WO2013053480A1

WO2013053480A1 - Composition for introducing nucleic acids into cells

Info

Publication number: WO2013053480A1
Application number: PCT/EP2012/004265
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: EP2765983A1

Abstract

The subject matter of the present application are pharmaceutical and/or cosmetic compositions which comprise at least one organic solvent in which complexes of at least one nucleic acid and organic cations are present in dissolved form. The compositions are advantageously suitable for use in the introduction of nucleic acid into cells and/or tissue, in particular of a mammal.

Description

Composition for introducing nucleic acids into cells

The present application relates to pharmaceutical and / or cosmetic compositions comprising at least one organic solvent in which complexes of at least one nucleic acid and organic cations are present dissolved. The compositions are useful e.g. advantageously for use in introducing nucleic acid into cells and / or tissue, especially a mammal.

State of the art

The therapeutic use of nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) has been extensively researched by the pharmaceutical industry for a long time. The aim of the effort is to develop a new class of nucleic acid-based therapeutics, which show a highly selective activity in the control of numerous diseases.

A fundamental problem with the development of nucleic acid-based therapeutics is that they usually 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 art Usually viral vector systems are used. The vector systems used in these methods 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 encoding 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 is complexed with organic cations in a certain way and dissolved in organic solvents, 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 composition comprising at least one organic solvent in which complexes of at least one nucleic acid and at least one organic cation are present in solution for use in introducing nucleic acid into cells or tissue, in particular into cells or tissue of a mammal. Likewise, the compositions can be used for transepithelial administration. The compositions of the present invention are particularly preferably suitable for application to the skin.

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 at least a first organic solvent. The first organic solvent contains dissolved therein one or more complexed with one or more organic cations nucleic acids. The nucleic acids are usually so complexed 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 zero Is 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 greater than or equal to 0.95 is 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 nucleic acid can be, for example, 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 are in the range 0.9 - 1, 1 lie, im Range from 0.91 to 1.10, range from 0.92 to 1.08, range from 0.93 to 1.70, range from 0.94 to 1.06, range from 0.95 to 1 , Lie in the range 0.96 - 1, 04, lie in the range 0.96 - 1, 04, in the range 0.97 - 1, 03 lie, in the range 0.98 - 1, 02 lie, in the range 0,99 - 1, 01 are in the range 0,995 - 1, 005, or even equal to 1. The latter value is achieved in particular if, in the preparation (see later), cations are present in excess in the precipitation of the nucleic acid and the precipitate of organic cation and nucleic acid is washed with water (see below).

It has been found that this form of complexation makes it possible to introduce large amounts of nucleic acids, which are otherwise insoluble in organic solvents, into such solvents. 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 been shown that the complexed nucleic acids dissolved in organic solvents can be effectively absorbed into cells and / or tissue, e.g. of a mammal. In particular, the dermal, transepithelial and / or transdermal incorporation of the nucleic acids complexed according to the invention (i.e., for example, via the skin of a mammal) has proved 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.

"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.

"At least one organic solvent" means that at least one organic solvent is present, but there may also be solvent mixtures of two or more, three or more, four or more, five or more, etc., of different organic solvents. The nucleic acids are then dissolved accordingly in these solvent mixtures.

"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.

The at least one nucleic acid in the compositions of the present invention dissolves in the organic solvents 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, in the compositions of the present invention, complexes of at least one nucleic acid and at least one organic cation are dissolved, wherein the complexes are not located inside a micelle, a liposome or similar nanoparticulate structures. In some embodiments, the compositions of the present invention even contain no micelles, liposomes, or similar nanoparticulate structures.

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 forming an insoluble complex in the aqueous solution, followed by incorporation of the precipitate in at least one organic solvent.

Such a method may include 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 at least one organic solvent (second liquid) e) optionally mixing the second liquid with a third liquid.

In a particular embodiment, the organic solvent (second liquid) is not an oil or fat. However, waxes, fats and / or oils but also aqueous liquids are particularly suitable as the third liquid.

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 the formula NR ₄ X may be used as the source of organic cations, each R independently being a hydrocarbon radical having from 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 hydrocarbon 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 C10 to C20, in particular 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- (spermidinecarboxamido) ethyl] -N-N-dimethyl-1-propaninium trifluoroacetate (DOSPA), diheptadecylamidoglycylspermidine (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 insoluble in an aqueous medium and, when prepared in aqueous media, may be an insoluble precipitate be separated. 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 g, preferably 15,000 g. If necessary, this process can be carried out several times after re-inclusion of the precipitate precipitated in water. 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.

In the course of complexing the at least one nucleic acids, it is of course also possible, as mentioned above, to use various organic cations by adding mixtures of different organic cations to the nucleic acid-containing aqueous liquid in order to precipitate the nucleic acid complexes.

The complexes obtained by precipitation can then be dissolved in a wide variety of organic solvents (including those generally considered to be aqueous solution precipitants). After incorporation into a solvent, these substantially 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 solvent is substantially identical to the number of negative charges of the nucleic acid.

Organic solvents which are suitable for receiving the complexed nucleic acid include in particular mono- or polyhydric alcohols or partially etherified alcohols. The solvent 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-R1 -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 suitable, such as 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, saturated or unsaturated hydrocarbons, such as pentane, hexane or heptane, or benzene-based aromatic hydrocarbons, such as styrene, can be used, ie in particular C1-C3-substituted benzenes. Alternatively, halogen-containing solvents such as chloroform, dichloromethane or carbon tetrachloride or other heteroatom-containing solvents such as dimethylformamide or tetrahydrofuran may be used. DMSO can also be used.

In particular, when using lower alcohols such as ethanol, propanol or butanol as a solvent, concentrations of complexed nucleic acid can be achieved, which are otherwise achieved only in neutral or aqueous media. Thus, in solution of herring sperm DNA, which was complexed in a charge stoichiometric ratio of about 1: 1 with CTAB, in the solvents methanol, ethanol, 1-butanol, 2-propanol, 1-pentanol and ethylene glycol monobutyl ether each a solubility of more than 10 mg of DNA per ml of solvent can be achieved.

In solvents which showed a lower solubility for the nucleic acids complexed according to the invention, it is likewise possible to achieve a concentration of dissolved nucleic acid of more than 1 mg / ml by first mixing the nucleic acid in a first solvent with high solubility (such as, for example, methanol, ethanol, Butanol, 2-propanol, 1-pentanol, ethylene glycol monobutyl ether or mixtures thereof) is dissolved, and the first solvent containing the complexed nucleic acids is then mixed with a second solvent having a lower solubility for the complexed according to the invention nucleic acids. In the oils walnut oil, wheat germ oil, linseed oil, safflower oil, rapeseed oil and almond oil, a concentration of 10 μg / ml could be achieved. Other usable oils are given below.

The dissolved in organic solvents nucleic acid complexes are characterized by an extremely high stability. This is essentially due to the fact that nucleases are inactive in non-aqueous environment. This is especially true when using Ribonucleic acid (RNA) of importance, 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 nucleic acids complexed according to the invention is also increased by the fact that no acid or alkaline environment is present in the organic solvent used. This avoids any acidic or alkaline hydrolysis of the nucleic acids.

After dissolving the nucleic acids in the at least one organic solvent, for example, the organic solvent may be mixed with any other water-immiscible liquids, e.g. with another of the above-mentioned organic solvents.

Preferably, in the compositions to be used according to the invention, the at least one organic solvent accounts for less than 70% (v / v), preferably less than 50% (v / v), more preferably less than 20% (v / v). The at least one organic solvent may also be present in a concentration of more than 1% (v / v), preferably more than 5% (v / v), more preferably more than 10% (v / v). For example, the organic solvent may be in a range of 1-70% (v / v), 5-50%, or even 10-20% (v / v).

The water content, in particular of the phase comprising the nucleic acid complexes, or else of the total compositions to be used according to the invention, is preferably less than about 50% (v / v), more preferably less than about 30% (v / v), even more preferably less than about 10% (v / v). A higher water content leads to the formation of larger aggregates which do not possess optimal penetration properties.

Preferably, the average diameter of the complexes of nucleic acid and organic cation in the compositions to be used according to the invention is smaller than 200nm, preferably less than 100nm, more preferably less than 50nm, most preferably less than 1m.

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 a therapeutically effective nucleic acid, i. 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, such as e.g. to be a Molecular Beacon.

For a cosmetic composition, the nucleic acid is a cosmetically active nucleic acid, i. a nucleic acid which, after administration to an individual, does not generally 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 one of the above-described organic solvents or solvent mixtures are brought into direct contact with the cells or tissues intended for cosmetic and / or therapeutic treatment, for example by dripping or brushing on Nucleic acid-containing solvent or solvent mixture. Preferably, an organic solvent is used which is compatible with respect to the cells and / or tissue to be treated. Unless the cosmetic and / or For example, if the therapeutic compositions of the present invention are intended for the treatment of the skin, eg, human skin, the corresponding organic solvent will be selected such that no toxic or skin integrity properties are associated with the solvent. Numerous organic solvents are known in the art which are useful as pharmaceutical carriers, especially for dermal administration.

It is of course also possible to use the liquid e.g. process with water to an emulsion and apply in this form to the skin. Other common dosage forms for the compositions of the invention include lotions, creams, ointments, gels and pastes. In a number of embodiments of the invention, however, the compositions to be used according to the invention are not emulsions and / or lotions, creams, ointments, gels and pastes.

If advantageous for the chosen administration, the nucleic acid complexes dissolved in an organic solvent may be mixed with a pharmaceutical carrier suitable for the particular administration. For example, the pharmaceutical carrier can be any water-immiscible lipophilic compound which can be mixed with the organic solvent comprising the nucleic acid complexes. Preferably, the pharmaceutical carrier is a liquid compound, e.g. one (possibly additional) oil.

In preferred embodiments of the present invention, the compositions to be used according to the invention comprise at least one oil and / or at least one wax and / or at least one 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.

Suitable vegetable oils are, for example, nut oils and seed oils. Suitable vegetable oils include, in particular, peanut oil, walnut oil, almond oil, hazelnut oil, coconut oil, soybean oil, olive oil, poppy seed 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 seed oils include, for example, 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, candelilla wax and Japan wax.

In addition to vegetable oils, waxes and fats, animal fats, waxes and oils, e.g. 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 include 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.

Preferably, the compositions of the present invention, when present in an oil, wax or fat, comprise at least one other solvent which is not an oil, wax or fat. In other words, particularly preferably, the compositions to be used according to the invention comprise, in addition to a (conventional) organic solvent such as ethanol, ethylene glycol monobutyl ether, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, and / or DMSO, an oil, fat or wax. A particularly preferred combination is a combination of DMSO with at least one oil, fat and / or wax.

The compositions of the invention may have an oil, wax and / or fat content greater than or equal to 25% (v / v), for example greater than or equal to about 30% (v / v), greater than or equal to about 40% (v / v ), greater than 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), or greater than or equal to about 85% (v / v). In particularly preferred embodiments, the (pharmaceutical or cosmetic) to be used according to the invention Compositions have an oil, wax and / or fat content of greater than about 90%, preferably about 91% (v / v) or greater, about 92% (v / v) or greater, about 93% (v / v) or greater , about 94% (v / v) or more, about 95% (v / v) or more, about 96% (v / v) or more, about 97% (v / v) or more, about 98% (v / v) or more, about 99% (v / v) or more.

In addition to a pharmaceutical carrier, the compositions according to the invention may contain further auxiliaries. Common auxiliaries include diluents, dyes, preservatives, emulsifiers, thickeners, fragrances, vitamins, and other agents known in the pharmaceutical arts. It is, for example, advantageous to add to the compositions of the invention cell-permeable substances which are known to increase the permeability of the cell membrane and thus facilitate the penetration of the nucleic acid complexes into the cells or tissues. Examples of substances which facilitate the entry of DNA or RNA into cells are in particular fatty acids, fatty acid esters, glycerol diesters, glycerol triesters, glycerol monoesters and urea. Another example of a substance useful in the present invention which increases the permeability of the cell membrane is dimethyl sulfoxide (DMSO). At the same time, as mentioned, DMSO can also be used as organic solvent in the context of the invention. In a particularly preferred embodiment, therefore, the organic solvent used to dissolve the complexed nucleic acid is DMSO. If another organic solvent has been used to take up the complexed nucleic acid, it may be mixed with DMSO to increase the amount and / or rate of uptake of the complexed nucleic acid by the cells and / or tissues. The amount of DMSO in the composition of the invention is preferably from about 1 to about 50% by volume, with a level of from about 5 to about 30% by volume, e.g. 10% by volume is particularly preferred.

Other suitable excipients include preservatives known in the pharmaceutical and / or cosmetic art, such as linear aliphatic 1, 3-diols, phenoxyethanol and chlorphenesin. By adding these compounds, the shelf life of the compositions of the present invention can be increased without causing microbial growth which would render the compositions unusable. Other auxiliaries customary in cosmetic and pharmaceutical preparations are readily known to the person skilled in the art. It has proved to be particularly advantageous if the compositions to be used according to the invention are in the form of a liquid which has a lower vapor pressure than water. This can prevent the liquid composition from evaporating after application to the cells and / or tissues such as the skin. Complete evaporation of the solvents of the composition could theoretically result in precipitation of the complexed nucleic acid contained in the fluid even before effective penetration into the cells and / or tissues can occur. Liquid compositions having complexed nucleic acids having a higher vapor pressure than water may be prepared by the type and amount of the particular solvent or solvents of the compositions. Thus, the properties of the pharmaceutical and / or cosmetic compositions of the invention can be influenced accordingly by using solvents having a higher vapor pressure than water, such as DMSO or higher alcohols such as butanol, pentanol, etc.

Another way to obtain nucleic acid-containing fluids having a higher vapor pressure than water is to mix a solution of complexed nucleic acids in a first solvent having a lower vapor pressure than water (such as ethanol) with a second solvent which has a higher vapor pressure than water.

It has proven particularly suitable in this connection to introduce an oil or fat into the compositions according to the invention. Initially, as described above, the complexed nucleic acids are incubated in a first solvent, e.g. in butanol, dissolved. Subsequently, the nucleic acid-containing solvent is mixed with one or more oils, so that a liquid is formed, which consists of complexed nucleic acids, solvents and oil.

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 understood to mean a nucleic acid whose nucleosides are linked to one another by phosphodiester bonds, the phosphate residues participating in the phosphodiester bonds being negatively charged, as is the case with naturally occurring DNA or RNA the charged nucleic acids to polyanionic acids (polyelectrolytes), in which the anionic groups by the negatively charged oxygen radicals of the phosphate groups which form the Phosphordiesterbindungen be formed. In principle, the residues of the nucleic acids carrying the negative charges may also be the negative residues of phosphorothioates or phosphorodithioates or also 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 chloroform, etc. A nucleic acid is considered to be dissolved in the sense of the invention if it differs Do not centrifuge for 5 minutes at 15000 xg.

The nucleic acids to be introduced may be single-stranded or double-stranded nucleic acids, e.g. single-stranded or double-stranded DNA or RNA. A double-stranded nucleic acid may be 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. It was shown that both oligonucleotides with a chain length of 10 to 100 nucleotides, as well as linear and circular plasmids with a size of several kb (for example, with a size of 1-10 kb) could be efficiently introduced into human and murine skin cells. Thus, the methods and compositions of the present invention open up new opportunities both in the field of gene therapy, in which nucleic acids encoding certain polypeptides are introduced into cells of a living mammal, as well as in the field of RNA interference, in which the targeted Expression of certain genes is affected.

In a particular embodiment of the invention, the nucleic acid to be introduced is an oligonucleotide, i. single-stranded or double-stranded DNA or RNA (or DNA / RNA hybrids) having a chain length of from about 5 to about 150 nucleotides, preferably from about 7 to about 100 nucleotides, preferably from about 10 to about 80 nucleotides, more preferably from 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 act 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.

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 introduction into tissue of a living mammal according to the invention include 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 as described herein are introduced into the cells or tissues. The vectors or plasmids preferably comprise a eukaryotic promoter that is active in the mammal.

applications

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

The present invention discloses formulations of nucleic acids with which after administration, eg after application to the skin of humans and animals, transport the nucleic acids in different cell types is possible. In particular, nucleic acids can be introduced into epithelial cells, endothelial cells, hair cells (such as hair root cells and / or hair follicle cells), muscle cells, fat and connective tissue cells and capillary cells of living mammals with the described compositions. As far as human cells are concerned, these are preferably not embryonic stem cells.

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.

As will be apparent from the examples below, the compositions are useful not only for delivery of nucleic acid into cells or tissues but also, in particular, for transepithelial administration. That is, the compositions of the present invention are capable of penetrating epithelial layers and reaching adjacent, deeper tissue layers. This allows treatment by a non-invasive route, e.g. without the use of syringes.

The pharmaceutical composition is for this purpose 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 where a longer residence time is set may, in particular, involve the introduction of nucleic acids into deeper skin layers, such as 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 plot will be 2, 3, 4, 5, 10, 15 or 20 times, e.g. repeated in the course of a month until the intracellular concentration of the nucleic acid in the cells or in the cell nuclei is sufficiently high.

As shown in the examples, complexed nucleic acid could be introduced into epidermal, dermal and subcutaneous tissue cells by using a composition of the invention. In particular, it could be shown that the cell nuclei also had absorbed a significant amount of the nucleic acids. The period of treatment before histological examination of the tissue was 1 to 4 days. During this time, the animals were vital, and no behavioral or morphological changes could be observed. The treatment with the compositions according to the invention thus shows no side effects.

The amount of complexed nucleic acid and the duration of the treatment are determined by the cells or tissues into which the complexed nucleic acids are to be introduced. In the context of the present invention, it was possible by experiments with mice to show that the nucleic acids were already present in the cells of the subcutis after three days.

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 be administered in principle in any manner 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 a further aspect, the present invention relates to a method of treating 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 or (tissue) of a patient.

The patient may be a human or an animal, in particular a mammal.

For certain applications, it is particularly advantageous to administer the complexed nucleic acids continuously to the respective cells and / or tissue over several days. This can e.g. by administering the complexed nucleic acid by means of a patch which is adhered to the skin. For this purpose, the nucleic acid complexed in the organic solvent may be applied to an absorbent material, such as e.g. a nonwoven or a cellulose matrix, to be dropped. The absorbent material is then sealed by covering with a suitable fixative, e.g. with a self-adhesive film, fixed on the surface of the individual to be treated (e.g., a mammal).

Fixing agents are preferably used which prevent leakage of the compositions to be used according to the invention on the side of the plaster facing away from the tissue surface. For high oil content compositions, e.g. Sheets of thermoplastic materials, such as e.g. Polyethylene terephthalate, polyethylene, polyvinyl chloride or polypropylene use find. However, it is sufficient to protect only the area of the patch, which is the outer boundary of the absorbent material, with an impermeable barrier, while other portions of the cover are air and fluid permeable.

The dosage of the complexed nucleic acid can be effected via the volume of the organic solvent and via the concentrations of the nucleic acid in the solvent. By failure of suitable dimensions of the patch, the surface to be treated, for example the skin surface, can be agreed. The administration of the compositions according to the invention by means of a plaster also has the advantage that abrasion or smearing of the applied composition is prevented. Thus, it can be ensured that the complexed nucleic acid can actually act on the area of the skin to be treated for the predetermined period of time.

By using patches, the tissue surface to be treated can be precisely adjusted. The patch may be targeted to the surface to be treated by selecting a suitable size of the absorbent material which will release the complexed nucleic acid. Thus, when using a patch, other mammalian body surfaces not intended for treatment with the compositions of the invention will be prevented from contacting the complexed nucleic acids.

An advantage of using patches to apply the compositions of the present invention is the ability to prefabricate large numbers of patches and store them under suitable conditions (e.g., at 4 ° C or at room temperature). Such prefabricated patches can be used quickly and easily when needed. Methods of making suitable patches are described, for example, in U.S. Patents 4,983,395 and 4,849,224.

Until used, the patches can be sealed in a tight-fitting material. As a result, contamination of the nucleic acid-containing compositions or other undesirable changes, e.g. by evaporation, be prevented. In this form, the plasters are easily stable for several months, in particular for up to about 6 months, 12 months, 18 months, 24 months, 30 months or 36 months, without significant changes in the nucleic acid-containing compositions.

Specific embodiments

Specific embodiments of the present invention include in particular:

A (pharmaceutical or cosmetic) composition comprising at least one organic solvent in which dissolved complexes of at least one nucleic acid and CTAB cations are present (wherein the nucleic acid is preferably complexed such that the negative charges of the nucleic acid by CTAB cations substantially fully compensated) for use in introducing nucleic acid into human cells or tissue. A (pharmaceutical or cosmetic) composition comprising at least one organic solvent in which dissolved complexes of a nucleic acid and at least one organic cation are present (wherein the nucleic acid is preferably complexed such that the negative charges of the nucleic acid by the positive charges of the organic Cations are substantially completely compensated) for use in introducing nucleic acid into cells of the human skin, in particular for introducing the nucleic acid into the cell nuclei of cells of the epidermis, dermis and / or subcutis.

A (pharmaceutical or cosmetic) composition comprising at least one organic solvent in which dissolved complexes of at least one nucleic acid and at least one organic cation are present, wherein the nucleic acid is siRNA or miRNA (wherein the nucleic acid is preferably complexed in that the negative charges of the nucleic acid are substantially completely compensated by positive charges of the organic cations, for use in the introduction of nucleic acid into cells of the human skin, whereby the nucleic acid enters the cell nuclei of cells of the epidermis, dermis and / or subcutis.

A pharmaceutical or cosmetic composition comprising at least one organic solvent in which dissolved complexes of at least one nucleic acid and CTAB cations are present, wherein the nucleic acid is siRNA or miRNA, which is preferably complexed such that the negative charges the nucleic acid is substantially completely compensated by CTAB cations, for use in the introduction of nucleic acid into cells of the human skin, in particular for introducing the nucleic acid into the cell nuclei of cells of the epidermis, dermis and / or subcutis.

Brief description of the figures

Fig. 1 shows the thin section of human skin after treatment with fluorescent oligonucleotides from Example 3. In Fig. 1, the cell nuclei by staining with Bisbenzimid shown and were visible in the blue channel of the fluorescence microscope (light areas) ..

Fig. 2 shows the same section as Figure 1, wherein the green channel is shown. The green fluorescence (bright areas) indicate the fluorescence of the applied FAM oligonucleotide. The fluorescence is observed on the surface of the skin (right margin) in the underlying cells. The bright areas behind the skin surface coincide with the areas of the cell nuclei in FIG. 1.

Fig. 3 shows the longitudinal section of a hair from a skin portion of a mouse after treatment with fluorescent oligonucleotides from Example 9. Shown are the cell nuclei by staining with bisbenzimide (Figure 3A) as well as the green fluorescence (bright areas) of the applied FAM oligonucleotide on (Figure 3B).

4 shows the cross section of a hair from a section of the skin of a mouse after treatment with fluorescent oligonucleotides from Example 9. Shown are the cell nuclei by staining with Bisbenzimid (Figure 4A) and the green fluorescence (bright areas) of the applied FAM oligonucleotide ( Figure 4B).

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

Preparation of 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 / mole) was labeled with the fluorescent dye TAMRA (oligonucleotide 1). Oligonucleotide 2 (molecular weight 5938.82 g / mole) having the same sequence was labeled with the fluorescent dye fluorescein 6FAM to allow detection of the oligonucleotide in the skin cells.

The oligonucleotides were obtained from Biolegio (6545 CG Nijmegen, The Netherlands).

Example 2

For the preparation of CTAB oligonucleotide complexes, 1 mg of the oligonucleotides were dissolved in 200 .mu.l water and mixed with 400 .mu.10m CTAB. The sample was incubated on ice for 30 min and the resulting precipitate was centrifuged for 5 min at 10,000 xg. The supernatant was removed and the precipitate was suspended with 200μΙ water and centrifuged again. The precipitate was dried overnight in the Speedvac. Example 3

Introducing DNA into human skin cells

After drying, the CTAB-nucleic acid complexes prepared in Example 2 were weighed and ethylene glycol monoethyl ether (EGE) or butanol was taken up in the organic solvent, the final concentration of the complexed nucleic acid being approximately 1 mg / ml. The CTAB-nucleic acid complexes dissolved in ethylene glycol monoethyl ether were then mixed with almond oil in a volume ratio of 9: 1 ecethylene glycol monoethyl ether (90% (v / v) oil, 10% (v / v) EGE). The final concentration of the complexed nucleic acid in the respective mixture was thus about 100 μg / ml.

Each 5 μΙ of the above mixture of oil and EGE or butanol was applied to pieces of non-woven paper (2 mm x 2 mm, Whatman) having a thickness of 1 mm. The nonwoven paper pieces soaked with the nucleic acid-containing solvent were applied to human skin fragments (about 1 cm x 4 cm) obtained from a fresh abdominal operation. The pieces of paper were fixed separately in different areas of the skin fragment with adhesive tape. As a control, a mixture of 90% (v / v) oil, 10% (v / v) EGE, and butanol was used.

The appropriately prepared skin fragment was transferred to a Petri dish with 5 ml Ringer's solution and incubated overnight at 37 ° C in the C0 ₂ -lnkubator. Subsequently, the skin fragment was carefully dissected with a scalpel so that partial fragments were obtained, each having a nonwoven paper on their surfaces. The partial fragments were frozen in liquid nitrogen for further storage. The frozen partial fragments were used for the production of thin sections with a thickness of about 8 pm. A nuclear staining with bisbenzimide (5 min, 1 μg / ml bisbenzimide in PBS) was carried out by fluorescence microscopy.

The results of fluorescence microscopy are shown in Figures 1-2. The fluorescence emitted by the labeled oligonucleotides could be detected in the epithelial cells as well as in the underlying connective tissue cells. The cell nuclei also showed fluorescence, indicating that the labeled oligonucleotides were able to penetrate the cell nuclei of the skin cells. FIG. 1 shows a fluorescence micrograph of a thin section of a skin fragment treated as described above. Clearly visible are the cell nuclei visualized by bisbenzimide staining, which appear as blue colored areas. FIG. 2 shows the same thin section as FIG. 1, in which green channel (fluorescence of the FAM oligos) is shown. The bright areas show the fluorescent oligonucleotide. It can be seen that the bright areas coincide with the blue areas of FIG. This correspondence shows that the fluorescent oligonucleotides were able to penetrate into the cell nuclei of the skin cells.

Skin fragments treated with TAMRA-labeled oligonucleotide showed a corresponding red fluorescence of the cell nuclei. Skin fragments that had been treated only with the control (without oligonucleotide) showed no fluorescence of nuclei in the green or red channel.

Example 4

Introduction of DNA into skin cells of mice

CTAB complexes of oligonucleotide 1 were prepared as in Example 2 and dissolved in ethylene glycol monobutyl ether (EGE) to a final concentration of approximately 1 pg / μl. Subsequently, the solvent containing the complexes was diluted 1:10 with almond oil (90% (v / v) oil, 10% EGE) to give a final concentration of 100 pg / ml CTAB nucleic acid.

Mice were prepared for dermal application of the composition by shaving a fur area in the neck. Once 50μΙ of the composition was applied to the shaved neck area.

The preparation of the treated skin area was carried out after 0, 1, 2 or 3 days. The preparations were frozen in liquid nitrogen and thin sections approximately 8 μm thick were prepared for fluorescence microscopy. The nuclear staining was carried out as described in Example 3 with bisbenzimide. Evaluation of the fluorescence microscopy showed after 3 days cell nuclei of epidermal cells, which appeared green by the fluorescein-labeled oligonucleotide. Example 5

Introduction of DNA into murine skin cells

CTAB complexes of oligonucleotide 1 were prepared as in Example 2 and dissolved in to give a final concentration of about 1 β Ι in butanol. Subsequently, the solvent mixture containing the complexes was diluted 1:10 with almond oil or walnut oil (90% (v / v) oil, 10% butanol to give a final concentration of CTAB-nucleic acid complexes of 100 g / ml.

Mice were prepared for dermal application of the composition by shaving a fur area in the neck. In each case 40 μΙ of the composition was applied once a day to the shaved neck area for three days.

The preparation of the treated skin area was done after 3 days after the last treatment. The preparations were frozen in liquid nitrogen and thin sections approximately 8 μm thick were prepared for fluorescence microscopy. The nuclear staining was carried out as described in Example 3 with bisbenzimide. The evaluation of the fluorescence microscopy showed after 3 days cell nuclei of epidermal cells and of cells of the hair follicle and bulge region, which appeared green by the fluorescein-labeled oligonucleotide.

Example 6

Introduction of DNA into murine skin cells

CTAB complexes of oligonucleotide 1 were prepared as in Example 2 and dissolved in ethylene glycol monobutyl ether (EGE) to a final concentration of approximately 0.5 pg / μΙ. Subsequently, the solvent containing the complexes was mixed 1: 1 with almond oil (50% (v / v) oil, 50% EGE) to give a final concentration of CTAB-nucleic acid complexes of 250pg / ml.

Mice were prepared for dermal application of the composition by shaving a fur area in the neck. In each case 40 μΙ of the composition was applied once a day to the shaved neck area for three days. The preparation of the treated skin area was done after 3 days after the last treatment. The preparations were frozen in liquid nitrogen and thin sections approximately 8 μm thick were prepared for fluorescence microscopy. The nuclear staining was carried out as described in Example 3 with bisbenzimide. Evaluation of fluorescence microscopy revealed cell nuclei of epidermal cells and cells of the hair follicle and bulge region, muscle cells, fat cells, connective tissue cells and capillary cells which appeared green by the fluorescein-labeled oligonucleotide.

Example 7

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

200 μl of 1% (w / v) salmon sperm DNA - equivalent to 2 mg - in water were poured into a 2 ml 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 means of 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 8

Preparation of Lipophilic Nucleic Acid Complexes with Castor Oil

5 mg of the fluorescent DNA oligonucleotide 2 was dissolved in 1 ml of water and mixed with 300 μl of 100 mM cetyltrimethylammonium bromide solution. The result was a yellowish turbidity, which was precipitated by centrifugation at 10,000 xg for 5 min. The supernatant was discarded and the precipitate suspended in 1 ml of water and again centrifuged as above. The supernatant was discarded and the precipitate dried under vacuum. The precipitate was taken up in 1 ml of ethanol. The result was a yellow, clear solution. 100μΙ of the fluorescent DNA was mixed in a plastic tube with 1 ml of castor oil. The result was a homogeneous, viscous fluid containing 90% (v / v) rinin citrus oil and 10% (v / v) ethanol and dissolved DNA.

Example 9

Incorporation of fluorescent DNA into hair cells by application of the lipophilic nucleic acid complexes to the skin of mice

Three mice (C27 / Black) were prepared for dermal application of the composition by shaving a fur area in the neck. In each case 10 .mu.Ι, 20 .mu.Ι and 40 .mu.Ι of the composition according to Example 8 were applied to the shaved neck area for each mouse. After three Days were the mice killed and the treated skin areas cut out. As a control skin areas were used, which were not treated with nucleic acid complexes. The preparations were fixed by overnight incubation at 4 ° C in 10% (w / v) formaldehyde in phosphate buffered saline (PBS) and frozen in liquid nitrogen. From the skin areas, thin sections approximately 8 μm thick were made on slides for fluorescence microscopy. Nuclear staining was carried out by incubation for 5 minutes in 2 μg / ml bisbenzimide in PBS. The thin sections were coated with Fluoromount and examined by fluorescence microscopy. The thin sections of the areas treated with fluorescent DNA oligonucleotide 2 showed a green fluorescence within the skin which was detectable particularly in the cell nuclei. No fluorescence was observed in thin sections of untreated skin areas. Figures 3 and 4 show in the longitudinal (Figure 3) and cross-section (Figure 4) hair, wherein the cell nuclei of hair cells were colored green by the fluorescein-labeled oligonucleotide 2. Hair cells from areas that were not treated with fluorescent DNA did not show any green fluorescence within the cells.

Example 10

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 11

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 600μΙ 10mM Cetyltrimethylammonium bromide solution mixed. The result was a white turbidity, which was deposited by centrifugation for 5 min at 10,000 xg. 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 12

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 13

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 unshaven head or back skin, each 20 μl of the siRNA-1 dissolved in dimethyl sulfoxide / propylene glycol (samples 1 d, 1 f and 1 g from Example 1 2). 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 1 2). As further controls, uncomplexed siRNA-1 was immediately dissolved in sterile TE (10 mM TrisCl, pH 8, 2 mM EDTA) or TE with 20% (v / v) dimethylsulfoxide to a final concentration of siRNA-1 of 2pg / μl and 20μΙ of each Solution also applied to each one mouse. 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 variation was compared with the siRNA-1 (tyrosinase-specific) treated mice observable to the control group. Over the entire period, the mice were vital and showed no pathological changes.

Example 14

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 fixed overnight 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 . A pharmaceutical and / or cosmetic composition comprising at least one organic solvent in which complexes of at least one nucleic acid and at least one organic cation are present in solution, for use in a method of therapeutic treatment of the human or animal body or for use in a diagnostic method on the human or animal body, the method comprising the introduction of the nucleic acid into cells or tissues of the human or animal body, in particular the introduction into cells or tissue of a mammal.

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 at least one organic cation in Essentially 1: 1.

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 pg / ml, more preferably more than 100 g / 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. A composition according to any one of the preceding claims further comprising at least one synthetic, vegetable or animal oil; and / or synthetic, vegetable or animal fat; and / or synthetic, vegetable or animal wax.

A composition according to claim 5 which has an oil, wax or fat content of 30% or more, preferably 50% or more, more preferably 80% or more.

A composition according to any one of the preceding claims wherein the organic solvent is selected from one or more members of the group consisting of:

a) alcohol, such as ethanol, 1-butanol, 2-propanol, 1-pentanol,

b) DMSO, or

c) glycol ethers, e.g. Ethylene glycol.

8. The composition according to claim any one of the preceding claims, wherein the at least one solvent is mixed with at least one further organic solvent.

9. Composition according to one of the preceding claims, wherein the at least one organic solvent in a concentration of less than 70% (v / v), preferably, less than 50% (v / v), more preferably less than 20% (v / v) is present.

Composition according to any one of the preceding claims, wherein the organic solvent is present in a concentration of more than 1% (v / v), preferably more than 5% (v / v), more preferably more than 10% (v / v) ,

A composition according to any one of the preceding claims, wherein the water content in the composition is less than 50% (v / v), preferably less than 30% (v / v), more preferably less than 10% (v / v).

12. The composition according to any one of the preceding claims, wherein the average diameter of the nucleic acid-organic cation complexes in the composition is less than 200 nm, preferably less than 10 nm, more preferably less than 50 nm, most preferably less than 10 nm.

13. The composition according to any one of the preceding claims, wherein the complexes are present dissolved in a homogeneous, coherent phase.

14. A composition according to any one of the preceding claims, wherein the complexes are not present within liposomes.

Composition according to any one of the preceding claims, wherein the cells are cells of the epidermis, dermis and / or subcutis.

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

7. The composition according to claim 1, wherein the nucleic acid is DNA, RNA, or DNA / RNA hybrids, in particular wherein the nucleic acid is DNA, RNA, or DNA / RNA hybrids of a size of 1. 100 b or bp acts.

18. A transdermal patch comprising a composition according to any one of claims 1-7.