| Description: |
RO1138452 is a potent and selective IP (prostacyclin) receptor antagonist. RO1138452 displays high affinity for IP receptors. In human platelets, pKi is 9.3±0.1; in a recombinant IP receptor system, pKi is 8.7±0.06. |
| In Vivo: |
RO1138452 is a potent and selective antagonist for both human and rat IP receptors and that is possesses analgesic and anti-inflammatory potential. RO1138452 (1-10 mg/kg, i.v.) significantly reduces acetic acid-induced abdominal constrictions. RO1138452 (3-100 mg/kg, p.o.) significantly reduces carrageenan-induced mechanical hyperalgesia and edema formation. One hour after administration of RO1138452 (5 mg/kg, i.v.) to rats, the total plasma concentration is 0.189 μg/mL, whereas the free plasma concentrations is calculated to be 0.009 μg/mL (28 nM)[1]. |
| In Vitro: |
RO1138452 is IP receptor antagonist. The pIC50 values of RO1138452 in attenuating cAMP accumulation is 7.0±0.07. Functional antagonism of RO1138452 is studied by measuring inhibition of carbaprostacyclin-induced cAMP accumulation in CHO-K1 cells stably expressing the human IP receptor. The antagonist affinity (pKi) of RO1138452 is 9.0±0.06. Selectivity profiles for RO1138452 are determined via a panel of receptor binding and enzyme assays. RO1138452 displays affinity at imidazoline2 (I2) (8.3) and platelet activating factor (PAF) (7.9) receptors[1]. RO1138452 (10 pM-10 μM) added to cells concurrently with a fixed concentration of Taprostene (1 μM) prevents, in a concentration-dependent manner, the inhibition of CXCL9 and CXCL10 release, with p[A]50 (molar) values of -8.73±0.11 and -8.47±0.16 (p>0.05), respectively[2]. |
| Kinase Assay: |
Selectivity is determined by the ability of RO1138452 (10 μM) to displace specific binding of standard radioligands at 51 receptors. When significant displacement of radioligand is observed (>70% for RO1138452), complete concentration-dependent displacement curves (in triplicate) are constructed to generate IC50 values. Displacement binding at the EP3 receptor is performed. Enzyme inhibition assays are also conducted. RO1138452 is evaluated at 10 μM in triplicate for inhibition of COX isoforms: COX-1 (ram seminal vesicle), COX-2 (sheep placenta and human umbilical vein). Arachidonic acid is used as a substrate and PGE2 accumulation is detected[1]. |
| Cell Assay: |
BEAS-2B cells are incubated for 30 min at 37°C in supplement-free keratinocyte serum-free medium (KSFM) in the absence and presence of 100 nM RO1138452. Cells are washed with supplement-free KSFM, incubated in the same medium for defined periods, and exposed to 1 μM Taprostene. Four hours later, cells are harvested in reporter lysis buffer, and luciferase activity is measured. The viability HAECs and BEAS-2B cells is determined colorimetrically by measuring the reduction of the tetrazolium salt MTT to formazan, by mitochondrial dehydrogenases[2]. |
| Animal Administration: |
Rats[1] Male Sprague-Dawley rats (n=3) are administered RO1138452 (5 mg/kg, i.v.). At various times after dose administration, the rats are anesthetized by halothane (5%), blood is collected by orbital bleed into a heparinized syringe and a plasma fraction is obtained by centrifugation of the blood at 2600× g for 5 min in a clinical centrifuge. The level of RO1138452 in each sample is determined by high-performance liquid chromatography with detection by mass spectrometry[1]. |
| References: |
[1]. Bley KR, et al. RO1138452 and RO3244794: characterization of structurally distinct, potent and selective IP (prostacyclin) receptor antagonists. Br J Pharmacol. 2006 Feb;147(3):335-45.
[2]. Ayer LM, et al. 4,5-Dihydro-1H-imidazol-2-yl)-[4-(4-isopropoxy-benzyl)-phenyl]-amine (RO1138452) is a selective, pseudo-irreversible orthosteric antagonist at the prostacyclin (IP)-receptor expressed by human airway epithelial cells: IP-receptor-mediated inhibition of CXCL9 and CXCL10 release. J Pharmacol Exp Ther. 2008 Feb;324(2):815-26. |
