AU756675B2 - Compositions for applying active substances to or through the skin - Google Patents

Description

DESCRIPTION

1 COMPOSITIONS FOR APPLYING ACTIVE SUBSTANCES TO OR THROUGH THE SKIN 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 phospholipid, 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 plospholipid 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 hydrogenated PC, phosphatidic acid (PA), phosphatidylserine phosphatidylethanolamine (PE), phosphatidylglycerol (PPG), phosphatidylinositol hydrogenated PC "nd others], ethanol (or other short chain alcohols), water and propylene glycol (or other glycols).

The novel composition enables the deliver)' of higher concentrations of active agent to/through 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 a number of enhancing agents, some used in research and some on the market, for promoting and increasing the amount of drug delivered to/through the skin. Exapmlcs of these enhancing agents are: DMSO, pyrrolidone derivatives, n-decylMSO, some surfactants, oleic acid, ethanol, AzoneP, and others(Barry, 1989).

Phospholipids are known for their broad use in liposoma! systems as -well as emulsifiers in the preparation of emulsions. All these systems used for pharmaceutical or cosmetic purposes are aqueous systems with7 smail if aay concentration Of alcohol andlOr glYcol for preservationi and/or improving texture of the formulation- Emnulsionis containing phosphoflpids are prepared by mixin .g an aqueous and an oily phase, in some !zases followed by use of an homogenizer- Preparation of liposomal systemns involves the use of organic solvents such ;S chloroform, alcohols arnd others- The prior art teaches away fromn high ioncentrations of alcohol in the Final liposorl-l preparations- In some %'&Acl of prpaail anognc ospholipid solutioni is evaporated to formn d lipidic film, which is then hydrated to give an aqueous vesicular syste.a( lRiaz et al.,1988). In alternative methods, liposomres arc :prepared by injecting an ethanolic solution of lipid into an aqueous solution, resulting in a di'lute ethanolic solution ethanol)(Batzri et.

or by dilution of proliposomreS (Leigh, 1991).. The alcohol,. is then rerrc" 'ed by different means such as dialysis (Krermer et al, 1977) or" is dilat-.d. The alcohol, if present is in low concentrations only, less Ehan aboic; 20% in the final product Kremer et al, 1977, Leigh,199l).

EP-A-0 582 239 relates -to a pharmaceutical and/or cos:: !tic composition for topical use comprising lirioleic: acid as active ingredienlt and a carrier. The h-ighest concentration of e&lcohol used in this composition is up to WO 91/11993 relates to an alcoholic aqueous gel-type phospholipid composition. The alcohol is ethanol, 1-propaniol OV or 2 -propanol. The composition is an aqueous solution of a.

liposomal gel consisting of 15.00 to 30.00 parts by weight of a p!.ospholipid concentrate, 14.00 to 20.00 parts by weight of alc.hol, the remainder being 50 to 71.00 parts by weight of an aqueous solution. Also disclosed are the use of this pho-spholipid composition to prepare liposomal solutions by dilution with an aqueous solution, and topical preparations containing i.L WO 92/18103 relates to a method of forming liposoimes, bilayered structures or a film formed by the collapse of suchi stzu'.ctures which comprises removing a proportion of the organlic liquid from a composition comprising: a) at least one biJlayer forming lipid; b) at least one volatile watermis'cible organic liquid; and c) a hydroph-ilic medium, whereupon the compostion spontaneously forms liposomes or bil;lyered structures. The hydrophilic medium may be water, glyfyerol, propylene glycol, a polyethylene glycol or a mix ure of two or more thereof. A composition for use in this met od is also described, together with liposomes or biluyered structures formed therefrom- The composition may inc,ude biologically active ingredients, such as medicaments and/or fragrances.

Brief Description of the Invention The invention relates to compositions which are hydro/alcoholic or hydro/alcoholic/glycolic phospholipid systems in which the concentration of cohol or its combination with polyol is relatively high. We call these cor.: positions: Ethosomal systems. The systems described in this invention are pharmaceutical, cosmetic, phytopharmaceutical or veterinary cor: positions for application to the skin (or other tissues) comprised of cor:binations 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 "soft" 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 Tables 3-5) and on the phospholipid corn entration. On the skin, the ethosomes change their size by fusing tog.A:her as a result of the change in solvent ratio. In the preparation, the vesi :le size does not change since the ratio between the solvents is cort :ant. Penetration and evaporation of the alcolhol following application to e 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 2a nature, depending on the ratio of the components and the chemical structure of the phospholipids, can be comprised of very small entities (nm's) up to larger vesicles (Pm's) (see Tables High alcoholic (organic solvent) concentration favours the production of ethosomes in nm's range which high aqueous and phospholipid concentrations favour the formation of large size ethosomes. As examples, formulation 509 (Table 4) containing 60% organic solvent and 38% water has a mean population of tens of nm's, while formulation 510 containing 50% organic solvent and 48% water has a mean population of 1mm. In system 509 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: phosphatidylcholine, (P phosphatidylserine (P phosphatidic acid (P phosphatidylethanolamine (P phosphatidylglycerol (P P hydrogenated PC and others. Some preferred phospholipids are soya phospholipids such as Phospholipon 90 (PL-90). The concentration of phospholipid ranges between about is 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 popylene 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- 20 52). Cationic lipids like cocoamide, Poe alkyl amines, dodecylamine, cetrimide, and like.

L o 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 25 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 larger ones, finally resulting in film formation. IN contrast to the present state of the art where "tough" liposomes accomplished by addition of different substances like cholesterol to the phospholipids and in absence of alcohol, this invention relates to "soft" 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 3 the skin surface as a result of solvent evaporation taking place after the application.

[RA\LIBZZ]06603.doc:nss These carriers can be used to deliver various active agents such as: peptides, antiaging, tanning agents, vitamins, antiviral drugs, psoriasis treatment agents, hormones, medicinally active components of plants such as oleoresins, volatile oils, glycosides, alkaloids, terpenes and others.

Examples of other 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, prazosin, papaverine, miconazole nitrate, ketoconazole and other antifungals, enzymes like SOD; peptides and amino acids.

Accordingly, a first aspect of the present invention provides a composition for medical or cosmetic use, for topical application to the skin, resulting in the enhanced transdermal passage or introduction of an active ingredient into the skin, which composition contains vesicles in the size range from the nm range to the plm range, which composition comprises from 0.6% ww to 10% w/w phospholipids, from 20% w/w to w/w of a C 2 to C 4 alcohol, from 0 to 30% w/w glycol, at least 20% w/w water and at least one active ingredient where the combined alcohol and glycol content does not exceed 70% w/w.

0 20 According to one embodiment the invention relates to a liposomal composition as defined hereinbefore wherein the penetration through the skin of the composition is at least four times higher than the penetration through the skin of a similar liposome composition containing less than 5% w/w alcohol.

2A preferred liposomal composition is one where the alcohol compound is ethanol, 25 and where the mixture of such compounds comprises ethanol and propylene glycol, where the ethanol concentration in the final preparation is between 20 and 50 weight% of the final product, the content of water being at least about 20% w/w.

Moreover, in a second aspect the invention relates to a process for the production of a cosmetic or medical composition for topical application to the skin, for rapidly introducing into the skin, or for enhanced penetration through the skin, which contains an active ingredient in a liposomal composition as defined hereinbefore which comprises mixing a phospholipid, a C 2 to C 4 -alcohol, or such alcohol and lower glycol, water, and STR4q/ an active ingredient so as to form a colloid system containing vesicles.

SzA third aspect of the present invention provides a cosmetic or medical composition produced by the process of the second aspect of the present invention described above.

[R:\LIBZZ]06603.doc:nss Methods In vitro skin penetration/permeation measurements Skin permeation experiments were carried out in our laboratory as previously described (Touitou, 1986; Touitou and Fabin, 1988).

Full thickness skin The animals used in these investigations were hairless mice obtainedfrom 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.

.o 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 cm2 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 expeirment, 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 "Transderm" computer program (Touitou and Fabin,1988). Each experiment was tetraplicated. The twotailed, paired Wilcoxon test was used for determining the statistical significance of the effect of enhancers on the permeability coefficient changes. For these analyses, the "Balance" (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 0 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 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: 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 and heating to dispersion at 60-70 0

C

The dispersion was then cooled (ice bath) with constant mixing for minutes. To the above dispersion a solution of 2% Minoxidil in ethanolpropylene 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.

S The preparation may be passed through a homogenizer (like Gaoline).

S concentrations of ingredients as in 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 f) Composition and preparation as described in a-c but containing 0.2 cholesterol in addition.

0 6 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, adhesives, creams, Pluronic F127 or Tetronic gels and the like, cellulose derivatives gels, plant extract gels (aloe vera gel etc), and the like.

*o Table 1: Examples of skin permeation enhancing syslems containing DYPI-11 DYPI-12 MI D200 SOD Immune IC -1 MM33 various drugs Mfv39 MMI2 MM43 -MM~E1I[M DYPI IYHINE ACYCLOVin

DJCLOFENAC

SOD

co fOQUINIMEX M INOX IDIL mI-c -to/ 1i 7 1tC ihrnI 2-0 2% 71c i/ml CI IOt.E-STEROL

PG

DUJN

[lOllI 5% 2% 5% 47% 471/ 2 8. 2%01 4 7%0 19.6% 490/10 29.,1,,0 31.2%, 19 46%. 2 3 1.2% 4 2% 2%o 2%1 6% 19.2%/4 19.5%/ 10.6%/ 3.4 28.8% 48% 48.8% 274.9%.

8 2 8 80 2 9 3 1 2 7 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 parts B) Carbopol

DDW

TEA

SOD

Cl) PL-90

DDW

C2) EtOH 3

PG

934P 1% 98% 1% 6% 2% 46% 27.6% 18.4% r r Gel preparation: 1. Preparation of 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 0 C with constant mixing An EtOH:PG mixture (C2) was prepared and added to Cl 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 Gel 46.5% PL-90 DDW (Cl) 22% PG EtOH(C2) 9 Example II- IMMUNE AGENT (ROQUINIMEX) ethosomal preparation A) Pluronic F127 DDW B) ROQUINIMEX 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% 4.2% S. EtOH 51.7% PG 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.

•o* o Example IV- Minoxidil ethosomal preparation A) Carbopol 934P 0.75% Ethonieen C/15 0.75% DDW 73.5% EtOH B) Minoxidil 4% EtOH 95% PG DDW 31% Tocopherol 0.02% Final concentrations: Carbopol 934 P 0.38% Ethomeen C/15 0.38% Minoxidil 2% EtOH 32.5% PG DDW 52.3% Carbopol gel was prepared by dispersing Carbopol 934P 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 0 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 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 and DDW (25-49%)can be changed.

The PL-90 concentration can be raised to about Example VIpreparation DICLOFENAC SODIUM ethosomal Diclofenac sod.

Carbopol 934 Ammonium 10% soln.

EtOH

DDW

PG

1% 1% 0.9% 1.8% 2 1.9% 68.9% 4.16% Example VII- Acyclovir Ammonium 10% Carbopol 934

DDW

EtOH

PC

ACYCLOVIR ethosomal preparation soin. 1.66% 0.83% 58.3% 27 4.16%

S.

S S

S

S. S

S*

SS

Other Eamples Sys. No. Ingrdients, dn Method* IL-90 DDW EtOl PG Other 500 2 48 30 20 Hot 501 2 48 30 20 Cc!d 502 2 30 48 20 $Hot 503 2 30 48 20 Cold 50,1 5 45 30 20 Hot 505 5 '15 30 20 Cold 506 5 45 20 Hot 507 5 30 45 20 Cold 508 2 30 38 30 Cold 509 2 30 30 30 Coid 510 2 48 20 30 Cold 511 1.8 30 38 30 Cold 512 5 30 45 20 Cold 513 5 35 30 30 Cold 514 2 33 30 30 5(rij52) Cold 515 17 26 40 1i Cold 516 9.3 4I1.4 27.7 18.5 Cold 517 5 20 40 40 5(Brij52) Cold 510 2 47 30 20 1(Co) Cold 525 10 011 26 0 Hot 529 1.7 55.1 34.3 8.6 ICold a a.

a..

a.

a a. a a a.

a.

a a a a a IIL-90: Cul: ph us pliulipid DDW: ler; Etoh: e'innol PG: propylenie qlycl I-Meni[Iiol Colclicirie Seo "Hu" or "Cold ellods described below.

"Cold" 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 0 C in a water bath. The water heated to 30 0 C in a separate vessel is added to the mixture which is then stiired for 5 minutes in a covered vessel.

"Hot" method: The phospholipid is dispersed in water by heating in a water bath at 400C until a colloidal solution is obtained. In a separate vessel ethanol and propylene glycol are mixed and heated to 400C. Once both mixtures reach 40 0 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; Qs quantity of drug in the skin at the end of the experiment) Kp reflects the permeation through the skin and Qs reflects the reservoir formation in the skin.

Example VIII MM33= MM36 vs. blank MM (MinoxitrimR, Trima, Israel) (see Table 1) minoxidil) Kp= 1.36 x 10-2 cm*hr- 1 8.84 x 10- 4 cm*hr-1 Qskin=0.658 mg/cm 2 0.0479 mg/cm 2 The ethosomal system showed an increse of 15.4 times in Kp and 13.7 e.

*Kp=1.96 x 10-3 cm*hr-1 5.75 x 10-5 cm*hr- Qskin =0.138 mg/cm 2 0.0378 mg/cm 2 se* The ethosomal system showed an increse of 34 times in Kp and 3.7 times .es in Qs.

Example. IX MM39 (see Table 1) MM blank(as above) Kp=1.96 x 10-3 cm*hr-I 5.75 x 10-5 cm*hr-1 Qskin =0.138 mg/cm 2 0.0378 mg/cm 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 (l:1) Kp =7.2 x 10-3 cm*hr-l 2.03 x 10-3 cm*hr-1 The ethosomal system showed an increse of 3.5 times in Kp. No significant changes in other kinetic parameters were observed Example XI (as in ex. IV) MM blank Qreceiver =0.135 g/cm 2 0.023 ig/cm 2 (24 hours) The ethosomal system showed an increase of 5.9 times in Kp Example XII ACVII (as in ex VII) ZoviraxR Qreceiver 2.9 1.57 ig/cm 2 3.065 0.38 pg/cm 2 (6 hours) Qskin 81.35 5.58 jg/cm 2 18.35 8.56 pg/cm 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% Minoxidil 2%PL-90, 2% Minoxidil 2% Minoxidil 48% EtOH 95, 28% DDW, in EtOH in EtOH 20% PG Qrec=3672 378 g/cm 2 217.5+ 118.5pg/cm 2 66.9+22.1ig/cm 2 (24 hours) Qskin =570 130.5 pg/cm 2 139.1 25.3 jig/cm 2 53.5 _2 pg/cm 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 SS.* solution (blank These results clearly indicate that the ethosomal system is a much more effective penetration enhancing system than ethanol or an ethanolic solution of phospholipid.

0O Example XIV MND200 Kp= 4.27 x 10-3 cm*hr-l Qskin 2.75 x 10-5 mg/cm 2 lag time 0:41 hrs The ethosomal system showed an increase change in Qs and a decrease of 5 times in Blank MND (only solvents) 1.57 x 10-3 cm*hr-l 2.35 x 10- 5 mg/cm 2 2:30 hrs of 2.7 times in Kp no significant lag time.

Example XV Differential Scanning Calorimetric (DSC) data on a system containing in water and an ethosomal system No. 525 (see Table 2 for exact composition) are presented in Figs.l 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's fluidity.

Example XVI: Figures 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.

Example XVII There were prepared: Caffeine ethosomes (liposohmal systems containing 20.9 and 35% ethanol) versus Caffeine liposomes with ethanol.

All three preparations contain the same concentration of caffeine and the same phospholipid (Phospholipon 90) at 5% concentration, without propylene glycol.

Methods In vitro skin penetration/permeation measurements.

Skin permeation experi: -ent was carried out in our laboratory by using the Valia-Chien cell assembly to perform the experiment.

The skin of 5-7 week old male mice was used in this investigation (nude mice obtained from the Weizmann Institute, Rehovot, Israel.). The skin was examined for integrity and cut for mounting on diffusion cell assembly. The skin from the back site was used.

The skin was mounted in cells with a surface area of 0.64 cm 2 and halfcell volume of 3 ml. The receiver compartments contained water for ensuring pseudo-sink conditions. Samples were directly assayed by HPLC (Touitou et al, 1994). The experiment was run for 24 hours.

Each formulation was tested in three cells.

Caffeine Ethosomes A) Caffeine Sod. Salicylate 4.8% SDistilled Water 52.2% B) Phospholipon-90 Ethanol 35.0% Example XVIII A composition was prepared as set out on page 6, b.

A) Dissolve Caffeine and Sod. Salicylate in the water.

B) Dissolve the phospholipid in ethanol. Add A to B with vigorous stirring.

Caffeine Liposomes Caffeine Sod. Salicylate Ethanol Distilled Water 4.8% 82.2% a a Preparation as above.

Results Qce quantity of drug in the receiver compartment of the diffusion cell at the end of the experiment.

Caffeine Liposomes ethanol) 86.8+17.1 pg/cm 2 Caffeine Ethosomes (20.9% ethanol) 496.8+71 pg/cm 2 Caffeine Ethosomes (35% ethanol) 4794.3+812 pg/cm 2 These results clearly indicate that the ethosomal system according to the invention containing 35% ethanol enabled an enhanced delivery of caffeine through the skin of 53 times higher than the liposomes containing caffeine with 5% ethanol. The above proves the outstanding improvement resulting from a high content of alcohol in the liposomal system, in the presence of liposomes.

An increase of skin penetration from 87 g/cm 2 to about 4794 lig/cm 2 an increase by a factor of about 53 times as large, demonstrates a dramatic and unexpected result of the novel liposomes with a high ethanol content, termed "ethosomes". A 53-fold skin penetration could not be expected at all on the basis of the prior art, which clearly teaches away from the present invention, i.e. that a high ethanol content is detrimental for liposomal preparations, and that the ethanol content of the final liposome preparation ought to be reduced so as to remove a large part of the initial ethanol content or by dilution.

Example XIX Further experiments were carried out with Minoxidil, comparing liposomes with a high ethanol content with Minoxidil in the vehicle.

The experiments were carried out as follows: The formulations tested were: Minoxidil ethosomes (liposomal systems containing ethanol) versu- Minoxidil in vehicle. Both preparations contain the same concentration of Minoxidil Methods S In vitro skin permeation measurements: Skin permeation experiment was carried out in our laboratory by using the Franz cell assembly to perform the experiment.

*.i 19 Frozen back side skin of a 5-7 week old male mice was used in this investigation (nude mice obtained from the Weizmann Institute, Rehovot, Israel). The skin was examined for integrity and cut for mounting on diffusion cell assembly.

Nude mouse skin was mounted in cells with a surface area of 1.77 cm 2 and receiver volume of about 7 ml. The receiver compartments contained 1/150M pH7 phosphate buffer for ensuring pseudo sink conditions. Samples were directly assayed by HPLC. The experiment was run for 12 hours. Each formulation was tested in three cells.

Minoxidil Ethosomes Minoxidil 1% Phospholipon 2% Ethanol 95% Distilled Water 57% Minoxidil vehicle Minoxidil 1% Ethanol 95% Distilled Water 59% Results Qlo= quantity in the receiver compartment of the diffusion cell at the end of experiment.

Minoxidil vehicle Minoxidil ethosomes 16.26+2.8 pg/cm 2 64.02+22.5 tg/cm 2 The above results, of about 16 pg/cm 2 versus about 64 pg/cm 2 skin penetration of the two preparations, demonstrates that the "ethosomes" of the invention resulted in an about 4-fold skin penetration compared with the penetration of the active substance in the vehicle only, i.e. not in liposome form. These ethosomes were without propylene glycol.

e oe Example XX The following experimental results, relate to various liposome systems of the invention containing 1% sodium diclofenac as model drug and in which various compositional factors have been changed: 1. the concentration of alcohol 2. the phospholipid 3. the type of alcohol.

The results demonstrate: 1. the cruciality of high concentrations of alcohol, and that the high skin permeation from ethosomal systems of the invention is still obtained: 2. with an additional example of phospholipid (Lipoid E 75-containing phosphatidyl ethanolamine and phosphatidyl choline isolated from egg, produced by Lipoid KG; Germany, 3. with isopropyl alcohol.

Methods: In vitro skin permeation measurements.

Skin permeation experiment was carried out in our laboratory by using the Valia Chien assembly to perform the experiments.

Frozen back side skin of a 5-7 week old mouse was used in these experiments (nude mice obtained from the Weizman Institute, Rehovot, Israel). The skin was examined for integrity and cut for mounting on diffusion cell assembly.

CC

The skin was mounted in cells with a surface area of 0.64 cm 2 and receiver volume of about 3 ml. The receiver compartments contained 1/150M pH7 phosphate buffer for ensuring pseudo sink conditions.

22 Samples were directly assayed by HPLC. The experiments were run for 17 hours. Each formulation was tested in triplicates.

FORMULATIONS:

XA XB, XB2

XC

w/w XD XE Sod.Diclofenac Phospholipon 90 Lipoid E Ethanol Isopropyl Alcohol Distilled Water 1 5 21 73 1 1 1 1 5 5 35 5 S 59 89 59 59 Results: quantity in the receiver compartment of the diffusion cell at the end of experiment.

XA

XB,

37.7+11 144.1+14 Qrec, Pg/cm 2

XB

2

XC

300.2+29 57.6+25 XD XE 285.2+37 1558.8+778 These results clearly indicate that: 1. The ethosomal systems of the hydrophilic drug diclofenac sodium, containing a high concentration of ethanol give an enhanced delivery of drug through the skin of eight times higher than the preparation containing only 5% ethanol; 2. The enhancing effect is also obtained with isopropyl alcohol; 3. The enhancing effect is obtained when the composition of phospholipids is changed; The above results demonstr'e the improvement in permeation with novel liposimal ("Ethosomal") rystems of the invention.

Remark: all these systems are without propylene glycol (PG) showing the noncriticallity of PG.

The invention io illustrated with reference to the enclosed Figures and photos, in which: Fig. 1 is a graph showing Differential Scanning Calorimetric data of an ethosomal system containing 5% PL-90 in water.

Fig. 2 is a graph showing Differential Scanning Calorimetric lata of ethosomal system No. 525 (See Table 2).

Fig. 3 is a photograph of ethosomal systemt No. 510 (See Table 2).

**i Fig. 4 is a photograph of ethosomal system No. 529 (See Table 2).

ee 99' 9. 9. 9 9 999 TABLE No. 3 MALVERN AUTOSIZER II c for ethosomal system No. 501 (see Table 2) Sample: 501; Vortex t 22.6; Aperture: 200 Data from accumulation file sum of 5 blocks Temperature: 22.60C; Viscosity: 4.1800; Refractie Index: 1.365; Angle: 90.0 Particle size. distributliori nm. Graph o Dji.stri.buLtion oC mass 10.0 12.1 i 12.1 14. 6 14.6 17.7 4- 17.7 21.5 21.5 26.0 I 26.0 31.5 31.5 38.2 S38 .2 46.2 46.2 56.0 56 .0 67 .9 67.9 82.2 2 8202 99 .6 1 99.6 120.7 1.

126.7 146 3 I.

146 .3 177 2 I- 177.2 214 7 I- 214 .7 260.1 t 260.1 315.1 315 .1 301. 7 346 .7 46 5 462 .5 560 3 560.3 678 .8 678. 8 822.4 4- 822 .4 996.4 9** 0* 9 i 9** p 9000 9 0 0 9 0 0 0000 .9 0* 0 0 00 9 I I -WC TABLE No. 4 MALVERN AUTOSIZER 11 c, lr othosomal systern No, 509 (see Table 2) Sample: 509; Vortex US; Non diluted Data from accumulation file sumn of 8 blocks Temperature: 22.60C; Viscosity: 4.8200; Refractive Index: 1.376; Angle: 90.0 Particle size dis~ribuLion- fin. Graph of Distributio n of mass 5.4 5.4 V~ 6.4 7 1 7 .7 9 .2 9 .2 11 .0 11.0 13.2- 15.8 .8 8 .9 -I 10.9 22.6 22.6 27A.1- 27.1 32.4 32.4 38.0 38.8 46.4 4-6.4 55.6 55.6 66.6 -1 66.6 79.7 1 79.7 95.5 1.

95.5 114.3 t 114.A 1..6.8 I.

136.8 163.8 1 163.8 196.2 1 196.2 234.9 1 234.9 2U1.2 201 .2 336 .7 1 9* i i 9* 0% 00* TABLE No. MALVERN- AUTOSIZER 11 c for ethosomal systeOm NQ.- 510 (see Tabl 2 Sample: 510; Vortex US; Non diluted Data from accumulaticn. file sum of 6 blocks Temperature: 22.6OC-, Viscosity: 4.6400; Refractive Index: 1.374; Angle: 90.0 Particle size distribUtioti nrn. Graph oE Distribution oE mass 17.4 21.1i 21.1 25.6 4 25.6 .A.C 4 31- 37.6 376 55 .1 55.1 66.8

I

6b .8 .9 98.0 119.8 113.9 174 .3 211.2 255 .9 310 .0 375. 6 455 .0 51 3 667 .9 809 .2 980. 3 1187 .7 14 39. 9 80U.9 99 -143.9 -174.1 -211.2 -255 .9 310 .0 375.6 551.3 -667.9 -809.2 -980.3 1107.7 1438 9 1743.3 4

I.

References 1. Barry, Optimizing percutaneous absorption. In Bronaugh, R.L. and Maibach, M.I. (Eds), Percutaneous Absorption, Dekker, New York, 1989, pp. 531-565.

2. Batzri, S. and Korn, E.D. Single bilayer liposomes prepared without sonication. Biochim. Biophys. Acta 298 1973) 1015-1019.

3. Kremer, J. M. v. d. Eskcr, M. W. Pathmamanoharan, C. and Wiersema, P. H. Vesicles of variable diameter prepared by a modified injection method. Biochemistry 16 (1977) pp.3932-3935.

4.Leigh, Pro-liposome compositions,United States Patent No.5,004,611, April 2, 1991.

Riaz, Weiner, Martin, F. In Pharmaceutical Dosage Forms.

Disperse Systems, Vol. 2; Lieberman, Rieger, Banker, Eds, Marcel Dekker, Inc. New York and Basel, 1988 pp 567- 602.

6. Touitou, E. and Fabin, B. Altered skin permeation of a highly lipophilic molecule: tetrahydrocannabino Int. J. Pharm. 43 (1988) 17-22.

C

7. Touitou, Levi-Schaffer, Shico-Ezra, Ben-Yossef, Ramy and Fabin, B. Enhanced permeation of theophylline through the skin and its effect on fibroblast proliferation. Int. J. Pharm. (1991) 159-166.

Claims (9)

1. A liposomal composition for medical or cosmetic use, for topical application to the skin, resulting in the enhanced transdermal passage or introduction of an active ingredient into the skin, which composition contains vesicles in the size range from the nm range to the [tm range, which composition comprises from 0.5% ww to 10% w/w phospholipids, from 20% w/w to 50% w/w of a C 2 to C 4 alcohol, from 0 to 30% w/w glycol, at least 20% w/w water and at least one active ingredient where the combined alcohol and glycol content does not exceed 70% w/w.

2. A composition according to claim 1 wherein the penetration through the skin of said composition is at least four times higher than the penetration through the skin of a similar composition comprising less than 5% w/w alcohol.

3. A composition according to any of claims 1 or 2, where the phospholipids comprise at least one member selected from the group consisting of phosphatidylcholine, (P hydrogenated P C, phosphatidic acid (P phosphatidylserine (P phosphatidyl- ethanolamine (P phosphatidylglycerol (P P phosphatidylinositol (P I hydrogenated P C and similar compounds.

4. A composition according to any one of claims 1 to 3, where the alcohol compound is ethanol, and where the combined alcohol and the glycol content comprises .ethanol and propylene glycol, where the ethanol concentration in the final preparation is 20 between 20 and 50 weight of the final product, the content of water being at least about w/w.

5. A composition according to any one of claims 1 to 4, in which the active ingredient is a member selected from the group consisting of peptides, enzymes, hormones, anti-aging agents, tanning agents, vitamins, antiviral drugs, plant extracts, 25 glycosides, alkaloids, anxiolytics, antiepileptics, antifungals, non-steroidal anti- inflammatory drugs, antihypertensive agents, corticosteroids, minoxidil, cannabinoids, antibiotics, hydroxy acids, antimitotics, antimycotics, retinoic acid, diclofenac and acyclovir.

6. A composition according to any one of claims 1 to 5, where the alcohol is selected from ethanol and isopropyl alcohol, where the glycol is propylene glycol or ethyl diglycol, or a mixture of any of the alcohols with any of the glycols, or each alcohol by itself. <ST 7. A composition according to any one of claims 1 to 6, comprising 22 to Z /w of a combination of the alcohol and propylene glycol, and more than 20% w/w [R:\LIBZZ]06603.doc:nss

8. A composition according to any one of claims 1 to 7, where the alcohol is S ethanol, and where the combined alcohol and the glycol content comprises ethanol and propylene glycol, where the amount of ethanol is between 20 and 50 weight-% of the composition, the content of water being at least about 20% w/w.

9. A liposomal composition for medical or cosmetic use, for topical application to the skin, resulting in the enhanced transdermal passage or introduction of an active ingredient into the skin, which composition contains vesicles in the size range from the nm range to the pm range, which composition comprises from 0.6% ww to 10% w/w phospholipids, from 20% w/w to 50% w/w of a C 2 to C 4 alcohol, from 0 to 30% w/w glycol, at least 20% w/w water and at least one active ingredient where the combined alcohol and glycol content does not exceed 70% w/w, substantially as hereinbefore described with reference to the Examples but excluding comparative Example 19. A process for the production of a cosmetic or medical composition for topical application to the skin, for rapidly introducing into the skin, or for enhanced penetration through the skin, which contains an active ingredient in a carrier defined in any of claims 1 to 9 which comprises mixing a phospholipid, a C 2 -to C 4 -alcohol, or such alcohol and glycol, water, and an active ingredient so as to form a colloid system containing vesicles.

11. A process for the production of a cosmetic or medical composition for topical o application to the skin, for rapidly introducing into the skin, or for enhanced penetration 20 through the skin, which contains an active ingredient in a carrier defined in any of claims 1 to 9 which comprises mixing a phospholipid, a C 2 -to C 4 -alcohol, or such alcohol and *a: glycol, water, and an active ingredient so as to form a colloid system containing vesicles, substantially as hereinbefore described with reference to the Examples but excluding comparative Example 19. 25 12. A cosmetic or medical composition when produced according to the process *to i* of claim 10 or claim 11. Dated 27 March, 2002 Elka Touitou Yissum Research Development Company of the Hebrew University of Jerusalem 7 Patent Attorneys for the Applicants/Nominated Persons SPRUSON FERGUSON [R:\LIBZZ]06603.doc:nss