Using Nuclear Magnetic Resonance to Troubleshoot a Stability Issue in a Real-World Formulation Chassis-Application to Consumer Oral Healthcare.

With direct application to current and future consumer healthcare products, this research sheds light on the importance of packaging and its potential effects on both Active Pharmaceutical Ingredient (API) delivery and stability. Industrially sourced, proprietary experimental formulations (PEFs), specifically oral cleansers, based on salicylic acid and hydrogen peroxide, discolored over time at different rates, depending on packaging type used. This discoloration stemmed from an interplay of two factors, involving both spontaneous formulation degradation and the interaction of both degradants and salicylic acid with the internal surface of the packaging. This manuscript reports on the investigation to uncover the origins of discoloration. To investigate this real-world, industrial pipeline problem, we exploited the high dimensionality and simple sample preparation uniquely afforded by NMR. Using a combination of 1D/2D NMR and diffusion-ordered NMR spectroscopy (DOSY) to leverage molecular mass estimations from, we not only quickly confirmed the identities of these degradants, but also assessed their formation as a function of temperature and pH, providing insight into the mechanisms underlying their formation. We were able to identify catechol as the main source of discoloration over a period of several weeks, being formed at the ppm level. Furthermore, we evaluated the formulation-container interaction, employing NMR, ICP-MS, and ATR-IR. Despite this comprehensive analysis, the root causes of discoloration could only tentatively be assigned to a surface Ti complex of salicylic acid and other hydroxy carboxylic acids. Through the understanding of formulation degradation pathways, we were able to support further toxicology assessment, vital to both consumer safety and the manufacturer. This work underscores the invaluable role of NMR in the analysis of intricate proprietary mixtures with a consumer-centric purpose. Our findings demonstrate that conventional analytical techniques falter in the face of such complexity, requiring extensive preparation and pre-analytical processing, highlighting the novelty and crucial relevance of NMR research to manufacturers and consumers. Such an analysis is of value in the pursuit of materials within the consumer-healthcare space, which meet the requirements for successful recycling or re-use.

A 3D Printed Device for In Vitro Generation of Stratified Epithelia at the Air-Liquid Interface.

Air-liquid interface (ALI) cultures are used to produce stratified epithelial tissues in vitro, notably for the production of oral mucosal equivalents. Currently, there are few purpose-built devices, which aim to enhance the ease and reproducibility of generating such tissue. Most ALI cultures utilize stainless steel grids or cell culture inserts to elevate the matrix or scaffold to the surface of the culture media. In this study, a novel buoyant epithelial culture device (BECD) was designed to both contain a fibroblast-seeded collagen hydrogel and float in culture media, thereby automatically maintaining the ALI without further user intervention. BECDs aim to mitigate several issues associated with ALI culture; reducing the chance of media flooding the epithelial layer from physical disturbance, reducing technique sensitivity for less-experienced users, and improving the reproducibility of the epithelia generated. H400 oral squamous cell carcinoma cells cultured in BECDs for 7, 14, and 21 days showed continuous increase in epithelial tissue thickness with expected localization of epithelial differentiation markers: cytokeratin 5, involucrin, and E-cadherin. Fused filament fabrication three-dimensional printing with polypropylene used in BECD production allows for rapid turnover and design iteration, presenting a versatile, adaptable, and useful tool for application in in vitro cell culture.

Investigation of the Formation of Squalene Oligomers Exposed to Ultraviolet Light and Changes in the Exposed Squalene as a Potential Skin Model.

UV-induced oligomerisation of squalene was undertaken to indicate the potential for squalene-containing biological systems to exhibit rheology changes. DOSY NMR enabled the determination of the molecular weight (MW) range using Stokes-Einstein Gierer-Wirtz Estimation (SEGWE Calculator, University of Manchester). This approach was validated by Atmospheric Solids Analysis Probe Time of Flight Mass Spectrometry (ASAP TOF MS). To demonstrate the principle, both benzoyl peroxide and AIBN were used, separately, to initiate rapid, radical oligomerisation. Subsequent experiments in the absence of initiators compared the influence of UV wavelength and time on the resulting oligomer formation. To further model a relevant biological implication of this potentially chaotic UV oligomerisation, both saturated and unsaturated free fatty acids were added to squalene and exposed to UV at 285 nm and 300 nm to determine if cross oligomerisation could be observed. This representation of sebum evidenced the formation of a distribution of higher MW oligomers. Internal viscosity was normalised using the DMSO solvent, but to confirm that changes in rheology did not affect diffusion, a final experiment where fresh squalene was added to our oligomer mixture, representative of sebum, showed that unchanged squalene possessed the anticipated monomeric diffusion coefficient and hence MW. This work suggests, at least qualitatively, that UV-induced squalene oligomerisation can occur over time and that this may have a role in the behaviour of squalene on the skin.

Characterizing the nanomechanical properties of microcomedones after treatment with sodium salicylate ex vivo using atomic force microscopy.

OBJECTIVE: The treatment of acne presents a major clinical and dermatological challenge. Investigating the nanomechanical properties of the microcomedone precursor lesions using atomic force microscopy (AFM) may prove beneficial in understanding their softening, dissolution and prevention. Although the exact biochemical mechanism of NaSal on microcomedones is not fully understood at present, it appears to exhibit a significant exfoliation effect on the skin via corneodesmosome dissolution. METHODS: Therefore, to support this exploration, sodium salicylate (NaSal), a common ingredient employed in skin care products, is applied ex vivo to microcomedones,collected by nose strip adhesive tape, and their nanomechanical properties are assessed using AFM. Although the exact biochemical mechanism of NaSal on microcomedones is not fully understood at present, it appears to exhibit a significant exfoliation effect on the skin via corneodesmosome dissolution. RESULTS: Herein, our findings demonstrate that when microcomedones are treated with 2% NaSal, samples appeared significantly more compliant ('softer') ((1.3 ± 0.62) MPa) when compared to their pre-treated measurements ((7.2 ± 3.6) MPa; p = 0.038). Furthermore, elastic modulus maps showed that after 2% NaSal treatment, areas in the microcomedone appeared softer and swollen in some, but not in all areas, further proving the valuable impact of 2% NaSal solution in altering the biomechanical properties and morphologies in microcomedones. CONCLUSION: Our results are the first of their kind to provide qualitative and quantitative mechanobiological evidence that 2% NaSal decreases the elastic modulus of microcomedones. Therefore, this study provides evidence that NaSal can be beneficial as an active ingredient in topical treatments aimed at targeting microcomedones.

OBJECTIF: Le traitement de l'acné présente un défi clinique et dermatologique majeur. L'étude des propriétés nanomécaniques des lésions précurseurs en tant que microcomédons à l'aide de la microscopie à force atomique (AFM) peut s'avérer bénéfique pour comprendre leur ramollissement, leur dissolution et leur prévention. MÉTHODES: Par conséquent, pour soutenir cette exploration, le salicylate de sodium (NaSal), un ingrédient couramment utilisé dans les produits de soins de la peau, est appliqué ex vivo aux microcomédons et leurs propriétés nanomécaniques sont évaluées à l'aide de l'AFM. Bien que le mécanisme biochimique exact du NaSal sur les microcomédons ne soit pas entièrement compris à l'heure actuelle, il semble présenter un effet exfoliant significatif sur la peau via la dissolution des cornéodesmosomes. RÉSULTATS: Ici, nos résultats démontrent que lorsque les microcomédons sont traités avec 2% de NaSal, les échantillons semblaient significativement plus conformes ("plus doux") ((1.3 ± 0.62) MPa) par rapport à leurs mesures pré-traitées ((7.2 ± 3.6) MPa ; P = 0,03826). De plus, les cartes du module d'élasticité ont montré qu'après un traitement à 2 % de NaSal, les zones du microcomédon semblaient plus molles et gonflées dans certaines zones, mais pas dans toutes, prouvant ainsi l'impact précieux d'une solution de NaSal à 2 % dans la modification des propriétés biomécaniques et de la morphologie des microcomédons. CONCLUSION: Nos résultats sont les premiers du genre à fournir des preuves mécanobiologiques qualitatives et quantitatives que 2% de NaSal diminue le module d'élasticité des microcomédons. Par conséquent, cette étude fournit des preuves que NaSal peut être bénéfique en tant qu'ingrédient actif dans les traitements topiques visant à cibler les microcomédons.

Topical delivery of niacinamide: Influence of neat solvents.

Niacinamide (NIA) has been widely used in cosmetic and personal care formulations for several skin conditions. Permeation of topical NIA has been confirmed in a number of studies under infinite dose conditions. However, there is limited information in the literature regarding permeation of NIA following application of topical formulations in amounts that reflect the real-life use of such products by consumers. The aim of the present work was therefore to investigate skin delivery of NIA from single solvent systems in porcine skin under finite dose conditions. A secondary aim was to probe the processes underlying the previously reported low recovery of NIA following in vitro permeation and mass balance studies. The solubility and stability of NIA in various single solvent systems was examined. The solvents investigated included Transcutol® P (TC), propylene glycol (PG), 1-2 hexanediol (HEX), 1-2 pentanediol (1-2P), 1-5 pentanediol (1-5P), 1-3 butanediol (1-3B), glycerol (GLY) and dimethyl isosorbide (DMI). Skin permeation and deposition of the molecule was investigated in full thickness porcine skin in vitro finite dose Franz-type diffusion experiments followed by mass balance studies. Stability of NIA for 72 h in the solvents was confirmed. The solubility of NIA in the solvents ranged from 82.9 ± 0.8 to 311.9 ± 4.5 mg/mL. TC delivered the highest percentage permeation of NIA at 24 h, 32.6 ± 12.1% of the applied dose. Low total recovery of NIA after mass balance studies was observed for some vehicles, with values ranging from 55.2 ± 12.8% to 106.3 ± 2.3%. This reflected the formation of a number of NIA degradation by-products in the receptor phase during the permeation studies. Identification of other vehicles for synergistic enhancement of NIA skin delivery will be the subject of future work.

Scope and limitations of nuclear magnetic resonance techniques for characterisation and quantitation of vitamin D in complex mixtures.

BACKGROUND: The accurate determination of vitamin D in skin is of considerable importance in evaluating penetration of skin health products through the different layers of the skin. OBJECTIVE: We report on the characterisation and quantitation of vitamin D in an idealised sample and in complex mixtures which mimic that of a typical skin cream, using qNMR, 2D NMR and DOSY techniques. METHODS: The characterisation and quantitation conditions were acquired over several heterogeneous samples, allowing for analysis of how the dynamic range and complexity of the different sample mixtures affect the limits of detection (LOD) and limits of quantitation (LOQ) of vitamin D. NMR is of particular value to this task as it is non-destructive, uses a primary ratio method for quantification, and tolerates a wide variety of hydrophilic and hydrophobic components within a given matrix. RESULTS: In this investigation, we have attained a trueness level <10%, repeatability values of <1% and brought the limit of quantitation down to 100 nmol/L (≈limit of baseline range of vitamin D2 and D3 per litre seen in vivo), commenting on the limitations observed, such as peak overlap and sensitivity limits. CONCLUSIONS: Pure shift optimised sequences allow us to reduce peak overlapping, allowing further characterisation of individual compounds and the separation of complex mixtures using NMR.

3-O-ethyl-l-ascorbic acid: Characterisation and investigation of single solvent systems for delivery to the skin.

l-ascorbic acid (AA), commonly known as vitamin C, has been widely used in topical formulations for many years as an antioxidant and anti-aging ingredient. However, the physicochemical properties of AA are not optimal for skin uptake and the molecule is also unstable, readily undergoing oxidation on exposure to air. The compound 3-o-ethyl-l-ascorbic acid (EA) has been developed as a stable vitamin C derivative and has been used in topical products. The aims of this work were to conduct a comprehensive characterisation of physicochemical properties of EA as well as to investigate the influence of various neat solvents on EA skin delivery. Nuclear magnetic resonance (NMR), mass spectroscopy, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were used to characterise the molecule. The pKa of the compound and the partition coefficient logP(o/w) were experimentally determined. A new HPLC method for analysis of the molecule was also developed and validated. A number of solvents for topical preparations were selected based on their wide use as excipients in topical formulations, their potential to act as skin penetration enhancers and their favourable safety profiles. The solubility and stability of EA was examined. Skin permeation of the molecule in full thickness porcine skin in vitro was investigated using Franz-type diffusion cells. The melting point, log P(o/w) value and pKa value of EA were determined to be 114.39 ±â€¯0.5 °C, -1.07 ±â€¯0.03 and 7.72 ±â€¯0.01 respectively. Skin penetration of EA was evident for the following vehicles 1,2 hexanediol (HEX), glycerol (GLY), propylene glycol (PG), 1,2 pentanediol (1-2P), isopropyl alcohol (IPA), propylene glycol monolaurate (PGML) and propylene glycol monocaprylate (PGMC). Skin uptake but no permeation through the skin was observed for Transcutol® (TC) and dipropylene glycol (DiPG), while no penetration was observed for the solvents 1,5 pentanediol (1-5P) and tripropylene glycol (TriPG). The findings of the permeation experiments confirm the potential of simple formulations to deliver EA to the skin. Studies are ongoing to identify complex vehicles for synergistic enhancement of EA skin penetration. To our knowledge this is the first study to conduct a comprehensive characterization of EA and examine its skin uptake and permeation properties in porcine skin.

In vitro studies investigating the dentin tubule-occlusion properties of an experimental anhydrous stannous fluoride dentifrice.

PURPOSE: To evaluate in vitro the ability for an experimental anhydrous 0.454% w/w stannous fluoride dentifrice to occlude dentin tubules. METHODS: Scanning electron microscopy (SEM) was used to assess tubule occlusion and the resistance of the occlusion to dietary acid in a 4-day model, where bovine dentin was treated with product twice daily with acid challenges administered on Days 3 and 4. The reduction in hydraulic conductance of the dentin following treatment was also measured. RESULTS: The experimental stannous fluoride dentifrice was effective at reducing the hydraulic conductance of dentin by occluding tubules. The occlusion was resistant to challenge with grapefruit juice.

Rhabdomyolysis associated with cerivastatin: six cases within 3 months at one hospital.

Rhabdomyolysis is an uncommon complication associated with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. We observed six cases of cerivastatin-associated rhabdomyolysis: two patients developed acute renal failure requiring hemodialysis, and one patient died. Four of the patients had impaired renal function, and five were prescribed drugs with the potential to interact with cerivastatin. Also, five of the six patients presented with symptoms of rhabdomyolysis 3-4 weeks after starting cerivastatin therapy.