A phase 1b/2b multicenter study of oral panobinostat plus azacitidine in adults with MDS, CMML or AML with ⩽30% blasts.

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.

Cloning, expression, functional coupling and pharmacological characterization of the rat dopamine D4 receptor.

It has been difficult to observe functional coupling of the D4 receptor to second messenger systems and a robust functional assay system for this receptor is still lacking. In the present study, the rat dopamine D4 receptor was cloned from rat retina. Sequence comparison revealed identity with the published sequence of Ashgari and co-workers, including the two amino acid insertions (V-Q) at position 92 which are not present in the published sequence of O'Malley and coworkers. The rat dopamine D4 receptor was stably expressed in Chinese hamster lung fibroblast CCL39 cells. [3H]spiperone saturation binding yielded a Bmax of 2,370+/-546 fmol/mg protein and a pKD of 8.74+/-0.14 (n=4). Forskolin-stimulated cAMP accumulation was inhibited by dopamine (Emax 61+/-1% inhibition of forskolin-stimulated levels, pEC50 7.33+/-0.06, n=23). A similar concentration-dependent inhibition was observed with the dopamine D2-like receptor agonists quinpirole and 7-OH-DPAT which elicited nearly the same Emax as dopamine. By contrast, apomorphine and a number of compounds with reported affinity for human dopamine D4 receptors (PD168077, U-101958, SDZ GLC 756, L-745,870 and NGD 94-1) behaved as partial agonists (Emax ranging between 26% and 56% of that of dopamine). The agonist effect of dopamine was completely blocked by preincubation with pertussis toxin, no further accumulation of cAMP above the forskolin-stimulated levels being observed. Antagonist pKB-values obtained against dopamine in this system were: 8.55+/-0.19 (n=3) for the partial agonist L-745,870, 8.38+/-0.23 (n=5) for spiperone, 7.18+/-0.17 (n=4) for haloperidol, 7.04+/-0.13 (n=4) for clozapine and <6 for raclopride. Other functional assays applicable were stimulation of [35S]GTPgammaS binding, extracellular acidification rate and a serum-responsive element using luciferase expression as a reporter gene. However, the receptor did not couple to phosphatidylinositol turnover or to intracellular Ca2+. Thus, expression of the rat dopamine D4 receptor in CCL39 cells provided several functional assay systems, of which inhibition of cAMP appeared to be the most robust one. These functional models can be used to evaluate the activity of compounds at the rat dopamine D4 receptor.

Receptor density as a factor governing the efficacy of the dopamine D4 receptor ligands, L-745,870 and U-101958 at human recombinant D4.4 receptors expressed in CHO cells.

1. The relationships between the density of dopamine D4.4 receptors and the agonist efficacies 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-a min e) were investigated in Chinese hamster ovary (CHO) cells, after treatment with the gene expression enhancer, sodium butyrate. 2. In CHO cells expressing D4.4 receptors (CHO/D4 cells), dopamine inhibited forskolin-stimulated cyclic AMP accumulation (Emax 56+/-1% inhibition, pEC50 7.4+/-0.1, n=10). U-101958 behaved as a partial agonist (39+/-7% the efficacy of dopamine, pEC50 8.1+/-0.3, n=4), whereas L-745,870 had no detectable agonist effect. 3. Receptor density, as estimated by [3H]-spiperone saturation binding was 240+/-30 fmol mg-1 protein (n=8) in CHO/D4 cell homogenates. It reached 560+/-150 (n=6), 1000+/-190 (n=4) and 840+/-120 (n=4) fmol mg-1 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 effects (increased Emax and pEC50 values) of dopamine. The efficacy of U-101958 (relative to dopamine) doubled and L-745,870 was turned into a partial agonist (efficacy 49% relative to dopamine, pEC50 8. 6+/-0.2, n=6, after 48 h treatment with sodium butyrate). These agonist effects of U-101958 and L-745,870 could be antagonized by spiperone (0.1 microM) but not by raclopride (10 microM). 5. The results show that U-101958 and L-745,870 are partial agonists at human dopamine D4.4 receptors expressed in CHO cells. Their efficacy is governed by receptor density. Agonist effects of these two compounds in vivo cannot be excluded under circumstances of increased receptor levels.

Functional, endogenously expressed corticotropin-releasing factor receptor type 1 (CRF1) and CRF1 receptor mRNA expression in human neuroblastoma SH-SY5Y cells.

Corticotropin-releasing factor (CRF) is a hypothalamic 41-amino acid peptide which stimulates corticotropin (ACTH) release from the anterior pituitary and is also involved in the body response to stress. CRF1 receptors represent a potential target for novel antidepressant/anxiolytic drugs. The aim of the present study was to search for a human cell line expressing native, functional CRF1 receptors as a starting material for screening purposes. We identified CRF1 receptors functionally coupled to cAMP formation in human neuroblastoma SH-SY5Y cells. CRF induced concentration-dependent increases in cAMP accumulation in SH-SY5Y cells (maximal increase 6.9 +/- 0.9 fold over basal values, n = 14). This effect was mimicked by related peptides with similar potencies: (mean pEC50 value) human/rat CRF (8.63), rat urocortin (9.32), sauvagine (8.97), urotensin I (8.93), ovine CRF (8.81). The efficacies of these agonists were nearly the same, with the exception of ovine CRF which was slightly less efficacious (75% the Emax of CRF). The responses to CRF were competitively antagonised by the following peptide fragments (mean pKB value): alpha-helical-CRF (9-41) (7.54), [D-Phe12,Nle21,38,C alpha MeLeu37]CRF (12-41) (8.36) and [D-Tyr12]astressin (9.49) and by the selective, non-peptidic CRF1 receptor antagonists, CP-154,526 (7.76) and antalarmin (9.19). Estimation of receptor density by [125I]Tyr0-ovine CRF saturation binding yielded a modest number of binding sites (Bmax 12 fmol/mg protein, KD 0.2 nM). Analysis of mRNA by reverse transcription-polymerase chain reaction clearly revealed the presence of mRNA for CRF1 receptors in SH-SY5Y cells. A slight signal for CRF2 receptor mRNA was also observed. We conclude that neuroblastoma SH-SY5Y cells are endowed with native CRF1 receptors positively coupled to cAMP formation. They therefore constitute a useful functional model for the search of CRF1 selective compounds with potential anxiolytic/antidepressant activity.

NGD 94-1 as an agonist at human recombinant dopamine D4.4 receptors expressed in HEK293 cells.

The atypical antipsychotic, clozapine has some selectivity for dopamine D4 receptors and is a silent antagonist at these receptors. NGD 94-1 (2-phenyl-4(5)-[4-(2-pyrimidinyl)-piperazin-1-yl-methyl]-imidazole ) is a highly selective dopamine D4 receptor ligand recently introduced as a putative antipsychotic mimicking the dopamine D4 receptor antagonist effects of clozapine. We show that NGD 94-1 is not silent. It behaved as an agonist in human embryonic kidney 293 cells expressing human recombinant dopamine D4.4 receptors. This questions the clinical use of NGD 94-1.

Functional alpha2C-adrenoceptors in human neuroblastoma SH-SY5Y cells.

The alpha2-adrenoceptor mediating inhibition of forskolin-stimulated cyclic AMP accumulation in human neuroblastoma SH-SY5Y cells was further characterized. The alpha2-adrenoceptor agonists, UK 14,304 (5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline), oxymetazoline, guanfacine, (-)-noradrenaline and clonidine concentration-dependently decreased cyclic AMP accumulation in this cell line (Emax ca. 50% inhibition). Agonist pEC50 values ranged between 6.7 and 7.8. Clonidine was a partial agonist. The effects of UK 14,304 were blocked after a pertussis toxin treatment. The concentration-response curves of UK 14,304 were shifted to the right in a parallel manner by the following antagonists (mean pK(B) values): yohimbine (8.17), idazoxan (7.63), prazosin (6.66), 2-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2 H,4H) isoquinolindione (ARC 239; 7.12) and 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB-4101; 8.12). The relatively high pKB values of prazosin and ARC 239 point to a non-alpha2A-adrenoceptor-mediated effect. The relatively high pK(B) value of WB-4101 further characterizes the alpha2-adrenoceptor in SH-SY5Y cells as being of the alpha2C subtype. The analysis of the expression of alpha2-adrenoceptor subtypes by reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the exclusive presence of alpha2C-adrenoceptor mRNA in SH-SY5Y cells. We propose that inhibition of forskolin-stimulated cAMP accumulation in SH-SY5Y cells be used as a functional model of human, native alpha2C-adrenoceptors.

The agonist activities of the putative antipsychotic agents, L-745,870 and U-101958 in HEK293 cells expressing the human dopamine D4.4 receptor.

1. Dopamine D4 receptor antagonists are being developed by several pharmaceutical companies as putative novel antipsychotics, possibly with low propensity to side-effects. Two such compounds, L-745,870 and U-101958 have been recently introduced. 2. The radioligand binding and functional activities of L-745,870 and U-101958 were investigated in human embryonic kidney (HEK)293 cells expressing the human recombinant dopamine D4.4 receptor (HEK293/D4 cells). [3H]-spiperone binding experiments were performed and inhibition of forskolin-stimulated cyclic AMP accumulation was used as the functional response. 3. [3H]-spiperone was found to label a homogeneous and saturable population of specific binding sites in HEK293/D4 cell homogenates (Bmax 505+/-90 fmol mg(-1) protein, pK(D) 9.5+/-0.1, n=3). Inhibition of specific [3H]-spiperone binding was observed with spiperone (pKi 9.6+/-0.1, n=3), clozapine (pKi 7.4+/-0.1, n=4), L-745,870 (pKi 8.5+/-0.1, n=3) and U-101958 (pKi 8.9+/-0.1, n=3). By contrast, raclopride was very weak (pKi < 5, n=3). 4. Dopamine inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D4 cells in a concentration-dependent fashion (Emax 71+/-2% inhibition of forskolin-stimulated levels, pEC50 8.7+/-0.1, n=10). This effect was mimicked by the dopamine D2-like receptor agonists, quinpirole and 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT). 5. L-745,870 and U-101958 also inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D4 cells in a concentration-dependent way. L-745,870 was less efficacious than dopamine (71% the efficacy of dopamine), whereas U-101958 behaved as a full agonist compared to dopamine. Potencies (pEC50) values of L-745,870 and U-101958 were 9.0+/-0.2 (n=4) and 8.7+/-0.3 (n=3), consistent with pKi values determined in radioligand binding studies. 6. Dopamine, L-745,870 and U-101958 (up to 1 microM) were devoid of effect on forskolin-stimulated cyclic AMP accumulation in control, non-transfected HEK293 cells. 7. The agonist effects of dopamine, L-745,870 and U-101958 in HEK293/D4 cells could be antagonized by spiperone (pK(B) 8.2-8.8) and clozapine (pK(B) 7.1), but not by raclopride (pK(B) < 5). None of these antagonists had any significant agonist activity at concentrations up to 10 microM. 8. These results show that the putative dopamine D4 receptor antagonists, L-745,870 and U-101958 are not devoid of intrinsic activity at human recombinant dopamine D4.4 receptors. Therefore, they may not represent the most appropriate drugs for testing the benefit of D4 receptor antagonism in schizophrenic patients, if agonism should translate in vivo.