Synthesis and in vitro pharmacological evaluation of a new series of 5-HT1A 5-HT2A and 5-HT2C receptor ligands containing a norbornene nucleus.

A series of 4-substituted piperazine derivatives bearing a norbornene nucleus have been prepared and their affinity for serotonin 5-HT1A, 5-HT2A and 5-HT2C receptors has been evaluated. Compounds showing the highest affinity have been selected and evaluated on dopaminergic (D1 and D2) and adrenergic (alpha1 and alpha2) receptors. The combination of structural elements (heterocyclic nucleus, oxyalkyl chain and 4-substituted piperazine) known to be critical in order to have affinity on serotonin receptors and the proper selection of substituents led to compounds with higher receptor specificity and affinity. In binding studies, several molecules showed affinity in nanomolar range towards 5-HT1A, 5-HT2A and 5-HT2C receptors and moderate to no affinity for other relevant receptors (D1, D2, alpha1 and alpha2). Compound 2q 4-[2-[4-(3,4-dichlorophenyl)piperazin-1-yl]ethoxy]-4-aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (Ki = 1.13 nM), was the most active and selective derivative for the 5-HT2C receptor with respect to other serotonin, dopaminergic and adrenergic receptors. Moreover, compound 3p showed mixed 5-HT2A/5-HT2C activity with affinity values in nanomolar range.

Intracellular oxidative activity and respiratory burst of leukocytes isolated from multiple sclerosis patients.

Oxidative damage induced by free radicals and reactive oxygen species (ROS) have been suggested to play an important role in the development of autoimmune diseases such as multiple sclerosis (MS) disease and it has been hypothesised that oxidative injury could mediate demyelination and axonal injury in MS subjects. In our study, we compared intracellular oxidative activity and the respiratory burst activity in MS patients (n=20) and healthy controls (n=15) using leukocytes as cellular model. At this purpose, intracellular ROS levels were evaluated by fluorometric assay using the 2'-7'-dichlorodihydrofluorescin diacetate probe (H(2)DCFDA) in untreated or in leukocytes stimulated with phorbol-12-myristate-13-acetate (PMA). Our results demonstrate that the intracellular spontaneous ROS production in leukocytes from MS patients was higher with respect to cells from control subjects (p<0.001). PMA addition induced a higher formation of ROS both in leukocytes from MS patients and controls (p<0.001). The PMA-induced production of ROS was significantly higher in leukocytes from MS with respect to controls (p<0.001). Significant positive correlations were established between intracellular spontaneous or PMA-induced production of ROS in leukocytes isolated from MS patients and the clinical parameters used to evaluate disease disability such as expanded disability status scale (EDSS), brain lesions evaluated by MRI and visual evoked potential (VEP) (p<0.001). In conclusion, our results demonstrate higher levels of intracellular ROS in untreated or in PMA-treated leukocytes isolated from MS patients with respect to healthy subjects confirming the role of oxidative stress in multiple sclerosis.

Increased levels of lipid hydroperoxides in plasma of patients with multiple sclerosis: a relationship with paraoxonase activity.

Paraoxonase, an enzyme associated with high density lipoproteins (HDL), plays an important role in the anti-oxidant and anti-inflammatory properties exerted by HDL. Increasing evidence supports a role of free radicals and oxidative stress in the inflammatory processes and in the pathogenesis of multiple sclerosis (MS). The aim of this study was to further investigate the relationship between oxidative damage and MS; therefore we compared the paraoxonase activity and levels of cholesteryl ester hydroperoxides (CE-OOH), as marker of lipid peroxidation, in plasma isolated from healthy subjects (n = 89) and from MS patients (n = 24) in the early stage disability (EDSS<3.5). Our results demonstrated for the first time that the activity of paraoxonase in the plasma of MS subjects was significantly lower with respect to controls (P <0.001). Moreover, our results showed a significant increase in the levels of CE-OOH in plasma from MS subjects (P<0.001). CE-OOH are biologically active substances derived from the oxidation of cholesteryl ester localized in the hydrophobic core of plasma lipoproteins (HDL, LDL). Therefore, our study demonstrates alterations of lipoprotein peroxidation in MS and provides further evidence that oxidative stress and impairment of the anti-oxidant system may play a role in MS.

Contribution of cervical cord MRI and brain magnetization transfer imaging to the assessment of individual patients with multiple sclerosis: a preliminary study.

This study was performed to assess how established diagnostic criteria for brain magnetic resonance imaging (MRI) interpretation in cases of suspected multiple sclerosis (MS) (Barkhofs criteria) would perform in the distinction of MS from other diseases and whether other MR techniques (cervical cord imaging and brain magnetization transfer imaging [MTI]), might help in the diagnostic work-up of these patients. We retrospectively identified 64 MS and 59 non-MS patients. The latter group included patients with systemic immune-mediated disorders (SID; n=44) and migraine (n=15). All patients had undergone MRI scans of the brain (dual echo and MTI) and of the cervical cord (fast short-tau inversion recovery). The number and location of brain T2-hyperintense lesions and the number and size of cervical cord lesions were assessed. Brain images were also postprocessed to quantify the total lesion volumes (TLV) and to create histograms of magnetization transfer ratio (MTR) values from the whole of the brain tissue. Barkhofs criteria correctly classified 108/123 patients, thus showing an accuracy of 87.8%. "False negative" MS patients were 13, while 2 patients with systemic lupus erythematosus (SLE) were considered as "false positives". Using brain T2 TLV, nine of the "false negative" patients were correctly classified. Correct classification of 10 MS patients and both the SLE patients was possible based upon the presence or absence of one cervical cord lesion. Two MS patients with negative Barkhofs criteria and no cervical cord lesions were correctly classified based on their brain MTR values. Overall, only one MS patient could not be correctly classified by any of the assessed MR quantities. These preliminary data support a more extensive use of cervical cord MRI and brain MTI to differentiate between MS and other disorders in case of incondusive findings on T2-weighted MRI scans of the brain.

Cervical cord magnetic resonance imaging findings in systemic immune-mediated diseases.

Brain magnetic resonance imaging (MRI) findings in patients with systemic immune-mediated diseases (SID) affecting the central nervous system (CNS) are heterogeneous and lack diagnostic specificity. In the present study, we evaluated the potential role of cervical cord MRI for increasing confidence when making a diagnosis of SID. Sagittal, T2-weighted images of the cervical cord were obtained in patients affected by systemic lupus erythematosus (SLE) with (NSLE, n=9) and without clinical CNS involvement (n=15), Behçet disease (BD, n=5), Wegener granulomatosis (WG, n=9), antiphospholipid antibody syndrome (APLAS, n=6) and multiple sclerosis (MS, n=10). Spinal cord hyperintense lesions were found in 9/10 MS patients, while no lesions were visible in the cervical cord of any patient with SID, regardless of the presence or absence of brain abnormalities. Cervical cord MRI can be useful in diagnosing patients suspected of having a SID.

The contribution of fast-FLAIR MRI for lesion detection in the brain of patients with systemic autoimmune diseases.

Fast fluid-attenuated inversion recovery (fFLAIR) is more sensitive that conventional or fast spin echo T2-weighted magnetic resonance imaging (MRI) for detecting lesions in the brain of patients with ischemic, inflammatory, or demyelinating diseases of the CNS. We investigated whether the use of fFLAIR also increases the sensitivity of brain MRI assessment in patients with systemic autoimmune disorders. Turbo spin echo (TSE) dual-echo and fFLAIR scans of the brain were obtained from patients affected by systemic lupus erythematosus (SLE) with (NSLE, n = 9) and without clinical CNS involvement (n = 15), Behçet disease (n = 5), Wegener granulomatosis (n = 9), and antiphospholipid antibody syndrome (n = 6). Brain hyperintense lesions were counted and classified according to their size and their location by two observers by consensual agreement. The total lesion volume was measured using a semiautomated technique for lesion segmentation on both TSE and fFLAIR scans. The imaging modalities showed brain hyperintense lesions in all 9 SLE patients with CNS involvement, 5 of 15 SLE patients without CNS involvement, 5 of 9 patients with Wegener granulomatosis, 1 of 5 with Behçet disease, and 3 of 6 with antiphospholipid antibody syndrome. A total of 342 lesions were seen on both sequences; 88 were seen only on TSE and 54 only on fFLAIR scans. The average number of brain lesions per scan was higher on TSE than on fFLAIR, since significantly more discrete (P<0.002) and small (P = 0.004) lesions were seen on TSE than on fFLAIR. The median total lesion volume, however, was similar on TSE and fFLAIR. Our study indicates that the use of fFLAIR does not improve the sensitivity of fast dual-echo MRI for detecting brain abnormalities in patients with systemic autoimmune disorders.

Brain involvement in systemic immune mediated diseases: magnetic resonance and magnetisation transfer imaging study.

OBJECTIVE: Magnetisation transfer imaging (MTI) provides information about brain damage with increased pathological specificity over conventional MRI and detects subtle abnormalities in the normal appearing brain tissue, which go undetected with conventional scanning. Brain MRI and MTI findings were compared in patients with multiple sclerosis (MS) and systemic immune mediated diseases (SIDs) affecting the CNS to investigate their roles in understanding the nature of brain damage in these diseases. METHODS: Brain dual echo, T1 weighted and MTI scans were obtained in patients affected by systemic lupus erithematosus (SLE) with (NSLE, n=9) and without clinical CNS involvement (n=15), Behçet's disease (BD) (n=5), Wegener's granulomatosis (WG) (n=9), and antiphospholipid antibody syndrome (APLAS) (n=6). Ten patients with clinically definite MS and 15 healthy controls also underwent the same scanning protocol. Brain MRI and MT ratio (MTR) images of the same subject were coregistered and postprocessed to obtain MTR histograms of the whole brain and of the NABT. RESULTS: Brain hyperintense lesions were found in all patients with MS and with NSLE and in 5/15 patients with SLE, 5/9 with WG, 1/5 with BD, and 3/6 with APLAS. The lesion burden in the brain was significantly higher in patients with MS compared with all the other disease groups. All MTR histogram parameters were significantly different among patient subgroups. Patients with MS had significantly lower average MTR than all except patients with NSLE and significantly lower peak height and location than patients with SLE. patients with NSLE had significantly lower average MTR than patients with SLE. CONCLUSIONS: Microscopic brain tissue damage is relevant in patients with MS, but, apart from patients with NSLE, it seems to be absent in systemic immune mediated diseases, even in the presence of macroscopic MRI lesions or clinical evidence of CNS involvement.

A magnetization transfer histogram study of normal-appearing brain tissue in MS.

OBJECTIVE: To evaluate 1) the ability of magnetization transfer ratio (MTR) histogram analysis to detect the extent of changes occurring outside MS lesions seen on conventional scans, 2) whether such changes vary in the different MS clinical phenotypes, 3) whether the changes are associated with the extent and severity of the macroscopic lesion load, and 4) the contribution to brain atrophy. METHODS: Dual-echo, T1-weighted, and MT scans of the brain were obtained from 77 patients with varying MS courses and 20 age- and sex-matched control subjects. To create MT histograms of the normal-appearing cerebral tissue, MS lesions were segmented from dual-echo scans, superimposed automatically, and nulled out from the coregistered and scalp-stripped MTR maps. Average MTR, peak height, and peak position were considered. T2 and T1 lesion loads, average lesion MTR, and brain volume were also measured. RESULTS: Average histogram MTR (p<0.0001) and peak position (p<0.0001) from patients with relapsing-remitting MS (RMMS) were lower than those from control subjects. Patients with primary progressive MS (PPMS) had lower average histogram MTR (p = 0.002) and histogram peak height (p = 0.01) than control subjects. Patients with secondary progressive MS (SPMS) had a lower peak height (p = 0.05) than those with RRMS. Average lesion MTR (p<0.0001) correlated highly with the histogram MTR. Average histogram MTR (p<0.0001) and T2 lesion load (p = 0.001) correlated highly with brain volume. CONCLUSIONS: The amount of microscopic changes account for an important fraction of the lesion load in MS. They may contribute to the development of brain atrophy and tend to be more evident in patients with secondary progressive MS.

Multiple sclerosis: interobserver agreement in reporting active lesions on serial brain MRI using conventional spin echo, fast spin echo, fast fluid-attenuated inversion recovery and post-contrast T1-weighted images.

Previous studies have addressed the question of the precision in assessing multiple sclerosis (MS) activity by counting enhancing lesions on gadolinium enhanced brain magnetic resonance imaging (MRI). However, counting the active lesions on serial unenhanced MRI obtained by various pulse sequences has not been yet considered. We compared the interobserver levels of agreement in reporting active MS lesions on serial enhanced and unenhanced MRI to assess whether the use of various unenhanced techniques may change the degree of interobserver measurement reproducibility. Dual-echo conventional spin echo (CSE), dual-echo fast spin echo (FSE), fast fluid-attenuated inversion recovery (FLAIR) and Gd-enhanced T1-weighted brain MRI were obtained from five MS patients at baseline and monthly for 2 months. Six experienced observers independently identified and counted active MS lesions on the two follow-up MRI scans. Active lesions were considered to be all the enhancing lesions and any new or enlarging lesion on enhanced and unenhanced scans. Interobserver levels of agreement were calculated by weighted kappa values. Very good agreement was reached only for counting total and new Gd-enhancing lesions. Good agreement was achieved for counting new lesions on the three unenhanced techniques, whereas the agreement for counting enlarging lesions was poor with all the MRI techniques. The level of agreement was significantly heterogeneous for various MRI techniques but not for various lesion sites. These results confirm that counting enhancing lesions is the most reliable method for assessing MS activity, but the use of any of the available unenhanced MRI techniques did not result in different levels of interobserver agreement when reporting new and enlarging MS lesions on serial scans.