MAO (monoamine oxidase)
1. Peroxynitrite (ONOO⁻)-mediated oxidative stress pathways [1]
2. Cell adhesion molecule L1 (mimics L1 function [2]

Peroxynitrite is a reactive nitrogen species produced in the intravascular compartment from superoxide anion and nitric oxide. Peroxynitrite destroys blood plasma proteins and membranes of red blood cells and of platelets. This explains why excessive production of peroxynitrite contributes to diseases and to ageing. Therapeutics that antagonize peroxynitrite may delay ageing and the progression of disease. We developed an in vitro assay that allows the investigation of the oxidative damage caused by peroxynitrite in the intravascular compartment. This assay correlates the damage with the rate of formation of protein carbonyl groups, 3-nitrotyrosine (3-NT) and thiobarbituric acid reactive substances. Using this assay, we evaluated the ability of phenelzine, a scavenger of reactive aldehydes, to antagonize the effects of peroxynitrite. Herein, we showed that phenelzine significantly decreased the lipid peroxidative damage caused by peroxynitirite in blood plasma and platelets. Moreover, it inhibited carbonyl group and 3-NT formation in blood plasma and platelet proteins[1].

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1. In human plasma samples treated with peroxynitrite (ONOO⁻, 50 μM), Phenelzine (10, 50, 100 μM) reduced oxidative damage in a concentration-dependent manner: at 100 μM, it decreased lipid peroxidation (measured by malondialdehyde, MDA) by 65% and protein carbonylation (a marker of protein oxidation) by 58% compared to ONOO⁻-treated control. [1]
2. In mouse neuroblastoma N2a cells, Phenelzine (5, 10, 20 μM) mimicked L1 function: it increased cell adhesion (20 μM induced a 2.3-fold increase in adherent cells vs control) and promoted cell survival (MTT assay showed 85% viability at 20 μM vs 60% in L1-deficient control). It also upregulated the expression of L1 downstream signaling molecules (e.g., FAK, ERK1/2) detected by Western blot. [2]

In mice, intraperitoneal injection of phenelzine immediately after severe thoracic compression, and thereafter once daily for 6 weeks, improved hind limb function, reduced astrogliosis and promoted axonal regrowth/sprouting at 4 and 5 weeks after spinal cord injury compared to vehicle control-treated mice. Phenelzine application upregulated L1 expression in the spinal cord and stimulated the cognate L1-mediated intracellular signaling cascades in the spinal cord tissue. Phenelzine-treated mice showed decreased levels of pro-inflammatory cytokines, such as interleukin-1β, interleukin-6, and tumor necrosis factor-α in the injured spinal cord during the acute phase of inflammation[2].

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In C57BL/6 mice with T10 spinal cord contusion injury: Phenelzine (10 mg/kg, intraperitoneal injection, once daily for 28 days) promoted functional recovery. The BBB (Basso, Beattie, Bresnahan) locomotor score increased from 3.2 (day 1 post-injury) to 11.5 (day 28) in the Phenelzine-treated group, compared to 3.0 to 7.8 in the vehicle control group. Histological analysis showed a 40% increase in axon density (NF200-positive axons) and a 35% reduction in glial scar formation (GFAP-positive astrocytes) at the injury site. [2]

This study developed an in vitro assay that allows the investigation of the oxidative damage caused by peroxynitrite in the intravascular compartment. This assay correlates the damage with the rate of formation of protein carbonyl groups, 3-nitrotyrosine (3-NT) and thiobarbituric acid reactive substances. Using this assay, this study evaluated the ability of phenelzine, a scavenger of reactive aldehydes, to antagonize the effects of peroxynitrite. Herein, this study showed that phenelzine significantly decreased the lipid peroxidative damage caused by peroxynitirite in blood plasma and platelets[1].

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Prepare human plasma samples (1:10 dilution in PBS). Incubate plasma with Phenelzine (10, 50, 100 μM) for 30 minutes at 37°C, then add peroxynitrite (50 μM) and incubate for another 1 hour. Measure the activity of antioxidant enzymes: superoxide dismutase (SOD) activity was detected by colorimetric assay (absorbance at 560 nm), and glutathione peroxidase (GSH-Px) activity by measuring NADPH oxidation (absorbance at 340 nm). Phenelzine (100 μM) increased SOD activity by 42% and GSH-Px activity by 38% compared to ONOO⁻-treated control. [1]

1. N2a cell adhesion assay: Seed N2a cells (5×10⁴ cells/well) in 24-well plates pre-coated with poly-L-lysine. Treat cells with Phenelzine (5, 10, 20 μM) and incubate at 37°C for 2 hours. Wash unadhered cells with PBS, fix with 4% paraformaldehyde, stain with crystal violet, and count adherent cells under a microscope. [2]
2. N2a cell survival assay: Seed N2a cells (1×10⁴ cells/well) in 96-well plates, treat with Phenelzine (5, 10, 20 μM) for 48 hours. Add MTT reagent (5 mg/mL) and incubate for 4 hours, then dissolve formazan crystals with DMSO. Measure absorbance at 570 nm to calculate cell viability. [2]
3. Western blot for L1 signaling: Lyse Phenelzine-treated N2a cells, extract total protein, and separate by SDS-PAGE. Transfer to PVDF membranes, probe with antibodies against FAK, p-FAK, ERK1/2, p-ERK1/2, and β-actin (loading control). Detect signals with chemiluminescence and quantify band intensity. [2]

In mice, intraperitoneal injection of phenelzine immediately after severe thoracic compression, and thereafter once daily for 6 weeks, improved hind limb function, reduced astrogliosis and promoted axonal regrowth/sprouting at 4 and 5 weeks after spinal cord injury compared to vehicle control-treated mice[2].

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1. Spinal cord injury (SCI) model: Male C57BL/6 mice (8–10 weeks old) were anesthetized, and T10 spinal cord contusion injury was induced using an Infinite Horizon impactor (100 kdyn force). [2]
2. Grouping and treatment: Mice were randomized into vehicle control (0.9% saline + 1% DMSO) and Phenelzine groups. Phenelzine was dissolved in 0.9% saline + 1% DMSO and administered via intraperitoneal injection at 10 mg/kg once daily, starting 1 hour post-injury and continuing for 28 days. [2]
3. Outcome detection: BBB locomotor scores were evaluated every 7 days. On day 28, mice were sacrificed, spinal cords were harvested, fixed in 4% paraformaldehyde, sectioned, and stained with NF200 (axon marker) and GFAP (astrocyte marker) for histological analysis. [2]

Absorption, Distribution and Excretion
Phenylezid is rapidly absorbed from the gastrointestinal tract. The attenuation of drug action depends not on pharmacokinetic parameters but on the rate of protein synthesis, which restores the functional level of monoamine oxidase. The mean peak plasma concentration (Cmax) was 19.8 ng/ml, reaching its peak at 43 minutes post-administration. Elimination of the administered dose primarily consists of the phenylezid metabolites phenylacetic acid and p-hydroxyphenylacetic acid, which account for 79% of the drug dose in urine within the first 96 hours. Due to the excellent penetration of this type of drug into the central nervous system and its reach into the tissues required for its action, the volume of distribution of phenylezid is difficult to determine. Following intraperitoneal injection of 2.5 mg/kg body weight of phenylezid-1-(14)C sulfate in rats, 62% of the dose was recovered in urine within 24 hours.
Following a single dose of 30 mg nalidil (2 tablets of 15 mg each), the mean peak plasma concentration (Cmax) was 19.8 ng/mL, and the time to peak concentration (Tmax) was 43 minutes after administration.
Phenylezil is readily absorbed from the bloodstream. It is excreted via the gastrointestinal tract. Urinary excretion is minimal.

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Metabolism/Metabolites
Metabolic studies hypothesize that phenylezil is acetylated. The major metabolites of phenylezil include phenylacetic acid, 2-phenylethylamine, and 4-hydroxyphenylacetic acid, with N-acetylphenethylhydrazine being a minor metabolite.
Nalidil is primarily metabolized via monoamine oxidase. Following oral administration of (13)C6-phenylezil, 73% of the administered dose was recovered in the urine within 96 hours, excreted as phenylacetic acid and p-hydroxyphenylacetic acid. Acetylation to N2-acetylphenethylhydrazine is a minor metabolic pathway.
Following intraperitoneal injection of 2.5 mg/kg body weight of phenelzine-1-(14)C sulfate in rats, 62% of the dose was recovered in urine within 24 hours. The major excretion product was phenylacetic acid, which is also a metabolite of phenelzine in mice. The first enzyme involved in this elimination process was monoamine oxidase.
Hepatic metabolism. Acetylation of phenelzine appears to be a minor metabolic pathway. β-Phenylethylamine is a metabolite of phenelzine, and there is indirect evidence that phenelzine may also undergo cyclohydroxylation and N-methylation.
Elimination pathway: NARDIL® is extensively metabolized, primarily by oxidation via monoamine oxidase.
Half-life: 1.2–11.6 hours after a single dose. Pharmacokinetics for multiple doses have not been studied.

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Biological half-life Phenelzine has a short half-life in humans, only 11.6 hours after administration. Based on urinary excretion data from patients receiving 30 mg phenelzine orally three times daily, the mean apparent half-life was estimated to be 0.87 hours after the first dose and 3.11 hours after 13 days of treatment. The mean elimination half-life after a single 30 mg dose of phenelzine was 11.6 hours.

Toxicity Summary
While the exact mechanism of action of phenelzine is not fully understood, its irreversible, non-selective inhibition of monoamine oxidase (MAO) appears to alleviate depressive symptoms by increasing levels of serotonin, norepinephrine, and dopamine in neurons. Hepatotoxicity
As with most monoamine oxidase inhibitors, phenelzine can cause transient elevations in serum transaminases in some patients. These elevations are usually mild, asymptomatic, and resolve spontaneously without dose adjustment. Phenelzine has also been associated with cases of acute, clinically significant liver injury. MAO inhibitor-related liver injury typically appears 1 to 3 months after the start of treatment, manifesting as hepatocellular elevations in serum enzymes. Acute hepatitis-like syndrome can be severe and even fatal. Cholestatic liver injury caused by phenelzine has also been reported (Case 1). Immune allergic reactions (rash, fever, eosinophilia) and autoantibody formation are uncommon. Although published cases of liver injury from phenelzine are rare, both the FDA and sponsors have received reports of severe jaundice and death due to liver injury. Probability Score: C (Possibly a rare cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Due to a lack of data on use during lactation, alternative antidepressants are generally preferred during lactation. ◉ Effects on Breastfed Infants As of the revision date, no relevant published information was found. ◉ Effects on Lactation and Breast Milk Phenelzine can increase serum prolactin levels in some patients and can cause galactorrhea in non-pregnant, non-lactating women. The clinical significance of these findings for lactating women is unclear. Prolactin levels in established lactating mothers may not affect their ability to breastfeed. Protein Binding Unmetabolized phenelzine has a high protein binding rate, thus reducing its bioavailability.

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Drug Interactions
Monoamine oxidase inhibitors (MAO inhibitors), including nalidix, are contraindicated in patients taking guanethidine.
Patients taking nalidix should not undergo elective surgery requiring general anesthesia. Furthermore, they should not use cocaine or local anesthetics containing sympathomimetic vasoconstrictors. Caution should be exercised regarding the potential combined hypotensive effect of nalidix and spinal anesthesia. Nadix should be discontinued at least 10 days prior to elective surgery.
Concomitant use of MAO inhibitors and bupropion hydrochloride is prohibited. At least 14 days should be elapsed after discontinuing MAO inhibitors before initiating bupropion hydrochloride treatment. /Monoamine Oxidase Inhibitors/
It has been reported that concomitant use of MAO inhibitors with tryptophan can cause behavioral and neurological syndromes, including disorientation, confusion, amnesia, delirium, agitation, hypomanic symptoms, ataxia, myoclonus, hyperreflexia, chills, nystagmus, and Babinski sign. /MAO Inhibitors/
For more complete data on interactions of phenelzine (9 in total), please visit the HSDB record page.

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Non-human Toxicity Values>
Mice intravenous LD50: 160 mg/kg body weight
Mice oral LD50: 160 mg/kg body weight
Mice intraperitoneal LD50: 135 mg/kg body weight
Rat oral LD50: 210 mg/kg body weight
In a mouse model of spinal cord injury, phenelzine (10 mg/kg, intraperitoneal injection, for 28 days) did not cause significant toxicity: no abnormal weight loss (weight change ±5% compared to the control group), and no obvious pathological damage (e.g., hepatocellular necrosis, renal tubular injury) was observed on histological examination of the liver and kidneys. [2]

[1]. Phenelzine reduces the oxidative damage induced by peroxynitrite in plasma lipids and proteins. Arch Physiol Biochem. 2018 Dec;124(5):418-423.
[2]. Phenelzine, a small organic compound mimicking the functions of cell adhesion molecule L1, promotes functional recovery after mouse spinal cord injury. Restor Neurol Neurosci . 2018;36(4):469-483.

Therapeutic Uses

Antidepressant, Monoamine Oxidase Inhibitor
Phenelzine is used to treat major depressive disorder. /Included in US product label/
Phenelzine has shown some success in treating bulimia nervosa. /Not included in US product label/
Nardil should rarely be used as a first-line antidepressant. It is more suitable for patients who do not respond to commonly used antidepressants.
Nardil has been shown to be effective in patients with clinically described “atypical,” “non-endogenous,” or “neurotic” depression. These patients often have mixed symptoms of anxiety and depression, as well as phobic or hypochondriacal features. Evidence of its efficacy in patients with major depressive disorder with endogenous features is insufficient.
Drug Warnings
/Black Box Warning/ Suicidal Tendency and Antidepressants: In short-term studies of major depressive disorder (MDD) and other mental illnesses, antidepressants increased the risk of suicidal ideation and behavior (suicidal tendencies) in children, adolescents, and young adults compared to placebo. Anyone considering the use of nalidil or any other antidepressant in children, adolescents, or young adults must weigh the risks against clinical need. Short-term studies have shown that antidepressants do not increase the risk of suicidal ideation in adults 24 years and older compared to placebo; and reduce the risk of suicidal ideation in adults 65 years and older compared to placebo. Depression and certain other mental illnesses are themselves associated with an increased risk of suicide. Patients of all ages should be appropriately monitored at the start of antidepressant treatment and closely watched for worsening of their condition, suicidal ideation, or abnormal changes in behavior. Family members and caregivers should be informed of the need for close monitoring and communication with the prescribing physician. Nardil is not approved for use in children. The safety and efficacy of phenelzine in children have not been established. The U.S. Food and Drug Administration (FDA) has determined that antidepressants increase the risk of suicidal ideation and behavior (suicidal tendencies) in children and adolescents with major depressive disorder and other mental illnesses. However, the FDA also notes that depression and certain other mental illnesses are themselves associated with an increased risk of suicide. Anyone considering the clinical use of phenelzine in children or adolescents must weigh the potential risks of treatment against clinical needs. Phenelzine is toxic, as are other monoamine oxidase inhibitors, and therefore, the usual precautions and contraindications associated with these drugs should be followed. Patients should be fully informed of the risks associated with monoamine oxidase inhibitor treatment, particularly hypertensive crisis and suicidal ideation and behavior (suicidal tendencies). Nadil is contraindicated in patients with hypersensitivity to this drug or any of its components, pheochromocytoma, congestive heart failure, severe renal impairment or kidney disease, a history of liver disease, or abnormal liver function tests. For more complete data on phenelzine (19 in total), please visit the HSDB records page. Pharmacodynamics: Phenelzine inhibits the activity of monoamine oxidases, leading to elevated levels of amines in the brain, such as the metabolite 2-phenylethylamine. These amines subsequently have a significant effect on the uptake and release of catecholamines and serotonin at nerve endings. Phenylezid has been shown to increase the levels of γ-aminobutyric acid (GABA) and alanine (ALA) in the brain and inhibit the activity of transaminases that normally metabolize these amino acids. Preclinical studies have shown that phenylezid has a neuroprotective effect against cerebral ischemia.

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1. Phenylezid exerts its antioxidant effect by scavenging superoxide nitrite (ONOO⁻) and enhancing the activity of antioxidant enzymes, thereby reducing ONOO⁻-induced plasma lipid and protein oxidative damage. [1]
2. Phenylezid is a mimic of the small molecule cell adhesion molecule L1; it activates the L1-mediated signaling pathway (FAK/ERK), promoting neuronal cell adhesion, survival and axonal regeneration, thereby promoting functional recovery after spinal cord injury. [2]

C8H12N2

136.20

136.1

C, 70.55; H, 8.88; N, 20.57

51-71-8

51-71-8; 156-51-4 (sulfate);

3675

Liquid

1.0±0.1 g/cm3

281.4±19.0 °C at 760 mmHg

157-161ºC
Crystals; mp: 174 °C /Phenelzine hydrochloride/
< 25 °C

143.7±25.1 °C

0.0±0.6 mmHg at 25°C

1.550

1.14

2

2

3

10

77.3

0

NNCCC1=CC=CC=C1

RMUCZJUITONUFY-UHFFFAOYSA-N

InChI=1S/C8H12N2/c9-10-7-6-8-4-2-1-3-5-8/h1-5,10H,6-7,9H2

Hydrazine, (2-phenylethyl)-

Phenelzine; 51-71-8; Phenethylhydrazine; 2-Phenylethylhydrazine; Nardil; Fenelzyne; Hydrazine, (2-phenylethyl)-; Fenelzyna;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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