Effects of Ceramide and Dihydroceramide Stereochemistry at C-3 on

Ceramides (Cer) are key components of the skin permeability barrier. Sphingosine-based ... Ceramides (Cer) are the key building blocks of the skin lip...
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Effects of Ceramide and Dihydroceramide Stereochemistry at C-3 on the Phase Behavior and Permeability of Skin Lipid Membranes Andrej Kovacik, Petra Pullmannová, Jaroslav Maixner, and Katerina Vavrova Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03448 • Publication Date (Web): 11 Dec 2017 Downloaded from http://pubs.acs.org on December 18, 2017

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Effects of Ceramide and Dihydroceramide Stereochemistry at C-3 on the Phase Behavior and Permeability of Skin Lipid Membranes Andrej Kováčik†, Petra Pullmannová†, Jaroslav Maixner§, and Kateřina Vávrová†* †

Skin Barrier Research Group, Charles University, Faculty of Pharmacy in Hradec Králové,

Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic §

University of Chemistry and Technology in Prague, Faculty of Chemical technology, Technická

5, 166 28 Prague, Czech Republic

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ABSTRACT. Ceramides (Cer) are key components of the skin permeability barrier. Sphingosine-based CerNS and dihydrosphingosine-based CerNdS (dihydroCer) have two chiral centers; however, the importance of the correct stereochemistry in the skin barrier Cer is unknown. We investigated the role of the configuration at C-3 of CerNS and CerNdS in the organization and permeability of model skin lipid membranes. Unnatural L-threo-CerNS and Lthreo-CerNdS with 24-C acyl chains were synthesized and, along with their natural D-erythro isomers, incorporated into membranes composed of major stratum corneum lipids (Cer, free fatty acids, cholesterol and cholesteryl sulfate). The membrane microstructure was investigated by Xray powder diffraction and infrared spectroscopy, including deuterated free fatty acids. Inversion of the C-3 configuration in CerNS and CerNdS increased phase transition temperatures, had no significant effects on lamellar phases, but also decreased the proportion of orthorhombic packing and decreased lipid mixing in the model membranes. These changes in membrane organization resulted in membrane permeabilities that ranged from unchanged to five-fold higher (depending on the permeability markers; namely, water loss, electrical impedance, flux of theophylline and flux of indomethacin) compared to membranes with natural CerNS/NdS isomers. Thus, the physiological D-erythro stereochemistry of skin Cer and dihydroCer appears to be essential for their correct barrier function.

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INTRODUCTION Ceramides (Cer) are the key building blocks of the skin lipid barrier. Along with free fatty acids (FFAs), cholesterol (Chol) and cholesteryl sulfate (CholS), Cer form multiple lipid membranes in the intercellular space of the stratum corneum (SC), the outermost layer of the mammalian skin. Cer participate in the maintenance of a stable internal environment, i.e., preventing penetration of unwanted environmental substances and the loss of body water.1-3 Cer are simple sphingolipids composed of a sphingoid base and amide-linked fatty acid. The most common sphingoid bases in the human body are the amino diol sphingosine (with a transdouble bond at C-4) and its saturated counterpart dihydrosphingosine. Sphingosine-based Cer (CerNS in shorthand nomenclature for skin Cer4) and dihydrosphingosine-based Cer (dihydroCer, CerNdS) share the common D-erythro configuration (i.e., (2S,3R)-configuration; Figure 1).5 The (2S)-configuration is determined biosynthetically by condensation of L-serine and palmitoyl coenzyme A by serine palmitoyl transferase, whereas the (3R)-hydroxyl is formed by ketosphinganine reductase.3 The effects of unnatural L-threo sphingolipids (i.e., with (2S,3S)configuration), particularly L-threo-dihydrosphingosine (safingol),6 have been explored in various cellular processes. However, the importance of the correct Cer stereochemistry for SC barrier properties is unknown. Only a single study has considered the behavior of L-threosphingolipids in the skin barrier, and this study revealed different lamellar organizations of Lthreo-CerNdS and D-erythro-CerNdS in model SC lipid mixtures with Chol and palmitic acid.7 In this work, we aimed to determine the effects of unnatural stereochemistry at C-3 in sphingosine-based CerNS and dihydrosphingosine-based CerNdS on the barrier properties and biophysical behavior of model SC lipid membranes. The main motivation for this study was to

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better understand the structure-permeability relationships of skin Cer. In addition, knowledge of the importance of the (R)-configuration at C-3 has practical implications in the design of barrier repair agents because creating this stereochemistry is synthetically challenging,8 in particular at a large scale. Hence, unnatural L-threo-CerNS ((2S,3S)-CerNS) and L-threo-CerNdS ((2S,3S)-CerNdS or Lthreo-dihydroCer), both with 24-carbon acyl chains, were synthesized and incorporated in model SC lipid membranes. These membranes were compared to those with natural D-erythro-CerNS and D-erythro-CerNdS, i.e., (2S,3R)-isomers (Figure 1). We used X-ray powder diffraction (XRPD) and Fourier-transform infrared spectroscopy (FTIR) to provide insight into the microstructure and thermotropic behavior of the model lipid membranes. The barrier properties of the model membranes were studied using four permeability markers – water loss through the membrane, electrical impedance, and absorption of two model permeants (theophylline (TH) and indomethacin (IND)).

Figure 1. Structures of the Studied CerNS and CerNdS with Natural (D-erythro) and Unnatural (L-threo) Stereochemistry. Unnatural stereochemistry at C-3 is highlighted in red.

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MATERIALS AND METHODS Chemicals. L-threo-sphingosine (synthetic, over 99% stereochemically pure, i.e., (2S,3S,4E)), Lthreo-dihydrosphingosine (synthetic, over 99% stereochemically pure, i.e., (2S,3S)), D-erythroCerNS (synthetic, over 99% stereochemically pure, i.e., (2S,3R,4E)) and D-erythro-CerNdS (synthetic, over 99% stereochemically pure, i.e., (2S,3R)) were purchased from Avanti Polar Lipids (Alabaster, USA). Deuterated free fatty acids (d-FFAs) were obtained from C/D/N isotopes (Pointe-Claire, Canada). FFAs, Chol, CholS and all other chemicals and solvents were from Sigma-Aldrich (Schnelldorf, Germany). Water was deionized, distilled, and filtered through a Millipore Q purification system. Synthesis of Unnatural CerNS and CerNdS. The appropriate sphingoid base (0.053 mmol), lignoceric acid (0.058 mmol) and 1-hydroxybenzotriazole hydrate (HOBt; 0.196 mmol) were dissolved in 5 mL of dry tetrahydrofuran under an argon atmosphere and cooled to 0°C. Next, 1ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC; 0.106 mmol) was slowly added and stirred at 0°C for 0.5 h, and the reaction mixture was allowed to warm to room temperature and stirred for 24 h. The reaction was monitored by TLC (Merck (Darmstadt, Germany) TLC aluminum sheets with silica gel 60 F254) using CHCl3/MeOH (10:1, v/v) as a mobile phase. For TLC visualization, ammonium molybdate with ceric sulfate in sulfuric acid was used. Next, the reaction mixture was evaporated and residue (50-55 mg) was purified by column chromatography (glass column; volume 100 mL, diameter 1.7 cm, length 20 cm) on silica gel (Merck Kieselgel 60, 0.040–0.063 mm) using 50:1 CHCl3/MeOH (v/v) as the mobile phase with approximately 1.5 mL/min flow rate.

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Melting points were measured on a Kofler apparatus and are uncorrected. The optical activity was measured using a Krűss optronic P3000 polarimeter (Krüss GmbH, Hamburg, Germany). 1

H and

13

C NMR spectra were recorded on a Varian Mercury Vx BB 300 or a VNMR S500

NMR spectrometer (Palo Alto, CA, USA). Chemical shifts were reported as δ values in parts per million (ppm) and were indirectly referenced to tetramethylsilane (TMS) via the solvent signal. Infrared spectra were measured on a Nicolet 6700 in the ATR mode (Thermo Scientific, Waltham, MA, USA). Mass spectrometry was performed with an LCQ Advantage Max (Thermo Finnigan, San Jose, USA) equipped with an APCI source. N-((2S,3S,4E)-1,3-dihydroxyoctadec-4-en-2-yl)-tetracosanamide (L-threo-CerNS). Yield = 62%, white crystals, Rf = 0.47 (CHCl3/MeOH, 10:1). M. p. = 84 – 87°C. [α]25D = –14.2 (c 0.99; CH3Cl/MeOH, 2:1). 1H NMR (500 MHz, CDCl3/MeOD, 10:1): δ = 5.69 (dt, J = 14.4, 6.3 Hz, 1H), 5.38 (dd, J = 15.8, 5.0 Hz, 1H), 4.34 – 4.27 (m, 1H), 4.07 – 3.98 (m, 2H), 3.85 – 3.74 (m, 1H), 2.20 (t, J = 6.7 Hz, 2H), 2.01 – 1.95 (m, 2H), 1.77 – 1.51 (m, 4H), 1.40 – 1.11 (m, 60H), 0.83 (t, J = 6.7 Hz, 6H) ppm.

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C NMR (125 MHz, CDCl3/MeOD, 10:1): δ = 173.7, 133.4,

128.9, 71.2, 62.6, 54.7, 36.5, 33.9, 32.2, 31.8, 29.6, 29.6, 29.5, 29.5, 29.4, 29.3, 29.3, 29.2, 29.1, 29.0, 22.6, 13.9 ppm. IR (ATR): ν = 3534, 2917, 2849, 1643, 1468, 721 cm-1. MS (APCI+): m/z 633.1 (M+H+ – H2O). N-((2S,3S)-1,3-dihydroxyoctadecan-2-yl)-tetracosanamide

(L-threo-CerNdS).

Yield = 68%,

white crystals, Rf = 0.48 (CHCl3/MeOH, 10:1). M. p. = 93 – 95°C, [α]25D = +2.8 (c 0.71; CH3Cl/MeOH, 2:1). 1H NMR (300 MHz, CDCl3/MeOD, 10:1): δ = 6.26 (d, J = 8.4 Hz, 1H), 4.03 – 3.85 (m, 2H), 3.83 – 3.66 (m, 1H), 3.29 – 2.99 (m, 1H), 2.22 (t, J = 6.8 Hz, 2H), 1.74 – 1.61 (m, 2H), 1.44 (m, 2H), 1.36 – 1.17 (m, 64H), 0.87 (t, J = 6.7 Hz, 6H) ppm.

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C NMR (75

MHz, CDCl3/MeOD, 10:1): δ = 173.4, 70.4, 64.4, 54.7, 34.1, 31.8, 29.6, 29.4, 29.3, 29.2, 29.1,

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29.0, 28.9, 28.8, 28.7, 25.0, 22.6, 14.0 ppm. IR (ATR): ν = 2915, 2848, 1643, 1557, 1472, 719 cm-1. MS (APCI+): m/z 653.1 (M+H+). Preparation of Lipid Membranes. Model SC lipid membranes were prepared in a similar manner as described previously9 as equimolar mixtures of Cer (D-erythro-Cer NS, or L-threoCerNS, or D-erythro-CerNdS, or L-threo-CerNdS), Chol, and FFAs, with 5 wt% of CholS.10 FFAs were mixed at a molar percentage that corresponds to the native composition of human skin FFAs, i.e., 1.8% hexadecanoic acid (C16:0), 4.0% octadecanoic acid (C18:0), 7.6% eicosanoic acid (C20:0), 47.8% docosanoic acid (C22:0) and 38.8% lignoceric acid (C24:0).11 The lipids were dissolved in 2:1 hexane/96% EtOH (v/v) at 4.5 mg/mL (note: 96% aq. EtOH is necessary to dissolve CholS). For permeability studies, these lipid solutions (3 × 100 µL per cm2) were slowly sprayed on Nuclepore polycarbonate filters with 15 nm pores (Whatman, Kent, UK) under a stream of nitrogen using a Linomat V (Camag, Muttenz, Switzerland) equipped with additional y-axis movement. The supporting filters did not significantly contribute to the barrier properties of the membranes.12 These lipid films were annealed at 90°C (a temperature that is above the main phase transitions of all the studied membranes) for 10 min and slowly (~3 h) cooled to room temperature. The membranes were equilibrated at 32°C and 30% air humidity for 24 h. The lipid films for FTIR experiments were prepared in the same way. X-Ray Powder Diffraction (XRPD). The lipid mixtures for the XRPD measurements were prepared in the same manner as those for permeation experiments, but the lipids were sprayed onto a 22 mm × 22 mm cover glass instead of polycarbonate filters. The XRPD data were collected at ambient temperature with an X’Pert PRO θ-θ powder diffractometer (PANalytical B.V., Almelo, Netherlands) with parafocusing Bragg-Brentano geometry using CuKα radiation (λ = 1.5418 Å, U = 40 kV, I = 30 mA) in modified sample holders over the angular range 0.6-30°

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(2θ). Data were scanned with an ultrafast detector X’Celerator with a step size of 0.0167° (2θ) and counting time of 20.32 s/step. For further details, see Ref.13 Fourier-Transform Infrared Spectroscopy (FTIR). Infrared spectra of the model SC lipid membranes were collected on a Nicolet 6700 spectrometer (Thermo Scientific, USA) equipped with a single-reflection MIRacle attenuated total reflectance ZnSe crystal (PIKE technologies, Madison, USA). A clamping mechanism with constant pressure was used. The spectra were generated by co-addition of 256 scans collected at a resolution of 2 cm-1. The temperature dependence of the IR spectra was studied from 28–100°C with 2°C steps using a temperature control module (PIKE technologies, Madison, USA).13 Permeation Experiments. The lipid membranes were mounted into Franz diffusion cells (a diffusion area of 0.5 cm2). The acceptor compartment of the cell (6.5 mL) was filled with phosphate-buffered saline (PBS, containing 10 mM phosphate buffer, 137 mM NaCl and 2.7 mM KCl) at pH 7.4 with 50 mg/L gentamicin. The acceptor phase was stirred at 32°C throughout the experiment. After a 12-h equilibration, the electrical impedance and water loss of the model membranes were measured, see below. Next, 100 µL of the model permeant—either 5% theophylline (TH) or 2% indomethacin (IND) suspensions in 60% propylene glycol—was applied to the membrane. Propylene glycol had no adverse effects on the membranes.12, 14-15 This setup ensured sink conditions for the selected compounds. Samples of the acceptor phase (300 µL) were withdrawn every 2 h over 10 h to reach steady state and were replaced with the same volume of PBS. TH and IND were analyzed by HPLC using a Shimadzu Prominence instrument (Kyoto, Japan) using previously validated methods.15

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Water Loss Measurement. Water loss through the model membranes [g/h/m2] was measured using a Tewameter® TM 300 probe and Multi Probe Adapter Cutometer® MPA 580 (CK electronic GmbH, Kӧln, Germany). The upper part of the Franz diffusion cell was removed and the probe was placed on the membrane holder containing a cylindrical hole, with a diameter of 0.8 cm (0.5 cm2), 0.6 cm from the membrane surface. Because the use of the membrane holder affects the measured values, we performed calibration measurements over various water/propylene glycol mixtures using an empty Nuclepore filter with and without the membrane holder.14 The environmental conditions were comparable during all measurements: ambient air temperature of 26-28°C and relative air humidity of 39-41%. Electrical Impedance. The electrical impedance (normalized to an area of 1 cm2; [kΩ × cm2]) of the model SC lipid membranes was recorded using an LCR meter 4080 (Conrad Electronic, Hirschau, Germany) operated in parallel mode with an alternating frequency of 120 Hz.16 To record the membrane impedance, the donor compartment of the Franz diffusion cell was filled with 0.5 mL of PBS, and the tips of the stainless-steel probes were carefully immersed in PBS in the donor and acceptor compartments of the diffusion cell.17 Data Analysis. The cumulative amount of drug that penetrated the lipid membrane (corrected for the acceptor phase replacement; [µg/cm2]) was plotted against time [h], and the steady-state flux [µg/h/cm2] was calculated from the linear region of the plot. Data are presented as the mean ± standard error of mean (SEM), and the number of replicates is given in the pertinent figure. Two groups of data were compared using unpaired t-tests; p