Decahydroisoquinoline derivatives as novel non-peptidic, potent and subtype-selective somatostatin sst(3) receptor antagonists.

Starting from non-peptidic sst(1)-selective somatostatin receptor antagonists, first compounds with mixed sst(1)/sst(3) affinity were identified by directed structural modifications. Systematic optimization of these initial leads afforded novel, enantiomerically pure, highly potent and sst(3)-subtype selective somatostatin antagonists based on a (4S,4aS,8aR)-decahydroisoquinoline-4-carboxylic acid core moiety. These compounds can efficiently be synthesized and show promising PK properties in rodents.

Discovery of novel non-peptidic beta-alanine piperazine amide derivatives and their optimization to achiral, easily accessible, potent and selective somatostatin sst1 receptor antagonists.

Structural simplification of the core moieties of obeline and ergoline somatostatin sst(1) receptor antagonists, followed by systematic optimization, led to the identification of novel, highly potent and selective sst(1) receptor antagonists. These achiral, non-peptidic compounds are easily prepared and show promising PK properties in rodents.

Ergoline derivatives as highly potent and selective antagonists at the somatostatin sst 1 receptor.

Non-peptidic compounds containing the octahydro-indolo[4,3-fg]quinoline (ergoline) structural element have been optimized into derivatives with high affinity (pK(d) r sst(1)>9) and selectivity (>1000-fold for h sst(1) over h sst(2)-h sst(5)) for the somatostatin sst(1) receptor. In functional assays, these ergolines act as antagonists at human recombinant sst(1) receptors. Pharmacokinetic studies in rodents reveal good oral bioavailability and brain penetration for some of these compounds.

Identification and SAR of potent and selective non-peptide obeline somatostatin sst1 receptor antagonists.

A novel class of non-peptide somatostatin receptor ligands bearing the octahydrobenzo[g]quinoline (obeline) structural element has been identified. SAR studies have been performed that led to the discovery of derivatives with high affinity (pK(d) r sst(1) > or = 9) and selectivity (> or = 150-fold for h sst(1) over h sst(2)-h sst(5)) for somatostatin receptor subtype sst(1). In a functional assay, the compounds act as antagonists at human recombinant sst(1) receptors.

Serotonin 5-HT7 receptors coupled to induction of interleukin-6 in human microglial MC-3 cells.

Brain serotonin 5-HT(7) receptors are known to be expressed in neurons and astrocytes. We now report the presence of these receptors in a third type of cell, microglial cells. 5-Hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) induced concentration-dependent stimulations of cAMP accumulation in the human microglial MC-3 cell line. The maximal effect of 5-HT was 3.4+/-0.3-fold stimulation (mean+/-S.E.M., n=5) above basal levels. The rank order of agonist potency (pEC50 values) was 5-CT (7.09)>5-HT (6.13)>or=5-MeOT (5.78)>>8-OH-DPAT (ca. 5). The effect of 5-CT was inhibited in a concentration-dependent manner by the selective 5-HT7 receptor antagonist SB-269970 (pA2 value 9.03). Western blot analysis revealed the presence of immunoreactive bands corresponding to the human 5-HT7 receptor in extracts of MC-3 cells. The presence of two splice variants of the 5-HT7 receptor (5-HT7(a/b)) was visualized by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis with specific primers. In real-time PCR studies, the mRNA for interleukin-6 (IL-6) was found to be increased by 2.5-fold in MC-3 cells after 1 h incubation with 5-CT (1 microM) and this effect was fully blocked by the 5-HT7 receptor antagonist SB-269970 (1 microM). These data show that functional 5-HT7 receptors are present in human microglial MC-3 cells, suggesting that they are involved in neuroinflammatory processes.

Binding and functional properties of the novel somatostatin analogue KE 108 at native mouse somatostatin receptors.

Clinically used somatostatin (SRIF) analogs, octreotide and lanreotide, act primarily by binding to SRIF receptor subtype 2 (sst2). In contrast, the recently described multiligand SOM230 binds with high affinity to sst(1-3) and sst5 and KE 108 is characterised as a high affinity ligand for all five SRIF receptors. In tumoural mouse corticotrophs (AtT-20 cells) and in mouse hippocampus, binding and functional features of KE 108 were examined and compared to SRIF-14, octreotide and SOM230. In AtT-20 cells, KE 108 bound with high affinity at [125I]LTT-SRIF-28-labelled sites similarly to SRIF-14, octreotide and SOM230. At the functional level, all four ligands increased guanosine-5'-O-(3-[35S]thio)-triphosphate binding and decreased cAMP accumulation or intracellular Ca2+ concentration through G(i/o) proteins. In hippocampal slices, KE 108, octreotide and SOM230 also bound with high affinity at [125I]LTT-SRIF-28-labelled sites similarly to SRIF-14, but KE 108, octreotide or SOM230 did not influence spontaneous epileptiform activity which was, in contrast, inhibited by SRIF-14. In conclusion, this study demonstrates that KE 108 has high affinity for native mouse SRIF receptors. Functionally, KE 108 mediates SRIF action at sst(2/5) in corticotrophs whereas it does not mimic the SRIF-induced inhibition of hippocampal excitation suggesting that the high potency and efficacy of a synthetic ligand to all known SRIF receptors may not reproduce entirely the effects of the natural SRIF.

Distinct functional properties of native somatostatin receptor subtype 5 compared with subtype 2 in the regulation of ACTH release by corticotroph tumor cells.

In a series of human corticotroph adenomas, we recently found predominant mRNA expression of somatostatin (SS) receptor subtype 5 (sst5). After 72 h, the multiligand SS analog SOM230, which has a very high sst5 binding affinity, but not Octreotide (OCT), significantly inhibited basal ACTH release. To further explore the role of sst5 in the regulation of ACTH release, we conducted additional studies with mouse AtT-20 cells. SOM230 showed a 7-fold higher ligand binding affinity and a 19-fold higher potency in stimulating guanosine 5'-O-(3-thiotriphosphate) binding in AtT-20 cell membranes compared with OCT. SOM230 potently suppressed CRH-induced ACTH release, which was not affected by 48-h dexamethasone (DEX) pretreatment. However, DEX attenuated the inhibitory effects of OCT on ACTH release, whereas it increased the inhibitory potency of BIM-23268, an sst5-specific analog, on ACTH release. Quantitative PCR analysis showed that DEX lowered sst(2A+2B) mRNA expression significantly after 24 and 48 h, whereas sst5 mRNA levels were not significantly affected by DEX treatment. Moreover, Scatchard analyses showed that DEX suppressed maximum binding capacity (B(max)) by 72% when 125I-Tyr3-labeled OCT was used as radioligand, whereas B(max) declined only by 17% when AtT-20 cells were treated with [125I-Tyr11]SS-14. These data suggest that the sst5 protein, compared with sst2, is more resistant to glucocorticoids. Finally, after SS analog preincubation, compared with OCT both SOM230 and BIM-23268 showed a significantly higher inhibitory effect on CRH-induced ACTH release. In conclusion, our data support the concept that the sst5 receptor might be a target for new therapeutic agents to treat Cushing's disease.

Functional activity of the multiligand analog SOM230 at human recombinant somatostatin receptor subtypes supports its usefulness in neuroendocrine tumors.

Functional gastroenteropancreatic tumors express all 5 somatostatin receptor subtypes (sst) in different quantities. Octreotide and lanreotide treat patients with these tumors by binding preferentially to sst2 and, to a lesser extent, to sst3 and sst5 receptors, thereby controlling prominent symptoms caused by hormone hypersecretion (diarrhea and flushing). Although symptoms initially improve in most patients, a loss of response occurs in about 50% during continuous treatment. The functional activity at sst receptors of SOM230, a new multiligand somatostatin analog, has been described and compared with that of somatostatin (SRIF-14) and octreotide. These data show that SOM230 is a full agonist with nanomolar potency at sst(1,2,3) and sst5 receptors. The multiligand activity profile of SOM230, together with its nondesensitizing inhibitory effect on growth hormone and insulin-like growth factor-I secretion in rats, may underlie its successful use in clinical trials and its potential for use in refractory patients with carcinoid tumors.

Functional expression of the serotonin 5-HT7 receptor in human glioblastoma cell lines.

Serotonin 5-HT(7) receptors are present in astrocytes. Understanding their role in this type of cell would greatly benefit from the identification of astroglial cell lines expressing this receptor type. The aim of the present study was to assess the expression of native 5-HT(7) receptors and 5-HT(7) receptor mRNA in a number of human glioblastoma cell lines, by means of cAMP measurements, Western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. 5-Hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) induced concentration-dependent stimulations of cAMP accumulation in the human glioblastoma cell lines, U-373 MG, U-138 MG, U-87 MG, DBTRG-05MG, T98G, H4, CCF-STTG1 and Hs 683. The rank order of potency was 5-CT>5-HT=5-MeOT>>8-OH-DPAT. The effect of 5-CT was inhibited in a concentration-dependent manner by the selective 5-HT(7) receptor antagonist SB-269970 in all human glioblastoma cells. Schild analyses yielded slope factors close to unity (0.89-1.13) and pA(2) values of 8.69-9.05. Western blot analysis revealed the presence of immunoreactive bands corresponding to the human 5-HT(7) receptor in extracts of all human glioblastoma cell lines. The presence of the three splice variants of the 5-HT(7) receptor (5-HT(7(a/b/d))) was visualized by RT-PCR analysis with specific primers in all human glioblastoma cell lines. In conclusion, human glioblastoma cell lines express functional 5-HT(7) receptors and the three splice variants of the corresponding mRNA. These cell lines could serve as model systems of native 5-HT(7) receptors in glial cells to investigate their putative role in processes like release of neurotrophic factors or inflammatory cytokines.

Differential inverse agonist efficacies of SB-258719, SB-258741 and SB-269970 at human recombinant serotonin 5-HT7 receptors.

Recombinant 5-hydroxytryptamine 5-HT7 receptors are known to express constitutive, i.e., agonist-independent activity. Nonselective ligands, like methiothepin, ritanserin or clozapine behave as full inverse agonists at 5-HT7 receptors. The aim of the present study was to evaluate the degree of inverse agonist activity of three selective 5-HT7 receptor antagonists ((R)-3,N-dimethyl-N-[1-methyl-3-(4-methyl-piperidin-1-yl)propyl]benzene sulfonamide or SB-258719, R-(+)-1-(toluene-3-sulfonyl)-2-[2-(4-methylpiperidin-1-yl)ethyl]-pyrrolidine or SB-258741 and (R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)-pyrrolidine-1-sulfonyl)-phenol or SB-269970) in the same model. cAMP accumulation was measured in intact Chinese hamster ovary (CHO) cells expressing human recombinant 5-HT7a receptors. In these cells, 5-HT stimulated cAMP levels and a series of ligands antagonized the effect of 5-HT with a 5-HT7 receptor-like profile. SB-258719 had no inverse agonist activity, SB-258741 behaved as a partial inverse agonist and SB-269970 was a quasi-full inverse agonist (as compared to methiothepin). The inverse agonist effect of SB-269970 was antagonized in a concentration-dependent manner by SB-258719. The widespread spectrum of inverse agonist activities shown by these compounds should help assessing the physiological relevance of constitutive 5-HT7 receptor activity in native tissues.

Functional characterization of the novel antipsychotic iloperidone at human D2, D3, alpha 2C, 5-HT6, and 5-HT1A receptors.

Iloperidone has demonstrated an interesting monoamine receptor profile in radioligand binding studies, with nanomolar affinity for certain noradrenaline, dopamine, and serotonin receptors. In this study, the agonist/antagonist activity of iloperidone was determined in cell lines expressing recombinant human D(2A), D(3), alpha(2C), 5-HT(1A), or 5-HT(6) receptors. With the exception of 5-HT(6) receptors, these receptors are negatively coupled to cyclase. Thus, after stimulation with forskolin, the agonists dopamine (at D(2A) and D(3)), noradrenaline (at alpha(2C)), or 8-OH-DPAT (at 5-HT(1A)) induced a reduction in cAMP accumulation. Conversely, activation of the 5-HT(6) receptor by 5-HT led to an increase in cAMP accumulation. Iloperidone alone was devoid of significant agonist activity but inhibited the agonist response in all 5 cell lines in a surmountable and concentration-dependent fashion. Iloperidone was most potent at D(3) receptors (pK(B) 8.59 +/- 0.20; n = 6), followed by alpha(2C) (pK(B) 7.83 +/- 0.06; n = 15), 5-HT(1A) (pK(B) 7.69 +/- 0.18; n = 10), D(2A) (pK(B) 7.53 +/- 0.04; n = 11) and 5-HT(6) (pK(B) 7.11 +/- 0.08; n = 11) receptors.

Functional characterisation of the putative somatostatin sst2 receptor antagonist CYN 154806.

The two forms (DTyr8 and LTyr8) of the putative somatostatin sst2 receptor antagonist CYN 154806 (Ac-4NO2-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-D/LTyr-NH2) were investigated on recombinant human somatostatin receptors and endogenous guinea-pig ileum receptors. In radioligand binding studies using the agonist radioligands [125I]LTT-SRIF-28, [125I][Tyr10]cortistatin-14, [125I]CGP 23996 and [125I][Tyr3]octreotide in Chinese hamster lung fibroblast (CCL39) and Chinese hamster ovary (CHO) cells expressing human somatostatin receptors (hsst1-5), CYN 154806 binds to sst2 receptors with nanomolar affinity (pKD=8.14-8.89), 40- to 4500-fold higher than for sst1, sst3 or sst4. High affinity was also demonstrated for sst5 receptors, particularly for LTyr8CYN 154806 where the sst5 affinity was higher than for sst2 receptors when using [125I]CGP 23996 and [125I][Tyr3]octreotide. Functional properties of the compounds were examined in Chinese hamster ovary (CHO) cells expressing human sst2 receptors, in (1) inhibition of forskolin-stimulated adenylate cyclase, (2) stimulation of serum response element-driven luciferase expression and (3) [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPS) binding. L- and DTyr8CYN 154806 showed full agonism at inhibition of forskolin-stimulated cAMP accumulation (pEC50=7.73 for both, Emax 104% and 78%, respectively), partial agonism at luciferase expression (pEC50=7.85 and 8.16, Emax=50% and 29%, respectively) and behaved as apparently silent antagonists at [35S]GTPS binding (no agonism observed, pKB=6.88 and 7.50, respectively). The agonist potential was confirmed in isolated guinea-pig ileum preparations via measurement of SRIF-induced inhibition of neurotransmission, where the L-isoform had marked agonism (pEC50=8.23, Emax=32%) whereas the D-isoform was apparently devoid of agonism. The present data suggest that CYN 154806 should be used with caution as an sst2 receptor antagonist tool, since it possesses intrinsic activity at sst2, and high affinity for both sst2 and sst5 receptors. The DTyr form, having lower intrinsic activity, especially in natural tissues, and greater selectivity for sst2 receptors, may be more reliable than LTyr CYN 154806.

Antagonist effect of pseudohypericin at CRF1 receptors.

St. John's wort (Hypericum perforatum L.) is widely used for the treatment of mild to moderately severe depression. However, the nature of its active principles and the exact mode of antidepressant action are still unknown. It has been suggested repeatedly in preclinical and clinical studies that the content of the acylphloroglucinol hyperforin decisively contributes to the antidepressant efficacy of St. John's wort extracts. Experimental studies in vivo also indicate that the naphthodianthrone hypericin may reduce the activity of the hypothalamic-pituitary-adrenal axis. Exacerbated hypothalamic-pituitary-adrenal activity has often been associated with depressive states in patients. Corticotropin-releasing factor (CRF) seems to be a major determinant in the regulation of the hypothalamic-pituitary-adrenal activity via activation of CRF(1) receptors. In the present study, we investigated the CRF(1) receptor antagonist activity of three main constituents of St. John's wort (hypericin, pseudohypericin and hyperforin) by measuring their effect on CRF-stimulated cAMP formation in recombinant Chinese hamster ovary (CHO) cells. As a selectivity test, the compounds were also tested against calcitonin in the same cells. Of the three compounds tested, only pseudohypericin selectively antagonised CRF (K(B) 0.76 microM). Hypericin and hyperforin affected both CRF and calcitonin with similar potencies and the same type of behaviour (competitive antagonism for hypericin, noncompetitive for hyperforin). It is concluded that pseudohypericin is the only real CRF(1) receptor antagonist of the three constituents tested. In addition, evidence is provided that beside hyperforin, both pseudohypericin and hypericin are implicated in the antidepressant efficacy of St. John's wort.

Toward selective ERbeta agonists for central nervous system disorders: synthesis and characterization of aryl benzthiophenes.

In an effort to identify selective ligands for the estrogen receptor subtype ERbeta, a series of aryl benzthiophenes was synthesized. In a radioligand binding assay and reporter gene assays in HeLa and SH-SY5Y cells, compounds were characterized as ERbeta-selective agonists. By targeting ERbeta in the brain, these compounds could lead to drugs able to separate the beneficial effects of estrogens on mood, learning, and memory from side effects such as the stimulation of endometrial and breast cancer.

Pharmacological and endocrinological characterisation of stress-induced hyperthermia in singly housed mice using classical and candidate anxiolytics (LY314582, MPEP and NKP608).

The stress-induced hyperthermia test is a paradigm developed several years ago to model the expression of autonomic hyperactivity in anxiety. Whereas in the classical stress-induced hyperthermia, cohort removal was used, in a recently described modification of the stress-induced hyperthermia model singly housed mice rather than groups of mice were used. The modification of this model can be summarized as follows: rectal temperature is recorded in singly housed animals at two consecutive time-points (T1 and T2) which are interspaced by a defined time-interval (15 min). Since the value at the second temperature-recording exceeds the value of the initial measure it is the difference between these two core-temperatures which reflects stress-induced hyperthermia. In the present study, the stress-induced hyperthermia paradigm, in its modified design, was evaluated in OF1/IC mice. By comparing the effect of various compounds in both the modified as well as the classical (cohort removal) stress-induced hyperthermia paradigm, a very high correlation was found for the pharmacological sensitivity of the two paradigms. Furthermore, it was demonstrated that other anxiolytics, all known to be active in the classical stress-induced hyperthermia paradigm, such as the benzodiazepines chlordiazepoxide (0.3, 1, 3, 10 mg/kg, p.o.), diazepam (0.1, 0.3, 1, 3 mg/kg, p.o.), clobazam (5 or 10 mg/kg, p.o.) and oxazepam (5 or 10 mg/kg, p.o.) as well as the non-benzodiazepines buspirone (7.5 or 15 mg/kg, p.o.) and ethanol (15% or 30%, 10 ml/kg, p.o.), showed a marked reduction in stress-induced hyperthermia in the modified design. New candidate anxiolytics, i.e. the metabotropic glutamate (mGlu) receptor group 2 agonist LY314582 (1 or 10 mg/kg, p.o.; racemic mixture of LY354740 ((2S,4S)-2-amino-4-(4,4-diphenylbut-1-yl)-pentane-1,5-dioic acid), the metabotropic glutamate 5 receptor antagonist MPEP (1, 7.5, 15 or 30 mg/kg, p.o.; 2-methyl-6-(phenylethynyl)pyridine) and the neurokinin 1 (NK1) receptor antagonist NKP608 (0.01 or 0.1 mg/kg, p.o.; quinoline-4-carboxylic acid [trans-(2R,4S)-1-(3,5-bis-trifluoromethyl-benzoyl)-2-(4-chloro-benzyl)-piperidin-4-yl]-amide) also reduced stress-induced hyperthermia in the modified paradigm clearly indicating anxiolytic-like activity for these compounds. Finally, the effects of the classical benzodiazepine chlordiazepoxide (10 mg/kg, p.o.), in parallel with its effect on stress-induced hyperthermia, were also investigated for its effect on plasma concentrations of the two stress hormones, adrenocorticotropin (ACTH) and corticosterone. It was shown that all three parameters were significantly increased 15 min after T1 in vehicle-treated mice whereas the increase was significantly attenuated following pre-treatment with chlordiazepoxide. In conclusion, all the data presented here indicate that the modified version of the stress-induced hyperthermia-paradigm is a valid and interesting alternative to the classical stress-induced hyperthermia test.