Abstract:
A cosmetic or medical composition for topical application to the skin. It results in the transdermal passage of an active ingredient, or in the introduction of such agent into the skin. The essential components of such compositions are a phospholipid, a lower aliphatic alcohol of two to four carbon atoms, optionally with propylene glycol, water and a compatible active ingredient. The alcohol content is generally from 20 to 50%, and when propylene glycol is present, the combined percentage of alcohol and glycol being up to about 70%. The composition are suitable for the topical application of a wide variety of cosmetic and pharmaceutical compounds.

Description:
FIELD OF THE INVENTION 
     The invention relates to novel compositions containing phospholipids, short chain alcohols (C2-C4) and water. These compositions may also contain polyols. Prefered compositions contain phospholipids, ethanol (EtOH), water (DDW), and propylene glycol (PG). 
     This invention relates to pharmaceutical, cosmetic, veterinary or phytopharmaceutical compositions for delivery to skin, membranes, or tissues, which enhance the delivery of the active agent at the site of application, where the agent may form a reservoir in the skin (membrane, tissue) or may be absorbed systemically into the blood circulation. 
     The compositions are hydro-alcoholic or hydro/alcoholic/glycolic phospholipid systems in which the concentration of alcohols, glycols, or their combination is relatively high. The main components of these systems are: phospholipids [with various chemical structures like phosphatidylcholine (PC), hydrogenated PC, phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PPG), phosphatidylinositol (Pl), hydrogenated PC and others, ethanol (or other short chain alcohols), water and propylene glycol (or other glycols). 
     The novel compositions enable the delivery of higher concentrations of active agent to/through a membrane (skin). The delivery can be modulated by changes in alcohol:water or alcohol-polyol:water ratio. 
     STATE OF THE PRIOR ART 
     In drug delivery, there are known a number enhancing agents, some used in research and some on the market, for promoting and increasing the amount of drug delivered to/through the skin. Examples of these enhancing agents are: DMSO, pyrrolidone derivatives, n-decylMSO, some surfactants, oleic acid, ethanol, Azone®, and others (Barry, 1989). 
     Phospholipids are known for their broad use in liposomal systems as well as emulsifiers in the preparation of emulsions. All these systems used for pharmaceutical or cosmetic purposes are aqueous systems with small if any concentration of alcohol and/or glycol for preservation and/or improving texture of the formulation. 
     Emulsions containing phospholipids are prepared by mixing an aqueous and an oily phase, in some cases followed by use of an homogenizer. 
     Preparation of liposomal systems involves the use of organic solvents such as chloroform, alcohols and others. In some methods of preparation, an organic phospholipid solution is evaporated to form a lipidic film, which is then hydrated to give an aqueous vesicular system (Riaz et al, 1988). In an alternative method, liposomes are prepared by injecting an ethanolic solution of lipid into an aqueous solution, resulting in a dilute ethanolic solution (2.5-7.5% ethanol) (Batzri et al, 1973). The alcohol, is then removed by different means such as dialysis (Kremer et al, 1977). The alcohol, if present is in low concentrations only, less than about 20% in the final product (e.g. 7.5%, Kremer et al, 1977). 
     SUMMARY OF THE INVENTION 
     The invention relates to compositions which are hydro/alcoholic or hydro/alcoholic/glycolic phospholipid systems in which the concentration of alcohol or its combination with polyol is relatively high. We call these compositions: Ethosomal systems. 
     The systems described in this invention are pharmaceutical, cosmetic, phytopharmaceutical or veterinary compositions for application to the skin (or other tissues) comprised of combinations of phospholipids, alcohols, water and glycol (polyols), as major components. 
     An ethosomal system is a skin permeation enhancing system which has the potential to form ethosomes, which are &#34;soft&#34; vesicles formed from phospholipid in the presence of water and ethanol (alcohol) and sometimes glycols (polyols). The size of the vesicles depends on the water: alcohol ratio.(see appendixes 1-6) and on the phospholipid concentration. On the skin, the ethosomes change their size by fusing together as a result of the change in solvent ratio. In the preparation, the vesicle size does not change since the ratio between the solvents is constant. Penetration and evaporation of the following application to the skin allows the transition from small to large vesicles, which grow in size until a film is formed. 
     An important characteristic of ethosomes is enhanced membrane permeability for various compounds. Ethosomal systems, vesicular in nature, depending on the ratio of the components and the chemical structure of the phospolipids, can be comprised of very small entities (nm&#39;s) up to larger vesicles (mm&#39;s) (see appendixes 1-6). High alcoholic (organic solvent) concentration favors the production of ethosomes in nm&#39;s range while high aqueous and phospholipid concentrations favorize the formation of large size ethosomes. As examples formulation 503 (Table 2) containing 68% organic solvent and 30% water has a mean population of less than 10 nm&#39;s, while formulation 510 containing 50% organic solvent and 48% water has a mean population of 1 mm. In system 503 the concentration of ethanol was 48% while in formulation 510 the ethanol concentration is only 20%, showing that the alcohol concentration is of great importance in determining vesicle size. The phospholipids which can be used are: phosphatidylserine (PC), phsophatidylserine (PS), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycol (PPG), hydrogenated PC and others. Some prefered phospholipids are soya phospholipids such as Phospholipon 90 (PL-90). The concentration of phospholipid ranges between about 0.5-10% w/w. Cholesterol at concentrations ranging between about 0.1-1% can also be added to the preparation. Examples of alcohols which can be used are: ethanol and isopropyl alcohol. Examples of glycols are propylene glycol and Transcutol®. The source of the phospholipids can be egg, soybean, semi-synthetics, and synthetics. Non ionic surfactants can be combined with the phospholipids in these preparations e.g. PEG-alkyl ethers (Brij-52). Cationic lipids like cocoamides, POE alkyl amines, dodecylamine, cetrimide and like. 
     The concentration of alcohol (EtOH etc.) in the final product ranges from about 20-50%. The concentration of the non-aqueous phase (alcohol and glycol combination) may range between about 22 to 70%. The rest of the carrier contains water and possible additives. 
     Vesicle formation is dependent on the water: alcohol ratio. This ratio is kept constant in the product, therefore, no changes in the entities population occur. Nevertheless, penetration and evaporation of the alcohol following application to the skin allows the transition from small vesicles to a larger ones, finally resulting in film formation. 
     In contrast to the present state of the art where &#34;tough&#34; liposomes accomplished by addition of different substances like cholesterol to the phospholipids and in absence of alcohol, this invention relates to &#34;soft&#34; vesicles, that can be easily formed in a hydroalcoholic medium. One of the important properties of these systems is that small entities can penetrate into the skin, while larger vesicles can form a reservoir in the skin and a film on the skin surface as a result of solvent evaporation taking place after the application. 
     These carriers can be used to deliver various active agents such as: Peptides, Anti-aging agents, tanning agents, and vitamins, Antiviral drugs, Psoriasis treatment agents, Hormones, Medicinally active componenets of plants such as oleoresins, volatile oils, glycosides, alkaloides, terpenes and others. 
     Examples of active agents which can be used with these systems are as follows: drugs like nicotine, nitroglycerine, estradiol (or like), testosterone (or like), progesterone, nifedipine, minoxidil, tetrahydrocannabinol (THC) or other cannabinoids, xanthines, anxiolytics (diazepam and others), antiepileptic (valnoctamide and others), diclofenac (and other NSAIDs), Antibiotics, corticosteroids, tocopherol, 5-FU, acyclovir, colchicine and other antimytotic agents, prazosin and other antihypertensive agents, papaverine, miconazole nitrate, ketoconazole and other antifungals, enzymes like SOD; peptides, hydroxy acids and amino acids. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a graph showing Differential Scanning Calorimetric data of an ethosomal system containing 50% PL-90 in water (See Table 2). 
     FIG. 2 is a graph showing Differential Scanning Calorimetric Data of an ethosomal system No. 525 (See Table 2). 
     FIG. 3 is a photograph of an ethosomal system No. 510 (See Table 2). 
     FIG. 4 is a photograph of an ethosomal system No. 529 (See Table 2). 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     In Vitro Skin Penetration/Permeation Measurements 
     Skin permeation experiments were carried out in our laboratory as previously described (Touitou and Fabin, 1988; Touitou et al, 1991). 
     Full Thickness Skin 
     The animals used in these investigations were hairless mice obtained from the Weizmann Institute, Rehovot, Israel. The skin of 5-7 week old male mice was excised before the experiment, examined for integrity and cut for diffusion cell assembly. In most of the experiments, the skin from the abdominal site was used. 
     In Vitro Permeation Experiments in Horizontal Diffusion Cells 
     The Valia-Chien cell assembly was used to perform the experiments on permeation of the drugs from various donor compositions through hairless mouse skin and the concentration in the skin. Mouse skin treated as described above was mounted in cells with a surface area of 0.64 cm 2  and half-cell volume of 3 ml. The receiver compartments contained a medium (hydroalcoholic, water, or buffer solutions) for ensuring pseudo-sink conditions. During the experiment, samples were withdrawn and mixed with scintillation cocktail (Packard, USA) and assayed in the Kontron Betamatic Scintillation Counter (Lumitron Scientific Industries) or directly assayed by HPLC. The sample volumes were replaced with fresh solution. The results were treated using the &#34;Transderm&#34; computer program (Touitou and Fabin, 1988). Each experiment was tetraplicated. The two-tailed, paired Wilcoxon test was used for determining the statistical significance of the effect of enhancers on the permeability coefficient changes. For these analyses, the &#34;Balance&#34; (IBM) computer program was used. 
     Skin Permeation Determination in Franz Cells 
     The permeation flux of drugs and the quantity of drug in the skin from the test system was measured in vertical cells (Franz diffusion cells). The experimental system conditions were: receiver temperature 37° C. and receiver volume 4-8 ml. The samples were assayed either by scintillation counting or by HPLC. The results are presented as a mean of 4 experiments and were analyzed by Transderm and by Balance (IBM) computer programs. 
     Size Distribution of Liposomes 
     The size distribution of ethosomes was measured in a Malvern autosizer (Malvern). (See appendixes 1-4). 
     Method of Preparation and Compositions 
     Examples of compositions are given in Tables 1 and 2. 
     a) Ethosomal system containing a relatively high concentration of Ethanol or Ethanol and Propylene Glycol was obtained as follows: 
     A lipophilic drug(10%) and PL-90(5%) were dissolved in an EtOH-PG(62.4%) mixture at room temperature. DDW(22.6%) was then added with vigorous stirring. 
     See example II-Immune agent. 
     b) Ethosomal systems containing a relatively high concentration of Ethanol (20-50%) or Ethanol and Propylene Glycol (22-70%) were obtained as follows: 
     PL-90 (0.5-10%) was dissolved in the EtOH-PG mixture. Hydrophilic drugs were dissolved in DDW and added to the PL-90 solution. 
     c) Ethosomal system was prepared by mixing (Heidolph mixer) PL-90 and water in concentrations as in &#34;b&#34; and heating to dispersion at 60°-70° C. The dispersion was then cooled (ice bath) with constant mixing for 30 minutes. To the above dispersion a solution of 2% Minoxidil in ethanol-propylene glycol (concentration as above) was added with vigorous mixing. A vesicular system was obtained. The preparation may be passed through a homogenizer (like Gaoline). 
     d) Minoxidil ethosomes were prepared by gently heating or at room temperature, a solution of soybean lecithin (Phospholipon 90) and Minoxidil in a propylene glycol ethanol mixture. Distilled water or buffer solution was added to the above system. A vesicular system was formed. The preparation may be passed through a homogenizer (like Gaoline). concentrations of ingredients as in &#34;c&#34;. 
     e) A vesicular system containing a relatively high concentration of Ethanol or Ethanol and Propylene Glycol was obtained as follows: A dispersion containing soya phospholipid (Phospholipon 90), Minoxidil, Ethanol, propylene glycol, double distilled water or buffer solution, is passed through a homogenizer in order to reduce particle size. concentrations of ingredients as in &#34;c&#34;. 
     f) Composition and preparation as described in a-c but containing 0.2% cholesterol in addition. 
     g) As in a-d where mixtures of phospholipids are used. 
     h) The vesicular systems can be incorporated in various carriers such as: PVP/VA (gels, membranes, solutions), PVP (gels, membranes, solutions) carbomer gels, polaxomer (gels, solutions), emulsions, creams, Pluronic F127 or Tetronic gels and the like, cellulose derivatives gels, PL-90ant extract gels (aloe vera gel etc), and the like. 
     
                                           TABLE 1__________________________________________________________________________Examples of skin permeation enhancing systems Containing various drugs    Systems    DYPH1         DYPH2              MND200                   SOD Immune                            THC 1                                 MM33                                     MM39                                         MM42                                             MM43__________________________________________________________________________COMPONENTSDYPHYLLINE     1%   1%ACYCLOVIRDICLOFENAC         7 μCi/mlSOD                      6%ROQUINIMEX                  10%MINOXIDIL                              2%  2%  2%  2%THC                              7 μci/mlPL-90     5%   5%   2%   2%  5%   2%   2%  2%  5%  5%CHOLESTEROL    --   --   --   --  --   --   --  --  --   0.1%PG       18.8%         --   19.6%                   18.4%                       31.2%                            19.6%                                 19.2%                                     19.2%                                         19.5%                                             18.6%DDW      47%  47%  49%  46% 22.6%                            29.4 28.8%                                     48% 48.8%                                             27.9%EtOH     28.2%         47%  29.4%                   27.6%                       31.2%                            49%  48% 28.8%                                         29.3%                                             46.5%__________________________________________________________________________ 
    
     Examples of Compositions and Preparation Methods 
     Example I--SOD Ethosomal Preparation 
     
         ______________________________________A)        Amerchol L-101 57.1%     4 parts     Amerlate P     14.3%     1 part     Brij 52        28.6%     2 partsB)        Carbopol 934P  1%     DDW            98%     TEA            1%     SOD            6%C1)       PL-90          2%     DDW            46%C2)       EtOH 3         27.6%     PG             18.4%______________________________________ 
    
     Gel Preparation 
     1. Preparation of &#34;A&#34; by melting the ingredients in a water bath. Cool while stirring. 
     2. Disperse Carb 934 P in DDW at room temperature. 
     3. Mix A and B in the ratio of 7% of A with 93% B and add TEA. 
     System Preparation 
     4. SOD was dissolved in a PL-90 dispersion in DDW [obtained by heating in a water bath of 70° C. with constant mixing (C1)]. 
     5. An EtOH:PG mixture (C2) was prepared and added to C1 with vigorous mixing and cooling in an ice bath. 
     
         ______________________________________Final formulation:6. Mix gel with the system in a 1:1 ratio.______________________________________Drug                    3%Lipid phase (A)         3.5%Gel (B)                 46.5%PL-90 + DDW (C1)        22%PG + EtOH(C2)           25%______________________________________ 
    
     Example II--IMMUNE AGENT (ROQUINIMEX)Ethosomal Preparation 
     
         ______________________________________A)        Pluronic F127     40%     DDW               60%B)        ROQUINIMEX        10%     PL-90             5%     EtOH 95%          31.2%     PG                31.2%     DDW               22.6%______________________________________ 
    
     Prepare a Pluronic gel of 40% in DDW. Dissolve Roquinimex and PL-90 in the EtOH-PG mixture. Add DDW to the latter with vigorous stirring. 
     Add B to A with vigorous stirring, at a ratio of 1:1. 
     Example III--THC Ethosomal Preparation 
     
         ______________________________________THC                    1%PL-90                  4.2%EtOH                   51.7%PG                     15%PVP-VA S-630           12.5%DDW                    16.6%______________________________________ 
    
     THC and PL-90 were dissolved in an EtOH-PG mixture with gentle heating while mixing. DDW was added with continuous stirring. PVP-VA was slowly added while stirring. The preparation was sonicated in 3 cycles of 5 minutes with 5 minutes rest between each cycle. 
     Example IV--Minoxidil Ethosomal Preparation (MM50-G) 
     
         ______________________________________A)        Carbopol 934P    0.75%     Ethomeen C/15    0.75%     DDW              73.5%     EtOH             25%B)        Minoxidil        4%     PL-90            5%     EtOH 95%         40%     PG               20%     DDW              31%     Tocopherol       0.02%MM50-G, Final concenrations:Carbopol 934 P         0.38%Ethomeen C/15          0.38%Minoxidil              2%PL-90                  2.5%EtOH (95%)             32.5%PG                     10%DDW                    52.3%______________________________________ 
    
     Carbopol gel was prepared by dispersing Carbopol 934 P in DDW and Ethomeen was added. Ethanol was added to obtain an hydroalcoholic gel. The drug, antioxidant, and PL-90 were dissolved in the EtOH-PG mixture with gentle heating (30° C.). DDW was added with vigorous stirring to obtain the system. 
     The system may be homogenized. 
     B was added to A at a 1:1 ratio. 
     The mean size of vesicles was found to be 35 nm 
     Example V--Minoxidil Ethosomal Preparation (MM) 
     As in example IV (MM50-G) with the following variations: 
     The gel:system ratio may be changed to 1:2, 1:3, or 1:4. 
     The ratio between solvents can be changed: EtOH (25-49%), PG (0-20%), and DDW (25-49%)can be changed. 
     The PL-90 concentration can be raised to about 10%. 
     Example VI--DICLOFENAC SODIUM Ethosomal Preparation 
     
         ______________________________________Diclofenac sod.         1%PL-90                   1%Carbopol 934            0.9%Ammonium 10% soln.      1.8%EtOH                    21.9%DDW                     68.9%PG                      4.16%______________________________________ 
    
     Example VII--ACYCLOVIR Ethosomal Preparation 
     
         ______________________________________Acyclovir               5%Ammonium 10% soln.      1.66%Carbopol 934            0.83%PL-90                   2.5%DDW                     58.3%EtOH                    27.5%PG                      4.16%______________________________________ 
    
     
                       TABLE 2______________________________________Other ExamplesSys  Ingredient, % w/wNo.  PL-90   DDW      EtOH  PG    Other  Method*______________________________________500  2       48       30    20            Hot501  2       48       30    20            Cold502  2       30       48    20            Hot503  2       30       48    20            Cold504  5       45       30    20            Hot505  5       45       30    20            Cold506  5       30       45    20            Hot507  5       30       45    20            Cold508  2       30       38    30            Cold509  2       38       30    30            Cold510  2       48       20    30            Cold511  1.8     30       38    30    0.2(M(-)*)                                     Cold512  5       30       45    20            Cold513  5       35       30    30            Cold514  2       33       30    30    5(Brij52)                                     Cold515  17      26       40    17            Cold516  9.3     44.4     27.7  18.5          Cold517  5       20       40    40    5(Brij52)                                     Cold518  2       47       30    20    1(Col**)                                     Cold525  10      64       26    0             Hot529  1.7     55.4     34.3  8.6           Cold______________________________________ PL-90: phospholipid; DDW: water; Etoh: ethanol; PG: propylene glycol *M(-): lMenthol **Col: Colchicine *See &#34;Hot&#34; or &#34;Cold&#34; methods described below. 
    
     &#34;Cold&#34; method 
     Phospholipid is dissolved in ethanol at room temperature by vigorous stirring with the use of Heidolph mixer. Propylene glycol is added during the stirring. The mixture is heated to 30° C. in a water bath. The water heated to 30° C. in a separate vessel is added to the mixture which is then stirred for 5 minutes in a covered vessel. 
     &#34;Hot&#34; method 
     The phospholipid is dispersed in water by heating in a water bath at 40° C. until a colloidal solution is obtained. In a seperate vessel ethanol and propylene glycol are mixed and heated to 40° C. Once both mixtures reach 40° C. the organic phase is added to the aqueous one. 
     During the preparation process the drug is dissolved in water or in ethanol depending on its hydrophilic/hydrohpobic properties. 
     EXAMPLES OF SKIN PERMEATION PARAMETERS OF VARIOUS DRUGS FROM THESE SYSTEMS (Kp=permeability coefficient;Q skin  =quantity of drug in the skin at the end of the experiment;Q rec  =quantity of drug in the receiver compartment of the cell at the end of the experiment) Kp reflects the permeation through the skin and Qs reflects the reservoir formation in the skin. 
     Example VIII 
     
         ______________________________________             blank MM (= Minoxitrim.sup.R,MM33= MM36 vs.    Trima, Israel)(see Table 1)     2% minoxidil______________________________________Kp=   1.36 × 10.sup.-2 cm*hr.sup.-1                 8.84 × 10.sup.-4 cm*hr.sup.-1Q.sub.skin= 0.658 mg/cm.sup.2                 0.0479 mg/cm.sup.2______________________________________ 
    
     The ethosomal system showed an increse of 15.4 times in Kp and 13.7 times in Qs. 
     Example IX 
     
         ______________________________________  MM39 (see Table 1)                MM blank(as above)______________________________________Kp=      1.96 × 10.sup.-3 cm*hr.sup.-1                    5.75 × 10.sup.-5 cm*hr.sup.-1Q.sub.skin=    0.138 mg/cm.sup.2                    0.0378 mg/cm.sup.2______________________________________ 
    
     The ethosomal system showed an increse of 34 times in Kp and 3.7 times in Qs. 
     Example X 
     
         ______________________________________THC (as in ex. III)             THC in PG:EtOH (1:1)______________________________________Kp=     7.2 × 10.sup.-3 cm*hr.sup.-1                 2.03 × 10.sup.-3 cm*hr.sup.-1______________________________________ 
    
     The ethosomal system showed an increse of 3.5 times in Kp. No significant changes in other kinetic parameters were observed 
     Example XI 
     
         ______________________________________    MM50G (as in ex. IV)                 MM blank______________________________________Q.sub.rec =      0.135 mg/cm.sup.2                     0.023 mg/cm.sup.2(24 hours)______________________________________ 
    
     The ethosomal system showed an increase of 5.9 times in Kp 
     Example XII 
     
         ______________________________________   ACV11 (as in ex VII)                Zovirax.sup.R______________________________________Q.sub.rec  2.9 ± 1.57 μg/cm.sup.2                    3.065 ± 0.38 μg/cm.sup.2(6 hours)Q.sub.skin     81.35 ± 5.58 μg/cm.sup.2                     18.35 ± 8.56 μg/cm.sup.2______________________________________ 
    
     The ethosomal system showed no significant change in Kp and an increase of 4.4 times in Qs. 
     Example XIII 
     
         ______________________________________MM122       MM122 blank a  MM122 blank b______________________________________2% PL-90, 2% Min-       2% PL-90,      2% Minoxidiloxidil48% EtOH 95,       2% Minoxidil   in EtOH 9528% DDW     in EtOH 95and 20% PGQrec= 3672 ± 378       217.5 ± 118.5 μg/cm.sup.2                      66.9 ± 22.1 μg/cm.sup.2μg/cm.sup.2 (24 hours)Q.sub.skin = 570 ± 130.5       139.1 ± 25.3 μg/cm.sup.2                      53.5 ± 12 μg/cm.sup.2μg/cm.sup.2______________________________________ 
    
     The ethosomal system showed an increase of 17 times in Kp and 4.1 times in Qs relative to a 2% phospholipid solution in ethanol (blank a) and an increse of 55 times in Kp and 11 times in Qs relative to an ethanolic solution (blank b). These results clearly indicate that the ethosomal system is a much more effective penetration enhancing system than ethanol or an ethanolic solution of phospholipid. 
     Example XIV 
     
         ______________________________________MND200             Blank MND (only solvents)______________________________________Kp=     4.27 × 10.sup.-3 cm*hr.sup.-1                  1.57 × 10.sup.-3 cm*hr.sup.-1Q.sub.skin =   2.75 × 10-5 mg/cm.sup.2                  2.35 × 10-5 mg/cm.sup.2lag time =   0:41 hrs       2:30 hrs______________________________________ 
    
     The ethosomal system showed an increse of 27 times in Kp no significant change in Qs and a decrease of 5 times in lag time. 
     Example XV 
     Differential Scanning Calorimetric (DSC) data on a system containing 5% PL-90 in water and an ethosomal system No. 525 (see Table 2 for exact composition) are presented in FIGS. 1 and 2, respectively. The thermograms clearly show a decrease in Tm (transition temperature) value for the ethosomal system which indicates an increase in phospholipid vesicle&#39;s fluidity. 
     Example XVI 
     FIGS. 3 and 4 present photographs of vesicles in ethosomal systems 510 and 529 (Table 2) as seen by means of a computerized image analyser connected to a light microscope (Cue 2 Galai- Axioscope Zeiss). These photos clearly show the presence of vesicles (ethosomes) in systems containing 20 and 34.3% ethanol, respectively. 
     
                                           TABLE 3__________________________________________________________________________MALVERN AUTOSIZER II c for ethosomal system No. 501 (see Table__________________________________________________________________________2)sample: 501vortex t = 22.6aperture 200Data from accumulation file sum of 5 blocksTemperature  22.6  Viscosity  4.1800  Refractive Index  1.365  Angle90.0Particle size distribution (nm.) Graph of Distribution of mass10.0-12.1 +12.1-14.6 +14.6-17.7 +17.7-21.5 +21.5-26.0 +26.0-31.5 +31.5-38.2 +38.2-46.2 +46.2-56.0 +56.0-67.9 +67.9-82.2 +82.2-99.6 + 99.6-120.7 +120.7-146.3146.3-177.2 +177.2-214.7+214.7-260.1+260.1-315.1+315.1-381.7+381.7-462.5+462.5-560.3+560.3-678.8+678.8-822.4+822.4-996.4+__________________________________________________________________________ 
    
     
                                           TABLE 4__________________________________________________________________________MALVERN AUTOSIZER II c for ethosomal system No. 503 (see Table__________________________________________________________________________2)sample: 503vortex22.6 tempData from accumulation file sum of 8 blocksTemperature  22.7  Viscosity  3.9100  Refractive Index  1.373  Angle90.0Particle size distribution (nm.) Graph of Distribution of mass1.6-1.8+1.8-2.12.1-2.4+2.4-2.8+2.8-3.2+3.2-3.7+3.7-4.3+4.3-4.9+4.9-5.7+5.7-6.5+6.5-7.5+7.5-8.6+ 8.6-10.0+10.0-11.5+11.5-13.2+13.2-15.2+15.2-17.6+17.6-20.2+20.2-23.3+23.3-26.8+26.8-30.9+30.9-35.6+35.6-41.1+41.1-47.3+__________________________________________________________________________ 
    
     
                                           TABLE 5__________________________________________________________________________MALVERN AUTOSIZER II c for ethosomal system No. 509 (see Table__________________________________________________________________________2)sample: 509vortex + USnon dilutedData from accumulation file sum of 8 blocksTemperature  22.6  Viscosity  4.8200  Refractive Index  1.376  Angle90.0Particle size distribution (nm.) Graph of Distribution of mass4.5-5.4 +5.4-6.46.4-7.7+7.7-9.2+ 9.2-11.0+11.0-13.2+13.2-15.8+15.8-18.9+18.9-22.6+22.6-27.1+27.1-32.4+32.4-38.8+38.8-46.4+46.4-55.6+55.6-66.6+66.7-79.7+79.7-95.5 + 95.5-114.3 +114.3-136.8 +136.8-163.8 +163.8-196.2 +196.2-234.9 +234.9-281.2 +281.2-336.7 +Size Class   % by Intensity   % by mass   % by number__________________________________________________________________________ 
    
     
                                           TABLE 6__________________________________________________________________________MALVERN AUTOSIZER II c for ethosomal system No. 510 (see Table__________________________________________________________________________2)sample: 510vortex + USnon dilutedData from accumulation file sum of 68 blocksTemperature  22.6  Viscosity  4.6400  Refractive Index  1.374  Angle90.0Particle size distribution (nm.) Graph of Distribution of mass17.4-21.1  +21.1-25.6  +25.6-31.0  +31.0-37.6  +37.6-45.545.5-55.1+55.1-66.8  +66.8-80.9  +80.9-98.0  + 98.0-118.8  +118.8-143.9  +143.9-174.3  +174.3-211.2  +211.2-255.9+255.9-310.0  +310.0-375.6  +375.6-455.0+455.0-551.3+551.3-667.9+667.9-809.2+809.2-980.3+ 980.3-1187.7+1187.7-1438.9+1438.9-1743.3+__________________________________________________________________________