CA2065579A1

CA2065579A1 - Stable small particle liposome preparations, their production and use in topical cosmetic and pharmaceutical compositions

Info

Publication number: CA2065579A1
Application number: CA002065579A
Authority: CA
Inventors: Edgar Mentrup; Christoph Michel; Thomas Purmann
Original assignee: Individual
Current assignee: Merz and Co GmbH and Co KG
Priority date: 1991-04-12
Filing date: 1992-04-08
Publication date: 1992-10-13
Also published as: DE4111982A1; DE59203259D1; EP0509338A1; ES2077904T3; DK0509338T3; JPH0748247A; GR3017147T3; ATE126430T1; EP0509338B1; DE4111982C2

Abstract

ABSTRACT OF THE DISCLOSURE

The invention relates to liposome preparations with controllable vesicle size and type and high stability without increased permeability, the vesicle membrane of the liposomes containing lecithin with 16-90% fatty acid-esterified collagen hydrolysate, especially OHAP, plus traditional jellifying agents, adjuvants, and pharmaceuti-cal agents as desired, and the production and use thereof in cosmetic and pharmaceutical compositions.

Description

2~57.'~

STABLE SMALL PARTICLE LIPOSOME PREPARATIONS, THEIR PRODUCTION AND USE IN TOPICAL COSMETIC
AND PHARMACEUTICAL COMPOSITIONS
As is generally known, liposomes are small vesicles having a bilayer structure. They are used in the pharmaceu-tical area as drug vehicles, e.g., for parenteral or topical application.
The most varied agents with different chemico-physical properties can be incorporated into liposomes. Water-soluble agents can be located in the core and between the vesicles' bilayers. Lipophilic substances, on the con-trary, are incorporated in the bilayer. Therefore, the incorporation of greater amounts of such agents into liposomes can result in a marked increase in vesicular diameter. In this case, pharmacokinetic and therefore also therapeutic properties can change considerably. Small liposomes afford more advantages because they are not identified by the RES (reticulo-endothelial system). In consequence, they are not eliminated from circulation through hepatic or renal filtration, resulting in higher plasma levels over prolonged periods of time. For example, for such liposomes half-lives of over 20 hours were determined (c~. J. Senior et al., Biochem. Biophys. Acta 839, 1 (1985)). This means greater availability of an encapsulated active agent at the potential site of action, e.g., a tumor, because the circulating liposome deposit is very slowly degraded by enzymatic decomposition and ; - 1 - MERZ 20/dlk , - - , , .

.: ,;
. ~ . , .
. . .: , . .

7t9 interactions with other blood constituents. During this process the encapsulated active substance is released and is available for thPrapeutic utilization.
Large vesicles, on the contrary, are identified by the RES (hepatic Xupffer cells) as foreign bodies and de-stroyed. Even a few minutes after the i.v. application of such particles, 90~ of these particles is eliminated. A~ter the application of 400 nm liposomes, a half-life of about 10 min. was ~ound; with particles of 1000 nm it was reduced to only about 5 min (cf. J. Senior et al. and P~0. Lelkes, Liposome Technology, Vol. 1). Also after topical applica-tion, small vesicles containing active ingr~dients ma~
possibly exert a better effect through improved penetra-tion, ~or example, along the hair follicles. Additionally, an interaction may be possible with the natural bilayer structures in the intercellular gaps (cf. W. Curatolo, Pharm. Res. 4, 271-341 (1987), G. Grubauer et al., J. Lipid Res. 30, 89-96 (1989)). Furthermore, from a statistical point of view, the interaction of unilamellar vesicles with other cells is likely to be greater than that of multi-lamellar vesicles. This facilitates the transfer of membrane constituents, e.g., bilayer-bound ingredients.
In FR-A 2 591 105, pharmaceutical compositions based on lipid-containing multilamellar vesicles for dermatologi-cal or cosmetic application are described which contain a retinoid or an analogue thereof. This combination is supposed to prevent skin irritations. The vesicular constituents used are hydrated lipids and, for example, cholesterol and sitosterol. However, the vesicles of the resulting liposomes are large in particle size, and are accompanied by disadvantages. Therefore, it is more reasonable to strive for small-size liposomes.
In addition to the particle size, the stability of the - 2 - MERZ 20/dlk :
- ~ , ~ , ,:-", ' '~ , ' ' , , . .

g ~3 ?~ ~

liposome preparations is also decisively important for their therapeutic or cosmetic use.
From a yalenical point of view, the therapeutic application of the liposome preparations is often problem-atic and limited because of their instability and the active ingredient incorporated. One of the main problems is a degradation of the active ingredient. Route and rate of decomposition are specific and vary from substance to substance. Oxidation processes can be reduced by using suitable antioxidants such as butyl-hydroxytoluene or vitamin E. pH-Dependent hydrolysis can be reduced by suitable buffers. In the case o~ substances senæitive to this process, hydrolysis can still be increased through intensified contact with water. However, they can be stabilized by sufficient incorporation in the bilayer.
Decisive for the stability of the active ingredient are the decomposition reaction and the physico-chemical properties of the substance.
Besides decomposi~ion of the active ingredient, as de-scribed above, the liposomes themselves can be affected by lipid oxidation, lipid hydrolysis, and aggregation. Lipid oxidation can be reduced through addition of antioxidants as described by A.A. Hunt, S. Tsang, in Int. J. Pharm. 8, 101 (1981) or by A.W.T. Koninss in Liposome Technology, Vol. I. Alternatively, there is the possibility of using lecithins with non-oxidizable saturated fatty acid esters which cannot be oxidized (cf. P. Kibat, Dissertation, Univer~ity of Heidelberg, 1987). Hydrolysis of lecithins to lysolecithins can be reduced by buffering the system within a pH range between 6-7 (cf. S. Froeckjear et al., Opt~miza-tion of Drug Delivery, A. Benzon Symposium 17, Kopenhagen (1982)).
Moreover, especially with small unilamellar vesicles, - 3 - MERZ 20/dlk ~ 7~3 7 r~

undesirable particle aggregation can occur already after a few weeks. During this process, the properties of the vesicles change during the shelf-life of a product. The measures known so ~ar to combat such changes comprise the use of hydrated (satura~ed) lipids or cholesterol up to 50 mol-% in relation to the total lipid amount (cf. P. Kibat).
The increase in particle size occurring when using hydrated lipids, however, turned out as a disadvantage. In addition, the phase transition temperature for hydrated lipids is between 50 and 60C, as compared to -15 to 30~C for common lecithins, e.g., phosphatidyl cholines (cf. B.D. Ladbrooke, D. Chapman, Chem. Phys. Lipids 3, 304 (1969). Because of the fluid state condition required for the manufacture of liposomes, the working temperature must be above the phase transition temperature. Therefore, the technical processing of hydrated lipids is more difficult. Furthermore, the synthetic hydrated lipids are more problematic from a toxicological point of view than native lecithins from eggs and soy beans.
When using cholesterol for stabilization purposes, the reduced bilayer-binding of lipophilic constituents is often problematic because of a competitive displacement through cholesterol (cf. Mentrup, Dissertation, University of Heidelberg, 1988). Depending on the characteristics of the active ingredients, the bilayer-binding of therapeutically effective amounts may be impossible.
The aim of the present invention, therefore, is to manufacture liposome preparations of small, mainly constant particle size and overall stability in which there is no decomposition of a possibly bound active ingredient.
Furthermore, toxic skin reactions are a~oided both with topical therapeutic and cosmetic application.
According to our investigations, this problem can be _ 4 _ MERZ 20/dlk '.
~ ' .

2 ~

solved by selecting a leci-thin as starting material for liposome production which contains a 16 to 90~ fatty acid-esterified collagen hydrolysate.
The best suited building elements for liposome preparations are soy or egg lecithin. In addition to these two native lecithins, other synthetic or native C16 - C20 phosphatidyl cholines with saturated or unsaturated fatty acid chains can also be used. In our invention, preferably 20 ~o 80~ of the ~atty acid-esterified collagen hydrolysate is incorporated. Especially preferred is the oleic acid--esterified collagen hydrolysate (OHAP or oleyl hydrolyzed animal protein). This material is an ampiphilic tenside present in solid orm. Although it has been used as stabilizer in pharmaoeutical and cosmetic creams, it has so far not been used to stabilize liposomes. Such fatty-acid esterified collagen hydrolysates are prepared by the condensation of hydrolisation products of animal protein with the chloride of the selected fatty acid, which may be besides oleic acid, for example, palmitic, stearic, linoleic, ricinoleic, coconut, abietic, or linolenic acids.
The total concentration of lecithin and fatty-acid esterified collagen hydrolysate in the resultant aqueous dispersion is between 5 and 350 mg/ml, preferably between 20 and 180 mg/ml. This corresponds to about 0.5 to 35% or preferably 2 - 18% lecithin and fatty-acid esterified collagen hydrolysate by weight in the liposome preparation.
The addition of fatty-acid esterified collagen hydrolysate, especially of OHAP, produces liposomes much smaller in size than would be obtained without this additive, which affords the above-described advantages. This effect has so far not been obtained with liposomes containing active ingredients.
Only with substance-free liposomes has a reduction in vesicular size been achieved by addition of a few other - 5 - MERZ 20/dlk -~

:

7 ~

substances with ampiphilic character. The following Table 1 shows the influence of various substances commonly used in liposome technology on the particle size of the lipo-somes produced in a high-pressure homogenizer, i.e., according to the same process. When producing large liposomes from soy lecithin by the hand shake method (cf.
A.D. Bangham et al., J. Mol. Biol. 13, 238 (1965)), the liposomes containing an ampiphilic substance (e~g., cholesterol) are much larger than the original vesicles.
The addition of fatty-acid esterified collagen hydrolysate, preferably of OHAP, according to the present invention, however, does effect a marked reduction in particle size, as described in the following. Therefore, the influence on vesicle size is independent of the process, and only lS substance-determined.

- 6 - MERZ 20/dlk .

. . .

2 ~

Table 1 Influence of various subs~ances on the size of substan^e-free lipid vesicles (Process: High-pressure homogenizer, 30 min. 1.4 . 108 Pa) . = _ _ ~...... --_ . .
¦ Concentration Lipid compo- Concentration Ve~icle ~ize oy iecithin nent lipid (mg/ml) component (nm) (mg/ml) I _ ~; ~ ~--- . ~1 100 ___ ___ 40 ~ 10 O~AP 20 25 ~ 4 medium-chain 20 71 + 15 triglyceride~
Squalene 20 57 ' 12 Chole~terol 20 58 1 14 Polyoxyethy~n~- 20 34 1 7 Polysorbate 80 20 24 + 4 96 Polysorbate 80 4 25 ~ 5 _ _ . _ ---~ ._---_ .. ~

Polysorbate 80 is the only tenside which, in substance-free liposomes, effects a marked reduction in vesicle size (cf. B. Kronberg et al., ~. Pharm~ Sci., 79 ~8), 667-71 (l990)). In liposomes containing substances such as toco-pherol nicotinate or hexachlorophene, the addition of poly-sorbate 80 (e.g. 20 ~) leads to a marked increase in part-icle size with inhomogeneous distribution. Therefore, this substance is not suited for the manufacture of pharmaceu-tically-stable products.

As already mentioned, the use of fatty acid-esterified collagen hydrolysate according to the invention effects a reduction of vesicle size also in the presence of lipo-philic therapeutics. Lipophilic agents suitable for - 7 - MERZ 20/dlk ' J ~ it r9 bilayer-binding are, for example, acne therapeutics such as hexachlorophene, tretinoin, or minocycline. Other topical agents such as a-tocopherol nicotinate, tromantadine base, croconazole, minocycline, meclocycline, cyproterone, cyproterone acetate, corticoids, 2-tert.-butyl-4-cyclo-hexylphenylnicotinate-N-oxide,corticosteroids,androgens, ethinyl estradiol, non-steroid antiphlogistics, dihydro-pyridines, spironolactone, erythromycin esters, lipophilic local anaesthetics, estradiol esters, or lipophilic anti-histaminics can also be incorporated. The amount of active substance can be varied in dependence upon th therapeutic requirements. For example, 10 mg to 50 g of active ingredi-ent can be used per 100 g of lipid-containing fatty acid-esterified collagen hydrolysate or OHAP-mixed lipid.
This affords the possibility of incorporating such agents into liposomes with very small vesicle size. Based on the general vesicle size, an increase in therapeutic activity is possible due to the small diameter.
A special advantage of adding fatty acid-esterified collagen hydrolysate to lecithin liposomes is that, by the addition of varying amounts thereof, which are incorporated into the vesicle membrane, the desired vesicle size can be controlled within a wide range as is shown in the following Table 2.

- 8 - MERZ 20/dlk ~ ~ 3 ~ 7 ~

Table 2 Influence ~of OHAP ~ercentaae on the vesicle size of toco~herol nicoklnate-containin~-liposomes (Process: Hiqh-pressure homoqenization -3Q min. 1.4 ._lo8 PaL

_= r . _ ~ _ __ _ _ _ ___ _ ._ _~
Concentration Concentration Vesicle size soy lecithin OHAP
(mg/ml) (mg/ml) (~m) , . _ . _ .__ 11 100 ___ 51 + 14 50 42 + 17 ~0 26 + 5 __. - ~ _ ___ _ ~
Tha following Table 3 shows that by the addition of OHAP
the particle size of larger liposomes obtained by ethanol injection can also be controlled.

Table 3 Influence of_~AP ~erce~ Le on the vesicle size of su~ance-free liposomes (Process: Ethanol in~ction accordin~ to ~xample 6) . ._ , Concentration Concentration Vesicle size Soy lecithin OHAP
¦ (mg/ml) (mg/ml) (nm) I _ ._ .. . _ I
32 ___ 637 i 309 32 8 175 + 106 24 12 109 ~ 26 24 2~ 99 + 26 . . --_ _ _ __ , . ~

_ g - ~RZ20/dlk : ~ ' , ;
- . ~ :

.

7 ~

As can be seen from Tables 2 and 3, the extent of vesicle size reduction in substance-loaded liposomes depends on the active ingredient. This is particularly apparent from Table 2.
An essential fac~or for the large-scale application of liposomes is the liposomes' storage stability already described. The most important physical paramster of the storage stability is the slze of the vesicles. Vesicle aggregation which especially occurs with lecithins can be markedly reduced by the addition of a suitable fatty acid-esterified collagen hydrolysate as shown in Table 4 according to Example 2. These liposomes according to the invention are therefore small~r and stable under storage conditions. A loss in therapeu~ic effect occurring as a result of aggregation can thus be prevented. In addition, possible chemical liposome instability can be reduced by reduction of the lecithin portion (due to addition of OHAP). It is only the lecithin that can be affected by the destabilizing formation of lysolecithin. Thus, the associ-ated toxic effect can be aonsiderably reduced.
The following Table 4 shows a comparison of the stability of the preparations according to the invention.

- 10 - MERZ 20/dlk 2 ~

Table 4 Stability o oH P-~nntaining tocopherol nicotlnate liposomes I =
¦ L~pid Tota~ ~ipid Vesicle size tnml after tlon 0 4 1Z 24 ~mg/ml) ~weeks) . _ _ _ _ . . I - !
Soy lecithin ~compari-son) 100 90 ~ 19 96 ~ 23 196 ~ 188 Z74 ~ 191 Soy leci-thin/OHAP
8 2 accord-xample 2 nvention) 100 49 ~ 14 45 ~ 1444 ~ 10 4Z ~ 11 ¦
~ = _ ~~ n_ ~ ~ _ -- ~= _____ ,= _ _ ~
The Table shows that the storage stability of the liposomes according to the invention is much better than that of comparable liposomes not containing OHAP~

The stabilization of liposomes by polysorbate described by B. Kronberg et al. can be demonstrated over only a short storage period. As already described, particularly sub-stance-containing liposomes are markedly larger and more inhomogeneous than liposomes made of soy lecithin and fatty acid-esterified collagen hydrolysate or OHAP. After a 4-week storage period, the liposomes develop consider-able sedimentation which is not acceptable for a pharma-ceutical product. Therefore, polysorbate as an additive to substance-containing liposomes is not suited for vesicle size reduction and stabilization.

Liposome preparations ~or topical application are usually mixed with polymer jellifying agents to increase viscosity and improve application. For this purpose, especially - 11 - MERZ 20/dlk 2 ~ ~ r~

polyacrylic acid (0.2 - 1.5 %), cellulose derivatives (0.2 - 3 ~) and sodium salts of acrylic acid/acrylamide copoly-merisates (CTFA nomenclature - 1 - 4 ~); commercially available as Hostacerin PN73~) in the concentrations indicated are used. When adding such jellifying agents to liposome preparations made o~ pure lecithin, the vesicles frequently aggregate as a result of in~eractions with the poiymer. In contrast to that, the fatty acid-esterified collagen hydrolysata-containing vesicles according to the invention surprisingly show only a slight change in vesicle size after having been made into polymer gels. This is documented in the following Table 5.
Table 5 Chanae of vesicle size after incorporation of llposomes .. _ _ ,, ~ ~ . _ ¦ Lipld Tot~ llpld Vaslcb slz~ V~iclo siz~ Vo~ br ~ d Cont~nt conc~n~ra- b~for~ lelli~ lelll d ~cUvo ~on nc~on n~on S 12 24 32 ~u~nco (mg/ml~ Inm) (nm) h~) db~ 32 . . - . . _ .___ __ .
l SL 100 I~S + 1~ 10~ 421 110 ~24 117 +23 124 ~23 142 + 31 82~#
l ._ _ , _ _, _ __ _ . _ , . _~
SVOHAP 100 40 ~ 10 40 4 1~ 42 + 1O 45 i: 10 42 + 11 41 ~ 100#
__ ,_ _ ___ ~__ . ~ _... _ ____ ..

SL = soy lecithin The liposomes according to the invention can be prepared by dispersing a mixture of lecithin, preferably egg or soy lecithin, and 16-90%, preferably 20-80~, fatty acid esterified collagen hydrolysate, preferably OHAP, and, as required, a pharmaceutical agent described above and the traditional auxiliary agents in an aqueous phase having an - 12 - MERZ 20/dlX

~ J3~;~

appropriate pH value, preferably about 5-7~ The resul-ting dispersion can be reduced in particle size and homogenized, preferably by high-pressure homogenization.
In addition, the above mixture of lecithin, collagen hydrolysate and auxiliary agents and, if required, pharma-ceutical agent, is preferably injected as an ethanolic solution into an aqueous phase (ethanol injection~.
~ubsequently, the dispersion can be mixed with the common amounts of a suitable jellifying agent, preferably one of those described above. The gel is prepared according to commonly known procedure.
Especially preferred in the production of the prepara-tions according to the invention are mixtures of egg or soy lecithins with fatty-acid esterified collagen hydrolysate, especially OHAP, the described jellifying agents and active substances, which are homogenized by high-pressure homogs-nization procedure or by ethanol injPction.
Alternatively, common adjuvants such as, for example, an antioxidant, e.g., vitamin E or but~lhydro~y toluene (BHT), and preservatives such as phenoxethol, sorbic acid, Kathon CG~ (Merck Index 11, No. 6677), or parabens can be added in usual amounts.
The vesicle si~e of the resultant substance-containing liposome preparations is about 20 to 150 nm, preferably 20 to 70 nm. Depending on the active ingredient used, they are especially suited for topical application as dermato-logicals or cosmetics. As compared to traditional products, an increase in therapeutic activity has been observed which is due to an intensified interaction of the vesicles with the skin cells because of their reduced diameter. Addition-ally, because of the product's higher stability, long-term efficacy without toxic side effects is ensured.
Furthermore, the preparations according to the - 13 - MERZ 20/dlk 2~ }7~

invention are easy to produce. During this process the described advantages may be utilized without hydrated lipids becoming necessary.
The invention is further illustrated by the following examples, which are not to be construed as limiting.
Example 1 Preparation of an OHAP-containinq liposome gel bY hiah-~ressure homogenization Introduce 80g of soy lecithin, 9g of phenoxyethanol and 45g of ethanol into a suitable vessel and heat to 50C
under stirring. To this clear homogenous liquid add 400 mg of butylhydroxy toluene, 20g of OHAP and 745.6g of hypotonic buffer (pH 6.5). Treat the lipid dispersion (9OOg) in a high-pressure homogenizer (1.1.108 Pa; 40 min.) and subsequently filter through a membrane (0.45 lum).
The gel is prepared by dispersing 5g of polyacrylic acid (e.g. Carbopol~ 984), lg of phenoxyethanol and 9.8g of TRIS buffer in a mixture of 5g of ethanol and 80.2g of phosphate buffer (pH 6.5). The gel concentrate is allowed to swell for one day, and then admixed into the liposome dispersion described above.
Example 2 Preparation of an OHAP-containing toco~herol nicotinate liposome ael by hiqh-pressure homoaenization Introduce 80g of soy lecithin, 20g of -tocopherol nicotinate, 7.5g of phenoxyethanol and 37.5g of ethanol into a suitable vessel and heat to 50C under stirring. To the clear homogeneous liquid add 400 mg of butylhydroxy toluene, 20g of OHAP and 8?9.5g of hypotonic phosphate buffer (pH 6.5). The resultant llpid dispersion (99Og) is treated in a high-pressure homogenizer (1.1 . 108 Pa; 40 min) and subsequently filtered through a membrane (0.45 ,um ) .

- 14 - MERZ 20/dlk 2 ~ 3 r~ 9 The gel is produced by sprinkling lOg of carboxy-methylcellulose onto 990g of liposome dispersion and allowed to swell for 24 hours. The liposomes are much stabler under storage conditions than comparable liposomes not containing OHAP. After 1- year storage at 21C, the active content is 96.9~. Within this period the vesicle size changed from 27.3 + 3.8 to 29.5 + 6.5 nm.
Example 3 Preparation of an OHAP-containina cvproterone li~osome ael bY high-pressure homoqenization Introduce 80g of soy lecithin, 100 mg of cyproterone, lOg of phenoxy ethanol and 50g of ethanol into a suitable vessel and heat to 50C under stirring. To the clear homogeneous liquid add 400 mg of butYlhydroxy toluene, 20g of OHAP and 829.5g of hypotonic phosphate buffer. Treat the resulting lipid dispersion (99Og) by high-pressure homoge-nization (1.1 . Io8 Pa; 40 min.) and subsequently filter -through a membrane (0.45 ~m). The gel is prepared by spreading lOg of carboxymethyl cellulose onto 990g of liposomal dispersion under stirring, and allowed to swell for 24 hours. The storage stability of the resulting liposomes is much better than that of comparable liposomes not containing OHAP. After l-year storage at 21C, the active content is 96.9~. Within this period the particle size changes from 27~3 + 3.8 to 29.5 + 6.5 nm.
Example 4 Preparation of an OHAP-containing tretinoin liposome ael bY hiah-Pressure homoaenization Introduce 200 mg of tretinoin and 400 mg of butyl hydroxy toluene into a suitable vessel and dissolve in 50g ethanol under stirring. Disperse 80g of lecithin, 20g of OHAP and lOg of phenoxy ethanol in isotonic phosphate buffer (pH 6.5; 802g). Pour the ethanolic solution into the - 15 - MERZ 20/dlk 2 ~ Y~ ~

aqueous phase under Ultraturraxtm treatmen-t. Treat the resulting dispersion (962.5g) with a high-pressure homoge-nizer (1.1 , 108 Pa; 40 min) and subsequently filter through a membrane (0~45 ~m)O The liposomes have a vesicle size of 23 ~ 4 nm.
The gel is prepared by dispersing 12.5g of polyacrylic acid (Carbomer 941~) and 25g TRIS in the liposome disper-sion under stirring, and allowed to swell for 24 hours.
The residual tretinoin content after 1 year (storage at 4C) was 95.2~, the vesicle size being 29.2 + 6.4 nm.
Example 5 Preparation of an OHAP-containing croconazole liposom~ qel by hiqh-pressure homoqenization Introduce lg of croconazole into a suitable vessel and dissolve in 10g of ethanol. Under Ultraturrax~n treatment, pour the ethanolic solution into the aqueous phase which contains the lecithin, OHAP, phenoxethol. Treat the resulting dispersion by hi~h-pressure homogenization (1.1 . 103 Pa; 40 min) and subsequently filter through a membrane (0.45 ~m). The gel is prepared by dispersing 12.5g of polyacrylic acid (Carbomer 941~) and 25g TRIS in the liposome dispersion under stirring, and allowed to swell for 24 hours. The pH value of the gel is between 7 and 8.
The vesicle size of the liposomes is 22 + 3 nm.
Example 6 PreEaration of an OHAP-containinq liposome dispersion by ethanol injection Dissolve 2.4g of soy lecithin and 2.4g of OHAP in 20 ml of ethanol and inject into 80 ml isotonic buffer (pH
6.5) by means of a suitable syringe. Inject the solution in an ultrasonic bath to achieve a better dispersion of the ethanolic solution. Then filter the solution through a membrane (0.45 ,um). The ~inal concentration is 4.8~ lipid - 16 - MERZ 20/dlk and 20% ethanol. The resultant vesicle size is much smaller than in the case of pure soy lecithin liposomes (cf. Table 3).
Example 7 Preparation of hexachlorophene-containing liPosomes bv ethanol in~ection Dissolve l.Og of hexachlorophene, 0.2g of tocopherol, 2.5g of soy lecithin and 2.5g of OHAP in 20g of ethanol and inject, using a suitable syringe, into 74 ml of citrata/phosphate buffer (pH 5.5; containing 0.2~ sorbic acid/potassium sorbate) under ultrasonic treatment. Filter the dispersion through a membrane (0.45 ,um). The final concentration is 1% hexachlorophene, 5.0~ lipid and 20~
ethanol, the vesicle size being 65 ~ 11.4 nm. Use this liposome dispersion for preparing a gel with lg of poly-acrylic acid (Carbopol 98 ~).
The residual active content after l-year storage at 4lC is 99.4%, the vesicle size being 66.3 + 10.3 nm.
Example 8 Preparation of OHAP-containina liPosomes for the encapsu-lation of hydro~hilic substances by high-pressure homoqe-nization Introduce 42g of soy lecithin and 8g of OHAP into a round-bottom flask and dissolve in ethanol. Remove the solvent under reduced pressure using a rotary evaporator.
Redisperse the resulting film with 450 ml of quinoline yellow- (2 mg/ml) or Na-carboxyfluoresceine- (2 mmolar) containing isotonic phosphate buffer (pH 6.5). Treat the resulting lipid dispersion by means of high-pressure homogenization (1.1 . 108 Pa; 20 min) and subsequently filter through a membrane (0.45 ,um).

- 17 - MERZ 20/dlk .

' 2 ~ ;~ t~

The following Table shows the initial vesicle size and encapsulation rate as well as the stability o~ these two parameters after 1-month storage at 4C:

I - _ . __ _ _ _ _ I
¦ Encapsulat- Vesicle size ~n~) Encapsulation rate I ed substance I ._ _ ., . _ __ ¦ Substance initial valueafter 1 ~onth initial value after 1 month lI _ j _ _ _ .
Quinoline 35.6 ~ 8.4 33.1 ~ 8~6 3.5 % 3.4 %
yello~ l _ . _ _ ~
Na-carboxy- 33.4 ~ 8.6 33.0 ~ 8.7 3.0 X 3.1 X
fluoresceine _ _ ~= _ . ~

*rate=percentage by weight of active ingredient incorporated into the liposomes to total weight.

- 18 - MERZ2~/dlk , .

2 ~ r~ ~

Cosmetic and Pharmaceutical ComPosi~ions or Formulations Innumerable cosmetic and pharmaceutical compositions and preparations, pre~era~ly for topical application, embodying liposomes according ~o the present invention and cosmetically- and/or pharmaceu~ically-aCtive ingredients, in effective amounts, can bP prepared in conventional manner and may incorporate any such active ingredient, preferably but not necessarily a lipid-soluble ingredient, those previously mentioned herein being representative of the active ingredients which may be employed, together with a cosmetically- or pharmaceutically-acceptable carrier or diluent, preferably a topically-acceptable carrier or diluent.
Representative examples of such compositions or preparations follow:
Composition of_a Cosmetic Topical Product N~me ot In~r~dl~nts I p~rc~nta0e formLIl~ (%~
j- ;-, . ~. . .- _=_ __ ¦ Soy l~cithin 10,000 1~ _ .
¦ Oleic acici e~terified collagen 10,000 hydrolysate (O~IAP) - - ~ .- .
dl-~ ocoph~rolnlaotinata ~10 I _ . ...
i3uty1at~d Hydroxytoluol (BHT) 0,008 I . , . ,._.,~. I
I Ethanol i5,000 I . . .. ~ . . I
~odlum Phosphat9 ~ 2H2a û,85~
Di80diumi~h~sphate 2H20 0,634 . - . .
Ho tacerin PN 73 1,760 ~ . , _ . . . . ~ ...
l Purifi~d Water 59l5~2 . . - ~
Perfume 0,200 , 100,000 r ~

- 19 - MERZ 20/dlk :.
.

~ 3~ ~

~anu~a~turing Procedure:
In an external container sodium phosphate and disodium phosphate are dissolved in purified water (I).
In an external container placed in a water bath soy l~cithin~ OHAP, tocopherol nicotinate and BHT are dissolved in ethanol at moderate temperature (II).
(I) is poured into (II) under stirring and the resulting dispersion finally homogenized using a high pressure homogenizer (III).
Th~ gel is produced by sprinkling the Hostacerin on~o the stirred liposome dispersion (III) and allowed to swell for 24 hours (IV).
Finally the perfume is mixed under stirring into the liposome gel (IV).
The foregoing cosmetic composition may be topically applied and, when so applied, provides an increased suppl.y of blood and nu~riments ~o the skin, has a po~itive influence upon skin regeneration, improves the barrier function and moisture content of the skin, and reduces the rate of skin aging.
Com~osition of a Pharmaceutical Topical Product Nam~ o~in~redi~n~ I p~ro~n~a~ ~o~mula (~) ~oy lecithln 8,000 Oi~ic acid-esterified colla~en 2,00 I hydrolys~te I
. _ . ., ..._~ ~
tlnQin ~,060 i _ . _ ~utylatad Hydroxyt~luol (BHT) 0,00 ., ............. ..

Ethanoi 5,000 .. ._ , . , ... .
Sodium Phosphat~ l 120 Di~odium Pho~phat~ 0.~30 . -__ _ ....... . _ Trom~thamine (U5PXXII)(buffer) 2,~00 l ~_ Carbopo!TM 934 (Polyaorylic a~id) Purified Water 77,~4B
I lao,ooo - 20 - MERZ~0/dlk ,,, , ': ' , . , , ~J'~i3 tJ ~ 7 ~

Manufacturin~ Procedure:
In an external container sodium phosphate, disodium phosphate and pheno~yethanol are dissolved in purified water (I).
Carbopoltm 934 and tromethamine are mixed, dispersed in 30~ (I) and stirred to complete swelling (II).
In an external container soy lecithin and OHAP are dispersed under stirring in the remaining amount of (I).
Avoiding exposure to light tretinoin and BHT are dissolved in ethanol and poured into the lecithin/OHAP dispersion under homogenization with an Ultraturraxbn for 5 min.
Finally this dispersion is homogenized using a high pressure homogenisator (III).
(II) and (III) are mixed and stirred until complete swelling (IV). These preparation steps are also carried out under avoidance of light exposure.
The foregoing pharmaceutical composition may be topically applied for the treatment o acne, promotes a homogenous and long-lasting distribution of the drug tretinoin in the skin, and promotes an improved toleration of the tretinoin therapy by the skin.
The liposome compositions of the present invention have the advantage of stability, that i5, an important reduced tendency to aggregate and/or to grow in size upon passage of time without an increase in permeability, as sho~n in the foregoing comparative Examples, especially when the liposome compositions of the invention consist essentially of "SUV", namely, small unilamellar (one bilayer) vesicles, in contrast to usual vesicles of the prior art and those vesicles of the prior art having less than the required amount of the important ingredient fatty acid-esterified collagen hydrolysate, especially the ingredient OHAP, which impart important characteristics to - 21 - MERZ 20/dlk 2 ~3~, 7~

the vesicles of the presen~ invention, including the afore-mentioned stability and resistance to growth in size over a period of time. Such SUV according to the present invention are, as previously disclosed, small unilamellar (one bilayer) vesicles having a particle size between abcut 20 and 150 nm, preferably between about 20 and 70 nm, and which contain between about 16% and 90% percent by weight of fatty acid-esterified collagen hydrolysate, especially of OHAP, and preferably between about 20% and 80~ by wsight of the aforesaid essential characteristic-enhancing ingredient. Such S W are produced by the procedure of Examples 1-5 and 8, as shown by freeze-fracture micro-graphs, whereas the vesicles produced by ethanol injection in Examples 6 and 7 are multilamellar (more than one bilayer). Thus, depending upon the technique employed, the production of unilamellar or multilamellar vesicles can be selected, and the size of the vesicles can in any case be controlled by variation in the fatty-acid esterified collagen hydrolysate, especially the OHAP, content of the vesicle membrane.
It is accordingly seen from the foregoing that the present invention provides novel liposome compositions consisting essentially of small vesicles, preferably but not limited to unilamellar (one bilayer) vesicles, compris-ing between about 16 and 90 percent by weight of fatty acid-esterified collagen hydrolysate, preferably between about 20 and 80 percent, especially such percentages of OHAP, and a method for the preparation thereof, as well as a method for the use thereof in the preparation of cosmetic and pharmaceutical preparations or compositions, and such compositions ~hemselves containing effective amounts of cosmetically-effectiveorpharmaceutically-effectiveactive ingredients, which are preferably but not necessarily - 22 - MERZ 20/dlk , : ~ ~
' -' . . ~ ' ~ 3~

lipid-soluble active ingredients, with all of the attendant advantages as set forth and illustrated in the foregoing, especially the important aspect of stability against undesirable and intolerable size increases in individual vesicle dimensions over time (which shortcoming has charac-terized previous similar prior art liposome vesicles and compositions containing the same), without an increase in permeability, and consequently an importantly improved storage stability, all of which advantages are attributable to the high percentage of fatty-acid esterified collagen hydrolysate, especially O~AP, in the vesicle membrane wall.
It is to be understood that the present invention is not to be limited to the exact details of operation, or to the exact compounds, compositions, methods, procedures, or embodiments shown and described, as various modifications and equivalents will be apparent to one skilled in the art, wherefore the present invention is to be limited only by the full scope which can be legally accorded to the appended claims.

- 23 - MERZ 20/dlk

Claims

1. Liposome preparation, wherein vesicles have a stable, small particle size and a vesicle membrane which contains a mixture of one or several lecithins with 16 to 90% by weight of fatty acid-esterified collagen hydrolysate.

2. Liposome preparation according to claim 1, wherein the vesicle membrane contains 20-80% by weight of fatty acid-esterified collagen hydrolysate.

3. Liposome preparation according to claim 1, wherein the fatty acid-esterified collagen hydrolysate is oleic acid-esterified collagen hydrolysate.

4. Liposome preparation according to claim 1, wherein the particle size of the vesicles is 20 to 150 nm.

5. Liposome preparation according to claim 1, wherein the lecithin is soy or egg lecithin and the particle size of the vesicles is 20 to 70 nm.

6. Liposome preparation according to claim 1, which additionally contains a jellification agent.

7. Liposome preparation according to claim 6, wherein the jellification agent is selected from polyacrylic acid, a cellulose derivative, and a sodium salt of an acrylic acid-acrylamide copolymerisate.

1 MERZ 20/dlk

8. Liposome preparation according to claim 1, which additionally contains a therapeutic agent.

9. Liposome preparation according to claim 8, wherein the therapeutic agent is selected from hexachlorophene, tretinoin, minocycline, meclocycline, .alpha.-tocopherol nicotinate, tromantadine base, croconazole, cyproter-one, cyproterone acetate, corticoids, 2-tert.-butyl-4-cyclohexylphenyl nicotinate-N-oxide, aorticoster-oids, androgens, ethinyl estradiol, non-steroid antiphlogistics, dihydropyridines, spironolactone, erythromycin esters, lipophilic local anaesthetics, estradiol esters, and lipophilic antihistaminics.

10. Liposome preparation of Claim 4, wherein the vesicles are multilamellar.

11. Liposome preparation of Claim 5, wherein the vesicles are unilamellar.

12. Process for the manufacture of a liposome preparation according to claim 1, wherein a) the lecithin is dispersed with 16 to 90% by weight of fatty acid-esterified collagen hydro-lysate; and b) the dispersion produced according to step a) is mixed with a jellifying agent.

13. Process according to claim 12, wherein the dispersion obtained according to step a) is reduced in particle size to 20-150 nm and homogenized.

14. Process of Claim 13, wherein the vesicles are unila-mellar.

15. Process according to claim 12, wherein in step a) a mixture of lecithin, fatty acid-esterified collagen hydrolysate, and ethanol is injected into an aqueous phase.

2 MERZ 20/dlk

16. Process of Claim 15, wherein the vesicles are multi-lamellar.

17. Process according to claim 12, wherein the lecithin is soy or egg lecithin, the amount of fatty acid esteri-fied collagen hydrolysate is 20-80% by weight, and the vesicle particle size is reduced to 20-70 nm.

18. Process according to claim 12, wherein the fatty acid-esterified collagen hydrolysate is oleic acid-esteri-fied collagen hydrolysate.

19. Process according to claim 12, wherein in step a) an active ingredient selected from hexachlorophene, tretinoin, minocycline, meclocycline, .alpha.-tocopherol nicotinate, tromantadine base, croconazole, cyproter-one, cyproterone acetate, corticoids, 2-tert.-butyl-4-cyclohexylphenyl nicotinate-N-oxide, corticoster-oids, androgens, ethinyl estradiol, non-steroid antiphlogistics, dihydropyridines, spironolactone, erythromycin esters, lipophilic local anaesthetics, estradiol esters, and lipophilic antihistaminics, is included in the preparation.

20. Process according to claim 12, wherein in step b) polyacrylic acid, a cellulose derivative, or a sodium salt of an acrylic acid-acrylamide copolymerisate is employed as jellifying agent.

21. Process of Claim 12, wherein the concentration of the lecithin and fatty-acid-esterified collagen hydroly sate in the resulting dispersion from step (a) is between about 5 and 350 mg/ml.

22. Process of Claim 12, wherein the concentration of the lecithin and fatty-acid-esterified collagen hydroly-sate in the resulting dispersion from step (a) is between about 20 and 180 mg/ml.

3 MERZ 20/dlk

23. Process of Claim 12, wherein the percentage of leci-thin and fatty-acid-esterified collagen hydrolysate in the resulting dispersion from step (a) is between about 0.5 and 35 percent by weight.

24. Process of Claim 12, wherein the percentage of leci-thin and fatty-acid-esterified collagen hydrolysate in the resulting dispersion from step (a) is between about 2 and 18 percent by weight.

25. A cosmetic composition suitable for topical applica-tion to humans comprising a liposome preparation of Claim 1, an effective amount of a cosmetically- and topically-effective active ingredient, and a cosmetic-ally-acceptable carrier or diluent.

26. A pharmaceutical composition suitable for topical application to humans comprising a liposome prepara-tion of Claim 1, an effective amount of a pharma-ceutically- and topically-effective active ingredient, and a pharmaceutically-acceptable carrier or diluent.

27. Method of preparing a dermatological or cosmetic composition suitable for topical application to humans comprising the step of admixing together a liposome preparation of Claim 1, an effective amount of a cosmetically- or pharmaceutically-effective active ingredient, and a cosmetically- or pharmaceutically-acceptable carrier or diluent.

28. Use of a liposome preparation according to claim 1 for manufacture of a cosmetic or dermatological prepa-ration for topical application to humans.

4 MERZ 20/dlk