Estrogen receptor (IC50 = 3.3 nM); CRISPR/Cas9
The primary target of trans-4-Hydroxytamoxifen is the estrogen receptor (ER), with an IC50 of 3.3 nM for binding to [3H]oestradiol. It acts as an antagonist in breast tissues and as an agonist in other tissues such as the endometrium. It also targets estrogen-related receptors (ERR). In addition, trans-4-Hydroxytamoxifen induces the CRISPR/Cas9 system based on ER-mediated nuclear translocation.

4-Hydroxytamoxifen, also known as Monohydroxytamoxifen, functions as a selective antagonist of estrogen receptors. It binds to [3H]oestradiol with an IC50 of 3.3 nM. The human 8S estrogen receptor is inhibited from binding [3H]oestradiol by 4-hydroxytamoxifen (10, 100 nM) [1]. 4-Hydroxytamoxifen decreases off-target CRISPR-mediated gene editing and activates intein-linked inactive Cas9. Conditionally activated Cas9 is 25 times more selective than wild-type Cas9 at altering target genomic locations in human cells [2].
In vitro, trans-4-Hydroxytamoxifen functions as a selective antagonist of estrogen receptors and binds to [3H]oestradiol with an IC50 of 3.3 nM. It inhibits the binding of the human 8S estrogen receptor to [3H]oestradiol at 10 and 100 nM concentrations. In T47D human breast cancer cells, fixed ring-trans-4-hydroxytamoxifen is a potent antiestrogen with an IC50 of approximately 4 × 10⁻⁸ to 2 × 10⁻⁷ M. It decreases off-target CRISPR-mediated gene editing and activates intein-linked inactive Cas9.

In immature rats, 4-Hydroxytamoxifen (0.2, 1, and 5 μg/d) reduces uterine wet weight in a dose-related manner [1]. 4-The methamphetamine-induced nigrostriatal dopamine depletion in both sexes of intact and gonadectomized C57BL/6 J mice is significantly attenuated by hydroxytamoxifen (6 μg/0.1 mL sesame oil, once daily subcutaneous injection). 4-The striatum's dopamine levels are unaffected by 4-hydroxytamoxifen [3].
In vivo, trans-4-Hydroxytamoxifen (0.2, 1, and 5 μg/d) reduces uterine wet weight in immature rats in a dose-related manner. It significantly attenuates methamphetamine-induced nigrostriatal dopamine depletion in both sexes of intact and gonadectomized C57BL/6J mice at a dose of 6 μg/0.1 mL sesame oil, administered as a once-daily subcutaneous injection. Trans-4-hydroxytamoxifen has been percutaneously administered to breast cancer patients before surgery.

Cytosol (200 μL) is incubated for 30 min at 4°C with different concentrations of oestradiol, tamoxifen and (4-Hydroxytamoxifen) or dihydroxytamoxifen administered in 10 μL methanol. Control tubes are incubated with 10 μL methanol alone and non-specific binding is determined in a parallel incubation of cytosol (200 μL) with methanol (10 μL) containing DES (5 × 106 M). [2,4,6,7-3H]Oestradiol solution (50 μL) in TED buffer is added to each tube to give a final concentration of 2 × 10-9 M. Incubation is continued for 4 h (4°C) and then 400 μL of a suspension of dextran-coated charcoal (250 mg % Norit A, 2.5 mg % dextran) in TED buffer are added and allowed to stand for 20 min. Tubes are centrifuged at 800 g for 10 min (4°C) and 400 μL samples of the supernatant are added to 10 mL tritium scintillator (6 g butyl PBD, 135 mL toluene, 720 ml dioxan, 100 g naphthalene, 45 mL absolute methanol). Samples are counted for 10 min in a liquid scintillation spectrometer. Counting efficiency is determined by external standardization (35-36 %). Results are represented as a percentage of the specifically bound radioactivity (c.p.m.) in the control tubes[1].

Modification of genomic GFP[2]
HEK293-GFP stable cells, which constitutively express Emerald GFP, served as the reporter cell line. Cells were maintained in “full serum media”: Dulbecco’s Modified Eagle’s Media plus GlutaMax with 10% (vol/vol) FBS and penicillin/streptomycin (1×). 5 × 104 cells were plated on 48-well collagen-coated Biocoat plates. 16–18 h after plating, cells were transfected with Lipofectamine 2000 according to the manufacturer’s protocol. Briefly, 1.5 µL of Lipofectamine 2000 was used to transfect 650 ng of total plasmid: 500 ng Cas9 expression plasmid, 125 ng sgRNA expression plasmid, and 25 ng near-infrared iRFP670 expressing plasmid. 12 h after transfection, the media was replaced with full serum media, with or without 4-HT (1 µM). The media was replaced again 3–4 days after transfection. Five days after transfection, cells were trypsinized and resuspended in full serum media and analyzed on a C6 flow cytometer with a 488-nm laser excitation and 520-nm filter with a 20-nm band pass. Transfections and flow cytometry measurements were performed in triplicate.

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High-throughput DNA sequencing of genome modifications[2]
HEK293-GFP stable cells were transfected with plasmids expressing Cas9 (500 ng) and sgRNA (125 ng) as described above. For treatments in which a reduced amount of wild-type Cas9 expression plasmid was transfected, pUC19 plasmid was used to bring the total amount of plasmid to 500 ng. 4-HT (1 µM final), where appropriate, was added during transfection. 12 h after transfection, the media was replaced with full serum media without 4-HT. Genomic DNA was isolated and pooled from three biological replicates 60 h after transfection using a previously reported protocol with a DNAdvance Kit. 150 ng or 200 ng of genomic DNA was used as a template to amplify by PCR the on-target and off-target genomic sites with flanking HTS primer pairs described previously. PCR products were purified using RapidTips and quantified using the PicoGreen dsDNA Assay Kit. Purified DNA was PCR amplified with primers containing sequencing adaptors, purified with the MinElute PCR Purification Kit and AMPure XP PCR Purification. Samples were sequenced on a MiSeq high-throughput DNA sequencer , and sequencing data was analyzed as described previously.

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Western blot analysis of intein splicing[2]
HEK293-GFP stable cells were transfected with 500 ng Cas9 expression plasmid and 125 ng sgRNA expression plasmid. 12 h after transfection, the media was replaced with full serum media, with or without 4-HT (1 µM). Cells were lysed and pooled from three technical replicates 4, 8, 12, or 24 h after 4-HT treatment. Samples were run on a Bolt 4–12% Bis-Tris gel. An anti-FLAG antibody (Sigma-Aldrich F1804) and an anti-mouse 800CW IRDye (LI-COR) were used to visualize the gel on an Odyssey IR imager.
In vitro cell-based assays are performed using estrogen-responsive breast cancer cell lines such as T47D and MCF-7. Cells are incubated at 37°C for 50 min with 1 nM [3H]estradiol in the absence or presence of increasing amounts (0.1 nM - 10 μM) of trans-4-Hydroxytamoxifen. Cell proliferation is measured to determine the antiestrogenic activity. The compound's effects on cell growth and gene expression are evaluated in ZR-75-1 human breast cancer cells.

The present study was undertaken to assess the ability of 4-hydroxytamoxifen (4-OHT) to alter methamphetamine-induced nigrostriatal dopaminergic toxicity. Three daily doses of 4-OHT (6 micro g/day) effectively attenuated methamphetamine-induced nigrostriatal dopamine depletions in both sexes of intact and gonadectomized C57BL/6 J mice. 4-OHT alone did not alter the dopamine content levels in the striatum. Both male and female mice exhibited similar Cu, Zn-superoxide dismutase protein levels in the striata whether after gonadectomy or 4-OHT treatment. Furthermore, basal body temperature and methamphetamine-induced hyperthermia were not affected by 4-OHT treatment in either sex of mice. Using a lucigenen-derived chemiluminescence assay, we found that 4-OHT by itself can serve as a potent superoxide anion radical scavenger in vitro. The protective effects of 4-OHT against methamphetamine-induced nigrostriatal dopamine depletion can be, in part, due to its antioxidative characteristics. The free radical-scavenging ability of 4-OHT calls for further investigations for its uses in clinical practice.[3]
Animals of each sex are divided into two groups: one group receives 4-Hydroxytamoxifen [6 μg/0.1 mL sesame oil/day, subcutaneously (s.c.) starting at 06.00 h] injections for three consecutive days, while the other group receives an equivalent amount of sesame oil injection for 3 days. Four hours following the third injection, each group is then subdivided into two groups: one receives four cumulative doses of methamphetamine hydrochloride (10 mg/kg, s.c.), and the other receives a comparable volume of saline at 2-h intervals. Bilateral gonadectomy is performed under pentobarbital anesthesia (50 mg/kg, intraperitoneally). Five weeks after surgery,gonadectomized mice of each sex are randomly divided into six groups. Five groups of each sex receive three daily injections ofvarious concentrations of 4-Hydroxytamoxifen (0, 1.5, 3.0, 6.0, and 12.0 μg/0.1 mL sesame oil/day). Four hours following the third injection, mice receive four doses of methamphetamine (MA, 10 mg/kg) at 2-h intervals. The remaining group of each sex receives sesame oil pretreatment for three consecutive days, followed by saline injections, and serves as the control group[3].
In vivo animal experiments are conducted using immature female rats for uterine weight assays. trans-4-Hydroxytamoxifen is administered at doses of 0.2, 1, and 5 μg/d, and uterine wet weight is measured. In C57BL/6J mice, the compound is administered as a subcutaneous injection (6 μg/0.1 mL sesame oil, once daily) to study its effects on dopamine depletion. Percutaneous administration studies are performed in breast cancer patients before surgery.

Absorption, Distribution and Excretion
Absorbed after topical application. Metabolisms/Metabolites Known human metabolites of 4-hydroxytamoxifen include 3,4-dihydroxytamoxifen, tamoxifen 4-O-sulfate, tamoxifen 4-O-glucuronide, and endorphins. 4-Hydroxytamoxifen is a known human metabolite of tamoxifen.
Pharmacokinetic (PK) properties of trans-4-Hydroxytamoxifen indicate that it is the active metabolite of tamoxifen formed primarily by CYP2D6 and CYP3A4. It can be administered percutaneously, passing through the skin and concentrating in receptor structures of breast tissue, thus avoiding hepatic metabolism. The compound has a molecular weight of 387.51 and a molecular formula of C26H29NO2. It is soluble in DMSO and ethanol. Storage at -20°C is recommended for long-term stability.

Toxicology (toxicology) data for trans-4-Hydroxytamoxifen are similar to those of tamoxifen. As a SERM, it can have both estrogenic and anti-estrogenic effects depending on the tissue. It may cause endometrial proliferation due to its agonist activity in the endometrium. Common side effects include hot flashes, nausea, and vaginal discharge. Long-term use may increase the risk of endometrial cancer and thromboembolic events.

[1]. A monohydroxylated metabolite of tamoxifen with potent antioestrogenic activity. J Endocrinol. 1977 Nov;75(2):305-16.

[2]. Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nat Chem Biol. 2015 May;11(5):316-8.

[3]. 4-Hydroxytamoxifen attenuates methamphetamine-induced nigrostriatal dopaminergic toxicity in intact and gonadetomized mice. J Neurochem. 2003 Dec;87(6):1436-43.

[4]. Drug Inducible CRISPR/Cas Systems. Comput Struct Biotechnol J. 2019;17:1171-1177.

Afimoxifene is a tertiary amine compound, belonging to the triphenylamine class. Its structure is similar to tamoxifen, except that the phenyl group with a Z-configuration to the ethyl substituent is hydroxylated at the para-position. It is a triphenylamine compound and also the active metabolite of tamoxifen. It possesses multiple functions, including antitumor activity, estrogen receptor antagonism, and metabolic activity. Afimoxifene belongs to the tertiary amine class and is also a phenolic compound. Functionally, it is related to tamoxifen. Afimoxifene (4-hydroxytamoxifen, trade name TamoGel) is a novel estrogen inhibitor currently under investigation for its efficacy in treating various estrogen-dependent diseases, including cyclical breast pain and gynecomastia. TamoGel is formulated using enhanced hydroalcoholic gel (EHG) technology. This technology enables transdermal delivery of drugs that cannot be administered orally. This drug was developed by Ascend Therapeutics. 4-hydroxytamoxifen has been reported to exist in Penicillium aurantiacobrunneum, and relevant data are available.
Afimoxifene is a metabolite of tamoxifen and has dual estrogenic and anti-estrogenic effects. Afimoxifene has a higher affinity for estrogen receptors than tamoxifen and acts as an antagonist in breast cancer cells.
4-Hydroxytamoxifen (afimoxifene) is a metabolite of tamoxifen. Afimoxifene (4-hydroxytamoxifen) is a selective estrogen receptor modulator and the active metabolite of tamoxifen. Afimoxifene is a transdermal gel formulation developed by Ascend Therapeutics under the trademark TamoGel. (Wikipedia)
Drug Indications

Used to treat menstrual cycle-related breast pain, fibrocystic breast disease, breast disease, gynecomastia, and keloids.
Mechanism of Action

Afimoxifene binds to the estrogen receptor (ER), inducing conformational changes in the receptor. This leads to the inhibition or alteration of estrogen-dependent gene expression.
Other information: trans-4-Hydroxytamoxifen is also known as (Z)-4-Hydroxytamoxifen, ICI 79280, and Monohydroxytamoxifen. It is a major metabolite of tamoxifen, a well-known antiestrogen used in breast cancer treatment. The compound is used in research to study estrogen receptor signaling and as a tool for conditional gene expression systems such as CRISPR/Cas9. Its CAS number is 68047-06-3.

C26H29NO2

387.52

387.219

C, 80.59; H, 7.54; N, 3.61; O, 8.26

68047-06-3

(E/Z)-4-Hydroxytamoxifen;68392-35-8;(E)-4-Hydroxytamoxifen;174592-47-3

449459

Typically exists as white to off-white solids at room temperature

1.1±0.1 g/cm3

514.4±50.0 °C at 760 mmHg

105-107ºC

264.9±30.1 °C

0.0±1.4 mmHg at 25°C

1.597

7.34

1

3

8

29

493

0

O(C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])C1C([H])=C([H])C(=C([H])C=1[H])/C(/C1C([H])=C([H])C(=C([H])C=1[H])O[H])=C(\C1C([H])=C([H])C([H])=C([H])C=1[H])/C([H])([H])C([H])([H])[H]

TXUZVZSFRXZGTL-QPLCGJKRSA-N

InChI=1S/C26H29NO2/c1-4-25(20-8-6-5-7-9-20)26(21-10-14-23(28)15-11-21)22-12-16-24(17-13-22)29-19-18-27(2)3/h5-17,28H,4,18-19H2,1-3H3/b26-25-

4-[(Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-2-phenylbut-1-enyl]phenol

ICI 79280; 4-Hydroxytamoxifen; trans-4-Hydroxytamoxifen; (Z)-4-Hydroxytamoxifen

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 : ~50 mg/mL (~129.03 mM)
Ethanol : ~20 mg/mL (~51.61 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.37 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 (5.37 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 (5.37 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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Solubility in Formulation 4: 5 mg/mL (12.90 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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