EP2765982A1 - Diméthylsulfoxyde utilisé comme solvant pour des acides nucléiques - Google Patents

Diméthylsulfoxyde utilisé comme solvant pour des acides nucléiques

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Publication number
EP2765982A1
EP2765982A1 EP12772077.9A EP12772077A EP2765982A1 EP 2765982 A1 EP2765982 A1 EP 2765982A1 EP 12772077 A EP12772077 A EP 12772077A EP 2765982 A1 EP2765982 A1 EP 2765982A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
dmso
complexes
composition
composition according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP12772077.9A
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German (de)
English (en)
Inventor
Marcus WEICHERT
Hans Kosak
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Individual
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Individual
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Priority to EP12772077.9A priority Critical patent/EP2765982A1/fr
Publication of EP2765982A1 publication Critical patent/EP2765982A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/541—Organic ions forming an ion pair complex with the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • A61K8/41—Amines
    • A61K8/416—Quaternary ammonium compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/46—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing sulfur
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/04—Preparations for care of the skin for chemically tanning the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/24—Vectors characterised by the absence of particular element, e.g. selectable marker, viral origin of replication

Definitions

  • 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.
  • DMSO dimethyl sulfoxide
  • 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.
  • nucleic acid-based drugs typically exert their action inside cells (e.g., in the cytosol or in the nucleus).
  • cells e.g., in the cytosol or in the nucleus.
  • diffusion of the nucleic acid through the cell membrane, which confines the cells to the outside takes place only to a very limited extent.
  • 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.
  • 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.
  • 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.
  • these systems are unsatisfactory in their efficiency since only small amounts of nucleic acid are taken up by the corresponding target cells.
  • nucleic acid which are complexed in a certain way with organic cations, are outstandingly soluble in dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • 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.
  • the present invention thus relates in a first aspect to a pharmaceutical or cosmetic composition
  • a pharmaceutical or cosmetic composition comprising:
  • DMSO dimethyl sulfoxide
  • 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.
  • 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.
  • nucleic acids which are otherwise insoluble in organic solvents, in high amounts in DMSO.
  • Nucleic acids e.g. DNA or RNA
  • DMSO dimethyl methacrylate
  • nucleic acids dissolved in DMSO can be effectively absorbed into cells and / or tissue, e.g. of a mammal.
  • the dermal or transdermal incorporation of the complexed nucleic acids according to the invention 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.
  • 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.
  • 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.
  • 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.
  • water eg demineralized water
  • Such a method may include the following steps:
  • such a method may comprise the following steps:
  • compositions may, of course, be done at any convenient time during the process.
  • Suitable organic cations in the context of the present invention are, for example, cationic detergents, lipids and nitrogen compounds with quaternary nitrogen, for example organic ammonium salts.
  • 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.
  • the hydrocarbon radical preferably contains only C and H atoms.
  • 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.
  • 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.
  • 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.
  • 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).
  • R does not comprise aromatic and / or non-aromatic ring systems
  • the at least one organic cation is one or more quaternary amines, with CTAB being particularly preferred.
  • CTAB is one or more quaternary amines
  • 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-prop
  • the complexing agent is added to the first liquid in step b) in dissolved form.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • R 1 and R 2 are each a hydrocarbon radical having 1 to 100 carbon atoms.
  • 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.
  • 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.
  • saturated or unsaturated hydrocarbons for example benzene-based aromatic hydrocarbons, such as styrene, ie in particular benzenes substituted by C1 to C3 radicals.
  • halogen-containing solvents such as chloroform, dichloromethane or carbon tetrachloride or other heteroatom-containing solvents such as dimethylformamide or tetrahydrofuran may be used.
  • the composition of the invention does not comprise halogenated hydrocarbons.
  • solvent mixtures of DMSO with ethanol, glycerol, ethylene glycol and / or propylene glycol are particularly preferred.
  • 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.
  • a high concentration of DMSO may be undesirable, as higher concentrations may cause skin irritation.
  • the compositions of the invention 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
  • 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.
  • 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.
  • RNA ribonucleic acid
  • 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.
  • compositions of the present invention are excellently formulated as cosmetic and / or therapeutic compositions.
  • 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.
  • 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.
  • the nucleic acid may be a diagnostically useful nucleic acid, for example a so-called molecular beacon.
  • 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.
  • nucleic acid will not have sequences of a (human) genome or (human) gene.
  • nucleic acids can be used as a source of allantoin.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • lactose 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.
  • 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.
  • humectants are e.g. Water, ethanol, isopropanol, glycerin, propylene glycol, acetone, glycerol triacetyl ester, oleyl oleyl ester, isopropyl myristate, sorbitol, etc.
  • 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 s
  • solubilizers are, for example, polyethylene glycols (PEG, macrogols), polyethylene oxides (PEO), polysorbates, sodium dioctylsulfosuccinate (and potassium or calcium salts thereof), etc.
  • wetting agents are, for example, polyethylene glycols (PEG, macrogols), polyethylene oxides (PEO), polysorbates, sodium dodecylsulfate, sodium cetylstearylsulfate, etc.
  • antifoaming agents are e.g. Dimethyl polysiloxanes, etc.
  • salt formers are e.g. Tris (hydroxymethyl) aminomethane, tris (hydroxyethyl) amine, etc.
  • buffers are e.g. Sodium dihydrogen phosphate, tris (hydroxymethyl) aminomethane, citric acid, tartaric acid, glycine, sodium bicarbonate, etc.
  • gelling agents are e.g. Pectin, tragacanth, polyacrylic acids, polyvinylpyrrolidone, (fumed silica), carboxymethylcellulose, etc.
  • thickening agents are e.g. Pectin, tragacanth, polyacrylic acids, polyvinylpyrrolidone, fumed silica, carboxymethylcellulose, gelatin, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylalcohol, bentonite, etc.
  • film formers are e.g. Ethyl cellulose, hydroxypropyl methyl cellulose phthalate, cellulose acetate phthalate, polyacrylates, etc.
  • 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.
  • Starches e.g., corn starch, rice starch, potato starch, or wheat starch
  • tragacanth e.g., tragacanth
  • cellulose e.g., cellulose, cellulose ethers (methylcellulose, ethylcellulose, hydroxypropylcellulose, carboxymethylcellulose), polyvinylpyrrolidone, sucrose, mannitol, gelatin, calcium hydrogen phosphate, gum arabic, polyethylene glycol, etc.
  • 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, hydroxypropylcellulose,
  • 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.
  • adsorbents are e.g. Bolus alba (kaolin), (fumed silica), etc.
  • lubricants are e.g. Polyethylene glycols (PEG, macrogols), polyethylene oxides (PEO), talc, stearic acid (e.g., magnesium stearate), etc.
  • lubricants are e.g. Polyethylene glycols (PEG, macrogols), polyethylene oxides (PEO), talc, stearic acid (e.g., as magnesium stearate), dimethylpolysiloxanes, etc.
  • mold release agents are e.g. Polyethylene glycols (PEG, macrogols), polyethylene oxides (PEO), talc, stearic acid (e.g., as magnesium stearate), dimethylpolysiloxanes, etc.
  • flow control agents are e.g. (fumed silica), etc.
  • antioxidants are e.g. Butylhydroxytoluene, all-rac-a-tocopherol, etc.
  • 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.
  • sweeteners are e.g. Sucrose, sorbitol, sweeteners such as saccharin sodium and cyclamate; Flavors, aspartame, fructose, lactose, glucose, mannitol, xylitol, etc.
  • flavor correcting agents are e.g. Sucrose, sorbitol, sweeteners such as saccharin sodium and cyclamate; Flavors, etc.
  • scent remedies are usually fragrances.
  • colorants eg titanium dioxide.
  • Another conceivable adjuvant for the compositions of the present invention is urea.
  • 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.
  • the excipient is an oil; a wax; and / or a fat.
  • 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.
  • suitable waxes or fats comprise synthetic, animal and / or vegetable waxes or synthetic, animal and / or vegetable fats.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the complexed nucleic acids are first dissolved in DMSO.
  • the nucleic acid-containing DMSO solution is mixed with one or more oils to form a liquid comprising complexed nucleic acids, DMSO and oil.
  • the nucleic acid-containing DMSO may be dissolved in the oil or form an emulsion with the oil.
  • 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.
  • 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.
  • CTAB quaternary ammonium compounds
  • the organic cations complexed with the nucleic acid are not identical to the pharmaceutically or cosmetically acceptable adjuvant.
  • 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.
  • plastics in particular, no synthetic artificial (i.e., non-naturally occurring) organic polymers such as those described e.g. from materials technology.
  • biological polymers such as nucleic acids are not covered by this.
  • 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.
  • compositions according to the invention preferably do not comprise sunflower oil, rapeseed oil, wheat germ oil, olive oil and / or coconut fat.
  • castor oil is used as the DMSO miscible oil.
  • 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.
  • 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.
  • DMSO dimethyl sulfoxide
  • nucleic acid to be introduced into the cells or tissue according to the invention is a charged nucleic acid.
  • 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.
  • 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.
  • 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.
  • 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.
  • the nucleic acid is not siRNA.
  • the nucleic acids may contain at least part of a sequence of a (human) genome or gene.
  • the nucleic acids do not contain a sequence of a (human) genome or gene.
  • 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.
  • the viral vectors are those that are no longer able to replicate in the cells transfected therewith.
  • 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.
  • 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.
  • 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.
  • 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.
  • Epithelial tissue e.g. Epithelial tissue, connective tissue and muscle tissue.
  • the introduction into the skin in particular in the epidermis, dermis and / or subcutis is preferred.
  • introduction via other types of epithelium e.g., intestinal epithelium
  • 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.
  • 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.
  • the composition is applied to the cells and / or tissues, e.g. by dripping or painting.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • nucleic acid can be introduced by using a composition of the invention, for example, in tissue cells of the epidermis, dermis and subcutis.
  • compositions 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.
  • 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,
  • 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,
  • 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
  • / 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.
  • 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:
  • composition of the present invention a composition of the present invention with cells and / or tissue (s) of a patient.
  • the patient can be a human or an animal.
  • the animal may be a mammal.
  • Fig. 1 shows the solubility of CTAB DNA in DMSO / water mixtures of different ratios.
  • 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.
  • 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.
  • Fig. 4 thin layer of the mouse skin after treatment with FAM-oligo in DMSO. Three days after application of 10 ⁇ 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.
  • CTAB DNA particle size 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.
  • 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%.
  • 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 Berlinberg).
  • oligonucleotides were intended for transdermal administration to human skin or mouse skin.
  • 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).
  • oligonucleotide complexes For the preparation of CTAB oligonucleotide complexes, 1 mg of the oligonucleotides were dissolved in 200 ⁇ water and mixed with 400 ⁇ 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 200 ⁇ water and centrifuged again. The precipitate was dried overnight in Speedvac and then taken up in 200 ⁇ DMSO. Assuming 100% precipitation efficiency, the DMSO-CTAB oligonucleotide solution contains 5 ⁇ l / ⁇ oligonucleotide.
  • 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.
  • mice were prepared for dermal application of the compositions by shaving a fur area in the neck. In each case 10 ⁇ 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 ⁇ m 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.
  • siRNAs were obtained as double-stranded products with 3 'deoxythymidine overhangs (dTdT) from Eurofins MWG Operon (85560 Ebersberg, Germany).
  • siRNA-1 tyrosinase-specific
  • siRNA-2 nonspecific control
  • siRNA oligonucleotide SEQ ID NO: 2 (tyrosinase-specific) and 2 (nonspecific control; SEQ ID NO: 3) were each dissolved in 200 ⁇ water and mixed with 600 ⁇ 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 200 ⁇ water and again centrifuged as above. The supernatants were discarded and the precipitates dried under vacuum. 200 ⁇ ethanol was added to the precipitates and the suspension was vortexed. The suspension remained cloudy. In 200 ⁇ steps more ethanol was added and mixed.
  • the complexed siRNA was completely dissolved.
  • 50 ⁇ , 10 ⁇ , 200 ⁇ of the complexed siRNA in ethanol were mixed with 950 ⁇ , 900 ⁇ and 800 ⁇ castor oil (total volume 1 ml each) by pipetting up and down. Clear solutions were generated with siRNAs.
  • siRNA oligonucleotide 1 SEQ ID NO: 2
  • 2 SEQ ID NO: 3
  • SEQ ID NO: 3 2 mg
  • the precipitate was taken up in 200 ⁇ dimethyl sulfoxide. 10 ⁇ , 20 ⁇ and 50 ⁇ of the complexed siRNA in dimethyl sulfoxide were mixed with 90 ⁇ , 80 ⁇ and 50 ⁇ propylene glycol (total volume 100 ⁇ each). Clear solutions with dissolved siRNAs were generated.
  • 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 ⁇ l of the siRNA-1 dissolved in dimethyl sulfoxide / propylene glycol (samples 1 d, 1 f and 1 g from example 12).
  • 20 ⁇ 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.
  • mice were in the same places per 20 ⁇ 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).
  • 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 ⁇ M each of this solution applied to one mouse each.
  • mice were kept ad libitum with water and dry diet.
  • 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.
  • the hair grew again with siRNA-1 (tyrosinase-specific), so that the effect was completely abolished after approx. 7 weeks after application.
  • 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.
  • tyrosinase protein in the skin of the treated mice was examined in thin sections using anti-tyrosinase antibodies.
  • 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.
  • 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 ⁇ m 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 ⁇ g / 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.

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Abstract

La présente invention concerne des compositions pharmaceutiques ou cosmétiques comprenant des complexes, constitués d'au moins un acide nucléique et d'au moins un cation organique, présents sous forme dissoute dans du diméthylsulfoxyde (DMSO), et comprenant au moins un adjuvant pharmaceutiquement ou cosmétiquement acceptable. La présente invention concerne également l'utilisation de ces compositions pour l'introduction d'acides nucléiques dans des cellules et/ou des tissus humains par exemple, ainsi que des procédés pour la préparation de ces compositions.
EP12772077.9A 2011-10-11 2012-10-11 Diméthylsulfoxyde utilisé comme solvant pour des acides nucléiques Withdrawn EP2765982A1 (fr)

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EP2508530A1 (fr) 2011-03-28 2012-10-10 Rheinische Friedrich-Wilhelms-Universität Bonn Purification d'oligonucléotides triphosphorylés au moyen d'étiquettes de capture
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BR112020011670A2 (pt) 2017-12-15 2020-11-17 Flagship Pioneering Innovations Vi, Llc. composições compreendendo polirribonucleotídeos circulares e seus usos
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