EP1737480A2

EP1737480A2 - Pharmaceutical combined preparation containing a therapeutic protein

Info

Publication number: EP1737480A2
Application number: EP05714189A
Authority: EP
Inventors: Seth Hallström; Harald Gasser
Original assignee: Individual
Current assignee: Individual
Priority date: 2004-03-29
Filing date: 2005-03-24
Publication date: 2007-01-03
Also published as: US20140051634A1; WO2005092362A3; WO2005092362A2; US20070238641A1; US8518869B2

Abstract

The invention relates to a pharmaceutical combined preparation containing a therapeutic protein with SH groups that are nitrosated, and containing a compound comprising thiol groups and having an average molecular weight of no greater than 10,000.

Description

Combination pharmaceutical preparation containing a therapeutic protein

The present invention relates to a pharmaceutical combination preparation which contains a therapeutic protein with SH groups which are nitrosated.

Nitrogen oxide (NO) is a gaseous molecule that is synthesized, among other things, in endothelial cells under normal physiological conditions. The endothelium-dependent relaxation of vascular smooth muscles is mainly due to NO. NO is therefore essential for the regulation of vascular tone. NO is also involved in a number of physiological and pathophysiological processes. For example, NO deficiency in ischemia / reperfusion for vasoconstriction and edema formation (Huk et al., Circulation 96 (1997), 667-675; Hallström et al., Circulation 105 (2002), 3032-3038).

In preparations of proteins with potentially free thiol groups, only 20-35% are actually in the free, reduced SH form. The remaining 65 to 80% are blocked, particularly in the case of protein preparations which are derived from blood or which are contacted with plasma or plasma derivatives in the course of their production process, mostly through mixed SS bonds with small compounds carrying small thiol groups, for example free L-cysteine or glutathione (Katachalski et al., J. Am. Chem. Soc. 79 (1957), 4096-4099; DeMaster et al, 1995, Biochemistry 34, 11494-11499).

In general, a distinction must be made in the sulfur-containing groupings in proteins between those that are in firmly bound or associated form, for example as intramolecular saturated disulfide bonds and are crucial for the conformation of the proteins, and those that contain the potentially free ( n) represent thiol group (s). The latter are a known quantity for the respective protein. Human serum albumin, for example, has a single potentially free thiol group per molecule in its native state, namely the cysteine in position 34. However, these potentially free thiol groups tend to form intermolecular disulfides, which is why they are also called mixed disulfides. Up to 80% of these thiol groups are present in the plasma as mixed disulfides and are therefore not immediately available as free thiol groups.

Reactions of the sulfhydryl group of low molecular weight and high molecular weight thiol compounds with NO, NO ₂ , NO ⁺ or NO ^" in the presence of oxygen lead to Formation of S-nitroso compounds, so-called S-nitrosothiols. S-nitrosothiols form a group of potent, bioactive compounds that stabilize physiologically formed NO and potentiate its biological effects. NO therefore does not work in biological systems per se, but also via biologically active redox adducts of NO, such as S-nitrosoproteins, S-nitrosoamino acids or other S-nitrosothiol compounds.

The experts suspect that the in vivo synthesis of S-nitrosothiol compounds takes place through nitrosation of endogenous thiol-containing molecules, such as, for example, reduced glutathione, L-cysteine, and serum albumin (Stamler et al., PNAS 89 (1992a), 7674- 7677; Stamler et al., PNAS 89 (1992b), 444-448). The reversible S-nitrosation of these molecules could be an important cellular regulatory mechanism. Significant effects on biological functions for some thiol-containing molecules have actually been attributed to the S-nitrosations. Among other things, the protective blocking of the N-methyl-D-aspartate receptor in exitatory neurons (Lei et al., Neuron 8 (1992), 1087-1099; Lipton et al., Nature 364 (1993), 626-632 ), Inactivation of protein kinase C (Gopalakrishna et al., J. Biol. Chem. 268 (1993), 27180-27185) and certain properties of hemoglobin on S-nitrosations (Stamler et al., Science 276 (1997), 2034- 2037; Gow and Stamler, Nature 391 (1998), 169-173). However, the molecular mechanism for in vivo S-nitrosation is largely unknown.

At least four possible mechanisms for the S-nitrosation of free thiol group-containing compounds by NO are currently being discussed in the literature. The first mechanism is an electrophilic attack by a reactive NO species, the nitrosonium cation (NO ⁺ ), on the nucleophilic sulfur atom (Stamler et al., Science 258 (1992d), 1898-1902). A second, controversial, mechanism is that S-nitrosation occurs through NO via peroxynitrite (ONOO ^" ) or NO ₂ (Pryor et al, J. Org. Chem. 47 (1982), 156-159; Mohr et al., FEBS Lett. 348 (1994), 223-227; Wu et al., Am. J. Physiol. 266 (1994), H2108-2113. The third, newer mechanism describes that S-nitrosations of thiol compounds (albumin, reduced glutathione ) is caused by dinitrosyl iron complexes (Boese et al., J. Biol. Chem. 270 (1995), 29244-29249). The fourth mechanism is an S-transnitrosation reaction or S-nitroso exchange reaction in which an NO- Group is transferred from an S-nitroso compound to a second thiol compound in exchange for an H group (Feelisch et al, J. Cardiovasc. Pharmacol. 17 (1991), Suppl. 3, S25-S33; Field et al., JCS Chem Commun. 6: 249-250 (1978). This reaction occurs rapidly in vitro and the formation of S-nitrosoglutathione is kinetically significantly preferred under physiological conditions. (Feelisch et al., (1991); Meyer et al, FEBS Lett. 345 (1994), 177-180; Singh et al., J. Biol. Chem. 271 (1996), 18596-18603; Tsikas et al, Anal. Biochem. 270: 231-241 (1999).

S-transnitrosation reactions are believed to be largely responsible for the biological effects of S-nitrosoglutathione, and it is believed that this process further leads to S-nitrosation of thiol-containing proteins (Mohr et al., FEBS Lett. (1994) It has been shown that S-transnitrosation reactions between S-nitrosoproteins and low molecular weight thiol compounds (e.g. L-cysteine and N-acetyl-L-cysteine) also occur in vivo (Scharfstein et al., J. Clin. Invest. 94 ( 1994), 1432-1439) However, direct evidence {in vivo) of the potentiation of the action, for example on the drop in blood pressure, has not been described.

The object of the present invention is to increase the physiological effect of proteins which contain nitrosated SH groups.

The object is achieved according to the invention by a pharmaceutical combination preparation which contains a therapeutic protein with SH groups which are nitrosated and a thiol group-containing compound with an average molecular weight of at most 10,000. The term "thiol groups" means sulfhydrile groups (-SH) and disulfide groups (-S-S-).

A preferred embodiment of the combination preparation according to the invention consists in that at least 90% of the SH groups present are nitrosated.

In the pharmaceutical combination preparation according to the invention, S-nitroso-albumin, S-nitroso-orosomucoid, S-nitroso-plasminogen activator, S-nitroso-fibrinogen, S-nitroso-Lys-plasminogen or S is particularly preferred as therapeutic protein with nitrosated SH groups -Nitroso hemoglobin included.

Reduced glutathione, L-cysteine, N-acetyl-cysteine, L-cysteinylglycine, γ-glutamylcysteine, penicillamine, penicillamide are particularly preferred as the thiol group-containing compound. Contain N-acetyl-penicillamine, N-acetyl-penicillamide, homocysteine, captopril, dehydroliponic acid and / or their oxidized form, which is reduced after administration in vivo.

It has been shown that a further preferred embodiment of the pharmaceutical combination preparation according to the invention contains reduced glutathione as therapeutic protein with nitrosated SH groups S-nitroso-albumin and as a compound containing thiol groups.

Furthermore, as a thio group-containing compound is a human one. Compound occurring in blood and tissue, particularly reduced glutathione, L-cysteine, L-cysteinylglycine, γ-glutamylcysteine or dehydroliponic acid is particularly preferred.

A further embodiment of the pharmaceutical combination preparation according to the invention consists in that it contains a therapeutic protein obtained by nitrosation, in which the degree of nitrosation is composed of at least 90% S-nitrosation and a maximum of 10% N, O, C-nitrosation.

In principle, the pharmaceutical preparation according to the invention for the protein component can contain any proteins with a _" free" thiol group, but proteins which can be used therapeutically for the purposes of the present invention are preferred, with physiological or blood-derived human proteins, such as albumin, orosomucoid and plasminogen activator (eg t-PA), fibrinogen, Lys-plasminogen or hemoglobin or also mixtures of such nitrosable or nitrosated proteins according to the invention are to be regarded as particularly preferred.

Accordingly, the pharmaceutical preparation according to the invention for the low-molecular thiol component can contain any low-molecular thiol compound, such as reduced glutathione, L-cysteine, N-acetyl-L-cysteine, L-cysteinylglycine, γ-glutamylcysteine, penicillamine or amides, N-acetyl Penicillamine or amides, homocysteine, captopril (D-2-methyl-3-mercaptopropanoyl-L-proli) and reduced thioctic acid (dehydroliponic acid), but preferred are low molecular weight thiol compounds in the blood (plasma) such as reduced glutathione, L-cysteine , L-cysteinylglycine, γ-glutamylcysteine and dehydroliponic acid. The production of nitrosated thiol group-containing proteins is described, for example, in EP-A 0 853 944.

The nitrosation is preferably carried out in such a way that only the freely available thiol groups are nitrosated and external nitrosations are avoided (equimolar nitrosation). This succeeds because primarily free SH groups are preferentially nitrosated and foreign nitrosations of the N, O, C atoms of the proteins take place only when there is an excess of nitrosating agent. For example, N- and C-nitroso compounds are suspected of being carcinogenic and also have different NO group release kinetics than S-nitroso compounds (see Zhang et al, J. Biol. Chem. 271 (24) (1996), 14271-14279), which is why in a preferred embodiment of the preparation according to the invention an N, 0, C-nitrosation degree of the proteins in the preparation of at most 10% is provided.

Although it is possible for the protein component to also subject crude fractions, such as plasma or early plasma fractions or culture fluids, to the equimolar nitrosation, the proteins are preferably made available in purified form. The degree of purity should preferably be such that the proteins can be administered pharmaceutically. Therefore, a protein for nitrosation with a degree of purity of at least 80%, in particular at least 90% (w / w%), based on protein, is provided for the protein component of the preparation according to the invention. Of course, higher values are also preferred. This purified protein can of course be formulated with other proteins into a pharmaceutical preparation.

The protein component in the preparation according to the invention can thus be a mixture of different equimolar nitrosated proteins, but it can also be a mixture of a non-nitrosated and an equimolar nitrosated protein. The pharmaceutical preparation preferably contains S-nitroso human serum albumin as a protein component and reduced glutathione. For a mixed form of the protein component, the pharmaceutical preparation preferably contains S-nitroso human serum albumin and hemoglobin. For the preparation according to the invention, the protein component is preferably made available in a higher purity than the non-nitrosated protein component. For example, human serum albumin is often administered in a purity of at least 80% of total protein as a pharmaceutical preparation. On an analog or higher degree of purity is also preferred for the S-nitrosated protein in the preparation. Therefore, after the nitrosation of the protein component for the nitrosated protein, an additional cleaning step is added.

In the combination preparation according to the invention, the low molecular weight thiol compounds are preferably made available in purified form. The degree of purity should preferably be suitable such that the low molecular weight thiol compound can be administered pharmaceutically in the preparation. Therefore, a degree of purity of at least 90%, in particular at least 95% w / w, based on the low molecular weight thiol compound is provided for the low molecular weight thiol compound of the preparation according to the invention. Of course, higher values are also preferred.

The pharmaceutical preparation according to the invention is preferably formulated in a pharmaceutically acceptable buffer solution, optionally with pharmaceutically acceptable stabilizers. For example, sodium caprylate and / or sodium acetyltryptophanate is used as a stabilizer. As a rule, one will be able to use formulations such as those used for the use of the protein component as a non-nitrosated product. The preparations can also be provided as a spray or in a form suitable for topical use. In particular, the preparation is provided in a form suitable for intravenous administration. With regard to the protein component, an IV-compatible preparation is characterized in particular by a low aggregate content or is free of aggregates.

Of course, precautions must often also be taken for the storage of the preparation according to the invention, so that this preparation remains stable over a longer period of time. The preparation according to the invention is therefore preferably in frozen or lyophilized form for storage purposes, in which it has a sufficiently long shelf life. Both the low-molecular thiol compound and the nitrosated protein of the preparation according to the invention can be stored in a common or separate form.

It has been shown that, for example, for proteins derived from plasma or blood, in particular for albumin or hemoglobin, the stability in solution at a pH between about 6 and 7 is greatest in a suitable buffer system (e.g. Ringer's solution). On the other hand, for the low molecular weight thiol compounds, in particular for reduced glutathione and L-cysteine, it has been shown that the stability in solution is greatest at a pH below 7.

The pharmaceutical preparation based on a protein with nitrosated thiol groups and a low molecular weight thiol compound is stable when stored separately in the lyophilized state.

In contrast to the known method (eg: Stamler et al., (1992a), the nitrosation can preferably be carried out in such a way that only after determining the “free” thiol content of the proteins is nitrosated to this proportion of “free” thiol groups and at the same time a low molecular weight Both native thiol-containing proteins and thiol-containing proteins in which the “free” thiol groups are de-blocked by a specific method can be considered as the protein component.

The reactants or the reaction products are separated off after the nitrosation reaction and preferably to a quantitative extent or to a value below the detection limit.

In a further preferred embodiment, the preparation according to the invention is also distinguished by a low aggregate content of the protein component. In particular, the proportion of aggregates in the pharmaceutical preparation is below 20%, preferably below 10%, most preferably below 5%.

The nitrosation of the thiol-containing proteins is carried out under aerobic conditions, especially when working with acidic sodium nitrite.

The nitrosation is preferably carried out with an agent selected from HNO ₂ , HNO, NOC1, NO ⁺ , RNO ₂ , N ₂ O ₃ , N ₂ O ₄ , NO ₂ - and NO radical, and in an acidic medium. Organic NO donors can also be used. In order to keep the degree of N- and C-nitrosation as low as possible, the nitrosation should be carried out with an agent related to the release of NO equimolar to the "free" thiol group content of the protein. Of course, a smaller ratio of agent for the nitrosation can also be used based on the thiol group content of the protein, but a ratio of 1: 1 is preferred. Since the S-nitrosation is preferred and proceeds much faster than the N- and C-nitrosations, an equimolar nitrosation means a minimal N- and The degree of nitrosation of the protein is guaranteed, and the duration of the nitrosation should be as short as possible. The nitrosation is therefore preferably carried out for a period of from 2 minutes to several hours, preferably 30 minutes, at a temperature between 15-30 ° C., preferably at room temperature in an aqueous solution at pH 0.3 to 3.5, most preferably at pH 1.0 to 3.0, preferably in acid n range up to pH 1.5.

All types of protein fractions can be used as the starting material for the protein component, in particular also blood, plasma, serum, a plasma fraction or a purified protein fraction, but also culture supernatants or corresponding extracts. If, however, substances are contained in the starting material which can adversely affect the nitrosation step, such as, for example, low molecular weight thiol group-containing proteins or thiol group-containing compounds, these should preferably be separated off. Preferred plasma fractions are those after the Colin fractionation and in particular the Cohn II and III fraction or the Cohn IV fraction.

Within the scope of the method according to the invention, a whole series of further purification steps can also be provided in any points of the method for the protein component.

A further purification step, selected from precipitation, gel filtration, diafiltration, ultrafiltration and chromatographic purification, can be provided. For example, albumin is purified using ion exchange chromatography.

In particular, it can be provided that a purification step is carried out after the nitrosation of the protein, so that the substances used there do not influence one another and are also not present in the finished nitrosated protein component. This purification step is preferably carried out in the form of a chromatographic purification, in particular by means of gel permeation chromatography

A treatment for inactivating viruses is preferably carried out before the nitrosation, but it can also be carried out terminally, ie after the nitrosation.

After nitrosation, the protein component of the combination preparation according to the invention can be processed into a pharmaceutical-eutomatic preparation in a manner known per se. For the protein component, the Hegel adheres to the formulation instructions (see Pharmacopoeia) for the non-nitrosated protein preparation.

The low molecular weight thiol compound, preferably reduced glutathione or L-cysteine, is provided in a pharmaceutically administrable form as the pure substance b and is administered simultaneously with the purified, nitrosated protein component i.v. apply.

Preferred medical uses of this combination preparation according to the invention include the preparation of a combination preparation for improving perfusion or microcirculation, preferably in vital organs such as e.g. in the G-brain (cerebral ischemia, ischemic insult), in the heart (myocardial infarction), in the kidney or in the extremities or in the whole organism. The combination preparation according to the invention can thus generally be used to prevent or treat ischemia and reperfusion damage. The combination preparation according to the invention is also suitable for the treatment of shock, in particular traumatic, hypovolemic or hemorrhagic or neurogenic shock.

The combination preparation according to the present invention can be used in various surgical fields, for example in transplant surgery and in all surgical procedures with subsequent reperfusion. It is particularly suitable for the treatment and / or prophylaxis of restenosis after angioplasty. The combination preparation can also be used for the treatment and / or prophylaxis of thrombotic conditions, ie those which are associated with platelet adhesion / aggregation. In a preferred embodiment, the S-NO tissue plasminogen activator can be used as a thrombolytic agent.

The combination preparation is also used to relax the non-vascular, smooth muscles, e.g. the smooth muscles of the airways, usable. Therefore, the preparation according to the present invention can be used for the treatment and / or prophylaxis of respiratory diseases. It can also be useful for diagnosing and / or treating male erectile dysfunction.

A further, essentially preferred, medical use of the combination preparation according to the invention comprises the production of a combination preparation for the controlled reduction of blood pressure, e.g. in hypertensive crises (i.e. chronic or acute hypertension crises). As a rule, a higher dosage is used than for the prevention or treatment of ischemia and reperfusion damage.

The medical combination preparation is preferably provided with respect to the protein component in a dose which, except for albumin, corresponds to that of the non-nitrosated protein. With albumin as a protein component, the dose depends primarily on the medical indication. In the prevention or treatment of ischemia and reperfusion damage, depending on the S-nitrosograd of the albumin preparation, a dosage of 0.035-1.0 μmol / kg / h is recommended. When reducing blood pressure, higher doses should be used (e.g. up to 10 μmol / kg / h of S-NO albumin with an S-nitrosation level of ~ 26%). A dose of 12-140 μmol / kg / h is recommended for the low molecular weight thiol compound (e.g. reduced glutathione). The amount or dose to be administered depends on the requirements of the patient, for example on parameters such as hematocrit, oxygenation, mean arterial and venous blood pressure or pulmonary arterial pressure, and can vary considerably from case to case. Significantly lower doses can also be used for the platelet adhesion / anti-aggregation effect.

A particular advantage of the combination preparation according to the invention is that at least the same efficiency is achieved with proteins containing thiol groups as with Monopreparations of thiol group-containing proteins, in which the freely available thiol group was increased to 90% free SH groups per mole of protein by reductive pretreatment. In terms of process engineering, the reductive pretreatment is avoided. Equimolar nitrosation to the freely available thiol group guarantees the same degree of purity of the active component of the protein preparation (N-, C-, O-nitrosierang <5%). Both albumin (4-5 g / dL plasma) and reduced L-glutathione (<5 μmol / L) are naturally occurring plasma components. The naturally occurring transnitrosation reaction is limited by the physiologically occurring reduced L-glutathione level in the plasma. By providing reduced L-glutathione, the release of the active substance NO of the S -nitrosized protein component can even be controlled in a dose-dependent manner from the reduced L-glutathione.

Of course, the combination preparation according to the invention can of course also be used for any indication of the non-nitrosated proteins in general, since their physiological effect is retained despite nitrosation. In addition, however, conditions which require the provision of an increased NO content are preferred indications for the combination preparations according to the invention.

Production of equimolar to the freely available thiol group nitrosated human serum albumin and provision of reduced glutathione:

a) Determination of the ratio of free SH groups per mole of protein before nitrosation.

The ratio of free SH groups per mole of protein was determined using the Ellman reagent (Ellman G.L., Arch. Biochem. Biophys. 82 (1959), 70-77) according to Sedlak and Lindsay, Anal. Biochem. 25: 192-205 (1968).

An SH group content of 26% (mol / mol, SH group content for protein) has been determined for human albumin 20% (manufacturer: Baxter). After reductive pretreatment of another human albumin preparation (AT 405 135; US 6 124 255 and US 6 358 918), values of up to 95% can be achieved and demonstrated. b) Equimolar nitrosation to the free SH group content of the respective human albumin preparation

The nitrosation of the albumin preparations was carried out equimolar to the content of the free SH groups (ratio: 1: 1 to a maximum of 1: 1, 2 molar to the determined value) with NaNO ₂ in 0.2 mol / L HC1 at pH 1.5-2. 5 for a period of 15-30 min at room temperature. The mixture was then neutralized with 1 mol / L NaOH. To separate unwanted reactants, preparative gel permeation chromatography was carried out using a stationary phase of beads with a heteroporous, swollen network of a Toyopearl TSK HW 40 (F) gel. Elution was carried out with bidistilled water at 4 ° C. The purified fraction containing S-nitroso-albumin (S-NO-albumin / albumin) was then lyophilized.

S-nitroso degree determination of the protein fraction with HPLC using the Saville and Griess reaction

The analysis can be carried out before or after the purification by means of preparative gel permeation chromatography. With this method, if available, excess nitrosating agent and buffer substances are separated from the S-NO albumin / albumin using a gel permeation column (Toyopearl TSK HW-40-S). Subsequently, in a post-column derivatization process using the Saville reaction (Saville B., Analyst 83 (1958), 670-672), the NO group is selectively split off from the RS-NO by Hg ²⁺ . Simultaneously, the nitrite formed is photometrically detected at 541 nm by a color reaction (Griess reaction; Griess, Ber.Dtsch. Chem. Ges. 12 (1897), 426-428). The chromatograms (FIG. 4) show equimolar nitrosated S-NO-HSA preparations with different S-nitroso degrees: a) human albumin nitrosated equimolar to the free SH group with a free SH group content per mol protein of 26%; b) human albumin nitrosated equimolar to the free SH group, which had a free SH group content per mole protein of 74% by reductive pretreatment; c) analogous to b), the nitrosation was not carried out equimolar but with a 6-fold molar excess of nitrosation agent.

The percentages given are the actual S-nitrosation levels on the protein according to the Saville principle. When determining the degree of S-nitrosation before preparative cleaning results in a possible second peak in the chromatogram of the excess nitrosating agent (chromatogram c). In the case of albumin preparations nitrosated equimolar to the free SH group, only traces of nitrite can be detected.

Providing reduced glutathione

Reduced glutathione produced by peptide synthesis with a purity of at least 95% was made available as a low molecular weight thiol compound.

The effect of the combination preparation according to the invention is described below in a preferred embodiment.

example 1

In example 1, the lowering of the mean arterial pressure by application of a combination preparation according to the invention consisting of a nitrosed serum albumin preparation and reduced glutathione is shown on the rabbit model. For comparison, the effect of a nitrosated serum albumin preparation is shown, which was applied without reduced glutathione.

The rabbit was anesthetized, initiating anesthesia with ketaset (50 mg / kg; bolus) and xylasin (5 mg / kg; bolus) and with continuous infusion via the vena auricularis of Ketaset (35 mg / kg / h) and 5 mg Maintain xylasin (5 mg / kg / h) dissolved in physiological saline (5 mL / h). After tracheotomy and intubation, the rabbits were connected to the ventilator (ventilator Harvard Apparatus-INSPIRA ASV) (tidal volume = 0.0062 x body weight (kg) ¹ ' ⁰ , respiration rate = 53.5 x body weight (kg) ^"0 ' ²⁶ ).

The mean arterial pressure (MAP) was measured using a pressure transducer in the femoral artery (amplifier unit - TAM-A and Isotec pressure transducer; Hugo Sachs Elektronik). The results are shown in Figures la-f. FIG. 1 a shows the dose-dependent comparison of the lowering of the mean arterial pressure (MAP) in the rabbits after bolus administration of nitrosed serum albumin with and without simultaneous continuous infusion of reduced glutathione (2.2 μmol / kg / min). Infusion: Vena femoralis (n = 3 per data point; mean ± standard deviation).

Fig. 1b is a representative example of the lowering of the mean arterial pressure with a bolus infusion of 0.1 μmol / kg S-NO-HSA with and without continuous infusion of reduced glutathione (2.2 μmol / kg / min).

Figures lc and ld show the lowering of the mean arterial pressure at two different doses of a nitrosated serum albumin preparation with a high degree of nitrosation (70%) and a native serum albumin with a low nitrosation level (26%) nitrosated to the freely available thiol group with simultaneous infusion of reduced glutathione (n = 3 per data point; mean ± standard deviation).

FIG. 1e is a representative example of the lowering of the mean arterial pressure by bolus injections of 0.1 μmol / kg S-NO-HSA with variable concentrations of GSH in vivo. Infusion: femoral vein.

FIG. 1f is a representative example of the lowering of the mean arterial pressure by simultaneous, continuous infusion of 0.05 μmol / kg / min S-NO-HSA with increasing concentration of reduced glutathione (a: 0.0 μmol GSH / kg / min , b: 0.1 μmol GSH / kg / min, c: 0.3 μmol GSH / kg / min). S-NO-HSA was infused through the jugular vein and reduced glutathione through the second auricular vein (or vena femoralis).

Example 2

Example 2 shows the potentiation of the NO release of a nitrosated serum albumin preparation by providing a low-molecular thiol compound using the example of reduced glutathione. The NO concentration was measured in vitro.

FIG. 2a shows the concentration-dependent potentiation of the NO release of a nitrosized serum albumin preparation by reduced glutathione, measured with a porphyrinic microsensor in vitro (S-NO-HSA: 30 μmol / L; n = 6, mean ± standard deviation; * P <0.05 vs S-NO-HSA; ⁿ P <0.05 vs 25 μmol / L GSH) ,

FIG. 2b shows a representative example of the in vitro measurement of the potentiation of the NO release of a nitrosated serum albumin preparation by reduced glutathione.

Example 3

Example 3 shows the effect of a preferred embodiment of the combination preparation according to the invention on platelet aggregation.

The platelet aggregation was carried out with human platelet-rich plasma (TRP) in a dual-channel chronolog aggregometer in principle according to the method of Born (1969). At the beginning of each experiment, the exact dose of collagen for which collagen-induced aggregation (~ lμg collagen / mL TRP) was determined (95-100% inhibition of collagen-induced aggregation by 300 μmol / L acetylsalicylic acid). In the experiments, increasing concentrations of reduced glutathione were primarily pre-incubated with TRP in the aggregometer for one minute and S-NO-HSA (2-4 μmol / L; concentration that causes 20% inhibition of collagen-induced aggregation) was added after one minute. After another minute, the aggregation was induced with collagen. Control experiments with collagen alone were carried out after every second to third measurement. The results are presented as% aggregation relative to the collagen-induced aggregation (100%) (mean ± standard error). The final concentrations in the aggregation vessel are shown in the figures. In experiments with other thiol- and non-thiol-containing substances (N-acetyl-L-cysteine, ascorbic acid, DL-homocysteine, taurine, L-cysteine), analogous to the experiments with reduced glutathione, primary preincubation with these substances.

FIG. 3 a shows the dose-dependent potentiation of the inhibition of collagen-induced platelet aggregation by S-NO-HSA (2-4 μmol / L) with reduced glutathione (n = 6; * P <0.05 versus S-NO-HSA). FIG. 3b shows the effect of N-acetylcysteine (1 mmol / L), ascorbic acid (Vit.C; 200 μmol / L), reduced glutathione, homocysteine, taurine and cysteine (all 1 mmol / L) on the inhibition of collagen induced Platelet aggregation by S-NO-HSA (2-4 µmol / L) n> 5 (taurine, n = 3); * P <0.01 versus S-NO-HSA.

Claims

claims:

1. Pharmaceutical combination preparation containing a therapeutic protein with SH groups, which are nitrosated, and a thiol group-containing compound with an average molecular weight of at most 10,000.

2. Pharmaceutical combination preparation according to claim 1, characterized in that at least 90% of the SH groups present are nitrosated.

3. Pharmaceutical combination preparation according to one of claims 1 or 2, characterized in that S-nitroso-albumin, S-nitroso-oromomucoid, S-nitroso-plasminogen activator, S-nitroso-fibrinogen as therapeutic protein with nitrosated SH groups, S-nitroso-lys-plasminogen or S-nitroso-hemoglobin is included.

4. Pharmaceutical combination preparation according to one of claims 1 or 2, characterized in that as the thiol group-containing compound reduced glutathione, L-cysteine, N-acetyl-cysteine, L-cysteinylglycine, γ-glutamylcysteine, penicillamine, penicillamide, N- acetyl Penicillamine, N-acetyl-penicillamide, homocysteine, captopril, dehydroliponic acid and / or their oxidized form, which is reduced after administration in vivo, is contained.

5. Pharmaceutical combination preparation according to one of claims 3 or 4, characterized in that the therapeutic protein containing nitrosated SH groups is S-nitroso-albumin and the compound containing thiol groups is reduced glutathione.

6. Pharmaceutical combination preparation according to claim 4, characterized in that a thio group-containing compound is a compound found in human blood and tissue, in particular reduced glutathione, L-cysteine, L-cysteinylglycine, γ-glutamylcysteine or dehydroliponic acid.

7. Pharmaceutical combination preparation according to one of claims 1 to 6, characterized in that it contains a therapeutic protein obtained by nitrosation, in which the degree of nitrosation is at least 90% from S-nitrosation and at most 10% from N, O, C- Nitrosation.