CYP2C19 helps amitriptyline convert to nortriptyline, which is its active metabolite. More so than serotonin, nortriptyline inhibits nortriptyline refeeding [1]. In a concentration- and time-stable manner, nortriptyline (6.25-100 μM; 24-72 hours) dramatically lowers both bladder MBT-2 bladder viability and TCCSUP [3]. In TCCSUP and MBT-2 cells, nortriptyline (12.55-100 μM; 24 hours) stimulates cell cycle signaling and cell bladder [3]. These inner and outer vaginal cells are induced by TCCSUP and MBT-2 cells at concentrations of 25 μM, 50 μM, or 100 μM (TCCSUP; 12.55-100 μM; 24 hours). In these bladder cancer cells, exposure to 12.5 μM, 25 μM, or 50 μM (MBT-2 cells) for a whole day caused cell cycle arrest. TCCSUP and MBT-2 cells undergo apoptosis when exposed to 25 μM, 50 μM, or 100 μM (TCCSUP); 12.5 μM, 25 μM, or 50 μM (MBT-2 cells). This apoptotic response lasts for 24 hours and increases the levels of Fas, FasL, FADD, Bax, Bak, and caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. decreases the expression of survivin, X-linked apoptosis protein inhibitor, BH3 interaction domain death agonist, Bcl-2, and Bcl-xL.

In MBT-2 cells, nortriptyline (10–20 mg/kg) administered intraperitoneally once a day for three weeks reduces the formation of bladder tumors [3].

Cell Viability Assay[3]
Cell Types: Human TCCSUP and Mouse MBT-2 Bladder Cancer Cells
Tested Concentrations: 6.25 μM, 12.5 μM, 25 μM, 50 μM and 100 μM
Incubation Duration: 24, 48 or 72 hrs (hours)
Experimental Results: Cells exhibit Toxic effects on TCCSUP and MBT-2 cells.

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Cell cycle analysis[3]
Cell Types: human TCCSUP and mouse MBT-2 Bladder cancer cell
Tested Concentrations: 25 μM, 50 μM or 100 μM (TCCSUP); 12.5 μM, 25 μM or 50 μM (MBT-2 cells)
Incubation Duration: 24 hour
Experimental Results: Caused cell cycle arrest in these bladder cancer cells.

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Apoptosis analysis[3]
Cell Types: human TCCSUP and mouse MBT-2 Bladder cancer cells
Tested Concentrations: 25 μM, 50 μM or 100 μM (TCCSUP); 12.5 μM, 25 μM or 50 μM (MBT-2 cells)
Incubation Duration: 24 hrs (hours)
Experimental Results: Induction of apoptosis in TCCSUP and MBT-2 cells.

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Western Blot Analysis[3]
Cell Types: human TCCSUP and mouse MBT-2 Bladder cancer cells
Tested Concentrations: 25 μM, 50 μM or 100 μM (TCCSUP); 12.5 μM, 25 μM or 50 μM (MBT-2 cells)
Incubation Duration: 24 hrs (hours)
Experimental Results: Increased expression of Fas, Fa

Animal/Disease Models: Adult male C3H/HeN mice (25-30 g; 2-3 months old) were injected with MBT-2 cells [3]
Doses: 10 or 20 mg/kg
Route of Administration: intraperitoneal (ip) injection; daily; three Week.
Experimental Results: Tumor growth was inhibited in mice vaccinated with MBT-2 cells.

Absorption, Distribution and Excretion
Nortriptyline is readily absorbed in the gastrointestinal tract, but plasma concentrations vary from patient to patient. It undergoes first-pass metabolism, reaching plasma concentrations within 7 to 8.5 hours after oral administration. The bioavailability of nortriptyline varies considerably, ranging from 45% to 85%. Nortriptyline and its metabolites are primarily excreted in the urine, with only a small amount (2%) excreted unchanged. Approximately one-third of a single oral dose is excreted in the urine within 24 hours. A small amount is excreted in the bile. The estimated apparent volume of distribution (Vd) β after intravenous administration is 1633 ± 268 L, ranging from 1460 to 2030 L (21 ± 4 L/kg). Nortriptyline crosses the placenta and is present in breast milk. It is distributed in the heart, lungs, brain, and liver. A study in healthy volunteers showed a mean plasma clearance of 54 L/h for nortriptyline. The mean systemic clearance of nortriptyline was 30.6 ± 6.9 L/h, ranging from 18.6 to 39.6 L/h. /Breast Milk/ Nortriptyline is distributed into breast milk. The concentration of nortriptyline in breast milk appears to be similar to or slightly higher than that in maternal serum.
Peak plasma concentrations occur within 7–8.5 hours after oral administration. Optimal efficacy appears to be associated with plasma concentrations of 50–150 ng/mL.
Clinical pharmacokinetics of amitriptyline were studied in four volunteers after oral administration of 75 mg. Peak plasma concentrations of amitriptyline ranged from 10.8 to 43.7 ng/mL. Its elimination was biphasic and conformed to first-order kinetics. The mean elimination half-life was 36.1 hours. The mean first-pass metabolism of amitriptyline was estimated to be 60%. Despite peak concentrations of the metabolite nortriptyline ranging from only 5.9 to 12.3 ng/mL, a significant amount of metabolite was generated.
Although tricyclic antidepressants (TCAs) are widely accepted for the treatment of pregnancy-related depression, their pharmacokinetic characteristics during pregnancy remain unclear. This study aimed to investigate the transplacental transport of amitriptyline (AMI) and its major active metabolite nortriptyline (NOR) in ex vivo perfused human placentas. Nine full-term human placentas were immediately obtained with the mothers' consent, and individual placental villi were perfused non-circulatingly for 2 hours. AMI (200 ng/ml) and NOR (150 ng/ml) were added to the maternal reservoir, and their presence in fetal circulation was monitored for 2 hours using antipyrine as a reference compound. The concentrations of AMI and NOR were determined by high-performance liquid chromatography (HPLC), and the concentration of antipyrine was determined by spectrophotometry. The mean (standard deviation) transplacental transport rates (TPT(SS)%) for AMI and NOR were 8.2 (2.3)% and 6.5 (1.8)%, respectively, calculated as the ratio of the steady-state concentration on the fetal venous side to the maternal arterial side. The TPTs for AMI and NOR were 81% and 62% of the freely diffused antipyrine, respectively. The absolute dose fraction (TPT(A)) across the placenta was slightly higher in the AMI group (7.7%) than in the NOR group (5.7%) (P=0.037). In all perfusion experiments, the AMI group reached fetal steady-state within 30 minutes, while the NOR group reached steady-state within 50 minutes. During the 2-hour perfusion, placental activity was maintained, the pH of the perfusion solution remained constant, the fetal arterial perfusion pressure was stable, and antipyrine transport was stable. Both AMI and NOR can cross the human placenta. However, the fetal exposure of NOR may be slightly lower than that of AMI, possibly due to the higher lipophilicity of AMI.

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Metabolism/Metabolites
Noretriptyline is metabolized in the liver via demethylation and hydroxylation, followed by conjugation with glucuronic acid. CYP2D6 plays a crucial role in the metabolism of noretriptyline, with CYP1A2, CYP2C19, and CYP3A4 also involved. The major active metabolite is 10-hydroxynortriptyline, existing in both cis and trans forms, with the trans form being more potent. 10-hydroxynortriptyline is the most common metabolite in plasma. Most other metabolites are conjugated metabolites with lower potency.
This study investigated the biotransformation of amitriptyline to its demethylated product, noretriptyline, in vitro using human liver microsomes from four different donors. These donors were pre-screened to reflect different metabolic rates. The relationship between reaction rate and substrate concentration conformed to an S-type Vmax model. Vmax ranged from 0.42 to 3.42 nmol/mg/min, and Km ranged from 33 to 89 μM amitriptyline. Ketoconazole is a potent inhibitor of N-demethylation, with an average Ki value of 0.11 ± 0.013 μM… while the CYP2D6 inhibitor quinidine (at concentrations up to 50 μM) and the CYP1A2 inhibitor α-naphthylflavonoid (at concentrations up to 5 μM, effective only at low concentrations) showed no effect. All tested selective serotonin reuptake inhibitors inhibited the formation of nortriptyline, with mean Ki values of 4.37 (± 3.38) uM for sertraline, 5.46 (± 1.95) uM for norsertraline, 9.22 (± 3.69) uM for fluvoxamine, 12.26 (± 5.67) uM for norfluoxetine, 15.76 (± 5.50) uM for paroxetine, and 43.55 (± 18.28) uM for fluoxetine. A polyclonal rabbit antibody targeting rat liver CYP3A1 inhibited N-demethylation of amitriptyline in antibody/microsomal protein ratios ranging from 1:1 to 10:1, with an asymptotic maximum of 60%. It has been reported that the metabolic distribution of antidepressants and antipsychotics is significantly influenced by cytochrome P450 (CYP) 2D6 isoenzymes. The two most studied antidepressants are amitriptyline and imipramine. Studies have shown that the conversion of amitriptyline to nortriptyline and the metabolism of nortriptyline to its 10-hydroxy metabolite are both influenced by the 2D6 isoenzyme. This study also investigated the stability of amitriptyline, nortriptyline, desipramine, and imipramine in formalin-fixed human liver tissue and formalin solution. The contents of tricyclic drugs and their major demethylated metabolites in frozen liver were determined and compared with those in formalin-fixed and preserved liver. The results showed that in the formalin environment, some secondary amine nortriptyline was methylated to the corresponding tertiary amine amitriptyline, and desipramine was methylated to imipramine. Nortriptyline was not detected in most cases, suggesting that its degradation rate may be faster than that of desipramine. There was no consistent proportional relationship between drug concentrations in frozen liver tissue, formalin-preserved liver tissue, or formalin solution. The methylation rate of secondary amines could not be quantified. Preserving liver tissue in formalin solution at room temperature can cause drug leaching into the formalin solution. The test drug can be detected in formalin-fixed liver and formalin culture medium for up to 22 months. Known metabolites of nortriptyline include E-10-hydroxynortriptyline and normethylnortriptyline. Nortriptyline is a known metabolite of amitriptyline. Like other tricyclic antidepressants (TCAs), nortriptyline is metabolized via the same hepatic metabolic pathway. Elimination pathway: Approximately one-third of a single oral dose is excreted in the urine within 24 hours. A small amount of the drug is excreted in the bile. Half-life: 16 to over 90 hours. The mean half-life of nortriptyline in the plasma of healthy volunteers is approximately 26 hours, but is reportedly in the range of 16 to 38 hours. One study mentioned a mean half-life of approximately 39 hours. The plasma half-life of nortriptyline ranges from 16 to over 90 hours. Clinical pharmacokinetics were studied in four volunteers after oral administration of 75 mg amitriptyline. ...The mean elimination half-life was 36.1 hours. ...

Toxicity Summary
Identification and Uses: Nortriptyline is a tricyclic antidepressant. Human Exposure and Toxicity: Overdose/poisoning symptoms include: arrhythmias, bundle branch block, cardiac arrest, hypotension, circulatory failure, dilated pupils, blurred vision, tachycardia, vasodilation, urinary retention, decreased gastrointestinal motility, decreased bronchial secretions, dry mucous membranes and skin, hypothermia, respiratory depression, seizures, abnormal tendon reflexes, disorientation, agitation, myoclonus, coma, and pyramidal tract signs. A woman with chronic obstructive pulmonary disease experienced decreased carbon dioxide sensitivity and impaired ventilatory compensation after taking nortriptyline, while exercise tolerance increased and dyspnea lessened. In other words, nortriptyline exhibits an inhibitory effect on respiratory control. Nortriptyline increases the risk of sudden death in the general population, especially in the presence of genetic and/or non-genetic factors (e.g., blocking cardiac sodium channels and reducing cardiac excitability). Short-term studies have shown that antidepressants increase the risk of suicidal ideation and behavior (suicidal tendencies) in children, adolescents, and young adults, with these studies focusing on major depressive disorder (MDD) and other mental illnesses. Animal studies: Dogs given oral nortriptyline hydrochloride for twelve months tolerated doses up to 20 mg/kg/day. However, high oral doses (40 mg/kg/day) led to symptoms of depression and ataxia, and death occurred at the end of the first month if treatment continued. In chronic toxicity studies, rats tolerated dietary concentrations equivalent to 150 mg/kg/day of nortriptyline hydrochloride for one year. Rats exhibited some growth retardation but no visceral damage. The genotoxicity of nortriptyline was assessed using the Drosophila wing cell somatic mutation and recombination assay (SMART). The drug did not show genotoxicity at concentrations up to 100 mM. Nortriptyline is believed to inhibit the reuptake of the neurotransmitter serotonin on neuronal membranes or act on β-adrenergic receptors. Tricyclic antidepressants do not inhibit monoamine oxidase or affect dopamine reuptake. Toxicity Data
LD50 (oral administration in rats) Interactions This case illustrates the pharmacokinetic interaction between the tricyclic antidepressant nortriptyline and the antituberculosis drug rifampin. During rifampin administration, the patient required a higher-than-expected dose of nortriptyline to achieve therapeutic concentrations. Upon discontinuation of rifampin, the patient experienced drowsiness, and serum nortriptyline concentrations rapidly increased to toxic levels. A previous report described a type 2 diabetic patient taking chlorpropamide (250 mg/day) who developed significant hypoglycemia after adding nortriptyline (125 mg/day). The pharmacokinetic interactions between the antipsychotics zoclothiasol and perphenazine and tricyclic antidepressants were investigated using data from a therapeutic drug monitoring database. Of the 290 patients monitored for amitriptyline and the 611 patients monitored for nortriptyline, 77 were concurrently taking perphenazine and 50 were concurrently taking zoclothiasol. Concomitant use with perphenazine increased the median steady-state serum concentration-to-dose ratio (C/D) of nortriptyline by 30-45%, while the median C/D of amitriptyline was unaffected. Conversely, the median C/D of both nortriptyline and amitriptyline was unaffected by concomitant use with zoclothiasol. This article describes two cases of adverse reactions due to the interaction between valproic acid (valproate) and nortriptyline, one a 33-year-old woman and the other a 36-year-old man. The first patient was taking 25 mg of nortriptyline daily; within one week of adding valproic acid (at doses up to 1000 mg/day), she developed significant hand and finger tremors. The plasma concentration of nortriptyline was 393 ng/mL, and the concentration of valproic acid was 105 mg/L. Both drugs were discontinued, and the tremor subsided over the next two days. The second patient was taking nortriptyline 75 mg/day and valproic acid, with a maximum dose of 1250 mg/day. The plasma concentration of nortriptyline was 345 ng/mL. The dose of nortriptyline was gradually reduced to 25 mg/day, followed by a concentration of 82 ng/mL. Thioridazine and loxapine were also being administered concurrently.
For more complete data on drug interactions of nortriptyline (31 in total), please visit the HSDB record page.

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Non-human toxicity values
Mouse intravenous LD50 17 mg/kg
Mouse intraperitoneal LD50 70 mg/kg
Mouse oral LD50 387 mg/kg
Rat intravenous LD50 22 mg/kg

[1]. eds. Medical Genetics Summaries. Bethesda (MD): National Center for Biotechnology Information (US); March 23, 2017.

[2]. Repurposing Autophagy Regulators in Brain Tumors [published online ahead of print, 2022 Feb 18]. Int J Cancer. 2022;10.1002/ijc.33965.

[3]. Nortriptyline induces mitochondria and death receptor-mediated apoptosis in bladder cancer cells and inhibits bladder tumor growth in vivo. Eur J Pharmacol. 2015 Aug 15:761:309-20.

Therapeutic Uses

Adrenergic reuptake inhibitors; tricyclic antidepressants
/Clinical Trials/ ClinicalTrials.gov is a registry and results database that lists human clinical studies funded by public and private institutions worldwide. The website is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each record on ClinicalTrials.gov includes a summary of the study protocol, including: the disease or condition; the intervention (e.g., the medical product, behavior, or procedure under investigation); the title, description, and design of the study; participation requirements (eligibility criteria); the location of the study; contact information for the study location; and links to relevant information from other health websites, such as the NLM's MedlinePlus (for providing patient health information) and PubMed (for providing citations and abstracts of academic articles in the medical field). The database includes nortriptyline.
Noretriptyline hydrochloride is indicated for the relief of depressive symptoms. Endogenous depression is more easily relieved than other depressive states. /Included in US product label/
Tricyclic antidepressants have been used to treat attention deficit hyperactivity disorder (ADHD). /Tricyclic antidepressants; not included in US product label/
For more complete data on the therapeutic uses of nortriptyline (one of 13), please visit the HSDB record page.

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Drug Warning
/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 nortriptyline hydrochloride or any other antidepressant in children, adolescents, or young adults must weigh this risk against clinical need. Short-term studies have shown that antidepressants did not increase the risk of suicide in adults 24 years and older compared to placebo; however, they did reduce the risk of suicide 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 starting antidepressant treatment should be appropriately monitored for worsening of their condition, suicidal tendencies, or unusual behavioral changes. Family members and caregivers should be informed of the necessity for close monitoring and communication with the prescribing physician. Nortriptyline hydrochloride is not approved for use in children. Many antidepressants (including nortriptyline hydrochloride) cause mydriasis, which may induce angle-closure glaucoma attacks in patients with narrow anatomical structures who have not undergone iridectomy. …These medications should be used with caution in patients with urinary retention, glaucoma, diabetes, impaired liver function, asthma, or a history of epilepsy. /Tricyclic Antidepressants/ …Many patients taking tricyclic antidepressants and/or antipsychotics have a higher risk of suicide. Antipsychotics have a relatively high safety margin, while tricyclic antidepressants have a much lower safety margin. Generally, the dosage of tricyclic antidepressants should be limited to a one-week supply. /Tricyclic Antidepressants/
For more complete data on novilin (32 total), please visit the HSDB records page.

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Pharmacodynamics
Notiline may exert its antidepressant effect by inhibiting the reuptake of serotonin and norepinephrine on neuronal cell membranes. It also exerts its antimuscarinic effect by acting on acetylcholine receptors.

C19H21N

263.37674

263.167

72-69-5

Nortriptyline hydrochloride;894-71-3

4543

White to off-white solid powder

1.084 g/cm3

403.4ºC at 760 mmHg

194.9ºC

1.633

4.217

1

1

3

20

307

0

PHVGLTMQBUFIQQ-UHFFFAOYSA-N

InChI=1S/C19H21N/c1-20-14-6-11-19-17-9-4-2-7-15(17)12-13-16-8-3-5-10-18(16)19/h2-5,7-11,20H,6,12-14H2,1H3

N-methyl-3-(2-tricyclo[9.4.0.03,8]pentadeca-1(15),3,5,7,11,13-hexaenylidene)propan-1-amine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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.)