DE102006060375A1 - New aprotinin variants, in which the amino acid lysine is interchanged against another amino acid, after the natural sequence, useful for e.g. reducing blood loss during polytrauma, shock, inflammation and cancer - Google Patents

Abstract

Aprotinin variants at 15th position, after the natural sequence, the amino acid lysine is interchanged against an another amino acid and additionally one or more of the naturally occurring amino group is modified with a non-protein-polymer, is new. An independent claim is included for the preparation of aprotinin variants. ACTIVITY : Vasotropic; Antiinflammatory; Cytostatic; Antibacterial; Immunosuppressive; Anticoagulant; Antiasthmatic; Antiarthritic; Antirheumatic; Analgesic; Cerebroprotective. MECHANISM OF ACTION : Bovine pancreatic trypsin inhibitor; Serine protease inhibitor; Elastase inhibitor.

Description

The The present invention relates to aprotinin variants having improved enzyme inhibitory, immunological and pharmacokinetic properties and their manufacture and use.

aprotinin, also called bovine pancreatic trypsin inhibitor (BPTI) is heard to the family of Kunitz-type serine protease inhibitors. The spectrum The inhibitable serine proteases include e.g. Trypsin, chymotrypsin, Plasmin and plasma kallikrein (W. Gebhard, H. Tschesche and H. Fritz, Proteinase Inhibitors, Barrett and Salvesen (eds.), Elsevier Science Publ. BV 375-387, 1986).

aprotinin consists of 58 amino acids. The three-dimensional structure of the protein was determined by X-ray analysis and NMR spectroscopy elucidated (Wlodawer et al., J. Mol. Biol. 198 (3), 469-480, 1987; Wagner et al. Biol. 196 (1), 227-231, , 1987; Berndt et al., Biochemistry 32 (17), 4564-4570, 1993).

Under the trade name Trasylol ^® natural aprotinin is originally used for the treatment of pancreatitis. Trasylol ^® is used today in cardiac surgery after clinical studies have shown that treatment with aprotinin significantly reduces the need for transfusion in such operations and reduces rebleeding (Royston, D., Cardiothorac, Vasc., Anesth., 6; 100, 1992).

It it could be shown that the exchange of the specifying inhibitory specificity amino acid in position 15 to valuable aprotinin variants with improved Inbihitoreigenschaften leads (DE-PS 3 339 693). ever after the imported amino acid can be generated in this way potent Inihibitoren, z. B. the Inhibit elastase from pancreas or leukocytes.

Furthermore, it has been shown that the inhibitory properties of aprotinin and the variants produced by replacement in position 15 are also determined by further amino acid residues in the contact region between the target protease to be inhibited and the inhibitor molecule. These include, in particular, the additional amino acid residues in positions 14, 16, 17, 18, 19, 34, 38 and 39. Aprotinin variants with improved properties by exchange of one or more of these amino acid residues in the region of the contact region have been described by way of example in the following patent applications: WO 89/01968, WO 89/10374, EP 0 307 592 . EP 683,229 ,

The abovementioned aprotinin variants can be prepared by genetic engineering. Both the production in E. coli (Auerswald et al., Biol. Chem. Hoppe Seyler, 369, 27-35, 1988) and the production in the yeast ( EP 0419878 ) are described, wherein in the exemplary Hefeverfahren the N-terminus has been modified for technical reasons. A yeast expression system expressing aprotinin variants with correct N-terminus is described in U.S. Patent Nos. 4,969,369 and 4,249,247 EP 0 683 229 described.

The Pegylation of aprotinin or aprotinin variants is also described in the literature. However, this pegylation leads to a change the inhibitory property of the molecule. This is how Stassen et al. (Thrombosis and Haemostasis, 74 (2), 646-54, 1995) pegylation an aprotinin variant in which, on average, one PEG molecule is coupled per aprotinin molecule is. This modification leads et al to an increase of the ki value for Trypsin by a factor of 4. Another example of the pegylation of aprotinin is described by Shin (Pharm Pharmacol Commun 4: 57-260, 1998). This modification leads even to a 29-fold increase of the IC50 value for the trypsin inhibition.

aprotinin is a protease inhibitor from bovine lungs, which is used in the application on People in some cases to antibody formation led against this protein Has. In such cases it may cause anaphylactic re-application of aprotinin Schock (Diefenbach et al., Anestesia and Analgesia, 80 (4), 830-831, 1995).

Desirable are therefore aprotininvarianten, in addition to an equal or higher efficacy against certain target enzymes in addition a lower immunogenicity exhibit. Desirable would also be that these variants do not react with antibodies due to an earlier one Treatment with aprotinin in the body available. The presence of such a variant would be at life-saving surgeries reuse this drug possible without the risk of anaphylactic shock.

These Properties can in proteins can be achieved by combining them with non-protein polymers, e.g. hydroxyethyl starch ("Hesylation") or polyethylene glycol Linked (PEG) (Abuchowski et al., J. Biol. Chem., 252, 3578-3581, 1977). However, as mentioned above, to lead these links often that the protein is active loses. This increases the likelihood of reducing activity with increasing degree of modification and with increasing molecular weight of used non-protein polymer, since these polymers interact of the protein with its target protein (e.g., enzyme or receptor).

Surprisingly we now found that aprotinin variants, in which to improve of the inhibitory properties the lysine was modified in position 15, without loss of activity to a high degree of the natural in aprotinin occurring amino groups, especially at the amino groups naturally can be modified in aprotinin occurring lysines. Even Modifications with non-protein polymers compared to Aprotinin have a very high molecular weight, such as polyethylene glycol with a molecular weight of about 20KD, lead to no loss of activity in the trypsin inhibition. Furthermore The aprotinins that are pegylated in this way do not react with antibodies that with the course occurring aprotinin react.

Aprotinin variants according to the invention

• • 1. Austausch des Lysins in Position 15 gegen eine andere Aminosäure, bevorzugt gegen eine Aminosäure, die zu einer Verbesserung der Aktivitätseigenschaften führt.
• • 2. Modifikation der Aminogruppen der Aminosäuren in Position 26, 41 und 46 mit Nicht-Protein-Polymeren, wie z.B. Polyethylenglycol zur Verbesserung der immunologischen und pharmakokinetischen Eigenschaften.

The aprotinin variants described in the present patent application are characterized by the following features:

• 1. Exchange of the lysine in position 15 for another amino acid, preferably against an amino acid, which leads to an improvement in the activity properties.
• 2. Modification of the amino groups of the amino acids in position 26, 41 and 46 with non-protein polymers, such as polyethylene glycol for the improvement of immunological and pharmacokinetic properties.

In addition, these can Aprotininvarianten be modified by further amino acid substitutions, the the activity characteristics change the molecule. Prefers Here are the positions 14, 16, 17, 18, 19, 34, 38 and 39.

Besides, these can Aprotininvarianten be modified at the N- or C-terminus.

In 1 is exemplified a Aprotinvariante. In this variant, based on the natural sequence in position 15, there is an arginine and the proline in position 2 is deleted. This will shift all amino acids by one position. In addition, this variant is pegylated on the lysines and on the N-terminus.

However, the aprotinin variants of the invention are not limited to that in the 1 example mentioned.

The The invention also relates to medicaments containing these aprotinin variants.

Of the described novel proteinase inhibitor is suitable for the treatment of disease states, where there are - too due to complex surgical procedures, e.g. in cardiac surgery or in alloarthroplasty joint replacement in transplantation medicine - for activation the plasmatic enzyme systems by extensive or intensive Foreign surface contact of the blood is coming.

Of the Inhibitor reduced blood loss in operations with a increased Bleeding risk (e.g., heart surgery, bone and joint surgery) are connected. They are suitable for therapy in shock, polytrauma and skull and brain trauma, sepsis, disseminated intravascular coagulopathy (DIC), Multi-organ failure, inflammatory Diseases involving the kallikrein system such as rheumatic Joint disease and asthma. It prevents invasive tumor growth and metastasis by inhibition of plasmin. He is suitable also for pain and edema therapy by inhibition of bradykinin synthesis and for the treatment of stroke.

He is also useful in dialysis therapy and artificial organs to reduce inflammation and to prevent coagulation and to increase the risk of bleeding Reduce.

A.) Preparation of the aprotinin variants according to the invention

1. Preparation of non-pegylated Aprotinin variants

For the production the inventive aprotinin variant is it cheap to use genetic engineering methods. For this purpose, with common molecular biological Methods into a suitable microbial expression organism the genetic information for introduced the synthesis of the respectively considered aprotinin variant. Of the recombinant microorganism is fermented; by choosing appropriate Conditions, the heterologous genetic information is brought to expression. The expressed aprotinin variant is subsequently obtained from the culture broth.

suitable Host organisms for the production of the aprotinin variants according to the invention can Be bacteria, yeasts or fungi. Expression may be intracellular or below Use of suitable secretion systems extracellularly done. The aprotinin variant can be correctly processed or fused Peptides or proteins are expressed.

A suitable system for expressing aprotinin variants is disclosed in the patent application US 5,231,010 described. This application also describes the fermentation of the microorganisms and the purification of the product.

In the patent application US 5,707,831 is also described production, expression, fermentation and purification of aprotinin variants.

2. Production the pegylated of the aprotinin variants

These Aprotinvarianten are then with polyethylene glycol Implemented (PEG).

By way of example, the reaction of DesPro2-Arg15-aprotinin with mPEG-SPA-2000 mPEG-SPA-5000 or mPEG-SPA-20000 (Nectar) is mentioned:
Solution A: 750 mg Na ₂ HPO ₄ are dissolved in 80 ml H ₂ O. The pH is adjusted to pH 9.0 with phosphoric acid. It is then made up to 100 ml with H ₂ O.
Solution B: Dissolve 106 mg DesPro2-Arg15-aprotinin in 13.5 ml H ₂ O.
Solution C: 3 ml of solution B and 9 ml of solution A are added together (protein concentration 8 mg / ml)
Solution D: 18 mg lysine are dissolved in 10 ml H ₂ O.

Execution of the reaction:

approach 1: 48 mg mPEG-SPA-2000 (from Nectar) are dissolved in 2 ml of solution C.

approach 2: 124 mg mPEG-SPA-5000 (Nectar) are dissolved in 2 ml of solution C.

approach 3: 258 mg mPEG-SPA-20000 (company Nectar) are dissolved in 2 ml of solution C.

The approaches are incubated at room temperature. To follow the reaction be at certain intervals Samples were drawn and the reaction stopped in these samples. These Samples are then analyzed by SDS-PAGE.

To End of reaction (120 minutes) will be the remaining volumes in the relationship 1: 1 with solution D diluted to stop the reaction. These samples are tested in the trypsin inhibition assay and their reaction with anti-aprotinin antibodies.

B.) analytics of the pegylated aprotinin

1. SDS gel electrophoresis

SDS gel electrophoresis was performed on the conditions of Laemmli (UK Laemmli, Nature 227, 680-685, 1970) on a polyacrylamide gel. The analysis of batch 1 was carried out on a 15% gel, the analysis of batches 2 and 3 on a gradient gel 5-17.5%). The preparation and implementation of electrophoresis, as well as the subsequent staining with Coomassie are described in the book Elektrophoresepraktikum (R. Westermeier, VCH 1990, ISBN 3-527-28172-X). There are 25μg each Gelatin applied. After staining, the gels are evaluated densitometrically. An example of this evaluation is in 2 ,

2. Activity (trypsin Inhibition test, titrimetric)

The activity is determined by a modified F.I.P. trypsin inhibition assay. Aprotinin inhibits the trypsin-catalyzed hydrolysis of Na-benzoyl-L-arginine ethyl ester (BAEE). The released in the reaction carboxyl groups determined by the alkali titration. The residual trypsin activity is one Measure of the inhibitory activity of the active ingredient.

The protein is adjusted to a concentration of 1 mg / ml and a dilution series prepared. To 1 ml of sample solution, 2 ml of buffer (15 mM borate buffer, pH 8.0 with 200 mM CaCl ₂ ) and 0.8 ml of trypsin solution (2 mg / ml) are added and incubated at 25 ° C for 5 minutes. Subsequently, 0.2 ml of BAEE solution (6.8 mg / ml) is added and the KOH consumption is measured over 5 minutes.

Result:

C.) Reaction with anti-aprotinin antibodies

To the Use comes a rabbit serum. The rabbit was treated with aprotinin treated so that antibody are detectable against aprotinin in the serum.

In a microtiter plate is 100 μl DesPro2-Arg15-aprotinin as well as material from approaches 1, 2 and 3 in a concentration series of 1 mg / ml, 100 ug / ml, 10 ug / ml and 1 μg / ml pipetted and over Night at 4 ° C attached. The addition takes place in PBS, pH 7.4. The plate will washed with PBS and blocked for 1 h with 1% BSA / PBS. The plate will washed again. Subsequently 100 μl Rabbit serum (diluted 1: 100 in 1% BSA / PBS) was added and 1.5 h at 37 ° C incubated. After washing the plate, 100 μl of biotinylated anti-rabbit IgG antibody (1: 1000 in 1% BSA / PBS) and incubated at 37 ° C for 1.5 h. After another wash 100 μl HRP-streptavidin (1: 75,000 in 1% BSA / PBS) was added and 1.5 h at 37 ° C incubated. The plate is washed and 200 μl substrate solution (TMB) added and Incubated for 15 min at room temperature. The plate is at 450 nm evaluated.

Result:

During DesPro2-Arg15 Aprotinin a concentration-dependent Reaction with the rabbit serum shows the pegylated react Derivatives not with the serum.

It follows a sequence listing according to WIPO St. 25. This can from the official publication platform downloaded from the DPMA.

Claims (7)

Aprotininvarianten characterized in that in position 15 after the natural sequence, the amino acid lysine is replaced by another amino acid and are additionally modified at several of the naturally occurring amino groups with a non-protein polymer. Aprotininvarianten according to claim 1, characterized that the variants at the positions 14, 16, 17, 18, 19, 34, 38 and 39 one or more additional ones amino acid changes wear. Aprotininvarianten according to any one of claims 1 and 2 characterized in that the aprotinin variants in addition to N- or C-terminus are modified. Aprotininvarianten according to claims 1 to 3, characterized that the non-protein polymer is polyethylene glycol is. Process for the preparation of Aprotinnvarianten after the claims 1 to 4, characterized in that the aprotinin variants modified with a non-protein polymer reagent. Process for the preparation of Aprotinnvarianten after Claim 4, characterized in that the aprotinin variants modified with a pegylating reagent. Use of the aprotinin variants according to claims 1 to 4 for the manufacture of medicaments, for use in surgery, to reduce blood loss, in polytrauma, shock, inflammation and Cancer.