Pouteria macrophylla Fruit Extract Microemulsion for Cutaneous Depigmentation: Evaluation Using a 3D Pigmented Skin Model.

Here, we verify the depigmenting action of Pouteria macrophylla fruit extract (EXT), incorporate it into a safe topical microemulsion and assess its effectiveness in a 3D pigmented skin model. Melanocytes-B16F10- were used to assess the EXT effects on cell viability, melanin synthesis, and melanin synthesis-related gene transcription factor expression, which demonstrated a 32% and 50% reduction of intra and extracellular melanin content, respectively. The developed microemulsion was composed of Cremophor EL®/Span 80 4:1 (w/w), ethyl oleate, and pH 4.5 HEPES buffer and had an average droplet size of 40 nm (PdI 0.40 ± 0.07). Skin irritation test with reconstituted epidermis (Skin Ethic RHETM) showed that the formulation is non-irritating. Tyrosinase inhibition was maintained after skin permeation in vitro, in which microemulsion showed twice the inhibition of the conventional emulsion (20.7 ± 2.2% and 10.7 ± 2.4%, respectively). The depigmenting effect of the microemulsion was finally confirmed in a 3D culture model of pigmented skin, in which histological analysis showed a more pronounced effect than a commercial depigmenting formulation. In conclusion, the developed microemulsion is a promising safe formulation for the administration of cutite fruit extract, which showed remarkable depigmenting potential compared to a commercial formulation.

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.

Versatile chromatographic method for catechin determination in development of topical formulations containing natural extracts.

Catechin is found in several natural sources, as Eugenia dysenterica and Syzygium cumini extracts. Its antioxidant and UV-protective properties suggest a potential use in cosmetic and dermatological formulations. A simple analytical method capable of giving support to experiments performed along the development of topical formulations containing this natural substance (i.e. drug assay, skin permeation and stability studies), however, is still needed. Thus, this work aimed to develop and validate a selective HPLC method for catechin determination during the development of topical formulations. Separation was achieved using an RP-C18 column (300 × 3.9 mm; 10 µm), with a mobile phase of methanol-phosphoric acid 0.01 m (15: 85, v/v), a flow rate of 0.8 mL/min, temperature set at 40°C and UV detection at 230 nm. The method was linear in a range from 0.5 to 10.0 µg/mL (r = 0.9998), precise with an overall variation coefficient of 5.5% and accurate with catechin recovery from the skin layers >85%. Additionally, the method was sensitive (limit of detection, 0.109 µg/mL; limit of quantification, 0.342 µg/mL) and selective against plant extracts, skin matrices and formulation interferents, as well as catechin degradation products. It was also robust regarding both methodology parameters and analytical stability.

Validation of a simple chromatographic method for naringenin quantification in skin permeation experiments.

Naringenin is a flavonoid that can be found in citrus fruits (e.g., Citrus sp). This natural compound is known for its antioxidant activity, antitumor, and neuroprotective potential, also acting directly in controlling the inflammatory response. Topical and transdermal routes are attractive alternatives that could circumvent the low oral bioavailability. However, a simple analytical method capable of determining naringenin in skin layers is still demanded. Thus, this work aimed to validate a selective and straightforward chromatographic method for naringenin determination in skin permeation studies. The developed method uses a reversed-phase C18 column as stationary phase and a mobile phase composed of methanol/phosphoric acid 0.01 M (65:35, v/v), eluted at a flow rate of 0.6 mL/min with detection at 290 nm. The method was linear (r2 > 0.99) in a broad concentration range of 0.5-10.0 µg/mL, precise with an overall variation coefficient lower than 2%, and accurate with naringenin recovery from the skin layers higher than 85%. Additionally, the method was sensitive (LD = 0.10 µg/mL, LQ = 0.20 µg/mL), selective against skin matrices as well as naringenin degradation products, and robust regarding methodology parameters. Therefore, the method was suitable to be used in skin permeation studies employing naringenin.

Follicular-targeted delivery of spironolactone provided by polymeric nanoparticles.

This study proposes developing a topical formulation based on poly-ε-caprolactone (PCL) or methacrylic acid/methyl methacrylate copolymer (EL100) nanoparticles to enable a safer and more effective therapy of alopecia and acne with spironolactone. The effect of the size of the nanoparticle on follicular-targeted drug delivery is also verified. Compatibility studies based on thermal analyses and complementary techniques showed a small interaction of the drug with excipients, which may not compromise the drug stability. PCL nanoparticles of 180.0 ±â€¯1.6 and 126.8 ±â€¯1.0 nm, and EL100 nanoparticles of 102.7 ±â€¯7.1 nm were then prepared. All nanoparticles entrapped more than 75 % of spironolactone, were physically stable, and stabilized the drug for at least 90 days. They were also non-irritant according to HET-CAM tests. Drug release from the nanoparticles was reduced in aqueous buffer media but fast when in contact with oil. Finally, in vitro skin penetration experiments revealed the largest nanoparticles (of 180 nm) targeted drug delivery to the hair follicles 5-fold (p < 0.05) more than the control solution, 2.1-fold (p < 0.05) more than nanoparticles produced with the same polymer (PCL) but with smaller size (123 nm), and 4.9-fold (p < 0.05) more than the 102-nm E100 nanoparticles. In conclusion, follicular targeting can be adjusted according to nanoparticle size, and this work succeeded in obtaining polymeric nanoparticles adequate to enable topical treatment of acne and alopecia with spironolactone.

Phonophoretic application of a glucosamine and chondroitin nanoemulsion for treatment of knee chondropathies.

Aim: This study was performed to assess the effect of the phonophoretic application of a nanoemulsion incorporating glucosamine and chondroitin sulfate (NANO-CG) associated with kinesiotherapy on the reduction of pain and stiffness in knee chondropathy. Materials & methods: NANO-CG was tested in vitro and in vivo prior to being applied in a randomized and controlled clinical trial. Results: Cell viability and hen's egg test-chorionallantonic membrane tests indicated the NANO-CG is safe for topical application. Permeation tests showed NANO-CG enhances drug permeation through the skin. There was no statistical significance between treated groups in this preliminary study, however, pain reduction and complete recovery of articular cartilage were observed in some patients treated with NANO-CG. Conclusion: We demonstrate that NANO-CG may be a promising candidate for the therapy of knee chondropathy.

Besifloxacin liposomes with positively charged additives for an improved topical ocular delivery.

Topical ophthalmic antibiotics show low efficacy due to the well-known physiological defense mechanisms of the eye, which prevents the penetration of exogenous substances. Here, we aimed to incorporate besifloxacin into liposomes containing amines as positively charged additives and to evaluate the influence of this charge on drug delivery in two situations: (i) iontophoretic and (ii) passive treatments. Hypothesis are (i) charge might enhance the electromigration component upon current application improving penetration efficiency for a burst drug delivery, and (ii) positive charge might prolong formulation residence time, hence drug penetration. Liposomes elaborated with phosphatidylcholine (LP PC) or phosphatidylcholine and spermine (LP PC: SPM) were stable under storage at 6 ºC for 30 days, showed mucoadhesive characteristics, and were non-irritant, according to HET-CAM tests. Electron paramagnetic resonance spectroscopy measurements showed that neither the drug nor spermine incorporations produced evident alterations in the fluidity of the liposome's membranes, which retained their structural stability even under iontophoretic conditions. Mean diameter and zeta potential were 177.2 ± 2.7 nm and - 5.7 ± 0.3 mV, respectively, for LP PC; and 175.4 ± 1.9 nm and + 19.5 ± 1.0 mV, respectively, for LP PC:SPM. The minimal inhibitory concentration (MIC) and the minimal bactericide concentration (MBC) of the liposomes for P. aeruginosa showed values lower than the commercial formulation (Besivance). Nevertheless, both formulations presented a similar increase in permeability upon the electric current application. Hence, liposome charge incorporation did not prove to be additionally advantageous for iontophoretic therapy. Passive drug penetration was evaluated through a novel in vitro ocular model that simulates the lacrimal flow and challenges the formulation resistance in the passive delivery situation. As expected, LP PC: SPM showed higher permeation than the control (Besivance). In conclusion, besifloxacin incorporation into positively charged liposomes improved passive topical delivery and can be a good strategy to improve topical ophthalmic treatments.

Iontophoresis enhances voriconazole antifungal potency and corneal penetration.

Strategies to enhance corneal penetration of voriconazole (VOR) could improve the treatment of fungal keratitis. Here, we evaluated the use of iontophoresis for ocular VOR delivery from either: (i) a cyclodextrin inclusion complex (CD VOR), (ii) a liposome (LP VOR), and (iii) a chitosan-coated liposome (LP VOR CS). LP VOR CS presented mean diameter of 139.2 ±â€¯1.3 nm and zeta potential equal to + 3.3 ±â€¯1.5 mV compared to 134.6 ±â€¯1.7 and -8.2 ±â€¯3.0 mV of LP VOR, which, together with mucin mucoadhesion study, confirmed chitosan-coating. Both drug and liposomal formulations were stable under the influence of an applied electric current. Interestingly, in vitro studies in Candida glabrata culture indicated a decrease in VOR MIC values following iontophoresis (from 0.28 to 0.14 µg/mL). Iontophoresis enhanced drug penetration into the cornea. After 10 min of a 2 mA/cm2 applied current, corneal retained amounts were 45.4 ±â€¯11.2, 30.4 ±â€¯2.1 and 30.6 ±â€¯2.9 µg/cm2 for, respectively, CD VOR, LP VOR, and LP VOR CS. In conclusion, iontophoresis increases drug potency and enhances drug penetration into the cornea, showing potential to be used as "an emergency burst delivery approach".

Mixture design applied in compatibility studies of catechin and lipid compounds.

The aim of this work was to evaluate the compatibility of (+)-catechin (CA) and excipients commonly used to prepare micro and nanoemulsions using thermal analysis along with complementary assays. Lipid compounds labrasol, plurol and ethyl oleate were combined with CA according to a simplex centroid mixture design and possible interactions between them were determined. Differential scanning calorimetry and thermogravimetric analyses were carried out together with Fourier transform infrared spectroscopy (FTIR) and morphologic characterization of the samples. A quantitative evaluation of thermal events involved in CA melting peak and initial sample decomposition temperature were performed. FTIR evaluation suggested an initial decomposition of CA mixtures exposed to a thermal aging depending on their composition corroborated by the darkening of these samples. The multiple regression analysis considering the thermal data revealed a thermal interaction compromising CA stability in multicomponent samples. Mixtures containing ethyl oleate exhibited a negative synergic action of this fatty acid with the others two lipid compounds (negative coefficients for two-factor and three-factor interaction terms). Indeed, samples decomposition was anticipated by at least 10°C in the case of ternary and quaternary mixtures containing ethyl oleate. In conclusion, CA formulations produced with lipid components must have their stability closely monitored and production process involving heating should be avoided, especially in formulations containing ethyl oleate.

Liquid chromatography-mass spectrometry for simultaneous determination of spironolactone and canrenone in plasma samples

Abstract n our study, we aimed to validate a method based on liquid chromatography-mass spectrometry (LC-MS) to quantify spironolactone (SPI) and its active metabolite canrenone (CAN) simultaneously in plasma samples to support in vivo experiments. Compounds were separated by using a C18 column with the isocratic elution of a mobile phase composed of 0.1% (v/v) formic acid in methanol-water (60:40 v/v) at a flow rate of 0.4 mL min−1. SPI and CAN were detected in na electrospray interface operating in a positive ionization mode and quantified using the selective ion mode monitoring of mass-charge ratios (m/z) of 439.0 for SPI and 363.1 for CAN. After calculating the matrix effect using theoretical equations, we observed the strong interference of plasma in the equipment-generated signal, which required creating analytical curves using the matrix as a solvent. The method was nevertheless linear (r 2 > 0.999) in a concentration range of 0.4-5.0 µg mL−1, as well as precise, with a coefficient of variation less than 5%. SPI's and CAN's recovery rates from the plasma ranged from 87.4% to 112.1%, while their limits of detection (i.e., 0.07 µg mL−1 and 0.03 µg mL−1, respectively) and quantification (i.e., 0.20 µg mL−1 and 0.08 µg mL−1, respectively) in the presence of plasma contaminants were low. Therefore, the bioanalytical method seems to be feasible for quantifying SPI and CAN in plasma