EP/DP receptor
EP1 (Ki = 1217 ± 98 nM) [1]
EP2 (Ki = 1150 ± 36 nM) [1]
EP3‑III (Ki = 1597 ± 140 nM) [1]
DP (Ki = 1415 ± 104 nM) [1]
TP (Ki = 4325 ± 232 nM) [1]

AH 6809 (1 μM; 30 min) suppresses the production of cAMP and IL-1β in macrophages that is stimulated by T. serrulatus venom (TsV) and amplified by PGE₂[4]. AH 6809 (30-300 μM) inhibits PGD2's anti-aggregatory activity in whole blood with an apparent pA₂ of 5.35[5].

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AH6809 is described as a weak EP1 antagonist with pA2 values of 6.4‑7.0 reported in the literature. It is non‑selective, showing similar affinity for EP2, EP3 and DP receptors. It also has weak blocking activity at EP2. [1]

AH 6809 (5 mg/kg; i.p.) reduces TsV-induced mortality, PGE₂ and IL-1β production, and neutrophil infiltration in the lungs of mice[4].

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1. Eight types and subtypes of the mouse prostanoid receptor, the prostaglandin D (DP) receptor, the prostaglandin F (FP) receptor, the prostaglandin I (IP) receptor, the thromboxane A (TP) receptor and the EP1, EP2, EP3 and EP4 subtypes of the prostaglandin E receptor, were stably expressed in Chinese hamster ovary cells. Their ligand binding characteristics were examined with thirty two prostanoids and their analogues by determining the Ki values from the displacement curves of radioligand binding to the respective receptors. 2. The DP, IP and TP receptors showed high ligand binding specificity and only bound their own putative ligands with high affinity such as PGD2, BW245C and BW868C for DP, cicaprost, iloprost and isocabacyclin for IP, and S-145, I-BOP and GR 32191 for TP. 3. The FP receptor bound PGF2 alpha and fluprostenol with Ki values of 3-4 nM. In addition, PGD2, 17-phenyl-PGE2, STA2, I-BOP, PGE2 and M&B-28767 bound to this receptor with Ki values less than 100 nM. 4. The EP1 receptor bound 17-phenyl-PGE2, sulprostone and iloprost in addition to PGE2 and PGE1, with Ki values of 14-36 nM. 16,16-dimethyl-PGE2 and two putative EP1 antagonists, AH6809 and SC-19220, did not show any significant binding to this receptor. M&B-28767, a putative EP3 agonist, and misoprostol, a putative EP2/EP3 agonist, also bound to this receptor with Ki values of 120 nM. 5. The EP2 and EP4 receptors showed similar binding profiles. They bound 16,16-dimethyl PGE2 and 11-deoxy-PGE1 in addition to PGE2 and PGE1. The two receptors were discriminated by butaprost, AH-13205 and AH-6809 that bound to the EP2 receptor but not to the EP4 receptor, and by 1-OH-PGE1 that bound to the EP4 but not to the EP2 receptor. 6. The EP3 receptor showed the broadest binding profile, and bound sulprostone, M&B-28767, GR63799X, 11-deoxy-PGE1, 16,16-dimethyl-PGE2 and 17-phenyl-PGE2, in addition to PGE2 and PGE1, with Ki values of 0.6-3.7 nM. In addition, three IP ligands, iloprost, carbacyclin and isocarbacyclin, and one TP ligand, STA2, bound to this receptor with Ki values comparable to the Ki values of these compounds for the IP and TP receptors, respectively. 7. 8-Epi-PGF2 alpha showed only weak binding to the IP, TP, FP, EP2 and EP3 receptor at 10 microM concentration[2].

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Radioligand binding assays were performed to determine the inhibition constants (Ki) of AH6809 at eight human prostanoid receptors stably expressed in HEK 293(EBNA) cells. Membrane preparations from cells expressing each receptor were incubated in a final volume of 0.2 mL containing 10 mM MES/KOH (pH 6.0) for EP subtypes, FP and TP, or 10 mM HEPES/KOH (pH 7.4) for DP and IP, with 1 mM EDTA, 10 mM MgCl2 (EP subtypes) or 10 mM MnCl2 (DP, FP, IP, TP), and the respective radioligand: [3H]PGE2 (0.5‑1.0 nM) for EP subtypes, [3H]PGD2 (0.7 nM) for DP, [3H]PGF2α (0.95 nM) for FP, [3H]iloprost (5 nM) for IP, and [3H]SQ‑29548 (1.8 nM) for TP. EP3 assays also contained 100 μM GTPγS. The reaction was initiated by adding membrane protein (e.g., ~30 μg for EP1, ~20 μg for EP2, ~2 μg for EP3, ~10 μg for EP4, ~60 μg for FP, ~30 μg for DP, ~10 μg for IP, ~10 μg for TP). AH6809 was added in dimethylsulfoxide (final concentration 1% v/v). Non‑specific binding was determined with 1 μM of the corresponding non‑radioactive prostanoid. Incubations were conducted for 60 min (EP subtypes, FP, IP) or 30 min (DP, TP) at 30 °C (EP subtypes, DP, FP, TP) or room temperature (IP). Incubations were terminated by rapid filtration through 96‑well Unifilter GF/C filters prewetted in assay buffer without EDTA at 4 °C. Filters were washed with 3‑4 mL of the same buffer, dried for 90 min at 55 °C, and residual radioactivity was determined by scintillation counting. Specific binding represented 85‑95% of total binding. Ki values were calculated from competition curves. [1]

In vitro pharmacological treatments
J774.1 macrophages were plated at the density of 2 × 105 cells per well in 200 μl of serum-free RPMI supplemented with antibiotics. The cells were then cultured at 37 °C in 5% CO2 for 2 h. Next, the supernatants were removed, and the cells were treated or not with specific inhibitors/antagonists for 30 min: indomethacin (10 μM); AH6809 (1 μM); AH23848 (1 μM); U-75302 (0.1 and 1 μM); and NFκB Activation Inhibitor (20 nM). H89 dihydrochloride hydrate (25 μM) was added for 2 h in the cell culture medium before stimulation. AH6809 and U-75302 from ethanol stock solutions were diluted in cell culture medium and the same concentration of ethanol (maximum 0.1%) was added to the medium only (control). The AH23848 and NFκB inhibitor from DMSO stock solutions were diluted in the cell culture medium and the same concentration of DMSO (maximum 0.1%) was added to the medium only (control). All compounds were diluted in 200 μl of serum-free DMEM, and the same solution with solvent diluents was used as control. After treatment, the cells were stimulated with TsV (50 μg ml−1) under the same experimental conditions and after 24 h at 37 °C in a humidified atmosphere 5% of CO2, the supernatants were collected for IL-1β quantification.[4]

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1. The effect of AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) has been studied upon the anti-aggregatory and aggregatory actions of various agents on human platelets in whole blood. 2. Prostaglandin D2 (PGD2), BW245C, 9 alpha, 11 beta-PGF2, PGI2 and 5'-N-ethylcarboxamide adenosine (NECA) all inhibited ADP-induced platelet aggregation in whole blood. The anti-aggregatory activity of PGD2, BW245C and 9 alpha, 11 beta-PGF2 but not PGI2 or NECA was antagonized by AH6809. NECA was antagonized by AH6809. 3. The antagonism of the anti-aggregatory activity of PGD2 by AH6809 was concentration-related and could be overcome by increasing the concentration of PGD2. Analysis of the data yielded an apparent pA2 for AH6809 of 5.35. 4. At approximately 10 fold higher concentrations than those required to antagonize the action of PGD2, AH6809 also antagonized the aggregatory effect of U-46619 in whole blood (pA2 = 4.45). However, concentrations of AH6809 up to 300 microM were without effect upon either ADP- or platelet activating factor (Paf)-induced aggregation (pA2 less than 3.5). 5. The potency of AH6809 against PGD2 and U-46619 was increased in a resuspended platelet preparation suggesting that the drug is extensively bound to plasma proteins. However, in resuspended platelets the specificity of AH6809 relative to that seen in whole blood was reduced since aggregation by ADP and Paf was also slightly antagonized. 6. In conclusion, AH6809 appears to be a weak but specific DP-receptor blocking drug on human platelets and should prove to be a useful drug tool for defining the involvement of endogenous PGD2 in platelet aggregation and classifying the mode of action of anti-aggregatory prostanoids[5].

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Human blood was collected by venepuncture and anti-coagulated with trisodium citrate. Aliquots (0.5 ml) of whole blood were placed in plastic tubes, flushed with 5% CO2 in air, capped, and incubated in a shaking water bath at 38 ± 0.5°C for 30 min. Aspirin (2 mM final concentration) was added to prevent endogenous prostaglandin production. AH6809 was preincubated for 10 min before addition of an aggregating agent. Platelet aggregation was quantified by counting the fall in single platelet count. Aggregation was induced with ADP, U-46619, or Paf. Peak aggregation was seen at 1 min with ADP and Paf, and at 5 min with U-46619. The maximum fall in platelet count was expressed as a percentage of the control count. EC50 values were measured as the concentration at which a 50% fall in platelet count was obtained. [5]
Human resuspended platelets were prepared by centrifugation of citrated whole blood containing creatine phosphate and creatine phosphokinase. Platelet-rich plasma was centrifuged in the presence of prostacyclin. The platelet pellet was resuspended in Krebs-Henseleit solution containing CP, CPK, and heparin to a final platelet count of 300-400 × 10^6 ml^-1. Aliquots (0.25 ml) were placed in tubes containing aspirin (20 μM), flushed with 5% CO2 in air, and stored at approximately 10°C. Human fibrinogen (100 μg ml^-1) was added before aggregation was initiated. AH6809 was preincubated for 10 min before addition of an aggregating agent. [5]

IL-1r−/−, Casp1/11−/− and C57Bl/6 (WT) mice without treatment were inoculated with a sublethal or lethal dose of TsV (or PBS) as described above. Alox5−/− mice and 129sv mice were pre-treated or not with IL-1 receptor antagonist (IL-1Ra) at 10 mg kg−1, i.p., 1 h before and again 1 h after the sublethal or lethal TsV injection. IL-1Ra was kindly provided by Dr Stephen Poole, from the National Institute for Biological Standards and Control. In a specific experiment, the mice were either treated or not treated with MK886 (5-LO inhibitor, 5 mg kg−1 i.p., in 200 μl of 1% alcohol in water), indomethacin (COX1/2 inhibitor, 2 mg kg−1 i.p. in 200 μl of Tris[hydroxymethyl]aminomethane-HCl; TRIS-HCl, pH 8.2), SC-560 (selective COX1 inhibitor, 3 mg kg−1 i.p., in 200 μl of PBS), celecoxib (COX2 inhibitor, 5 mg kg−1 i.p., in 200 μl of water) or EP2 antagonist (AH6809, 5 mg kg−1 i.p., in 200 μl of PBS)67. The drugs (MK886 or indomethacin) or vehicles were administered four times, at 4 h and 0.5 h before and again 4 and 8 h after the lethal dose of TsV. The others drugs (SC-560, celecoxib, and EP2 antagonist) or vehicles were administered 1 day and again 1 h before the i.p. injection of lethal dose of TsV (180 μg kg−1). In other experiments, the Alox5−/− mice were treated or not with LTB4 (50 ng per mice, intranasal (i.n.) administration, in 20 μl of PBS). The LTB4 or vehicle (PBS+0.05% of ethanol) were administered 2 h and 0.5 h before the dose lethal of TsV (180 μg kg−1). The lungs were excised immediately after death or from mice survivors that were killed 8–12 h after the injection of TsV or vehicle. In some sets of experiments, two groups of mice were inoculated with PBS or a sublethal (120 μg kg−1) dose of TsV and, in only one, BAL fluids were collected 4 h later, to count the total cell number and neutrophils, as described previously47. In the other group, without BAL, the lungs were excised and weighed and 2 mg of tissue was homogenized in 2 ml of incomplete RPMI. After centrifugation (400g for 10 min), the supernatants were transferred to new tubes, split into two samples of 1 ml and stored at −80 °C until use. One sample was used for IL-1β and protein quantification analysis and the other for PGE2 and LTB4 measurement. For analysis of MPO activity, one lobule of a lung was cut out, immediately frozen in liquid nitrogen, and stored at −80 °C until use. In the therapeutic protocol, the lethal dose (180 μg kg−1) and superdose (360 μg kg−1) of TsV were injected and indomethacin (2 mg kg−1 i.p.) or vehicle were administered either 15 or 30 min after and again 4 and 8 h later. Mice survivors were killed 12 h after the envenomation.[4]

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C57BL/6 mice (6–8 weeks old, both sexes, matched by sex and age) were inoculated intraperitoneally with a lethal dose of Tityus serrulatus venom (TsV) at 180 µg/kg in 200 µl PBS. AH6809 was administered intraperitoneally at 5 mg/kg in 200 µl PBS, given 24 hours and again 1 hour before TsV injection. Control mice received vehicle (PBS) only. Survival was monitored for 8–12 hours after envenomation. Lungs were excised immediately after death or from surviving mice killed at 8–12 hours, then protein content, PGE2 release, IL-1β production, LTB4 release, and MPO activity were quantified in lung parenchyma.[4]

The potency of AH6809 against PGD2 and U-46619 was increased in a resuspended platelet preparation compared to whole blood, suggesting that the drug is extensively bound to plasma proteins. [5]

In resuspended platelets, the specificity of AH6809 relative to that seen in whole blood was reduced, since aggregation by ADP and Paf was also slightly antagonized. [5]

[1]. The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs. Biochim Biophys Acta. 2000 Jan 17;1483(2):285-93.

[2]. Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptors expressed in Chinese hamster ovary cells. Br J Pharmacol. 1997 Sep;122(2):217-24.

[3]. 6-Isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809), a human EP2 receptor antagonist. Biochem Pharmacol. 1995 Nov 9;50(10):1731-3.

[4]. Opposing roles of LTB4 and PGE2 in regulating the inflammasome-dependent scorpion venom-induced mortality. Nat Commun. 2016 Feb 23;7:10760.

[5]. AH6809, a prostaglandin DP-receptor blocking drug on human platelets. Br J Pharmacol. 1988 Jul;94(3):745-54.

9-O-6-prop-2-oxy-2-xanthonic acid is a member of the xanthonone class of compounds.

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AH6809 has been used as an experimental tool to study prostanoid receptor pharmacology. It is among the few compounds reported to have weak blocking activity at EP2, although no selective EP2 antagonists are available. The compound is a xanthone derivative. 1]

C17H14O5

298.29006

298.084

C, 68.45; H, 4.73; O, 26.82

33458-93-4

119461

White to off-white solid powder

1.3±0.1 g/cm3

514.2±50.0 °C at 760 mmHg

192.9±23.6 °C

0.0±1.4 mmHg at 25°C

1.618

3.91

1

5

3

22

446

0

O=C1C2=C(C=CC(C(O)=O)=C2)OC3=CC(OC(C)C)=CC=C31

AQFFXPQJLZFABJ-UHFFFAOYSA-N

InChI=1S/C17H14O5/c1-9(2)21-11-4-5-12-15(8-11)22-14-6-3-10(17(19)20)7-13(14)16(12)18/h3-9H,1-2H3,(H,19,20)

9-oxo-6-propan-2-yloxyxanthene-2-carboxylic acid

AH-6809; AH6809; AH 6809; 33458-93-4; AH 6809; 6-Isopropoxy-9-oxoxanthene-2-carboxylic acid; AH-6809; AH6809; 6-isopropoxy-9-oxo-9h-xanthene-2-carboxylic acid; 9-oxo-6-propan-2-yloxyxanthene-2-carboxylic acid; 6-(1-Methylethoxy)-9-oxo-9H-xanthene-2-carboxylic acid;

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: ~25 mg/mL (~83.8 mM)
H2O: ~0.1 mg/mL (~0.3 mM)

Solubility in Formulation 1: 2.5 mg/mL (8.38 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (8.38 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.)