabolites
Mintazapine is extensively metabolized in the human body. Demethylation, hydroxylation, and subsequent glucuronide conjugation are the main metabolic pathways of mirtazapine. In vitro human liver microsomal studies show that cytochrome 2D6 and 1A2 generate the 8-hydroxy metabolite of mirtazapine. The CYP3A enzyme metabolizes the drug into N-demethyl and N-oxide metabolites. The drug also has several other unconjugated metabolites that are pharmacologically active but have limited concentrations in the blood.
Known metabolites of mirtazapine include mirtazapine N-oxide, N-demethylmirtazapine, and 8-hydroxymirtazapine.
Mitazapine is primarily metabolized via demethylation and hydroxylation, followed by glucuronide conjugation. Cytochrome P450 2D6 and cytochrome P450 1A2 are involved in the formation of the 8-hydroxymirtazapine metabolite, while cytochrome P450 3A4 is responsible for the formation of the N-demethyl and N-oxide metabolites. Multiple metabolites possess pharmacological activity, but plasma concentrations are extremely low.
Elimination pathway: This drug is known to be primarily excreted via the kidneys (75%).
Half-life: 20-40 hours

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Biological half-life
20-40 hours

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Oral absorption: In healthy volunteers, after oral administration of mirtazapine (15 mg), the peak plasma concentration (Cmax) was 30-50 ng/mL, and the time to peak concentration was 2-4 hours (Tmax); the oral bioavailability was approximately 50% (due to first-pass metabolism)[1]
-Distribution: The volume of distribution (Vd) in the human body is 3-7 L/kg, indicating its extensive tissue penetration; the drug concentration in the brain is approximately 10 times that in plasma[1]
-Metabolism: mirtazapine is mainly metabolized in the liver via CYP1A2, CYP2D6 and CYP3A4; the main metabolites are N-demethylmirtazapine (active, approximately 50% of the original drug) and 8-hydroxymirtazapine (inactive)[1]
- Elimination: The half-life (t1/2) in the human body is 20-40 hours; approximately 75% of the dose is excreted in the urine (as metabolites) and approximately 15% in the feces.[1]

Toxicity Summary
Mirtazapine, as an antagonist of presynaptic α2 receptors in the central nervous system, inhibits negative feedback in presynaptic neurons, leading to increased norepinephrine (NE) release. Blocking heterologous α2 receptors present in serotonergic neurons enhances serotonin (5-HT) release and increases the interaction between 5-HT and 5-HT_sub>1 receptors, thereby enhancing mirtazapine's anxiolytic effect. Mirtazapine also acts as a weak antagonist of 5-HT_sub>1 receptors, and a potent antagonist of 5-HT_sub>2 (especially subtypes 2A and 2C) and 5-HT_sub>3 receptors. Blockage of these receptors may explain the low incidence of adverse reactions such as anxiety, insomnia, and nausea. Mirtazapine also exhibits significant antagonistic effects on H1 receptors, thus producing a sedative effect. Mirtazapine has no effect on the reuptake of norepinephrine (NE) or serotonin (5-HT), and its activity on dopaminergic and muscarinic receptors is extremely low.

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Toxicity Data
LD50: 600-720 mg/kg (oral, mouse) (L1855)
LD50: 320-490 mg/kg (oral, rat) (L1855)

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Plasma protein binding: mirtazapine has a plasma protein binding rate of 85-90% in humans (as determined by balanced dialysis); renal or hepatic impairment does not affect its binding[1]
-Adverse reactions: Common side effects in clinical studies include drowsiness (due to H1 receptor blockade), increased appetite and weight gain; no significant hepatotoxicity or nephrotoxicity was observed at therapeutic doses (15–45 mg/day)[1]
-High-dose toxicity in mice: No death was observed from a single oral dose of up to 200 mg/kg of mirtazapine; mice experienced transient sedation, but recovered within 24 hours. Histopathological examination of the liver and kidneys revealed no abnormal lesions [3]
- Drug interactions: Mirtazapine may increase plasma concentrations of warfarin (CYP2C9 substrate) and diazepam (CYP3A4 substrate) by inhibiting hepatic CYP enzymes; it is contraindicated for use with monoamine oxidase inhibitors (risk of serotonin syndrome) [1]

[1]. A review of the pharmacological and clinical profile of mirtazapine. CNS Drug Rev. Fall 2001;7(3):249-64.

[2]. Neurochemical and autonomic pharmacological profiles of the 6-aza-analogue of mianserin, Org 3770 and its enantiomers. Neuropharmacology. 1988 Apr;27(4):399-408.

[3]. The Antidepressant Mirtazapine Inhibits Hepatic Innate Immune Networks to Attenuate Immune-Mediated Liver Injury in Mice. Front Immunol. 2019 Apr 12;10:803.

Pharmacodynamics
General Effects and Suicide Risk Warnings: Mirtazapine is effective in treating moderate to severe depression and can alleviate many associated symptoms. These symptoms may include sleep disturbances, loss of appetite, anhedonia, and anxiety. It is important to note that, as with other antidepressants, suicidal ideation and behavior may occur or worsen while taking mirtazapine. This risk is particularly pronounced in young adults. Patients, healthcare professionals, and families should closely monitor patients for suicidal thoughts, worsening depression, anxiety, agitation, sleep changes, irritability, aggression, impulsivity, restlessness, and other unusual behaviors when administering this medication or adjusting the dosage. Do not give mirtazapine to children. The increased risk of suicidal ideation and behavior, especially in young adults, should be carefully considered when deciding whether to prescribe this medication. Effects on Appetite and Weight Gain: In addition to the effects described above, mirtazapine also has an appetite-stimulating effect and has been used to increase appetite and reduce nausea in cancer patients. Some studies and case reports have shown that when used in combination with psychotherapy and/or other psychotropic medications, this medication can improve eating habits and increase weight in patients with anorexia nervosa. In a clinical trial, female patients with depression who received mirtazapine for 6 weeks experienced clinically significant average increases in weight, fat mass, and leptin concentration, without affecting glucose homeostasis. Regarding its effects on sleep, mirtazapine is used to treat sleep disorders due to its drowsiness-inducing properties, a common side effect in patients taking the drug. Studies have shown that the sedative effect of mirtazapine can improve sleep latency, sleep duration, and sleep quality. Insomnia is common in patients with depression, and mirtazapine has been shown to be effective in treating insomnia.

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Mirtatazapine is a tetracyclic antidepressant and a derivative of mianserin (6-aza analogue, Org3770). Its unique mechanism of action involves dual regulation of the norepinephrine and serotonin systems: blocking α2-adrenergic autoreceptors/heterogeneous receptors to enhance neurotransmitter release and antagonizing 5-HT2A/3 receptors to reduce serotonin-related side effects (e.g., insomnia, sexual dysfunction) [1][2] - Clinical indications include major depressive disorder (MDD), generalized anxiety disorder (GAD), and insomnia associated with depression. Mirtazapine is often used to treat patients with loss of appetite or weight loss due to its appetite-stimulating effect [1]. Literature [3] found that mirtazapine has a novel pharmacological effect: protecting the liver from immune-mediated damage by inhibiting the NF-κB inflammatory pathway. This suggests that mirtazapine may be used to treat liver diseases associated with excessive inflammation [3]. Unlike selective serotonin reuptake inhibitors (SSRIs), mirtazapine has a faster onset of action (usually 1-2 weeks, compared to 2-4 weeks for SSRIs) and a lower risk of serotonin syndrome when used alone [1].

C17H19N3

265.35

265.157

C, 76.95; H, 7.22; N, 15.84

85650-52-8

(S)-Mirtazapine; 61337-87-9; (S)-Mirtazapine-d3; (R)-Mirtazapine; 61364-37-2; Mirtazapine-d3; 1216678-68-0; Mirtazapine-d4; 1215898-55-7; (R)-Mirtazapine-d3; 85650-52-8; 61337-67-5 (deleted); 1448014-35-4 (HCl); 207516-99-2 (HCl); 207516-99-2 (2HCl); 868363-97-7 (HBr); 868528-74-9 (HBr); 341512-89-8 (hemihydrate)

4205

White to off-white solid powder

1.2±0.1 g/cm3

432.4±45.0 °C at 760 mmHg

114-116 °C ; 114 - 116 °C

215.3±28.7 °C

0.0±1.0 mmHg at 25°C

1.668

2.75

0

3

0

20

345

0

N1C2N3C(C4C(CC=2C=CC=1)=CC=CC=4)CN(C)CC3

RONZAEMNMFQXRA-UHFFFAOYSA-N

InChI=1S/C17H19N3/c1-19-9-10-20-16(12-19)15-7-3-2-5-13(15)11-14-6-4-8-18-17(14)20/h2-8,16H,9-12H2,1H3

5-methyl-2,5,19-triazatetracyclo[13.4.0.02,7.08,13]nonadeca-1(15),8,10,12,16,18-hexaene

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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 (9.42 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 (9.42 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 (9.42 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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 (Please use freshly prepared in vivo formulations for optimal results.)