Androgen Receptor/AR

At the cellular level, testosterone, or its active metabolite dihydrotestosterone, exerts its effects by binding to the androgen receptor. Studies show that testosterone significantly promotes DNA, total protein, and β-glucuronidase synthesis in mouse kidney cell lines, with a half-maximal response concentration of approximately 2 × 10⁻⁹ M. Furthermore, human hepatic cell lines such as C3A and HepaRG metabolize testosterone into various products, including androstenedione and hydroxylated derivatives, making it a useful probe for assessing the activity of drug-metabolizing enzymes like CYP3A4.

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Testosterone is a steroid sex hormone indicated to treat primary hypogonadism and hypogonadotropic hypogonadism. Testosterone antagonizes the androgen receptor to induce gene expression that causes the growth and development of masculine sex organs and secondary sexual characteristics. Testosterone was isolated from samples and also synthesized in 1935.

Testosterone replacement therapy demonstrates clear clinical benefits in hypogonadal men. Meta-analyses indicate that oral testosterone therapy significantly improves bone mineral density (mean increase of 3.1% over 12 months) and reduces fat mass (mean reduction of 1.4 kg). Additionally, potential cognitive benefits, including improvements in memory and executive function, have been reported. Animal studies also confirm that subcutaneous testosterone injections effectively stimulate prostate and seminal vesicle growth in castrated male rats.

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Testosterone antagonizes the androgen receptor to induce gene expression that causes the growth and development of masculine sex organs and secondary sexual characteristics. The duration of action of testosterone is variable from patient to patient with a half life of 10-100 minutes. The therapeutic index is wide considering the normal testosterone levels in an adult man range from 300-1000ng/dL. Counsel patients regarding the risk of secondary exposure of testosterone topical products to children.

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- Essential for the growth and development of male organs and secondary sexual characteristics, and maintains the functions of testis, accessory structures, prostate, and seminal vesicles in adult men, as well as sexual desire [1]
- Promotes protein synthesis, inhibits protein breakdown, and is involved in the regulation of body water and electrolyte balance, causing sodium and water retention [1]
- Promotes the synthesis of erythropoietin in the kidney, stimulates erythropoiesis, and affects bone growth and epiphyseal closure [1]

Absorption, Distribution and Excretion
The AUC of a single topical application of 100 mg testosterone is 10425 ± 5521 ngh/dL, and the Cmax is 573 ± 284 ng/dL. The bioavailability of topical testosterone is approximately 10%. 90% of the intramuscular dose is excreted in the urine, primarily as glucuronide and sulfate conjugates. 6% is excreted in the feces, primarily as unconjugated metabolites. The volume of distribution of testosterone in elderly men is 80.36 ± 24.51 L. The mean metabolic clearance rate in middle-aged men is 812 ± 64 L/day. After topical application of gels or transdermal preparations, testosterone can be absorbed into the systemic circulation through the skin. When testosterone hydroalcoholic gel preparations (AndroGel, Testim) are applied topically to the skin, the gel dries rapidly on the skin surface, forming a reservoir of hormones and continuously releasing hormones into the systemic circulation. After topical application of a 1% concentration of testosterone gel, approximately 10% of the testosterone dose is absorbed percutaneously into the systemic circulation. The manufacturer of AndroGel states that serum testosterone levels rise within 30 minutes of topical application of a 100 mg dose of the 1% gel, and most patients reach physiological concentrations (pre-treatment concentrations not described) within 4 hours; percutaneous absorption continues throughout the 24-hour dosing interval. Serum testosterone concentrations approach steady-state levels at the end of the initial 24 hours and reach steady-state on the second or third day after using the 1% gel. With daily topical application of 1% gel (AndroGel), serum testosterone concentrations typically remain within the normal range at 30, 90, and 180 days after the start of treatment. With daily use of 10 g or 5 g of AndroGel, the mean daily serum testosterone concentrations on day 30 were 794 ng/dL and 566 ng/dL, respectively. After discontinuation of topical treatment, serum testosterone concentrations remain within the normal range for 24–48 hours, but return to pre-treatment levels by the fifth day after the last dose. When applying transdermal formulations topically, the degree of transdermal testosterone absorption varies depending on the application site. This may be due to regional differences in skin permeability, skin blood flow, and/or the degree of adhesion between the transdermal system and the skin. In one study, researchers applied transdermal patches to the abdomen, back, chest, calves, thighs, or upper arms. The results showed that serum hormone levels were qualitatively similar across sites, but steady-state serum concentrations differed significantly, decreasing sequentially from back to thighs, upper arms, abdomen, chest, and calves. Applying Androderm transdermal patches to the abdomen, back, thighs, or upper arms yielded similar serum testosterone concentration profiles; therefore, rotating these sites during long-term treatment is recommended. Nighttime application of Androderm transdermal patches (around 10 PM) mimics the endogenous circadian rhythm of healthy young men in terms of serum testosterone concentration profiles. One study showed that showering 3 hours after application of Androderm transdermal patches reduced peak plasma testosterone concentrations by 0.4% compared to not showering 3 hours after application. Furthermore, showering 3 hours after applying the transdermal patch did not significantly alter systemic testosterone exposure. Following topical application of the transdermal testosterone patch, the hormone is absorbed through the skin into systemic circulation. Although there are individual differences in transdermal testosterone absorption, serum testosterone concentrations typically reach normal levels on the first day of use with the recommended dose and remain stable throughout continued use without accumulation. Patients receiving Androderm have reported a mean daily serum testosterone concentration of 498 ng/dL at steady state. The mean testosterone to dihydrotestosterone ratio is within the normal range. Testosterone esterification typically produces compounds with lower polarity. Testosterone enanthate is slowly absorbed from the lipid tissue phase at the intramuscular injection site, reaching peak serum concentrations approximately 72 hours after intramuscular injection; therefore, the duration of action of this preparation is relatively long after intramuscular injection (i.e., up to 2–4 weeks). Due to the local irritation caused by intramuscular injection of testosterone esters, their absorption rate may be unstable. /Testosterone Esters/
For more complete data on the absorption, distribution, and excretion of testosterone (9 types), please visit the HSDB record page.

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Metabolism/Metabolites
Testosterone is metabolized to 17-ketosteroids via two distinct pathways. The main active metabolites are estradiol and dihydrotestosterone (DHT). Testosterone can be hydroxylated at multiple sites by CYP3A4, CYP2B6, CYP2C9, and CYP2C19; it can be glucuroninated by UGT2B17; it can be sulfated; it can be converted to estradiol by aromatase; testosterone is converted to dihydrotestosterone (DHT) by 5α-reductase; it is metabolized to androstenedione by CYP3A4, CYP2C9, and CYP2C19; or it can be converted to DHT glucuronide. Androstenedione is metabolized to estrone by aromatase, and estrone can undergo a reversible reaction to produce estradiol. Androstenedione can also be converted to 5α-androstanedione by 5α-reductase, which can be further metabolized to 5α-androsone. DHT can undergo glucuronidation or sulfation, or be metabolized to 5α-androstanedionol, androstan-3α,17β-diol, or androstan-3β,17β-diol. Dihydrotestosterone (DHT) can also be reversibly converted to 5α-androstanedione. Extensive reductive metabolism of testosterone occurs not only in the liver but also in various extrahepatic tissues, especially in the target organs of sex hormones; ultimately, effective physiological androgens are formed in these target tissues. Testosterone metabolism occurs not only in the prostate and seminal vesicles but also in the rat uterus, rabbit placenta, rodent testes, and primate brain. In rats, the small intestine also metabolizes testosterone. In target organs such as the prostate, sebaceous glands, and seminal vesicles, testosterone is converted to 5α-dehydrotestosterone; only the latter can bind to androgen receptors in these target organs. Significant quantitative differences exist in testosterone metabolism between female and male rats. This phenomenon occurs because many steroid-metabolizing enzymes in rats are either androgen-dependent or estrogen-dependent; therefore, sex hormones function in an inducible or inhibitory manner. Testosterone esters, such as propionate, heptaate, cyclovalerate, valerate, isovalerate, heptaate, and undecanoate, are partially cleaved in the body to release the parent compound. This was confirmed by oral administration of testosterone undecanoate in an oily solution to rats: most of the compound is converted in the intestinal wall, the first step being the partial cleavage of the fatty acid moiety. However, the unmetabolized portion, along with the metabolite 5α-dihydrotestosterone undecanoate, is absorbed through the lymphatic system and is available to exert androgenic effects. /Testosterone Esters/
For more information on the metabolism/metabolites (complete) data of testosterone (6 metabolites), please visit the HSDB record page.
Known human metabolites of testosterone include 2β-hydroxytestosterone, androstenedione, 16β-hydroxytestosterone, 6α-hydroxytestosterone, 15β-hydroxytestosterone, 15-hydroxytestosterone, testosterone sulfate, 16-hydroxytestosterone, and 2α-hydroxytestosterone. Testosterone is metabolized into 17-ketosteroids via two distinct pathways. The main active metabolites are estradiol and dihydrotestosterone (DHT). Excretion pathways: After intramuscular injection, approximately 90% of the dose is excreted in the urine as glucuronic acid and sulfate conjugates; approximately 6% is excreted in the feces, primarily in unconjugated form. Half-life: 10-100 minutes. The half-life of testosterone varies considerably, ranging from 10 to 100 minutes. The plasma half-life of testosterone has been reported to be 10-100 minutes. The plasma half-life after intramuscular injection of testosterone propionate is approximately 8 days. After removal of the Androdum transdermal patch, plasma testosterone concentration decreased, with an apparent half-life of approximately 70 minutes…

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- Most testosterone in the blood is bound to sex hormone-binding globulin (approximately 65%), a portion is bound to plasma albumin or cortisol-binding protein (approximately 33%), and only a small amount (approximately 2%) exists in free form [1]
- It is mainly metabolized and inactivated in the liver, and the final metabolites are excreted in the urine [1]

Toxicity Summary
Identification and Uses: Testosterone is a systemic anabolic steroid. It consists of tasteless or nearly tasteless crystals or crystalline powder. Natural anabolic steroids are synthesized in the testes, ovaries, and adrenal glands. Anabolic steroids are classified as Schedule III controlled substances. Human Exposure and Toxicity: The main risks associated with testosterone are androgen overdose: menstrual irregularities and virilization in women, and impotence, premature cardiovascular disease, and benign prostatic hyperplasia in men. Both men and women taking oral anabolic steroids containing substituted 17-α-carbons may experience liver damage. Mental changes may occur during or after use of these drugs. Acute overdose can cause nausea and gastrointestinal upset. Long-term use is thought to lead to increased muscle mass and may exacerbate male characteristics and androgen-related effects. There is currently no clear evidence that anabolic steroids improve overall athletic performance. Early-onset prostate cancer has been reported following long-term anabolic steroid abuse. There are also case reports of liver cancer associated with anabolic steroid abuse. Because testosterone can cause masculinization in female fetuses, its use by pregnant women may harm the fetus. In female offspring born to women who took androgens during pregnancy, androgenic effects have been observed, including clitoral hypertrophy, fusion of the labia majora to form a scrotum-like structure, vaginal abnormalities, and persistent urogenital sinuses. The degree of masculinization is related to the dosage of the drug taken by the pregnant woman and the gestational age of the fetus; female fetuses are most susceptible to masculinization when exposed to androgens in early pregnancy. Animal studies: Testosterone's effect on the prostate of castrated rats showed a significant increase in prostate weight, which occurred after 6 weeks of testosterone treatment. In female mice that received subcutaneous injections of 25 micrograms of testosterone daily for the first five days after birth, 7 out of 9 mice developed proliferative epithelial lesions resembling epidermoid carcinoma at approximately 71 weeks of age. Long-term testosterone treatment in rats reduced the incidence of prostate cancer. Only long-term use of testosterone after administration of a carcinogen could lead to an increased incidence of prostate cancer. Subcutaneous injections of 0.5 to 80 mg of testosterone daily for 4 to 8 consecutive days from day 10 to 20 of gestation, and subcutaneous injections of 1 to 55 mg of testosterone propionate daily from day 12 to 19 of gestation, both resulted in embryonic resorption, necrosis, death, postpartum death, and varying degrees of masculinization in female offspring. Testosterone norpropionate (testosterone cyclopentylpropionate) exhibits genotoxicity and cytotoxicity in mice. Testosterone has both mitogenic and genotoxic effects in L929 cells. Testosterone is considered an anabolic steroid. It plays a crucial role in the development of male reproductive tissues (such as the testes and prostate) and promotes the development of secondary sexual characteristics, such as increased muscle mass, bone mass, and body hair growth. High levels of testosterone can lead to masculinization in females or precocious puberty in males. Long-term high levels of testosterone in adults can lower high-density lipoprotein cholesterol (good cholesterol) levels and raise low-density lipoprotein cholesterol (bad cholesterol) levels, thereby increasing the risk of heart attack, stroke, and blood clots. Long-term, high-dose use of anabolic steroids (such as testosterone) appears to lead to cardiomyopathy and weakened left ventricular function. Gynecomastia (often caused by excessively high circulating estradiol levels) is due to aromatase promoting the conversion of testosterone to estradiol. Men may also experience decreased libido and temporary infertility. The mechanism of action of testosterone is as follows: Free testosterone is transported to the cytoplasm of target tissue cells, where it can bind to androgen receptors or be reduced to 5α-dihydrotestosterone (DHT) by the cytoplasmic enzyme 5α-reductase. DHT binds more strongly to the same androgen receptor than testosterone, thus its androgenic potency is approximately five times that of testosterone. Once bound, the ligand-receptor complex undergoes a structural change, allowing it to enter the cell nucleus and bind directly to specific nucleotide sequences of chromosomal DNA. These binding regions are called hormone response elements (HREs), and they influence the transcriptional activity of certain genes, thereby producing androgenic effects.

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Effects during pregnancy and lactation
◉ Overview of medication use during lactation
Limited data suggest that subcutaneous implantation of a low-dose (100 mg) extended-release testosterone in lactating mothers does not appear to significantly increase testosterone levels in breast milk. Subcutaneous injection of testosterone propionate does increase testosterone levels in breast milk. However, due to the extensive first-pass metabolism of testosterone and its low oral bioavailability, it does not appear to increase serum testosterone levels in breastfed infants. Breastfed infants do not appear to be adversely affected by testosterone treatment by the mother or transgender father. High doses of testosterone can suppress lactation.
◉ Effects on breastfed infants
A postpartum mother received a subcutaneous implantation of a 100 mg extended-release testosterone tablet, and her infant (age not specified) was breastfed (feeding extent not specified). No adverse reactions were observed in the infant during the 5-month observation period.
A transgender man began weekly subcutaneous injections of 50 mg testosterone propionate at 13.75 months postpartum. One month later, the dose was increased to 80 mg per week. His male infant received partial "breast feeding" (the extent of which was not specified) until spontaneous weaning 137 days after the start of testosterone use (at 18 months of age). During this period, the infant's pediatrician did not observe any adverse events or signs of masculinization. The infant's growth and development were normal.
◉ Effects on Lactation and Breast Milk
Whether caused by tumors or exogenous testosterone, excessively high serum testosterone levels can reduce milk production in postpartum women. Testosterone alone reduces serum prolactin levels; however, when used in combination with estrogen and progesterone, serum prolactin levels do not decrease significantly. Testosterone has previously been used to suppress lactation, usually in combination with estrogen.
Protein Binding
40% of testosterone is bound to sex hormone-binding globulin, 2% exists in free form, and the remainder is bound to albumin and other proteins.
Interactions
In recent years, there has been an increase in the concurrent use of anabolic androgenic steroids and cocaine. However, the long-term effects of adolescent exposure to these substances on cardiovascular function remain unclear. This study investigated the effects of 10 consecutive days of treatment with testosterone and cocaine, alone or in combination, on baseline cardiovascular parameters, baroreflex activity, hemodynamic responses induced by vasoactive drugs, and cardiac morphology in adolescent rats. Testosterone alone increased arterial blood pressure, decreased heart rate, and exacerbated baroreflex-mediated tachycardia. Animals in the cocaine-treated group exhibited resting bradycardia, but arterial blood pressure and baroreflex activity remained unchanged. Combined treatment with testosterone and cocaine did not affect baseline arterial blood pressure and heart rate, but reduced baroreflex-mediated tachycardia. None of the treatments affected arterial blood pressure responses induced by vasoconstrictors or vasodilators. Furthermore, drug treatments did not alter the heart-body ratio or left and right ventricular wall thickness. However, histological analysis of left ventricular sections from animals treated with testosterone and cocaine alone or in combination revealed increased myocardial fiber spacing, vasodilation, and fibrosis. These data suggest that significant changes occur in the cardiovascular system of adolescent rats after testosterone treatment. However, results showed that the effects of cocaine use alone or in combination during puberty on cardiovascular function were minimal. Topical application of 0.1% triamcinolone cream before applying a transdermal testosterone patch did not alter testosterone absorption; however, pre-application of triamcinolone ointment significantly reduced testosterone absorption. One study showed that intramuscular injection of testosterone propionate led to increased propranolol clearance. It is currently unclear whether topical application of testosterone gel produces this interaction. Testosterone propionate: Testosterone may enhance the effects of oral anticoagulants, leading to bleeding in some patients. When patients taking oral anticoagulants begin testosterone therapy, the dose of the anticoagulant may need to be reduced to prevent excessive hypoprothrombinemia. For patients receiving both testosterone and anticoagulants, more frequent monitoring of the international normalized ratio (INR) and prothrombin time is recommended, especially during the start or discontinuation of treatment. For more complete data on testosterone interactions (10 items in total), please visit the HSDB records page.

[1]. https://pubchem.ncbi.nlm.nih.gov/compound/6013
[2]. Br J Clin Pharmacol. 2012 Jul;74(1):3-15.
[3]. J Biol Chem. 1964 May:239:1569-77.

Therapeutic Uses
Androgens / Clinical Trials / 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 includes a summary of 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 (for providing patient health information) and PubMed (for providing citations and abstracts of academic articles in the medical field). The database includes testosterone. In men, testosterone is used to treat congenital or acquired primary hypogonadism, such as after orchiectomy or testicular failure caused by cryptorchidism, bilateral testicular torsion, orchitis, or vanishing testis syndrome. Testosterone is also used to treat congenital or acquired hypogonadism in men, such as hypogonadism caused by idiopathic gonadotropin or gonadotropin-releasing hormone (luteinizing hormone-releasing hormone) deficiency or hypogonadism caused by pituitary-hypothalamic damage due to tumors, trauma, or radiation. If any of these conditions occur before puberty, androgen replacement therapy is required during puberty to promote the development of secondary sexual characteristics, and long-term treatment is needed to maintain these characteristics. Other men who develop testosterone deficiency after puberty also require long-term androgen therapy to maintain sexual characteristics. /US product label includes/
After diagnosis, testosterone can be used to stimulate puberty in rigorously screened men with delayed puberty. These men typically have a family history of delayed puberty and it is not caused by a pathological disease. If these men do not respond to psychological support, short-term treatment with conservative doses of androgens may be considered. Because androgens may have adverse effects on skeletal maturation in these pre-pubertal men, this potential risk should be fully discussed with the patient and their parents before initiating androgen therapy. If these pre-pubertal males begin androgen therapy, wrist and hand X-rays should be performed every 6 months to determine the effect of treatment on the epiphyseal center. Testosterone has been designated an orphan drug by the U.S. Food and Drug Administration (FDA) for the treatment of this condition. /Included on U.S. product label/
For more complete data on the therapeutic uses of testosterone (17 types), please visit the HSDB record page.

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Drug Warning
/Black Box Warning/ Warning: Indirect exposure to testosterone. There have been reports of masculinizing symptoms in children after indirect exposure to testosterone gel. Children should avoid contact with unwashed or unclothed areas where men have applied testosterone gel. Healthcare providers should advise patients to strictly follow the recommended instructions for use.
Postmarketing surveillance data have shown cardiovascular events, including myocardial infarction or stroke, with the testosterone transdermal patch (Androderm). Testosterone should be used with caution in patients at high risk of cardiovascular disease (e.g., older men, people with diabetes, or obese individuals). Patients should be informed that they should immediately report any symptoms suggestive of myocardial infarction or stroke (e.g., chest pain, shortness of breath, unilateral limb weakness, difficulty speaking). Post-marketing surveillance data shows that venous thromboembolic events, including deep vein thrombosis (DVT) and pulmonary embolism (PE), have occurred with testosterone preparations (including the Androderm transdermal testosterone patch). If a patient experiences symptoms such as lower extremity pain, edema, fever, and erythema, or acute respiratory distress, they should be evaluated for DVT or PE, respectively. If venous thromboembolism is suspected, testosterone treatment should be discontinued immediately, and appropriate evaluation and treatment should be initiated. Because sodium and water retention can lead to edema, testosterone should be used with caution in patients with cardiac, renal, and/or hepatic dysfunction. Edema, whether or not accompanied by congestive heart failure, can be a serious complication in patients with a pre-existing cardiac, renal, and/or hepatic disease. If edema occurs during testosterone treatment and is considered a serious complication, the drug should be discontinued; diuretic treatment may also be necessary. For more complete data on drug warnings regarding testosterone (34 in total), please visit the HSDB record page.

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Pharmacodynamics
Testosterone antagonizes androgen receptors, inducing gene expression, thereby promoting the growth and development of male sex organs and secondary sexual characteristics. The duration of action of testosterone varies from person to person, with a half-life of 10-100 minutes. Given that normal testosterone levels in adult men range from 300-1000 ng/dL, its therapeutic index is broad. Patients should be informed that children may be at risk of secondary exposure when using topical testosterone products.

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- Testosterone is a natural androgen and steroid hormone, mainly produced by the testes in men, and also secreted by the ovaries and adrenal glands in women. It can also be synthesized artificially[1].
- It can be used to assess the function of the hypothalamus-pituitary-gonadal axis and is associated with the diagnosis of precocious puberty, sexual dysfunction, male testicular insufficiency, female virilization, adrenal cortex or ovarian tumors[1].
- Clinically, it is mainly used for androgen replacement therapy, perimenopausal syndrome and dysfunctional uterine bleeding, advanced breast cancer, anemia, weakness and prevention of benign prostatic hyperplasia[1].

C19H28O2

288.43

288.208

58-22-0

6013

White needles from dilute acetone
Needles from dilute acetone
White or slightly cream-white crystals or crystalline powder

1.1±0.1 g/cm3

432.9±45.0 °C at 760 mmHg

152-156 °C

184.7±21.3 °C

0.0±2.3 mmHg at 25°C

1.560

3.47

1

2

0

21

508

6

C[C@@]12CCC(=O)C=C2CC[C@H]3[C@@H]4CC[C@@H]([C@@]4(C)CC[C@@H]31)O

MUMGGOZAMZWBJJ-DYKIIFRCSA-N

InChI=1S/C19H28O2/c1-18-9-7-13(20)11-12(18)3-4-14-15-5-6-17(21)19(15,2)10-8-16(14)18/h11,14-17,21H,3-10H2,1-2H3/t14-,15-,16-,17-,18-,19-/m0/s1

(8R,9S,10R,13S,14S,17S)-17-hydroxy-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-3-one

Primotest; Homosteron; Testosterone; testosterone; 58-22-0; Testosteron; Androderm; Testim; Homosterone; Virosterone; Testiculosterone;

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: >40 mg/ml
Acetonitrile: ~1 mg/ml
Ethanol: ~1 mg/ml
Methanol: ~1 mg/ml

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.)