PGD2, produced by mast cells, has been detected in high concentrations at sites of allergic inflammation. It can stimulate vascular and other inflammatory responses by interaction with D prostanoid receptor (DP) and chemoattractant receptor-like molecule expressed on Th2 cells (CRTH2) receptors. A significant role for PGD2 in mediating allergic responses has been suggested based on the observation that enhanced eosinophilic lung inflammation and cytokine production is apparent in the allergen-challenged airways of transgenic mice overexpressing human PGD2 synthase, and PGD2 can enhance Th2 cytokine production in vitro from CD3/CD28-costimulated Th2 cells. In the present study, we investigated whether PGD2 has the ability to stimulate Th2 cytokine production in the absence of costimulation. At concentrations found at sites of allergic inflammation, PGD2 preferentially elicited the production of IL-4, IL-5, and IL-13 by human Th2 cells in a dose-dependent manner without affecting the level of the anti-inflammatory cytokine IL-10. Gene transcription peaked within 2 h, and protein release peaked ∼8 h after stimulation. The effect of PGD2 was mimicked by the selective CRTH2 agonist 13,14-dihydro-15-keto-PGD2 but not by the selective DP agonist BW245C, suggesting that the stimulation is mediated by CRTH2 and not DP. Ramatroban, a dual CRTH2/thromboxane-like prostanoid receptor antagonist, markedly inhibited Th2 cytokine production induced by PGD2, while the selective thromboxane-like prostanoid receptor antagonist SQ29548 was without effect. These data suggest that PGD2 preferentially up-regulates proinflammatory cytokine production in human Th2 cells through a CRTH2-dependent mechanism in the absence of any other costimulation and highlight the potential utility of CRTH2 antagonists in the treatment of allergic diseases.

Several agonists were tested for their ability to stimulate [ 35 S]-GTPgS binding to membranes coexpressing the receptor and various G proteins. All the compounds tested showed agonist activity in preparations expressing G i3 and G o . However, for G i2 and G i1 preparations, compounds such as S-(7)-3-PPP and p-tyramine were unable to stimulate [ 35 S]-GTPgS binding. 4 Most of the compounds showed higher relative e cacies (compared to dopamine) and higher potencies in the preparation expressing G o . Comparison of the e ects of di erent agonists in the di erent preparations showed that each agonist di erentially activates the four G proteins. 5 We conclude that the degree of selectivity of G protein activation by the D 2L receptor can depend on the conformation of the receptor stabilised by an agonist.

D prostanoid receptor 2 (DP 2 ) [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 D 2 (PGD 2 ). (5-Fluoro-2-methyl-3-quinolin-2-ylmethylindo-1-yl)-acetic acid (OC000459) is an indole-acetic acid derivative that potently displaces [ 3 H]PGD 2 from human recombinant DP 2 (K i ϭ 0.013 M), rat recombinant DP 2 (K i ϭ 0.003 M), and human native DP 2 (Th2 cell membranes; K i ϭ 0.004 M) but does not interfere with the ligand binding properties or functional activities of other prostanoid receptors (prostaglandin E 1-4 receptors, D prostanoid receptor 1, thromboxane receptor, prostacyclin receptor, and prostaglandin F receptor). OC000459 inhibited chemotaxis (IC 50 ϭ 0.028 M) of human Th2 lymphocytes and cytokine production (IC 50 ϭ 0.019 M) by human Th2 lymphocytes. OC000459 competitively antagonized eosinophil shape change responses induced by PGD 2 in both isolated human leukocytes (pK B ϭ 7.9) and human whole blood (pK B ϭ 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-PGD 2 (DK-PGD 2 ) in this species (ED 50 ϭ 0.04 mg/kg p.o.) and airway eosinophilia in response to an aerosol of DK-PGD 2 in guinea pigs (ED 50 ϭ 0.01 mg/kg p.o.). These data indicate that OC000459 is a potent, selective, and orally active DP 2 antagonist that retains activity in human whole blood and inhibits mast cell-dependent activation of both human Th2 lymphocytes and eosinophils.

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1 The relationships between the density of dopamine D 4.4 receptors and the agonist e cacies of L-745,870 (3-(4-[4-chlorophhenyl]piperazin-1-yl)-methyl-1H-pyrrolo [2,3-b]pyridine) and U-101958 ((1-benzyl-piperidin-4-yl)-(3-isopropoxy-pyridin-2-yl)-methyl-amine) were investigated in Chinese hamster ovary (CHO) cells, after treatment with the gene expression enhancer, sodium butyrate. 2 In CHO cells expressing D 4.4 receptors (CHO/D 4 cells), dopamine inhibited forskolin-stimulated cyclic AMP accumulation (E max 56+1% inhibition, pEC 50 7.4+0.1, n=10). U-101958 behaved as a partial agonist (39+7% the e cacy of dopamine, pEC 50 8.1+0.3, n=4), whereas L-745,870 had no detectable agonist e ect. 3 Receptor density, as estimated by [ 3 H]-spiperone saturation binding was 240+30 fmol mg 71 protein (n=8) in CHO/D 4 cell homogenates. It reached 560+150 (n=6), 1000+190 (n=4) and 840+120 (n=4) fmol mg 71 protein after treatment with sodium butyrate (5 mM) for 6, 18 and 48 h, respectively. 4 The increase in receptor density was associated with a gradual enhancement of the agonist e ects (increased E max and pEC 50 values) of dopamine. The e cacy of U-101958 (relative to dopamine) doubled and L-745,870 was turned into a partial agonist (e cacy 49% relative to dopamine, pEC 50 8.6+0.2, n=6, after 48 h treatment with sodium butyrate). These agonist e ects of U-101958 and L-745,870 could be antagonized by spiperone (0.1 mM) but not by raclopride (10 mM). 5 The results show that U-101958 and L-745,870 are partial agonists at human dopamine D 4.4 receptors expressed in CHO cells. Their e cacy is governed by receptor density. Agonist e ects of these two compounds in vivo cannot be excluded under circumstances of increased receptor levels.

1 Dopamine D 4 receptor antagonists are being developed by several pharmaceutical companies as putative novel antipsychotics, possibly with low propensity to side-eects. Two such compounds, L-745,870 and U-101958 have been recently introduced. ]-spiperone binding was observed with spiperone (pK i 9.6+0.1, n=3), clozapine (pK i 7.4+0.1, n=4), L-745,870 (pK i 8.5+0.1, n=3) and U-101958 (pK i 8.9+0.1, n=3). By contrast, raclopride was very weak (pK i 55, n=3). 4 Dopamine inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D 4 cells in a concentration-dependent fashion (E max 71+2% inhibition of forskolin-stimulated levels, pEC 50 8.7+0.1, n=10). This eect was mimicked by the dopamine D 2 -like receptor agonists, quinpirole and 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT).

Treatment with azacitidine (AZA), a demethylating agent, prolonged overall survival (OS) vs conventional care in patients with higher-risk myelodysplastic syndromes (MDS). As median survival with monotherapy is <2 years, novel agents are needed to improve outcomes. This phase 1b/2b trial (n=113) was designed to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D) of panobinostat (PAN)+AZA (phase 1b) and evaluate the early efficacy and safety of PAN+AZA vs AZA monotherapy (phase 2b) in patients with higher-risk MDS, chronic myelomonocytic leukemia or oligoblastic acute myeloid leukemia with <30% blasts. The MTD was not reached; the RP2D was PAN 30 mg plus AZA 75 mg/m2. More patients receiving PAN+AZA achieved a composite complete response ([CR)+morphologic CR with incomplete blood count+bone marrow CR (27.5% (95% CI, 14.6–43.9%)) vs AZA (14.3% (5.4–28.5%)). However, no significant difference was observed in the 1-year OS rate (PAN+AZA, 60% (50–80%); AZA, 70% (50–80%)) or time to progression (PAN+AZA, 70% (40–90%); AZA, 70% (40–80%)). More grade 3/4 adverse events (97.4 vs 81.0%) and on-treatment deaths (13.2 vs 4.8%) occurred with PAN+AZA. Further dose or schedule optimization may improve the risk/benefit profile of this regimen.

Prostaglandin D2 (PGD2) acting at the CRTH2 receptor (chemoattractant receptor-homologous molecule expressed on Th2 cells) has been linked with a variety of allergic and other inflammatory diseases. We describe a family of indole-1-sulfonyl-3-acetic acids that are potent and selective CRTH2 antagonists that possess good oral bioavailability. The compounds may serve as novel starting points for the development of treatments of inflammatory disease such as asthma, allergic rhinitis, and atopic dermatitis.