WO2013053481A1

WO2013053481A1 - Dimethyl sulfoxide as solvent for nucleic acids

Info

Publication number: WO2013053481A1
Application number: PCT/EP2012/004266
Authority: WO
Inventors: Hans Kosak; Marcus WEICHERT
Original assignee: Secutech International Pte. Ltd.
Priority date: 2011-10-11
Filing date: 2012-10-11
Publication date: 2013-04-18
Also published as: EP2765982A1

Abstract

The subject matter of the present application are pharmaceutical or cosmetic compositions, comprising complexes dissolved in dimethyl sulfoxide (DMSO) and comprising at least one nucleic acid and at least one organic cation; and comprising at least one pharmaceutically or cosmetically compatible auxiliary. The present invention further relates to the use of said compositions for introducing nucleic acid into, for example, human cells or tissue and to methods for producing such compositions.

Description

Dimethyl sulfoxide as a solvent for nucleic acids

The present application relates to pharmaceutical or cosmetic compositions comprising complexes of at least one nucleic acid and at least one organic cation dissolved in dimethyl sulfoxide (DMSO) and at least one pharmaceutically or cosmetically acceptable adjuvant. Furthermore, the present invention relates to the use of these compositions for introducing nucleic acid into human cells or tissue and to methods for producing these compositions.

State of the art

The use of nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) has been extensively researched by the pharmaceutical and cosmetic industries for a long time.

A fundamental problem with the development of nucleic acid-based drugs is that they typically exert their action inside cells (e.g., in the cytosol or in the nucleus). However, due to the charge of the nucleic acids, diffusion of the nucleic acid through the cell membrane, which confines the cells to the outside, takes place only to a very limited extent. To overcome this barrier, a variety of different methods have been developed to introduce nucleic acid into cells. These methods include, for example, electroporation, the use of liposomes and synthetic particles, or the direct injection of naked nucleic acid into the blood for the purpose of subsequent uptake by the corresponding target cells.

In addition, numerous methods for introducing nucleic acid into cells in the context of gene therapy approaches are known in the prior art, whereby viral vector systems are usually used. The used in these methods Vector systems are developed from naturally occurring viruses, which have typically been modified so that they are no longer able to replicate in the cells. The viral vectors are produced by special packaging cell lines and can be purified from the culture supernatant of appropriate cell cultures. Concerns about the safety of such vector systems are due, among other things, to possible recombination with naturally occurring viruses. It is not excluded that such a recombination could give rise to recombinant pathogenic viruses which pose a risk to the therapeutically treated organism.

In addition to viral vector systems, non-viral vectors are also common in gene therapy. Suitable non-viral vectors are prepared by recombinant DNA techniques and introduced either in the form of "naked DNA" or in complexed form into the respective target cell. The nucleic acids are coupled to polylysine for transport via the cell membrane, for example. However, these systems are unsatisfactory in their efficiency since only small amounts of nucleic acid are taken up by the corresponding target cells.

The possibility of incorporation of naked nucleic acid has long been explored intensively in the context of DNA vaccination. Thus, it has been shown that gene expression in the living organism can be achieved by direct intramuscular injection of RNA or DNA vectors which code for different enzymes (Wolff et al. (1990), Science, 247, 1465). 1468).

Despite the developments outlined above, there continues to be a need for a simple and inexpensive method of introducing nucleic acid into cells or tissues of living organisms. The method should preferably be associated with a low health risk and if possible renounce viral vector systems and similar aids. It has now surprisingly been found that nucleic acid, which are complexed in a certain way with organic cations, are outstandingly soluble in dimethyl sulfoxide (DMSO). Nucleic acids dissolved in DMSO can be administered directly into the cell interior, for example through the skin of a living mammal. As can be determined by means of fluorescent labeling, the nucleic acids can surprisingly penetrate into the cell nuclei of the skin cells. Summary of the invention

The present invention thus relates in a first aspect to a pharmaceutical or cosmetic composition comprising:

a) complexes of at least one nucleic acid and at least one organic cation dissolved in dimethyl sulfoxide (DMSO); and

b) at least one pharmaceutically or cosmetically acceptable excipient.

Further preferred embodiments of the invention are disclosed in the following description and the dependent claims.

Detailed Description of the Invention Compositions and their preparation

The present invention provides compositions comprising dimethylsulfoxide as an organic solvent. One or more nucleic acids complexed with one or more organic cations are dissolved therein. The nucleic acids are usually complexed in such a way that the negative charges of the nucleic acids are substantially completely compensated by positive charges of the organic cations. "Substantially completely compensated" here means, in particular, that the ratio of the number of positive charges of the cations to negative charges of the nucleic acid is preferably greater than or equal to 0.5, preferably greater than or equal to 0.6, preferably greater than or equal to 0.7 is preferably greater than or equal to 0.8, preferably greater than or equal to 0.9, preferably greater than or equal to 0.91, preferably greater than or equal to 0.92, preferably greater than or equal to 0.93, preferably greater than or equal to is 0.94, preferably greater than or equal to 0.95, preferably greater than or equal to 0.96, preferably greater than or equal to 0.97, preferably greater than or equal to 0.98, preferably greater than or equal to 0.985, preferably is greater than or equal to 0.99, preferably greater than or equal to 0.995, or even equal to 1. The ratio of the number of positive charges of the cations to negative charges of the nuclei For example, acid can be in the range 0.5-2, for example in the range 0.6-1.8, in the range 0.7-1.3, in the range 0.8-1.2, in the range 0 , 9 - 1, 1, are in the range 0.91 - 1, 09, in the range 0.92 - 1, 08 lie, in the range 0.93 - 1, 07 lie, in the range 0.94 - 1, 06 lie in the area 0,95 - 1, 05, lying in the range 0,96 - 1, 04, lying in the range 0,96 - 1, 04, lying in the range 0,97 - 1, 03, in the range 0,98 - 1, 02, are in the range 0.99 - 1, 01, in the range 0.995 - 1.005 are, or even equal to 1. The latter value is achieved, in particular, when cations are present in the precipitation of the nucleic acid in excess during the preparation (see later) and the precipitate of organic cations and nucleic acid is washed with water.

It has been found that this form of complexation makes it possible to solubilize nucleic acids, which are otherwise insoluble in organic solvents, in high amounts in DMSO. Nucleic acids, e.g. DNA or RNA, under physiological conditions, are anionic polymers that have a high number of negatively charged phosphate groups. Due to the charge of the polymers, these are readily soluble in aqueous solution, while the solubility in organic solvents is extremely low. In the context of the present invention, it has also been shown that the complexed nucleic acids dissolved in DMSO can be effectively absorbed into cells and / or tissue, e.g. of a mammal. In particular, the dermal or transdermal incorporation of the complexed nucleic acids according to the invention (i.e., for example, via the mammalian skin) has proven to be highly efficient. Just a few hours after application to the skin, correspondingly labeled nucleic acids could be detected in various skin cells and in the connective tissue underneath. The nucleic acids complexed according to the invention are therefore particularly suitable as a constituent of cosmetic and / or pharmaceutical compositions for use in the introduction of nucleic acid into cells and / or tissue, e.g. in cells and / or tissue of the skin.

"At least one organic cation" means that at least one type of organic cation is present (eg, CTAB), but there may also be cation mixtures of two or more, three or more, four or more, five or more, etc. different organic cations ( For example, CTAB besides DDAB.) Usually, the at least one organic cation is simply positively charged, and it will be apparent to those skilled in the art that the term is not indicative of the amount of substance.

The phrase "dimethylsulfoxide-dissolved complexes" as used herein is intended to mean that the complexes are dissolved in DMSO In addition to the solution in DMSO alone, this also includes the cases in which solvent mixtures (ie homogeneous phase) of DMSO with one or more, two or more, three or more, four or more, five or more, etc. of other organic solvents. The nucleic acids are then dissolved accordingly in these DMSO-containing solvent mixtures. In the presence of a nonhomogeneous mixture with several phases, the nucleic acid complexes according to the invention are present - at least predominantly - in the DMSO-containing phase.

"At least one nucleic acid" means that at least one nucleic acid is present, but there may also be nucleic acid mixtures of two or more, three or more, four or more, five or more, etc. different nucleic acids. eg DNA in addition to RNA) and / or the sequence of the nucleic acids (length and / or base sequence).

The at least one nucleic acid in the compositions of the present invention dissolves in DMSO due to charge compensation in the complexes formed. The solution of the nucleic acid therefore does not require the formation of micelles, liposomes or similar nanoparticulate structures. Usually, therefore, complexes of at least one nucleic acid and at least one organic cation are dissolved in the compositions of the present invention, wherein the complexes are not located inside a micelle, a liposome or similar synthetic nanoparticulate structures. In some embodiments, the compositions of the present invention even contain no micelles, liposomes, or synthetic nanoparticles at all.

A suitable process for preparing complexes of nucleic acid and organic cation which are suitable according to the invention comprises dissolving at least one nucleic acid in an aqueous liquid, in particular water (eg demineralized water), precipitating the at least one nucleic acid by adding at least one organic cation containing the nucleic acid forms insoluble complex in the aqueous solution, and subsequent uptake of the precipitate in DMSO or DMSO-containing solvent mixture.

Such a method may include the following steps:

a) providing complexes of at least one nucleic acid and at least one organic cation, b) dissolving the complexes in DMSO or DMSO-containing solvent mixture, and c) adding a pharmaceutically or cosmetically acceptable excipient to the DMSO solution or the DMSO-containing solvent mixture having the complexes of at least one nucleic acid and at least one organic cation dissolved therein the DMSO or the DMSO-containing solvent mixture from b) does not already contain the pharmaceutically or cosmetically acceptable excipient.

In particular, such a method may comprise the following steps:

a) providing a solution with at least one nucleic acid dissolved in an aqueous first liquid,

b) precipitating the at least one nucleic acid by adding at least one organic cation (complexing agent) which forms insoluble complexes with the nucleic acid in the first liquid to the first liquid,

c) separating the complexes from the first liquid,

d) dissolving the complexes in DMSO or DMSO-containing solvent mixture (second liquid), and

e) adding the pharmaceutically or cosmetically acceptable excipient to the second liquid with the dissolved complexes of at least one nucleic acid and at least one organic cation, as far as the DMSO or the DMSO-containing solvent mixture from d) does not already contain the pharmaceutically or cosmetically acceptable excipient.

The addition of the pharmaceutically or cosmetically acceptable excipient may, of course, be done at any convenient time during the process.

Suitable organic cations in the context of the present invention (or sources for the cations) are, for example, cationic detergents, lipids and nitrogen compounds with quaternary nitrogen, for example organic ammonium salts. In particular, one or more compounds of formula NR ₄ X may be used as the source of organic cations, each R independently being a hydrocarbon radical of 1 to 20 carbon atoms which may be branched or unbranched, and X is a halogen selected from the group consisting of from chlorine, bromine, iodine, in particular chlorine or bromine, preferably bromine. In particular, however, the hydrocarbon radical preferably contains only C and H atoms. According to further embodiments of the invention, the source of the at least one organic cation may be one or more compounds of the formula NR ¹ R ² R ³ R ⁴ X, where R ¹ , R ² , R ³ and R ^{4 are} organic radicals, in particular carbon hydrogen radicals as defined above, and wherein at least R ^{1 is} a C ¹ radical and R ² to R ^{4 are} longer-chain radicals, or

R ¹ and R ^{2 are} each a C ¹ radical and R ³ and R ^{4 are} longer chain radicals, or

R ¹ , R ² and R ^{3 are} each a C 1 radical and R ^{4 is} a longer chain radical.

In particular, the radicals R ¹ , R ² , R ³ and R ^{4 are} each and independently of one another as defined below for "R".

R in the compounds of the formula NR ₄ X is preferably an alkyl radical having 1 to 20 carbon atoms, which may be branched or unbranched. In particular, one or more, especially two or three, of R may be relatively short chain radicals such as C1 to C3, such as methyl, ethyl and propyl, while an R may be a longer radical such as C8 or CI O to C20, especially C10 to C16 such as octyl , Nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, cetyl, heptadecyl, icosanyl, stearyl or nonadecyl. A preferred combination of radicals R is three methyl radicals and one long-chain radical, in particular a C10 to C20 hydrocarbon radical as defined above, in particular a cetyl radical as in cetyltrimethylammonium bromide (CTAB). Preferably, R does not comprise aromatic and / or non-aromatic ring systems

Preferably, the at least one organic cation (or source of the cation) is one or more quaternary amines, with CTAB being particularly preferred. Other compounds which are suitable according to the invention for complexing the nucleic acids include benzethonium chloride, benzalkonium, benzalkonium chloride, didecyldimethylammonium bromide (DCAB), dodecyltrimethylammonium bromide (DCTAB), DOTAP, lipofectin, lipofectamine N- [1- (2,3-dioleyloxy) propyl] - N, N, N-trimethylammonium chlorides (DOTMA), dimethyldioctadecylammonium bromide (DDAB), dioleyldimethylammonium chlorides (DODAC), 2,3-dioleoyloxy-N- [2- (spermidinecarboxylic amido) ethyl] -N-dimethyl-1-propaninium trifluoroacetate (DOSPA), diheptadecylamidoglycyl spermidine (DHGS) and the like. Preferably, the organic cation is not polylysine and / or TC-chol.

Preferably, the complexing agent is added to the first liquid in step b) in dissolved form. This allows a faster precipitation than when adding an undissolved complexing agent. The nucleic acid is thereby preferably complexed with the at least one organic cation such that a ratio of substantially 1: 1 (ie substantially completely compensated) between the negative charges (ie the anionic groups) of the at least one nucleic acid and the charges of the at least one organic cation is present. In particular, organic cations such as those mentioned above, e.g. CTAB, are able to approximate the positively charged group in a sufficiently small distance to the negatively charged oxygen atom of the nucleic acid, so that a sufficient (local) shielding of the total charge of both ions is effected.

An advantage of the setting of such a ratio is that the total of organic cations and nucleic acid is approximately charge neutral, that is essentially completely complexed. The complexed nucleic acid is therefore largely insoluble in an aqueous medium and can be separated in the preparation in aqueous media as an insoluble precipitate. This property thus facilitates the production of the desired, stoichiometrically complexed nucleic acid. The separation of the complexes according to step c) therefore preferably takes place by means of centrifugation or filtration. These methods are a particularly simple and efficient way of separating the precipitated complexes. For example, the precipitate may be separated by centrifugation for 1 to 10 minutes, preferably 5 minutes, at 10,000 to 20,000 xg, preferably 15,000 xg. Optionally, this process can be performed several times after re-inclusion of precipitated precipitate in water, thereby, for example, an excess of free quaternary amines can be separated. An advantage of setting such a ratio is further that upon contact of the thus complexed nucleic acid with a living cell or an organ of a living organism no or only a few free organic cations can interact with the cells or the organism. As a result, the organic cations can not elicit cytotoxic effects or other metabolic disorders in the cells, which otherwise can be common. Of course, as mentioned above, in the course of complexing the at least one nucleic acid, various organic cations can also be used by adding mixtures of different organic cations to the nucleic acid-containing aqueous liquid to precipitate the nucleic acid complexes.

The complexes obtained by precipitation are dissolved in DMSO or DMSO-containing solvent mixtures. The nucleic acid complexes can be directly dissolved in DMSO, dissolved in DMSO, and then mixed with another solvent, or else first into another solvent, e.g. Ethanol and then mixed with DMSO. In any case, the dissolved complexes are according to the invention, at least substantially, in the DMSO-containing phase. After incorporation into DMSO or DMSO-containing solvent mixtures, these essentially essentially do not contain any organic cations which go beyond the amount required to compensate for the negative charges of the nucleic acid. This means that the number of total positive charges of the organic cations in the DMSO or DMSO-containing solvent mixture is substantially identical to the number of negative charges of the nucleic acid.

Organic solvents which are particularly suitable for mixing with DMSO include in particular monohydric or polyhydric alcohols or partially etherified alcohols. The additional solvent (s) therefore preferably contains at least one ether group and / or at least one hydroxyl group. Particularly suitable as solvents (amphiphilic) compounds can be described by the formula HO-1 -O-R2. In this case, R 1 and R 2 are each a hydrocarbon radical having 1 to 100 carbon atoms. In particular, C1 to C5 alcohols are suitable, such as, for example, methanol, ethanol, 1-propanol or 2-propanol, butanol, such as 1-butanol, or pentadiol, such as 1-pentadiol. Further, polyhydric alcohols, such as ethylene glycol or partially etherified derivatives thereof, such as ethylene glycol, which is etherified with methanol, ethanol, propanol or butanol, such as ethylene glycol ethers, in particular ethylene glycol monobutyl ether, ethyl ether or methyl ether, usable. Alternatively, it is possible to use saturated or unsaturated hydrocarbons, for example benzene-based aromatic hydrocarbons, such as styrene, ie in particular benzenes substituted by C1 to C3 radicals. Alternatively, halogen-containing solvents such as chloroform, dichloromethane or carbon tetrachloride or other heteroatom-containing solvents such as dimethylformamide or tetrahydrofuran may be used. Especially however, for pharmaceutical and cosmetic applications it is preferred that the composition of the invention does not comprise halogenated hydrocarbons. Particular preference is given to solvent mixtures of DMSO with ethanol, glycerol, ethylene glycol and / or propylene glycol.

The compositions of the invention may in principle comprise any proportion of DMSO. They may have a DMSO content of about 1% (v / v) or greater, from about 5% (v / v) or greater, from about 10% (v / v) or greater, from about 15% (v / v ) or more, of about 20% (v / v) or more, of about 30% (v / v) or more, of about 40% (v / v) (v / v) or more, of about 50% ( v / v) or more, from 60% (v / v) or greater, from about 70% (v / v) or greater, from about 80% (v / v) or greater, from about 90% (v / v ) or more, or even about 95% (v / v) or more. For immediate therapeutic, diagnostic or cosmetic use on the body, a high concentration of DMSO may be undesirable, as higher concentrations may cause skin irritation. In particular, in such cases of immediate therapeutic, diagnostic or cosmetic application of the composition to the patient, e.g. when applied to the skin, it may be desirable for the compositions of the invention to have a DMSO content of about 60% (v / v) or less, a DMSO content of about 55% (v / v) or less, a DMSO Proportion of about 50% (v / v) or less, a DMSO content of about 45% (v / v) or less, a DMSO content of about 40% (v / v) or less, a DMSO content of about 35% (v / v) or less, a DMSO content of about 30% (v / v) or less, a DMSO content of about 25% (v / v) or less, a DMSO level of about 20 % (v / v) (v / v) or less, a DMSO content of about 15% (v / v) or less, a DMSO content of 10% (v / v) or less, or even just a DMSO Content of about 5% (v / v) or less. In the case of compositions according to the invention which are applied to the skin, for example, a proportion of 20% to 60% DMSO can be present.

Furthermore, it is advantageous, for example, for the pharmaceutical or cosmetic application, if the water content in the phase containing the nucleic acid complexes is less than 50% (v / v), preferably less than 30% (v / v), more preferably less than 20% (v / v). A higher water content leads to the formation of larger aggregates which do not possess optimal cell and tissue penetration properties. In particular for therapeutic, diagnostic and cosmetic applications, the average diameter of the complexes of nucleic acid and organic cation in the compositions to be used according to the invention is preferably less than 200 nm, preferably less than 100 nm, more preferably less than 50 nm, very particularly preferably less than 10 nm.

The nucleic acid complexes dissolved in DMSO are characterized by an extraordinarily high stability. This is essentially due to the fact that nucleases are inactive in non-aqueous environments. This is especially important when using ribonucleic acid (RNA), since in all aqueous systems with a high RNAse activity must be expected. Outside of an RNAse-free environment, which can be realized practically only under special laboratory conditions, is expected in principle with a rapid degradation of the RNA, which in particular significantly hampers the use of therapeutically effective RNA as a pharmaceutical agent. This problem is solved by complexing the nucleic acid to the complexes described herein.

Furthermore, the stability of the complexed nucleic acids according to the invention is also increased by the fact that no acidic or alkaline environment is present in DMSO. This avoids any acidic or alkaline hydrolysis of the nucleic acids.

Pharmaceutical / cosmetic compositions

The compositions of the present invention are excellently formulated as cosmetic and / or therapeutic compositions.

For a pharmaceutical composition, the nucleic acid is preferably a therapeutically active nucleic acid, ie, a nucleic acid which, upon administration to an individual, exerts a biological function and thereby provides a therapeutic benefit. In the prior art, a variety of nucleic acids have been described as therapeutically effective. Frequently, in such cases, the nucleic acid will have sequences of a genome or gene. Likewise, the nucleic acid may be a diagnostically useful nucleic acid, for example a so-called molecular beacon. For a cosmetic composition, the nucleic acid is a cosmetically active nucleic acid, ie a nucleic acid, which, after administration to an individual, generally does not lead to any therapeutic but to a cosmetic benefit. Usually, the nucleic acid will not have sequences of a (human) genome or (human) gene. For example, nucleic acids can be used as a source of allantoin. In addition to uric acid, allantoin is the final product of the breakdown of nucleic acids, especially of purine bases, in various animal species, especially mammals. Allantoin is used in cosmetics in skin creams, sunscreens, shampoos, toothpaste and anti-sweating (hyperhidrosis) and skin irritants. It accelerates cell building, cell formation or cell regeneration and calms the skin.

According to a simple embodiment of the present invention, the nucleic acid complexes which are present dissolved in DMSO or DMSO-containing solvent mixture are directly contacted with the cells or tissues intended for cosmetic and / or therapeutic treatment, e.g. by dripping or brushing on the nucleic acid-containing DMSO solution. DMSO is particularly suitable for this because DMSO is cell- and tissue-compatible.

It is of course also possible to dissolve the complexes dissolved in dimethyl sulfoxide (DMSO) from at least one nucleic acid and at least one organic cation, e.g. with an oil to form an emulsion and e.g. Apply to the skin in this form. Other common dosage forms for the compositions of the invention include, for example, lotions, creams, ointments, gels and pastes. However, in many embodiments of the invention, the compositions of the invention are not an emulsion, lotion, cream, ointment, gel and / or paste.

According to the invention, in the pharmaceutical or cosmetic compositions of the present invention, the nucleic acid complexes dissolved in DMSO are mixed with a pharmaceutically or cosmetically acceptable excipient which is suitable, for example, for the respectively planned administration. Preferably, the at least one adjuvant is selected from the group consisting of: fillers, solvents, humectants, emulsifiers, solubilizers, wetting agents, antifoams, salt formers, buffers, gelling agents, thickeners, film formers, binders, coating agents, disintegration accelerators, Adsorbents, lubricants, lubricants, mold release agents, flow control agents, antioxidants, preservatives, sweeteners, flavor correctors, odor control agents, and colorants.

Examples of fillers are e.g. Lactose, cellulose, starches (e.g., corn starch, rice starch, potato starch, or wheat starch), sucrose; Paraffin; Hard fat; Polyethylene glycols (e.g., macrogols, PEG), polyethylene oxides (PEO), lactose, glucose, mannitol, glycine, calcium hydrogen phosphate, calcium carbonate, etc.

Examples of solvents as adjuvants are e.g. Ethanol, isopropanol, glycerin, propylene glycol, acetone, glycerol triacetyl ester, oleyl oleate, and isopropyl myristate, macrogols, polyethylene glycols, polyethylene oxides, synthetic, vegetable or animal oils, synthetic, vegetable or animal fats, etc.

Examples of humectants are e.g. Water, ethanol, isopropanol, glycerin, propylene glycol, acetone, glycerol triacetyl ester, oleyl oleyl ester, isopropyl myristate, sorbitol, etc.

Examples of emulsifiers are e.g. Cetyl alcohol, stearyl alcohol, cetylstearyl alcohol, glycerol monostearate, lecithins, fatty acid esters of sorbitan (eg sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan trioleate, sorbitan tristearate, sorbitan monolaurate), polyoxyethylene sorbitan (polysorbates, eg polysorbate 20, 40, 60 or 80), or polyoxyethylene (eg macrogol stearate 400, polyoxyl 40 stearate, polyoxyl 50 stearate), polyoxyethylene fatty alcohol ethers (eg, polyoxyl 23 lauryl ether, polyoxyl 20 ceto stearyl ether, polyoxyl 10 oleyl ether), sodium dodecyl sulfate, sodium cetyl stearyl sulfate, sodium dioctyl sulfosuccinate (and potassium or calcium salts thereof ), Cholesterol, polyoxyethylene fatty acid glycerides (eg, macrogol-1 500-glycerol triricinoleate, macrogol glycerol hydroxystearate), macrogol 1000 glycerol monofatty acid ester, macrogol 1000 glycerol monolaurate, macrogol 1000 glycerol monostearate, macrogol 1000 glycerol monooleate, poloxamer, etc.

Examples of solubilizers are, for example, polyethylene glycols (PEG, macrogols), polyethylene oxides (PEO), polysorbates, sodium dioctylsulfosuccinate (and potassium or calcium salts thereof), etc. Examples of wetting agents are, for example, polyethylene glycols (PEG, macrogols), polyethylene oxides (PEO), polysorbates, sodium dodecylsulfate, sodium cetylstearylsulfate, etc.

Examples of antifoaming agents are e.g. Dimethyl polysiloxanes, etc.

Examples of salt formers are e.g. Tris (hydroxymethyl) aminomethane, tris (hydroxyethyl) amine, etc.

Examples of buffers are e.g. Sodium dihydrogen phosphate, tris (hydroxymethyl) aminomethane, citric acid, tartaric acid, glycine, sodium bicarbonate, etc.

Examples of gelling agents are e.g. Pectin, tragacanth, polyacrylic acids, polyvinylpyrrolidone, (fumed silica), carboxymethylcellulose, etc.

Examples of thickening agents are e.g. Pectin, tragacanth, polyacrylic acids, polyvinylpyrrolidone, fumed silica, carboxymethylcellulose, gelatin, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylalcohol, bentonite, etc.

Examples of film formers are e.g. Ethyl cellulose, hydroxypropyl methyl cellulose phthalate, cellulose acetate phthalate, polyacrylates, etc.

Examples of binders are e.g. Starches (e.g., corn starch, rice starch, potato starch, or wheat starch), tragacanth, cellulose, cellulose ethers (methylcellulose, ethylcellulose, hydroxypropylcellulose, carboxymethylcellulose), polyvinylpyrrolidone, sucrose, mannitol, gelatin, calcium hydrogen phosphate, gum arabic, polyethylene glycol, etc.

Examples of wrapping agents are e.g. Sucrose (e.g., for sugar draging); Gelatin (e.g., in capsules); Gelatin polysuccinate (e.g., "soft capsules"), polyacrylates, ethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose,

Carboxymethyl cellulose sodium, hydroxypropyl methyl cellulose phthalate, macrogols, polyethylene glycols, polyethylene oxides, etc. Examples of disintegrants (disintegrants) are, for example, starches (for example maize starch, rice starch, potato starch, or wheat starch); croscarmellose; Sodium hydrogencarbonate eg in combination with citric acid, etc.

Examples of adsorbents are e.g. Bolus alba (kaolin), (fumed silica), etc.

Examples of lubricants are e.g. Polyethylene glycols (PEG, macrogols), polyethylene oxides (PEO), talc, stearic acid (e.g., magnesium stearate), etc.

Examples of lubricants are e.g. Polyethylene glycols (PEG, macrogols), polyethylene oxides (PEO), talc, stearic acid (e.g., as magnesium stearate), dimethylpolysiloxanes, etc.

Examples of mold release agents are e.g. Polyethylene glycols (PEG, macrogols), polyethylene oxides (PEO), talc, stearic acid (e.g., as magnesium stearate), dimethylpolysiloxanes, etc.

Examples of flow control agents are e.g. (fumed silica), etc.

Examples of antioxidants are e.g. Butylhydroxytoluene, all-rac-a-tocopherol, etc.

Examples of preservatives are e.g. PHB esters, benzalkonium chloride, benzyl alcohol, thiomersal, benzoic acid, methyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate, citric acid, linear aliphatic 1, 3-diols, phenoxyethanol and chlorphenesin etc.

Examples of sweeteners are e.g. Sucrose, sorbitol, sweeteners such as saccharin sodium and cyclamate; Flavors, aspartame, fructose, lactose, glucose, mannitol, xylitol, etc.

Examples of flavor correcting agents are e.g. Sucrose, sorbitol, sweeteners such as saccharin sodium and cyclamate; Flavors, etc.

Examples of scent remedies are usually fragrances.

An example of colorants is eg titanium dioxide. Another conceivable adjuvant for the compositions of the present invention is urea.

Preferably, the excipient is not water, even though the composition may of course also comprise water in addition to the excipient.

The adjuvant can be, for example, any lipophilic compound which is immiscible with water and which can be mixed with DMSO or else forms a 2-phase system with DMSO. Preferably, the excipient is an oil; a wax; and / or a fat. In other embodiments of the present invention, at least one further adjuvant is present in addition to the oil, wax or fat.

Suitable oils which can be used to prepare the compositions of the present invention include synthetic, animal and vegetable oils. Likewise suitable waxes or fats comprise synthetic, animal and / or vegetable waxes or synthetic, animal and / or vegetable fats.

However, it is particularly preferred if the oils are not synthetic oils, but e.g. animal and / or vegetable oils. Suitable vegetable oils are e.g. Nut oils and seed oils. Suitable vegetable oils include, in particular, peanut oil, walnut oil, almond oil, hazelnut oil, soybean oil, olive oil, poppy oil, hemp oil, pumpkin seed oil, sunflower seed oil, sesame seed oil, cottonseed oil, thistle oil, linseed oil, rapeseed oil, castor oil and the like. Germ oil derived from grain can also be used herein as a carrier. Preferred germ oils include e.g. those derived from corn, wheat, oats, rye, rice and triticale. For example, coconut fat or cocoa butter may be used as suitable vegetable fats. Possible vegetable waxes include, for example, sugarcane wax, carnauba wax, jojoba oil, Candeli Ilawachs and Japan wax.

In addition to vegetable oils, waxes and fats, it is also possible to use animal fats, waxes and oils, such as fish oils or oils and fats derived from mink or deer. Cod liver oil and whale oil (such as spermaceti) are examples of fish oils that may be used herein. Possible sources of animal waxes are for example, spermaceti, wool wax and beeswax. Suitable methods for obtaining pure oils of animal origin are known in the art.

Possibly. It is also possible to use pharmaceutically or cosmetically compatible synthetic oils, fats and waxes. Suitable synthetic oils and fats are based, for example, on silicone or paraffin compounds. An example is Vaseline. Soya wax can be obtained by hydrogenation from soy and is an example of a synthetic wax.

For example, the compositions of the invention may have an oil or fat content greater than 25% (v / v), for example greater than or equal to about 30% (v / v), greater than or equal to about 40% (v / v), greater or equal to about 50% (v / v), greater than or equal to about 60% (v / v), greater than or equal to about 70% (v / v), greater than or equal to about 80% (v / v), etc.

Preferably, the compositions of the present invention do not comprise petroleum, mineral oil, mineral oil distillate, light oil, heavy oil, toluene, benzene, or fuels, such as diesel fuel, and / or gasoline.

It has proven particularly suitable in this connection to additionally introduce an oil or fat into the compositions according to the invention. Initially, as described above, the complexed nucleic acids are first dissolved in DMSO. Subsequently, the nucleic acid-containing DMSO solution is mixed with one or more oils to form a liquid comprising complexed nucleic acids, DMSO and oil. Depending on the oil, the nucleic acid-containing DMSO may be dissolved in the oil or form an emulsion with the oil.

If the compositions comprise an emulsifier or surfactants as auxiliary substances, they must be functionally distinguished from the organic cations which are obligatory according to the invention and may possibly also be considered as surfactant / emulsifier. These adjuvants are thus in addition to the obligatory required cations and are not used to complex with the nucleic acid but fulfill another function, such as the emulsification of two liquids. A non-limiting example of such a case is an emulsion of water and a mixture of oil and DMSO containing the nucleic acid complexes in the oil-DMSO phase. Also in all other cases, where organic cations are theoretically understood as an adjuvant in the context of the present invention can be distinguished whether the corresponding substance is now present in complex with the nucleic acid, or whether he perceives the function as an excipient and is not present in complex with the nucleic acid. Benzalkonium chloride is, for example, a quaternary ammonium compound which is frequently used as a preservative. However, other quaternary ammonium compounds such as CTAB are sometimes more suitable for complexing with nucleic acids. Preferably, therefore, in the present invention, the organic cations complexed with the nucleic acid are not identical to the pharmaceutically or cosmetically acceptable adjuvant.

Preferably, the compositions of the invention do not comprise plastics, in particular, no synthetic artificial (i.e., non-naturally occurring) organic polymers such as those described e.g. from materials technology. Of course, biological polymers such as nucleic acids are not covered by this.

In certain embodiments of the invention, the compositions according to the invention preferably do not comprise in parallel triglycerides, phospholipids and cholesterol (or alternatively cholesterol esters) or even no triglyceride, phospholipid, cholesterol and / or cholesterol esters.

In certain embodiments of the invention, the compositions according to the invention preferably do not comprise sunflower oil, rapeseed oil, wheat germ oil, olive oil and / or coconut fat.

In a preferred embodiment, castor oil is used as the DMSO miscible oil.

In certain embodiments of the invention, the complexes of at least one nucleic acid and at least one organic cation dissolved in dimethylsulfoxide (DMSO) are preferably not located inside a micelle, a liposome or a nanoparticle.

In certain embodiments of the invention, in the compositions according to the invention, the complexes of at least one nucleic acid and at least one organic cation dissolved in dimethyl sulfoxide (DMSO) are preferably not adsorbed on the surface of a micelle, a liposome and / or a nanoparticle. The nucleic acid

The nucleic acid to be introduced into the cells or tissue according to the invention is a charged nucleic acid. According to the invention, the term "nucleic acid" is to be understood as meaning a nucleic acid whose nucleosides are linked to one another by phosphate groups, the phosphate residues involved usually carrying a negative charge, as is the case with naturally occurring DNA or RNA, in other words polyanionic acids (polyelectrolytes) in which the anionic groups are formed by the negatively charged oxygen radicals of the phosphate groups which form the phosphodiester bonds. [. Grund] In principle, the radicals of the nucleic acids carrying the negative charges may also be the negative radicals of phosphorothioates or phosphorodithioates or else other negatively charged groups of nucleic acids.

Usually, due to their charge, nucleic acids dissolve well in water, but not in water-immiscible organic liquids, such as hydrocarbons or other organic solvents, such as ethanol, etc. A nucleic acid is considered to be dissolved in the sense of the invention if it is replaced by a Do not centrifuge for 5 minutes at 15000 xg.

The nucleic acids in the compositions of the invention may be single-stranded or double-stranded nucleic acids, e.g. single-stranded or double-stranded DNA or RNA. A double-stranded nucleic acid can be present, for example, in the form of two separate single strands or as a hairpin loop structure. The size of the nucleic acid to be introduced is not critical according to the invention.

In a particular embodiment of the invention, the nucleic acid is an oligonucleotide, ie single-stranded or double-stranded DNA or RNA (or DNA / RNA hybrids) having a chain length of from about 2 to about 15 nucleotides, preferably from about 5 to about 100 nucleotides , preferably about 10 to about 80 nucleotides, more preferably about 20 to about 60 nucleotides. The oligonucleotide is preferably a synthetically produced oligonucleotide of known sequence. The nucleic acids may be, for example, DNA, cDNA, mRNA, sRNA, ribozymes, antisense DNA, RNA, antisense RNA, siRNA, decoys, tRNA, rRNA, snRNA, snoRNA, or miRNA. In a particular embodiment, the nucleic acid is not siRNA. For a therapeutic application, it is particularly advantageous if the nucleic acids contain at least part of a sequence of a (human) genome or gene. For the cosmetic application, it is again advantageous if the nucleic acids do not contain a sequence of a (human) genome or gene.

In a particular embodiment of the invention, the nucleic acid to be introduced is a vector or a plasmid with a size of more than 1 kb (1000 nucleotides). The size of the vector or plasmid will typically be greater than 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 1k, 12kb, 13kb, 14 kb, 15 kb or even more than 20 kb. It may be with the nucleic acid to be introduced into the cells, e.g. is a gene therapy vector which is administered for expression in a mammal. Examples of gene therapy vectors suitable for delivery into tissue of a living mammal according to the invention include, for example, viral and non-viral vectors. Viral vectors are derived from various viruses, e.g. of retroviruses, herpesviruses, adenoviruses or adeno-associated viruses (AAV), see Lundstrom, Trends Biotechnol. (2003), 21 (3): 1 1 7-22. Preferably, the viral vectors are those that are no longer able to replicate in the cells transfected therewith. In addition to the viral vectors, non-viral vectors, e.g. eukaryotic expression vectors into which cells or tissues are introduced. The vectors or plasmids preferably comprise a eukaryotic promoter that is active in the mammal.

applications

According to the invention, the compositions according to the invention are advantageously suitable, for example, for use in medicine, for example for therapy or diagnostics or in cosmetics, in particular when nucleic acids have to be introduced into cells or tissue in the methods. Preferably, the cells or tissues to be treated by the incorporation of the nucleic acid complexes are cells and / or tissues of a mammal, eg, cells and / or tissues of human, rat, hamster, guinea pig, dog, Cat, horse, cattle, pig, sheep, goat and others. In In a particularly preferred embodiment, the composition serves to introduce nucleic acid into human cells and / or tissue.

Tissues into which nucleic acids can be introduced according to the invention include e.g. Epithelial tissue, connective tissue and muscle tissue. For the introduction, the introduction into the skin, in particular in the epidermis, dermis and / or subcutis is preferred. Also, introduction via other types of epithelium (e.g., intestinal epithelium) is preferred. Particularly preferred is the introduction of the nucleic acids into cells involved in the formation of hair. As far as human cells are concerned, these are preferably not embryonic stem cells.

The pharmaceutical or cosmetic composition according to the invention is brought into contact with the cells and / or tissues to be treated. In this case, the contact with the complexed nucleic acids preferably takes place without injury to the respective tissue surface, such as e.g. the skin of the mammal. In a simple embodiment of the invention, the composition is applied to the cells and / or tissues, e.g. by dripping or painting. In a particularly preferred embodiment, the tissue intended for uptake of the complexed nucleic acid according to the invention is epithelial tissue of a mammal, for example the skin of a mammal.

Depending on the cells or tissues in which the complexed nucleic acids are to be incorporated, as well as the size and amount of the nucleic acids, the residence time of the (pharmaceutical or cosmetic) composition on the cells or tissues will generally be in the range of minutes or hours. However, in some applications it may also be advantageous to provide a residence time of several days, especially if it is desired to introduce a large amount of nucleic acid into the cells or tissues. Applications in which a longer residence time can be adjusted, in particular, involve the incorporation of nucleic acids into deeper skin layers, e.g. into the dermis or subcutis.

When introducing the complexed nucleic acid into the cells and / or tissue, in particular into the skin, it may be necessary or expedient to repeat the application of the composition according to the invention once or even several times. Preferably, the application is at least 2, 3, 4, 5, or at least 7 times, eg in Repeated one week until the intracellular concentration of the nucleic acid in the cells or in the cell nuclei is sufficiently high. However, a single application may also be sufficient.

A particularly suitable application form of the complexed nucleic acid dissolved in DMSO are dermal or transdermal patches. Through the use of patches, which have a depot for the complexed nucleic acid dissolved in DMSO, contact with the treated skin can be achieved over a relatively long period of time, thus causing a continuous uptake of the nucleic acids.

Complexed according to the invention nucleic acid can be introduced by using a composition of the invention, for example, in tissue cells of the epidermis, dermis and subcutis.

In the course of the good incorporation of the compositions of the present invention into various cell and tissue types, the composition of the present invention may, in principle, be administered by any means known to those skilled in the art. The compositions can be administered, for example, parenterally, enterally, and / or topically. Routes of administration therefore include, for example, intravenous, intramuscular, subcutaneous, intradermal or transdermal administration, oral or rectal administration, epicutaneous, dermal, inhalative, nasal, or intranasal administration. Particularly preferred are topical, dermal, and / or transdermal administration.

In preferred embodiments of the present invention, one or more or all of the following conditions are satisfied with respect to the compositions:

a) the composition does not include petroleum, mineral oil, mineral oil distillate, light oil, heavy fuel oil, toluene, benzene, mineral fat, fuels, diesel fuel, and / or gasoline,

b) the organic cations in the complex with the nucleic acid are not identical to the pharmaceutically or cosmetically acceptable adjuvant, c) the composition does not comprise any synthetic artificial organic polymers,

d) the composition does not comprise in parallel a triglyceride, phospholipid, cholesterol and / or cholesterol ester, e) are the complexes of at least one nucleic acid and at least one organic cation dissolved in dimethyl sulfoxide (DMSO) not inside a micelle, a liposome or a nanoparticle, and / or f) are dissolved in dimethyl sulfoxide (DMSO) complexes of at least one Nucleic acid and at least one organic cation are not adsorbed on the surface of a micelle, a liposome and / or a nanoparticle.

In a further aspect, the present invention relates to a pharmaceutical or cosmetic method of treatment on a patient or a method for introducing at least one nucleic acid into cells and / or tissue of a patient, the method comprising the following step:

a) contacting a composition of the present invention with cells and / or tissue (s) of a patient.

The patient can be a human or an animal. In particular, the animal may be a mammal.

characters

Fig. 1 shows the solubility of CTAB DNA in DMSO / water mixtures of different ratios.

Figure 2 EtBr-CTAB DNA in DMSO is completely soluble in one phase in each tested mixing ratio (9-67%) with ethylene and propylene glycol. Left side: Propylene glycol; Right side: ethylene glycol. Above: room light shot; Down UV light. The DMSO fraction increases from left to right (9%, 1%, 33%, 50% and 67% (v / v)).

Fig. 3 thin layer of the mouse skin after treatment with FAM-oligo in DMSO-propylene glycol.

Three days after application of 10μ1 of the composition. Upper green channel (FAM oligo fluorescence). Top, left Overview The green color (bright dots) indicates the invaded FAM oligo. Above, right detail of the cross-cut hair root with cell nuclei that have taken up the FAM oligo. Below blue channel (Bisbenz Fluorescence nuclear staining). Below, left Overview The blue color (bright dots) indicates the cell nuclei. Below, right detail of the cross-cut hair root with cell nuclei.

Fig. 4 thin layer of the mouse skin after treatment with FAM-oligo in DMSO. Three days after application of 10μΙ of the composition. Green channel (FAM oligo fluorescence). Top, left Overview: the green color indicates the invaded FAM oligo. Detail of several longitudinally cut hairs. In the lower part of the hair shaft (light rods) show concentric bright points. These are the nuclei of hair root cells in which the green-fluorescent oligonucleotide has penetrated.

Fig. 5 Photograph of a mouse four weeks after treatment with siRNA-1 (tyrosinase) (SEQ ID NO: 2) in DMSO-propylene glycol formulation.

Fig. 6 Photograph of a mouse four weeks after treatment with siRNA-1 (tyrosinase) (SEQ ID NO: 2) in DMSO-propylene glycol formulation.

Fig. 7 Photograph of a mouse three weeks after treatment with siRNA-1 (tyrosinase) (SEQ ID NO: 2) in DMSO-ethanol castor formulation.

Fig. 8 Photograph of a mouse three weeks after treatment with siRNA-1 (tyrosinase) (SEQ ID NO: 2) in DMSO-ethanol castor formulation.

Fig. 9 Photograph of a mouse five weeks after treatment with siRNA-1 (tyrosinase) (SEQ ID NO: 2) in DMSO-ethanol castor formulation.

Fig. 10 Fluorescence microscopic images of a thin section of a skin area five weeks after treatment with siRNA-1 (SEQ ID NO: 2) in dimethyl sulfoxide / propylene glycol. A skin area is prepared showing the transition between the hairless and the hairy area. Flg. 10A: Representation of cell nuclei with bisbenzimide. Fig. 10B: Representation of tyrosinase by means of anti-tyrosinase antibodies. The following embodiments illustrate further preferred embodiments of the present invention, but the present invention is not limited to these specific embodiments.

example 1

Comparison of solubility of CTAB DNA in various organic solvents

250 ml of a 1% (w / v) salmon sperm DNA - corresponding to 2.5 mg - were introduced into 1, 7 ml plastic tubes in water. The DNA was precipitated with an equal volume of 100 mM CTAB and the precipitate precipitated by centrifugation (5 min, 10,000 × g). The pellet was suspended in 500μl water and recentrifuged. The resulting pellet was dried in Speedvac overnight. Each volume was filled with 1 ml of different solvents and the CTAB DNA pellet shaken overnight (800 rpm). The tubes were recentrifuged and the absence of precipitate was assessed as evidence of very good solubility of the CTAB DNA in the appropriate solvent.

Solubility of CTAB DNA in various solvents

Example 2

Comparison of solubility of CTAB-DNA in DMSO / water mixtures

In 11 1, 7ml plastic tubes 250μΙ of a 1% (w / v) salmon sperm DNA - equivalent to 2.5mg - was filled into water. The DNA was precipitated with an equal volume of 100 mM CTAB and the precipitate precipitated by centrifugation (5 min, 10,000 × g). The pellet was suspended in 500μl water and recentrifuged. The resulting pellet was dried in Speedvac overnight. In each tube, a volume of 1 ml of different mixture of DMSO was filled with water and the CTAB DNA pellet shaken overnight (800 rpm). The tubes were recentrifuged and the DNA in the supernatant determined by measuring absorbance at 260nm. It was found that the solubility in water is low and increases greatly from a DMSO concentration of 80% (v / v). % DMSO 0 10 20 30 50 60 70 80 90 95 100

260nm 0,218 0,235 0,222 0,227 0,145 0,128 0,144 0,655 2,862 3,003 2,896

% of max 7.5 8.1 7.7 7.9 5.0 4.4 5.0 22.6 98.8 103.7 100.0

Example 3

Determination of CTAB DNA particle size in DMSO-water mixtures

For the introduction of the nucleic acids into mammalian cells via the skin, the particle size of the CTAB DNA particles is important. Smaller particles can easily penetrate the skin and be absorbed by the cell membrane. Therefore, CTAB DNA particle size was determined by dynamic light scattering.

200μΙ of a 1% (w / v) salmon sperm DNA - equivalent to 2mg - in water were placed in a 2ml tube. The DNA was precipitated with an equal volume of 100 mM CTAB and the precipitate precipitated by centrifugation (5 min, 10,000 × g). The pellet was suspended in 500μl water and recentrifuged. The resulting pellet was dried in Speedvac overnight. A volume of 2 ml DMSO was placed in the tube and the CTAB DNA pellet shaken overnight (800 rpm). There was a clear solution. The CTAB DNA solution in DMSO was diluted 1:10 with DMSO and water to produce various DMSO-water mixtures in the concentration range with a DMSO content (v / v) of between 10-100%. 0.5 ml each of the different CTAB-DNA solutions in the DMSO-water mixtures was transferred to plastic cuvettes and the particle size was measured by dynamic light scattering with a Zetasizer (Malvern Instruments GmbH, 71083 Herrenberg).

At high DMSO concentrations greater than 80% (v / v) DMSO, a mean particle diameter of about 10 nm was observed, which increased more than 10-fold with increasing water content. DMSO H20

Concentration of medium concentration

Vol. Conc. Particle Diameter Vol. Conc.

% (v / v) Nm (SD)% (v / V)

10,227 (6,7) 90

20,139 (7.1) 80

33 154 (5.5) 66

40 183 (6.2) 60

50 168 (4.0) 50

60 8 (3) 40

60 10 (3,1) 40

70 60 (4.5) 30

80 7 (1, 5) 20

90 13 (2,1) 10

100 10 (1, 2) 0

Example 4

Determination of solubility of CTAB DNA in DMSO-Glycol mixtures

20 mg salmon sperm DNA was dissolved in 10 ml of water in a 50 ml tube. The resulting DNA solution was spiked with the same volume of 100 mM CTAB containing 2 mg ethidium bromide (EtBr). A red precipitate formed, which was centrifuged off by centrifugation at 5000 × g for 15 minutes. The supernatant was discarded and the precipitate washed by suspension in 10 ml of water and re-centrifugation. The precipitate (EtBr-CTAB-DNA) was dried overnight in the Speedvac and dissolved in 10 ml DMSO. To determine the miscibility of CTAB-DNA in DMSO with glycols, 500 .mu.l ethylene glycol or propylene glycol was placed in five 1, 7ml tubes and mixed with different volumes of EtBr-CTAB-DNA in DMSO, mixed and centrifuged for 5 min at 10,000 xg. The samples were photographed in room light and UV light. It was found that the DMSO-dissolved EtBr-CTAB DNA was miscible with ethylene glycol and propylene glycol in all tested mixing ratios. Neither a phase separation nor a precipitation was observed.

Example 5

Preparation of CTAB oligonucleotides

The following oligonucleotides were intended for transdermal administration to human skin or mouse skin.

An oligonucleotide (3'-GAT CCT GCA TAT GGT AGT G -5 ', SEQ ID NO: 1) molecular weight 5938.82 g / mol) was incubated with the fluorescent dye fluorescein (6FAM) (oligonucleotide 1) or TAMRA (oligonucleotide 2). marked to allow detection of the oligonucleotide in the skin cells. The oligonucleotides were obtained from Biolegio (Nijmegen, The Netherlands).

For the preparation of CTAB oligonucleotide complexes, 1 mg of the oligonucleotides were dissolved in 200 μΙ water and mixed with 400 μΙ 10 mM CTAB. The sample was incubated on ice for 30 min and the resulting precipitate was centrifuged for 5 min at 10,000 x g. The supernatant was removed and the precipitate was suspended with 200μΙ water and centrifuged again. The precipitate was dried overnight in Speedvac and then taken up in 200μΙ DMSO. Assuming 100% precipitation efficiency, the DMSO-CTAB oligonucleotide solution contains 5μl / μΙ oligonucleotide.

For the application of CTAB oligonucleotides, the following dilutions were prepared: Mixture 1: 1 μβ μΙ CTAB oligonucleotide in 10% DMSO with 20% ethanol, 70% ethylene glycol

Mixture 2 1 g μΙ CTAB oligonucleotide in 50% DMSO with 50% propylene glycol

Mixture 3 1 g μΙ CTAB oligonucleotide in 33% DMSO with 67% castor oil

Mixture 4 1 μ ^ μΐ CTAB oligonucleotide in 50% DMSO with 50% ethanol

Mixture 5 1 pg / pl CTAB oligonucleotide in 5% DMSO with 15% ethanol, 80% Riz

The percentages are given in (v / v).

Note: The excipient may provide two particular properties in the context of this invention: A) it dilutes the DMSO, which can cause skin irritation in high concentration; and B) it enhances the uptake of the CTAB complexes into the skin and skin cells. Oils, glycols and ethanol meet these features.

Example 6

Introduction of DNA into murine skin cells

Mice were prepared for dermal application of the compositions by shaving a fur area in the neck. In each case 10 μΙ of the composition were applied to an area of about 1 cm ² . In some cases, the application was repeated after two days.

The preparation of the treated skin area was made 2 days after the last application. , The preparations were frozen in liquid nitrogen and thin sections approximately 8 μm thick were prepared for fluorescence microscopy. In some cases, nuclear staining was performed with bisbenzimide (5 min, 1 pg / ml in PBS). Evaluation of fluorescence microscopy showed after 2 days cell nuclei of epidermal cells and cells of the hair follicle and bulge region which appeared green by the fluorescein-labeled oligonucleotide. After a single application, the labeled nucleotide was already detectable in the skin tissue and skin cells.

The fluorescence-labeled oligonucleotide DNA was detected in the following cell types: hairy and hair root cells, skin connective tissue cells, capillary cells, muscle cells, epidermal cells. Example 7

Production of oligonucleotides for influencing hair growth and pigmentation of the skin

siRNAs were obtained as double-stranded products with 3 'deoxythymidine overhangs (dTdT) from Eurofins MWG Operon (85560 Ebersberg, Germany). siRNA-1 (tyrosinase-specific) (5'-CUA UCU GAA UCA GAU UCU A dTdT-3 '; SEQ ID NO:

2), with two deoxythymidines at the 3 'end and corresponding complementary RNA counterstrand also with two deoxythymidines at the 3' end. siRNA-2 (nonspecific control) (5'-GCU GAC CCU GAA GUU CAU C dTdT-3 '; SEQ ID NO:

3), with two deoxythymidines at the 3 'end and corresponding complementary RNA counterstrand also with two deoxythymidines at the 3' end.

Example 8

Production of complexed siRNA in ethanol / castor oil

2 mg of the siRNA oligonucleotide (SEQ ID NO: 2) (tyrosinase-specific) and 2 (nonspecific control; SEQ ID NO: 3) were each dissolved in 200μΙ water and mixed with 600μΙ 10mM cetyltrimethylammonium bromide solution. The result was a white turbidity which was precipitated by centrifugation at 10,000 xg for 5 min. The supernatants were discarded and the precipitates suspended in 200μΙ water and again centrifuged as above. The supernatants were discarded and the precipitates dried under vacuum. 200μΙ ethanol was added to the precipitates and the suspension was vortexed. The suspension remained cloudy. In 200μΙ steps more ethanol was added and mixed. With the addition of a total of 1200μΙ ethanol, the complexed siRNA was completely dissolved. 50μΙ, 10ΟμΙ, 200μΙ of the complexed siRNA in ethanol were mixed with 950μΙ, 900μΙ and 800μΙ castor oil (total volume 1 ml each) by pipetting up and down. Clear solutions were generated with siRNAs.

Example 9

Preparation of complexed siRNA in dimethyl sulfoxide / propylene glycol

2 mg of siRNA oligonucleotide 1 (SEQ ID NO: 2) and 2 (SEQ ID NO: 3) were each dissolved in 200μΙ water and mixed with 600μΙ 10mM Cetylammoniumbromid solution. There was a white turbidity which was precipitated by centrifugation at 10,000 xg for 5 min. The supernatant was discarded and the precipitate suspended in 200μΙ water and again centrifuged as above. The supernatant was discarded and the precipitate dried under vacuum. The precipitate (cetylammonium siRNA) was taken up in 200μΙ dimethyl sulfoxide. 10μΙ, 20μΙ and 50μΙ of the complexed siRNA in dimethyl sulfoxide were mixed with 90μΙ, 80μΙ and 50μΙ propylene glycol (total volume 100μΙ each). Clear solutions with dissolved siRNAs were generated.

Example 10

Effect of dermal application of lipophilic siRNA-1 (tyrosinase-specific) complexes in mice

14 mice were used to study the effect of lipophilic siRNA-Tyr complexes. 3 mice were applied to the unshaved head or back skin per 20 μl of the siRNA-1 dissolved in dimethyl sulfoxide / propylene glycol (samples 1 d, 1 f and 1 g from example 12). 20 μΙ of the dissolved in ethanol / castor oil siRNA-1 (samples 1 a, 1 b and 1 c of Example 1 1) were applied to the unshaven head or back skin. 3 further mice were in the same places per 20μΙ of dissolved in ethanol / castor oil siRNA 2 (nonspecific controls, samples 2a, 2b and 2c of Example 1 1) and 3 other mice dissolved in dimethylsulfoxide / propylene glycol siRNA 2 (nonspecific control, samples 2d, 2f and 2g of Example 12). As further controls, uncomplexed siRNA-1 was immediately dissolved in sterile TE (10 mM TrisCl, pH8, 2 mM EDTA) or TE with 20% (v / v) dimethylsulfoxide to a final concentration of siRNA-1 of 2 g and 20 μM each of this solution applied to one mouse each. Thereafter, the mice were kept ad libitum with water and dry diet. After about 2 weeks, the hairs of the mice treated with siRNA-1 (tyrosinase-specific) turned white and precipitated. The siRNA-2 control hairs as well as the uncomplexed siRNA-1 treated mice remained dense and black. After a further 3 weeks, the hair grew again with siRNA-1 (tyrosinase-specific), so that the effect was completely abolished after approx. 7 weeks after application. Apart from the hair alteration, no deviation of the siRNA-1 (tyrosinase-specific) treated mice was observable compared to the control group. Over the entire period, the mice were vital and showed no pathological changes.

example 1

Detection of reduction of tyrosinase in thin sections of siRNA-1 treated skin areas To demonstrate the specific response of the tyrosinase-specific siRNA-1, the tyrosinase protein in the skin of the treated mice was examined in thin sections using anti-tyrosinase antibodies. To this end, a mouse treated with complexed siRNA-1 in dimethyl sulfoxide / propylene glycol (20%: 80%) was sacrificed at 5 weeks and cut out of a skin area that included both part of a hairless and part hairy area of the skin. As a control, a skin area was used which had not been treated with complexed siRNA-1. The skin fragments were over Fixed at 4 ° C in PBS with 10% formaldehyde, frozen in liquid nitrogen and cut into 8 μm thin sections. The sections were incubated in 1 to 200 rabbit anti-tyrosinase antibodies diluted in PBS (Antibody-online.com, Atlanta, GA 30346, USA) for 1 hr at room temperature. The sections were rinsed in PBS and incubated with rhodamine-coupled anti-rabbit antibodies for 1 hour at room temperature. The sections were then rinsed with PBS and incubated for nuclear staining with 2 μg / ml bisbenzimide in PBS. The sections were then rinsed again with PBS and capped with a drop of Fluoromount. The thin sections were evaluated by means of a fluorescence microscope. The controls, which had not been treated with complexed siRNA-1, showed a clear red fluorescence in the area of the hair cells and epithelial cells. The red fluorescence indicates the presence of tyrosinase in the thin sections. Skin areas treated with complexed siRNA-1 showed no or greatly reduced red fluorescence.

Claims

claims

1 . Pharmaceutical or cosmetic composition comprising:

b) at least one pharmaceutically or cosmetically acceptable excipient.

2. Composition according to claim 1, wherein the nucleic acid is complexed so that the negative charges of the nucleic acid are substantially completely compensated by organic cations, in particular wherein the molar ratio between the anionic charges of the at least one nucleic acid and the charges of the organic cations is substantially 1 : 1 is.

3. Composition according to one of the preceding claims, wherein the composition has a nucleic acid concentration of more than 1 pg / ml, preferably more than 10 g / ml, more preferably more than 100 pg / ml.

A composition according to any one of the preceding claims, wherein the organic cations comprise quaternary amines, in particular wherein the organic cations comprise cetyltrimethylammonium bromide (CTAB).

5. The composition according to claim 1, wherein the at least one adjuvant is selected from the group consisting of fillers, solvents, humectants, emulsifiers, solubilizers, wetting agents, antifoams, salt formers, buffers, gelling agents, thickeners, film formers, binders, wrapping agents, Disintegrants, adsorbents, lubricants, lubricants, mold release agents, flow control agents, antioxidants, preservatives, sweeteners, flavor correctors, scent-correctors, and colorants.

6. The composition according to any one of the preceding claims, wherein the excipient consists of a primary alcohol, in particular ethanol.

7. The composition according to any one of claims 1 to 5, wherein the excipient consists of a polyhydric alcohol, in particular glycerol, ethylene glycol, propylene glycol or a polyethylene glycol.

A composition according to any one of claims 1 to 5, wherein the excipient consists of an oil, wax or fat, in particular wherein the excipient is castor oil.

Composition according to any one of the preceding claims, wherein the composition has an oil, wax or fat content of more than 30%, preferably more than 50%, more preferably more than 80% (v / v).

10. A composition according to any one of the preceding claims, wherein the DMSO content of the composition is between 2% to 95%, preferably between 5% to 90% (v / v), more preferably between 10% and 60% (v / v) ,

1 1. A composition according to any one of the preceding claims, wherein:

a) the composition does not comprise petroleum, mineral oil, mineral oil distillate, light oil, heavy fuel oil, toluene, benzene, mineral fat, fuels, diesel fuel, and / or gasoline,

b) the organic cations in the complex with the nucleic acid are not identical with the pharmaceutically or cosmetically acceptable adjuvant, c) the composition does not comprise any synthetic artificial organic polymers,

d) the composition does not comprise a triglyceride, phospholipid, cholesterol and / or cholesterol ester,

e) the complexes of at least one nucleic acid and at least one organic cation dissolved in dimethyl sulfoxide (DMSO) are not located inside a micelle, a liposome or a nanoparticle, and / or f) the complexes of at least one nucleic acid dissolved in dimethylsulfoxide (DMSO) and at least one organic cation are not adsorbed to the surface of a micelle, liposome and / or nanoparticle.

A composition according to any one of the preceding claims, wherein the composition has a water content of about 50% (v / v) or less, preferably about 40% (v / v) or less, preferably about 30% (v / v ) or less, preferably from about 20% (v / v) or less, preferably from about 15% (v / v) or less, preferably from about 10% (v / v) or less, preferably from about 5% (v / v) or less, or more preferably about 0%.

13. The composition according to any one of the preceding claims, wherein the complexes have an average diameter of less than 200nm, preferably less than 100nm, more preferably less than 50nm.

14. Composition according to one of the preceding claims for use in the introduction of nucleic acid into cells or tissue, in particular for use in the introduction of nucleic acid into cells of the epidermis, dermis and / or subcutis.

15. A process for preparing a composition comprising complexes dissolved in dimethyl sulfoxide (DMSO) from at least one nucleic acid and at least one organic cation, and at least one pharmaceutically or cosmetically acceptable excipient, in particular for the preparation of one of the compositions according to any one of the preceding claims, wherein the method following steps include:

a) providing complexes of at least one nucleic acid and at least one organic cation,

b) dissolving the complexes in DMSO or DMSO-containing solvent mixture, and c) adding a pharmaceutically or cosmetically acceptable excipient to the DMSO solution or the DMSO-containing solvent mixture having dissolved therein complexes of at least one nucleic acid and at least one organic cation, insofar the DMSO-containing solvent mixture according to b) does not already contain the pharmaceutically or cosmetically acceptable excipient.