Ülo Langel

PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation

Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne’s muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine™ 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.

Galanin Receptors and Ligands

The neuropeptide galanin was first discovered 30 years ago. Today, the galanin family consists of galanin, galanin-like peptide (GALP), galanin-message associated peptide (GMAP), and alarin and this family has been shown to be involved in a wide variety of biological and pathological functions. The effect is mediated through three GPCR subtypes, GalR1-3. The limited number of specific ligands to the galanin receptor subtypes has hindered the understanding of the individual effects of each receptor subtype. This review aims to summarize the current data of the importance of the galanin receptor subtypes and receptor subtype specific agonists and antagonists and their involvement in different biological and pathological functions.

Galanin-induced inhibition of insulin secretion from rat islets : effects of rat and pig galanin and galanin fragments and analogues

The 29-amino acid neuropeptide galanin occurs in intrapancreatic nerves and inhibits insulin secretion. To study the structure-activity relations of galanin, we examined the effects of pig and rat galanin, three galanin fragments (galanin-(1-11), galanin-(1-16) and rat galanin-(17-29) and four galanin analogues ([Ala2]pig galanin, [Ala2]rat galanin, [D-Trp2]rat galanin and [D-Trp2]galanin-(1-16] on glucose-stimulated insulin secretion from isolated rat islets. Pig and rat galanin and galanin-(1-11) equipotently inhibited glucose-stimulated (8.3 mM) insulin secretion at and above 10(-7) M (P less than 0.05), whereas galanin-(1-16), inhibited insulin secretion at 10(-6) M (P less than 0.01). In contrast, the C-terminal rat galanin-(17-29) and the galanin analogues did not influence insulin secretion. Thus, rat and pig galanin are equipotent in inhibiting glucose-stimulated insulin secretion from rat islets. The active site resides in the N-terminal part of the molecule. Furthermore, the binding of galanin to its receptor depends on structural characteristics governed by the N-terminal position and in particular by the Trp2 residue.

N-Terminally elongated fragments of galanin(1–16) inhibit insulin secretion from isolated mouse islets

The neuropeptide galanin inhibits insulin secretion and has been suggested to be an adrenergic co-transmitter in the endocrine pancreas. Recently, N-terminally elongated forms of galanin have been identified in both porcine brain and adrenals. Whether these elongated peptides show galanin-like biological effects is not known. We therefore synthesized two N-terminally elongated fragments of galanin(1-16), which contains the active site of galanin. The synthesized peptides were galanin(-9-16) and galanin(-7-16), which correspond to amino acids 24-61 and 26-61 in the galanin precursor molecule. Both these peptides were found to potently inhibit glucose-(11.1 mM)-stimulated insulin secretion from isolated mouse islets of Langerhans in all concentrations studied (1-1000 nM) (P < 0.0001). The potency of the peptides was not different from that of synthetic rat galanin. Thus, at 100 nM, insulin secretion was inhibited by galanin(-7-16) by 83 +/- 7% and by galanin(-9-16) by 71 +/- 17% and by rat galanin by 93 +/- 4% (not statistically different). Furthermore, the galanin receptor antagonist, M35 (10 nM), prevented the inhibitory action of the two N-terminally galanin fragments. This study thus shows that N-terminally elongated galanin-fragments as entire galanin inhibits insulin and thus indicates that the effect of galanin on insulin secretion is not dependent on a free amino-terminus.

Galanin/GALP Receptors and CNS Homeostatic Processes

Galanin is a 29/30 amino acid peptide neurotransmitter that is widely distributed throughout the central nervous system and periphery. There are three well-characterized G-protein coupled galanin receptors subtypes (GalR1-3). A more recently discovered 60 amino acid galanin-like peptide (GALP) shares amino acid sequence homology with galanin (1-13) in position 9-21 and has high binding affinity for GalR1-3, with highest affinity for GalR3. Considerable evidence has accumulated that implicates both galanin and GALP as playing important roles in regulating food and water intake behavior and related neuroendocrine functions. Pharmacological tools are emerging that will allow dissociation of specific roles for the peptides and their associated receptor subtypes in mediating the homeostatic processes of energy and fluid balance.