Jarmila Zbytovská

Amphiphilic transdermal permeation enhancers: structure-activity relationships.

Transdermal drug delivery offers numerous advantages over conventional routes of administration; however, poor permeation of most drugs across the skin barrier constitutes a serious limitation of this methodology. One of the approaches used to enlarge the number of transdermally-applicable drugs uses permeation enhancers. These compounds promote drug permeation through the skin by a reversible decrease of the barrier resistance. Enhancers can act on the stratum corneum intracellular keratin, influence desmosomes, modify the intercellular lipid domains or alter the solvent nature of the stratum corneum. Even though, hundreds of substances have been identified as permeation enhancers to date, yet our understanding of the structure-activity relationships is limited. In general, enhancers can be divided into two large groups: small polar solvents, e.g. ethanol, propylene glycol, dimethylsulfoxide and amphiphilic compounds containing a polar head and a hydrophobic chain, e.g. fatty acids and alcohols, 1-dodecylazepan-2-one (Azone), 2-nonyl-1,3-dioxolane (SEPA 009), and dodecyl-2-dimethylaminopropanoate (DDAIP). In this review we have focused on structure-activity relationships of amphiphilic permeation enhancers, including the properties of the hydrophobic chains, e.g. length, unsaturation, and branching, as well as the polar heads characteristics, e.g. hydrogen bonding ability, lipophilicity, and size. We present over 180 examples of enhancers with different polar head to illustrate the structural requirements and the possible role of the polar head. We have given an overview of the methods used for investigation of the mechanisms of permeation enhancement, namely differential scanning calorimetry (DSC), infrared (IR) and Raman spectroscopy, X-ray diffraction and future perspectives in this field. Furthermore, biodegradability and chirality of the enhancers are discussed.

Effect of ceramide acyl chain length on skin permeability and thermotropic phase behavior of model stratum corneum lipid membranes

Stratum corneum ceramides play an essential role in the barrier properties of skin. However, their structure-activity relationships are poorly understood. We investigated the effects of acyl chain length in the non-hydroxy acyl sphingosine type (NS) ceramides on the skin permeability and their thermotropic phase behavior. Neither the long- to medium-chain ceramides (8-24 C) nor free sphingosine produced any changes of the skin barrier function. In contrast, the short-chain ceramides decreased skin electrical impedance and increased skin permeability for two marker drugs, theophylline and indomethacin, with maxima in the 4-6C acyl ceramides. The thermotropic phase behavior of pure ceramides and model stratum corneum lipid membranes composed of ceramide/lignoceric acid/cholesterol/cholesterol sulfate was studied by differential scanning calorimetry and infrared spectroscopy. Differences in thermotropic phase behavior of these lipids were found: those ceramides that had the greatest impact on the skin barrier properties displayed the lowest phase transitions and formed the least dense model stratum corneum lipid membranes at 32°C. In conclusion, the long hydrophobic chains in the NS-type ceramides are essential for maintaining the skin barrier function. However, this ability is not shared by their short-chain counterparts despite their having the same polar head structure and hydrogen bonding ability.

Ceramides in the skin lipid membranes: length matters.

Ceramides are essential constituents of the skin barrier that allow humans to live on dry land. Reduced levels of ceramides have been associated with skin diseases, e.g., atopic dermatitis. However, the structural requirements and mechanisms of action of ceramides are not fully understood. Here, we report the effects of ceramide acyl chain length on the permeabilities and biophysics of lipid membranes composed of ceramides (or free sphingosine), fatty acids, cholesterol, and cholesterol sulfate. Short-chain ceramides increased the permeability of the lipid membranes compared to a long-chain ceramide with maxima at 4-6 carbons in the acyl. By a combination of differential scanning calorimetry, Fourier transform infrared spectroscopy, X-ray diffraction, Langmuir monolayers, and atomic force microscopy, we found that the reason for this effect in short ceramides was a lower proportion of tight orthorhombic packing and phase separation of continuous short ceramide-enriched domains with shorter lamellar periodicity compared to native long ceramides. Thus, long acyl chains in ceramides are essential for the formation of tightly packed impermeable lipid lamellae. Moreover, the model skin lipid membranes are a valuable tool to study the relationships between the lipid structure and composition, lipid organization, and the membrane permeability.

l-Serine and glycine based ceramide analogues as transdermal permeation enhancers: polar head size and hydrogen bonding

Novel transdermal permeation enhancers related to stratum corneum ceramides were investigated. The synthesis of a series of simple compounds based on two selected amino acids, L-serine and glycine, and their enhancement activities are reported. Glycine derivative 3 enhanced the permeation of theophylline through human skin in vitro 12.5+/-0.5 times. The relationships between properties of the polar head of the compounds and their activity are discussed.

Effects of sphingomyelin/ceramide ratio on the permeability and microstructure of model stratum corneum lipid membranes

The conversion of sphingomyelin (SM) to a ceramide (Cer) by acid sphingomyelinase (aSMase) is an important event in skin barrier development. A deficiency in aSMase in diseases such as Niemann-Pick disease and atopic dermatitis coincides with impaired skin barrier recovery after disruption. We studied how an increased SM/Cer ratio influences the barrier function and microstructure of model stratum corneum (SC) lipid membranes. In the membranes composed of isolated human SC Cer (hCer)/cholesterol/free fatty acids/cholesteryl sulfate, partial or full replacement of hCer by SM increased water loss. Partial replacement of 25% and 50% of hCer by SM also increased the membrane permeability to theophylline and alternating electric current, while a higher SM content either did not alter or even decreased the membrane permeability. In contrast, in a simple membrane model with only one type of Cer (nonhydroxyacyl sphingosine, CerNS), an increased SM/Cer ratio provided a similar or better barrier against the permeation of various markers. X-ray powder diffraction revealed that the replacement of hCer by SM interferes with the formation of the long periodicity lamellar phase with a repeat distance of d=12.7nm. Our results suggest that SM-to-Cer processing in the human epidermis is essential for preventing excessive water loss, while the permeability barrier to exogenous compounds is less sensitive to the presence of sphingomyelin.

Influence of aggregation on interaction of lipophilic, water-insoluble azaphthalocyanines with DOPC vesicles.

Dioleoylphosphatidylcholine unilamellar vesicles made by extrusion technique (LUVETs) were studied as the delivery system for lipophilic water‐insoluble potential photosensitizers for photodynamic therapy (PDT). Two azaphthalocyanines (AzaPcs) with hydrophobic substituents only and two also possessing two charged amino groups were introduced into the study. All compounds are insoluble in water and form aggregates in PBS with tetrahydrofuran as cosolvent. The size of these aggregates depends on the concentration of AzaPc in solution. AzaPcs with tert‐butyl substituents were found to be incorporated into the lipid bilayer of vesicles in the monomeric form even at high concentrations. The stability of LUVETs with incorporated AzaPc was excellent for at least 4 weeks. Therefore, they are suitable for use as a delivery system for these water‐insoluble photosensitizers. Very low amount of AzaPc with n‐octyl substituents incorporated into LUVETs due to its stronger self‐aggregation. Values of binding constants determined for all AzaPcs showed inverse order than expected from their lipophilicities. However, the binding constants followed the order of the strength of aggregation forces. Aggregation of AzaPcs in water medium plays a very important role in the interaction of AzaPcs with LUVETs.

Effect of lipid nanoparticle formulations on skin delivery of a lipophilic substance

The aim of this study was to follow the skin penetration of a model lipophilic compound (Nile red) delivered by nanoparticulate carriers, the so-called lipid nanocapsules. The nanocapsules consisting of an oil core stabilized by amixture of surfactants were prepared by the phase inversion temperature method. Varying the particle composition (the oil/surfactant ratio) nanoparticles of different size were prepared and characterized. The penetration profile of Nile red delivered into the porcine skin by the nanoparticles compared to non-particulate samples was determined using fluorescence microscopy combined with a novel, statistically robust quantitative image analysis method. This study demonstrated that lipid nanoparticles promoted the skin penetration of encapsulated Nile red in comparison with all the non-particulate samples. Nile red delivered by the lipid-based nanoparticles was able to diffuse across the stratum corneum and partition itself uniformly in the epidermis. No relationship between Nile red penetration into the skin and the particle size was found. Moreover, the presence of sodium chloride in the water phase had a negative impact on the Nile red penetration into the skin. The results indicate that the physico-chemical circumstances of the nanoparticulate formulation play the major role in the penetration of lipophilic substances into the skin.

Permeability Barrier and Microstructure of Skin Lipid Membrane Models of Impaired Glucosylceramide Processing

Ceramide (Cer) release from glucosylceramides (GlcCer) is critical for the formation of the skin permeability barrier. Changes in β-glucocerebrosidase (GlcCer’ase) activity lead to diminished Cer, GlcCer accumulation and structural defects in SC lipid lamellae; however, the molecular basis for this impairment is not clear. We investigated impaired GlcCer-to-Cer processing in human Cer membranes to determine the physicochemical properties responsible for the barrier defects. Minor impairment (5–25%) of the Cer generation from GlcCer decreased the permeability of the model membrane to four markers and altered the membrane microstructure (studied by X-ray powder diffraction and infrared spectroscopy), in agreement with the effects of topical GlcCer in human skin. At these concentrations, the accumulation of GlcCer was a stronger contributor to this disturbance than the lack of human Cer. However, replacement of 50–100% human Cer by GlcCer led to the formation of a new lamellar phase and the maintenance of a rather good barrier to the four studied permeability markers. These findings suggest that the major cause of the impaired water permeability barrier in complete GlcCer’ase deficiency is not the accumulation of free GlcCer but other factors, possibly the retention of GlcCer bound in the corneocyte lipid envelope.

Physical Compatibility of Propofol-Sufentanil Mixtures.

BACKGROUND: Combined infusions of propofol and sufentanil preparations are frequently used in clinical practice to induce anesthesia and analgesia. However, the stability of propofol emulsions can be affected by dilution with another preparation, sometimes leading to particle coalescence and enlargement. Such unwanted effects can lead to fat embolism syndrome after intravenous application. This study describes the physical stability of 5 commercially available propofol preparations mixed with sufentanil citrate solutions. METHODS: Two common markers of emulsion stability were used in this study; namely, the zeta potential and size distribution of the emulsion droplets. Both were measured using dynamic light scattering. The data for the pure propofol preparations and their mixtures with sufentanil citrate solution were compared. RESULTS: The absolute value of zeta potential decreased in 4 of the 5 propofol preparations after they had been mixed with sufentanil citrate. This effect indicates a lowering of repulsive interactions between the emulsion droplets. Although this phenomenon tends to cause agglomeration, none of the studied mixtures displayed a substantial increase in droplet size within 24 hours of blending. However, our long-term stability study revealed the instability of some of the propofol–sufentanil samples. Two of the 5 studied mixtures displayed a continual increase in particle size. The same 2 preparations showed the greatest reductions in the absolute value of zeta potential, thereby confirming the correlation of both measurement methods. The increase in particle size was more distinct in the samples stored at higher temperatures and with higher sufentanil concentrations. CONCLUSIONS: To ensure the microbial stability of an emulsion infusion preparation, clinical regulations require that such preparations should be applied to patients within 12 hours of opening. In this respect, we can confirm that during this period, none of the studied propofol–sufentanil mixtures displayed any physical instability that could lead to particle enlargement; thus, fat embolism should not be a risk after their intravenous application. However, our long-term stability study revealed differences between commercially available preparations containing the same active ingredient; some of the mixtures showed an increase in particle size and polydispersity over a longer period. Although our results should not be generalized beyond the particular propofol–sufentanil preparations and concentrations studied here, they do suggest that, as a general principle, a compatibility study should be performed for any preparation before the first intravenous application to exclude the risk of droplet aggregation.

Simplified stratum corneum model membranes for studying the effects of permeation enhancers

The activity of transdermal permeation enhancers is usually evaluated in vitro on human or animal skin, but skin samples can be hard to source and highly variable. To provide a more consistent basis for evaluating the activity of permeation enhancers, we prepared relatively simple and inexpensive artificial membranes that imitate the stratum corneum (SC) lipid matrix. Our membranes were composed of stearic acid, cholesterol, cholesterol sulfate and a ceramide (CER) component consisting of N-2-hydroxystearoyl phytosphingosine (CER[AP]) and/or N-stearoyl phytosphingosine (CER[NP]). First, the permeation of theophylline (TH) and indomethacin (IND) through these membranes was compared with their permeation through porcine skin. Because the mixed CER[AP]/[NP] membrane gave the closest results to skin, this membrane was then used to test the effects of two permeation enhancers: N-dodecyl azepan-2-one (Azone) and (S)-N-acetylproline dodecyl ester (L-Pro2). Both enhancers significantly increased the flux of TH and IND through the skin and, even more markedly, through the lipid membrane, L-Pro2 having a stronger effect than Azone. Thus, our simplified model of the SC lipid membrane based on phytosphingosine CERs appears to be suitable for mimicking skin permeation.