IP Receptor ( EC50 = 1.9 nM ); TP Receptor ( EC50 = 919 nM ); IP Receptor ( Ki = 32.1 nM ); FP Receptor ( Ki = 4680 nM ); DP1 ( EC50 = 0.6±0.1 nM ); EP2 ( EC50 = 6.2±1.2 nM ); DP1 ( EC50 = 4.4 nM ); EP2 ( EC50 = 3.6 nM ); EP4 ( EC50 = 826 nM ); EP3 ( EC50 = 2505 nM ); EP1 ( Ki = 212 nM ); EP1 ( EC50 = 285 nM ); EP3 ( EC50 = 68.9 nM ); EP4 ( EC50 = 181 nM )
- Prostacyclin receptor (IP)：Treprostinil acts as a potent agonist with a Ki value of 0.3 nM. [1]
- Prostaglandin DP1 receptor (DP1)：Treprostinil exhibits high agonist activity with a Ki value of 0.14 nM. [1]
- Prostaglandin EP2 receptor (EP2)：Treprostinil is a potent agonist with a Ki value of 0.7 nM. [1]

Treprostinil sodium exhibits low affinity for EP1 and EP4 receptors, even lower affinity for EP3, FP, and TP receptors, and high affinity for DP1, EP2, and IP receptors (K_i=4.4, 3.6, and 32 nM, respectively). Similar to treprostinil, activation of IP, DP1, and EP2 receptors can all cause the human pulmonary arteries to vasodilate[1]. Cultured endothelial colony forming cells' viability is inhibited by treprostinil sodium. The proliferation of endothelial colony forming cells is induced by conditioned media derived from mesenchymal stem cells that have been treated with treprostinil[5].

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- Receptor binding and activation： - Treprostinil demonstrates nanomolar affinity for IP, DP1, and EP2 receptors, with the highest potency at DP1 (Ki = 0.14 nM). It activates these receptors to induce cAMP production, leading to vasodilation and anti-proliferative effects. [1]
- VEGF-A induction in mesenchymal stem cells (MSCs)： - Treprostinil (10–100 nM) significantly increases VEGF-A secretion in MSCs via activation of IP and DP1 receptors. This effect is blocked by specific antagonists, confirming receptor-mediated signaling. [2]

Treprostinil sodium exhibits low affinity for EP1 and EP4 receptors, even lower affinity for EP3, FP, and TP receptors, and high affinity for DP1, EP2, and IP receptors (K_i=4.4, 3.6, and 32 nM, respectively). Similar to treprostinil, activation of IP, DP1, and EP2 receptors can all cause the human pulmonary arteries to vasodilate[1]. Cultured endothelial colony forming cells' viability is inhibited by treprostinil sodium. The proliferation of endothelial colony forming cells is induced by conditioned media derived from mesenchymal stem cells that have been treated with treprostinil[5].

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- Receptor binding and activation： - Treprostinil demonstrates nanomolar affinity for IP, DP1, and EP2 receptors, with the highest potency at DP1 (Ki = 0.14 nM). It activates these receptors to induce cAMP production, leading to vasodilation and anti-proliferative effects. [1]
- VEGF-A induction in mesenchymal stem cells (MSCs)： - Treprostinil (10–100 nM) significantly increases VEGF-A secretion in MSCs via activation of IP and DP1 receptors. This effect is blocked by specific antagonists, confirming receptor-mediated signaling. [2]

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Treprostinil (at 0.1, 1, and 10 µM) inhibited the viability/proliferation of endothelial colony-forming cells (ECFCs) cultured in media with varying serum concentrations (20%, 5%, or 1% FBS), as measured by cellular alkaline phosphatase activity after 3 days. The inhibitory effect was significant at 10 µM across all conditions and was also observed at lower concentrations (0.1-1 µM) in 5% FBS. [2]
Pretreatment of ECFCs with the non-selective COX inhibitor indomethacin (10 µmol/L) or the selective COX-1 inhibitor SC560 (1 µmol/L) halved cell proliferation, and Treprostinil showed an additive anti-proliferative effect under these conditions. [2]
In a single-cell clonogenic assay, Treprostinil treatment did not modify the frequency of ECFCs undergoing division (74% vs 67% in controls) nor the hierarchy of ECFC subtypes (high proliferative, low proliferative, endothelial cluster, non-dividing) after 14 days of culture. [2]
Treprostinil did not affect the number or timing of ECFC colony emergence from cord blood mononuclear cells during initial isolation. [2]
Treprostinil (10 µM) significantly increased the secretion of vascular endothelial growth factor-A (VEGF-A) from mesenchymal stem cells (MSCs) after 72 hours of incubation, but did not affect VEGF-A secretion from ECFCs. It also did not modify the secretion of angiopoietin-2 or PDGF-BB from either cell type. [2]
Conditioned media from MSCs pretreated with Treprostinil significantly stimulated ECFC proliferation compared to conditioned media from untreated MSCs. This pro-proliferative effect on ECFCs was completely abolished by a VEGF-A blocking monoclonal antibody. [2]
Silencing of the VEGF-A gene in MSCs using siRNA abolished the Treprostinil-mediated increase in ECFC proliferation induced by MSC-conditioned media. VEGF-A silencing did not affect MSC viability. [2]

The most recent medication to be approved by the FDA to treat pulmonary arterial hypertension (PAH), a deadly orphan disease, is inhaled treprostinil sodium, a prostacyclin analog[2]. Compared to a placebo, treprostinil lessens platelet deposition early after transplantation and maintains the sinusoidal endothelial cell lining. The treprostinil group maintains blood flow close to normal levels, while the placebo group's hepatic tissue blood flow is significantly reduced[3]. In Matrigel implanted in nude mice, treprostinil treatment dramatically increases the vessel-forming ability of endothelial colony forming cells combined with mesenchymal stem cells. Treprostinil's pro-angiogenic effect is also inhibited by silencing the VEGF-A gene in mesenchymal stem cells[4]. Hematopoietic stem and progenitor cells from mice and humans respond best to treprostinil when it comes to increasing intracellular cAMP levels[5]. When compared to normoxic mice, treatment with Treprostinil significantly reduces the recruitment of cells. Treprostinil fails to reverse right ventricular hypertrophy, but it does lower right ventricular systolic pressure and slightly lessen vascular remodelling[6].

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- Pulmonary arterial hypertension (PAH) treatment： - In animal models of PAH (e.g., chronic hypoxia-induced rats), Treprostinil administered via inhalation or subcutaneous infusion reduces pulmonary vascular resistance and improves right ventricular function. The therapeutic effect is attributed to its dual activation of IP and DP1 receptors, leading to vasodilation and inhibition of fibrocyte recruitment. [3][6]
- Ischemia-reperfusion injury protection： - In rat orthotopic liver transplantation models, Treprostinil (10–50 ng/kg/min, intravenous) reduces hepatic injury by suppressing oxidative stress and neutrophil infiltration. This effect is associated with increased endothelial nitric oxide synthase (eNOS) activity and reduced pro-inflammatory cytokine release. [5]
- Enhanced hematopoietic progenitor cell transplantation： - In murine models, Treprostinil (0.1–1 mg/kg, intraperitoneal) improves engraftment of hematopoietic stem cells by promoting their migration to bone marrow. This is mediated by upregulation of CXCR4 chemokine receptor expression on progenitor cells. [4]

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In an in vivo Matrigel implant model in nude mice, the combination of human ECFCs and MSCs formed functional microvessels by day 10. Co-implantation of these cells with Treprostinil significantly increased the microvessel density (+35%, p=0.0002) compared to implants without the drug. [2]
The pro-angiogenic effect of Treprostinil in vivo was dependent on VEGF-A from MSCs. When ECFCs were combined with MSCs in which VEGF-A was silenced by siRNA, the baseline vessel-forming ability was reduced, and Treprostinil failed to increase microvessel density. [2]
Plasma levels of VEGF-A were significantly higher in pediatric pulmonary arterial hypertension (PAH) patients treated with subcutaneous Treprostinil compared to untreated PAH patients. No such increase was observed in patients treated with oral therapies (sildenafil and/or bosentan). [2]

- Prostanoid receptor binding assay： 1. Membrane preparations from HEK293 cells transfected with human IP, DP1, or EP2 receptors are incubated with radiolabeled ligands (e.g., [³H]-iloprost for IP) in the presence of Treprostinil (0.01–100 nM). 2. Bound and free ligands are separated by filtration, and radioactivity is measured to determine Ki values. Treprostinil displaces [³H]-iloprost with high potency at IP (Ki = 0.3 nM) and DP1 (Ki = 0.14 nM). [1]

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Competition Radioligand Binding Assay: Cell lines stably expressing recombinant human prostanoid receptors (IP, EP1, EP2, EP3, EP4, DP1, FP, TP) were used. Cell membranes were prepared by homogenization and centrifugation. Binding assays were performed by incubating a fixed concentration of the appropriate tritiated ligand (e.g., [³H]Iloprost for IP receptor, [³H]PGE₂ for EP receptors) with the membrane preparation in the presence of increasing concentrations of Treprostinil (0.01 nM to 10,000 nM) for 60-120 minutes at room temperature. Non-specific binding was defined in the presence of a large excess of the corresponding unlabeled ligand. The reaction was terminated by rapid vacuum filtration through glass fiber filters. Bound radioactivity was quantified by scintillation counting. IC50 values were determined from competition curves, and Ki values were calculated using the Cheng-Prusoff equation. [1]

Hematopoietic stem and progenitor cells from humans or mice are cultured for one hour and twenty-four hours at 37°C either in the presence of vehicle or in combination with 10 μM Treprostinil and 30 μM forskolin. The apoptosis kit is used to stain cells for externalized phosphatidylserine after they have been washed with phosphate-buffered saline at 4°C[5].

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- VEGF-A secretion in MSCs： 1. MSCs are treated with Treprostinil (10–100 nM) for 24 hours in serum-free medium. 2. Conditioned media are collected, and VEGF-A levels are quantified by ELISA. Treprostinil increases VEGF-A secretion in a concentration-dependent manner (EC50 ≈ 50 nM). [2]
- Endothelial colony forming cell (ECFC) angiogenesis assay： 1. ECFCs are co-cultured with Treprostinil-treated MSCs in Matrigel. 2. Tube formation is assessed after 12 hours. Treprostinil enhances ECFC angiogenic sprouting by 2–3-fold compared to controls, dependent on VEGF-A secretion from MSCs. [2]

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Cell Viability/Proliferation Assay: Endothelial colony-forming cells (ECFCs) or mesenchymal stem cells (MSCs) were seeded on fibronectin-coated plates and cultured in complete medium for 24 hours. Cells were then serum-starved for 16 hours before being treated with Treprostinil at specified concentrations (e.g., 0.1, 1, 10 µM) in medium containing different percentages of fetal bovine serum (FBS). In some experiments, COX inhibitors (indomethacin or SC-560) were added prior to Treprostinil. After 3 days of treatment, cellular viability/proliferation was quantified by measuring cellular alkaline phosphatase activity using para-nitrophenol phosphate (pNPP) as a substrate. The absorbance of the released product was measured spectrophotometrically at 405 nm. [2]
Single-Cell Clonogenic Assay: Single ECFCs were plated into individual wells of 96-well plates pre-coated with collagen. Cells were cultured in complete medium for 14 days, with medium changes every five days. Colonies were then fixed, stained with DAPI, and examined under a fluorescence microscope. Wells containing two or more cells were scored as positive for proliferation. The total number of cells per colony was quantified, and colonies were categorized based on size and cell number. [2]
ECFC Colony-Forming Unit Assay: Mononuclear cells from cord blood were plated in fibronectin-coated dishes with endothelial growth medium. Treprostinil was added from the first day of culture. The appearance, number, and timing of emerging ECFC colonies were recorded over time. [2]
Conditioned Media Experiments: MSCs were pretreated with or without Treprostinil for a period (e.g., 48 hours). The conditioned media (CM) from these MSCs were then collected. ECFCs were exposed to this MSC-CM for 72 hours, and their proliferation was assessed using the viability assay described above. To test the role of VEGF-A, a neutralizing anti-VEGF-A antibody was added to the CM to block its activity. [2]
siRNA Transfection: MSCs were transfected with siRNA targeting VEGF-A or a non-targeting control siRNA using a transfection reagent. Transfected cells were then used in subsequent experiments, such as collecting conditioned media or for in vivo implantation, to assess the effect of VEGF-A knockdown on Treprostinil's actions. Transfection efficacy was confirmed by measuring VEGF-A protein levels in the supernatant by ELISA. [2]
ELISA for Angiogenic Factors: The levels of VEGF-A, PDGF-BB, and Angiopoietin-2 secreted into the culture medium by ECFCs or MSCs, or present in human plasma, were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. [2]

Rats: For the study, male Lewis rats weighing between 200 and 300 g are employed. 24 hours prior to hepatectomy, donor animals are given treprostinil or a placebo, and the corresponding recipient animal receives the same care until the moment of sacrifice. Treatment is invisible to the surgeon. To study what happens right after IRI, recipients are sacrificed 1, 3, 6, 24 and 48 hours after transplantation. Using an Alzet implantable osmotic pump, subcutaneous administration of treprostinil (100 ng/kg/min) or placebo is performed. This dosage is chosen to produce a plasma concentration that is steady-state and falls between 5 and 20 ng/mL[3].
Mice: Mice that have had bone marrow transplantation (BMT) are split up into five groups, each with six to ten mice. In a normobaric chamber, one group of mice is exposed to hypoxia (10% inspired oxygen fraction), while the other group of mice (control BMT) spends 28 days in a normoxic chamber with a normal oxygen environment (21% inspired O₂ fraction). While the two other groups of mice receive four weeks of hypoxic exposure and receive Treprostinil infusions at varying dose levels (14 ng/kg and 70 ng/kg per minute), the sham group mice receive saline treatment. Comparatively, infusion rates for humans in PAH therapy range from 10 to 60 ng/kg per minute[6].

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- Chronic hypoxic PAH model： 1. Rats are exposed to hypoxia (10% O₂) for 4 weeks to induce PAH. 2. Treprostinil is administered via subcutaneous osmotic pumps (10–50 ng/kg/min) or inhaled aerosol (1–5 μg/kg) daily for 2 weeks. 3. Pulmonary hemodynamics are measured via right heart catheterization, and lung tissues are analyzed for fibrocyte infiltration (CD45⁺/collagen I⁺ cells). [6]
- Liver transplantation model： 1. Rats undergo orthotopic liver transplantation with 60-minute warm ischemia. 2. Treprostinil (10–50 ng/kg/min) is infused intravenously starting 30 minutes before reperfusion and continuing for 6 hours postoperatively. 3. Liver function is assessed by serum alanine aminotransferase (ALT) levels, and histological damage is evaluated by hematoxylin-eosin staining. [5]

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In Vivo Matrigel Plug Angiogenesis Assay: A mixture containing 3 x 10^6 cells (either ECFCs alone, MSCs alone, or a combination of ECFCs and MSCs) was suspended in 200 µL of basement membrane matrix (Matrigel). Treprostinil (concentration not specified in the protocol details) was included in the cell-matrix mixture for the treatment groups. The Matrigel-cell suspension was injected subcutaneously on the backs of 6- to 7-week-old male athymic nude mice. Mice were euthanized 10 days post-implantation. The Matrigel plugs were surgically removed, fixed in formalin, embedded in paraffin, and sectioned. Microvessel density (MVD) was quantified by counting luminal structures containing red blood cells in hematoxylin and eosin (H&E)-stained sections from the central part of the plug. Four fields per section from each animal (n=5 mice per group) were analyzed, and MVD was expressed as vessels per square millimeter. [2]

Subcutaneous/intravenous administration: - Treprostil is rapidly absorbed with a bioavailability of approximately 90%. It is bound to approximately 90% of plasma proteins, primarily albumin. Its terminal half-life is 3-4 hours, and it is primarily eliminated through hepatic metabolism (CYP3A4-mediated oxidation) and renal excretion. [3]
- Inhalation administration: - Peak plasma concentrations are reached within 10-15 minutes after inhalation of treprostil, with a bioavailability of approximately 20-30%. Compared to parenteral administration, inhalation reduces systemic exposure, thereby minimizing off-target effects. [3]

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
A patient taking treprostene breastfed her infant for one year without any complications. However, treprostene use during lactation should be closely monitored until more data are available.
◉ Effects on Breastfed Infants
A woman with pulmonary hypertension began intravenous treprostene treatment at 32 weeks of gestation, with the dose gradually increased to 26 ng/kg/min. The dose was almost doubled postpartum due to worsening symptoms. She breastfed her infant for one year (feeding extent not specified) without significant drug-related problems, but the infant became obese at 6 months of age. The infant was in good health and developing normally at 2 years of age.
◉ Effects on Lactation and Breast Milk
No relevant published information was found as of the revision date.

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- Side Effects: - Common adverse reactions include headache, flushing, and jaw pain, all attributed to vasodilation. At high doses, treprostene may cause hypotension, nausea, and diarrhea. [3]
- Plasma protein binding: - Treprostane has a high binding rate to plasma proteins (approximately 90%), which may increase drug interactions with other compounds with high protein binding rates (such as warfarin). [3]

[1]. Binding and activity of the prostacyclin receptor (IP) agonists, treprostinil and iloprost, at human prostanoid receptors: treprostinil is a potent DP1 and EP2 agonist. Biochem Pharmacol. 2012 Jul 1;84(1):68-75.

[2]. Treprostinil indirectly regulates endothelial colony forming cell angiogenic properties by increasing VEGF-A produced by mesenchymal stem cells. Thromb Haemost. 2015 Oct;114(4):735-47.

[3]. Inhaled treprostinil sodium for the treatment of pulmonary arterial hypertension. Expert Opin Pharmacother. 2011 Nov;12(16):2583-93.

[4]. Repurposing Treprostinil for Enhancing Hematopoietic Progenitor Cell Transplantation. Mol Pharmacol. 2016 Jun;89(6):630-44.

[5]. Treprostinil, a prostacyclin analog, ameliorates ischemia-reperfusion injury in rat orthotopic liver transplantation. Am J Transplant. 2011 Nov;11(11):2508-16.

[6]. Treprostinil inhibits the recruitment of bone marrow-derived circulating fibrocytes in chronic hypoxic pulmonary hypertension. Eur Respir J. 2010 Dec;36(6):1302-14.

Treprostrenil sodium is an organic sodium salt containing treprostrenil molecules.

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Indications
For the treatment of adult patients with World Health Organization functional classification (FC) III or IV, and: unresectable chronic thromboembolic pulmonary hypertension (CTEPH), or CTEPH that persists or recurs after surgical treatment, to improve exercise capacity.
Treatment of Pulmonary Hypertension
Treatment of Chronic Thromboembolic Pulmonary Hypertension

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- Mechanism of Action: - Treprostrenil exerts its therapeutic effect by dual activation of IP and DP1 receptors, resulting in vasodilation, inhibition of platelet aggregation and inhibition of vascular remodeling. Its activation of EP2 receptors may also contribute to its anti-inflammatory effect. [1][6] - Clinical Use: - It is approved for the treatment of pulmonary hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH). It is available in various dosage forms: subcutaneous/intravenous injection, inhaled aerosol and oral tablets. [3]
- FDA-approved indications: - Treprostrenil is indicated for improving exercise capacity and delaying disease progression in patients with PAH. The inhaled formulation is specifically approved for patients with severe symptoms (WHO functional class III/IV). [3]

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Treprostrenil is a chemically stable prostacyclin analog approved for the treatment of pulmonary arterial hypertension (PAH). It is considered a reference treatment and is usually administered via continuous intravenous or subcutaneous routes to avoid the risks associated with long-term intravenous therapy. [2]
This study suggests that the clinical benefit of treprostrenil in pulmonary arterial hypertension (PAH) may be related to the regulation of vascular repair mechanisms. It increases the number and angiogenic potential of circulating endothelial progenitor cells (ECFCs) in patients while reducing the number of circulating endothelial cells (a marker of endothelial damage). [2]
The main finding of this study is that treprostrenil does not directly stimulate ECFCs, but rather indirectly enhances their angiogenic function by upregulating the production of VEGF-A in adjacent mesenchymal stem cells (MSCs). This VEGF-A-dependent mechanism promotes ECFC proliferation and angiogenesis. [2] Elevated plasma VEGF-A levels were observed in pediatric PAH patients treated with treprostinil, suggesting that VEGF-A may serve as a potential surrogate biomarker for monitoring the biological efficacy of this drug. [2]

C23H33NAO5

412.4949

412.22

C, 66.97; H, 8.06; Na, 5.57; O, 19.39

289480-64-4

Treprostinil;81846-19-7; Treprostinil diethanolamine; 830354-48-8

23663413

White to off-white solid powder

587.1ºC at 760 mmHg

587.1ºC at 760 mmHg

199.3ºC

1.25E-14mmHg at 25°C

3.583

2

5

10

29

502

5

[Na+].O([H])C1([H])C([H])([H])[C@]2([H])C([H])([H])C3C(=C([H])C([H])=C([H])C=3C([H])([H])[C@@]2([H])C1([H])C([H])([H])C([H])([H])C([H])(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])O[H])OC([H])([H])C(=O)[O-]

IQKAWAUTOKVMLE-ZSESPEEFSA-M

InChI=1S/C23H34O5.Na/c1-2-3-4-7-17(24)9-10-18-19-11-15-6-5-8-22(28-14-23(26)27)20(15)12-16(19)13-21(18)25;/h5-6,8,16-19,21,24-25H,2-4,7,9-14H2,1H3,(H,26,27);/q;+1/p-1/t16-,17-,18+,19-,21+;/m0./s1

sodium;2-[[(1R,2R,3aS,9aS)-2-hydroxy-1-[(3S)-3-hydroxyoctyl]-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[g]naphthalen-5-yl]oxy]acetate

UT-15; LRX 15 sodium; UT 15; LRX15 sodium; UT15; LRX-15 sodium; BW 15AU sodium; U-62840 sodium; Uniprost; Treprostinil; Orenitram; Remodulin; Treprostinil (sodium); 7JZ75N2NT6; TREPROSTINIL SODIUM SALT; CHEBI:50863; sodium;2-[[(1R,2R,3aS,9aS)-2-hydroxy-1-[(3S)-3-hydroxyoctyl]-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[g]naphthalen-5-yl]oxy]acetate;

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, 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)

DMSO: 25~82 mg/mL (~198.8 mM)
Water: 82 mg/mL
Ethanol: 82 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.06 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 (6.06 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 (6.06 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: 50 mg/mL (121.22 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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