Synthesis, Biological, and Structural Explorations of New Zwitterionic Derivatives of 14- O-Methyloxymorphone, as Potent µ/δ Opioid Agonists and Peripherally Selective Antinociceptives.

Herein, the synthesis and pharmacological characterization of an extended library of differently substituted N-methyl-14- O-methylmorphinans with natural and unnatural amino acids and three dipeptides at position 6 that emerged as potent µ/δ opioid receptor (MOR/DOR) agonists with peripheral antinociceptive efficacy is reported. The current study adds significant value to our initial structure-activity relationships on a series of zwitterionic analogues of 1 (14- O-methyloxymorphone) by targeting additional amino acid residues. The new derivatives showed high binding and potent agonism at MOR and DOR in vitro. In vivo, the new 6-amino acid- and 6-dipeptide-substituted derivatives of 1 were highly effective in inducing antinociception in the writhing test in mice after subcutaneous administration, which was antagonized by naloxone methiodide demonstrating activation of peripheral opioid receptors. Such peripheral opioid analgesics may represent alternatives to presently available drugs for a safer pain therapy.

Morphinans and isoquinolines: acetylcholinesterase inhibition, pharmacophore modeling, and interaction with opioid receptors.

Following indications from pharmacophore-based virtual screening of natural product databases, morphinan and isoquinoline compounds were tested in vitro for acetylcholinesterase (AChE) inhibition. After the first screen, active and inactive compounds were used to build a ligand-based pharmacophore model in order to prioritize compounds for biological testing. Among the virtual hits tested, the enrichment of actives was significantly higher than in a random selection of test compounds. The most active compounds were biochemically tested for their activity on mu, delta, and kappa opioid receptors.

Binding of the chemokine SLC/CCL21 to its receptor CCR7 increases adhesive properties of human mesangial cells.

BACKGROUND: Adherence of human mesangial cells to the surrounding matrix contributes to glomerular homeostasis and is important for the maintenance of glomerular architecture and function in normal adult human kidney. The expression of chemokines and corresponding chemokine receptors on adjacent intrinsic renal cells indicates a novel chemokine/chemokine receptor function on nonimmune cells important for glomerular homeostasis. A constitutive expression of the chemokine SLC/CCL21 on human podocytes and of its corresponding receptor CCR7 on mesangial cells was shown before. SLC/CCL21 has a positive effect on proliferation and migration of mesangial cells and leads to increased cell survival in Fas-induced apoptosis. In leukocytes chemokines mediate integrin-dependent firm adhesion. Therefore, we examined the influence of chemokine receptor CCR7 activation by SLC/CCL21 on adhesive properties of human mesangial cells to matrix molecules. METHODS: Adhesion assays, mechanical detachment assays, and evaluation of integrin activation by integrin-linked kinase activity were performed. Changes in the cytoskeletal F-actin were illustrated by phalloidin immunofluorescence staining. RESULTS: SLC/CCL21 stimulation enhanced adhesiveness to fibronectin in a time- and concentration-dependent manner. SLC/CCL21 also increased the firmness of mesangial cells adhesion as judged by detachment assays. Furthermore activation of integrin-linked kinase occurred with SLC/CCL21 addition to mesangial cells, resulting in increased phosphorylation of glycogen synthase kinase-3 (GSK-3) and protein kinase B (PKB/Akt). Exposure of mesangial cells to SLC/CCL21 also resulted in F-actin rearrangements with membrane ruffling and extensions leading to bridging between mesangial cells. CONCLUSION: Activation of CCR7 on mesangial cells by SLC/CCL21 enhances the degree and firmness of cell adhesion and increases cell spreading and the formation of cell-cell contacts. This includes integrin-linked kinase activation and F-actin rearrangements. Thus, local chemokine generation and chemokine receptor expression on mesangial cells may play an important role in the maintenance of glomerular homeostasis and in local remodeling processes.

Regulation of glucose transporters in human peritoneal mesothelial cells.

BACKGROUND: Risk factors for peritoneal fibrosis and mesothelial cell (MsC) injury in CAPD are infections and bioincompatibility of the dialysate, including high glucose concentrations. To study a potential link between dialysate and glucose toxicity in MsC, we investigated the expression of facilitative glucose transporters (GLUT), which could contribute to glucose toxicity. METHODS: After induction of cell differentiation, MsC were incubated in regular medium or medium with 60 mM D-glucose, 30 mM glucose plus 30 mM mannitol, 60 mM mannitol, PD effluent, or with a cytokine mix. Expression of GLUT1, GLUT3, SGLT and GAPDH/L32 was studied by RNase protection assay. MsC were incubated under identical conditions with 14C-fluoro-deoxy-glucose for 30 minutes and glucose uptake was measured. To estimate Vmax and Km, 14C-fluoro-deoxy-glucose uptake rates were determined over a range of 0.6 to 10 mM unlabeled glucose. RESULTS: The cytokine mix significantly stimulated GLUT1 expression (3-fold) and GLUT3 (1.7-fold). There was a 1.4-fold increase in GLUT1 (p<0.05) and a 1.7-fold increase in GLUT3 (p<0.05) after incubation in high glucose but not in mannitol or PD-effluent controls. Glucose uptake studies confirmed this increase after incubation in 30 mM (p<0.05) and 60 mM glucose solutions. Kinetic studies showed the Km was approximately 3.7 mM for this transport. CONCLUSIONS: GLUT mRNA expression and glucose uptake are induced by high ambient glucose concentrations and cytokines. Unlike many other cells, MsC are not able to protect themselves from increased glucose concentrations by downregulation of GLUTs. The intracellular glucose concentration may therefore increase during CAPD, affecting growth factor expression and glycosylation, and contributing to glucose toxicity.

Acceptance of gene therapy by the heart surgery patient.

OBJECTIVE: The aim of the present study was to evaluate the attitude of cardiac surgery patients towards gene therapeutic approaches to heart disease. METHODS: One hundred and fifty patients having undergone coronary artery bypass grafting (n=97), valvular operations (n=40) or combined procedures (n=13) were personally interviewed prior to discharge using a self designed questionnaire. RESULTS: Seventeen percent of the surveyed patients were unable to complete the interview because of total non-understanding of the topic. Of the remaining patients, 33% could basically define the term 'gene'. After explanation of gene therapy principles 70% believed that gene therapy protocols had already been started clinically. Fifty-two percent would accept enrollment in a clinical trial (85% in cases of otherwise incurable disease). If clinical gene transfer would be carried out 73% of patients would accept adenovirus as a vector, 94 and 80%, respectively, would accept catheter-based intervention or surgery for performance of gene therapy. Fifty-four percent would agree to a prophylactic gene therapy protocol. CONCLUSIONS: We conclude that the general attitude of heart surgery patients regarding gene therapy is positive. A considerable number of patients are unable to understand the basic principles of gene therapy. These data can be useful for planning of clinical gene therapy trials and show potential difficulties in obtaining informed consent.