GW 501516 (also known as endurobol; GW-501516; GSK-516) is a novel and potent PPARδ agonist with an EC50 of 1.1 nM. Endurobol was withdrawn from clinical development in 2007 due to evidence from animal experiments that the medication caused cancer to spread quickly throughout multiple organs. Endurobol was initially being studied as a potential treatment for metabolic and cardiovascular disorders. When GW501516, a particular PPARβ/δ selective agonist, activated the PPARβ/δ ligand, it significantly reduced colony formation and cell proliferation in EBV positive undifferentiated NPC C666-1 cells, inducing a G2/M phase arrest in comparison to control cells. Furthermore, GW501516 induced apoptosis in C666-1 cells in a way that required both BAX and caspase. GW501516 significantly reduced the ectopic NPC xenograft tumorigenicity in BALB/c nu/nu mice, which was derived from the C666-1 NPC cells, in line with the in vitro result. Its suppression of integrin-linked kinase (ILK) gene and protein expression via AMPKα-dependent signaling pathways is closely linked to this effect. All together, we demonstrated that the degree of differentiation in NPC cell lines is inversely correlated with PPARβ/δ expression, and we demonstrated how GW501516 inhibits tumor growth in NPC cells by activating AMPKα. According to this study, PPARβ/δ targeting molecules may be helpful for chemoprevention of poorly differentiated NPCs in particular.

Physicochemical Properties

Molecular Formula	C21H18F3NO3S2
Molecular Weight	453.497733592987
Exact Mass	453.068
Elemental Analysis	C, 55.62; H, 4.00; F, 12.57; N, 3.09; O, 10.58; S, 14.14
CAS #	317318-70-0
Related CAS #	317318-70-0
PubChem CID	9803963
Appearance	White to off-white solid powder
Density	1.4±0.1 g/cm3
Boiling Point	584.5±60.0 °C at 760 mmHg
Melting Point	134-136°C
Flash Point	307.3±32.9 °C
Vapour Pressure	0.0±1.7 mmHg at 25°C
Index of Refraction	1.619
LogP	6.29
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	7
Heavy Atom Count	30
Complexity	572
Defined Atom Stereocenter Count	0
SMILES	OC(COC1=CC=C(C=C1C)SCC2=C(N=C(S2)C3=CC=C(C=C3)C(F)(F)F)C)=O
InChi Key	YDBLKRPLXZNVNB-UHFFFAOYSA-N
InChi Code	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)
Chemical Name	2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid
Synonyms	Endurobol; GSK516; GW1516; GW501516; GW 1516; GSK 516; GW-501516; GW-1516; GSK-516; Cardarine; GW 501516
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	PPARδ (EC50 = 1.1 nM)
ln Vitro	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].
ln Vivo	Peroxisome 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] Exercise 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] Activation 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] GW 501516 impairs bone formation, which lowers BMD and deteriorates bone properties in OVX rats[2]. GW 501516 reduces both proximal tubular cell damage and interstitial inflammation in a protein-overload mouse nephropathy model[3]. GW 501516 treatment increases running endurance and the percentage of muscle fibers that are positive for succinate dehydrogenase (SDH) in trained and untrained mice[4].
Cell Assay	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].
Animal Protocol	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]. 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]
References	[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.
Additional Infomation	GW 501516 is an aromatic ether that is phenoxyacetic acid in which the phenyl group is substituted at position 2 by a methyl group and at position 4 by a (1,3-thiazol-5-ylmethyl)sulfanediyl group, and in which the 1,3-thiazolyl group is substituted at positions 2 and 4 by p-trifluoromethylphenyl and methyl groups, respectively. It has a role as a PPARbeta/delta agonist and a carcinogenic agent. It is a monocarboxylic acid, a member of 1,3-thiazoles, an organofluorine compound, an aryl sulfide and an aromatic ether. Cardarine (GW-501516) is a peroxisome proliferator-activator receptor-delta agonist for the potential treatment of dyslipidemia. Cardarine has been investigated for the treatment of Obesity, Lipid Disorders, and Cardiovascular Disease. Drug Indication Investigated for use/treatment in hyperlipidemia. Mechanism of Action This drug regulates fatty acid oxidation in several tissues, such as skeletal muscle and adipose tissue. Overexpression of PPARdelta using a transgenic murine model promotes an increase of muscle oxidative capability. It also plays a major role in the metabolic adaptations to western diet characterized by an excessive amount of saturated fat.

Solubility Data

Solubility (In Vitro)

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

Solubility (In Vivo)

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. 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. 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.. Solubility in Formulation 4: 2% DMSO+40% PEG 300+2% Tween 80+ddH2O: 6mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.2051 mL	11.0254 mL	22.0507 mL
	5 mM	0.4410 mL	2.2051 mL	4.4101 mL
	10 mM	0.2205 mL	1.1025 mL	2.2051 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.