EP2187735A2 - Substances for the protection of cells and/or tissues - Google Patents

Abstract

The invention relates to substances that are suitable for protecting cells and/or tissues.

Description

 Substances for the protection of cells and / or tissues

Patent application

Field of the invention

The invention relates to substances which are suitable for the protection of cells and / or tissue.

Background of the invention

Mammalian organs and tissues or other eukaryotic cells can be exposed to a variety of damaging influences. Especially the cells of higher organisms are particularly susceptible, especially damage then occurs frequently when cells or tissues are dissolved out of the organism, such as in cell cultures or in transplants. Furthermore, even if the original environment of the cells or organs, e.g. due to surgical intervention or pathological processes.

Particularly severe damage to cells or tissues of a mammal occurs under ischemic conditions. As ischemia is called the pathologically reduced or reversed blood flow of a tissue due to lack of arterial supply of blood, resulting in oxygen deficiency of the cells or tissue. Despite the diminished oxygen supply, paradoxical oxidative damage to the cells or tissue is frequently found here. Ischemia-induced damage to cells or tissues can often occur in surgical procedures and is responsible for high complication rates. To counteract this risk is an object of the invention.

Particularly important is the protection of cells and tissue in the field of transplant surgery. From the removal of an organ to its use in the receiver, it is important to protect its functions as much as possible.

Since cells or tissues rarely can be transplanted immediately in place, it will always be necessary to preserve the cells or tissue. Frequently, the tissue is stored after removal at low temperatures, whereby the metabolism is reduced. However, this can cause serious damage to the tissue due to the effects of the cold itself. This concerns in particular internal organs, which are cold-stored. An example is the kidneys, where the cold damages the endothelial cells of the kidneys, resulting in the loss of barrier function, which is associated with a greatly increased risk of immunological complications or dysfunctions. Although the active ingredients used hitherto for the prevention of cold damage in the context of studies or experimentally used, such as dopamine or dobutamine, have a protective effect, a very high concentration is required for this purpose. When applied to animals or humans, therefore, after a short time a very strong hemodynamic effect, which usually leads to complications and is therefore undesirable. When dopamine or dobutamine is applied to cell cultures, the metabolism is significantly altered, so that the cells no longer have their proper functioning and are therefore unsuitable for transplantation.

Another possibility for the preservation of cells or tissue, in particular during transplantation, is the perfusion of the tissue or the cells with Preservative-containing solutions. Thus, solutions for increasing the lifetime of transplantation tissues containing PHB and PHB folic acid antagonists in combination are described. Further, DE 295 04 589 IM describes for this purpose the use of benzoic acid and its derivatives, optionally in combination with other active substances. Elsewhere, reference is made to the use of adrenaline or carvedilol.

An optimal substance, on the one hand sufficiently protects the cells or the tissue against ischemic damage and on the other hand achieves the desired protective effect in low concentration, so that no haemodynamic effects occur, the substance is neither harmful nor environmentally harmful, has not yet been found.

The object of the invention was therefore to find a substance which cells and tissues, in vivo but especially during storage and transport, i. ex-vivo, protects, in particular tissue to be transplanted or removed cells from ischemic damage or cold damage preserved or reduced. Furthermore, the substance should be able to be used in low concentration and have no hemodynamic or otherwise undesirable activity.

These objects are achieved by a substance with the features according to claim 1. The subclaims have advantageous developments to the content.

Detailed description of the invention

Surprisingly, it has now been found that an aromatic system containing at least one aromatic ring having two reducing substituents Ri and R _Σ , and another substituent R ₃ , so that the log P of the molecule is at least 2.5, cells or tissues can protect. The substance according to the invention is represented in the following general formula (1):

wherein the double circle is an aromatic system having 6 to 18 carbon atoms and carrying at least the substituents R ₁ , R ₂ and R ₃ , wherein R 1 and R _{2 are} each selected from the group consisting of OH, SH and NH ₂ , the may also be in protected form, and R _{3 is} a hydrophobic group, where log P of the substance is at least 2.5.

The aromatic system may be composed of both aromatic rings carrying exclusively carbons as ring atoms and those having heteroatoms as long as they are biocompatible. Carbazoles and derivatives thereof are, for example, aromatic rings containing heteroatoms in the aromatic system; only aromatics containing carbon are preferred.

The aromatic system has one or more aromatic rings which may be fused together. Preferred examples are phenyl, naphthalene and anthracene. The aromatic system may carry, in addition to the substituents R 1, R ₂ and R _3, further substituents which are inert with respect to the desired properties and may optionally stabilize or activate the system. Apart from R 1, R ₂ and R _3, no further substituents are preferably bonded.

The aromatic system which carries at least the substituents R 1, R ₂ and R ₃ is preferably characterized by containing 5-, 6- or 7-membered rings. Rings in a size of 5 to 7 atoms have a high ring stability, so have reduced internal stresses, even at high degree of substitution of the aromatic ring. In addition, such aromatic systems are readily available, well studied and therefore safe, in terms of not harmful or harmful to the environment.

More preferred is an aromatic system having 1 to 3 rings. Although in principle compounds can also be used whose aromatic system contains more rings, it has been found that in particular smaller aromatic systems with only 1 to 3 rings can better penetrate through the cell wall due to their small size.

The substituents R 1 and R ₂ are selected from the group consisting of OH, SH and NH ₂ , optionally in protected form, wherein any combination of these radicals may be present. Preferably, Ri and R _{2 are} each OH. The groups may be protected with a protecting group to protect them from harmful reactions during storage.

The radicals Ri and R ₂ are each bound in the aromatic system to an aromatic ring, either in ortho or para position to each other. It is believed that the reducing effect of the two functional groups and thus the protection of the tissue from oxidative damage is enhanced precisely by this selective position of the two substituents Ri and R ₂ to one another. It can be assumed that due to the conformation of the aromatic ring at the ortho or para position, the two functional groups, ie the substituents R ₁ and R ₂ , point in the same direction, and thus their function is synergistically enhanced. The aromatic system is particularly preferably derived from 2,5-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 2,5-dihydroxyphenethylamine or 3,4-dihydroxyphenethylamine.

However, the substance according to the invention can protect cells or tissues from damaging influences only if the substance is formed such that the log P of the substance is at least 2.5, log P is an empirically calculated parameter and can be mathematically calculated from the structure of a substance where P is the partition coefficient of the considered substance between n-octanol and water, so represents a measure of the hydrophobicity of a substance. Small values of the log P mean an increased hydrophilicity of the molecule, while large values mean increased lipophilicity.

It has been found that a molecule with a log P of at least 2.5 has such a good lipophilicity or hydrophobicity that the molecule can migrate better through the cell wall into cells than conventional substances with a lower log P in order to protect it unfold. On the other hand, highly hydrophilic molecules with a log P of less than 2.5 do not pass through the semipermeable cell wall, so that their effect is reduced. With a log P value of 2.5, a threshold appears to be reached which refers to substances that are capable of penetrating the cells of a tissue to prevent ischemic damage to the tissue by reducing or preventing oxidative factors.

In order to set the log P value, the preparation according to the invention has, in addition to the two reducing substituents R 1 and R ₂ , another substituent R ₃ . This serves to set the application properties of the substance targeted and is varied in particular to adjust the log P value accordingly. In particular, the constitution of R _{3 is} not limited as long as it is biocompatible and contributes to hydrophobicity. Thus, R ₃ can be both a homoalkyl radical, as well as a heteroalkyl radical, straight-chain or branched. The definition of R ₃ includes substituted, such as unsubstituted, homoatomic or heteroatomic "radicals" in the chemical sense In a preferred embodiment, the substituent R _{3 is} an alkyl substituent having a chain length of C6 to C26 and preferably C 8 to C 18. In other words, R is ₃ preferably represents a saturated alkyl radical consisting of carbon atoms which may be linear or branched and comprises 6 to 26 and preferably 8 to 18 carbon atoms Among the alkyl radicals, preference is given to linear versus branched alkyl chains It is believed that such alkyl substituents have a carbon chain of 6 to 26 carbon atoms on the aromatic ring which carries the more hydrophilic substituents Ri and R ₂ , the hydrophobicity of the aromatic ring significantly raises, so that the penetration of the cells is in turn facilitated, and thus the substance according to the invention, these cells and thus the tissue or the organ, can protect. The hydrophilic, strongly reducing substituents R 1 and R ₂ are thus "masked" and the substances are introduced into the cell where they take effect, but this lipophilic-enhancing effect can only occur from a chain length of at least 6 carbon atoms and is the strongest with a hydrocarbon radical of 8 to 18 carbon atoms From a carbon number of more than 26 carbon atoms, the substituent R _{3 has} too strong a shielding effect, so that the substance can not exert its protective effect in the cell, since the active strongly reducing functional groups R 1 and R ₂ sterically hindered.

The substituent R ₃ may be bonded directly to the aromatic system. In a preferred embodiment, the bond is via a bridging member, which may be the chemical moiety Y-NHCO, where Y is either a direct bond between the aromatic system and the NHCO group, or an alkyl group with a carbon chain of C1 to C8 and preferred from C1 to C3. R ₃ is then attached to the carbonyl carbon. Together with R ₃ , this thus represents an amide.

Amides, ie substances that have a peptide bond, are common in nature. They are building blocks of the polypeptides, the proteins. It is therefore estimated that substances which have an amide group can in principle immigrate very well into cells.

The inventors have now found that amides which have an alkyl radical having 1 to 8 carbon atoms and preferably 1 to 3 carbon atoms on the nitrogen atom and an alkyl chain having 2, preferably 6 to 26, carbon atoms on the carbonyl carbon are well suited to the permeability of the invention Substance according to the invention to increase through the cell wall into the cell interior, and thus to facilitate the entry into the cell interior, so that the substance can develop their cell-protecting or organ-protective effect in place very well. Thus, for bonds via a bridging member, the alkyl chain of R _{3 may be} shorter since the chain is extended by the bridging member. Although longer alkyl chains on the nitrogen atom would also be suitable, it has been found that especially the short alkyl chains, ie those which comprise a maximum of 3 carbon atoms, are particularly well suited. It is believed that this is related to the steric arrangement on the aromatic ring, or with the electron cloud of the lone pair of electrons on the nitrogen atom, which can cause a shielding effect. The same applies to the alkyl radical which is bonded to the carbonyl carbon. However, here the effect of the steric shielding is no longer so great, because already by the nitrogen, the essential shielding is effected, so that in principle longer carbon chains of up to 26 C-atoms are possible.

In a further preferred embodiment, R _{3 is} bonded via a group having the structure Y-COO, where Y again represents a direct bond between the aromatic system and the grouping COO, but also a C1 to C8 alkyl group, preferably a C1 to C3 alkyl group can. R ₃ , ie preferably an alkyl radical having a chain length of C ₂ to C 26, is bonded to an oxygen atom and in this embodiment gives an ester grouping.

For such an ester grouping similar to the previously employed, steric considerations apply, as already detailed for the amides. Esters and amides also have a similar polarity so that they can be used alternatively or in combination. However, the peptide bond contributes to a slightly improved acceptance in the cell or tissue compared to the ester. Furthermore, esters are not as stable chemically as peptides and split even with slightly changed pH values in acid and alcohol, whereby the effect of the substance according to the invention is at least partially lost.

In a further preferred embodiment, R _{3 is} bonded via a group having the structure Y-CH ₂ O, where Y in turn represents a direct bond between the aromatic system and the grouping CH ₂ O or else a C1 to C8 alkyl group, preferably a C1 to C3 alkyl group can be. R _3, ie preferably an alkyl radical having a chain length of C ₂ to C 26, is bonded to the oxygen, which in this embodiment leads to an ether grouping. Ethers with the formula given above can also be used as substituent R ₃ . As a result of the ether group, the molecule acquires a proportion of hydrophilicity which, however, is markedly reduced with respect to the esters or amides. However, free ethers are much less abundant in the body of a mammal, and the acceptance of such substances is therefore somewhat reduced compared to amides and esters. However, this effect is at least partially counterbalanced by the markedly increased lipophilicity, so that ethers in the abovementioned constitution likewise represent an alternative for the substituent R ₃ .

In a further preferred embodiment, at least one of the two substituents Ri or R ₂ bears a protective group. Functional group protecting groups are used in chemistry whenever a particular functional group is to be prevented from premature reaction. After cleavage of the protecting group then the reactive functional group is free again and can react as desired. Suitable protective groups are the protective groups customarily used in organic chemistry for OH, SH and NH ₂ groups, which are well known to the person skilled in the art. As is well known to those skilled in the art, the protecting groups must sufficiently bind to the functional groups R 1 and R ₂ to protect them during storage, but the bond must be designed to redissolve the protecting group in a physiological environment.

Suitable protecting groups for OH are acyl groups, preferably the acetyl group or succinyl group or else phosphate groups. The substance according to the invention is accordingly reacted with desired protection of one of the radicals R 1 or R ₂ with a suitable acid, such as, for example, acetic acid or phosphoric acid. This produces either an ester, an amide or a thioester, depending on the reducing substituent. Protecting groups of this type can be split off again very easily, usually even under slightly different conditions inside the cell, for example when the pH is changed. The cleavage of the protecting group recovers the functional, strongly reducing group, ie either OH, SH or NH ₂ , which protects it from oxidative damage inside the cell. Particularly suitable are acetyl protecting groups. They are readily available, well known, do not split any harmful substances when the protecting group is removed, and are also inexpensive.

Alternatively usable are succinyl protective groups or phosphate protecting groups. They are obtained by reaction of the substituent Ri and / or R ₂ with succinic acid or phosphoric acid. The succinyl group or phosphate group can easily be cleaved off again under conditions which are present in the interior of a cell, so that the reducing effect of the OH, SH or NH ₂ groups reappears. Succinic acid and phosphoric acid, which are recovered after cleavage of the protective group, are harmless to the body, which can easily be flushed out again.

Without wishing to be bound by theory, it is believed that an aromatic system bearing on a ring at least two substituents R 1 and R ₂ selected from OH, SH and NH _{2 has a} strong reducing action and therefore oxidative damage to cells or tissues as counteracted by ischemic conditions, counteracts.

To prevent this damage, a very low concentration is sufficient, i. a concentration of the substance of about 0.5 to 200 uM, preferably 1 to 100 uM.

In order to be able to develop the desired protective effect, the substance according to the invention can be administered in a variety of ways. All modes of administration are suitable here, such as parenteral or oral administration, with parenteral administration being preferred. It is essential that the substance enters the bloodstream of the tissue to be protected or the cells to be protected, so that there can be an accumulation of the active substance in sufficient quantity. This is usually achieved by injection or infusion into the bloodstream of the donor. The substance according to the invention is particularly suitable in the form of an injectable preparation for administration to a donor. The preparation consists at least of the substance according to the invention and at least one pharmaceutically acceptable carrier. The carrier may be water in the simplest case. In most cases, the substance is predissolved in suitable pharmaceutically acceptable solvents such as PEG derivatives or the like and then administered after processing either as a solution or dispersion or in the form of liposomes or micelles. Also biologically and physiologically compatible surfactants can be used for better processing. For this purpose, the surfactants also used for pharmaceutical products are suitable, for example substances which are marketed under the name "Pluronic".

The preparation is suitable for being injected into a donor and can preferably be used as a rinsing solution through which the relevant organ to be transplanted flows so that the substance according to the invention reaches all cells of the organ. An almost complete flush is achieved after about 30 minutes to 2 hours. The preparation preferably contains the substance at 0.5 to 20 μM, since there is thus a sufficient, effective concentration of the substance according to the invention which protects the cells or the organs.

The substance according to the invention described above is used to protect cells or even tissues and organs. In particular, the protection relates to damage by oxygen deficiency (ischemic conditions) of the cells / tissue, especially in tissue or cells to be transplanted. The substance according to the invention is used in very low concentration and shows no hemodynamic activity. It is highly compatible with this and prolongs the life of cells or tissue intended for transplantation, in order to reduce or completely prevent damage to the tissue prior to transplantation, thus significantly increasing the chances of successful transplantation ,

Embodiments of the invention will be described in more detail below The active substances are synthesized as described below. The effect of the substances on the cold-induced damage of cells is quantified in a model system. For this purpose, endothelial cells, for example cells of the endothelium of the human umbilical vein, are cultivated in culture. The cells are incubated with different concentrations of the test substances for variable periods of time, after which the medium is replaced by fresh medium without test substance. Subsequently, the cells are incubated at ⁰ ° C. for, for example, 24 hours. At the end of the incubation period, the laktatdehydrogenase released is determined in the supernatant of the culture vessels by known methods, the concentration of which is a measure of the cell damage. The effectiveness of each compound is determined by the concentration at which 50% of the release of lactate dehydrogenase is inhibited (EC50).

Example 1 N-Octanoyl-dopamine

1 gram of N-octanoic acid is dissolved in 10 ml of tetrahydrofuran and 0.90 grams of N-ethyldiisopropylamine are added. While stirring, 0.75 grams (0.658 ml) of ethyl chloroformate is added slowly. After 3 hours, the mixture is mixed with 15 ml of ethyl acetate and 10 ml of water. The organic phase is separated and dried over magnesium sulfate.

Under a nitrogen atmosphere, 1, 24 grams of dopamine hydrochloride is dissolved in 10 ml of dimethylformamide. To this is added a stoichiometric amount of ethoxycarbonyl octanoate dissolved in ethyl acetate with stirring. The resulting turbidity dissolves again after addition of the stoichiometric amount of N-ethyldiisopropylamine. After stirring with exclusion of light overnight, 20 ml of an aqueous solution with 5% sodium bicarbonate / 1% sodium sulfite are added and the organic phase is separated off. The aqueous phase is extracted again with 10 ml of ethyl acetate. The combined organic phases are washed successively with 10 ml sat. Saline, 10 ml of 0.5 M sulfuric acid and 10 ml of brine. The organic phase is over Dried magnesium sulfate and the solvent removed in vacuo (rotary evaporator). This gives 1.74 grams (96%) of a very tough, nearly colorless oil.

Example 2 O-succinyl-N-octanoyl-dopamine

0.66 grams of N-octanoyl-dopamine are dissolved under nitrogen in 3 ml of tetrahydrofuran, to which 236 mg of succinic anhydride in 4 ml of tetrahydrofuran are added. After stirring overnight, the solvent is removed in vacuo and the solid residue taken up in 5 ml of 5% sodium bicarbonate and 5 ml of ethyl acetate. The organic phase is discarded, the aqueous phase is mixed with 10 ml of ethyl acetate and acidified with 10 ml of 0.5 M sulfuric acid. The organic phase is washed with sat. Washed brine, dried over sodium sulfate and the solvent removed in vacuo. This gives crude O-succinyl-N-octanoyl-dopamine, which is further purified by recrystallization.

Example 3 N-decanoyl-dopamine

1, 72 grams of n-decanoic acid is dissolved in 10 ml of tetrahydrofuran, 1.2 grams of thionyl chloride added. After addition of a drop of dimethylformamide is heated with stirring to reflux. After 5 h, the solvent is distilled off. 0.95 grams of dopamine hydrochloride are dissolved in 6 ml of dimethylformamide and the stoichiometric amount of decanoic acid chloride is slowly added dropwise in an ice bath under nitrogen. After 3 hours, the procedure is as in Example 1.

Example 4 N-octadecanoyl-dopamine

1.42 grams of stearic acid is dissolved in 10 ml of tetrahydrofuran and 0.57 g of N-hydroxysuccinimide and 1.03 grams of dicyclohexylcarbodiimide are added. After stirring overnight, the precipitate is separated by filtration, with Washed tetrahydrofuran and the combined filtrates freed of solvents in vacuo. Under a nitrogen atmosphere, the N-octanoyloxysuccinimide thus obtained is reacted with the stoichiometric amount of dopamine hydrochloride and triethylamine (dissolved in dimethylformamide). After stirring overnight with exclusion of light, N-octanoyldopamine is obtained after working up.

Example 5 2,5-Dihydroxybenzoylamidooctane

From 2.38 grams of 2,5-dihydroxybenzoic acid, the acid chloride is prepared in a known manner by means of phosphorus trichloride. To this, dissolved in 20 ml of tetrahydrofuran, the stoichiometric amount of N-octylamine is slowly added under a nitrogen atmosphere in an ice bath with vigorous stirring. After completion of the addition, the ice bath is removed and stirred overnight with exclusion of light. The solvent is removed in vacuo, the organic phase washed successively with sodium bicarbonate / sodium sulfite solution, water, dilute phosphoric acid and brine and finally dried over molecular sieves. After removal of the solvent gives 2,5-Dihydroxybenzoylamidooctan as an almost white solid.

Example 6 3,4-Dihydroxybenzoylamidooctane

1.19 grams of 3,4-dihydroxybenzoic acid are dissolved in 10 ml of tetrahydrofuran and under nitrogen 0.57 grams of N-hydroxysuccinimide, 1.03 grams of dicyclohexylcarbodiimide and 0.65 grams of octylamine are added. After stirring overnight with exclusion of light, the precipitate is filtered off and the organic phase is diluted with 15 ml of ethyl acetate and washed with 10 ml of 5% sodium bicarbonate / 1% sodium sulfite. After shaking with brine, 0.5 M sulfuric acid and brine, the organic phase was dried over sodium sulfate and freed from the solvent. This gives 1.44 g (83%) of a beige solid. Example 7 2,5-Bisacetoxybenzoylamidohexane

From 2,5-dihydroxybenzoic acid is prepared by known methods with acetic anhydride and sodium acetate 2,5-bisacetoxybenzoic acid. From 1.19 grams of this compound dissolved in 10 ml of diethyl ether, the active ester is synthesized by adding 0.68 grams of N-hydroxybenzotriazole and 0.96 grams of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide. After stirring overnight, the solvent is removed and the residue taken up in 10 ml of ethyl acetate and 10 ml of water. The organic phase is dried and 0.5 grams of hexylamine are added. After stirring overnight, it is washed successively with sodium bicarbonate solution, brine and dilute phosphoric acid and the organic phase is dried. After removal of the solvent, 1, 3 grams (81%) of crude 2,5-Bisacetoxy- benzoylamidohexan.

The protective effect of some substances according to the invention is shown by their EC50 values (dopamine, adrenaline, norepinephrine and dobutamine for comparison only):

Example 8

0.5 grams of N-octanoyl-dopamine is taken up in 9.5 grams of a mixture of 60% (v / v) 1, 2-propylene glycol and 40% (v / v) water and mixed. The result is a clear, durable solution, which is suitable for sterilization according to the recognized pharmaceutical rules for parenteral use in mammals.

Claims

claims 1. substance for the protection of cells or tissue, characterized in that the substance has at least one aromatic ring of the formula 1 with two substituents Ri and R ₂ , selected from the group consisting of OH, SH and NH ₂ , wherein Ri and R ₂ each other in ortho or para position and another substituent R ₃ , which brings the log P of the molecule to at least 2.5. Formula 1) 2. Substance according to claim 1, characterized in that the aromatic system has 1 to 3 rings, which may be condensed. 3. Substance according to claim 1, characterized in that the aromatic system contains aromatic rings with 5, 6 or 7 carbons. 4. Substance according to claim 1 to 3, characterized in that R _{3 is} a substituted or unsubstituted alkyl radical having a chain length of C6 to C26, preferably C8 to C18. 5. Substance according to one of claims 1 to 4, characterized in that R _{3 is} linked via Y-CH ₂ O, Y-COO or Y-NHC (O) -, wherein Y is a direct bond or a C1 to C8 Alkyl group, preferably a C1 to C3 alkyl group. 6. Substance according to one of the preceding claims, characterized in that at least one of the two substituents Ri or R ₂ carries a protective group. 7. Substance according to Claim 6, characterized in that the protective group is an acyl group, preferably an acetyl group, phosphate group or a succinyl group. 8. A preparation comprising an effective amount of substance according to any one of claims 1 to 7, dissolved in a physiologically acceptable carrier, wherein the carrier is a water or an organic solvent-based carrier, and optionally a surfactant. 9. A preparation according to claim 8, characterized in that the carrier is water and that the substance was optionally pre-dissolved in a solubilizer. 10. A preparation according to any one of claims 8 or 9, characterized in that it contains the substance to 0.5 to 200 uM. 11. A preparation according to any one of claims 8 to 10, characterized in that it is in injectable form. 12. A preparation according to any one of claims 8 to 11, characterized in that it is in the form of a dispersion and the substance is contained in the form of micelles or liposomes. 13. Use of a substance according to any one of claims 1 to 7 for the preparation of an injection solution for organ protection.