SPHINGOID BASES A MAJOR COMPONENT OF HUMAN SKIN

15 May

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).

CONCLUSIONS

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.

ACKNOWLEDGMENT

The authors wish  to thank Dr. Kees Korevaar for his  helpful contribution to the manuscript.

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