Estrogen receptor (IC50 = 3.3 nM); CRISPR/Cas9

4-Hydroxytamoxifen，也称为单羟基他莫昔芬，作为雌激素受体的选择性拮抗剂。它与 [3H]雌二醇结合，IC50 为 3.3 nM。 4-羟基他莫昔芬 (10, 100 nM) 可抑制人 8S 雌激素受体与 [3H]雌二醇的结合 [1]。 4-Hydroxytamoxifen 减少脱靶 CRISPR 介导的基因编辑并激活内含肽连接的非活性 Cas9。条件激活的 Cas9 在改变人类细胞中的靶基因组位置方面的选择性比野生型 Cas9 高 25 倍 [2]。

在未成熟大鼠中，4-Hydroxytamoxifen（0.2、1 和 5 μg/d）以剂量相关的方式降低子宫湿重 [1]。 4-羟基他莫昔芬（6 μg/0.1 mL 芝麻油，每天一次皮下注射）显着减弱了完整和去腺切除的两性 C57BL/6 J 小鼠中甲基苯丙胺诱导的黑质纹状体多巴胺耗竭。 4-纹状体的多巴胺水平不受 4-羟基他莫昔芬的影响 [3]。

将胞质溶胶 (200 μL) 与不同浓度的雌二醇、他莫昔芬和 (4-羟基他莫昔芬) 或二羟基他莫昔芬在 10 μL 甲醇中一起在 4°C 下孵育 30 分钟。对照管仅与 10 μL 甲醇一起孵育，并在细胞质 (200 μL) 与含有 DES (5 × 106 M) 的甲醇 (10 μL) 平行孵育中测定非特异性结合。将 TED 缓冲液中的 [2,4,6,7-3H]雌二醇溶液 (50 μL) 添加到每个管中，得到 2 × 10-9 M 的终浓度。继续孵育 4 小时 (4°C)，然后然后添加 400 μL 葡聚糖包被的木炭（250 mg% Norit A、2.5 mg% 葡聚糖）在 TED 缓冲液中的悬浮液，并静置 20 分钟。将管在 800 g 下离心 10 分钟 (4°C)，并将 400 μL 上清液样品添加到 10 mL 氚闪烁体中（6 g 丁基 PBD、135 mL 甲苯、720 ml 二恶烷、100 g 萘、45 mL 无水甲醇） ）。样品在液体闪烁光谱仪中计数 10 分钟。计数效率由外部标准化确定（35-36%）。结果表示为对照管中特异性结合放射性 (cpm) 的百分比[1]。

基因组GFP的修饰[2]
HEK293-GFP稳定细胞，其组成性表达Emerald GFP，用作报告细胞系。将细胞维持在“全血清培养基”中：Dulbecco改良的Eagle培养基加上含有10%（vol/vol）FBS和青霉素/链霉素（1×）的GlutaMax。将5×104个细胞接种在48孔胶原包被的生物涂层板上。电镀后16-18小时，根据制造商的方案，用Lipofectamine 2000转染细胞。简言之，使用1.5µL Lipofectamine 2000转染650 ng总质粒：500 ng Cas9表达质粒、125 ng sgRNA表达质粒和25 ng近红外iRFP670表达质粒。转染后12小时，用全血清培养基替换培养基，无论是否加入4-HT（1µM）。转染后3-4天再次更换培养基。转染后5天，对细胞进行胰蛋白酶消化并重悬于全血清培养基中，并在C6流式细胞仪上用488nm激光激发和520nm带通滤光片进行分析。转染和流式细胞术测量一式三份。

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基因组修饰的高通量DNA测序[2]
如上所述，用表达Cas9（500ng）和sgRNA（125ng）的质粒转染HEK293-GFP稳定细胞。对于转染减少量的野生型Cas9表达质粒的处理，使用pUC19质粒使质粒的总量达到500纳克。在转染过程中酌情添加4-HT（1µM最终）。转染后12小时，用不含4-HT的全血清培养基代替培养基。转染后60小时，使用先前报道的DNAdvanceKit方案从三个生物复制品中分离并汇集基因组DNA。使用150ng或200ng的基因组DNA作为模板，用前面描述的侧翼HTS引物对通过PCR扩增靶上和靶外基因组位点。使用RapidTips纯化PCR产物，并使用PicoGreen dsDNA测定试剂盒进行定量。用含有测序衔接子的引物对纯化的DNA进行PCR扩增，用MinElute PCR纯化试剂盒和AMPure XP PCR纯化进行纯化。在MiSeq高通量DNA测序仪上对样品进行测序，并如前所述分析测序数据。

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内含子剪接的蛋白质印迹分析[2]
用500ng Cas9表达质粒和125ng sgRNA表达质粒转染HEK293-GFP稳定细胞。转染后12小时，用全血清培养基替换培养基，无论是否加入4-HT（1µM）。在4-HT处理后4、8、12或24小时裂解并汇集来自三个技术重复的细胞。样品在Bolt 4-12%Bis-Tris凝胶上进行。使用抗FLAG抗体（Sigma-Aldrich F1804）和抗小鼠800CW IRDye（LI-COR）在Odyssey IR成像仪上观察凝胶。

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].

Absorption, Distribution and Excretion
Absorbed following topical application.

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Metabolism / Metabolites
4-Hydroxytamoxifen has known human metabolites that include 3,4-Dihydroxy-Tamoxifen, Tamoxifen 4-O-sulfate, Tamoxifen 4-O-glucuronide, and Endoxifen.
4-Hydroxytamoxifen is a known human metabolite of tamoxifen.

[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 amino compound that is tamoxifen in which the phenyl group which is in a Z- relationship to the ethyl substituent is hydroxylated at the para- position. It is the active metabolite of tamoxifen. It has a role as an antineoplastic agent, an estrogen receptor antagonist and a metabolite. It is a tertiary amino compound and a member of phenols. It is functionally related to a tamoxifen.
Afimoxifene (4-Hydroxytamoxifen, trade name TamoGel) is a new estrogen inhibitor under investigation for a variety of estrogen-dependent conditions, including cyclic breast pain and gynecomastia. TamoGel is formulated using Enhanced Hydroalcoholic Gel (EHG) Technology. This technology enables percutaneous delivery of drugs that cannot be delivered orally. It is being developed by Ascent Therapeutics.
4-Hydroxytamoxifen has been reported in Penicillium aurantiacobrunneum with data available.
Afimoxifene is a tamoxifen metabolite with both estrogenic and anti-estrogenic effects. Afimoxifene has a higher affinity for the estrogen receptor than tamoxifen, and functions as an antagonist in breast cancer cells.
4-Hydroxytamoxifen (Afimoxifene) is a metabolite of Tamoxifen. Afimoxifene (4-hydroxytamoxifen) is a selective estrogen receptor modulator which is the active metabolite of tamoxifen. Afimoxifene is a transdermal gel formulation and is being developed by Ascend Therapeutics, Inc. under the trademark TamoGel. (Wikipedia)

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Drug Indication
For the potential treatment of menstrual-cycle related mastalgia, fibrocystic breast disease, breast disease, gynecomastia and Keloid scarring.

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Mechanism of Action
Afimoxifene binds to estrogen receptors (ER), inducing a conformational change in the receptor. This results in a blockage or change in the expression of estrogen dependent genes.

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

32.7

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 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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (5.37 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (5.37 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (5.37 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 5 mg/mL (12.90 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。