IP Receptor (Ki = 260 nM)
Selexipag (active metabolite: ACT-333679) is a highly selective and long-acting oral prostacyclin (PGI₂) receptor (IP receptor, Ptgir) agonist. [2]

The long-acting oral prodrug selexipag (NS-304) is an IP receptor agonist whose active form, MRE-269, is very selective for IP receptors. [3H]Iloprost binding to human and rat IP receptors is inhibited by selexipag (NS-304) in a concentration-dependent manner. The rat IP receptor has a Ki of 2100 nM and the human IP receptor has a Ki of 260 nM. With an EC50 of 177nM, intracellular cAMP levels in hIP-CHO cells rose in a concentration-dependent manner upon treatment with selenium (NS-304). With IC50 values of 5.5 and 3.4 μM, respectively, selexipag (NS-304) similarly prevents platelet aggregation in humans and monkeys, but not in dogs (IC50 > 100 μM) [1].

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Gene Expression Analysis (qPCR): In the lungs of nitrofen-induced CDH rat pups, the mRNA expression of the IP receptor gene (Ptgir) was significantly increased compared to controls. This increase showed only a trend towards improvement after antenatal treatment with NS-304. Conversely, the expression of prostacyclin synthase (Ptgis) was significantly decreased in CDH lungs, and this did not improve with NS-304 treatment. The expression of phosphodiesterase-3 (Pde3) showed no difference between control and CDH groups, but its expression was significantly decreased after NS-304 treatment in both control and CDH pups. [2]
- Protein Expression Analysis (Western Blot): Western blot analysis on whole lung extracts showed that the protein levels of the IP receptor (Ptgir) were reduced in CDH samples. Treatment with a combination of sildenafil and NS-304 restored Ptgir expression to nearly control levels. [2]
- Immunohistochemistry: Immunohistochemical staining was performed to visualize the expression and localization of the IP receptor (Ptgir) in the lungs of control and CDH rat pups, with and without NS-304 treatment. [2]

After NS-304 was given orally to rats, the Cmax of MRE-269 (active form of Selelexipag/NS-304) was 1.1 μg/mL and in dogs, it was 9.0 μg/mL. In anesthetized rats, selexipag (NS-304) administered intradually at doses of 1 or 3 mg/kg enhances FSBF during a 4-hour period. Specifically, Selelexipag (NS-304) at 3 mg/kg increased FSBF over time, reaching a maximal rise of 93% in FSBF within an hour of dosing [1].

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The plasma concentrations of MRE-269 (active form of Selelexipag/NS-304) remained near peak levels for more than 8 h after oral administration of NS-304 to rats and dogs, and NS-304 increased femoral skin blood flow in rats in a long-lasting manner without affecting the hemodynamics. These findings indicate that NS-304 acts as a long-acting IP receptor agonist in vivo. The continuous vasodilation evoked by NS-304 was not attenuated by repeated treatment, indicating that NS-304 is unlikely to cause severe desensitization of the IP receptor in rats. Moreover, a microdose pharmacokinetic study in which NS-304 was orally administered to healthy male volunteers showed conversion of NS-304 to MRE-269 and a long plasma elimination half-life for MRE-269 (7.9 h). In conclusion, NS-304 is an orally available and long-acting IP receptor agonist prodrug, and its active form, MRE-269, is highly selective for the IP receptor. Therefore, NS-304 is a promising drug candidate for various vascular diseases, especially pulmonary arterial hypertension and arteriosclerosis obliterans.[1]

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In a Sugen 5416/hypoxia rat model of PAH, selexipag significantly improved pulmonary artery obstruction, decreased right ventricular systolic pressure, decreased right ventricular hypertrophy and improved survival rate.[3]

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Effects on Lung Morphology (Air Saccules): In the nitrofen-induced CDH rat model, antenatal treatment with NS-304 alone did not significantly improve the abnormal airspace morphology (characterized by increased air saccule density and decreased airspace size) as measured by D₂-score, mean linear intercept (Lm), or number of air saccules. The combination of NS-304 with sildenafil also showed no significant improvement and appeared to counteract the positive effects of sildenafil monotherapy. [2]
- Effects on Pulmonary Vasculature (Vascular Remodeling): Antenatal treatment with NS-304 significantly reduced the increased thickening of the smooth muscle layer in small pulmonary vessels (25-50 μm) observed in CDH pups. It also normalized the increased number of Ki-67/Sma double-positive (proliferating) cells in these vessels. However, NS-304 did not improve the decreased vascular branching and total vasculature volume in CDH lungs. [2]
- Effects on Cardiovascular Defects (Right Ventricular Hypertrophy): Right ventricular hypertrophy, an indication of pulmonary hypertension, was present in CDH pups. Antenatal treatment with NS-304 significantly improved this hypertrophy, reducing the thickness of the right ventricular wall. [2]
- General Effects: Treatment with NS-304 increased the lung-to-kidney weight ratio (LW/KW) in both control and CDH pups, and increased the lung-to-body weight ratio (LW/BW) in CDH pups. [2]

Prostacyclin (PGI2) and its analogs are useful for the treatment of various vascular disorders, but their half-lives are too short for widespread clinical application. To overcome this drawback, we have synthesized a novel diphenylpyrazine derivative, 2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide (NS-304), a prodrug of the active form {4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic acid (MRE-269). NS-304 is an orally available and potent agonist for the PGI2 receptor (IP receptor). The inhibition constant (Ki) of MRE-269 for the human IP receptor was 20 nM; in contrast, the Ki values for other prostanoid receptors were >2.6 μM. MRE-269 was therefore a highly selective agonist for the IP receptor[1].

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Radioligand binding assays for human prostanoid receptors and rat IP receptor were performed using membrane preparations from CHO cells stably expressing each receptor. Membrane protein (50–200 μg depending on receptor) was incubated with the corresponding ³H‑labeled ligand (e.g., 5 nM ³H‑iloprost for IP, 5 nM ³H‑PGD₂ for DP, 5 nM ³H‑PGE₂ for EP₁/EP₂, 1 nM ³H‑PGE₂ for EP₃/EP₄, 1 nM ³H‑PGF₂α for FP, 5 nM ³H‑SQ‑29548 for TP) in binding buffer (25 mM Tris‑HCl, pH 7.4, containing 10 mM MgCl₂, 1 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride) at 25°C (DP, EP₁, FP) or 37°C (EP₂, EP₃, EP₄, IP, TP) for 1–2 hours. Non‑specific binding was determined in the presence of >500‑fold excess unlabeled ligand. Bound ligand was collected by rapid filtration on glass filters, washed four times with ice‑cold 50 mM Tris‑HCl buffer (pH 7.4), and radioactivity measured by liquid scintillation counting. The dissociation constant (Kd) and maximal binding sites (Bmax) were calculated. The IC50 (concentration giving 50% inhibition of specific binding) was estimated by linear regression, and the inhibition constant Ki was calculated using the equation Ki = IC50 / (1 + [L]/Kd), where [L] is the concentration of ³H‑labeled ligand [1].

CHO cells expressing the human IP receptor (hIP-CHO cells) are seeded at 1×105 cells/well in a 24-well plate and cultured for 48 h. The cells are washed with Dulbecco's phosphate-buffered saline without divalent cations, preincubated in the medium for 1 h at 37°C, and then incubated for 15 min at 37°C with medium containing each drug in the presence of 500 μM 3-isobutyl-1-methylxanthine. The medium is removed, and perchloric acid solution is added to terminate the reaction. Intracellular cAMP levels are measured by enzymelinked immunosorbent assay[1].

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Intracellular cAMP measurement: hIP‑CHO cells were seeded at 1×10⁵ cells/well in 24‑well plates and cultured for 48 hours. Cells were washed with divalent cation‑free Dulbecco’s phosphate‑buffered saline, preincubated for 1 hour at 37°C, then incubated for 15 minutes at 37°C with medium containing test compound in the presence of 500 μM 3‑isobutyl‑1‑methylxanthine (IBMX). The reaction was terminated by adding perchloric acid, and intracellular cAMP levels were measured by enzyme‑linked immunosorbent assay (ELISA) [1].
Platelet aggregation assay: Blood freshly collected from humans, monkeys, dogs, or rats was mixed with 3.8% trisodium citrate (1:10 v/v). Platelet‑rich plasma was prepared by centrifugation at 150g for 10 minutes at room temperature. Platelet aggregation was induced by 10 μM ADP and monitored at 37°C with an aggregometer in the presence or absence of test compound, following the method of Born (1962) [1].

Pregnant Sprague-Dawley rats received either 100 mg nitrofen dissolved in 1 ml olive oil or just olive oil by gavage on gestational age day (E) 9.5. Administration of nitrofen at exactly this time point induces mainly left-sided CDH in ~70% of the offspring, whereas all pups have PH. This study included only pups with an observable diaphragmatic defect. Pregnant rats were divided into eight groups: control, nitrofen (CDH), control + sildenafil, nitrofen + sildenafil (CDH+sildenafil), control + NS-304 , nitrofen + NS-304 (CDH+NS-304), control + sildenafil/NS-304, and nitrofen + sildenafil/NS-304 (CDH+sildenafil/NS-304). Sildenafil (100 mg·kg−1·day−1) and NS-304 (1 mg·kg−1·day−1) were dissolved in 0.8% ethanol in water and administered via oral gavage for 4 consecutive days from E17.5 to E20.5. At E21 pups were delivered by caesarean section and euthanized by lethal injection of pentobarbital (Fig. 1). The dosage of sildenafil was based on our previous study, whereas for NS-304 a dose study was performed.[2]

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Male Sprague-Dawley rats, cynomolgus monkeys, and male beagle dogs are used. Selexipag (NS-304) is orally administered to rats at 10 mg/kg and to dogs at 3 mg/kg, and blood samples are collected at various times and centrifuged to obtain plasma. The plasma concentrations of Selexipag (NS-304) and MRE-269 after oral administration of Selexipag (NS-304) to each animal are determined by high performance liquid chromatography coupled to mass spectrometry (LC/MS), and their pharmacokinetic parameters are calculated.Rats are orally administered Selexipag (NS-304) at 3 mg/kg twice daily for 1, 2, 3, or 4 weeks as a pretreatment. On the day after the final administration in the pretreatment, rats are anesthetized with urethane, and the FSBF is measured with a laser Doppler flowmeter after intraduodenal administration of Selexipag (NS-304) at 3 mg/kg[1].

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Effects on Lung Morphology (Air Saccules):** In the nitrofen-induced CDH rat model, antenatal treatment with NS-304 alone did not significantly improve the abnormal airspace morphology (characterized by increased air saccule density and decreased airspace size) as measured by D₂-score, mean linear intercept (Lm), or number of air saccules. The combination of NS-304 with sildenafil also showed no significant improvement and appeared to counteract the positive effects of sildenafil monotherapy. [2]
- **Effects on Pulmonary Vasculature (Vascular Remodeling):** Antenatal treatment with NS-304 significantly reduced the increased thickening of the smooth muscle layer in small pulmonary vessels (25-50 μm) observed in CDH pups. It also normalized the increased number of Ki-67/Sma double-positive (proliferating) cells in these vessels. However, NS-304 did not improve the decreased vascular branching and total vasculature volume in CDH lungs. [2]
- **Effects on Cardiovascular Defects (Right Ventricular Hypertrophy):** Right ventricular hypertrophy, an indication of pulmonary hypertension, was present in CDH pups. Antenatal treatment with NS-304 significantly improved this hypertrophy, reducing the thickness of the right ventricular wall. [2]
- **General Effects:** Treatment with NS-304 increased the lung-to-kidney weight ratio (LW/KW) in both control and CDH pups, and increased the lung-to-body weight ratio (LW/BW) in CDH pups. [2]

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Effects on Lung Morphology (Air Saccules): In the nitrofen-induced CDH rat model, antenatal treatment with NS-304 alone did not significantly improve the abnormal airspace morphology (characterized by increased air saccule density and decreased airspace size) as measured by D₂-score, mean linear intercept (Lm), or number of air saccules. The combination of NS-304 with sildenafil also showed no significant improvement and appeared to counteract the positive effects of sildenafil monotherapy. [2]
- Effects on Pulmonary Vasculature (Vascular Remodeling): Antenatal treatment with NS-304 significantly reduced the increased thickening of the smooth muscle layer in small pulmonary vessels (25-50 μm) observed in CDH pups. It also normalized the increased number of Ki-67/Sma double-positive (proliferating) cells in these vessels. However, NS-304 did not improve the decreased vascular branching and total vasculature volume in CDH lungs. [2]
- Effects on Cardiovascular Defects (Right Ventricular Hypertrophy): Right ventricular hypertrophy, an indication of pulmonary hypertension, was present in CDH pups. Antenatal treatment with NS-304 significantly improved this hypertrophy, reducing the thickness of the right ventricular wall. [2]
- General Effects: Treatment with NS-304 increased the lung-to-kidney weight ratio (LW/KW) in both control and CDH pups, and increased the lung-to-body weight ratio (LW/BW) in CDH pups. [2]

Absorption, Distribution and Excretion
Following oral administration, peak concentrations of selexipag and its metabolites were observed to be reached at 1–3 hours and 3–4 hours, respectively. Food affects absorption, leading to a delayed time to peak concentration and a reduction in peak plasma concentration of approximately 30%. However, food has no significant effect on drug exposure. 93% is excreted in feces, and 12% in urine. The mean excretion rate is 35 L/h. Metabolism/Metabolites The active metabolite of selexipag is produced by the hydrolysis of acylsulfonamides by hepatic carboxylesterase 1. Oxidative metabolism catalyzed by CYP3A4 and CYP2C8 produces hydroxylated and dealkylated products. UGT1A3 and UGT2B7 are involved in the glucuronidation of the active metabolite. Other metabolites circulating besides the active metabolite do not exceed 3% of the total drug-related substances. Biological Half-Life The terminal half-life of selexipag is 0.8–2.5 hours. The terminal half-life of the active metabolite is 6.2–13.5 hours.

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CYP Interaction and Clearance: The study discusses the potential for drug-drug interactions, noting that sildenafil and selexipag may both induce the activation of the CYP3A4 enzyme, which is involved in the clearance of both compounds. This could lead to increased clearance and reduced exposure to the active metabolite, potentially explaining the lack of synergistic effect when they are combined. [2]
- Placental Transfer: The study suggests that the positive effects of NS-304 on the fetal pulmonary vasculature indicate that the compound, or its active metabolite, successfully entered the fetal circulation following maternal administration. [2]

Hepatotoxicity
Selexipag was associated with a low incidence (0% to 3%) of elevated serum transaminases, similar to the placebo group in clinical trials. These elevations were typically mild (rarely exceeding 3 times the upper limit of normal), transient, and asymptomatic. No cases of elevated serum enzymes with jaundice were reported in these pre-registration clinical trials. Since its approval and widespread use, there have been no reports of clinically visible liver injury with jaundice caused by selexipag, but its use is limited. Probability Score: E (Unlikely to be the cause of clinically visible liver injury). Protein Binding Selexipag and its active metabolites have high protein binding rates, approximately 99%.

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Embryotoxicity and Malformations: In the dose-finding study, pups of mothers treated with NS-304 (at doses up to 10 mg/kg/day) did not show any malformations of the face, palate, limbs, or other organs. Histological examination of lungs and kidneys revealed no abnormalities. The study also references embryotoxicity studies in rats and rabbits which showed no malformations or irregularities after the use of selexipag during pregnancy. [2]
- Hepatic Steatosis: Initial use of the compound dissolved in 8% ethanol resulted in steatosis (fatty liver) in the pups. This adverse effect was resolved after the ethanol percentage in the vehicle was adjusted from 8% to 0.8%. [2]

[1]. 2-[4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy]-N-(methylsulfonyl)acetamide (NS-304), an orally available and long-acting prostacyclin receptor agonist prodrug. J Pharmacol Exp Ther. 2007 Sep;322(3):1181-8.

[2]. Treatment of rat congenital diaphragmatic hernia with sildenafil and NS-304, selexipag's active compound, at the pseudoglandular stage improves lung vasculature. Am J Physiol Lung Cell Mol Physiol. 2018 May 10.

Selexipag belongs to the pyrazine class of compounds, with the chemical name N-(methanesulfonyl)-2-{4-[(propyl-2-yl)(pyrazin-2-yl)amino]butoxy}acetamide, containing two phenyl substituents at the 5 and 6 positions of the pyrazine ring. It is an orphan drug used to treat pulmonary arterial hypertension (PAH) and a prodrug of ACT-333679 (free carboxylic acid). Selexipag possesses various pharmacological activities, including orphan drug, prostacyclin receptor agonist, platelet aggregation inhibitor, vasodilator, and prodrug. It is a monocarboxylic acid amide, ether, pyrazine compound, aromatic amine, tertiary amine compound, and N-sulfonylformamide. It is functionally related to ACT-333679. On December 22, 2015, the U.S. FDA approved Selexipag for the treatment of pulmonary arterial hypertension (PAH) to slow disease progression and reduce the risk of hospitalization. PAH is a relatively rare disease with a generally poor prognosis, requiring more treatment options to prolong long-term efficacy. Selexipag and its active metabolite ACT-333679 (MRE-269), marketed by Actelion Pharmaceuticals under the brand name Uptravi, act as prostacyclin receptor agonists, increasing pulmonary vasodilation and reducing elevated pressure in the blood vessels supplying the lungs. Selexipag is a prostacyclin receptor agonist. Selexipag's mechanism of action is as a prostacyclin receptor agonist. Selexipag is a prostacyclin receptor agonist that causes pulmonary vasodilation and is used to treat pulmonary arterial hypertension (PAH). Elevated serum enzyme levels during selexipag treatment are rare, but have not been found to be associated with clinically significant cases of acute liver injury. Drug Indications Selexipag is indicated for the treatment of pulmonary arterial hypertension (PAH) to slow disease progression and reduce the risk of hospitalization.
FDA Label
Uptravi is indicated for the long-term treatment of adult patients with pulmonary arterial hypertension (PAH) of WHO functional classification (FC) II-III, as a combination therapy for patients whose PAH is poorly controlled by endothelin receptor antagonists (ERAs) and/or phosphodiesterase type 5 (PDE-5) inhibitors, or as monotherapy for patients who are not suitable for these therapies. It has been shown to be effective in PAH populations including idiopathic and hereditary PAH, connective tissue disease-related PAH, and corrected simple congenital heart disease-related PAH.
Treatment of Pulmonary Arterial Hypertension
Mechanism of Action

Celepag is a selective prostacyclin (IP, also known as PGI2) receptor agonist. A key characteristic of pulmonary arterial hypertension is a reduction in pulmonary prostacyclin and prostacyclin synthase (an enzyme that helps produce prostacyclin). Prostacyclin is a potent vasodilator with antiproliferative, anti-inflammatory, and antithrombotic effects; therefore, the use of IP receptor agonists for treatment has a sound theoretical basis. Celepag has a unique chemical structure; it is not PGI2 or a PGI2 analog and is highly selective for the IP receptor. It is metabolized by carboxylesterase 1 to produce an active metabolite (ACT-333679), which is approximately 37 times more potent than celepag. Both celepag and its metabolites are selective, targeting the IP receptor rather than other prostaglandin receptors.

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Pharmacodynamics
At the maximum tolerated dose of 1600 mcg twice daily, celepag did not prolong the QT interval to a clinically significant degree. Both celepag and its metabolites inhibited platelet aggregation in vitro in a concentration-dependent manner, with IC50 values of 5.5 µM and 0.21 µM, respectively. However, at clinically relevant concentrations, platelet aggregation parameters were not affected after repeated administrations of celepag to healthy subjects.

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Background: Selexipag is a novel, highly selective, long-acting oral PGI₂ receptor agonist that was recently approved for the treatment of pulmonary hypertension in adults. Its active compound, NS-304, is hydrolyzed by the liver to its active metabolite ACT-333679, which has an even higher affinity for the PGI₂ receptor. [2]
- Clinical Context: The study is the first to investigate the antenatal use of selexipag (targeting the PGI₂ pathway) in combination with sildenafil (targeting the NO pathway) in a rat model of congenital diaphragmatic hernia (CDH). The treatment was initiated at E17.5, a phase of lung development comparable to 20 weeks of gestation in humans, which is when CDH is typically detected by ultrasound. [2]

C26H32N4O4S

496.62

496.214

C, 62.88; H, 6.50; N, 11.28; O, 12.89; S, 6.46

475086-01-2

Selexipag-d7;1265295-21-3;Selexipag-d6;1265295-92-8

9913767

Typically exists as White to yellow solids at room temperature

1.2±0.1 g/cm3

1.579

4.56

1

7

12

35

730

0

S(C([H])([H])[H])(N([H])C(C([H])([H])OC([H])([H])C([H])([H])C([H])([H])C([H])([H])N(C1=C([H])N=C(C2C([H])=C([H])C([H])=C([H])C=2[H])C(C2C([H])=C([H])C([H])=C([H])C=2[H])=N1)C([H])(C([H])([H])[H])C([H])([H])[H])=O)(=O)=O

QXWZQTURMXZVHJ-UHFFFAOYSA-N

InChI=1S/C26H32N4O4S/c1-20(2)30(16-10-11-17-34-19-24(31)29-35(3,32)33)23-18-27-25(21-12-6-4-7-13-21)26(28-23)22-14-8-5-9-15-22/h4-9,12-15,18,20H,10-11,16-17,19H2,1-3H3,(H,29,31)

2-[4-[(5,6-diphenylpyrazin-2-yl)-propan-2-ylamino]butoxy]-N-methylsulfonylacetamide

NS 304; ACT293987; NS-304; ACT 293987; NS304; Uptravi; ACT-293987; Selexipag; 475086-01-2; NS-304; Uptravi; ACT-293987; NS 304; ACT 293987; 2-(4-((5,6-diphenylpyrazin-2-yl)(isopropyl)amino)butoxy)-N-(methylsulfonyl)acetamide;

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 (~100.68 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.03 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 (5.03 mM) 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; with ultrasonication.
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 (5.03 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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 (Please use freshly prepared in vivo formulations for optimal results.)