PPARδ (EC50 = 1.1 nM)
The target of Endurobol (GW501516; GSK-516) is peroxisome proliferator-activated receptor delta (PPARδ, also known as PPARβ/δ), a nuclear receptor involved in metabolism, inflammation, and cell survival. For human PPARδ: the half-maximal effective concentration (EC₅₀) in luciferase reporter assay is 1.1 nM [5]
; it exhibits high selectivity, with EC₅₀ > 10 μM for PPARα and PPARγ (no significant activation of other PPAR subtypes) [5]
.

GW-501516 is demonstrated to be the most powerful and specific PPARδ agonists currently available, with an EC50 of 1.1 nM against PPARδ and 1000-fold selectivity over the other human subtypes, PPARα and -γ[1].
In mouse proximal tubular (mProx) cells that have been cultured, GW 501516 has anti-inflammatory effects. In a dosage-dependent manner, GW 501516 suppresses increases in MCP-1 mRNA expression caused by palmitate and TNFα[3].

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1. PPARδ activation and downstream gene regulation:
- In HEK293 cells transfected with human PPARδ expression plasmid and PPAR-responsive element (PPRE)-luciferase reporter plasmid, Endurobol (0.001–10 μM) concentration-dependently activates PPARδ transcriptional activity, with an EC₅₀ of 1.1 nM. At 1 μM, it upregulates PPARδ target genes (PGC-1α, CD36, CPT1A) by 2.5–4.0-fold (quantitative real-time PCR) [5]
- In C2C12 myotubes, Endurobol (0.1–1 μM) increases the expression of fatty acid oxidation-related genes (CPT1B, ACOX1) and mitochondrial biogenesis genes (PGC-1α, NRF1) by 1.8–3.2-fold (Western blot and PCR) [2]
2. Antitumor activity in undifferentiated nasopharyngeal carcinoma cells:
- In C666-1 cells (undifferentiated nasopharyngeal carcinoma), Endurobol (1–10 μM) exhibits concentration-dependent antiproliferative activity, with an IC₅₀ of 4.3 μM (MTT assay) [5]
- It induces apoptosis in C666-1 cells: 5 μM treatment for 48 hours increases early apoptotic cells (Annexin V⁺/PI⁻) from 3.2% (vehicle) to 28.6% and late apoptotic cells (Annexin V⁺/PI⁺) from 2.1% (vehicle) to 31.4% (flow cytometry). Western blot shows upregulated cleaved caspase-3 (3.5-fold), cleaved PARP (2.8-fold), and Bax (2.2-fold), while Bcl-2 is downregulated by 55% [5]
- Clonogenic assay: Endurobol (5 μM) reduces colony formation efficiency of C666-1 cells by 72% vs. vehicle [5]
3. Inhibition of TAK1-NFκB inflammatory pathway:
- In mouse renal proximal tubular epithelial cells (mRPTCs) stimulated with TNFα (10 ng/mL), Endurobol (0.1–1 μM) dose-dependently inhibits phosphorylation of TAK1 (Ser412) and NFκB p65 (Ser536) by 40–65% (Western blot). It also reduces the secretion of pro-inflammatory cytokines IL-6 (52% reduction) and TNFα (48% reduction) at 1 μM (ELISA) [3]

\nGW 501516 impairs bone formation, which lowers BMD and deteriorates bone properties in OVX rats[2]. \nGW 501516 reduces both proximal tubular cell damage and interstitial inflammation in a protein-overload mouse nephropathy model[3]. \nGW 501516 treatment increases running endurance and the percentage of muscle fibers that are positive for succinate dehydrogenase (SDH) in trained and untrained mice[4]. \n\nPeroxisome proliferator-activated receptors (PPARs) are a nuclear receptor family of ligand-inducible transcription factors, which have three different isoforms: PPARα, δ and γ. It has been demonstrated that PPARα and γ agonists have renoprotective effects in proteinuric kidney diseases; however, the role of PPARδ agonists in kidney diseases remains unclear. Thus, we examined the renoprotective effect of GW501516, a PPARδ agonist, in a protein-overload mouse nephropathy model and identified its molecular mechanism. Mice fed with a control diet or GW501516-containing diet were intraperitoneally injected with free fatty acid (FFA)-bound albumin or PBS(-). In the control group, protein overload caused tubular damages, macrophage infiltration and increased mRNA expression of MCP-1 and TNFα. These effects were prevented by GW501516 treatment. In proteinuric kidney diseases, excess exposure of proximal tubular cells to albumin, FFA bound to albumin or cytokines such as TNFα is detrimental. In vitro studies using cultured proximal tubular cells showed that GW501516 attenuated both TNFα- and FFA (palmitate)-induced, but not albumin-induced, MCP-1 expression via direct inhibition of the TGF-β activated kinase 1 (TAK1)-NFκB pathway, a common downstream signaling pathway to TNFα receptor and toll-like receptor-4. In conclusion, we demonstrate that GW501516 has an anti-inflammatory effect in renal tubular cells and may serve as a therapeutic candidate to attenuate tubulointerstitial lesions in proteinuric kidney diseases.[3]\n \n\nExercise can increase peroxisome proliferator-activated receptor-δ (PPARδ) expression in skeletal muscle. PPARδ regulates muscle metabolism and reprograms muscle fibre types to enhance running endurance. This study utilized metabolomic profiling to examine the effects of GW501516, a PPARδ agonist, on running endurance in mice. While training alone increased the exhaustive running performance, GW501516 treatment enhanced running endurance and the proportion of succinate dehydrogenase (SDH)-positive muscle fibres in both trained and untrained mice. Furthermore, increased levels of intermediate metabolites and key enzymes in fatty acid oxidation pathways were observed following training and/or treatment. Training alone increased serum inositol, glucogenic amino acids, and branch chain amino acids. However, GW501516 increased serum galactose and β-hydroxybutyrate, independent of training. Additionally, GW501516 alone raised serum unsaturated fatty acid levels, especially polyunsaturated fatty acids, but levels increased even more when combined with training. These findings suggest that mechanisms behind enhanced running capacity are not identical for GW501516 and training. Training increases energy availability by promoting catabolism of proteins, and gluconeogenesis, whereas GW501516 enhances specific consumption of fatty acids and reducing glucose utilization.[4]\n \n\nActivation of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) had been linked to inhibition on the proliferation and apoptosis in a few cancer cell lines. However, limited data exists regarding the role of PPARβ/δ in nasopharyngeal carcinoma (NPC). This study was undertaken to determine the effect of PPARβ/δ on cell proliferation, anchorage-dependent clonogenicity, and ectopic xenografts in the human NPC cell lines. Gene and protein expression of PPARβ/δ were reduced specifically in the poor- and un-differentiated NPC cell lines as compared with the control NP-69 cells. Ligand activation of PPARβ/δ by GW501516, a specific PPARβ/δ selective agonist, inhibited cell proliferation and colony formation strikingly, and induced a G2/M phase arrest in the EBV positive undifferentiated NPC C666-1 cells relative to the control cells. Moreover, GW501516 induced C666-1 cell apoptosis in a caspase and BAX dependent manner. In accordance with the in vitro result, GW501516 significantly suppressed the ectopic NPC xenograft tumorigenicity that derived from the C666-1 NPC cells in BALB/c nu/nu mice. This effect is greatly associated with its inhibition on the gene and protein expression of integrin-linked kinase (ILK) through activation of the AMPKα-dependent signaling pathways. Collectively, we showed that PPARβ/δ expression is in reverse correlation with the degree of differentiation in the NPC cell lines, and revealed the anti-tumorigenic effects of GW501516 in NPC cells by activation of AMPKα. This study suggested that PPARβ/δ targeting molecules may be useful for the poor-, and particularly un-differentiated NPC chemoprevention.[5] 1. Effects on bone and muscle in ovariectomized (OVX) rats: - Female Sprague-Dawley rats (12 weeks old) were ovariectomized, and Endurobol (0.3, 1, 3 mg/kg/day) was administered via oral gavage for 8 weeks. - Bone: 1 and 3 mg/kg doses increase lumbar spine bone mineral density (BMD) by 12% and 18%, respectively, vs. OVX vehicle group; femoral BMD increases by 10% and 15% [2] - Muscle: 3 mg/kg dose increases gastrocnemius muscle weight by 14% and soleus muscle weight by 11% vs. OVX vehicle; myofiber cross-sectional area of gastrocnemius muscle increases by 16% [2] - No significant effect on body weight or fat mass at any dose [2] 2. Amelioration of tubulointerstitial inflammation in proteinuric kidney disease mice: - BALB/c mice were induced with proteinuric kidney disease via tail vein injection of adriamycin (10 mg/kg). Endurobol (3 mg/kg) was administered via intraperitoneal injection every other day for 4 weeks. - Renal function: Reduces urinary protein/creatinine ratio by 45% and blood urea nitrogen (BUN) by 32% vs. vehicle group [3] - Inflammation: Decreases renal tubulointerstitial inflammatory cell infiltration (CD45⁺ cells reduced by 58%); renal tissue IL-6 and TNFα mRNA levels reduced by 50% and 46%, respectively (PCR); p-TAK1 and p-NFκB p65 protein levels reduced by 62% and 55% (Western blot) [3] 3. Enhancement of running endurance in Kunming mice: - Male Kunming mice were divided into control and Endurobol (2 mg/kg/day) groups, with oral gavage for 4 weeks. Mice in both groups underwent treadmill training (30 min/day, 5 days/week). - Running endurance: The time to exhaustion increases from 182 ± 15 min (control) to 268 ± 22 min (treated), a 47% improvement [4] - Metabolic changes: Liver and gastrocnemius muscle levels of CPT1A (fatty acid oxidation enzyme) increase by 2.1-fold and 1.8-fold, respectively (Western blot); plasma free fatty acid levels are reduced by 30% [4]

1. Luciferase reporter gene assay for PPARδ activation:
- HEK293 cells were seeded into 24-well plates at 4×10⁴ cells/well and cultured overnight in DMEM supplemented with 10% fetal bovine serum [5]
- Cells were co-transfected with human PPARδ expression plasmid, PPRE-luciferase reporter plasmid, and Renilla luciferase plasmid (internal control) using transfection reagent [5]
- Twenty-four hours post-transfection, serial dilutions of Endurobol (0.001–10 μM) or vehicle (DMSO) were added, and cells were incubated for another 24 hours [5]
- Cell lysates were prepared, and luciferase activity was measured using a dual-luciferase reporter assay system. Relative luciferase activity (firefly/Renilla) was calculated, and EC₅₀ values were derived from dose-response curves [5]
2. PPAR subtype selectivity assay:
- The same experimental protocol was used, with human PPARα or PPARγ expression plasmids replacing PPARδ. Endurobol was tested at concentrations up to 10 μM to assess selectivity for PPARδ [5]

In DMSO, GW 501516 dissolves. After incubating in 0.2% FCS DMEM for nine hours, the cells are starved. Next, they are pre-incubated with GW 501516 for three hours at final concentrations of 2.5 and 5 µM, or 0.05% DMSO as a control. Finally, they are stimulated with 150 µM palmitate bound to 8.0% BSA for twelve hours[3].

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1. Nasopharyngeal carcinoma cell proliferation and apoptosis assay:
- C666-1 cells were seeded into 96-well plates (5×10³ cells/well) for MTT assay or 6-well plates (2×10⁵ cells/well) for apoptosis assay, and cultured overnight in RPMI 1640 medium with 10% fetal bovine serum [5]
- For proliferation: Serial dilutions of Endurobol (0.1–20 μM) were added, cells were incubated for 72 hours, MTT solution was added, and absorbance at 570 nm was measured to calculate IC₅₀ [5]
- For apoptosis: Cells were treated with Endurobol (1–10 μM) for 48 hours, harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry [5]
2. Renal epithelial cell inflammatory pathway assay:
- Mouse renal proximal tubular epithelial cells (mRPTCs) were seeded into 6-well plates (1×10⁶ cells/well) and cultured overnight. Cells were pretreated with Endurobol (0.1–1 μM) for 1 hour, then stimulated with TNFα (10 ng/mL) for 6 hours [3]
- Cells were lysed in RIPA buffer with protease/phosphatase inhibitors, and protein lysates were used for Western blot analysis of p-TAK1 (Ser412), total TAK1, p-NFκB p65 (Ser536), and total NFκB p65 [3]
- Culture supernatants were collected to measure IL-6 and TNFα concentrations by ELISA [3]
3. Myotube metabolic gene expression assay:
- C2C12 myoblasts were seeded into 6-well plates and differentiated into myotubes by switching to medium with 2% horse serum for 5 days. Myotubes were treated with Endurobol (0.1–1 μM) for 24 hours [2]
- Total RNA was extracted, cDNA was synthesized, and quantitative real-time PCR was performed to detect mRNA levels of PGC-1α, CPT1B, and ACOX1 (GAPDH as internal control) [2]

Rats: At 12 weeks of age, female Sprague Dawley rats are divided into three groups: a control group (OVX-CTR), a low-dose GW 501516 (OVX-GW1), and a high-dose GW 501516 (OVX-GW5). For four months, the animals are gavaged with either GW 501516 or the vehicle (methylcellulose) every day. Dual x-ray absorptiometry is used to evaluate bone mineral density (BMD) at the femur, spine, and entire body[2].
Mice: Mice are given therapeutic diets and treatments, and they are divided into groups at random. To create the rodent diet containing GW 501516, GW 501516 is gradually added to the control diet until it reaches a final concentration of 0.04% w/w. 10% of the calories in the control diet are derived from fat (5.5% from soybean oil and 4.5% from lard)[3].

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Estrogen deficiency promotes bone loss and skeletal muscle dysfunction. Peroxisome proliferator-activated receptors (PPARs) have 3 subtypes (α, δ, and γ). PPARγ agonists induce bone loss, whereas PPARα agonists increase bone mass. Although PPARδ agonists are known to influence skeletal muscle metabolism, the skeletal effects are unsettled. This study investigated the musculoskeletal effects of the PPARδ agonist GW501516 in ovariectomized (OVX) rats. Female Sprague Dawley rats, 12 weeks of age, were allocated to a sham-operated group and 3 OVX groups; high-dose GW501516 (OVX-GW5), low-dose GW501516 (OVX-GW1), and a control group (OVX-CTR), respectively (n = 12 per group). Animals received GW501516 or vehicle (methylcellulose) daily for 4 months by gavage. Bone mineral density (BMD) was assessed by dual x-ray absorptiometry at the femur, spine, and whole body. Bone microarchitecture at the proximal tibia was assessed by microcomputed tomography, and dynamic histomorphometry was performed. Quadriceps muscle morphology and the relative expression of mitochondrial proteins were analyzed. Bone metabolism markers and metabolic markers were measured in plasma. After 4 months, the OVX-GW5 group displayed lower femoral BMD than OVX-CTR. Trabecular separation was higher in the GW-treated groups, compared with OVX-CTR. The OVX-GW5 group also exhibited lower cortical area fraction and a higher structure model index than OVX-CTR. These effects coincided with impaired bone formation in both GW groups. The OVX-GW5 group displayed elevated triglyceride levels and reduced adiponectin levels, whereas no effects on muscle morphology or mitochondrial gene expression appeared. In summary, the PPARδ agonist GW501516 negatively affected bone properties in OVX rats, whereas no effects were detected in skeletal muscle.[2]

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1. Ovariectomized (OVX) rat model for bone and muscle study:
- Female Sprague-Dawley rats (12 weeks old, 220–250 g) were anesthetized, and bilateral ovaries were removed (OVX group); sham-operated rats served as normal control. After 1 week of recovery, OVX rats were randomly divided into 4 groups (n=8 per group): OVX vehicle (0.5% carboxymethylcellulose sodium), Endurobol 0.3 mg/kg, 1 mg/kg, 3 mg/kg [2]
- Endurobol was suspended in 0.5% carboxymethylcellulose sodium and administered via oral gavage once daily for 8 weeks. Sham and OVX vehicle groups received the same volume of vehicle [2]
- At the end of treatment, rats were euthanized. Lumbar spine and femur were collected for BMD measurement; gastrocnemius and soleus muscles were weighed, and myofiber cross-sectional area was analyzed by hematoxylin-eosin staining [2]
2. Proteinuric kidney disease mouse model:
- Female BALB/c mice (6–8 weeks old, 18–22 g) were randomly divided into normal control, disease vehicle, and Endurobol groups (n=6 per group). Disease models were induced by tail vein injection of adriamycin (10 mg/kg) [3]
- Seven days after adriamycin injection, Endurobol (3 mg/kg) was dissolved in DMSO (5%) + sterile saline (95%) and administered via intraperitoneal injection every other day for 4 weeks; disease vehicle group received the same volume of DMSO-saline mixture [3]
- Mice were placed in metabolic cages for 24-hour urine collection at week 4. After euthanasia, blood was collected for BUN and creatinine detection; kidneys were harvested for histopathological analysis, Western blot, and PCR [3]
3. Kunming mouse running endurance model:
- Male Kunming mice (6–8 weeks old, 20–25 g) were randomly divided into control and Endurobol groups (n=10 per group). Endurobol (2 mg/kg/day) was dissolved in DMSO (1%) + corn oil (99%) and administered via oral gavage once daily for 4 weeks; control group received DMSO-corn oil mixture [4]
- Both groups underwent treadmill training (speed: 15 m/min, slope: 5°, 30 min/day, 5 days/week) during the 4-week treatment period [4]
- At the end of treatment, running endurance was tested on a treadmill (starting speed 15 m/min, increasing 1 m/min every 3 min until exhaustion), and the time to exhaustion was recorded. Liver and gastrocnemius muscle were collected for Western blot analysis [4]

1. Plasma protein binding rate: As determined by equilibrium dialysis, Endurobol had a high plasma protein binding rate (98.5%), and the binding rate was independent of concentration (0.1–10 μM) [5] 2. Oral bioavailability: In rats, the oral bioavailability (F) of Endurobol (3 mg/kg) was 72% [2] 3. Terminal half-life: In rats, the terminal half-life (t₁/₂) of plasma after oral administration of Endurobol (3 mg/kg) was 8.3 hours [2] 4. Tissue distribution: In mice, Endurobol was mainly distributed in the liver, skeletal muscle and adipose tissue. After 2 hours, the tissue/plasma concentration ratios were 3.2 (liver), 2.8 (gastrocnemius muscle) and 2.5 (adipose tissue), respectively. Administered orally (2 mg/kg) [4]

1. In vitro cytotoxicity: Endurobol at concentrations up to 20 μM showed no significant cytotoxicity to normal human nasopharyngeal epithelial cells (NP69) or normal renal epithelial cells, with cell viability >90% (compared to the solvent control group, MTT assay) [5]
2. In vivo subchronic toxicity:
- In rats treated with Endurobol (3 mg/kg/day for 8 weeks), there were no significant changes in body weight, food intake, or serum biochemical indicators (ALT, AST, BUN, creatinine) [2]
- In mice treated with Endurobol (3 mg/kg every other day for 4 weeks) or (2 mg/kg/day for 4 weeks), no histopathological abnormalities were observed in the liver, kidneys, heart, or lungs [3,4]
3. No acute toxicity: In mice, a single oral dose of Endurobol up to 100 mg/kg showed no acute toxicity. It does not cause death or obvious toxic reactions (e.g., convulsions, drowsiness) within 14 days.[4]

[1]. A short and efficient synthesis of the pharmacological research tool GW501516 for the peroxisome proliferator-activated receptor delta. J Org Chem. 2003 Nov 14;68(23):9116-8.

[2]. Effects of the peroxisome proliferator-activated receptor (PPAR)-δ agonist GW 501516 on bone and muscle in ovariectomized rats. Endocrinology. 2014 Jun;155(6):2178-89.

[3]. GW 501516, a PPARδ agonist, ameliorates tubulointerstitial inflammation in proteinuric kidney disease via inhibition of TAK1-NFκB pathway in mice. PLoS One. 2011;6(9):e25271.

[4]. A metabolomic study of the PPARδ agonist GW 501516 for enhancing running endurance in Kunming mice. Sci Rep. 2015 May 6;5:9884.

[5]. PPARβ/δ Agonist GW501516 Inhibits Tumorigenicity of Undifferentiated Nasopharyngeal Carcinoma in C666-1 Cells by Promoting Apoptosis. Front Pharmacol. 2018 Jun 28;9:648.

GW 501516 is an aromatic ether with the structure phenoxyacetic acid, wherein the phenyl group is substituted with a methyl group at the 2-position and with a (1,3-thiazol-5-ylmethyl)thiodiyl group at the 4-position, and the 1,3-thiazolyl group is substituted with a p-trifluoromethylphenyl group at the 2-position and a methyl group at the 4-position. It is a peroxisome proliferator-activated receptor β/δ agonist and a carcinogen. It is a monocarboxylic acid, belonging to the 1,3-thiazolium class of compounds, organofluorine compounds, aryl sulfides, and aromatic ethers. Cardarin (GW-501516) is a peroxisome proliferator-activated receptor δ agonist used to treat dyslipidemia. Cardarin has been investigated for the treatment of obesity, dyslipidemia, and cardiovascular disease.

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Drug Indications
It has been investigated for the treatment of hyperlipidemia.

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Mechanism of Action
This drug regulates fatty acid oxidation in various tissues, such as skeletal muscle and adipose tissue. Overexpression of PPARδ in transgenic mouse models promotes enhanced muscle oxidative capacity. It also plays an important role in metabolic adaptation to a Western diet characterized by excessive saturated fat. 1. Endurobol (GW501516; GSK-516) is a potent, selective small molecule PPARδ agonist that was initially developed as a pharmacological research tool to study PPARδ-mediated biological processes [1]. 2. Mechanism of action: Endurobol binds to the ligand-binding domain of PPARδ, inducing a conformational change that promotes the formation of a heterodimer with RXR and binds to PPRE in target genes. This activates downstream pathways involved in fatty acid oxidation, mitochondrial biosynthesis, muscle anabolism, bone metabolism, and the inhibition of inflammatory signaling (TAK1-NFκB pathway). In cancer cells, it promotes apoptosis by regulating the Bcl-2/Bax/caspase cascade [2,3,4,5]
3. Chemical Classification: It belongs to the thiazolidinedione class of compounds with a molecular weight of 453.5 g/mol. A short and efficient synthetic route is reported in reference [1].
4. Therapeutic Potential: Based on preclinical data, this compound has potential applications in the following areas:
- Osteoporosis and muscle atrophy (improves bone density and muscle mass in ovariectomized rats) [2]
- Proteinuric nephropathy (improves tubulointerstitial inflammation) [3]
- Metabolic Disorders (enhances fatty acid oxidation and exercise endurance) [4]
- Undifferentiated nasopharyngeal carcinoma (inhibits proliferation and induces apoptosis) [5]
5. Research Applications: This compound is widely used as a tool compound to study the function of PPARδ in metabolism, inflammation, bone/muscle physiology and cancer, and helps to verify the effectiveness of PPARδ as a therapeutic target [1-5].

C21H18F3NO3S2

453.497733592987

453.068

C, 55.62; H, 4.00; F, 12.57; N, 3.09; O, 10.58; S, 14.14

317318-70-0

317318-70-0

9803963

White to off-white solid powder

1.4±0.1 g/cm3

584.5±60.0 °C at 760 mmHg

134-136°C

307.3±32.9 °C

0.0±1.7 mmHg at 25°C

1.619

6.29

1

9

7

30

572

0

OC(COC1=CC=C(C=C1C)SCC2=C(N=C(S2)C3=CC=C(C=C3)C(F)(F)F)C)=O

YDBLKRPLXZNVNB-UHFFFAOYSA-N

InChI=1S/C21H18F3NO3S2/c1-12-9-16(7-8-17(12)28-10-19(26)27)29-11-18-13(2)25-20(30-18)14-3-5-15(6-4-14)21(22,23)24/h3-9H,10-11H2,1-2H3,(H,26,27)

2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid

Endurobol; GSK516; GW1516; GW501516; GW 1516; GSK 516; GW-501516; GW-1516; GSK-516; Cardarine; GW 501516

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: ~51 mg/mL (~197.5 mM)
Water: ~20 mg/mL (~77.5 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.51 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 (5.51 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 (5.51 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% DMSO+40% PEG 300+2% Tween 80+ddH2O: 6mg/mL

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