EP1002083A1 - Variantes d'aprotinine a proprietes ameliorees et bikunines de variantes d'aprotinine - Google Patents

Variantes d'aprotinine a proprietes ameliorees et bikunines de variantes d'aprotinine

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Publication number
EP1002083A1
EP1002083A1 EP98930775A EP98930775A EP1002083A1 EP 1002083 A1 EP1002083 A1 EP 1002083A1 EP 98930775 A EP98930775 A EP 98930775A EP 98930775 A EP98930775 A EP 98930775A EP 1002083 A1 EP1002083 A1 EP 1002083A1
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European Patent Office
Prior art keywords
arg
ala
thr
leu
cys
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EP98930775A
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German (de)
English (en)
Inventor
Werner Schröder
Heiner Apeler
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Bayer AG
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Bayer AG
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8114—Kunitz type inhibitors
    • C07K14/8117—Bovine/basic pancreatic trypsin inhibitor (BPTI, aprotinin)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to aprotinin variants and bikunins of aprotinin variants with improved enzyme-inhibitory, immunological and pharmacokinetic properties and their production.
  • Aprotinin also known as a bovine pancreatic trypsin inhibitor (BPTI) belongs to the Kunitz-type serine protease inhibitor family.
  • the spectrum of inhibitable serine proteases includes 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 is a single chain, 58 amino acid polypeptide with the following sequence:
  • Trasylol ® natural aprotinin is originally used for the treatment of pancreatitis. Trasylol is used today in cardiac surgery - 2 -
  • potent inhibitors can be generated in this way, e.g. inhibit pancreatic or leukocyte elastase.
  • inhibitory properties of aprotinin and the variants produced by exchange 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, above all, the additional amino acid residues in positions 14, 16, 17, 18, 19, 34, 38 and 39. Variants of aprotinin with improved properties by exchanging one or more of these amino acid residues in the region of the contact region were u. a. Described by way of example in the following patent applications: WO 89/01968, WO 89/10374, EP 307 592, EP 683 229, DE 19 62 998.2, WO 94/01461.
  • aprotinin and its variants could be improved by amino acid exchanges, which determine the physicochemical properties of the substance. So it was possible to significantly lower the kidney binding by lowering the positive net charge of the molecule.
  • Such variants have been described in patent application WO 92/06111.
  • N-terminally modified aprotinin variants have been described in patent application EP 419 878.
  • Bikunins are protease inhibitors, e.g. the inter-alpha-trypsin inhibitor, which contain two Kunitz domains which are separated by a spacer from several amino acids (J.-P.Salier, TIBS 15; 435-439, 1990). As a rule, however, only one of the two Kunitz domains is inhibitory.
  • aprotinin variants each containing only one of the features mentioned, have been described in the patent applications cited above.
  • the aprotinin variants according to the invention now combine in their molecular structure two or three of the features mentioned above or represent protease inhibitors from several Kunitz domains.
  • the amino acid sequences are shown by way of example for some variants or bikunins in FIGS. 1 and 2.
  • the aprotinin variants according to the invention are not limited to the examples mentioned in FIGS. 1 and 2.
  • the aprotinin variants according to the invention also include variants with the N-terminal extension Ala (-2) - Gln (-1), with the natural amino acid residue proline in position 2, with the exchange of other amino acids which carry a positive charge against neutral or negatively charged ones Amino acid residues, or with the exchange of other neutral amino acids for negatively charged amino acid residues, as well as with amino acid exchanges in the spacer, which ensure individual activity of the individual Kunitz domains, as well as connections that result from cleavage of the Kex protease in the leader.
  • the selection of exchanges of amino acid residues in the aprotinin variant follows the principle of producing a substance which has a reduced positive net charge at physiological pH, preferably in the range from +2 to -2.
  • the changes mentioned in the amino acid sequence including the N-terminal extensions or shortenings or deletions can be used in any combination with one another.
  • the aprotinin variants according to the invention thus include all compounds which have a combination of the abovementioned features and have a positive net charge which is reduced at a physiological pH.
  • the amino acids in the spacer can be combinations of natural amino acids that ensure the alignment of the individual domains so that they are individually active.
  • the aprotinin variants for the linkage are described inter alia in EP 307 592, WO 92/06111, EP 419 878, WO 89/01968, WO 89/10374, EP 0307592, EP 683 229, DE 196 29 982. - 5 -
  • the present invention relates to aprotinin variants with a net charge of +3 to -3 at pH 7, wherein in the binding region the amino acid residue is 10 Ser, the amino acid residue is 13 Ile, Phe or Leu, the amino acid residue is 15 Arg, the amino acid residue is 17 Tyr, Leu or Arg and the amino acid residue is 19 Thr or Lys.
  • aprotinin variants with a net charge of +2 to -2 are preferred, those with +1 to -1 being particularly preferred.
  • Aprotinin variants can have an altered N-terminal and / or C-terminal sequence. This means aprotinin variants with an N-terminal extension or shortening or with deleted amino acids in the N-terminus. - 6 -
  • X 2 is Pro or a bond
  • X 13 is Ile
  • Phe or Leu
  • X 17 is Tyr Leu or Arg
  • X 24 is Asp or Asn
  • X 31 is Glu or Gin is X 39 Arg or Leu
  • X 46 Lys or Leu is or polyethylene glycol derivatives thereof.
  • DesPro2-Ser10-lle13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin DesPro2-Ser10-lle13-Arg15-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin are particularly preferred , DesPro2-Ser10-lle13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Leu39-Asn41-Glu53-Aprotinin, DesPro2-Ser10-lle13-Arg15-Thr19-Asp24-Thr26-Glu31 -Leu39-Asn41 -Glu53-Aprotinin , DesPro2-Ser10-Phe13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-Aprotinin, DesPro2-Ser10-Phe13-Arg15-T
  • aprotinin variants mentioned above are suitable for inhibiting plasma kallikrein, plasmin and factor Xa.
  • the invention further relates to bikunins comprising two aprotinin variants linked by a spacer with several amino acid residues, one of the arpotinin variants a) being an aprotinin variant in which the amino acid residue is 13, Ile, Phe or Leu, the amino acid residue 15 is Arg, Val or Leu, the amino acid residue 17 is Tyr, Leu or Ala, the amino acid residue 19 is Thr or Lys, the amino acid residue 39 is Arg or Leu, and the amino acid residue 46 is Leu or Lys, or b) is an aprotinin variant according to the invention defined above. - 7 -
  • the spacer preferably has 10 to 40, in particular 10 to 20, amino acid residues. Sequences with 13 amino acid residues are particularly preferred as spacers, in particular the following sequences: Asn-Ala-Asn-Arg-Ile-Ile-Lys-Thr-Thr-Leu-Gln-Gln-Glu, Asn-Ala-Asn-Arg-Leu -Leu-Lys-Thr-Thr-Leu-Gln-Gln-Glu and Gln-Ala-Gln-Arg-lle-lle-Lys-Thr-Thr-Leu-Gln-Gln-Glu.
  • bikunins can have an altered N-terminal sequence. This means Bikunine with an N-terminal extension or shortening or with deleted amino acids in the N-terminus. These bikunines can also contain exchanges and extensions or shortenings of the preferred spacer sequences.
  • the Bikunine are suitable for the inhibition of plasma kallikrein, factor Xa, plasmin and elastase.
  • This invention also relates to medicaments containing one or more of these aprotinin variants or bikunins.
  • novel protease inhibitors described are suitable for the treatment of disease states in which - also as a result of complex surgical processes such as e.g. in cardiac surgery or joint replacement or in transplantation medicine, in artificial organs - the plasmatic enzyme systems are activated by extensive or intensive contact with foreign surfaces and / or by cellular components of the blood.
  • the inhibitors reduce blood loss during operations that are associated with an increased risk of bleeding (e.g. heart operations, bone and joint surgery). They can also be used to treat thromboembolic conditions such as those that may occur after surgery, in hypercoagulable blood conditions, after accidents or after thrombolysis treatment. They are suitable for therapy for shock, polytrauma, sepsis, disseminated intravascular coagulation (DIC), multi-organ failure (MOF), for unstable angina, for heart attack, stroke, embolism and deep vein thrombosis, for reocclusion, perfusion damage, thrombosis and bleeding after the To prevent thrombolysis. You can use rheumatic like inflammatory diseases involving the kallikrein system - 9 -
  • Joint diseases and asthma can be used. They prevent invasive tumor growth and metastasis by inhibiting fibrinolysis. They are also suitable for pain and edema therapy, e.g. in traumatic brain injury, by inhibiting tissue and plasma kallikrein. By inhibiting the intrinsic and extrinsic blood coagulation pathways, they can be used to prevent activation of hemostasis in dialysis treatment.
  • the invention further relates to DNA sequences which code for one of the aprotinin variants or bikunins according to the invention, microorganisms which contain such a DNA sequence, and a method for producing the aprotinin variants and bikunins according to the invention using the microorganisms.
  • Figures 1 and 2 show the preferred aprotinin variants and bikunins of aprotinin variants of the present invention.
  • the bold amino acid residues represent mutations of natural aprotinin.
  • D represents a deleted amino acid residue.
  • the spacers of the bikunins are amino acid residues 59 to 71.
  • the genetic information ie a corresponding DNA sequence, which codes the aprotinin variants and bikunins according to the invention, is introduced into a suitable microbial expression organism for the synthesis of the aprotinin variant in each case using conventional molecular biological methods.
  • the recombinant microorganism is fermented; the heterologous genetic information is expressed by choosing suitable conditions.
  • the expressed aprotinin variant or the bikunin is then obtained from the culture broth.
  • Suitable host organisms for the production of the aprotinin variants and bikunins according to the invention can be bacteria, yeasts or fungi. Expression can be carried out intracellularly or extracellularly using suitable secretion systems.
  • the aprotin variant or the bikunin can be processed correctly or fused to peptides or proteins.
  • Enzymes The enzymes used (restriction endonucleases, alkaline phosphatase from calf intestine, T4 polynucleotide kinase and T4 DNA ligase) were obtained from Boehringer Mannheim and GIBCO / BRL and used according to the manufacturers' instructions.
  • Oligonucleotides for 'site directed mutagenesis' experiments and primers for PCR and sequencing reactions were produced using the '380 A DNA Synthesizer' from Applied Biosystems.
  • the mutagenesis experiments were carried out by a method by Deng and Nickoloff (Deng et al., Anal. Biochem. 200, 81-88, 1992) using a kit from Pharmacia Biotech ('Unique Site Elimination Mutagenesis'). All vector constructions and mutagenesis experiments - 11 -
  • yeast cells e.g. strain JC34.4D (MAT ⁇ , ura3-52, suc2) were dissolved in 10 ml YEPD (2% glucose; 2% peptone; 1% Difco
  • Yeast extract Yeast extract
  • the cells were washed with 5 ml of solution A (1 M sorbitol; 10 mM bicin pH 8.35; 3% ethylene glycol), resuspended in 0.2 ml of solution A and stored at -70 ° C.
  • Plasmid DNA (5 ⁇ g) and Carrier DNA (50 ⁇ g DNA from herring sperm) were added to the frozen cells. The cells were then thawed by shaking at 37 ° C for 5 min. After adding 1.5 ml of solution B (40% PEG 1000; 200 mM
  • Feeding was started after a fermentation time of 7 hours.
  • Samples were taken from the fermenter at regular intervals and cell growth was determined by measuring the optical density at 700 nm. In addition, the concentration of the protease inhibitors in the supernatant was determined by activity measurement.
  • the pH was lowered to 3.0 by adding 50% (w / v) citric acid and the fermenter was heated to 70 ° C. for 10 min.
  • the cells were then separated by centrifugation at 7500 x g and the supernatant was released for protein purification.
  • the sequence analyzes were carried out using a Model 473A protein sequencer from Applied Biosystems (Forster City, U.S.A.). The standard sequencing program was used. The sequencer, the various sequencing programs and the PTH detection system are described in detail in the operating manual (User's manual protein sequencing System model 473A (1989) Applied Biosystems Forster City, CA 94404, U.S.A.).
  • the reagents for operating the sequencer and the HPLC column for PTH detection were obtained from Applied Biosystems.
  • HPLC analyzes were carried out with an HP1090 HPLC system from Hewlett Packard (D-Waldbronn).
  • An RP-18 HPLC column 250 mm x 4.6 mm, 5 ⁇ - - 15 -
  • Model 270A-HT capillary electrophoresis was from Applied Biosystems (Forster City, CA 94404, U.S.A.). The samples were usually injected hydrodynamically over different time intervals.
  • the capillary column used 50 ⁇ m x 72 cm was from Applied Biosystems.
  • the amino acid analyzes were carried out with an amino acid analyzer LC3000 from Eppendorf Biotronik (D-Maintal). A slightly modified standard separation program from Biotronik was used. The separation programs and the function of the analyzer are described in detail in the device manual.
  • the molecular weights were determined using a MALDI I system from Kratos / Shimadzu (D-Duisburg).
  • the SDS electrophoresis was carried out with an electrophoresis system from Pharmacia (D-Freiburg).
  • the kinetic data were determined using the microplate reader from SLT (D-Crailsheim). The microtiter plates were washed with a washing machine from Dynatec (D-Denkendorf).
  • Enzymes and substrates were from Calbiochem (D-Bad Soden). All other chemicals and reagents were from Merck (D-Darmstadt) or Sigma (D-Deisenhofen). 96 well plates were purchased from Greiner.
  • the rabbit polyclonal anti-aprotinin antibodies were raised in the rabbit by immunization with aprotinin.
  • the human polyclonal anti-aprotinin antibodies come from patients treated with aprotinin. - 16 -
  • N-terminal sequence analysis 1-3 nmol of protease inhibitor dissolved in water was loaded onto a sequencer sheet which had been preincubated with Polybren ® . The protein was sequenced using the almost normal sequencer cycle. The PTH amino acids were identified by online HPLC using a 50 pmol PTH standard.
  • Amino acid analysis 200 ⁇ g protein were dissolved in 200 ⁇ l 6N HCl and hydrolyzed at 166 ° C. for 1 h. Approximately 1 nmol of the sample was placed on the amino acid analyzer. The amount of amino acid was determined using a 5 nmol standard. Cysteine was determined after performic acid oxidation of the protein (C.H.W. Hirs, Methods Enzymol. 11, 59-62) as described above.
  • SDS gel electrophoresis was carried out according to the conditions of Laemmli. 10 ⁇ g of the protease inhibitor were analyzed with a 10-20% SDS gel and visualized by means of silver staining (Merril et.al.). (U.K. Laemmli, Nature 227, 680-685 (1970). And C.R.Merril, M.LDunau, D.Goldman, Anal.Biochem. 110: 201-207 (1981)).
  • Capillary electrophoresis 8 ng of the protease inhibitor were examined by capillary electrophoresis on a glass column (length 72 cm, inner diameter 50 ⁇ m). Conditions: current 65 ⁇ A, column temperature 30 ° C, 100 mM phosphate buffer pH 3.0, detection 210 nm, application under pressure for 1 s.
  • Reverse phase chromatography 5 nmol of the protease inhibitor was chromatographed on a Bakerbond RP-18 HPLC column (5 ⁇ material, 4.6 mm ⁇ 250 mm, 30 nm (300 angstroms) pore size). An acetonitrile / TFA gradient was used as eluent. Conditions: flow 0.7 ml / min, column temperature 40 ° C., detection 210 nm, solvent A: 0.1% TFA, solvent B: 0.1% TFA / 60% acetonitrile; Gradient: 0 min. 0% B, 10 min. 0% B, 70 min. 100% B, 80 min. 0% B. - 17 -
  • Molecular weight determination 1 ⁇ g of the protease inhibitor was analyzed using the MALDI technique. Sinapic acid was used as the matrix. The standard proteins for mass calibration were melittin (2847 Da), bovine insulin (5734 Da), cytochrome C (12327 Da) and myoglobin (16951 Da).
  • Protein content The protein content of the samples was determined using amino acid analysis or by measuring the OD280 nm.
  • Trypsin inhibition test (titrimetric): The trypsin-inhibitory activity of the protease inhibitors in the fermentation broths was determined via the trypsin-catalyzed hydrolysis of N ⁇ -benzoyl-L-arginine ethyl ester (BAEE). The number of carboxyl groups released in the reaction, which are determined by an alkali titration, is a measure of the tryptic inhibitory activity of the inhibitors.
  • BAEE N ⁇ -benzoyl-L-arginine ethyl ester
  • Determination of the cross-reaction of the protease inhibitors with polyclonal rabbits or human anti-aprotinin antibodies 0.5-10 ng protease inhibitor or aprotinin dissolved in the coupling buffer were bound to a microtiter plate at 4 ° C. overnight. The wells were washed 4 times with 300 ⁇ l washing buffer and then 100 ⁇ l of the blocking solution was added. The plate was covered and incubated at 37 ° C for one hour.
  • the polyclonal rabbit anti-aprotinin antibody (0.2 ⁇ g / ml in 1% BSA in PBS buffer) or the polyclonal human antibody (20 ⁇ g / ml in 1% HSA in PBS buffer) was added .
  • the plate was covered, incubated at 37 ° C for one hour and then washed as described above.
  • 100 ⁇ l of biotinylated anti-rabbit or anti-human antibody 25 ⁇ l + 10 ml of 1% BSA or 1% HSA in PBS buffer were then added and incubated at 37 ° C. for one hour.
  • the plate was washed as described above and then 100 ul streptavidin-peroxidase complex (50 ul + 10 ml 1% BSA or 1% HSA in PBS buffer) was added to each well. The plate was covered and incubated at 37 ° C for one hour and then washed as described above. - 18 -
  • the substrate reaction was carried out with TMB substrate + peroxidase solution (1 + 1; 100 ⁇ l per well). After 10 min, the reaction was stopped with 100 ⁇ l / well of 2 M phosphoric acid and the absorption was measured at 450 nm (reference 570 nm).
  • Coupling buffer 15 mM Na 2 CO 3 , 35 mM NaHCO 3 , pH 9.6
  • Sample buffer The samples were dissolved in a suitable concentration in 1% BSA or HSA in PBS buffer pH 7.
  • Blocking buffer 3% (w / v) BSA or HSA in PBS.
  • the substrates were chromozyme PL for plasmin, HD-Pro-Phe-Arg-pNA for factor XI, S-2444 for trypsin, Suc-Phe-Leu-Phe-pNA for chymotrypsin, Bz-III-Glu-Gly-Arg-pNA for FXa, HD-Val-Leu-Arg-pNA for urinary kallikrein and HD-Pro-Phe-Arg-pNA.
  • Example 1 Preparation of a yeast expression vector for the secretion of recombinant Ser10-Ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin
  • a Ser10-Arg15-Ala17-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin gene was used as a starting material for the production of the Ser10-Ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin gene modified pYES2 vector (plU28.11.L).
  • the GAL1 promoter present on the vector pYES2 (Invitrogen) and the f1 ori are replaced in this vector by the MF ⁇ 1 promoter.
  • the vector plU28.11.L was digested with EcoRI and partially with Xbal and then filled in with Klenow polymerase and dNTPs.
  • the vector resulting from this cloning (plU17.4.M) was subjected to a double-strand mutagenesis reaction using the Mutagenesis Primer A using the USE method (Pharmacia Biotech), the restriction being able to be carried out using Xhol, since the mutagenesis resulted in the singular Xhol interface in the aprotinin gene is removed.
  • the mutagenesis primer A had the following sequence:
  • This primer generates the mutations Ile13, Tyr17 and Thr19 in the Ser10-Arg15-Ala17-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin gene.
  • the clones were analyzed by restriction digestion with the enzymes Xbal and Xhol. The desired sequence was also confirmed by DNA sequencing of the clone pIU2.7.M.
  • Yeast cells (JC34.4D) were transformed with the vector plU2.7.M.
  • the expression vector plU2.7.M no longer contains any ⁇ -factor per sequence, so that the processing of the Ser10-Ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin takes place exclusively and independently of the signal peptidase is from the cleavage by the Kexll protease.
  • Example 2 Preparation of a yeast expression vector for the secretion of recombinant Ser10-Ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Leu39-Asn41-Glu53-aprotinin
  • a Ser10-Arg15-Ala17-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin gene was used as the starting material for the production of the Ser10-Ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Leu39-Asn41-Glu53-Aprotinin gene in the vector plU17.4.M (see example 1).
  • the vector plU17.4.M was subjected to a double-strand mutagenesis reaction with the mutagenesis primers A (see Example 1) and B using the USE method (Pharmacia Biotech), the restriction being able to be carried out with Xhol, since - 20 -
  • mutagenesis with primer A removes the unique Xhol site in the aprotinin gene.
  • the mutagenesis primer B had the following sequence:
  • Primer A generates the mutations Ile13, Tyr17 and Thr19 and primer B the mutation Leu39 in the Ser10-Arg15-Ala17-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin gene.
  • the clones were analyzed by restriction digestion with the enzymes Xbal and Xhol. The desired sequence was also confirmed by DNA sequencing of the clone pES8.4.O.
  • Yeast cells (JC34.4D) were transformed with the vector pES8.4.O.
  • Example 3 Preparation of a yeast expression vector for the secretion of recombinant He13-Arg15-Tyr17-Thr19-Leu39-Leu46-aprotinin with the natural N-terminal sequence 'Arg-Pro-Asp'
  • the MF ⁇ 1 promoter with the ⁇ factor was first used pre-sequence and the 5 ° end of the aprotinin gene (up to the recognition sequence of the restriction enzyme Xhol) were amplified by PCR and cloned.
  • the primers used had the following sequences:
  • Primer C (the EcoRV recognition sequence is underlined): 5 'GGG .
  • AI CTATTGATAAGATTTAAAGGTATTTGACAAG 3 '.
  • the PCR mixture contained 200 ng pA202 plasmid DNA, 0.2 ⁇ M primer C, 0.2 ⁇ M primer D, 200 ⁇ M dNTPs, 1 x PCR reaction buffer 11 (Stratagene, Opti- Prime® ) and 2.5 U Taq DNA polymerase (Perkin bucket) in a total volume of 50 ⁇ l.
  • the " cycle " conditions were: 1 min at 94 ° C, 30 cycles of 1 min each at 94 ° C, 1 min at 50 ° C and 2 min at 72 ° C and a subsequent 5 min incubation at 72 ° C.
  • the PCR mixture was diluted 1: 5 and ligated with the vector pCRH (Invitrogen). E. coli DH5 ⁇ cells were transformed with the ligation mixture. Positive clones were identified after restriction digestion with the enzyme EcoRI and several clones were sequenced. The clone plU20. 11.L was used for further work.
  • the E. coli / yeast swing vector pYES2 (Invitrogen) was used to construct a yeast secretion vector in which the Ile13-Arg15-Tyr17-Thr19-Leu39-Leu46-aprotinin sequence was directly linked to the yeast alpha-factor pre-sequence is.
  • the vector pYES2 was cut with the restriction enzymes Sspl and BamHI, dephosphorylated and gel-cleaned.
  • the GAL1 promoter present on the vector pYES2 and the f1 ori are removed in the process. An approx.
  • 1030 bp DNA fragment was cut out of the vector plU20.11.L with EcoRV and Xhol, purified by agarose gel electrophoresis and together with an approx. 180 bp Xhol and BamHI fragment from the vector pEM24.3.L in Vector pYES2 clipped with Sspl and BamHI.
  • E. coli DH5 ⁇ cells were transformed. After a restriction digest, positive clones were identified with the enzyme Xhol and sequenced. Yeast cells (JC34.4D) were transformed with the vector pEM1.4.L resulting from this cloning.
  • Example 4 Preparation of a yeast expression vector for the secretion of recombinant DesPro2-Ill13-Arg15-Tyr17-Thr19-Leu39-Leu46 spacer-Arg15-Ala17-bikunin
  • Tyr17-Thr19-Leu39-Leu46-Spacer-Arg15-Ala17-Bikunin genes served one DesPro2-He13-Arg15-Tyr-17-Thr19-Leu-39-Leu46 gene, one Arg15-Ala17 gene and two - 22 -
  • Each aprotinin variant gene was coupled to one of the spacer oligonucleotides by means of a PCR reaction and ligated into a PCR cloning vector.
  • Spacer oligonucleotide 2 5 'GGA AGA TTG TAT GAG AAC CTG CGG TGG TGC TAA CGC TAA CAG AAT TAT GAA GAC TAC TTT GCA ACA AGA AAA GCC AGA TTT CTG TC 3'
  • the PCR mixture contained 570 ng pEM30.3.L plasmid DNA (coding for DesPro (2) - Ile (13) -Arg (15) -Tyr (17) -Thr (19) -Leu (39) -Leu (46 ) -Protinin), 20 pmol primer E (primer E binds in the ⁇ -factor leader and has the following sequence: 5 'AACGGGTTATTGTTTATA 3'), 60 pmol spacer oligonucleotide 1, 200 ⁇ M dNTP, 1x PCR reaction buffer II (Perkin Elmer), 2 mM MgCI 2 and 2.5 U Taq DNA polymerase (Perkin Elmer) in a total volume of 100 ⁇ l.
  • the Oycle ' conditions were: 3 min at 95 ° C, 25 cycles of 1 min each at 94 ° C, 1 min at 55 ° C and 1 min at 72 ° C and a subsequent 5 min incubation at 72 ° C.
  • the PCR mixture was diluted 1: 5 and ligated with the vector pCRII (invitrogen). With the ligation approach, E. coli DH5 ⁇ cells were transformed. Positive clones were identified via "blue-white screening" and after restriction digestion with the enzymes Xhol and BamHI. The clone pEM22.5.L contained the desired sequence and was used for further work.
  • Reaction conditions Heat test batch without Klenow fragment for 3 min at 95 ° C. After adding the Klenow fragment, the mixture was incubated at room temperature for 30 min. The mixture was heated to 70 ° C. and 2.5 U Taq DNA polymerase and 20 pmol reverse primer M13 were added.
  • the cycle conditions were: 3 min at 95 ° C, 30 cycles of 1 min each at 94 ° C, 1 min at 45 ° C and 1 min at 72 ° C and a subsequent 5 min incubation at 72 ° C.
  • Approximately 150 ng of the PCR fragment were ligated with the vector pCRH (Invitrogen). With the ligation approach, E. coli DH5 ⁇ cells were transformed. After a restriction digest, positive clones were identified with the enzyme EcoRI and sequenced. The clone pEM12.6.L contained the desired sequence and was used for the further work.
  • a 182 bp DNA fragment was cut out of the vector pEM22.5.L with Hind III and BspMI and a 240 bp DNA fragment with BamHI and BspMI from the vector pEM 12.6.L, purified by agarose gel electrophoresis and overnight ligated with T4 DNA ligase.
  • the ligation product was cloned into the vector pUC 19 via the restriction sites Hind III and BamHI.
  • the resulting clone (pEM16.6.L) had the desired sequence.
  • coli / yeast swing vector pA202 was used for the construction of a yeast secretion vector in which the DesPro2-Ile13-Arg15-Tyr17-Thr19-Leu39-Leu46-Spacer-Arg15-Ala17-Bikunin sequence with the yeast alpha factor pre -pro- linked.
  • the vector pA202 carries an ampicillin resistance gene (bla) and a URA3 gene as selectable marker genes for E. coli and yeast.
  • Col E1 the Col E1 and the 2 ⁇ origin of replication (ori).
  • the REP3 locus is also in this region.
  • a 1200 bp EcoRI-Hindlll fragment carries the MF ⁇ 1 promoter and the N-terminal pre-pro sequence of the yeast alpha factor precursor protein (Kurjan and Herskowitz, Cell 30, 933-943, 1982).
  • Yeast vector cloning A 422 bp fragment is cut out of the clone pEM16.6.L with the restriction enzymes HindIII and BamHI. This is purified on an agarose gel and also cloned into the vector pA202 cut with HindIII and BamHI. The resulting clone pEM21.6.L is used for the transformation of yeast cells (JC34.4D).
  • E. coli / yeast swing vectors with different promoters such as the constitutive GAPDH or the inducible GAL10 promoter can be produced in a similar manner and also lead to the secretion of the DesPro2-Ile13-Arg15-Tyr17-Thr19-Leu39-Leu46-spacer-Arg15-Ala17-bikunin.
  • swing vectors with other origins of yeast replication e.g. use the chromosomally autonomously replicating segment (ars).
  • Suitable selectable marker genes are, in addition to the URA3 gene, those genes which help an auxotrophic yeast mutant to prototrophy, such as e.g. the LEU2, HIS3 or TRP1 genes. In addition, genes whose products are resistant to various antibiotics, such as e.g. mediate the aminoglycoside G418.
  • yeasts such as After transformation with suitable vectors, the methylotrophic yeasts Pichia pastoris or Hansenula polymorpha are also able to produce DesPro2-Ile13-Arg15-Tyr17-Thr19-Leu39-Leu46-Spacer-Arg15-Ala17-Bikunin.
  • a recombinant Saccharomyces cerevisae expression strain was used for the fermentation (see: Methods for Carrying Out the Invention). - 25 -
  • Example 6 Cleaning of aprotinin variants without a net charge at neutral pH and of bikunins
  • the cell-free supernatant is adjusted to pH 3 by adding concentrated citric acid.
  • the solution must be suitably diluted with purified water to achieve a conductivity of less than 8 mS / cm.
  • the solution is then applied to a cation exchange column which has previously been equilibrated with an acid buffer. Unbound material is removed by washing extensively with start buffer.
  • the product is eluted using a salt gradient.
  • the fractions obtained are examined for their product content by means of reverse phase high pressure liquid chromatography (RP-HPLC) and a biological activity test which determines the protease inhibition. Fractions containing the product are pooled and applied directly to a preparative RP-HPLC column.
  • RP-HPLC reverse phase high pressure liquid chromatography
  • the column was previously equilibrated with an acid buffer. Unbound protein is removed by washing the column with start buffer. The product is eluted using an organic solvent gradient. As already described above, the fractions are examined again for the product content and those fractions which contain product are combined. Depending on the purity of the product, it may be necessary to purify the pooled fractions on a second RP-HPLC column. The conditions are essentially the same as previously described. The product solution obtained is diluted with water for injections and filled into suitable portions and freeze-dried. - 26 -
  • the contents of the fermenter were adjusted to pH 3 with concentrated citric acid and heated to 70 ° C. for 10 minutes.
  • the cells were then removed by centrifugation (15 minutes, 7500 ⁇ g, Heraeus centrifuge) and the supernatant obtained was filtered (8 ⁇ m to 0.2 ⁇ m, Millipore, Germany). At this stage, the supernatant can be stored by freezing at -18 ° C until further use.
  • the solution was then applied by addition of purified water to a conductivity of less than 8 mS / cm and applied to an SP-Sepharose ® FF column (Pharmacia, Sweden). The column had previously been equilibrated with 50 mM citrate-NaOH buffer, pH 3.
  • Unbound protein was removed by intensive washing with the same buffer.
  • the product was then eluted using a salt gradient (1 M NaCl).
  • the fractions obtained were examined for the product content by means of reverse phase high pressure liquid chromatography (RP-HPLC, C4) and by testing the protease inhibition activity.
  • the protein was taken up in 150 mM NaCl / 50 mM phosphate buffer pH 7.3 and chromatographed on a Superdex 30. The fractions obtained were examined again for the product content using the methods described above and those containing product were combined.
  • Material from 10 l fermentations was purified by the following procedure. After the fermentation was complete, the contents of the fermenter were adjusted to pH 3 with concentrated citric acid and heated to 70 ° C. for 10 minutes. The cells were then removed by centrifugation (15 minutes, 7500 xg, Heraeus centrifuge) and the supernatant obtained was filtered (8 ⁇ m to 0.2 ⁇ m, Millipore, Germany). At this stage, the supernatant can be stored by freezing at -18 ° C until further use. The solution was then applied by addition of purified water to a conductivity of less than 8 mS / cm and applied to an SP-Sepharose ® FF column (Pharmacia, Sweden).
  • the column had previously been equilibrated with 50 mM citrate-NaOH buffer, pH 3. Unbound protein was removed by intensive washing with the same buffer. The product was then eluted using a salt gradient (1 M NaCl). The obtained fractions were chromatographed after adjusting to less than 8 mS / cm with a Sepharose ® HP. The column had previously been equilibrated with 20 mM Hepes-NaOH buffer, pH 6. Unbound protein was removed by intensive washing with the same buffer. The product was then eluted using a salt gradient (1 M NaCl).
  • the product solution was then applied directly to the first RP-HPLC column (Source 15 RPC, Pharmacia, Sweden), which had previously been equilibrated with 0.1% trifluoroacetic acid / water. Unbound protein was removed by intensive washing with the same buffer. The product was eluted using a linear acetonitrile gradient (0-70%). The fractions obtained were again checked for product content using the methods described above, and those containing product were combined and lyophilized.
  • the protein was chromatographed on a Source S30 using a pH 6 Hepes / NaCl gradient. The fractions obtained were examined again for the product content using the methods described above and those containing the product were combined and lyophilized.
  • Example 7 Determination of the Ki value of human plasmin with Ser10-Ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31 -Asn41 -Glu53-aprotinin
  • the enzyme / inhibitor solution was preincubated for 4 h at room temperature, then 180 ⁇ l of each solution was added to the well of a microtiter plate and 20 ⁇ l of substrate solution were added. The change in absorption was measured at 405 nm for 10 min. The speed of the enzyme reactions was determined and the Ki value calculated therefrom by the method of Bieth (Biochemical Medicine 32: 387-97 (1984) or Meth. In Enzym. 248: 59-84 (1995)).
  • Substrate solution 1x10 3 M Chromozym PL in assay buffer - 29 -
  • Assay buffer 0.2 M tris (hydroxymethyl) aminomethane
  • the kinetic constants of the complexation with the enzymes kallikrein, factor Xa, trypsin and chymotrypsin were determined using the same procedure.
  • the substrates were HD-Pro-Phe-Arg-pNA for plasma kallikrein, S-2444 for trypsin, Suc-Phe-Leu-Phe-pNA for chymotrypsin and Bz-III-Glu-Gly-Arg-pNA for FXa.
  • the kinetic constants of the bikunins, the single domains of the bikunins and the PEG conjugates were determined using the same method.
  • Example 8 Results of the protein chemical characterization of Ser10-Ile13-Arg 15-Tyr17-Thr19-Asp24-Thr26-Glu31 -Asn41 -Glu53-aprotinin
  • protease inhibitor Ser10-lle13-Arg 15-Tyr17-Thr19-Asp24-Thr26-Glu31 -Asn41 - Glu53-aprotinin was produced by secretion using a genetically modified yeast organism. It was purified from the yeast supernatant to homogeneity by various chromatographic methods. The identity of the inhibitor with the cloned sequence is shown in the protein analysis below.
  • N-terminal sequence analysis The protease inhibitor was completely sequenced over 58 steps. The following list shows the particular protein sequence that is identical to the cloned sequence: 1
  • Amino acid analysis represents an important quantitative parameter for the characterization of a protein. In addition to the protein content, the number of individual amino acids is determined if the primary structure is known. The amino acid analysis of Ser10-Ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin is in good agreement with the theoretical values from the primary structure (Tab. 1).
  • Cysteine was determined after performic acid oxidation.
  • Reverse phase chromatography In HPLC chromatography of proteins on chemically bound reverse phases, the hydrophobic interaction of the proteins leads to binding to the phase used. The proteins are displaced by organic solvents (mobile phase) according to the strength of their binding to the stationary phase. For this reason, this method is one - 31 -
  • protease inhibitor Ser10-Ill13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31 -Asn41 -Glu53-aprotinin eluted as a slender peak from an RP-18 phase.
  • CE Chromatography Capillary electrophoresis allows the separation of peptides and proteins based on their charge in the electric field. The quality of the separation depends on the buffer, the pH value, the temperature and the additives used. So-called “fused silica” columns with an inner diameter of 50 to 100 ⁇ m are used as capillaries. The protease inhibitor Ser10-Ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin was separated on a "fused silica" column in an electric field. The electropherogram shows a slender peak.
  • Molecular weight determination The molecular weight of Ser10-Ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin was determined using the MALDI technique to 6425Dalton. The molecular weight determined is in good agreement with the theoretical value of 6408 daltons within the scope of the accuracy of the measurement method. Sinapic acid was used as the matrix.
  • SDS gel electrophoresis Ser10-Ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin was analyzed by SDS electrophoresis under reducing and non-reducing conditions. The gels show a band in the range of approximately 6.5 kD.
  • Example 9 Determination of the Ki values for the complexation of enzymes with Ser10-Ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41 -Glu53-aprotinin
  • the inhibitory constants of Ser10-ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin were determined for various enzymes.
  • the Ki values are shown in Table 2. - 32 -
  • Example 10 Interaction of the protease inhibitors with polyclonal rabbits or human anti-aprotinin antibodies
  • protease inhibitors were examined for their cross-reactivity with polyclonal rabbits or human anti-aprotinin antibodies. It was found that the different protease inhibitor variants show only a very weak cross-reactivity towards the polyclonal rabbits or human anti-aprotinin antisera used.
  • Example 11 Determination of the Ki value of human urinary kallikrein with DesPro2-Ile13-Arg 15-Tyr17-Thr19-Leu39-Leu46 spacer-Arg 15-Ala17-bikunin
  • the Ki value was determined using human urinary kallikrein as described in Example 7.
  • the inhibitor concentration used was 1.1 ⁇ g / ⁇ l. 33 -
  • Assay buffer 0.05 M tris- (hydroxymethyl) aminomethane, 0.1 M NaCl, 0.05% Tween ® -20; pH 7.5; 1 ml benzyl alcohol / l.
  • the kinetic constants of the complexation with the enzymes kallikrein, factor Xa, chymotrypsin and plasmin were determined using the same procedure.
  • the substrates were HD-Pro-Phe-Arg-pNA for plasma kallikrein, Suc-Phe-Leu-Phe-pNA for chymotrypsin, Bz-III-Glu-Gly-Arg-pNA for FXa and Chromozym PL for plasmin.
  • Example 12 Results of the protein chemical characterization of DesPro2-Ile13-Arg 15-Tyr17-Thr19-Leu39-Leu46 spacer-Arg 15-Ala17-bikunin
  • the protease inhibitor DesPro2-lle13-Arg 15-Tyr17-Thr19-Leu39-Leu46-Spacer-Arg15-Ala17-Bikunin was produced by secretion using a genetically modified yeast organism. It was purified from the yeast supernatant to homogeneity by various chromatographic methods. The identity of the inhibitor with the cloned sequence is shown in the protein analysis below.
  • the protease inhibitor was completely sequenced.
  • the sequencing was carried out N-terminally, via fragments of the bikunin which were isolated from the culture supernatant and via cyanogen bromide.
  • the following list shows the specific protein sequences of the fragments: N-terminal sequencing (underlined), fragment of bikunin from the culture supernatant of the yeast fermentation (bold), bromine cyanogen fragment (italic).
  • the sequence found is identical to the cloned sequence. - 34 -
  • Amino acid analysis represents an important quantitative parameter for the characterization of a protein. In addition to the protein content, the number of individual amino acids is determined if the primary structure is known. The amino acid analysis of DesPro2-lle13-Arg 15-Tyr17-Thr19-Leu39-Leu46-Spacer-Arg15-Ala17-Bikunin is in good agreement with the theoretical values from the primary structure (Table 3).
  • Cysteine and methionine were determined after performic acid oxidation (Met as methionine sulfone).
  • Reverse phase chromatography In HPLC chromatography of proteins on chemically bound reverse phases, the hydrophobic interaction of the proteins leads to binding to the phase used. The proteins are displaced by organic solvents (mobile phase) according to the strength of their binding to the stationary phase. For this reason, this method is a good criterion for assessing the purity of a protein. DesPro2-Ile13-Arg15-Tyr17-Thr19-Leu39-Leu46-Spacer-Arg15-Ala17-Bikunin eluted as a clean peak from the RP-18 phase.
  • Example 13 Determination of the Ki values for the complexation of enzymes with DesPro2-III13-Arg 15-Tyr17-Thr19-Leu39-Leu46 spacer-Arg 15-Ala17-bikunin
  • Table 4 Inhibitory constants of the complexation of DesPro2-lle13-Arg15-Tyr17-Thr19-Leu39-Leu46-Spacer-Arg15-Ala17-Bikunin with the enzymes urinary kallikrein, factor Xa, bovine chymotrypsin, plasma kallikrein and plasmin.
  • Example 14 Results of the protein chemical characterization of Thr-Thr-Leu-Gln-Gln-Glu-Lys-DesArg 1 -Arg 15-Ala17-aprotinin
  • protease inhibitor Thr-Thr-Leu-Gln-Gln-Glu-Lys-DesArg1-Arg15-Ala17-aprotinin was isolated from the yeast supernatant along with other cleavage products of bikunin by various chromatographic methods.
  • Reverse phase chromatography In HPLC chromatography of proteins on chemically bound reverse phases, the hydrophobic interaction of the proteins leads to binding to the phase used. The proteins are displaced by organic solvents (mobile phase) according to the strength of their binding to the stationary phase. For this reason, this method is a good criterion for assessing the purity of a protein. Thr-Thr-Leu-Gln-Gln-Glu-Lys-DesArg1-Arg15-Ala17-aprotinin eluted as a clean peak from the RP-18 phase.
  • Molecular weight determination The molecular weight of Thr-Thr-Leu-Gln-Glu-Glu-Lys-DesArg1-Arg15-Ala17-aprotinin was determined using the MALDI technique as Na + -lon to 7154 daltons. The molecular weight determined is in good agreement with the theoretical value of the Na + ion of 7150 daltons within the scope of the accuracy of the measurement method. Sinapic acid was used as the matrix. - 38 -
  • Aprotinin was analyzed by SDS electrophoresis under reducing and non-reducing conditions.
  • the gels show a band in the range of approx. 7kD.
  • Example 15 Determination of the Ki values for the complexation of enzymes with Thr-Thr-Leu-Gln-Gln-Glu-Lys-DesArg1-Arg15-Ala17-aprotinin
  • the inhibitory constants of Thr-Thr-Leu-Gln-Gln-Glu-Lys-DesArg1-Arg15-Ala17-aprotinin were determined for various enzymes.
  • the Ki values are shown in Table 5.
  • Table 5 Inhibitory constants of the complexation of Thr-Thr-Leu-Gln-Gln-Glu-Lys-DesArg1-Arg15-Ala17-aprotinin with the enzymes urinary kallikrein, factor Xa, chymotrypsin, plasma kallikrein and plasmin.
  • Chymotrypsin 1x10 "8 - 39 -
  • Example 16 Results of the protein chemical characterization of Asp (-3) -Lys (- 2) -Arg (-1) -DesPro2-Ile13-Arg15-Tyr17-Thr19-Leu39-Leu46-aprotinin-Asn59-Ala60-Asn61
  • the protease inhibitor Asp (-3) -Lys (-2) -Arg (-1) -DesPro2-Ill13-Arg15-Tyr17-Thr19-Leu39-Leu46-aprotinin-Asn59-Ala60-Asn61 was obtained from the yeast supernatant along with other cleavage products of bikunin different chromatographic methods isolated.
  • the protease inhibitor corresponds to the front part of the bikunin. It contains the LKunitz domain. It was cleaned to homogeneity. Belonging to Bikunin is shown in the following protein analysis tests.
  • Reverse phase chromatography In HPLC chromatography of proteins on chemically bound reverse phases, the hydrophobic interaction of the proteins leads to binding to the phase used. The proteins are displaced by organic solvents (mobile phase) according to the strength of their binding to the stationary phase. For this reason, this method is a good criterion for assessing the purity of a protein.
  • the protease inhibitor Asp (-3) -Lys (-2) -Arg (-1) -DesPro2-Ill13-Arg15-Tyr17-Thr19-Leu39-Leu46-aprotinin-Asn59-Ala60-Asn61 eluted as a clean peak from the RP-18 - phase. - 40 -
  • Capillary electrophoresis The protease inhibitor Asp (-3) -Lys (-2) -Arg (-1) -DesPro2-lle13-Arg15-Tyr17-Thr19-Leu39-Leu46-aprotinin-Asn59-Ala60-Asn61 was analyzed using capillary electrophoresis. The inhibitor shows a slim peak at approx. 12 min.
  • Example 17 Determination of the Ki values for the complexation of enzymes with Asp (- 3) -Lys (-2) -Arg (-1) -DesPro2-Ile13-Arg15-Tyr17-Thr19-Leu39-Leu46-Aprotinin-Asn59- Ala60-Asn61
  • the inhibitory constants of Asp (-3) -Lys (-2) -Arg (-1) -DesPro2-Ile13-Arg15-Tyr17-Thr19-Leu39-Leu46-aprotinin-Asn59-Ala60-Asn61 were determined for various enzymes.
  • the Ki values are shown in Table 6.
  • Table 6 Inhibitory constants of the complexation of Asp (-3) -Lys (-2) -Arg (-1) - DesPro2-Ile13-Arg15-Tyr17-Thr19-Leu39-Leu46-aprotinin-Asn59-Ala60-Asn61 with the enzymes Factor Xa, plasma kallikrein and plasmin.
  • Example 18 Synthesis and purification of Ser10-Ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin-PEG-5000
  • the SC-PEG-5000 (succinimidyl carbonate, polyethylene glycol monomethyl ether-5000; average molecular weight 5 kD) was obtained from Calbiochem, D-Bad Soden.
  • Example 19 Results of the protein chemical characterization of PEG-Ser10-He13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin
  • protease inhibitor PEG-Ser10-Ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin was separated from the starting products after the reaction by gel filtration and used for characterization.
  • Example 20 Determination of the Ki values for the complexation of enzymes with PEG-Ser10-Ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin
  • Table 7 Inhibitory constants of the complexation of PEG-Ser10-Ile13-Arg15-Tyr17-Thr19-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin with the enzymes factor Xa, plasma kallikrein and plasmin.

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Abstract

L'invention concerne des variantes d'aprotinine ayant une charge nette comprise entre +3 et -3 pour un pH de 7, dans la zone de la liaison, le reste aminoacide 10 désigne Ser, le reste aminoacide 13 désigne Ile, Phe ou Leu, le reste aminoacide 15 désigne Arg, le reste aminoacide 17 désigne Tyr, Leu ou Arg et le reste aminoacide 19 désigne Thr ou Lys. L'invention concerne en outre des bikunines comprenant deux variantes d'aprotinine liées avec plusieurs restes aminoacide par un espaceur. Une des variantes d'aprotinine est a) une variante d'aprotinine dans laquelle le reste aminoacide 13 désigne Ile, Phe ou Leu, le reste aminoacide 15 désigne Arg, Val ou Leu, le reste aminoacide 17 désigne Tyr, Leu ou Ala, le reste aminoacide 19 désigne Thr ou Lys, le reste aminoacide 39 désigne Arg ou Leu, et le reste aminoacide 46 désigne Leu ou Lys, ou b) une variante d'aprotinine obtenue selon l'invention et définie précédemment. L'invention concerne en outre: des médicaments contenant une ou plusieurs de ces variantes d'aprotinine ou des bikunines; des séquences d'ADN codant une de ces variantes d'aprotinine ou ces bikunines; des micro-organismes contenant une telle séquence d'ADN; et un procédé de préparation de ces variantes d'aprotinine et de ces bikunines à l'aide de micro-organismes.
EP98930775A 1997-06-13 1998-06-02 Variantes d'aprotinine a proprietes ameliorees et bikunines de variantes d'aprotinine Withdrawn EP1002083A1 (fr)

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DE19725014A DE19725014A1 (de) 1997-06-13 1997-06-13 Aprotininvarianten mit verbesserten Eigenschaften und Bikunine von Aprotininvarianten
DE19725014 1997-06-13
PCT/EP1998/003259 WO1998056916A1 (fr) 1997-06-13 1998-06-02 Variantes d'aprotinine a proprietes ameliorees et bikunines de variantes d'aprotinine

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GB0411145D0 (en) * 2004-05-19 2004-06-23 Imp College Innovations Ltd Protease inhibitors and their therapeutic applications
CN101102819A (zh) * 2004-12-23 2008-01-09 Csl百灵有限公司 血栓的形成和/或稳定化的预防
WO2008086858A1 (fr) * 2006-12-22 2008-07-24 Bayer Schering Pharma Aktiengesellschaft Production et utilisation de variantes non naturelles du domaine 2 de la bikunine du placenta humain conçues par évolution moléculaire dirigée
WO2008110301A1 (fr) * 2007-03-13 2008-09-18 Bayer Schering Pharma Aktiengesellschaft Variants d'aprotinine à propriétés améliorées
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GB2208511A (en) * 1987-08-07 1989-04-05 Bayer Ag Variants of bovine pancreatic trypsin inhibitor produced by recombinant dna technology
US5591603A (en) * 1987-08-28 1997-01-07 Novo Nordisk A/S Process for preparing aprotinin and aprotinin analogs in yeast cells
DK225488D0 (da) * 1988-04-26 1988-04-26 Novo Industri As Polypeptid
DE3930522A1 (de) * 1989-09-13 1991-03-21 Bayer Ag Rekombinante aprotinin-varianten - gentechnisches verfahren zur mikrobiellen herstellung von homogen prozessierten aprotinin-varianten sowie die therapeutische anwendung derselben
IL99585A0 (en) * 1990-10-01 1992-08-18 Novo Nordisk As Aprotinin analogues,their production and pharmaceutical compositions containing them
US5747449A (en) * 1992-07-13 1998-05-05 Corvas International, Inc. Bovine pancreatic trypsin inhibitor derived inhibitors of factor XA
CA2145517A1 (fr) * 1992-09-25 1994-04-14 Ignace Lasters Inhibiteurs du complexe facteur viia-facteur tissulaire derives de l'inhibiteur de la trypsine pancreatique bovine
CA2180950C (fr) * 1994-01-11 2005-03-29 William Markland Proteines de "domaine de kunitz" a activite antikallikreine, et leurs analogues
US5589359A (en) * 1994-08-05 1996-12-31 Chiron Corporation Chimeric proteins
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