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.

Absorption increases in the fasting state and decreases in malabsorption states (e.g., celiac disease); absorption may also decrease with age. American Association of Health-System Pharmacists, 2015; Drug Information, 2015. Bethesda, MD, 2015, p. 3230.
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, which involves continuous deiodination, in which approximately 80% of circulating T3 originates from peripheral T4. The liver is the primary site of degradation of T4 and T3, and deiodination of T4 also occurs in other sites, including the kidneys and other tissues. The elimination of T4 and T3 involves the liver binding them with glucuronic acid and sulfate. These hormones undergo enterohepatic circulation, and their conjugates are hydrolyzed and reabsorbed in the intestine. 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 generated in rabbits and mice / from Table / Goodwin, BL, Handbook of Intermediate Metabolism of Aromatic Compounds. New York: Wiley, 1976, p. T-14.
3,3',5-triiodo-L-thyroxine is generated in humans, mice, dogs, and rabbits. / from Table / Goodwin, BL, Handbook of Intermediate Metabolism of Aromatic Compounds. New York: Wiley, 1976, p. T-14.
L-thyroxine-4'-β-D-glucuronide is generated in dogs, humans, and rats. L-thyroxine-4'-sulfate is generated in dogs. / from Table / Goodwin, BL, Handbook of Intermediate Metabolism of Aromatic Compounds. New York: Wiley, 1976, p. T-14.
In rats, 3,3',5,5'-tetraiodothyronine is produced. In rats, L-thyroxine is produced. /Excerpt from Table/ Goodwin, BL, Handbook of Intermediate Metabolism of Aromatic Compounds. New York: Wiley, 1976, p. T-14. T-14
In dogs, 3,3'-diiodo-1-thyroxine is produced. In humans and rats, 3,3',5,5'-tetraiodothyronine is produced. /Excerpt from Table/ Goodwin, BL, Handbook of Intermediate Metabolism of Aromatic Compounds. New York: Wiley, 1976, p. T-14.

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Biological Half-Life
T₄Half-life is 6 to 7 days. T₃Half-life 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. Plumb DC, Veterinary Medicine Handbook. 8th Edition (Pocket Edition). Ames, Iowa: Wiley-Blackwell, 2015, p. 14. 842
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.

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 is limited, but there is currently no evidence of adverse effects on infants. The American Thyroid Association recommends levothyroxine treatment for subclinical and clinical hypothyroidism in breastfeeding women planning to breastfeed. Adequate levothyroxine treatment during lactation may help hypothyroid breastfeeding mothers with insufficient milk production to restore normal milk production. For patients with Hashimoto's thyroiditis, the required dose of levothyroxine postpartum may be higher than before pregnancy.
◉ Effects on Breastfed Infants
There are no reports on the effects of maternal use of exogenous thyroid hormones on their infants. There have been reports that breastfeeding appears to reduce cretinism symptoms in hypothyroid infants, but the levels of thyroid hormones in breast milk are not ideal, so this result is 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). These mothers reported no adverse reactions in their infants. One mother who had undergone thyroidectomy took 100 mcg of levothyroxine daily, along with calcium carbonate and calcitriol. Her breastfed infant was reported to be "developing well" at 3 months of age. A woman with propionic acidemia took 50 mcg of levothyroxine sodium daily, along with biotin, carnitine, and multiple amino acids, while exclusively breastfeeding her infant for 2 months, followed by 10 months of mixed feeding. At that time, the infant's growth and development were normal. ◉ Effects on Lactation and Breast Milk Adequate serum thyroid hormone levels are necessary for normal lactation. Thyroid hormone supplementation can improve milk production caused by hypothyroidism. Supraphysiological doses are not expected to further improve lactation.

[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 levorotatory isomer of thyroxine (T4), structurally similar to endogenous hormones produced by the thyroid gland. In peripheral tissues, levothyroxine is deiodinated by 5'-deiodinase to form triiodothyronine (T3). T3 enters cells and binds to thyroid hormone receptors in the nucleus; the activated hormone-receptor complex then triggers gene expression, producing proteins required for cellular respiration, thermoproduction, cell growth and differentiation, and protein, carbohydrate, and lipid metabolism. T3 also has a cardiac excitatory effect. The main hormone of the thyroid gland is thyroxine. Thyroxine is synthesized through the iodination of tyrosine (monoiodotyrosine) and the coupling of iodotyrosine (diiodotyrosine) in thyroglobulin. Thyroxine is released from thyroglobulin via proteolytic hydrolysis and secreted into the bloodstream. Thyroxine is deiodinated in peripheral tissues to form triiodothyronine, which has a broad stimulatory effect on cellular metabolism.

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

23665037

Off-white to light brown solid powder

576.3ºC at 760 mmHg

207 °C

302.3ºC

3.922

7

10

5

30

426

1

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.)