Determining topical product bioequivalence with stimulated Raman scattering microscopy.

Generic drugs are essential for affordable medicine and improving accessibility to treatments. Bioequivalence (BE) is typically demonstrated by assessing a generic product's pharmacokinetics (PK) relative to a reference-listed drug (RLD). Accurately estimating cutaneous PK (cPK) at or near the site of action can be challenging for locally acting topical products. Certain cPK approaches are available for assessing local bioavailability (BA) in the skin. Stimulated Raman scattering (SRS) microscopy has unique capabilities enabling continuous, high spatial and temporal resolution and quantitative imaging of drugs within the skin. In this paper, we developed an approach based on SRS and a polymer-based standard reference for the evaluation of topical product BA and BE in human skin ex vivo. BE assessment of tazarotene-containing formulations was achieved using cPK parameters obtained within different skin microstructures. The establishment of BE between the RLD and an approved generic product was successfully demonstrated. Interestingly, within the constraints of the current study design the results suggest similar BA between the tested gel formulation and the reference cream formulation, despite the differences in the formulation/dosage form. Another formulation containing polyethylene glycol as the vehicle was demonstrated to be not bioequivalent to the RLD. Compared to using the SRS approach without a standard reference, the developed approach enabled more consistent and reproducible results, which is crucial in BE assessment. The abundant information from the developed approach can help to systematically identify key areas of study design that will enable a better comparison of topical products and support an assessment of BE.

Confocal Raman Spectroscopy for Assessing Bioequivalence of Topical Formulations.

The evaluation of bioequivalence (BE) for topical dermatological drug products is challenging, and there has been significant interest from regulatory authorities in developing new BE methodologies in recent years. Currently, BE is demonstrated by comparative clinical endpoint studies; these are costly and time-consuming and often lack sensitivity and reproducibility. Previously, we reported excellent correlations between in vivo Confocal Raman Spectroscopy in human subjects and in vitro skin permeation testing (IVPT) with the human epidermis for skin delivery of ibuprofen and a number of excipients. The aim of the present proof-of-concept study was to evaluate CRS as a method to assess BE of topical products. Two commercially available formulations, Nurofen Max Strength 10% Gel and Ibuleve Speed Relief Max Strength 10% Gel, were selected for evaluation. Delivery of ibuprofen (IBU) to the skin was determined in vitro and in vivo by IVPT and CRS, respectively. The formulations examined were found to deliver comparable amounts of IBU across the skin over 24 h in vitro (p > 0.05). Additionally, the formulations resulted in similar skin uptake values measured with CRS in vivo, either at 1 h or 2 h after application (p > 0.05). This is the first study to report the capability of CRS for the demonstration of BE of dermal products. Future studies will focus on the standardisation of the CRS methodology for a robust and reproducible pharmacokinetic (PK)-based evaluation of topical BE.

Dermal Delivery of Diclofenac Sodium-In Vitro and In Vivo Studies.

Previously, we reported the use of confocal Raman spectroscopy (CRS) as a novel non-invasive approach to determine drug disposition in the skin in vivo. Results obtained by CRS were found to correlate with data from the well-established in vitro permeation test (IVPT) model using human epidermis. However, these studies used simple vehicles comprising single solvents and binary or ternary solvent mixtures; to date, the utility of CRS for monitoring dermal absorption following application of complex marketed formulations has not been examined. In the present work, skin delivery of diclofenac sodium (DFNa) from two topical dermatological drug products, namely Diclac® Lipogel 10 mg/g and Primofenac® Emulsion gel 1%, was determined by IVPT and in vivo by both CRS and tape stripping (TS) methodologies under similar experimental conditions. The in vivo data were evaluated against the in vitro findings, and a direct comparison between CRS and TS was performed. Results from all methodologies showed that Diclac promoted significantly greater DFNa delivery to the skin (p < 0.05). The cumulative amounts of DFNa which permeated at 24 h in vitro for Diclac (86.5 ± 9.4 µg/cm2) were 3.6-fold greater than the corresponding amounts found for Primofenac (24.4 ± 2.7 µg/cm2). Additionally, total skin uptake of DFNa in vivo, estimated by the area under the depth profiles curves (AUC), or the signal intensity of the drug detected in the upper stratum corneum (SC) (4 µm) ranged from 3.5 to 3.6-fold greater for Diclac than for Primofenac. The shape of the distribution profiles and the depth of DFNa penetration to the SC estimated by CRS and TS were similar for the two methods. However, TS data indicated a 4.7-fold greater efficacy of Diclac relative to Primofenac, with corresponding total amounts of drug penetrated, 94.1 ± 22.6 µg and 20.2 ± 7.0 µg. The findings demonstrate that CRS is a methodology that is capable of distinguishing skin delivery of DFNa from different formulations. The results support the use of this approach for non-invasive evaluation of topical products in vivo. Future studies will examine additional formulations with more complex compositions and will use a wider range of drugs with different physicochemical properties. The non-invasive nature of CRS coupled with the ability to monitor drug permeation in real time offer significant advantages for testing and development of topical dermatological products.

Dermal Delivery of Niacinamide-In Vivo Studies.

In vivo human studies are considered to be the "gold standard" when investigating (trans)dermal delivery of actives. Previously, we reported the effects of a range of vehicles on the delivery of niacinamide (NIA) using conventional Franz cell studies. In the present work, dermal delivery of NIA was investigated in vivo in human subjects using confocal Raman spectroscopy (CRS) and tape stripping (TS). The vehicles investigated included propylene glycol (PG), Transcutol® P (TC), binary combinations of PG with oleic acid (OA) or linolenic acid (LA) and a ternary system comprising of TC, caprylic/capric triglyceride (CCT) and dimethyl isosorbide (DMI). For the CRS studies, higher area under curve (AUC) values for NIA were observed for the PG:LA binary system compared with PG, TC and TC:CCT:DMI (p < 0.05). A very good correlation was found between the in vitro cumulative permeation of NIA and the AUC values from Raman intensity depth profiles, with a Pearson correlation coefficient (R2) of 0.84. In addition, an excellent correlation (R2 = 0.97) was evident for the signal of the solvent PG and the active. CRS was also shown to discriminate between NIA in solution versus crystalline NIA. The findings confirm that CRS is emerging as a powerful approach for dermatopharmacokinetic studies of both actives and excipients in human.

In Vitro-In Vivo Correlation in Dermal Delivery: The Role of Excipients.

The composition of topical and transdermal formulations is known to determine the rate and the extent of drug delivery to and through the skin. However, to date, the role of excipients in these formulations on skin delivery of actives has received little attention from scientists in the field. Monitoring skin absorption of both drug and vehicle may provide insights into the mechanism by which excipients promote permeation and may facilitate the design of effective and safer products. Previously, we have investigated the use of quantitative Confocal Raman Spectroscopy (CRS) to investigate the delivery of an active to the skin, and we also reported the first fully quantitative study that compared this method with the well-established in vitro permeation test (IVPT) model. To further explore the potential of quantitative CRS in assessing topical delivery, the present work investigated the effects of commonly used excipients on the percutaneous absorption of a model drug, ibuprofen (IBU). Permeation of IBU and selected solvents following finite dose applications to human skin was determined in vitro and in vivo by Franz diffusion studies and quantitative CRS, respectively. The solvents used were propylene glycol (PG), dipropylene glycol (DPG), tripropylene glycol (TPG), and polyethylene glycol 300 (PEG 300). Overall, the cumulative amounts of IBU that permeated at 24 h in vitro were similar for PG, DPG, and TPG (p > 0.05). These three vehicles outperformed PEG 300 (p < 0.05) in terms of drug delivery. Concerning the vehicles, the rank order for in vitro skin permeation was DPG ≥ PG > TPG, while PEG 300 did not permeate the skin. A linear relationship between maximum vehicle and IBU flux in vitro was found, with a correlation coefficient (R2) of 0.95. When comparing in vitro with in vivo data, a positive in vitro-in vivo (IVIV) correlation between the cumulative permeation of IBU in vitro and the total amount of IBU that penetrated the stratum corneum (SC) in vivo was observed, with a Pearson correlation coefficient (R2) of 0.90. A strong IVIV correlation, R2 = 0.82, was found following the linear regression of the cumulative number of solvents permeated in vitro and the corresponding skin uptake in vivo measured with CRS. This is the first study to correlate in vivo permeation of solvents measured by CRS with data obtained by in vitro diffusion studies. The IVIV correlations suggest that CRS is a powerful tool for profiling drug and vehicle delivery from dermal formulations. Future studies will examine additional excipients with varying physicochemical properties. Ultimately, these findings are expected to lead to new approaches for the design, evaluation, and optimization of formulations that target actives to and through the skin.

Laparoscopic removal of a fishbone migrating from the gastrointestinal tract to the pancreas.

Laparoscopic surgery can be performed safely for the removal of a foreign body embedded in the pancreas and should be preferred instead of open surgery, whenever possible.

A comparison of the in vitro permeation of 3-O-ethyl-l-ascorbic acid in human skin and in a living skin equivalent (LabSkin™).

OBJECTIVES: The safety assessment of personal care products often entails determining dermal absorption of their ingredients. Such experiments are typically performed in human or animal skin in vitro; however, ethical and safety considerations are associated with obtaining these tissues. Several human skin equivalent models (HSEs) have been developed as alternatives to human tissue. The barrier function of such models however, is normally less developed than human skin. Here, we examine the permeability of the HSE LabSkinTM to a model compound, 3-O-ethyl-l-ascorbic acid (EA) compared with human skin. METHODS: Skin uptake and permeation of EA was investigated in vitro using heat-separated human epidermis and LabSkinTM . Finite dose (5 µL cm-2 ) Franz-diffusion studies were conducted using 2 % (w/w) EA in a ternary solvent mixture comprising propylene glycol (PG), propylene glycol monolaurate (PGML), and isopropyl myristate (IPM). These excipients are commonly used in cosmetic products and they have been reported to promote permeation of EA in a different model, namely porcine skin. RESULTS: Permeation of EA through LabSkinTM was evident from 2 h; however, EA permeation in human skin was not detected until 5 h. Similar amounts of EA permeated through the two membranes at time points 8, 10, 12 and 24 h (p > 0.05). The cumulative amounts of EA delivered through LabSkinTM at 24 h were 41.3 ± 2.0 µg cm-2 , corresponding to 55.1 ± 1.8 % of the applied dose. Similar amounts permeated across human skin, 49.4 ± 4.1 µg cm-2 , accounting for 58.0 ± 4.2 % of the dose applied (p > 0.05). CONCLUSION: The permeation of EA in LabSkinTM compared well with results for human epidermis in terms of the permeation profiles and the cumulative amounts of EA that permeated. The data suggest that the skin barrier of the two models was similar with regard to their overall permeability to the hydrophilic active EA. The findings are promising for the use of LabSkinTM as a surrogate for human skin in permeability testing. Future studies will focus on exploring the reproducibility and robustness of LabSkinTM for delivery of other actives that span a range of physicochemical properties.

OBJECTIFS: L'évaluation de la sécurité des produits de soins personnels implique souvent de déterminer l'absorption cutanée de leurs ingrédients. Ces expérimentations sont généralement réalisées in vitro sur la peau humaine ou animale ; cependant, des considérations éthiques et de sécurité sont associées à l'obtention de ces tissus. Plusieurs modèles équivalents de peau humaine (Human Skin Equivalent, HSE) ont été développés comme alternatives au tissu humain. La fonction barrière de ces modèles est cependant normalement moins développée que la peau humaine. Ici, nous examinons la perméabilité du HSE LabSkin™ à un composé modèle, l'acide 3-O-éthyl-l-ascorbique (EA) en le comparant à la peau humaine. MÉTHODES: L'absorption cutanée et la perméation de l'EA ont été étudiées in vitro à l'aide d'épiderme humain séparé par la chaleur et de LabSkin™. Des études de diffusion de Franz à dose limitée (5 µL cm-2 ) ont été réalisées en utilisant 2 % (p/p) d'EA dans un mélange de solvant ternaire contenant du propylène glycol (PG), du propylène glycol monolaurate (PGML) et du myristate d'isopropyle (IPM). Ces excipients sont fréquemment utilisés dans les produits cosmétiques et il a été rapporté qu'ils favorisent la perméation de l'EA dans un modèle différent, à savoir la peau porcine. RÉSULTATS: La perméation de l'EA par LabSkin™ était évidente dès 2 h ; cependant, la perméation de l'EA dans la peau humaine n'a pas été détectée avant 5 h. Des quantités similaires d'EA ont pénétré les deux membranes aux points temporels 8, 10, 12 et 24 h (p > 0,05). Les quantités cumulées d'EA délivrées par LabSkin™ à 24 h étaient de 41,3 ± 2,0 µg cm-2 , correspondant à 55,1 ± 1,8 % de la dose appliquée. Des quantités similaires ont pénétré la peau humaine, 49,4 ± 4,1 µg cm-2 , représentant 58,0 ± 4,2 % de la dose appliquée (p > 0,05). CONCLUSION: La perméation de l'EA dans LabSkin™ a bien soutenu la comparaison quant aux résultats concernant l'épiderme humain en termes de profils de perméation et de quantités cumulées d'EA qui ont pénétré. Les données suggèrent que la barrière cutanée des deux modèles était similaire en ce qui concerne leur perméabilité globale à l'EA hydrophile actif. Les résultats sont prometteurs pour l'utilisation de LabSkin™ en tant que substitut de la peau humaine dans les tests de perméabilité. Les études futures se concentreront sur l'exploration de la reproductibilité et de la robustesse de LabSkin™ pour la délivrance d'autres principes actifs qui couvrent un éventail de propriétés physicochimiques.

Franz Cell Diffusion Testing and Quantitative Confocal Raman Spectroscopy: In Vitro-In Vivo Correlation.

Previously, we reported the use of Confocal Raman Spectroscopy (CRS) to investigate the topical delivery of actives and excipients. We have also correlated the results from CRS with findings from in vitro diffusion studies in human skin. However, until now CRS has only been used as a semi-quantitative method of determining the skin uptake of molecules, with results expressed as arbitrary units of signal intensity. Clearly, this posed challenges for using CRS to determine skin delivery and to assess the drug bioavailability and bioequivalence of topical formulations. In the present work, the permeation of niacinamide (NIA) from various formulations in human skin was studied in vitro using conventional Franz cells and in vivo using a quantitative CRS method under finite dose conditions. The selection of NIA was based on its wide use in pharmaceutical and personal care formulations for many years. This is the first fully quantitative study to compare these methods. The vehicles investigated were neat Transcutol® P (TC); binary combinations of propylene glycol (PG) with propylene glycol monolaurate (PGML); and ternary mixtures of PG, PGML, and isopropyl myristate (IPM). These solvents were selected to encompass a range of physicochemical properties. NIA permeation was evident from all formulations in vitro and in vivo. The vehicles PG:PGML and PG:PGML:IPM delivered comparable amounts across the skin in vitro at 24 h (100.3-106.7 µg/cm2, p > 0.05) that were significantly higher compared with those of TC (1.3 µg/cm2, p < 0.05). An excellent in vitro in vivo correlation (R2 = 0.98) was found following the linear regression of the cumulative amounts of NIA permeated in vitro and the amounts of NIA at 2 µm in the skin measured with CRS. A very good correlation between the cumulative permeation of NIA in vitro and the total amount of NIA that penetrated the stratum corneum (SC) per unit of surface area (µg/cm2) in vivo was also observed, with a Pearson correlation coefficient (R2) of 0.94. The findings support the use of CRS for the quantitative measurement of actives delivered to the skin in vivo. Future studies will focus on exploring the reproducibility and reliability of the method by investigating the delivery of different actives from a wider range of vehicles. Additionally, quantitative CRS will be evaluated further as a method for assessing the bioequivalence of topical formulations.

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.

Preparation, Characterisation, and Topical Delivery of Terbinafine.

Terbinafine (TBF) is commonly used in the management of fungal infections of the skin because of its broad spectrum of activity. Currently, formulations containing the free base and salt form are available. However, there is only limited information in the literature about the physicochemical properties of this drug and its uptake by the skin. In this work, we conducted a comprehensive characterisation of TBF, and we also examined its percutaneous absorption in vitro in porcine skin. TBF-free base was synthesised from the hydrochloride salt by a simple proton displacement reaction. Both the free base and salt form were further analysed using Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA). Delivery of TBF-free base in excised porcine skin was investigated from the following solvents: Isopropyl myristate (IPM), propylene glycol monolaurate (PGML), Transcutol® (TC), propylene glycol (PG), polyethylene glycol 200 (PEG 200), oleic acid (OL), ethanol (EtOH), and isopropyl alcohol (IPA). Permeation and mass balance studies confirmed that PG and TC were the most efficacious vehicles, delivering higher amounts of TBF-free base to the skin compared with a commercial gel (p < 0.05). These preliminary results are promising and will inform the development of more complex formulations in future work.

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.