Peroxisome proliferator-activated receptor γ (PPARγ) – IC50 for inhibition of troglitazone-induced PPARγ transcriptional activity = 140 μM [1].

After six days, BRL 49653 and hormone-induced adipocyte clearance in 3T3-L1 cells are strongly inhibited by mifobate (100-400 μM; 24 hours rest) in a dose-dependent manner [1]. The agonist-coupled response of TZD-stimulated coactivator dormant coactivator-like 1 (SRC-1) is inhibited by mifobate (SR-202). Mifobate inhibits the release of fat when thiazolidinediones or a combination of insulin, dexamethasone, and 3-cell isotetra-1-methylxanthine (IBMX) are used [1].

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In HeLa cells transfected with PPARγ and a PPRE-containing reporter, SR-202 alone (up to 400 μM) did not alter basal transcriptional activity, but dose-dependently inhibited troglitazone-induced PPARγ activity (IC50 = 140 μM). It did not inhibit PPARα, PPARβ, or FXR activities at concentrations up to 400 μM [1].

In a coactivator-dependent receptor ligand assay (CARLA) using the PPARγ ligand-binding domain and SRC-1, SR-202 alone did not promote SRC-1 recruitment. Co-incubation of 0.5 μM BRL 49653 (rosiglitazone) with increasing concentrations of SR-202 (50–400 μM) dose-dependently decreased SRC-1 recruitment; at 400 μM, BRL 49653-stimulated SRC-1 binding was reduced by 75%. SR-202 did not affect PPARα or PPARβ interaction with SRC-1 [1].

In 3T3-L1 preadipocytes, SR-202 (50–400 μM) inhibited both BRL 49653 (25 nM)-induced and hormone (dexamethasone/IBMX/insulin)-induced adipocyte differentiation, as shown by Oil Red O staining and decreased aP2 mRNA levels. The anti-adipogenic effect was dose-dependent and reversible by high concentrations of BRL 49653 (1 μM). Cytotoxicity was excluded by LDH release (<6% of total cellular LDH after 36 h at 400 μM) and unchanged cell numbers [1].

In WT mice, treatment with SR-202 (400 mg/kg in food for 10 wk from weaning) decreased body weight gain, white adipose tissue (WAT) mass, brown adipose tissue (BAT) mass, and adipocyte size under both standard diet (SD) and high-fat diet (HFD). It also reduced mRNA levels of PPARγ target genes (LPL, aP2, CD36, SREBP-1c) in WAT. Plasma levels of leptin and TNFα were significantly decreased. Under HFD, SR-202 prevented hyperinsulinemia, reduced free fatty acids (FFA), and improved insulin sensitivity [1].

In ob/ob mice (8 wk old, treated for 20 d with SR-202 at 400 mg/kg in food), SR-202 prevented the time-dependent increase in fed glucose (to 67% of untreated) and insulin (to 70% of untreated). Glucose tolerance and insulin tolerance tests showed enhanced glucose disposal and insulin sensitivity. SR-202 also reduced LPL, CD36, and aP2 mRNA levels in WAT of ob/ob mice [1].

In ob/ob mice, mifobate (400 mg/kg; given for 20 days) enhances insulin resistance[1].

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In wild-type mice, SR-202 (400 mg/kg as food admixture from weaning for 10 wk) significantly reduced body weight gain, WAT mass, and BAT mass under both SD and HFD. Adipocyte size was decreased, and the number of adipocytes per microscopic field increased. SR-202 prevented HFD-induced adipocyte hypertrophy and insulin resistance, as evidenced by lower plasma insulin and FFA levels. It also decreased PPARγ target gene expression (LPL, aP2, CD36, SREBP-1c) in WAT [1].

In ob/ob mice (8 wk old, treated for 20 d with SR-202 at 400 mg/kg in food), SR-202 reduced fed glucose and insulin levels, improved glucose tolerance and insulin sensitivity, and decreased expression of LPL, CD36, and aP2 in WAT. Body weight gain was comparable to untreated controls [1].

Coactivator-dependent receptor ligand assay (CARLA): The ligand-binding domain of human PPARγ was incubated with radiolabeled SRC-1 in the presence of increasing concentrations of SR-202 (0–400 μM) with or without 0.5 μM BRL 49653. SR-202 alone did not promote SRC-1 binding. In the presence of BRL 49653, SR-202 dose-dependently inhibited SRC-1 recruitment, as quantified by densitometry of pull-down gels. No effect was seen on PPARα or PPARβ [1].

3T3-L1 preadipocyte differentiation: Cells were pretreated with SR-202 (50, 100, 200, or 400 μM) or vehicle for 24 h, then induced to differentiate with either BRL 49653 (25 nM) + insulin (5 μg/mL) or a mixture of dexamethasone (1 μM), insulin (10 μg/mL), and IBMX (0.5 mM) for 2–6 days. Differentiation was assessed by Oil Red O staining for lipid accumulation and by ribonuclease protection assay for aP2 mRNA (adipocyte marker). LDH release was measured to rule out cytotoxicity [1].

Transactivation assay: HeLa cells were cotransfected with full-length PPARα, PPARβ, PPARγ, or FXR expression plasmids and a reporter plasmid containing a PPRE or FXRE. Cells were treated with specific ligands (Wy14643 for PPARα, bezafibrate for PPARβ, troglitazone for PPARγ, GW1358 for FXR) and increasing concentrations of SR-202 (0–400 μM) for 36 h. CAT activity was measured and normalized to β-galactosidase activity [1].

Animal/Disease Models: Eightweeks old male ob/ob mice[1]
Doses: 400 mg/kg
Route of Administration: Feed (food mixture Maintenance for 20 days)
Experimental Results: Prevented increase in glucose concentration over time.

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Wild-type and PPARγ heterozygous mice (mixed sv129/C56Bl6 background) – at 3 wk of age, separated by sex, genotyped, and fed either standard diet (SD) or high-fat diet (HFD; 45 kcal% fat) supplemented or not with SR-202 (400 mg/kg as food admixture) for 10 wk. Mice were weighed weekly. At the end, tissues were removed, weighed, and frozen. Blood was collected from the orbital sinus for plasma metabolite measurements [1].

Ob/ob mice (8 wk old, male) were fed SD with or without SR-202 (400 mg/kg in food) for 20 d. Blood was withdrawn from the tail tip for glucose and insulin measurements at fed state on indicated days. Glucose tolerance test: 6-h fasted mice injected i.p. with 2 mg/g body weight glucose, blood taken at 0, 15, 30, 60, 120 min. Insulin tolerance test: injected i.p. with 0.75 mU/g insulin, blood taken similarly [1].

SR-202 was not cytotoxic to HeLa cells at 400 μM for 36 h (LDH release <6% of total). In 3T3-L1 cells, LDH release after 48 h at 400 μM was only 5% of total cellular LDH. Cell numbers remained unchanged after differentiation protocols [1].

In WT mice treated with 400 mg/kg SR-202 for 10 wk, no adverse effects on general health or body weight gain (compared to untreated controls under SD/HFD) were reported. In ob/ob mice, body weight gain was comparable between treated and untreated groups [1].

[1]. A new selective peroxisome proliferator-activated receptor gamma antagonist with antiobesityand antidiabetic activity. Mol Endocrinol. 2002 Nov;16(11):2628-44.

[(4-Chlorophenyl)-dimethoxyphosphorylmethyl]dimethyl phosphate is a trialkyl phosphate ester.

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SR-202 (dimethyl α-dimethoxyphenyl)-p-chlorobenzyl phosphate is a synthetic phosphonophosphate compound identified as a selective PPARγ antagonist. It inhibits TZD-stimulated recruitment of the coactivator SRC-1 to PPARγ and TZD-induced transcriptional activity. In vitro, it blocks both TZD- and hormone-induced adipocyte differentiation. In vivo, it reduces adiposity, prevents high-fat diet-induced insulin resistance, and improves glucose homeostasis in ob/ob mice. The compound has both antiobesity and antidiabetic effects. It is a low-affinity antagonist (IC50 140 μM in transactivation assay). Treatment with SR-202 reproduces the phenotype of PPARγ heterozygous mice (reduced PPARγ activity) [1].

C11H17CLO7P2

358.6475

358.014

76541-72-5

60910

White to off-white solid powder

1.355g/cm3

436.6ºC at 760 mmHg

350.8ºC

1.494

4.242

0

7

8

21

395

0

VQHUQHAPWMNBLP-UHFFFAOYSA-N

InChI=1S/C11H17ClO7P2/c1-15-20(13,16-2)11(19-21(14,17-3)18-4)9-5-7-10(12)8-6-9/h5-8,11H,1-4H3

Phosphoric acid, (4-chlorophenyl)(dimethoxyphosphinyl)methyl dimethyl ester

SR 202 SR-202Mifobate

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 : ~100 mg/mL (~278.82 mM)
H2O : ~35.87 mg/mL (~100.01 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.97 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 2: ≥ 2.5 mg/mL (6.97 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 3: 50 mg/mL (139.41 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.)