AT-56 is an orally active and selective inhibitor of lipocalin-type prostaglandin D synthase (L-PGDS) [1]. It inhibits human and mouse L-PGDSs in a concentration-dependent manner but does not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and microsomal PGE synthase-1 (m-PGES-1) [1].
AT-56 inhibits L-PGDS activity in a competitive manner against the substrate PGH₂ with a Kᵢ value of 75 μM. The Kₘ value of L-PGDS for PGH₂ is 14 μM [1].

In human medulloblastoma TE-671 cells expressing L-PGDS, AT-56 (1-30 μM; 10 min) dose-dependently suppresses PGD2 synthesis with an IC50 of roughly 3 μM [1].

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AT-56 inhibited the PGDS activity of both human CSF L-PGDS (β-trace) and recombinant mouse L-PGDS C89A/C186A mutant in a concentration (10-250 μM)-dependent manner. At 250 μM, L-PGDS activity was inhibited to 30% of control, with an IC₅₀ value of approximately 95 μM [1].
AT-56 up to 250 μM did not significantly affect the activities of COX-1, COX-2, m-PGES-1, or H-PGDS [1].
Kinetic analysis revealed that AT-56 inhibited recombinant L-PGDS in a competitive manner against PGH₂. The Vₘₐₓ remained unchanged while Kₘ increased with increasing AT-56 concentrations (0-120 μM) [1].
NMR titration analysis showed that AT-56 binds to the catalytic pocket of L-PGDS containing the catalytic center Cys⁶⁵, but does not bind to the retinoid-binding pocket. Large chemical shift changes (>0.08 ppm) were observed at residues Ser⁵², Thr⁸⁰, Met⁹⁴, and His¹¹⁶ upon AT-56 binding [1].
Fluorescence quenching studies demonstrated that AT-56 binds near Trp⁵⁴ in the AB-loop of L-PGDS but not to Trp⁴³ in the retinoid-binding pocket. Fluorescence intensity decreased to about 60% in the presence of 10 μM AT-56 [1].
AT-56 inhibited PGD₂ production by L-PGDS-expressing human TE-671 cells after stimulation with Ca²⁺ ionophore (5 μM A23187) with an IC₅₀ value of approximately 3 μM, without affecting their production of PGE₂ and PGF₂α. AT-56 had no effect on PGD₂ production by H-PGDS-expressing human megakaryoblastic MEG-01S cells [1].

AT-56 (1-30 mg/kg; oral) reduces PGD2 production in the stab-injured brain [1]. AT-56 (1-10 mg/kg; oral) suppresses L-PGDS-mediated allergic airway inflammation in mice [1]. AT-56 (10 mg/kg; oral) has a Cmax (2.15 μg/ml), half-life (1.71 hours), and excellent oral bioavailability (82%) [1].

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In H-PGDS knockout mice (which express only L-PGDS), oral administration of AT-56 (1-30 mg/kg) 1 hour before stab wound injury decreased PGD₂ production in the brain in a dose-dependent manner. At 30 mg/kg, PGD₂ levels were reduced to 40% of control, while PGE₂ and PGF₂α levels were not significantly affected [1].
In human L-PGDS transgenic mice with ovalbumin-induced lung inflammation, oral administration of AT-56 (1 and 10 mg/kg) 1 hour before and 24 hours after antigen exposure dose-dependently reduced total cells, eosinophils, and monocytes in bronchoalveolar lavage fluid. At 10 mg/kg, total cells were reduced to 23%, eosinophils to 6%, and monocytes to 41% of vehicle-treated control levels [1].

L-PGDS activity was measured with 10 μM [1-¹⁴C]PGH₂ as substrate in 100 mM Tris-HCl (pH 8.0) containing 1 mM GSH, 0.1 mg/ml IgG, and 10% DMSO. Reactions were incubated at 25°C for 30 seconds. Products were separated by thin layer chromatography and quantified using an imaging plate system [1].
COX-1 and COX-2 activities were measured with 50 μM [1-¹⁴C]arachidonic acid as substrate in 100 mM Tris-HCl (pH 8.0) containing 2 μM hematin, 5 mM L-tryptophan, 0.1 mg/ml IgG, and 10% DMSO [1].
For kinetic analysis, recombinant mouse L-PGDS C89A/C186A mutant was incubated with various concentrations of PGH₂ (3-20 μM) in 100 mM Tris-HCl (pH 8.0) and 1 mM dithiothreitol in the presence of 0, 40, 100, or 120 μM AT-56 in 10% DMSO. Lineweaver-Burk plots were prepared to determine kinetic constants [1].

TE-671 cells (L-PGDS-expressing human medulloblastoma) and MEG-01S cells (H-PGDS-expressing human megakaryoblastic) were cultured in DMEM with 10% FBS. MEG-01S cells were differentiated with TPA to express H-PGDS and COX-1. Cells were pretreated with AT-56 (0-100 μM) for 10-15 minutes, then stimulated with 5 μM A23187 at 37°C for 15 minutes. Culture media were collected and PGD₂, PGE₂, and PGF₂α were quantified by enzyme immunoassay [1].
For radiolabeling experiments, cells were prelabeled with [1-¹⁴C]arachidonic acid (3.7 kBq/well) for 12 hours before assay. After stimulation, radioactive metabolites were extracted, separated by TLC, and analyzed by autoradiography [1].

Animal/Disease Models: H-PGDS KO mice with stab brain injury (14-16 weeks, 25-30 g, C57BL/6 strain) [1]
Doses: 0, 1, 3, 10, 30 mg/kg administered Method: Po 1 hour before stabbing
Experimental Results:Inhibited L-PGDS response in the brain. Using 30 mg/kg AT-56 diminished the total amount of PGD2 in the brain to 40%.

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Animal/Disease Models: Human L-PGDS overexpressing TG mice (male, 14-16 weeks, 25-30 g) [1]
Doses: 0, 1, 10 mg/kg
Route of Administration: 1 hour before and 24 hrs (hrs (hours)) after antigen exposure Hourly oral
Experimental Results: Prevention of eosinophil infiltration by inhibition of transgenic human L-PGDS.

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Animal/Disease Models: Male C57BL/6 mice (7 weeks, 22-26 g) [1]
Doses: 10 mg/kg orally, 2 mg/kg intravenously (iv) (iv)(iv) (pharmacokinetic/PK/PK analysis) Dosing methods: oral and intravenous (iv) (iv)injection
Experimental Results: Oral bioavailability (82%); Cmax (2.15 μg/ml); T1/2 (1.71 hrs (hrs (hours)), oral); T1/2 (2.35 hrs (hrs (hours)), intravenous (iv) (iv)injection).

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For stab wound brain injury: H-PGDS KO mice (14-16 weeks old, C57BL/6 strain) received oral administration of AT-56 (1, 3, 10, and 30 mg/kg body weight) dissolved in 0.5% methylcellulose, 1 hour before injury. Under pentobarbital anesthesia (50 mg/kg), a 25-gauge needle was inserted into the frontal cortex (2 mm caudal to bregma, 2 mm lateral to sagittal suture, 1 mm deep). Brains were harvested 10 minutes after wounding, frozen in liquid nitrogen, and stored at -80°C until PG measurement [1].
For lung inflammation model: Human L-PGDS transgenic mice (FVB strain) were sensitized by intraperitoneal injection of 10 μg ovalbumin in 0.2 ml aluminum hydroxide gel on days 0 and 14. On day 21, mice were exposed to aerosolized ovalbumin (50 mg/ml in sterile saline) for 20 minutes. AT-56 (1 and 10 mg/kg) was orally administered 1 hour before and 24 hours after antigen exposure. Bronchoalveolar lavage fluid was collected 48 hours after challenge for cell counting [1].

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For stab wound brain injury: H-PGDS KO mice (14-16 weeks old, C57BL/6 strain) received oral administration of AT-56 (1, 3, 10, and 30 mg/kg body weight) dissolved in 0.5% methylcellulose, 1 hour before injury. Under pentobarbital anesthesia (50 mg/kg), a 25-gauge needle was inserted into the frontal cortex (2 mm caudal to bregma, 2 mm lateral to sagittal suture, 1 mm deep). Brains were harvested 10 minutes after wounding, frozen in liquid nitrogen, and stored at -80°C until PG measurement [1].
For lung inflammation model: Human L-PGDS transgenic mice (FVB strain) were sensitized by intraperitoneal injection of 10 μg ovalbumin in 0.2 ml aluminum hydroxide gel on days 0 and 14. On day 21, mice were exposed to aerosolized ovalbumin (50 mg/ml in sterile saline) for 20 minutes. AT-56 (1 and 10 mg/kg) was orally administered 1 hour before and 24 hours after antigen exposure. Bronchoalveolar lavage fluid was collected 48 hours after challenge for cell counting [1].

Male C57BL/6 mice (7 weeks old) received a single oral dose of 10 mg/kg AT-56 or a single intravenous dose of 2 mg/kg AT-56. Plasma concentrations were determined by HPLC-MS at various time points [1].
After oral administration, plasma AT-56 reached maximum concentration (Cₘₐₓ = 2.15 μg/ml) within 30 minutes (tₘₐₓ = 0.5 h) and decreased with time, falling below detection limit (0.4 ng/ml) at 12 hours. The half-life (t₁/₂) was 1.71 hours for oral administration and 2.35 hours for intravenous administration [1].
The area under the concentration-time curve (AUC) was 1.28 μg·h/ml for intravenous (2 mg/kg) and 8.95 μg·h/ml for oral (10 mg/kg) administration. Bioavailability (BA) was calculated to be 82%, indicating good oral absorption in mice [1].

No acute toxic effects of AT-56 were detected after oral administration even at a dose of 100 mg/kg [1].
In the CAM assay, AT-56 at doses up to 200 ng per egg did not cause necrosis or any visible adverse effects on chicken embryos [1].
The study notes that AT-56 selectively inhibits PGD₂ production without affecting the production of other prostanoids (PGE₂, PGF₂α), suggesting it may avoid side effects caused by suppression of cytoprotective and anti-inflammatory PGs [1].

[1]. Biochemical, functional, and pharmacological characterization of AT-56, an orally active and selective inhibitor of lipocalin-type prostaglandin D synthase. J Biol Chem. 2009 Mar 20; 284(12): 7623-30.

AT-56 (4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine) is a derivative of HQL-79 (an H-PGDS inhibitor) and was found to have inhibitory activity against L-PGDS [1].
PGD₂ is a lipid mediator involved in sleep regulation and inflammatory responses. It acts via DP₁ and DP₂ receptors. L-PGDS contributes to PGD₂ production in the central nervous system, ocular tissues, cardiovascular system, and male genital organs, and is involved in sleep regulation, sex determination, atherosclerosis protection, and adipogenesis [1].
L-PGDS is unique among lipocalin family members as the only enzyme. It also binds and transports various lipophilic substances including retinoic acid, retinal, biliverdin, bilirubin, gangliosides, and amyloid β peptides with high affinities (Kd = 20-200 nM), suggesting it may act as a transporter protein and endogenous chaperone [1].
AT-56 is the first reported competitive inhibitor of L-PGDS against PGH₂. Its selectivity and oral bioavailability make it a useful prototypic molecule for developing selective L-PGDS inhibitors that may act as anti-somnolence or anti-inflammatory drugs [1].

397.226

C, 75.54; H, 6.85; N, 17.62

162640-98-4

11741525

White to off-white solid powder

1.2±0.1 g/cm3

620.4±65.0 °C at 760 mmHg

329.0±34.3 °C

0.0±1.8 mmHg at 25°C

1.646

5.84

1

4

5

30

591

0

LQNGMDUIRLSESZ-UHFFFAOYSA-N

InChI=1S/C25H27N5/c1-3-9-22-19(7-1)12-13-20-8-2-4-10-23(20)25(22)21-14-17-30(18-15-21)16-6-5-11-24-26-28-29-27-24/h1-4,7-10,12-13H,5-6,11,14-18H2,(H,26,27,28,29)

1-[4-(2H-tetrazol-5-yl)butyl]-4-(2-tricyclo[9.4.0.03,8]pentadeca-1(15),3,5,7,9,11,13-heptaenylidene)piperidine

AT-56 AT56 AT 56

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~100 mg/mL (~251.56 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.29 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 25.0 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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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.29 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.29 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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