Microdialysis on Ex Vivo Porcine Ear Skin Can Validly Study Dermal Penetration including the Fraction of Transfollicular Penetration-Demonstrated on Caffeine Nanocrystals.

Common ex vivo methods for penetration investigations often fail to monitor transfollicular penetration appropriately. In the present investigation, the validity of dermal microdialysis on the ex vivo porcine ear skin to investigate penetration kinetics, including transfollicular penetration, was studied. In setup A, a caffeine nanocrystal formulation was compared to a non-particular caffeine gel formulation. In setup B, two caffeine nanocrystal formulations of different sizes (200 nm, 700 nm) were compared to each other. Microdialysis samples were collected for 46 h. After sampling, the skin layers were separated, homogenized, and caffeine was quantified in all samples. In setup A the area under the curve (AUC) after crystal gel formulation application was 12 times higher than after non-particular formulation application. Setup B showed an increased AUC of 42% in the microdialysis data when the 700 nm caffeine crystals were applied compared to the 200 nm crystals. The microdialysis data was supported by the separation, homogenization and extraction data. Microdialysis performed on ex vivo porcine ear skin is a novel experimental setup. It is of high interest for further investigations since it is able to also capture the impact of follicular and transfollicular penetration kinetics as no other ex vivo setup can.

Dermal smartPearls - Optimized silica particles for commercial products & mechanistic considerations.

The amorphous state of actives can be long-term stabilized by incorporation into mesoporous particles, thus the increase in the saturation solubility by amorphicity can be exploited to improve the bioavailability. In this study 5 different silica particles were investigated regarding loading capacity and long-term stability of the amorphous form. Five different silica were used ranging in pore mean size from 3 to 25 nm, pore volume 0.4 to 1.8 mL/g, and BET surface from 740 to 320 m2/g. As model active avobenzone was used, because it is a challenging molecule by its high crystallisation tendency. To be industrially feasible, a loading capacity of about 50% pore volume was investigated. The particles were loaded by an immersion evaporation method, being able to be used in industrial production. A theory of the active precipitation in the pores was developed based on the Ostwald-Miers range. The 25 nm pore-sized particles showed a crystalline fraction directly after loading, the 3 nm and 17 nm pore-sized particles after 1 month of storage. Long-term stability of 1 year had the silica with 6 nm and 10 nm pore size, thus being ideal for products. By nitrogen sorption studies, primarily filling of the pores from bottom to top was identified as loading mechanism. HPLC analysis showed some active remaining in the pores due to strong interaction with the pore surface, which needs to be considered when developing dermal products. Interestingly, the increase in saturation solubility Cs - determined in carrageenan gels - remained also for silica particles showing a minor partial crystalline avobenzone fraction. Thus, limited crystallinity does not impair the shelf-life and performance of dermal formulations.

BergaCare SmartLipids: commercial lipophilic active concentrates for improved performance of dermal products.

SmartLipids are the latest generation of dermal lipid nanoparticles with solid particle matrix. Their characteristic properties resulting from the "chaotic" and disordered particle matrix structure are reviewed. These properties are high loading and firm inclusion of active agents, physical stability of the particle matrix lipid modification (primarily α, ß'), and related to these three properties the improved chemical stabilization of labile active agents. Exemplarily data for these effects are shown and underlying mechanisms are discussed. Further, general properties of lipid nanoparticles, which are also exhibited by the SmartLipids, are reviewed. These include the restauration of the protective lipid skin barrier (anti-pollution effect), penetration enhancement by occlusion (invisible patch effect) and the option to control the release of active agents for optimized biological effect and reduction of side effects (e.g., skin irritation through sensitizing active agents), which improves the skin tolerability. Regulatory aspects, such as submicron particle status, excipients, and certifications, are also discussed.

Phenylethyl resorcinol smartLipids for skin brightening - Increased loading & chemical stability.

Phenylethyl resorcinol (PER, 4-(1-phenylethyl)1,3-benzenediol) is a very potent tyrosinase inhibitor with clinically proven effectiveness at already 0.5%. A major challenge of incorporating PER into dermal products is its high sensitivity against light. Previously, by incorporating PER in nanostructured lipid carriers (NLC), chemical stability and tyrosinase activity could be increased by 29% and 67%, respectively. Despite this, degradation still occurred accompanied with reddish discoloration of the formulation - not acceptable for market products. In this study PER was incorporated into smartLipids, the 3rd generation of lipid carriers. Compared to NLC, the smartLipids formulation had a higher PER loading, was PEG-free and used ECOCERT-certified Lanette N. For PER stabilization, 14 additives from three groups were investigated: UV blockers, antioxidants and chelating agents. The UV blockers Tinosorb S and Oxynex ST liquid, as well as the chelating agents ethylenediaminetetraacetic acid and phytic acid completely prevented reddish discoloration under test conditions (3 months in the dark and 7 days at light exposure). Investigating the stabilizing mechanisms, UV absorbers with high absorption in UV-A range were most effective (Tinosorb S, Oxynex liquid). They showed good stabilization in dark and at light exposure. Chelating agents had mainly an effect via pH shift to pH 2, thus are not suitable for dermal products requiring pH around 5. Antioxidants were less effective. The antioxidants propyl gallate and BHT showed best stabilization at storage in dark and slightly less at light exposure, not impairing the physical stability. Some antioxidants even accelerated discoloration (e.g. Tinogard TT). In general, low standard electrode potential (<0.4 mV) seems to be favorable for PER stabilization. In conclusion, Tinosorb S proved to be the best stabilizer; combination with an antioxidant is optional. By combination of smartPearls technology and the Tinosorb S stabilization, PER market products without discoloration are feasible.

Skin Metabolism: Relevance of Skin Enzymes for Rational Drug Design.

Transdermal therapeutic systems (TTS) have numerous pharmacological benefits. Drug release, for example, is independent of whether a patient is in a fed or a fasted state, and lower doses can be given as gastrointestinal and hepatic first-pass metabolism is avoided. In addition, inter- and intrapatient variability is minimized as the release of the drug is mainly controlled by the system. This makes TTS interesting as alternative systems to the most common dosage form of oral tablets. The difficulty with the dermal administration route is transporting the drug through the skin, since the skin is an efficient barrier against foreign bodies. Various strategies have been reported in the literature of how drug penetration can be improved. Most of them, however, focus on overcoming the stratum corneum as the first (mechanical) skin barrier. However, penetration is much more complex, and the skin's barrier function does not only depend on the stratum corneum; what has been underestimated is the second (biological) skin barrier formed of enzymes. Compared to the stratum corneum, very little is known about these enzymes, e.g., which enzymes are present in the skin and where exactly they are localized. Hence, very few strategies can be found for how to bypass or even use the skin enzyme barrier for TTS development. This review article provides an overview of the skin enzymes considered to be relevant for the biotransformation of dermally applied drugs. Also, we discuss the use of dermal prodrugs and soft drugs and give the stereoselectivity of skin metabolism careful consideration. Finally, we provide suggestions on how to make use of the current knowledge about skin enzymes for rational TTS design.

Azithromycin nanocrystals for dermal prevention of tick bite infections.

Azithromycin was optimized as nanocrystals with a drug content of 10.0 % (w/w) and a surfactant D-α -tocopheryl polyethylenglycol 1000 succinate (TPGS) content of 1.0 % (w/w) using bead milling for 10 min. The photon correlation spectroscopy (PCS) diameter of the bulk population was 189 nm, laser diffraction (LD) diameter 90 % was 370 nm. Spherical morphology of the optimal nanocrystals was observed by transmission electron microscope (TEM). They were stable over 1 year of storage at 4 °C with the particle size within the nanometer range which was confirmed by PCS, LD and light microscope. An acceptable physical stability of 2 years was also obtained when stored at 4 °C. No microbial attack to the nanocrystals was observed before 3 years storage at 4 °C. The saturation solubility of the nanocrystals was up to triple compared to the raw drug powder (RDP) in water. When incorporated into the gel base, highest penetration efficacy was achieved by the optimal nanocrystals compared to 1) the clinically effective ethanol-solution-gel, 2) the gel with propylene glycol and 3) the gel with RDP in the ex vivo porcine ear penetration study. Even though propylene glycol improved saturation solubility of nanocrystals, it could not bring benefit to nanocrystals in the penetration study. Based on these optimized azithromycin nanocrystals, topical administration for enhanced dermal bioavailability of azithromycin seems to be feasible.

Maximum Loaded Amorphous Azithromycin Produced Using the Wetness Impregnation Method with Fractional Steps for Dermal Prophylaxis Against Lyme Disease.

Azithromycin was loaded onto the µm-sized mesoporous silica Davisil® SP53D-11920 using the wetness impregnation method with fractional steps (WIFS) and further incorporated into a 5 % hydroxypropyl cellulose gel to prevent Lyme disease. Maximum loadings (32.0 % w/w and 33.2 % w/w) were produced by different concentrated loading solutions and determined by X-ray diffraction (XRD). A total of 24 months stability of the amorphous azithromycin state in the silica (33.2 % loading) and 18 months stability in the gel (33.2 % loading) at 4 °C were also confirmed by XRD. The higher kinetic solubility at 40 min (1,300 µg/mL versus 93 µg/mL) and higher porcine ear skin penetration compared to the raw drug powder indicated higher dermal bioavailability of the azithromycin-loaded silica (32.0 % loading), even when compared to the "gold standard" nanocrystals and another clinical effective azithromycin formulation with ethanol. In summary, maximum loaded silicas with azithromycin by WIFS is a promising dermal formulation for prophylaxis against Lyme disease.

Glabridin smartPearls - Silica selection, production, amorphous stability and enhanced solubility.

Glabridin, a compound in the root extract of Glycyrrhiza glabra, has been identified as an effective tyrosinase inhibitor. Applied on skin, melanin synthesis is inhibited, making glabridin an interesting candidate for skin whitening or for the treatment of age spots. However, main obstaclefor its practical use is its low dermal bioavailability, caused by its poor water solubility. In this work smartPearls technology was used to increase the glabridins water solubility. smartPearls consist of silica particles with mesopores in which actives can be loaded. By this, actives are stabilized in amorphous state and simultaneously finely distributed in nm-range. Both amorphization and nanoization are well known approaches to increase saturation solubilities. In smartPearls these approaches are combined. In the first step, glabridin smartPearls formulation was developed, screening systematically the suitability of 4 different silicas varying in their pore sizes (3, 6, 10, 17 nm). Also, most suited filling level of glabridin was determined (25, 50, 80% referred to total pore volume of respective silica). Silica loading was performed by the immersion-evaporation method, resulting in pores filled with glabridin from bottom to top. By light microscopy, dynamic scanning calorimetry and X-ray diffraction the sample with 6 nm pore size and filling levels of 25% and 50% have been verified to be completely amorphous. Highest physical storage stability of 7 months up to now was obtained for the 25% filled sample. In the next step, concept of increased saturation solubility for smartPearls was proven. Dissolution profiles were recorded in situ for glabridin smartPearls and compared to glabridin raw drug powder. Both saturation solubility and dissolution velocity were remarkably improved. The water solubility for example increased by a factor of more than 4. This makes glabridin smartPearls promising for creating skin products with improved dermal bioavailability.

smartPearls® for dermal bioavailability enhancement - Long-term stabilization of suspensions by viscoelasticity.

smartPearls® are a novel dermal delivery system based on mesoporous (pores 2-50 nm) particles, developed in 2014. Their pores can be loaded with active which is long-term stabilized in its amorphous state. The increased saturation solubility by the amorphous state leads to an increased dermal bioavailability of poorly soluble actives. To avoid sedimentation of the porous particles (3-50 µm) in dermal formulations, viscoelastic gels were developed using ι-carrageenan, polyacrylate and the viscoelastic Kühne salad dressing as a reference from food industry. Silica particles (company Grace/US, 50 and 150 µm) were loaded into the gels and long-term stability was assessed by a VIS sedimentation test. Furthermore, the gels were characterized by analytical centrifugation (LUMiSizer®) to assess the critical rpm/g values, allowing to order them after their absolute viscoelastic stabilizing ability. Characterization was complemented by rotation rheology, amplitude sweep and a frequency sweep analysis for the determination of elastic and viscous moduli G' and G'' at varying conditions. Based on the throughout characterization, polymers can be selected to sufficiently stabilize dermal formulations even with large sized smartPearls® - the prerequisite for using this delivery system in dermal products.

smartPearls - Novel physically stable amorphous delivery system for poorly soluble dermal actives.

Dermally applied poorly soluble actives whether in cosmetics or pharmaceuticals show insufficient skin penetration. Especially actives being insoluble in both phases of dermal vehicles, i.e. water and oil have no or less real effect. An approach to overcome this obstacle is the use of amorphous actives instead of the crystalline ones. The higher saturation solubility creates an increased concentration gradient between the formulation and skin. Thus, the diffusive flux into the skin is improved. However, the amorphous state of actives is highly labile, and the durability of such formulations would be too short for a marketable product. smartPearls is a novel technology efficiently long-term stabilize the amorphous state. They consist of µm sized particles with mesopores (e.g. silica: SYLOID®, AEROPERL®, Neusilin®), in which the active can be loaded and preserved in amorphous state. Due to the tightness of the pores, not enough space is given for re-crystallization. In this work, the skin penetration of poorly soluble actives loaded in smartPearls is compared to the present "gold standards" in dermal delivery, e.g. amorphous microparticles, amorphous nanoparticles and nanocrystals. The performance was at least similar or even better than the gold standards, explainable by the increased saturation solubility of active due to a) amorphous state and b) nanostructure inside the µm-sized particles. Sedimentation investigations showed, that the physical stabilization of very dense smartPearls in semi-solid vehicles is possible by viscoelastic repulsion. Also, the technical, regulatory and marketing aspects for the use of smartPearls technology in products are discussed, e.g. status of excipients used, and advantages of not being a nanoparticle, but being as efficient as them. Overall, smartPearls proved to be a promising dermal delivery technology for poorly soluble actives with a high market potential.

Lipid-drug-conjugate (LDC) solid lipid nanoparticles (SLN) for the delivery of nicotine to the oral cavity - Optimization of nicotine loading efficiency.

Nicotine, obtained from tobacco leaves, has been used to promote the cessation of smoking and reduce the risk of COPD and lung cancer. Incorporating the active in lipid nanoparticles is an effective tool to minimize its irritation potential and to use the particles as intermediate to produce final products. However, as a hydrophilic active, it is a challenge to prepare nicotine loaded lipid nanoparticles with high drug loading. In this study, lipid-drug-conjugates (LDC) were formed by nicotine and different fatty acids to enable the production of sufficiently loaded nicotine lipid nanoparticles. The encapsulation efficiency of nicotine in LDC-containing SLN was about 50%, which increased at least fourfold compared to the non-LDC formulations (around 10%) due to the increased lipophilicity of nicotine by strong interactions between positively charged nicotine and negatively charged fatty acids (formation of LDCs). The z-average of all formulations (150-350 nm) proved to be in the required submicron size range with a narrow size distribution. In summary, nicotine loaded LDC lipid nanoparticles with high drug loading were successfully developed with Kolliwax® S and stearic acid as counter-ion forming the LDC and hydrogenated sunflower oil (HSO) as lipid particle matrix.

Dermal miconazole nitrate nanocrystals - formulation development, increased antifungal efficacy & skin penetration.

Miconazole nitrate nanosuspension was developed to increase its antifungal activity and dermal penetration. In addition, the nanosuspension was combined with the synergistic additive chlorhexidine digluconate. The production was performed by wet bead milling and both production and formulation parameters were optimized. A stabilizer screening revealed poloxamer 407 and Tween 80 both at 0.15% as the most effective stabilizers for miconazole nanosuspensions at 1.0%. The nanocrystals were incorporated into a hydroxypropyl cellulose gel base. Short-term stability (3months) of the nanocrystal bulk population could be shown at room temperature and fridge. Besides the stable bulk nanocrystals, some longitudinal crystal growth to needle like crystals occurred. The addition of ionic compounds as the chlorhexidine digluconate often destabilizes suspensions. Surprisingly here, the addition minimized the crystal growth. An underlying mechanism is proposed. An inhibition zone assay was performed using Candida albicans (ATCC® 10231™). When comparing the nanocrystals in suspension and in gel to µm-sized miconazole nitrate formulations and two market products, the increase in inhibition zone diameter for the nanosuspension formulations was most pronounced in the chlorhexidine digluconate free formulations. These nanocrystal formulations were closely or similarly effective as the microsuspensions and the market products containing the synergistic chlorhexidine digluconate, showing the potential of the nanosuspension formulation. Nanosuspension performance was even further increased when chlorhexidine digluconate was added. Ex-vivo skin penetration studies on porcine ears revealed distinctly less remaining miconazole nitrate on the skin surface for nanocrystals (e.g., 76-86%) compared to market products (e.g. 94%). Also, penetration was increased e.g. in skin depth of 5-10µm from <1.0/1.7% to e.g. 3.3-6.2% for nanocrystals.

Mucoadhesive tetrahydrocannabinol-loaded NLC - Formulation optimization and long-term physicochemical stability.

Tetrahydrocannabinol (THC) is used to treat pain in cancer patients. On the market there are mainly oral formulations. Especially to treat the problematic breakthrough pain in cancer, an easy applicable formulation with fast onset is desired. This formulation was developed as an aqueous nasal spray using nanostructured lipid carriers (NLC). The NLC were prepared with cetyl palmitate, having good miscibility with the oily THC and yielding particles with 1year physical long-term stability. To make the particles mucoadhesive, small particles with diameters of about 200nm were produced and additionally their surface positively charged using a cationic stabilizer. Optimal NLC suspensions contained 1% particles (lipid:THC ratio 7:1) stabilized with 0.05% cetylpyridinium chloride (CPC), and 2% particles with a mixture of 0.05% CPC, and 0.05% Tween® 80. The particle size remained unchanged during spraying using commercial spray bottle, and PARI BOY. A strong interaction with negatively charged mucin was shown by a sharp decrease of the positive NLC zeta potential and fast charge reversal in the mucin solution test. The solid matrix of the NLC had a stabilizing effect on THC. 91% THC remained after 6months storage at 4°C, and 79% under stress conditions at 40°C. By adding additional chemical stabilizers, and producing under protective conditions, a commercial formulation for patient seems feasible.

A novel concept for the treatment of couperosis based on nanocrystals in combination with solid lipid nanoparticles (SLN).

For the post laser treatment of couperosis a new dermal formulation was developed combining three actives: vitamin K1, A1 and rutin, where both vitamins were incorporated into solid lipid nanoparticles (SLN) and the poorly soluble antioxidant rutin formulated as nanocrystal. All three formulations were stable over 6 months either on their own or after their incorporation into a hydrogel. Vitamin A1 at 0.3% in emulsions shows local skin irritation due to very rapid release. By forming SLN, prolonged release with less irritation potential but deeper penetration was achieved in porcine ear skin. Due to the nanosized rutin, the new hydrogel showed clearly increased antioxidant activity, representing a stronger protection potential against reactive oxygen species (ROS), compared to marketed anti-redness products with rutin as raw drug powder or water-soluble derivative. In addition, rutin nanocrystals showed up to 5 times pronounced penetration compared to µm-sized raw drug powder. The orientating in-vivo case study revealed a three to six times faster recovery after laser treatment of couperosis by twice daily application of the new hydrogel, regarding scabbed-over areas and erythema. Continued use of the new gel also showed preventive properties against recurrences of veins for at least 8 month.