Effect of Water on Molecular Mobility and Physical Stability of Amorphous Pharmaceuticals.

We investigated the influence of sorbed water concentration on the molecular mobility and crystallization behavior in a model amorphous drug and a solid dispersion. The temperature scaling (Tg/T) allowed us to simultaneously evaluate the effects of water content and temperature on the relaxation time. In the supercooled dispersions, once scaled, the relaxation times of the systems with different water content overlapped. Thus, the observed increase in mobility could be explained by the "plasticization" effect of water. This effect also explained the decrease in crystallization onset temperature brought about by water. That is, plasticization is the underlying mechanism governing the observed increase in mobility and physical instability in the supercooled state. Similar results were observed in the glassy drug substance. A single linear relationship was observed between crystallization time (time for 0.5% crystallization) and Tg/T in both dry and water containing systems. Since fragility is unaffected by modest amounts of water, much like crystallization time, the mobility in the glass is expected to scale with Tg.

Correlation between Molecular Mobility and Physical Stability in Pharmaceutical Glasses.

We investigated a possible correlation between molecular mobility and physical stability in glassy celecoxib and indomethacin and identified the specific mobility mode responsible for physical instability (crystallization). In the glassy state, because the structural relaxation times are very long, the measurement was enabled by time domain dielectric spectroscopy. However, the local motions in the glassy state were characterized by frequency domain dielectric spectroscopy. Isothermal crystallization was monitored by powder X-ray diffractometry using either a laboratory source (supercooled state) or synchrotron source (glassy state). Structural (α) relaxation time correlated well with characteristic crystallization time in the supercooled state. On the other hand, a stronger correlation was observed between the Johari-Goldstein (ß) relaxation time and physical instability in the glassy state but not with structural relaxation time. These results suggest that Johari-Goldstein relaxation is a potential predictor of physical instability in the glassy state of these model systems.

The role of polymer concentration on the molecular mobility and physical stability of nifedipine solid dispersions.

We investigated the influence of polymer concentration (2.5-20% w/w) on the molecular mobility and the physical stability in solid dispersions of nifedipine (NIF) with polyvinylpyrrolidone (PVP). With an increase in polymer concentration, the α-relaxation times measured by broadband dielectric spectroscopy were longer, which reflects a decrease in molecular mobility. In the supercooled state, at a given temperature (between 55 and 75 °C), the relaxation time increased linearly as a function of polymer concentration (2.5-20% w/w). The temperature dependence of the relaxation time indicated that the fragility of the dispersion, and by extension the mechanism by which the polymer influences the relaxation time, was independent of polymer concentration. The time for NIF crystallization also increased as a function of polymer concentration. Therefore, by using molecular mobility as a predictor, a model was built to predict NIF crystallization from the dispersions in the supercooled state. The predicted crystallization times were in excellent agreement with the experimental data.

The role of drug-polymer hydrogen bonding interactions on the molecular mobility and physical stability of nifedipine solid dispersions.

We investigated the influence of drug-polymer hydrogen bonding interactions on molecular mobility and the physical stability in solid dispersions of nifedipine with each of the polymers polyvinylpyrrolidone (PVP), hydroxypropylmethyl cellulose (HPMCAS), and poly(acrylic acid) (PAA). The drug-polymer interactions were monitored by FT-IR spectroscopy, the molecular mobility was characterized using broadband dielectric spectroscopy, and the crystallization kinetics was evaluated by powder X-ray diffractometry. The strength of drug-polymer hydrogen bonding, the structural relaxation time, and the crystallization kinetics were rank ordered as PVP > HPMCAS > PAA. At a fixed polymer concentration, the fraction of the drug bonded to the polymer was the highest with PVP. Addition of 20% w/w polymer resulted in ∼65-fold increase in the relaxation time in the PVP dispersion and only ∼5-fold increase in HPMCAS dispersion. In the PAA dispersions, there was no evidence of drug-polymer interactions and the polymer addition did not influence the relaxation time. Thus, the strongest drug-polymer hydrogen bonding interactions in PVP solid dispersions translated to the longest structural relaxation times and the highest resistance to drug crystallization.

Influence of molecular mobility on the physical stability of amorphous pharmaceuticals in the supercooled and glassy States.

We investigated the correlation between molecular mobility and physical stability in three model systems, including griseofulvin, nifedipine, and nifedipine-polyvinylpyrrolidone dispersion, and identified the specific mobility mode responsible for instability. The molecular mobility in the glassy as well as the supercooled liquid states of the model systems were comprehensively characterized using dynamic dielectric spectroscopy. Crystallization kinetics was monitored by powder X-ray diffractometry using either a laboratory (in the supercooled state) or a synchrotron (glassy) X-ray source. Structural (α-) relaxation appeared to be the mobility responsible for the observed physical instability at temperatures above Tg. Although the direct measurement of the structural relaxation time below Tg was not experimentally feasible, dielectric measurements in the supercooled state were used to provide an estimate of the α-relaxation times as a function of temperature in glassy pharmaceuticals. Again, there was a strong correlation between the α-relaxation and physical instability (crystallization) in the glassy state but not with any secondary relaxations. These results suggest that structural relaxation is a major contributor to physical instability both above and below Tg in these model systems.

Ultrasonication as a potential tool to predict solute crystallization in freeze-concentrates.

PURPOSE: We hypothesize that ultrasonication can accelerate solute crystallization in freeze-concentrates. Our objective is to demonstrate ultrasonication as a potential predictive tool for evaluating physical stability of excipients in frozen solutions. METHODS: The crystallization tendencies of lyoprotectants (trehalose, sucrose), carboxylic acid buffers (citric, tartaric, malic, and acetic) and an amino acid buffer (histidine HCl) were studied. Aqueous solutions of buffers, lyoprotectants and mixtures of the two were cooled from room temperature to -20°C and sonicated to induce solute crystallization. The crystallized phases were identified by X-ray diffractometry (laboratory or synchrotron source). RESULTS: Sonication accelerated crystallization of trehalose dihydrate in frozen trehalose solutions. Sonication also enhanced solute crystallization in tartaric (200 mM; pH 5), citric (200 mM pH 4) and malic (200 mM; pH 4) acid buffers. At lower buffer concentrations, longer annealing times following sonication were required to facilitate solute crystallization. The time for crystallization of histidine HCl progressively increased as a function of sucrose concentration. The insonation period required to effect crystallization also increased with sucrose concentration. CONCLUSIONS: Sonication can substantially accelerate solute crystallization in the freeze-concentrate. Ultrasonication may be useful in assessing the crystallization tendency of formulation constituents used in long term frozen storage and freeze-drying.

Quantification, mechanism, and mitigation of active ingredient phase transformation in tablets.

Model tablet formulations containing thiamine hydrochloride [as a nonstoichiometric hydrate (NSH)] and dicalcium phosphate dihydrate (DCPD) were prepared. In intact tablets, the water released by dehydration of DCPD mediated the transition of NSH to thiamine hydrochloride hemihydrate (HH). The use of an X-ray microdiffractometer with an area detector enabled us to rapidly and simultaneously monitor both the phase transformations. The spatial information, gained by monitoring the tablet from the surface to the core (depth profiling), revealed that both DCPD dehydration and HH formation progressed from the surface to the tablet core as a function of storage time. Film coating of the tablets with ethyl cellulose caused a decrease in both the reaction rates. There was a pronounced lag time, but once initiated, the transformations occurred simultaneously throughout the tablet. Thus the difference in the phase transformation behavior between the uncoated and the coated tablets could not have been discerned without the depth profiling. Incorporation of hydrophilic colloidal silica as a formulation component further slowed down the transformations. By acting as a water scavenger it maintained a very "dry" environment in the tablet matrix. Finally, by coating the NSH particles with hydrophobic colloidal silica, the formation of HH was further substantially decelerated. The microdiffractometric technique not only enabled direct analyses of tablets but also provided the critical spatial information. This helped in the selection of excipients with appropriate functionality to prevent the in situ phase transformations.

Response of the cell membrane-cytoskeleton complex to osmotic and freeze/thaw stresses. Part 2: The link between the state of the membrane-cytoskeleton complex and the cellular damage.

In an earlier paper [35], we examined the mutual interaction between the actin cytoskeleton and the cell membrane and explored the role this interaction plays during freeze/thaw. In this follow-up paper, we investigate the physical and chemical stresses induced by freeze/thaw and explore the different mechanisms of damage caused by these stresses. Our results showed that changes in cell volume during freeze/thaw and the unfrozen water content in the solution alter the cytoskeleton stiffness, and the available membrane material. Combined with unfavorable ice-membrane interactions and increasing membrane stiffness, increased de-structuring of the membrane (such as bleb and microvilli formation) synergistically act on the membrane-cytoskeleton system generating irreversible damage.

Response of the cell membrane-cytoskeleton complex to osmotic and freeze/thaw stresses.

In order to develop successful cryopreservation protocols a better understanding of the freeze- and dehydration-induced changes occurring in the cell membrane and its underlying support, the actin cytoskeleton, is required. In this study, we compared the biophysical response of model mammalian cells (human foreskin fibroblasts) to hyperosmotic stress and freeze/thaw. Transmitted light, infrared spectroscopy, fluorescence- and cryo-microscopy were used to investigate the changes in the cell membrane and the actin cytoskeleton. We observed that a purely hyperosmotic challenge at room temperature resulted in bleb formation. A decrease in temperature abrogated the blebbing behavior, but was accompanied by a decrease in viability. These results suggested that cell survival depended on the availability of the membrane material to accommodate the volumetric expansion back to the original cell volume at isotonic conditions. Our data also showed that freeze/thaw stresses altered the cell membrane morphology resulting in a loss of membrane material. There was also a significantly lower incidence of blebbing after freeze/thaw as compared to isothermal osmotic stress experiments at room temperature. Significant depolymerization of the actin cytoskeleton was seen in cells whose membranes had been compromised by freeze/thaw stresses. Actin depolymerization using cytochalasin D affected the stability of the membrane against mechanical stress at isothermal conditions. This study shows that both the membrane and cytoskeleton, as a system, are involved in the osmotic and freeze/thaw-induced responses of the mammalian cells.

Roles of membrane structure and phase transition on the hyperosmotic stress survival of Geobacter sulfurreducens.

Geobacter sulfurreducens is a delta-proteobacterium bacteria that has biotechnological applications in bioremediation and as biofuel cells. Development of these applications requires stabilization and preservation of the bacteria in thin porous coatings on electrode surfaces and in flow-through bioreactors. During the manufacturing of these coatings the bacteria are exposed to hyperosmotic stresses due to dehydration and the presence of carbohydrates in the medium. In this study we focused on quantifying the response of G. sulfurreducens to hyperosmotic shock and slow dehydration to understand the hyperosmotic damage mechanisms and to develop the methodology to maximize the survival of the bacteria. We employed FTIR spectroscopy to determine the changes in the structure and the phase transition behavior of the cell membrane. Hyperosmotic shock resulted in greatly decreased membrane lipid order in the gel phase and a less cooperative membrane phase transition. On the other hand, slow dehydration resulted in increased membrane phase transition temperature, less cooperative membrane phase transition and a small decrease in the gel phase lipid order. Both hyperosmotic shock and slow dehydration were accompanied by a decrease in viability. However, we identified that in each case the membrane damage mechanism was different. We have also shown that the post-rehydration viability could be maximized if the lyotropic phase change of the cell membrane was eliminated during dehydration. On the other hand, lyotropic phase change during re-hydration did not affect the viability of G. sulfurreducens. This study conclusively shows that the cell membrane is the primary site of injury during hyperosmotic stress, and by detailed analysis of the membrane structure as well as its thermodynamic transitions it is indeed possible to develop methods in a rational fashion to maximize the survival of the bacteria during hyperosmotic stress.

Heterogeneity in desiccated solutions: implications for biostabilization.

Biopreservation processes such as freezing and drying inherently introduce heterogeneity. We focused on exploring the mechanisms responsible for heterogeneity in isothermal, diffusively dried biopreservation solutions that contain a model protein. The biopreservation solutions used contained trehalose (a sugar known for its stabilization effect) and salts (LiCl, NaCl, MgCl2, and CaCl2). Performing Fourier transform infrared spectroscopy analysis on the desiccated droplets, spatial distributions of the components within the dried droplet, as well as their specific interactions, were investigated. It was established that the formation of multiple thermodynamic states was induced by the spatial variations in the cosolute concentration gradients, directly affecting the final structure of the preserved protein. The spatial distribution gradients were formed by two competing flows that formed within the drying droplet: a dominant peripheral flow, induced by contact line pinning, and the Marangoni flow, induced by surface tension gradients. It was found that the changes in cosolute concentrations and drying conditions affected the spatial heterogeneity and stability of the product. It was also found that trehalose and salts had a synergistic stabilizing effect on the protein structure, which originated from destructuring of the vicinal water, which in turn mediated the interactions of trehalose with the protein. This interaction was observed by the change in the glycosidic CO, and the CH stretch vibrations of the trehalose molecule.

Dielectric spectroscopy of small molecule pharmaceuticals-effect of sample configuration.

In dielectric spectroscopy, a technique traditionally used to characterize molecular mobility in polymers, the sample is usually analyzed as a thin film. In recent years, the technique has been extended to characterize both drug substances and drug products and has revealed a correlation between molecular mobility and stability. However, for pharmaceutical systems, analysis of powders is strongly preferred over films. Therefore, the dielectric behavior of several compounds of pharmaceutical interest-nifedipine, indomethacin, itraconazole, and griseofulvin-obtained using powder and film samples were compared. The magnitudes of the intrinsic dielectric properties were affected by the sample configuration with the powder samples consistently yielding lower values. The use of effective medium theories enabled us to account for the effect of air in the powder samples. The relaxation time, a property of immense importance to the pharmaceutical community, was not influenced by the sample configuration.

Characterization of the effect of NaCl and trehalose on the thermotropic hysteresis of DOPC lipids during freeze/thaw.

This study characterizes the freeze/thaw behavior of large unilamellar DOPC vesicles in the presence of 0.5 or 4 M NaCl and 73 to 146 mM trehalose using Fourier transform infrared (FTIR) spectroscopy. Differences in lipid hydration between cooling and heating caused hysteresis in lipid phase change behavior and altered post-thaw lipid chain order. Lipids transitioned to a more ordered state during cooling. However, with heating, there was further conversion to a more ordered state at temperatures lower than the chain melting temperature. This conversion was more pronounced at higher concentrations of NaCl due to the formation of NaCl dihydrate crystals and the resulting changes in lipid hydration. Trehalose was shown to be capable of abrogating the severe dehydration effect at sufficiently high trehalose/NaCl concentrations by suppressing the formation of the NaCl dihydrate crystals. Moreover, trehalose enhanced recovery of prefreeze membrane structure.