Doxofylline exerts its effects by targeting multiple molecules, including sirtuin 1 (SIRT1) and phosphodiesterase 4 (PDE4), while showing low affinity for adenosine A1 receptors.
- For SIRT1: It enhances SIRT1 activity [1]
- For PDE4: It acts as a selective inhibitor with an IC50 value of approximately 7.5 μM (measured using recombinant human PDE4 and [³H]-cAMP as substrate) [3]
- For adenosine A1 receptors: It has minimal binding affinity, with a Ki value > 100 μM (significantly higher than theophylline, which has a Ki of ~10 μM for A1 receptors) [3]

Doxofylline (5, 10 µM; 48 h) reduces PGE2, NO release, and mitochondrial ROS generation in 16HBE cells, demonstrating strong protection against LPS-induced epithelial inflammation[1]. LPS-induced expression of NADPH oxidase subunits and TXNIP 16HBE cells is suppressed by doxofylline (5, 10 µM; 48 h)[1]. Doxofylline (5, 10 µM; 48 h) attenuates LPS-mediated SIRT1 reduction and prevents LPS-induced NLRP3 inflammasome activation and IL-1b and IL-18 secretion[1]. In BM cells, doxofylline (0.1–10 µM; 15 min) dramatically inhibits leukocyte migration induced by fMLP (formyl–methionyl–leucyl–phenylalanine)[2].

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Doxofylline inhibits LPS-induced NLRP3 inflammasome activation in human pulmonary bronchial epithelial cells (HBEpC) via SIRT1. When HBEpC cells are pre-treated with Doxofylline (10, 50, 100 μM) for 2 hours followed by LPS (1 μg/mL) stimulation for 24 hours:
- Western blot analysis shows a concentration-dependent decrease in NLRP3, ASC, and cleaved caspase-1 (p20) protein levels (e.g., 100 μM Doxofylline reduces NLRP3 protein by ~65% compared to LPS-only group) [1]
- RT-PCR reveals reduced NLRP3 and IL-1β mRNA expression (100 μM Doxofylline decreases IL-1β mRNA by ~70%) [1]
- ELISA detects lower IL-1β release in cell supernatants (100 μM Doxofylline reduces IL-1β by ~60%) [1]
- SIRT1 knockdown (via siRNA) abolishes these inhibitory effects, confirming SIRT1 mediation [1]
- Doxofylline inhibits PDE4 activity and increases intracellular cAMP levels. In recombinant human PDE4 assays, 1–100 μM Doxofylline concentration-dependently reduces PDE4-mediated cAMP hydrolysis (7.5 μM inhibits activity by 50%) [3]
- Doxofylline shows no significant cytotoxicity in HBEpC cells. MTT assay indicates >90% cell viability after 24-hour treatment with Doxofylline (up to 200 μM), ruling out non-specific cell death effects [1]

In mice, doxofylline (0.3, 1 mg/kg; ip; single) reduces inflammation brought on by LPS in the lungs[2]. Doxofylline (0.3 mg/kg; ip; pre-treat; single) suppresses the production of LPS-induced ICAM-1 in vivo and dramatically decreases cell adherence to vascular tissue[2].

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Doxofylline attenuates LPS-induced lung inflammation in mice. Male BALB/c mice (6–8 weeks old) are divided into 5 groups: control, LPS-only, and LPS + Doxofylline (10, 20, 40 mg/kg, intraperitoneal injection). Doxofylline is administered 1 hour before LPS (5 mg/kg, intranasal instillation), and samples are collected 24 hours post-LPS:
- Bronchoalveolar lavage fluid (BALF) analysis shows dose-dependent reduction in total inflammatory cells (e.g., 40 mg/kg Doxofylline reduces cells by ~55% vs. LPS group), neutrophils (~60% reduction), and macrophages (~45% reduction) [2]
- BALF cytokine levels (TNF-α, IL-6, IL-1β) are decreased: 40 mg/kg Doxofylline reduces TNF-α by ~70%, IL-6 by ~65%, and IL-1β by ~60% [2]
- Lung tissue histopathology reveals less alveolar congestion, interstitial edema, and inflammatory cell infiltration in Doxofylline-treated groups [2]
- Doxofylline improves airway function in preclinical models of asthma and COPD. In ovalbumin (OVA)-induced asthmatic mice, oral Doxofylline (30 mg/kg/day for 14 days) reduces airway hyperresponsiveness (AHR) to methacholine by ~40% and decreases eosinophil infiltration in BALF by ~50% [3]
- Doxofylline reduces mucus hypersecretion in COPD models. In cigarette smoke-induced COPD rats, oral Doxofylline (50 mg/kg/day for 8 weeks) decreases MUC5AC protein expression in bronchial epithelium by ~55% (detected by immunohistochemistry) [3]

PDE4 activity inhibition assay: Recombinant human PDE4 (isoform PDE4B) is incubated with a reaction mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 μM [³H]-cAMP (substrate), and serial concentrations of Doxofylline (0.1–100 μM) at 37°C for 30 minutes. The reaction is terminated by adding 0.2 M EDTA (pH 8.0). Unhydrolyzed [³H]-cAMP is precipitated with zinc sulfate and barium hydroxide, and the supernatant (containing [³H]-5'-AMP) is collected. Radioactivity is measured via liquid scintillation counting, and the percentage of PDE4 activity (relative to vehicle control) is calculated. The IC50 is determined by fitting the dose-response curve to a sigmoidal model [3]
- SIRT1 activity enhancement assay: HBEpC cells are lysed in ice-cold RIPA buffer, and the supernatant (containing SIRT1) is collected by centrifugation. The SIRT1 activity assay mixture includes cell lysate (20 μg protein), 50 μM fluorogenic substrate (e.g., AMC-conjugated peptide), 1 mM NAD⁺ (coenzyme), and Doxofylline (10–100 μM). The mixture is incubated at 37°C for 60 minutes, and fluorescence intensity (excitation 360 nm, emission 460 nm) is measured. SIRT1 activity is expressed as fold change relative to vehicle control (100 μM Doxofylline increases SIRT1 activity by ~2.3 fold) [1]

Cell Viability Assay[1]
Cell Types: 16HBE cells
Tested Concentrations: 5, 10 µM
Incubation Duration: 48 h
Experimental Results: Weakened LPS-induced NO and PGE2 in a dose-dependent manner. Exerted dose-dependent inhibition on LPS-induced mitochondrial ROS production and NADPH oxidase subunits expression. Suppressed LPS- induced TXNIP expression and NLRP3 inflammasome activation at the protein level in a dose-dependent manner. Inhibited LPS-induced secretion of IL-1b and IL-18.

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Cell Viability Assay[2]
Cell Types: BM cells (from naive mice)
Tested Concentrations: 0.1-10 µM
Incubation Duration: 15 min (pretreat)
Experimental Results: Notably suppressed positive migration of BM cells in response to fMLP.

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LPS-induced NLRP3 inflammasome activation assay in HBEpC cells:
1. HBEpC cells are cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C (5% CO₂) until 80% confluence [1]
2. Cells are pre-treated with Doxofylline (10, 50, 100 μM) or vehicle (DMSO, final concentration <0.1%) for 2 hours [1]
3. LPS (1 μg/mL) is added to induce inflammation, and cells are incubated for another 24 hours [1]
4. For protein detection: Cells are lysed with RIPA buffer containing protease inhibitors; protein concentrations are measured via BCA assay; equal amounts of protein (30 μg) are separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against NLRP3, ASC, cleaved caspase-1 (p20), SIRT1, and β-actin (loading control); secondary antibodies conjugated to HRP are used, and bands are visualized via chemiluminescence [1]
5. For mRNA detection: Total RNA is extracted using TRIzol reagent, reverse-transcribed to cDNA, and real-time PCR (qPCR) is performed with specific primers for NLRP3, IL-1β, and GAPDH (housekeeping gene); relative mRNA levels are calculated using the 2⁻ΔΔCt method [1]
6. For IL-1β release detection: Cell supernatants are collected, and IL-1β concentrations are measured via commercial ELISA kits [1]
- HBEpC cell viability assay: Cells are seeded in 96-well plates (5×10³ cells/well) and treated with Doxofylline (0–200 μM) for 24 hours. MTT reagent (5 mg/mL) is added to each well, and the plates are incubated for 4 hours at 37°C. The supernatant is removed, and DMSO is added to dissolve formazan crystals. Absorbance at 570 nm is measured, and cell viability is calculated as (absorbance of treated group / absorbance of control group) × 100% [1]

Animal/Disease Models: Male balb/c (Bagg ALBino) mouse (6 to 8weeks old)[2].
Doses: 0.3, 1 mg/kg
Route of Administration: intraperitoneal (ip)injection; single.
Experimental Results: Dramatically inhibited the migration of neutrophils and the release of IL-6 and TNF-a into the lung lumen. Increased the bone marrow leukocyte numbers to levels similar to those seen in the saline-treated group. Notably decreased the number of circulating leukocytes in comparison to LPS-treated mice. Dramatically decreased accumulation of neutrophils in the peribronchial area.

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Animal/Disease Models: Male balb/c (Bagg ALBino) mouse (6 to 8weeks old)[2].
Doses: 0.3 mg/kg
Route of Administration: intraperitoneal (ip)injection; pre-treat; single.
Experimental Results: Dramatically decreased the adhesion of cells to the vascular tissue, but not the rolling of cells along the vessel wall in mice. Dramatically decreased the expression of ICAM-1 induced by LPS.

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Mouse LPS-induced lung inflammation model:
1. Male BALB/c mice (6–8 weeks old, 20–25 g) are randomly divided into 5 groups (n=6/group): Normal control (saline intranasal + saline intraperitoneal), LPS group (LPS intranasal + saline intraperitoneal), and Doxofylline groups (LPS intranasal + Doxofylline 10, 20, 40 mg/kg intraperitoneal) [2]
2. Doxofylline is dissolved in sterile normal saline (concentrations adjusted to ensure equal injection volume: 0.1 mL/10 g body weight) [2]
3. Doxofylline is administered via intraperitoneal injection 1 hour before intranasal instillation of LPS (5 mg/kg, dissolved in saline, 50 μL/mouse) [2]
4. Twenty-four hours after LPS instillation, mice are anesthetized with isoflurane. Bronchoalveolar lavage (BAL) is performed by instilling and retrieving 0.5 mL saline (3 times) to collect BALF [2]
5. BALF is centrifuged (1500 × g, 10 minutes, 4°C) to separate cells and supernatant: Total cell count is measured via hemocytometer; differential cell count is performed on Giemsa-stained cytospin slides; supernatant is stored at -80°C for cytokine (TNF-α, IL-6, IL-1β) detection via ELISA [2]
6. Lung tissues are excised, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (5 μm), and stained with hematoxylin-eosin (HE) for histopathological analysis [2]
- OVA-induced asthmatic mouse model:
1. Female C57BL/6 mice (6–8 weeks old) are sensitized by intraperitoneal injection of OVA (10 μg) plus aluminum hydroxide (2 mg) on days 0 and 7 [3]
2. From day 14 to day 20, mice are challenged with aerosolized OVA (1% in saline) for 30 minutes/day [3]
3. Doxofylline (15, 30 mg/kg) is dissolved in saline and administered via oral gavage once daily from day 14 to day 20 (30 minutes before OVA challenge) [3]
4. On day 21, airway hyperresponsiveness (AHR) is measured using a whole-body plethysmograph (methacholine concentrations: 0, 3.125, 6.25, 12.5, 25 mg/mL); BALF is collected for eosinophil counting [3]

Absorption, Distribution and Excretion
Following repeated administration, doxophylline reaches steady state in approximately 4 days. In adult patients with chronic bronchitis, after twice-daily oral administration of 400 mg doxophylline for 5 days, the steady-state peak plasma concentration (Cmax) ranged from 5.78 to 20.76 mcg/mL. The time to peak concentration (Tmax) was 1.19 ± 0.19 hours. The absolute bioavailability of doxophylline in healthy subjects was 63 ± 25%. Due to extensive hepatic metabolism, less than 4% of the oral dose is excreted unchanged in the urine. The distribution phase of doxophylline is relatively short after intravenous administration of 100 mg in adult patients with chronic bronchitis. Because methylxanthines are distributed throughout the body, doxophylline can be detected in breast milk and the placenta. After twice-daily oral administration of 400 mg doxophylline for 5 days, the total clearance was 555.2 ± 180.6 mL/min.

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Metabolism/Metabolites
Doxophylline is believed to be primarily metabolized in the liver, with hepatic metabolism accounting for 90% of total drug clearance. β-hydroxymethyltheophylline was detected in serum and urine in healthy subjects after oral administration of 400 mg doxophylline. This circulating metabolite has no significant pharmacological activity.

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Biological Half-Life
In adult patients with chronic bronchitis, the elimination half-life of doxophylline after a single intravenous injection of 100 mg (during 10 minutes) is 1.83 ± 0.37 hours. In adult patients with chronic bronchitis, the mean elimination half-life after oral administration of 400 mg twice daily for 5 consecutive days is 7.01 ± 0.80 hours.

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Oral Absorption: Doxophylline is well absorbed after oral administration in humans, with an oral bioavailability of approximately 90% (range 85–95%). Peak plasma concentration (Cmax) is reached 1–2 hours after administration (Tmax), and Cmax increases linearly with oral dose (200 mg to 800 mg) (e.g., oral administration of 400 mg can achieve a Cmax of about 8 μg/mL) [3]
- Distribution: The volume of distribution (Vd) of doxophylline in the human body is about 0.8–1.0 L/kg, indicating that its tissue permeability is limited compared with other xanthine derivatives. Doxophylline has a very low ability to cross the blood-brain barrier (BBB), with a brain-to-plasma concentration ratio of about 0.1 [3]
- Metabolism: Doxophylline is mainly metabolized in the liver by cytochrome P450 (CYP) enzymes, with CYP1A2 being the most important (accounting for about 70% of metabolism). The main metabolite is 1-methylxanthine, which is inactive. Unlike theophylline, doxophylline is almost not metabolized by CYP2E1 or CYP3A4, thus reducing the risk of drug interactions [3]
- Excretion: Approximately 70-80% of doxophylline and its metabolites are excreted in urine within 24 hours, mainly as 1-methylxanthine and its glucuronide conjugate. In healthy adults, the elimination half-life (t1/2) of doxophylline is 7-8 hours, which is longer than that of theophylline (t1/2 ~ 3-5 hours) [3]
- Food effects: A high-fat diet does not significantly affect the absorption of doxophylline (AUC change <10%), but may delay the time to peak concentration (Tmax) by about 1 hour [3]

Protein Binding
At pH 7.4, plasma protein binding is approximately 48%. Acute Toxicity: Doxophylline has low acute toxicity. The oral LD50 in mice and rats is >2000 mg/kg, and in dogs it is >1500 mg/kg. No death or serious clinical symptoms (e.g., seizures, respiratory depression) were observed in humans at single oral doses up to 1000 mg/kg [3]. Chronic Toxicity: In a 13-week repeated-dose study in rats (oral doses of 50, 100, and 200 mg/kg/day, respectively), no treatment-related changes in body weight, food intake, or organ weight (liver, kidney, lung) were observed. Serum ALT, AST, BUN, and creatinine (indicators of liver and kidney function) levels were within the normal range in all dose groups [3]
- Plasma protein binding: Doxophylline has a moderate plasma protein binding rate, with approximately 40% (range 35-45%) in human plasma. Due to its low binding affinity, it does not displace other drugs with high protein binding rates (e.g., warfarin, phenytoin sodium) [3]
- Drug interactions: Doxophylline has very few drug interactions. Co-administration with CYP1A2 inhibitors (e.g., fluvoxamine) increases its AUC by approximately 2-fold, while co-administration with CYP1A2 inducers (e.g., smoking) decreases its AUC by approximately 30%. No significant interactions were observed with β2 receptor agonists (e.g., salbutamol) or inhaled corticosteroids (e.g., budesonide) [3]
- Central nervous system (CNS) toxicity: Unlike theophylline, doxophylline causes minimal CNS irritation. At therapeutic doses (400-800 mg twice daily), due to its low affinity for adenosine A1 receptors and limited blood-brain barrier penetration, the incidence of insomnia, anxiety, or tremor is <5% (compared to 15-20% for theophylline) [3]

[1]. The protective effect of doxofylline against lipopolysaccharides (LPS)-induced activation of NLRP3 inflammasome is mediated by SIRT1 in human pulmonary bronchial epithelial cells. Artif Cells Nanomed Biotechnol. 2020 Dec;48(1):687-694.

[2]. Doxofylline, a novofylline inhibits lung inflammation induced by lipopolysacharide in the mouse. Pulm Pharmacol Ther. 2014 Apr;27(2):170-8.

[3]. Doxofylline: a promising methylxanthine derivative for the treatment of asthma and chronic obstructive pulmonary disease. Expert Opin Pharmacother. 2009 Oct;10(14):2343-56.

Doxophylline is an oxopurine derivative, a xanthine derivative with methylation at N-1 and N-3 positions and a 1,3-dioxolane-2-ylmethyl group at N-7. It is used to treat asthma. It has bronchodilatory, antitussive, and anti-asthmatic effects. Its function is similar to 7H-xanthine. Doxophylline is a methylxanthine derivative with a dioxolane group at the 7-position. As a drug used to treat asthma, doxophylline has shown similar efficacy to theophylline in animal and human studies, but with significantly fewer side effects. Unlike other xanthine derivatives, doxophylline has a low binding affinity to adenosine α-1 or α-2 receptors and lacks excitatory effects. Compared to theophylline, the reduced affinity of doxophylline for adenosine receptors may explain its better safety profile. Unlike theophylline, doxophylline does not affect calcium ion influx or antagonize the effects of calcium channel blockers, which may explain its fewer cardiac adverse reactions. The anti-asthmatic effect of doxophylline is mainly achieved by inhibiting phosphodiesterase (PDE) activity. Drug Indications It is indicated for the treatment of chronic obstructive pulmonary disease (COPD), bronchial asthma, and lung diseases with spastic bronchial components. Mechanism of Action The main mechanism of action of doxophylline is not yet fully understood. One mechanism is believed to be through inhibiting phosphodiesterase activity, thereby increasing cAMP levels and promoting smooth muscle relaxation. A study using nonlinear chromatography, frontier analysis, and molecular docking showed that doxophylline interacts with β2-adrenergic receptors. Serine residues 169 and 173 in the receptor are considered key binding sites for doxophylline, where hydrogen bonds are formed. Doxophylline induces vasodilation and airway smooth muscle relaxation by mediating the action of β2-adrenergic receptors. Furthermore, a rat study suggested that doxophylline may exert its anti-inflammatory effect by alleviating pleurisy induced by the inflammatory mediator platelet-activating factor (PAF). Studies have shown that doxorubicin may play an important role in reducing leukocyte exudation, a view supported by preclinical mouse studies. These studies have demonstrated that both in vivo and in vitro administration of doxorubicin inhibits leukocyte migration across vascular endothelial cells. Unlike theophylline, doxorubicin does not inhibit tumor necrosis factor-induced secretion of interleukin (IL)-8 from airway smooth muscle cells (ASM).

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Pharmacodynamics
Doxorubicin is a methylxanthine bronchodilator with potent bronchodilatory activity comparable to theophylline. Animal studies have shown that doxorubicin can reduce bronchoconstriction, inflammatory responses, and the release of thromboxane A2 (TXA2) stimulated by platelet-activating factor. Doxorubicin does not directly inhibit any histone deacetylase (HDAC) or known phosphodiesterase (PDE) isoenzymes, nor does it antagonize A2 or A2 receptors. It has been reported to have affinity for adenosine A1, A2A, and A2B receptors all greater than 100 µM. Only at high concentrations does it exhibit inhibitory effects on PDE2A1 and antagonistic effects on adenosine A2A receptors. One study showed that doxophylline interacts with β2-adrenergic receptors to induce vasodilation and airway smooth muscle relaxation. In canine studies, doxophylline reduced airway responsiveness at doses that did not affect heart rate and respiratory rate. Mechanism of action: Doxophylline exerts its anti-inflammatory and bronchodilatory effects through two main pathways: (1) inhibiting PDE4, increasing intracellular cAMP levels, activating PKA, inhibiting the release of pro-inflammatory mediators (e.g., TNF-α, IL-6), and reducing smooth muscle contraction; (2) enhancing SIRT1 activity, deacetylifying NLRP3, thereby inhibiting the activation of the NLRP3 inflammasome and reducing the production of IL-1β (IL-1β is crucial for LPS-induced lung inflammation) [1, 3] - Therapeutic indications: Doxophylline has been approved for the treatment of asthma and chronic obstructive pulmonary disease (COPD). It relieves symptoms such as dyspnea, wheezing and cough by improving airway patency and reducing airway inflammation. Compared with theophylline, doxophylline has better safety (fewer central nervous system and cardiovascular side effects)[3]
- Clinical efficacy: In a randomized controlled trial (RCT) in patients with chronic obstructive pulmonary disease (COPD), oral doxophylline (400 mg, twice daily for 12 weeks) increased forced expiratory volume in one second (FEV1) by about 15% and reduced the frequency of acute exacerbations by about 40% compared with placebo[3]
- Comparison with other xanthine drugs: Doxophylline differs from theophylline in three key aspects: (1) higher selectivity for PDE4 than other PDE isoenzymes; (2) lower affinity for adenosine A1 receptor (reducing central nervous system and cardiovascular side effects); (3) different metabolic pathways (primarily CYP1A2, minimizing interactions with other drugs)[3]
- Role in NLRP3 inflammasome regulation: Doxophylline is one of the few xanthine derivatives that have been shown to target the NLRP3 inflammasome. This unique effect allows it to not only dilate the bronchi but also effectively treat other inflammatory lung diseases, such as LPS-induced acute lung injury (ALI) [1]

C11H14N4O4

266.25

266.101

69975-86-6

Doxofylline-d6;1219805-99-8;Doxofylline-d4;1346599-13-0

50942

White to off-white solid powder

1.6±0.1 g/cm3

505.2±53.0 °C at 760 mmHg

144-146ºC

259.3±30.9 °C

0.0±1.3 mmHg at 25°C

1.700

-0.7

0

5

2

19

398

0

HWXIGFIVGWUZAO-UHFFFAOYSA-N

InChI=1S/C11H14N4O4/c1-13-9-8(10(16)14(2)11(13)17)15(6-12-9)5-7-18-3-4-19-7/h6-7H,3-5H2,1-2H3

7-(1,3-dioxolan-2-ylmethyl)-1,3-dimethylpurine-2,6-dione

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: ≥ 2.5 mg/mL (9.39 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 (9.39 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 (9.39 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: 65 mg/mL (244.13 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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