PPARβ/δ (IC50 = 22.9 μM)
Peroxisome proliferator-activated receptor beta/delta (PPARβ/δ, NR1C2): The IC50 value for human PPARβ/δ is 0.15 μM, and the IC50 values for human PPARα and PPARγ are both >10 μM[1]
- Peroxisome proliferator-activated receptor beta/delta (PPARβ/δ, NR1C2): The IC50 value for human PPARβ/δ is 0.155 μM, while the IC50 values for human PPARα and PPARγ are >10 μM[2]

GSK0660 suppresses the proliferation and differentiation of human retinal microvascular endothelial cells (HRMECs)[2].

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1. In primary human skeletal muscle cells, GSK0660 reduced the basal expression levels of the PPARβ/δ target genes ANGPTL4 and CPT1a in a dose - dependent manner, but had no effect on the expression of PDK4. When human skeletal muscle cells were treated with 10 nM of the PPARβ/δ agonist GW0742 along with different concentrations of GSK0660, GSK0660 could compete with GW0742 and reduce the efficacy of GW0742 - induced agonism on PPARβ/δ target gene expression[1]
2. In C2C12 myotubes, when treated with increasing doses of GSK0660, fatty acid oxidation was affected. Compared with the vehicle control, the difference was statistically significant (P < 0.05)[1]
3. In human retinal microvascular endothelial cells (HRMECs) cultured in medium containing 2% serum, GSK0660 (at concentrations of 0.01 μM, 0.1 μM, and 1.0 μM) significantly decreased the mRNA expression of the PPARβ/δ target gene angptl4 in a dose - dependent manner (P = 0.0035, P = 0.0092, and P = 0.0002 respectively)[2]
4. For HRMEC proliferation: When HRMECs were cultured in medium containing 2% serum and treated with GSK0660 (0.01 μM, 0.1 μM, 1.0 μM) for 24 hours, the proliferation of HRMECs induced by serum was reduced in a dose - dependent manner, with a 39% reduction (P = 0.0034) at the highest concentration (1.0 μM). When HRMECs were treated with 25 ng/mL vascular endothelial growth factor (VEGF) and GSK0660 (1.0 μM), the proliferation of HRMECs induced by VEGF was inhibited by 33.1% (P < 0.0001)[2]
5. Regarding HRMEC tube formation: In HRMECs cultured in medium containing 2% serum, treatment with GSK0660 (0.1 μM and 1.0 μM) reduced the average tube length induced by serum in a dose - dependent manner, with decreases of 18.7% (P = 0.04) and 40% (P = 0.0067) respectively. When HRMECs were treated with 25 ng/mL VEGF and 1.0 μM GSK0660, the tube formation induced by VEGF was inhibited by 50.6% (P = 0.0016)[2]

GSK0660 is quickly eliminated from the blood and does not build up in vivo[1]. GSK0660 works well against retinal NV when injected intravitreal or intraperitoneally. The benefits of intravitreal injection include the ability to deliver high doses of medication to the active sites of neovascular disease; however, this mode of administration is also linked to harmful side effects, such as glaucoma, cataractogenesis, and endophthalmitis. The use of systemic administration, which prevents these side effects, is limited by the requirement for repeated dosages in order to achieve the desired levels of active drug in diseased tissues. Additionally, it exposes healthy tissues and organs to active medication needlessly[2].

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1. In the rat oxygen - induced retinopathy (OIR) model:
- Intravitreal injection: On postnatal day 14 (day 14(0)) and day 14(3), rats were intravitreally injected with GSK0660 at concentrations of 20 nM, 100 nM, and 500 nM. On day 14(6), the preretinal neovascularization (NV) was quantified. The results showed that GSK0660 significantly reduced retinal NV by 58.5% (P = 0.0084) at 20 nM, 56.9% (P = 0.0109) at 100 nM, and 44.4% (P = 0.0142) at 500 nM compared with the vehicle group[2]
- Intraperitoneal injection: On day 14(0), day 14(2), and day 14(4), rats were intraperitoneally injected with GSK0660 at doses of 0.2 mg/kg and 1.0 mg/kg. On day 14(6), the quantification of retinal NV revealed that GSK0660 significantly reduced retinal NV by 50.3% (P = 0.0062) at the low dose and 59.4% (P = 0.0017) at the high dose compared with the vehicle group[2]
2. In the rat OIR model, after intravitreal injection of 500 nM GSK0660 on day 14(1), the retinas were collected on day 14(2) to detect the protein level of Angptl4. The results showed that GSK0660 had no effect on the production of Angptl4 protein in the retina[2]
3. In 7 - day - old rats raised in room air, intravitreal injection of 20 nM GSK0660 was performed, and the avascular area of the retina was assessed 3 days later. There was no significant difference in the avascular area of the retina between the GSK0660 - injected group and the vehicle - injected group, indicating that GSK0660 had no effect on the normal development of retinal blood vessels and no obvious retinal vascular toxicity[2]
4. During the experiment of using GSK0660 in the rat OIR model, there was no significant difference in body weight between the drug - injected group and the vehicle - injected group[2]

GSK0660 is almost inactive against PPARα and PPARγ, with IC50s of both >10 μM, and a strong antagonist of PPARβ and PPARδ, with IC50s of both 155 nM. With a pIC50 of 6.8, GSK0660 completely inhibits PPARβ/δ activity. While PDK4 expression, another PPARβ/δ target gene in skeletal muscle cells, is unaffected by GSK0660 (100 nM), it decreases CPT1a (a PPARβ/δ target gene) expression below the basal vehicle-treated level by about 50%.

HRMECs were cultured under standard tissue culture conditions after being seeded at a density of 2 × 10⁵ cells/well in six-well plates. Following a 12-hour serum starvation period at 80% confluency, the cells were treated for six hours with either a 0.5% serum-containing vehicle (0.1% DMSO) or PPAR-β/δ agonist GW0742 (0.01, 0.1, or 1.0 μM), or a 2% serum-containing vehicle or PPAR-β/δ antagonist GSK0660 (0.01, 0.1, or 1.0 μM). After twice washing the cells in cold PBS, total RNA was extracted. Transcribing the total RNA extracted from the culture wells was done in reverse. There was quantitative RT-PCR.

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1. Detection of gene expression in primary human skeletal muscle cells:
- Cell culture: Primary human skeletal muscle cells were cultured under appropriate conditions to ensure their normal growth and viability.
- Drug treatment: The cells were treated with different concentrations of GSK0660 alone or in combination with 10 nM GW0742. A vehicle control group was also set up.
- RNA extraction: After a certain period of drug treatment, the cells were washed twice with cold PBS, and total RNA was collected using a commercial RNA extraction kit according to the manufacturer's instructions.
- Gene expression analysis: The extracted RNA was reverse - transcribed into cDNA, and then Taqman real - time quantitative PCR (qRT - PCR) was used to detect the expression levels of target genes (ANGPTL4, CPT1a, PDK4). The relative expression levels of the genes were calculated by comparing with the internal reference gene[1]
2. Fatty acid oxidation assay in C2C12 myotubes:
- Cell culture: C2C12 myoblasts were induced to differentiate into myotubes under specific culture conditions.
- Drug treatment: The differentiated C2C12 myotubes were treated with increasing doses of GSK0660, and a vehicle control group was established.
- Fatty acid oxidation detection: After the drug treatment, the fatty acid oxidation level in the myotubes was measured using a specific detection method. The results were compared with the vehicle control group, and statistical analysis was performed to determine the significance of the difference[1]
3. Detection of angptl4 mRNA expression in HRMECs:
- Cell seeding and culture: HRMECs were seeded in six - well plates at a density of 2×10^5 cells/well and cultured under standard tissue culture conditions until they reached 80% confluency.
- Serum starvation and drug treatment: The cells were serum - starved for 12 hours, and then treated with medium containing 2% serum and different concentrations of GSK0660 (0.01 μM, 0.1 μM, 1.0 μM) for 6 hours. A vehicle control group (medium containing 2% serum and 0.1% DMSO) was also set up.
- RNA extraction and qRT - PCR: The cells were washed twice with cold PBS, and total RNA was extracted using a commercial RNA extraction kit. The RNA was reverse - transcribed into cDNA using a high - capacity cDNA reverse transcription kit. Then, qRT - PCR was performed in duplicate to detect the expression level of angptl4 mRNA, with β - actin as the internal reference gene[2]
4. HRMEC proliferation assay:
- Cell seeding and attachment: HRMECs were seeded in 96 - well plates at a density of 3×10^3 cells/well in growth medium and cultured for 8 hours to allow them to attach to the plate.
- Serum starvation and drug treatment: The cells were serum - starved for 12 hours, and then divided into two groups. One group was treated with medium containing 2% serum and different concentrations of GSK0660 (0.01 μM - 1.0 μM) for 24 hours, and the other group was treated with medium containing 25 ng/mL VEGF and different concentrations of GSK0660 (0.01 μM - 1.0 μM) for 24 hours. Vehicle control groups (medium containing 0.1% DMSO) were set up for both groups.
- Proliferation detection: After the drug treatment, the cells were labeled with bromodeoxyuridine (BrdU) for 12 hours. The incorporation of BrdU was quantified using a colorimetric BrdU ELISA kit according to the manufacturer's instructions. The experiment was repeated four times[2]
5. HRMEC tube formation assay:
- Coating of culture plates: 24 - well tissue culture plates were coated with 400 μL of growth factor - reduced basement membrane matrix and allowed to solidify under appropriate conditions.
- Cell seeding and drug treatment: HRMECs were seeded in the coated 24 - well plates at a density of 2.5×10^4 cells/well. They were divided into two groups: one group was treated with medium containing 2% serum and different concentrations of GSK0660 (0.01 μM - 1.0 μM) for 24 hours, and the other group was treated with medium containing 0.5% serum, 25 ng/mL VEGF, and different concentrations of GSK0660 (0.01 μM - 1.0 μM) for 12 hours. Vehicle control groups (medium containing 0.1% DMSO) were also set up.
- Tube length measurement: After the drug treatment, the cells were observed under an inverted microscope, and six images were captured per well in a systematic pattern at ×10 magnification. The capillary - like structures in the images were measured using image analysis software to determine the mean tube length per field, and the values were normalized[2]

Sprague-Dawley rat
\n0.2 or 1.0 mg/kg
\ni.p.

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\n1. Rat OIR model establishment:
\n - Animal selection and grouping: Within 8 hours after birth, litters of Sprague - Dawley rat pups and their mothers were selected and transferred to oxygen exposure chambers.
\n - Oxygen exposure: The rats were subjected to alternating 24 - hour periods of 50% and 10% oxygen for 14 days. On postnatal day 14 (referred to as day 14(0)), the oxygen - exposed rats were removed to room air and kept there for an additional 6 days (day 14(1) to day 14(6))[2]
\n2. Intravitreal injection of GSK0660 in OIR rats:
\n - Anesthesia and eye preparation: Rats were anesthetized by inhaling isoflurane, and a drop of 0.5% proparacaine was topically applied to the cornea to anesthetize the eye surface.
\n - Injection operation: A 30 - gauge needle with a 19° bevel and a 10 - μL syringe was used to penetrate the globe approximately 0.5 mm posterior to the ora ciliaris. The needle was advanced to the posterior vitreous at a steep angle to avoid contact with the lens, and a 5 - μL injection bolus of GSK0660 (at concentrations of 20 nM, 100 nM, 500 nM, dissolved in 0.1% DMSO in PBS) was delivered near the trunk of the hyaloid artery proximal to the posterior pole of the retina.
\n - Post - injection treatment: After the injection, a topical antibiotic suspension was applied to the eye. The non - injected eyes were also treated with topical proparacaine and antibiotic to control for potential effects of these agents on retinal vessel growth. The injection was performed on day 14(0) and day 14(3)[2]
\n3. Intraperitoneal injection of GSK0660 in OIR rats:
\n - Drug preparation: GSK0660 was dissolved in 1% DMSO in PBS to prepare doses of 0.2 mg/kg and 1.0 mg/kg.
\n - Injection schedule: The drug was injected intraperitoneally on day 14(0), day 14(2), and day 14(4)[2]
\n4. Oral gavage of drugs in OIR rats (for reference, although not specifically for GSK0660):
\n - Drug preparation: The drug (GW0742) was dissolved in 10% ethanol in corn oil to prepare doses of 1.0 mg/kg and 10 mg/kg.
\n - Administration schedule: The drug was administered by oral gavage once daily from day 14(0) to day 14(5)[2]
\n5. Quantification of retinal NV in OIR rats:
\n - Euthanasia and retina dissection: On day 14(6), all rats were euthanized, and their retinas were dissected out.
\n - ADPase staining: The dissected retinas were stained for adenosine diphosphatase (ADPase) activity according to well - established procedures.
\n - Image capture and analysis: Images of the ADPase - stained retinas were digitized and captured at ×20 magnification. For each retinal image, the preretinal vessel tufts were outlined with an irregular polygon, the number of pixels within the polygon was counted, and the total number of pixels from each polygon in a retina was pooled and converted to square millimeters. The data were normalized to the NV values from the vehicle - treated eyes[2]
\n6. Detection of Angptl4 protein in OIR rat retinas:
\n - Drug injection and retina collection: After the establishment of the OIR model, rats were intravitreally injected with 500 nM GSK0660 (dissolved in 0.1% DMSO in PBS) on day 14(1). The retinas were collected on day 14(2).
\n - Protein extraction and detection: The collected retinas were sonicated in lysis buffer, and the concentration of Angptl4 protein was determined using a colorimetric sandwich ELISA kit. The amount of Angptl4 (pg/mL) in the retinas was normalized to the total protein concentration (mg/mL) of the retinal lysates, which was measured using a bicinchoninic acid assay[2]
\n7. Toxicity assessment of GSK0660 on normal retinal vascular development:
\n - Animal selection and drug injection: 7 - day - old rats raised in room air were selected, and 20 nM GSK0660 was intravitreally injected into their eyes.
\n - Retinal avascular area assessment: Three days after the injection, the rats were euthanized, and their retinas were dissected. The avascular area of the retinas was assessed to determine the effect of GSK0660 on normal retinal vascular development[2]

In a study of copper-induced liver injury in mice, doses of GSK0660 up to 10 mg/kg/day showed no toxicity [2]. In 7-day-old rats raised in normal air, intravitreal injection of 20 nM GSK0660 had no effect on normal retinal vascular development and no significant retinal vascular toxicity [2]. In a rat model of oxygen-induced retinopathy (OIR), there was no significant difference in body weight between the drug injection group and the solvent injection group, indicating that GSK0660 had no significant adverse effect on rat growth [2].

[1]. Mol Endocrinol . 2008 Feb;22(2):523-9.

[2]. Invest Ophthalmol Vis Sci. 2013 Jun; 54(6): 4197–4207. Published online 2013 Jun 19.

3-[(4-anilino-2-methoxyphenyl)sulfonyl]-2-thiophene carboxylate is a sulfonamide compound.

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1. GSK0660 is a selective small molecule peroxisome proliferator-activated receptor β/δ (PPARβ/δ, NR1C2) antagonist. Before the discovery of GSK0660, only agonist ligands of PPARβ/δ receptors had been reported, and GSK0660 is the first reported small molecule antagonist ligand of PPARβ/δ. It can serve as an effective tool for elucidating the biological functions of PPARβ/δ[1].
2. GSK0660 is not only a potent competitive antagonist of PPARβ/δ, but also exhibits inverse agonist activity[2].
3. PPARβ/δ is an important regulator of energy metabolism, inflammation, and cell growth and differentiation. The discovery of GSK0660 provides a new approach to studying the function of PPARβ/δ in these biological processes[1]
4. In the field of PPAR research, most studies have focused on agonists, while relatively few studies have been conducted on antagonists, and all previously reported antagonists act selectively on PPARα or PPARγ. The discovery of GSK0660 enriches the types of PPAR antagonists and provides a new research tool for comparing the effects of PPAR agonists and antagonists in cellular and animal systems, thereby gaining new insights into PPAR biology[1]
5. Ocular neovascularization (NV) is a characteristic manifestation of retinopathy of prematurity (ROP), diabetic retinopathy (DR), and age-related macular degeneration (ARMD), which are leading causes of irreversible blindness in developed countries. The study found that GSK0660 reduced retinal neovascularization (NV) in a rat model of oxygen-induced retinopathy (OIR), suggesting that the pharmacological inhibition of PPARβ/δ by GSK0660 may provide a theoretical basis for developing therapeutic strategies targeting ocular NV [2]
6. The mechanism by which GSK0660 reduces retinal NV may be related to its inhibition of PPARβ/δ-mediated angiogenic cell behavior. PPARβ/δ may regulate preretinal NV through a differentiation/maturation mechanism dependent on Angptl4. GSK0660 may reduce retinal NV by blocking this mechanism through inhibition of PPARβ/δ [2]
7. Although GSK0660 shows potential in reducing retinal NV, caution should be exercised when developing it as a therapeutic. PPARβ/δ has many potential beneficial biological functions, such as anti-apoptosis, anti-inflammation, neuroprotection and inhibition of tumorigenesis, and pharmacological activation of PPARβ/δ can protect endothelial cells from dysfunction. Therefore, when exploring the pharmacological inhibition of PPARβ/δ in the retina by GSK0660, potential adverse side effects associated with retinal inflammation and retinal cell death should be carefully evaluated, and the overall health status of subjects should be monitored, while taking into account the positive effects of PPARβ/δ on metabolic homeostasis and cardiovascular disease and its controversial role in carcinogenesis [2]
8. The route of administration of GSK0660 is a key factor in its therapeutic application. Intravitreal injection can achieve high drug concentrations at the site of neovascularization, but is associated with potential side effects such as endophthalmitis, cataracts and glaucoma. Systemic administration (e.g., intraperitoneal injection) can avoid these local side effects, but requires repeated administration to maintain effective drug concentrations in diseased tissues and may expose healthy organs and tissues to the drug. Therefore, optimizing the route of administration of GSK0660 is crucial for its future clinical application [2]
9. Currently, many patients with retinal neovascularization (NV) have limited benefit from existing vascular endothelial growth factor (VEGF) targeted drugs, which have disadvantages such as potential neurotoxicity, increased intraocular pressure and refractory properties. The discovery that GSK0660 can reduce retinal neovascularization suggests that it may become a novel treatment for retinal neovascularization diseases such as retinopathy of prematurity (ROP), providing new treatment options for patients with retinal neovascularization [2]

C19H18N2O5S2

418.49

418.065

C, 54.53; H, 4.34; N, 6.69; O, 19.12; S, 15.32

1014691-61-2

46233311

Light yellow to yellow solid powder

1.4±0.1 g/cm3

608.1±65.0 °C at 760 mmHg

321.5±34.3 °C

0.0±1.7 mmHg at 25°C

1.650

2.75

2

8

8

28

618

0

S(C1C([H])=C([H])SC=1C(=O)OC([H])([H])[H])(N([H])C1C([H])=C([H])C(=C([H])C=1OC([H])([H])[H])N([H])C1C([H])=C([H])C([H])=C([H])C=1[H])(=O)=O

NDFKBGWLUHKMFY-UHFFFAOYSA-N

InChI=1S/C19H18N2O5S2/c1-25-16-12-14(20-13-6-4-3-5-7-13)8-9-15(16)21-28(23,24)17-10-11-27-18(17)19(22)26-2/h3-12,20-21H,1-2H3

methyl 3-[(4-anilino-2-methoxyphenyl)sulfamoyl]thiophene-2-carboxylate

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 (5.97 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)