ER/estrogen-receptor; Clomiphene binds to muscarinic cholinergic receptors and calcium channel blocker binding sites in cell membranes [1]

In membrane preparations from various tissues, Clomiphene inhibits the binding of radiolabeled muscarinic ligands (e.g., quinuclidinyl benzilate) and calcium channel blockers (e.g., nitrendipine) to their respective binding sites. The inhibition is concentration-dependent, indicating competitive or non-competitive interaction with these sites [1]

Perinatal treatment of male mice with Clomiphene (administered to pregnant mice at a dose of 100 μg/day from gestational day 12 to postnatal day 10) alters sexual orientation, as evidenced by changes in mating behavior and preference tests. Treated male mice show reduced preference for female mice compared to controls [2]
In clinical settings, Clomiphene is used for ovulation induction in women with anovulatory infertility. It stimulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release from the pituitary, leading to follicular development and ovulation. Ovulation rates of 70-80% are reported, with pregnancy rates of 30-40% [3]

Membrane fractions from tissues (e.g., brain, heart) are prepared and incubated with radiolabeled muscarinic ligands or calcium channel blockers in the presence of varying concentrations of Clomiphene. After incubation, bound and free radioligands are separated by filtration or centrifugation. The amount of bound radioligand is measured, and inhibition curves are generated to assess the interaction of Clomiphene with the binding sites [1]

For perinatal studies, pregnant mice receive Clomiphene via subcutaneous injection at a dose of 100 μg/day from gestational day 12 to postnatal day 10. Control groups receive vehicle injections. Offspring male mice are tested for sexual orientation using behavioral assays (e.g., preference for female mice vs. male mice) after reaching adulthood [2]

Absorption, Distribution and Excretion
Based on early studies of clomiphene citrate labeled with 14C, this drug is readily absorbed orally in humans. Based on early studies of clomiphene citrate labeled with 14C, this drug is readily absorbed orally in humans and is primarily excreted via feces. The average urinary excretion rate is approximately 8%, and the fecal excretion rate is approximately 42%. Subcutaneous injection of clomiphene citrate labeled with 14C…distributed in the tissues of female newborn guinea pigs…estrogen-responsive tissues showed a high affinity for 14C. (14)C levels remained stable in the uterus…decreased in the ovaries and plasma…increased in the adrenal glands. /clomiphene citrate/ Approximately half of the ingested dose was excreted within five days; trace amounts of the drug remained in the feces for up to six weeks after administration. /Clomiphene Citrate/
Clomiphene is well absorbed after oral administration. The drug and its metabolites are primarily excreted in feces, with a small amount excreted in urine. The relatively long plasma half-life (approximately 5 to 7 days) is mainly due to plasma protein binding, enterohepatic circulation, and accumulation in adipose tissue. Long-lived active metabolites may also be produced.

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Metabolism/Metabolites
Liver
Incubation of the nonsteroidal anti-estrogenic clomiphene with rat liver microsomes resulted in the formation of 4-hydroxy, N-deethyl, and N-oxide metabolites, in stark contrast to previous similar experiments using rabbit microsomes, where only the first two metabolites were detected. No urinary excretion of the drug or its metabolites was detected after oral administration of clomiphene. 4-hydroxyclomiphene was the only detectable elimination product in the fecal extract.
Liver

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Biological Half-Life
5-7 days

Toxicity Summary
Clomiphene possesses both estrogenic and anti-estrogenic properties, but its exact mechanism of action remains unclear. Clomiphene appears to stimulate the release of gonadotropins (follicle-stimulating hormone (FSH) and luteinizing hormone (LH)), thereby promoting follicular maturation, ovulation, and corpus luteum development and function, ultimately leading to pregnancy. The release of gonadotropins may be due to direct stimulation of the hypothalamic-pituitary axis, or it may be due to clomiphene competing with endogenous estrogens from the uterus, pituitary gland, or hypothalamus, thereby reducing the inhibitory effect of estrogen on the hypothalamic-pituitary axis. Clomiphene has no significant progesterone, androgenic, or anti-androgenic effects and does not appear to interfere with the function of the pituitary-adrenal or pituitary-thyroid axis. Toxicity Data
The acute oral LD50 of clomiphene is 1700 mg/kg in mice and 5750 mg/kg in rats. The toxic dose in humans is unknown. There are no reports of acute overdose of clomiphene. During clomiphene treatment, if the dose exceeds the recommended dose, overdose symptoms may occur, including nausea, vomiting, vasomotor flushing, blurred vision, spots or flashes of light in front of the eyes, scotomas, enlarged ovaries, and pelvic or abdominal pain. Hepatotoxicity: Information on serum transaminase levels during clomiphene treatment is limited because clomiphene is usually taken only in low doses for short periods. Although there are a few reports of mild serum enzyme elevations in patients taking clomiphene, there is no conclusive evidence that it causes specific, clinically significant liver damage. Drugs used to treat female infertility often work by stimulating ovarian follicles, which can lead to ovarian hyperstimulation syndrome (OHSS), sometimes accompanied by elevated serum enzymes and even jaundice. This syndrome usually appears 4 to 14 days after ovarian stimulation with gonadotropins or clomiphene and is characterized by abdominal pain, abdominal distension with ascites, enlarged ovaries, and ovarian cysts. Patients may experience significant fluid shifts, leading to hemoconcentration and rapid development of severe ascites and pleural effusion. Liver dysfunction is present in 25% to 40% of patients with ovarian hyperstimulation syndrome (OHSS), typically manifested as mild to moderate elevations in alanine aminotransferase (ALT) and aspartate aminotransferase (AST), but with minimal or no elevation in serum bilirubin and alkaline phosphatase levels. Liver dysfunction usually resolves as OHSS subsides, typically within 2 to 3 weeks of onset. In severe cases, OHSS can be fatal, but death is usually due to dehydration, shock, and sepsis, rather than liver failure. In typical cases of abnormal liver enzymes, liver histology shows nonspecific changes, including sinusoidal dilatation, mild fatty deposits, and focal inflammatory infiltration dominated by macrophages and lymphocytes. The incidence of OHSS is lower with clomiphene citrate compared to ovulation induction with human chorionic gonadotropin (hCG).
Probability Score: C (Possibly the cause of clinically significant liver damage in ovarian hyperstimulation syndrome).

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Effects during pregnancy and lactation
◉ Summary of medication use during lactation
A small amount of clomiphene was detected in the breast milk of a woman. Multiple studies have found that clomiphene can suppress lactation in women who do not wish to breastfeed. Its mechanism of action appears to be through reducing serum prolactin levels, especially the peak serum prolactin level after stimulation. Clomiphene may interfere with lactation in breastfeeding mothers.
◉ Effects on breastfed infants
A woman taking 2.04 mg/kg clomiphene daily breastfed part-time. She did not observe any adverse reactions in her infant.
◉ Effects on Lactation and Breast Milk
A double-blind study compared the effects of clomiphene daily at 50 mg for 10 days (n = 110), clomiphene daily at 100 mg for 5 days (n = 26), and placebo (n = 41) on inhibiting lactation and relieving pain and breast engorgement in non-lactating postpartum mothers. According to women's reports, both doses of clomiphene were superior to placebo, but the 100 mg daily dose was slightly superior to the 50 mg daily dose.
A study compared the effects of clomiphene daily at 100 mg (n = 60) for 5 days with placebo (n = 30) on inhibiting lactation and relieving breast engorgement symptoms. Physicians observed that starting clomiphene within 12 hours postpartum was superior to starting clomiphene at 12 hours or longer postpartum in all indicators; both treatments were more effective than mechanical measures alone (e.g., breast binding). A randomized trial compared the effects of four treatment regimens on reducing postpartum serum prolactin levels and decreasing lactation: clomiphene 50 mg twice daily for 14 days (n = 15); bromocriptine 2.5 mg twice daily for 14 days (n = 15); diethylstilbestrol 5 mg three times daily for 14 days (n = 15); testosterone propionate 75 mg intramuscularly once (n = 15); and placebo orally three times daily (n = 15). After three days of treatment, serum prolactin levels in the clomiphene group decreased to 65% of baseline, while those in the bromocriptine group decreased to only 35%. Clomiphene was also less effective than bromocriptine in suppressing lactation and relieving breast engorgement. Another study compared the effects of starting clomiphene 100 mg/day on the first postpartum day for 7 days (n = 10) with placebo (n = 12). The results showed that clomiphene was no more effective than placebo in suppressing lactation or lowering serum prolactin levels. Women who did not wish to breastfeed during the first week postpartum were either treated with clomiphene 50 mg/day twice daily (n=10) or a placebo (n=10). Women taking clomiphene did not experience elevated serum prolactin levels above baseline during breast pump use; women taking the placebo experienced normal post-stimulation increases in serum prolactin. A total of 80 postpartum women participated in the study. Forty women took clomiphene 50 mg/day twice daily for five consecutive days starting from day one postpartum; 20 women took clomiphene 50 mg twice daily for five consecutive days starting from day four postpartum; and the remaining 20 women received a placebo. All women taking clomiphene experienced lactation suppression, breast engorgement, discomfort, and decreased serum prolactin levels. For subjects 1 day postpartum, serum prolactin concentrations were significantly lower than baseline levels on day 3; for subjects 4 days postpartum, serum prolactin concentrations were significantly lower than baseline levels on day 5. Placebo did not suppress lactation or lower serum prolactin levels. Adverse Reactions: Some reported adverse reactions to clomiphene include headache, dizziness, exacerbation of psychiatric illness, gynecomastia, testicular tumors, vasomotor flushing, gastrointestinal discomfort, and breast pain. Other common adverse reactions include nausea, vomiting, ovarian enlargement, blurred vision, scotoma, abnormal uterine bleeding, pelvic pain, and hypertriglyceridemia. Some serious adverse reactions to clomiphene include multiple pregnancy, thrombocytopenia, pancreatitis, increased risk of ovarian cancer with long-term use, increased risk of malignant melanoma, severe visual impairment, and liver damage. Ovarian hyperstimulation syndrome (OHSS) has been reported in patients treated with clomiphene citrate for ovulation induction. OHSS can progress rapidly (within 24 hours) and may develop into a medical emergency.

[1]. Interaction of antiestrogens with binding sites for muscarinic cholinergic drugs and calcium channel blockers in cell membranes. Cancer Chemother Pharmacol. 1990;26(4):310-2;
[2]. [Perinatal clomiphene citrate treatment changes sexual orientations of male mice]. Dongwuxue Yanjiu. 2013 Oct;34(5):487-92.
[3]. Ovulation induction with clomifene: a primary care perspective. J Fam Plann Reprod Health Care. 2012;38(1):48-52.

Therapeutic Uses
Female Fertility Agents Veterinary Drugs: Used to induce ovulation in anovulatory female animals and to induce the restoration of normal menstrual cycles in animals with amenorrhea or oligomenorrhea. Multiple Pregnancy Rate: Approximately 8% of pregnancies are induced, six times the normal rate, but lower than with human menopausal gonadotropin (HMG). Multiple pregnancies are almost always twins; large multiple pregnancies are reported in rare cases. About 20% of pregnancies induced by clomiphene citrate result in spontaneous abortion (mostly early abortion), a rate that may be only slightly higher than normal but not higher than in infertile individuals. Clomiphene Citrate Clomiphene citrate has also been used to treat male oligospermia…but the value of this treatment has not been determined. Clomiphene Citrate For more complete data on the therapeutic uses of clomiphene (10 types), please visit the HSDB record page.

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Drug Warnings
Objective signs are rare, but there have been reports of decreased vision, definable scotoma, and altered retinal cell function. Clomiphene Citrate
Other adverse reactions include…headache, breast tenderness, and abdominal distension. Symptoms disappear after discontinuation of the drug. Clomiphene Citrate
Some physicians consider visual abnormalities a contraindication to continued use, while others continue treatment with lower doses. …Clomiphene should not be given to pregnant women; there is no indication for clomiphene treatment once pregnancy occurs. Clomiphene Citrate
Sulfobromophthalein retention may increase. High doses may increase norepinephrine levels.
For more complete data on drug warnings for clomiphene (21 in total), please visit the HSDB records page.

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Pharmacodynamics
Clomiphene (formerly known as clomiphene) is an oral nonsteroidal ovulation-inducing drug belonging to the class of selective estrogen receptor modulators (SERMs). Clomiphene can cause polyovulation, thus increasing the risk of twins. Clomiphene has a lower incidence of ovarian hyperstimulation syndrome compared to purified follicle-stimulating hormone (FSH). There may be risks of ovarian cancer and weight gain. Clomiphene can interact with tissues containing estrogen receptors, including the hypothalamus, pituitary gland, ovaries, endometrium, vagina, and cervix. Clomiphene may compete with estrogen for estrogen receptor binding sites and may delay the replenishment of intracellular estrogen receptors. Clomiphene initiates a series of endocrine events that ultimately lead to a pre-ovulatory gonadotropin surge and subsequent follicle rupture. The first endocrine event in clomiphene treatment is an increase in pituitary gonadotropin release. This initiates steroid production and follicle development, leading to ovarian follicle growth and elevated circulating estradiol levels. After ovulation, plasma progesterone and estradiol levels rise and fall as in a normal ovulatory cycle. Clomiphene is a styrene derivative with anti-estrogenic properties and is commonly used in reproductive medicine. Its ability to interact with muscarinic receptors and calcium channel binding sites suggests that, in addition to its major role in inducing ovulation, there may be potential off-target effects [1][3]

C26H28NOCL

405.95962

405.186

C, 76.92; H, 6.95; Cl, 8.73; N, 3.45; O, 3.94

911-45-5

Clomiphene citrate;50-41-9;Clomifene hydrochloride;57049-00-0;Clomifene-d5 hydrochloride;1346606-66-3

1548953

Typically exists as solid at room temperature

1.104g/cm3

509ºC at 760mmHg

117.25°C

261.6ºC

1.588

6.562

0

2

9

29

481

0

Cl/C(C1=CC=CC=C1)=C(C2=CC=C(OCCN(CC)CC)C=C2)/C3=CC=CC=C3

GKIRPKYJQBWNGO-OCEACIFDSA-N

InChI=1S/C26H28ClNO/c1-3-28(4-2)19-20-29-24-17-15-22(16-18-24)25(21-11-7-5-8-12-21)26(27)23-13-9-6-10-14-23/h5-18H,3-4,19-20H2,1-2H3/b26-25+

2-[4-[(E)-2-chloro-1,2-diphenylethenyl]phenoxy]-N,N-diethylethanamine

clomiphene; Clomiphene B; 911-45-5; Chlomaphene; Chloramifene; Clomifen; 2-(p-(2-Chloro-1,2-diphenylvinyl)phenoxy)triethylamine; 1HRS458QU2;

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.)