Transnasal Delivery of the Peptide Agonist Specific to Neuromedin-U Receptor 2 to the Brain for the Treatment of Obesity.

Obesity and metabolic syndrome are threats to the health of large population worldwide as they are associated with high mortality, mainly linked to cardiovascular diseases. Recently, CPN-116 (CPN), which is an agonist peptide specific to neuromedin-U receptor 2 (NMUR2) that is expressed predominantly in the brain, has been developed as a new therapeutic candidate for the treatment of obesity and metabolic syndrome. However, treatment with CPN poses a challenge due to the limited delivery of CPN to the brain. Recent studies have clarified that the direct anatomical connection of the nasal cavity with brain allows delivery of several drugs to the brain. In this study, we confirm the nasal cavity as a promising CPN delivery route to the brain for the treatment of obesity and metabolic syndrome. According to the pharmacokinetic study, the clearance of CPN from the blood was very rapid with a half-life of 3 min. In vitro study on its stability in the serum and cerebrospinal fluid (CSF) indicates that CPN was more stable in the CSF than in the blood. The concentration of CPN in the brain was higher after nasal administration, despite its lower concentrations in the plasma than that after intravenous administration. The study on its pharmacological potency suggests the effective suppression of increased body weight in mice in a dose-dependent manner due to the direct activation of NMUR2 by CPN. This results from the higher concentration of corticosterone in blood after nasal administration of CPN as compared to nasal application of saline. In conclusion, the above findings indicate that the nasal cavity is a promising CPN delivery route to the brain to treat obesity and metabolic syndrome.

Magnesium binding sites of the active conformation pf Hammerhead ribozyme.

The modeling of the active conformation was done by using modeling program InsightII and Discover from the previous NMR, chemical modification and mutation data. This model structure has the reverse Watson-Crick GC base pair between C17 at the cleavage site and G5 at the core region. Two magnesium ions bind to this ribozyme. One binds to the pro-Rp oxygen of the P9 phosphate and the N7 nitrogen of G10.1, similarly to the crystal structure, the other to N3 of C3, the 2'O of C17 and the Sp-O of the scissile phosphate, while the Rp-O of that binds to NH2 of C3.

NMR studies of the effect of GA-AG base pairs to the active conformation of hammerhead ribozyme.

The effect of the conserved bases (GA-AG base pairs) in the core region to active conformation of hammerhead ribozyme was studied by NMR. This ribozyme has two conformers in the solution. One is the same as the crystal structure, which has sheared GA-AG base pairs and Y-shape conformation, while the other has Watson-Crick GA-AG base pairs and the extend conformation between the stems I and II. On addition of magnesiun ions, the peaks around GA-AG base pairs and cleavage site in only Y-shape conformation, shift or became broadening, following to produce the peaks of product by the cleavage reaction.

Effect of the conserved bases G10.1C11.1 in the stem II on Y-shape conformation of Hammerhead ribozyme.

The effect of the conserved bases in the stem II on Y-shape conformation of Hammerhead ribozyme was studied by NMR. On wild type, Hammerhead ribozyme forms Y-shape conformation, similar to the crystal structure, while on the mutation of G10.1C11.1 in the stem II by C10.1G11.1, that form the different conformation. This come from stability of the shared GA-AG base pairs, which affect the stacking between stem III and A14U7 base pair. On the mutation, this stacking is weak, so U4 turn does not occur, consequently stems II and I are linear.

NMR studies of the alternative conformation of hammerhead ribozyme in the solution.

The conformation of the hammerhead ribozyme was studied by NMR. In the absence of magnesium ion, the U turn does not exist at the core region. Consequently, stem II extends to stem I. G5 in the core region forms a base pair with C17 at the cleavage site. On the mutation of G16.2C15.2 in stem III by C16.2G15.2, G5 and U4 at the core region interact with C16.2 and U16.1, respectively, where stem III becomes shorter, while stem I does longer. On the addition of magnesium ions, U4 turn occurs in wild type, but does not in the mutation.

The new loop-loop interactions between the peripheral domains and three-dimensional model of Tetrahymena group I intron.

The long-range interactions between the peripheral domains of Tetrahymena group I intron were studied by NMR. The 20mer RNA contained the 9.1a loop region (AUGCAA) and the 17mer RNA contained the 2.1 loop region (AGAUUGC) were synthesized and studied by NMR. They form both hairpin structures at low NaCl concentration. The 20mer RNA forms dimer by loop-loop interaction, while the 17mer RNA is monomer. On the addition of the 17mer to the 20mer, the new imino proton signals induced by the interaction between the 9.1a and 2.1 loops were not observed. It is found that the 9.1a loop-loop interaction (GCAA-GCAA) with two base pairs prefers to that between the 9.1a and 2.1 loops (GCAA-UUGC) with four base pairs. By my model of intact folding structure containing A-minor tertiary interaction and the compared with sequence, it is presumed that the 9.1a loop does not bind to the 2.1 loop, but to the 5c loop (UGCAA), while the 2.1 loop binds to 3' exon (UAA).