Degeneration of cholinergic basal forebrain nuclei after focally evoked status epilepticus.

Status epilepticus (SE) of limbic onset might cause degenerative phenomena in different brain structures, and may be associated with chronic cognitive and EEG effects. In the present study SE was evoked focally by microinfusing picomolar doses of cyclothiazide+bicuculline into the anterior extent of the piriform cortex (APC) in rats, the so-called area tempestas, an approach which allows to evaluate selectively the effects of seizure spreading through the natural anatomical circuitries up to secondary generalization. In the brain of rats submitted to SE we analyzed neuronal density, occurrence of degenerative phenomena (by Fluoro-Jade B-FJB- staining) and expression of heat shock protein-70 (HSP-70) in the piriform cortex, the hippocampus and ventromedial thalamus. We further analyzed in detail, the loss of cholinergic neurons, and the presence of FJB- and HSP-70 positive neurons in basal forebrain cholinergic areas, i.e. the medial septal nucleus (MSN, Ch1), the diagonal band of Broca (DBB, Ch2 and Ch3) and the Nucleus basalis of Meynert (NBM, Ch4). In fact, these nuclei are strictly connected with limbic structures, and play a key pivotal role in different cognitive functions and vigilance. Although recent studies begun to investigate these nuclei in experimental epilepsy and in persons with epilepsy, conflicting results were obtained so far. We showed that after severe and long-lasting, focally induced limbic SE there is a significant cell loss within all of the abovementioned cholinergic nuclei ipsi- and contra-laterally to the infusion site. In parallel, these nuclei show also FJB and heat shock protein-70 expression. Those effects vary depending on the single nucleus assessed and on the severity of the SE seizure score. We also showed the occurrence of cell loss and degenerative phenomena in limbic cortex, hippocampus and limbic thalamic areas. These novel findings show direct evidence of SE-induced neuronal damage which is solely due to seizure activity ruling out potential confounding effects produced by systemic pro-convulsant neurotoxins. A damage to basal forebrain cholinergic nuclei, which may underlie cognitive alterations, is documented for the first time in a model of SE triggered focally.

Synthesis and characterisation of neem leaf extract, 2, 3-dehydrosalanol and quercetin dihydrate mediated silver nano particles for therapeutic applications.

The utility of green silver nanoparticles (AgNPs) in veterinary medicine is steadily increasing as they have many therapeutic applications against pathogens and arthropods of livestock. In this study, green AgNPs using neem (N-AgNPs), 2,3-dehydrosalanol (2,3-DHS-AgNPs) and quercetin dihydrate (QDH-AgNPs) were synthesised and characterised. Synthesised compounds were characterised by UV-Vis spectroscopy and the peak absorbance was recorded at 370 nm for neem extract. For N-AgNPs, 2,3-DHS-AgNPs and QDH-AgNPs, the maximum absorbance peaks were at 430, 230 and 220 nm, respectively. The FTIR analysis confirmed the synthesis of green AgNPs. The XRD pattern of N-AgNPs showed the peaks corresponding to whole spectra of 2 θ values ranging from 10-80. The relatively higher intensity of (111, 222) planes in face centred cubic crystalline structure supports the formation of synthesised AgNPs. In DLS analysis, the hydrodynamic diameter of neem leaf extract was found to be 259.8 nm, followed by 5.3, 6.7 and 261.8 nm for 2,3-DHS-AgNPs, N-AgNPs and QDH-AgNPs, respectively. Based on the transmission electron microscopy and scanning electron microscopy image analyses, confirmed the formation of N-AgNPs, 2,3-DHS-AgNPs and QDH-AgNPs. These eco-friendly phyto-AgNPs may be of use as an effective alternative to chemical control methods against the arthropods of livestock.

Subacute and subchronic toxicity of Avalon(®) mixture (bentazone+dicamba) to rats.

Subacute and subchronic toxicity of the herbicide Avalon(®), a mixture of bentazone and dicamba, were tested on rats. Avalon(®) was administered at dose levels of 250, 500 and 1000mg/kg body weight/day for 28 and 90 days. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activities were monitored together with biochemistry parameters. The results showed that the mixture caused increases in the activities of ALT, AST and ALP, elevated concentrations of sodium, albumin and albumin/globulin ratio in males. In females, ALT activity, cholesterol and phosphate levels were increased. The changes generally were dose related and, in most cases, females exhibited lower susceptibility than males. The effects of a mixture are, in the most cases, different from the effects of the individual substances. The effects of bentazone were not prevalent which would be expected taking the composition of the mixture into account.

Raphe AMPA receptors and nicotinic acetylcholine receptors mediate ketamine-induced serotonin release in the rat prefrontal cortex.

Several lines of evidence indicate that ketamine has a rapid antidepressant-like effect in rodents and humans, but underlying mechanisms are unclear. In the present study, we investigated the effect of ketamine on serotonin (5-HT) release in the rat prefrontal cortex by in vivo microdialysis. A subcutaneous administration of ketamine (5 and 25 mg/kg) significantly increased the prefrontal 5-HT level in a dose-dependent manner, which was attenuated by local injection of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) antagonists into the dorsal raphe nucleus (DRN). Direct stimulation of AMPARs in the DRN significantly increased prefrontal 5-HT level, while intra-DRN injection of ketamine (36.5 nmol) had no effect. Furthermore, intra-DRN injection of an α 4 ß 2-nicotinic acetylcholine receptor (nAChR) antagonist, dihydro-ß-erythroidine (10 nmol), significantly attenuated the subcutaneous ketamine-induced increase in prefrontal 5-HT levels. These results suggest that AMPARs and α 4 ß 2-nAChRs in the DRN play a key role in the ketamine-induced 5-HT release in the prefrontal cortex.

Effects of bentazone on lipid peroxidation and antioxidant systems in human erythrocytes in vitro.

Bentazone, a benzothiadiazole herbicide, is widely used for a variety of crops including cereals, maize, peas, rice and soy beans. The concern for human health is stil very high because bentazone is continuously monitored in environment and several studies to evaluate its potential carcinogenic effects when chronic and high doses were administered to animals. We aimed to investigate the possible effects of bentazone on lipid peroxidation, levels of glutathione and activities of antioxidant enzymes in human erythrocytes in vitro. For that, erythrocyte were incubated with bentazone in different concentrations (0-50 nM) at 37 °C for 1 hr. Bentazone showed significant increase in the levels of malondialdehyde (MDA) at the highest concentration in erythrocytes as an index of lipid peroxidation. Besides, alterations in the levels of reduced glutathione (GSH) and activities of glutathione peroxidase (GSH-Px) were observed while the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GSH-Rd) were unchanged. In conclusion, findings from this study indicate that in vitro toxicity of bentazone may be associated with oxidative stress and this work warrants further in vivo investigations.

A physiologically based modeling strategy during preclinical CNS drug development.

Physiologically based pharmacokinetic (PBPK) modeling of the central nervous system (CNS) provides the opportunity to predict the relevant drug concentrations at the therapeutic target site during preclinical and clinical development. In order to successfully interpret model results, and to provide confidence in the subsequent human predictions, it is essential that an appropriate model structure is chosen at the preclinical stage which takes into account both physiological and drug-specific knowledge. However, the models published to date in the literature show significant variation in the approaches applied by different authors, which can lead to difficulties in the interpretation of model parameter estimates. We aimed to develop a coherent PBPK modeling approach in the rat, which would also be adaptable depending on the quantity and quality of in vivo data obtained during drug development. Based on a sensitivity analysis of the model parameters, and using three CNS drugs as case studies (atomoxetine, acetaminophen, and S 18986), we proposed a decision tree to aid in the appropriate parametrization and structure of the model according to the data available. We compared our parameter estimates to those originally published, and considered the impact of the respective approaches on the mechanistic interpretation of the parameter values. Since the measurement of brain extracellular fluid (ECF) concentrations using microdialysis is not routinely performed in the industrial environment, we also evaluated the bottom-up scaling of in vitro permeability data from the Caco-2 cell line to predict BBB passive permeability in the absence of measured ECF concentrations. Our strategy demonstrates the value of PBPK as a prediction tool throughout the development process of CNS-targeting drugs.

S 18986 reverses spatial working memory impairments in aged mice: comparison with memantine.

RATIONALE: Normal or pathological ageing is characterized by working-memory dysfunction paired with a marked reduction in several neurotransmitters activity. The development of therapeutic strategy centered on the glutamatergic system known to bear a critical role in cognitive functions, is therefore of major importance in the treatment of mild forms of AD or age-related memory dysfunctions. OBJECTIVES: In Experiment 1, we investigated the effects of ageing on spatial working memory measured by sequential alternation (SA). Thus, the decay of alternation rates over a series of trials separated by varying intertrial temporal intervals (ITI, from 5 sec to 180 sec) was studied in mice of different age groups. In Experiment 2, we investigated the memory-enhancing potential of S 18986--a modulator of AMPA receptors--on age-related SA impairments, in comparison with memantine--an antagonist of NMDA receptors--. RESULTS: In Experiment 1, aged mice responded at chance with shorter ITI's and exhibited greater levels of interference in the SA task as compared to young adult mice. In Experiment 2, (1) S 18986 at 0.03 and 0.1 mg/kg reversed the memory deficit in aged mice but did not modify performance in young adult mice; (2) memantine at 10 mg/kg also increased SA rates in aged mice but did not improve performance in young adult mice. CONCLUSION: The SA task is a useful tool to reveal age-induced time-dependent working memory impairments. As compared to memantine, S 18986--a compound targeting AMPA receptors--contributes a valuable therapy in the treatment of age-related cognitive dysfunctions or mild forms of AD.

Epileptiform response of CA1 neurones to convulsant stimulation by cyclothiazide, kainic acid and pentylenetetrazol in anaesthetized rats.

We have previously reported that cyclothiazide (CTZ) evokes epileptiform activities in hippocampal neurons and induces seizure behavior. Here we further studied in vivo the sensitivity of the hippocampal CA1 neurons in response to CTZ in epileptogenesis in comparison with two other classic convulsants of kainic acid (KA) and pentylenetetrazol (PTZ). CTZ administered intracerebral ventricle (i.c.v.) induced epileptiform activities from an initial of multiple evoked population spikes, progressed to spontaneous spikes and finally to highly synchronized burst activities in hippocampal CA1 neurons. PTZ, when given by subcutaneously, but not by intracerebral ventricle injection, evoked similar progressive epileptiform activities. In contrast, KA given by i.c.v. induced a quick development of epileptiform burst activities and then shortly switched to continuous high frequency firing as acute status epilepticus (ASE). Pharmacologically, alprazolam, a high-potency benzodiazepine ligand, inhibited CTZ and PTZ, but not KA, induced epileptiform burst activities while GYKI 53784, an AMPA receptor antagonist, suppressed CTZ and KA but not PTZ evoked epileptiform activities. In conclusion, CTZ and PTZ induced epileptiform activities are most likely to share a similar progressive pattern in hippocampus with GABAergic mechanism dominant in epileptogenesis, while CTZ model involves additional glutamate receptor activation. KA induced seizure in hippocampus is different to that of both CTA and PTZ. The results from this study indicate that hippocampal neurons respond to various convulsant stimulation differently which may reflect the complicated causes of the seizure in clinics.

The AMPA receptor positive allosteric modulator, S18986, is neuroprotective against neonatal excitotoxic and inflammatory brain damage through BDNF synthesis.

Brain lesions induced in newborn mice by the glutamatergic agonists ibotenate (acting on NMDA and metabotropic receptors) or S-willardiine (acting on AMPA-kainate receptors) mimic some aspects of periventricular white matter lesions and neocortical grey matter damage observed in human neonates at risk for developing cerebral palsy. The neonatal mouse brain can be sensitized to excitotoxic damage by IL-1beta exposure similar to that observed in the human situation. Positive modulators of AMPA receptors have received increasing attention as potential neuroprotective agents in a number of neurodegenerative disorders of the adult. However whether they can also act as a neuroprotectant in neonatal brain damage has yet to be defined. Therefore the present study uses a well-defined rodent model of neonatal excitotoxic brain lesions to assess the neuroprotective effects of S18986, a positive allosteric modulator of AMPA receptors, as well as its mechanisms of action. In this model, S18986 provided a dose-dependent and long-lasting protection of developing white matter and cortical grey matter against an excitotoxic insult and also when this was combined with a sensitizing inflammatory insult. Neuroprotective effects of S18986 in cortical grey matter involved decreased necrotic and apoptotic cell death. S18986-induced neuroprotection against NMDA receptor-mediated brain lesions was blocked by inhibitors of ERK and PI3 kinase-Akt pathways. S18986 effects were abolished by a neutralizing anti-BDNF antibody and real time PCR confirmed the stimulation by S18986 of BDNF production in the neonatal brain. The present study provides strong experimental support for the role of S18986 as a candidate molecule for therapy in cases of excitotoxic perinatal brain lesions and identifies BDNF as a key mediator of this S18986-mediated neuroprotection.

Detection of urinary markers for thiazide diuretics after oral administration of hydrochlorothiazide and altizide-relevance to doping control analysis.

In sports, thiazide diuretics are used to flush out previously taken prohibited substances with forced diuresis and in sports where weight classes are involved to achieve acute weight loss. Thiazide diuretics include compounds which are very unstable and hydrolyse in aqueous media. Because information regarding the urinary detection of the hydrolysis products is limited, urinary excretion profiles for the hydrolysis product 4-amino-6-chloro-1,3-benzenedisulphonamide were established in 6 healthy volunteers after oral administration of altizide (15 mg per tablet) and hydrochlorothiazide (25mg per tablet). Additionally, the excretion profile of chlorothiazide, a metabolite of altizide and hydrochlorothiazide, was also determined. A quantitative liquid-chromatographic tandem mass spectrometric method to detect the 4 substances was developed and validated. The result of this work shows that altizide is eliminated within 48 h in urine whereas hydrochlorothiazide was detectable after 120 h. Chlorothiazide was determined to be a minor metabolite of altizide and hydrochlorothiazide and could be detected up to 120 h. The hydrolysis product, 4-amino-6-chloro-1,3-benzenedisulphonamide, was detectable 120 h after administration, with concentrations at least 10 times higher than the parent drug. Concentrations ranged between 41-239 and 60-287 ng/mL after altizide and hydrochlorothiazide administration, respectively. The study shows that 4-amino-6-chloro-1,3-benzenedisulphonamide is an important target compound for the long time detection of thiazide diuretics in urine.

Cyclothiazide: a subunit-specific inhibitor of GABAC receptors.

We tested the effects of cyclothiazide (CTZ), an agent used to block desensitization of AMPA-type glutamate receptors, on heterologously expressed GABA(C) receptors formed by homomeric rho subunits. CTZ inhibition of GABA(C) receptors was subunit specific; it produced a dose-dependent reduction of the GABA-elicited current on homomeric rho2 receptors with an IC(50) of about 12 microm, but had no significant effect on homomeric rho1 receptors. This differential sensitivity was attributable to a single amino acid located on the second transmembrane domain of the rho subunits. Mutating the residue at this position from serine to proline on the rho2 subunit eliminated CTZ sensitivity, whereas switching proline to serine on the rho1 subunit made the receptor CTZ sensitive. The inhibitory properties of CTZ were consistent with its action as a channel blocker on the receptors formed by rho2 subunits. The effect showed a small degree of voltage dependence, and was due mainly to a non-competitive mechanism that reduced the maximum response elicited by GABA. In addition, the prominent membrane current rebound when co-application of GABA and CTZ was terminated suggests that the binding site for CTZ on the GABA(C) receptor is distinct from that for GABA, and that CTZ acts as a non-competitive antagonist on the GABA(C) receptor. CTZ inhibited the open channel of the GABA(C) receptor with a time constant of about 0.4 s, but the kinetics were approximately 10-fold slower when GABA is absent. The ability of CTZ to interact with various types of neurotransmitter receptors indicates that the drug has multiple actions in the CNS.

Behavioral and biological effects of chronic S18986, a positive AMPA receptor modulator, during aging.

AMPA receptors are a major subtype of ionotropic receptors that respond to glutamate. Positive allosteric modulators of AMPA receptors selectively enhance fast excitatory neurotransmission in the brain and increase overall neuronal excitability. In addition to enhancing cognitive performance, S18986 (Servier, France) and other AMPA receptor modulators have also been shown to be neuroprotective. A particularly relevant context for AMPAR modulator studies is during aging because of increased neuronal vulnerability. It is currently unknown if chronic AMPAR modulator treatment can alter the course of brain aging, a process characterized by impairment of cognitive function, reduced neuronal excitability, and increased inflammation in the brain. We examined the behavioral and some relevant CNS effects of chronic S18986 in rats from 14 to 18 months of age. Here we show that chronic, oral administration of S18986 increases locomotor activity and performance in a spatial memory task in aged rodents. In addition, chronic S18986 treatment retards the decline of forebrain cholinergic neurons by roughly 37% and midbrain dopaminergic neurons by as much as 43% during aging and attenuates the age-related increase in the expression of a microglial marker in the hippocampus. These results provide a framework for further studies of the potentially beneficial effects of AMPAR modulators on brain aging.

Comparison of single vs. multiple administrations of the AMPA receptors modulator S 18986 in the object recognition task in rats.

The present study aimed at defining the best scheme of administration of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-positive modulator (S)-2,3-dihydro-[3,4]-cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide (S 18986) [once daily (o.d.) administration of 1 mg/kg for 3 days vs. three times daily (t.i.d.) administration of 0.3 mg/kg for 3 days] to get an optimal procognitive activity in the object recognition task in rats. Memory performance [Recognition Index (RI)] of rats was significantly improved 1 h (RI = 41%, P < 0.01) and 3 h (RI = 46%, P < 0.001) following oral administration of S 18986 (1 mg/kg, o.d.) when compared with animals receiving the vehicle (RI = 6%). When the interval between administration and testing was increased to 6 h and 9 h, no statistically significant improvement in memory performance was observed (RI = 42% for 6 h and RI = 18% for 9 h vs. 20% for the vehicle group). When S 18986 was administered at 0.3 mg/kg t.i.d., no statistically significant improvement in memory performance was observed (RI = 36%). These findings show a long-lasting efficacy of the AMPA receptor allosteric modulator in the object recognition task despite a short half-life in plasma and in brain (approximately 1 h). Accordingly, multiple administrations of S 18986 are not required to obtain a maximal efficacy in this paradigm, because a o.d. schedule of administration leads to a powerful procognitive activity.

Targeting AMPA receptor gating processes with allosteric modulators and mutations.

Allosteric modulators and mutations that slow AMPAR desensitization have additional effects on deactivation and agonist potency. We investigated whether these are independent actions or the natural consequence of slowing desensitization. Effects of cyclothiazide (CTZ), trichlormethiazide (TCM), and CX614 were compared at wild-type GluR1 and "nondesensitizing" GluR1-L497Y mutant receptors by patch-clamp recording with ultrafast perfusion. CTZ, TCM, or L/Y mutation all essentially blocked GluR1 desensitization; however, the effects of L/Y mutation on deactivation and glutamate EC50 were three to five times greater than for modulators. CTZ and TCM further slowed desensitization of L/Y mutant receptors but paradoxically accelerated deactivation and increased agonist EC50. Results indicate that CTZ and TCM target deactivation and agonist potency independently of desensitization, most likely by modifying agonist dissociation (koff). Conversely, CX614 slowed desensitization and deactivation without affecting EC50 in both wild-type and L/Y receptors. The S750Q or combined L497Y-S750Q mutations abolished all CTZ and TCM actions without disrupting CX614 activity. Notably, the S/Q mutation also restored L/Y deactivation and EC50 to wild-type levels without restoring desensitization, further demonstrating that desensitization can be modulated independently of deactivation and EC50 by mutagenesis and possibly by allosteric modulators.

Cuticular uptake of xenobiotics into living plants. Part 2: influence of the xenobiotic dose on the uptake of bentazone, epoxiconazole and pyraclostrobin, applied in the presence of various surfactants, into Chenopodium album, Sinapis alba and Triticum aestivum leaves.

This study has determined the uptake of three pesticides, applied as commercial or model formulations in the presence of a wide range of surfactants, into the leaves of three plant species (bentazone into Chenopodium album L. and Sinapis alba L., epoxiconazole and pyraclostrobin into Triticum aestivum L.). The results have confirmed previous findings that the initial dose (nmol mm(-2)) of xenobiotic applied to plant foliage is a strong, positive determinant of uptake. This held true for all the pesticide formulations studied, although surfactant concentration was found to have an effect. The lower surfactant concentrations studied showed an inferior relationship between the amount of xenobiotic applied and uptake. High molecular mass surfactants also produced much lower uptake than expected from the dose uptake equations in specific situations.

Cyclothiazide induces robust epileptiform activity in rat hippocampal neurons both in vitro and in vivo.

Cyclothiazide (CTZ) is a potent blocker of AMPA receptor desensitization. We have recently demonstrated that CTZ also inhibits GABA(A) receptors. Here we report that CTZ induces robust epileptiform activity in hippocampal neurons both in vitro and in vivo. We first found that chronic treatment of hippocampal cultures with CTZ (5 microM, 48 h) results in epileptiform activity in the majority of neurons (80%). The epileptiform activity lasts more than 48 h after washing off CTZ, suggesting a permanent change of the neural network properties after CTZ treatment. We then demonstrated in in vivo recordings that injection of CTZ (5 micromol in 5 microl) into the lateral ventricles of anaesthetized rats also induces spontaneous epileptiform activity in the hippocampal CA1 region. The epileptogenic effect of CTZ is probably due to its enhancing glutamatergic neurotransmission as shown by increasing the frequency and decay time of mEPSCs, and simultaneously inhibiting GABAergic neurotransmission by reducing the frequency of mIPSCs. Comparing to a well-known epileptogenic agent kainic acid (KA), CTZ affects neuronal activity mainly through modulating synaptic transmission without significant change of the intrinsic membrane excitability. Unlike KA, which induces significant cell death in hippocampal cultures, CTZ treatment does not result in any apparent neuronal death. Therefore, the CTZ-induced epilepsy model may provide a novel research tool to elucidate the molecular and cellular mechanisms of epileptogenesis without any complication from drug-induced cell death. The long-lasting epileptiform activity after CTZ washout may also make it a very useful model in screening antiepileptic drugs.

Neuropharmacokinetics of a new alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) modulator, S18986 [(S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide], in the rat.

The aim of our study was to determine the neuropharmacokinetics of S18986 [(S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide], a new positive allosteric modulator of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type receptors, in the rat. We focused on its blood-brain barrier (BBB) uptake and on its brain intra- and extracellular fluid (bICF-bECF) partitioning. BBB transport of S18986 was measured using the in situ brain perfusion technique. bECF concentrations were determined by microdialysis in the two effector areas, i.e., frontal cortex (FC) and dorsal hippocampus (DH), and blood samples were collected simultaneously through a femoral catheter. Cerebrospinal fluid and brain tissue concentrations were determined using a conventional pharmacokinetic approach. Using all the experimental data, pharmacokinetic modeling was applied to describe the S18986 blood-brain disposition. The brain uptake clearance of S18986 was found to be high, about 20 mul s(-1) g(-1). Terminal half-lives were similar in plasma and brain, at around 1 h. Experimental and predicted blood and brain concentrations were a good fit with the pharmacokinetic model, which assumed first-order rate constants at each interface. Ratios of bECF to the unbound plasma area under the curve (AUC) were 0.24 in FC and 0.25 in DH, whereas ratios of bICF/plasma AUC were 1 in FC and 1.5 in DH. We conclude that despite the ratio of bECF/plasma AUC below 1, there is nevertheless an elevated BBB uptake of S18986. This can be explained by the S18986 nonhomogenous bECF/bICF partitioning, since S18986 mainly distributes into hippocampal bICF. This illustrates the importance of taking bECF/bICF partitioning into account when interpreting the neuropharmacokinetics of a drug.

Piperidine carboxylic acid derivatives of 10H-pyrazino[2,3-b][1,4]benzothiazine as orally-active adhesion molecule inhibitors.

Novel piperidine carboxylic acid derivatives of 10H-pyrazino[2,3-b][1,4]benzothiazine were prepared and evaluated for their inhibitory activity on the upregulation of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1). Replacement of the methanesulfonyl group on the piperidine ring of previously prepared derivatives with a carboxylic acid-containing moiety resulted in a number of potent adhesion molecule inhibitors. Of these, (anti) [3-(10H-pyrazino[2,3-b][1,4]benzothiazin-8-yl)methyl-3-azabicyclo[3.3.1]non-9-yl]acetic acid 2q (ER-49890), showed the most potent oral inhibitory activities against neutrophil migration in an interleukin-1 (IL-1) induced paw inflammation model using mice, and leukocyte accumulation in a carrageenan pleurisy model in the rat, and therapeutic effect on collagen-induced arthritis in rats.

Anti-HIV-1 activity of benzothiadiazine dioxide.

Antiviral assays carried out on the potent benzothiadiazine dioxide (BTD) human cytomegalovirus (HCMV) inhibitors have led us to find marginal but selective anti-HIV-1 activity. Specific pharmacological studies, such as time of addition experiments and assays on specific viral strains with mutations on its reverse transcriptase, have indicated that BTD compounds act as non-nucleoside reverse transcriptase inhibitors. Theoretical calculations showed a butterfly conformation for the active derivatives that are compatible with their mechanism of action. Therefore, BTD derivatives can be considered as potential lead compounds for the treatment of opportunistic HCMV infections in immunocompromised individuals such as AIDS patients.

Characterization of audiogenic-like seizures in naive rats evoked by activation of AMPA and NMDA receptors in the inferior colliculus.

The role of glutamate receptors in the inferior colliculus (IC) in audiogenic and audiogenic-like seizures was investigated in adult rats with transient neonatal hypothyroidism by 0.02% propylthiouracil (PTU) treatment through mother's milk (PTU rats) and in naive rats treated intracisternally with N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid (AMPA), or cyclothiazide, an inhibitor of rapid AMPA receptor desensitization. All rats showed audiogenic or audiogenic-like seizures characterized by running fit (RF) and generalized tonic-clonic seizures (GTCS). While systemically administered MK-801 inhibited GTCS, intracisternally administered NBQX inhibited RF and GTCS in both audiogenic and audiogenic-like seizures. Auditory stimulation shortened the latency to GTCS induced by AMPA, but not NMDA, at a subclinical dose and further elongated the shortened duration of RF, but not GTCS, induced by MK-801 pretreatment. Furthermore, Northern blot analysis was used to evaluate the expression of the immediate-early gene c-fos in the IC following induction of audiogenic or audiogenic-like seizures. The significant induction of c-fos mRNA by audiogenic seizures in PTU rats or by AMPA- or cyclothiazide-induced seizures in naive rats was prominent in the IC. MK-801 suppressed c-fos mRNA expression in the IC induced by audiogenic seizures in PTU rats or by AMPA-induced seizures in naive rats. NBQX suppressed the expression of c-fos mRNA in the IC induced by AMPA-induced seizures but did not suppress c-fos mRNA in PTU rats or rats with cyclothiazide-induced seizures. Auditory stimuli failed to affect c-fos mRNA induction by AMPA. The present study suggests that audiogenic-like seizures can be reproduced by glutamate receptor agonists in which AMPA receptors are primarily linked to the initiation of audiogenic seizures (RF) while NMDA receptors presumably located within the IC are involved in the propagation of GTCS in audiogenic seizures.