Composition for the preservation and fixation of organs, tissues, whole body specimens and corpses

Abstract

Use of a composition containing a 5-oxo-pyrrolidine-2-carboxylic acid derivative with formula IIa for the fixation and optional preservation of biological organs and / or tissues and / or whole body specimens and / or corpses.

Description

[0001] The present invention relates to a composition for preserving and fixing organs, tissues, whole body specimens and corpses, and a compound contained therein.

[0002] The modulation, and in particular the inhibition, of protein activity is of central importance in various fields of science, medicine, and technology.

[0003] In cell and tissue preservation, it is crucial for the long-term storage of such preparations that the activity of proteins with functional or catalytic roles is completely inactivated to prevent tissue degradation. At the same time, structural proteins must remain largely intact to maintain the integrity and shape of the biological material. Similarly, in the preservation of pharmaceutical preparations, food, and feed products, the inhibition of functional and non-structural proteins is essential for long-term shelf life.

[0004] Furthermore, the inactivation of functional proteins plays a central role in the field of technical fermentation and bioprocess engineering, life and agricultural sciences, as well as in medical applications, for example in connection with the inactivation of toxins, hormones or inflammatory mediators.

[0005] In the prior art, aldehydes such as formaldehyde or detergents with proven denaturing effects on proteins, such as sodium dodecyl sulfate (SDS) or other ionic surfactants with a chain length of C are used to modulate or inhibit the activity of non-structural proteins, especially enzymes. 12 Aldehydes are used. However, they exhibit high acute toxicity. They are considered carcinogenic, mutagenic, and highly sensitizing. Therefore, aldehydes have recently come under renewed scrutiny. Furthermore, aldehydes not only have a significant effect on proteins with enzymatic activity but also on structural proteins, altering their structure through cross-linking. SDS and ionic surfactants with a chain length of C 12 They tend to precipitate and thus lose their effectiveness in environments with increased electrolyte concentration, especially in combination with metal ions such as Ca ++ , Mg ++ or K +They are therefore unsuitable for use in complex biological systems. Furthermore, strongly denaturing detergents are considered highly sensitizing and thus harmful to health. Detergents that are not strongly denaturing, such as fatty alcohols like Triton X100, are not suitable for inactivating functional proteins and are therefore used for protein insolubilization. The following documents and sources should also be cited as state of the art: Römpp Lexikon Chemie, entry: “Non-structural protein” (URL: https: / / roempp.thieme.de [accessed on: 05 / 06 / 15]); B. MEYER; C. KLUIN: Efficiency of Glucoprotamin®-containing disinfectants against different species of atypical Mycobacteria. In: J. Hosp. Infect. Vol. 42(2), 1999, pp. 151-154; F. von RHEINBABEN; B. MEYER: Glucoprotamine. In: A. KRAMER and O. ASSADIN (eds.), Wallhäußer's Practice of Sterilization, Disinfection, Antisepsis and Preservation, 1999, pp. 786-787, 6.Edition, Stuttgart, Georg Thieme Verlag KG, DE 196 03 977 A1, WO 2012 / 028 196 A1, JP 51- 035 433 A, JP 09- 315 946 A, JP 2009- 215 267 A, US 4 053 588 A and EP 0 156 275 A2.

[0006] Against this background, it is an object of the present invention to provide a new composition or active ingredient that is suitable for the preservation and fixation of organs, tissues, whole body preparations and corpses, but does not have the disadvantages of the compositions or compounds currently used in the prior art.

[0007] This task is solved by using a composition containing a 5-oxo-pyrrolidine-2-carboxylic acid derivative of formula II for the preservation and / or fixation of biological organs and / or tissues and / or whole body specimens and / or corpses.

[0008] This problem is further solved by the use of a 5-oxo-pyrrolidine-2-carboxylic acid derivative with the formula (II) given above.

[0009] As the inventors discovered, a 5-oxo-pyrrolidine-2-carboxylic acid derivative exhibits excellent properties for modulating the activity of non-structural proteins. At the same time, the 5-oxo-pyrrolidine-2-carboxylic acid derivative is significantly less hazardous to health than the aldehydes, detergents, or surfactants currently in use.

[0010] A particular advantage is that the observed effects are dose-dependent. The modulation of the activity of non-structural proteins can therefore be precisely controlled to the desired strength and duration, thus opening up a wide range of applications.

[0011] 5-Oxo-pyrrolidine-2-carboxylic acid derivatives can be produced using methods generally known in the prior art, such as the method described in DE 3 410 956. Furthermore, the inventors were able to develop a new method for producing 5-oxo-pyrrolidine-2-carboxylic acid derivatives that involves even less production effort.

[0012] Non-structural proteins are defined as proteins that do not serve as structural components in the tissues or cells of living organisms. Therefore, non-structural proteins include, in particular, proteins with a catalytic function, such as enzymes, but also peptides, peptide hormones, receptors, cytokines, peptide toxins, etc.

[0013] According to the inventors, the invention is also suitable for modulating the catalytic activity of nucleic acid molecules, such as ribozymes, aptamers, siRNA, etc., as well as the activity of steroid hormones.

[0014] The term "modulation" refers to a change. Therefore, "modulation of the activity of non-structural proteins" refers to the targeted modification of the function of proteins as they function in their natural environment.

[0015] The features, properties, advantages and further developments of the composition according to the invention apply accordingly to the 5-oxo-pyrrolidine-2-carboxylic acid derivative.

[0016] The problem underlying the invention is hereby completely solved.

[0017] Modulation can involve inhibition, preferably deactivation.

[0018] This measure has the advantage that protein activities, for example of an enzymatic or catalytic nature, are selectively reduced or even switched off, while structural proteins remain able to give cells their shape and tissues their strength and elasticity. This is of crucial importance, particularly in the field of cell and tissue preservation.

[0019] The non-structural protein could be an enzyme.

[0020] This measure also has the advantage that, with the composition according to the invention or the 5-oxo-pyrrolidine-2-carboxylic acid derivative according to the invention, catalytic activities are specifically inhibited or switched off, whereas the structural proteins can still exert their biomechanical and physical properties.

[0021] The enzyme can be selected from the group consisting of: oxidoreductases, such as alcohol dehydrogenases; hydrolases, such as alkaline phosphatase, endoproteases, RNases, DNases, lipases; transferases; lyases; isomerases; ligases.

[0022] This measure has the advantage that it specifically inhibits or completely eliminates enzymes that are of particular importance in the field of preservation or medical implications.

[0023] The 5-oxo-pyrrolidine-2-carboxylic acid derivative is glucoprotamine with formula II:

[0024] This measure involves the use of a 5-oxo-pyrrolidine-2-carboxylic acid derivative, which, according to current technology, has primarily been used as a surface disinfectant in everyday clinical practice. Glucoprotamine is a multi-component substance. The two most important components and main active substances of glucoprotamine are (2S)-pyrrolidine-5-oxo-carboxylic acid amide, N-3-(dodecylamino)propyl, and (2S)-pyrrolidine-5-oxo-carboxylic acid amide, N-3-(tetradecylamino)propyl. Glucoprotamine (CAS No. 164907-72-6) is also known as the amine N-C12-14-alkylpropylenedi-,L-glutamate. In formula (II), the brackets indicate that the alkyl groups can be longer- or shorter-chain, in particular C12 (IIa) and C14 (IIb), which are present in a mixture. With a C12 group, glucoprotamine has the structural formula C 20 H 39 N3O2 and the molecular weight 353.55, with a C14 residue the structural formula C 22 H 43N3O2 and the molecular weight 381.61.

[0025] Glucoprotamine is characterized by its insensitivity to high protein loads, low toxicity and ecotoxicity, and rapid and complete biodegradability. It is readily water-soluble and stable over long periods. In one study, no loss of efficacy was observed even after eight years of storage. It is distributed, for example, by Ecolab Deutschland GmbH, Düsseldorf.

[0026] The suitability of glucoprotamine discovered according to the invention was surprising and not expected. Prior art postulates the destruction of the cytoplasmic membrane of bacterial cells and the envelope of enveloped viruses as the mechanism of disinfection; Meyer and Kluin (1999), Efficiency of glucoprotamine-containing disinfectants against different species of atypical microbacteria, J. Hosp. Infect. 42(2), pp. 151-154. The efficacy against naked lipophilic viruses is explained by an interaction with lipophilic groups in the protein capsid of the particles; cf. von Rheinbaben and Meyer (2008), Glucoprotamine. In: Kramer and Assadin (eds.), Wallhäußer's Practice of Sterilization, Disinfection, Antisepsis and Preservation, 6th edition, Stuttgart, New York: Georg Thieme Verlag KG, pp. 786-787.

[0027] The suitability of glucoprotamine according to the invention is neither described nor suggested in the prior art.

[0028] The composition according to the invention, or the 5-oxo-pyrrolidine-2-carboxylic acid derivative, is intended for use as a preservative and / or fixative.

[0029] The inventors have demonstrated that the composition, specifically the 5-oxo-pyrrolidine-2-carboxylic acid derivative, is suitable for the aldehyde-free preservation of tissues, organs, and whole-body specimens. The comprehensive and simultaneous inactivation of several enzyme classes involved in tissue lysis enables a novel method of preserving biological material. In their experiments, the inventors were able to show that both organ and whole-body specimens retain their structural integrity virtually unchanged after treatment with the composition according to the invention. Even after several months, whole-body specimens could be preserved with a realistic feel and unprecedented quality.

[0030] The property recognized by the inventors makes the composition, or rather the 5-oxo-pyrrolidine-2-carboxylic acid derivative, ideal as a preservative for cosmetic, food or pharmaceutical products.

[0031] The inhibitory effect on non-structural proteins, as observed by the inventors, makes the composition, or rather the 5-oxo-pyrrolidine-2-carboxylic acid derivative, particularly suitable for use as a pharmaceutical. The composition according to the invention, or rather the 5-oxo-pyrrolidine-2-carboxylic acid derivative, can be used to specifically inhibit the activity of hormones, antibodies, cytokines, interferons, interleukins, chemokines, growth factors, colony-stimulating factors, tumor necrosis factors, receptors, ribozymes, and other disease-mediating enzymes.

[0032] The invention can be used specifically in the treatment of chronic inflammatory diseases, for example by creating a deinflammatory environment, such as in chronic rheumatoid arthritis. This can be achieved, for example, by intra-articular injection of the composition according to the invention or of the 5-oxo-pyrrolidine-2-carboxylic acid derivative, or by lavage with it. Further examples include chronic inflammatory bowel diseases such as Crohn's disease or ulcerative colitis, chronic obstructive pulmonary disease, chronic skin and mucous membrane diseases, or autoimmune diseases.

[0033] The invention is also used in cases of acute inflammation, for example in the context of wound debridement or lavage.

[0034] The composition according to the invention or the 5-oxo-pyrrolidine-2-carboxylic acid derivative can also be used in the fields of oncology, immunology and allergology, for example for cytokine or chemokine inactivation or for the inactivation of hormones.

[0035] The composition according to the invention, or the 5-oxo-pyrrolidine-2-carboxylic acid derivative, can also be used in the fields of cosmetics, personal care, and anti-aging. For example, it can be used to inactivate odor-causing enzymes and thus as a component of deodorants. Furthermore, the composition according to the invention, or the 5-oxo-pyrrolidine-2-carboxylic acid derivative, can be used to selectively inactivate elastases and / or collagenases, thereby preventing signs of skin aging. The composition according to the invention, or the 5-oxo-pyrrolidine-2-carboxylic acid derivative, can therefore be used as a component of creams, ointments, lotions, or other topically applied formulations.

[0036] The composition or the 5-oxo-pyrrolidine-2-carboxylic acid derivative can be used as a biocide, preferably selected from the group consisting of: insecticide, ovicide, acaricide, molluscicide, nematicide, anthelmintic, herbicide, algicide, graminicide and arboricide.

[0037] This measure has the advantage that the composition, or rather the 5-oxo-pyrrolidine-2-carboxylic acid derivative, can be used for targeted applications in life sciences and agriculture, where it can exert a specific effect due to its modulating or inhibitory effect on non-structural proteins, such as enzymes. Through the targeted inhibition of its non-structural proteins, the organism's metabolic activity is suppressed, and it thus loses its damaging properties.

[0038] The composition according to the invention or the 5-oxo-pyrrolidine-2-carboxylic acid derivative according to the invention can be used as a fermentation aid.

[0039] This measure enables the targeted application of the invention in food and beverage production. The targeted inhibition of fermentation enzymes allows for precise control of the fermentation process. Use in industrial fermentation and bioprocess engineering is also possible, particularly in bioethanol, chemical, or pharmaceutical production, silage technology, biogas plants, or bioreactor systems.

[0040] The composition according to the invention, or the 5-oxo-pyrrolidine-2-carboxylic acid derivative, can also be used in the field of tissue engineering to create an enzyme-free deinflammatory environment.

[0041] The composition, or the 5-oxo-pyrrolidine-2-carboxylic acid derivative, can be formulated for use as a surface coating agent.

[0042] For example, functional groups such as OH, SH, or photoreactive groups can be attached to positions 3 and 4 of the pyrrolidine ring. This allows the use of the 5-oxo-pyrrolidine-2-carboxylic acid derivative or the composition according to the invention as a surface coating in medical and biotechnological product development and application. In this context, coated medical devices such as suture materials, stents, joint implants, and wound dressings are particularly relevant. The coating can be achieved, for example, via adhesion, but also via covalent bonding or immobilization of the 5-oxo-pyrrolidine-2-carboxylic acid derivative.

[0043] It is understood that the features mentioned above and those to be explained below can be used not only in the combination specified, but also in other combinations or on their own, without leaving the scope of the present invention.

[0044] The present invention will now be explained in more detail with reference to exemplary embodiments, from which further properties, features, and advantages will become apparent. These exemplary embodiments are purely illustrative and do not limit the scope of the invention.

[0045] Reference is made to the attached illustrations, which depict the following: Fig. 1: Reaction sequence of the alcohol dehydrogenase (ADH) reaction. ADH is completely inhibited by glucoprotamine (squares; n=3; error bars=SEM). The kinetics of the control without glucoprotamine (circles; n=3; error bars=SEM) show that the substrate / coenzyme used in the test was present at a saturating concentration. Fig. 2: Concentration-dependent modulation of alkaline phosphatase (ALP) by glucoprotamine. Compared to a positive control without glucoprotamine (ALP positive), a 20-minute incubation with glucoprotamine (GP) leads to inactivation of ALP depending on its concentration (n=3; error bars=SEM). Fig. 3: Inactivation of endoproteases by glucoprotamine compared to formalin and alcohol / glycerol. Treatment of various rat organs (Wistari rat; postnatal, 3 days) with glucoprotamine leads to inactivation of endoproteases; as with formalin treatment, no normalized relative fluorescence activity (RFU / min / mg protein) could be measured. With alcohol / glycerol treatment, endoprotease activity could still be determined in the kidney and colon (n=3). Fig. 4: Real-time determination of RNase inactivation by glucoprotamine. (A) Treatment with glucoprotamine leads to inactivation of RNase A (squares; n=3, error bars=SEM) compared to untreated RNase A (circles, n=3, error bars=SEM). (B) Glucoprotamine inactivates the RNases (squares, n=3, error bars=SEM) of a contaminated work surface (positive control; circles, n=3, error bars=SEM). This inactivation is comparable to the decontamination effect of commercially available solutions: (C) RNaseZap (triangles, n=3, error bars=SEM) and (D) RNase-ExitusPlus (triangles, n=3, error bars=SEM). (B)-(D) Positive control (circles) work surface treated with nuclease-free water. Fig. 5: Real-time determination of lipase inactivation by glucoprotamine. (A) Glucoprotamine inactivates bovine lipoprotein lipase (squares, n=3, error bars = SEM), while in the positive control, lipase activity increases relatively over time (circles, n=3, error bars = SEM). (B) Glucoprotamine inactivates human lipases from subcutaneous adipose tissue (squares, n=3, error bars = SEM). The activity of the lipases extracted from human adipose tissue increases relatively over time (circles, n=3, error bars = SEM). Fig. 6: Preservation of rat organs (Wistar, postnatal, 31 days) with glucoprotamine compared to preservation with formalin. Fig. 7: Opened thorax situs of a body donor after transarterial infusion with glucoprotamine, 7 months post mortem. Fig. 8: Opened left ventricle of a body donor after transarterial infusion with glucoprotamine, 7 months post mortem. Examples of implementation: 1. Preparation of the 5-oxo-pyrrolidine-2-carboxylic acid derivative

[0046] The production of the 5-oxo-pyrrolidine-2-carboxylic acid derivative is described using the example of glucoprotamine in DE 34 10 956, the contents of which are incorporated by reference into the present application.

[0047] Furthermore, the inventors have developed an improved process for producing a 5-oxo-pyrrolidine-2-carboxylic acid derivative.

[0048] The reaction takes place with the starting materials. I. 5-Oxo-pyrrolidine-2-carboxylic acid derivatives (preferably the S-enantiomer, but also the R-enantiomer or racemate) and II. N-substituted monoamines (2.3) and / or diamines (2.2) and / or fatty amides (2.3) instead of. a) Reaction of 5-oxo-pyrrolidine-2-carboxylic acid derivatives with N-substituted monoamides Definition of residues: R1 = linear alkyl group with C 1-6; preferably C1; R2 = linear alkyl group with chain length C 2-22 , where monounsaturated and polyunsaturated alkyl groups are included. Scheme 1: Reaction of the 5-oxo-pyrrolidine-2-carboxylic acid derivative with N-substituted monoamides

[0049] The reaction requires only a preferred temperature of 60°C, a preferred reaction time of 60 minutes, and a preferred pressure of 300-350 mbar. Methanol is distilled off. b) Reaction of 5-oxo-pyrrolidine-2-carboxylic acid derivatives with N-substituted diamines. Definition of the residues: n = 1-6, R1 = linear alkyl group with C 1-6 ; preferably C1; R2 = linear alkyl or acyl group with chain length C 2-22 , including monounsaturated and polyunsaturated alkyl and acyl groups. Scheme 2: Reaction of 5-oxo-pyrrolidine-2-carboxylic acid derivatives with N-substituted diamines

[0050] The reaction requires only a preferred temperature of 60°C, a preferred reaction time of 60 minutes, and a preferred pressure of 300-350 mbar. Methanol is distilled off. c) Reaction of 5-oxo-pyrrolidine-2-carboxylic acid derivatives with N-substituted fatty amides. Definition of the residues: R1 = linear alkyl group with C 1-6 ; preferably C1 R2 = linear alkyl group with chain length C 2-24 , where monounsaturated and polyunsaturated alkyl groups are included. Scheme 3: Reaction of 5-oxo-pyrrolidine-2-carboxylic acid derivatives with N-substituted fatty amides

[0051] The reaction requires only a preferred temperature of 60°C, a preferred reaction time of 60 minutes, and a preferred pressure of 300-350 mbar. Methanol is distilled off.

[0052] Using this new method, the inventors were able to successfully produce the 5-oxo-pyrrolidine-2-carboxylic acid derivative called glucoprotamine.

[0053] The two active substances (2S)-pyrrolidine-5-oxo-carboxylic acid amide, N-3-(dodecylamino)propyl and (2S)-pyrrolidine-5-oxo-carboxylic acid amide, N-3-(tetradecylamino)propyl, subsumed under the active ingredient glucoprotamine, are not produced in the process according to the invention by reaction of the linear starting material L-glutamic acid or its ester derivatives and the fatty amine mixture dodecyl / tetradecylpropylidenediamine, also known as cocospropylene-1,3-diamine, but by reaction of the already cyclically present starting material 5-oxo-pyrrolidine-2(S)-carboxylic acid methyl ester, also known as L-pyrroglutamate methyl ester, with cocospropylene-1,3-diamine at only about 60°C, about 60 min. and about 300 to 350 mbar. Distillation of methanol. Scheme 4: Synthesis of glucoprotamine from 5-oxo-pyrrolidine-2(S)-carboxylic acid methyl ester and cocopropylene-1,3-diamine

[0054] For this purpose, 251 g (1 mol) of cocospropylene-1,3-diamine (CAS No. 6171-63-7) (70 mol% dodecylpropylidenediamine, 30 mol% tetradecylpropylidenediamine) were melted in a water bath at 60°C. Subsequently, 143.14 g (1 mol) of methyl 5-oxo-pyrrolidine-2(S)-carboxylate were added and the reaction was carried out in a rotary evaporator at 60°C for 1 hour at a reduced pressure of 330 mbar. The methanol (32 g) produced during the reaction was distilled off. The reaction product was liquid-viscous at 60°C and solidified at room temperature to a beige-yellow, waxy paste. The melting point of the reaction product is 60–70°C.

[0055] Analysis of the synthesized product confirms the substance glucoprotamine. High-resolution mass spectrometry results showed a mass deviation of only 0.01 to 0.04 ppm of the test substance from the theoretical masses of [M+H]+ = 253 g / mol (2S)-pyrrolidine-5-oxo-carboxylamide, N-3-(dodecylamino)propyl and [M+H]+ = 286 g / mol (2S)-pyrrolidine-5-oxo-carboxylamide, N-3-(tetradecylamino)propyl.

[0056] A 1H and 13C NMR structural analysis showed agreement with the theoretically predicted spectra (Scifinder / ChemDraw 13.0).

[0057] Due to its mild reaction conditions, the new manufacturing process allows the use of long-chain and / or unsaturated, and therefore vulnerable, carbon chains without the risk of decomposition or oxidation during the synthesis reaction. Furthermore, the new process allows the use of pre-prepared chemical starting materials. Even complex chemical reactions are possible without interfering with the synthesis reaction. d) Modification of 5-oxo-pyrrolidine-2-carboxylic acid derivatives

[0058] The starting materials 5-oxo-pyrrolidine-2-carboxylic acid derivatives and N-substituted monoamine, diamine and fatty amide derivatives are to be modified with the aim of influencing the drug kinetics, drug dynamics and drug performance of the synthesis products.

[0059] The starting substance 5-oxo-pyrrolidine-2-carboxylic acid can be modified at positions 3 and 4 of the pyrrolidine ring.

[0060] For example, the attachment of protected hydroxyl or sulfhydryl groups to positions 3 and / or 4 of the pyrrolidine ring enables the coupling of the active substance to the surfaces of various materials. This allows the manufactured active substances to be used as surface coatings.

[0061] Scheme 5: Modification of the 5-oxo-pyrrolidine-2-carboxylic acid derivative by attachment of (a) hydroxyl and / or (b) sulfhydryl groups. An example of a hydroxyl group at position 4 and a sulfhydryl group at position 3 is shown. R1 represents a linear alkyl group with C1-6; preferably C1.

[0062] The attachment of, for example, photoreactive groups can enable a conditioned modification of the active substance.

[0063] Scheme 6: Modification of the 5-oxo-pyrrolidine-2-carboxylic acid derivative by attachment of photoreactive groups. Two examples are shown at positions 3 and 4. R1 represents a linear alkyl group with C1-6; preferably C1.

[0064] The mild new manufacturing process also allows, for example, the use of alkyl groups of different chain lengths or even in unsaturated form.

[0065] Condition. Modifying the chain length can lead to an altered drug profile. (1) diamines, (2) monoamines, (3) fatty amides with R2 = • linear alkyl group with a chain length of C 2-22 • mono- and / or polyunsaturated alkyl groups • in (1) also singly and / or polyunsaturated acyl groups • n = 1-6 Scheme 7: Representation of modified monoamine, diamine, and fatty amide groups. 2. Modulation / Inactivation of representatives of various enzyme classes by glucoprotamine. 2.1 Oxidoreductases (enzyme class 1; EC1) Inactivation of alcohol dehydrogenase (ADH; EC1). 1.1.1

[0066] Measurement principle: The enzyme alcohol dehydrogenase (ADH) catalyzes the reversible conversion of ethanol to acetaldehyde with simultaneous reduction of nicotinamide adenine dinucleotide (NAD). +) to NADH. While the conversion of alcohol to aldehyde is not directly detectable, the formation of NADH can be monitored optically using a photometer. The increase in absorbance at 340 nm is a direct measure of the alcohol conversion and thus of ADH activity. To test the inactivation of ADH by glucoprotamine, a commercially available NAD-ADH reagent (NAD-ADH reagent multiple test vial; Sigma-Aldrich; Germany) was mixed with 2.6% glucoprotamine and incubated for 10 minutes at 37°C. Subsequently, 1% ethanol was added to the mixture as a substrate, and the ADH activity at 340 nm was immediately determined using an Infinite M200 microplate reader (Tecan, Switzerland). The resulting ADH kinetics were plotted against a control without glucoprotamine ( Fig. 1) The results of the colorimetric analysis presented in the ADH kinetics show that glucoprotamine completely inactivates ADH. 2.2 Hydrolases (Enzyme class 3; EC3) Inactivation of alkaline phosphatase (EC3.1.3.1)

[0067] Measurement principle: Alkaline phosphatase (ALP) catalyzes the hydrolysis of phosphate esters under alkaline conditions to organic radicals and inorganic phosphates. ALP activity is detected by the cleavage of p-nitrophenyl phosphate (pNPP). The formation of the yellow nitrophenolate can be directly monitored by measuring the absorbance at 405 nm.

[0068] To test the inactivation of ALP by glucoprotamine, a commercially available test system (Alkaline Phosphatase Assay Kit; Abcam, USA) was used. For this purpose, the ALP contained in the kit was incubated in four different batches with 2.6%, 5.2%, 7.8%, and 10.4% glucoprotamine for 20 minutes at 37°C. Simultaneously, a positive control was prepared with the same enzyme concentration but without glucoprotamine and incubated and measured in the same way as the other batches. After the addition of 1 mM pNPP, the batches were incubated for a further 30 minutes, and the absorbance at 405 nm was then determined using an Infinite M200 microplate reader. Fig. 2) The values ​​from this end-time determination show an inactivation of ALP by glucoprotamine, which is concentration-dependent. Inactivation of endoproteases

[0069] Measurement principle: The activity of endoproteases was also determined using a fluorescence resonance energy transfer (FRET) peptide library containing over 2.5 million peptides (Kapprell et al. (2011), Assay and Drug Development Technologies). The principle of this protease detection is based on the MCA fluorophore as a donor and the 2,4-dinitrophenyl residue as a quencher, which are coupled to the peptides. As soon as the protease cleaves the peptides, the donor and the quencher are separated, resulting in a strong fluorescence signal. The protease activity is thus directly proportional to the increase in the relative fluorescence intensity.

[0070] To determine the inactivation of endoproteases by glucoprotamine, nine different rat organs (Wistar rats, postnatal, day 3) were incubated (three of each) with either 2.6% glucoprotamine, 4% formalin, or an alcohol / glycerol solution (70% alcohol / 30% glycerol). These organs were then homogenized in Tris buffer (pH 7.4). Unfixed, freshly prepared rat organs (n=3) served as positive controls. After determining the protein concentration (Qubit Protein Assay, Life Technologies, Germany), the samples were centrifuged, and 10 µl of the supernatant was mixed with 80 µl of Tris buffer and 10 µM of the FRET-based peptide library. The relative fluorescence intensity (RFU) was immediately determined using an Infinite M200 microplate reader.The relative fluorescence intensity per unit time (slope), normalized to the protein concentration, shows inactivation of endoproteases by glucoprotamine, similar to the inactivation of endoproteases by formalin. In contrast, alcohol / glycerol fixation in the kidney and colon did not lead to complete inactivation of endoproteases, but only to a reduction in protease activity. Fig. 3) In the positive control, endoprotease activity was detected in all organs. Inactivation of RNases

[0071] Measurement principle: The activity of RNases can be determined using a cleavable, fluorescently labeled RNase substrate according to the FRET principle (RNaseAlert Lab Test Kit; Applied Biosystems, Germany). The substrate is a modified RNA oligonucleotide that emits green fluorescence when cleaved by RNases. The RNase activity is therefore directly proportional to the increase in fluorescence intensity.

[0072] Two different procedures were chosen to determine the inactivation of RNases by glucoprotamine. First, 5 µl of RNase A (approx. 2 pg) from a commercial test system (RNaseAlert Lab Test Kit) was mixed with 2.6% glucoprotamine according to the manufacturer's instructions and incubated for 10 minutes at 37°C. The positive control was treated with nuclease-free water instead of glucoprotamine and otherwise identically. After adding the fluorescently labeled substrate, the RNase activity was determined in real time using an Infinite M200 microplate reader. Over the 20-minute measurement period, no increase in relative fluorescence intensity (RFU) was detected in the glucoprotamine-treated sample, thus demonstrating the inactivation of RNase A by glucoprotamine. Fig. 4A). In the positive control, a constant substrate-saturated RNase activity was observed.

[0073] As a next step, a laboratory work surface was contaminated with human sweat and saliva. The work surface was then divided into four equally sized areas, and these were treated differently: nuclease-free water (positive control), 2.6% glucoprotamine ( Fig. 4B), RNaseZap (commercial RNase decontamination solution from Blyde Biosystems; Fig. 4C) and RNase-ExitusPlus (commercial RNase decontamination solution from AppliChem, Germany; Fig. 4D). After a 10-minute incubation, the treated surfaces were rinsed twice with 500 ml of nuclease-free water. After the surfaces had dried, 500 µl of nuclease-free water was evenly distributed on each work surface, incubated for one minute, and then removed using a pipette. 45 µl of these four different preparations were labeled with the fluorescently labeled substrate, and the change in fluorescence intensity, and thus the RNase activity, was determined in real time using an Infinite M200 Microplate Reader (Tecan, Switzerland). Fig. 4 BD). Treating work surfaces with glucoprotamine leads to the inactivation of RNases present in sweat and saliva, thus decontaminating the work surface. This complete decontamination is comparable to that achieved with commercially available decontamination solutions. On work surfaces treated only with nuclease-free water...

[0074] In contrast, the positive control showed an increase in fluorescence intensity over time, indicating high RNase activity. Inactivation of lipases

[0075] Measurement principle: The lipase hydrolyzes arachidonoyl-1-thioglycerol to arachidonic acid and thioglycerol. Thioglycerol reacts with the thiofluorometric detector to form a strongly fluorescent product, which can be analyzed at an excitation wavelength of 380 to 390 nm and an emission wavelength of 510 to 520 nm.

[0076] To test the inactivation of lipases by glucoprotamine, a commercial test system was used according to the manufacturer's instructions (Lipase Activity Assay, Cayman Chemical Company, USA). Two different test procedures were performed. In the first, 10 µl of bovine milk lipoprotein lipase was mixed with 2.6% glucoprotamine, and assay buffer and thiol detector were added according to the manufacturer's instructions. In the positive control, glucoprotamine was omitted. Both solutions were incubated for 15 minutes at 37°C. Subsequently, the lipase substrate was added, and the lipase activity was determined in real time by detecting the change in fluorescence intensity (RFU). Fig. 5A). Bovine milk lipoprotein lipases are inactivated by glucoprotamine. In a second test procedure, 200 mg of human gluteal subcutaneous adipose tissue was harvested post-mortem and homogenized with ice-cold PBS using a Precellys ceramic kit 1.4 / 2.1 mm (PeqLab, Germany) in a minilyse workstation homogenizer (PeqLab) for 4 x 10 seconds at 5,000 rpm. The homogenate was then centrifuged for 10 minutes at 10,000 x g, and 10 µl of the middle phase was withdrawn. This sample was either treated with 2.6% glucoprotamine or added directly (positive control) to the assay buffer and the thiol detector. Both solutions were incubated for 15 minutes at 37°C, and the fluorescence intensity was determined in real time as described above. Fig. 5B). Glucoprotamine inactivates human lipases from subcutaneous adipose tissue. 3. Preservation of rat organs

[0077] To demonstrate the preservative effect of glucoprotamine, organs were harvested from adult Wistar rats (postnatal, 31 days) and photographed. The organs were then incubated for 7 days in either 2.6% glucoprotamine or 4% formalin. Afterward, the organs were stored uncovered at room temperature for a further 25 days without preservative solutions and then photographed and evaluated again. Fig. 6) Glucoprotamine-preserved organs exhibited comparable preservation to formalin-fixed organs, but retained their tissue elasticity. 4. Preservation of a human whole-body specimen

[0078] After obtaining the results of the investigations at the individual enzyme level as well as the results of the organ fixation in the animal model (immersion fixation after organ removal), in accordance with the practice of anatomical body donation (Ethics Committee vote 237 / 2007B01 and Burial Act Baden-Württemberg of July 21, 1970) and analogous to formalin fixation, the body of a female body donor was infused intra-arterially via the femoral artery with 17 liters of a 2.5% glucoprotamine / 20% ethanol solution at a pump capacity of 1 bar.

[0079] The whole-body specimen was wrapped in a moist cloth soaked in the aforementioned solution and sealed in foil. Tissue consistency and condition were checked after 2 weeks, 1 month, 3 months, and 7 months. Inspection after 2 weeks and 1 month

[0080] There were no signs of lysis, the tissue structure remained intact, and the haptics, unlike those obtained with formalin fixation, are realistic. Invasive examination after 3 months

[0081] A diagnostic laparoscopy was performed, followed by open surgical examination via a midline laparotomy. Results: The gastrointestinal tract appeared intact with no signs of prior lysis. A microbiological swab examination revealed no sterility in the intraperitoneal space of the colon and the abdominal recesses.

[0082] As part of an orthopedic surgery course, an arthroscopy of the shoulder joint was performed. The joint space was found to be realistically preserved, and the ligament and cartilage structures felt realistic to the touch. Invasive examinations after 7 months

[0083] After 7 months, an anatomical lid dissection was performed ( Fig. 7 and Fig. 8) to open and inspect the entire abdominal and thoracic cavities. All organs appeared intact. Even organs with a naturally high content of digestive enzymes, such as the pancreas, retained their morphological integrity. The left side of the heart was opened and the endocardial space inspected ( Fig. 8) Even fine structures, e.g., valves, chordae tendineae, were preserved in a realistic manner.

[0084] Even after 7 months, good structural integrity was evident, with no signs of lysis having taken place. 5. Conclusion

[0085] The inventors have developed a versatile active ingredient, a 5-oxo-pyrrolidine-2-carboxylic acid derivative such as glucoprotamine, which can be used to specifically modulate, and preferably inhibit, the activity of non-structural proteins. This substance is significantly less harmful to health than currently used aldehydes, detergents, and surfactants.

Claims

[1] Use of a composition containing a 5-oxo-pyrrolidine-2-carboxylic acid derivative having formula IIa for the fixation and optional preservation of biological organs and / or tissues and / or whole body specimens and / or corpses. [2] Use of a composition containing a 5-oxo-pyrrolidine-2-carboxylic acid derivative having formula IIb for the fixation and optional preservation of biological organs and / or tissues and / or whole body specimens and / or corpses. [3] Use of a 5-oxo-pyrrolidine-2-carboxylic acid derivative with formula IIa for the fixation and optional preservation of biological organs and / or tissues and / or whole body specimens and / or corpses. [4] Use of a 5-oxo-pyrrolidine-2-carboxylic acid derivative with formula IIb for the fixation and optional preservation of biological organs and / or tissues and / or whole body specimens and / or corpses.

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