The effect of pressurized carbon dioxide as a temporary plasticizer and foaming agent on the hot stage extrusion process and extrudate properties of solid dispersions of itraconazole with PVP-VA 64.

The aim of the current research project was to explore the possibilities of combining pressurized carbon dioxide with hot stage extrusion during manufacturing of solid dispersions of itraconazole and polyvinylpyrrolidone-co-vinyl acetate 64 (PVP-VA 64) and to evaluate the ability of the pressurized gas to act as a temporary plasticizer as well as to produce a foamed extrudate. Pressurized carbon dioxide was injected into a Leistritz Micro 18 intermeshing co-rotating twin-screw melt extruder using an ISCO 260D syringe pump. The physicochemical characteristics of the extrudates with and without injection of carbon dioxide were evaluated with reference to the morphology of the solid dispersion and dissolution behaviour and particle properties. Carbon dioxide acted as plasticizer for itraconazole/PVP-VA 64, reducing the processing temperature during the hot stage extrusion process. Amorphous dispersions were obtained and the solid dispersion was not influenced by the carbon dioxide. Release of itraconazole from the solid dispersion could be controlled as a function of processing temperature and pressure. The macroscopic morphology changed to a foam-like structure due to expansion of the carbon dioxide at the extrusion die. This resulted in increased specific surface area, porosity, hygroscopicity and improved milling efficiency.     

Serotonin-binding proteins in the bovine cerebral cortex: interaction with serotonin and catecholamines.

The soluble serotonin-binding proteins (SBP) present in bovine frontal cortex are very similar to those reported in rat brain. Binding of [3H]serotonin to SBP, present in ammonium sulphate-precipitated proteins from bovine cortex, requires Fe2+ but not Fe3+. In the presence of an optimal concentration of Fe2+ (0.1 mM), bovine SBP behave as a single class of non-cooperative sites for [3H]serotonin binding (Bmax = 120 +/- 12 pmol/mg protein, KD = 0.12 +/- 0.04 microM, n = 3). Binding of [3H]serotonin is decreased by nucleotides and by reagents which modify sulfhydryl groups and reduce disulfide bonds and by metal ion chelators. Serotonin analogs possessing an hydroxyl group on the indole ring and catecholamine analogs possessing an intact catechol moiety are effective competitors (Ki from 0.1 to 0.3 microM). In both cases, the aliphatic amino group does not contribute to the binding, but the affinity is strongly decreased if aromatic hydroxyl groups are methoxylated. Catecholamine-SBP interactions can also be demonstrated directly by binding experiments. Binding of [3H]dopamine is greatly enhanced by Fe2+, Cu2+ and Mn2+, but not by Fe3+. The Fe(2+)-dependent binding component of [3H]dopamine is saturable (Bmax = 279 +/- 64 pmol/mg protein, KD = 0.19 +/- 0.02 microM, n = 3), and possesses the same physicochemical properties as SBP: it elutes immediately after the void volume on a Sephacryl S100 HR (1.6 x 140 cm) gel filtration column (reflecting aggregation) and it migrates with an apparent molecular weight of 57-58 kDa on native polyacrylamide gel electrophoresis. Whereas the serotonin-storing role of SBP in serotonergic neurons has already been well documented, the present data advocate that these proteins may also possess catecholamine-storing properties.     

T-cell vaccination in multiple sclerosis: update on clinical application and mode of action

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Autoreactive T cells specific for myelin antigens are considered to play a prominent role in the initiation of the local inflammatory response, ultimately leading to myelin damage. Several studies indicate that autoreactive T cells are not completely deleted in the thymus, but are part of the normal T cell repertoire. Accidentally activated autoreactive T cells, however, may not automatically lead to autoimmune disease. Several reports support the existence of peripheral regulatory networks that prevent the activation and expansion of pathogenic T cells. Anti-idiotypic and anti-ergotypic T cells are part of this regulatory network and are thought to control autoreactive T cells by recognition of certain clonotypic and ergotypic determinants. These clonotypic networks may not function properly in patients with MS. Immunization with attenuated autoreactive T cells, termed T cell vaccination (TCV), may enhance or restore the regulatory networks to specifically suppress the autoreactive T cells as shown in experimental autoimmune encephalomyelitis (EAE), a commonly used animal model for MS. In the past decade, TCV has been tested for MS in several clinical trails. This review summarizes these clinical trails and updates our current knowledge on the mode of action of T cell vaccination.     

Clonal expansion of myelin basic protein-reactive T cells in patients with multiple sclerosis: Restricted T cell receptor V gene rearrangements and CDR3 sequence

Myelin basic protein (MBP)-reactive T cells are thought to play an important role in the pathogenesis of multiple sclerosis (MS). In some patients with MS, these autoreactive T cells display a limited heterogeneity in their epitope recognition and T cell receptor (TCR) variable (V) gene usage. These individual-dependent properties of MBP-reactive T cells have led to the speculation that they may represent clonal expansion in vivo in some MS patients. In the present study, 51 MBP-reactive T cell clones derived from patients with MS and healthy individuals were examined for their epitope recognition and the TCR Vα and Vβ gene rearrangements. The V gene junctional region sequences of identified α and β genes were further analyzed to probe their clonal origins, as the sequences are unique for individual clones. Our data showed that 26 clones derived from nine patients with MS shared a predominant reactivity to the immunodominant regions of MBP, 84–102, 110–129 and 143–168, and used various TCR Vα and Vβ rearrangements. The V gene usage of the clones was restricted to certain Vα Vβ combination(s) in a given MS patient, but varied among different patients. The sequence analysis revealed that the clones generated from a given patient shared a limited or a single junctional region sequence pattern(s), indicating their oligoclonal or monoclonal origin(s). In contrast, 25 MBP-reactive T cell clones derived from normal individuals exhibited unfocused epitope recognition and V gene usage. Thus, the limited heterogeneity of MBP-reactive T cells in their structural and functional charactertistics reflects their clonal expansion in vivo in some patients with MS.     

Short-term treatment of gastroesophageal reflux disease

OBJECTIVE: To investigate the efficacy of acid suppressant drugs in the empirical treatment of gastroesophageal reflux disease (GERD) and in the treatment of endoscopy-negative reflux disease (ENRD). DESIGN: medline, embase, and the Cochrane Controlled Trials Register were searched. Bibliographies were reviewed. SETTING: Studies were eligible that compared the short-term use of proton pump inhibitors (PPIs) and histamine-2 receptor antagonists (H2RAs) with each other or with placebo in adults with GERD who were enrolled irrespective of endoscopic findings (empirical cases) or in whom endoscopy showed no signs of esophagitis (endoscopy-negative cases). MEASUREMENTS: Of 1,408 studies, only 13 could be included for meta-analysis. Data on 3,433 patients empirically treated for GERD and 2,520 patients treated for ENRD were extracted. The primary endpoint was relief of heartburn. MAIN RESULTS: In the empirical treatment of GERD, the summary relative risk (sRR) for symptom relief from H2RAs versus placebo was 0.77 (95% confidence interval [95% CI], 0.60 to 0.99). RR in the only placebo-controlled PPI trial was 0.35 (95% CI, 0.26 to 0.46). The sRR for standard dose PPIs versus H2RAs was 0.55 (95% CI, 0.44 to 0.68). In treatment of ENRD, both PPIs (sRR, 0.64; 95% CI, 0.52 to 0.79) and H2RAs (sRR, 0.78; 95% CI, 0.62 to 0.97) were superior to placebo, and PPIs were superior to H2RAs (sRR, 0.81; 95% CI, 0.70 to 0.95). CONCLUSIONS: Acid suppressant therapy (with a PPI or an H2RA) is more effective than placebo for short-term relief of heartburn in patients with persistent symptoms who are treated empirically for GERD and in those in whom esophagitis was excluded after endoscopy. The benefit of PPIs compared with H2RAs is more pronounced in patients treated empirically.     

Osteoprotegerin and receptor activator of nuclear factor-kappaB ligand mRNA expression in patients with rheumatoid arthritis and healthy controls

OBJECTIVE: To further understand the role of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANK-L) in rheumatoid arthritis (RA), we studied the levels of RANK-L and OPG mRNA in peripheral blood mononuclear cells (PBMC) and synovial tissue of patients with RA and controls. METHODS: RANK-L and OPG mRNA levels were measured in PBMC and CD4+/CD8+ T cell subsets of patients with chronic RA, osteoarthritis (OA), and healthy controls, using quantitative real-time polymerase chain reaction. OPG and RANK-L mRNA levels were measured in paired blood and synovial tissue samples of patients with early, untreated RA at 2 timepoints with an interval of 16 weeks. RESULTS: RANK-L mRNA levels were significantly higher in PBMC of patients with early and chronic RA compared to healthy controls. Contrary to healthy controls, RANK-L mRNA levels in patients with chronic RA were mainly of CD4+ T cell origin. OPG mRNA was observed in the blood of all (17/17) early RA patients, but could not be detected in chronic RA patients (0/14) or in patients with OA (0/8). Three out of 17 healthy controls showed measurable levels of OPG mRNA. The OPG/RANK-L ratio tended to be higher in the synovium than in the PBMC of early RA patients. RANK-L mRNA in synovial tissue was mainly of non-T cell origin. CONCLUSION: Since RANK-L and OPG mRNA levels are elevated in PBMC of RA patients, and CD4+ T cells are the major contributors to RANK-L mRNA expression, mononuclear cells in patients with RA may be involved in the pathways that regulate bone metabolism.