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

Levothyroxine treatment generates an abnormal uterine contractility patterns in an in vitro animal model[2]
Screening of thyroid function confirmed a hypothyroid state for all rats under iodine-free diet to which T4 was subsequently administered to counterbalance hypothyroidism. Results demonstrate that hypothyroidism significantly decreased contractile duration (-17%) and increased contractile frequency (+26%), while high doses of T4 increased duration (+200%) and decreased frequency (-51%). These results thus mimic the pattern of abnormal contractions previously observed in uterine tissue from T4-treated hypothyroid pregnant women. Conclusion: Our data suggest that changes in myometrial reactivity are induced by T4 treatment. Thus, in conjunction with our previous observations on human myometrial strips, management of hypothyroidism should be improved to reduce the rate of C-sections in this group of patients[2].

Adrenaline (cortogen) is converted to active adrenal cortex by the enzyme deiodinase (DIO), and the levels of TSH, the catalytic adrenaline, are correlated with this response. The adrenal cortex gets activated by DIO1 and DIO2, and gets deactivated by DIO3. In the negative feedback regulation of pituitary TSH, the actions of DIO1 and DIO2 are decisive [1]. The modulation of ion channels, pumps, and regulatory contractions is well-established for thyroxine (T3) and L-thyroxine (T4). Furthermore, it has been demonstrated that androgens influence charging excitation, calcium replenishment, contractile mortality, and the regulation of drug control and feeding by L-thyroxine and triiodothyronine. Significantly reduced levels of triiodothyronine and L-thyroxine were detected in the cohort fed an iodine-free diet for 12 weeks, as compared to the control group fed a regular diet (p<0.001). A rise in L-thyroxine levels (p=0.02) was noted in the group receiving low-dose L-thyroxine treatment, although triiodothyronine levels (p=0.19) remained nearly uniform with headache severity. Increases in circulation concentrations of triiodothyronine and L-thyroxine were observed after treatment with high-dose L-thyroxine as compared to the hypothyroid group that did not receive treatment (p<0.001 and p=0.004, each). Comparing the levels of thyroid hormone to the control values, there was a significant rise (p=0.03).

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Thyroid hormones play a vital role in the human body for growth and differentiation, regulation of energy metabolism, and physiological function. Hypothyroidism is a common endocrine disorder, which generally results from diminished normal circulating concentrations of serum thyroxine (fT4) and triiodothyronine (fT3). The primary choice in hypothyroidism treatment is oral administration of levothyroxine (L-T4), a synthetic T4 hormone, as approximately 100-125 μg/day. Generally, dose adjustment is made by trial and error approach. However, there are several factors which might influence bioavailability of L-T4 treatment. Genetic background could be an important factor in hypothyroid patients as well as age, gender, concurrent medications and patient compliance. The concentration of thyroid hormones in tissue is regulated by both deiodinases enzyme and thyroid hormone transporters. In the present study, it was aimed to evaluate the effects of genetic differences in the proteins and enzymes (DIO1, DIO2, TSHR, THR and UGT) which are efficient in thyroid hormone metabolism and bioavailability of L-T4 in Turkish population. According to our findings, rs225014 and rs225015 variants in DIO2, which catalyses the conversion of thyroxine (pro-hormone) to the active thyroid hormone, were associated with TSH levels. It should be given lower dose to the patients with rs225014 TT and rs225015 GG genotypes in order to provide proper treatment with higher effectivity and lower toxicity.[1]

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.

Female non-pregnant Sprague-Dawley rats (N = 22) were used and divided into four groups: 1) control, 2) hypothyroidism, 3) hypothyroidism treated with low T4 doses (20 μg/kg/day) and 4) with high T4 doses (100 μg/kg/day). Hypothyroidism was induced by an iodine-deficient diet. Isometric tension measurements were performed in vitro on myometrium tissues in isolated organ baths. Contractile activity parameters were quantified (amplitude, duration, frequency and area under the curve) using pharmacological tools to assess their effect.[2]

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Sprague–Dawley female rats (N = 22) were used. Non-pregnant rats were divided into four groups: 1) control, 2) hypothyroidism, 3) hypothyroidism treated with low doses of Levothyroxine (T4) (20 μg/kg/day) and 4) with high doses of T4 (100 μg/kg/day). Control rats (group 1) were fed with standard diet (TD.120461, Harlan laboratories, Madison, WI) while the intervention rats were fed with iodine-free diet for 12 weeks to induce hypothyroidism (groups 2–4) which was continued for four more weeks to allow screening of hypothyroid status and T4-treatment. Food and water (iodine-free diet) were available ad libitum. The hypothyroid group treated with low (group 3) or high doses of T4 (group 4) were injected intraperitoneally every 24 h with respectively 20 μg/kg/day and 100 μg/kg/day as previously described by Medeiros. Blood samples were collected for thyroid function screening at week 12 and 16 following the initiation of either the control or iodine-free diet. Hysterectomy was performed under general anesthesia (isoflurane 2%) at the end of the treatment and the two uterine horns were placed in physiological Krebs' solution until isometric tension measurements within no more than 1 h.[2]

Absorption, Distribution and Excretion
Oral levothyroxine (T4) is absorbed in the gastrointestinal tract at a rate of approximately 40% to 80%, with most doses absorbed via the jejunum and upper ileum. Fasting increases T4 absorption, while malabsorption syndrome and certain foods (such as soy, milk, and dietary fiber) decrease it. T4 absorption may also decrease with age. Furthermore, many medications can affect T4 absorption, including bile acid sequestrants, sucralfate, proton pump inhibitors, and minerals such as calcium (including calcium in yogurt and dairy products), magnesium, iron, and aluminum supplements. To prevent the formation of insoluble chelates, levothyroxine should generally be taken on an empty stomach, at least 2 hours before a meal, and at least 4 hours apart from any interacting medications. Thyroid hormones are primarily excreted through the kidneys. A portion of the conjugated hormone reaches the colon unchanged and is excreted in the feces. Approximately 20% of T4 is excreted in the feces. The amount of T4 excreted in urine decreases with age. Over 99% of circulating thyroid hormones are bound to plasma proteins, including thyroxine-binding globulin (TBG), thyroxine-binding prealbumin (TBPA), and albumin (TBA). These proteins have varying binding affinity and affinity for each hormone. TBG and TBPA have a higher affinity for T4, which partly explains the higher serum levels of T4 compared to T3, its lower metabolic clearance, and its longer half-life. Protein-bound thyroid hormones are in an inverse equilibrium with a small amount of free hormone. Only the free hormone possesses metabolic activity. Many drugs and physiological conditions can affect the binding of thyroid hormones to serum proteins. Thyroid hormones do not readily cross the placental barrier. Levothyroxine sodium for injection is administered intravenously. After administration, synthetic levothyroxine is indistinguishable from endogenously secreted natural hormone. Oral T4 absorption in the gastrointestinal tract ranges from 40% to 80%. Most of the levothyroxine dose is absorbed in the jejunum and upper ileum. Compared to an equivalent dose of oral levothyroxine sodium solution, the relative bioavailability of Euthyrox tablets is approximately 93%. Fasting can increase T4 absorption, while malabsorption syndrome and certain foods (such as soy-based infant formula) can decrease T4 absorption. Dietary fiber can also decrease T4 bioavailability. T4 absorption may also decrease with age. Furthermore, many medications and foods can affect T4 absorption. The absorption rate of levothyroxine in the gastrointestinal tract varies among individuals (range: 40-80%). In animal studies, levothyroxine is absorbed in the proximal and mid-jejunum; the drug is not absorbed by the stomach or distal colon, and absorption in the duodenum is minimal (if any). Human studies have shown that levothyroxine is absorbed in the jejunum and ileum, with a small amount also absorbed in the duodenum. The extent of levothyroxine absorption in the gastrointestinal tract depends on the product formulation and the type of intestinal contents, including plasma proteins and soluble dietary factors, which may bind to thyroid hormones, preventing their diffusion. In addition, concurrent oral administration of infant formula, soy flour, cottonseed flour, walnuts, fiber-rich foods, ferrous sulfate, antacids, sucralfate, calcium carbonate, cation exchange resins (e.g., sodium polystyrene sulfonate), simethicone, or bile acid sequestrants may reduce levothyroxine absorption. Levothyroxine absorption increases in the fasting state and decreases in malabsorption states (e.g., celiac disease). Absorption may also decrease with age. For more complete data on the absorption, distribution, and excretion of levothyroxine (7 types), please visit the HSDB record page.

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Metabolism/Metabolites
Approximately 70% of secreted T4 is deiodinated to produce an equal amount of T3 and reverse triiodothyronine (rT3), the latter of which does not produce calories. T4 is slowly metabolized to T3 via its primary metabolic pathway, a process completed through continuous deiodination, with approximately 80% of circulating T3 originating from peripheral T4. The liver is the primary site of degradation for both T4 and T3, and the deiodination of T4 also occurs in other sites, including the kidneys and other tissues. Elimination of T4 and T3 involves the liver conjugating them with glucuronic acid and sulfate. These hormones undergo enterohepatic circulation, where their conjugates are hydrolyzed and reabsorbed in the intestines. The conjugates reaching the colon are hydrolyzed and excreted in feces as free compounds. Several other minor T4 metabolites have also been identified. L-tyrosine is produced in rabbits and mice (data from tables). 3,3',5-triiodo-L-thyroxine is produced in humans, mice, dogs, and rabbits. Levothyroxine-4'-β-D-glucuronide is produced in dogs, humans, and rats. Levothyroxine-4'-sulfate is produced in dogs. 3,3',5,5'-tetraiodothyropyruvate is produced in rats. Levothyroxine is produced in rats. /Table/
3,3'-Diiodo-levothyroxine is generated in dogs. 3,3',5,5'-Tetraiodothyroacetic acid is generated in humans and rats. /Table/

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Biological Half-Life
The half-life of T4 is 6 to 7 days. The half-life of T3 is 1 to 2 days.
Compared to humans, the elimination half-life of orally administered levothyroxine in dogs is relatively short. The serum half-life is approximately 12-16 hours.
The plasma half-lives of levothyroxine and triiodothyronine are typically 6-7 days and approximately 1-2 days, respectively. The plasma half-lives of levothyroxine and triiodothyronine are shortened in patients with hyperthyroidism, while they are prolonged in patients with hypothyroidism.

Toxicity Summary
Identification and Uses: Levothyroxine exists in crystalline or needle-like forms. As a medicine, levothyroxine sodium is used as a replacement or supplemental treatment for congenital or acquired hypothyroidism. It is also used to treat or prevent various types of normal-thyroid goiter, including thyroid nodules, subacute or chronic lymphocytic thyroiditis (Hashimoto's thyroiditis), multinodular goiter, and as an adjunct to surgery and radioactive iodine therapy for the treatment of thyroid-stimulating hormone-dependent well-differentiated thyroid carcinoma. Injectable 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 overdose are the same as those of hyperthyroidism. In addition, confusion and disorientation may occur. There have been reports of cerebral embolism, shock, coma, and death. Studies have shown that extreme caution must be exercised when initiating levothyroxine sodium administration in very low birth weight infants. Children taking levothyroxine generally have a good prognosis, but overdose can lead to serious complications, including seizures and arrhythmias, requiring close monitoring. In a genotoxicity study, researchers tested the ability of levothyroxine to induce sister chromatid exchange and micronuclei in cultured human lymphocytes. Results showed that levothyroxine only exhibited a weak chromosome breakage effect at high concentrations. Animal studies: A single acute overdose in small animals is less likely to cause severe thyrotoxicosis compared to chronic overdose. Dogs and cats may experience symptoms such as vomiting, diarrhea, a shift from hyperactivity to lethargy, hypertension, tachycardia, tachypnea, dyspnea, and abnormal pupillary light reflex. Clinical symptoms in dogs may appear within 1–9 hours of ingestion. Four pregnant New Zealand white rabbits received intramuscular injections of 250 μg/kg of levothyroxine on days 25 and 26 of gestation. Concentrations of free levothyroxine in maternal and fetal plasma were higher than in the control group, reaching their peak on day 14 of the neonatal period. Treatment resulted in fetal hyperglycemia and decreased fetal liver glycogen levels. Animal studies have not been conducted to evaluate the carcinogenicity, mutagenicity, or effects on fertility of levothyroxine.

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Effects during pregnancy and lactation
◉ Overview of medication use during lactation
Levothyroxine (T4) is a normal component of human breast milk. Data on exogenous levothyroxine supplementation during lactation are limited, but no adverse effects on infants have been found. The American Thyroid Association recommends that women planning to breastfeed should use levothyroxine to treat subclinical and clinical hypothyroidism. Adequate levothyroxine treatment during lactation may restore normal milk production in lactating mothers with hypothyroidism and insufficient milk production. Patients with Hashimoto's thyroiditis may require an increased dose of levothyroxine postpartum compared to pre-pregnancy levels.
◉ Effects on breastfed infants
There are no reports on the effects of maternal use of exogenous thyroid hormones on infants. There have been reports that breastfeeding may reduce cretinism in infants with hypothyroidism, but the levels of thyroid hormones in breast milk are not ideal, and the results are controversial. The levels of thyroid hormones in the breast milk of mothers of extremely premature infants appear insufficient to affect the infant's thyroid function. The levels of thyroid hormones in breast milk are clearly insufficient to interfere with the diagnosis of hypothyroidism. In a telephone follow-up study, five breastfeeding mothers reported taking levothyroxine (dosage not specified). The mothers reported that their infants experienced no adverse reactions. One mother who had undergone thyroidectomy took 100 mcg of levothyroxine sodium tablets daily, along with calcium carbonate and calcitriol. Her breastfed infant was reported to be "growing well" at 3 months of age. A woman with propionic acidemia took 50 mcg of levothyroxine sodium tablets daily, along with biotin, carnitine, and multiple amino acids, and exclusively breastfed her infant for 2 months, followed by mixed feeding for 10 months. At that time, the infant's growth and development were normal.
◉ Effects on Lactation and Breast Milk
Adequate serum thyroid hormone levels are essential for normal lactation. Thyroid hormone supplementation can improve insufficient milk production caused by hypothyroidism. Supraphysiological doses are not expected to further improve lactation.

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Protein Binding
Circulating thyroid hormones are bound to plasma proteins exceeding 99%, including thyroxine-binding globulin (TBG), thyroxine-binding prealbumin (TBPA), and albumin (TBA). TBG and TBPA have a higher affinity for T4, which partly explains the higher serum levels of T4 compared to T3, its slower metabolic clearance, and longer half-life. Protein-bound thyroid hormones are in an inverse equilibrium with a small amount of free hormones; only free hormones are metabolically active.

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Interactions
Antacids (e.g., aluminum hydroxide, magnesium hydroxide, calcium carbonate), simethicone, and sucralfate can bind to thyroid drugs in the gastrointestinal tract, thereby delaying or preventing their absorption. Calcium carbonate can form an insoluble chelate with levothyroxine, leading to decreased levothyroxine absorption and increased serum thyroid-stimulating hormone (TSH) concentrations. In vitro studies have shown that levothyroxine can bind to calcium carbonate under acidic pH conditions. To minimize or avoid this interaction, some clinicians recommend that these medications and thyroid drugs be taken approximately 4 hours apart when taken simultaneously.
Serum digitalis concentrations may be lower in patients with hyperthyroidism or hypothyroidism whose thyroid function has returned to normal. Therefore, the efficacy of digitalis in these patients may be reduced.
Drugs that induce hepatic microsomal enzymes (e.g., carbamazepine, phenytoin sodium, phenobarbital, rifampin) can accelerate the metabolism of thyroid drugs, leading to increased thyroid drug dosage requirements. Phenytoin sodium and carbamazepine also reduce the serum protein binding rate of levothyroxine, potentially decreasing total T4 and free T4 levels by 20-40%, but most patients have normal serum TSH concentrations and clinically normal thyroid function. /Thyroid Medications/
Bile acid sequestrants (e.g., cholestyramine resin, colestipol) can bind to thyroid medications in the gastrointestinal tract, significantly reducing their absorption. In vitro studies have shown that this binding is not easily reversed. To minimize or avoid this interaction, when these medications must be taken simultaneously, they should be taken at least 4 hours apart.
For more complete data on drug interactions of levothyroxine (14 types), please visit the HSDB record page.

[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

Therapeutic Uses
ClinicalTrials.gov is a registry and results database that lists human clinical studies funded by public and private institutions worldwide. The website is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each record on ClinicalTrials.gov contains summary information about the study protocol, including: the disease or condition; the intervention (e.g., the medical product, behavior, or procedure being investigated); the title, description, and design of the study; participation requirements (eligibility criteria); the location of the study; contact information for the study location; and links to relevant information from other health websites, such as the NLM's MedlinePlus (which provides patient health information) and PubMed (which provides citations and abstracts of academic articles in the medical field). The database includes levothyroxine. Levothyroxine sodium is used to treat congenital or acquired hypothyroidism of various etiologies as a replacement or complementary therapy, but not for transient hypothyroidism during the recovery phase of subacute thyroiditis. Specific indications include: primary (thyroidal), secondary (pituitary), tertiary (hypothalamic) hypothyroidism, and subclinical hypothyroidism. Primary hypothyroidism may be caused by insufficiency, primary atrophy, partial or complete congenital absence of the thyroid gland, or the effects of surgery, radiation, or medication, with or without goiter. /US Product Label Content/
Levothyroxine sodium is used to treat or prevent various types of euthyroid goiter, including thyroid nodules, subacute or chronic lymphocytic thyroiditis (Hashimoto's thyroiditis), multinodular goiter, and as an adjunct to surgery and radioactive iodine therapy for the treatment of thyroid-stimulating hormone-dependent well-differentiated thyroid carcinoma. /US Product Label Content/
Levothyroxine sodium for injection is indicated for the treatment of myxedema coma. Important Usage Limitations: The relative bioavailability of levothyroxine sodium for injection compared to oral levothyroxine products has not been established. Caution should be exercised when switching patients from oral levothyroxine products to levothyroxine sodium for injection because accurate dose conversion has not been studied. /Included in US Product Label/
For more complete data on the therapeutic uses of levothyroxine (6 types), please visit the HSDB record page.

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Drug Warning
/Black Box Warning/ Warning: Not for the treatment of obesity or weight loss. Thyroid hormones, including levothyroxine sodium for injection, should not be used to treat obesity or weight loss. High doses may produce serious and even life-threatening toxicities.
/Black Box Warning/ Warning: Thyroid hormones, including levothyroxine, whether used alone or in combination with other treatments, should not be used to treat obesity or weight loss. In patients with normal thyroid function, doses within the daily hormone requirement range are ineffective for weight loss. Higher doses may produce serious and even life-threatening toxicities, especially when used in combination with sympathomimetic drugs (such as appetite suppressants). Overdose of levothyroxine sodium for injection (more than 500 micrograms) has been associated with cardiac complications, especially in older adults and patients with underlying heart disease. Potential adverse events associated with high-dose injectable levothyroxine sodium include arrhythmias, tachycardia, myocardial ischemia and infarction, or exacerbation of congestive heart failure and death. Caution should be exercised in these populations, including using lower doses within the recommended dose range. Close monitoring of patients is recommended after levothyroxine sodium injection.
To date, studies have not identified pediatric-specific problems that limit the use of thyroid hormones in children. However, caution must be exercised when interpreting neonatal thyroid function test results because serum T4 concentrations can transiently increase or decrease, and the pituitary gland in infants is relatively insensitive to negative feedback from thyroid hormones. /Thyroid Hormones/
For more complete data on drug warnings for levothyroxine (17 in total), please visit the HSDB record page.

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Pharmacodynamics
Oral levothyroxine is a synthetic hormone with the same physiological effects as endogenous T4, thus maintaining normal T4 levels in cases of T4 deficiency. Levothyroxine has a narrow therapeutic index, requiring dose adjustments to maintain normal thyroid function and keep thyroid-stimulating hormone (TSH) levels within the therapeutic range of 0.4–4.0 mIU/L. Both overdose and underdose of levothyroxine can negatively impact growth and development, cardiovascular function, bone metabolism, reproductive function, cognitive function, mood, gastrointestinal function, and glucose and lipid metabolism. Levothyroxine dosage should be adjusted slowly and cautiously, with close monitoring of the patient's dose-response to avoid these adverse effects. TSH levels should be monitored at least annually to prevent levothyroxine overdose, which can lead to hyperthyroidism (TSH <0.1 mIU/L) and symptoms such as tachycardia, diarrhea, tremor, hypercalcemia, and fatigue. Because many cardiac functions, including heart rate, cardiac output, and systemic vascular resistance, are closely related to thyroid status, levothyroxine overdose can lead to tachycardia, myocardial wall thickening, and increased myocardial contractility, and may induce angina or arrhythmias, especially in patients with cardiovascular disease and elderly patients. For any group with pre-existing heart conditions, the starting dose of levothyroxine should be lower than the recommended dose for younger individuals or those without heart disease. Patients taking levothyroxine concurrently with sympathomimetic medications should be monitored for signs and symptoms of coronary artery insufficiency. If cardiac symptoms occur or worsen, the dose of levothyroxine should be reduced, or the medication should be discontinued for one week and then restarted at a lower dose. Excessive levothyroxine replacement therapy may lead to increased bone resorption and decreased bone mineral density, especially in postmenopausal women. Increased bone resorption may be associated with elevated serum calcium and phosphorus levels and increased urinary calcium and phosphorus excretion, elevated bone alkaline phosphatase, and decreased serum parathyroid hormone levels. To mitigate this risk, the lowest effective dose of levothyroxine that achieves the expected clinical and biochemical response should be administered. Adding levothyroxine to diabetic patients may worsen glycemic control, leading to increased dosages of hypoglycemic agents or insulin. Glycemic control should be closely monitored after starting, changing, or discontinuing levothyroxine.

C15H11I4NO4

776.8700

776.686

C, 23.19; H, 1.43; I, 65.34; N, 1.80; O, 8.24

51-48-9

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

5819

Crystals
Needles

2.6±0.1 g/cm3

576.3±50.0 °C at 760 mmHg

235 °C

302.3±30.1 °C

0.0±1.7 mmHg at 25°C

1.795

5.93

3

5

5

24

420

1

IC1C(=C(C([H])=C(C=1[H])C([H])([H])[C@@]([H])(C(=O)O[H])N([H])[H])I)OC1C([H])=C(C(=C(C=1[H])I)O[H])I

XUIIKFGFIJCVMT-LBPRGKRZSA-N

InChI=1S/C15H11I4NO4/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)/t12-/m0/s1

(S)-2-amino-3-(4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl)propanoic acid

L-Thyroxin, L Thyroxin, T4, Levothyroxine sodium, Levothyroxine sodium pentahydrate, Thyroxine; L-thyroxine; 51-48-9; thyroxine; thyroxin; Levothyroxin; Tetraiodothyronine; 3,3',5,5'-Tetraiodo-L-thyronine;

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)

DMSO : ~250 mg/mL (~321.80 mM)
1M NaOH : 5 mg/mL (~6.44 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (2.68 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 20.8 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: ≥ 2.08 mg/mL (2.68 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 20.8 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: ≥ 2.08 mg/mL (2.68 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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