Emollients in dermatological creams: Early evaluation for tailoring formulation and therapeutic performance.

The targeted choice of the emollient of a cream determines its physicochemical properties and clinical effectiveness. This work researched the effects of emollient properties on the final characteristics and potential performance of oil-in-water dermatological creams. Seven emollients with different chemical characteristics and structures (alkane, triglyceride, ether, silicone, vegetable oils, and mineral oil) were tested in a model formulation. Early stability, pH, droplet size distribution, rheology, tackiness, adhesivity, spreadability, tribology, and release profile of a lipophilic substance model (in Franz cells, through a synthetic membrane, for six hours) were assessed. The creams had acid epicutaneous pH and a "shear-thinning" "solid-like" viscoelastic behavior. Among the seven emollients' properties, polarity, density, and viscosity were the most influential. Droplet parameters were the most impacted, pH and release were moderately affected, and the textural properties were lowly to moderately impacted. The emollient substitution in the model formulation affected the experimental parameters differently, allowing formulation optimization and tailoring its potential therapeutic performance regarding drug release, coadjutant effects, and dwell time on the skin. By looking at the creams' characteristics, it was possible to select the best-suited emollients for releasing a lipophilic drug, applying on painful skin, and formulation in wash-off products or leave-on protective barrier creams.

Design of minocycline-containing starch nanocapsules for topical delivery.

Pharmaceutical research has been focussed on developing improved delivery systems while exploring new ways of using approved excipients. The present work investigated the potential of starch nanocapsules (StNC) as a topical delivery platform for hydrophilic antimicrobial drugs using minocycline hydrochloride (MH) as a model drug. Thus, a quality by design approach was used to assess the role of different factors that affect the main pharmaceutical properties of StNC prepared using an emulsification-solvent evaporation method. Full characterisation was performed in terms of particle size, encapsulation efficiency, morphology and physical stability at 5 ± 3 °C. Results show the surfactant and lipid contents play a major role in StNC particle size distribution. The MH loading only promoted minor changes upon StNC properties. Formulations were stable without variations on physicochemical properties. All tested formulations presented a zeta-potential of +33.6 ± 6.7 mV, indicating a good physical stability and evidencing that StNC are suitable nanocarriers for topical use.

Starch nanocapsules containing a novel neutrophil elastase inhibitor with improved pharmaceutical performance.

Psoriasis and atopic dermatitis patients show an excessive amount of elastase in peripheral blood neutrophils due to an imbalance between this proteolytic enzyme and its endogenous inhibitors, the search for new human neutrophil elastase (HNE) inhibitors are required. The HNE is an attractive therapeutic target and inhibitors with new molecular architectures have been extensively investigated. In this context a promising novel synthetic human neutrophil elastase inhibitor (ER143) was associated to a starch-based nanoparticulate system (StNC) with improved pharmaceutical performance, using a quality by design approach to support product development and optimization. The resulting formulation was characterized in terms of and in vitro release, permeation and retention studies in newborn pig skin, using Franz diffusion cells revealing the StNC have the ability to control the drug release rate and contribute to a high skin retention and/or permeation profiles. The anti-inflammatory activity accessed in vivo using the croton oil-induced ear inflammation model in mice showed that erythema and edema were attenuated in 98% following local application. These observations suggest the association of ER143 to the StNC promotes a deeper skin penetration and retention, also confirming StNC as a potential topical delivery system.

Safety assessment of starch-based personal care products: Nanocapsules and pickering emulsions.

The safety profile of the ingredients used in topical dosage forms and its evaluation is an issue of utmost importance. A suitable equilibrium between safety and efficacy is crucial before promoting a dermatological product. The aim of this work was to assess the safety and biological effects of starch-based vehicles (St-BV) used in such products. The hazard, exposure and dose-response assessment were used to characterize the risk of each ingredient. The EpiSkin™ assay and human repeat insult patch tests were performed to compare the theoretical safety assessment to in vitro and in vivo data. The efficacy of the St-BV was studied using biophysical measurements in human volunteers during 28 days, showing that all ingredients and their combinations were safe for the consumer. Tissue viability determined using the EpiSkin™ testing reached values between 84.0 ±â€¯5.0% and 98.0 ±â€¯8.6% after application of St-BV, which were considered as non-irritant to the skin. These observations were confirmed by the in vivo studies where the St-BV did not induce any sensitization on the volunteers, being safe for human use. Moreover, St-BV increased skin hydration and microcirculation, emerging as an attractive alternative to chemical raw materials.

Design of novel starch-based Pickering emulsions as platforms for skin photoprotection.

Green coffee oil and modified starch were recently found to have an enhanced protection effect against UV radiation. Therefore, this work aimed to develop an innovative sunscreen formulation based on Pickering emulsions concept, i.e., surfactant-free emulsions stabilized by physical UV filters associated natural oils as a key strategy for prevention against UV-induced skin damage. The Pickering emulsions of different compositions were characterized in terms of pH, mechanical, physical and microbiological stability by a thorough pharmaceutical control. In addition, the sun protection factor (SPF) as well as the in vitro and in vivo biological properties of the final formulations, including Episkin®, HRIPT and sunscreen water resistance. Formulation studies demonstrated the addition of physical UV filters was beneficial, leading to the inclusion of ZnO and TiO2 to ensure a high SPF against UVA and UVB, respectively. Although starch particles presented no intrinsic photoprotection properties, they proved to be a SPF promoter by a synergistic effect. Green coffee oil was the selected natural oil due to the highest SPF, when compared to other natural oils tested. Besides the excellent sunscreen activity confirmed by in vitro and in vivo results, the final formulations proved to be also suitable for topical use according to the rheological assessment and stability throughout the study period (3months). In conclusion, the combination of three multifunctional solid particles and green coffee oil, contributed to achieve a stable and effective innovative sunscreen with a wide range of UV radiation protection.

A Quality by design (QbD) approach on starch-based nanocapsules: A promising platform for topical drug delivery.

Exploring novel applications for approved excipients with a history of safe use in therapeutics is a smart strategy to obtain improved pharmaceutical products. The present study aimed at developing a novel starch-based nanoparticulate carrier system (StNC) for topical delivery of lipophilic bioactive molecules. The role of the different factors that affect the particle size distribution and zeta potential of StNC prepared by the emulsification-solvent evaporation method was assessed using a quality by design approach. An optimal formulation was selected and fully characterized in terms of molecular interactions (DSC and FTIR), morphology (TEM and AFM), as well as in vitro and in vivo biological properties, including biological sensitivity/irritation studies performed in human volunteers. Results show the surfactant and lipid contents play a major role in StNC particle size distribution. In addition, all tested formulations presented a zeta potential of ca. +33.6±6.7 mV, indicating a good physical stability, while revealing an excellent compromise between stability, safety and cosmeticity, evidencing that StNC are suitable nanocarriers for topical use. Finally, the design planning methodology has clearly shown its usefulness for optimizing the formulation, being also crucial for the understanding of StNC formation process. The StNC proved to be a promising formulation strategy and a potential nanocarrier for topical lipophilic bioactive molecules.

Starch-based Pickering emulsions for topical drug delivery: A QbD approach.

Pickering emulsions are stabilized by solid particles instead of surfactants and have been widely investigated in pharmaceutical and cosmetic fields since they present less adverse effects than the classical emulsions. A quality by design (QbD) approach was applied to the production of w/o emulsions stabilized by starch. A screening design was conducted to identify the critical variables of the formula and the process affecting the critical quality properties of the emulsion (droplet size distribution). The optimization was made by establishing the Design Space, adjusting the concentration of starch and the quantity of the internal aqueous phase. The emulsion production process was, in turn, adjusted by varying the time and speed of stirring, to ensure quality and minimum variability. The stability was also investigated, demonstrating that an increase in starch concentration improves the stability of the emulsion. Rheological and mechanical studies indicated that the viscosity of the emulsions was enhanced by the addition of starch and, to a higher extent, by the presence of different lipids. The developed formulations was considered non-irritant, by an in vitro assay using human cells from skin (Df and HaCat) with the cell viability higher than 90% and, with self-preserving properties. Finally, the QbD approach successfully built quality in Pickering emulsions, allowing the development of hydrophilic drug-loaded emulsions stabilized by starch with desired organoleptic and structural characteristics. The results obtained suggest that these systems are a promising vehicle to be used in products for topical administration.

Elafin drives poor outcome in high-grade serous ovarian cancers and basal-like breast tumors.

High-grade serous ovarian carcinoma (HGSOC) and basal-like breast cancer (BLBC) share many features including TP53 mutations, genomic instability and poor prognosis. We recently reported that Elafin is overexpressed by HGSOC and is associated with poor overall survival. Here, we confirm that Elafin overexpression is associated with shorter survival in 1000 HGSOC patients. Elafin confers a proliferative advantage to tumor cells through the activation of the MAP kinase pathway. This mitogenic effect can be neutralized by RNA interference, specific antibodies and a MEK inhibitor. Elafin expression in patient-derived samples was also associated with chemoresistance and strongly correlates with bcl-xL expression. We extended these findings into the examination of 1100 primary breast tumors and six breast cancer cell lines. We observed that Elafin is overexpressed and secreted specifically by BLBC tumors and cell lines, leading to a similar mitogenic effect through activation of the MAP kinase pathway. Here too, Elafin overexpression is associated with poor overall survival, suggesting that it may serve as a biomarker and therapeutic target in this setting.

Primary ex vivo cultures of human fallopian tube epithelium as a model for serous ovarian carcinogenesis.

Recent studies suggest that some serous ovarian carcinomas (SOCs) arise from the fallopian tube (FT) epithelium rather than the ovarian surface epithelium. This hypothesis places emphasis on the FT secretory epithelial cell as a cell-of-origin. Herein, we report the development of a novel ex vivo primary human FT epithelium culture system that faithfully recapitulates the in vivo epithelium, as shown by morphological, ultrastructural and immunophenotypic analyses. Mass spectrometry-based proteomics reveal that these cultures secrete proteins previously identified as biomarkers for ovarian cancer. We also use this culture system to study the response of the FT epithelium to genotoxic stress and find that the secretory cells exhibit a distinct response to DNA damage when compared with neighboring ciliated cells. The secretory cells show a limited ability to resolve the damage over time, potentially leaving them more susceptible to accumulation of additional mutagenic injury. This divergent response is confirmed with in situ studies using tissue samples, further supporting the use of this ex vivo culture system to investigate FT epithelial pathobiology. We anticipate that this novel culture system will facilitate the study of SOC pathogenesis, and propose that similar culture systems could be developed for other organ site-specific epithelia.

Photocatalytic decolouration of Orange II by ZnO active layers screen-printed on ceramic tiles.

In this work ZnO layers have been deposited by screen-printing in common ceramic tiles. These layers were characterized and tested for the photocatalytic degradation of the organic dye Orange II in aqueous solutions, using a batch photoreactor either under visible light provided by a Philips ML-160 W lamp or under direct exposure to sunlight. For sake of comparison, ZnO suspensions have also been evaluated for similar reacting conditions. The influence of experimental parameters such as (i) firing temperature of the printed layer; (ii) layer thickness; and (iii) operation time have been investigated. Screen-printed ZnO layers obtained in optimal processing conditions showed photocatalytic activity comparable to aqueous ZnO suspensions. The maximal attenuation degree is over 70% and decolourisation rate, assuming that reaction kinetics follows a pseudo-first order rate law, is over 0.015 min(-1). Thus these ZnO-layered ceramic tiles can be regarded as an alternative to photocatalytic suspensions of the same material with the advantage of avoiding the removal of the photocatalyst.

Identification of cyclin B1 as a shared human epithelial tumor-associated antigen recognized by T cells.

We eluted peptides from class I molecules of HLA-A2.1(+) breast adenocarcinoma and loaded reverse phase high-performance liquid chromatography (HPLC) fractions onto dendritic cells to prime naive CD8(+) T cells. Fractions that supported growth of tumor-specific cytotoxic T lymphocytes were analyzed by nano-HPLC micro-ESI tandem mass spectrometry. Six HLA-A2.1-binding peptides, four 9-mers (P1-P4) differing in the COOH-terminal residue, and two 10-mers (P5 and P6) with an additional COOH-terminal alanine, were identified in one fraction. Peptide sequences were homologous to cyclin B1. We primed CD8(+) T cells from another HLA-A2.1(+) healthy donor with synthetic peptides and generated P4-specific responses. We also detected memory T cells specific for one or more of these peptides in patients with breast cancer and squamous cell carcinomas of the head and neck (SCCHN). T cells from one patient, restimulated once in vitro, could kill the tumor cell line from which the peptides were derived. Immunohistochemical analysis of tumor lines and tissue sections showed cyclin B1 overexpression and aberrant localization in the cytoplasm instead of the nucleus. Sequencing genomic DNA and cDNA corresponding to P1-P6 region showed that differences in COOH-terminal residues were not due to either DNA mutations or errors in transcription, suggesting a high error rate in translation of cyclin B1 protein in tumors.

Differences in the expression of human class I MHC alleles and their associated peptides in the presence of proteasome inhibitors.

We have studied the contributions of proteasome inhibitor-sensitive and -insensitive proteases to the generation of class I MHC-associated peptides. The cell surface expression of 13 different human class I MHC alleles was inhibited by as much as 90% or as little as 40% when cells were incubated with saturating concentrations of three different proteasome inhibitors. Inhibitor-resistant class I MHC expression was not due to TAP-independent expression or preexisting internal stores of peptides. Furthermore, it did not correlate with the amount or specificity of residual proteasome activity as determined in in vitro proteolysis assays and was not augmented by simultaneous incubation with multiple inhibitors. Mass spectrometry was used to directly characterize the peptides expressed in the presence and absence of proteasome inhibitors. The number of peptide species detected correlated with the levels of class I detected by flow cytometry. Thus, for many alleles, a significant proportion of associated peptide species continue to be generated in the presence of saturating levels of proteasome inhibitors. Comparison of the peptide-binding motifs of inhibitor-sensitive and -resistant class I alleles further suggested that inhibitor-resistant proteolytic activities display a wide diversity of cleavage specificities, including a trypsin-like activity. Sequence analysis demonstrated that inhibitor-resistant peptides contain diverse carboxyl termini and are derived from protein substrates dispersed throughout the cell. The possible contributions of inhibitor-resistant proteasome activities and nonproteasomal proteases residing in the cytosol to the peptide profiles associated with many class I MHC alleles are discussed.

Identification and modulation of a naturally processed T cell epitope from the diabetes-associated autoantigen human glutamic acid decarboxylase 65 (hGAD65).

T cell recognition of autoantigens is critical to progressive immune-mediated destruction of islet cells, which leads to autoimmune diabetes. We identified a naturally presented autoantigen from the human islet antigen glutamic acid decarboxylase, 65-kDa isoform (GAD65), by using a combination of chromatography and mass spectrometry of peptides bound by the type I diabetes (insulin-dependent diabetes mellitus, IDDM)-associated HLA-DR4 molecule. Peptides encompassing this epitope-stimulated GAD65-specific T cells from diabetic patients and a DR4-positive individual at high risk for developing IDDM. T cell responses were antagonized by altered peptide ligands containing single amino acid modifications. This direct identification and manipulation of GAD65 epitope recognition provides an approach toward dissection of the complex CD4(+) T cell response in IDDM.

Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI fourier transform ion cyclotron resonance mass spectrometry.

Subfemtomole peptide sequence analysis has been achieved using microcapillary HPLC columns, with integrated nanoelectrospray emitters, coupled directly to a Fourier transform ion cyclotron resonance mass spectrometer. Accurate mass (+/-0.010 Da) peptide maps are generated from a standard six-protein digest mixture, whose principle components span a concentration dynamic range of 1000:1. Iterative searches against approximately 189000 entries in the OWL database readily identify each protein, with high sequence coverage (20-60%), from as little as 10 amol loaded on-column. In addition, a simple variable-flow HPLC apparatus provides for on-line tandem mass spectrometric analysis of tryptic peptides at the 400-amol level. MS/MS data are searched against approximately 280000 entries in a nonredundant protein database using SEQUEST. Accurate precursor and product ion mass information readily identifies primary amino acid sequences differing by asparagine vs aspartic acid (deltam = 0.98 Da) and glutamine vs lysine (deltam = 0.036 Da).

Immunodominance among EBV-derived epitopes restricted by HLA-B27 does not correlate with epitope abundance in EBV-transformed B-lymphoblastoid cell lines.

Using synthetic peptides, the HLA-B27-restricted CTL response to EBV in asymptomatic virus carriers has been mapped to four epitope regions in EBV latent cycle Ags. One of these peptide-defined epitopes (RRIYDLIEL) tends to be immunodominant and is recognized in the context of all three B27 subtypes studied, B*2702, B*2704, and B*2705. The other peptide-defined epitopes induce responses only in the context of one subtype, the immunogenic combinations being RRARSLSAERY/B*2702, RRRWRRLTV/B*2704, and FRKAQIQGL/B*2705. We used immunoaffinity chromatography to isolate the naturally presented viral peptides associated with these MHC class I molecules on the surface of EBV-transformed B-LCL. Using CTL reconstitution assays in conjunction with mass spectrometry, we established that the naturally processed and presented peptides are identical with the previously identified synthetic sequences. Despite the subtype-specific immunogenicity of three of the four epitopes, all four epitope peptides were found in association with each of the three different HLA-B27 subtypes. Indeed, those peptides that failed to induce a response in the context of a particular HLA-B27 subtype were frequently presented at greater abundance by that subtype than were the immunogenic peptides. Furthermore, among the peptides that did induce a response, immunodominance did not correlate with epitope abundance; in fact the immunodominant RRIYDLIEL epitope was least abundant, being present at less than one copy per cell. The relationship of this unexpected finding to the persistence of EBV is discussed.

Serum protein immunogenicity: implications for liver xenografting.

The objectives of this study were threefold: (i) assess immunogenicity of donor plasma proteins following hepatic xenotransplantation, (ii) identify potential immunogens, and (iii) consider the implications of antibody formation against these plasma proteins in xenograft survival. We studied liver and heart xenografts in a concordant combination, hamster to rat. All grafts were examined at necropsy for evidence of rat immunoglobulin G (IgG) deposition. Cardiac xenografts were placed in recipients who had, or had not, been sensitized with hamster serum. Hepatic xenografts were placed in naive recipients to see if antibodies to hamster serum proteins could be eluted from the rejecting organ. Sera of immunized rats were examined for the presence of anti-hamster antibodies by immunoelectrophoresis and by Western blotting following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of hamster serum. Antibodies in sera of immunized rats were compared with those eluted from rejecting livers. Candidate antigens were identified by tandem mass spectrometry, sequence analysis, and reference to protein databases. Results showed that sera of immunized rats recognized a minimum of four different antigens in hamster serum by immunoelectrophoresis, and a minimum of seven by the more sensitive SDS-PAGE Western blot. IgG eluted from rejecting livers bound three of seven candidate antigens recognized by sera of the immunized animals. Sequence analysis searches revealed proteinase inhibitors in each of the three SDS-PAGE bands common to the above samples. All of these candidate proteinase inhibitor immunogens share a common catabolic fate, uptake via the lipoprotein-related protein (LRP/alpha 2-macroglobulin receptor (CD91). Sensitization to hamster serum proteins hastened cardiac xenograft rejection in 30-50% of recipients (depending on sensitization protocol). Vascular deposition of rat IgG occurred in all rejecting xenografts. Antibody binding to proteinase inhibitors could disturb their functional activity and contribute to the pathogenesis of delayed xenograft rejection.

Cutting edge: the HLA-A*0101-restricted HY minor histocompatibility antigen originates from DFFRY and contains a cysteinylated cysteine residue as identified by a novel mass spectrometric technique.

In this report, we describe the use of novel mass spectrometry instrumentation to identify a male-specific minor histocompatibility Ag restricted by HLA-A*0101 (A1-HY). This Ag has the sequence IVDC*LTEMY, where C* represents a cysteine disulfide bonded to a second cysteine residue. The core peptide sequence is found in the protein product of DFFRY, a Y chromosome gene not previously identified as the source of an HY Ag. The male-specific form of the peptide differs from its X chromosomal counterpart by the substitution of serine for the C* residue. Both peptides are expressed on the cell surface at 30 or fewer copies per cell. However, A1-HY-specific CTL recognize the DFFRY-derived peptide at a 1500-fold lower dose than the female homologue. Thus, these studies have identified a new source of HY epitopes and provide additional information about the influence of posttranslational modifications of class I-associated peptides on T cell recognition.

Proteasomes can either generate or destroy MHC class I epitopes: evidence for nonproteasomal epitope generation in the cytosol.

Proteasomes have been implicated in the production of the majority of peptides that associate with MHC class I molecules. We used two different proteasome inhibitors, the peptide aldehyde N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL) and the highly specific inhibitor lactacystin, to examine the role of proteasomes in generating peptide epitopes associated with HLA-A*0201. Neither LLnL nor lactacystin was able to completely block the expression of the HLA-A*0201. Furthermore, the effects of LLnL and lactacystin on the expression of different categories of specific epitopes, TAP independent vs TAP dependent and derived from either cytosolic or membrane proteins, were assessed. As predicted, presentation of two TAP-dependent epitopes was blocked by LLnL and lactacystin, while a TAP-independent epitope that is processed in the endoplasmic reticulum was unaffected by either inhibitor. Surprisingly, both LLnL and lactacystin increased rather than inhibited the expression of a cytosolically transcribed and TAP-dependent peptide from the influenza A virus M1 protein. Mass spectrometric analyses of in vitro proteasome digests of a synthetic 24 mer containing this epitope revealed no digestion products of any length that included the intact epitope. Instead, the major species resulted from cleavage sites within the epitope. Although cleavage at these sites was inhibitable by LLnL and lactacystin, epitope-containing species were still not produced. We conclude that proteasomes may in some cases actually destroy epitopes that would otherwise be destined for presentation by class I molecules. These results suggest that some epitopes are generated by nonproteasomal proteases in the cytosol.

Electrospray ionization Fourier transform ion cyclotron resonance at 9.4 T.

We present the first results from a new electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer operated at a magnetic field of 9.4 T (i.e. > or = 2.4 T higher than for any prior FTICR instrument). The 9.4 T instrument provides substantially improved performance for large molecules (> or = 50% increase in mass resolving power) and complex mixtures (> or = 100% increase in dynamic range) compared to lower-field (< or = 6 T) instruments. The higher magnetic field makes possible larger trapped-ion population without introduction of significant space--charge effects such as spectral peak shift and/or distortion, and coalescence of closely-spaced resonances. For bovine ubiquitin (8.6 kDa) we observe accurate relative isotopic abundances at a signal-to-noise ratio greater than 1000:1, whereas a complete nozzle-skimmer dissociation electrospray ionization (ESI) FTICR mass spectrum of bovine carbonic anhydrase (29 kDa) is achieved from a single scan with a signal-to-noise ratio of more than 250:1. Finally, we are able to obtain mass resolving power, m/delta m > 200,000, routinely for porcine serum albumin (67 kDa). The present performance guides further modifications of the instrument, which should lead to significant further improvements.

An external source 7 T Fourier transform ion cyclotron resonance mass spectrometer with electrostatic ion guide.

An external source 7 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer offers three main novel features. First, a 9-way ion-source cross allows for mounting of up to three ionization sources simultaneously, thereby minimizing 'downtime' for changing ion sources. Second, an electrostatic (wire-in-cylinder) ion guide transports the ions approximately 1.5 m from the ion source to the ion trap for mass analysis, through a large magnetic field gradient. Third, the system operates from a modular data system described elsewhere in this issue. Luteinizing hormone-releasing hormone (LH-RH) matrix-assisted laser desorption/ionization (MALDI) FTICR positive-ion mass spectra exhibit signal-to-noise ratio greater than 1000:1 and mass resolving power, m/delta m 50% > 100,000. Laser-induced fragmentation of bradykinin demonstrates the ability of the ion guide to transmit both molecular and fragment ions simultaneously. Ultra-high resolution (average resolving power approximately 400,000) was achieved for poly(ethylene glycol) of specified number-average molecular weight, Mn approximately 3400. Future installation of an electrospray source to the ion-source cross should allow for better characterization of the performance of the ion guide.