Search

Abstract Amphipathic arginine-rich peptide, A2-17, exhibits moderate perturbation of lipid membranes and the highest cell penetration among its structural isomers. We investigated the direct cell-membrane penetration mechanism of the A2-17 peptide while focusing on structural flexibility. We designed conformationally constrained versions of A2-17, stapled (StpA2-17) and stitched (StchA2-17), whose α-helical conformations were stabilized by chemical crosslinking. Circular dichroism confirmed that StpA2-17 and StchA2-17 had higher α-helix content than A2-17 in aqueous solution. Upon liposome binding, only A2-17 exhibited a coil-to-helix transition. Confocal microscopy revealed that A2-17 had higher cell penetration efficiency than StpA2-17, whereas StchA2-17 remained on the cell membrane without cell penetration. Although the tryptophan fluorescence analysis suggested that A2-17 and its analogs had similar membrane-insertion positions between the interface and hydrophobic core, StchA2-17 exhibited a higher membrane affinity than A2-17 or StpA2-17. Atomic force microscopy demonstrated that A2-17 reduced the mechanical rigidity of liposomes to a greater extent than StpA2-17 and StchA2-17. Finally, electrophysiological analysis showed that A2-17 induced a higher charge influx through transient pores in a planer lipid bilayer than StpA2-17 and StchA2-17. These findings indicate that structural flexibility, which enables diverse conformations of A2-17, leads to a membrane perturbation mode that contributes to cell membrane penetration.

Background: The combination of multiple polymers is anticipated to serve as a means to diversify the physical properties and functionalities of dissolving microneedles. The mixing state of components is considered as a crucial factor in determining their suitability. Objectives: The purpose of this study was to elucidate whether thermal analysis of frozen aqueous solutions can appropriately predict the miscibility of hyaluronic acid (HA) and other polymers used for dissolving microneedles prepared by a micromolding method. Methods: Aliquots of aqueous polymer solutions were applied for thermal analysis by heating the samples from −70 °C at 5 °C/min to obtain the transition temperature of amorphous polymers and/or the crystallization/melting peaks of polymers (e.g., polyethylene glycol (PEG)). Films and dissolving microneedles were prepared by air-drying of the aqueous polymer solutions to assess the polymer miscibility in the solids. Results: The frozen aqueous single-solute HA solutions exhibited a clear Tg′ (the glass transition temperature of maximally freeze-concentrated solutes) at approximately −20 °C. The combination of HA with several polymers (e.g., dextran FP40, DEAE-dextran, dextran sulfate, and gelatin) showed a single Tg′ transition at temperatures that shifted according to their mass ratio, which strongly suggested the mixing of the freeze-concentrated solutes. By contrast, the observation of two Tg′ transitions in a scan strongly suggested the separation of HA and polyvinylpyrrolidone (PVP) or HA and polyacrylic acid (PAA) into different freeze-concentrated phases, each of which was rich in an amorphous polymer. The combination of HA and PEG exhibited the individual physical changes of the polymers. The polymer combinations that showed phase separation in the frozen solution formed opaque films and microneedles upon their preparation by air-drying. Coacervation occurring in certain polymer combinations was also suggested as a factor contributing to the formation of cloudy films. Conclusions: Freezing aqueous polymer solutions creates a highly concentrated polymer environment that mimics the matrix of dissolving microneedles prepared through air drying. This study demonstrated that thermal analysis of the frozen solution offers insights into the mixing state of condensed polymers, which can be useful for predicting the physical properties of microneedles.

Dissolving microneedles (MNs) are novel transdermal drug delivery systems that can be painlessly self-administered. This study investigated the effects of experimental conditions on the mechanical characterization of dissolving MNs for quality evaluation. Micromolding was used to fabricate polyvinyl alcohol (PVA)-based dissolving MN patches with eight different cone-shaped geometries. Axial force mechanical characterization test conditions, in terms of compression speed and the number of compression needles per test, significantly affected the needle fracture force of dissolving MNs. Characterization using selected test conditions clearly showed differences in the needle fracture force of dissolving MNs prepared under various conditions. PVA-based MNs were divided into two groups that showed buckling and unbuckling deformation, which occurred at aspect ratios (needle height/base diameter) of 2.8 and 1.8, respectively. The needle fracture force of PVA-based MNs was negatively correlated with an increase in the needle’s aspect ratio. Higher residual water or higher loading of lidocaine hydrochloride significantly decreased the needle fracture force. Therefore, setting appropriate methods and parameters for characterizing the mechanical properties of dissolving MNs should contribute to the development and supply of appropriate products.

Abstract We previously developed an amphipathic arginine-rich peptide, A2-17, which has high ability to directly penetrate across cell membranes. To understand the mechanism of the efficient cell-penetrating ability of the A2-17 peptide, we designed three structural isomers of A2-17 having different values of the hydrophobic moment and compared their membrane interaction and direct cell penetration. Confocal fluorescence microscopy revealed that cell penetration efficiency of peptides tends to increase with their hydrophobic moment, in which A2-17 L14R/R15L, an A2-17 isomer with the highest hydrophobic moment, predominantly remains on plasma cell membranes. Consistently, Trp fluorescence analysis indicated the deepest insertion of A2-17 L14R/R15L into lipid membranes among all A2-17 isomers. Electrophysiological analysis showed that the duration and charge flux of peptide-induced pores in lipid membranes were prominent for A2-17 L14R/R15L, indicating the formation of stable membrane pores. Indeed, the A2-17 L14R/R15L peptide exhibited the strongest membrane damage to CHO-K1 cells. Atomic force microscopy quantitatively defined the peptide-induced membrane perturbation as the decrease in the stiffness of lipid vesicles, which was correlated with the hydrophobic moment of all A2-17 isomers. These results indicate that optimal membrane perturbation by amphipathic A2-17 peptide is critical for its efficient penetration into cells without inducing stabilized membrane pores.

In order to assess and predict the bioequivalence (BE) of oral drug products, a new in vitro system “BE checker” was developed, which reproduced the environmental changes in the gastrointestinal (GI) tract by changing the pH, composition, and volume of the medium in a single chamber. The dissolution and membrane permeation profiles of drugs from marketed products were observed in the BE checker under various conditions reflecting the inter-patient variations of the GI physiology. As variable factors, initial gastric pH, gastric emptying time, and GI agitation strength were varied in vitro. Dipyridamole, a basic drug, showed rapid and supersaturated dissolution when the paddle speed in the donor chamber was 200 rpm, which corresponds to the high agitation strength in the stomach. In contrast, supersaturated dissolution disappeared, and the permeated amount decreased under the conditions with a slow paddle speed (100 and 50 rpm) and short gastric emptying time (10 min). In those conditions, disintegration of the formulation was delayed, and the subsequent dissolution of dipyridamole was not completed before the fluid pH was changed to neutral. Similar results were obtained when the initial gastric pH was increased to 3.0, 5.0, and 6.5. To investigate that those factors also affect the BE of oral drug products, dissolution and permeation of naftopidil from its ordinary and orally disintegrating (OD) tablets were observed in the BE checker. Both products showed the similar dissolution profiles when the paddle speed and gastric emptying time were set to 100 rpm and 10 or 20 min, respectively. However, at a low paddle speed (50 rpm), the dissolution of naftopidil from ordinary tablets was slower than that from the OD tablets, and the permeation profiles became dissimilar. These results indicated the possibility of the bioinequivalence of some oral formulations in special patients whose GI physiologies are different from those in the healthy subjects. The BE checker can be a highly capable in vitro tool to assess the BE of oral drug products in various populations.

Abstract The rapidly growing medical expense going with aging population in Japan requires the use of generic products derived from off-patent active pharmaceutical ingredients (APIs) formulations to reduce the financial burden for the national health insurance system, while at the same time avoiding undermining the quality of medical care. This article provides an overview of the regulatory approaches available to confirm the quality of generic products and gain their greater acceptance by patients. Among several approaches taken by the Ministry of Health, Labor, and Welfare (MHLW), designing systems to supply higher quality products, and providing scientific information to patients and healthcare professionals are key elements to promote the voluntary choice of the generic product. The revision of bioequivalence guidelines and the enhancement of good manufacturing practice (GMP) requirements facilitate the rational development and manufacturing control of generic formulations. A program termed Quality Reevaluation of Ethical Drugs was carried out from 1997 to 2012 using dissolution tests to avoid any significant bioINequivalence between originators and generic oral formulations. The evaluation of product quality and the assessment of the literature information by the Expert Committee on Quality of Generic Drug Products have provided a unique science-based and patient-focused approach for the distribution of reliable generic products. Some current and future issues regarding complex generic drugs are also discussed.