FORM - 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL SPECIFICATION
(See Section 10 and Rule 13)
NOVEL PSEUDOCERAMIDES
HINDUSTAN UNILEVER LIMITED, a company incorporated under
the Indian Companies Act, 1913 and having its registered office
at 165/166, Backbay Reclamation, Mumbai -400 020, Maharashtra, India
The following specification describes the invention

FIELD OF INVENTION
The present invention relates to Pseudoceramides and a process for making the same.
The invention has been developed primarily for use in cosmetic compositions and will be described hereinafter with reference to this application. However, it will be appreciated that the invention is not limited to this particular field of use.
BACKGROUND AND RELEVANT ART
Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.
The top layer of human skin or the epidermis is composed of many different cell types including keratinocytes, melanocytes and Langerhans cells. Keratinocytes are the major cell type of the epidermis (75-80% of the total number of cells in the human epidermis). Within the epidermis, the keratinocyte resides in four distinct stages of differentiation. The basal layer rests in the basal lamina separating epidermis from the dermis. These cells are largely columnar, rapidly proliferating cells. These basal cells migrate upward within the epidermis, initiated by the process of differentiation. The topmost layer of the skin, the stratum corneum, is formed from the granular layer by the destruction of the cell organelles. The cells in the stratum corneum; corneocytes, contain extensively cross-

linked proteins, surrounded by a highly resistant cell envelope. The corneocytes are embedded in a bed of specific lipid structures mainly consisting of glycolipids, cholesterol, free fatty acids and Ceramides. Among them, the Ceramides play an important role in maintaining well-balanced water content which is involved in skin elasticity, appearance and the protective barrier for the skin. The outermost layer of the corneocytes is peeled off from the skin during the normal process of desquamation and upper layer is constantly being regenerated. This loss at the surface of the skin is compensated by the migration of cells from the basal layer to the surface of the epidermis. It entails the perpetual renewal of the skin. Skin aging or damage caused by detergents which remove the lipids essential for the barrier function may disturb the lipids and reduce Ceramide content in the stratum cornuem. Thus, cell cohesion may be weakened and the stratum corneum may not serve as a protective barrier. The skin may lose elasticity. As the Ceramide content decreases, trans-epidermal water loss increases, resulting into dry skin-condition, direct exposure to the exterior irritation such as UV radiation or chemicals and peeling off the stratum cornuem; thereby making the skin rough and damaged.
It has been reported that the external application, such as in cosmetic or pharmaceutical base, of Ceramides can recover the lamellar structure disturbed by skin aging, or the damage of the stratum corneum. Thus stratum corneum can fully function as a protective barrier.
Ceramides are class of lipid molecules. It is composed of Sphingosine and a fatty acid. Ceramides are the major

lipidic components of skin barrier. They are amides of fatty acids with long-chain di-or trihydroxy bases, the commonest in animals being "Sphingosine" and in plants being "Phytosphingosine". The acyl part of Ceramides is generally a long chain saturated or monosaturated fatty acid. The most frequent fatty acids found in animal Ceramides are 18:0, 24:0 and 24:1. In addition, long-chain hydroxyl fatty acids are also found.
A representative structure of Ceramide where R=H is given below:

Stratum corneum intercellular lipids play an important role in regulation of skin water barrier homeostasis and water holding capacity. Lipids in general are not hygroscopic molecules, and therefore are unable to bind water. Due to presence of polar -OH (hydroxyl) head groups, the Ceramides are capable of forming hydrogen bonds with water molecules, thereby creating a favourable environment for movement of water into the skin. Thus, Ceramides play a critical role in water holding capacity of stratum corneum.
Pseudoceramides, generally have structures that differ more clearly from the basic structure of natural Ceramides and are often designed for cosmetics application. Skin, when exposed to factors such as extreme cold, sunrays, low relative humidity and repeated washing and contact with organic solvents loses its barrier properties. Ceramides

have been used in cosmetics, in order to solve this problem. Natural sources of Ceramides are generally not fit for use in cosmetics, because of inter-alia, bacterial contamination, conservation difficulty and BSE (bovine spongiform encephalopathy). To overcome these problems, synthetic Ceramides have been proposed. These compounds, when used in cosmetics formulations, have shown to bring moisturizing effect which has been found to control or prevent drying of skin.
Examples of some reported synthetic Ceramides are given below, which have been described in Journal of Lipid Research, Vol. 45, 923-932, 2004.

Representative prior arts on Pseudoceramides include
EP1480947 (COREANA COSMETICS CO LTD, 2007) and WO2007/147370 (UNIVERZITA KARLOVA V PRAZE FAR, et al)
Pseudoceramides have similar effect on lamellar structure as in all cases similar to that of natural Ceramides.


OBJECT OF THE INVENTION
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art.
It is an object of this invention to provide novel Pseudoceramides.
It is an object of the present invention to provide Pseudoceramides that would give moisturizing effect and induce keratinocyte differentiation.
The present inventors have developed a novel class of Pseudoceramides, which has a Cholic acid base. Cholic acid is a molecule from the class of bile acids. The bile acids are a family of natural products consisting of a facial amphiphilic steroid molecule with a polar side chain. It has been found by the present inventors that the novel compound is able to induce keratinocyte differentiation, and hence it provides a novel and useful cosmetic ingredient.


SUMMARY OF THE INVENTION
According to a first aspect, there is provided a compound of the general formula:
R2

where R is either -H, -CH3 or -C2H5, and atleast two of the stereo bonds have "a" configuration; R1 is a linear or branched C8-C22 Carbon chain, R2 is either -H, -CH3 or -C2H5 and where n= 1 to 8.

According to the second aspect, the present invention provides A process for making a compound according to the first aspect, comprising a step of reacting a compound of the formula (I) with a compound of the formula (II) in the presence of a coupling agent, an agent for activation of carboxylic group of (I), a solvent, and a base, at temperature in the range of 40 °C to 100 °C.

where R is either -H, -CH3 or -C2H5, and atleast two of the
stereo bonds have "a" configuration; R1 is a linear or

branched C8-C22 Carbon chain, R2 is either -H, -CH3 or -C2H5 and where n= 1 to 8.
According to the third aspect, the present invention provides a cosmetic composition comprising 0.0001% by weight to 20% by weight compound according to the first aspect, in a cosmetically acceptable carrier.
According to the fourth aspect, the present invention provides a method of treating skin, comprising applying to the skin, a cosmetic composition comprising the compound according to the first aspect.
According to the fifth aspect, the present invention provides use of a compound according to the first aspect for epidermal keratinocyte differentiation.
According to the sixth aspect, the present invention provides multivesicular liposomes of the compound according to the first aspect of the invention.
According to the seventh aspect, the present invention provides a cosmetic composition comprising multivesicular liposomes of the compound according to the first aspect of the invention.
DETAILED DESCRIPTION OP THE INVENTION
According to the first aspect, the present invention relates to a compound of the general formula:

where R is either -H, -CH3 or -C2H5, and atleast two of the stereo bonds have "a" configuration; R1 is a linear or branched C8-C22 Carbon chain, R2 is either -H, -CH3 or -C2H5 and where n= 1 to 8.
In another aspect, the present invention provides a process for making a compound according to the first aspect, comprising a step of reacting a compound of the formula (I) with a compound of the formula (II) in the presence of a coupling agent, an agent for activation of carboxylic group of (I), a solvent, and a base at temperature in the range of 40 °C to 100 °C.

R-
(I) (ID
where R is either -H, -CH3 or -C2H5, and atleast two of the stereo bonds have "a" configuration; R1 is a linear or branched C8-C22 Carbon chain, R2 is either -H, -CH3 or -C2H5 and where n= 1 to 8.


The phrase "Carbon chain" is meant herein to include saturated and unsaturated chains. It is preferred that R1 is an alkyl group, more preferably a linear C10-C18 alkyl group, and most preferably R1 is a linear C12 alkyl group. It has been observed by the present inventors that when R1 is C12 then the phase transition temperatures of the resulting Pseudoceramides are within the range of phase transition temperatures of skin Ceramides therefore making it compatible for the skin applications.
Preferably, atleast 2 of the R is -H, thereby making the compound facial amphiphilic. It is highly preferred that each of the R is -H, thereby making all three groups as -OH (hydroxyl) groups. It is known for cooperative hydrogen bonding which aids bimolecular aggregate formation.
Preferably, all three stereo bonds have "a" configuration. This helps in making the Cholic acid moiety (i.e the compound I in the process according to the invention, which eventually forms a part of the compounds according to the invention) facially amphiphilic. Without wishing to be bound by theory, it is believed that the presence of Cholic acid residue, represented as (I) above, in the backbone of Pseudoceramides may assist the formation of water filled region in the stratum corneum. It is further believed that Cholic acid molecules when anchored to the backbone can form channels in model membranes like phosphotidylcholines.
It is preferred that each of the R2 is -H, which is commonly present among natural Ceramides.


It is preferred that n= 1 to 4 and it is highly preferred that n= 1 or 2. In a most preferred aspect, n= 2.
It is preferred that the agent for activation of carboxylic group of (I) is selected from N-HydroxyBenzotriazenone, N, N-dimethyl-4-aminopyridine) , N-Hydroxy-benzotriazole, N-hydroxybenzotriazine, N-hydroxysuccinimide or 2-hydroxypyridine. This is used for making insitu activated ester of Cholic acid. The more preferred agent is N-HydroxyBenzotriazenone.
The preferred temperature of the process is in the range of 60 °C to 80 °C.
The preferred solvent for carrying out the reaction between (I) and (II) is selected from dry Dimethylformamide, Dimethylsulphoxide or Dimethylacetamide, and more preferably, dry Dimethylformamide.
While several bases selected from the group consisting of Triethylamine, Diisopropylamine, Pyridine, Sodium hydroxide, Potassium hydroxide, Sodium carbonate or Potassium carbonate can be used, it is preferred that the base is Triethylamine.
The preferred coupling agent is selected from Dicylcohexyl carbodiimide (DCC) or l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, with Dicylohexylcarbodiimide (DCC) being more preferred.
A highly preferred compound according to the present invention has the following structure.

The present invention also provides a cosmetic composition comprising 0.0001% by weight to 20% by weight compound according to the first aspect of the invention in a cosmetically acceptable carrier.
The invention also provides a method of treating skin, comprising applying to the skin, a cosmetic composition comprising the compound according to the first aspect of the invention.
The invention also relates to use of a compound according to the first aspect of the invention for the treatment of skin conditions.
It has been observed by the present inventors that the compound according to the first aspect of the invention is able to form multivesicular liposomes. Accordingly the invention, in another aspect, relates to multivesicular liposomes of the compound of the first aspect. The benefits of the multivesicular liposomes are as follows. Multivesicular liposomes (MVLs) are uniquely distinct from conventional unilamellar liposomes in composition, structure, and size and are the only class of commercial liposomes that have demonstrated depot delivery of both


biologically active small molecule and protein/peptide drugs. These MVLs are characterized by the presence of a continuous bilayer membrane, with numerous internal aqueous compartments that are contiguous and separated by bilayer septums. In contrast to multilamellar liposomes, the multiple aqueous chambers of multivesicular liposomes are non-concentric. As a result of their larger size (median diameter typically 10-30 |om) , these MVLs are not rapidly cleared by tissue macrophages and can act as a drug/cosmetic depot providing slow release of drugs/cosmetic actives delivered through different routes of administration. Moreover, the biocompatibility and biodegradability of the MVL lipid matrix allows for the sustained delivery of biologically active molecules in desired areas of application. The unique architecture of MVLs provides high molecule loading of water-soluble biologically active molecules, reasonable stability during storage, and control over the active molecules release-rate.
The present invention also relates to a cosmetic composition comprising the multivesicular liposomes of the compound in accordance with the first aspect of the invention.
In the cosmetic compositions comprising the novel compounds (Pseudoceramides) according to the invention, the Pseudoceramide is preferably present in an amount of 0.0001 % by weight to 20 % by weight, on the total weight of the composition. The preferred amount of the Pseudoceramide is 0.0005 % to 10 % by weight, and more preferably, 0.005 % to 10 % by weight. If the amount is less than 0.0001%, a moisturizing maintenance effect may be negligible; and in the case of exceeding 20 wt%, the increase of moisturizing


maintenance effect in parallel with the increase of amount may be rarely represented. Similarly, the novel multivesicular liposomes of the novel Pseudoceramides according to the invention may be formulated into cosmetic compositions, and it is preferred that the liposomes are present from 0.0001 % by weight to 10 % by weight, based on the total weight of the composition, more preferably from 0.0005% to 10%, and most preferably from 0.005% to 10% by weight.
The cosmetic compositions of this invention may be formulated in a wide variety of forms, for example, including a solution, a suspension, an emulsion, a paste, an ointment, a gel, a cream, a lotion, a powder, soap, a surfactant-containing cleanser, an oil, a powder foundation, an emulsion foundation, a wax foundation and a spray.
The cosmetically acceptable carrier contained in the present cosmetic composition, may be varied depending on the type of the formulation. For example, the formulation of ointment, pastes, creams or gels may comprise animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silica, talc, zinc oxide or mixtures of these substances. In the formulation of powder or spray, it may comprise lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder and mixtures of these substances. Spray may additionally comprise the customary propellants, for example, chlorofluorohydrocarbons, propane/butane or dimethyl ether.


The formulation of solution and emulsion may comprise solvent, solubilizer and emulsifier, for example water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol, oils, in particular cottonseed oil, groundnut oil, maize germ oil, olive oil, castor oil and sesame seed oil, glycerol fatty esters, polyethylene glycol and fatty acid esters of sorbitan or mixtures of these substances. The formulation of suspension may comprise liquid diluents, for example water, ethanol or propylene glycol, suspending agents, for example ethoxylated isosteary alcohols, polyoxyethylene sorbitol esters and poly oxyethylene sorbitan esters, micocrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth or mixtures of these substances.
The formulation of soap may comprise alkali metal salts of fatty acids, salts of fatty acid hemiesters, fatty acid protein hydrolyzates, isethionates, lanolin, fatty alcohol, vegetable oil, glycerol, sugars or mixtures of these substances.
Furthermore, the cosmetic compositions according to an aspect of this invention may contain auxiliaries. The non-limiting examples of auxiliaries include preservatives, antioxidants, stabilizers, solubilizers, vitamins, colorants, odour improvers or mixtures of these substances.
The invention will now be described with reference to the following non-limiting exemplary embodiments.


EXAMPLES
EXAMPLE-1
Synthesis of a compound according to the invention
Dodecyl amine was obtained from Aldrich, Glycidol from Fluka, Cholic acid from S.D. Fine chemicals India and N-hydroxybenzotriazenone from Aldrich. Following instruments were utilised for spectral and other studies.
lH Nuclear Magnetic Resonance (NMR): Bruker (400/500 MHz)
IR spectroscopy: PerkinElmer
MASS Spectrum : Bruker Daltonics
Differential Scanning Calorimetry (DSC): Perkin-Elmer
CryoSEM (Scanning Electron Microscopy): Hitachi
The novel compound (Pseudoceramide, Compound-A) was prepared in a two-step manner.
Step-1
Synthesis of n- (2, 3-dihydroxypropyl) -n-Dodecylamine
(indicated as (II) , as in the process according to the invention)
Dodecylamine (1000 mg, 5.4 mmoles) was taken in 150 ml dry Toluene, AR. Glycidol (450 ul, 6.7 mmoles) was taken in 100 ml of dry Toluene, AR, added dropwise and heated for 8 hours at 60 °C. Then toluene was evaporated under vacuum on rotavap evaporator. After evaporation, a white solid was obtained, which was recrystallized from hot hexane three times to


separate from the product from the di-adduct by-product. The reaction scheme can be represented as follows:



H H

+

H

Dodecylamine

Glycidol

t

Dry Toluene, Nitrogen atmosphere 60 °c, 8 hours

O-H

(ID
Synthesis of (Compound-A)
Cholic acid (788.0 mg, 1.93 mmoles Ex. Himedia) was dissolved in 20 ml dry Dimethylformamide as solvent (DMF)
(Ex. Spectrochem, India) . The solvent was dried over molecular sieves of 4A from Merck. Triethylamine, HPLC grade
(TEA) Ex. Spectrochem India (300 ul, 2.1 mmoles) as the base, Dicyclohexylcarbodiimide (DCC) as the coupling agent, Ex. Aldrich (509.0 mg, 2.1 mmoles) and 3-hydroxy-l,2,3-benzotriazin-4(3H)-one (N-Hydroxybenzotriazenone Ex. Aldrich
(426.0 mg, 2.6 mmoles) as the agent for activation of the carboxylic group of Cholic acid, were added to the mixture


and stirred for an hour. To this mixture 500 mg (1.93 mmoles of Compound-1 was added and stirred at 4 8 °C for 6 hours. DMF was evaporated under vacuum using rota- evaporator. The reaction was carried out under ultrapure grade Nitrogen atmosphere.
The yellow residue was dissolve in 3 ml Methanol and the solution was poured onto an aqueous solution prepared from 15 ml of saturated NaHC03 and 5 ml of sat Na2C03 (3:1). The precipitate was purified by column chromatography (Si02, CHCl3/MeOH; 19:1 v/v) to give the pure Pseudoceramide (Compound-A) having Rf ~ 0.79.
The schematic representation of the above synthesis is given below:



O-H
N I
DCC, TEA, Dry DMF

H'

o P-H

Compound-A

Spectral data of Compound-A
The Mass spectrum showed M+ peak at 647.9, corresponding to the Molecular formula (C39H70NO6) and Molecular weight = 648.9 +MS at 672.2 corresponding to the Molecular formula (C39H7oN06Na) . Figure-1 is the Mass spectrum of Compound-A.
The IR spectrum showed a peak at 1617 cm-1 for amide C-0 stretching, a broad band at 3315 cm-1 for 0-H stretching a peak at 1078 cm-1,1045 cm-1 for C-0 stretching of alcohol group and peaks at 1307 cm-1, 1375 cm-1 for O-H plane bending. Figure-2 is the IR spectrum of Compound-A.
The 1H NMR, on the other hand, showed peaks at (CDC13 SOLVENT, 400 MHz in 5 ppm) 0.67 ( 3H, s), 0.87 ( 6H, s) 0.94 ( 3H, s), 1.01-1.88 ( 38H, m) , 2.4 ( 2H, m), 2.8 ( 1H, br.s), 3.2 - 3.7 ( 8H, m) , 3.8 ( lH,s) and 4.0 ( lH,s). Figure-3 is the NMR spectum of Compound-A.
Differential scanning calorimetry data (DSC)
DSC is a thermo-analytical technique in which the difference in the amount of heat required to increase the temperature of the sample and reference is measured as a function of temperature. Both the sample and reference are maintained at same temperature through out the experiment. The basic principle underlying this technique is that when sample undergoes physical transformation such as phase transitions, more (or less) heat will need to flow to it than the reference to maintain at same temperature. Whether more or less heat must flow to the sample depends whether the process is exothermic or endothermic.


Two endothermic peaks were observed; at 37.1 °C and 58.9 °C which might be resulting from breakage of Hydrogen bonding between the -OH groups of cholic acid and breaking of Hydrogen bonding between the -OH groups of head group. The melting behaviour of compound-A shows that it is well-compatible with phase transition behaviours of human stratum corneum therefore can be favourably accepted by the skin. Figure-4 is the DSC thermogram of Compound-A.
EXAMPLE-2
Study of bio-activity of Compound-A and its effect on keratinocyte differentiation
Background and Principles:
In this study, the effect of novel Pseudoceramide (Compound-A) on keratinocyte differentiation was investigated. Involucrin is the main precursor involved in the formation of corneocyte envelope during stratum corneum formation while keratin-10 is a marker of differentiation status. The appearance of keratin-10 in keratinocytes marks the early stage, and involucrin protein, the late stage of keratinocyte differentiation.
Procedure
Immunofluoresence staining of keratinocytes for Involucrin
Method: HaCaT Keratinocytes (procured from Dr Fusnig, USA)
were plated in acid washed coverslips in Keratinocyte growth
media (KGM) , procured from Gibco containing 0.02 mM Ca+2
(CaC12), and were cultured in humidified incubator at 37°C


in an atmosphere of 5% C02. After 48 hours, some of the wells were treated with Magnesium acetate (MgAc, 20 mM) or Ca+2 (2 mM) to induce differentiation. Other wells were treated with different doses of Compound-A (1 to 10 ng/ml; 1.54 nM - 15.4 nM). After 48 hours of incubation, the cells were drained of media and washed with phosphate buffered saline (PBS, Salts procured from Merck), fixed with 3% Paraformaldehyde and permeabilized with 0.2% Triton X100, a commercial surfactant, in PBS. After washing with PBS, the cells were blocked with 3% Bovine serum albumin (Sigma, USA). The cells were then incubated with antibodies specific for human Involucrin (1:20) (Ex. Santacruz Biotechnology INC, USA) overnight at 4 °C. The next day the cells were washed and incubated with secondary antibody conjugated to Fluoresein isothiocynate (FITC) (1:40) for 1 hour. The cells were again washed with PBS and then observed under fluorescence microscope after mounting.
Results: Keratinocytes grown in low calcium media showed a monolayer of cells of even size forming a mosaic like pattern which did not show much staining for Involucrin. Incubation of cells with MgAc resulted in large differentiated keratinocytes which were also brightly stained for Involucrin. The treated cells (treated with varying doses of compound-A) also showed dose response related reactivity to Involucrin antibody. More number of differentiated keratinocytes with higher Involucrin staining was observed with higher non toxic doses of Compound-A.


Reverse Transcription-Polymerase Chain Reaction (RT-PCR) of keratinocytes for Keratin-10 and Involucrin mRNA expression
Method: Keratinocytes were seeded in 24 well plates in KGM and containing 0.02 mM Ca+2 were cultured in a humidified incubator at 37°C in an atmosphere of 5% C02. After 48 hours of culture, the cells were treated with Ca+2 (2 mM) , MgAc (20 mM) and Compound-A (1-10 ng/ml; 1.54 nM - 15.4 nM) Cells were then further incubated for 4 hours and RNA was extracted using Trizol (Ex. Sigma) using suppliers instructions. Cellular RNA was subjected to reverse transcription-polymerase chain reaction (RT-PCR) for Involucrin and Keratin-10 mRNA expression using specific primers. All experiments were performed in triplicate.
Results: Semi quantitative RT-PCR using specific primers was carried out and the PCR product was run on a 2% agarose gels (Ex. Himedia). The level of mRNA transcipts for Involucrin and keratin-10 was higher in both Ca+2 and MgAc treated keratinocytes compared to untreated keratinocytes. Compound-A also increased the levels of mRNA transcripts in treated cells compared to control. The table shows representative data of one experiment from a set of three. Data is presented as the ratio of mRNA of Involucrin/keratin-10 to GAPDH (Glyceraldehyde 3 phosphate dehydrogenase) (internal control) mRNA levels, in table-1 below.
Table-1

Involucrin Keratin-10
Untreated 0.34 0.25
Compound-A 1.07 1.87
Ca+Z 0.48 2.71
MgAc 0.70 1.70


These results indicate that the compound-A induced significant keratinocyte differentiation. Keratinocyte differentiation is an important requirement for proper barrier function which keeps the skin naturally hydrated and protects it from environmental abuses.
Multivesicular liposomes of compound-A
It has been found by the present inventors that the Pseudoceramide (Compound-A) forms multivesicular liposomes in ethanolic water system. The liposomes of Compound-A efficiently encapsulate hydrophilic and hydrophobic biologically active molecules, and therefore can function as a delivery vehicle for hydrophobic and hydrophilic actives for skin care.
Liposomes are closed vesicular structures comprising a bilayer membrane as a hydrophobic compartment and an aqueous core. They are spontaneously formed when natural or synthetic amphiphatic molecules are added to an aqueous medium. As liposomes have hydrophilic as well as lipophilic features, they can encapsulate hydrophilic substances in their inner compartment and encapsulate lipophilic substances in their bilayer membrane. Liposomes may vary in sizes, lamellarity, surface charge, and membrane lipid composition, thus allowing optimization to a plethora of different applications.
Liposomes, especially multivesicular are promising carriers for topical delivery of hydrophilic and hydrophobic molecules.


Liposomes were prepared by dispersion of ethanolic solution of Compound-A in aqueous solution. One percent (1%) ethanolic solution of Compound-A was prepared, and liposomes were prepared with final concentration of the compound, between 500-5000 ppm in ethanol-water mixture. The aggregation property of Compound-A was studied using Cryo-SEM (Scanning Electron Micrography). Cryo-SEM studies revealed that the Compound-A is capable of forming multivesicular liposomes with diameter ranging from 0.lum -30um. The Cyro-SEM images are included as figure 5 (which is an image of liposomes formed when the concentration of Compound-A in ethanol-water was 1000 ppm) and figure 6 (which is an image of liposomes formed when the concentration of Compound-A in ethanol-water was 2000 ppm)
Encapsulation Efficiency of the multivesicular liposomes
Liposomal dispersion was prepared in the presence of niacinamide as hydrophilic molecule. The total niacinamide content of the dispersion (A) was measured by HPLC. The dispersion was incubated for 1 hour and then centrifuged several times until all the liposomes separated as pellet. The supernatant was injected into the HPLC to measure the un-encapsulated Niacinamide content (B).
The encapsulation efficiency (EE) was determined by EE% = (A-B)/A * 100.
At 3000 ppm concentration of the liposomes in dispersion, the EE% for Niacinamide was found to be 20±5%. Similarly, for acetaminophen, as the hydrophobic molecule, the EE% was found to be 15±5%. Therefore, the multivesicular liposomes


are capable of serving as efficient delivery vehicles for biologically active molecules in cosmetic compositions.
Example-3
The Pseudoceramide of the present invention is useful as a cosmetic active ingredient. Therefore it can be incorporated in a cosmetic composition, which can increase keratinocyte differentiation, moisture retention, skin tonicity and recuperation, and thereby improve skin appearance and defer skin ageing.
Cosmetic cream composition; A vanishing cream composition was made by using the following ingredients. The formulation is presented in table-2 below.
Table-2

Ingredients % by weight
Phase A
Potassium hydroxide 0.56
Water 75.0
Glycerine 1.00
Phase-B
Hysteric acid 17.90
Cetyl alcohol 0.53
DC-200(350 est)Ex. Dow Corning 0.50
Compound-A 1.00
Phase C
Isopropyl myristate 0.75
Micronized Ti02 0.20
Parsol MCX® 0.75
Parsol 1789® 0.40
Phase D
Niacinamide Ex. Veer Chemie, India 1.00
Minors q. s.


Hysteric acid is a commercially available product Ex. Godrej India, which is a 55:45 mixture of Palmitic acid and Stearic acid.
Parsol MCX® and Parsol 1789® are commercially available suncreens.
Procedure
Phase-A ingredients were heated to 75 °C. Phase-B was heated to 75 °C in a container, separate from that of Phase-A. Phase B was added to phase A at 75 °C and stirred for 20 minutes followed by the addition of Phase-C at 70 °C. Thereafter, the phases were combined, while mixing, with the heat being turned off. Phase-D was heated to 50 °C and mixed into phases A and B. The mixture was cooled to 40 °C and then the minor ingredients were added. The formulation was cooled to 35 °C and packed.
It will be appreciated that the illustrated example, provides for a novel Pseudoceramide, which provides keratinocyte differentiation.
Although the invention has been described with reference to specific embodiments, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.