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 with extensive variation in plasma levels, depending on the patient. This drug undergoes first-pass metabolism and its plasma concentrations are attained within 7 to 8.5 hours after oral administration. The bioavailability of nortriptyline varies considerably and ranges from 45 to 85%.
Nortriptyline and its metabolites are mainly excreted in the urine, where only small amounts (2%) of the total drug is recovered as unchanged parent compound. Approximately one-third of a single orally administered dose is excreted in urine within 24 hours. Small amounts are excreted in feces via biliary elimination.
The apparent volume of distribution (Vd)β, estimated after intravenous administration is 1633 ± 268 L within the range of 1460 to 2030 (21 ± 4 L/kg). Nortriptyline crosses the placenta and is found in the breast milk. It distributes to the heart, lungs, brain, and the liver.
The average plasma clearance of nortriptyline in a study of healthy volunteers was 54 L/h. The average systemic clearance of nortriptyline is 30.6 ± 6.9 L / h, within the range of 18.6 to 39.6 L/hour.
/MILK/ Nortriptyline is distributed into milk. Nortriptyline concentrations in milk appear to be similar to or slightly greater than those present in maternal serum.
Peak plasma concentrations occur within 7-8.5 hours after oral administration. Optimal response to the drug appears to be associated with plasma concentrations of 50-150 ng/mL.
The clinical pharmacokinetics of amitriptyline were studied in four volunteers after the oral administration of 75 mg. Peak amitriptyline plasma concentrations ranged from 10.8 to 43.7 ng/mL. The disappearance was biphasic and followed first-order kinetics. The mean elimination half-life was 36.1 hours. The mean estimated first-pass metabolism of amitriptyline was 60 per cent. Significant quantities of the metabolite, nortriptyline, were produced although peak concentrations ranged from only 5.9 to 12.3 ng/mL.
Although tricyclic antidepressants (TCAs) have gained wide acceptance for use in the treatment of depression in pregnant women, their pharmacokinetics during pregnancy have been poorly characterized. The aim of the present study was to investigate the transplacental transfer of amitriptyline (AMI) and its main active metabolite nortriptyline (NOR) in isolated perfused human placenta. Nine term human placentae were obtained immediately after delivery with maternal consent and a 2-h non-recirculating perfusion of a single placental cotyledon was performed. AMI (200 ng/ml) and NOR (150 ng/ml), with antipyrine as a reference compound, were added to the maternal reservoir and their appearance to the fetal circulation was followed for 2 h. AMI and NOR concentrations were measured by high performance liquid chromatography (HPLC) and antipyrine concentrations spectrophotometrically. The mean (SD) transplacental transfers (TPT(SS)%) for AMI and NOR were 8.2 (2.3)% and 6.5 (1.8)%, respectively, calculated as the ratio between the steady-state concentrations in fetal venous and maternal arterial sides. The TPTs of AMI and NOR were 81% and 62% of the freely diffusable antipyrine. The absolute fraction of the dose that crossed the placenta (TPT(A)) was moderately, but significantly higher for AMI (7.7%) than for NOR (5.7%) (P=0.037). In all perfusions, steady state at the fetal side was reached by 30 min for AMI and by 50 min for NOR in the fetal side. The viability of the placentae was retained during the 2-h perfusion, as evidenced by unchanged pH of the perfusate and by stable perfusion pressures in fetal artery and stable antipyrine transfer. Both AMI and NOR cross the human placenta. However, the fetal exposure with NOR may be somewhat smaller compared with AMI, probably due to the higher lipophilicity of AMI.

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Metabolism / Metabolites
Nortriptyline is metabolized via demethylation and hydroxylation in the liver followed by glucuronic acid conjugation. CYP2D6 plays a large role in nortriptyline metabolism, with contributions from CYP1A2, CYP2C19 and CYP3A4. The main active metabolite is 10-hydroxynortriptyline exists in both cis and a trans form, with the trans form is higher in potency. 10-hydroxynortriptyline is the most frequently found in the plasma. Most of the other metabolites are conjugated, and are less potent.
Biotransformation of amitriptyline to its demethylated product nortriptyline was studied in vitro with human liver microsomes from four different donors, preselected to reflect a range of metabolic rates. Reaction velocity versus substrate concn was consistent with a sigmoid Vmax model. Vmax varied from 0.42 to 3.42 nmol/mg/min, Km from 33 to 89 uM amitriptyline. Ketoconazole was a highly potent inhibitor of N-demethylation, with a mean Ki value of 0.11 + or - 0.013 uM ... whereas quinidine (up to 50 uM), a CYP2D6 inhibitor, and alpha-naphthoflavone (up to 5 uM), a CYP1A2 inhibitor only at low concn, showed no effect. All selective serotonin reuptake inhibitors tested had an inhibitory effect on the formation of nortriptyline, with mean Ki values of 4.37 (+ or - 3.38) uM for sertraline, 5.46 (+ or - 1.95) uM for desmethylsertraline, 9.22 (+ or - 3.69) uM for fluvoxamine, 12.26 (+ or - 5.67) uM for norfluoxetine, 15.76 (+ or - 5.50) uM for paroxetine, and 43.55 (+ or - 18.28) uM for fluoxetine. A polyclonal rabbit antibody against rat liver CYP3A1, in antibody/microsomal protein ratios varying from 1:1 to 10:1, inhibited N-demethylation of amitriptyline to an asymptotic max of 60%.
The metabolic disposition of the antidepressants and antipsychotics has been reported to be significantly influenced by the cytochrome P450 (CYP) 2D6 isozyme. The two most studied antidepressants are amitriptyline and imipramine. Amitriptyline conversion to nortriptyline and nortriptyline metabolism to its 10-hydroxymetabolite were shown to be influenced by the 2D6 isozyme.
The stability of amitriptyline, nortriptyline, desipramine and imipramine in formalin-fixed human liver tissue and formalin soln was investigated. The levels of the tricyclic and its primary demethylated metabolite in the frozen liver were determined and compared with levels obtained in the formalin-fixed liver and formalin soln in which the liver was stored. It was obvious that some methylation of the secondary amine, nortriptyline, to the corresponding tertiary amine, amitriptyline, and of desipramine to imipramine took place in the formalin environment. Nortriptyline was not detected in most cases, suggesting that it may degrade more rapidly than desipramine. There was no consistent ratio between the concn of the drug in the frozen liver tissue versus formalin-preserved tissue or versus formalin soln. The methylation rates of the secondary amines could not be quantitated. Storage of the liver tissue in formalin at room temp resulted in leaching of the drugs into the formalin soln. The drugs tested may be detected for up to 22 mo in the formalin-fixed liver and in the formalin medium.
Nortriptyline has known human metabolites that include E-10-hydroxynortriptyline and demethylnortriptyline.
Nortriptyline is a known human metabolite of amitriptyline.
Undergoes hepatic metabolism via the same pathway as other TCAs.
Route of Elimination: Approximately one-third of a single orally administered dose is excreted in urine within 24 hours. Small amounts are excreted in feces via biliary elimination.
Half Life: 16 to 90+ hours

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Biological Half-Life
The average plasma half-life of nortriptyline in healthy volunteers is about 26 hours, but is said to range from 16 to 38 hours. One study mentions a mean half-life of about 39 hours.
The plasma half-life of nortriptyline ranges from 16 to more than 90 hours.
The clinical pharmacokinetics of amitriptyline were studied in four volunteers after the oral administration of 75 mg. ... The mean elimination half-life was 36.1 hours. ...

Toxicity Summary
IDENTIFICATION AND USE: Nortriptyline is a tricyclic antidepressant. HUMAN EXPOSURE AND TOXICITY: Symptoms of overdose/poisoning include the following: arrhythmias, bundle branch block, cardiac arrest, hypotension, circulatory collapse, mydriasis, 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, myoclonic jerks, coma and pyramidal signs. A woman with chronic obstructive pulmonary disease receiving nortriptyline experienced depression of CO2 sensitivity and ventilatory load compensation with a concomitant increase in exercise tolerance with decreased dyspnea. In other words, nortriptyline demonstrated a depressant effect on ventilatory control. Nortriptyline increases the risk for sudden cardiac arrest in the general population, particularly in the presence of genetic and/or non-genetic factors that decrease cardiac excitability by blocking the cardiac sodium channel. Antidepressant have been shown to increase the risk of suicidal thinking and behavior (suicidality) in children, adolescents, and young adults in short-term studies of major depressive disorder (MDD) and other psychiatric disorders. ANIMAL STUDIES: Dogs receiving nortriptyline hydrochloride orally for twelve months tolerated as much as 20 mg/kg/day. However, a large oral dose (40 mg per kg daily) caused signs of depression and ataxia and, with continued treatment, death at the end of the first month. In chronic toxicity studies, rats tolerated a concentration of nortriptyline hydrochloride in the diet equivalent to 150 mg/kg/day for one year. Rats showed some growth retardation but no visceral damage. Nortriptyline was tested for genotoxicity using the somatic mutation and recombination test (SMART) in wing cells of Drosophila melanogaster. The drug was not genotoxic at concentrations up to 100 mM.
It is believed that nortriptyline either inhibits the reuptake of the neurotransmitter serotonin at the neuronal membrane or acts at beta-adrenergic receptors. Tricyclic antidepressants do not inhibit monoamine oxidase nor do they affect dopamine reuptake.

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Toxicity Data
LD₅₀=mg/kg (orally in rat)

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Interactions
This case illustrates a pharmacokinetic interaction between the tricyclic antidepressant, nortriptyline, and the antituberculosis drug, rifampin. Higher than expected doses of nortriptyline were required to obtain a therapeutic drug level while the patient was receiving rifampin. Following the discontinuation of rifampin, the patient became drowsy and the serum nortriptyline levels rose precipitously into the toxic range.
A case of significant hypoglycemia has been reported in a type II diabetic patient maintained on chlorpropamide (250 mg/day), after the addition of nortriptyline (125 mg/day).
Using data from a therapeutic drug monitoring database, kinetic interactions between the neuroleptics zuclopenthixol and perphenazine and tricyclic antidepressives were studied. Out of 290 patients monitored for amitriptyline and 611 patients monitored for nortriptyline, 77 patients were comedicated with perphenazine and 50 patients with zuclopenthixol. Comedication with perphenazine incr the median steady-state serum concn to daily dose ratio (C/D) of nortriptyline by 30-45%, whereas the median C/D of amitriptyline was unaffected. On the contrary, median C/D values of nortriptyline and amitriptyline were not significantly influenced by comedication with zuclopenthixol.
Two cases are described in which a 33-yr-old woman and a 36-yr-old man developed adverse effects resulting from an interaction between valproic acid (valproate) and nortriptyline. The first patient was taking 25 mg/day nortriptyline and within 1 wk of the addition of valproic acid in doses up to 1000 mg/day, she developed marked tremulousness of her hands and fingers. The plasma level of nortriptyline was 393 ng/mL and that of valproic acid was 105 mg/L. Both drugs were stopped and tremulousness subsided over the next 2 days. The second patient was receiving 75 mg/day nortriptyline and up to 1250 mg/day valproic acid. A plasma level of nortriptyline was 345 ng/mL. The nortriptyline dosage was titrated down to 25 mg/day and a subsequent drug level was 82 ng/mL. Thioridazine and loxapine were given concomitantly.
For more Interactions (Complete) data for NORTRIPTYLINE (31 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Mouse iv 17 mg/kg
LD50 Mouse ip 70 mg/kg
LD50 Mouse oral 387 mg/kg
LD50 Rat iv 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 Uptake Inhibitors; Antidepressive Agents, Tricyclic
/CLINICAL TRIALS/ ClinicalTrials.gov is a registry and results database of publicly and privately supported clinical studies of human participants conducted around the world. The Web site is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each ClinicalTrials.gov record presents summary information about a study protocol and includes the following: Disease or condition; Intervention (for example, the medical product, behavior, or procedure being studied); Title, description, and design of the study; Requirements for participation (eligibility criteria); Locations where the study is being conducted; Contact information for the study locations; and Links to relevant information on other health Web sites, such as NLM's MedlinePlus for patient health information and PubMed for citations and abstracts for scholarly articles in the field of medicine. Nortriptyline is included in the database.
Nortriptyline hydrochloride is indicated for the relief of symptoms of depression. Endogenous depressions are more likely to be alleviated than are other depressive states. /Included in US product label/
Tricyclic antidepressants have been used for the treatment of attention deficit hyperactivity disorder (ADHD). /Tricyclic antidepressant; NOT included in US product label/
For more Therapeutic Uses (Complete) data for NORTRIPTYLINE (13 total), please visit the HSDB record page.

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Drug Warnings
/BOXED WARNING/ Suicidality and Antidepressant Drugs. Antidepressants increased the risk compared to placebo of suicidal thinking and behavior (suicidality) in children, adolescents, and young adults in short-term studies of major depressive disorder (MDD) and other psychiatric disorders. Anyone considering the use of nortriptyline hydrochloride or any other antidepressant in a child, adolescent, or young adult must balance this risk with the clinical need. Short-term studies did not show an increase in the risk of suicidality with antidepressants compared to placebo in adults beyond age 24; there was a reduction in risk with antidepressants compared to placebo in adults aged 65 and older. Depression and certain other psychiatric disorders are themselves associated with increases in the risk of suicide. Patients of all ages who are started on antidepressant therapy should be monitored appropriately and observed closely for clinical worsening, suicidality, or unusual changes in behavior. Families and caregivers should be advised of the need for close observation and communication with the prescriber. Nortriptyline hydrochloride is not approved for use in pediatric patients.
The pupillary dilation that occurs following use of many antidepressant drugs including nortriptyline hydrochloride may trigger an angle-closure attack in a patient with anatomically narrow angles who does not have a patent iridectomy.
... They should be used with caution in patients with urinary retention, glaucoma, diabetes, impaired liver function, asthma, and a history of convulsive seizures. / Tricyclic antidepressants/
... Many patients who take tricyclics and/or antipsychotic medications have a high risk for suicide. The margin of safety for antipsychotics is relatively high. For tricyclics, the margin is much less favorable. A rule of thumb is that the quantity of tricyclic antidepressants dispensed should be limited to a one week supply. /Tricyclic Antidepressants/
For more Drug Warnings (Complete) data for NORTRIPTYLINE (32 total), please visit the HSDB record page.

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Pharmacodynamics
Nortriptyline exerts antidepressant effects likely by inhibiting the reuptake of serotonin and norepinephrine at neuronal cell membranes. It also exerts antimuscarinic effects through its actions on the acetylcholine receptor.

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.)