'Progressive MS - macro views': The need for novel clinical trial paradigms to enable drug development for progressive MS.

This article outlines the principal challenges to establish a standard phase-2 approach for progressive multiple sclerosis (PMS) and presents referring strategies to accelerate the registration process via a guidance approved by regulatory agencies. Accordingly, the contribution of 'big datasets' for a better understanding of the natural history of primary-progressive multiple sclerosis (PPMS) and secondary-progressive multiple sclerosis (SPMS) and of their prognostic factors and the value of novel biomarkers are discussed. The establishment of new industry-academic initiatives, such as independent consortia under the umbrella of Progressive MS Alliance (PMSA), with the endorsement of MS organizations and Scientific Societies (e.g. European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS)) may be crucial to overcome some of the current challenges. Within this frame, the continuous interaction with regulatory agencies is instrumental for the formal validation of the many developments suitable to improve clinical trialling in PMS.

The Italian Neuroimaging Network Initiative (INNI): enabling the use of advanced MRI techniques in patients with MS.

Magnetic resonance imaging (MRI) is an important paraclinical tool to diagnose and monitor multiple sclerosis (MS). Conventional MRI measures lack of pathological specificity and are weakly correlated with MS clinical manifestations. Advanced MRI techniques are improving the understanding of the mechanisms underlying tissue injury, repair, and functional adaptation in MS; however, they require careful standardization. The definition of standardized methods for the collection and analysis of advanced MRI techniques is central not only to improve the understanding of disease pathophysiology and evolution, but also to generate research hypotheses, monitor treatment, increase cost-effectiveness and power of clinical trials. We promoted the Italian Neuroimaging Network Initiative (INNI), involving centers and investigators with an International recognized expertise, with the major goal to determine and validate novel MRI biomarkers to be utilized as predictors and/or outcomes in future MS studies. The INNI initiative supported the creation of a centralized repository, where advanced structural and functional MRI scans available at the participating sites, with the related clinical and neuropsychological data, are collected. These data will be used to perform research studies to identify clinical, neuropsychological and imaging biomarkers characteristics of the entire spectrum of MS. INNI will be instrumental to help to define standardized MRI and clinical protocols towards an increasing uptake of personalized interventions for people with MS at a national and international level. Upon approval of the INNI Steering Committee, the data collected in the online database will be shared with any research center detailing specific research proposals on disease pathophysiology or treatment effects.

Progressive MS Alliance Industry Forum: Maximizing Collective Impact To Enable Drug Development.

The Progressive MS Alliance Industry Forum describes a new approach to address barriers to developing treatments for progressive multiple sclerosis (MS). This innovative model promises to facilitate robust collaboration between industry, academia, and patient organizations and accelerate research towards the overarching goal of developing safe and effective treatments for progressive MS.

Development and validation of the multiple sclerosis questionnaire for the evaluation of job difficulties (MSQ-Job).

OBJECTIVE: Multiple sclerosis (MS) affects young adults of working age. Difficulties in work-related activities are usually ascribed to MS symptoms, while the impact of workplace features is underestimated. This article presents the Multiple Sclerosis Questionnaire for Job Difficulties (MSQ-Job), designed to assess working difficulties due to MS symptoms and workplace features. METHODS: A sample of employed MS patients completed the MSQ-Job, the WHO-Disability Assessment Schedule (WHODAS 2.0) and the 54-items MS Quality of Life Questionnaires (MSQOL-54); the expanded disability status scale (EDSS) was used to define MS severity. Factor structure was evaluated using principal component extraction and Oblimin rotation; internal consistency was assessed with Cronbach's alpha; construct and discriminant validity using t-test (EDSS 0-2 vs >2; patients self-reporting need for support vs patients reporting no needs; full-time vs part-time employees); and Pearson's correlation with WHODAS 2.0 and MSQOL-54. RESULTS: The MSQ-Job is a 42-item questionnaire with six scales and an overall factor. Scores range on a 0-100 scale (higher scores indicate more and more severe difficulties); patients with EDSS>2 and self-reporting support needs had worse scores than those with EDSS 0-2 and without needs. Correlations with WHODAS 2.0 and MSQOL-54 were generally significant (P < 0.0007) and below 0.70. CONCLUSIONS: The MSQ-Job jointly measures the impact of respondents' symptoms and workplace features on work activities and enables to assess the effects of clinical and occupational interventions and better describe the impact of MS indirect costs.

Observational case-control study of the prevalence of chronic cerebrospinal venous insufficiency in multiple sclerosis: results from the CoSMo study.

BACKGROUND: Chronic cerebrospinal venous insufficiency (CCSVI) has been proposed as a possible cause of multiple sclerosis (MS). OBJECTIVES: The CoSMo study evaluated the association between CCSVI and MS. METHODS: The primary end-point of this multicentric, case-control study was to compare the prevalence of CCSVI between patients with MS, patients with other neurodegenerative diseases (ONDs) and healthy controls (HCs). Color-coded duplex sonography was performed by a sonologist and the images were sent to one of three central sonologists for a second reading. Agreement between local and central sonologists or, in case of disagreement, the predominant judgment among the three central readers, was required for a diagnosis of CCSVI. All readings, data collection and analysis were blinded. RESULTS: The study involved 35 MS centers across Italy and included 1874 subjects aged 18-55. 1767 (94%) were evaluable: 1165 MS patients, 226 patients with ONDs and 376 HCs. CCSVI prevalence was 3.26%, 3.10% and 2.13% for the MS, OND and HC groups, respectively. No significant difference in CCSVI prevalence was found amongst the three cohorts (MS versus HC, OR = 1.55, 95%CI = 0.72-3.36, p = 0.30; OND versus HC, OR = 1.47, 95%CI = 0.53-4.11, p = 0.46; MS versus OND, OR = 1.05, 95%CI = 0.47-2.39, p = 0.99). High negative and low positive agreement was found between the local and centralized readers. CONCLUSIONS: CCSVI is not associated with MS.

Macrophages and liposomes in inflammatory disease: friends or foes?

Liposome-encapsulated corticosteroids have shown to exert strong beneficial effects in inflammatory diseases, such as arthritis and cancer. To extend the clinical applicability of these potent nanomedicines, the therapeutic effect of dexamethasone phosphate loaded long-circulating liposomes (LCL-DXP) was evaluated in animal models of multiple sclerosis (MS) and Crohn's disease (CD). In mice with experimental autoimmune encephalitis (EAE), a model for MS, treatment with LCL-DXP, but not free DXP, resulted in a decrease in disease activity when compared to PBS treated mice. In contrast, in mice with chronic DSS-induced colitis, a model for CD, treatment with LCL-DXP did not induce an improvement, but in fact worsened the fecal blood loss after treatment, indicating an aggravation of the disease. It is hypothesized that modulation of macrophage polarization towards a M2 phenotype underlies the efficacy of corticosteroid-based drug delivery systems, which is supported by the presented data. On the one hand, M1 polarized macrophages are part of the pathogenesis of MS; the modulation to M2-polarization by LCL-DXP is therefore beneficial. On the other hand, M1-polarized intestinal macrophages fulfill a protective and inflammation-suppressing role in intestinal homeostasis; changing their phenotype to M2 causes reduced protection to invading microorganisms, leading to a more severe intestinal inflammation. These findings therefore indicate that the interplay between the specific phenotype of macrophages and the specific inflammatory context of the inflammatory disease in question may be an important determining factor in the therapeutic applicability of liposomal corticosteroids in inflammatory disease.

Variable effects of cyclophosphamide in rodent models of experimental allergic encephalomyelitis.

In this study, we have evaluated the effects of cyclophosphamide on the development of experimental allergic encephalomyelitis (EAE) in four EAE rodent models: monophasic EAE in Lewis rats, protracted relapsing (PR)-EAE in DA rats, myelin oligodendrocyte protein (MOG)-induced EAE in C57Bl/6 mice and proteolipid protein (PLP)-induced EAE in Swiss/Jackson Laboratory (SJL) mice. Cyclophosphamide, administered either prophylactically or therapeutically, suppressed most strongly the clinical symptoms of PR-EAE in DA rats. Treated rats in this group also exhibited the lowest degree of inflammatory infiltration of the spinal cord, as well as the lowest levels of nuclear factor kappa B, interleukin-12 and interferon-gamma. Cyclophosphamide prophylactically, but not therapeutically, also delayed significantly the onset of EAE in Lewis rats. In contrast, regardless of the treatment regimen used, was unable to influence the clinical course of EAE in either MOG-induced EAE in C57Bl/6 mice or PLP-induced EAE in SJL mice. This heterogeneous pharmacological response to cyclophosphamide suggests that significant immunopathogenic differences exist among these EAE rodent models that must be considered when designing preclinical studies. In addition, the effectiveness of cyclophosphamide in dark Agouti (DA) rats with PR-EAE suggests that this may be a particularly useful model for studying novel therapeutic approaches for refractory and rapidly worsening multiple sclerosis in human patients.

Administration of a monomeric CCL2 variant to EAE mice inhibits inflammatory cell recruitment and protects from demyelination and axonal loss.

Based on gene expression data, we tested the P8A-CCL2 variant of the chemokine CCL2, able to interfere with the chemotactic properties of the parental molecule, in relapsing-remitting (RR)-EAE SJL. Only preventive treatment significantly delayed disease onset in a dose dependent manner. P8A-CCL2 administration, however, decreased demyelination, axonal loss and number of CNS infiltrating T cells and macrophages. Immunological analysis revealed that P8A-CCL2 does not act on Ag-specific T cell proliferation and does not interfere with the differentiation of IFNgamma-releasing effectors T cells. These results suggest that the therapeutic mechanism of P8A-CCL2 may rely on interference with immune cell recruitment.

Beneficial effects of r-h-CLU on disease severity in different animal models of peripheral neuropathies.

Clusterin is a protein involved in multiple biological events, including neuronal cytoprotection, membrane recycling and regulation of complement-mediated membrane attack after injury. We investigated the effect of recombinant human clusterin in preclinical models of peripheral neuropathies. Daily treatment with clusterin accelerated the recovery of nerve motor evoked potential parameters after sciatic nerve injury. Prophylactic or therapeutic treatment of experimental autoimmune neuritis rats with clusterin also accelerated the rate of recovery from the disease, associated with remyelination of demyelinated nerve fibers. These data demonstrate that clusterin is capable of ameliorating clinical, neurophysiological and pathological signs in models of peripheral neuropathies.

TNF-alpha modulates angiopoietin-1 expression in rheumatoid synovial fibroblasts via the NF-kappa B signalling pathway.

Angiopoietin-1 (Ang-1) is one of a family of ligands for the Tie-2 receptor which has been demonstrated to be involved in angiogenesis. Little is known about the regulation of Ang-1 gene expression. We have previously demonstrated that TNF-alpha is able to up-regulate the expression of Ang-1 mRNA in synovial fibroblasts. This present study investigated the signal transduction pathways involved in the TNF-alpha induced expression of Ang-1. TNF-alpha signals primarily through the p38, JNK, MAP kinase, and IKK pathways resulting in the activation of the transcription factors AP-1 and NF-kappa B. Experiments with inhibitors and siRNA for these various signal transduction pathways revealed that TNF-alpha stimulation of Ang-1 expression occurs via the NF-kappa B signal transduction pathway.

Activity of N-(phenethyl)phenylethanolamines at beta(1) and beta(2) adrenoceptors: structural modifications can result in selectivity for either subtype.

A series of phenylethanolamines bearing a 2-[1-phenylpropyl] substituent on the nitrogen atom was evaluated in vitro for activity at beta(1)- and beta(2)-adrenoceptors. As previously observed, the presence of 3,4-dihydroxy substitution on phenylethanolamine is required for potent activation of both subtypes, whereas the 3,5-dihydroxy analog showed selectivity for the beta(2)-subtype. Replacement by a carboxyl group of the 4-hydroxyl group on the aralkyl nitrogen substituent produced only a small reduction in beta(1) potency (5-fold), whereas beta(2) potency was reduced by more than 100-fold. Hence this structural class includes agonists having either a beta(1), nonselective beta(1)/beta(2) or beta(2) selectivity profile.

NK3 receptor blockade prevents hyperalgesia and the associated spinal cord substance P release in monoarthritic rats.

Previous studies in vitro have shown that NK3 receptors exist on primary afferent terminals in rat spinal cord and mediate potentiation of the depolarisation-evoked substance P (SP) release. In the present study we have investigated the role of the NK3 receptor-mediated SP release system in a model of inflammatory pain. Monoarthritis was induced in rats by unilateral injection of complete Freund's adjuvant (CFA); withdrawal latencies to a thermal stimulus were subsequently measured at various times following CFA. The CFA-treated paw displayed hyperalgesia as early as 4 h after CFA injection and hyperalgesia was maintained until day 4 but had disappeared by day 21. The thermal hyperalgesia was associated with an increase in basal SP release from spinal cord synaptosomes. The possible involvement of endogenous neurokinin B acting at NK3 receptors was tested by using SB 223412-A [(S)-(-)-N-(alpha-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carbo xamide hydrochloride], a novel, potent (Ki=30 nM) and selective (Ki>10,000 nM for NK1 and NK2 receptors), non-peptidic NK3 receptor antagonist. In vitro SB 223412-A antagonised the potentiation of SP release produced by senktide in spinal cord synaptosomes. Administered systemically to monoarthritic rats (50 mg/kg, p.o., b.i.d., for 4 days), the NK3 receptor antagonist SB 223412-A significantly reduced thermal hyperalgesia and normalised the basal release of SP from spinal cord synaptosomes. The data suggest that neurokinin B acting at NK3 receptors that mediate SP release within the spinal cord play a role in inflammation. These NK3 receptors may represent, therefore, appropriate targets in the therapy of inflammatory pain.

Comparative effects of selective kappa-opioid receptor agonists on dopamine levels in the dorsal caudate of freely moving rats.

Microdialysis was utilized to evaluate the effects of selective kappa-opioid receptor agonists on dopamine levels in the dorsal caudate of conscious rats. Subcutaneous administration of equivalent antinociceptive doses of spiradoline--(+/-)-(5 alpha, 7 alpha, 8 beta)-3, 4-dichloro-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl] benzeneacetamide--(U62066; 12 mg/kg), BRL 52656--(2S)-1-[(4- trifluoromethylphenyl)acetyl]-2-[(1-pyrrolidinyl)methyl]piperidine--(2 mg/kg) and enadoline--(-)-(5 beta, 7 beta, 8 alpha)-N-methyl-N-[7-(1- pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]benzo[b]furan-4-acetamide-- (CI-977; 0.1 mg/kg) produced similar, statistically significant decreases in dorsal caudate dopamine levels; BRL 53001--(2S)-2- (dimethylaminomethyl)-1-[(5,6,7,8-tetrahydro-5-oxo-2-naphthyl)ace tyl] piperidine--(12 mg/kg) was, however, without effect. At a higher dose (36 mg/kgP, BRL 53001 also caused a significant reduction in dopamine levels. BRL 52974--4-(1-pyrrolidinylmethyl) 5-[(3,4-dichlorophenyl)acetyl]-4,5,6,7-tetrahydroimidazo[4,5-c] pyridine, a selective kappa-opioid receptor agonist with limited ability to cross the blood brain barrier or produce antinociceptive effects, had no effect on dopamine levels at 10 mg/kg s.c. Overall, these findings suggest that selective kappa-opioid receptor agonists decrease dopamine levels in the dorsal caudate of rats via a central locus of action. Furthermore, compared to other kappa-opioid receptor agonists, BRL 53001 appears to have a reduced propensity to decrease dopamine levels at equianalgesic doses.

Methylazoxymethanol-induced microencephaly: persistent increase of cortical somatostatin-like immunoreactivity.

Treatment of pregnant rats with methylazoxymethanol (MAM) at day 15 of gestation induces a marked microencephaly in the offspring, characterized by impaired formation of interneurons in the areas affected. We have measured somatostatin immunoreactivity in the cortex, striatum and hippocampus of the offspring of pregnant rats treated with graded doses of MAM. A long-lasting increase in the cortical levels of this peptide was found, suggesting that somatostatinergic interneurons might be selectively spared by administration of the cytotoxic agent at this gestational age.

Gas chromatographic-mass spectrometric determination of levodropropizine plasma levels in healthy volunteers.

A gas chromatographic-mass spectrometric method for the qualitative and quantitative analysis of levodropropizine (S(-)-3-(4-phenyl-piperazin-1-yl)-propane-1,2-diol, DF 526) in plasma is described. The method proved to be highly selective and sensitive. Drug concentrations as low as 5 ng/ml could be measured. Levodropropizine plasma levels were measured in 6 healthy volunteers after administration of an acute 60 mg dose. Peak concentrations were reached between 40 and 60 min and measurable amounts of drug were present till 8 h after administration.

Adenosine modulates the dopaminergic function in the nigro-striatal system by interacting with striatal dopamine dependent adenylate cyclase.

Behavioral and pharmacological evidences suggest that dopaminergic mechanisms in striatum might be counteracted by adenosine or potentiated by its pharmacological antagonists methylxanthines. To test whether adenosine modulation of the dopaminergic function could be, at least in part, due to an interaction at the level of the adenylate cyclase complex, we studied the effects of the adenosine analog R-Phenyl-isopropil-adenosine (R-PIA) on basal and dopamine-sensitive adenylate cyclase in rat striatum. R-PIA, which interacts with both adenosine A1-inhibitory and A2-stimulatory receptors, dose-dependently inhibited the stimulation induced by dopamine, and seemed to utilize the same pool of enzyme linked to dopaminergic D1 receptors. Two experimental approaches leading to supersensitivity of striatal dopaminergic receptors, (i.e., 6-hydroxy-dopamine injection in substantia nigra and reserpine administration) also induced upregulation of adenosine-dependent adenylate cyclase in striatum, and altered R-PIA modulation of dopamine-sensitive adenylate cyclase. Conversely, after subchronic treatment with neuroleptics such as haloperidol or sulpiride, upregulation of 3H-Spiroperidol binding in striatum was not associated with changes of R-PIA dependent adenylate cyclase in this area. It is concluded that adenosine might modulate post-synaptic responses to dopamine via adenosine receptors which functionally interact with dopaminergic D1 receptors in striatum.