Treatment of canine sinonasal aspergillosis with clotrimazole infusion in patients with cribriform plate lysis.

OBJECTIVE: To describe the treatment of sinonasal aspergillosis with topical 1% clotrimazole solution in dogs with cribriform plate lysis. MATERIALS AND METHODS: This retrospective study includes data retrieval from medical records of dogs with sinonasal aspergillosis and cribriform plate lysis that underwent topical treatment with 1% clotrimazole solution. RESULTS: Five dogs with sinonasal aspergillosis, cribriform plate lysis diagnosed on CT scans, and normal neurologic examinations were treated with a single (n=3) or multiple (n=2) infusions of clotrimazole solution. No dogs developed clinical neurologic disease after therapy. CLINICAL SIGNIFICANCE: In this study, a topical clotrimazole solution was not associated with adverse neurologic effects in neurologically normal dogs with sinonasal aspergillosis and cribriform plate lysis.

Building comparative gene expression databases for the mouse preimplantation embryo using a pipeline approach to UniGene.

To understand early mammalian development there is a need to compare profiles of gene expression from different stages of the preimplantation mouse embryo. We describe here a method that uses gene expression data held in the UniGene database of the National Institutes of Health (NIH). The full mouse UniGene database (build #151) contains 43,104 gene clusters generated from approximately 4.1 million sequences. The Expressed Sequence Tags (EST) used to build UniGene are derived from cDNA libraries that are archived separately in the database of Expressed Sequence Tags (dbEST) database, with their own catalogue numbers. The mouse dbEST database contains 32 non-normalized dbEST libraries constructed from preimplantation stages (unfertilized oocyte, fertilized oocyte, 2-, 4-, 8- and 16-cell embryo and blastocyst). These libraries contain 219,852 EST sequences mapping to 15,731 UniGene clusters. We have developed a computational pipeline approach that imports and aggregates inventories of gene expression contained in these dbEST libraries. It uses these data to build an annotated web-based database of preimplantation gene expression with an in-built capacity for comparison of expression profiles. Comparison of gene expression profiles obtained for each developmental stage show statistically significant changes in gene expression during preimplantation development. These in silico-generated profiles were validated using RT-PCR.

Genetic knockout and pharmacological blockade studies of the 5-HT7 receptor suggest therapeutic potential in depression.

The affinity of several antidepressant and antipsychotic drugs for the 5-HT7 receptor and its CNS distribution suggest potential in the treatment of psychiatric diseases. However, there is little direct evidence of receptor function in vivo to support this. We therefore evaluated 5-HT7 receptors as a potential drug target by generating and assessing a 5-HT7 receptor knockout mouse. No difference in assays sensitive to potential psychotic or anxiety states was observed between the 5-HT7 receptor knockout mice and wild type controls. However, in the Porsolt swim test, 5-HT7 receptor knockout mice showed a significant decrease in immobility compared to controls, a phenotype similar to antidepressant treated mice. Intriguingly, treatment of wild types with SB-258719, a selective 5-HT7 receptor antagonist, did not produce a significant decrease in immobility unless animals were tested in the dark (or active) cycle, rather than the light, adding to the body of evidence suggesting a circadian influence on receptor function. Extracellular recordings from hypothalamic slices showed that circadian rhythm phase shifts to 8-OH-DPAT are attenuated in the 5-HT7 receptor KO mice also indicating a role for the receptor in the regulation of circadian rhythms. These pharmacological and genetic knockout studies provide the first direct evidence that 5-HT7 receptor antagonists should be investigated for efficacy in the treatment of depression.

Basic fibroblast growth factor expression in isolated small human ovarian follicles.

Basic fibroblast growth factor (bFGF) is involved in cell proliferation, differentiation, and angiogenesis. It has long been known that bFGF acts as a powerful mitogen for various mammalian granulosa cells in culture. To investigate the possible involvement of bFGF expression in follicle initiation and growth in vivo, we performed nested RT-PCR on ovarian cortical biopsies and quantitative PCR on human follicle populations isolated by laser capture microdissection. Using morphological criteria, follicles were characterized as putative non-growing, primary, or small secondary. RNA was extracted from samples, reverse-transcribed, and relative gene expression levels determined with TaqMan real-time PCR, using 18S rRNA as the endogenous control. Results confirmed bFGF expression in human adult ovarian cortex, and in the isolated follicles a down-regulation of bFGF mRNA was evident as small follicles develop. This study demonstrates a possible relationship between bFGF mRNA expression and follicle development.

The hypothermic effect of 5-CT in mice is mediated through the 5-HT7 receptor.

The 5-HT(7) receptor is a recent addition to the 5-HT receptor family and to date there is no clear idea as to its potential role in the CNS. The receptor has been mapped by in situ hybridization and 5-HT(7)-like immunoreactivity and has been detected in discrete areas of the brain including the hypothalamus (Oliver et al., 1999). This suggests the receptor may be involved in temperature regulation and have shown that a selective 5-HT(7) receptor antagonist reverses the hypothermic effect of 5-CT in guinea-pigs. The current study confirmed that the 5-HT(7) receptor antagonists, SB-269970 (1-30 mg/kg, i.p.) and SB-258719 (5-20 mg/kg, i.p.), but not the 5-HT(1A) receptor antagonist, WAY 100635(0.1-1 mg/kg, s.c.), or the 5-HT(1B/D) antagonist, GR127935 (1.25-5 mg/kg, i.p.), reversed the hypothermic effect of 5-CT in mice. In addition the effect of 5-CT on body temperature was examined on 5-HT(7) receptor null mutant mice. 5-CT (0.1-1 mg/kg, i.p.) significantly reduced rectal temperature in wildtype but not 5-HT(7) receptor knockout mice. This suggests that the hypothermic effects of 5-CT are mediated through the 5-HT(7) receptor. All procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act (1986).

Edg2 receptor functionality: gialpha1 coexpression and fusion protein studies.

Recombinant receptor cell lines are widely used in G-protein-coupled receptor selectivity studies. To unequivocally interpret the results of such studies, it is essential that the host cell line does not endogenously express the receptor of interest and in addition is unresponsive to the receptor's natural ligand. Here we describe an approach to overcome such difficulties associated with orphan receptors or, as in the present case, receptors whose endogenous ligand ubiquitously affects mammalian cells. The functional heterologous assay system described is for the hEdg2 receptor, which uses lysophosphatidic acid as its endogenous ligand. Once activated, this receptor mediates its effects via multiple secondary messenger pathways, including a Gi-coupled pathway. We have transiently expressed a pertussis toxin-insensitive hEdg2 receptor-ratGialpha1 fusion protein into human embryonic kidney cells and have monitored the ability of compounds to stimulate [(35)S]GTPgammaS binding in membranes prepared from these cells after pretreatment with toxin. Because the assay conditions used favor Gi-mediated responses and because endogenous Gialpha subunits are rendered inactive, the response measured is, by definition, fusion protein-mediated. Consequently, we have developed an assay that monitors definitively Edg2 receptor-mediated responses in a mammalian cell line. A limited structure activity relationship study suggests that the lysophospholipid carbon chain has a role in receptor activation and in addition indicates that certain modifications to the phosphate group are tolerated.

EDG receptors as a therapeutic target in the nervous system.

EDG receptors are a family of closely related G-protein-coupled receptors, so-called since the first family member to be cloned is encoded by an endothelial differentiation gene. Of the six family members identified, five use lysophospholipids as their endogenous ligands. The sixth receptor, EDG-6, remains an orphan. These receptors activate multiple secondary-messenger pathways involving coupling to Gi, Gq/11, and G12/13 trimeric guanine nucleotide-binding proteins and are thought to play an important role in cell growth, development and maintenance, and cytoskeletal-dependent changes. EDG receptors are expressed in most mammalian cells and tissues, each subtype having a distinct distribution pattern, raising the possibility of tissue-specific biological roles that could be explored in drug-discovery programs. In this study the distribution of EDG-receptor mRNA within the nervous system has been investigated. As seen in peripheral tissues, these receptors appear to be discretely localized within specific brain regions and cell types. For example, EDG-1, -3, -4 receptors are confined to neuronal cells, EDG-2 receptors to white matter tracts, while EDG-5 receptors appear to be expressed in various cell types, including neuronal cells, white matter tracts, and ependymal cells. EDG-6-receptor mRNA was not detected in the nervous system. Speculation as to the role of these receptors in physiological/pathophysiological processes, particularly those involving cell development, proliferation, maintenance, migration, differentiation, plasticity, and apoptosis can be made from such distribution studies. EDG receptors located in brain neuronal cells might, for example, influence apoptosis and be involved in cell rescue following ischemic damage or during the early stages of progressive neurodegenerative diseases. Those restricted to oligodendrocytes might play a crucial role in myelination and offer a potential target in the treatment of demyelinating diseases, such as multiple sclerosis. In order to explore the role of these receptors, it is necessary to identify selective compounds. To this end we have developed an agonist-induced [35S]GTP gamma S binding assay using an HEK cell line expressing a pertussis-toxin-insensitive human-EDG-2-receptor-rat-Gi alpha 1-fusion protein. Such as assay system overcomes the problems associated with the almost ubiquitous responsiveness of mammalian cells to lysophospholipid. This assay lends itself to high throughput application, opening up the possibility of identifying compounds to further probe the therapeutic potential of EDG receptor manipulation.

Molecular phenotype of the human oocyte by PCR-SAGE.

Consecutive application of PCR and serial analysis of gene expression (SAGE) was used to generate a catalog of approximately 50, 000 SAGEtags from nine human oocytes. Matches for known genes were identified using the National Institutes of Health SAGEtag database. This database links directly to the UniGene database, providing rapid discrimination between SAGEtags that match known genes and expressed sequence tags and those that currently have no match. Matches in the oocyte SAGE catalog were found for surface receptors, second-messenger systems, and cytoskeletal, apoptotic, and secreted proteins. Many of these proteins were not previously known to be expressed in mammalian oocytes. The relative abundances of transcripts for cytoskeletal proteins and proteins known to be in oocytes are consistent with their documented expression, suggesting an absence of representational distortion by the PCR step. The expression profile of the human oocyte may help identify factors that reprogram somatic cell nuclei to totipotency.

4-Hydroxy-1-[3-(5-(1,2,4-triazol-4-yl)-1H-indol-3-yl)propyl]piperidines: selective h5-HT1D agonists for the treatment of migraine.

A series of 4-hydroxy-1-[3-(5-(1,2,4-triazol-4-yl)-1H-indol-3-yl)propyl] piperidines was investigated as potential selective h5-HT1D agonists for the treatment of migraine. The 4-[(N-benzyl-N-methyl)amino]methyl analog 12a was found to be a full agonist at the h5-HT1D receptor with good binding selectivity over the h5-HT1B receptor.

3-[3-(Piperidin-1-yl)propyl]indoles as highly selective h5-HT(1D) receptor agonists.

Several 5-HT(1D/1B) receptor agonists are now entering the marketplace as treatments for migraine. This paper describes the development of selective h5-HT(1D) receptor agonists as potential antimigraine agents which may produce fewer side effects. A series of 3-[3-(piperidin-1-yl)propyl]indoles has been synthesized which has led to the identification of 80 (L-772,405), a high-affinity h5-HT(1D) receptor full agonist having 170-fold selectivity for h5-HT(1D) receptors over h5-HT(1B) receptors. L-772,405 also shows very good selectivity over a range of other serotonin and nonserotonin receptors and has excellent bioavailability following subcutaneous administration in rats. It therefore constitutes a valuable tool to delineate the role of h5-HT(1D) receptors in migraine. Molecular modeling and physical properties have been utilized to postulate the binding conformation of these compounds in the receptor cavity.

2,7-diazabicyclo[3.3.0]octanes as novel h5-HT receptor agonists.

The conformational restriction of a (benzylamino)methyl substituted pyrrolidine to form 2,7-diazabicyclo[3.3.0]octanes has led to a series of compounds with high affinity at the h5-HT1D receptor as well as dramatically increased concentrations in the hepatic portal vein following oral administration.

Fluorination of 3-(3-(piperidin-1-yl)propyl)indoles and 3-(3-(piperazin-1-yl)propyl)indoles gives selective human 5-HT1D receptor ligands with improved pharmacokinetic profiles.

It has previously been reported that a 3-(3-(piperazin-1-yl)propyl)indole series of 5-HT1D receptor ligands have pharmacokinetic advantages over the corresponding 3-(3-(piperidin-1-yl)propyl)indole series and that the reduced pKa of the piperazines compared to the piperidines may be one possible explanation for these differences. To investigate this proposal we have developed versatile synthetic strategies for the incorporation of fluorine into these ligands, producing novel series of 4-fluoropiperidines, 3-fluoro-4-aminopiperidines, and both piperazine and piperidine derivatives with one or two fluorines in the propyl linker. Ligands were identified which maintained high affinity and selectivity for the 5-HT1D receptor and showed agonist efficacy in vitro. The incorporation of fluorine was found to significantly reduce the pKa of the compounds, and this reduction of basicity was shown to have a dramatic, beneficial influence on oral absorption, although the effect on oral bioavailability could not always be accurately predicted.

3-(Piperazinylpropyl)indoles: selective, orally bioavailable h5-HT1D receptor agonists as potential antimigraine agents.

Clinically effective antimigraine drugs such as Sumatriptan have similar affinity at h5-HT1D and h5-HT1B receptors. In the search for a h5-HT1D-selective agonist as an antimigraine agent, a novel series of 3-(propylpiperazinyl)indoles have been synthesized and evaluated at h5-HT1D and h5-HT1B receptors. This class of compounds has provided subnanomolar, fully efficacious h5-HT1D agonists with up to 200-fold selectivity for the h5-HT1D receptor over the h5-HT1B receptor. Unlike other h5-HT1D-selective series, several propylpiperazines demonstrate good oral bioavailability. The optimum compound was 1-(3-[5-(1,2, 4-triazol-4-yl)-1H-indol-3-yl]propyl)-4-(2-(3-fluorophenyl)ethyl)p ipe razine (7f) which has excellent selectivity for h5-HT1D receptors over other 5-HT receptor subtypes and good oral bioavailability in three species. Compound 7f has been selected for further investigation as a potential development candidate in the treatment of migraine.

Synthesis and serotonergic activity of 3-[2-(pyrrolidin-1-yl)ethyl]indoles: potent agonists for the h5-HT1D receptor with high selectivity over the h5-HT1B receptor.

The design, synthesis, and biological evaluation of a novel series of 3-[2-(pyrrolidin-1-yl)ethyl]indoles with excellent selectivity for h5-HT1D (formerly 5-HT1Dalpha) receptors over h5-HT1B (formerly 5-HT1Dbeta) receptors are described. Clinically effective antimigraine drugs such as Sumatriptan show little selectivity between h5-HT1D and h5-HT1B receptors. The differential expression of h5-HT1D and h5-HT1B receptors in neural and vascular tissue prompted an investigation of whether a compound selective for the h5-HT1D subtype would have the same clinical efficacy but with reduced side effects. The pyrrolidine 3b was initially identified as having 9-fold selectivity for h5-HT1D over h5-HT1B receptors. Substitution of the pyrrolidine ring of 3b with methylbenzylamine groups gave compounds with nanomolar affinity for the h5-HT1D receptor and 100-fold selectivity with respect to h5-HT1B receptors. Modification of the indole 5-substituent led to the oxazolidinones 24a,b with up to 163-fold selectivity for the h5-HT1D subtype and improved selectivity over other serotonin receptors. The compounds were shown to be full agonists by measurement of agonist-induced [35S]GTPgammaS binding in CHO cells expressed with h5-HT receptors. This study suggests that the h5-HT1D and h5-HT1B receptors can be differentiated by appropriate substitution of the ligand in the region which binds to the aspartate residue and reveals a large binding pocket in the h5-HT1D receptor domain which is absent for the h5-HT1B receptor. The compounds described herein will be important tools to delineate the role of h5-HT1D receptors in migraine.

Pharmacological characterisation of a cloned dog 5-HT1B receptor cell line.

In this study, the binding of [3H]5-HT to the cloned dog 5-hydroxytryptamine1B (dog 5-HT1B) receptor, stably expressed in Chinese hamster ovary cells (ATCC CCL 61)(CHO-K1), was characterised and its pharmacology compared with that of the cloned human and rat 5-HT1B receptors. [3H]5-HT specifically labeled, with high affinity, an apparently homogeneous population of binding sites in the dog 5-HT1B receptor cell line yielding a pKd of 8.1. [3H]5-HT inhibition and agonist-induced [35S] guanosine 5'[gamma-thio] triphosphate ([35S]GTPgammaS) binding studies revealed comparable results with the human but not the rat 5-HT1B receptor. In all three recombinant receptor cell lines, methiothepin displayed inverse agonism and GR127935 (N-[4-methoxy-3-(4-methyl-1-piperizinyl)phenyl]-2'-methyl-4'-(5-me thyl-1,2,4-oxadiazole-3-yl)[1,1'-biphenyl]-carboxamide) weak partial agonism.