1、Hotline: 400-820-3792Inhibitors Agonists Screening Librarieswww.MedChemEPitolisantCat. No.: HY-12199CAS No.: 362665-56-3Synonyms: Tiprolisant分式: CHClNO分量: 295.85作靶點(diǎn): Histamine Receptor作通路: GPCR/G Protein; Immunology/Inflammation; NeuronalSignaling儲(chǔ)存式: Please store the product under the recommended c

2、onditions inthe COA.BIOLOGICAL ACTIVITY物活性 Pitolisant種有效的選擇性的咪唑類重 組組胺 H3 受體反相激動(dòng)劑，Ki 為 0.16 nM。IC50 & Target Ki: 0.16 nM (H3 receptor) 1EC50: 1.5 nM (H3 receptor) 1體外研究 On the stimulation of guanosine 5-O-(3-35Sthio)triphosphate binding to this receptor, Pitolisant (BF2.649)behaves as a competitive a

3、ntagonist with a Ki value of 0.16 nM and as an inverse agonist with an EC50value of 1.5 nM and an intrinsic activity 50% higher than that of ciproxifan. Pitolisant displaces125Iiodoproxyfan binding from mouse brain cortical membranes with an IC50 value of 26.44.5 nM. Takinginto account the Kd value

4、of the radioligand (1619 pM), the deduced Ki value for Pitolisant is 141 nM.Pitolisant displaces 125Iiodoproxyfan binding from membranes of rat glioma C6 cells stably expressing thehuman H3 receptor with an IC50 value of 4.20.2 nM. Taking into account the Kd value of the radioligand(504 pM), the ded

5、uced Ki value for Pitolisant is 2.70.5 nM. Pitolisant progressively reverses this responsewith a Hill coefficient close to unity and an IC50 value of 33068 nM, leading to a Ki value of 174 nM.Pitolisant elicits a dose-dependent decrease of the basal-specific 35SGTPS binding to membranes with amaxima

6、l effect corresponding to 751% of the basal-specific binding and an EC50 value of 1.50.1 nM 1.體內(nèi)研究The administration of Pitolisantat a single dose of 10 mg/kg 30 min before a single dose of Olanzapine (2mg/kg b.w.) also significantly affects immobility time in the FST. Subsequent administration of t

7、heaforementioned drug sequence in mice statistically significantly increases the duration of immobility incomparison to the time determined in the control group in the FST. It decreased locomotor activity as well. In1/3 Master of Small Molecules 您邊的抑制劑師www.MedChemEcontrast, the results obtained in s

8、ubchronic treatment after fifteen administrations of both drugs (Pitolisant 10mg/kg b.w., and after 30 min Olanzapine 2 mg/kg b.w., and again after 4 h Olanzapine 2 mg/kg b.w.) showthat the administration of Pitolisant followed by that of Olanzapine equalized the locomotor activity in mice; incompar

9、ison to the level of motility in the control group, to which only Pitolisant is administered. Moreimportantly, this combination of drugs significantly reduces immobility time to the level obtained in the controlgroup in the forced swim test in mice one-way ANOVA; F (3,20)=4.226,P=0.0181 2. Rats give

10、n Pitolisant(10 mg/kg) during the conditioning phase stayed 50294 s on the paired texture, a value not statisticallydifferent from that of controls, indicating that Pitolisant did not support place preference 3.PROTOCOLKinase Assay 1 35SGTPS binding assays are performed. CHO-K1 cells stably expressi

11、ng the human H3 receptor (400fmol/mg protein) are homogenized in ice-cold buffer (50 mM Tris/HCl, pH 7.4). Homogenates are centrifugedtwice (20,000g for 10 min at 4C), and the final pellet is resuspended in 50 volumes of buffer. Membranes(550 g of protein) are pretreated with adenosine deaminase (1

12、U/mL) and incubated for 60 min at 25C with0.1 nM 35SGTPS and the drugs to be tested in a final volume of 1 mL of assay buffer (50 mM Tris/HCl, 50mM NaCl, 5 mM MgCl2, 10 M GDP, and 0.02% bovine serum albumin, pH 7.4). The nonspecific binding isdetermined using 10 M nonradioactive GTPS. Incubations ar

13、e stopped by rapid filtration under vacuumthrough GF/B glass fiber filters. After washing with ice-cold water, the radioactivity trapped on filters iscounted by liquid scintillation spectrometry. A similar assay is used to assess competitive antagonism. Inbrief, membranes (10 g of protein) of HEK-29

14、3 cells stably expressing the human H3 receptor (600fmol/mg protein) are preincubated in presence of Pitolisant in the buffer (50 mM Tris/HCl, pH 7.4, 10 mMMgCl2, 100 mM NaCl, and 10 M GDP) in a 96-well microplate under gentle agitation at room temperature(19-20C) for 30 min before the addition of 0

15、.1 nM 35SGTPS (final volume 200 L). The nonspecificbinding is determined using a 10 M concentration of nonradioactive GTPS. After 30 min, incubationsperformed in triplicate are stopped by rapid filtration under vacuum on a Multiscreen MAFCOB50 microplate.Radioactivity trapped on filters is counted b

16、y liquid scintillation spectrometry 1.MCE has not independently confirmed the accuracy of these methods. They are for reference only.Animal Mice 2Administration 23 Adult female Albino Swiss mice weighing 20-22 g are used in the study. Olanzapine or Pitolisant aresuspended in 1 % Tween 80. The compou

17、nds or vehicle are administered intraperitoneally (i.p.) 30 min priorto the acute experiment. In the Pitolisant+Olanzapine group, Pitolisant is administered 15 min beforeOlanzapine. Subchronic treatment is done at about 9:00 am (0.2 mL Tween to control group, Pitolisant-10mg/kg b.w. to Pitolisant gr

18、oup, Olanzapine-2 mg/kg b.w. to Olanzapine group, Pitolisant-10 mg/kg b.w. andOlanzapine after 15 min-2 mg/kg b.w. to Pitolisant+Olanzapine group) and at about 1:00 pm (Olanzapinegroup and Pitolisant+Olanzapine group).Rats 3Male Wistar rats (220-300 g) receive vehicle (methylcellulose 1%, p.o.), Pit

19、olisant (10 mg/kg, p.o.) or D-amphetamine (2.5 mg/kg, i.p. in saline). Ninety minutes later, they are killed by decapitation and nucleusaccumbens are dissected out, weighed, frozen in liquid nitrogen and stored at -80C. Tissues arehomogenized in 1 mL of a 0.4 N perchloric acid/2.7 mM EDTA solution.

20、After centrifugation (8000 rpm, 20min, 4C), supernatants are analysed by HPLC coupled to electrochemical detection. Tissue concentrations2/3 Master of Small Molecules 您邊的抑制劑師www.MedChemEof dopamine (DA), dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA) are determined andthe corresponding ratios (DOPAC/DA, HVA/DA) are calculated.MCE has not independently confirmed the accuracy of these methods. They are for reference only.REFERENCES1. Ligneau X, et al. BF2.649 1-3-3-(4-Chlorophenyl)propoxypropylpiperidine, hydrochloride, a nonimidazole inverse agon