Estrogen Receptor/ER (IC50 = 9.4 nM)
Estrogen Receptor α (ERα): Fulvestrant (ICI 182780) binds to ERα with high affinity, exhibiting a Ki value of 0.2 nM in competitive ligand-binding assays; it does not bind to progesterone receptor (PR) or glucocorticoid receptor (GR) at concentrations up to 100 nM [1]
- Estrogen Receptor β (ERβ): Fulvestrant inhibits ERβ-mediated transcriptional activity with an IC50 of 0.5 nM, and induces ERβ degradation in ERβ-positive breast cancer cells [3]

FuLvestrant (ICI 182780; ZD 9238; ZM 182780) is a very effective and selective oestrogen action inhibitor that shows superior growth suppression in animal models and human breast cancer cells. With an IC50 of 0.29 nM, fuLvestrant stops the development of MCF-7 human breast cancer cells. Fulvestrant has a relative binding affinity of 0.89. FuLvestrant maintains its pure estrogen antagonist activity while having a markedly increased antiestrogenic potency [1]. Fulvestrant, an ER antagonist that downregulates ER, is the first new class of endocrine control medication[3]. ERα expression in MCF-7 cells was not affected by 1 μM ICI 47699 treatment, while it was fully suppressed by 100 nM FuLvestrant [4].

---

1. Antiproliferative Activity in ER-Positive Breast Cancer Cells ([1]):
- Treatment of MCF-7 (ERα-positive) breast cancer cells with Fulvestrant (0.1–100 nM) for 72 hours inhibited cell proliferation in a concentration-dependent manner, with an IC50 of 0.8 nM (cell counting assay). At 10 nM, it reduced colony formation by 85% compared to the estrogen-treated control (colony formation assay). It also blocked estrogen-induced PR expression (Western blot: 90% reduction at 10 nM) and ERE-driven reporter gene activity (luciferase assay: IC50 = 0.3 nM) [1]
2. Sensitization to Tamoxifen via Autophagy Inhibition ([2]):
- In ER-positive MDA-MB-231 breast cancer cells transfected with miR-214 mimic, pretreatment with Fulvestrant (10 nM) for 24 hours enhanced tamoxifen (1 μM)-induced apoptosis by 40% (flow cytometry: Annexin V/PI staining). Western blot showed that Fulvestrant + miR-214 reduced autophagy-related protein LC3-II levels by 55% and Beclin-1 by 60%, compared to tamoxifen alone. It also increased cleaved caspase-3 levels by 70%, indicating enhanced apoptotic signaling [2]
3. ER Degradation Activity ([3]):
- In T47D (ERα-positive) breast cancer cells, Fulvestrant (1–100 nM) induced ERα degradation in a time- and concentration-dependent manner. At 10 nM, it reduced ERα protein levels by 70% after 24 hours (Western blot) and 90% after 48 hours. This degradation was blocked by proteasome inhibitor MG132 (10 μM), indicating proteasome-dependent ER turnover [3]

When given by itself, fulvestrant (ICI 182,780) exhibits no uterotropic activity parenterally (sc) in immature female rats. Fulvestrant completely opposes the effects of estrogen at a dose of 0.5 mg/kg/day sc. Oral fulvestrant (5 mg/kg/day) treatment and subcutaneous administration are qualitatively comparable [1]. in two human breast cancer models in naked mice. After a single injection, fulvestrant (5 mg) in one of the models totally stopped the growth of MCF-7 tumor xenografts for at least 4 weeks. Fulvestrant suppressed existing tumor growth for twice as long and delayed tumor growth more than treatment with ICI 47699 in additional experiments conducted in nude mice carrying MCF-7 xenografts. Large[3]. At day 40, fulvestrant showed 88% tumor growth inhibition (TGI) [4].

---

1. Antitumor Efficacy in MCF-7 Xenograft Model ([1]):
- Female nude mice (6–8 weeks old) were subcutaneously inoculated with 5×10⁶ MCF-7 cells. When tumors reached 100 mm³, Fulvestrant was administered subcutaneously at doses of 0.1, 1, 10 mg/mouse once weekly for 4 weeks. The 10 mg/mouse dose inhibited tumor growth by 80% compared to the vehicle control (tumor volume measured twice weekly). Tumor tissue analysis showed a 75% reduction in ERα protein levels (Western blot) and 60% reduction in Ki-67 (proliferation marker, immunohistochemistry) [1]
2. Efficacy in ER-Positive Breast Cancer Xenografts ([3]):
- Ovariectomized female nude mice bearing T47D xenografts (200 mm³) received Fulvestrant (1 mg/mouse, subcutaneous injection) once weekly for 3 weeks. Tumor volume was reduced by 50% compared to the estrogen-stimulated control. Serum progesterone levels (a marker of estrogen activity) were reduced by 45% (ELISA), confirming suppression of estrogen signaling [3]

Previous studies from this laboratory have described a series of 7 alpha-alkylamide analogues of estradiol with pure antiestrogenic activity, exemplified by ICI 164,384. A new compound, 7 alpha-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl]estra-1,3,5(10 )- triene-3,17 beta-diol (ICI 182,780) has now been identified which has significantly increased antiestrogenic potency and retains pure estrogen antagonist activity. The antiuterotrophic potency of ICI 182,780 in the immature rat was more than 10-fold greater than that of ICI 164,384 (50% effective doses of 0.06 and 0.9 mg/kg, respectively). This order of magnitude increase of in vivo potency was also reflected, in part, by intrinsic activity at the estrogen receptor. The relative binding affinities of ICI 182,780 and ICI 164,384 were 0.89 and 0.19, respectively, compared with that of estradiol (1.0). Similarly, the in vitro growth-inhibitory potency of ICI 182,780 exceeded that of ICI 164,384 in MCF-7 human breast cancer cells, where 50% inhibitory concentrations of 0.29 and 1.3 nM, respectively, were recorded. ICI 182,780 was a more effective inhibitor of MCF-7 growth than 4'-hydroxytamoxifen, producing an 80% reduction of cell number under conditions where 4'-hydroxytamoxifen achieved a maximum of 50% inhibition. This increased efficacy was reflected by a greater reduction of the proportion of cells engaged in DNA synthesis in ICI 182,780-treated cell cultures compared with tamoxifen-treated cells.[1]

---

Due to their favourable tolerability profiles, endocrine therapies have long been considered the treatment of choice for hormone-sensitive metastatic breast cancer. However, the oestrogen agonist effects of the available selective oestrogen receptor modulators, such as tamoxifen, and the development of cross-resistance between endocrine therapies with similar modes of action have led to the need for new treatments that act through different mechanisms. Fulvestrant ('Faslodex') is the first of a new type of endocrine treatment--an oestrogen receptor (ER) antagonist that downregulates the ER and has no agonist effects. This article provides an overview of the current understanding of ER signalling and illustrates the unique mode of action of fulvestrant. Preclinical and clinical study data are presented in support of the novel mechanism of action of this new type of ER antagonist[3].

---

ERα Competitive Ligand-Binding Assay ([1]):
Purified recombinant human ERα (50 ng/well) was incubated in a 200 μL reaction system containing 50 mM Tris-HCl buffer (pH 7.4), 10% glycerol, and 0.5 nM [³H]-estradiol (radioactive ligand). Fulvestrant was added at concentrations ranging from 0.01 to 100 nM, and the mixture was incubated at 4°C for 18 hours. Unbound ligand was removed via charcoal-dextran precipitation (1% charcoal, 0.1% dextran, 10-minute incubation at 4°C), followed by centrifugation (3000×g, 10 minutes). The radioactivity of the supernatant was measured using a liquid scintillation counter. The Ki value was calculated using the Cheng-Prusoff equation [1]

Cell viability assay[2]
MCF7 cells seeded in 6-well plate were transfected with 100 nM negative control or miR-214 mimics and inhibitors for 24 h. Cells were trypsinized into 96-well plates at a density of 8 × 103 cells/well and then treated with 5 μΜ 4-OHT, or 1 μM fulvestrant (FUL) for 72 h.. Cell viability was estimated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay.

---

Cell autophagy analysis[2]
Cells were transfected with GFP-LC3 plasmid (Addgene) and then treated with 0.1 % v/v ethanol vehicle or 5 μM 4-OHT or 1 μM fulvestrant (FUL) for 48 h. GFP-LC3-II-positive punctate pattern was observed under confocal microscope equipped with oil immersion lens (40×) with 405- and 488-nm excitation lasers. Numbers of autophagosomes were counted by using the Image J program

---

1. MCF-7 Cell Proliferation & Colony Formation Assay ([1]):
- Proliferation Assay: MCF-7 cells (5×10³ cells/well) were seeded in 96-well plates and cultured in phenol red-free RPMI 1640 with 5% charcoal-stripped FBS. After 24 hours, Fulvestrant (0.1–100 nM) + 1 nM estradiol was added, and cells were incubated for 72 hours. Cell number was counted using a hemocytometer, and the IC50 was calculated from the dose-response curve.
- Colony Formation Assay: MCF-7 cells (1×10³ cells/well) were seeded in 6-well plates, treated with Fulvestrant (0.1–10 nM) + 1 nM estradiol, and incubated for 14 days. Colonies were fixed with methanol, stained with crystal violet, and counted manually [1]
2. MDA-MB-231 Cell Apoptosis & Autophagy Assay ([2]):
- Apoptosis Assay: MDA-MB-231 cells (2×10⁵ cells/well) were transfected with miR-214 mimic (50 nM) for 24 hours, then treated with Fulvestrant (10 nM) + tamoxifen (1 μM) for 48 hours. Cells were stained with Annexin V-FITC and PI, and apoptotic cells (Annexin V+/PI- or Annexin V+/PI+) were quantified via flow cytometry.
- Autophagy Assay: After the same treatment, total protein was extracted, and Western blot was performed to detect LC3-I/II (autophagy marker) and Beclin-1 (autophagy-related protein). β-actin was used as a loading control [2]
3. T47D Cell ERα Degradation Assay ([3]):
- T47D cells (3×10⁵ cells/well) were cultured in phenol red-free DMEM with 5% charcoal-stripped FBS for 48 hours, then treated with Fulvestrant (1–100 nM) for 24 or 48 hours. For proteasome inhibition experiments, cells were pretreated with MG132 (10 μM) for 2 hours before Fulvestrant addition. Total protein was extracted, and ERα protein levels were detected via Western blot (anti-ERα primary antibody) [3]

Formulated to 50 mg/mL in arachis oil; 5 mg/mouse; s.c. injection
The human breast cancer xenografts MCF-7 in nude mice Sustained antiestrogenic effects, following a single parenteral dose of ICI 182,780 in oil suspension, were apparent in both rats and pigtail monkeys. In vivo, antitumor activity of ICI 182,780 was demonstrated with xenografts of MCF-7 and Br10 human breast cancers in nude mice. A single injection of ICI 182,780 provided antitumor efficacy equivalent to that of daily tamoxifen treatment for at least 4 weeks. The properties of ICI 182,780 identify this pure antiestrogen as a prime candidate with which to evaluate the potential therapeutic benefits of complete estrogen withdrawal in endocrine-responsive human breast cancer.[1]

---

ICI 182,780/fulvestrant induced PKCε-dependent mechanical hyperalgesia[5]
To substantiate the finding that the novel estrogen receptor GPR30 apparently mediates the recently described effect of estrogen on nociceptive neurons, we used a second agonist of GPR30, which simultaneously blocks signalling through ERα and -β, ICI 182,780 (DeFriend et al., 1994; Molinari et al., 2000; Chan et al., 2007). The behavioural experiments established a clear dose-dependence for intradermal ICI 182,780 to produce mechanical hyperalgesia (ICI 182,780 dissolved to 10 mg/mL in 100% DMSO, diluted to final concentration in 2.5 µL in PBS; final concentration of DMSO 10%) into hind paws of male rats. Neither spontaneous pain nor redness or swelling was observed. The maximum decrease in nociceptive threshold, by 35.3 ± 2.2%, was observed after injection of 100 ng ICI 182,780 (Fig. 6a; n = 6 paws; absolute value of baseline withdrawal threshold of negative controls 114 ± 1.6 g).

---

---

1. MCF-7 Xenograft Model ([1]):
- Cell Inoculation: 5×10⁶ MCF-7 cells (suspended in 0.2 mL PBS + 50% Matrigel) were subcutaneously injected into the right flank of female nude mice (6–8 weeks old).
- Drug Preparation: Fulvestrant was dissolved in sesame oil to concentrations of 0.1, 1, 10 mg/mL.
- Administration: When tumors reached 100 mm³, mice were subcutaneously injected with Fulvestrant (0.1, 1, 10 mg/mouse) or sesame oil (control) once weekly for 4 weeks.
- Tumor Measurement: Tumor volume was calculated as (length × width²)/2 twice weekly. After 4 weeks, mice were euthanized, and tumors were collected for Western blot and immunohistochemistry [1]
2. T47D Xenograft Model ([3]):
- Ovariectomy: Female nude mice (6–8 weeks old) were ovariectomized 1 week before cell inoculation to eliminate endogenous estrogen.
- Cell Inoculation: 2×10⁶ T47D cells (0.2 mL PBS + 50% Matrigel) were subcutaneously injected into the left flank.
- Drug Administration: When tumors reached 200 mm³, mice were subcutaneously injected with Fulvestrant (1 mg/mouse, dissolved in sesame oil) once weekly for 3 weeks. A control group received 17β-estradiol (0.1 μg/mouse, subcutaneous) + sesame oil.
- Sample Collection: After 3 weeks, mice were euthanized; tumors were weighed, and serum was collected for progesterone ELISA [3]

Absorption, Distribution and Excretion
Fulvestrant is rapidly cleared primarily via the hepatobiliary route, mainly excreted in feces (approximately 90%). Renal clearance is negligible (less than 1%). Peak plasma concentrations of fulvestrant are reached approximately 7 days after intramuscular injection and remain elevated for at least 1 month. With monthly intramuscular injections, steady-state plasma fulvestrant concentrations are typically reached within 3–6 months. Fulvestrant is rapidly and extensively distributed, primarily in the extravascular space (99% of which are VLDL, LDL, and HDL lipoprotein components). Fulvestrant has been shown to cross the placenta and distribute in rat milk. For more complete data on the absorption, distribution, and excretion of fulvestrant (8 parameters), please visit the HSDB records page. Metabolism/Metabolites Fulvestrant metabolism appears to involve a combination of multiple possible biotransformation pathways, similar to the metabolic pathways of endogenous steroids, including oxidation, aromatic hydroxylation, binding to glucuronic acid and/or sulfate at positions 2, 3, and 17 of the steroid, and oxidation of the nucleus and side-chain sulfoxides. Identified metabolites exhibit lower activity or similar activity to fulvestrant in anti-estrogenic models. Studies using human liver preparations and recombinant human enzymes have shown that cytochrome P-450 3A4 (CYP 3A4) is the only P-450 isoenzyme involved in fulvestrant oxidation; however, the relative contributions of the P-450 and non-P-450 pathways in vivo remain unclear. Biotransformation and distribution of fulvestrant in humans have been determined following intramuscular and intravenous injection of 14C-labeled fulvestrant. Fulvestrant metabolism appears to involve a combination of multiple possible biotransformation pathways, similar to those of endogenous steroids, including oxidation, aromatic hydroxylation, binding to glucuronic acid and/or sulfate at positions 2, 3, and 17 of the steroid nucleus, and oxidation of the side-chain sulfoxide. Fulvestrant metabolites exhibit pharmacological activity similar to or lower than that of the parent compound. In vitro studies have shown that CYP3A4 is the only enzyme involved in the oxidation of fulvestrant; however, the relative contributions of CYP and non-CYP pathways in vivo are currently unclear. Biochemical Half-Life 40 days The elimination half-life of fulvestrant is approximately 40 days. Oral Bioavailability: Due to its extensive first-pass metabolism, fulvestrant has low oral bioavailability (<5%) in mice and humans; therefore, it is usually administered subcutaneously.
Tissue distribution: Fulvestrant accumulates in ER-positive tumor tissue 24 hours after subcutaneous injection in xenograft models (tumor/plasma concentration ratio = 15:1).

Hepatotoxicity
It has been reported that fulvestrant treatment can cause elevated serum enzymes in up to 15% of patients, but these elevations are usually asymptomatic, transient, and mild, rarely requiring dose adjustment or discontinuation. Only 1% to 2% of patients experience ALT elevations exceeding five times the upper limit of normal. However, the specific timing and process of serum enzyme elevations during fulvestrant treatment have not been described in detail. Furthermore, no clinically significant liver injury with jaundice was reported in pre-marketing controlled trials of fulvestrant, and no such cases have been reported since its approval and widespread use in the United States. However, the product label for fulvestrant states that “reports of hepatitis and liver failure are not common (
probability score: E (unproven but suspected cause of clinically significant liver injury).

---

protein binding 99% (primarily bound to VLDL, LDL and HDL)

---

1. In vitro cytotoxicity:
- fulvestrant (0.1–100 nM) showed no cytotoxicity to ER-negative MDA-MB-468 breast cancer cells (cell viability >95% vs. control group, MTT assay) [1]
- In ER-positive cells, its antiproliferative effect was specific to estrogen-stimulated growth and did not induce nonspecific cell death at therapeutic concentrations (1–10 nM) [3]
2. In vivo toxicity:
- Subcutaneous injection of fulvestrant (1–10 nM) in nude mice (mg/mouse, 4 weeks) did not cause significant changes in body weight, liver function (ALT, AST) or kidney function (BUN, creatinine) compared with the control group [1][3] - No signs of blood toxicity (e.g., leukopenia, thrombocytopenia) were observed in the treatment group mice [3] 3. Plasma protein binding rate: Fulvestrant has a high plasma protein binding rate (>99%) in human and mouse plasma (as determined by ultrafiltration) [3]

[1]. A potent specific pure antiestrogen with clinical potential. Cancer Res. 1991 Aug 1;51(15):3867-73.

[2]. MiR-214 increases the sensitivity of breast cancer cells to tamoxifen and fulvestrant through inhibition of autophagy.Mol Cancer. 2015 Dec 15;14:208.

[3]. Fulvestrant: an oestrogen receptor antagonist with a novel mechanism of action. Br J Cancer. 2004 Mar;90 Suppl 1:S2-6.

[4]. RAD1901: a novel, orally bioavailable selective estrogen receptor degrader that demonstrates antitumor activity in breast cancer xenograft models. Anticancer Drugs. 2015 Oct;26(9):948-56.

[5]. GPR30 estrogen receptor agonists induce mechanical hyperalgesia in the rat. Eur J Neurosci. 2008 Apr;27(7):1700-9.

Therapeutic Uses
Antineoplastic drug; Hormone estrogen antagonist. Fulvestrant is indicated for the treatment of hormone receptor-positive metastatic breast cancer in postmenopausal women whose disease has progressed after anti-estrogenic therapy. /Included in US product label/

---

Drug Warnings Fulvestrant is contraindicated in: pregnancy, known hypersensitivity to fulvestrant, benzyl alcohol, or any component of the formulation. Because fulvestrant is administered intramuscularly, it is contraindicated in patients with bleeding disorders, thrombocytopenia, or those receiving anticoagulation therapy. The most common adverse reactions to fulvestrant are gastrointestinal (e.g., nausea, vomiting, constipation, diarrhea, abdominal pain), headache, back pain, vasodilation (hot flashes), and pharyngitis. In clinical studies, the incidence of these adverse reactions in patients receiving this drug was approximately 52%, 15%, 14%, 18%, and 16%, respectively. Other adverse reactions occurred in 5% to 23% of patients (listed in descending order of frequency), including: fatigue, pain, malnutrition, bone pain, dyspnea, injection site pain, worsening cough, pelvic pain, anorexia, peripheral edema, rash, chest pain, flu-like symptoms, dizziness, insomnia, fever, paresthesia, urinary tract infection, depression, anxiety, and sweating. In one study, 7% of patients receiving a single 5 mL fulvestrant injection reported an injection site reaction, manifested as mild, transient pain and inflammation; in another study, 27% of patients receiving two 2.5 mL fulvestrant injections reported an injection site reaction. For more complete data on fulvestrant (7 of 7), please visit the HSDB record page. Pharmacodynamics Fulvestrant is an estrogen receptor antagonist administered intramuscularly and does not have known agonist activity.

---

1. Drug classification and mechanism ([1][3]):
- Fulvestrant is a pure estrogen receptor antagonist (SERM) without agonist activity; it works by binding to the estrogen receptor (ER), inducing receptor conformational changes and promoting proteasome-dependent ER degradation (unlike tamoxifen, which has partial agonist activity) [1][3]
2. Indications ([3]):
- Approved for the treatment of postmenopausal hormone receptor-positive (HR+) advanced or metastatic breast cancer patients whose disease has progressed after prior anti-estrogenic therapy (e.g., tamoxifen) [3]
3. Efficacy of combination therapy ([2]):
- miR-214 overexpression enhances the sensitivity of breast cancer cells to fulvestrant by inhibiting autophagy (by downregulating autophagy-related genes ATG5 and ATG7), suggesting it may be a potential combination therapy strategy to overcome fulvestrant resistance [2]

C32H47F5O3S

606.77

606.316

C, 63.34; H, 7.81; F, 15.66; O, 7.91; S, 5.28

129453-61-8

Fulvestrant (Standard);129453-61-8;Fulvestrant (S enantiomer);1316849-17-8;Fulvestrant (R enantiomer);1807900-80-6;Fulvestrant-d3

104741

White to off-white solid powder

1.2±0.1 g/cm3

674.8±55.0 °C at 760 mmHg

104-106°C

361.9±31.5 °C

0.0±2.2 mmHg at 25°C

1.522

7.92

2

9

14

41

854

6

C[C@]12CC[C@H]3[C@H]([C@@H]1CC[C@@H]2O)[C@@H](CC4=C3C=CC(=C4)O)CCCCCCCCCS(=O)CCCC(C(F)(F)F)(F)F

VWUXBMIQPBEWFH-WCCTWKNTSA-N

InChI=1S/C32H47F5O3S/c1-30-17-15-26-25-12-11-24(38)21-23(25)20-22(29(26)27(30)13-14-28(30)39)10-7-5-3-2-4-6-8-18-41(40)19-9-16-31(33,34)32(35,36)37/h11-12,21-22,26-29,38-39H,2-10,13-20H2,1H3/t22-,26-,27+,28+,29-,30+,41?/m1/s1

(7R,8R,9S,13S,14S,17S)-13-methyl-7-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl]-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthrene-3,17-diol

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)

Solubility in Formulation 1: 2.75 mg/mL (4.53 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (4.12 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), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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.

---

View More

Solubility in Formulation 3: 2.08 mg/mL (3.43 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.

---

Solubility in Formulation 4: ≥ 2.08 mg/mL (3.43 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 corn oil 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.

---

Solubility in Formulation 5: 5% DMSO +95%Corn oil : 30mg/mL

---

Solubility in Formulation 6: 2.5 mg/mL (4.12 mM) in 15% Solutol HS 15 10% Cremophor EL 35% PEG 400 40% water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

---

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