Description of an original molecular ordering process into a disordered crystalline form: the atypical low-temperature transformation of the disordered form III of linezolid.

Form III of linezolid was prepared by heating the commercial form above 150 °C and subsequently analyzed upon cooling down to -160 °C, by low- and high-frequency Raman spectroscopy, differential scanning calorimetry and powder X-ray diffraction (PXRD). It was observed that form III was preserved down to 0 °C. At lower temperatures a soft mode was clearly detected by low-frequency Raman spectroscopy associated with the detection of additional Raman bands distinctive of additional intermolecular H-bond interactions. Raman spectroscopy investigations performed in a wide frequency range revealed a continuous transformation characterized by both displacive and order-disorder signatures. By contrast, PXRD highlighted the absence of symmetry breaking, Bragg peaks being still indexed in the same unit cell from room temperature down to -160 °C. Additionally, a significant broadening of Bragg peaks was observed with decreasing temperature interpreted as being a consequence of a distribution of frozen molecular conformations.

Analysis of Co-Crystallization Mechanism of Theophylline and Citric Acid from Raman Investigations in Pseudo Polymorphic Forms Obtained by Different Synthesis Methods.

Designing co-crystals can be considered as a commonly used strategy to improve the bioavailability of many low molecular weight drug candidates. The present study has revealed the existence of three pseudo polymorphic forms of theophylline-citric acid (TP-CA) co-crystal obtained via different routes of synthesis. These forms are characterized by different degrees of stability in relation with the strength of intermolecular forces responsible for the co-crystalline cohesion. Combining low- and high-frequency Raman investigations made it possible to identify anhydrous and hydrate forms of theophylline-citric acid co-crystals depending on the preparation method. It was shown that the easiest form to synthesize (form 1'), by milling one hydrate with an anhydrous reactant, is very metastable, and transforms into the anhydrous form 1 upon heating or into the hydrated form 2 when it is exposed to humidity. Raman investigations performed in situ during the co-crystallization of forms 1 and 2 have shown that two different types of H-bonding ensure the co-crystalline cohesion depending on the presence of water. In the hydrated form 2, the cohesive forces are related to strong O-H … O H-bonds between water molecules and the reactants. In the anhydrous form 1, the co-crystalline cohesion is ensured by very weak H-bonds between the two anhydrous reactants, interpreted as corresponding to π-H-bonding. The very weak strength of the cohesive forces in form 1 explains the difficulty to directly synthesize the anhydrous co-crystal.

Identification of incommensurability in L-leucine: can lattice instabilities be considered as general phenomena in hydrophobic amino acids?

L-Leucine is an essential amino acid which has been focusing a lot of investigations on its phase transition sequence for more than fifty years. Combining Raman spectroscopy and X-ray diffraction experiments provides a new interpretation of the second order phase transition extending between 270 and 360 K as a displacive incommensurate-normal phase transition. A soft mode was clearly detected from low-frequency Raman investigations which exhibits the temperature dependence (A·(TC-T)1/2) typical of the temperature behavior of the amplitudon, an excitation specific to incommensurate phases. Simultaneously to the softening of the amplitudon, several very weakly intense X-ray reflections vanish upon heating at 360 K, and thereby are interpreted as satellite reflections. This incommensurability was described as resulting from the freezing of thermally activated hydrophobic side-chain rotations upon cooling in disordered orientations. Raman investigations were also performed on the isomeric amino acid L-norleucine previously identified as undergoing a normal-incommensurate phase transition around 200 K. Comparison of both studies suggests that the temperature behavior of thermally activated local motions generates lattice instabilities. Loss of periodicity can result from the freezing of rotations of molecular moieties in disordered orientations, or from the enhancement of anharmonicity of these rotations. This could be a general phenomenon in hydrophobic amino acids with direct consequences on their applications in the life science area.

Revisiting the phase transition sequence in L-methionine: Description of the disordering mechanism in an essential amino acid.

Raman spectroscopy investigations on L-methionine (L-Met) performed in a large temperature range (170-420 K) and in a wide spectral window (5-3600 cm-1) have revealed an extended disordering mechanism triggered by thermally activated motions of the terminal side-chain atoms, from 250 up to 390 K. This very progressive disordering process is characterized by two thermodynamic features, the first corresponding to a broad endotherm (250 → 310 K) marking the beginning of the process, while the second ending the disordering transformation is a sharp endothermic peak at 390 K. These thermodynamic events are correlated with the softening of lattice vibrations and the increase of the quasielastic scattering, considered as the signatures of displacive phase transitions. The amorphous-like band-shape of the low-frequency Raman spectrum collected above 390 K, resulting from the strong anharmonicity of local motions, is contrasting with the detection of additional Bragg peaks above 390 K by x-ray diffraction, consistent with the Cp jump accompanying the endothermic peak. These observations suggest that L-Met is progressively dynamically disordered adopting additional configurations in the crystalline lattice through rotations of CH3 and the side-chain flexibility not clearly detected by x-ray diffraction. These results should be crucial for considering the stability of dried proteins composed of methionine residues.

A detailed description of the devitrification mechanism of d-mannitol.

The devitrification mechanism of d-mannitol was carefully investigated using micro calorimetry experiments and Raman spectroscopy, in order to understand the phase transformation of the undercooled liquid into an apparently amorphous state, called phase X. It was found from micro spectroscopy analyses that the formerly assigned "phase X" observed during the devitrification of undercooled d-mannitol results from a surface crystallization accompanied by a very slow bulk crystallization into the α form. Such a phenomenon can be more easily identified by analyzing microscopic samples obtained upon slow heating runs from the glassy state.

Study of the phase transition in lysozyme crystals by Raman spectroscopy.

BACKGROUND: Recently, it has been revealed that tetragonal lysozyme crystals show a phase transition at 307 K upon heating. The underlying mechanisms of the phase transition are still not fully understood. Here we focus on the study of high-frequency vibrational modes arising from the protein and their temperature evolution in the vicinity of Tph as well as on the detailed study of crystalline water dynamics near Tph. METHODS: Raman experiments have been performed at temperatures 295­323 K including Tph. The low-frequency modes and the modes of fingerprint region, CH- and OH-stretching regions have been analyzed. RESULTS AND CONCLUSIONS: In spite of the absence of noticeable rearrangements in protein structure, the high-frequency vibrational modes of lysozyme located in the fingerprint region have been found to exhibit the features of critical dynamics near Tph. Pronounced changes in the dynamics of α-helixes and Tyr residues exposed on the protein surface point to the important role of H-bond rearrangements at the phase transition. Additionally the study of temperature evolution of OH-stretching modes has shown an increase in distortions of tertahedral H-bond network of crystalline water above Tph. These changes in water dynamics could play a crucial role in the mechanisms of the phase transition. GENERAL SIGNIFICANCE: The present results shed light on the mechanisms of the phase transition in lysozyme crystals.

Relationship between ß-relaxation and structural stability of lysozyme: microscopic insight on thermostabilization mechanism by trehalose from Raman spectroscopy experiments.

Raman investigations were carried out in the low-frequency and amide I regions on lysozyme aqueous solutions in absence and presence of trehalose. Raman spectroscopy gives the unique opportunity to analyze the protein and solvent dynamics in the low-frequency range while monitoring the unfolding process by capturing the spectrum of the amide I band. From the analysis of the quasielastic intensity, a dynamic change is firstly observed in a highly hydrated protein, around 70 °C, and interpreted in relation with the denaturation mechanism of the protein. The use of heavy water and partly deuterated trehalose gives clear information on protein-trehalose interactions in the native state of lysozyme (at room temperature) and during the thermal denaturation process of lysozyme. At room temperature, it was found that trehalose is preferentially excluded from the protein surface, and has a main effect on the tetrahedral local order of water molecules corresponding to a stiffening of the H-bond network in the solvent. The consequence is a significant reduction of the amplitude of fast relaxational motions, inducing a less marked dynamic transition shifted toward the high temperatures. Upon heating, interaction between trehalose and lysozyme is detected during the solvent penetration within the protein, i.e., while the native globular state softens into a molten globule (MG) state. Addition of trehalose reduces the protein flexibility in the MG state, improving the structural stability of the protein, and inhibiting the protein aggregation.

Relationship between synthesis method-crystal structure-melting properties in cocrystals: the case of caffeine-citric acid.

The influence of the crystal synthesis method on the crystallographic structure of caffeine-citric acid cocrystals was analyzed thanks to the synthesis of a new polymorphic form of the cocrystal. In order to compare the new form to the already known forms, the crystal structure of the new cocrystal (C8H10N4O2·C6H8O7) was solved by powder X-ray diffraction thanks to synchrotron experiments. The structure determination was performed using `GALLOP', a recently developed hybrid approach based on a local optimization with a particle swarm optimizer, particularly powerful when applied to the structure resolution of materials of pharmaceutical interest, compared to classical Monte-Carlo simulated annealing. The final structure was obtained through Rietveld refinement, and first-principles density functional theory (DFT) calculations were used to locate the H atoms. The symmetry is triclinic with the space group P-1 and contains one molecule of caffeine and one molecule of citric acid per asymmetric unit. The crystallographic structure of this cocrystal involves different hydrogen-bond associations compared to the already known structures. The analysis of these hydrogen bonds indicates that the cocrystal obtained here is less stable than the cocrystals already identified in the literature. This analysis is confirmed by the determination of the melting point of this cocrystal, which is lower than that of the previously known cocrystals.

Co-Amorphous Versus Deep Eutectic Solvents Formulations for Transdermal Administration.

Transdermal administration can be considered as an interesting route to overcome the side-effects inherent to oral intake. Designing topical formulations with maximum drug efficiency requires the optimization of the permeation and the stability of the drug. The present study focuses on the physical stability of amorphous drugs within the formulation. Ibuprofen is commonly used in topical formulations and then was selected as a model drug. Additionally, its low Tg allows easy, unexpected recrystallization at room temperature with negative consequence on skin penetration. In this study, the physical stability of amorphous ibuprofen was investigated in two types of formulations: (i) in terpenes-based deep eutectic solvents (DES) and (ii) in arginine-based co-amorphous blends. The phase diagram of ibuprofen:L-menthol was mainly analyzed by low-frequency Raman spectroscopy, leading to the evidence of ibuprofen recrystallization in a wide range of ibuprofen concentration. By contrast, it was shown that amorphous ibuprofen is stabilized when dissolved in thymol:menthol DES. Forming co-amorphous arginine-ibuprofen blends by melting is another route for stabilizing amorphous ibuprofen, while recrystallization was detected in the same co-amorphous mixtures obtained by cryo-milling. The mechanism of stabilization is discussed from determining Tg and analyzing H-bonding interactions by Raman investigations in the C=O and O-H stretching regions. It was found that recrystallization of ibuprofen was inhibited by the inability to form dimers inherent to the preferential formation of heteromolecular H-bonding, regardless of the glass transition temperatures of the various mixtures. This result should be important for predicting ibuprofen stability within other types of topical formulations.

Mechanism for Stabilizing an Amorphous Drug Using Amino Acids within Co-Amorphous Blends.

Designing co-amorphous formulations is now recognized as a relevant strategy for improving the bioavailability of low-molecular-weight drugs. In order to determine the most suitable low-molecular-weight excipients for stabilizing the drug in the amorphous state, screening methods were developed mostly using amino acids as co-formers. The present study focused on the analysis of the thermal stability of co-amorphous blends prepared by cryo-milling indomethacin with several amino acids in order to understand the stabilization mechanism of the drug in the amorphous state. Combining low- and mid-frequency Raman investigations has provided information on the relation between the physical properties of the blends and those of the H-bond network of the amorphous drug. This study revealed the surprising capabilities of L-arginine to stiffen the H-bond network in amorphous indomethacin and to drastically improve the stability of its amorphous state. As a consequence, this study suggests that amino acids can be considered as stiffeners of the H-bond network of indomethacin, thereby improving the stability of the amorphous state.

Low-Frequency Raman Spectroscopy: An Exceptional Tool for Exploring Metastability Driven States Induced by Dehydration.

The use of low-frequency Raman spectroscopy (LFRS; ω < 150 cm-1) is booming in the pharmaceutical industry. Specific processing of spectra is required to use the wealth of information contained in this spectral region. Spectra processing and the use of LFRS for analyzing phase transformations in molecular materials are detailed herein from investigations on the devitrification of ibuprofen. LFRS was used to analyze the dehydration mechanism of two hydrates (theophylline and caffeine) of the xanthine family. Two mechanisms of solid-state transformation in theophylline were determined depending on the relative humidity (RH) and temperature. At room temperature and 1% RH, dehydration is driven by the diffusion mechanism, while under high RH (>30%), kinetic laws are typical of nucleation and growth mechanism. By increasing the RH, various metastability driven crystalline forms were obtained mimicking successive intermediate states between hydrate form and anhydrous form achieved under high RH. In contrast, the dehydration kinetics of caffeine hydrate under various RH levels can be described by only one master curve corresponding to a nucleation mechanism. Various metastability driven states were achieved depending on the RH, which can be described as intermediate between forms I and II of anhydrous caffeine.

Silicone matrices for controlled dexamethasone release: toward a better understanding of the underlying mass transport mechanisms.

Dexamethasone-loaded silicone matrices offer an interesting potential as innovative drug delivery systems, e.g. for the treatment of inner ear diseases or for pacemakers. Generally, very long drug release periods are targeted: several years/decades. This renders the development and optimization of novel drug products cumbersome: experimental feedback on the impact of the device design is obtained very slowly. A better understanding of the underlying mass transport mechanisms can help facilitating research in this field. A variety of silicone films were prepared in this study, loaded with amorphous or crystalline dexamethasone. Different polymorphic drug forms were investigated, the film thickness was altered and the drug optionally partially/completely exchanged by much more water-soluble dexamethasone 'phosphate'. Drug release studies in artificial perilymph, scanning electron microscopy, optical microscopy, differential scanning calorimetry, X-ray diffraction and Raman imaging were used to elucidate the physical states of the drugs and polymer, and of the systems' structure as well as dynamic changes thereof upon exposure to the release medium. Dexamethasone particles were initially homogeneously distributed throughout the systems. The hydrophobicity of the matrix former very much limits the amounts of water penetrating into the system, resulting in only partial drug dissolution. The mobile drug molecules diffuse out into the surrounding environment, due to concentration gradients. Interestingly, Raman imaging revealed that even very thin silicone layers (<20 µm) can effectively trap the drug for prolonged periods of time. The physical state of the drug (amorphous, crystalline) did not affect the resulting drug release kinetics to a noteworthy extent.

Milling-Assisted Loading of Drugs into Mesoporous Silica Carriers: A Green and Simple Method for Obtaining Tunable Customized Drug Delivery.

Mesoporous silica (MPS) carriers are considered as a promising strategy to increase the solubility of poorly soluble drugs and to stabilize the amorphous drug delivery system. The development by the authors of a solvent-free method (milling-assisted loading, MAL) made it possible to manipulate the physical state of the drug within the pores. The present study focuses on the effects of the milling intensity and the pore architecture (chemical surface) on the physical state of the confined drug and its release profile. Ibuprofen (IBP) and SBA-15 were used as the model drug and the MPS carrier, respectively. It was found that decreasing the milling intensity promotes nanocrystallization of confined IBP. Scanning electron microscopy and low-frequency Raman spectroscopy investigations converged into a bimodal description of the size distribution of particles, by decreasing the milling intensity. The chemical modification of the pore surface with 3-aminopropyltriethoxisylane also significantly promoted nanocrystallization, regardless of the milling intensity. Combined analyses of drug release profiles obtained on composites prepared from unmodified and modified SBA-15 with various milling intensities showed that the particle size of composites has the greatest influence on the drug release profile. Tuning drug concentration, milling intensity, and chemical surface make it possible to easily customize drug delivery.

Confinement of molecular materials using a solid-state loading method: a route for exploring new physical states and their subsequent transformation highlighted by caffeine confined to SBA-15 pores.

Using the innovative solid-state loading (milling-assisted loading, MAL) method to confine caffeine to cylindrical pores (SBA-15, ∅ = 6 nm) gives the opportunity to explore the original physical states of caffeine and their subsequent transformation using low-frequency Raman spectroscopy, powder X-ray diffraction and microcalorimetry investigations. It was shown that MAL makes possible the loading of the selected form in the polymorphism of caffeine. While form II has similar structural and dynamics properties in confined and bulk forms, the confined rotator phase (form I) exhibits clear differences with the bulk form inherent to its orientational disorder. Interestingly, the two confined forms of caffeine undergo an exothermic disordering transformation upon heating into a physical state at the border between a nanocrystallized orientationally disordered phase and an amorphous state, not existing in the bulk form. The melting of this new physical state was observed at 150 °C, i.e. 85 degrees below the melting temperature of the bulk form I, thus demonstrating the confinement of caffeine. It was also found that the liquid confined to pores of 6 nm mean diameter recrystallizes upon cooling, which can be explained by the very disordered nature of the recrystallized state.

Influence of urea and guanidine hydrochloride on lysozyme stability and thermal denaturation; a correlation between activity, protein dynamics and conformational changes.

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.

Contribution of low-frequency Raman spectroscopy to determine the solubility curves of drugs in polymers: The case of paracetamol/PVP.

A new method for determining solubility lines of drugs in polymers, based on low-frequency Raman spectroscopy measurements, is described and the results obtained by this method are compared with those obtained using a more classical method based on differential scanning calorimetry investigations. This method was applied to the paracetamol/PVP system using molecular and crystalline dispersions (MCD) rather than usual physical mixtures to reach faster the equilibrium saturated states and make the determination of the solubility line more rapid.

Trehalose or Sucrose: Which of the Two Should be Used for Stabilizing Proteins in the Solid State? A Dilemma Investigated by In Situ Micro-Raman and Dielectric Relaxation Spectroscopies During and After Freeze-Drying.

The bioprotective properties of 2 disaccharides (sucrose and trehalose) were analyzed during the freeze-drying (FD) process and at the end of the process, to better understand the stabilization mechanisms of proteins in the solid state. In situ Raman investigations, performed during the FD process, have revealed that sucrose was more efficient than trehalose for preserving the secondary structure of lysozyme during FD, especially during the primary drying stage. The lower bioprotective effect of trehalose was interpreted as a consequence of a stronger affinity of this disaccharide to water, responsible for a severe phase separation phenomenon during the freezing stage. Dielectric spectroscopy investigations on the freeze-dried state of protein formulations have shown the capabilities of trehalose assisted by residual water to reduce the molecular mobility of the vitreous matrix, suggesting that trehalose is more efficient to preserve the protein structure during long-term storage.

Kinetics and mechanism of polymorphic transformation of sorbitol under mechanical milling.

In this paper, we present a kinetic investigation of the polymorphic transformation γ â†’ α of sorbitol under milling in the objective to identify the microscopic mechanisms that govern this type of solid-state transformation. The milling was performed with a high energy planetary mill and the milled material was analysed by DSC, PXRD and Raman spectrometry. The transformation kinetics was found to be sigmoidal with a noticeable incubation time. Moreover, this incubation time was shown to shorten rapidly when seeding the initial form γ with the final form α. The origin of the incubation period and its evolution upon seeding are puzzling as polymorphic transformations induced by milling are not expected to occur through a nucleation and growth process. To explain these puzzling kinetic features, we propose a two-step transformation mechanism involving local amorphisations due to the mechanical impacts, immediately followed by rapid recrystallizations of the amorphized fractions. The key point of the mechanism is that recrystallizations are oriented towards the forms γ or α, depending on the crystalline form of neighbouring crystallites. This mechanism has been validated by numerical simulations which were able to reproduce all the experimental kinetic features of the polymorphic transformation (kinetic law and effects of seeding) upon milling.

Thermostabilization mechanism of bovine serum albumin by trehalose.

Thermal denaturation of bovine serum albumin (BSA) is analyzed from differential scanning calorimetry (DSC) and Raman spectroscopy investigations. DSC curves exhibit a marked dependence on protein concentration. BSA thermal denaturation becomes broader and bimodal, and the temperature of denaturation increases with increasing protein concentration. Raman scattering investigations simultaneously carried out in the low-frequency range (10-350 cm(-1)) and in the amide I band region (1500-1800 cm(-1)) indicate that the denaturation process is described as a biphasic process independent of protein concentration. The dependence of the protein stability upon the protein concentration can be interpreted from the coupling of protein and solvent dynamics. The confrontation of previous results obtained from Raman investigations on lysozyme (LYS) and the present study of BSA brings out significant information on protein dynamics and the coupling of protein and hydration-water dynamics in relation with the solvent accessible surface area. Contrary to LYS, the modification of the dynamics of hydration water by the protein is clearly observed on BSA. The influence of trehalose on the protein dynamics was analyzed. We found that trehalose reduces the dynamic fluctuations of polar side chains at the protein-solvent interface. The mechanism of thermostabilization by trehalose is related to the reduction of the exposure of hydrophobic groups of BSA to the water molecules, and to a strengthening of intermolecular O-H interactions in the hydrogen-bond network of water, leading to the stabilization of the tertiary structure.

Polymorphism versus devitrification mechanism: Low-wavenumber Raman investigations in sulindac.

The polymorphism of sulindac was investigated by Raman investigations, mainly in the low-wavenumber region in order to analyze the influence of the amorphization method on recrystallization and crystalline form stability. By devitrification of the quenched liquid, it was found that the undercooled liquid crystallizes into Form I, and a polymorphic transformation by cooling Form I toward Form IV, was clearly revealed. The low-wavenumber spectra of polymorphic forms are direct fingerprints of crystals, indicating a degree of disorder of Form IV intermediate between those of the ordered Form II (commercial form) and the relatively disordered Form I. This study has shown the enantiotropic relationship between Forms I and IV and that both the temperature of crystallization and the physical stability of Form I prepared is dependent on the technique used for preparing amorphous sulindac.