Patients' contribution to drug safety in Catalonia: the interest of personal feelings on adverse drug reactions.

PURPOSE: There are few studies on the personal view retrieved by patients in the spontaneous reports' free-text section of suspected adverse drug reactions. METHODS: We analysed the suspected adverse drug reactions (ADRs) spontaneous reports sent to the Catalan Centre of Pharmacovigilance between 2013 and 2017. The information provided in the free-text section was classified as (1) temporal sequence, (2) description of symptoms, (3) description of psychological impact, (4) withdrawal effects, (5) alternative causes, and (6) rechallenge. The concordance level between the perceived severity by the reporter and the pharmacovigilance team was assessed by the Kappa index (ƙ). Usual descriptive statistics were used to describe variables. RESULTS: Nationally, 190 spontaneous reports described 383 ADRs, which 28.6% were unknown or poorly known in the literature, and 52.1% were serious. The most frequent ADRs were gastrointestinal (19.3%) and neurological (19.1%), and among the most common 213 suspected medicines, there were those used for nervous system conditions (18.8%). The agreement on the perception of ADRs' severity between citizens and centre's technicians was 'good' (K = 0.62 (0.51-0.72)). An analysis of the free-text section of reports showed that one-quarter of the reports provided useful additional data, like the psychobiosocial impact, which could explain the discrepancy between patients and health professionals in the classification of the severity of some ADRs. CONCLUSIONS: Patients' report free-text section provides relevant information, mainly about symptoms description, psychobiosocial impact and feelings. Therefore, it is a section to be enhanced and analysed. These findings should encourage the strengthening of citizens reporting.

Highlights from the Fifth International Meeting on Metabotropic Glutamate Receptors, held September 18-23, 2005, in Taormina, Sicily, Italy. Metabotropic glutamate receptors as therapeutic targets.

The study of metabotropic glutamate receptors (mGluRs) is one of the fastest growing areas of neuropharmacology. The Fifth International Meeting on Metabotropic Glutamate Receptors, held September 18-23, 2005, in Taormina, Sicily, Italy, encompassed more than 80 oral presentations and 70 poster presentations. The role of mGluRs in various physiological and pathological conditions, such as learning and memory, modulation of sensory inputs, control of movement, schizophrenia, anxiety, seizures, addiction to drugs, neurodegeneration and developmental regulation of synaptic circuits, was discussed.

Botulinum neurotoxin inhibits depolarization-stimulated protein phosphorylation in pure cholinergic synaptosomes.

Botulinum neurotoxin, a strong blocker of acetylcholine release at peripheral cholinergic synapses, inhibits depolarization-stimulated protein phosphorylation in pure cholinergic synaptosomes isolated from the electric organ of Torpedo marmorata. Moreover, tetrodotoxin has the same effect on protein phosphorylation when cholinergic synaptosomes are depolarized by veratridine. Correlation between presynaptic protein phosphorylation and acetylcholine release is suggested by the fact that botulinum neurotoxin blocks specifically neurotransmitter release without affecting membrane depolarization and calcium fluxes in our synaptosomal preparation.

Regulation of ionotropic glutamate receptor subunits in different rat brain areas by a preferential sigma(1) receptor ligand and potential atypical antipsychotic.

The effect of chronic administration of the putative atypical antipsychotic E-5842, a preferential sigma(1) receptor ligand, on ionotropic glutamate receptor subunit levels of mRNA and protein, was studied. The repeated administration of E-5842 differentially regulated levels of the NMDA-2A and of GluR2 subunits in a regionally specific way. Levels of immunoreactivity for the NMDA-2A subunit were up-regulated in the medial prefrontal cortex, the frontoparietal cortex, the cingulate cortex, and in the dorsal striatum, while they were down-regulated in the nucleus accumbens. Levels of the GluR2 subunit of the AMPA receptor were up-regulated in the medial prefrontal cortex and the nucleus accumbens and down-regulation was observed in the dorso-lateral striatum. Regulation of the levels of mRNA for the different subunits was also observed in some cases. The results show that E-5842, through a mechanism still unknown, is able to modify levels of several glutamate receptor subunits and these changes could be related to its antipsychotic activity in pre-clinical tests.

GABAA receptor modulation by the novel intravenous general anaesthetic E-6375.

E-6375 (4-butoxy-2-[4-(2-cyanobenzoyl)-1-piperazinyl] pyrimidine hydrochloride) is a new intravenous general anaesthetic with an anaesthetic potency, in mice, comparable to propofol, or etomidate. Here, we examined the effect of E-6375 upon the GABAA receptor, a putative target of intravenous anaesthetic action. E-6375 reversibly enhanced GABA-evoked currents mediated by recombinant GABAA (alpha1beta2gamma2L) receptors expressed in Xenopus laevis oocytes, with little effect on NMDA- and kainate-evoked currents mediated by NR1a/NR2A and GluR1o/GluR2o glutamate receptors, respectively. E-6375 prolonged the decay of GABA-evoked miniature inhibitory postsynaptic currents recorded from rat Purkinje neurones demonstrating the anaesthetic also enhanced the activity of synaptic GABAA receptors. The GABA enhancing action of E-6375 on recombinant GABAA receptors was unaffected by the subtype of the alpha isoform (i.e. alphaxbeta2gamma2L; x=1-3) within the receptor, but was increased by the omission of the gamma2L subunit. Receptors incorporating beta2, or beta3, subunits were more sensitive to modulation by E-6375 than those containing the beta1 subunit. The selectivity of E-6375 was largely governed by the identity (serine or asparagine) of a single amino acid residue within the second transmembrane domain of the beta-subunit. The various in vivo actions of general anaesthetics may be mediated by GABAA receptor isoforms that have a differential distribution within the CNS. The identification of agents, such as E-6375, that discriminate between GABAA receptor subtypes may augur the development of general anaesthetics with an improved therapeutic profile.

Effect of acute administration of the 5-HT1A receptor ligand, lesopitron, on rat cortical 5-HT and dopamine turnover.

1. The involvement of presynaptic 5-hydroxytryptamine1A (5-HT1A) autoreceptors in the anxiolytic-like properties of lesopitron (E-4424) (2-(4-[4-(4-chloro-1-pyrazolyl)butyl]-1- piperazinyl)pyrimidine) was studied. Brain microdialysis was used to examine the effect of the drug on the release of 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the frontal cortex of awake, freely moving rats. Moreover, extracellular cortical 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were also studied to assess the possible participation of dopaminergic systems. 2. Lesopitron administered at a dose which induces anxiolytic behavior in rats (30 micrograms kg-1, i.p.) markedly reduced 5-HT levels (to 45% of the basal value) in cortical perfusates, having no effect on 5-HIAA, DOPAC and HVA. The effects of lesopitron were compared with those produced by the anxiolytic, and structurally related compound, buspirone. 3. Buspirone administered at a dose inducing anxiolytic-like effects in rats (5 mg kg-1, i.p.) produced a marked decrease in cortical 5-HT levels (to 20% of the basal value), but in contrast to lesopitron, buspirone produced a pronounced increase in cortical DOPAC (to 300% of the basal value) and HVA (to 400% of the basal value) levels. Buspirone administered at a low dose (30 micrograms kg-1, i.p.) was unable to affect cortical 5-HT levels. 4. To test the hypothesis that the 5-HT decreasing effect of lesopitron could be due to 5-HT1A autoreceptor (somatodendritic)-mediated inhibition of 5-HT neurotransmission, lesopitron was administered locally into the raphe nuclei. Intraraphe administration of 10 micro M lesopitron caused a decrease incortical 5-HT levels (the effect being of the same order as that obtained after systemic injection), with no effect on 5-HIAA, DOPAC and HVA. Raphe 5-HT extracellular levels were not modified afterintraraphe administration of lesopitron, indicating the absence of 5-HT reuptake blocking properties.5 We concluded that lesopitron, at an anxiolytic dose produced a marked inhibition of 5-HT release in the frontal cortex of awake, freely moving rats. This effect was observed after systemic administration as well as after intraraphe administration of the drug, suggesting an agonistic action at raphe 5-HTIA autoreceptors controlling 5-HT release in the projecting areas. In contrast to buspirone, lesopitrontreatment had no effect on cortical DOPAC or HVA levels.

Decrease of adenylyl cyclase activity and expression by a sigma1 receptor ligand and putative atypical antipsychotic.

We examined whether changes in the adenylyl cyclase system could be induced by the administration of the sigma1 receptor ligand and putative atypical antipsychotic 4-[4-fluorophenyl]-1,2,3,6-tetrahydro-1-[4-[1,-2,4-triazol-1-il]butyl]pyridine citrate) (E-5842). Repeated (21 days) but not acute (2 h) treatment with E-5842 induced a significant decrease in adenylyl cyclase type I immunoreactivity and adenylyl cyclase activity in rat frontal cortex membranes, with less or no effect in other brain regions such as the hippocampus or the striatum. Changes in immunoreactivity were not observed in other adenylyl cyclases (type V/VI). The reported changes, observed only after a chronic treatment, could be related to the mechanism of action of sigma receptor ligands in general or to that of E-5842 in particular and should be taken into account, given the long duration of treatment in psychiatric patients.

Identification of MARPP (14-20), morphine- and cyclic AMP-regulated phosphoproteins of 14-20 kDa, as myelin basic proteins: evidence for their acute and chronic regulation by morphine in rat brain.

MARPP(14-20), morphine- and cyclic AMP-regulated phosphoproteins of 14, 17-18, and 20 kDa, were identified originally by one-dimensional electrophoresis as a group of proteins whose state of phosphorylation was decreased by acute morphine and increased by chronic morphine in the rat locus coeruleus. We now show that MARPP(14-20) represent myelin basic proteins based on biochemical and immunochemical criteria. First, MARPP(14-20) were found to have isoelectric points of about 11 based on their migration on non-equilibrium pH gradient electrophoresis. Second, MARPP(14-20) were greatly enriched in myelin fractions of brain, and were not detectable, or present at very low levels, in other subcellular fractions of brain. Third, analysis of phosphorylated MARPP(14-20) by one- and two-dimensional gel electrophoresis demonstrated precise comigration with immunolabeled myelin basic proteins. In contrast, MARPP(14-20) were distinguished from histones, another group of low molecular weight, highly basic phosphoproteins, in these subcellular fractionation and immunochemical studies. Finally, we confirmed using two-dimensional electrophoresis, that changes observed previously by one-dimensional electrophoresis in the phosphorylation of MARPP(14-20) in response to acute and chronic morphine, and to acute forskolin, occur in myelin basic proteins. It was also found that changes in the state of phosphorylation of myelin basic proteins in response to chronic morphine occur without a change in the total amount of the proteins as determined by immunoblot analysis. The results demonstrate that the phosphorylation of myelin basic proteins is regulated by morphine in the nervous system, and raise the possibility that regulation of these proteins contributes to mechanisms underlying some of the acute and chronic actions of opiates in brain.

Dopaminergic brain reward regions of Lewis and Fischer rats display different levels of tyrosine hydroxylase and other morphine- and cocaine-regulated phosphoproteins.

We studied cyclic AMP-dependent protein phosphorylation in the mesolimbic and nigrostriatal dopamine systems of two genetically inbred rat strains, Lewis (LEW) and Fischer (F344) rats. These strains represent genetically divergent populations of rats that have been used to study possible genetic factors involved in a variety of biological processes. We found striking differences in levels of tyrosine hydroxylase, and several other phosphoproteins, in the mesolimbic, but not the nigrostriatal, dopamine system between the two rat strains. Interestingly, in Sprague-Dawley rats, these same phosphoproteins are altered by chronic morphine and chronic cocaine specifically in the mesolimbic dopamine system, generally thought to be a brain reward pathway that mediates some of the reinforcing actions of many drugs of abuse. As LEW and F344 rats have been reported to show different levels of preference for several types of drugs of abuse, the results are consistent with the possibility that these phosphoproteins may mediate aspects of drug reinforcement and contribute to individual differences in vulnerability to drug addiction.

Lewis and Fischer rat strains display differences in biochemical, electrophysiological and behavioral parameters: studies in the nucleus accumbens and locus coeruleus of drug naive and morphine-treated animals.

In previous studies, we demonstrated that tyrosine hydroxylase and neurofilament proteins are regulated by chronic morphine and chronic cocaine treatments in the ventral tegmental area in Sprague-Dawley rats and that the inbred Lewis and Fischer 344 rat strains, under drug-naive conditions, show different levels of these proteins specifically in this brain region. In the current study, we compared Lewis and Fischer rats with respect to levels of adenylate cyclase, cyclic AMP-dependent protein kinase and G-proteins in the nucleus accumbens (NAc) and locus coeruleus (LC), brain regions in Sprague-Dawley rats where these proteins are regulated by chronic exposure to morphine or to cocaine. We found that levels of adenylate cyclase and cyclic AMP-dependent protein kinase activity are higher in the NAc and LC of Lewis rats compared to Fischer rats, whereas levels of Gi alpha and G beta were lower. These strain differences were not seen in several other brain regions analyzed and no strain differences were detected in levels of other G-protein subunits. Lewis and Fischer rats also differed in the ability of chronic morphine to regulate adenylate cyclase and cyclic AMP-dependent protein kinase in the NAc and LC. In the NAc, chronic morphine increased levels of the two enzymes in the Fischer strain only, whereas in the LC chronic morphine increased levels of the enzymes in both strains, with more robust effects seen in the Lewis rat. To understand possible physiological consequences of these strain differences in the cyclic AMP pathway, we studied LC neuronal activity under basal and chronic morphine-treated conditions. LC neurons of Lewis rats showed higher spontaneous firing rates in brain slices in vitro than those of Fischer rats and also showed greater morphine-induced increases in responsiveness to bath-applied 8-bromo-cyclic AMP. These electrophysiological findings are generally consistent with the biochemical observations. Moreover, Lewis and Fischer rats displayed very different opiate withdrawal syndromes, with different types of behaviors elicited upon precipitation of opiate withdrawal with the opiate receptor antagonist, naltrexone. The possible relationship between these behavioral findings and the biochemical and electrophysiological data is discussed. These studies provide further support for the possibility that Lewis and Fischer rat strains provide a useful model system in which some of the genetic factors that contribute to drug-related behaviors can be investigated.

The effect of E-5842, a sigma receptor ligand and potential atypical antipsychotic, on Fos expression in rat forebrain.

We examined the ability of E-5842 (4-(4-fluorophenil)-1,2,3,6- tetrahydro-1-[4-(1,2,4-triazol-1-il)butyl] pyridine citrate), a sigma receptor ligand, to increase Fos protein expression in regions of rat forebrain. An acute administration of E-5842 increased levels of Fos in the medial prefrontal cortex and the nucleus accumbens, without affecting the levels of the protein in the striatum, an effect very similar to that of clozapine. Our results suggest that E-5842 may be an atypical antipsychotic with low propensity to produce extrapyramidal side-effects.

Electrophysiological effects of E-5842, a sigma1 receptor ligand and potential atypical antipsychotic, on A9 and A10 dopamine neurons.

Extracellular single unit recording techniques were used to study the effects of the novel potential atypical antipsychotic E-5842, (4-(4-fluorophenyl)-1,2,3,6-tetrahydro-1-[4-(1,2,4-triazol-1-il)bu tyl]pyridine citrate), a preferential sigma1 receptor ligand, on the activity of dopamine cells in substantia nigra pars compacta (A9) and ventral tegmental area (A10) in anesthetized rats. Acute i.v. administration of E-5842 (up to 3.2 mg kg(-1)) did not change the spontaneous activity of the dopamine neurons, which still responded to the inhibitory effect of a subsequent administration of high dose of apomorphine. Acute administration of E-5842 (20 mg kg(-1), i.p.) did not change the number of spontaneously active A9 or A10 dopamine cells. Chronic administration of E-5842 (20 mg kg(-1) day(-1) x 21 days, s.c.) decreased the number of spontaneously active A10 but not A9, dopamine neurons. This effect was reversed by the administration of apomorphine, thus, indicating a possible depolarization inactivation phenomenon. Our results suggest an influence of E-5842 on dopaminergic neurotransmission, although the exact mechanism remains unknown. The effect of E-5842 on A10 is similar, in some ways, to the effects observed with several atypical antipsychotics and suggest the atypicality of the compound and that E-5842 may exert its antipsychotic effects without causing significant extrapyramidal side effects.

Depolarization-stimulated protein phosphorylation in pure cholinergic nerve endings.

Cholinergic synaptosomes obtained from the electric organ of Torpedo marmorata have been used to study chemical stimulation-stimulated protein phosphorylation. Cholinergic synaptosomes were exposed to elevated K+0 concentrations or other chemical depolarizing agents such as gramicidin or secretagogues as the calcium ionophore A23187. During depolarization several synaptosomal proteins increase their state of phosphorylation. This phenomenon depends on the presence of Ca2+ in the external medium. These results suggest that stimulation of protein phosphorylation may be implicated in the acetylcholine release process and could represent a modulation mechanism in the neurotransmitter release machinery at this cholinergic synapse.

Changes in phosphoinositide signalling activity and levels of the alpha subunit of G(q/11) protein in rat brain induced by E-5842, a sigma(1) receptor ligand and potential atypical antipsychotic.

Changes in the phosphoinositide (PPI) signal transduction system induced by E-5842, a new sigma(1) (sigma(1)) receptor ligand and potential atypical antipsychotic, were studied in the rat frontal cortex, hippocampus and striatum. Acute treatment with E-5842 increased phospholipase C (PLC) activity in the striatum and the hippocampus. Chronic treatment with E-5842 induced an increase in the activity of PLC in the frontal cortex and the striatum. Similar up-regulation of the activity of the enzyme was also observed in rat frontal cortex membranes in presence of GTPgammaS. After chronic treatment with E-5842, it was also observed a significant increase of the immunoreactivity levels of G(q/11)alpha in the frontal cortex. Our results suggest that part of the antipsychotic effects of E-5842 could be related to the regulation of the PPI signal transduction pathway, especially after a prolonged treatment.

Up-regulation of sigma(1) receptor mRNA in rat brain by a putative atypical antipsychotic and sigma receptor ligand.

Sigma(1) (sigma(1)) receptor mRNA expression was studied in the prefrontal cortex, striatum, hippocampus and cerebellum of rat brain by northern blot and in situ hybridization. The effects of a chronic treatment with antipsychotic drugs (haloperidol and clozapine), and with E-5842, a sigma(1) receptor ligand and putative atypical antipsychotic on sigma(1) receptor expression were examined. A significant increase in the levels of sigma(1) receptor mRNA in the prefrontal cortex and striatum after E-5842 administration was observed, while no apparent changes were seen with either haloperidol or clozapine. Our results suggest a long-term adaptation of the sigma(1) receptor at the level of mRNA expression in specific areas of the brain as a response to a sustained treatment with E-5842.

Second messenger and protein phosphorylation mechanisms underlying opiate addiction: studies in the rat locus coeruleus.

We have studied the role of second messenger and protein phosphorylation pathways in mediating changes in neuronal function associated with opiate addiction in the rat locus coeruleus. We have found that chronic opiates increase levels of the G-protein subunits Gi alpha and Go alpha, adenylate cyclase, cyclic AMP-dependent protein kinase, and a number of phosphoproteins (including tyrosine hydroxylase) in this brain region. Electrophysiological data have provided direct support for the view that this up-regulation of the cyclic AMP system contributes to opiate tolerance, dependence, and withdrawal exhibited by these neurons. As the adaptations in G-proteins and the cyclic AMP system appear to occur at least in part at the level of gene expression, current efforts are aimed at identifying the mechanisms, at the molecular level, by which opiates regulate the expression of these intracellular messenger proteins in the locus coeruleus. These studies will lead to an improved understanding of the biochemical basis of opiate addiction.

Ultrastructural changes induced by 12-O-tetradecanoylphorbol 13-acetate in pure cholinergic synaptosomes of Torpedo electric organ.

We have studied the morphological changes induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment on pure cholinergic synaptosomes from Torpedo electric organ. These changes were studied by both ultrathin sections and freeze-fracture techniques. We found that after a treatment with TPA, a redistribution of synaptic vesicles inside the nerve endings and exocytotic images could be observed. Also, TPA, under conditions that induced the acetylcholine release, did not change the density of intramembrane particles at the synaptosomal protoplasmic hemimembrane leaflet. Similar results were found when calcium was not present in the extrasynaptosomal medium, and our results suggest that acetylcholine release induced by phorbol ester is probably mediated by exocytosis of synaptic vesicles.

Phorbol esters induce neurotransmitter release in cholinergic synaptosomes from Torpedo electric organ.

The effect of phorbol esters and so the involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C;PKC) in the release of acetylcholine (ACh) was studied using Torpedo electric organ synaptosomes. 12-O-Tetradecanoylphorbol 13-acetate (TPA), a known activator of PKC, induced neurotransmitter release in a concentration-dependent manner and increased the potassium-evoked release of ACh. The effect of TPA was shown to be independent of the extrasynaptosomal calcium concentration. TPA-induced ACh release was reversed by H-7, an inhibitor of PKC activity. This drug showed no effect on potassium-evoked ACh release. Botulinum toxin, a strong blocker of potassium-induced ACh release in that synaptosomal preparation, showed no inhibitory effect on the TPA-induced ACh release. Our results suggest that activation of PKC potentiates the release of an ACh pool that is not releasable by potassium depolarization, independently of the extracellular calcium concentration.