WO2011039144A1

WO2011039144A1 - Substances containing disulfide groups for self-assembly carriers for controlled release of an active ingredient

Info

Publication number: WO2011039144A1
Application number: PCT/EP2010/064272
Authority: WO
Inventors: Holger Egger; Axel Eble; Juergen Liebscher; Andreas Herrmann; Martin Loew; Anna Arbuzova; Nicolai Brodersen
Original assignee: Bayer Technology Services Gmbh
Priority date: 2009-09-29
Filing date: 2010-09-27
Publication date: 2011-04-07
Also published as: DE102009043342A1; EP2482801A1

Abstract

The present invention relates to novel substances for production of self-assembly active ingredient carriers in the form of a liposome comprising a disulfide group for controlled release of an active ingredient present therein, and to a method for releasing active ingredients using the aforementioned carrier.

Description

DISULPHIDE GROUPS CONTAINING SUBSTANCES FOR SELF-ORGANIZED SUPPLEMENTS UR CONTROLLED RELEASE OF AN ACTIVE SUBSTANCE

The present invention relates to novel substances for the preparation of self-assembled drug carriers in the form of a liposome comprising a disulfide group for the controlled release of a drug contained therein, as well as a method for the release of drugs using the aforementioned carrier.

Drug release drug delivery systems based on liposome carriers of about 100 nm in size are generally known in the medical arts. They are generally regarded as advantageous if a substance which is toxic to certain organs but which can be used as a medicinal agent in other places of the human body should only be released at those desired sites.

Furthermore, in the course of transport of the active ingredient to the site of action, decomposition or inactivation of the active ingredient may occur, for example, by degradation or attack by the immune system, so that such carrier systems are also to be regarded as advantageous in this regard if they are on the way to Protect site of action from premature release or decomposition. Among the aforementioned liposome carriers is generally understood a substance group consisting of non-toxic phospholipids. Frequently such polymers, such as polyethylene glycols, which are characterized by improved stability in the circulation of treated patients in such liposome carriers, since the aforementioned polyethylene glycols form a form of steric protection around the actual liposome carrier. This gives so-called lipopolymers.

The aforementioned statements regarding liposome carriers are also described by J. Davidsen et al. in "Secreted phospholipase A2 as a novel enzymatic trigger mechanism for localized liposomal drug release and absorption in diseased tissue" published in Biochimica et Biophysica Acta 1609 (2003) 95 - 101. Further, J. Davidsen et al Drug carrier in the form of the above-mentioned liposome carrier can depend in particular on their release behavior of a variety of chemical and physical parameters whose interaction is not fully understood.

J. Davidsen et al. In this regard, they disclose their studies on a liposome formulation consisting of a 100 nm diameter liposome carrier. The liposome carrier can be prepared from l, 2-hexadecanoyl-sn-glycero-3-phosphocholine (DPPC) and / or 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (DPPE - PEG2000) and / or 1-0-hexadecanoyl-2-hexanodecnoyl-sn-glycero-3-phosphocholine (1-O-DPPC) and / or 1 -0-hexadecanoyl-2-hexanodecnoyl-sn-glycero-3-phospho ethanolamine-N- [methoxy (Polyethylene glycol) -350] (1-O-DPPE-PEG350). The aforementioned substances are referred to as lipids, which can also form multilamellar vesicles and which belong to the substance group of phospholipids.

It is further disclosed that in the liposome formulation comprising 1-O-DPPC and 10 mol% of 1-O-DPPE-PEG350, calcein can be included. Calcein is referred to as a model substance for drugs. Release of calcein may be as disclosed by J. Davidsen et al. by treatment of this liposome formulation with phospholipase A2 (PLA2). Release is by hydrolytic cleavage of either DPPC or 1-O-DPPC, or 1-O-DPPE-PEG350, which is catalyzed by PLA2 to give a lysolipid and a fatty acid.

Thus, J. Davidsen et al. no cleavage into a hydrophobic and a hydrophilic portion. In particular, J. Davidsen et al. not that the liposome carrier can comprise a disulfide bond.

By Meng et al. is disclosed in "Reduction-sensitive polymers and bioconjugates for biomedical applications" published in Biomaterials 30 (2009) 2180-2198 that certain polymeric materials having disulfide bridges either in the polymeric strand, or in one of its side chains, are suitable to entrap agents However, substances always have a polymeric character as they are formed by particles, ie solids, in which the potential active substances are to be included, so that they are in no case substances with a lipid character which would be suitable for the production of liposomes.

The disclosed mode of action of the materials is based on the possibility of bond cleavage of the disulfide group, which either dissolves the polymers or separates the polyethylene glycol side chains attached to the polymers with the disulfide group, thereby recognizing and degrading the particle after its application in the patient becomes. Accordingly, the release from the drug carrier according to the disclosure of Meng et al. either upon dissolution of a solid by cleavage of polymeric bonds or through degradation of the solid by the patient's immune system after the particle has become recognizable to them. In no case will the drug be released directly. In WO 2000/059474 AI lipid compositions are disclosed which consist of a hydrophobic "tail group" and a hydrophilic "head group", wherein the latter hydrophilic "head group" is covalently attached to the hydrophobic "tail group". Within the hydrophilic "head group" a distinction is made between a first and a second region, wherein these two regions are linked by a disulfide group which can be cleaved, such as by glutathione. The first region of the hydrophilic "head group" carries a positive charge at physiological pH according to WO 2000/059474 AI and the second region carries a negative charge at physiological pH. The first region is covalently attached to the hydrophobic "tail group".

The chemical formula of the abovementioned lipid compositions generally disclosed in WO 2000/059474 A1 is X - Y - S - S - Z. Here, the chemical group X forms the abovementioned hydrophobic "tail group" and Y and Z the first or second region According to the further disclosure of WO 2000/059474 A1, in the abovementioned chemical formula, the chemical group X as a hydrophobic "tail group" can be a group which is substantially similar to the glycerol core of a fat and to which the chemical group Y is attached another chemical group W is attached.

The chemical group W can be selected from the list consisting of CHR ₃ , NR ₃ , N ⁺ (R ₃ ) ₂ , O, S, C (O) NH, NH (CO), OC (O) NH and OP (0 ) (OR ₃ ) 0. The chemical group R 3 further contained in the chemical group W may be hydrogen or a C ₁ -C 4 alkyl radical.

The chemical group Y can be a C ₁ -C ₁₂ alkyl radical, a C ₂ -C ₁₂ alkenyl radical or a C ₂ -C ₁₂ alkynyl radical having substituents in the form of alkyl radicals, amino radicals, aminoalkyl radicals, guanidine radicals, guadinealkyl radicals, amido radical or amidoalkyl radical, where the said alkyl, alkenyl, or alkynyl radicals of chemical group Y may be further interrupted by NR ₃ , N ⁺ (R ₃ ) ₂ , C (O), NHC (NH), C (NH) NH, NHC (NH) NH. Alternatively, the chemical group Y may also be an amino acid residue or a peptide.

According to the disclosure of WO 2000/059474 A1, the chemical group Z can be a C ₁ -C ₁₂ -alkyl radical, alkenyl radical or an alkynyl radical which in turn is substituted by alkyl radicals, carboxyl radicals, carboxyalkyl radicals, amino acid radicals, peptides, oligonucleotides or so-called "target molecule radicals" can be.

However, the abovementioned chemical groups X, Y and Z must in any case be designed so that the groups X and Y in their entirety have a positive charge at physiological pH and the group Z has a negative charge at physiological pH. Accordingly, the cleavage of substances of the formula X - Y - S - S - Z on the disulfide group according to WO 2000/059474 A1 results in a group of the type X - Y - S which is positively charged and has a chemical group of the type S - Z, which is negatively charged. In any case, the group of the species X-Y-S, in that it still comprises the "hydrophobic tail group" and a positive charge, is a chemical group having amphiphilic properties, ie, being hydrophobic in one region and hydrophilic in a region The group of the type S - Z is by its negative charge and by the fact that it comprises a C 1 -C 12 alkyl radical, alkenyl radical or an alkynyl radical either also amphiphilic or even - in the case of particularly short alkyl radicals, alkenyl radicals or alkynyl radicals - only hydrophilic ,

WO 2000/059474 AI further discloses that the abovementioned substances are suitable for the preparation of liposome carriers, by means of which a targeted release of substances at one site of action can take place. The release is based on the chemical cleavage of the disulfide group. However, WO 2000/059474 A1 does not disclose that a cleavage of a previously amphiphilic substance into a hydrophilic and a hydrophobic fraction can be achieved.

However, such a division into a hydrophilic and a hydrophobic fraction would allow a particularly efficient destabilization and dissolution of the liposome carrier constructed with such substances. In addition to the compositions described above, it is also well known that specific enzymes in the body of a mammal attack and cleave specific binding of substances. In this connection, for example, D. Mustacich et al. in Biochem. J. (2000) 346, 1-8, "Thioredoxin reductase" that such an enzyme is the thioredoxin reductase (short (TrxRs).) It is further disclosed that this enzyme is present at least ten times more, especially in tumor tissues, than in healthy tissue ,

In addition, specific binding of substances in biological systems can be split by the fact that in the immediate vicinity of the cell environment in which the effect of the drug is to be achieved, the reduction potential changes significantly and suddenly. For example, it is known that glutathione is a reducing agent that is particularly found in the vicinity of tissue in which a significant number of cells are exposed to a stressful situation, such as that caused by disease of the tissue in question.

In the treatment of cancers, it is often necessary to use pharmaceutical agents, which in addition to their primary effect on the tumor tissue also have significant side effects on the healthy tissue of the patient. In the course of such treatments, the patient is often treated with a larger dose of the pharmaceutical agent than would require the actual tumor disease, since it must be ensured that the amount of drug administered to the patient the site of action (the Tumor) is still reached in sufficiently large dosage. So it would be desirable if the drug would be released only at the site of action and would only unfold its effect there. On the one hand, this would lead to a reduction in the dose and, on the other hand, to a lesser negative impact on the patient. However, in many cases it must be ensured at the same time that the release of the active substance at the potential site of action takes place immediately and as quantitatively as possible and not in a creeping manner.

However, the above-mentioned objects are not or only inadequately solved using the substances disclosed in the above-mentioned prior art for the construction of a liposome carrier. It is therefore an object to provide materials for the preparation of excipients in the form of a liposome, e.g. can be cleaved enzymatically on a disulfide group contained in these substances, so that a rapid and complete release of the active ingredients contained is possible. There is the further object to provide a self-organized drug carrier in the form of a liposome using such substances, which can be destabilized as by enzymes and which releases the active substance contained in it quickly and as quantitatively as possible in the cleavage.

Another object is to provide a method for drug release using the aforementioned drug carrier, which makes it possible to produce a medicine which is suitable for the targeted treatment of tumor diseases, for example, by the drug only in the immediate vicinity of the site of action of the human body and as quantitative as possible the medicine is released.

The first subject of the present invention, therefore, substances suitable for the preparation of drug carriers in the form of a liposome according to the formula (I)

characterized in that the substances according to formula (I) have in X on one side of the disulfide group a hydrophilic chemical group comprising at least five carbon atoms and comprising at least one ether group and / or amine group or ammonium group, and wherein (b) on the other side of the disulfide group is a hydrophobic group in which n and m are independently of one another a natural number from 1 to 30, but the sum of n and m is at least 16. The substances according to the invention of the formula (I) are particularly advantageous because they have in the radicals having 1 to 30 carbon atoms chemical groups which have hydrophobic properties and at the same time cause sterically that when introducing the substances according to the formula (I) in a polar medium these substances arrange themselves so that it comes to the formation of spherical structures. This usually results in so-called lipid bilayers, wherein the hydrophilic chemical groups X of the substances according to the formula (I) both outside and inside towards the polar solvent that may also be located inside the aforementioned spherical structures, while the hydrophobic chemical groups Both layers are oriented inside the lipid bilayer. This spherical arrangement can be selectively resolved on the disulfide group also provided in the compounds of the formula (I) by selectively cleaving them so that hereinafter a hydrophilic chemical group consisting of the chemical groups X and a sulfur radical and a hydrophobic chemical group comprising the radicals having 1 to 30 carbon atoms. Thus, for the first time, the separation of a previously amphiphilic substance, suitable for the construction of a drug carrier in the form of a liposome, into a hydrophilic and a hydrophobic moiety via the cleavage of a disulfide bond is possible with the novel substances.

The radicals with n, m carbon atoms according to the formula (I) are usually saturated hydrocarbon radicals, as also shown in the formula (I).

The radicals with n, m carbon atoms can also be monounsaturated or polyunsaturated radicals. Thus, the radicals with n, m carbon atoms can also have one or more double and / or triple bonds.

Accordingly, according to the present invention, the formula (I) is not to be understood in terms of the hydrocarbon radicals having n, m carbon atoms shown therein, in that these radicals are exclusively saturated hydrocarbon radicals with n, m carbon atoms. The chemical group X in preferred embodiments comprises at least eight carbon atoms. It is likewise preferred if the chemical group X also comprises at least one carboxylic acid ester group in addition to the at least one ether group and / or amine group or ammonium group.

It is particularly preferred if the chemical group X comprises either at least two ether groups or at least one amine group or ammonium group and at least two carboxylic acid ester groups.

It is also particularly preferred if the at least one amine group or ammonium group of the chemical group X of the substances according to the formula (I) is a quaternary ammonium group. In such embodiments, the substance according to the formula (I) carries at least one positive charge and is present in combination with at least one single or multiple negatively charged counterion. Such counterions may be, for example, halogen ions, such as chloride, bromide and / or iodide, but also any other counterions with negative charges.

It is very particularly preferred if the chemical group X of the substances according to the formula (I) comprises at least three ether groups. Here, it is preferable that the chemical group X is attached via a carboxylic acid ester group to a chemical group Y according to the further preferred embodiment described below.

Within this very particularly preferred embodiment of the substances according to the formula (I), the chemical group X, which then more preferably contains only one amine group or ammonium group, is also preferably attached via this amine group or ammonium group to the rest of the substance according to the formula (I) , This embodiment is particularly advantageous because the amine group or ammonium group and the at least three ether groups in their entirety result in a particularly hydrophilic property of the chemical group X.

In a further preferred embodiment of the substances according to the formula (I), the chemical group X also comprises a phosphate group. In yet another preferred embodiment of the compounds according to the formula (I), a chemical group Y is attached to the chemical group X, the chemical group Y being a chemical group selected from the list consisting of biotin, protein, peptide, nucleic acid nucleoside and glycoal is. Non-limiting examples of glyco groups according to the present invention are, for example, mannose groups or glucose groups.

The substances of this further preferred embodiment are substances according to the formula (I *).

The chemical group Y is biotin in preferred embodiments of the present invention.

In the preferred embodiment, where the Y chemical group is biotin, it may be attached to the X chemical group by either an amide bond or via a carboxylic acid ester group.

In this further preferred embodiment, the chemical group Y can be present at the chemical group X terminal or as a side group. In all embodiments of the substances according to the formula (I *), the chemical group Y is the (bio) chemical identification of the substances according to formula (I).

By the group Y is recognized in the case of using biotin according to the preferred embodiment (bio) chemically clear by streptavidin, can attach to this another chemical and / or biological group, the substance according to the formula (I *) more gives (bio-) chemical properties. For example, an antibody conjugated to streptavidin may be bound to the biotin which then enables the compound of formula (I *) to preferentially accumulate on target molecules via the action of the antibody.

Particularly preferred substances according to the formula (I *) are those according to the formula (Ia)

or according to the formula (Ib)

In a further very particularly preferred embodiment of the substances of the formula (I *), the chemical group X comprises two carboxylic acid ester groups, a first amine group or ammonium group and a second amine group or ammonium group, so that the chemical group Y to the chemical group X via an amide bond is connected.

Within that further very particularly preferred embodiment of the substances of the formula (I *), the substance according to the formula (Ic)

especially preferred.

In a preferred further development of the substances according to the invention, the chemical group X comprises a further chemical fluorescent dye group X 'as its constituent, which is covalently bound to the chemical group X.

Within this preferred development, the fluorescent fluorescent dye group X 'comprises a dansyl group. This is already present, for example, in the particularly preferred substances according to the formulas (Ib) and (Ic).

Another object of the present invention is a process for the preparation of the substances according to the invention according to the formula (I), which is characterized in that a substance according to the formula

with a substance of the formula

or a substance of the formula

* 1 FG ₃

(IIb), with a substance of the formula

or a substance of the formula

(He), with a substance of the formula

or a substance of formula (IIb) is reacted with a substance of formula (IIIc) in a solvent (LM).

The chemical groups FGi, FG ₂ , FG ₃ , FG ₄ indicated in the above formulas (IIa), (IIIa), (IIb) and (IIIc) are groups selected from the list consisting of hydrogen, halogen, phthalimido, succinimido , a sulfonyloxy group such as benzenesulfonyloxy, tosylate, and triflate.

The chemical groups FGi, FG ₂ and FG ₄ are preferably hydrogen and the chemical group FG ₃ is tosyl. The chemical groups X _! Given in the above formulas (IIb), (IHb), (IIc) and (IIIc) _. and X ₂ are each components of the chemical group X according to formulas (I), (Ia), (Ib) and / or (Ic). The covalent bond between Xi and X ₂ with elimination of FG ₃ in the case of the chemical reaction between the substances of the formula (IIb) and (IIIb) thus leads to the formation of the chemical group X as a constituent of the substances according to the formulas (I), (I *), (Ia), (Ib) and / or (Ic). The same applies in connection with the reactions of substances (IIc) and (IIIc), as well as (IIb) and (IIIc) also for the chemical group FG ₄ .

In preferred embodiments of the process according to the invention, in the chemical group Xi or X _{2 there is} an amine group via which the bond between the chemical groups Xi and X ₂ takes place covalently to obtain the chemical group XCH ₂ .

In the reaction of substances (IIb) and (IIIb), as well as (IIc) and (IIIc) or (IIb) and (IIIc) usually a substance according to formula (I) is obtained which carries a positive charge within the chemical group X. and forms a salt together with the chemical group FG ₃ and / or FG ₄ , which then carries a negative charge by cleavage. In a first further development of the process according to the invention, the substances of the formulas (IIIb) and (IIIc) are obtained from substances of the formula (IIIa) by adding an azodicarbonyl compound, preferably 4-phenyl-1,2,4-triazoline-3,5 -dione to which substance according to the formula (IIIa) is added in a solvent (LM) and thereafter to the resulting reaction solution a thioalcohol, preferably a substance selected from the list consisting of cysteinol, 2-aminoethanethiol and 2-dimethylaminoethanethiol and cysteine, preferably 2-dimethylaminoethanethiol is added.

In the further development of the process according to the invention, it is preferred to carry out the addition of an azodicarbonyl compound at temperatures of 10.degree. C. to 40.degree. C., preferably at room temperature (23.degree. C.) and thereafter the resulting reaction solution for a period of about two hours to 40.degree to warm up to 60 ° C. The addition of a thioalcohol is usually carried out subsequently again at temperatures of 10 ° C to 40 ° C, preferably at room temperature (23 ° C).

It is also preferred within the preferred development of the process according to the invention if, after the above reaction with the addition of an azodicarbonyl compound and subsequent addition of a thioalcohol, a purification step is provided for separating off any by-products formed.

Such purification steps may be, for example, the preparative chromatographic methods well known to those skilled in the art, such as thin film, or column chromatography methods. The solvent (LM) in which the aforesaid reactions are carried out is selected, for example, from the list consisting of acetonitrile (MeCN), halogenated hydrocarbons, non-halogenated hydrocarbons and alcohols, without being limited thereto.

Preferred halogenated hydrocarbons are dichloromethane (DCM) and trichloromethane (TCM). The reaction of the substances (IIa) and (IIIa) or (IIb) and (IIIb) usually takes place at temperatures from room temperature (23 ° C.) to 80 ° C. Preferably at temperatures of 40 ° C to 80 ° C.

In a further development of the method according to the invention, a further chemical fluorescent dye group X 'as its constituent, as defined above, is covalently bound to the chemical group X. This bonding of the chemical fluorescent dye group X 'usually takes place before the reaction of the substances of the formula (IIa) and (IIIa) or (IIb) and (IIIb). It may be necessary, especially when the attachment of the fluorescent fluorescent dye group X 'is carried out after the reaction of the substances of the formula (IIa) and (IIIa) or (IIb) and (IIIb), that contain chemically functional groups in the chemical Group X, such as amino or carboxyl groups must be previously protected with protecting groups prior to reaction with the chemical fluorescent dye group X '. Such a protecting group is, for example, a tert-butyl-diphenyl-silyl radical.

The chemical fluorescent dye group X 'as a constituent of the chemical group X may in the context of the process according to the invention also contain more chemical groups than those which are responsible for the actual fluorescence property. In particular, fractions of the groups contained later in the chemical group X of the substance according to the formula (I) can first be introduced by linking the chemical fluorescent dye group X 'into this chemical group X of the substances according to the formula (I).

The above process according to the invention can be carried out in a preferred embodiment by using substances of the formula (IIa *)

or according to the formula (IIb *)

or according to the (IIc *) Y-

(IIc *) in place of the substances according to the formulas (IIa), (IIb) or (IIc).

Preferred embodiments of the process according to the invention are those in which the substances according to the formula (IIa) or (IIa *) are reacted with those of the formula (IIIa) or in which compounds of the formula (IIb) or (IIb *) be reacted with those of formula (IHb).

These substances are used when the substances according to the formula (I *) are to be obtained as a product of the process according to the invention.

A further subject of the present invention is a self-organized active substance carrier in the form of a liposome, characterized in that this active substance carrier comprises at least one substance according to the formula (I)

wherein the at least one substance according to the formula (I)

(a) in X have a chemical group comprising at least five carbon atoms and comprising at least one ether group and / or amine group or ammonium group, wherein

(b) n and m independently of each other is a natural number from 1 to 30, but where the sum of n and m is at least 16 found.

By the property of the substances according to the formula (I) that they have in the radicals with 1 to 30 carbon atoms chemical groups which possess hydrophobic properties and at the same time lead sterically to the fact that when introducing the substances according to the formula (I) into a polar Medium these substances arrange themselves so that these hydrophobic chemical groups are arranged inside, away from the polar solvent, these form optionally with other substances of the same or similar properties liposomes when introduced into polar solvents, such as water. At the same time, the substances according to the formula (I) are selectively cleavable in such a liposome, but at the envisaged disulfide group, so that hereinafter a hydrophilic chemical group consisting of the chemical group X and a sulfur radical, and a hydrophobic chemical group comprising the radicals with 1 arise up to 30 carbon atoms. In preferred embodiments of the self-assembled active substance carriers in the form of a liposome, this active substance carrier may also contain at least one substance according to the formula (I *)

in which the chemical group Y is a chemical group selected from the list consisting of biotin, protein, peptide, glyco group, nucleic acid, nucleoside or glyco group. In addition to the substances of the formula (I) and / or (I *), the abovementioned active substance carrier according to the invention usually comprises at least one further substance with at least one chemical group having lipophilic properties and at least one chemical group having hydrophilic properties. Such further substances are preferably lipids.

Such lipids are usually those from the class of phospholipids, in particular phosphatidylcholine and / or phosphatidylethanolamine.

Preferred lipids are those selected from the list consisting of La-phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, ammonium (l, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-lisamine rhodamine B sulfonyl).

The aforementioned further substances arrange themselves when introduced into a polar medium in the same way as the substances according to the formula (I).

The active substance carrier according to the invention which comprises the abovementioned further substances usually contains these at a molar proportion of at least 50%, preferably of at least 70%, particularly preferably of 80 to 90%.

In preferred embodiments of the active substance carrier according to the invention, this comprises two lipids as further substances. Within this preferred embodiment of the active ingredient carrier according to the invention, one of the two lipids is preferably La-phosphatidylcholine. Particularly preferred is the La-phosphatidylcholine that La-phosphatidylcholine, which can be obtained from eggs of chickens.

In a first particularly preferred embodiment of the active substance carrier according to the invention, this consists of a molar fraction of La-phosphatidylcholine between 70 and 90%, a molar proportion of ammonium (l, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamin-N-lissamine rhodamine B sulfonyl) between 0.01 and 2% and a proportion of a substance of formula (I), so that a total of 100% molar fraction are obtained.

In a second particularly preferred embodiment of the active ingredient carrier according to the invention, this consists of a molar fraction of La-phosphatidylcholine between 10 and 30%, a molar fraction of l, 2-dioleoyl-sn-glycero-3-phosphoethanolamin between 50 and 65% and a proportion a substance according to the formulas (I), (I *), so that a total of 100% molar fraction are obtained.

The active substance carriers according to the invention are liposomes which comprise so-called lipid bilayers, the hydrophilic chemical groups X of the substances according to the formulas (I), (I *) which are part of these active compound carriers being oriented toward the polar solvent, whereas the hydrophobic chemical groups X. Groups of substances according to the formulas (I), (I *) or the above-described lipids of both layers are oriented inside the lipid bilayer. A final object of the present invention is a process for the release of an active substance from the active substance carriers according to the invention in the form of a liposome, characterized in that amphiphilic substances according to the formula (I) or (I *) at their disulfide group chemically in a hydrophobic and a hydrophilic portion be split.

The chemical cleavage according to the method of the invention may be an enzymatically catalyzed chemical cleavage or may be a non-enzymatically catalyzed reduction in which the disulfide group of the compounds of formula (I) and / or (I *) is cleaved as a result of their reduction.

Preferably, the chemical cleavage, an enzymatically catalyzed cleavage. The enzyme which catalyzes such a chemical cleavage is preferably a thioreductase, thiooxidase, thiooxireductase or thidisulfide oxidoreductase.

Also preferred is the non-enzymatically catalyzed cleavage of the disulfide group by reduction, wherein as the reducing agent a substance selected from the list consisting of glutathione, Cysteine, tris (2-carboxyethyl) phosphine (TCEP) dithiothreitol (DTT) and 1,4-dithioerythritol (DTE). Preferably, this reducing agent is tris (2-carboxyethyl) phosphine (TCEP).

The abovementioned reducing agents are particularly advantageous because they usually have a reduction potential which is only sufficient to reduce at least one of the two sulfur atoms of the disulfide group of the compounds of formula (I) and / or (I *), thereby cleaving their bond. At the same time, however, the reduction potential of the abovementioned reducing agents does not suffice to reduce other chemical groups of the substances according to the formula (I) and / or (I *) or groups of the further substances of the active substance carriers according to the invention in the form of a liposome. This ensures in particular that a cleavage of the amphiphilic substances according to the formula (I) or (I *) takes place in a hydrophobic and a hydrophilic portion, whereby the release according to the invention is particularly advantageous.

Glutathione and cysteine are substances which also occur in particularly high concentrations in the body of mammals, in particular humans, at sites which are changed in an inflammatory manner, as a result of which targeted release is made possible, in particular with the present invention.

The method according to the invention for the release of an active substance is based on the surprising finding that it is sufficient to cleave the substances contained in the active substance carriers according to the formulas (I) and / or (I *) into a hydrophilic part and a hydrophobic part, which without Being bound to a theory results in the liposome being destabilized to break up and release the active ingredient contained in it.

The present invention will be elucidated with reference to the following examples and figures, but without being limited thereto.

Fig. 1 shows experimental results according to Example 10. Shown is the fluorescence intensity (F) over the measured wavelength λ in nm of the composition VII according to Example 9 at the time t = 0 (A), i. directly after their preparation, at time t = 2 h (B), and immediately after addition of Triton X 100 (C).

FIG. 2 shows a part of the test results according to Example 11. Shown is the relative fluorescence intensity (F *) at a wavelength of 520 nm over the time (t) from the addition of TCEP to the solution of the composition III according to Example 9 at temperatures of 30 ° C (purple), 35 ° C (Illb) and 40 ° C (IIIc).

FIG. 3 shows a part of the test results according to Example 11. Shown is the relative fluorescence intensity (F *) at a wavelength of 520 nm over time (t) from the addition of TCEP according to Example 9 at temperatures of 30 ° C (IVa), 35 ° C (IVb)

The present invention will be further illustrated by the following examples, without being limited thereto.

Examples

Example 1: Preparation of a substance according to the formula (IIIa)

3.2 g (29 mmol) of 3-mercaptopropane-1,2-diol were dissolved under ice-cooling (0 ° C.) in 35 ml of ethanol and 28 ml of a 2 molar NaOH solution. With stirring, 5.1 ml (44 mmol) of benzyl chloride was added slowly (within 1 h). The course of the reaction was monitored by thin-layer chromatography (eluent: CHCl ₃ / MeOH; 5: 1, R _f = 0.4), and after one hour of stirring at 0 ° C., the reaction mixture was neutralized with 2 molar hydrochloric acid.

With toluene, the solvent was then distilled off azeotropically and the residue was taken up in CHCl ₃ and filtered. The chloroform was then distilled off and the resulting oil was purified by column chromatography (eluent: CH 2 Cl 2 / MeOH; 5: 1). The combined fractions were freed of solvent and 5.47 g (28 mmol) of 3- (benzylthio) propane-1,2-diol were obtained as a colorless oil. This corresponded to a yield of 95%.

From the thus obtained 3- (benzylthio) propane-l, 2-diol, 3 g (15.1 mmol) were dissolved together with 11.1 g (33.2 mmol) of 1-octadecyl bromide in 40 ml of toluene and stirred at room temperature (23 ° C) added 2.2 g (39.8 mmol) of potassium hydroxide. The reaction mixture was subsequently reacted at 1150C for 27 hours under reflux. From the cooled mixture, the toluene was removed by distillation and the solid residue taken up in diethyl ether. The suspension was extracted once with a saturated ammonium chloride solution and twice with water, and the separated organic phase was dried over magnesium sulfate. The mixture was then filtered and the filtrate was concentrated on a rotary evaporator. There was thus obtained 10.6 g (15.1 mmol) of 3- (benzylthio) -1,2-bis-O- (octadecyl) -propane as a colorless oil, which corresponded to a quantitative yield.

10.6 g of the obtained 3- (benzylthio) -l, 2-bis-O- (octadecyl) -propane were dissolved at 40 ° C in 160 ml of dry isopropanol. Thereafter, 6 g (261 mmol) of metallic sodium were added in small pieces to the stirred solution. After completion of the addition, the mixture was reacted at 91 ° C for 24 h at reflux. Then the reaction mixture was allowed to cool to 50 ° C and transferred the suspension in 200 g of ice. Under ice cooling, the suspension was acidified with 2 molar hydrochloric acid solution to give a pH of 2 and 400 ml of chloroform was added. The organic phase was then separated and extracted with 500 ml of saturated sodium chloride solution. The chloroform phase was separated again and dried over magnesium sulfate. The solids content was filtered off and the collected filtrate was freed of solvent in a drying oven. The residue obtained was purified by column chromatography (column material: silica gel, gradient: cyclohexane-> DCM / cyclohexane, 1: 1, R _f = 0.4) and the combined fractions were concentrated on a rotary evaporator and dried. There were thus obtained 3.31 g (5.4 mmol) of 3-mercapto-l, 2-bis-O- (octadecyl) -propane as a colorless solid, which corresponded to a yield of 36%. This 3-mercapto-l, 2-bis-O- (octadecyl) -propane corresponds to the substance according to the formula (IIIa), where n and m are each 18 and the chemical group FG _{2 is} hydrogen. Example 2 Preparation of a Substance According to Formula (Illb)

400 mg (0.66 mmol) of 3-mercapto l, 2-bis-O- (octadecyl) -propane, which were obtained from Example 1, were dissolved in 14 ml of dry dichloromethane (DCM) and 5.7 ml of dry acetonitrile and stirring at room temperature (23 ° C) were added 1 14 mg (0.66 mmol) of 4-phenyl-l, 2,4-triazoline-3,5-dione (PTAD). The resulting red solution was stirred under inert gas (argon) for half an hour at room temperature (23 ° C) and then heated to 50-55 ° C with an oil bath.

After the mixture was decolorized after about 2 hours reaction time, 243 mg (1.72 mmol) of 2-dimethylaminoethanethiol HCl was added and the reaction mixture was stirred at room temperature (23 ° C) for about 20 hours. The solvent was then removed by distillation and the residue obtained was purified by column chromatography (column material: silica gel, eluent: DCM / methanol, 12: 1, R _f = 0.6). The combined fractions were freed from the solvent and from this 322 mg (0.45 mmol) of 2 - ((2,3-bis (octadecyl) propyl) disulfanyl) -N, N-dimethylethanamine were obtained as a colorless solid. This corresponded to a yield of 68%. The obtained 2 - ((2,3-bis (octadecyl) propyl) disulfanyl) -N, N-dimethylethanamine corresponds to the substance according to the formula (IIIb), where n and m are each 18 and the chemical group Xi N, N- dimethylaminoethyl.

Example 3: Preparation of a Substance According to the Formula (IIb *)

2.74 g (68.5 mmol) of sodium hydroxide were dissolved in 15 ml of water. While stirring, 87.8 g (452 mmol) of tetraethylene glycol, which had previously been dissolved in 15 ml of tetrahydrofuran (THF), were added dropwise to the sodium hydroxide solution produced. The reaction mixture was cooled (0 ° C) and 8.3 g (43.7 mmol) of tosyl chloride, previously dissolved in 50 ml of THF, were added slowly over a period of 1.5 hours. The mixture was then allowed to react under ice-cooling for a further 2 hours with stirring. Subsequently, the viscous mass was poured onto 250 g of ice and the emulsion extracted with 300 ml of dichloromethane (DCM). The organic phase was washed three times with 50 ml of water and dried over magnesium sulfate. The desiccant was then filtered off and the filtrate was freed from the solvent on a rotary evaporator. After drying in vacuo (3 mbar, 40 ° C) 14.06 g (40 mmol) of 1-O-tosyl-tetraethylene glycol were obtained as a colorless oil. This corresponded to a yield of 92%. In 30 ml of dichloromethane (DCM), 1 g (4.1 mmol) of (+) - biotin, 1.04 g (4.1 mmol) of 1-O-tosyltetraethylene glycol, 1.01 g (4.9 mmol) of N, N'-dicyclohexylcarbodiimide (DCC) and 70 mg (0.57 mmol) of 4- (dimethylamino) -pyridine (DMAP) and stirred at room temperature (23 °) for 96 hours, then the solvent was removed on a rotary evaporator and purified by column chromatography. Column: silica gel, eluent: DCM / methanol, 10: 1, R _f = 0.3) The solvent of the fractions obtained was then removed by distillation and 1.03 g (1.7 mmol) of L-0-tosyl-2- This corresponds to a yield of 43%. The resulting 10-tosyl-2-O-biotinyl-tetraethylene glycol corresponds to the substance of the formula (IIb *), where 1-O-tosyl-2-methylbenzyl is obtained. corresponds to the chemical group FG ₃ , 2-O-Biotinyl- corresponds to the chemical group Y and the tetraethylene glycol is the chemical group X.

Example 4 Preparation of a Substance According to the Formula (I), in Particular According to the Formula (Ia)

In 1.7 ml of chloroform, 161 mg (0.23 mmol) of 2 - ((2,3-bis (octadecyl) propyl) disulfanyl) -N, N-dimethylethanamine from Example 2 and 332 mg (0.58 mmol) of 1 -0-tosyl-2-0'-biotinyl-tetraethylene glycol suspended from Example 3 and sealed under argon in a pressure vessel. The reaction mixture was warmed to 70 ° C and stirred for seven days. The mixture was then allowed to cool to room temperature (23 ° C) and the solvent was removed by distillation. The resulting residue was purified by column chromatography (column material: silica gel, eluent: DCM / methanol, 5: 1, R _f = 0.2), and 15 mg (0.09 mmol) of 2- (2,3-bis (octadecyloxy) propyl ) disulfanyl) .N, N-dimethyl-N- (2-O-biotinyl-tetraethyleneglycol-1-yl) ethanammonium tosylate as the tosylate salt of the substance of the formula (Ia) in the form of a yellow solid. This corresponds to a yield of 39%.

Example 5: Preparation of a Substance According to Formula (IIb *) Comprising a Chemical Group X '

31 1 mg (2 mmol) of serine methyl ester HCl were dissolved in 6 ml of a saturated sodium bicarbonate solution and added dropwise to a mixture of 540 mg (2 mmol) of dansyl chloride and 24 ml of acetone / water (2: 1). The reaction mixture was stirred for 20 hours at room temperature (23 ° C) and the solvent was then distilled off in vacuo. The residue was taken up in 100 ml of water and 140 ml of ethyl acetate. The organic phase was separated and dried over magnesium sulfate. After filtration, the filtrate was concentrated on a rotary evaporator and the residue was dried in vacuo (4 mbar, 40 ° C). Subsequent thin layer chromatography (eluent: DCM / methanol, 10: 1, R _f = 0.7) showed a pure product fraction. 568 mg (1.6 mmol) of 2-N-dansyl-serine-methyl ester were thus obtained as a yellow solid. 568 mg (1.6 mmol) of the 2-N-dansyl-serine methyl ester were dissolved in 12 ml of dry dichloromethane (DCM) and 394 mg (1.6 mmol) of (+) - biotin, 399 mg (1.9 mmol ) Ν, Ν'-dicyclohexylcarbodiimide (DCC) and 28 mg (0.23 mmol) of 4- (dimethylamino) -pyridine (DMAP). The suspension was stirred at room temperature (23 ° C) for nine days. The solvent was then removed by distillation, and the residue was purified by column chromatography (column material: silica gel, DCM / methanol, 20: 1, R _f = 0.5). This gave 725 mg (1.3 mmol) of 3-0-biotinyl-2-N-dansyl-serine methyl ester as a yellow solid.

The resulting 3-O-biotinyl-2-N-dansyl-serine methyl ester was suspended in 10 ml of dichloroethane (DCE) in its entirety, and ₁ g (5.5 mmol) of Me ₃ SnOH was added with stirring. The suspension was stirred under inert gas (argon) at 70 ° C for 1.5 hours. The solvent was then removed on a rotary evaporator and the residue was combined with 150 ml of ethyl acetate. The organic phase was washed 3 times with 150 ml of potassium hydrogen sulfate solution (0.01 N), separated and dried over magnesium sulfate. After filtration, the filtrate was concentrated and the residue dried in vacuo (3 mbar, 40 ° C). Subsequent thin-layer chromatography (mobile phase: DCM / methanol, 10: 1, R _f = 0.4) showed a pure product fraction. This gave 558 mg (0.99 mmol) of 3-0-biotinyl-2-N-dansyl-serine as a yellow solid.

The resulting 3-O-biotinyl-2-N-dansyl-serine was dissolved in its entirety in 8 ml of dry dichloromethane (DCM) and with stirring 345 mg (0.99 mmol) of 1-O-tosyl-tetraethylene glycol, as in the 245 mg (1.2 mmol) of N, N'-dicyclohexylcarbodiimide DCC and 20 mg (0.16 mmol) of 4- (dimethylamino) -pyridine (DMAP) were added. The reaction mixture was then stirred at room temperature for 72 hours. The solvent was then removed by rotary evaporation and the residue was purified by column chromatography (column material: silica gel, eluent: DCM / methanol, 13: 1, R _f = 0.4). This gave 292 mg (0.33 mmol) of 3-0-biotinyl-2-N-dansyl-serine (1-0-tosyl-tetraethylene glycol) ester as a yellow solid in a yield of 33%. The solid obtained corresponds to a substance of the formula (IIb *).

Example 6 Production of a Substance According to the Formula (I), in Particular According to the Formula (Ib) Comprising a Chemical Fluorescence Dye Group X '

292 mg (0.33 mmol) of the prepared according to Example 5 -0-biotinyl-2-N-dansyl-serine (l-0-tosyl-tetraethylene glycol) ester were together with 80 mg (0, 1 1 mmol) of the 2 - ((2,3-bis (octadecyl) propyl) disulfanyl) -N, N-dimethylethanamine prepared according to Example 2 in 1 ml of trichloromethane. The mixture was so for five days in a pressure vessel under argon at 70 ° C with stirring brought to reaction and then the solvent removed on a rotary evaporator. The residue was purified by column chromatography (column material: silica gel, eluent: DCM / methanol, 5: 1, R _f = 0.2) to give 88 mg (0.05 mmol) of 2 - ((2,3-bis (octadecyloxy) propyl ) disulfanyl) -N, N-dimethyl-N- (2-0- (3-O-biotinyl-2-N-dansyl-serin-1-yl) -tetraethyleneglycol-1-yl) ethanammonium tosylate according to the formula ( Ib) as a yellow solid. This corresponds to a yield of 50%.

Example 7: Preparation of a substance according to formula (IIa *)

5.75 g (29 mmol) of 3- (benzylthio) propane-1,2-diol were dissolved in 140 ml of dry dimethylformamide under argon. Thereafter, 2.55 g (37.8 mmol) of imidazole and 8.5 g (31 mmol) of tert-butyl-diphenyl-silyl chloride were added, and the reaction mixture was stirred at room temperature for 72 hours. Then, 30 ml of ethanol were added and the mixture was freed from the solvent by distillation. The residue was taken up in 200 ml of chloroform and washed twice with 100 ml of water. The separated organic phase was dried over magnesium sulfate, filtered and the filtrate was concentrated on a rotary evaporator. The resulting yellowish oil was purified by column chromatography (column material: silica gel, trichloromethane / methanol, 30: 1, R _f = 0.8) and the resulting fractions were freed from the solvent. This gave 12.37 g (28 mmol) of 1- (benzylthio) -3- (tert-butyldiphenylsilyloxy) propan-2-ol, which corresponded to a yield of 96%. The attached tert-butyldiphenylsilyloxy radical serves as a protective group in the following, in order to enable subsequent attachment of a chemical fluorescent dye group X '. 3.72 g (8.5 mmol) of the obtained 1- (benzythio) -3- (tert-butyldiphenylsilyloxy) propan-2-ol were taken together with 2.41 g (9.2 mmol) of triphenylphosphane and 1.39 g (9, 4 mmol) of phthalimide dissolved in 68 ml of dry tetrahydrofuran (THF). 2.09 g (10.3 mmol) of diisopropyl azodicarboxylate (DIAD) were slowly added dropwise to the stirred solution under argon atmosphere and at room temperature. The reaction mixture was allowed to react with stirring for 24 hours and then the solvent was removed by distillation. The residue was purified by column chromatography (column material: silica gel, eluent dichloromethane / cyclohexane, 1: 2, R _f = 0.4). The pooled fractions gave 3.2 g (5.66 mmol) of 2- (1- (benzylthio) -3- (tert-butyldiphenylsilyloxy) propan-2-yl) isoindoline-1,3-dione as a colorless oil. This corresponded to a yield of 68%. The resulting 2- (1- (benzylthio) -3- (tert -butyldiphenylsilyloxy) propan-2-yl) isoindoline-1,3-dione was dissolved in its entirety in 48 ml of ethanol (96%), and it was 23 ° C) and with stirring 0.4 ml (6.6 mmol) of hydrazine hydrate (51%) was added. The mixture was allowed to react at reflux for 2.5 hours and then reacted for three days at room temperature with stirring. The solvent was removed by distillation and the residue by means of Column chromatography (column material: silica gel, eluent dichloromethane / methanol, 25: 1, R _f = 0.3). The fractions obtained gave 1.40 g (3.2 mmol) of 1- (benzylthio) -3- (tert-butyldiphenylsilyloxy) propan-2-amine as a colorless oil, corresponding to a yield of 57%.

77 mg (0.18 mmol) of the obtained 1- (benzylthio) -3- (tert-butyldiphenylsilyloxy) propan-2-amine were dissolved together with 40 mg (0.16 mmol) of (+) - biotin in 1.7 ml of dry dimethylformamide , At room temperature, 38 ml (0.21 mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), 38 mg (0.25 mmol) of N-hydroxybenzotriazole (HOBt) and 31 ml (0.18 mmol ) Diisopropylethylamine (DIPEA) was added and the reaction stirred for 24 hours. Then 5 ml of toluene were added and the solvent was removed by distillation. The residue was dried in vacuo (5 mbar, 40 ° C.) and taken up in 20 ml of chloroform. The organic phase was washed three times with 20 ml of water and dried over magnesium sulfate. After filtration, the filtrate was freed from the solvent in a rotary evaporator and 124 mg (0, 18 mmol) of N- (l- (benzylthio) -3- (tert-butyldiphenylsilyloxy) propan-2-yl) biotinamide as a colorless solid in quantitative Yield. Under argon atmosphere, the N- (l- (benzylthio) -3- (tert-butyldiphenylsilyloxy) propan-2-yl) - biotinamide in 4 ml of dry tetrahydrofuran (THF) were dissolved and condensed with dry ice cooling 50 ml of liquid ammonia. With stirring, 100 mg (4.3 mmol) of sodium were added in small pieces in an argon countercurrent. The dark blue solution was stirred under dry ice cooling (-78 ° C) for a further 2.5 hours and then treated with 500 mg of ammonium chloride. From the colorless suspension, the ammonia was expelled in an argon stream and the residue obtained was mixed with 40 ml of water. The suspension was then acidified with molar hydrochloric acid solution to give a pH of 3, and 100 ml of chloroform was added. The organic phase was separated and freed from the solvent in a rotary evaporator. The resulting residue was purified by column chromatography (column material: silica gel, eluent: DCM / methanol, 10: 1, R _f = 0.3). There was obtained 95 mg (0.17 mmol) of 1-mercapto-3- (tert-butyldicyclohexa-2,5-dienylsilyloxy) propane-2-biotinylamide as a colorless oil in 55% yield.

The N- (1-mercapto-3- (tert-butyldicyclohexa-2,5-dienylsilyloxy) propan-2-yl) biotinamide corresponds to the substance of the formula (IIa *), where 2-biotinyl corresponds to the chemical group Y. , the chemical group FGi corresponds to hydrogen and the 3- (tert-butyldicyclohexa-2,5-dienylsilyloxy) propane (...) amide radical of the chemical group X comprising the protective group 3- (tert-butyldicyclohexa-2,5-dienylsilyloxy ) corresponds.

Example 8 Preparation of a Substance of the Formula (I), in Particular According to the Formula (Ic) 91 mg (0.15 mmol) of 3-mercapto-l, 2-bis-O- (octadecyl) -propane from Example 1 were dissolved under argon in 3.4 ml of dry dichloromethane and 1.2 ml of dry acetonitrile and stirred 26 mg (0.15 mmol) 4-phenyl-l, 2,4-triazoline-3,5-dione (PTAD) was added.

The red solution was stirred for 1.5 h at room temperature (23 ° C) and then the temperature was raised to 60 ° C. After the solution had decolorized, the 95 mg (0.17 mmol) of the N- (1-mercapto-3- (tert-butyldicyclohexa-2,5-dienylsilyloxy) propan-2-yl) biotinamide obtained in Example 7 were obtained added and the solution stirred at room temperature (23 ° C) for 24 hours. The solvent was removed by distillation and the resulting residue was purified by column chromatography (column material: silica gel, gradient: dichloromethane-dichloromethane / methanol 10: 1, R _f = 0.5). Thereby, 76 mg (0.064 mmol) of (3- (tert-butyldicyclohexa-2,5-dienylsilyloxy) propane-2-biotinylamido-1-yl) -3-dithio-1,2-bis-O- (octadecyl) -propane as a colorless solid with a yield of 38%.

This (3- (tert-butyldicyclohexa-2,5-dienylsilyloxy) propane-2-biotinylamido-1-yl) -3-dithiol, 2-bis-0- (octadecyl) propane already corresponds to the precursor of the substance according to Formula (Ic), but by the tert-butyldicyclohexa-2,5-dienylsilyloxy group still a protective group is present, which is still to be replaced by a chemical fluorescent dye group X '.

The obtained (3- (tert-butyldicyclohexa-2,5-dienylsilyloxy) propane-2-biotinylamido-1-yl) -3-dithio-1,2-bis-O- (octadecyl) -propane was dissolved in 2 ml of tetrahydrofuran and 50 mg (1.35 mmol) of ammonium fluoride were added with stirring. Subsequently, 1 ml (1 mmol) of a 1 molar tetra-n-butylammonium fluoride (TBAF) solution in tetrahydrofuran was added dropwise to the suspension, and the reaction mixture was stirred for three days at room temperature (23 ° C.). After the solvent was removed in vacuo, the residue was purified by column chromatography (column material: silica gel, gradient: dichloromethane-dichloromethane / methanol 12: 1, R _f = 0.3). The combined fractions were freed from the solvent and thus 40 mg (0.042 mmol) of (3-hydroxypropan-2-biotinylamid-1-yl) -3-dithio-1,2-bis-O- (octadecyl) -propane were obtained isolated as a colorless solid. This corresponded to a yield of 66%.

This replaced the aforesaid protecting group in favor of a hydrogen atom, so that the now existing hydroxy group is suitable for the further reaction with the chemical fluorescent dye group X '. The chemical fluorescent dye group X 'was obtained by first suspending 5 g (18.5 mmol) of dansyl chloride in 50 ml of dry pyridine and slowly adding 1.63 g (18.5 mmol) of N, N-dimethylaminoethyleneamine. The reaction mixture was stirred at room temperature for two hours and then the solvent was removed by distillation. Subsequently, the residue was taken up in 100 ml of chloroform and washed with 100 ml an ammonium hydroxide solution (2.5%). The organic phase was separated and dried over magnesium sulfate. After filtration, the filtrate was freed from the solvent and the resulting yellow oil dried in vacuo (4 mbar, 40 ° C). There was thus obtained 5.26 g (16.4 mmol) of 5- (dimethylamino) -N- (2- (dimethylamino) ethyl) naphthalene-1-sulfonamide in 89% yield. The 5- (dimethylamino) -N- (2- (dimethylamino) ethyl) naphthalene-1-sulfonamide was suspended in 40 ml of dry ethanol along with 2.95 g (21.3 mmol) of potassium carbonate. While stirring, 2.66 g (21.3 mmol) of 2-bromoethanol were added to this suspension. The reaction mixture was reacted for three days under reflux at 90 ° C and then the solvent removed in a rotary evaporator. The resulting residue was purified by column chromatography (column material: ALOX 50-200μ neutral, eluent CHCl ₃ / methanol / 25% NH ₃ , 10: 1: 0.1, R _f = 0.2) and the combined fractions gave 2.65 g ( 5.9 mmol) of 2- (5- (dimethylamino) naphthalene-1-sulfonamido) -N- (2-hydroxyethyl) -N, N-dimethylethanammonium bromide as an orange solid in a yield of 36%. 1 g of the resulting 2- (5- (dimethylamino) naphthalene-1-sulfonamido) -N- (2-hydroxyethyl) -N, N-dimethylethanammonium bromide was added together with 540 mg (5.4 mmol) of succinic anhydride and 15 mg (0.12 mmol) of 4 - (Dimethylamino) -pyridine (DMAP) suspended in 10 ml of dry pyridine. The reaction mixture was heated under inert gas (argon) to 70 ° C and stirred for three days. The solution was then cooled to room temperature (23 ° C) and 20 ml of toluene added. The solvent was distilled off in vacuo and the residue was purified by column chromatography (column material: silica gel, eluent: dichloromethane / methanol / formic acid, 5: 1: 0.2, R _f = 0.3). 1.1 g (2.0 mmol) of 2- (5- (dimethylamino) naphthalene-1-sulfonamido) -N- (2-O-succinyl-ethyl) -N, N-dimethylethanammonium bromide as a yellow-green, were obtained after removal of the solvent. Hydroscopic solid with a yield of 90% as chemical fluorescent dye group X '. 46 mg (0.084 mmol) of this chemical fluorescent dye group X 'in the form of 2- (5- (dimethylamino) naphthalene-1-sulfonamido) -N- (2-O-succinyl-ethyl) -N, N-dimethylethanammonium bromide were then added with 40 mg (0.042 mmol) of the previously prepared (3-hydroxy-propane-2-biotinylamid-1-yl) -3-dithio-l, 2-bis-0- (octadecyl) -propane dissolved in 1, 1 ml of dichloromethane and it Under argon, 181 mg (0.88 mmol) of dicyclohexylcarbodiimide (DCC) and 37 mg (0.30 mmol) of 4- (dimethylamino) -pyridine (DMAP) were added. The reaction mixture was sealed in a pressure vessel and stirred for 14 days at 40 ° C. After the solvent was removed by distillation, the residue was purified by column chromatography (column material: silica gel, eluent: dichloromethane / methanol, 5: 1, R _f = 0.2). The combined fractions were freed from the solvent and thus 34 mg (0.023 mmol) of L-O- (2-dansylamide-N, N-dimethylethanammonium bromide-N- (ethyl-0-succinyl)) - 2-N-biotinyl-3 - ((2,3-bis (octadecyloxy) propyl) disulfanyl) -2-aminopropanol according to the formula (Ic) as a yellow solid in a yield of 55%.

Example 9 Preparation of Active Ingredient Carriers According to the Invention Substances According to Formula (I) Stock solutions of the substances obtained from Examples 4, 6 and 8 were prepared by mixing the substances according to Examples 4 and 6 in a chloroform / methanol solution (5: 1 ) were dissolved, or the substance was dissolved in chloroform according to Example 8.

To these stock solutions were then added further substances according to Table 1 with the molar proportions listed there, wherein in the case of Composition I and II, the other substances were also dissolved in chloroform and in the case of compositions III to VIII, the other substances also in a chloroform / Methanol solution (5: 1) were dissolved.

The listed in Table 1 La-phosphatidylcholine (eggPC), the l, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and the ammonium (l, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N -lissamine rhodamine B sulfonyl) (Rh-DPPE) were each purchased (Avanti Polar Lipids, Alabaster, AL).

Table 1 further provides information in the last column as to whether or not an active substance has been incorporated into the respective unilamellar vesicles (active ingredient carriers) obtained from compositions I to VII. By way of example, in the experiments carried out here, calcein has been used as the water-soluble substance which can be easily detected spectroscopically in replacement of a pharmaceutically active substance.

The compositions I to VII were freed from the solvent (- mixture) in a rotary evaporator, so that the solid mixtures of the substances precipitated as a film on the wall of the glass flask.

Table 1: Compositions I to VII according to Example 9

Molar fraction [%]

# Fabric (Ia) Fabric (Ib) Fabric (Ic) eggPC DOPE Rh-Calcein

377 392 325 DPPE I 0 0 10 89,9 0 0,1 No

II 0 0 0 99.9 0 0, 1 no

III 20 0 0 30 50 0 Yes

IV 10 0 0 30 60 0 Yes

V 0 0 0 40 60 0 Yes

VI 0 10 0 90 0 0 no

VII 0 10 0 89 0 1 no

In the case of Compositions I and II, the precipitated film was reacted with a 176 mM sucrose solution containing 2- (4- (2-hydroxyethyl) -1-piperazinyl) -ethanesulfonic acid (HEPES) at 10 mM concentration and having a pH of 7.4 recorded at 40 ° C. The resulting suspension was subjected to five freezing and thawing operations. The freezing was carried out by immersing the glass flask in an isopropanol-dry ice mixture and thawing respectively by immersion in a water bath at 50 ° C. Thereafter, the suspension was passed 10 times through a polycarbonate filter with 100 nm pore diameter (Nucleopore GmbH) at a temperature of 40 ° C and the resulting unilamellar vesicles (drug carriers) were centrifuge for one hour at 100,000 x g. The supernatant was discarded and the pellet of the resulting drug carrier was resuspended in 100 mM potassium chloride solution with 10 mM HEPES at pH 7.4.

In the case of compositions III to VII, the precipitated films were taken up in cyclohexane (in the case of compositions III to V, cyclohexane with an additional 5% by volume of ethyl acetate), the solutions frozen in glass tubes at -80 ° C and lyophilized overnight.

The next day, to Compositions III-V, a 70 mM calcein solution prewarmed to 70 ° C with 10 mM HEPES, 1 mM ethylenediaminetetraacetate (EDTA) was added at pH 7.4. The resulting suspensions were subjected to five freezing and thawing operations in the same manner as those of Compositions I and II, except that the thawing was done with a 70 ° C water bath. In the same way, the suspension was passed through a polycarbonate filter 10 times. With the compositions VI and VII was analogous method, with the only difference that no calcein solution, but the o. Potassium chloride solution was used.

To the obtained from the compositions III to V unilamellar vesicles (excipients), which are loaded with calcein, of excess calcein, which is not inside the drug carriers After freeing, the permeate of the above-mentioned filtration was passed through a column located in a centrifuge unit column (Sephadex G 50, Quiagen, centrifugation: 3 minutes at 530 xg).

Example 10: Demonstration of the Formation of Unilamellar Vesicles (Active Ingredient Carriers) Without Active Ingredient The unilamellar vesicles (active ingredient carriers) obtained from the composition VII were introduced into the respective solutions in a temperature-controlled spectroscopic measuring cell with stirrer (Aminco type: Bowman series 2) and in Range of wavelengths from 350 to 650 nm under excitation at 330 nm with respect to the emitted fluorescent light measured.

The composition VII has in the used material (Ib) with the dansyl radical a fluorescent group and further in the further substance ammonium (l, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamin-N-lissamine rhodamine B sulfonyl ) (Rh-DPPE) with the rhodamine group another fluorescence-active group. These two fluorescent groups, when in close proximity to each other, can exhibit a so-called Forster resonance energy transfer (FRET) between the dansyl residue and the rhodamine, which causes the fluorescence of the dansyl to precipitate and disappear - via FRET - (only) the rhodamine fluorescence can be seen, which could be seen at 590 nm.

Accordingly, the fluorescence signals of the unilamellar vesicles (drug carrier) obtained from composition VII were measured and compared with the fluorescence signals of the same sample after incubation with 10 μΐ of 20% polyethylene glycol p- (1,1,3,3-tetramethylbutyl) - phenyl ether (Triton X 100) was added. Triton X 100 is a non-ionic detergent that generally dissolves unilamellar vesicles.

It can be seen from FIG. 1 that the fluorescence signals of the unilamelallaric vesicles (active ingredient carriers) obtained from the composition VII have an unchangeable measurement rash - and in particular no - at 590 nm directly after their production (A) and at least over a period of two hours (B) Measuring at 520 nm, as it would be unique for a dansyl radical - has. Immediately after the addition of Triton X 100, the separated fluorescence signals of the dansyl residue and the rhodamine (C) can be seen.

It is thus clear that prior to the addition of Triton X-100, unilamellar vesicles were present within the meaning of the active substance carriers according to the invention. Example 11: Release of active ingredient from unilamellar vesicles (active substance carriers)

The unilamellar vesicles (excipients) obtained from compositions III to IV were investigated in a similar manner as in the previous example 10, with the difference that that was excited not at 330 nm, but at 492 nm. This is the wavelength of the approximate excitation maximum of calcein.

Further, experiments were carried out at various temperatures (30 ° C, 35 ° C and 40 ° C) and tris (2-carboxyethyl) phosphine (TCEP) was used as the reducing agent for the disulfide group in place of the nonionic detergent. Thus, the actual release by cleavage of the disulfide group was examined.

The results of the experiments with the compositions III are shown in FIG. The results with compositions IV are shown in FIG. The relative fluorescence intensity (F *) over the measuring time (t) for the measurements at 30 ° C, 35 ° C and 40 ° C is plotted. Relative fluorescence intensity (F *) here means in each case the fluorescence intensity which was measured, based on a theoretical maximum fluorescence intensity value of the pure calcein in solution.

It can be seen in Fig. 2 that at a temperature of 30 ° C (purple) over a period of up to 800 s still no satisfactory release is achieved. At a temperature of 35 ° C (Illb), a spontaneous release takes place within the first 300 s. At a temperature of 40 ° C (IIIc) spontaneous release already takes place within the first 200 s.

It can be seen in FIG. 3 that at a temperature of 30 ° C. (IVa) no satisfactory release is achieved over a period of up to 1200 s. At a temperature of 35 ° C (IVb), release occurs approximately within the first 800 seconds. At a temperature of 40 ° C (IVc) spontaneous release already takes place within the first 500 s.

Since composition IV, unlike that according to III, contains only 10 mol% of the substance according to formula (Ia), it is also obvious that its presence is responsible for the improved, spontaneous release. In addition, in all cases, the release particularly positive at temperatures obtained, as they are common in the human body. The particularly rapid release takes place in the range of 35 ° C to 40 ° C.

Claims

claims

1 Substances suitable for the preparation of active substance carriers in the form of a liposome according to the formula (I)

characterized in that the substances according to formula (I)

(A) in X on one side of the disulfide group, a hydrophilic chemical group comprising at least five carbon atoms and comprising at least one ether group and / or amine group or ammonium group, and wherein

(b) on the other side of the disulfide group is a hydrophobic group in which n and m are independently of one another a natural number from 1 to 30, but the sum of n and m is at least 16.

2. Substances according to claim 1, characterized in that the radicals with n, m carbon atoms are monounsaturated or polyunsaturated radicals.

3. Substances according to claim 1, characterized in that the radicals with n, m carbon atoms are saturated hydrocarbon radicals.

4. Substances according to one of claims 1 to 3, characterized in that the chemical group X comprises at least eight carbon atoms.

5. Substances according to one of claims 1 to 4, characterized in that the chemical group X also comprises at least one carboxylic ester group.

6. Substances according to one of claims 1 to 5, characterized in that the chemical group X comprises either at least two ether groups or at least one amine group or ammonium group and at least two carboxylic acid ester groups.

7. Substances according to one of the preceding claims, characterized in that the at least one amine group or ammonium group of the chemical group X is a quaternary ammonium group.

8. Substances according to claim 7, characterized in that the substance according to the formula (I) carries a positive charge and is present in combination with at least one singly or multiply negatively charged counterion.

9. Substances according to one of the preceding claims, characterized in that the chemical group X comprises at least three ether groups.

10. Substances according to one of the preceding claims, characterized in that the chemical group X is attached to a chemical group Y, wherein the chemical group Y is a chemical group selected from the list consisting of biotin, protein, peptide, nucleic acid, nucleoside and glycoal is.

1 1. Substances according to claim 10, characterized in that the chemical group X is attached via a carboxylic acid ester group to the chemical group Y.

12. Substances according to claim 10, characterized in that the chemical group X comprises two carboxylic acid ester groups, a first amine group or ammonium group and a second amine group or ammonium group, wherein the chemical group Y is attached to the chemical group X via an amide bond.

13. Substances according to one of claims 10 or 11, characterized in that they have the chemical formula (Ia)

exhibit.

A substance according to claim 12, characterized in that it has the chemical formula (Ic)

having.

15. Self-assembled active substance carrier in the form of a liposome, characterized in that this active substance carrier comprises at least one substance according to formula (I)

wherein the at least one substance according to the formula (I)

(a) have in X a chemical group comprising at least five carbon atoms and comprising at least one ether group and / or amine group or ammonium group, wherein

(b) n and m are independently of one another a natural number from 1 to 30, but the sum of n and m is at least 16.

16. A self-assembled drug carrier according to claim 15, characterized in that it comprises at least one further substance, with at least one chemical group having lipophilic properties and at least one chemical group having hydrophilic properties.

17. A self-assembled drug carrier according to claim 16, characterized in that the further substance with at least one chemical group having lipophilic properties and at least one chemical group having hydrophilic properties is a lipid.

18. A self-assembled drug carrier according to claim 17, characterized in that the lipid is selected from the list consisting of La-phosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamin and ammonium (1, 2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-lisamine rhodamine B sulfonyl).

19. A process for the release of an active substance from the active substance carriers according to one of claims 16 to 18, characterized in that the amphiphilic substances contained in the active substance carrier according to one of claims 1 to 14 are cleaved at their disulfide group chemically into a hydrophobic and a hydrophilic portion.