The effect of urea and guanidine hydrochloride (GuHCl) on lysozyme stability has been investigated using activity measurements, microcalorimetry and Raman spectroscopy in the low-frequency and amide I regions. Raman investigations on lysozyme dissolved in H(2)O and D(2)O in the presence of up to 10 M denaturants have revealed direct binding between the protein and both denaturants. The analysis of isotopic exchanges in the amide I region allows the identification of binding sites as hydrophilic and hydrophobic groups, respectively, for urea and GuHCl. The weak loss of activity of lysozyme in the presence of urea (∼15% maximum) is mainly assigned to a transformation of the tertiary structure corresponding to a molten globule state without unfolding of α-helix structures, in contrast to GuHCl which clearly induces conformational changes, associated with a larger loss of activity (40% maximum). The denaturing power of urea and guanidine hydrochloride on lysozyme has been related to the solvent and protein dynamics, reflecting direct interaction between denaturants and protein. It clearly appears that solvent dynamics control protein dynamics, and the significant hardening of the dynamics of GuHCl aqueous solutions is considered responsible for its important denaturing power. The comparison between the low-frequency spectra of solvents and lysozyme aqueous solutions in the absence and presence of different types of additives (urea, GuHCl, trehalose) reveals the Raman signature of the hydration water dynamics. This comparison points out the exclusion of trehalose around the protein surface.

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The inhalation route is widely studied for many drug applications focusing on either local or systemic distributions. One matter of concern is the solubilization of hydrophobic drugs.We have studied the feasibility of using different cyclodextrins (CDs) to elaborate pharmaceutical formulations for the inhalation route and tested the short-term toxicity of such formulations administered by inhalation to C57BL/6 mice.We have shown that HP-β-CD, γ-CD, as well as RAMEB aqueous solutions can undergo aerosolization and that the resulting droplet-size ranges are compatible with pulmonary deposition. In vivo, we have demonstrated that short-term exposure to inhaled HP-β-CD, γ-CD and RAMEB solutions are non-toxic after assessing bronchoalveolar lavage (BAL), lung and kidney histology, bronchial responsiveness to methacholine and blood urea. The only change noted is a slight increase in lymphocyte count in the BAL after HP-β-CD and γ-CD inhalation.We conclude that CDs are useful in significantly enhancing the solubility of apolar drugs with a view to inhalation therapy although an increase in lymphocyte counts in the BAL after CDs inhalations needs further investigations.