The membranes of all eukaryotic cells contain numerous classes of glycerolipids and sphingolipids. In the past decade, the long-neglected ceramides have become one of the most attractive lipid molecules in molecular cell biology, because of their involvement in essential structures (stratum corneum) and processes (cell sig- naling). Long-chain ceramides are among the most hydrophobic molecules in nature; they are totally insoluble in water and they hardly mix with phospholipids in membranes, giving rise to ceramide-enriched domains. Most natural ceramides have a long N-acyl chain comprising 16 – 22 C atoms, but short N-acyl chain cera- mides with two to six C atoms also exist in nature. These molecules have been extensively used in experimentation, because they can be dispersed easily in water.
Ceramides play major roles in maintaining the epidermal barrier (34). Depletion of ceramides, associated with disrupted barrier function in the epider- mis, leads to the clinical manifestation of dryness and inflammation. Besides their contribution to the structural integrity of a cell, ceramides are sphingoid- based signaling molecules that regulate cell cycle arrest, proliferation, differen- tiation, and apoptosis (35,36). There are specific structural and stereochemical requirements for the production of biological responses by the activation of specific biochemical targets. Endogenous levels of ceramides are regulated by a balance between its de novo synthesis and the rate of its breakdown to the corresponding sphingoid bases.
The core structures are sphingoid bases and constitute a large group of struc- turally diverse, biologically important long-chain amino alcohols, possessing a
2-amino-1,3-diol moiety. The most common member of this group found in nature is (2S,3R)-D-erythro-2-amino-1,3-octadec-4E-ene-diol, dubbed sphingosine. Another member is phytosphingosine (PS), typically consisting of an 18-carbon chain that incorporates a 2-amino-1,3,4-triol moiety at one end. It is a bioactive lipid that displays various physiological activities and it also occurs widely in animals, plants, and yeasts (2).
PS is present in human skin, but only in low concentrations, since it is an enzymatic breakdown product of ceramides. It comprises about 40% of the epider- mal sphingoid bases and plays an important part in regulating the micro-flora of the skin, since it mediates a wide variety of activities, such as stimulating the bio- synthesis of new PS-based ceramides by keratinocytes. Furthermore, it acts as a potent anti-inflammatory antagonist to a number of inflammatory cytokines, which are expressed by epidermal keratinocytes.
Both PS and sphingosine originate from sphinganine as a common precursor either by action of a desaturase introducing a (4E)-double bond to yield sphingosine or by action of a hydroxylase that adds a third hydroxy group to the C-4 of dihydro- ceramide to yield PS. The metabolic pathway starts from ceramides, and sphingoid bases are released upon amidolysis of the core structures. Both have shown to exert potent growth-suppressive effects in various cell types. Since PS and its derivatives are available only in a limited amount from natural sources, there is a continuing interest in developing efficient synthesis routes.
CURRENT SYNTHESIS ROUTES FOR SPHINGOID BASES
Sphingolipids are natural substances. More than 300 kinds of sphingolipids are known, displaying a wide variety of biological activities. However, access to sphin- golipids is not very straightforward. Although these compounds are widespread in nature, it is difficult to isolate substantial quantities because the concentration of sphingolipids in natural products is very low. Because sphingolipids have a very specific and complex three-dimensional structure, they are not easy to synthesize via chemical routes. Chemical routes to sphingoid bases are usually an economical challenge because their synthesis involves multiple protection and deprotection steps, as well as low yields due to necessary purification steps because of unwanted side products.
Economically, sphingolipids can be produced only in large quantities in a biotechnological process. Large quantities of the sphingoid-based PS and cera- mides are being produced via fermentation for the personal care industry. Through enzymatic modification and/or organic chemistry, fatty acids can be added to the building block to create ceramides. Degussa has developed a patented biofermentation process for the large-scale production of PS. The process utilizes a natural, non-GM yeast and the product is chemically identical to PS, which is naturally present in the human skin because of the same stereoche- mical D-erythro configuration. Because of this, a wide range of sphingolipids including ceramides and sphingoid bases such as PS are nowadays commercially available.
PS serves also as a starting material for a better and more efficient way to produce the naturally occurring D-erythro-sphingosine. Sphingosine and its most
prominent derivative sphingosine-1-phosphate (S1P) have been initially described as intermediates in the metabolic pathway of long-chain sphingoid bases. Now, it is widely accepted that S1P is a unique bioactive lipid messenger and is involved in a variety of cellular functions, including vascular maturation, angionesis, tumor necrosis factor-alpha signaling, regulation of cell motility, and in signal transduc- tion pathways of platelet-derived growth factor. The addition of sphingosine has been claimed, for example, to suppress significantly DNA synthesis in human ker- atinocytes, and can regulate proliferation and survival intracellularly. Also, it serves as a ligand for G protein-coupled receptors of the Edg-I subfamily extracellularly. The effect of Sphingosine and its derivatives are specific and depend on the pre- sence of the D4-double bond.
Today, 50 years after the report describing the preparation of racemic D-erythro-sphingosine (37), an efficient, cost-effective, and regio-/stereoselective synthesis has been developed, starting from the commercially available and cheap D-ribo-PS (38). As shown in Figure 2, first attempts were undertaken to generate 5 directly from 2; however, the used reaction conditions resulted in the formation of 7 which easily transformed into 8. The alternative route via 9 – 12 resulted in 5 with high yield (82%). However, further examination of a direct transformation from 2 to 5 resulted in the successful reaction conditions h. Sub- sequent reactions via 13 and 6 resulted in the desired regio- and stereo-selectively correct product 14. Finally, D-erythro-sphingosine was generated in high yield (70%) over seven steps.
PHYTOSPHINGOSINE POSSESSES MANY PROPERTIES THAT ARE USEFUL FOR THE TREATMENT OF ACNE
Firstly, PS possesses antimicrobial activity (39). The skin’s microflora is very diverse; however, they exist in a healthy equilibrium. This balance is partly due to the free fatty acids that are released by the bacterial breakdown of triglycerides, having a limiting action on numbers of microorganisms. However, more impor- tantly, is the liberation of free sphingoid bases, which show growth inhibitory activity against gram-positive bacteria, yeast, and moulds. It is suggested that by topical application of PS, the growth of undesirable microorganisms will be inhibited.
To examine the antimicrobial activity of PS, solutions of various PS-concen- trations were prepared (39). The final PS test solutions contained 0.83% ethanol,
1.5% Tween 80, and 267 – 1066 mg/L PS. As described by Bibel et al. (40), ethanol inhibits the growth of microorganisms by itself. In our studies, however, we have used an ethanol concentration that is far below the inhibitory level. Table 1 shows the determined minimal inhibitory concentration of PS for different microorganisms.
Second, PS affects inflammation. The outcome of several different gene expression studies (41) on cultured primary human keratinocytes was as follows:
1. The expression of proinflammatory chemokines like IL-8, CXCL2, and endothelin-1 was significantly down regulated.
2. The regulation of various genes involved in cellular reactive oxygen species (ROS) metabolism was observed, indicating that these compounds may sub- stantially modulate the skin’s capacity to handle ROS, and thus also encounter inflammation.
3. The expression of differentiation markers like loricrin, involucrin, transglutami- nase1, and filaggrin was induced after treatment with PS. This leads to a shift from proliferation to differentiation and thus reduces the symptoms of hyperkeratinization.
Along this line, experiments with reconstituted human epidermis (SkinEthicTM) (42) were performed. The reconstituted human epidermis consists of a three-dimensional, multilayered keratinocyte structure grown on an air – liquid interphase, without any other cell type. Inflammatory conditions were simu- lated by the topical application of a 0.35% sodium dodecyl sulphate (SDS) solution for 40 minutes. Afterward, an O/W formulation containing 0.2% PS and a placebo formulation without PS were applied to the treated and untreated skin model for 28 hours. For the determination of general viability effects, an XTT assay was per- formed and the total protein amount was assigned using a Bradford test. Addition- ally, potential cytotoxicity of the sample was excluded by the measurement of lactate dehydrogenase release. The IL-1 amount was quantified using an ELISA- kit. The results demonstrated that IL-1 was down regulated on the protein level after treatment with PS. Under unstressed conditions, the release of IL-1 was decreased by 52% 24 hours after the topical application of 0.2% PS out of a basic cream formulation. The incubation of the reconstituted human epidermis with SDS prior to the experiment led to a 3.4, -fold increase in IL-1 secretion (12.8 –
42.9 pg/mL), mimicking an inflamed skin status. Under these conditions, the appli- cation of PS even decreased the release of IL-1 by 69%.
Additional benefits may be derived from the effect of topical PS, as it is known to act as a precursor to ceramides, and PS-based ceramides are reduced in the skin of acne subjects. It has been demonstrated by electrospray-ionization-mass spec- troscopy on extracts of the lipid phase of cultured keratinocytes (43) that PS can be taken up efficiently by the cells. Furthermore, PS can be metabolized and con- verted to glucosylceramides, which are the precursors of the different barrier cera- mides. In our experiments (43), the level of PS -derived barrier ceramides was increased, too. This indicates that the depleted PS-ceramide reservoirs that occur during acne can be filled up by the topical application of PS. However, the level of PS-derived ceramides was not increased by the upregulation of gene expression. Genes that are involved in the ceramide synthesis pathway, like fatty acid synthase, serine palmitoyl transferase, or UDP-glucose ceramide glycosyltransferase did not show a significantly increased expression profile (41).
Nevertheless, the best way to demonstrate the efficacy of PS is in a clinical study (44). Volunteers with moderate, inflamed acne on the face participated in a half-face study. Objective criteria for the studies were based on “Echelle d’e´ valu- ation clinique des lesions d’acne´ ”/ECLA/Clinical evaluation of acne injuries: the number of comedones, the number of pustules, and the number of papules.
The evaluated products were applied on one-half of the face. The evaluated pro- ducts included the commonly used compound BPO as the benchmark ingredient against acne. Since the combination of BPO and PS in a formulation has been shown to be unstable (chemical interaction between the active ingredients), the two compounds have to be separated from each other. Therefore, a two- chamber dispenser was used. The following trial combinations were chosen:
B Placebo versus 0.2% PS (15 volunteers aged 10 – 50 years)
B 4% BPO versus 0.2% PS þ 4% BPO formulated in a two-chamber dispenser
(30 volunteers aged 15 – 25 years)
The consultations of the volunteers with the dermatologist were on day 0, day 30, and day 60. Neither PS nor BPO caused cutaneous intolerance throughout the study period. However, PS had a strong effect on diminishing the number of papules, pustules, but not comedones. After 60 days of treatment with 0.2% PS, papules and pustules were decreased by 89% (Table 2). In addition, PS is perfectly tolerated by the skin and shows a very good action against inflammatory superficial acne. Despite a good cutaneous acceptance, the antibacterial potential of BPO was only able to decrease the observed papules and pustules, and comedones by 32% and 22% (Table 2), respectively, after 60 days of treatment. However, the combi- nation of 0.2% PS with 4% BPO resulted in a synergistic effect. Already, after 30 days of treatment, the number of papules plus pustules and comedones were diminished to 60% and 43%, respectively, compared with 25% and 26% (“ 2 ” corresponds to an increase!) respectively, with PS only, or to 10% and 15% with BPO only (Table 2). Although PS was not able to prevent the formation of come- dones, it was able to at least control the number of comedones that were induced by the placebo formulation alone.
Subjective efficacy was clear: the skin was less inflamed, and the volunteers noticed clearly a rapid improvement in the inflammatory lesions and a diminution of comedones. In most cases, the effect was noticed after one week of treatment. The use of PS resulted in a softening and “comfort” of the skin, which the volunteers had lost when they developed acne. Both PS and the combination product (PS and BPO) were considered to have the following characteristics: easy to use, good cosmetic quality, good cosmetic acceptability with ease of use, and nongreasy appearance. Even make-up could still be applied without problems.
Taken together, the effects as an anti-inflammatory and a bacteriostatic agent could be the reasons for the reduction of comedone formation compared with placebo (Fig. 3).
Acne is a common dermatological diagnosis that affects boys and girls during puberty and may persist throughout adulthood. Anti-acne therapies target: (i) the enhanced sebum production, (ii) the enhanced hyperkeratinization, (iii) the increased P. acnes colonization, and (iv) the inflammatory response. In addition, there is increasing evi- dence for the need of the treatment of the non-lesional sites to prevent comedogenesis and to improve skin barrier function. On the basis of the latest scientific findings, agents that possess anti-inflammatory effects are highly interesting because they seem to represent a useful adjunct to the established anti-acne armory.
PS is naturally occurring in human skin since it is an enzymatic breakdown product of ceramides. It comprises about 40% of the epidermal sphingoid bases and plays an important part in regulating the microflora of the skin. A commercial bio- fermentation process for PS has been established and is based on a non-GM yeast. PS obtained via this route is chemically identical to the material found in human skin because of the same stereochemical D-erythro configuration. This unique con- figuration is crucial for optimal performance since only the right three-dimensional structure makes favorable interactions with any stereoselective cellular targets.
Starting from PS, an economical and efficient chemical route for another sphingoid base, sphingosine, has been developed. The skin-identical sphingosine is generated by a seven-step chemical reaction cascade in high yield and represents a bioactive sphingoid base with yet unexplored profiles but should potentially be also an effective molecule to treat the blemished skin. First study results do point is this direction.
PS has been proven to be a clinically useful adjunct for the treatment of acne. The use of PS resulted in a softening and comfort of the skin, which the volunteers
had lost when they developed acne. Both PS and a combination product of PS and BPO were considered to be effective treatments and offer qualities such as good cos- metic acceptability with ease of use and non-greasy appearance.
Taken together, the effects of PS as an anti-inflammatory and a bacteriostatic agent could be considered a new effective way to treat acne-prone skin and might find its way into a number of skin products designed for this specific purpose.
The authors wish to thank Dr. Kees Korevaar for his helpful contribution to the manuscript.