D prostanoid receptor 2 (DP2 or CRTH2)

Timapiprant (OC000459) (0.0001 μM-10 μM; 5 hours) suppresses human Th2 cells' chemotaxis (IC50=0.028 μM) and their production of cytokines (IC50=0.019 μM)[1]. Timapiprant (OC000459) (1 μM) prevents Th2 cells and eosinophils from becoming activated in reaction to IgE/anti-IgE-stimulated human mast cell supernatants[1]. With an IC50 of 0.035 uM, timapiprant (OC000459) (1 nM–1000 nM; 16 hours) suppresses PGD2's anti-apoptotic action on Th2 cells[1].

In rats (ED50=0.04 mg/kg), timapiprant (OC000459) (gavage; 2 mg/kg, 10 mg/kg) reduces blood eosinophilia caused by 13,14-dihydro-15-keto-PGD2 (DK-PGD2)[1]. In reaction to an aerosol of DK-PGD2, timapiprant (OC000459) (gavage; 0.01 mg/kg, 0.1 mg/kg, 1.0 mg/kg) reduces airway eosinophilia in guinea pigs (ED50=0.01 mg/kg)[1].

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CRTH2 (chemoattractant receptor expressed on T-helper (Th) type 2 cells) is a G-protein-coupled receptor expressed by Th2 lymphocytes and eosinophils that mediates prostaglandin (PG)D(2)-driven chemotaxis. We studied the efficacy of the oral CRTH2 antagonist OC000459 in steroid-naïve asthmatic patients. A randomised, double-blind, placebo-controlled, two-way crossover study of 16 days' treatment with OC000459 (200 mg twice daily) on the late (LAR) and early (EAR) asthmatic responses to bronchial allergen challenge was conducted, with 16 subjects completing the study. There was a 25.4% (95% CI 5.1-45.6%) reduction in the LAR area under the curve (AUC) for change in forced expiratory volume in 1 s with OC000459 compared with placebo (p=0.018) but no effect on the EAR. Sputum eosinophil counts at 1 day post-allergen challenge were lower after OC000459 treatment (p=0.002). PGD(2)-induced blood eosinophil shape change ex vivo was assessed at day 7 (n=7). The AUC of eosinophil shift for OC000459 was lower than placebo; the mean difference was -33.6% (95% CI -66.8- -0.4%; p=0.048). OC000459 treatment inhibited LAR and post-allergen increase in sputum eosinophils. This CRTH2 antagonist appears to inhibit allergic inflammation in asthma. [2]

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During the first treatment period, treatment with OC000459 significantly reduced both nasal and ocular symptoms in allergic subjects compared with placebo after challenge with grass pollen. A significant effect was observed on the 2nd day of dosing which was increased on the 8th day of dosing. The therapeutic effects of OC000459 persisted into the second treatment period despite a 3-week washout phase. The safety profile of OC000459 was similar to that of placebo. Conclusion: Treatment with OC000459 was well tolerated and led to a significant and persistent reduction in the symptoms of rhinoconjunctivitis [3].

Effect of OC000459 on Binding of [3H]PGD2 to Human DP2.[1]
OC000459 inhibited the binding of [3H]PGD2 to membranes from CHO cells transfected with human DP2 with Ki of 0.013 ± 0.002 μM (n = 13 independent experiments) as shown in Fig. 2A. OC000459 also displaced [3H]PGD2 from membranes from human Th2 lymphocytes (Ki = 0.004 ± 0.001 μM; n = 3 independent experiments), indicating that the compound was active on the native receptor as shown in Fig. 2B. OC000459 was active on rat recombinant DP2 (0.003 ± 0.001 μM; n = 5 independent experiments) but did not...

Apoptosis Analysis[1]
Cell Types: Th2 Cells
Tested Concentrations: 0.0001 μM-10 μM
Incubation Duration: 16 hrs (hours)
Experimental Results: Inhibited the antiapoptotic effect of PGD2.

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 concentrations ± standard deviations of OC000459 in plasma before administration on day 8 (n=16) and day 15 (n=16) were 442 ± 236 ng·mL−1 and 560 ± 421 ng·mL−1, respectively. These OC000459 plasma concentrations did not differ with the order of administration (data not shown).

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Pharmacokinetic Analysis [2]
Blood samples were collected into 2 mL plastic (vacutainer) tubes containing heparin lithium pre-cooled to 4°C. Blood samples were placed on ice water and centrifuged at approximately 2,500 × g for 10 minutes at 4°C within 15 minutes of collection. The resulting plasma was transferred to polypropylene vials and immediately frozen below -20°C until delivery to BioDynamics Research Ltd. The concentrations of OC000459 in plasma were determined using a validated liquid chromatography-tandem mass spectrometry method. In summary, the analytical method involves liquid-liquid extraction followed by analysis of the extract using a liquid chromatography-tandem mass spectrometer equipped with a TurboIonspray interface. The mass spectrometer operates in multiple reaction monitoring (MRM) mode with positive ion detection. A stable isotope internal standard is used. The limit of quantification (LOQ) for OC000459 in plasma is 1 ng/mL⁻¹. Results below this value are reported as below the LQ. For samples with results above the calibration range (upper limit 400 ng/mL⁻¹), the sample is diluted 10-fold with plasma before reanalysis.

[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 being investigated for the treatment of severe eosinophilic asthma. OC000459 has also been investigated for the treatment of bronchial asthma.

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Prostaglandin receptor 2 (DP₂) [also known as the chemokine receptor homolog (CRTH2) expressed on T helper cell 2 (Th2) cells] is selectively expressed by Th2 lymphocytes, eosinophils, and basophils, and mediates the recruitment and activation of these cell types in response to prostaglandin D₂ (PGD₂) stimulation. (5-Fluoro-2-methyl-3-quinoline-2-ylmethylindole-1-yl)acetic acid (OC000459) is an indoleacetic acid derivative that effectively replaces [³H]PGD₂ in recombinant human DP₂ (K(i) = 0.013 μM), recombinant rat DP₂ (K(i) = 0.003 μM), and native human DP₂ (Th2 cell membrane; K(i) = 0.004 μM), without interfering with the ligand-binding properties or functional activity of other prostaglandin receptors (prostaglandin E₁₋₄ receptor, D prostaglandin receptor 1, thromboxane receptor, prostacyclin receptor, and prostaglandin F receptor). OC000459 inhibits the chemotaxis (IC₅₀ = 0.028 μM) and cytokine production (IC₅₀ = 0.019 μM) of human Th2 lymphocytes. OC000459 competitively antagonizes PGD₂-induced morphological changes in eosinophils in isolated human leukocytes (pK(B) = 7.9) and human whole blood (pK(B) = 7.5), but does not inhibit the response of eosinophil chemokines, 5-oxoeicosatetraenoic acid, or complement component C5a. OC000459 also inhibits the activation of Th2 cells and eosinophils by IgE/anti-IgE activated human mast cell supernatant. OC000459 has no significant inhibitory activity against 69 receptors, including cyclooxygenase 1 (COX1) and COX2, and 19 enzymes. Studies have shown that OC000459 has oral bioavailability in rats and can effectively inhibit eosinophilia induced by 13,14-dihydro-15-one-PGD₂ (DK-PGD₂) in rats (ED₅₀ = 0.04 mg/kg orally) and respiratory eosinophilia induced by inhalation of DK-PGD₂ aerosol in guinea pigs (ED₅₀ = 0.01 mg/kg orally). These data indicate that OC000459 is a highly effective, selective and orally available DP₂ antagonist that remains active in human whole blood and can inhibit mast cell-dependent activation of 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

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

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 0.4 mg/mL (1.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% 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 4.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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