Investigation of the molecular structure of the human stratum corneum ceramides [NP] and [EOS] by mass spectrometry

The aim of this study was to characterize the two ceramide (CER) subclasses CER[NP] and CER[EOS] of human stratum corneum and to identify the chemical structures of their subspecies. High-performance thin-layer chromatography and normal-phase high-performance liquid chromatography were used for the separation of CER fractions, whereas nanoelectrospray ionization tandem mass spectrometry was applied to investigate the chemical structures in detail. Thus, CER[EOS] fragmentation revealed that in addition to linoleic acid other esterified fatty acids occur in the ω-position. Of particular interest is the identification of a 17:2 fatty acid located in this part of the molecule. Several subspecies of CER[NP], including subspecies with odd numbers of carbon atoms in both chains, the non-α-hydroxylated fatty acid moiety (part N) and the phytosphingosine (part P), were identified. Furthermore, it was found that 12% of CER[NP] subspecies have an odd number of carbon atoms in both chains of the molecule. Similar results were obtained for CER[EOS]. Both the esterified fatty acid (part E) and the sphingosine base (part S) were found to contain odd-numbered chain lengths. These results underline the heterogeneity of the CER composition in the stratum corneum lipid bilayer.     

SC lipid model membranes designed for studying impact of ceramide species on drug diffusion and permeation, Part III: Influence of penetration enhancer on diffusion and permeation of model drugs

The impact of the lipophilic penetration enhancer, oleic acid (OA), on the barrier properties of stratum corneum (SC) lipid model membranes was investigated based on diffusion and permeation studies of model drugs covering a broad range of lipophilicities. Diffusion and permeation experiments of urea, caffeine and diclofenac sodium were conducted using Franz-type diffusion cells. HPLC and capillary electrophoresis techniques were employed to analyze the amount of permeated drug. An incorporation of OA to the SC lipid model membranes did not change the relation between the diffusion and permeation behavior of model drugs presented previously for SC lipid model membranes without OA. The fastest rate of diffusion through SC lipid model membranes occurred in the case of the most hydrophilic drug, urea. In the case of permeation studies of caffeine and diclofenac sodium across SC lipid model systems, the permeability parameters were either equal or slightly larger in favor of the most lipophilic drug, diclofenac sodium. OA had a pronounced impact on the barrier properties of SC lipid model membranes. It caused the impairment of the barrier function of the SC lipid model membrane with Cer [AP] (phytosphingosine-based ceramide), however, surprisingly improved the barrier properties of the SC lipid model system with Cer [EOS] (sphingosine-based acylceramide).     

pH-induced modifications to stratum corneum lipids investigated using thermal,spectroscopic, and chromatographic techniques

The effects of nonphysiological pH on stratum corneum lipid content and structure have been studied. Human stratum corneum samples were soaked in solutions at pH 1, 2, 6, 11, or 12 for up to 24 h. After removal of the stratum corneum, the buffer solutions were analyzed for lipid composition using thin-layer chromatography analysis and the stratum corneum sheets were examined using differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy. The results demonstrate that only buffers of pH 11 or higher affect the stratum corneum lipids. No large difference in the contents of ceramides and cholesterol extracted by buffers of varying pH was observed. In contrast, free fatty acid extraction was pH dependent; amounts removed by 24-h treatment with pH 11 or 12 buffers were comparable, and were similar to amounts extracted with a methanol-chloroform mixture for 15 min. No appreciable changes in DSC and FTIR spectra were detected between untreated stratum corneum and stratum corneum samples treated with buffers at pHs in the range 1-6. For tissue treated with pH 11 and 12, the position of the endothermal melting peak T2 shifted from 72 to 74 degrees C on the DSC thermograms. Small changes in the broadness of spectral peaks at 2855 cm(-1) [attributable to upsilon(CH(2)) stretching of stratum corneum lipids and 1655 cm(-1) upsilon(C=O) stretching amide I band] can be seen in the FTIR spectra from the treated stratum corneum samples, although no shifts in peak positions were observed. Intensity changes in peaks from extraneous lipids [upsilon(C=O) stretching mode at 1735 cm(-1)] were observed after buffer treatments. The changes provoked by the alkaline buffers are not dramatic and it may be concluded that the stratum corneum appears remarkably resilient to extended exposure in both highly acidic (pH 1) and highly alkaline (pH 12) environments.     

A Novel in Vitro Percutaneous Penetration Model: Evaluation of Barrier Properties with P-Aminobenzoic Acid and Two of Its Derivatives

Purpose The objective of this study was to evaluate the utility of a stratum corneum substitute (SCS) as a novel in vitro percutaneous penetration model. The SCS consists of synthetic stratum corneum (SC) lipids (cholesterol, free fatty acids, and specific ceramides) applied onto a porous substrate. The composition, organization, and orientation of lipids in the SCS bear high resemblance to that of the intercellular barrier lipids in SC. Methods The barrier integrity of the SCS was evaluated by means of passive diffusion studies, using three model compounds with different lipophilicities. The effects of lipid layer thickness, permeant lipophilicity, and altered lipid composition on the barrier properties were investigated, using isolated human SC as a control sample. Results For all three model compounds, the permeability characteristics of the SCS with a 12-μm-thick lipid layer closely resemble those of human SC. Modification of the lipid composition, generating an SCS that lacks the characteristic long periodicity phase as present in SC, was accompanied by a 2-fold increased permeability. Conclusions The SCS offers an attractive tool to predict solute permeation through human skin. Moreover, as its lipid composition can be modified, they may also serve as a suitable screening model for diseased skin.     

Synthesis of ceramides NS and NP with perdeuterated and specifically ω deuterated N‐acyl residues

The synthesis of 12 deuterated ceramides with either a deuteration at the last carbon atom of the amide bound fatty acid or a perdeuterated fatty acid chain is described. The ceramides were prepared starting from sphingosine or phytosphingosine and ω deuterated or perdeuterated fatty acids with PyBOP® as activating agent in high yields. For the synthesis of the specifically deuterated fatty acids, dicarboxylic acids were transformed into ω deuterated alkyl bromide, which was chain elongated with blocked ω bromo alcohols by copper catalyzed Grignard coupling. Oxidation of regenerated alcohol function yields the ω deuterated fatty acids.     

The influence of topical formulations on the permeation of 5-aminolevulinic acid and its n-butyl ester through excised human stratum corneum

The limited permeation of 5-aminolevulinic acid (ALA) through excised human stratum corneum could be improved by using 5-aminolevulinic acid-n-butyl ester (ABE). Furthermore drug permeation could be increased by choice of a permeation enhancing formulation. In this study, permeation of ALA and ABE was investigated from various formulations. In addition, differential scanning calorimetry (DSC) and wide angle X-ray diffraction (WAXD) experiments were performed in order to reveal an interaction between the tested formulations and stratum corneum lipid structure. Drug incorporation into Dolgit^® Mikrogel showed the highest increase in permeability with both ALA and ABE. Especially, ABE together with Dolgit^® Mikrogel was the most promising combination. Further permeation studies with poloxamer based ABE formulations, partially enriched with ibuprofen acid and medium chained triglycerides showed that both compounds promote permeation. The permeation coefficients of either drug from Excipial^® Creme and Basiscreme DAC were found to be very similar. These results were in accordance with those of DSC and WAXD experiments. Interaction between formulation and stratum corneum lipid structure resulting in an increased drug permeation only occurred after pretreatment with formulations enriched with ibuprofen acid. After pretreatment with Excipial^® Creme, Basiscreme DAC or Excipial^® Fettcreme stratum corneum structure and subsequently permeability remained unchanged. Nevertheless permeation of ALA from Excipial^® Fettcreme is slower than from the tested hydrophilic formulations and therefore believed to be influenced by the affinity of ALA to the vehicle and stratum corneum.     

Fourier transform infrared spectroscopy studies of lipid domain formation in normal and ceramide deficient stratum corneum lipid models

The current work describes thermotropic and kinetic Fourier transform infrared (FTIR) spectroscopy studies of lipid dynamics and domain formation in normal and ceramide (CER) deficient lipid samples designed as simple models of the stratum corneum (SC). For the first time, this work focuses on the time dependence of lipid reorganization and domain formation in CER deficient models. By utilizing deuterated fatty acid (FA) and simultaneously monitoring the methylene vibrational modes of both CER and FA chains these experiments follow the time evolution of lipid organization in these SC lipid models following an external stress. Kinetic and thermotropic experiments demonstrate differences in both CER and FA chain fluidity and ordered domain formation with decreased levels of CER. In the CER deficient model, the formation of CER orthorhombic domains is retarded compared to the normal model. Furthermore, there is little evidence of hexongally packed (or mixed) FA domains in the CER deficient model compared to the models of normal SC. These data demonstrate that barrier lipid organization, in terms of ceramide domain formation, is altered in the ceramide deficient model. This work highlights the successful development of an experimental methodology to study time dependent changes in lipid biophysics in simple SC model membranes and suggests this approach will prove useful for understanding some of the biophysical changes that underlie impaired physiological barrier function in diseased skin.     

Use of electroporation to accelerate the skin permeability enhancing action of oleic acid

Rat skin permeability after treatment by electroporation (newly developed frog type electrode, 100V, 10 pulses), oleic acid/propylene glycol (PG) and a combination of both were investigated using Fourier transformed infrared attenuated total reflectance (FT-IR/ATR) analysis. Electroporation immediately disordered the stratum corneum lipid structure up to a certain threshold level. This action lasted throughout the experiment. This may be attributed to the formation of long lifetime of metastable lipid structures, which may allow molecules to pass to the inside of the stratum corneum due to the electroporation-induced fluidized lipid membranes. Electroporation also altered the protein structure of the stratum corneum. When electroporation was combined with 0.05 M oleic acid/PG, uptake of oleic acid and PG into the stratum corneum was remarkably accelerated compared to the application of only 0.05 M oleic acid/PG to the skin. This indicates that electroporation enables oleic acid and PG to penetrate the stratum corneum easily by disrupting the structure of the latter. PG transfer into the dermis from the epidermis was accelerated, not because of the direct action of electroporation on the dermis, but because electroporation induced the rapidly disordering action of oleic acid on the stratum corneum. Lipid-soluble indomethacin permeated the skin more rapidly when the skin was treated with electroporation plus oleic acid/PG than with 0.05 M oleic acid/PG in vitro.     

Correlation between stratum corneum/water-partition coefficient and amounts of flufenamic acid penetrated into the stratum corneum

The stratum corneum of various donors differs in particular in the composition of the lipoidal phase. Considering the drug amounts penetrating into the stratum corneum a simple methodology to correlate these differences in the stratum corneum composition with the drug amounts detectable within the stratum corneum is desirable. Penetration experiments investigating several incubation times were carried out with three different skin flaps using the Saarbruecken penetration model and the lipophilic model drug flufenamic acid. The drug amounts within the stratum corneum were obtained with the tape-stripping technique, while the drug amounts present in the deeper skin layers were achieved by cryosectioning. The stratum corneum/water-partition coefficient was determined with the same three skin flaps to characterize the lipoidal stratum corneum phase in general, and the differences were attributed to the different amounts of ceramides and sterols. In addition, for the lipophilic drug flufenamic acid, a direct linear correlation was found between the stratum corneum/water-partition coefficients and the drug amounts penetrated into the stratum corneum for all investigated time intervals (correlation coefficients of r(30 min) = 0.998, r(60 min) = 0.998 and r(180 min) = 0.987). In contrast to the stratum corneum/water-partition coefficients, the determination of a corresponding relationship for the stratum corneum and the deeper skin layers failed due to the reason that steady-state conditions could not be achieved for the deeper skin layers during the investigated time intervals. In summary, the stratum corneum/water-partition coefficients offer the possibility to predict drug amounts within the stratum corneum of different donor skin flaps without a time consuming determination of the lipid composition of the stratum corneum.     

The influence of Span20 on stratum corneum lipids in langmuir monolayers: comparison with Azone

Recently we have proved that Span 20 has the same enhancer effect as Azone on in vitro percutaneous penetration of lipophilic compounds (logP(oct) from 1.34 to 2.33). The purpose of this work is to study the interactions of Span 20 with stratum corneum lipids monolayers and to compare them with Azone. The surface pressure-area characteristics of Span 20 in mixed monolayers with different model lipids (ceramides, cholesterol, free fatty acids and two mixtures of ceramides+cholesterol, and ceramides+cholesterol+free fatty acids) in similar proportions to that which exists in human stratum corneum lipids were recorded as compression isotherms at 25 degrees C. Azone was also investigated on monomolecular films of some of these lipids. The results indicate that the effect exerted upon lipid packing by the Span 20 correspond, as in the case of Azone, to increased fluidity within monolayers. To quantify and compare the effect of Span 20 and Azone, the compressibility of enhancer-lipid model mixed monolayers was calculated, and expressed as a function of mole fraction of enhancer present on the films. Statistical comparison of the results obtained from both enhancers shows that they are equally potent in their interaction with the lipid models assayed. These models, if restricted, seem to be good for predict the activity and potency of percutaneous enhancers on the fluididity of the lipidic structure of the stratum corneum.     

Dibenz[b,f]azepines,Part 7[1]: Synthesis of New,Potentially CNS Active Dibenz[b,f]azepine Derivatives

Reactions of 5-carboxamido-5H-dibenz[b,f]azepines ( 1a–1d ) with glyoxylic acid methylester methyl hemiacetal (GMHA) led to 5-(carboxamido-N-α-hydroxy-acetic acid methyl ester)-5H-dibenz[b,f]azepines ( 2a–2d ). The reactions with glycols yielded the oligoethylene glycol derivatives ( 3,4 ). The new compounds were screened as anticonvulsants and/or antidepressants (AD).     

Increase of skin-ceramide levels in aged subjects following a short-term topical application of bacterial sphingomyelinase from Streptococcus thermophilus

Several studies have demonstrated that ceramides play an essential role in both the barrier and water-holding functions of healthy stratum corneum, suggesting that the dysfunction of the stratum corneum associated with ageing as well that observed in patients with several skin diseases could result from a ceramide deficiency. In a previous study our group reported a significant increase in skin ceramide levels in healthy subjects after treatment in vivo with a cream containing a preparation of Streptococcus thermophilus. The presence of high levels of neutral sphingomyelinase activity in this organism was responsible for the observed increase of stratum corneum ceramide levels, thus leading to an improvement in barrier function and maintenance of stratum corneum flexibility. The aim of the present work is to investigate the effects of the topical treatment of a Streptococcus thermophilus-containing cream on ceramide levels of stratum corneum of healthy elderly women. The ceramide levels, transepidermal water loss and capacitance were evaluated on stratum corneum sheets from the forearms of 20 healthy female subjects treated with a base cream or the same cream containing a sonicated preparation of the lactic acid bacterium Streptococcus thermophilus. A 2-week topical application of a sonicated Streptococcus thermophilus preparation led to significant and relevant increase of stratum corneum ceramide levels. Moreover, the hydration values of the treated forearm of each subject was significantly higher than control sites. These results suggest that the experimental cream was able to improve the lipid barrier and to increase a resistance against ageing-associated xerosis.     

Investigating the barrier function of skin lipid models with varying compositions

The lipids in the uppermost layer of the skin, the stratum corneum (SC), play an important role in the barrier function. The main lipid classes in stratum corneum are ceramides, cholesterol, and free fatty acids. In previous publications, a lipid model was presented, referred to as the stratum corneum substitute (SCS), that closely mimics the SC lipid organization and SC barrier function. In the present study, we use the SCS to study the effect of changes in lipid organization on the lipid barrier function using benzoic acid as permeation compound. First, in the SCS, we increased the level of one of the three major lipid classes keeping the ratio between the other lipid classes constant. An increased cholesterol level resulted in an increase in phase-separated cholesterol and a reduction in the permeability. An increase in ceramide or free fatty acid level resulted in the formation of additional phases, but had no significant influence on the permeability. We also examined models that mimic selected changes in lipid composition reported for dry or diseased skin. The SCS that mimics the composition in recessive X-linked ichthyosis skin displayed a twofold increase in permeability. This increase is possibly related to the formation of an additional, less ordered phase in this model.     

A convenient synthesis of [11C]paraquat and other [N‐methyl‐11C]bisquaternary ammonium compounds

[¹¹C]Paraquat was synthesized by the reaction of [¹¹C]methyl triflate with the mono‐triflate salt of 1‐methyl‐[4,4′]bipyridinyl. The product was selectively separated from the precursor by a microcolumn of Chelex 100 ion exchange resin. The method was applied to the synthesis of a variety of [N‐methyl‐¹¹C]bisquaternary ammonium compounds. This is the first reported use of a chelating cation exchange resin for the selective purification of organic dications. Copyright © 2002 John Wiley & Sons, Ltd.     

Bioevaluation of a novel [32P]‐CP‐PLLA microparticle for pancreatic cancer treatment

A novel [³²P]‐CP‐PLLA ([³²P]‐chromic phosphate‐poly‐L ‐lactic acid) microparticle was designed and evaluated for the treatment of pancreatic carcinoma. The microparticle was prepared from [³²P] chromic phosphate and poly‐L ‐lactic acid. Bioelimination, biodistribution, SPECT image, and therapeutic effect were studied in mice bearing human pancreatic tumor xenografts. [³²P]chromic phosphate ([³²P]‐CP) colloid at the same radioactivity dose was compared as the control. High radioactivity (>95%ID) of [³²P]‐CP‐PLLA was retained at the tumor, and almost no radioactivity excretion (<1%ID) was observed in urine and feces for 14 days, while radioactivity of [³²P]‐CP colloid, was distributed to the liver, spleen, and lung (varied individual), and the excretion increased over 5%ID. Compared with controls, reduced tumor volumes were seen in the [³²P]‐CP‐PLLA microparticle treatment group (P<0.01). Dose dependence was seen histologically. [³²P]‐CP‐PLLA microparticle retained high activity and long‐term residence in tumor. With minimal distribution to normal organs [³²P]‐CP‐PLLA microparticle is superior to [³²P]‐CP colloid and is more suitable for brachytherapy in solid tumor. Drug Dev Res 71:364–370, 2010. © 2010 Wiley‐Liss, Inc.     

Condensed Heterocyclic Compounds: Synthesis and Antiinflammatory Activity of Novel Thiazolo[3,2-a]pyrimidines

In this study, thirty six new 2-benzylidene-7-methyl-3-oxo-5-phenyl-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic acid methyl esters were synthesized and characterized by spectral, crystallographic, and elemental analysis. The antiinflammatory activity of the compounds was tested by the carrageenan hind paw edema test. It was found that compound 6a having a 2-methoxyphenyl group at position 5 and a benzylidene group at position 2 was the most potent compound in this series. All the compounds that were tested for ulcer activity gave positive results.     

Stereoselective synthesis of L‐[2,3,4,5‐D4] ornithine

Synthesis of L ‐[2,3,4,5‐D₄]ornithine in which all of the diastereotopic hydrogens were stereoselectively labeled with deuterium was investigated. The chirally deuterated 3‐aminopropanal derivative, a key intermediate in this synthesis, was prepared by a catalytic deuteration of an unsaturated γ‐lactone derived for L ‐glutamic acid followed by several functional group interconversions. Condensation of the obtained deuterium‐labeled 3‐aminopropanal derivative with a chiral glycine template afforded unsaturated ornithine. The dehydroornithine was then subjected to a catalytic deuteration followed by deprotection to give the L ‐[2,3,4,5‐D₄]ornithine. Copyright © 2002 John Wiley & Sons, Ltd.     

Comparative studies on the effects of water, ethanol and water/ethanol mixtures on chemical partitioning into porcine stratum corneum and silastic membrane.

The effects of water and ethanol vehicles on stratum corneum and silastic membrane partitioning of 11 industrial and agricultural compounds were studied to aid in characterizing and assessing risk from skin exposure. Zero percent, 50% and 100% aqueous ethanol solutions were used as solvents for (14)C labeled phenol, 4-nitrophenol, pentachlorophenol, dimethyl parathion, parathion, chloropyrifos, fenthion, triazine, atrazine, simazine and propazine. Compound partitioning between the solvents and porcine stratum corneum/silastic membrane were estimated. Stratum corneum was exposed to aqueous ethanol ranging from 0% to 100% v/v ethanol in 20% increments and Fourier transform infrared spectroscopy (FT-IR) was used to obtain an index of lipid disorder. Gravimetry and FT-IR were used to demonstrate lipid extraction in aqueous ethanol solutions. Partitioning patterns in silastic membranes resembled those in stratum corneum and were correlated with octanol/water partitioning. Partitioning was highest in water and was higher from 50% ethanol than from 100% ethanol, except for parathion, 4-nitrophenol, atrazine and propazine. Correlation existed between molecular weight and partitioning in water, but not in ethanol and ethanol/water mixtures. Lipid order, as reflected in FT-IR spectra, was not altered. These studies suggest that stratum corneum partitioning of the compounds tested is primarily determined by relative compound solubility between the stratum corneum lipids and the donor solvent. Linear relationships existed between octanol/water partitioning and stratum corneum partitioning. Partitioning was also correlated with molecular weight in water solvent systems, but not in ethanol and ethanol/water mixtures. Ethanol and ethanol/water mixtures altered the stratum corneum through lipid extraction, rather than through disruption of lipid order.     

A novel no‐carrier‐added submicromolar scale radiosynthesis of [S‐methyl‐14C]‐florfenicol

In this paper is reported a novel reaction scheme for the no‐carrier‐added submicromolar scale radiosynthesis of [S‐methyl‐¹⁴C]‐florfenicol that has been newly designed, developed and employed by us successfully. The [¹⁴C]‐product was obtained in an overall radiochemical yield of 30% based on [¹⁴C]‐methyl iodide taken for the reaction with a radiochemical purity of more than 96%. The specific activity of the product was ~50 mCi (1.85 GBq)/mmol. Chlorosulfonation of compound I was followed by sodium salt formation in situ and it was succeeded by the introduction of [¹⁴C]‐methyl group by coupling with [¹⁴C]‐CH₃I. Subsequently, the oxazolidin‐2‐one protecting group was opened up by a reaction with sulfuric acid in dioxane and later, the amino group was dichloroacetylated with methyl‐2,2‐dichloroacetate in triethylamine to obtain [S‐methyl‐¹⁴C]‐florfenicol.     

Synthesis of [14C]‐radiolabelled entecavir

Radiolabelled [¹⁴C]entecavir, ( 1 ), was prepared in 12 steps from (1S,2R,3S,5R)‐3‐(benzyloxy)‐2‐(benzyloxymethyl)‐6‐oxa‐bicyclo[3.1.0]hexane 2 . The chemical yield of [¹⁴C]entecavir was 14% from the epoxide 2 . Introduction of [¹⁴C] radiolabel was achieved by elaboration of 4,5‐diaminopyrimidine 8 with triethyl[¹⁴C]orthoformate to purine derivative 9 . The radiochemical yield of [¹⁴C]entecavir from triethyl[¹⁴C]orthoformate was 11.3%. Radiochemical purity of [¹⁴C]entecavir determined by HPLC was 99.8%. The specific activity of [¹⁴C]entecavir was 108 µCi/mg (29.9 mCi/mmol). Copyright © 2005 John Wiley & Sons, Ltd.