Human Endogenous Metabolite; synthetic form of the thyroid hormone thyroxine (T4)

Thyroid stimulating hormone (TSH) levels are linked to deiodinases (DIOs), which catalyze the transformation of thyroxine (pro-hormone) to the active thyroid hormone. While DIO3 plays a role in inactivating the secretion, DIO1 and DIO2 catalyze the activation of thyroid hormone secretion. The negative feedback regulation of pituitary TSH secretion is largely dependent on the activities of DIO1 and DIO2[1]. Ionic channels, pumps, and regulatory contractile proteins are known to have their expression modulated by the hormones triiodothyronine (T3) and levothyroxine (T4). Additionally, it has been demonstrated that thyroid hormones affect the calcium flux and homeostasis that are in charge of excitation and contractility, with L-thyroxine and triiodothyronine influencing the pharmacological regulation and secretion of this process. Rats fed an iodine-free diet for 12 weeks showed a significant reduction in their levels of L-thyroxine and triiodothyronine compared to the control group fed a standard diet (p<0.001). L-thyroxine levels rise (p=0.02) in the group receiving low doses of the medication, but triiodothyronine levels essentially stay the same (p=0.19) as in the control group. Rats given large doses of L-thyroxine show a significant increase in circulating concentrations of both triiodothyronine and L-thyroxine relative to the hypothyroid group that was not treated (p<0.001 and p=0.004, respectively), as well as a significant increase in L-thyroxine levels relative to the control values (p=0.03)[2].

Thyroid-stimulating hormone (TSH) levels are correlated with the catalysis of thyroxine (prohormone) conversion to active thyroid hormone by deiodinase (DIO). Thyroid hormone secretion is activated by DIO1 and DIO2, whereas secretion is inactivated by DIO3. The regulation of pituitary TSH secretion by negative feedback is largely dependent on the actions of DIO1 and DIO2 [1]. The expression of ion channels, pumps, and regulating contractile proteins is regulated by the hormones triiodothyronine (T3) and L-thyroxine (T4). Moreover, it has been demonstrated that thyroid hormones affect calcium homeostasis and flux, which are in charge of excitation and contraction. Triiodothyronine and L-thyroxine are known to modify the pharmacological regulation and secretion of calcium. Triiodothyronine and L-thyroxine levels significantly decreased (p<0.001) in rats given an iodine-free diet for 12 weeks as compared to controls given a regular diet. Triiodothyronine levels were essentially comparable to those in the control group (p=0.19), but an increase in L-thyroxine was noted in the low-dose L-thyroxine treatment group (p=0.02). Rats treated with high-dose L-thyroxine showed significantly higher circulating concentrations of both triiodothyronine and L-thyroxine compared to the untreated hypothyroid group (p<0.001 and p=0.004, respectively), and L-thyroxine levels were significantly higher than the control value (p=0.03)[2].

Biochemical techniques[2] ELISA assays were performed using a standard rat Thyroxine (T4) and T3 ELISA kit according to the manufacturer's protocol. Western blot analysis was performed exactly as previously described.

Rats: The experiment uses 22 female Sprague-Dawley rats. There are four groups of non-pregnant rats: 1) No thyroid function, 2) hypothyroidism, 3) hypothyroidism treated with low doses of L-thyroxine (20 μg/kg/day), and 4) high doses of L-thyroxine (100 μg/kg/day). While the intervention rats (groups 2-4) are fed an iodine-free diet for 12 weeks to induce hypothyroidism, the control group (group 1) is fed a standard diet. This is followed by an additional 4 weeks of feeding to allow for L-thyroxine treatment and screening for hypothyroidism. You have unlimited access to food and water (iodine-free diet). Groups 3 and 4, which represent the hypothyroid group, receive intraperitoneal injections of 20 μg/kg and 100 μg/kg of L-thyroxine per day, respectively, every 24 hours. Within weeks 12 and 16 of starting the iodine-free or control diet, blood samples are taken for thyroid function screening. After treatment, a hysterectomy is performed under general anesthesia (isoflurane 2%), and the two uterine horns are kept in physiological Krebs' solution until isometric tension measurements are taken, which should take no longer than an hour.

bsorption is increased in the fasting state and decreased in malabsorption states (e.g., sprue); absorption also may decrease with age. American Society of Health-System Pharmacists 2015; Drug Information 2015. Bethesda, MD. 2015, p. 3230

For more Absorption, Distribution and Excretion (Complete) data for LEVOTHYROXINE (7 total), please visit the HSDB record page.

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Metabolism / Metabolites
Approximately 70% of secreted T4 is deiodinated to equal amounts of T3 and reverse triiodothyronine (rT3), which is calorigenically inactive. T4 is slowly eliminated through its major metabolic pathway to T3 via sequential deiodination, where approximately 80% of circulating T3 is derived from peripheral T4. The liver is the major site of degradation for both T4 and T3, with T4 deiodination also occurring at a number of additional sites, including the kidney and other tissues. Elimination of T4 and T3 involves hepatic conjugation to glucuronic and sulfuric acids. The hormones undergo enterohepatic circulation as conjugates are hydrolyzed in the intestine and reabsorbed. Conjugated compounds that reach the colon are hydrolyzed and eliminated as free compounds in the feces. Other minor T4 metabolites have been identified.

Yields l-tyrosine in rabbit, in rat /From table/ Goodwin, B.L. Handbook of Intermediary Metabolism of Aromatic Compounds. New York: Wiley, 1976., p. T-14

Yields 3,3',5-triiodo-L-thyronine in man, rat, dog, rabbit. /From table/ Goodwin, B.L. Handbook of Intermediary Metabolism of Aromatic Compounds. New York: Wiley, 1976., p. T-14

Yields l-thyroxine-4'-beta-d-glucuronide in dog, in man, in rat. Yields l-thyroxine-4'-sulfate in dog. /From table/ Goodwin, B.L. Handbook of Intermediary Metabolism of Aromatic Compounds. New York: Wiley, 1976., p. T-14

Yields 3,3',5,5'-tetraiodothyropyruvic acid in rat. Yields l-thyronine in rat. /From table/ Goodwin, B.L. Handbook of Intermediary Metabolism of Aromatic Compounds. New York: Wiley, 1976., p. T-14

Yields 3,3'-diiodo-l-thyronine in dog. Yields 3,3',5,5'-tetraiodothyroacetic acid in man, in rat. /From table/ Goodwin, B.L. Handbook of Intermediary Metabolism of Aromatic Compounds. New York: Wiley, 1976., p. T-14

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Biological Half-Life
T₄ half-life is 6 to 7 days. T₃ half-life is 1 to 2 days.

In dogs orally administered levothyroxine has relatively ... short elimination half life when compared to humans. ... The serum half life is approximately 12-16 hours. Plumb D.C. Veterinary Drug Handbook. 8th ed. (pocket). Ames, IA: Wiley-Blackwell, 2015., p. 842

The usual plasma half-lives of thyroxine and triiodothyronine are 6-7 days and approximately 1-2 days, respectively. The plasma half-lives of thyroxine and triiodothyronine are decreased in patients with hyperthyroidism and increased in those with hypothyroidism.

Drug Warnings
/BOXED WARNING/ WARNING: NOT FOR TREATMENT OF OBESITY OR FOR WEIGHT LOSS. Thyroid hormones, including Levothyroxine Sodium for Injection, should not be used for the treatment of obesity or for weight loss. Larger doses may produce serious or even life threatening manifestations of toxicity.

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Toxicity Summary
IDENTIFICATION AND USE: Levothyroxine occurs as crystals or needles. As a drug, levothyroxine sodium is used as replacement or supplemental therapy in congenital or acquired hypothyroidism. It is also used in the treatment or prevention of various types of euthyroid goiters, including thyroid nodules, subacute or chronic lymphocytic thyroiditis (Hashimoto's thyroiditis), multinodular goiter and, as an adjunct to surgery and radioiodine therapy in the management of thyrotropin-dependent well-differentiated thyroid cancer. The injection form of levothyroxine sodium is indicated for the treatment of myxedema coma. Levothyroxine has also been used in veterinary medicine. HUMAN EXPOSURE AND TOXICITY: The signs and symptoms of levothyroxine overdosage are those of hyperthyroidism. In addition, confusion and disorientation may occur. Cerebral embolism, shock, coma, and death have been reported. Studies indicate that careful attention is necessary when initiating the administration of levothyroxine sodium to very-low-birth-weight infants. Ingestion of levothyroxine in children typically follows a benign course, but overdose can result in significant complications, including seizures and arrhythmias, both of which should be monitored for. In one genotoxicity study, the ability of thyroxine to induce sister chromatid exchange and micronuclei was tested in cultured human lymphocytes. Thyroxine exhibited weak clastogenic effects only at high concentrations. ANIMAL STUDIES: A single acute overdose in small animals is less likely to cause severe thyrotoxicosis than chronic overdosage. Vomiting, diarrhea, transition from hyperactivity to lethargy, hypertension, tachycardia, tachypnea, dyspnea, and abnormal pupillary light reflexes may be noted in dogs and cats. In dogs, clinical signs may appear within 1-9 hours after ingestion. Four pregnant New Zealand white rabbits received intramuscular levothyroxine at 250 ug/kg on days 25 and 26 of gestation. Maternal and fetal plasma-free levothyroxine concentration was higher than in controls, with the highest concentration noted at 14 days of neonatal period. Treatment resulted in fetal hyperglycemia and depletion of fetal liver glycogen content. Animal studies have not been performed to evaluate levothyroxine carcinogenic potential, mutagenic potential or effects on fertility.

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Protein Binding
Circulating thyroid hormones are greater than 99% bound to plasma proteins, including thyroxine-binding globulin (TBG), thyroxine-binding prealbumin (TBPA) and albumin (TBA). The higher affinity of both TBG and TBPA for T4 partially explains the higher serum levels, slower metabolic clearance and longer half-life of T4 compared to T3. Protein-bound thyroid hormones exist in reverse equilibrium with small amounts of free hormone where only unbound hormone is metabolically active.

[1]. Arici M, et al. Association between genetic polymorphism and levothyroxine bioavailability in hypothyroid patients. Endocr J. 2018 Mar 28;65(3):317-323.
[2]. Corriveau S, et al. Levothyroxine treatment generates an abnormal uterine contractility patterns in an in vitro animalmodel. J Clin Transl Endocrinol. 2015 Sep 9;2(4):144-149.

Levothyroxine Sodium is the sodium salt of levothyroxine, a synthetic levoisomer of thyroxine (T4) that is similar to the endogenous hormone produced by the thyroid gland. In peripheral tissues, levothyroxine is deiodinated by 5'-deiodinase to form triiodothyronine (T3). T3 enters the cell and binds to nuclear thyroid hormone receptors; the activated hormone-receptor complex in turn triggers gene expression and produces proteins required in the regulation of cellular respiration; thermogenesis; cellular growth and differentiation; and the metabolism of proteins, carbohydrates and lipids. T3 also exhibits cardiostimulatory effects.

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The major hormone derived from the thyroid gland. Thyroxine is synthesized via the iodination of tyrosines (MONOIODOTYROSINE) and the coupling of iodotyrosines (DIIODOTYROSINE) in the THYROGLOBULIN. Thyroxine is released from thyroglobulin by proteolysis and secreted into the blood. Thyroxine is peripherally deiodinated to form TRIIODOTHYRONINE which exerts a broad spectrum of stimulatory effects on cell metabolism.

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L-thyroxine is the L-enantiomer of thyroxine. It has a role as a thyroid hormone, an antithyroid drug, a human metabolite and a mouse metabolite. It is a thyroxine, an iodophenol, a 2-halophenol, a L-phenylalanine derivative and a non-proteinogenic L-alpha-amino acid. It is a conjugate acid of a L-thyroxine(1-). It is an enantiomer of a D-thyroxine. It is a tautomer of a L-thyroxine zwitterion.

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Levothyroxine is a synthetically produced form of thyroxine, a major endogenous hormone secreted by the thyroid gland. Also known as L-thyroxine or the brand name product Synthroid, levothyroxine is used primarily to treat hypothyroidism, a condition where the thyroid gland is no longer able to produce sufficient quantities of the thyroid hormones T4 (tetraiodothyronine or thyroxine) and T3 (triiodothyronine or [DB00279]), resulting in diminished down-stream effects of these hormones. Without sufficient quantities of circulating thyroid hormones, symptoms of hypothyroidism begin to develop such as fatigue, increased heart rate, depression, dry skin and hair, muscle cramps, constipation, weight gain, memory impairment, and poor tolerance to cold temperatures. In response to Thyroid Stimulating Hormone (TSH) release by the pituitary gland, a normally functioning thyroid gland will produce and secrete T4, which is then converted through deiodination (by type I or type II 5′-deiodinases) into its active metabolite T3. While T4 is the major product secreted by the thyroid gland, T3 exerts the majority of the physiological effects of the thyroid hormones; T4 and T3 have a relative potency of ~1:4 (T4:T3). T4 and T3 act on nearly every cell of the body, but have a particularly strong effect on the cardiac system. As a result, many cardiac functions including heart rate, cardiac output, and systemic vascular resistance are closely linked to thyroid status. Prior to the development of levothyroxine, [DB09100] or desiccated thyroid, used to be the mainstay of treatment for hypothyroidism. However, this is no longer recommended for the majority of patients due to several clinical concerns including limited controlled trials supporting its use. Desiccated thyroid products contain a ratio of T4 to T3 of 4.2:1, which is significantly lower than the 14:1 ratio of secretion by the human thyroid gland. This higher proportion of T3 in desiccated thyroid products can lead to supraphysiologic levels of T3 which may put patients at risk of thyrotoxicosis if thyroid extract therapy is not adjusted according to the serum TSH.

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Thyroxine is a metabolite found in or produced by Escherichia coli (strain K12, MG1655). Levothyroxine is a l-Thyroxine.

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Levothyroxine has been reported in Homo sapiens and Bos taurus with data available. Levothyroxine is a synthetic levoisomer of thyroxine (T4), similar to the endogenous hormone produced by the thyroid gland. Thyroxine is de-iodinated to form triiodothyronine (T3) in the peripheral tissues. T3 enters the cell and binds to nuclear thyroid hormone receptors, and the hormone-receptor complex in turn triggers gene expression and produces proteins required in the regulation of cellular respiration, thermogenesis, cellular growth and differentiation, and metabolism of proteins, carbohydrates and lipids. T4 and T3 also possess cardiac stimulatory effect.

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Levothyroxine Sodium is the sodium salt of levothyroxine, a synthetic levoisomer of thyroxine (T4) that is similar to the endogenous hormone produced by the thyroid gland. In peripheral tissues, levothyroxine is deiodinated by 5'-deiodinase to form triiodothyronine (T3). T3 enters the cell and binds to nuclear thyroid hormone receptors; the activated hormone-receptor complex in turn triggers gene expression and produces proteins required in the regulation of cellular respiration; thermogenesis; cellular growth and differentiation; and the metabolism of proteins, carbohydrates and lipids. T3 also exhibits cardiostimulatory effects.

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Thyroxine is a hormone synthesized and secreted by the thyroid gland containing four iodine atoms and is converted to triiodothyronine (T3) in the body, influencing metabolism and organ function. The major hormone derived from the thyroid gland. Thyroxine is synthesized via the iodination of tyrosines (MONOIODOTYROSINE) and the coupling of iodotyrosines (DIIODOTYROSINE) in the THYROGLOBULIN. Thyroxine is released from thyroglobulin by proteolysis and secreted into the blood. Thyroxine is peripherally deiodinated to form TRIIODOTHYRONINE which exerts a broad spectrum of stimulatory effects on cell metabolism.

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Drug Indication
Levothyroxine is indicated as replacement therapy in primary (thyroidal), secondary (pituitary) and tertiary (hypothalamic) congenital or acquired hypothyroidism. It is also indicated as an adjunct to surgery and radioiodine therapy in the management of thyrotropin-dependent well-differentiated thyroid cancer.

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Therapeutic Uses
/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.

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Pharmacodynamics
Oral levothyroxine is a synthetic hormone that exerts the same physiologic effect as endogenous T4, thereby maintaining normal T4 levels when a deficiency is present. Levothyroxine has a narrow therapeutic index and is titrated to maintain a euthyroid state with TSH (thyroid stimulating hormone) within a therapeutic range of 0.4–4.0 mIU/L. Over- or under-treatment with levothyroxine may have negative effects on growth and development, cardiovascular function, bone metabolism, reproductive function, cognitive function, emotional state, gastrointestinal function and glucose and lipid metabolism. The dose of levothyroxine should be titrated slowly and carefully and patients should be monitored for their response to titration to avoid these effects. TSH levels should be monitored at least yearly to avoid over-treating with levothyroxine which can result in hyperthyroidism (TSH <0.1mIU/L) and symptoms of increased heart rate, diarrhea, tremor, hypercalcemia, and weakness to name a few. As many cardiac functions including heart rate, cardiac output, and systemic vascular resistance are closely linked to thyroid status, over-treatment with levothyroxine may result in increases in heart rate, cardiac wall thickness, and cardiac contractility and may precipitate angina or arrhythmias, particularly in patients with cardiovascular disease and in elderly patients. In populations with any cardiac concerns, levothyroxine should be initiated at lower doses than those recommended in younger individuals or in patients without cardiac disease. Patients receiving concomitant levothyroxine and sympathomimetic agents should be monitored for signs and symptoms of coronary insufficiency. If cardiac symptoms develop or worsen, reduce the levothyroxine dose or withhold for one week and restart at a lower dose. Increased bone resorption and decreased bone mineral density may occur as a result of levothyroxine over-replacement, particularly in post-menopausal women. The increased bone resorption may be associated with increased serum levels and urinary excretion of calcium and phosphorous, elevations in bone alkaline phosphatase and suppressed serum parathyroid hormone levels. Administer the minimum dose of levothyroxine that achieves the desired clinical and biochemical response to mitigate this risk. Addition of levothyroxine therapy in patients with diabetes mellitus may worsen glycemic control and result in increased antidiabetic agent or insulin requirements. Carefully monitor glycemic control after starting, changing or discontinuing levothyroxine.

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Mechanism of Action
Levothyroxine is a synthetically prepared levo-isomer of the thyroid hormone thyroxine (T4, a tetra-iodinated tyrosine derivative) that acts as a replacement in deficiency syndromes such as hypothyroidism. T4 is the major hormone secreted from the thyroid gland and is chemically identical to the naturally secreted T4: it increases metabolic rate, decreases thyroid-stimulating hormone (TSH) production from the anterior lobe of the pituitary gland, and, in peripheral tissues, is converted to T3. Thyroxine is released from its precursor protein thyroglobulin through proteolysis and secreted into the blood where is it then peripherally deiodinated to form triiodothyronine (T3) which exerts a broad spectrum of stimulatory effects on cell metabolism. T4 and T3 have a relative potency of ~1:4. Thyroid hormone increases the metabolic rate of cells of all tissues in the body. In the fetus and newborn, thyroid hormone is important for the growth and development of all tissues including bones and the brain. In adults, thyroid hormone helps to maintain brain function, food metabolism, and body temperature, among other effects. The symptoms of thyroid deficiency relieved by levothyroxine include slow speech, lack of energy, weight gain, hair loss, dry thick skin and unusual sensitivity to cold. The thyroid hormones have been shown to exert both genomic and non-genomic effects. They exert their genomic effects by diffusing into the cell nucleus and binding to thyroid hormone receptors in DNA regions called thyroid hormone response elements (TREs) near genes. This complex of T4, T3, DNA, and other coregulatory proteins causes a conformational change and a resulting shift in transcriptional regulation of nearby genes, synthesis of messenger RNA, and cytoplasmic protein production. For example, in cardiac tissues T3 has been shown to regulate the genes for α- and β-myosin heavy chains, production of the sarcoplasmic reticulum proteins calcium-activated ATPase (Ca2+-ATPase) and phospholamban, β-adrenergic receptors, guanine-nucleotide regulatory proteins, and adenylyl cyclase types V and VI as well as several plasma-membrane ion transporters, such as Na+/K+–ATPase, Na+/Ca2+ exchanger, and voltage-gated potassium channels, including Kv1.5, Kv4.2, and Kv4.3. As a result, many cardiac functions including heart rate, cardiac output, and systemic vascular resistance are closely linked to thyroid status. The non-genomic actions of the thyroid hormones have been shown to occur through binding to a plasma membrane receptor integrin aVb3 at the Arg-Gly-Asp recognition site. From the cell-surface, T4 binding to integrin results in down-stream effects including activation of mitogen-activated protein kinase (MAPK; ERK1/2) and causes subsequent effects on cellular/nuclear events including angiogenesis and tumor cell proliferation.

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Methods of Manufacturing
The thyroid hormones thyroxine (L-3,5,3',5'-tetraiodothyronine, T4) and 3,5,3'-triiodothyronine, T3, are iodinated derivatives of thyronine and are formed by oxidative coupling of the precursors 3-monoiodotyrosine and 3,5-diiodotyrosine.

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Formulations / Preparations
Levothyroxine Sodium Powder (Veterinary): 0.22% (1 g of T4 in 454 g of powder): One level teaspoonful contains 12 mg of T4. Available in 1 lb and 10 lb containers; many trade name products may be available and include: Equine Thyroid Supplement, Thyrozine Powder, Levoxine Powder; Thyro-L, Throxine-L Powder, Thyrosyn Powder, Thyrokare Powder; (Rx). Labeled for use in horses.

C15H12I4NNAO5

816.87

816.679

C, 22.06; H, 1.48; I, 62.14; N, 1.71; Na, 2.81; O, 9.79

25416-65-3

Thyroxine sulfate;77074-49-8;L-Thyroxine sodium salt pentahydrate;6106-07-6;L-Thyroxine sodium;55-03-8;L-Thyroxine-13C6-1;1217780-14-7;Biotin-(L-Thyroxine);149734-00-9;Biotin-hexanamide-(L-Thyroxine);2278192-78-0;Thyroxine hydrochloride-13C6;1421769-38-1;L-Thyroxine-13C6;720710-30-5;L-Thyroxine-13C6,15N;1431868-11-9

23667619

Typically exists as solid at room temperature

576.3ºC at 760 mmHg

207 °C

302.3ºC

3.922

C1=C(C=C(C(=C1I)OC2=CC(=C(C(=C2)I)O)I)I)C[C@@H](C(=O)[O-])N.O.O.O.O.O.[Na+]

JMHCCAYJTTWMCX-QWPJCUCISA-M

InChI=1S/C15H11I4NO4.Na.5H2O/c16-8-4-7(5-9(17)13(8)21)24-14-10(18)1-6(2-11(14)19)3-12(20)15(22)23;;;;;;/h1-2,4-5,12,21H,3,20H2,(H,22,23);;5*1H2/q;+1;;;;;/p-1/t12-;;;;;;/m0....../s1

sodium;(2S)-2-amino-3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]propanoate;pentahydrate

25416-65-3; Levothyroxine sodium; Levothyroxine sodium monohydrate; L-Thyroxine sodium xhydrate; Levothyroxine sodium hydrate; L-Thyroxine sodium hydrate; Monosodium L-thyroxine hydrate; 31178-59-3;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

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

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

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

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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

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

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

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