Hiroshi Ueda

Proteomics, a powerful approach allowing the broad analysis of protein expression levels in samples, has been used to characterize mesenchymal stromal cells (MSCs) used in regenerative medicine, focusing on the influence of their tissue of origin, culture media, and passage number. However, the proteomic signatures arising from comprehensive alterations in culture conditions remain to be characterized. Here, we evaluated the effects of major culture parameters, including cell seeding density, detachment methods, media composition, and passage number, on both the cellular and supernatant proteomic profiles of human MSCs. Among the evaluated parameters, the 15-passage condition exerted the most significant impact on the cell samples, with the most significant proteome remodeling, including decreased DNA replication-related proteins and increased lipid metabolism-related proteins. Regarding the supernatant samples, significant differences were found between xeno-free media and serum-containing media, with serum-containing media showing higher secretion of the insulin growth factor-binding protein (IGFBP) family. Further, functional changes included reduction in thrombospondin-1, which is associated with MSC migration. These findings demonstrate that culture conditions significantly influence the characteristics of MSCs used in regenerative medicine, and that a wide proteomics evaluation of both the cells and supernatant is crucial for understanding these characteristics.

This highlight reviews traditional and advanced approaches to characterizing pharmaceutical crystals, covering PXRD, thermal analysis, spectroscopy, SCXRD, and NMR crystallography, with particular emphasis on pharmaceutical applications of MicroED.

This study aimed to investigate solid dispersion formulations of itraconazole (ITZ) by using carboxymethyl ethylcellulose (CMEC) as an enteric cellulose derivative to improve stability, dissolution, and permeation profiles. Polyvinylpyrrolidone covinyl acetate (PVPVA) and hydroxypropyl methylcellulose acetate succinate (HPMCAS) were used as traditional carriers. Ball-milling successfully induced the formation of solid dispersions of ITZ with the used polymers, resulting in single-glass transition temperatures. The infrared spectra suggested the formation of hydrogen bonding between ITZ and each polymer. Water sorption of CMEC was comparable to that of HPMCAS, which was significantly lower than that of PVPVA, enabling the maintenance of the amorphous state of the solid dispersion under 40 °C/75% relative humidity. The dissolution-permeation profiles of amorphous ITZ were improved in the solid dispersions with PVPVA or HPMCAS, and further enhancement was obtained through combination with CMEC. The antiprecipitation effect of the polymers on the supersaturated ITZ was in the same order as the dissolution-permeation test. The improvement mechanisms of the dissolution-permeation and antiprecipitation effect of CMEC could be explained by the prevention effect on the growth of the size of the drug-rich colloids of ITZ via the supersaturated condition. We conclude that CMEC has big potential as a solid dispersion carrier.

Macrocyclic peptides have garnered increasing attention due to their therapeutic potential. However, the low cell membrane permeability of macrocyclic peptides limits their access to intracellular targets. This study aimed to apply live-cell Raman imaging to detect intracellular macrocyclic peptides as a potential therapeutic modality. Alkyne-tags with specific Raman bands were attached to several types of macrocyclic peptides originally designed as HIV protease inhibitors. Membrane permeability was measured by using the traditional penetration test. Variations in permeability were observed among the compounds, with the alkyne group showing a minimal effect on the results. Macrocyclic peptides with an alkyne-tag, which exhibited no permeability in the penetration test, were added to the live cells and evaluated using Raman imaging. Two-dimensional (2D) and three-dimensional (3D) Raman imaging detected the macrocyclic peptides in the intracellular region. However, the alkyne signal was not observed in cells treated with the macrocyclic peptides, showing cell penetration in the penetration test. These results are consistent with the observed pharmacological activities and suggest that the traditional penetration test may be insufficient for effective drug design of macrocyclic peptides. We conclude that combining emerging techniques, such as Raman imaging, with traditional methods can provide a more detailed evaluation of the permeability of macrocyclic peptides.

Crystal engineering is important for controlling polymorphs. Several polymorphs are known for indomethacin (IMC), with recent discoveries of new polymorphs. The aim of this study is to investigate a new polymorph of IMC. Amorphous IMC was stirred in a low pH suspension for one day, followed by observing the generation of X-ray diffraction peaks. This pattern did not agree with those of previously reported forms, which was subsequently assigned to a κ-form. This crystal maintained its form for two months at 40 °C and 75% relative humidity. Thermal analysis of the κ-form showed an original melting point followed by trans-formation to the γ form. In spectroscopic analyses, the patterns of the new crystal could be distinguished from those of traditional polymorphs. The crystal structure of the κ-form was investigated via Microcrystal electron diffraction (MicroED) because the crystal size was under 1 μm. The κ-form had repeating motifs consisting of carboxylic acid dimers., The construction was com-posed of only one crystallographically independent IMC molecule, similar to the γ-form. However, the dihedral angle between indole ring and p-chlorobenzoyl group, as well as the mode of the hydrogen bonding of amide carbonyl to other functional groups, was significantly different. We successfully discovered a new polymorph κ-form, revealing its crystal structure via MicroED.