No abstract

The antimicrobial peptide magainin 2 isolated from the skin of the African clawed frog Xenopus laevis crosses lipid bilayers by transiently forming a peptide-lipid supramolecular complex pore inducing membrane permeabilization and flip-flop of membrane lipids [Matsuzaki, K., Murase, O., Fujii, N., and Miyajima, K. (1996) Biochemistry 35, 11361-11368]. In contrast, the antimicrobial peptide buforin 2 discovered in the stomach tissue of the Asian toad Bufo bufo gargarizans efficiently crosses lipid bilayers without inducing severe membrane permeabilization or lipid flip-flop, and the Pro(11) residue plays a key role in this unique property [Kobayashi, S, Takeshima, K., Park, C. B., Kim, S. C., and Matsuzaki, K. (2000) Biochemistry 39, 8648-8654]. To elucidate the translocation mechanism, the secondary structure and the orientation of the peptide in lipid bilayers as well as the effects of the peptide concentration, the lipid composition, and the cis-trans isomerization of the Pro peptide bond on translocation efficiency were investigated. The translocation efficiencies of F10W-buforin 2 (BF2), P11A-BF2, and F5W-magainin 2 (MG2) across egg yolk L-alpha-phosphatidyl-DL-glycerol (EYPG)/egg yolk L-alpha-phosphatidylcholine (1/1) bilayers were dependent supralinearly on the peptide concentration, suggesting that the translocation mechanisms of these peptides are similar. The incorporation of the negative curvature-inducing lipid egg yolk L-alpha -phosphatidylethanolamine completely suppressed the translocation of BF2, indicating the induction of the positive curvature by BF2 on the membrane is related to the translocation process, similarly to MG2. In pure EYPG, where the repulsion between polycationic BF2 molecules is reduced, membrane permeabilization and coupling lipid flip-flop were clearly observed. Structural studies by use of Fourier transform infrared-polarized attenuated total reflection spectroscopy indicated that BF2 assumed distorted helical structures in EYPG/EYPC bilayers. A BF2 analogue with an alpha-methylproline, which fixed the peptide bond to the trans configuration, translocated similarly to the parent peptide, suggesting the cis-trans isomerization of the Pro peptide bond is not involved in the translocation process. These results indicate that BF2 crosses lipid bilayers via a mechanism similar to that of MG2. The presence of Pro(11) distorts the helix, concentrating basic amino acid residues in a limited amphipathic region, thus destabilizing the pore by enhanced electrostatic repulsion, enabling efficient translocation.

The pressurized metered dose inhaler (MDI) is a convenient and promising technology for drug delivery to the respiratory tract. For example, steroid MDIs have been applied to the topical therapy instead of oral administration which causes severe systemic side effects such as notable suppression of the hypothalamic-pituitary-adrenal axis, nephrosis, gastric ulcer, moon face and so on. As the topical therapy with MDI improves the therapeutic index by about 10 to 50 times, i.e., increase the local concentration and decrease the systemic content, the patients can reduce the dose or stop oral intake. 1-3)However, these MDIs contain chlorofluorocarbons (CFCs) as a propellant, which are known to contribute to ozone layer depletion.4) Therefore, many aerosol products using non-CFC propellants have been developed. 5) As a propellant for pharmaceutical use, two candidates named hydrofluoroalkanes (HFAs), HFA-134a and HFA-227, are available at this moment.Meanwhile, Stmerin ® D (Astellas Pharma Inc., Tokyo, Japan) was a MDI for the treatment of asthma, which contained isoproterenol sulfate (bronchodilator, b-adrenergic stimulant), atropinemethylbromide (bronchodilator, anticholinergic agent) and dexamethasone (anti-inflammatory drug). This commercial product also contained mixed CFCs (CFC-11/CFC-12/CFC-114) as propellants. So the CFC replacement was carried out due to the environmental problem. Generally the CFC replacement is considered to be very difficult because of the physicochemical differences between CFCs and HFAs.6,7) One of the main reasons is CFC-11, which has been used as a solvent in the aerosol formulations, is able to dissolve many suspending agents or surfactants and is also easily mixed with other CFCs, but the HFAs hardly dissolve such suspending agents. This makes it difficult to disperse the drugs in HFAs.From screening of large amount of suspending agents, we have recently found out that middle chain fatty acid triglyceride (MCT) has potential to suspend the drugs in HFAs. Two preparations with HFA-134a and HFA-227 were prepared.The objective of this study is to evaluate the stability of suspension type aerosol preparations with HFAs as propellants. For such kinds of aerosol preparations, the moisture content in the canister is known to influence the stability of spray performance.8) So the relationship between the moisture content in the canisters and spray performance is also discussed. ExperimentalMaterials Isoproterenol sulfate (Boehringer Ingelheim, Germany) and atropine-methylbromide (Boehringer Ingelheim, Germany) are used after pulverized using the jet mill. Dexamethasone micronised is purchased from Roussel uclaf (Paris, France). These active ingredients are of pharmaceutical grade and used without further purification.HFA-134a (1,1,1,2-tetrafluoroethane, Mistui Dupont Fluoro Chemicals, Japan) and 1,1,2,3,3, Solvey, Germany) are used as a propellant. Middle chain fatty acid triglyceride (MCT, Miglyol 812, Mitsuba, Japan) is used and functions as both a suspending agent and a lubricant for th...