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.

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.

Autoradiographic localization of 5-CT-insensitive 5-HT1-like recognition sites in guinea pig and rat brain.

Quantitative autoradiographic studies, with [3H]5-HT, were used to investigate the distribution of 5-CT-insensitive 5-HT1-like (5-HT1E/1F) recognition sites in rat and guinea pig brain. For comparison and control purposes the distribution of the closely related 5-HT1D binding site, which is abundant in the guinea pig but not the rat, was also investigated, as well as total specific [3H]5-HT binding. Results from this study confirm the previously described regional distribution of the 5-HT1D binding site and also revealed a predominance of 5-CT-insensitive 5-HT1-like 5-HT1E/1F) recognition sites in the olfactory tubercle, caudate putamen, nucleus accumbens and substantia nigra of both species. Interestingly 5-CT-insensitive 5-HT1-like (5-HT1E/1F) recognition sites were particularly dense in the claustrum of the guinea pig, but not the rat.

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).

Synthesis and biological activity of 3-[2-(dimethylamino)ethyl]-5-[(1,1-dioxo-5-methyl-1,2,5-thiadiazolidin- 2-yl)-methyl]-1H-indole and analogues: agonists for the 5-HT1D receptor.

A novel series of 5-(1,1-dioxo-1,2,5-thiadiazolidin-2-yl)tryptamines was designed, synthesized, and evaluated as 5-HT1D receptor agonists. Compounds such as 8d,f,k were identified which had comparable affinity, potency, and receptor selectivity to that of the antimigraine drug sumatriptan. Both 8d,k were found to be well absorbed in the rat with oral bioavailabilities of 66% and 62%, respectively. Additionally, 8d was found to be selective over other non-serotonergic receptors and exhibited relatively low central nervous system penetration.

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.

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.

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.

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.

L-694,247: a potent 5-HT1D receptor agonist.

1. The 5-hydroxytryptamine (5-HT) receptor binding selectivity profile of a novel, potent 5-HT1D receptor agonist, L-694,247 (2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl ]- 1H-indole-3-yl]ethylamine) was assessed and compared with that of the 5-HT1-like receptor agonist, sumatriptan. 2. L-694,247 had an affinity (pIC50) of 10.03 at the 5-HT1D binding site and 9.08 at the 5-HT1B binding site (sumatriptan: pIC50 values 8.22 and 5.94 respectively). L-694,247 retained good selectivity with respect to the 5-HT1A binding site (pIC50 = 8.64), the 5-HT1C binding site (6.42), the 5-HT2 binding site (6.50) and the 5-HT1E binding site (5.66). The pIC50 values for sumatriptan at these radioligand binding sites were 6.14, 5.0, < 5.0 and 5.64 respectively. Both L-694,247 and sumatriptan were essentially inactive at the 5-HT3 recognition site. 3. L-694,247, like sumatriptan, displayed a similar efficacy to 5-HT in inhibiting forskolin-stimulated adenylyl cyclase in guinea-pig substantia nigra although L-694,247 (pEC50 = 9.1) was more potent than sumatriptan (6.2) in this 5-HT1D receptor mediated functional response. L-694,247 (pEC50 = 9.4) was also more potent than sumatriptan (6.5) in a second 5-HT1D receptor mediated functional response, the inhibition of K(+)-evoked [3H]-5-HT release from guinea-pig frontal cortex slices. 4. The excellent agreement observed for L-694,247 between the 5-HTlD radioligand binding affinity and the functional potency confirm that the two functional models (the inhibition of forskolin-stimulated adenylyl cyclase in guinea-pig substantia nigra and the inhibition of K+-evoked [3H]-5-HT release from guinea-pig frontal cortex) do indeed reflect 5-HTID-mediated events.5. L-694,247 is a novel, highly potent 5-HTID/5-HTIB receptor ligand which should prove useful for the exploration of the physiological role of these receptors in animals.

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.

Characterisation of a cloned human 5-HT1A receptor cell line using [35S]GTP gamma S binding.

Compound potencies and efficacies depend upon receptor reserve and hence estimating this parameter in assay systems allows for a more meaningful interpretation of the data generated. This study describes a method whereby the degree of receptor reserve, with respect to 5-hydroxytryptamine (5-HT), was determined for a HeLa cell line expressing the human 5-HT1A receptor using the agonist-induced [35S]guanosine 5'[gamma-thio]triphosphate ([35S]GTP gamma S) binding assay, followed by a comparison of the potencies and relative efficacies of several compounds. Following irreversible antagonism with benextramine 5-HT yielded a pKA of 7.3, compared with a pKobs of 8.4 from saturation analysis, indicating the presence of high and low affinity state receptors. A 20% receptor occupancy elicited a half-maximal functional response consistent with the presence of receptor reserve. 5-HT, 5-carboxamidotryptamine (5-CT), 8-hydoxy-dipropylamino-tetralin (8-OH-DPAT), 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4-yl)-1 H-indole (RU24969), buspirone, gepirone, mesulergine and sumatriptan were equally efficacious. 1-(2-Methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine (NAN 190) displayed reduced relative efficacy and methiothepin inverse agonism.

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.

[3H]L-694,247 labels the 5-HT1D beta receptor in pig caudate membranes.

This study, carried out in pig caudate membranes, characterises the radioligand binding site labelled with [3H]L-694,247 (2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl ]-1H-indole-3 - yl]ethylamine). The affinities of 27 standard compounds are consistent with binding to a 5-HT1D recognition site. In addition the results indicate that, under the assay conditions described, [3H]L-694,247 specifically labels the 5-HT1D beta recognition site since ketanserin and ritanserin display a low affinity consistent with their activities at this subtype of the 5-HT1D receptor.

5-Carboxamidotryptamine-insensitive 5-HT1-like receptors are concentrated in guinea pig but not rat, claustrum.

5-CT (5-carboxamidotryptamine)-insensitive (5-HT1E/5-HT1F) 5-HT1-like recognition sites have been mapped autoradiographically in rat and guinea pig brain using [3H]5-HT in the presence of 5-CT and mesulergine to mask 5-HT1A, 5-HT1B, 5-HT1D and 5-HT2C binding sites. Binding was more dense in the guinea pig but in both species 5-CT-insensitive 5-HT1-like sites were located in the olfactory tubercle, interpeduncular nucleus, caudate putamen, nucleus accumbens, substantia nigra, frontal cortex and hippocampus. These receptors were particularly marked in the claustrum of the guinea pig but not the rat.

An investigation of the 5-HT1D receptor binding affinity of 5-hydroxytryptamine, 5-carboxyamidotryptamine and sumatriptan in the central nervous system of seven species.

The ability of three 5-HT1 receptor agonists, 5-HT (5-hydroxytryptamine), 5-CT (5-carboxyamidotryptamine) and sumatriptan to inhibit the binding of [3H]5-HT, in the presence of cyanopindolol and mesulergine, from cerebral cortical and/or caudate membranes in seven species (dog, guinea-pig, rabbit, pig, human, hamster and calf) has been investigated. Under the experimental conditions used, 5-CT and sumatriptan consistently yielded displacement curves best fit to a two-site model whereas 5-HT always gave a monophasic displacement curve. The pIC50 values obtained with 5-HT displacement gave a mean of 8.1 +/- 0.1 (mean +/- S.E.M.). In contrast the biphasic displacement curves for 5-CT and sumatriptan yielded high and low affinity pIC50 values of 8.3 +/- 0.1, 5.5 +/- 0.1 and 7.6 +/- 0.1, 5.0 +/- 0.1, respectively. These data indicate that under these experimental conditions the high affinity component labelled by [3H]5-HT is the same receptor subtype, previously denoted the 5-HT1D receptor, in all seven species.

Interaction of the atypical neuroleptic clozapine with 5-HT3 receptors in the cerebral cortex and superior cervical ganglion of the rat.

Clozapine, an atypical neuroleptic drug devoid of extrapyramidal side effects, was a moderately potent, competitive inhibitor of the binding of [3H]quaternised ICS 205-930 to 5-HT3 receptor sites in rat cortical membranes, possessing a pKi value of 7.0. In contrast, several other antipsychotic agents, including fluphenazine, alpha-flupenthixol, haloperidol, spiperone and (-)-sulpiride were essentially inactive. Clozapine also antagonised the 2-methyl 5-HT-induced depolarisation of the rat isolated superior cervical ganglion, a response known to be mediated via 5-HT3 receptors. Clozapine (0.1-1 microM) induced parallel displacements to the right of the dose-response curve to 2-methyl 5-HT in this tissue, possessing a pKb value of 7.3. These data suggest that the atypical antipsychotic profile of clozapine may be related, at least, in part to its ability to interact with central 5-HT3 receptor sites.

Induction of DNA breaks in SV40 by heavy ions.

Simian virus (SV40) DNA was used to study the induction of DNA strandbreaks by heavy ions varying in LET. DNA was exposed to X-rays and to accelerated particles either in dilute solution or in the presence of different radical scavengers. Relative proportions of the intact supercoiled DNA, nicked form arising from single strand breaks (SSB) and linear molecules produced by double strandbreaks (DSB) were quantified on the base of their electrophoretic mobility in agarose gels. Cross sections for the induction of SSBs and DSBs were calculated from the slope of dose effect curves. Mercaptoethanol was found to protect more efficiently against DNA strand breakage than Tris. When the biological efficiency, i.e. the number of strand breaks per unit dose and molecule weight was evaluated as a function of LET, curves for SSB induction always showed a continuous decrease. For DSB induction, an increase in the yield of DSBs with a maximum around 500 keV/micrometer was observed in the presence of radical scavenger. This peak of biological efficiency gradually disappeared when the radiosensitivity of the system was increased, and was no longer apparent in the dilute buffer system, where DNA showed a high susceptibility to strand breakage. When the relative biological efficiency was plotted versus LET, the curve for DSB induction observed in a low radical scavenging environment paralleled the curve obtained for SSB induction.