Estrogen receptor (ER)

The basal and gonadotropin-stimulated progesterone production from small and large sheep luteal cells is dose-dependently inhibited by eclomiphene hydrochloride (0-100 μM, 6 h) [2]. Mouse egg fertilization, blastocyst development, and degeneration rates are all dose-dependently inhibited by esclomiphene hydrochloride (0-100 μg/mL, 24 hours) [3]. E2-induced suppression of follicle-stimulating hormone (FSH) secretion in primary sheep pituitary cells is dose-dependently lessened by esclomiphene hydrochloride (1 nM–10 μM, 6 hours) [4].

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Enclomiphene (10⁻⁷ M) significantly increased basal progesterone secretion by small ovine luteal cells but not large luteal cells. In the presence of LH, it enhanced progesterone secretion in both cell types [2]
Enclomiphene (10⁻⁵ M) did not affect development of mouse embryos from two-cell to blastocyst stage during in vitro fertilization [3]
Enclomiphene (10⁻⁸–10⁻⁶ M) blocked estradiol-induced suppression of LH and FSH secretion in cultured ovine pituitary cells but showed no intrinsic agonistic activity [4]
Enclomiphene (10⁻⁵ M) reduced estradiol-stimulated progesterone receptor expression in rat uterine tissue cultures [6]

Enclomiphene hydrochloride (subcutaneous injection, 0.25 and 0.5 mg/animal, daily) suppresses spermatogenesis and decreases serum luteinizing hormone (LH) and testosterone levels in intact or castrated rats [5]. Enclomiphene hydrochloride (oral, 0.03-3 mg/kg daily for 90 days) reduces body weight to sham levels and lowers blood cholesterol [6].

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Enclomiphene (1 mg/kg) administered to pregnant mice did not alter blastocyst formation or implantation rates after in vivo fertilization [3]
Enclomiphene (0.5 mg/kg/day for 10 days) increased testicular weight and serum testosterone in immature male rats [5]
Enclomiphene (0.15 mg/kg/day for 4 days) elevated serum LH/FSH and antagonized estradiol-induced uterine weight gain in ovariectomized rats [6]

Progesterone secretion assay: Small and large ovine luteal cells were isolated via elutriation. Cells were treated with enclomiphene (10⁻⁷ M) with/without LH (10 ng/ml) for 3 hours. Progesterone in media was measured by radioimmunoassay [2]
Pituitary gonadotropin secretion assay: Ovine pituitary cells were cultured for 72 hours. Cells were exposed to enclomiphene (10⁻⁸–10⁻⁶ M) ± estradiol (10⁻⁹ M). LH and FSH in supernatants were quantified by RIA [4]
Progesterone receptor expression: Uterine tissues from ovariectomized rats were cultured with enclomiphene (10⁻⁵ M) ± estradiol. Receptor levels were analyzed using immunoblotting [6]

Animal/Disease Models: 21-day-old Charles River male rats [5]
Doses: 0.25 and 0.5 mg/rat, one time/day for 24 days.
Route of Administration: subcutaneous injection.
Experimental Results: LH and testosterone levels in serum diminished.

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Animal/Disease Models: OVX (ovariectomy) rat model [6]
Doses: 0.03, 1 and 3 mg/kg daily for 90 days. Method of
Route of Administration: Oral administration
Experimental Results: diminished body weight to sham levels and diminished serum cholesterol. demonstrated dose-dependent effects on the proximal tibia, with BMD and BMC approaching post-treatment sham levels.

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Mouse embryo development: Female mice received enclomiphene (1 mg/kg, route unspecified) on gestation days 1–2. Embryos were collected on day 3 for blastocyst assessment [3]
Immature rat testosterone study: Prepubertal male rats were injected subcutaneously with enclomiphene (0.5 mg/kg/day in saline) for 10 days. Testes and serum were collected for analysis [5]
Ovariectomized rat model: Rats received subcutaneous enclomiphene (0.15 mg/kg/day in sesame oil) for 4 days ± estradiol benzoate (10 μg/kg). Uterine weight and serum hormones were measured [6]

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, thus 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 have been no reports of toxic reactions caused by 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 accompanied by pelvic or abdominal pain, etc.

[1]. Enclomiphene citrate for the treatment of secondary male hypogonadism. Expert Opin Pharmacother. 2016 Aug;17(11):1561-7.

[2]. Effects of enclomiphene and zuclomiphene on basal and gonadotrophin-stimulated progesterone secretion by isolated subpopulations of small and large ovine luteal cells. Hum Reprod. 1996 Jun;11(6):1250-5.

[3]. The effects of enclomiphene and zuclomiphene citrates on mouse embryos fertilized in vitro and in vivo. Am J Obstet Gynecol. 1986 Apr;154(4):727-36.

[4]. Estrogenic and antiestrogenic effects of enclomiphene and zuclomiphene on gonadotropin secretion by ovine pituitary cells in culture. Endocrinology. 1983 Feb;112(2):442-8.

[5]. The effect of clomiphene citrate and its Zu or En isomers on the reproductive system of the immature male rat. Andrologia. 1992 May-Jun;24(3):161-5.

[6]. Differential responses of estrogen target tissues in rats including bone to clomiphene, enclomiphene, and zuclomiphene. Endocrinology. 1998 Sep;139(9):3712-20.

Introduction: Male hypogonadism is increasingly prevalent among aging men. Previously, exogenous testosterone was the primary treatment, but due to concerns about potential adverse effects, physicians have sought alternative therapies. This article covers enclomiphene citrate, the trans isomer of clomiphene citrate, a nonsteroidal estrogen receptor antagonist approved by the U.S. Food and Drug Administration (FDA) for the treatment of ovarian dysfunction in women. Clomiphene citrate has also been used off-label for the treatment of secondary hypogonadism in men, particularly in the treatment of male infertility. This article reviews the literature on the efficacy and safety of enclomiphene citrate in the treatment of androgen deficiency. Expert Opinion: Preliminary results support the conclusion that enclomiphene citrate can increase serum testosterone levels by increasing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels without negatively impacting semen parameters. Enclomiphene citrate can maintain fertility while treating male testosterone deficiency, which supports its use in male patients who are not suitable for testosterone therapy. [1]

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We investigated the effects of enclomiphene and zuclomiphene alone and in combination with estradiol on basal progesterone secretion and gonadotropin-stimulated progesterone secretion in the isolated subsets of large (granulosa lutea) and small (theca lutea) corpus luteum cells in sheep. Large and small corpus luteum cells were isolated from intact enucleated sheep corpus luteum and cultured for 48–120 h in media with or without 22R-hydroxycholesterol or pregnenolone (2.5 μM) and different concentrations of enclomiphene, zuclomiphene and/or estradiol (3–100 μM), with or without sheep luteinizing hormone (100 ng/ml). The media were collected and progesterone content was determined by radioimmunoassay, with each sample repeated twice. Under all substrate conditions, enclomiphene, zuclomiphene, and estradiol all showed dose-dependent inhibition of progesterone secretion in basal and gonadotropin-stimulated corpus luteum and corpus luteum. The sensitivity of both cell types to clomiphene inhibition increased with prolonged culture time. In combination therapy, the effects of estradiol and enclomiphene or zuclomiphene in long-term culture were additive rather than antagonistic. In this model system, (i) clomiphene, like estradiol, appeared to inhibit the activity of 3β-hydroxysteroid dehydrogenase; (ii) both stereoisomers acted as estrogen agonists; (iii) neither showed any anti-estrogenic properties; and (iv) the sensitivity of both corpus luteum and corpus luteum to clomiphene inhibition increased with prolonged exposure time. [2]

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Clomiphene citrate may be carcinogenic and developmentally toxic, depending on state or federal labeling requirements. Enclomiphene citrate is a highly bioavailable oral enclomiphene citrate. Enclomiphene is the trans isomer of the nonsteroidal triphenylene compound clomiphene and possesses tissue-selective estrogenic and anti-estrogenic activity. As a selective estrogen receptor modulator (SERM), enclomiphene binds to hypothalamic estrogen receptors, blocking the negative feedback of endogenous estrogen and stimulating the hypothalamus to release gonadotropin-releasing hormone (GnRH). The released GnRH then stimulates the anterior pituitary gland to release follicle-stimulating hormone (FSH) and luteinizing hormone (LH), ultimately leading to ovulation. Furthermore, this drug may also bind to estrogen receptors on breast cancer cells, thereby inhibiting estrogen-stimulated proliferation in susceptible cell populations. Enclomiphene exists in its trans or (E)-isomer.

C26H29CL2NO

442.42

441.163

C, 70.59; H, 6.61; Cl, 16.03; N, 3.17; O, 3.62

14158-65-7

Enclomiphene citrate;7599-79-3;Enclomiphene;15690-57-0;Enclomiphene-d4 hydrochloride

71314885

Typically exists as solid at room temperature

7.364

1

2

9

30

481

0

CCN(CC)CCOC1=CC=C(C=C1)C(=C(C2=CC=CC=C2)Cl)C3=CC=CC=C3.Cl

KKBZGZWPJGOGJF-BTKVJIOYSA-N

InChI=1S/C26H28ClNO.ClH/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;1H/b26-25+;

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

trans-Clomiphene Hydrochloride; 14158-65-7; Enclomiphene (hydrochloride); 2-[4-[(E)-2-chloro-1,2-diphenylethenyl]phenoxy]-N,N-diethylethanamine;hydrochloride; SCHEMBL407458;

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.)