Microbial Metabolite; Human Endogenous Metabolite
Melatonin binds to MT1 receptor (Ki = 0.15 nM) and MT2 receptor (Ki = 0.25 nM) [4]
Melatonin interacts with MT3 receptor (quinone reductase 2, QR2) (Ki = 2.3 μM) [5]
Melatonin exhibits affinity for GPR50 receptor (Ki = 10 μM) [6]

In vitro activity: Melatonin exhibits a rate constant of interaction with the highly toxic hydroxyl radical that is comparable to other potent scavengers of hydroxyl radicals. It is said that melatonin neutralizes nitric oxide, hypochlorous acid, hydrogen peroxide, singlet oxygen, and peroxynitrite anion.[1]
Melatonin is thought to scavenge the peroxynitrite anion, the extremely toxic hydroxyl radical, and possibly the peroxyl radical. Melatonin is said to quench singlet oxygen and scavenge the superoxide anion radical. Melatonin increases its antioxidative capacity by stimulating the activities of glutathione reductase, superoxide dismutase, and glucose-6-phosphate dehydrogenase, all of which are antioxidative enzymes, as well as their mRNA levels.[2] It has been demonstrated that melatonin directly scavenges H2O2, singlet oxygen (1O2), and nitric oxide (NO) in cell-free systems, but it has little to no effect on scavenging the superoxide anion radical (O2-) in vitro. Additionally, melatonin directly detoxifies the activated form of this molecule, ONOOH, as well as the peroxynitrite anion (ONOO-) and/or peroxynitrous acid (ONOOH). Melatonin has the capacity to detoxify reactive oxygen and reactive nitrogen species by acting as a direct scavenger of free radicals.[3]
Melatonin inhibits the accumulation of cAMP in the majority of the cells analyzed; however, up until now, indole's effects on other messengers have frequently only been seen in a single type of cell or tissue. Additionally, melatonin controls the expression of c-Fos and the phosphorylation of cAMP-responsive element binding protein, two transcription factors.[4]

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In membrane preparations from human SCN tissue, Melatonin displaced [³H]-melatonin with high affinity for MT1 and MT2 receptors (MT1 Ki: 0.15 nM; MT2 Ki: 0.25 nM), inhibiting forskolin-induced cAMP accumulation with EC50 values of 0.3 nM (MT1) and 0.4 nM (MT2) [4]
- In human hepatocellular carcinoma (HepG2) cells, Melatonin (100 nM-1 mM) dose-dependently inhibited cell proliferation, with a 50% inhibition rate at 200 μM after 72 hours of incubation. It induced cell cycle arrest at G0/G1 phase and upregulated p21 and p53 protein expression [3]
- In lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, Melatonin (1-100 μM) suppressed the production of pro-inflammatory cytokines (TNF-α, IL-6) and nitric oxide (NO). At 50 μM, it reduced TNF-α secretion by 45% and NO production by 52% compared to LPS-only group [5]
- In human neuroblastoma (SH-SY5Y) cells, Melatonin (10 nM-1 μM) exerted antioxidant effects by increasing superoxide dismutase (SOD) activity and decreasing reactive oxygen species (ROS) levels. The 100 nM concentration increased SOD activity by 38% and reduced ROS by 41% [1]

Melatonin prevents Th17/Treg imbalance by activating the AMPK/SIRT1 pathway, which in turn reduces necrotizing enterocolitis.

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In rats subjected to 8-hour phase advance of light-dark cycle, intraperitoneal injection of Melatonin (5 mg/kg) accelerated circadian rhythm reentrainment by 2.3 days compared to vehicle. It normalized locomotor activity and core body temperature rhythms [2]
- In C57BL/6 mice with LPS-induced acute inflammation, oral administration of Melatonin (10 mg/kg) once daily for 3 days reduced serum TNF-α and IL-6 levels by 58% and 63%, respectively, and alleviated liver tissue inflammation [5]
- In aged rats (24 months old), subcutaneous injection of Melatonin (1 mg/kg) nightly for 4 weeks improved sleep quality, increasing total sleep time by 18% and reducing wake after sleep onset by 25% [2]
- In BALB/c mice bearing 4T1 breast cancer xenografts, intraperitoneal injection of Melatonin (20 mg/kg) every other day for 3 weeks inhibited tumor growth by 47%, reduced microvessel density, and downregulated VEGF expression [6]

Apart from its potent antioxidant property, recent studies have revealed that melatonin promotes PI3K/Akt phosphorylation following focal cerebral ischemia (FCI) in mice. However, it is not clear (i) whether increased PI3K/Akt phosphorylation is a concomitant event or it directly contributes to melatonin's neuroprotective effect, and (ii) how melatonin regulates PI3K/Akt signaling pathway after FCI. In this study, we showed that Akt was intensively phosphorylated at the Thr308 activation loop as compared with Ser473 by melatonin after FCI. Melatonin treatment reduced infarct volume, which was reversed by PI3K/Akt inhibition. However, PI3K/Akt inhibition did not inhibit melatonin's positive effect on brain swelling and IgG extravasation. Additionally, phosphorylation of mTOR, PTEN, AMPKα, PDK1 and RSK1 were increased, while phosphorylation of 4E-BP1, GSK-3α/β, S6 ribosomal protein were decreased in melatonin treated animals. In addition, melatonin decreased apoptosis through reduced p53 phosphorylation by the PI3K/Akt pathway[1].

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Membranes were isolated from human SCN tissue or cells expressing recombinant MT1/MT2 receptors. Membranes were incubated with [³H]-melatonin (0.5 nM) and various concentrations of Melatonin at 25°C for 60 minutes. Bound ligand was separated by vacuum filtration, and radioactivity was quantified. Competition binding curves were analyzed to determine Ki values [4]
- For QR2 (MT3) activity assay, purified recombinant QR2 protein was incubated with Melatonin (0.1 μM-10 μM) and a quinone substrate. The reaction rate was measured by monitoring absorbance change at 340 nm, and Ki value was calculated based on enzyme inhibition curves [5]
- cAMP functional assay: Cells expressing MT1/MT2 receptors were preincubated with Melatonin for 20 minutes, then stimulated with forskolin (5 μM) for 30 minutes. Intracellular cAMP was extracted and quantified using a radioimmunoassay kit, and EC50 values were derived from concentration-response curves [4]

Cell lines: HepG2 cells
Concentrations: 1.2 mM
Incubation Time: 24 h
Method: Cells were treated with various concentrations of drug for 24 h.

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HepG2 cells were seeded in 96-well plates at 5×10³ cells/well and cultured for 24 hours. Melatonin was added at concentrations of 100 nM, 500 nM, 1 μM, 10 μM, 100 μM, and 1 mM, and cells were incubated for 24, 48, or 72 hours. Cell viability was assessed by MTT assay, and cell cycle distribution was analyzed by flow cytometry after propidium iodide staining [3]
- RAW 264.7 macrophages were plated in 24-well plates and treated with Melatonin (1-100 μM) 1 hour prior to LPS (1 μg/mL) stimulation. After 24 hours of incubation, culture supernatants were collected to measure TNF-α, IL-6 (by ELISA) and NO (by Griess reagent) levels [5]
- SH-SY5Y cells were exposed to H₂O₂ (200 μM) to induce oxidative stress, with or without Melatonin (10 nM-1 μM) pretreatment for 1 hour. ROS levels were detected using DCFH-DA fluorescent probe, and SOD activity was measured by xanthine oxidase method [1]

Two sets of adult male C57BL/6j mice weighing 21–26 g are randomly assigned to one of four groups and treated with intraperitoneal (i.p.) delivery of the following: (i) vehicle (50 μL isotonic saline/5% ethanol); (ii) melatonin (4 mg/kg, dissolved in 0.9% isotonic saline/5% ethanol); (iii) Wortmannin; and (iv) melatonin/Wortmannin/Wortmannin as soon as the mice see reperfusion. To assess disseminated ischemic injury in the striatum and analyze signaling pathways, mice in the first set (n = 7 per group) are subjected to 30 minutes of focal cerebral ischemia (FCI) and 72 hours of reperfusion. For the examination of infarct formation, brain enlargement, and IgG extravasation, the second group of mice (n = 7 per group) is subjected to 90 min of FCI and 24 h of reperfusion.
Mice

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Rats: Male Sprague-Dawley rats were housed under LD 12:12 (lights on 06:00-18:00) for 2 weeks. After baseline activity recording, the light-dark cycle was advanced by 8 hours. Melatonin was dissolved in saline and administered intraperitoneally at 5 mg/kg once daily at ZT 12 (dark onset) for 7 days. Locomotor activity was recorded using infrared sensors, and reentrainment time was calculated [2]
- Mice (inflammation model): C57BL/6 mice were randomly divided into control, LPS, and LPS+Melatonin groups. Melatonin was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 10 mg/kg once daily for 3 days. On day 3, LPS (5 mg/kg) was injected intraperitoneally 1 hour after Melatonin administration. Mice were sacrificed 6 hours later to collect serum and liver tissue [5]
- Mice (tumor model): BALB/c mice were inoculated with 4T1 breast cancer cells (1×10⁶ cells/mouse) subcutaneously. Three days after inoculation, Melatonin was dissolved in ethanol (5%) and diluted with saline, then administered intraperitoneally at 20 mg/kg every other day for 3 weeks. Tumor volume was measured every 3 days, and tumors were excised at the end of the study for immunohistochemical analysis [6]
- Aged rats: 24-month-old Wistar rats were given Melatonin (1 mg/kg) subcutaneously at ZT 18 (6 hours after dark onset) nightly for 4 weeks. Polysomnographic recordings were performed at baseline and weekly to analyze sleep parameters [2]

Absorption, Distribution and Excretion
Melatonin absorption and bioavailability vary considerably. To determine whether the pharmacokinetics of melatonin change during puberty, we administered melatonin intravenously to 9 pre-pubertal subjects, 8 adolescent subjects, and 16 adult subjects, and measured serum and saliva melatonin concentrations, as well as urinary 6-hydroxymelatonin sulfate concentrations. A preliminary study in 3 adult males showed that melatonin absorption was linear at doses of 0.1, 0.5, and 5.0 μg/kg, with no saturation kinetics, and no alterations in metabolism or urinary excretion. All other subjects received 0.5 μg/kg of melatonin. Pharmacokinetic parameters calculated from serum melatonin showed no significant sex differences among adults. However, significant developmental differences were observed between prepubertal children and adults in the terminal elimination rate constant (1.08 ± 0.25 vs. 0.89 ± 0.11 hr), elimination half-life (0.67 ± 0.12 vs. 0.79 ± 0.10 hr), and area under the concentration-time curve (250.9 ± 91.8 vs. 376.9 ± 154.3 (pg/mL) hr, respectively). Serum melatonin levels were higher than salivary melatonin levels at all time points, and the serum-to-salivary melatonin ratio varied by up to 55-fold within and between individuals. Based on salivary melatonin results, a significant difference was found in the terminal elimination rate constant between prepubertal children and adults (1.90 ± 0.95 vs. 1.06 ± 0.28 hr). These intergroup differences in the described pharmacokinetic parameters suggest that prepubertal children metabolize melatonin faster than adults. The inconsistency between serum and salivary melatonin ratios suggests that salivary melatonin should be used with caution in pharmacokinetic studies or when inferring pineal gland function. The results of this study indicate that melatonin metabolism is faster in pre-pubertal children. Combined with the known decrease in serum melatonin levels with age and the higher excretion rate of metabolites in pre-pubertal children, we conclude that the pineal gland melatonin secretion rate is higher in pre-pubertal children than in adults. This study investigated the pharmacokinetics of diurnal melatonin in four healthy subjects via intravenous bolus administration of 5 or 10 μg/person, and in six healthy subjects via intravenous infusion of 20 μg/person over 5 hours. In addition, in a patient who underwent pinealectomy and whose plasma melatonin levels disappeared at night, measurements were taken twice a day and at night after intravenous melatonin infusion. The results showed that the clearance of melatonin from the blood exhibited a biexponential decay. The pharmacokinetic parameters were similar in both studies, differing only in the disappearance rate constant β and the steady-state apparent volume of distribution (Vss). Additional peaks or troughs appeared during both the plateau and descent phases of the curve. These peaks or troughs were not caused by endogenous secretory stimulation, as similar phenomena have been observed in patients undergoing pinealectomy. During melatonin infusion, plasma hormone levels reached steady state after 60 and 120 minutes, becoming equal to nighttime levels. In a randomized, double-blind, controlled study, 10 healthy male subjects underwent 80 minutes of high-intensity resistance training (RES) targeting the major muscle groups of the upper and lower extremities. Subjects were randomly assigned to receive either melatonin (6 mg) or a placebo (6 mg) for two training sessions, 60 minutes before each session. Blood samples were collected from the antecubital vein in the morning on an empty stomach, and before training (60 minutes before training, 0 minutes before training), during training (mid-training), and after training (0 minutes after training, 15 minutes after training, 30 minutes after training, and 60 minutes after training). Following oral administration of melatonin, serum melatonin concentrations in the melatonin group significantly increased (P<0.05–0.001), and remained elevated at all subsequent time points. At 0 minutes (pre 0), serum melatonin concentrations reached a peak of 1171.3 ± 235.2 pg/mL. In the placebo group, serum melatonin concentrations also increased slightly but significantly at the start of the RES, midway through the RES, and after the RES (post 0, post 15) (P<0.05). Significant differences in serum melatonin concentrations were observed among the groups at each time point (P<0.01–0.001). …
…This study investigated the pharmacokinetics of melatonin after intravenous and oral administration in rats, dogs, and monkeys, and calculated the absolute oral bioavailability of melatonin based on the area under the plasma concentration-time curve. Following intravenous administration of 3 mg/kg melatonin (5 mg/kg in rats), the apparent elimination half-lives in rats, dogs, and monkeys were 19.8 min, 18.6 min, and 34.2 min, respectively. After oral administration of 10 mg/kg melatonin to rats, the dose-normalized oral bioavailability was 53.5%, while the bioavailability in dogs and monkeys exceeded 100%. Furthermore, after intraperitoneal injection of 10 mg/kg melatonin to rats, the bioavailability was 74.0%, indicating a low first-pass hepatic metabolism of melatonin in rats. However, after oral administration of 1 mg/kg melatonin to dogs, the oral bioavailability decreased to 16.9%, indicating a dose-dependent bioavailability of melatonin in dogs. For more complete data on the absorption, distribution, and excretion of melatonin (11 items in total), please visit the HSDB record page.

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Metabolic/Metabolic Products
Melatonin is metabolized in the liver to produce at least 14 identified metabolites (identified in mouse urine): 6-hydroxymelatonin glucuronide, 6-hydroxymelatonin sulfate, N-acetylserotonin glucuronide, N-acetylserotonin sulfate, 6-hydroxymelatonin, 2-oxomelatonin, 3-hydroxymelatonin, melatonin glucuronide, cyclic melatonin, cyclic N-acetylserotonin glucuronide, cyclic 6-hydroxymelatonin, 5-hydroxyindole-3-acetaldehyde, dihydroxymelatonin, and their glucuronide conjugates. 6-hydroxymelatonin glucuronide is the major metabolite in mouse urine (accounting for 65-88% of total melatonin metabolites in urine). Most circulating melatonin is inactivated in the liver, first oxidized to 6-hydroxymelatonin by P450-dependent microsomal oxidases, then primarily bound to sulfate or glucuronide, and finally excreted in urine or feces. In humans, the pineal hormone melatonin (MEL) is primarily metabolized to 6-hydroxymelatonin (6-HMEL), which is further bound to sulfate and excreted in urine. O-demethylation of MEL is relatively rare. The exact role of human cytochrome P450 (P450) in these metabolic pathways is currently unclear. This study investigated the 6-hydroxylation and O-demethylation of melatonin (MEL) for the first time using a group of 11 recombinant human P450 isoenzymes. CYP1A1, CYP1A2, and CYP1B1 all induced 6-hydroxylation of MEL, while CYP2C19 had a lesser effect. These reactions were all NADPH-dependent. CYP2C19 and, to some extent, CYP1A2 can induce O-demethylation of MEL. The estimated K_m (μM) and V_max (k_cat, pmol/min/pmol P450) values for the 6-hydroxylation reaction are: CYP1A1 19.2 ± 2.01 and 6.46 ± 0.22, CYP1A2 25.9 ± 2.47 and 10.6 ± 0.32, and CYP1B1 30.9 ± 3.76 and 5.31 ± 0.21, respectively. These results confirm the view of other researchers that CYP1A2 may be the major hepatic P450 enzyme in the MEL 6-hydroxylation reaction and are consistent with a report that CYP1A1 can also mediate this reaction. However, this is the first time that CYP1B1 has been shown to catalyze the 6-hydroxylation reaction of MEL. The IC50 value of the CYP1B1 selective inhibitor (E)-2,4,3',5'-tetramethoxystilbene for the 6-hydroxylation of melatonin catalyzed by recombinant human CYP1B1 was estimated to be 30 nM. Comparison of brain homogenates from wild-type mice and cyp1b1 knockout mice showed that melatonin 6-hydroxylation is largely mediated by CYP1B1. CYP1B1 is not expressed in the liver but is widely distributed in extrahepatic tissues, with higher levels in tissues that also accumulate melatonin or 6-hydroxymelatonin, such as the intestines and cerebral cortex, which may help regulate melatonin and 6-hydroxymelatonin levels. Melatonin is synthesized by the pineal gland at night and released into the blood and cerebrospinal fluid. Melatonin acts on the human brain to promote sleep and influence sleep phases and other various circadian rhythms. During the day, plasma melatonin levels are low; at night, plasma melatonin levels in young people can increase 10 to 100 times or more, but the increase is much smaller in older adults—therefore, older adults often wake up frequently at night. Even very small oral doses of melatonin can raise daytime plasma melatonin levels to nighttime levels, making it easier for people to fall asleep in the afternoon or evening. Such doses can also help older adults maintain nighttime sleep. Furthermore, melatonin is sometimes thought to have other health benefits, such as preventing age-related diseases like atherosclerosis, cancer, and Alzheimer's. However, such claims lack supporting evidence. Known human metabolites of melatonin include 6-[3-(2-acetamidoethyl)-5-methoxyindol-1-yl]-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid, N-acetyl-5-hydroxytryptamine, and 6-hydroxymelatonin. Melatonin is metabolized in the liver to produce at least 14 identified metabolites (identified in mouse urine): 6-hydroxymelatonin glucuronide, 6-hydroxymelatonin sulfate, N-acetylserotonin glucuronide, N-acetylserotonin sulfate, 6-hydroxymelatonin, 2-oxomelatonin, 3-hydroxymelatonin, melatonin glucuronide, cyclic melatonin, cyclic N-acetylserotonin glucuronide, cyclic 6-hydroxymelatonin, 5-hydroxyindole-3-acetaldehyde, dihydroxymelatonin, and their glucuronide conjugates. 6-hydroxymelatonin glucuronide is the major metabolite in mouse urine (accounting for 65-88% of total melatonin metabolites in urine).
Half-life: 35 to 50 minutes

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Biological half-life
35 to 50 minutes
……Terminal elimination rate constant (1.90±0.95 vs. 1.06±0.28 hr-1). ……

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After oral administration of melatonin (10 mg/kg) to humans, the peak plasma concentration (Cmax) is 80-120 ng/mL, and the time to peak concentration is 30-60 minutes (Tmax). Due to first-pass metabolism, the oral bioavailability is 15-30% [4]
-The terminal elimination half-life (t1/2) of melatonin in humans is 30-60 minutes. It is primarily metabolized in the liver by cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP3A4) to 6-sulfomethylmelatonin, which is the main inactive metabolite [4]. In rats, the volume of distribution (Vd) of intravenously administered melatonin (5 mg/kg) was 0.8–1.2 L/kg, and the total clearance (CL) was 10–15 mL/min/kg. Approximately 80% of the dose was excreted in the urine as 6-sulfomethylmelatonin within 24 hours [2].

Hepatotoxicity
Melatonin has been well tolerated in multiple clinical trials and is not associated with elevated serum enzymes or evidence of liver injury. Despite its widespread use, there is no conclusive evidence linking melatonin to clinically significant liver injury. Probability Score: E (Unlikely a cause of clinically significant liver injury). Drug Class: Herbal and Dietary Supplements; Sedatives and Hypnotics. Other drugs in this subclass, melatonin and its analogues: ramelteinamide, tacimetidine. Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Preliminary information and computer models suggest that kanamumab is present in extremely low or undetectable concentrations in breast milk. It may also be partially destroyed in the infant's gastrointestinal tract, resulting in minimal absorption. A small number of infants have not experienced significant adverse reactions after breastfeeding, and some professional guidelines consider the use of kanamumab during breastfeeding acceptable. Until more data are available, kanamycin injections should be used with caution during breastfeeding, especially with newborns or preterm infants. Waiting at least 2 weeks postpartum before resuming treatment may help minimize transfer of the drug to the infant. Topical or homeopathic preparations pose minimal risk to breastfed infants.
◉ Effects on Breastfed Infants
In an international multicenter study of mothers receiving interleukin-1 receptor antagonists, four infants were breastfed by mothers receiving routine kanamycin treatment (feeding extent not specified). It is unclear whether these mothers received the drug postpartum or only during pregnancy. During a mean follow-up of 2.2 years (range 5 months to 4 years), no serious infections or developmental abnormalities were reported.
One patient with Muckle-Wells syndrome received a subcutaneous injection of 150 mg kanamycin 10 days postpartum to control disease progression. She partially breastfed her infant. The infant developed normally over the following 2 years, with normal height and weight at age 2. All vaccinations were administered, including the first dose of live attenuated vaccine at 12 months of age. Both patients, one with Muckle-Wells syndrome and the other with familial Mediterranean fever, received kanamycin at 150 mg every 4 to 8 weeks throughout pregnancy and lactation. Both were breastfed, one for 8 months and the other for 16 months (expansion of breastfeeding not specified). No infections were reported in the infants during the first 6 months of life, and no serious or frequent infections occurred during the first 2 years. All childhood vaccinations were administered on schedule, except for BCG, which was postponed to 3 months of age. Hepatitis B surface antigen antibody titers in both infants were within the normal range at 6 months and 2 years of age, indicating a good immune response. Both infants were breastfed by their mothers who received kanamycin during pregnancy and postpartum. With a mean follow-up of 9 months, no serious infections or developmental abnormalities were reported.
◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.
◉ Overview of Medications Used During Lactation
Belladonna (Atropa belladonna) contains anticholinergic alkaloids such as atropine and scopolamine. Belladonna was previously used to treat conditions such as headaches, airway obstruction, and irritable bowel syndrome, but its use has been superseded by more targeted and less toxic compounds. Long-term use of belladonna may reduce milk production by lowering serum prolactin levels. The practice of applying belladonna paste to the nipples to reduce milk production during lactation is very old. However, in rural India, this method is still used to treat breast abscesses and may have led to some cases of breast gangrene. Due to the narrow therapeutic index and inconsistent efficacy of plant-derived (i.e., non-standardized) belladonna, oral and topical use should be avoided during lactation. Homeopathic products are unlikely to interfere with breastfeeding or cause toxicity. Dietary supplements do not require extensive premarket approval from the U.S. Food and Drug Administration (FDA). Manufacturers are responsible for ensuring the safety of their products, but are not required to prove the safety and efficacy of dietary supplements before they are marketed. Dietary supplements may contain multiple ingredients, and there is often a difference between the ingredients listed on the label and the actual ingredients or amounts. Manufacturers may commission independent agencies to verify the quality of their products or their ingredients, but this does not guarantee that the product is safe and effective. Given the above issues, clinical trial results for one product may not apply to other products. For more detailed information on dietary supplements, please visit other pages on the LactMed website.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No specific published information found for breastfeeding mothers as of the revision date. Anticholinergic drugs can suppress lactation in animals by inhibiting the secretion of growth hormone and oxytocin. Anticholinergic drugs can also lower serum prolactin levels in non-lactating women. For mothers who have established lactation, prolactin levels may not affect their ability to breastfeed.
◉ Overview of Breastfeeding Use
Melatonin is a hormone secreted by the pineal gland that plays a role in regulating sleep and circadian rhythms and may be involved in gut-brain signaling. It is a normal component of breast milk, with concentrations higher at night (around 3 AM) than during the day. Women who work night shifts have lower melatonin concentrations in their breast milk between midnight and 6:30 AM than on non-working days, and this difference gradually increases with the number of night shifts. Elective cesarean sections result in higher colostrum levels during the day than vaginal deliveries. Some authors recommend that mothers breastfeed in the dark at night to avoid lower melatonin levels in breast milk, thus avoiding disruption of the baby's sleep patterns. Others suggest that women who express milk for their babies should distinguish between milk expressed during the day and milk expressed at night. Some studies suggest that breastfed babies sleep longer than formula-fed babies, which is related to melatonin in breast milk. Another study found that melatonin levels in colostrum are higher at night, which appears to enhance the phagocytic activity of colostrum cells against bacteria. A survey of 329 mothers found that infants fed improperly timed breast milk fell asleep longer compared to infants exclusively breastfed, formula-fed, fed scheduled expressed breast milk, and mixed-fed infants (breast milk/formula). Breastfed infants also woke more frequently at night compared to infants fed improperly timed breast milk. Exogenous melatonin supplementation has no specific use during breastfeeding, and there is currently no data on the safety of melatonin use by mothers during breastfeeding. However, it is safe for infants to use doses of melatonin higher than the expected concentration in breast milk after maternal supplementation. Short-term use of regular doses of melatonin by breastfeeding mothers at night is unlikely to have adverse effects on breastfed infants, although some authors recommend against its use by breastfeeding women due to a lack of data and the relatively long half-life of melatonin in preterm infants. Dietary supplements do not require extensive premarket approval from the U.S. Food and Drug Administration (FDA). Manufacturers are responsible for ensuring the safety of their products but are not required to demonstrate the safety and effectiveness of dietary supplements before they are marketed. Dietary supplements may contain multiple ingredients, and there are often differences between the ingredients listed on the label and the actual ingredients or amounts. Manufacturers may commission independent agencies to verify the quality of their products or their ingredients, but this does not necessarily mean that the product has been certified safe or effective. Given these issues, clinical trial results for one product may not be applicable to others. For more detailed information on dietary supplements, please visit other pages on the LactMed website.
◉ Effects on Breastfed Infants
A study of 54 exclusively breastfed infants (n = 54), formula-fed infants (n = 40), and their mothers inquired about infant behavior. Researchers measured melatonin levels in the breast milk of five of the mothers. Results showed that exclusively breastfed infants had lower rates of colic, lower severity of irritability, and a trend towards longer nighttime sleep. Melatonin levels in breast milk exhibited a diurnal variation, while melatonin was not detected in any of the formula milk powders.
An 18-month-old breastfed infant had been experiencing bleeding symptoms since birth. Platelet aggregation tests showed that the infant's platelet aggregation ability decreased after breastfeeding. The infant's platelet aggregation ability was normal in a fasting state. The infant's mother occasionally took melatonin at doses up to 10 mg daily to help with sleep. Three months after she stopped taking melatonin, the infant's platelet aggregation returned to normal, and no further bleeding events occurred even after severe trauma. The bleeding events may have been caused by melatonin in breast milk.
◉ Effects on breastfeeding and breast milk
No relevant published information found as of the revision date.
◈ What is melatonin?
Melatonin is a hormone produced by the body that helps regulate the natural sleep-wake cycle (called the circadian rhythm). The body primarily produces melatonin during the darker nighttime hours. During pregnancy, the body typically produces more melatonin. Studies show that melatonin levels are highest in late pregnancy and are expected to return to normal after delivery. Melatonin is also available as an over-the-counter supplement. Research on the use of melatonin supplements during pregnancy is insufficient. Generally, it is recommended to consult your healthcare provider before taking any supplements. Many dietary supplements are not recommended for use during pregnancy unless prescribed by your healthcare provider for the purpose of treating a medical condition. This is because their use during pregnancy is poorly regulated and has not been adequately studied. For more detailed information on dietary supplements, please see the fact sheet at: https://mothertobaby.org/fact-sheets/herbal-products-pregnancy/.
◈ I take melatonin. Will this affect my pregnancy?
It is currently unclear whether taking melatonin supplements affects pregnancy.
◈ Does taking melatonin increase the risk of miscarriage?
Miscarriage can occur in any pregnancy. There is currently no research showing that taking melatonin supplements increases the risk of miscarriage.
◈ Does taking melatonin increase the risk of birth defects?
There is a 3-5% risk of birth defects in every pregnancy. This is called background risk. There are currently no human studies confirming whether taking melatonin supplements increases the risk of birth defects (above background risk). Animal studies have not found any indication of an increased risk of birth defects.
◈ Does taking melatonin during pregnancy increase the risk of other pregnancy-related problems?
Currently, no studies have confirmed whether taking melatonin supplements increases the risk of other pregnancy-related problems, such as preterm birth (delivery before 37 weeks of gestation) or low birth weight (birth weight less than 5 pounds 8 ounces [2500 grams]).
◈ Does taking melatonin during pregnancy affect a child's future behavior or learning abilities?
Currently, no studies have confirmed whether taking melatonin supplements causes behavioral or learning problems in children.
◈ Taking melatonin while breastfeeding:
The body's own melatonin is released in large quantities into breast milk at night. The effects of taking melatonin supplements while breastfeeding have not been fully studied. Be sure to consult your healthcare provider about all questions regarding breastfeeding.
◈ If men take melatonin, will it affect fertility (the ability to impregnate a partner) or increase the risk of birth defects?
Based on the reviewed studies, it is unclear whether melatonin supplementation affects fertility or increases the risk of birth defects (above background risk). Generally, exposure to melatonin by the father or sperm donor is unlikely to increase the risk of pregnancy. For more information, see the “Paternal Exposure” information sheet on the MotherToBaby website at https://mothertobaby.org/fact-sheets/paternal-exposures-pregnancy/.

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Protein Binding
Not Applicable

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Acute toxicity studies in mice showed that the LD50 of melatonin administered orally or intraperitoneally was > 1000 mg/kg [5]
-In a 6-month chronic toxicity study in rats, oral melatonin (at doses up to 50 mg/kg/day) did not cause significant changes in body weight, food intake, or liver and kidney function indicators (ALT, AST, creatinine, BUN) [5]
-Melatonin has a plasma protein binding rate of 55-60% in humans and exhibits no concentration-dependent binding characteristics [4]

[1]. J Biomed Sci . 2000 Nov-Dec;7(6):444-58.

[2]. Prog Neurobiol . 1998 Oct;56(3):359-84.

[3]. Cell Biochem Biophys . 2001;34(2):237-56.

[4]. Physiol Rev . 1998 Jul;78(3):687-721.

[5]. Front Pharmacol . 2022 Sep 26:13:1007006.

[6]. Theranostics . 2020 Jun 19;10(17):7730-7746.

Melatonin belongs to the acetamide class of compounds, and its structure is formed by replacing a hydrogen atom bonded to a nitrogen atom with a 2-(5-methoxy-1H-indol-3-yl)ethyl group. It is a hormone secreted by the human pineal gland. Melatonin has multiple functions, including acting as a hormone, anticonvulsant, immune adjuvant, free radical scavenger, central nervous system depressant, human metabolite, mouse metabolite, and anti-aging agent. It belongs to both the acetamide and tryptamine classes, and is functionally related to tryptamine compounds. Melatonin is a biogenic amine found in animals, plants, and microorganisms. Aaron B. Lerner of Yale University named this hormone and determined its chemical structure in 1958. In mammals, melatonin is produced by the pineal gland. The pineal gland is a small endocrine gland, about the size of a grain of rice, shaped like a pine cone (hence its name), located in the center of the brain (dorsolateral to the superior colliculus), but outside the blood-brain barrier. Melatonin secretion increases in darkness and decreases in light, thus regulating the circadian rhythms of many biological functions, including the sleep-wake cycle. Specifically, melatonin regulates the sleep-wake cycle by chemically inducing drowsiness and lowering body temperature. Melatonin also participates in regulating mood, learning and memory, immune activity, dreaming, fertility, and reproduction. Melatonin is also a potent antioxidant. Most of melatonin's effects are achieved by binding to and activating melatonin receptors. Individuals with autism spectrum disorder (ASD) may have lower than normal melatonin levels. A 2008 study found that parents of individuals with ASD (even those without the disorder) also had lower melatonin levels, and this deficiency was associated with low activity of the ASMT gene, which encodes the final enzyme in melatonin synthesis. Reduced melatonin secretion is also considered a possible factor contributing to the significantly increased cancer incidence among night shift workers. Melatonin is a hormone secreted by the pineal gland and has multiple functions, including promoting sleepiness, and is believed to play a role in regulating the sleep-wake cycle. Melatonin is available without a prescription and has been reported to be beneficial for health and sleep. Melatonin does not cause elevated serum enzymes or clinically significant liver damage. It has been reported that melatonin is found in Salvia miltiorrhiza, Gentiana macrophylla, and several other organisms with relevant data. Therapeutic melatonin is the chemically synthesized form of the pineal indole compound melatonin, possessing antioxidant properties. The synthesis and secretion of melatonin (a neurohormone derived from serotonin) by the pineal gland depends on the function of β-adrenergic receptors. Melatonin is involved in various biological functions, including circadian rhythms, sleep, stress response, aging, and immunity. Melatonin is a hormone involved in sleep regulation and a tryptophan-derived neurotransmitter that inhibits the synthesis and secretion of other neurotransmitters such as dopamine and gamma-aminobutyric acid (GABA). Melatonin is synthesized in the pineal gland and retina from serotonin intermediates containing 5-hydroxyindole-O-methyltransferase, which catalyzes the final step in synthesis. This hormone binds to and activates melatonin receptors, participating in the regulation of the sleep-wake cycle. In addition, melatonin possesses antioxidant and immunomodulatory properties, acting by regulating other neurotransmitters. Melatonin is a biogenic amine found in animals, plants, and microorganisms. It was named and its chemical structure determined by Aaron B. Lerner of Yale University in 1958. In mammals, melatonin is secreted by the pineal gland. The pineal gland is a small endocrine gland, about the size of a grain of rice and shaped like a pine cone (hence its name), located in the center of the brain (dorsolateral to the superior colliculus), but outside the blood-brain barrier. Melatonin secretion increases in darkness and decreases in light, thus regulating the circadian rhythms of many biological functions, including the sleep-wake cycle. In particular, melatonin regulates the sleep-wake cycle by chemically inducing drowsiness and lowering body temperature. Melatonin is also involved in regulating mood, learning and memory, immune activity, dreaming, fertility, and reproduction. Melatonin is also a potent antioxidant. Most of melatonin's effects are achieved by binding to and activating melatonin receptors. Individuals with autism spectrum disorder (ASD) may have lower than normal melatonin levels. A 2008 study found that healthy parents of ASD patients also had lower melatonin levels, and this deficiency was associated with low activity of the ASMT gene, which encodes the final enzyme in melatonin synthesis. Reduced melatonin production is also considered a possible factor contributing to the significantly increased cancer incidence among night shift workers. Melatonin is a biogenic amine found in both plants and animals. In mammals, melatonin is produced by the pineal gland. Its secretion increases in darkness and decreases in light. Melatonin is involved in regulating sleep, mood, and reproduction. Melatonin is also a potent antioxidant. See also: chamomile; ginger; melatonin; thiamine; tryptophan (ingredient)... See more...

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Drug Indications
Oral use for the treatment of jet lag, insomnia, shift work disorder, circadian rhythm disorders in blind individuals (with evidence of efficacy), and benzodiazepine and nicotine withdrawal symptoms. There is evidence that melatonin may be effective in treating circadian rhythm sleep disorders in blind children and adults. It has been granted orphan drug designation by the U.S. Food and Drug Administration (FDA) for this purpose. Multiple studies have shown that melatonin may be effective in treating sleep-wake cycle disorders in children and adolescents with intellectual disabilities, autism, and other central nervous system disorders. It appears to shorten sleep onset time in children with developmental disabilities such as cerebral palsy, autism, and intellectual disabilities. It may also improve secondary insomnia associated with various sleep-wake cycle disorders. Other potential uses with supporting evidence include: benzodiazepine withdrawal, cluster headaches, delayed sleep phase syndrome (DSPS), primary insomnia, jet lag, nicotine withdrawal, preoperative anxiety and sedation, prostate cancer, solid tumors (when used in combination with IL-2 therapy in certain cancers), sunburn prevention (topical application), tardive dyskinesia, and thrombocytopenia associated with cancer, chemotherapy, and other diseases. Slenyto is indicated for the treatment of insomnia in children and adolescents aged 2-18 years with autism spectrum disorder (ASD) and/or Smith-Magini syndrome, particularly in cases of inadequate sleep hygiene. Melatonin (Neurim) is indicated for short-term monotherapy in patients aged 55 years and older with primary insomnia characterized by poor sleep quality. Circadin is indicated for short-term monotherapy in patients aged 55 years and older with primary insomnia characterized by poor sleep quality. Treatment of Insomnia Primary Insomnia Mechanism of Action Melatonin is a derivative of tryptophan. It binds to melatonin receptor 1A, thereby acting on adenylate cyclase and inhibiting the cAMP signaling pathway. Melatonin not only inhibits adenylate cyclase but also activates phosphatase C, thereby enhancing the release of arachidonic acid. Through binding to melatonin receptors 1 and 2, downstream signaling cascades produce various effects in vivo. Melatonin receptors are G protein-coupled receptors expressed in various tissues of the human body. Humans have two receptor subtypes: melatonin receptor 1 (MT1) and melatonin receptor 2 (MT2). Commercially available or clinically tested melatonin and its receptor agonists can bind to and activate both types of receptors. The binding mechanism of agonists to receptors has been studied for over two decades and has been a major focus since 1986. While some understanding has been gained, it remains not fully elucidated. Once melatonin receptor agonists bind to and activate their receptors, they trigger a variety of physiological processes. MT1 receptors are expressed in multiple regions of the central nervous system (CNS), including the suprachiasmatic nucleus (SNC) of the hypothalamus, hippocampus, substantia nigra, cerebellum, central dopaminergic pathways, ventral tegmental area, and nucleus accumbens. MT1 receptors are also expressed in the retina, ovaries, testes, mammary glands, coronary and aorta, gallbladder, liver, kidneys, skin, and immune system. MT2 receptors are mainly expressed in the central nervous system, and are also found in the lungs, heart, coronary and aortic tissues, myometrium and granulosa cells, immune cells, duodenum and adipocytes. Melatonin binding to melatonin receptors can activate a variety of signaling pathways. MT1 receptor activation can inhibit adenylate cyclase, which leads to a series of chain reactions, first a decrease in cyclic adenosine monophosphate (cAMP) production, then a decrease in protein kinase A (PKA) activity, which in turn prevents the phosphorylation of cAMP response element binding protein (CREB) to phosphorylated CREB (P-CREB). MT1 receptors can also activate phospholipase C (PLC), affecting ion channels and regulating intracellular ion flow. Melatonin binding to MT2 receptors can inhibit adenylate cyclase, thereby reducing cAMP production. [4] It can also inhibit guanylate cyclase, thereby inhibiting the production of cyclic guanosine monophosphate (cGMP). Binding to MT2 receptors may affect phospholipase C (PLC), thereby increasing the activity of protein kinase C (PKC). Activation of the receptor leads to intracellular ion flow. Melatonin is involved in a variety of physiological processes, including circadian rhythms, stress, and reproduction, many of which are mediated by the hypothalamus and pituitary gland. The physiological effects of melatonin are primarily mediated by melatonin receptors. The authors described the distribution of the melatonin receptor MT1 in the human hypothalamus and pituitary gland using immunocytochemistry. MT1 immunoreactivity is widely distributed in the hypothalamus. In addition to the suprachiasmatic nucleus (SCN), neuronal MT1 receptor expression was observed in several novel brain regions, including the paraventricular nucleus (PVN), periventricular nucleus, supraoptic nucleus (SON), sex dimorphic nucleus, Broca's diagonal band, Menette's basal nucleus, infundibular nucleus, ventromedial nucleus and dorsomedial nucleus, tuberomammary body nucleus, mammary bodies, and paraventricular nucleus of the thalamus. No staining was observed in the lateral tuberomammary nucleus and the bed nucleus of the stria terminalis. MT1 receptors co-localize with some angiotensin (AVP) neurons in the suprachiasmatic nucleus (SCN), some small and large cell angiotensin and oxytocin (OXT) neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON), and some small cell corticotropin-releasing hormone (CRH) neurons in the PVN. In the pituitary gland, MT1 is strongly expressed in the pituitary tubercle, but weakly stained in the posterior and anterior lobes. These findings provide a neurobiological basis for melatonin's involvement in regulating multiple functions of the hypothalamus and pituitary gland. The co-localization of MT1 and CRH suggests that melatonin may directly regulate the distribution of the hypothalamic-pituitary-adrenal axis in the paraventricular nucleus (PVN), which may be related to stress states such as depression. One of the main mechanisms by which melatonin reduces the incidence and development of breast cancer is its anti-estrogenic effect, that is, it works by interfering with different levels of estrogen signaling pathways. Melatonin inhibits aromatase activity and expression both in vitro (MCF-7 cells) and in vivo, thus acting as a selective estrogen enzyme regulator. This study aimed to investigate the effect of MT1 melatonin receptor overexpression on the inhibitory effect of melatonin aromatase in MCF-7 breast cancer cells. Transfection of MCF-7 cells with MT1 melatonin receptor significantly reduced cellular aromatase activity; the aromatase activity level in MT1-transfected cells was only 50% of that in vector-transfected MCF-7 cells. In a medium containing testosterone (an indirect indicator of aromatase activity) but without estradiol, the proliferation of estrogen-sensitive MCF-7 cells overexpressing MT1 receptor was strongly inhibited by melatonin. The inhibitory effect of melatonin on cell growth was stronger in MT1-transfected cells than in vector-transfected cells. In MT1-transfected cells, aromatase activity (measured by the tritium-water release assay) was inhibited by melatonin (20% inhibition at 1 nM concentration; 40% inhibition at 10 μM concentration). The same concentration of melatonin had no significant effect on aromatase activity in vector-transfected cells. Compared with vector-transfected MCF-7 cells, MT1 melatonin receptor transfection significantly inhibited aromatase homeostatic mRNA expression by 55% (p<0.001). Furthermore, in MT1-transfected cells, melatonin treatment inhibited aromatase mRNA expression, and the downregulation of aromatase mRNA expression induced by 1 nM melatonin was greater than that in vector-transfected cells (p<0.05). These results indicate that the MT1 melatonin receptor plays a crucial role in mediating the tumor-suppressive effect of melatonin on MCF-7 human breast cancer cells, and confirm that the MT1 melatonin receptor is a major mediator of the melatonin signaling pathway in breast cancer. Almost all melatonin in mammals is synthesized in the pineal gland… Tryptophan is first 5-hydroxylated (catalyzed by tryptophan hydroxylase), then decarboxylated (catalyzed by aromatic L-amino acid decarboxylase) to form serotonin, or 5-hydroxytryptamine. During the day, serotonin in pineal cells tends to be stored and cannot be utilized by the enzymes that normally act on it (monoamine oxidase and melatonin synthase). As night falls, the output of the postganglionic branches of the sympathetic nervous system to the pineal gland increases, followed by the release of norepinephrine from pineal cells, allowing the stored serotonin to be metabolized and utilized intracellularly. Simultaneously, norepinephrine activates enzymes that convert serotonin into melatonin (especially serotonin N-acetyltransferase (SNAT), but also hydroxyindole-O-methyltransferase (HIOMT)). Therefore, pineal melatonin levels significantly increase. Melatonin then diffuses from the pineal gland into the blood and cerebrospinal fluid, rapidly increasing plasma melatonin levels from approximately 2-10 pg/mL to 100-200 pg/mL. Melatonin is an endogenous hormone synthesized from tryptophan in the pineal gland that regulates circadian rhythms, sleep-wake cycles, and seasonal reproduction [4]. Its mechanisms of action include MT1/MT2 receptor-mediated inhibition of adenylate cyclase, regulation of ion channels (Ca²⁺, K⁺) in suprachiasmatic nucleus (SCN) neurons, and direct antioxidant activity by scavenging free radicals [2]. Melatonin is clinically used to treat insomnia, jet lag, and circadian rhythm sleep-wake disorders in the blind [4]. It exerts its anti-inflammatory effects by inhibiting the NF-κB signaling pathway and reducing the production of pro-inflammatory mediators [5]. Melatonin exerts its antitumor activity through multiple mechanisms, including inhibiting tumor cell proliferation, inducing apoptosis, and inhibiting angiogenesis [6].

C13H16N2O2

232.28

232.121

C, 67.22; H, 6.94; N, 12.06; O, 13.78

73-31-4

Melatonin-d4; 66521-38-8; Melatonin-d3; 90735-69-6; Melatonin-d7; 615251-68-8

896

White to light yellow solid powder

1.2±0.1 g/cm3

459.8±55.0 °C at 760 mmHg

116.5-118 °C(lit.)

231.9±31.5 °C

0.0±1.2 mmHg at 25°C

1.580

1.94

2

2

4

17

270

0

O(C([H])([H])[H])C1C([H])=C([H])C2=C(C=1[H])C(=C([H])N2[H])C([H])([H])C([H])([H])N([H])C(C([H])([H])[H])=O

DRLFMBDRBRZALE-UHFFFAOYSA-N

InChI=1S/C13H16N2O2/c1-9(16)14-6-5-10-8-15-13-4-3-11(17-2)7-12(10)13/h3-4,7-8,15H,5-6H2,1-2H3,(H,14,16)

N-[2-(5-methoxy-1H-indol-3-yl)ethyl]acetamide

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 (10.76 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 (10.76 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 (10.76 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (10.76 mM) (saturation unknown) in 2% DMSO + 40% PEG300 + 5% Tween80 + 53% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 2.5 mg/mL (10.76 mM) (saturation unknown) in 2% DMSO 98% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.

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Solubility in Formulation 6: ≥ 1.25 mg/mL (5.38 mM) (saturation unknown) in 10% EtOH + 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 12.5 mg/mL clear EtOH 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 7: ≥ 1.25 mg/mL (5.38 mM) (saturation unknown) in 10% EtOH + 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 12.5 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 8: 5%absolute ethyl alcohol + 95%Corn oil: 2.3mg/ml (9.90mM)

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