Antisense oligonucleotide and thyroid hormone conjugates for obesity treatment

Using the principle of antibody-drug conjugates that deliver highly potent cytotoxic agents to cancer cells for cancer therapy, we here report the synthesis of antisense-oligonucleotides (ASO) and thyroid hormone T3 conjugates for obesity treatment. ASOs primarily target fat and liver with poor penetrance to other organs. Pharmacological T3 treatment increases energy expenditure and causes weight loss, but is contraindicated for obesity treatment due to systemic effects on multiple organs. We hypothesize that ASO-T3 conjugates may knock down target genes and enrich T3 action in fat and liver. Two established ASOs are tested. Nicotinamide N-methyltransferase (NNMT)-ASO prevents diet- induced obesity in mice. Apolipoprotein B (ApoB)-ASO is an FDA approved drug for treating familial hypercholesterolemia. NNMT-ASO and ApoB-ASO are chemically conjugated with T3 using a non- cleavable sulfo-SMCC linker. Both NNMT-ASO-T3 (NAT3) and ApoB-ASO-T3 (AAT3) enhance thyroid hormone receptor activity. Treating obese mice with NAT3 or AAT3 decreases adiposity and increases lean mass. ASO-T3 enhances white fat browning, decreases genes for fatty acid synthesis in liver, and shows limited effects on T3 target genes in heart and muscle. Furthermore, AAT3 augments LDL cholesterol-lowering effects of ApoB-ASO. Therefore, ASO and hormone/drug conjugation may provide a novel strategy for obesity and hyperlipidemia treatment

Exhaustive Sampling of Docking Poses Reveals Binding Hypotheses for Propafenone Type Inhibitors of P-Glycoprotein

Overexpression of the xenotoxin transporter P-glycoprotein (P-gp) represents one major reason for the development of multidrug resistance (MDR), leading to the failure of antibiotic and cancer therapies. Inhibitors of P-gp have thus been advocated as promising candidates for overcoming the problem of MDR. However, due to lack of a high-resolution structure the concrete mode of interaction of both substrates and inhibitors is still not known. Therefore, structure-based design studies have to rely on protein homology models. In order to identify binding hypotheses for propafenone-type P-gp inhibitors, five different propafenone derivatives with known structure-activity relationship (SAR) pattern were docked into homology models of the apo and the nucleotide-bound conformation of the transporter. To circumvent the uncertainty of scoring functions, we exhaustively sampled the pose space and analyzed the poses by combining information retrieved from SAR studies with common scaffold clustering. The results suggest propafenone binding at the transmembrane helices 5, 6, 7 and 8 in both models, with the amino acid residue Y307 playing a crucial role. The identified binding site in the non-energized state is overlapping with, but not identical to, known binding areas of cyclic P-gp inhibitors and verapamil. These findings support the idea of several small binding sites forming one large binding cavity. Furthermore, the binding hypotheses for both catalytic states were analyzed and showed only small differences in their protein-ligand interaction fingerprints, which indicates only small movements of the ligand during the catalytic cycle

Cysts of potentially harmful dinoflagellates, with emphasis on the genus Alexandrium, in Sepetiba Bay (Brazil) during a port survey of GloBallast

Sepetiba Bay in south-eastern Brazil has been selected as a pilot demonstration site within the Global Ballast Water Management Programme (GloBallast; IMO/PNUD/ GEF). As part of the port baseline survey, a series of 43 sediment cores was taken in triplicate in the bay in November 2001, by SCUBA divers, using PVC tubes. The top 60mm of each core was processed for dinoflagellate cyst analysis. Cysts of the potentially toxic Alexandriumcf. minutum were identified in 41% of the samples, but the highest density (22 cysts cm–3) was found in the port area, at the Iron Ore Terminal. Other Alexandrium-type cysts were observed in 37% of the samples and theirhighest density (26 cysts cm–3) was also associated with the port area. These results indicate that the northern area of the bay is unsuitable for the intake by ships of ballast water. This investigation highlights the importanceof cyst surveys to the management of ballasting and deballasting in port areas

Liposomal budesonide for dry powder inhaler: Preparation and stabilization

The purpose of the study was to prepare stable liposomally entrapped budesonide (BUD) for a dry powder inhaler (DPI) formulation. BUD liposomes composed of egg phosphatidyl choline and cholesterol were prepared by lipid film hydration technique and sonicated to have the desired size (<5 μm). A rapid method was used for separation of free drug by centrifugation at a lower centrifugal force (G value). Liposomal dispersion was subjected to lyophilization after blending BUD with cryoprotectant in varying bulk and mass ratios, and percent drug remaining entrapped after lyophilization was optimized. Comparative drug retention studies on storage of DPI formulations were carried out in accordance with International Conference on Harmonization guidelines. Critical relative humidity of the formulations was determined and reported as one of the manufacturing controls. Sucrose was found to be the most effective cryoprotectant when present on both sides of the lamellae of liposomes in a bulk strength of 500 mM and mass ratio of lipid:sugar; 1∶10. Blending of sorbolac before lyophilization showed better retention of encapsulated drug (95.59%). The respirable fraction of the product (20.69±1.50%) was comparable with that of the control (26.49±1.52%), suggesting that the liposomal BUD can be successfully delivered throughout the broncho-pulmonary tree. The findings demonstrate that liposome of BUD can be prepared with a high entrapment value, stabilized by lyophilization, and delivered as an aerosolized DPI. The stability studies of lyophilized product suggests a shelf-life of one year when stored under refrigeration (2°C–8°C)