Systematic review of disease-modifying therapies to assess unmet needs in multiple sclerosis: tolerability and adherence.

Reviews of therapeutic drugs usually focus on the highly selected and closely monitored patient populations from randomized controlled trials. The objective of this study was to review systematically the tolerability and adherence of multiple sclerosis disease-modifying therapies, using data from both randomized controlled trials and observational settings. Relevant literature was identified using predefined search terms, and adverse event and study discontinuation data were extracted and categorized according to study type (randomized controlled trial or observational) and study duration. A total of 151 papers were selected for analysis; 33% were classified as randomized controlled trials and 62% as observational studies. Most of the papers concerned interferon preparations and glatiramer acetate; the limited available information on mitoxantrone and natalizumab precluded extensive examination of these. The most common adverse events were flu-like symptoms (interferon therapies only) and injection-site reactions. Mean discontinuation rates ranged from 16% to 27%. There were no marked differences in tolerability or adherence data from randomized controlled trials and observational studies, but the incidence of adverse events remained high in lengthy studies and discontinuations accumulated with time. The present systematic review of randomized clinical trial and observational data highlights the tolerability and adherence issues associated with commonly used first-line multiple sclerosis treatments.

Fos expression in the brains of rats performing an attentional set-shifting task.

Impairments in executive function and cognitive control are a common feature of neuropsychiatric and neurodegenerative disorders. A promising behavioral paradigm for elucidating the neural mechanisms of executive function is extradimensional/intradimensional (ED/ID) shifting, which places demands on executive function by requiring the adjustment of behavioral responses based on affective or attentional information. To augment the understanding of the brain systems required for these aspects of executive function, we examined the induction of Fos protein in rats tested in the ED/ID paradigm. We found increased Fos-like immunoreactivity (Fos-LI) in several cortical areas, including medial and orbital frontal cortex (OFC), in rats performing affective or attentional shifts relative to rats performing control discriminations. However, increased Fos-LI was also present in rats that performed a yoked number of additional control discrimination trials, without affective or attentional shifting. These observations suggest that cortical networks required for affective and attentional shifting are also activated during comparable discrimination tasks that do not require shifting, consistent with a role for these networks in monitoring ongoing behavior even in situations in which adaptation to changing behavioral demands is not required.

Evaluation of the pro-cognitive effects of the AMPA receptor positive modulator, 5-(1-piperidinylcarbonyl)-2,1,3-benzoxadiazole (CX691), in the rat.

RATIONALE: Positive allosteric modulators of the glutamatergic alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) receptor do not stimulate AMPA receptors directly but delay deactivation of the receptor and/or slow its desensitisation. This results in increased synaptic responses and enhanced long-term potentiation. Thus, it has been suggested that such compounds may have utility for the treatment of cognitive impairment. OBJECTIVES: The objective of the study was to investigate the effect of an AMPA positive modulator, CX691, (1) in three rodent models of learning and memory, (2) on neurochemistry in the dorsal hippocampus and medial prefrontal cortex following acute administration, and (3) on brain-derived neurotrophic factor (BDNF) messenger RNA (mRNA) expression in the rat hippocampus following acute and sub-chronic administration. RESULTS: CX691 attenuated a scopolamine-induced impairment of cued fear conditioning following acute administration (0.1 mg/kg p.o.) and a temporally induced deficit in novel object recognition following both acute (0.1 and 1.0 mg/kg p.o.) and sub-chronic (bi-daily for 7 days) administration (0.01, 0.03, 0.1 mg/kg p.o.). It also improved attentional set-shifting following sub-chronic administration (0.3 mg/kg p.o.). Acute CX691 (0.1, 0.3 and 1.0 mg/kg, p.o.) increased extracellular levels of acetylcholine in the dorsal hippocampus and medial prefrontal cortex and dopamine in the medial prefrontal cortex. Sub-chronic administration of CX691 (0.1 mg/kg, p.o.) elevated BDNF mRNA expression in both the whole and CA(1) sub-region of the hippocampus (P < 0.05). CONCLUSIONS: Collectively, these data support the pro-cognitive activity reported for AMPA receptor positive modulators and suggest that these compounds may be of benefit in treating disorders characterised by cognitive deficits such as Alzheimer's disease and schizophrenia.

Medial prefrontal cortex volume loss in rats with isolation rearing-induced deficits in prepulse inhibition of acoustic startle.

Rearing rats in isolation produces perturbations in behavior and brain neurochemistry suggested to resemble those of schizophrenia. In particular, isolation-reared rats display deficits in prepulse inhibition of acoustic startle that in humans are associated with disorders including schizophrenia and are interpreted as abnormalities in sensorimotor gating. The prefrontal cortex is considered important in the regulation of prepulse inhibition of acoustic startle and postmortem studies suggest that neuropil and total volume, but not total number of neurons, are decreased in this region of the brains of schizophrenic patients. In this study we used design-based stereological techniques to examine the brains of Lister Hooded rats, reared in isolation and which displayed prepulse inhibition of acoustic startle deficits, for changes in morphology compared with the brains of their socially-reared littermates. Pooled data from three batches of animals revealed a significant 7% volume loss of the medial prefrontal cortex of isolation-reared rats whereas neuron number in this region was unchanged. In contrast, volume and total neuron number were unaffected in the rostral caudate putamen. The robust reduction in prefrontal cortical volume observed in isolation-reared rats, in the absence of reductions in total neuron number, suggest that there is a loss of volume of the neuropil. These changes parallel those reported in schizophrenia patients and therefore support the construct validity of this model.

The effects of ziprasidone on regional c-Fos expression in the rat forebrain.

RATIONALE: Typical and atypical antipsychotic drugs produce characteristic patterns of immediate early gene expression in rat forebrain that are considered to reflect their effects in schizophrenia subjects. OBJECTIVE: To use c-Fos immunohistochemistry to investigate the functional neuroanatomical profile of the newly introduced atypical agent ziprasidone. MATERIALS AND METHODS: c-Fos immunohistochemistry was performed on paraformaldehyde-fixed cryosections of rat brains obtained, initially, from animals 2, 4, or 6 h after oral administration of 10 mg/kg ziprasidone or vehicle and, subsequently, from animals 2 h after oral administration of 1, 3, or 10 mg/kg ziprasidone or vehicle. The density of immunoreactive nuclei was assessed in pre-determined forebrain regions. RESULTS: Ziprasidone induced a time-dependent increase in the density of c-Fos-positive nuclei that was maximal at 2 h. At the 2 h time-point, c-Fos expression was significantly (p<0.05) elevated in the shell and core of the nucleus accumbens, lateral and medial caudate putamen, and lateral septum. At 4 h post-dose, c-Fos expression was also significantly increased in the cingulate gyrus. Ziprasidone-induced c-Fos expression was dose-dependent with significant (p<0.05) c-Fos expression observed in the nucleus accumbens (shell and core) and caudate putamen (lateral and medial) at 3 and 10 mg/kg and in the lateral septum at 10 mg/kg. CONCLUSIONS: Increased c-Fos expression in the nucleus accumbens and lateral septum is considered to be predictive of activity against positive symptoms, in the caudate putamen of motor side effect liability, and in the cingulate gyrus of efficacy against negative symptoms. Thus, the observed pattern of c-Fos expression induced in rat brain by ziprasidone is consistent with its reported clinical effects, namely, efficacy against positive symptoms with a therapeutic window over motor side effects and with some activity against negative symptoms.

Measurement of cGMP and soluble guanylyl cyclase activity.

Soluble guanylyl cyclase is responsible for synthesis of guanosine 3'5'-cyclic monophosphate. The enzyme is activated by NO, so measurement of tissue levels of cGMP and the activity of guanylyl cyclase is important in studies of NO-cGMP signal transduction. The method of choice for measuring tissue levels of cGMP is a radioimmunoassay, but it can also be measured using a scintillation proximity assay. Guanylyl cyclase activity is determined by measuring the conversion of GTP to cGMP using a radioimmunoassay or by following the conversion of [³²P]GTP to [³²P]cGMP, with separation of the product by precipitation and chromatography on an alumina column.

Lamotrigine inhibits monoamine uptake in vitro and modulates 5-hydroxytryptamine uptake in rats.

Lamotrigine is a novel anticonvulsant drug which also stabilises mood in bipolar illness via an unknown mechanism. We report the concentration-dependent inhibition of 5-hydroxytryptamine (5-HT) uptake in both human platelets and rat brain synaptosomes (IC50s were 240 and 474 microM, respectively) by lamotrigine. Synaptosomal uptake of noradrenaline (IC50 239 microM) and dopamine (IC50 322 microM) was also inhibited. Tetrodotoxin failed to modulate 5-HT uptake suggesting that sodium channel blockade does not mediate the lamotrigine effect. Lithium, sodium valproate, zonisamide, and carbamazepine all possess anti-manic activity but only the latter inhibited 5-HT uptake. The inhibition of the p-chloroamphetamine-induced 5-HT syndrome in rats suggests that lamotrigine also inhibits 5-HT uptake in vivo. These effects probably reflect an affinity for biogenic amine transporters. However, at present, it remains uncertain whether, at clinically effective doses, these effects contribute significantly to the efficacy of lamotrigine in bipolar illness.

The nitric oxide-cyclic GMP pathway and synaptic plasticity in the rat superior cervical ganglion.

1. We have investigated the possibility that nitric oxide (NO) and soluble guanylyl cyclase, an enzyme that synthesizes guanosine 3':5'-cyclic monophosphate (cyclic GMP) in response to NO, contributes to plasticity of synaptic transmission in the rat isolated superior cervical ganglion (SCG). 2. Exposure of ganglia to the NO donor, nitroprusside, caused a concentration-dependent accumulation of cyclic GMP which was augmented in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine. The compound, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of soluble guanylyl cyclase, completely blocked this cyclic GMP response. 3. As assessed by extracellular recording, nitroprusside (100 microM) and another NO donor, S-nitrosoglutathione (30 microM) increased the efficacy of ganglionic synaptic transmission in response to electrical stimulation of the preganglionic nerve, an effect that was reversible and which could be replicated by the cyclic GMP analogue, 8-bromo-cyclic GMP. Ganglionic depolarizations resulting from stimulation of nicotinic receptors with carbachol were not increased by nitroprusside. The potentiating actions of the NO donors on synaptic transmission, but not that of 8-bromo-cyclic GMP, were inhibited by ODQ. 4. Brief tetanic stimulation of the preganglionic nerve resulted in a long-term potentiation (LTP) of synaptic transmission that was unaffected by ODQ, either in the absence or presence of the NO synthase inhibitor, NG-nitro-L-arginine (L-NOARG, 100 microM). A lack of influence of L-NOARG was confirmed in intracellular recordings of LTP of the excitatory postsynaptic potential. Furthermore, under conditions where tetanically-induced LTP was saturated, nitroprusside was still able to potentiate synaptic transmission, as judged from extracellular recording. 5. We conclude that NO is capable of potentiating ganglionic neurotransmission and this effect is mediated through the stimulation of soluble guanylyl cyclase and the accumulation of cyclic GMP. However, this potentiation is distinct from LTP of nicotinic synaptic transmission, in which neither NO nor soluble guanylyl cyclase appear to participate.

Nitric oxide-dependent long-term potentiation is blocked by a specific inhibitor of soluble guanylyl cyclase.

The diffusible second messenger, nitric oxide, is synthesised in central neurons in response to activation of glutamate receptors or other stimuli that increase cytosolic Ca2+ concentrations. Among the many roles suggested for nitric oxide in the central nervous system is that of mediating synaptic plasticity. For example, long-term potentiation in the CA1 region of the rat hippocampus was reported to be blocked by inhibitors of nitric oxide synthase and exogenous nitric oxide has been claimed to induce an enduring enhancement of synaptic strength under certain conditions. These findings, however, are controversial and even when a participation of nitric oxide is evident, the transduction mechanism is unclear. A well-known action of nitric oxide is to stimulate the soluble form of guanylyl cyclase, thereby evoking an accumulation of cyclic GMP in target cells but several other mechanisms have been proposed, including stimulation of ADP ribosyltransferase or cyclooxygenase, and nitrosylation of protein thiol residues. The identification of a selective inhibitor of soluble guanylyl cyclase, the oxadiazoloquinoxaline derivative, ODQ, provides, for the first time, the means to investigate the importance of the cyclic GMP pathway in nitric oxide signal transduction. We find that ODQ and the nitric oxide synthase inhibitor, nitroarginine, reduce hippocampal long-term potentiation in an equal and mutually exclusive manner, suggesting that the actions of nitric oxide in this phenomenon are entirely mediated through cyclic GMP. The experiments also show that there is a component of long-term potentiation that involves neither nitric oxide nor cyclic GMP.

Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one.

In brain and other tissues, nitric oxide (NO) operates as a diffusible second messenger that stimulates the soluble form of the guanylyl cylase enzyme and so elicits an accumulation of cGMP in target cells. Inhibitors of NO synthesis have been used to implicate NO in a wide spectrum of physiological and pathophysiological mechanisms in the nervous system and elsewhere. The function of cGMP in most tissues, however, has remained obscure. We have now identified a compound, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), that potently and selectively inhibits NO-stimulated guanylyl cyclase activity. In incubated slices of cerebellum, ODQ reversibly inhibited the NO-dependent cGMP response to glutamate receptor agonists (IC50 approximately nM) but did not affect NO synthase activity. The compound did not affect synaptic glutamate receptor function, as assessed in hippocampal slices, nor did it chemically inactivate NO. ODQ did, however, potentially inhibit cGMP generation in response to NO-donating compounds. An action on NO-stimulated soluble guanylyl cyclase was confirmed in studies with the purified enzyme. ODQ failed to inhibit NO-mediated macrophage toxicity, a phenomenon that is unrelated to cGMP, nor did it affect the activity of particulate guanylyl cyclase or adenylyl cyclase. ODQ is the first inhibitor that acts selectively at the level of a physiological NO "receptor" and, as such, it is likely to prove useful for investigating the function of the cGMP pathway in NO signal transduction.

The nitric oxide--cyclic GMP pathway and synaptic depression in rat hippocampal slices.

The ability of exogenous nitric oxide (NO) to modify synaptic transmission was investigated in area CA1 of the rat hippocampal slice. The NO donors S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (SNOG) depressed field excitatory postsynaptic potentials evoked by low frequency stimulation of the Schaffer collateral-commissural pathway. Upon washout of the NO donors, synaptic transmission rapidly returned to control levels. A similar reversible synaptic depression was produced by SNAP when tetanic stimulation (100 Hz; 1 s) was delivered in its presence. The effect of SNAP was not mimicked by its precursor or breakdown product and was blocked by haemoglobin, indicating that the effect involved NO. Roussin's black salt, a photolabile NO donor, also depressed transiently field excitatory postsynaptic potentials following photolysis. The depression was induced rapidly following a flash of UV light (20 s duration) focused onto the slice using a confocal microscope. The depressant effect of the NO donors on synaptic transmission was mimicked by zaprinast, a specific cGMP-phosphodiesterase inhibitor. Zaprinast depressed to a similar extent both the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and N-methyl-D-aspartate receptor-mediated components of excitatory postsynaptic currents without affecting passive membrane properties, indicating a presynaptic locus of action. SNAP, SNOG and zaprinast all elevated cGMP levels in rat hippocampal slices. Immunocytochemical staining revealed that the cGMP accumulation was mainly in a network of varicose fibres running throughout the CA1 region, consistent with a presynaptic site of action of NO. We conclude that NO, possibly through activation of guanylate cyclase, may be involved in transient presynaptic depression in the CA1 region of the hippocampus.

The NO-cGMP pathway in neonatal rat dorsal horn.

Incubation of slices of neonatal rat spinal cord with nitric oxide donor compounds produced marked elevations in cyclic guanosine 3',5' monophosphate (cGMP) levels. The excitatory amino acid receptor agonists N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) produced smaller increases, which were blocked by the nitric oxide synthase (NOS) inhibitor L-NG-nitroarginine (NOArg), indicating that these cGMP responses were mediated by nitric oxide. Immunocytochemistry revealed that, in response to NMDA, cGMP accumulated in a population of small cells and neuropil in laminae II and III of the dorsal horn. This area was also shown, by reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry, to contain NOS. These observations suggest that, in the rat spinal cord, NMDA receptor activation is linked to the formation of NO and, hence, of cGMP. This pathway is located selectively in the superficial dorsal horn, consistent with a role in the processing of nociceptive signals.

The nitric oxide-cyclic GMP signalling pathway in rat brain.

Nitric oxide is a novel signalling molecule in the brain and a potent activator of the cyclic GMP-synthesising enzyme, soluble guanylate cyclase. To determine if stimulation of cyclic GMP formation is a widespread mechanism of nitric oxide signal transduction, we have compared the distribution of the nitric oxide-generating enzyme (nitric oxide synthase) with that of nitric oxide-stimulated cyclic GMP accumulation, throughout the rat brain. The former was done using NADPH diaphorase histochemistry and the latter by cyclic GMP immunohistochemistry following perfusion of the nitric oxide donor, nitroprusside, in vivo. At a gross level, there was generally a good match when the two were compared in adjacent sections. Although the relative staining intensity varied from area to area, in no grey matter region did we observe cyclic GMP accumulation in the absence of nitric oxide synthase staining. In detail, the locations were complementary rather than identical. In some areas, nitric oxide synthase was found in postsynaptic structures and cyclic GMP accumulation in presynaptic elements and fibres; in others, the locations were reversed. Glial cells and their processes also accumulated cyclic GMP in the cerebellum. The results suggest that soluble guanylate cyclase is a major nitric oxide "receptor" throughout the brain. They also support the hypothesis that nitric oxide generated therein primarily functions as a mediator of cell-cell signaling rather than as a conventional second messenger acting within the cells in which it is produced. The types of communication subserved by nitric oxide appear to be extraordinarily diverse.

The intrinsic and vagal extrinsic innervation of the rat stomach contains nitric oxide synthase.

Nitric oxide synthase (NOS) associated with the intrinsic and vagal extrinsic innervation of the rat gastric corpus was studied in both control and neonatally capsaicin-treated animals. Nerve cell bodies and fibres of both the myenteric and sub-mucosal plexi of the gastric corpus was found to contain NOS, but were distinct from those containing VIP. NOS-positive fibres were seen innervating the circular smooth muscle layer. Stain accumulated both proximal and distal to a vagal ligature. Staining of the nucleus of the solitary tract (NTS) was associated with specific sub-nuclei. Neonatal capsaicin treatment did not alter the staining in the NTS, vagus nerve or stomach. The data presented here support the idea that NO is a non-adrenergic non-cholinergic transmitter associated with gastric function.

NADPH-diaphorase staining in autonomic and somatic cranial ganglia of the rat.

Using NADPH-diaphorase staining as a marker for the enzyme nitric oxide synthase (NOS) we have investigated the possible sites of nitric oxide (NO) synthesis in a number of cranial ganglia in the rat. Intense staining was found in the majority of neurones in the sphenopalatine ganglion, suggesting a major role for NO in postganglionic parasympathetic systems in the head. In contrast the neurones of the superior cervical ganglion were not stained by this histochemical procedure but were enveloped by a mesh of intensely staining fibres. As preganglionic sympathetic neurones in the intermediolateral horn of the spinal cord stain for NADPH-diaphorase, our results would suggest that NO acts as a neurotransmitter between pre- and post-ganglionic sympathetic neurones.

Sources and targets of nitric oxide in rat cerebellum.

Nitric oxide (NO) mediates cell-cell signalling in the brain and stimulates cyclic GMP (cGMP) production in target cells. We have used NADPH-diaphorase (reduced nicotinamide adenine dinucleotide phosphate-diaphorase) histochemistry to identify NO-producing neurones and cGMP immunohistochemistry to locate the targets of NO in rat cerebellum. NADPH-diaphorase staining was prominent in granule cells and in the molecular layer. cGMP immunostaining in cerebellar slices stimulated with the NO donors, nitroprusside and SIN-1, was found in granule cells, glomeruli, fibres, Bergmann glia and in other astrocytes. The results provide visible evidence that NO mediates neuron-neuron and neuron-glia communication.

Nitric oxide may act as a messenger between dorsal root ganglion neurones and their satellite cells.

The distribution of NADPH-diaphorase and cyclic GMP in neonatal dorsal root ganglia in vitro has been investigated under control conditions and in response to incubation with either sodium nitroprusside or N-methyl-D-aspartate. NADPH-diaphorase activity which reveals the distribution of nitric oxide (NO) synthase in neurons was found to be intense in some dorsal root ganglion neurones and present at a lower level in the majority. Basal levels of cGMP were found to be low but when stimulated by sodium nitroprusside were found to be selectively increased in satellite cells. The results suggest that NO may function as a signalling system between neurones and satellite cells in sensory ganglia.

Intercellular action of nitric oxide in adult rat cerebellar slices.

Nitric oxide (NO) is a novel messenger molecule that is produced following glutamate receptor activation and which stimulates cyclic GMP (cGMP) formation. To determine if the mode of action of NO is predominantly intra- or inter-cellular in intact brain tissue, we tested the ability of haemoglobin (Hb), a NO-binding protein that remains extracellular, to inhibit cGMP accumulation induced by glutamate receptor agonists in adult rat cerebellar slices. Responses to agonists acting selectively on N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors were inhibited by Hb (IC50 approximately 20 microM) suggesting that NO functions predominantly as a mediator of communication between cells. The effects of NO-donating drugs (e.g. nitroprusside) were also inhibited by Hb, implying that they yield NO extracellularly.

Comparative effects of some nitric oxide donors on cyclic GMP levels in rat cerebellar slices.

In the central nervous system, glutamate receptor activation and other stimuli can lead to the cellular production of nitric oxide (NO), an activator of the cyclic GMP-synthesising enzyme, soluble guanylate cyclase. Four 'nitrovasodilators' which yield NO were tested for their ability to elevate cGMP levels in rat cerebellar slices. Nitroprusside (NP), SIN-1, S-nitroso-N-penicillamine (SNAP) and hydroxylamine all caused very large (up to 300-fold) increments. Their threshold concentrations were between 1 and 30 microM. SNAP was the most potent (EC50 approximately 50 microM) followed by hydroxylamine (200 microM) and SIN-1 (1 mM), the latter compound having the highest efficacy. No maximal response to NP was evident at concentrations up to 10 mM. Slices could be challenged a second time with NP (300 microM) with no evidence of a change in sensitivity. The NO-donors are likely to be valuable for studying the functions of NO in brain tissue; however, the concentrations of NP, SNAP and SIN-1 required to elevate cGMP in the slices are orders of magnitude higher than those needed to stimulate guanylate cyclase activity in broken cell preparations, suggesting that rapid inactivation of NO takes place in the intact tissue.