Assessing anxiolytic-like effects of selective serotonin reuptake inhibitors and serotonin-noradrenaline reuptake inhibitors using the elevated plus maze in mice.

The anxiolytic-like effects of selective serotonin reuptake inhibitors (SSRIs; paroxetine, fluvoxamine) and serotonin-noradrenaline reuptake inhibitors (SNRIs; milnacipran, venlafaxine) were compared with those of benzodiazepines (diazepam, chlordiazepoxide) and tricyclic antidepressants (imipramine, amitriptyline) using the elevated plus maze in mice. Paroxetine and fluvoxamine had no significant effects on the time spent in open arms and the number of open arm entries, even at a dose of 20 mg/kg p.o. On the other hand, milnacipran and venlafaxine showed a dose-dependent increase in the time spent in open arms and the number of open-arm entries. Significant effects were observed at doses of 10 and 20 mg/kg p.o. for both drugs. Diazepam and chlordiazepoxide showed potent anxiolytic-like effects, whereas imipramine and amitriptyline caused no anxiolytic-like effects. Like diazepam and chlordiazepoxide, milnacipran and venlafaxine increased the distance moved in open arms at the same dose levels showing anxiolytic-like effects. From these results, it may be concluded that SNRIs caused anxiolyic-like effects similar to benzodiazepines.

E7070, a novel sulphonamide agent with potent antitumour activity in vitro and in vivo.

E7070 (N-(3-Chloro-7-indolyl)-1,4-benzenedisulphonamide) was selected from our sulphonamide compound collections via antitumour screening and flow cytometric analysis. Following treatment with E7070, the cell cycle progression of P388 murine leukaemia cells was disturbed in the G1 phase. The cell-killing effect on human colon cancer HCT116 cells was found to be time-dependent. In the panel of 42 human tumour cell lines, E7070 showed an antitumour spectrum that was distinct from those of other anticancer drugs used in clinic. Animal tests using human tumour xenograft models demonstrated that E7070 could cause not only tumour growth suppression, but also tumour regression in three of five colorectal and two of two lung cancers. In the HCT116 xenograft model, E7070 was shown to be superior to 5-FU, MMC and CPT-11 (irinotecan). Furthermore, complete regression of advanced LX-1 tumours was observed in 80% of E7070-treated mice. All of these observations have promoted this drug to clinical evaluation.

Cell cycle regulation in the G1 phase: a promising target for the development of new chemotherapeutic anticancer agents.

As a result of substantial advances in recent cancer biology, cell cycle regulation in the G1 phase has attracted a great deal of attention as a promising target for the research and treatment of cancer. Many of the important genes associated with G1 regulation have been shown to play a key role in proliferation, differentiation and oncogenic transformation and programmed cell death (apoptosis). Currently, a variety of "cytostatic" agents that affects G1 progression and/or G1/S transition are being evaluated in clinical trials. Flavopiridol is a potent inhibitor of cyclin-dependent kinases (CDKs). UCN-01 was originally found to be a PKC-selective protein kinase antagonist. More recent studies have revealed that this agent can also inhibit several CDKs and the checkpoint kinase CHK1. FR901228, MS-27-275 and SAHA are histone deacetylase inhibitors that induce changes in the transcription of specific genes via the hyperacetylation of histones. The proteasome inhibitor PS-341 disrupts the degradation process of intracellular proteins, including cell cycle regulatory proteins such as cyclins. R115777, SCH66336 and BMS-214662 are non-peptidic farnesyl transferase inhibitors that prevent p21 ras oncogene activation. Rapamycin derivative CCI-779 downregulates signals through S6 kinase and FRAP (FKBP-rapamycin associating protein), affecting the expression levels of mRNAs important for progression from G1 to S phase. 17-Allylaminogeldanamycin targets the Hsp-90 (heat shock protein-90) family of cellular chaperones regulating the function of signaling proteins. TNP-470 (AGM-1470), a fumagillin derivative shows antiangiogenic action through binding to MetAP-2 (methionine aminopeptidase-2). The antitumor sulfonamide E7070, causing a cellular accumulation in the G1 phase, has been shown to suppress the activation of CDK2 and cyclin E expression in HCT116 colorectal cancer cell line highly sensitive to the drug. With respect to several growth factor receptors such as EGFR, PDGFR, bFGFR and VEGFR, potent and specific inhibitors of receptor tyrosine kinases have been also examined as hopeful drug candidates. In this report, we review the current status of extensive efforts directed towards the discovery and development of new chemotherapeutic anticancer agents targeting cell cycle regulation in the G1 phase, with particular focus on the compounds undergoing clinical investigations.

Antitumor activity- and gene expression-based profiling of ecteinascidin Et 743 and phthalascidin Pt 650.

BACKGROUND: Ecteinascidin 743 (Et 743) is a potent antitumor marine alkaloid currently undergoing phase II clinical trials. The synthetic analog phthalascidin (Pt 650), a designed structural analog of Et 743 displays in vitro potency comparable to Et 743. In this study, we used a panel of 36 human cancer cell lines, flow cytometry and oligonucleotide microarrays to analyze further these two compounds in a parallel fashion with regard to both antitumor activity (phenotype) and gene expression (genotype) bases. RESULTS: The cancer panel experiment established that activity patterns of Et 743 and Pt 650 were essentially the same with their IC(50) values ranging from pM to low nM. By means of flow cytometric cell cycle analysis using HCT116 cells, they were shown to disrupt S phase progression after a 12-h treatment at 2.0 nM, eventually resulting in the late S and G2/M accumulation at the 24-h time point. Array-based gene expression monitoring also demonstrated that the Et 743 and Pt 650 profiles were highly similar in two distinct cancer cell lines, HCT116 colon and MDA-MB-435 breast. Characteristic changes were observed in subsets of genes involved in DNA damage response, transcription and signal transduction. In HCT116 carrying the wild-type p53 tumor suppressor gene, the up-regulation of several p53-responsive genes was evident. Furthermore, a subset of genes encoding DNA-binding proteins to specific promoter regions (e.g. the CCAAT box) was down-regulated in both cell lines, suggesting one potential mode of action of this series of antitumor agents. CONCLUSION: A combination of gene expression analysis using oligonucleotide microarrays and flow cytometry confirms an earlier finding that Et 743 and Pt 650 have remarkably similar biological activities.

A focused compound library of novel N-(7-indolyl)benzenesulfonamides for the discovery of potent cell cycle inhibitors.

A series of compounds containing an N-(7-indolyl)benzenesulfonamide pharmacophore was synthesized and evaluated as a potential antitumor agent. Cell cycle analysis with P388 murine leukemia cells revealed that there were two different classes of potent cell cycle inhibitors; one disrupted mitosis and the other caused G1 accumulation. Herein described is the SAR summary of the substituent patterns on this pharmacophore template.

A general method for acylation of indoles at the 3-position with acyl chlorides in the presence of dialkylaluminum chloride.

[reaction--see text] Indoles are selectively acylated at the 3-position in high yields on treatment with a wide variety of acyl chlorides in CH(2)Cl(2) in the presence of diethylaluminum chloride or dimethylaluminum chloride. The reaction proceeds under mild conditions and is applicable to indoles bearing various functional groups without NH protection.

An acoustically evoked short latency negative response in profound hearing loss patients.

In our auditory brainstem response (ABR) tests, a peculiar V-shaped negative wave at around 3 4 ms latency was observed. At present, this acoustically evoked short latency negative response (ASNR) is poorly understood. In order to study its appearance and origin, the ABR data of 3104 tests during 1980-98 were reviewed. The ASNR was found only in profound hearing loss ears under intense stimuli (80-120 dB nHL). Out of the total 653 profound hearing loss patients (981 ears), the click-evoked ASNRs were present in 80 patients (12.3%), 117 ears (11.9%). The age range was from 8 months to 70 years. Demographic outcome revealed significant higher appearance rates in young subjects especially in the 20-30 years group. The ASNR was excluded from an artifact by its reproducibility over time, equipment and institutes. Moreover, it became absent after external auditory canal occlusion, which simply blocked the air conduction without any influence upon scalp potentials or equipment. It had neural response characteristics that the latency and amplitude shortened and increased respectively in response to the increase of stimulus intensity. Because the peculiar V-shaped waveform obviously differs from ABR, the ASNR was not interpreted as a potential generated from the conventional auditory pathway. On the other hand, the ASNR individuals were of good vestibular function in sharp contrast with their poor hearing. This suggests the probable relation between the ASNR and the vestibular system. The saccule and vestibular nucleus are hypothesized to be the sense organ and the origin of the response respectively.

Discovery of novel antitumor sulfonamides targeting G1 phase of the cell cycle.

Described herein is the discovery of a novel series of antitumor sulfonamides targeting G1 phase of the cell cycle. Cell cycle control in G1 phase has attracted considerable attention in recent cancer research, because many of the important proteins involved in G1 progression or G1/S transition have been found to play a crucial role in proliferation, differentiation, transformation, and programmed cell death (apoptosis). We previously reported our first antitumor sulfonamide E7010 as a novel tubulin polymerization inhibitor. Interestingly enough, continuous research on structurally related compounds led us to the finding of another class of antitumor sulfonamides that block cell cycle progression of P388 murine leukemia cells in G1 phase, but not in M phase. Of the compounds examined, N-(3-chloro-7-indolyl)-1,4-benzenedisulfonamide (E7070) showed significant antitumor activity against HCT116 human colon carcinoma both in vitro (IC(50) 0.11 microg/mL in cell proliferation assay) and in vivo (not only growth suppression but also a marked reduction of tumor size in nude mice). Because of its promising efficacy against human tumor xenografts and its unique mode of action, E7070 is currently undergoing phase I clinical trials in European countries.

Phthalascidin, a synthetic antitumor agent with potency and mode of action comparable to ecteinascidin 743.

A series of totally synthetic molecules that are structurally related to the marine natural product ecteinascidin 743 (Et 743) has been prepared and evaluated as antitumor agents. The most active of these, phthalascidin, is very similar to Et 743 with regard to in vitro potency and mode of action across a variety of cell types. The antiproliferative activity of phthalascidin (IC50 = 0.1-1 nM) is greater than that of the agents Taxol, camptothecin, adriamycin, mitomycin C, cisplatin, bleomycin, and etoposide by 1-3 orders of magnitude, and the mechanism of action is clearly different from these currently used drugs. Phthalascidin and Et 743 induce DNA-protein cross-linking and, although they seem to interact with topoisomerase (topo) I (but not topo II), topo I may not be the primary protein target of these agents. Phthalascidin and Et 743 show undiminished potency in camptothecin- and etoposide-resistant cells. Phthalascidin is more readily synthesized and more stable than Et 743, which is currently undergoing clinical trials. The relationship of chemical structure and antitumor activity for this class of molecules has been clarified by this study.

Depudecin induces morphological reversion of transformed fibroblasts via the inhibition of histone deacetylase.

Depudecin is a fungal metabolite that reverts the rounded phenotype of NIH 3T3 fibroblasts transformed with v-ras and v-src oncogenes to the flattened phenotype of the nontransformed parental cells. The mechanism of detransformation induced by this agent had not been determined. Here, we demonstrate that depudecin inhibits histone deacetylase (HDAC) activity effectively both in vivo and in vitro. Depudecin induces similar morphological reversion in v-ras transformed NIH 3T3 cells as do other naturally occurring HDAC inhibitors such as trichostatin A or trapoxin. It competitively inhibits the binding of [3H]trapoxin in vitro and the nuclear binding of a trapoxin-coumarin fluorophore in vivo, suggesting that depudecin shares a nuclear binding protein and site on that protein with trapoxin. Furthermore, depudecin induces hyperacetylation of histones in a dose-dependent manner and at concentrations comparable with that required for detransformation. An in vitro histone deacetylase assay, using purified recombinant HDAC1, reveals that depudecin inhibits 50% of the enzyme activity at a concentration of 4.7 microM. These results demonstrate that depudecin is a novel HDAC inhibitor and suggest that its ability to induce morphological reversion of transformed cells is the result of its HDAC inhibitory activity.

A role for histone deacetylase activity in HDAC1-mediated transcriptional repression.

Treatment of mammalian cells with small molecule histone deacetylase (HDAC) inhibitors induces changes in the transcription of specific genes. These changes correlate directly with an increase in the acetylation levels of all four core histones in vivo. Antibodies directed against endogenous HDAC1, HDAC2, or HDAC3 immunoprecipitate histone deacetylase activity that is inhibited in vitro by the small molecule trapoxin (TPX), and all three HDACs are retained by a TPX-affinity matrix. HDAC1 and HDAC2 are associated in HeLa cells in a complex that is predominantly separate from an HDAC3 immune complex. Both Jurkat HDAC1 and HeLa HDAC1/2 immune complexes deacetylate all four core histones and recombinant HDAC1 deacetylates free and nucleosomal histones in vitro. Purified recombinant HDAC1 deacetylates core histones in the absence of protein cofactors. Site-directed mutagenesis was used to identify residues required for the enzymatic and structural integrity of HDAC1. Mutation of any one of four conserved residues causes deleterious effects on deacetylase activity and a reduced ability to bind a TPX-affinity matrix. A subset of these mutations also cause a decreased interaction with the HDAC1-associated proteins RbAp48 and mSin3A. Disruption of histone deacetylase activity either by TPX or by direct mutation of a histidine presumed to be in the active site abrogates HDAC1-mediated transcriptional repression of a targeted reporter gene in vivo.