PPARα

The peroxisome proliferator activated receptors PPARalpha, PPARgamma, and PPARdelta are ligand-activated transcription factors that play a key role in lipid homeostasis. The fibrates raise circulating levels of high-density lipoprotein cholesterol and lower levels of triglycerides in part through their activity as PPARalpha agonists; however, the low potency and restricted selectivity of the fibrates may limit their efficacy, and it would be desirable to develop more potent and selective PPARalpha agonists. Modification of the selective PPARdelta agonist 1 (GW501516) so as to incorporate the 2-aryl-2-methylpropionic acid group of the fibrates led to a marked shift in potency and selectivity toward PPARalpha agonism. Optimization of the series gave 25a, which shows EC50 = 4 nM on PPARalpha and at least 500-fold selectivity versus PPARdelta and PPARgamma. Compound 25a (GW590735) has been progressed to clinical trials for the treatment of diseases of lipid imbalance[1].

GW 590735 (0.5–5 mg/kg; PO twice daily for 5 days) raises HDL cholesterol and lowers LDLc and TG in an Apo-AI transgenic mouse model (human transgenic male C57BL/6 mice). APOA-II)(1). Cl, Vd, T1/2, and F% of GW 590735 (IV; 2.7 mg/kg; rat) were displayed after 2.4 hours, 1 L/kg, 5 mL/min/kg, and 47%, respectively [1]. The results of treatment with GW 590735 (IV; 2 mg/kg; dog) showed that Cl, Vd, T1/2, and F% were, respectively, 13 mL/min/kg, 2.8 L/kg, 2.6 hours, and 85% [1].

The protein was then diluted to 1 mg/mL with buffer C such that the final buffer composition was 220 mM ammonium acetate, 20 mM HEPES pH 7.5, 1 mM EDTA, and 1 mM DTT. The peptide SRC116 was added in a molar ratio of 1.5 as a 2 mg/100 μL DMSO stock. The ligand was then added in a 5:1 molar ratio as a 2 mg/100 μL DMSO stock and spun at 4 K for 20 min to clarify the solution before concentrating in Centriprep 10 filtration units. The solution containing the PPARα LBD-SRC1 complexes was concentrated to approximately 10 mg/mL with 80% yield[1].

The PPARα ligand binding domain (amino acids 192−468) with an N-terminal 6xHis tag was expressed using the T7 promoter of plasmid vector pRSETA. BL21(DE3) E. coli cells transformed with this expression vector were grown at 24 °C in shaker flasks for 66 h. The cells were harvested, resuspended, and lysed. The lysed cells were centrifuged, and the supernatant was loaded on a Ni-agarose column. The column was washed with 150 mL of buffer A (10% glycerol, 20 mM HEPES pH 7.5, 25 mM imidazole), and the protein was eluted with a 450 mL gradient of buffer B (10% glycerol, 20 mM HEPES pH 7.5, 500 mM imidazole). The protein, which eluted at 20% buffer B, was diluted with one volume of buffer C (20 mM HEPES, pH 7.5, 1 mM EDTA) and loaded on a 100 mL S-Sepharose column. The column was washed with 100 mL buffer C, and the PPARα LBD protein was eluted with a 200 mL gradient of buffer D (20 mM HEPES, pH 7.5, 10 mM DTT, 1 M ammonium acetate). The PPARα LBD eluted from the column at 43% buffer D. The protein yield was 9 mg/L of cells grown and was >95% pure, as determined by SDS-PAGE analysis[1].

Pharmacokinetics in Rat and Dog.[1]
Compound 25a was administered to Wistar rats (n = 15) by oral gavage at dose of 3 mg/kg in pH 7 buffer, 0.1% Tween80 and by intravenous injection via the penis vein (n = 30) at a dose of 2.7 mg/kg in 10% DMSO and PEG200. Compound 25a was administered orally to male beagle dogs (n = 3) by stomach intubation at a dose of 3 mg/kg in pH 7 buffer, 0.1% Tween80 and by intravenous injection via the cephalic vein at a dose of 2 mg/kg in 10% DMSO and PEG200. Blood samples were placed on wet ice, and plasma was collected after centrifugation. Plasma samples were stored frozen at −20 °C until time of analysis. Plasma samples (0.5 mL) were diluted with 1:1 buffer (NaH2PO4, 0.1 M, pH 4) and then extracted with ethyl acetate (5 mL). The ethyl acetate was evaporated, and the residue was resuspended in 200 μL of mobile phase (water/acetonitrile/TFA; 30v/70v/0.1%). Samples were analyzed by high-performance liquid chromatography spectrometric analysis (LC/MS/MS). Pharmacokinetic parameters were determined by SIPHAR.

---

Apo-A-I Transgenic Mouse Model. [1]
Male C57BL/6 mice transgenic for human ApoA-I were randomized into treatment groups of n = 5 animals. Twice a day oral administration of vehicle (0.5% HPMC/1% Tween80, pH = 7.0) or indicated doses of compound as a suspension began when animals were nine weeks old and lasted for 5 days. Animals were fasted overnight before blood samples were taken by intracardiac puncture. Whole liver was collected and weighed. Blood samples were left for 30 min at 37 °C to coagulate and centrifuged 10 min at 10 000 rpm. Total serum fraction was then collected and frozen at −20 °C until use. Total cholesterol and total TG were dosed using kits 61219 and 61236, respectively, following manufacturer instructions. After 10 min of incubation at 37 °C, the colorimetric reaction was read at 492 nm with an iEMS reader. Cholesterol HDL, LDL, and VLDL fractions were separated by HPLC. Samples were diluted 1/5 in phosphate buffer (Ca++ and Mg- free) and filtered on 0.45 μm to remove excess proteins before HPLC. All changes reported with an asterisk are statistically significant (p < 0.05) as determined by one-way ANOVA analysis.

[1]. Sierra ML, et al. Substituted 2-[(4-aminomethyl)phenoxy]-2-methylpropionic acid PPARalpha agonists. 1. Discovery of a novel series of potent HDLc raising agents. J Med Chem. 2007;50(4):685-695.

Pharmacokinetics in Rat and Dog.
Following intravenous administration of compound 25a (2.7 mg/kg) to the rat, distribution to the tissues was limited with the volume of distribution (1 L/kg) similar to that of total body water (0.6 L/kg). Total plasma clearance was low (5 mL/min/kg), representing about 6% of rat hepatic blood flow. The low clearance and moderate volume of distribution resulted in a plasma half-life of 2.4 h. Following a single oral dose of compound 25a at 3 mg/kg, the maximum concentration of compound in the plasma was 1461 ng/mL after 1.5 h. The bioavailability was high (47%).
Following intravenous administration of compound 25a to the dog at 2 mg/kg, distribution to the tissues was limited with the volume of distribution (2.8 L/kg) being greater than that of total body water (0.6 L/kg). Total plasma clearance was moderate (13 mL/min/kg), representing about 35% of hepatic blood flow in the dog. The moderate clearance and low volume of distribution resulted in a plasma half-life of 2.6 h. Following a single oral dose of compound 25a at 3 mg/kg, the maximum concentration of compound in the plasma was 1449 ng/mL. The bioavailability was high (85%).[1]

---

In Vivo Pharmacology.
Several compounds in this series demonstrated profound in vivo activity in animal models of dyslipidemia, as illustrated by compound 25a. The human Apo-A-I-transgenic mouse model has been proposed to be potentially relevant to human disease, because in this model, fibrates give upregulation rather than the repression of Apo-A-I seen in other rodent models. Compound 25a shows similar PPARα agonist potency and selectivity versus murine and human PPARs (murine PPAR EC50, α = 15 nM; δ = 1000 nM; γ > 10 000 nM). When administered orally twice a day for 5 days, 25a, prepared as a suspension in 0.5% HPMC 100/1% Tween80 at pH = 7.0, gave dose-related decreases in circulating TG, VLDLc, and LDLc and concomitant increases in HDLc (Table 7). The ED50 for the HDL effect was approximately 1 mg/kg. Circulating levels of Apo-A-I were also increased by the treatment (data not shown), consistent with the mechanism of action. As a reference, Fenofibrate was tested in human Apo-A-I transgenic mice at 50 mg/kg. It produced the expected profile with a decrease in plasma TG (−43%), VLDL cholesterol (−64%), and LDL cholesterol (−78%), as well as an increase in HDL cholesterol (+26%). The finding that 25a is able to lower LDLc and TG and increase HDL cholesterol in the Apo-A-I-transgenic mouse model suggests that it will deliver significant therapeutic benefit in the treatment of dyslipidemia and hypertriglyceridemia. Compound 25a (GW590735) has been progressed to clinical trials for the treatment of diseases of lipid imbalance.[1]

---

Starting from the selective PPARδ agonist 1, which showed weak potency on PPARα, we have developed a new series of highly potent and selective PPARα agonists, one of which, 25a, has progressed to clinical evaluation. The superior potency and PPAR subtype selectivity of 25a suggest that this compound offers the potential to deliver significantly improved therapeutic benefits over the fibrates in dyslipidemia and hypertriglyceridemia.

C23H21F3N2O4S

478.49

478.11741282

343321-96-0

343321-96-0

9956726

White to off-white solid powder

1.3±0.1 g/cm3

1.572

5.22

117Ų

CC1=C(SC(=N1)C2=CC=C(C=C2)C(F)(F)F)C(=O)NCC3=CC=C(C=C3)OC(C)(C)C(=O)O

ILUPZUOBHCUBKB-UHFFFAOYSA-N

InChI=1S/C23H21F3N2O4S/c1-13-18(33-20(28-13)15-6-8-16(9-7-15)23(24,25)26)19(29)27-12-14-4-10-17(11-5-14)32-22(2,3)21(30)31/h4-11H,12H2,1-3H3,(H,27,29)(H,30,31)

2-methyl-2-[4-[[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carbonyl]amino]methyl]phenoxy]propanoic acid

GW 590735; GW-590735;343321-96-0; 622402-22-6; GW590735; GW-590735; 2-methyl-2-(4-((4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carboxamido)methyl)phenoxy)propanoic acid; QKY617BBX5; CHEMBL219586; GW590735

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~250 mg/mL (~522.5 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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