Physicochemical properties of otic products for Canine Otitis Externa: comparative analysis of marketed products.

BACKGROUND: Otitis externa is a commonly diagnosed dermatological disorder in canines. The pathogens primarily involved in canine otitis externa (COE) include Staphylococcus pseudintermedius, Pseudomonas aeruginosa, Proteus mirabilis, and Malassezia pachydermatis. As COE tends to be superficial, medications delivered topically are often effective and practical in managing the condition. As such, there is a wide variety of approved topical products currently available in the market. The efficacy of topical dosage forms can be dependent on various factors such as the pharmacology of active constituents and the physicochemical properties of the formulation, including pH, viscosity, spreadability, and bio-adhesion. Currently, there is a lack of published literature available on the optimal properties of topical COE products. In this study, we compared the physicochemical properties of nine commercially available otic veterinarian products in Australia used clinically to manage COE. RESULTS: Based on our comparative analysis, the pH (6.26 ± 0.04) of an aqueous-based product was similar to a healthy dog's external auditory canal. Products containing polymers exhibited higher viscosity and bio-adhesion. Spreadability was inversely related to viscosity and Osurnia ® a product with high viscosity demonstrated the lowest spreadability. Aqueous-based otic products showed better syringebility whereas oil-based systems required higher force to expel the products. Variability in droplet size was noted. Derm Otic, Baytril Otic, and Aurizon Ear Drops had the lower standard deviation which indicates they would give a more consistent dose. CONCLUSIONS: Findings from this work provide considerations for industry researchers or formulation scientists working in the area of otic dosage formulations.

Insights into the degradation chemistry of tazarotene, a third generation acetylenic retinoid: LC-HRMS (Orbitrap), LC-MSn and NMR characterization of its degradation products, and prediction of their physicochemical and ADMET properties.

Tazarotene is a prodrug that belongs to the acetylenic class of retinoids. The drug was subjected to hydrolytic, oxidative and photolytic stress testing to establish its comprehensive degradation chemistry. The drug proved to be unstable under acidic and basic hydrolytic conditions, yielding tazarotenic acid, which is a known major degradation product (DP) and an active metabolite. Additionally, two DPs each were generated upon interaction of drug and tazarotenic acid with HCl, used as an acid stressor. These were experimentally proven as pseudo DPs, as they did not originate when H2SO4 was employed as the stressor. The drug was also unstable under oxidative and photolytic conditions, yielding six DPs. All the products were separated on reversed phase (C18) column, using mobile phase composed of 10 mM ammonium formate (pH 3.5) and acetonitrile, which was run in a gradient mode. The separated DPs were subjected to LC-HRMS and LC-MSn studies for their initial characterization. Seven hydrolytic and oxidative DPs that could be isolated using semi-preparative column were subjected to extensive 1D (1H, 13C and DEPT-135) and 2D (COSY, HSQC and HMBC) NMR studies to confirm their structures. In total, five novel DPs were characterized, apart from two previously reported DPs, viz., tazarotenic acid and tazarotene sulfoxide, and four additional pseudo DPs. The complete degradation pathway of the drug was established. In silico ADMET properties of the drug and its DPs were evaluated using ADMET Predictor™.

Profile of depigmenting cosmetics and dermatological products on the market

The skin is the largest and most exposed organ of the human body, therefore subject to diseases and alteration of its appearance. Among these alterations, the cutaneous hyperchromia may be cited. Currently, the market offers numerous products with depigmenting action to the treatment of such disorders. The aim of this work was to analyze depigmenting products commercialized in establishments in the city of Bento Gonçalves (RS, Brazil) and websites of cosmetic companies. It was found 45 products with depigmenting action and, from these, 59 different active agents were identified. The main active compounds found were kojic acid, arbutin, ascorbic acid, hydroquinone and glycolic acid. Another observed data was that in 78% of the studied products the active substances were being used in combination. The most used vehicles were also studied as a reference to the use of sunscreen in the treatment of cutaneous hyperchromia. The present work had identified in the market a variety of products with depigmentation action and, because of this, it aims to serve as a reference to the healthcare professionals, especially at the prescribing moment, looking for the best results, with regards to treatment efficiency and safety.(AU)

Stability-indicating analytical method of quantifying spironolactone and canrenone in dermatological formulations and iontophoretic skin permeation experiments.

A simple, stability-indicating, chromatographic method of quantifying spironolactone (SPI) and its metabolite, canrenone (CAN), in the presence of excipients typical in dermatological formulations and skin matrices in studies of passive and iontophoretic permeation was proposed and validated here. SPI and CAN were separated using a reversed-phase column with a mobile phase of methanol-water (60:40, v/v) at a flow rate of 1.0 mL/min. Data were collected with a UV detector at 238 and 280 nm, with retention times of 6.2 and 7.9 min for SPI and CAN, respectively. The method was precise, accurate and linear (r2 > 0.99) in a concentration range of 1-30 µg/mL, and recovery rates of SPI and CAN from the different skin layers exceeded 85%. The method was not only sensitive (LOD of 0.05 and 0.375 µg/mL and LOQ of 0.157 and 1.139 µg/mL for SPI and CAN, respectively) but also selective against skin matrices and highly representative components of topical formulations. The method moreover demonstrated SPI's degradation in iontophoresis by applying Pt-AgCl electrodes and its continued drug stability using Ag-AgCl electrodes. Altogether, the method proved valuable for quantifying SPI and CAN and may be applied in developing and controlling the quality of dermatological products.

Reliable Identification of Significant Differences in Differential Hydrogen Exchange-Mass Spectrometry Measurements Using a Hybrid Significance Testing Approach.

Differential hydrogen exchange-mass spectrometry (HX-MS) measurements are valuable for identification of differences in the higher order structures of proteins. Typically, the data sets are large with many differential HX values corresponding to many peptides monitored at several labeling times. To eliminate subjectivity and reliably identify significant differences in HX-MS measurements, a statistical analysis approach is needed. In this work, we performed null HX-MS measurements (i.e., no meaningful differences) on maltose binding protein and infliximab, a monoclonal antibody, to evaluate the reliability of different statistical analysis approaches. Null measurements are useful for directly evaluating the risk (i.e., falsely classifying a difference as significant) and power (i.e., failing to classify a true difference as significant) associated with different statistical analysis approaches. With null measurements, we identified weaknesses in the approaches commonly used. Individual tests of significance were prone to false positives due to the problem of multiple comparisons. Incorporation of Bonferroni correction led to unacceptably large limits of detection, severely decreasing the power. Analysis methods using a globally estimated significance limit also led to an overestimation of the limit of detection, leading to a loss of power. Here, we demonstrate a hybrid statistical analysis, based on volcano plots, that combines individual significance testing with an estimated global significance limit, that simultaneously decreased the risk of false positives and retained superior power. Furthermore, we highlight the utility of null HX-MS measurements to explicitly evaluate the criteria used to classify a difference in HX as significant.

A New HPLC-DAD Method for the Concurrent Determination of Hydroquinone, Hydrocortisone Acetate and Tretinoin in Different Pharmaceuticals for Melasma Treatment.

A new simple and robust HPLC-DAD method was developed for the concurrent analysis of hydroquinone (HQ), hydrocortisone acetate (HCA) and tretinoin (TRN) triple combination for the first time using an Inertsil ODS 3-C18 column (150 mm × 4.6 mm, 5 µm particle size) column with 0.05 M phosphate buffer (pH 5.0) and acetonitrile at a ratio of (10:90, v/v) as a mobile phase, eluted by an isocratic elution mode at a flow rate of 1.0 mL/min and detected at 265 nm. Mefenamic acid was used as an internal standard (I.S.). The method produced linear responses in the concentration range of 10-200, 5-100 and 1-40 µg/mL, with detection limits of 2.01, 1.13 and 0.28 × 10-3 and quantitation limits of 6.11, 3.41 and 0.87 × 10-3 µg/mL for HQ, HCA and TRN, respectively, and a correlation coefficient higher than 0.9998. All validation requirements were satisfied by proving its linearity, precision, accuracy, robustness and specificity. The method was extended for application in triple combination cream, HQ/TRN co-formulated cream and HQ and TRN single ingredient cream with a recovery >97%.

HPLC profiling of Mimosa pudica polyphenols and their non-invasive biophysical investigations for anti-dermatoheliotic and skin reinstating potential.

Plant-derived polyphenols are known to have promising biological activities including antioxidant and antityrosinase and may be a potential candidate for anti-dermatoheliotic remedy. The present study was performed to investigate the polyphenolic contents of Mimosa pudica (MP) seed extract and its anti-dermatoheliotic potential using non-invasive biophysical techniques after developing a stable topical emulgel formulation. Moreover, its in-vitro cytotoxicity was also evaluated using normal Human keratinocytes (HaCat cells) to rule out any cellular incompatibility. The results revealed that MP seed extract, constituted with a number of polyphenolic compounds, has very good antioxidant and anti-tyrosinase potential. There were significant positive effects (p ≤ 0.05) invoked by its topical emulgel formulation on various dermatoheliotic associated skin parameters like erythema, melanin, elasticity, hydration, and sebum as compared to placebo. In the meantime, it was also found to be biocompatible and did not show any effect on HaCat cell viability and structure. In conclusion, the topical emulgel preparation loaded with MP seed extract could be a great strategy to deal with dematoheliosis.

Comparison of indirubin concentrations in indigo naturalis ointment for psoriasis treatment: a randomized, double-blind, dosage-controlled trial.

BACKGROUND: Indigo naturalis and its refined formulation, Lindioil, are effective in treating psoriatic symptoms topically. Indirubin is the active ingredient in indigo naturalis. OBJECTIVES: To determine the efficacy and safety of different concentrations of indirubin in Lindioil ointment for treating psoriasis. METHODS: In this randomized, double-blind trial, adult patients presenting with chronic plaque psoriasis for > 1 year and with < 20% of the body surface area (BSA) affected were randomized to apply Lindioil ointment containing 200, 100, 50 or 10 µg g-1 of indirubin twice daily for 8 weeks followed by an additional 12-week safety/extension period. The primary end point was the mean percentage change in Psoriasis Area and Severity Index (PASI) score along with the proportion of participants achieving 75% and 90% reductions in PASI scores (PASI 75 and PASI 90, respectively) from baseline to week 8. RESULTS: The results from week 8 revealed that the 200 µg g-1 group had the greatest reduction in PASI score [69·2%, 95% confidence interval (CI) 55·5-82·8], followed by the 100 µg g-1 group (63·1%, 95% CI 52·8-73·5), the 10 µg g-1 group (53·4%, 95% CI 42·8-64·0) and the 50 µg g-1 group (50·3%, 95% CI 37·4-63·2), with a between-group comparison of P = 0·0445. The group with the highest proportion of the patients achieving PASI 75 (57%, P = 0·0474) and PASI 90 (30%, P = 0·0098) was the 200 µg g-1 group. No severe treatment-related adverse events were reported during the 20-week evaluation. CONCLUSIONS: An amount of 200 µg g-1 of indirubin in Lindioil ointment is the most effective concentration studied so far for treating psoriasis topically, and is safe.

Comparison of efficacy of products containing azelaic acid in melasma treatment.

BACKGROUND: Melasma is one of the most frequently diagnosed hyperpigmentation changes on the skin of women's faces. Nearly 30% of women using oral estrogen therapy struggle with this problem. A common way of reducing melasma is the application of azelaic acid products. AIM: Comparison of efficacy of three dermocosmetic products, containing azelaic acid, in the reduction in melasma for women aged 35-55. MATERIAL AND METHODS: A group of 60 women diagnosed with melasma were divided into three even, twenty-person subgroups. Each subgroup was assigned one dermocosmetic product containing azelaic acid. For 24 weeks, the patients applied the assigned product twice a day. The level of the colorant within the hyperpigmentation was marked before the treatment, after 1 month, after 3 months, and after 6 months of therapy. The pigmentation was measured using Mexameter(®) (Courage + Khazaka electronic, Germany). In addition, during each inspection, the patients' level of hydration, elasticity, and intensity of erythema was checked using Corneometer(®) , Reviscometer(®) . RESULTS: All dermocosmetics containing azelaic acid that were applied significantly contributed to the reduction in pigment in the pigmentary lesion. The largest decrease in the amount of pigment was observed in the first 3 months of use of the products. A combination containing 20% azelaic acid and mandelic acid, phytic acid, 4N-butyl resorcinol, and ferulic acid proved to be the most effective dermocosmetic III (Sesderma, Valencia, Spain). CONCLUSIONS: Dermocosmetics containing azelaic acid significantly contribute to the clearing of melasma. The effect depends on the treatment time, the acid concentration, and addition of other components.

Formulation considerations in the design of topical, polymeric film-forming systems for sustained drug delivery to the skin.

Polymeric film-forming systems (FFSs) are potential drug delivery systems for topical application to the skin. The FFSs form thin and transparent polymeric films in situ upon solvent evaporation. Their application convenience and cosmetic attributes, superior to conventional semi-solids, may offer improved patient compliance. This study represents the first phase of an investigation into the use of FFSs for prolonged dermal drug delivery. FFS formulations were distinguished based on their ability to sustain the release of betamethasone 17-valerate (BMV) in vitro over 72 h. The effect of film-forming polymer (hydrophilic: hydroxypropyl cellulose (Klucel™ LF); hydrophobic: polymethacrylate copolymers (Eudragit® NE and Eudragit® RS), and polyacrylate copolymer (Dermacryl® 79) was first determined, and then the impact of incorporation of plasticisers (triethyl citrate, tributyl citrate, and dibutyl sebacate) was examined. The Klucel film released a significantly higher amount of BMV than the hydrophobic FFS, 42 versus 4 µg/cm(2), respectively. The release was increased when a plasticiser was incorporated, and with higher enhancement ratios achieved with the more lipophilic plasticisers. In conclusion, the results show that FFSs can sustain drug release (hence representing useful systems for prolonged dermal therapy) and emphasise the importance of the formulation on drug delivery, with the type of polymer being of greatest significance.

DEVELOPMENT AND VALIDATION OF THE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE QUANTITATIVE DETERMINATION OF ERYTHROMYCIN IN DERMO-PREPARATIONS.

PURPOSE: For the analysis of erythromycin (ER) in topical pharmaceutical forms we developed a high performance liquid chromatography dosage method (HPLC) greatly improved over the method formalized by the European Pharmacopoeia 5th ed. MATERIALS AND METHODS: The work conditions for using a chromatography column with C18 stationary phase were established as follows: optimal column temperature, 45 degrees C; the mobile phase consisted of a mixture of 0.001 M disodium phosphate solution : acetonitrile in the ratio 20:80, volume of sample injected 20 µL, injection rate 1 mL/min and spectrum was recorded at λ = 200 nm. The method was validated by determining the following parameters: system precision, linearity, limit of detection (LOD), limit of quantification (LOQ) and accuracy. RESULTS: We determined linearity in the concentration range studied, 0.025 to 1.5 mg/mL. The equation of the regression line is: A = 53.7430 x C + 0.00203 with a regression coefficient of 0.9997. The method has LOQ = 0.0196 mg/mL and LOD = 0.005969. The accuracy of the method is appropriate, the ER recovery degree is in the range of 101.27 - 104.99%. CONCLUSIONS: The HPLC method with UV detection for the quantitative determination of ER is manageable, responsive, linear, precise and accurate and it can be used for the quantitative analysis of ER in topical pharmaceutical formulations. UV DETECTION.

HPLC method for identification and quantification of three active substances in a dermatological preparation--Viosept ointment.

The study was aimed at developing a HPLC method to identify and quantify domiphen bromide, tripelennamine hydrochloride and clioquinol in Viosept ointment. The tested substances were successfully separated using Inertsil ODS-3 (250 x 4.6 mm, 5 µm) as a stationary phase and a gradient elution. Detection at 310 nm wavelength was applied for tripelennamine hydrochloride and clioquinol, and at 215 nm wavelength for domiphen bromide. Methods of extraction of the tested substances were developed: domiphen bromide and clioquinol were extracted with acetone from heated solutions, and tripelennamine hydrochloride was extracted in a hexane-water system. Validation procedure confirmed the method to be sufficiently selective, precise and accurate. Correlation coefficients of calibration curves pointed out that they were linear within the examined concentration range.

Influence of the composition of monoacyl phosphatidylcholine based microemulsions on the dermal delivery of flufenamic acid.

Although microemulsions are one of the most promising dermal carrier systems, their clinical use is limited due to their skin irritation potential. Therefore, microemulsions based on naturally derived monoacyl phosphatidylcholine (MAPL) were developed. The influence of the water, oil and surfactant content on dermal delivery of flufenamic acid was systematically investigated for the first time. A water-rich microemulsion led to significantly higher in vitro skin penetration of flufenamic acid compared to other microemulsions. The superiority of the water-rich microemulsion over a marketed flufenamic acid containing formulation was additionally confirmed. Differences in drug delivery could be explained by alterations of the microemulsions after application. Evaporation of isopropanol led to crystal-like structures of MAPL on the skin surface from the surfactant- or oleic acid-rich microemulsions. In contrast, the formation of this additional barrier was hindered in case of the water-rich microemulsion. The skin penetration of MAPL was additionally analyzed by combined ATR-FTIR and tape stripping experiments, where MAPL itself penetrated only into the initial layers of the stratum corneum, independent of the microemulsion composition. Since a surfactant must penetrate the skin to cause irritation, MAPL can be presumed as a skin-friendly emulsifier with the ability to stabilize pharmaceutically acceptable microemulsions.

Inhibitory effect of a novel combination of Salvia hispanica (chia) seed and Punica granatum (pomegranate) fruit extracts on melanin production.

In recent years, dietary fatty acids have been extensively evaluated for nutritional as well as cosmetic benefits. Among the dietary fats, the omega-3 (ω3) and omega-6 (ω6) forms of polyunsaturated fatty acids (PUFAs) have been found to exhibit many biological functions in the skin such as prevention of transepidermal water loss, maintenance of the stratum corneum epidermal barrier, and disruption of melanogenesis in epidermal melanocytes. In this study, we examined the effect of chia seed extract, high in ω3 (linolenic acid) and ω6 (linoleic acid) PUFAs, for its capacity to affect melanogenesis. Chia seed extract was shown to inhibit melanin biosynthesis in Melan-a cells; however, linoleic and α-linolenic acids alone did not effectively reduce melanin content. Further investigation demonstrated that chia seed extract in combination with pomegranate fruit extract had a synergistic effect on the inhibition of melanin biosynthesis with no corresponding effect on tyrosinase activity. Investigation of the possible mechanism of action revealed that chia seed extract downregulated expression of melanogenesis-related genes (Tyr, Tyrp1, and Mc1r), alone and in combination with pomegranate fruit extract, suggesting that the inhibition of melanin biosynthesis by a novel combination of chia seed and pomegranate fruit extracts is possibly due to the downregulation of gene expression of key melanogenic enzymes.

Characterization of suspected illegal skin whitening cosmetics.

An important group of suspected illegal cosmetics consists of skin bleaching products, which are usually applied to the skin of the face, hands and décolleté for local depigmentation of hyper pigmented regions or more importantly, for a generalized reduction of the skin tone. These cosmetic products are suspected to contain illegal active substances that may provoke as well local as systemic toxic effects, being the reason for their banning from the EU market. In that respect, illegal and restricted substances in cosmetics, known to have bleaching properties, are in particular hydroquinone, tretinoin and corticosteroids. From a legislative point of view, all cosmetic products containing a prohibited whitening agent are illegal and must be taken off the EU market. A newly developed screening method using ultra high performance liquid chromatography-time off flight-mass spectrometry allows routine analysis of suspected products. 163 suspected skin whitening cosmetics, collected by Belgian inspectors at high risk sites such as airports and so-called ethnic cosmetic shops, were analyzed and 59% were classified as illegal. The whitening agents mostly detected were clobetasol propionate and hydroquinone, which represent a serious health risk when repeatedly and abundantly applied to the skin.

Application of HPLC with ELSD detection for the assessment of azelaic acid impurities in liposomal formulation.

In the course of research and development of a new pharmaceutical formulation of azelaic acid in the liposomal form, we developed a rapid and accurate method for the detection of impurities using high-performance liquid chromatography. A chromatographic column from Merck (Purospher Star RP C18, 250-4 mm (5 µm) was used in the assay, and the mobile phase gradient consisted of three phases: A--methanol : water (5 : 95) + 1.5% (v/v) acetic acid; B--water : methanol (5 : 95) + 1.5% (v/v) acetic acid; and C--chloroform. Detection of the impurities and the active substance was performed by an evaporative light-scattering detector. The method was validated for selectivity, system precision, method precision, limit of detection, and response rates. The proposed method can be used to detect impurities in the liposomal formulation of azelaic acid. The method enables separation of azelaic acid from the identified and unidentified impurities and from the excipients used in the drug form.

Suppressive effect of mangosteen rind extract on the spontaneous development of atopic dermatitis in NC/Tnd mice.

Atopic dermatitis (AD) is a chronic and relapsing skin disorder characterized by pruritic and dry skin lesions with allergic inflammation. Recent studies have revealed anti-inflammatory and anti-allergic effects of xanthones in mangosteen through regulation of the nuclear factor (NF)-κB signaling pathway. Activation of NF-κB signals is responsible for allergic inflammation in AD. To develop a new preventive therapy for AD, we examined the effects of the natural medicine, mangosteen rind extract (ME), on AD in a murine model. ME (250 mg/kg per day) was administrated to NC/Tnd mice, a model for human AD, for 6 weeks to evaluate its preventive effects on AD. We also confirmed the effects of ME on various immune cell functions. Oral administration of ME prevented the increase of clinical skin severity scores, plasma total immunoglobulin E levels, scratching behavior, transepidermal water loss and epidermal hyperplasia in NC/Tnd mice; moreover, no adverse effects were noted. We demonstrated that ME suppressed thymic stromal lymphopoietin and interferon-γ mRNA expression both in vitro and in vivo. Not only immunoglobulin E production from splenic B cells but also immunoglobulin E-mediated degranulation of bone marrow-derived cultured mast cells was significantly reduced by the addition of ME to the culture. In addition, mRNA expression levels of nerve growth factor were decreased in ME-administrated NC/Tnd mice compared with those of controls. Keratinocyte proliferation was well-controlled by ME application. Oral administration of ME exhibited its suppressive potential on the early development of AD by controlling inflammation, itch and epidermal barrier function.