Nasal residence time and rheological properties of a new bentonite-based thixotropic gel emulsion nasal spray - AM-301.

OBJECTIVE: The present work provides characterization of rheological properties of a new bentonite-based thixotropic gel emulsion nasal spray (AM-301), its nasal residence time, distribution, safety and tolerability. SIGNIFICANCE: The nasal epithelium is a portal of entry for allergens and primary infection by airborne pathogens. Non-pharmacological interventions, which enhance physical and biological barriers, protect against allergens and pathogens without drug-related side effects. AM-301 has shown promising efficacy and safety in the nasal epithelium against viruses (in vitro) and pollen (clinical). METHODS: Technical part (i) spray characterization was performed with a validated droplet size distribution method; evaluation of the rheological properties of the formulation was performed by a validated amplitude sweep method and a validated oscillation, rotation, oscillation; Clinical part (ii) nasal and oropharyngeal endoscopy were used to provide a semi-quantitative evaluation of distribution and residence time of fluorescein-labelled AM-301 in the nose and oropharynx of healthy volunteers; (iii) tolerability and safety. RESULTS: (i) The non-Newtonian rheological properties of the formulation allow AM-301 to be sprayed and then to revert to a gel to prevent run-off from the nasal cavity; (ii) the formulation remains on the inferior turbinate, septum and oropharynx of volunteers for up to 210 min and on the middle turbinate for up to 60 min; two nasal sprays provide no substantial benefit over a single application with regards to coverage or retention; (iii) the spray is well tolerated. CONCLUSIONS: Single dose spray delivery of AM-301 provides extended coverage of the nasal mucosa up to the inferior turbinates.

Investigation of the Compatibility of the Skin PAMPA Model with Topical Formulation and Acceptor Media Additives Using Different Assay Setups.

The Skin Parallel Artificial Membrane Permeability Assay (PAMPA) is a 96-well plate-based skin model with an artificial membrane containing free fatty acid, cholesterol, and synthetic ceramide analogs to mimic the stratum corneum (SC) barrier. The current study evaluates the compatibility of lipophilic solvents/penetration enhancer, topical emulsions containing different emulsifier systems, and organic acceptor media additives with the artificial membrane of the assay. Additionally, different assay setups (standard setup: donor in bottom plate versus modified setup: donor in top plate) were compared. Methylparaben (MP), ethylparaben (EP), and propylparaben (PP) were used as model permeants and internal standards for proper assay execution. The permeation order of the parabens (MP > EP > PP) remained the same with different lipophilic solvents, and the ranking of lipophilic solvents was comparable under standard and modified conditions (isopropyl myristate, IPM > dimethyl isosorbide, DMI ≥ propylene glycol, PG > diisopropyl adipate, DIPA). Pre-incubation of the Skin PAMPA plates with IPM, DIPA, and DMI, as well as with formulations that contain non-ionic emulsifiers, and acceptor solutions containing DMSO or EtOH (≤ 50%) for 4 h did not increase the percentage of permeated parabens in the main experiment, suggesting that those compounds do not make the artificial membrane more permeable. High-resolution mass spectrometry confirmed that acceptor solutions with ≤ 50% DMSO or EtOH do not extract stearic acid, cholesterol, and certramides at standard assay conditions. Hence, if certain constraints are considered, the Skin PAMPA model can be used as a pre-screening tool for topical formulation selection.

Rational Design of Topical Semi-Solid Dosage Forms-How Far Are We?

Specific aspects of semi-solid dosage forms for topical application include the nature of the barrier to be overcome, aspects of susceptibility to physical and chemical instability, and a greater influence of sensory perception. Advances in understanding the driving forces of skin penetration as well as the design principles and inner structure of formulations, provide a good basis for the more rational design of such dosage forms, which still often follow more traditional design approaches. This review analyses the opportunities and constraints of rational formulation design approaches in the industrial development of new topical drugs. As the selection of drug candidates with favorable physicochemical properties increases the speed and probability of success, models for drug selection based on theoretical and experimental approaches are discussed. This paper reviews how progress in the scientific understanding of mechanisms and vehicle-influence of skin penetration can be used for rational formulation design. The characterization of semi-solid formulations is discussed with a special focus on modern rheological approaches and analytical methods for investigating and optimizing the chemical stability of active ingredients in consideration of applicable guidelines. In conclusion, the combination of a good understanding of scientific principles combined with early consideration of regulatory requirements for product quality are enablers for the successful development of innovative and robust semi-solid formulations for topical application.

Topical Film-Forming Solid Solutions for Enhanced Dermal Delivery of the Retinoid Tazarotene.

Topical film-forming solutions (FFSs) show considerable potential for dermal delivery of an API. Through a mechanism of in situ film formation upon solvent evaporation, they may improve skin delivery by prolonging substantivity on the skin, by establishing a transient supersaturation, and/or by enhancing solubility through the formation of a solid dispersion in the resulting film. This work aimed at developing an FFS for topical application with enhanced skin delivery. The tested FFSs were composed of the lipophilic retinoid tazarotene and the hydrophobic polyamide-3 polymers. The residual films cast from FFSs were examined by DSC and their release mechanism was investigated. Additionally, ex vivo skin penetration of tazarotene was explored. In comparison to a physical mixture, the glass transition (Tg) was significantly increased (p < 0.01) in in-situ generated polyamide-3 (11,500 Da)/tazarotene films with ratios 5:1 and 10:1, indicating a molecular distribution of tazarotene within the polymer. Stress testing at 32°C and 40°C further indicated that these films were kinetically stabilized for at least two weeks. Tazarotene release from solid solution films was notably increased as compared to the crystalline and the amorphous tazarotene. A ten-times higher skin penetration of the ratio 10:1 film (containing 0.1% tazarotene) was observed as compared to a commercial 0.1% tazarotene cream. Hence, topical solid solutions may represent an option for improved dermal API delivery.

Influence of emulsifier concentration on partition behavior and chemical stability of betamethasone dipropionate in emulsion gels.

The partitioning of active pharmaceutical ingredients (API) in emulsions is influenced by various factors, such as composition of the phases, emulsifier type and concentration, and temperature. Therefore, the chemical stability of the API can be influenced by its partitioning into the aqueous phase as degradation is typically facilitated in water. With increasing emulsifier concentration from 0.15% to 5.0%, more betamethasone dipropionate (BDP) was solubilized in polysorbate 80 micelles leading to a small, but increased fraction of BDP exposed to the aqueous phase along with preferential partitioning of BDP to the aqueous phase and thus increased degradation. Similarly, by enhancing solubility and partitioning to the aqueous phase, the addition of polyethylene glycol 400 also led to increased BDP degradation. Due to pH dependent degradation of BDP, increasing emulsifier concentrations resulted in a more pronounced degradation of BDP at pH 8, which is beyond the stability optimum, whereas at pH 5 the API was sufficiently stable and no differences in concentration were detected within 12 weeks even under accelerated conditions. No significant differences were seen with the varying emulsifier concentrations regarding ex vivo skin penetration.

Development of a design of experiments optimized method for quantification of polysorbate 80 based on oleic acid using UHPLC-MS.

The aim was to develop a straightforward UHPLC-MS quantification method for polysorbate 80 using oleic acid as surrogate marker, which was the commonest substance within the emulsifier. However, hydrolysis of polysorbate 80 and subsequent analysis of fatty acids revealed a co-elution of oleic acid and an isomer while all the other fatty acids were successfully separated by varying retention times and mass-to-charge ratios. For identification and separation of the isomer a derivatization method was evaluated. Oxidation to the corresponding dihydroxystearic acids with potassium permanganate resulted in peak separation of cis/trans and structural isomers of the 18:1 fatty acids. Hydrolyzed and derivatized polysorbate 80 was quantified indirectly in the range of 0.046-5.83 µg/mL (R2 > 0.997) with a limit of detection of 11.4 ng/mL. Quantification of polysorbate 80 using oleic acid as a surrogate marker showed good reproducibility and linearity. As all isomers of the 18:1 fatty acids were successfully separated, the previously co-eluting peak was identified as elaidic acid and was found as a component in the mixture of the emulsifier polysorbate 80. Additionally, cis-vaccenic acid was separated as a second co-eluting isomer. Therefore, derivatization led to successful chromatographical separation of cis/trans and structural 18:1 fatty acid isomers.

Correlation of antimicrobial effects of phenoxyethanol with its free concentration in the water phase of o/w-emulsion gels.

Antimicrobial testing is a time consuming and cost-intensive but essential method for evaluation of newly developed pharmaceutical formulations for topical use. In this study the correlation between free preservative concentration in emulsion gels measured by equilibrium dialysis and the successful preservative effectiveness testing for Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Aspergillus brasiliensis (analyzed according to Ph. Eur. and USP) was investigated. The higher the lipophilicity of the oil phase and the lower the content of the aqueous phase with regard to dissolved ingredients the more preferably distributed is phenoxyethanol to the water phase and, consequently, the higher was the efficacy against the microbes. Increased emulsifier concentrations reduced the free amount of the preservative due to micellar interactions. Aspergillus brasiliensis was the most resistant and Staphylococcus aureus the most sensitive germ towards phenoxyethanol in o/w-emulsion gels.

Reviewing biophysical and cell biological methodologies in cell-penetrating peptide (CPP) research.

The discovery of cell-penetrating peptides (CPPs), which have the ability to translocate across the plasma membranes of mammalian cells, has led to widespread optimism for delivery of problematic therapeutic cargoes to cells. These cargoes include peptide, protein, and nucleic acid biopharmaceuticals and even nano-sized vectors such as liposomes and nanoparticles. Research on CPPs includes biophysical studies of membrane models to investigate fundamental principles of CPP-lipid membrane interactions as well as cell studies focusing on the efficiency of uptake, mechanisms of translocation, and toxicity. However, both lines of research have suffered from misinterpretation as well as premature extrapolations. In this review, we provide a critical evaluation of the potential and limitations of selected biophysical methodologies such as fluorescence spectroscopy, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, atomic-force microscopy (AFM), and non-spectroscopic methods. We include a discussion of the most important bilayer membrane models in CPP research. We then evaluate important cell biological methodologies, in particular confocal laser scanning microscopy (CLSM) and fluorescence-associated cell sorting (FACS) in combination with various techniques to distinguish between translocated and non-translocated CPPs. Moreover, we discuss the diverse methodologies for tracing the pathways of CPP translocation and their routes of intracellular trafficking.

Bilayer interaction and localization of cell penetrating peptides with model membranes: a comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43-58).

Cell-penetrating peptides (CPPs) are able to translocate problematic therapeutic cargoes across cellular membranes. The exact mechanisms of translocation are still under investigation. However, evidence for endocytic uptake is increasing. We investigated the interactions of CPPs with phospholipid bilayers as first step of translocation. To this purpose, we employed four independent techniques, comprising (i) liposome buffer equilibrium dialysis, (ii) Trp fluorescence quenching, (iii) fluorescence polarization, and (iv) determination of zeta-potentials. Using unilamellar vesicles (LUVs) of different phospholipid composition, we compared weakly cationic human calcitonin (hCT)-derived peptides with the oligocationic CPPs pVEC and penetratin (pAntp). Apparent partition coefficients of hCT-derived peptides in neutral POPC LUVs were dependent on amino acid composition and secondary structure; partitioning in negatively charged POPC/POPG (80:20) LUVs was increased and mainly governed by electrostatic interactions. For hCT(9-32) and its derivatives, D values raised from about 100-200 in POPC to about 1000 to 1500 when negatively charged lipids were present. Localization profiles of CPPs obtained by Trp fluorescence quenching were dependent on the charge density of LUVs. In POPC/POPG, hCT-derived CPPs were located on the bilayer surface, whereas pVEC and pAntp resided deeper in the membrane. In POPG LUVs, an increase of fluorescence polarization was observed for pVEC and pAntp but not for hCT-derived peptides. Generally, we found strong peptide-phospholipid interactions, especially when negatively charged lipids were present.

A custom tailored model to investigate skin penetration in porcine skin and its comparison with human skin.

Reliable models for the determination of skin penetration and permeation are important for the development of new drugs and formulations. The intention of our study was to develop a skin penetration model which (1) is viable and well supplied with nutrients during the period of the experiment (2) is mimicking human skin as far as possible, but still is independent from the problems of supply and heterogeneity, (3) can give information about the penetration into different compartments of the skin and (4) considers specific inter-individual differences in skin thickness. In addition, it should be quick and inexpensive (5) and without ethical implications (6). Using a chemically divers set of four topically approved active pharmaceutical ingredients (APIs), namely diclofenac, metronidazole, tazarotene, and terbinafine, we demonstrated that the model allows reliable determination of drug concentrations in different layers of the viable epidermis and dermis. For APIs susceptible for skin metabolism, the extent of metabolic transformation in epidermis and dermis can be monitored. Furthermore, a high degree of accordance in the ability for discrimination of skin concentrations of the substances in different layers was found in models derived from porcine and human skin. Viability, proliferation, differentiation and markers for skin barrier function were surveyed in the model. This model, which we call 'Hamburg model of skin penetration' is particularly suited to support a rational ranking and selection of dermatological formulations within drug development projects.

Correlation of hydrotropic solubilization by urea with logD of drug molecules and utilization of this effect for topical formulations.

Solubilization of drugs in aqueous phases of liquid and semisolid environment is typically achieved by co-solvents or surfactants. On contrast, solubilization by means of hydrotropic agents, i.e., small hydrophilic organic compounds like urea or citric acid, is little explored in the context of pharmaceutical formulations. Especially, with regard to topical dosage forms, however, hydrotropic solubilization can provide valuable alternatives to establish solubilization approaches. A difficulty of employing hydrotropic solubilization was that its extent could not be predicted for different drug molecules. Using a chemically heterogeneous set of 12 compounds relevant for dermatology (with overall 16 different logD values tested), we were able to demonstrate that hydrotropic effects of urea can be predicted by logD values of drugs. All compounds with logD values between 2 and 4.5 showed a solubility enhancement factor (EF) of >5 in 40% aqueous solutions of urea. For logD values below 2 or above 5, only EF<5 were found. For some compounds, e.g., diclofenac (pH 4) and prednicarbate could achieved only EF>5 at 5% urea and EF>250 at 20% urea.

The cell penetrating peptides pVEC and W2-pVEC induce transformation of gel phase domains in phospholipid bilayers without affecting their integrity.

The cell penetrating peptide (CPP) pVEC has been shown to translocate efficiently the plasma membrane of different mammalian cell lines by a receptor-independent mechanism without exhibiting cellular toxicity. This ability renders CPPs of broad interest in cell biology, biotechnology, and drug delivery. To gain insight into the interaction of CPPs with biomembranes, we studied the interaction of pVEC and W2-pVEC, an Ile --> Trp modification of the former, with phase-separated supported phospholipid bilayers (SPB) by atomic force microscopy (AFM). W2-pVEC induced a transformation of dipalmitoyl phosphatidylcholine (DPPC) domains from a gel phase state via an intermediate state with branched structures into essentially flat bilayers. With pVEC the transformation followed a similar pathway but was slower. Employing fluorescence polarization, we revealed the capability of the investigated peptides to increase the fluidity of DPPC domains as the underlying mechanism of transformation. Due to their tighter packing, sphingomyelin (SM) domains were not transformed. By combination, AFM observations, dynamic light scattering studies, and liposome leakage experiments indicated that bilayer integrity was not compromised by the peptides. Transformation of gel phase domains in SPB by CPPs represents a novel aspect in the discussion on uptake mechanisms of CPPs.

Membrane surface-associated helices promote lipid interactions and cellular uptake of human calcitonin-derived cell penetrating peptides.

hCT(9-32) is a human calcitonin (hCT)-derived cell-penetrating peptide that has been shown to translocate the plasma membrane of mammalian cells. It has been suggested as a cellular carrier for drugs, green fluorescent protein, and plasmid DNA. Because of its temperature-dependent cellular translocation resulting in punctuated cytoplasmatic distribution, its uptake is likely to follow an endocytic pathway. To gain insight into the molecular orientation of hCT(9-32) when interacting with lipid models, and to learn more about its mode of action, various biophysical techniques from liposome partitioning to high-resolution NMR spectroscopy were utilized. Moreover, to establish the role of individual residues for the topology of its association with the lipid membrane, two mutants of hCT(9-32), i.e., W30-hCT(9-32) and A23-hCT(9-32), were also investigated. Although unstructured in aqueous solution, hCT(9-32) adopted two short helical stretches when bound to dodecylphosphocholine micelles, extending from Thr10 to Asn17 and from Gln24 to Val29. A23-hCT(9-32), in which the helix-breaking Pro23 was replaced by Ala, displayed a continuous alpha-helix extending from residue 12 to 26. Probing with the spin label 5-doxylstearate revealed that association with dodecylphosphocholine micelles was such that the helix engaged in parallel orientation to the micelle surface. Moreover, the Gly to Trp exchange in W30-hCT(9-32) resulted in a more stable anchoring of the C-terminal segment close to the interface, as reflected by a twofold increase in the partition coefficient in liposomes. Interestingly, tighter binding to model membranes was associated with an increase in the in vitro uptake in human cervix epithelial adenocarcinoma cell line cells. Liposome leakage studies excluded pore formation, and the punctuated fluorescence pattern of internalized peptide indicated vesicular localization and, in conclusion, strongly suggested an endocytic pathway of translocation.