D prostanoid receptor 2 (DP2 or CRTH2)

Timipiprant (OC000459)（0.0001 μM-10 μM；5 小时）抑制人 Th2 细胞的趋化性 (IC50=0.028 μM) 及其细胞因子的产生 (IC50=0.019 μM)[1]。 Timipiprant (OC000459) (1 μM) 可防止 Th2 细胞和嗜酸性粒细胞在 IgE/抗 IgE 刺激的人肥大细胞上清液反应中被激活[1]。 timapiprant (OC000459)（1 nM–1000 nM；16 小时）的 IC50 为 0.035 uM，可抑制 PGD2 对 Th2 细胞的抗凋亡作用[1]。

在大鼠 (ED50=0.04 mg/kg) 中，timapiprant (OC000459)（灌胃；2 mg/kg、10 mg/kg）可减少由 13,14-二氢-15-酮-PGD2 (DK-PGD2) 引起的血液嗜酸性粒细胞增多[ 1]。在对 DK-PGD2 气雾剂的反应中，timapiprant (OC000459)（灌胃；0.01 mg/kg、0.1 mg/kg、1.0 mg/kg）可减少豚鼠气道嗜酸性粒细胞增多 (ED50=0.01 mg/kg)[1]。

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CRTH2（在T辅助（Th）2型细胞上表达的化学引诱剂受体）是一种由Th2淋巴细胞和嗜酸性粒细胞表达的G蛋白偶联受体，介导前列腺素（PG）D（2）驱动的趋化性。我们研究了口服CRTH2拮抗剂OC000459对类固醇幼稚哮喘患者的疗效。进行了一项随机、双盲、安慰剂对照、双向交叉研究，对OC000459（200mg，每日两次）治疗支气管过敏原激发的晚期（LAR）和早期（EAR）哮喘反应进行了16天的治疗，共有16名受试者完成了这项研究。与安慰剂相比，OC000459在1秒内用力呼气量变化的LAR曲线下面积（AUC）减少了25.4%（95%CI 5.1-45.6%）（p=0.018），但对EAR没有影响。OC000459治疗后1天的痰液嗜酸性粒细胞计数较低（p=0.002）。在第7天评估PGD（2）诱导的离体血液嗜酸性粒细胞形态变化（n=7）。OC000459的嗜酸性粒细胞偏移AUC低于安慰剂；平均差异为-33.6%（95%CI-66.8--0.4%；p=0.048）。OC000459治疗抑制了LAR和过敏原后痰液嗜酸性粒细胞的增加。这种CRTH2拮抗剂似乎可以抑制哮喘中的过敏性炎症。[2]

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在第一个治疗期，与安慰剂相比，在草花粉激发后，OC000459治疗显著减轻了过敏受试者的鼻部和眼部症状。在给药第2天观察到显著效果，在给药的第8天增加。尽管经过了3周的洗脱期，OC000459的治疗效果仍持续到第二个治疗期。OC000459的安全性与安慰剂相似。 结论：OC000459治疗耐受性良好，并导致鼻结膜炎症状显著和持续减轻[3]。

OC000459对[3H]PGD2与人DP2结合的影响。[1]
OC000459抑制了[3H]PGD2与转染有人DP2的CHO细胞膜的结合，Ki为0.013±0.002μM（n=13个独立实验），如图2A所示。OC000459还从人Th2淋巴细胞的膜上置换了[3H]PGD2（Ki=0.004±0.001μM；n=3个独立实验），表明该化合物对天然受体具有活性，如图2B所示。 OC000459对大鼠重组DP2有活性（0.003±0.001μM；n=5个独立实验），但没有。。。

细胞凋亡分析[1]
细胞类型： Th2 细胞
测试浓度： 0.0001 μM-10 μM
孵育时间： > 16小时
实验结果：抑制PGD2的抗凋亡作用。

This was a two-centre, double-blind, randomised, placebo-controlled, crossover study. Eligible subjects were randomised to receive OC000459 200 mg orally twice daily or matching placebo for 16 days, in addition to inhaled salbutamol as required. The washout period was 3 weeks between treatment periods. The measurement of vital signs (heart rate and blood pressure) and FEV1 during the treatment periods are shown in table 1; these measurements were performed pre-dose on days 1, 8, 15 and 16, while exhaled nitric oxide fraction (FeNO) was performed pre-dose on days 1, 8 and 15. Pre-dose blood sampling was performed for the measurements of biochemistry and haematology at days 1, 8 and 15, with pharmacokinetics also measured on days 8 and 15. Seven subjects provided blood samples for the measurement of eosinophil shape change up to 8 h post-dose on day 8. An inhaled allergen challenge was performed on day 15 at 3 h post-dose. On day 16, a methacholine challenge was performed at 3 h post-dose, followed by induced sputum; methacholine challenge and induced sputum were therefore performed 24 h post-allergen challenge. [2]

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Thirty-six healthy male subjects with a history of grass pollen allergic rhinoconjunctivitis were recruited for the study. All subjects were tested for skin prick positivity to grass pollen and levels of grass pollen–reactive IgE were measured by radioallergosorbent test. After giving informed consent, subjects were randomised to receive OC000459 then placebo or placebo then OC000459. Thirty-five subjects completed both treatment periods.

The study design is shown in Fig. 1. The study was a single centre, randomised, double-blind, placebo-controlled, two-way crossover trial conducted out of grass pollen season. Patients underwent screening at least 14 days prior to the first dosing to ensure they were eligible to take part in the study. There were two treatment periods, each 8 days in duration. Group A received OC000459 (200-mg bid, given as two 100-mg capsules) followed by matching placebo and Group B received placebo first followed by OC000459. Each treatment period was separated by a washout period of at least 7 days to ensure drug was no longer present at the start of the second period. In practice, the washout period was 19–23 days (20.9 ± 0.7 days, mean ± SD).[3]

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Dissolved in 10% DMSO/saline solution; 10 mg/kg; Oral administration

Sprague-Dawley rats

Pharmacokinetics [2]
The mean±sd plasma OC000459 concentrations were 442±236 ng·mL−1 at pre-dose on day 8 (n=16) and 560±421 ng·mL−1 at pre-dose on day 15 (n=16). There were no differences in these OC000459 plasma concentrations due to sequence (data not shown).

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Pharmacokinetic analysis [2]
Blood samples were taken into 2-mL plastic (Vacutainer) tubes containing lithium heparin pre-cooled to 4°C. Blood samples were kept on water ice and were centrifuged at about 2,500×g for 10 min at 4°C, within 15 min of collection. The resulting plasma was transferred to polypropylene vials and immediately frozen at < -20°C until transferred to BioDynamics Research Ltd. The concentration of OC000459 in plasma was determined using a validated liquid chromatography–tandem mass spectrometry bioanalytical method. In brief, the analytical method involved liquid–liquid extraction followed by analysis of the extracts using a liquid chromatograph–tandem mass spectrometer equipped with a TurboIonspray interface. The mass spectrometer was operated using multiple reaction monitoring in the positive ion detection mode. A stable isotope internal standard was used. The lower limit of quantification for OC000459 in plasma was 1 ng·mL−1. Values below this level were reported as below lower limit of quantification. Samples that gave results above the calibration range (upper limit 400 ng·mL−1) were diluted 10-fold with plasma prior to re-analysis.

[1]. Pharmacologic profile of OC000459, a potent, selective, and orally active D prostanoid receptor 2 antagonist that inhibits mast cell-dependent activation of T helper 2 lymphocytes and eosinophils. J Pharmacol Exp Ther. 2012.

[2]. Inhibition of the asthmatic allergen challenge response by the CRTH2 antagonist OC000459. Eur Respir J. 2013 Jan;41(1):46-52.

[3]. The CRTH2 antagonist OC000459 reduces nasal and ocular symptoms in allergic subjects exposed to grass pollen, a randomised, placebo-controlled, double-blind trial. Allergy. 2012 Dec;67(12):1572-9.

OC000459 is under investigation for the treatment of Severe Eosinophilic Asthma. OC000459 has been investigated for the treatment of Bronchial Asthma.

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D prostanoid receptor 2 (DP₂) [also known as chemoattractant receptor-homologous molecule expressed on T helper 2 (Th2) cells (CRTH2)] is selectively expressed by Th2 lymphocytes, eosinophils, and basophils and mediates recruitment and activation of these cell types in response to prostaglandin D₂ (PGD₂). (5-Fluoro-2-methyl-3-quinolin-2-ylmethylindo-1-yl)-acetic acid (OC000459) is an indole-acetic acid derivative that potently displaces [³H]PGD₂ from human recombinant DP₂ (K(i) = 0.013 μM), rat recombinant DP₂ (K(i) = 0.003 μM), and human native DP₂ (Th2 cell membranes; K(i) = 0.004 μM) but does not interfere with the ligand binding properties or functional activities of other prostanoid receptors (prostaglandin E₁₋₄ receptors, D prostanoid receptor 1, thromboxane receptor, prostacyclin receptor, and prostaglandin F receptor). OC000459 inhibited chemotaxis (IC₅₀ = 0.028 μM) of human Th2 lymphocytes and cytokine production (IC₅₀ = 0.019 μM) by human Th2 lymphocytes. OC000459 competitively antagonized eosinophil shape change responses induced by PGD₂ in both isolated human leukocytes (pK(B) = 7.9) and human whole blood (pK(B) = 7.5) but did not inhibit responses to eotaxin, 5-oxo-eicosatetraenoic acid, or complement component C5a. OC000459 also inhibited the activation of Th2 cells and eosinophils in response to supernatants from IgE/anti-IgE-activated human mast cells. OC000459 had no significant inhibitory activity on a battery of 69 receptors and 19 enzymes including cyclooxygenase 1 (COX1) and COX2. OC000459 was found to be orally bioavailable in rats and effective in inhibiting blood eosinophilia induced by 13,14-dihydro-15-keto-PGD₂ (DK-PGD₂) in this species (ED₅₀ = 0.04 mg/kg p.o.) and airway eosinophilia in response to an aerosol of DK-PGD₂ in guinea pigs (ED₅₀ = 0.01 mg/kg p.o.). These data indicate that OC000459 is a potent, selective, and orally active DP₂ antagonist that retains activity in human whole blood and inhibits mast cell-dependent activation of both human Th2 lymphocytes and eosinophils.[1]

C21H17FN2O2

348.37

348.127

C, 72.40; H, 4.92; F, 5.45; N, 8.04; O, 9.18

851723-84-7

Timapiprant sodium;950688-14-9

11462174

Light yellow to yellow solid powder

1.3±0.1 g/cm3

574.4±50.0 °C at 760 mmHg

301.2±30.1 °C

0.0±1.7 mmHg at 25°C

1.646

4.37

55.12

1

4

4

26

516

0

FATGTHLOZSXOBC-UHFFFAOYSA-N

InChI=1S/C21H17FN2O2/c1-13-17(11-16-8-6-14-4-2-3-5-19(14)23-16)18-10-15(22)7-9-20(18)24(13)12-21(25)26/h2-10H,11-12H2,1H3,(H,25,26)

2-(5-fluoro-2-methyl-3-(quinolin-2-ylmethyl)-1H-indol-1-yl)acetic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

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

配方 1 中的溶解度: ≥ 0.4 mg/mL (1.15 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 4.0 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80 +，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。