Targeted delivery of antisense oligonucleotides to pancreatic ß-cells.

Antisense oligonucleotide (ASO) silencing of the expression of disease-associated genes is an attractive novel therapeutic approach, but treatments are limited by the ability to deliver ASOs to cells and tissues. Following systemic administration, ASOs preferentially accumulate in liver and kidney. Among the cell types refractory to ASO uptake is the pancreatic insulin-secreting ß-cell. Here, we show that conjugation of ASOs to a ligand of the glucagon-like peptide-1 receptor (GLP1R) can productively deliver ASO cargo to pancreatic ß-cells both in vitro and in vivo. Ligand-conjugated ASOs silenced target genes in pancreatic islets at doses that did not affect target gene expression in liver or other tissues, indicating enhanced tissue and cell type specificity. This finding has potential to broaden the use of ASO technology, opening up novel therapeutic opportunities, and presents an innovative approach for targeted delivery of ASOs to additional cell types.

Robust anti-obesity and metabolic effects of a dual GLP-1/glucagon receptor peptide agonist in rodents and non-human primates.

AIMS: To characterize the pharmacology of MEDI0382, a peptide dual agonist of glucagon-like peptide-1 (GLP-1) and glucagon receptors. MATERIALS AND METHODS: MEDI0382 was evaluated in vitro for its ability to stimulate cAMP accumulation in cell lines expressing transfected recombinant or endogenous GLP-1 or glucagon receptors, to potentiate glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cell lines and stimulate hepatic glucose output (HGO) by primary hepatocytes. The ability of MEDI0382 to reduce body weight and improve energy balance (i.e. food intake and energy expenditure), as well as control blood glucose, was evaluated in mouse models of obesity and healthy cynomolgus monkeys following single and repeated daily subcutaneous administration for up to 2 months. RESULTS: MEDI0382 potently activated rodent, cynomolgus and human GLP-1 and glucagon receptors and exhibited a fivefold bias for activation of GLP-1 receptor versus the glucagon receptor. MEDI0382 produced superior weight loss and comparable glucose lowering to the GLP-1 peptide analogue liraglutide when administered daily at comparable doses in DIO mice. The additional fat mass reduction elicited by MEDI0382 probably results from a glucagon receptor-mediated increase in energy expenditure, whereas food intake suppression results from activation of the GLP-1 receptor. Notably, the significant weight loss elicited by MEDI0382 in DIO mice was recapitulated in cynomolgus monkeys. CONCLUSIONS: Repeated administration of MEDI0382 elicits profound weight loss in DIO mice and non-human primates, produces robust glucose control and reduces hepatic fat content and fasting insulin and glucose levels. The balance of activities at the GLP-1 and glucagon receptors is considered to be optimal for achieving weight and glucose control in overweight or obese Type 2 diabetic patients.

Enantiospecific reassessment of the pharmacokinetics and pharmacodynamics of oral eflornithine against late-stage Trypanosoma brucei gambiense sleeping sickness.

This study aimed to characterize the stereoselective pharmacokinetics of oral eflornithine in 25 patients with late-stage Trypanosoma brucei gambiense sleeping sickness. A secondary aim was to determine the concentrations of L- and D-eflornithine required in plasma or cerebrospinal fluid (CSF) for an efficient eradication of the T. brucei gambiense parasites. Patients were randomly allocated to receive either 100 (group I, n=12) or 125 (group II, n=13) mg/kg of body weight of drug every 6 h for 14 days. The concentrations of L- and D-eflornithine in the plasma and CSF samples were measured using a stereospecific liquid chromatographic method. Nonlinear mixed-effects modeling was used to characterize the plasma pharmacokinetics. The plasma concentrations of L-eflornithine were on average 52% (95% confidence interval [CI], 51, 54%; n=321) of the D-enantiomer concentrations. The typical oral clearances of L- and D-eflornithine were 17.4 (95% CI, 15.5, 19.3) and 8.23 (95% CI, 7.36, 9.10) liters/h, respectively. These differences were likely due to stereoselective intestinal absorption. The distributions of eflornithine enantiomers to the CSF were not stereoselective. A correlation was found between the probability of cure and plasma drug exposure, although it was not more pronounced for the L-enantiomer than for that of total eflornithine. This study may explain why oral treatment for late-stage human African trypanosomiasis (HAT) patients with racemic eflornithine has previously failed; the more potent L-enantiomer is present at much lower concentrations in both plasma and CSF than those of the D-enantiomer. Eflornithine stereoselective pharmacokinetics needs to be considered if an oral dosage regimen is to be explored further.

Determination of eflornithine enantiomers in plasma by precolumn derivatization with o-phthalaldehyde-N-acetyl-L-cysteine and liquid chromatography with UV detection.

A bioanalytical method for indirect determination of eflornithine enantiomers in 75 microL human plasma has been developed and validated. L- and D-eflornithine were derivatized with o-phthalaldehyde and N-acetyl-L-cysteine to generate diastereomers which were separated on two serially connected Chromolith Performance columns (RP-18e 100 x 4.6 mm i.d.) by a isocratic flow followed by a gradient flow for elution of endogenous compounds. The diastereomers were detected with UV (340 nm). The between-day precisions for L- and D-eflornithine in plasma were 8.4 and 2.3% at 3 microm, 4.0 and 5.1% at 400 microm, and 2.0 and 3.7% at 1000 microm. The lower limit of quantification was determined to be 1.5 microm, at which precision was 14.9 and 9.9% for L- and D-eflornithine, respectively.