Timapiprant (formerly known as OC000459; OC-000459) is a novel, potent, selective, and orally bioactive prostanoid receptor 2 (DP2- also known as CRTH2)) antagonist with IC50 of 13 nM. It reduces nasal and ocular symptoms in allergic subjects exposed to grass pollen, a randomised, placebo-controlled, double-blind trial. OC000459 inhibits mast cell-dependent activation of T helper 2 lymphocytes and eosinophils. As an antagonist of DP2, OC000459 can prevent PGD2 from binding to human DP2 and rat recombinant DP2 with Ki values of 4nM and 3nM. OC000459 treatment inhibited LAR and post-allergen increase in sputum eosinophils. OC000459 appears to inhibit allergic inflammation in asthma.

Physicochemical Properties

Molecular Formula	C21H17FN2O2
Molecular Weight	348.37
Exact Mass	348.127
Elemental Analysis	C, 72.40; H, 4.92; F, 5.45; N, 8.04; O, 9.18
CAS #	851723-84-7
Related CAS #	Timapiprant sodium;950688-14-9
PubChem CID	11462174
Appearance	Light yellow to yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	574.4±50.0 °C at 760 mmHg
Flash Point	301.2±30.1 °C
Vapour Pressure	0.0±1.7 mmHg at 25°C
Index of Refraction	1.646
LogP	4.37
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	4
Heavy Atom Count	26
Complexity	516
Defined Atom Stereocenter Count	0
InChi Key	FATGTHLOZSXOBC-UHFFFAOYSA-N
InChi Code	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)
Chemical Name	2-(5-fluoro-2-methyl-3-(quinolin-2-ylmethyl)-1H-indol-1-yl)acetic acid
Synonyms	OC000459; 851723-84-7; timapiprant; 2-(5-fluoro-2-methyl-3-(quinolin-2-ylmethyl)-1H-indol-1-yl)acetic acid; OC-000459; OC-459; UNII-04XB9TB8OL; OC-000459; OC 000459
HS Tariff Code	2934.99.9001
Storage	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.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	D prostanoid receptor 2 (DP2 or CRTH2)
ln Vitro	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].
ln Vivo	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]. 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] 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].
Enzyme Assay	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...
Cell Assay	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.
Animal Protocol	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] 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] Dissolved in 10% DMSO/saline solution; 10 mg/kg; Oral administration Sprague-Dawley rats
ADME/Pharmacokinetics	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). 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.
References	[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.
Additional Infomation	OC000459 is under investigation for the treatment of Severe Eosinophilic Asthma. OC000459 has been investigated for the treatment of Bronchial Asthma. 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]

Solubility Data

Solubility (In Vitro)

DMSO: 4 mg/mL (11.5 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility (In Vivo)

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.8705 mL	14.3526 mL	28.7051 mL
	5 mM	0.5741 mL	2.8705 mL	5.7410 mL
	10 mM	0.2871 mL	1.4353 mL	2.8705 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.