Translating chemometric analysis into physiological insights from in vivo confocal Raman spectroscopy of the human stratum corneum.

The superficial layer of the skin, the stratum corneum (SC), consists of corneocytes surrounded by lipid regions and acts as a protective barrier for the body against water loss, toxic agents and microorganisms. As most substances permeate the stratum corneum through the lipid regions, lipid organization is considered crucial for the skin barrier function. Here, we investigate the potential of in vivo confocal Raman spectroscopy to describe the composition and organization of the SC. Confocal Raman spectroscopy is finding increasing use in the characterization of skin in biomedical, pharmaceutical and cosmetic applications. In this work, we analyze the spectra using chemometric methods and obtain principal components that correspond to the primary skin constituents: protein (keratin), natural moisturizing factor (NMF), water and lipid contributions in both ordered (orthorhombic) and disordered structural organization. By identifying these important components of the SC, these results highlight the utility of this in vivo, non-invasive, and depth resolved tool at the forefront of skin research.

Transcriptional profiling of epidermal barrier formation in vitro.

BACKGROUND: Barrier function is integral to the health of epithelial tissues. Currently, there is a broad need to develop and improve our knowledge with regard to barrier function for reversal of mild skin irritation and dryness. However, there are few in vitro models that incorporate modulations of both lipids and epidermal differentiation programs for pre-clinical testing to aid in the understanding of barrier health. OBJECTIVE: We have generated a reconstituted epidermis on a decellularized dermis (DED) and characterized its barrier properties relative to human epidermis in order to determine its utility for modeling barrier formation and repair. METHODS: We followed the process of epidermal differentiation and barrier formation through immunocytochemistry and transcriptional profiling. We examined barrier functionality through measurements of surface pH, lipid composition, stratum corneum water content, and the ability to demonstrate topical dose-dependent exclusion of surfactant. RESULTS: Transcriptional profiling of the epidermal model during its formation reveals temporal patterns of gene expression associated with processes regulating barrier function. The profiling is supported by gradual formation and maturation of a stratum corneum and expression of appropriate markers of epidermis development. The model displays a functional barrier and a water gradient between the stratum corneum and viable layers, as determined by confocal Raman spectroscopy. The stratum corneum layer displays a normal acidic pH and an appropriate composition of barrier lipids. CONCLUSION: The epidermal model demonstrates its utility as an investigative tool for barrier health and provides a window into the transcriptional regulation of multiple aspects of barrier formation.

Identification and characterization of toxicity of contaminants in pet food leading to an outbreak of renal toxicity in cats and dogs.

This paper describes research relating to the major recall of pet food that occurred in Spring 2007 in North America. Clinical observations of acute renal failure in cats and dogs were associated with consumption of wet pet food produced by a contract manufacturer producing for a large number of companies. The affected lots of food had been formulated with wheat gluten originating from China. Pet food and gluten were analyzed for contaminants using several configurations of high-performance liquid chromatography (HPLC) and mass spectrometry (MS), which revealed a number of simple triazine compounds, principally melamine and cyanuric acid, with lower concentrations of ammeline, ammelide, ureidomelamine, and N-methylmelamine. Melamine and cyanuric acid, have been tested and do not produce acute renal toxicity. Some of the triazines have poor solubility, as does the compound melamine cyanurate. Pathological evaluation of cats and dogs that had died from the acute renal failure indicated the presence of crystals in kidney tubules. We hypothesized that these crystals were composed of the poorly soluble triazines, a melamine-cyanuric acid complex, or a combination. Sprague dawley rats were given up to 100 mg/kg ammeline or ammelide alone, a mixture of melamine and cyanuric acid (400/400 mg/kg/day), or a mixture of all four compounds (400 mg/kg/day melamine, 40 mg/kg/day of the others). Neither ammeline nor ammelide alone produced any renal effects, but the mixtures produced significant renal damage and crystals in nephrons. HPLC-MS/MS confirmed the presence of melamine and cyanuric acid in the kidney. Infrared microspectroscopy on individual crystals from rat or cat (donated material from a veterinary clinic) kidneys confirmed that they were melamine-cyanuric acid cocrystals. Crystals from contaminated gluten produced comparable spectra. These results establish the causal link between the contaminated gluten and the adverse effects and provide a mechanistic explanation for how two apparently innocuous compounds could have adverse effects in combination, that is, by forming an insoluble precipitate in renal tubules leading to progressive tubular blockage and degeneration.

Dehydration of risedronate hemi-pentahydrate: analytical and physical characterization.

Dehydration of hydrates of pharmaceutical active ingredients (pharmaceutical hydrates) may easily occur during storage or manufacturing. Loss of water may have little effect on the crystal lattice, produce less hydrated forms or possibly amorphous forms. Characterizing the effects of water loss on crystal hydrate forms is important for understanding the behavior of pharmaceutical hydrates throughout the manufacturing and storage processes. This study shows that exposure of the hemi-pentahydrate form of risedronate monosodium to gentle heating (60 degrees C) or conditions of low relative humidity (<10% RH) results in the loss of 1 mole of channel-type water. Upon removal of the channel-type water, the crystal lattice adjusts producing a distinct phase characterized by X-ray, thermal, IR, Raman, and NMR data. Adjustment of the crystal lattice appears to compromise crystal integrity and can result in reduced crystallite and particle sizes.

Application of on-line Raman spectroscopy for characterizing relationships between drug hydration state and tablet physical stability.

Experiments were conducted to elucidate the relationship between risedronate sodium (RS) hydration state and the physical stability of tablets containing RS. The RS crystal lattice contains channels occupied by water, which is removed by drying processes at temperatures below the boiling point of water, causing a reversible contraction of the crystal lattice. In this study, risedronate sodium was wet granulated followed by fluid bed drying to final granulation moisture contents between 1 and 7%, and then compressed into tablets. During drying, the RS solid-state form was continuously monitored using on-line Raman spectroscopy. Raman spectra acquired in these experiments enabled direct monitoring of changes in the RS crystal lattice, due to dehydration, which provided key information relating RS solid-state form characteristics to final granulation moisture content. Final granulation moisture was found to have a significant effect on the change in RS hydration state measured by Raman spectroscopy. As the final granulation moisture decreased, the amount of RS dehydrated form increased. The largest Raman spectral changes were in the CH stretching region and the region including the 3-picoline ring and PO2- stretches. These changes are indicative of substantial changes in the RS solid-state structure. Final granulation moisture also had a significant effect on the change in tablet thickness over time. Lower final granulation moisture caused a greater increase in tablet thickness as the RS rehydrated. In addition, the change in RS hydration state during fluid bed drying, measured by on-line Raman, was correlated to the increase in tablet thickness and subsequent loss of tablet integrity. Raman spectroscopy allowed direct RS hydration state monitoring, rather than inference from a bulk moisture measurement. Development of a Process Analytical Technology (PAT), specifically Raman, to monitor RS solid-state during drying enabled establishment of relationships between fundamental hydration dynamics associated with RS and final product performance attributes.

Application of Raman spectroscopy for on-line monitoring of low dose blend uniformity.

On-line Raman spectroscopy was used to evaluate the effect of blending time on low dose, 1%, blend uniformity of azimilide dihydrochloride. An 8 qt blender was used for the experiments and instrumented with a Raman probe through the I-bar port. The blender was slowed to 6.75 rpm to better illustrate the blending process (normal speed is 25 rpm). Uniformity was reached after 20 min of blending at 6.75 rpm (135 revolutions or 5.4 min at 25 rpm). On-line Raman analysis of blend uniformity provided more benefits than traditional thief sampling and off-line analysis. On-line Raman spectroscopy enabled generating data rich blend profiles, due to the ability to collect a large number of samples during the blending process (sampling every 20s). In addition, the Raman blend profile was rapidly generated, compared to the lengthy time to complete a blend profile with thief sampling and off-line analysis. The on-line Raman blend uniformity results were also significantly correlated (p-value < 0.05) to the HPLC uniformity results of thief samples.

Structural and analytical characterization of three hydrates and an anhydrate form of risedronate.

Four hydration states are reported for Risedronate monosodium. A single-crystal X-ray structure determination is provided as proof of assignment for the monohydrate, hemi-pentahydrate, and variable hydrate forms. The structure provided for the anhydrate form was determined through simulating annealing calculations and subsequent Reitveld refinement of a high-quality X-ray powder diffraction patterns Favorable comparisons of experimentally obtained X-ray powder patterns are made to those generated from the single crystal data. Characteristic infrared, Raman, and NMR spectra are provided and discussed for each form as are thermal analysis profiles. In addition, photomicrographs are provided for each of the forms isolated for this study. The hemi-pentahydrate is demonstrated to be the equilibrium form at room temperature and 37 degrees C, in the presence of water.