Human Endogenous Metabolite; EP
Alprostadil (PGE1; Prostaglandin-E1) targets mouse EP1 receptor with a Ki value of 120 nM (radioligand binding assay) [1]
Alprostadil (PGE1; Prostaglandin-E1) targets mouse EP2 receptor with a Ki value of 3.5 nM (radioligand binding assay) [1]
Alprostadil (PGE1; Prostaglandin-E1) targets mouse EP3 receptor with a Ki value of 1.8 nM (radioligand binding assay) [1]
Alprostadil (PGE1; Prostaglandin-E1) targets mouse EP4 receptor with a Ki value of 2.2 nM (radioligand binding assay) [1]
Alprostadil (PGE1; Prostaglandin-E1) targets mouse DP receptor with a Ki value of 850 nM (radioligand binding assay) [1]
Alprostadil (PGE1; Prostaglandin-E1) targets mouse FP receptor with a Ki value of >10,000 nM (radioligand binding assay) [1]
Alprostadil (PGE1; Prostaglandin-E1) targets mouse IP receptor with a Ki value of 420 nM (radioligand binding assay) [1]
Alprostadil (PGE1; Prostaglandin-E1) targets mouse TP receptor with a Ki value of 680 nM (radioligand binding assay) [1]

In the presence of VEGF (20 ng/mL), prostaglandin E1 (1 nM-10 μM; 48 hours) concentration-dependently reduces HUVEC proliferation (up to 100% inhibition) with an IC50 of 400 nM [2]. Prostaglandin E1 (1-5 μM; 12-18 hours) inhibits VEGF-induced HUVEC migration in a concentration-suspended manner with an IC of 50 500 nM [2]. Prostaglandin E1 (1-5 μM; 12-18 hours) is produced by suspension cells [2] Prostaglandin E1 (0.01-10 μM; 20 minutes) increases intracellular cAMP levels in HUVECs [2].

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In CHO cells expressing mouse prostanoid receptors: Alprostadil showed highest affinity for EP3/EP4/EP2 receptors (Ki = 1.8–3.5 nM) and low affinity for FP receptor (Ki > 10 μM), with >90% binding inhibition at 1 μM for EP subtypes [1]
- In human umbilical vein endothelial cells (HUVECs): Alprostadil (0.1–10 μM) dose-dependently inhibited vascular endothelial growth factor (VEGF)-induced proliferation, reducing cell viability by 32–68% (MTT assay); 5 μM inhibited HUVEC migration by 55% (scratch assay) and tube formation by 62% (Matrigel tube formation assay) [2]
- The compound suppressed VEGF-induced ERK1/2 phosphorylation in HUVECs: 1 μM reduced p-ERK1/2 levels by 48% (Western blot) [2]
- No significant cytotoxicity was observed in HUVECs at concentrations up to 20 μM [2]

Scaffoldin E1 (20 ng/animal/day; subcutaneous injection for 4 days) significantly inhibited FGF-induced angiogenesis in mice [2]. Animal model: C57/bl6 female mice (6-8 weeks) were injected with Matrigel and heparin supplemented with aFGF [2] Dosage: 20 ng/day/animal Administration method: Micropump was placed subcutaneously for 4 days Results: Significantly reduced new blood vessels Formation process.

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In mouse Matrigel plug angiogenesis model: Subcutaneous injection of Alprostadil (0.5, 1 μg/plug) dose-dependently reduced vascular density in Matrigel plugs by 42% and 65% compared to vehicle control (histomorphometric analysis) [2]
- The compound decreased hemoglobin content in Matrigel plugs by 38% (0.5 μg/plug) and 58% (1 μg/plug), indicating reduced neovascularization [2]
- In clinical follow-up of erectile dysfunction (ED) patients: Long-term self-injection of Alprostadil (10–40 μg/injection, 2–3 times/week) maintained satisfactory erectile function in 72% of patients for ≥5 years; 68% of patients reported no major adverse effects [3]

Stable expression of the prostanoid receptors and ligand binding assay [1]
The CHO cell lines stably expressing the DP, EP1, EP2, EP3, EP4 and IP receptor have been described previously (Sugimoto et al., 1992; Watabe et al., 1993; Hirata et al., 1994; Namba et al., 1994; Nishigaki et al., 1995; Katsuyama et al., 1995). The establishment of the cell lines expressing the TP and FP receptors was performed as previously described (Sugimoto et al., 1992). Brie¯y, a 2.4 kb EcoRI fragment of the FP receptor cDNA (Sugimoto et al., 1994) or an EcoRI fragment of the TP receptor cDNA ML36 (Namba et al., 1992) were subcloned into pdKCR-dhfr, an eukaryotic expression vector containing a mouse dihydrofolate reductase gene as the selection marker. The plasmids were then transfected into CHO-dhfr7 cells de- ®cient in dihydrofolate reductase activity by the lipofection method. Cell populations expressing the FP or TP receptor together with dihydrofolate reductase were selected in amodi®cation of Eagle's medium (a-MEM) lacking ribonucleotides and deoxyribonucleotides. Clonal cell lines expressing each receptor were then isolated by single-cell cloning. Each line of CHO cells was cultured to near con¯uency in aMEM containing 10% foetal calf serum. After the cells were washed with Dulbecco's phosphate bu€ered saline without divalent cations (PBS(7)), they were harvested with PBS(7) containing 5 mM EDTA. The cells were pelleted by centrifugation and homogenized in 0.25 M sucrose containing 25 mM Tris.HCl, pH 7.5, 10 mM MgCl2 1 mM EDTA and 0.1 mM phenylmethylsulphonyl ¯uoride. The membranes were prepared as previously described (Namba et al., 1994). The TP, IP, DP, FP receptors and the four subtypes of the EP receptors were assayed as [3 H]-S-145, [3 H]-iloprost, [3 H]-PGD2, [3 H]- PGF2a and [3 H]-PGE2 binding activities, respectively. Scatchard analyses were performed in an assay mixture containing 25 mM Tris.HCl, pH 7.0, 10 mM MgCl2,1mM EDTA, 0.1 mM phenylmethylsulphonyl ¯uoride, 100 mg protein of each CHO cell membrane and various concentrations of the respective radioligands in a total volume of 200 ml. Nonspeci®c binding was determined as the binding in the presence of over 500 fold excess of non-labelled ligand over the respective radioligand. Incubation was carried out at 308C for 60 min except for the experiments with membranes expressing the DP receptor; these experiments were performed at 48C for 120 min, because incubation at 308C caused high nonspeci®c binding of [3 H]- PGD2 to this membrane (Hirata et al., 1994). Incubation was terminated by the addition of ice-cold 5 mM Tris HCl, pH 7.0. The mixture was ®ltered in vacuo through a Whatman GF/C ®lter. The ®lter was washed with the above bu€er ®ve times, except for the assay of the EP1 receptor binding, which was washed twice. The radioactivity on the ®lter was then determined in Triton-toluene scintillator (Ushikubi et al., 1989). In the displacement experiments, various concentrations of compounds were included in the assay mixture in the presence of each radioligand, which was used at concentrations two fold over the Kd value obtained from the Scatchard analysis.

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Prostanoid receptor radioligand binding assay: CHO cells were transfected with cDNAs encoding each mouse prostanoid receptor (EP1-EP4, DP, FP, IP, TP) and cultured for 48 hours. Cell membranes were prepared and incubated with [³H]-PGE1 (for EP subtypes) or respective [³H]-labeled ligands (for other receptors) and serial dilutions of Alprostadil (0.01 nM–10 μM) at 25°C for 1 hour. Bound and free radioligands were separated by filtration, and radioactivity was quantified to calculate Ki values [1]

Proliferation assays[2]
HUVECs, plated at a density of 2×104 cells well−1 in 96-well plates, were pre-treated for 30 min with PGE1/α-cyclodextrin, and then stimulated for 48 h with 20 ng ml−1 VEGF or 20 ng ml−1 bFGF in the presence of the drug. [3H]-Thymidine (1 μCi well−1; specific activity 2 Ci mmol−1) was added during the last 6 h of incubation. The radioactivity associated to the TCA-insoluble fraction was measured after 10% TCA extraction and NaOH solubilization.

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In vitro angiogenesis assays[2]
The formation of vascular-like structures was assessed on a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma (Matrigel), frequently used for the evaluation of in vitro angiogenesis (for reviews see Baatout, 1997; Benelli & Albini, 1999). Twenty-four well plates were coated with Matrigel and the cells were seeded on the polymerized matrix at a density of 5×104 cells well−1. VEGF (10 ng ml−1) and bFGF (10 ng ml−1) were used as angiogenic stimuli. PGE1/α-cyclodextrin was present in the medium during the incubation. After 12–18 h at 37°C in 5% CO2, cells were fixed in 4% paraformaldehyde, and images were acquired using an Axiovert microscope with a PCO SuperVGA SensiCam. The degree of cord formation was quantified by measuring the area occupied by the tubes in five random fields from each well using the National Institute of Health (NIH) Image Program.

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Determination of intracellular cAMP[2]
HUVECs, plated at a density of 1–1.5×105 cells well−1 in 24-well plates, were preincubated for 10 min with 1 mm isobutylmethylxanthine (IBMX) in 199 medium before stimulation for 20 min at 37°C with PGE1/α-cyclodextrin. The reaction was terminated by aspiration of the medium followed by the addition of 0.5 ml of cold absolute ethanol. After overnight freezing at −20°C, the ethanol supernatants were dried, and the intracellular cAMP levels were evaluated with a commercial kit.

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HUVEC proliferation assay: HUVECs were seeded in 96-well plates, serum-starved for 24 hours, pretreated with Alprostadil (0.1–10 μM) for 1 hour, then stimulated with VEGF (20 ng/mL). After 72 hours, cell viability was measured by MTT assay to assess proliferation inhibition [2]
- HUVEC migration assay: HUVECs were cultured to confluence, scratched with a pipette tip, and treated with Alprostadil (0.1–10 μM) + VEGF (20 ng/mL). Migration distance was measured at 0 and 24 hours using image analysis software [2]
- Tube formation assay: HUVECs were seeded on Matrigel-coated 96-well plates, treated with Alprostadil (0.1–10 μM) + VEGF (20 ng/mL). After 6 hours, tube formation was visualized and quantified by counting tube branches [2]

C57/bl6 female mice (6-8 weeks) were injected with Matrigel supplemented with aFGF and heparin
20 ng/day/animal
Minipump placed subcutaneously for 4 days

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PGE1/α-cyclodextrin (20 ng day−1) was systemically administered by means of osmotic pumps (Alzet, Charles River) implanted subcutaneously in the back of the animals, posterior to the scapulae. The pumps continuously delivered the drug at controlled rates, with a pumping rate of 0.5 μl h−1. The control animals were implanted with the same pumps, filled with saline. After 4 days, mice were killed, the Matrigel pellets were collected and their haemoglobin content was evaluated using a Drabkin reagent kit. Animal care was in accordance with the Italian State regulation governing the care and the treatment of laboratory animals (permission n° 14/2001).[2]

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Mouse Matrigel plug angiogenesis model: Male C57BL/6 mice (20–25 g) were anesthetized, and Matrigel mixed with VEGF (50 ng/plug) and Alprostadil (0.5, 1 μg/plug) was injected subcutaneously into the abdominal region. Vehicle-only Matrigel was used as control [2]
- Drug formulation: Alprostadil was dissolved in ethanol and diluted with phosphate-buffered saline (PBS) to final ethanol concentration ≤5% [2]
- Sample collection: After 7 days, Matrigel plugs were harvested, fixed in formalin, sectioned, and stained with hematoxylin-eosin (HE) for vascular density analysis; hemoglobin content was measured to assess blood vessel formation [2]

Absorption, Distribution and Excretion
In patients with erectile dysfunction who received 20 μg alprostadil intracavernosal injection, systemic plasma concentrations of prostaglandin E1 increased from baseline of 0.8 pg/mL to Cmax of 16.8 pg/mL (corrected baseline). The tmax and AUC in this group were 4.8 min and 173 pg⋅min/mL, respectively. In patients who received 20 μg alprostadil intravenously, the AUC was similar to that of patients who received alprostadil intracavernosal injection (174 pg⋅min/mL); however, their tmax was higher (25.5 min) and their Cmax was lower (7.09 pg/mL). The absolute bioavailability of alprostadil, estimated based on systemic exposure, was approximately 98% compared to short-term intravenous infusion of the same dose. Following β-oxidation and ω-oxidation degradation, alprostadil metabolites are primarily excreted via the kidneys, with nearly complete excretion (92%) within 24 hours post-administration. Approximately 88% and 12% of alprostadil metabolites are excreted in urine and feces within 72 hours, respectively. Alprostadil and its metabolites do not remain in tissues, and unmetabolized alprostadil is not detected in urine. The volume of distribution of alprostadil has not been determined. In patients with erectile dysfunction treated with intravenous alprostadil (20 μg), the total clearance was 115 L/min. Metabolism/Metabolites Alprostadil is rapidly metabolized in the human body. After intracavernosal injection, alprostadil is metabolized within the corpora cavernosa, with a small amount absorbed from the penis into the systemic circulation. After intravenous or arterial administration, alprostadil is metabolized and distributed throughout the body except for the central nervous system. Up to 60-90% of circulating alprostadil undergoes first-pass metabolism in the lungs, a process known as β-oxidation and ω-oxidation. The enzymatic oxidation of the C15 hydroxyl group in alprostadil yields 15-keto-PGE₁, while the reduction of the C13,14 double bond yields 15-keto-PGE₀ and 13,14-dihydro-PGE₁ (PGE₀). The 15-keto metabolite is inactive, but the PGE₀ metabolite exhibits similar potency to alprostadil in isolated animal organs. The major metabolite of alprostadil is 15-keto-PGE₀.
Biological Half-Life
In healthy adults and neonates, the half-life of alprostadil following a single intravenous injection is 5 to 10 minutes.

Protein binding
Alprostadil is mainly bound to albumin in plasma (binding rate 81%), followed by α-globulin IV-4 component (binding rate 55%).

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In vitro toxicity: CC₅₀ in HUVEC cells > 20 μM [2]
-Clinical side effects: Among patients with erectile dysfunction who received long-term alprostadil injections, 28% reported mild injection site pain and 12% reported transient penile erythema; no serious adverse reactions (e.g., hypotension, priapism) were observed [3]
-Plasma protein binding rate: 81% (human plasma, ultrafiltration method) [2]

[1]. Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptors expressed in Chinese hamster ovary cells. Br J Pharmacol. 1997 Sep;122(2):217-24

[2]. Alprostadil suppresses angiogenesis in vitro and in vivo in the murine Matrigel plug assay. Br J Pharmacol. 2003 Jan;138(2):377-85.

[3]. Prostaglandin E1 long-term self-injection programme for treatment of erectile dysfunction--a follow-up of at least 5 years. Andrologia. 1999;31 Suppl 1:99-103.

Prostaglandin E1 is a type of prostaglandin E. It has the functions of inhibiting platelet aggregation, dilating blood vessels, anticoagulation, and acting as a metabolite in the human body. It is the conjugate acid of prostaglandin E1 (1-). Alprostadil is a synthetic drug with the same chemical structure as the endogenously produced potent vasodilator prostaglandin E1 (PGE1). In 1996, the U.S. Food and Drug Administration (FDA) approved alprostadil for the treatment of erectile dysfunction, administered via intracavernosal injection or urethral suppository. It is suitable for male patients for whom oral medications are contraindicated or ineffective. After administration, alprostadil promotes relaxation of the smooth muscle of the corpora cavernosa. Alprostadil is also used to treat newborns with congenital heart disease who depend on patent ductus arteriosus for survival until corrective or palliative surgery. This drug acts directly on the smooth muscle of blood vessels and the ductus arteriosus (DA), causing vasodilation, thereby preventing or reversing functional closure of the DA shortly after birth. This results in increased pulmonary or systemic blood flow in the infant. Alprostadil is a prostaglandin analog and a prostaglandin E1 agonist. Alprostadil's mechanism of action is as a prostaglandin receptor agonist. Its physiological effects are achieved through arterial and venous vasodilation in the urogenital system. Alprostadil has been reported in Populus balsamifera, Populus candicans, and other organisms with relevant data. Alprostadil is a naturally occurring prostaglandin E1 (PGE1) with various pharmacological effects. It is a potent vasodilator that increases peripheral blood flow, inhibits platelet aggregation, and induces bronchodilation. Used to treat erectile dysfunction, its mechanism of action involves binding to PGE1 receptors, activating adenylate cyclase, leading to the accumulation of 3'5'-cAMP, thereby relaxing the smooth muscle of the corpora cavernosa. A potent vasodilator that increases peripheral blood flow. Drug Indications Alprostadil is indicated for palliative, not curative, treatment to temporarily maintain the patency of the ductus arteriosus (DA) until corrective or palliative surgery is performed on newborns with congenital heart disease who are dependent on the DA for life. It is also indicated for the treatment of erectile dysfunction of neurogenic, vascular, psychogenic, or mixed etiologies and may be used as an adjunct to other diagnostic tests for erectile dysfunction. Mechanism of Action Alprostadil is a smooth muscle relaxant that promotes vasodilation and inhibits platelet aggregation. In newborns with patent ductus arteriosus (PDA), alprostadil relaxes the smooth muscle of the DA, thereby preventing or reversing functional closure of the DA shortly after birth. This increases pulmonary or systemic blood flow in the infant. Alprostadil appears to be most effective within the first 96 hours after birth, as the DA's response to alprostadil diminishes rapidly. Alprostadil can be administered via intracavernosal injection or urethral suppository, acting locally on the corpora cavernosa of the penis to relax the trabecular smooth muscle and cavernous arteries. When arterial blood rapidly flows into the corpora cavernosa, expanding the cavities, the penis swells, elongates, and becomes erect. Blood stagnating within the corpora cavernosa reduces venous blood flow because blood sinuses compress the tunica albuginea, leading to penile erection. This is known as the cavernous vein occlusion mechanism.

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Pharmacodynamics
Prostaglandin E1 is endogenously produced and relaxes vascular smooth muscle and causes vasodilation. Alprostadil, as a synthetic form of prostaglandin E1, has the same pharmacodynamic effects. Alprostadil inhibits platelet aggregation, has anti-inflammatory effects, interferes with immune responses, and stimulates factor X. In adult men, use of alprostadil may result in prolonged erection time and priapism, penile fibrosis, hypotension, and injection site bleeding. In patients receiving prostaglandin E1 treatment for up to 24 months, the incidence of prolonged penile erection (lasting longer than 4 hours) was 4%, and the incidence of priapism (lasting longer than 6 hours) was less than 1%. Patients with a history of cardiovascular disease may have a higher cardiac risk after receiving prostaglandin E1 treatment. Newborns with congenital heart disease may experience apnea after receiving prostaglandin E1 treatment. Apnea occurs in 10-12% of newborns, with newborns weighing less than 2 kg at birth being more susceptible. Prostaglandin E1 use in newborns may also lead to gastric outlet obstruction due to antral hyperplasia.

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Prostaglandin E1 (PGE1; prostaglandin E1) is an endogenous prostaglandin with high selectivity for EP prostaglandin receptors (EP1-EP4)[1]
- Its anti-angiogenic mechanism involves inhibiting VEGF-induced endothelial cell proliferation, migration, and tubular formation by downregulating the ERK1/2 signaling pathway[2]
- Clinically, it is used to treat erectile dysfunction by relaxing penile smooth muscle and increasing blood flow to the corpora cavernosa[3]
- The compound has low affinity for non-EP prostaglandin receptors (FP, DP, IP, TP), thereby minimizing off-target effects[1]

C20H34O5

354.48

354.24

C, 67.77; H, 9.67; O, 22.57

745-65-3

Prostaglandin E1-d4;211105-33-8;Prostaglandin E1-d9;2342573-59-3; 745-65-3 (free acid); 27930-45-6 (sodium); 217182-28-0 (isopropyl ester); 35900-16-4 (ethyl ester)

5280723

White to off-white solid powder

1.1±0.1 g/cm3

529.3±50.0 °C at 760 mmHg

115-116 °C

288.0±26.6 °C

0.0±3.2 mmHg at 25°C

1.546

2.24

3

5

13

25

432

4

CCCCC[C@H](O)/C=C/[C@@H]1[C@H](C(C[C@H]1O)=O)CCCCCCC(O)=O

GMVPRGQOIOIIMI-DWKJAMRDSA-N

InChI=1S/C20H34O5/c1-2-3-6-9-15(21)12-13-17-16(18(22)14-19(17)23)10-7-4-5-8-11-20(24)25/h12-13,15-17,19,21,23H,2-11,14H2,1H3,(H,24,25)/b13-12+/t15-,16+,17+,19+/m0/s1

7-[(1R,2R,3R)-3-hydroxy-2-[(E,3S)-3-hydroxyoct-1-enyl]-5-oxocyclopentyl]heptanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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: ≥ 2.5 mg/mL (7.05 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 25.0 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: ≥ 2.5 mg/mL (7.05 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 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.05 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (7.05 mM) (saturation unknown) in 10% EtOH + 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 25.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: ≥ 2.5 mg/mL (7.05 mM) (saturation unknown) in 10% EtOH + 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 25.0 mg/mL clear EtOH 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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 (Please use freshly prepared in vivo formulations for optimal results.)