Composition for the modulation of the activity of non-structure proteins

Abstract

The present invention relates to a composition for the modulation of the activity of non-structure proteins and a compound contained therein.

Claims

1. A method for the preservation and fixation of protein-containing subject-matter, comprising the step of contacting said subject-matter with a composition comprising a 5-oxo-pyrrolidine-2-carboxylic acid derivative of formula I ##STR00012## wherein R.sub.1 is selected from the group consisting of: linear alkyl residue with C.sub.1-C.sub.6, ##STR00013## R.sub.2 and R.sub.3 are each independent from each other selected from the group consisting of: H, XH, ##STR00014## with each: n=0-20, m=1-25, and X=O or S.

2. The method of claim 1, wherein said subject-matter is biological material.

3. The method of claim 2, wherein said biological material is selected from the group consisting of: tissues, organs, full bodies, dead bodies.

4. The method of claim 1, wherein said subject-matter is selected from the group consisting of: cosmetics, food products, animal feed products, and pharmaceutical products.

5. The method of claim 1, wherein said 5-oxo-pyrrolidine-2-carboxylic acid derivative comprises a compound of formula II: ##STR00015##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Reaction process of the reaction of alcohol dehydrogenase (ADH). The ADH is completely inhibited by glucoprotamin (squares; n=3; error bars=SEM). The kinetics of the control without glucoprotamin (circles; n=3; error bar=SEM) show that the substrate/coenzyme which were used in the test were present in saturating concentration.

(2) FIG. 2: Concentration-depending modulation of the alkalic phosphatase (ALP) by glucoprotamin. In comparison with the positive control without glucoprotamin (ALP positive) an incubation with glucoprotamin (GP) for 20 minutes results in an inactivation of ALP in dependence of its concentration (n=3; error bars=SEM).

(3) FIG. 3: Inactivation of the endoproteases by glucoprotamin in comparison with formalin and alcohol/glycerol. The treatment of various rat organs (Wistar rat; postnatal, 3 days) with glucoprotamin results in an inactivation of the endoproteases; no normalized, relative fluorescence activity (RFU/min/mg protein) could be measured like in the treatment with formalin. In the treatment with alcohol/glycerol still an activity of the endoproteases in the kidneys and the colon could be detected (n=3).

(4) FIG. 4: Realtime detection of the inactivation of the RNases by glucoprotamin. (A) The treatment with glucoprotamin results in an inactivation of the RNase A (squares; n=3, error bars=SEM) in comparison to untreated RNase A (circles, n=3, error bars=SEM). (B) Glucoprotamin inactivates RNAses (squares, n=3, error bars=SEM) of a contaminated work area (positive control; circles, n=3, error bars=SEM). This inactivation can be compared with 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 area treated with nuclease-free water.

(5) FIG. 5: Realtime detection of the inactivation of the lipases by glucoprotamin. (A) Glucoprotamin inactivates the bovine lipoprotein lipase (squares, n=3, error bars=SEM) whereas in the positive control the lipase activity relatively increases over the time (circles, n=3, error bars=SEM). (B) Glucoprotamin inactivates human lipases from subcutaneous fatty tissue (squares, n=3, error bars=SEM). The activity of the lipases obtained from the human fatty tissue relatively increases over the time (circles, n=3, error bars=SEM).

(6) FIG. 6: Preservation of organs from the rat (Wistar, postnatal, 31 days) with glucoprotamin in comparison to the preservation with formalin.

(7) FIG. 7: Opened thorax situs of a body donor after a transarterial infusion with glucoprotamin, 7 months post mortem.

(8) FIG. 8: Opened left heart ventricle of a body donor after transarterial infusion with glucoprotamin, 7 months post mortem.

EXAMPLES

1. Production of the 5-Oxo-Pyrrolidine-2-Carboxylic Acid Derivative

(9) The production of the 5-oxo-pyrrolidine-2-carboxylic acid derivative is described in the DE 34 10 956 by the way of the example of glucoprotamin, the content of this document is incorporated herein and made to the subject of the present application.

(10) In addition, the inventors have developed an improved method for the production of a 5-oxo-pyrrolidine-2-carboxylic acid derivative.

(11) The reaction takes place with the starting substances

(12) I. 5-oxo-pyrrolidine-2-carboxylic acid derivatives (preferably the S enantiomer, but also the R enantiomer or racemate) and

(13) II. N substituted monoamines (2.3) and/or diamines (2.2) and/or fatty amides (2.3).

(14) a) Conversion of 5-Oxo-Pyrrolidine-2-Carboxylic Acid Derivatives with N Substituted Monoamines

(15) ##STR00005##

(16) Definition of the Residues:

(17) R.sub.1=linear alkyl residue with C.sub.1-6; preferably C.sub.1;

(18) R.sub.2=linear alkyl residue with the chain length C.sub.2-22, wherein mono and polyunsaturated alkyl residues are included.

(19) Scheme 1: Conversion of the 5-Oxo-Pyrrolidine-2-Carboxylic Acid Derivatives with N Substituted Monoamines

(20) The reaction only requires a preferred temperature of 60 C., a preferred reaction time of 60 min and a preferred pressure of 300-350 mbar. Methanol is distilled off.

(21) b) Conversion of the 5-Oxo-Pyrrolidine-2-Carboxylic Acid Derivatives with N Substituted Diamines

(22) ##STR00006##

(23) Definition of the Residues:

(24) n=1-6,

(25) R.sub.1=linear alkyl residue with C.sub.1-6; preferably C.sub.1;

(26) R.sub.2=linear alkyl or acyl residue with the chain length C.sub.2-22, wherein mono and polyunsatured alkyl and acyl residues are included.

(27) Scheme 2: Conversion of the 5-Oxo-Pyrrolidine-2-Carboxylic Acid Derivatives with N Substituted Diamines

(28) The reaction only requires a preferred temperature of 60 C., a preferred reaction time of 60 min and a preferred pressure of 300-350 mbar. Methanol is distilled off.

(29) c) Conversion of the 5-Oxo-Pyrrolidine-2-Carboxylic Acid Derivatives with N Substituted Fatty Amides

(30) ##STR00007##

(31) Definition of the Residues:

(32) R.sub.1=linear alkyl residue with C.sub.1-6; preferably C.sub.1

(33) R.sub.2=linear alkyl residue with the chain length C.sub.2-24, wherein mono and polyunsaturated alkyl residues are included.

(34) Scheme 3: Conversion of the 5-Oxo-Pyrrolidine-2-Carboxylic Acid Derivatives with N Substituted Fatty Amides

(35) The reaction only requires a preferred temperature of 60 C., a preferred reaction time of 60 min and a preferred pressure of 300-350 mbar. Methanol is distilled off.

(36) With this new method the inventors succeeded in producing the 5-oxo-pyrrolidine-2-carboxylic acid derivative referred to as glucoprotamin.

(37) The two active substances subsumed under the active agent glucoprotamin, i.e. (2S)-pyrrolidine-5-oxo-carboxylic acid amid, N-3-(dodecylamino)propyl and (2S)-pyrrolidine-5-oxo-carboxylic acid amid, N-3-(tetradecylamino)propyl, in the method according to the invention are not generated by the reaction of the linear starting substance L-glutamic acid or its ester derivatives and the fatty amine mixture do-decyl/tetradecylpropylene diamine, also referred to as cocospropylene-1,3-diamine, but by the reaction of the already cyclic starting product 5-oxo-pyrrolidine-2(S)-carboxylic acid methylester, also referred to as L-pyroglutamic acid methyl ester, with cocospropylene-1,3-diamine only at approximately 60 C., approximately 60 min and approximately 300 to 350 mbar and by distilling off of methanol.

(38) ##STR00008##

(39) For this purpose, 251 g (1 mol) cocospropylene-1,3-diamine (CAS-No. 6171-63-7) (70 mol % dodecylpropylene diamine, 30 mol % tetradecylpropylene diamine) were melted in the water bath at 60 C. Then 143.14 g (1 mol) 5-oxo-pyrrolidine-2(S)-carboxylic acid methylester were added and brought to reaction at a reduced pressure of 330 mbar in a rotary evaporator for 1 hour at 60 C. The methanol generated in the reaction (32 g) was distilled off. The conversion product was liquid-viscose at 60 C. and solidified at room temperature to a beige-yellow waxy paste. The melting temperature of the conversion product is 60-70 C.

(40) The analysis of the synthesized product confirms the substance of glucoprotamin. The results of the high resolution mass spectrometry showed a mass deviation of the test substance of only 0.01 to 0.04 ppm of the theoretical masses of [M+H]+=253 g/mol (2S)-pyrrolidine-5-oxo-carboxylic acid amid, N-3-(dodecylamino)propyl and [M+H]+=286 g/mol (2S)-pyrrolidine-5-oxo-carboxylic acid amid, N-3-(tetradecylamino)propyl.

(41) A 1H and 13C NMR structure analysis showed the correspondence with the theoretically predicted spectra (Scifinder/ChemDraw 13.0).

(42) The new production method allows due to its mild reaction conditions the use of long chain and/or unsaturated and thus vulnerable carbon chains without running the risk of a disintegration or oxidation in the context of the synthesis reaction. Furthermore, in the new production method chemically assembled starting substances can be used. Also complex chemical reactions become possible without influencing the synthesis reaction.

(43) d) Modification of the 5-Oxo-Pyrrolidine-2-Carboxylic Derivatives

(44) The starting substances 5-oxo-pyrrolidine-2-carboxylic acid derivatives and N substituted monoamine, diamine and fatty amid derivatives should be modified with the objection to take influence on the kinetics of the active agent, the dynamics of the active agent, and the performance of the active agent of the synthesis products.

(45) The starting substance 5-oxo-pyrrolidine-2-carboxylic acid can be modified at the positions 3 and 4 of the pyrrolidine ring.

(46) The attachment of for example protected hydroxyl or sulfhydryl groups at the positions 3 and/or 4 of the pyrrolidine ring allows the coupling of the active substance to surfaces of various materials. This allows the use of the produced active agents as a surface coating.

(47) ##STR00009##

(48) The attachment of for example photoreactive groups can allow a conditioned modification of the active substance

(49) ##STR00010##

(50) The mild new production method also allows for example the use of alkyl residues with various chain lengths and also in unsaturated state. A modification of the chain length can result in a changed profile of the active agent.

(51) (1) Diamines, (2) monoamines, (3) fatty amides

(52) ##STR00011##

(53) with R.sub.2= linear alkyl residue with the chain length of C.sub.2-22 mono and/or polyunsaturated alkyl residues in (1) also mono- and/or polyunsaturated acyl residues n=1-6

(54) Scheme 7: Presentation of Modified Monoamine, Diamine and Fatty Amid Groups.

2. Modulation/Inactivation of Representatives of Various Enzyme Classes by Glucoprotamin

(55) 2.1 Oxidoreductases (Enzyme Class 1; EC1)

(56) Inactivation of the Alcohol Dehydrogenase (ADH; EC1.1.1.1)

(57) Measure principle: The enzyme alcohol dehydrogenase (ADH) catalyzes the reversible conversion of ethanol to acetaldehyde and simultaneously the 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 optically monitored at the photometer. The increase of the absorption at 340 nm is a direct measure of the conversion of the alcohol and thus for the activity of the ADH. For testing the inactivation of the ADH by glucoprotamin 2.6% of glucoprotamin was added to commercially available in NAD-ADH reagent (NAD-ADH reagent multiple test vial; Sigma Aldrich; Germany) and incubated for 10 minutes at 37 C. Then 1% ethanol was added to the mixture as substrate and the ADH activity was determined immediately at 340 nm in an Infinite M200 microplate reader (Tecan, Switzerland). The resulting ADH kinetics was plotted in relation to the control without glucoprotamin (FIG. 1). The results shown in the ADH kinetics of the colorimetric analysis show that glucoprotamin completely inactivates the ADH.

(58) 2.2 Hydrolases (Enzyme Class 3; EC3)

(59) Inactivation of the Alkaline Phosphatase (EC3.1.3.1)

(60) Measurement principle: The alkaline phosphatase (ALP) catalyzes the hydrolysis of phosphate ester under alkaline conditions to organic radicals and inorganic phosphates. The detection of the ALP activity is made via the cleavage of p-nitrophenylphosphate (pNPP). The formation of the yellow nitrophenolate can directly be monitored via the measurement of the absorption at 405 nm.

(61) For testing the inactivation of the ALP by glucoprotamin a commercially available test system (Alkaline Phosphatase Assay Kit; Abcam, USA) was employed. For this purpose, ALP contained in the kit system was incubated for 20 minutes at 37 C. with 2.6%, 5.2%, 7.8%, and 10.4% glucoprotamin. At the same time the positive control with the same enzyme concentration without glucoprotamin was used and incubated like the other samples and measured. After the addition of 1 mM pNPP the samples were incubated for further 30 minutes and then the absorption at 405 nm was determined in an Infinite M200 microplate reader (FIG. 2). The values of this end time measurement show an inactivation of the ALP by glucoprotamin, which is concentration-dependent.

(62) Inactivation of Endoproteases

(63) Measurement principle: The activity of endoproteases is also measured by means of a Fluorescence Resonance Energy Transfer (FRET) peptide library which contains more than 2.5 millions of 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 from each other and a strong fluorescent signal occurs. The protease activity is therefore directly proportional to the increase of the relative fluorescent intensity.

(64) For the determination of the inactivation of the endoproteases by glucoprotamin nine (three times each) different organs of the rat (Wistar rats, postnatal, day 3) were either incubated with 2.6% glucoprotamin, 4% formalin or an alcohol/glycerol solution (70% alcohol/30% glycerol). Then these organs were homogenized in Tris buffer (pH 7.4). Unfixed, freshly prepared organs of the rat (n=3) were used as positive control. After the determination of the protein concentration (Qubit Protein Assay, Life Technologies, Germany), the mixtures were centrifuged and 10 l of the supernatant was added to 80 l Tris buffer and 10 l of the FRET-based peptide library. The relative fluorescence intensity (RFU) was immediately determined by the aid of an Infinite M200 microplate reader. The relative fluorescence intensity per time unit (gradient) normalized to the protein concentration shows an inactivation of the endoproteases by glucoprotamin which is similar to the inactivation of the endoproteases by formalin. The alcohol/glycerol fixation in the kidney and in the colon did not result in a complete inactivation of the endoproteases but only to a reduction of the activity of the proteases (FIG. 3). In the positive control in all organs an activity of endoproteases could be identified.

(65) Inactivation of the RNases

(66) Measurement principle: The activity of RNases can be determined by the aid of a cleavable fluorescence labeled RNase substrate according to the FRET principle (RNaseAlert lab test kit; Applied Biosystems, Germany). The substrate is a modified RNA oligonucleotide which emits green fluorescence when it is cleaved by RNases. The RNase activity is thus directly proportional to the increase of the fluorescence intensity.

(67) For the determination of the inactivation of RNases by glucoprotamin two different approaches were chosen. At first 5 l RNase A (approximately 2 pg) from a commercial test system (RNaseAlert Lab Test Kit) were added to 2.6% glucoprotamin according to the information of the manufacturer and incubated for 10 minutes at 37 C. To the positive control instead of glucoprotamin nuclease free water was added but for the rest treated in the same way. After the addition of fluorescence labeled substrate, the RNase activity was determined by the aid of an Infinite M200 microplate reader in realtime. Over the measurement period of 20 minutes, no increase of the relative fluorescence intensity (RFU) could be detected in the mixture which was treated with glucoprotamin, which then shows the inactivation of the RNase A by glucoprotamin (FIG. 4A). In the positive control a consistent RNase activity saturated with substrate could be identified.

(68) In another approach a laboratory workplace was contaminated with human perspiration and saliva. Then the work area was separated in four areas of the same size and they were each differently treated: nuclease free water (positive control), 2.6% glucoprotamin (FIG. 4B), RNaseZap (commercial RNase decontamination solution of Blyde Biosystems; FIG. 4C) and RNase exitusPlus (commercial RNase decontamination solution of AppliChem, Germany; FIG. 4D). After incubation for 10 minutes the treated areas were rinsed two times with 500 ml nuclease free water. After drying of the areas 500 l each of nuclease free water were evenly distributed on the working areas, incubated for one minute and removed by the aid of a pipette. The fluorescence labeled substrate was added to 45 l of these four different mixtures and the change of the fluorescence intensity and therefore the RNase activity was determined by the aid of an Infinite M200 microplate reader (Tecan, Switzerland) in realtime (FIG. 4B-D). The treatment of the working places with glucoprotamin results in an inactivation of the RNases contained in perspiration and saliva and therefore to a decontamination of the working area. This complete decontamination can be compared to so far commercially available decontamination solutions. On the working place which was only treated with nuclease free water (positive control), however, an increase of the fluorescence intensity depending on the time and therefore a high RNase activity could be identified.

(69) Inactivation of the Lipases

(70) Measurement principle: The lipase hydrolyses arachidonoyl-1-thioglycerol to arachidonic acid and thioglycerol. Thioglycerol reacts with the thiofluometric detector to a strongly fluorescent product which can be analyzed at an excitation wave-length of 380 to 390 nm and an emission wavelength of 510 to 520 nm.

(71) For the testing of the inactivation of the lipases by glucoprotamin, a commercial test system was used according to the information of the manufacturer (Lipase Activity Assay, Cayman Chemical Company, USA). Two different test processes were performed. At first 10 l bovine milk lipoprotein lipase were supplemented with 2.6% of glucoprotamin and assay buffer and thiol detector were added according to the information of the manufacturer. In the positive control, glucoprotamin was omitted. Both solution mixtures were incubated for 15 minutes at 37 C. Then the lipase substrate was added and the lipase activity was determined by the detection of the change of fluorescence intensity (RFU) in realtime (FIG. 5A). The bovine milk lipoprotein lipases were inactivated by glucoprotamin. In a second test process, 200 mg of human, gluteal, subcutaneous fatty tissue was removed post mortem and homogenized with ice cold PBS by the aid of a Precellys ceramic kit 1.4/2.1 mm (PegLab, Germany) in a minilysis workplace homogenizer (PegLab) for 410 seconds at 5,000 rpm. The homogenate was then centrifuged for 10 minutes at 10,000g and 10 l of the mid phase were removed. This sample was either supplemented with 2.6% of glucoprotamin or directly (positive control) added to the assay buffer at the thiol detector. Both solution mixtures were incubated for 15 minutes at 37 C. and, as described above, the fluorescence intensity was measured in realtime (FIG. 5B). Glucoprotamin inactivates human lipases from subcutaneous fatty tissue.

3. Preservation of Organs from the Rat

(72) For the detection of the preservation activity of glucoprotamin, organs were removed from adult Wistar rats (postnatal, 31 days) and photodocumented. Then the organs were either incubated in 2.6% of glucoprotamin or in 4% of formalin for 7 days. Then the organs were stored for additional 25 days without preservation solutions, uncovered at room temperature and then again photodocumented and assessed (FIG. 6). Glucoprotamin-preserved organs show a preservation which is comparable to formalin fixed organs but they maintained their tissue elasticity.

4. Preservation of a Human Full Body Preparation

(73) After the availability of the examination results on the single enzyme level and the results of the organ fixation in the animal model (immersion fixation after organ removal) in compliance with the practice of the anatomic body donation (Ethics Committee Vote 237/2007601 and the Burial Law of Baden-Wrttemberg of 21 Jul. 1970) by analogy with the formalin fixation, the body of a body donor was infused with 17 l of a 2.5% glucoprotamin/20% ethanol solution intraarterial via the A. femoralis with a pump performance of 1 bar.

(74) The full body preparation was wrapped into a wet cloth with an above-mentioned solution and sealed in a foil. A control of the consistency of the tissue and of the state of the tissue was made after 2 weeks, 1 month, after 3 months and after 7 months.

(75) Inspection after 2 Weeks and 1 Month

(76) No signs of lysis could be found, the tissue structure remained un-changed, the haptics is close to reality as opposed to the formalin fixation.

(77) Invasive Examination after 3 Months

(78) The diagnostic laparoscopy and then an open surgical examination via a mediane laparotomy were performed. Result: The gastrointestinal tract was intact without signs of an occurred lysis. A performed microbiological smear test showed an asepsis of the intraperitoneal space in the area of the colon and the recessus of the abdominal cavity.

(79) In the context of an orthopedic surgery course an arthroscopy of the shoulder joint was carried out. The joint space was preserved close to reality, structures of ligaments and cartilages were close to reality with respect to haptics.

(80) Invasive Examinations after 7 Months

(81) After 7 months, an anatomical cover situs preparation was carried out (FIGS. 7 and 8) to open and inspect the entire abdominal area. All of the organs were preserved. Also the organs with a per se high content of digestive enzymes, such as the pancreas, maintained their morphological integrity. The left heart was opened and the endocardial space was inspected (FIG. 8). Even fine structures, e.g. atrioventricular valves, chordae tendineae, were preserved close to reality.

(82) Also after 7 months, a good preservation of structure could be found without signs of an occurred lysis.

5. Conclusion

(83) The inventors provide an active substance in form of a 5-oxo-pyrrolidine-2-carboxylic acid derivative, such as the glucoprotamin, which is versatilely applicable and by means of which the activity of non-structure proteins can be modulated, preferably inhibited. The substance is of significantly less health concerns than the currently used aldehydes, detergents and surfactants.