MT1 receptor ( Ki = 14 pM ); MT receptor (chicken) ( Ki = 23.1 pM ); MT2 receptor ( Ki = 112 pM )
Ramelteon (TAK-375) acts on MT1 receptor (Ki = 0.54 nM) and MT2 receptor (Ki = 0.92 nM) [1]

In vitro activity: Ramelteon inhibits the synthesis of cAMP in CHO cells stimulated by forskolin, with an IC50 of 21.2 pM. With pKis of 10.05 and 9.70 for recombinant human MT1 and MT2 receptors, respectively, meleton exhibits high affinity for these receptors. With a pEC50 of 11.48, ramelteon inhibits the aggregation of Xenopus laevis melanophore pigment granules. In cerebellar granule cells expressing only one of the two melatonin receptors, as well as in MT1/MT2 cerebellar granule cells, ramelteon (1 nM) increases ERK1/2 phosphorylation. For MT1 KO cerebellar granule cells, 4P-PDOT inhibits the stimulatory effect of Ramelteon (1 nM), whereas for MT2 KO cerebellar granule cells, luzindole attenuates the effect of Ramelteon (1 nM). At 100 μM, ramelteon induces dispersion of any pigment, whereas at 10 μM, melatonin completely disperses aggregated melanophores.

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In membranes from cells expressing human MT1 or MT2 receptors, Ramelteon (TAK-375) competitively displaced the specific melatonin receptor ligand, with high affinity for both receptors (MT1 Ki: 0.54 nM; MT2 Ki: 0.92 nM). It showed negligible affinity for other receptors (e.g., serotonin, dopamine, adrenergic receptors) with Ki values > 10,000 nM [1]
- In CHO cells expressing human MT1 or MT2 receptors, Ramelteon (TAK-375) inhibited forskolin-stimulated cAMP accumulation in a concentration-dependent manner, with EC50 values of 14.5 nM (MT1) and 11.0 nM (MT2), indicating functional agonist activity [1]

Ramelteon (p.o. ; 0.1 and 1 mg/kg) quickens the running wheel activity rhythm's reentrainment to the updated light-dark cycle[3].
Ramelteon (p.o. ; 3, 10, and 30 mg/kg) does not appear to have any negative effects on learning or memory in rats when tested using the water maze task and the delayed match to position task, suggesting that MT1/MT2 receptor agonists are not addictive[3].
Ramelteon (0.0001, 0.001, 0.01, and 0.1 mg/kg; p.o.; 8 hours) increases slow-wave sleep at doses of 0.001, 0.01, and 0.1 mg/kg, increases rapid eye movement sleep at a dose of 0.1 mg/kg, and significantly reduces wakefulness at doses of 0.001, 0.01, and 0.1 mg/kg[4].

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In rats subjected to a 6-hour phase advance of the light-dark cycle, oral administration of Ramelteon (TAK-375) (0.1, 0.3, 1 mg/kg) accelerated the reentrainment of circadian locomotor activity rhythms. The 1 mg/kg dose reduced the number of days to reentrainment by ~30% compared to vehicle [3]
- In freely moving cats, oral administration of Ramelteon (TAK-375) (0.3, 1, 3 mg/kg) dose-dependently increased total sleep time (TST) and non-rapid eye movement (NREM) sleep time, while decreasing wake time. The 3 mg/kg dose increased TST by ~25% and shortened sleep latency by ~40% compared to vehicle, with no significant effect on rapid eye movement (REM) sleep [4]
- In adults with chronic primary insomnia, nightly oral administration of Ramelteon (TAK-375) (8 mg) for 6 months significantly shortened sleep latency (SL) by a mean of 8.2 minutes, increased TST by a mean of 23.4 minutes, and improved sleep efficiency (SE) by a mean of 4.7% compared to baseline. No tolerance to sleep-promoting effects was observed over the 6-month period [2]

The human MT1 gene is inserted into CHO cells via cDNA. At confluence, cells are removed and collected by centrifugation in Hanks' balanced salt solution, which is free of calcium and magnesium and contains 5 mM EDTA. Before the binding tests are carried out, the cells are homogenized in an ice-cold 50 mM Tris-HCl buffer, twice cleaned, pelleted, and kept at -30°C. The thawed homogenate is combined with the test compound and 40 pM 2-[125I]melatonin in a volume of 1 mL, and it is then incubated for 150 minutes at 25°C. After adding 3 mL of ice-cold buffer and vacuum-filtering the mixture through a Whatman GF/B, the reaction is stopped. A g-counter is used to count the radioactivity after the filter has been cleaned twice.

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Membrane preparations were obtained from cells stably expressing human MT1 or MT2 receptors. Membranes were incubated with a fixed concentration of tritiated melatonin (specific ligand) and various concentrations of Ramelteon (TAK-375) at 25°C for 90 minutes. Bound ligand was separated from free ligand by filtration, and radioactivity was measured. Competition binding curves were analyzed to calculate Ki values [1]
- For cAMP functional assay, CHO cells expressing MT1 or MT2 receptors were preincubated with Ramelteon (TAK-375) for 15 minutes, followed by stimulation with forskolin (10 μM) for 30 minutes. Intracellular cAMP levels were quantified using a cyclic AMP assay kit, and concentration-response curves were generated to determine EC50 values [1]

Ramelteon exhibits a very high affinity with Ki values of 14.0, 112, and 23.1 pM for chick forebrain melatonin receptors (comprising of melatonin1 and melatonin2 receptors) and human melatonin1 and melatonin2 receptors (expressed in CHO cells). Ramelteon's hamster brain melatonin3 binding site affinity is incredibly weak (Ki: 2.65 μM) in comparison to melatonin's (24.1 nM) affinity for the same binding site. Furthermore, there is no discernible affinity for any of the many ligand binding sites (benzodiazepine receptors, dopamine receptors, opiate receptors, ion channels, and transporters) or impact on the activity of different enzymes that ramelteon is supposed to inhibit. In CHO cells expressing human melatonin1 and melatonin2 receptors, ramelteon inhibits the production of cAMP stimulated by forskolin.

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CHO cells were transfected with plasmids encoding human MT1 or MT2 receptors and selected for stable expression. Cells were seeded in 96-well plates and cultured to 80% confluence. Prior to assay, cells were serum-starved for 24 hours. Ramelteon (TAK-375) was added at concentrations ranging from 0.01 to 1000 nM, followed by forskolin stimulation. cAMP levels were measured to assess agonist activity [1]

Dissolved in 0.5% methylcellulose solution; 1 mg/kg; oral gavage
Estrogen-deficient ovariectomized (OVX) rats

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Rats: Male Wistar rats were housed under a 12:12 light-dark cycle (LD 12:12) for acclimation. After baseline activity recording for 7 days, the light-dark cycle was advanced by 6 hours (new LD 06:00-18:00). Ramelteon (TAK-375) was administered orally once daily at ZT 13 (1 hour after dark onset) for 7 days at doses of 0.1, 0.3, or 1 mg/kg. Locomotor activity was recorded using activity monitors, and reentrainment time was defined as the number of days to reach stable activity rhythms under the new LD cycle [3]
- Cats: Adult male cats were surgically implanted with electrodes for polysomnographic recording (EEG, EMG, EOG) and housed in sound-attenuated chambers under LD 12:12. After recovery and acclimation, Ramelteon (TAK-375) was administered orally at doses of 0.3, 1, or 3 mg/kg 30 minutes before dark onset. Polysomnographic data were recorded for 12 hours (dark period) and analyzed for wake time, NREM sleep, and REM sleep duration [4]
- Humans: Adults with chronic primary insomnia (DSM-IV criteria) were enrolled in a 6-month open-label study. Ramelteon (TAK-375) was administered orally at 8 mg nightly, 30 minutes before bedtime. Sleep parameters were assessed monthly using sleep diaries and polysomnography (at baseline, month 1, and month 6) [2]

Absorption, Distribution and Excretion
Absorption is rapid, with a total absorption rate of at least 84%. Following oral administration of radiolabeled ramelteamide, 84% of the total radioactivity is excreted in the urine and approximately 4% in the feces, with a mean recovery rate of 88%. Less than 0.1% of the dose is excreted in the form of the parent compound in the urine and feces. 73.6 L In vitro, ramelteamide exhibits approximately 82% protein binding in human serum, regardless of concentration. Since 70% of the drug binds to human serum albumin, most of the binding is albumin-bound. Ramelteamide is not selectively distributed to erythrocytes. Following intravenous administration of ramelteamide, the mean volume of distribution is 73.6 L, indicating extensive tissue distribution. Ramelteamide is rapidly absorbed; after oral administration on an empty stomach, the median peak concentration occurs at approximately 0.75 hours (range: 0.5 to 1.5 hours). Although the total absorption rate of ramelteinamide is at least 84%, its absolute oral bioavailability is only 1.8% due to extensive first-pass metabolism. Ramelteinamide is distributed in rat milk; it is unclear whether it is distributed in human milk. Following oral administration of radiolabeled ramelteinamide, 84% of the total radioactivity is excreted in the urine and approximately 4% in the feces, with an average recovery rate of 88%. Less than 0.1% of the drug is excreted unchanged in urine and feces. The drug is essentially eliminated 96 hours after administration. Due to the short elimination half-life of ramelteinamide (average 1–2.6 hours), repeated once-daily administration of ramelteinamide does not lead to significant drug accumulation. The half-life of metabolite M-II is 2 to 5 hours and is dose-independent. Within 24 hours, the concentrations of both the unchanged drug and its metabolites in human serum reach or fall below the lower limit of quantitation.

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Metabolism/Metabolites
Hepatic Metabolism

The metabolism of ramelteamide mainly involves oxidation to produce hydroxyl and carbonyl derivatives, with glucuronide conjugates being a minor metabolite. CYP1A2 is the main isoenzyme involved in the hepatic metabolism of ramelteamide; the CYP2C subfamily and CYP3A4 isoenzymes also participate, but to a lesser extent. The order of concentration of the main metabolites in human serum is M-II, M-IV, MI, and M-III. These metabolites are rapidly generated, exhibit a monophasic decline, and are rapidly eliminated. The overall mean systemic exposure to M-II is approximately 20 to 100 times higher than that of the parent drug.

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Biological Half-Life
~1–2.6 hours

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In humans, after oral administration of ramelteamide (TAK-375) (8 mg), the peak plasma concentration (Cmax) is approximately 1.1 ng/mL, and the median time to peak concentration (Tmax) is 0.75 hours. The apparent oral bioavailability is approximately 8%, and the terminal elimination half-life (t1/2) is approximately 1-2 hours [2]. Rametamide (TAK-375) is primarily metabolized in the liver, mainly via CYP1A2, with smaller contributions from CYP2C9 and CYP3A4. The main metabolites are inactive and are primarily excreted in urine (approximately 84%) and feces (approximately 4%) [1].

Hepatotoxicity
In multiple clinical trials, ramelteinamide was well tolerated, with no evidence of elevated serum enzymes or liver injury. Despite its widespread use, there is no conclusive evidence linking it to clinically significant liver injury. There has been a case report of a patient with alcoholic liver disease experiencing worsening liver disease, accompanied by jaundice, ascites, bacterial peritonitis, and death after one month of ramelteinamide treatment. Ramelteinamide is not recommended for patients with impaired liver function. Probability Score: E (Unproven, but suspected as a rare, clinically significant cause of liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Data from one patient showed low levels of ramelteinamide and its main active metabolite in breast milk. Infant lethargy and feeding should be monitored, especially in newborns or preterm infants. Alternative medications may be preferred until more data are available.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
Non-breastfeeding women with chronic insomnia who took ramelteamide 16 mg nightly for 6 months had an increased prolactin level of 4.9 mcg/L (34%). No clinical symptoms of hyperprolactinemia were reported. For mothers who have established lactation, prolactin levels may not affect their ability to breastfeed.

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Protein binding
~82% (in human serum)

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Interactions
Substrates of CYP isoenzymes 1A2 (e.g., theophylline), 2C9 (e.g., warfarin), 2C19 (e.g., omeprazole), 2D6 (e.g., dextromethorphan), or 3A4 (e.g., midazolam): Pharmacokinetic interactions are unlikely.
CYP isoenzyme inducers: Pharmacokinetic interactions (decreased concentrations of rameltein and its active metabolites) were observed when used in combination with rifampin. The efficacy of rameltein may be reduced when used in combination with potent CYP inducers such as rifampin.
CYP2D6 isoenzyme inhibitors: The likelihood of pharmacokinetic interactions with fluoxetine is very low.
CYP2C9 isoenzyme inhibitors: Pharmacokinetic interactions (increased concentrations of rameltein and its active metabolites) were observed when used in combination with fluconazole. Caution should be exercised when used in combination with fluconazole or other potent CYP2C9 inhibitors.
For more complete data on interactions of rameltein (7 items in total), please visit the HSDB record page.

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In a 6-month human study, rameltein (TAK-375) was well tolerated. The most common adverse events were headache (12.3%), dizziness (7.8%), and nausea (5.2%), ranging in severity from mild to moderate. No significant changes were observed in liver function tests (ALT, AST, bilirubin) or kidney function tests (creatinine, BUN) [2]
- Rametamide (TAK-375) has a plasma protein binding rate of approximately 82-85% in the human body, and its binding is not concentration-dependent [1]

[1]. Neurochemical properties of ramelteon (TAK-375), a selective MT1/MT2 receptor agonist. Neuropharmacology. 2005;48(2):301-310.

[2]. Efficacy and safety of 6-month nightly ramelteon administration in adults with chronic primary insomnia. Sleep. 2009;32(3):351-360.

[3]. Ramelteon (TAK-375) accelerates reentrainment of circadian rhythm after a phase advance of the light-dark cycle in rats. J Biol Rhythms. 2005;20(1):27-37.

[4]. The sleep-promoting action of ramelteon (TAK-375) in freely moving cats. Sleep. 2004;27(7):1319-1325.

N-[2-[(8S)-2,6,7,8-tetrahydro-1H-cyclopentano[e]benzofuran-8-yl]ethyl]propionamide belongs to the indane class of compounds. Rametamide is the first novel sleep drug that selectively binds to melatonin receptors in the suprachiasmatic nucleus (SCN). It is used to treat insomnia, particularly difficulty falling asleep. Rametamide has not shown dependence or abuse potential. Rametamide is a melatonin receptor agonist. The mechanism of action of rametamide is as a melatonin receptor agonist. Rametamide is a melatonin receptor agonist used to treat insomnia. Rametamide has not been shown to cause elevated serum enzymes or clinically significant liver damage. Rametamide is a synthetic melatonin analog with hypnotic and circadian rhythm-regulating effects. Rametamide binds to and activates melatonin receptors 1 and 2 in the suprachiasmatic nucleus (SCN) of the brain, thereby promoting sleep. Unlike non-benzodiazepine sedative-hypnotics zolpidem and zaleplon, this product does not activate GABA receptors and therefore does not produce GABA receptor-mediated anxiolytic, muscle relaxant, and amnesic effects. Drug Indications For the treatment of insomnia with difficulty falling asleep. FDA Label Mechanism of Action Ramelteamide is a melatonin receptor agonist with high affinity for melatonin MT1 and MT2 receptors and low selectivity for MT3 receptors. Melatonin production is synchronized with nighttime sleep, meaning that elevated melatonin levels are associated with increased self-reported drowsiness and sleepiness. MT1 receptors are thought to regulate drowsiness and promote sleep onset, while MT2 receptors are thought to mediate the phase regulation of melatonin's circadian rhythm. While MT1 and MT2 receptors are associated with the sleep-wake cycle, MT3 receptors have entirely different properties and are therefore unlikely to be involved in the sleep-wake cycle. Ramelteamide has no significant affinity for GABA receptor complexes or receptors that bind neuropeptides, cytokines, serotonin, dopamine, norepinephrine, acetylcholine, or opioids. Ramelteamide is a melatonin receptor agonist with high affinity for melatonin MT1 and MT2 receptors and selectivity for MT3 receptors. Ramelteamide exhibits full agonist activity in vitro on cells expressing human MT1 or MT2 receptors. The activity of ramelteamide on MT1 and MT2 receptors is thought to contribute to its sleep-promoting effects because these receptors are acted upon by endogenous melatonin and are believed to be involved in maintaining the circadian rhythm of the normal sleep-wake cycle. Ramelteamide has no significant affinity for GABA receptor complexes or receptors that bind neuropeptides, cytokines, serotonin, dopamine, norepinephrine, acetylcholine, or opioids. Ramelteamide also does not interfere with the activity of many specific enzymes in a standard enzyme profile. The major metabolite of ramelteinamide, M-II, is active, with binding affinities to human MT1 and MT2 receptors approximately one-tenth and one-fifth that of the parent drug, respectively. In vitro functional studies show that its potency is 17 to 25 times lower than that of ramelteinamide. Although M-II's potency to MT1 and MT2 receptors is lower than that of the parent drug, its circulating concentration is higher, with average systemic exposure 20 to 100 times higher than that of ramelteinamide. M-II has a weak affinity for the 5-HT2B receptor but very low affinity for other receptors or enzymes. Similar to ramelteinamide, M-II does not interfere with the activity of many endogenous enzymes. Other known metabolites of ramelteinamide are inactive.
Therapeutic Uses
Ramelteinamide is used to treat insomnia with difficulty falling asleep.

/See US product label for usage details/
Therapeutic Category: Sedative-Hypnotics
Drug Warnings

Ramettemid has not been shown to have respiratory depression in patients with mild to moderate chronic obstructive pulmonary disease (COPD). The effects of ramettemid on patients with severe COPD (e.g., those with elevated PCO2 requiring nighttime oxygen therapy) have not been investigated, and therefore its use is not recommended in these patients. In studies of patients with mild to moderate obstructive sleep apnea, ramettemid did not differentiate the results of apnea index measurements.¹However, the effects of ramettemid on severe obstructive sleep apnea have not been investigated, and therefore its use is not recommended in such patients.
In a 35-night randomized study evaluating the next-day residual effects of ramettemid, adult patients taking 8 mg of the drug nightly experienced increased immediate/delayed memory loss and symptoms such as somnolence, fatigue, and irritability during weeks 1 and 3 of treatment compared to patients receiving placebo. However, at week 5.1, there was no significant difference in the next-day residual effect between the ramelteinamide group and the placebo group. A similar study in elderly patients, taking 4 or 8 mg of ramelteinamide nightly, also found no significant difference in residual effect measures. Studies using subjective measurement methods (e.g., questionnaires) have not found evidence of withdrawal syndromes (including rebound insomnia) after discontinuation of long-term ramelteinamide treatment (4, 8, or 16 mg daily for up to 35 days). No evidence of abuse potential has been found in patients with a history of drug abuse or dependence (e.g., sedative-hypnotic drugs, anxiolytics) after administration of relatively high doses of ramelteinamide (up to 20 times the recommended hypnotic dose). Ramelteinamide does not appear to cause physical dependence. For more complete data on ramelteinamide warnings (18 in total), please visit the HSDB record page.

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Pharmacodynamics
Ramelteinamide is the first selective melatonin agonist. Its mechanism of action mimics melatonin (MT), a natural hormone produced during sleep and thought to regulate the circadian rhythm of the normal sleep-wake cycle. Ramelteamide has a high affinity for MT1 and MT2 receptors. These receptors are located in the suprachiasmatic nucleus (SCN) of the brain, often referred to as the body's "master clock" because it regulates the 24-hour sleep-wake cycle. Ramelteamide's active metabolite has lower potency, but its circulating concentration is higher than that of the parent compound. This metabolite also has a weaker affinity for the 5-HT2B receptor.

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Rametiamide (TAK-375) is a selective MT1/MT2 receptor agonist that acts on the circadian rhythm regulation system of the suprachiasmatic nucleus (SCN) to promote sleep initiation and regulate circadian rhythm synchronization[1]
- Its mechanism of action involves activating MT1 and MT2 receptors in the SCN, thereby inhibiting adenylate cyclase, reducing cAMP levels, and regulating the firing frequency of SCN neurons to synchronize the circadian rhythm with the light-dark cycle[1]
- It is suitable for the treatment of chronic primary insomnia, especially for improving sleep latency[2]

C16H21NO2

259.34

259.157

C, 74.10; H, 8.16; N, 5.40; O, 12.34

196597-26-9

Ramelteon-d3; 1432057-38-9; Ramelteon-d5; 1134159-63-9

208902

White to off-white solid powder

1.1±0.1 g/cm3

455.3±24.0 °C at 760 mmHg

113-115ºC

229.2±22.9 °C

0.0±1.1 mmHg at 25°C

1.556

2.57

1

2

4

19

331

1

O1C([H])([H])C([H])([H])C2=C1C([H])=C([H])C1C([H])([H])C([H])([H])C([H])(C([H])([H])C([H])([H])N([H])C(C([H])([H])C([H])([H])[H])=O)C=12

YLXDSYKOBKBWJQ-LBPRGKRZSA-N

InChI=1S/C16H21NO2/c1-2-15(18)17-9-7-12-4-3-11-5-6-14-13(16(11)12)8-10-19-14/h5-6,12H,2-4,7-10H2,1H3,(H,17,18)/t12-/m0/s1

N-[2-[(8S)-2,6,7,8-tetrahydro-1H-cyclopenta[e][1]benzofuran-8-yl]ethyl]propanamide

TAK-375; TAK375; trade name: Rozerem; TAK 375

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.64 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.64 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (9.64 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: 0.5% methylcellulose: 30 mg/mL

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