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WO2008110301A1 - Variants d'aprotinine à propriétés améliorées - Google Patents

Variants d'aprotinine à propriétés améliorées Download PDF

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Publication number
WO2008110301A1
WO2008110301A1 PCT/EP2008/001820 EP2008001820W WO2008110301A1 WO 2008110301 A1 WO2008110301 A1 WO 2008110301A1 EP 2008001820 W EP2008001820 W EP 2008001820W WO 2008110301 A1 WO2008110301 A1 WO 2008110301A1
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aprotinin
seq
bpti
amino acid
inhibition
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PCT/EP2008/001820
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German (de)
English (en)
Inventor
Axel Harrenga
Felix Oehme
Heiner Apeler
Frank Dittmer
Jürgen Franz
Michael Sperzel
Beatrix Stelte-Ludwig
Karl Ziegelbauer
Simone Greven
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Bayer Schering Pharma Aktiengesellschaft
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Publication of WO2008110301A1 publication Critical patent/WO2008110301A1/fr

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    • 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)

Definitions

  • the present invention relates to aprotinin variants with improved immunological and enzyme-inhibiting properties and their preparation and use.
  • Kunitz domains are polypeptides that inhibit a variety of different potency serine proteases. They usually contain three disulfide bridges, which stabilize the protein and determine its three-dimensional structure. The interaction with the respective serine protease occurs mainly via an approximately 9 amino acid residues long loop in the N-terminal region of the
  • Kunitz-domain This loop binds to the catalytic center of the protease and thus prevents cleavage of the corresponding protease substrates (Laskowski and Kato [1980], Bode and Huber [1992]).
  • Aprotinin also referred to as bovine pancreatic trypsin inhibitor (BPTI) (BPTI; Figure 1: SEQ ID NO: 1), is considered a prototype of the Kunitz domains (Fritz and Wunderer [1983]). It is a basic protein of 58 amino acid residues in length that can be isolated from various bovine organs (including pancreas, lung, liver and heart). Aprotinin is stabilized by three disulfide bridges (Cys 5 - Cys 55, Cys 14 - Cys 38, Cys 30 - Cys 51) and is among others. a potent inhibitor of trypsin, plasmin and plasma kallikrein.
  • BPTI bovine pancreatic trypsin inhibitor
  • X-ray diffraction analysis of the aprotinin-bovine trypsin complex revealed that the aprotinin contact region to the protease catalytic center is essentially formed by a loop consisting of amino acid residues 11 to 19 (see Bode and Huber [1992] and references therein).
  • the amino acid residue Lys 15 which is in particularly close contact with the catalytically active serine residue of the protease.
  • the amino acid residue Lys 15 is therefore designated by definition as Pl residue (Schechter and Berger [1967].) N-terminal of Lys-15 are the residues P2, P3, etc., while the amino acid residues are C-terminal of Lys-15 as Pl ', P2', etc. It has previously been shown that the inhibitory effect of aprotinin can be altered by targeted replacement of amino acid residues in the range from residue 11 to residue 19 (Otlewski et al., 2001, Apeler et al [2004], Krowarsch et al. [2005].) The amino acid residues 36-39 are also important for the effectiveness of aprotinin (Fritz and Wunder [1983], Krowarsch et al. [2005]).
  • aprotinin is sold under the trade name Trasylol is currently used mainly 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 [1992]) It is attributed to the inhibition of intrinsic blood coagulation (contact activation), the inhibition of fibrinolysis and the reduction of thrombin formation (Blauhut et al. [1991], Dietrich et al. [1995]). Thus, inhibition of both the plasma and plasma kallikrein is important for the hemostatic effect of aprotinin.
  • Kunitz domains within the meaning of this invention are homologs of the aprotinin having 55 to 62 amino acid residues, which usually contain six cysteine residues and three disulfide bridges, each between the positions Cys 5 - Cys 55; Cys 14 - Cys 38 and Cys 30 - Cys 51 (aprotinin numbering) are formed.
  • the amino acid residues are numbered according to the 58 amino acid residues of aprotinin as shown in Table 1.
  • Some Kunitz domains contain insertions or deletions in addition to the amino acid residues shown in Table 1.
  • Kunitz inhibitors have hitherto been found, inter alia, in various vertebrates (eg human, bovine) and in some invertebrates (nudibranch, sea anemone) (Laskowski and Kato [1980] and references therein). Such naturally occurring Kunitz domains are referred to in this application as "natural Kunitz domains.” Examples of natural Kunitz domains include aprotinin, human placental bikunin domain 1, human placental Bikunin domain 2 and human TFPI-I domain 1. The sequences of some natural Kunitz domains are listed in Table 2.
  • Non-natural Kunitz domains Various unnatural Kunitz domains have been described in the literature (Dennis et al., 1995, Markland et al., 1996a). , Markland et al [1996b], Apeler et al. [2004], EP 0307592) The sequences of some non-natural Kunitz domains are shown in Table 3.
  • Aprotinin is a potent inhibitor of plasmin and plasma kallikrein (Table 4), its clinical effect is attributed to the inhibition of intrinsic blood coagulation (contact activation), the inhibition of fibrinolysis and the reduction of thrombin formation (Blauhut et al., 1991, Dietrich et al., 1995) Formation of antibodies Repeated Trasylol may cause allergic reactions (anaphylactic shock), which may increase the possibility of multiple use of aprotinin restricted (Dietrich et al. [2001], Beierlein et al. [2005]).
  • the aim of the present invention is the production of aprotinin variants with an aprotinin comparable or improved efficacy (in terms of enzyme inhibition of plasmin and plasma kallikrein, inhibition of fibrinolysis, inhibition of coagulation and reduction of bleeding time) with less antigenicity and immunogenicity.
  • aprotinin surprisingly revealed positions of amino acid residues of aprotinin whose targeted replacement with corresponding amino acid residues of other natural or non-natural Kunitz domains yield aprotinin variants which show an immunologically improved profile. These amino acid residues serve as a starting point for targeted mutagenesis (formation of chimeras, see below).
  • amino acid residues of aprotinin Tyr-10, Arg-17, He-19, Ala-40, Lys-41 and Ala-48; Also important in this analysis are the amino acid residues of aprotinin Pro-9, Lys-26, Ala-27, Leu-29, Gln-31, Arg-39, Lys-46 and Asp-50 Considered as immunologically relevant.
  • Variants of aprotinin according to the invention are formed by formation of chimeras between aprotinin and other natural or non-natural Kunitz domains.
  • the formation of chimeras occurs by replacement of one or more amino acid residues that are considered immunologically relevant.
  • amino acid residues in the aprotinin active site become more resistant to the amino acid residues of other natural or non-natural Kunitz domains exchanged to improve the effectiveness.
  • Variants of aprotinin according to the invention have the general formula: X 1 X 2 DFCLEPPX 1 OTGPCX 15 X 16 XI 7 XI 8 XI 9 RYFYNAKAGX 29 CQTFVYGGCRX 4 OX 4 IRNNFKSX 48 EDCMRTCGGA
  • X n means an amino acid residue of a Kunitz domain according to the numbering shown in Table 1. This amino acid residue can be either aprotinin or another natural or non-natural Kunitz domain listed in Tables 2 and 3. Variants of the invention are produced by exchanging at least one amino acid residue of aprotinin for another amino acid residue of the corresponding natural or unnatural Kunitz domain.
  • Preferred variants of aprotinin according to the invention inhibit plasmin with an IC50 ⁇ 30 nM.
  • Plasma kallikrein is inhibited by preferred erf ⁇ ndungssiee variants with an IC50 ⁇ 30 nM (see Table 4).
  • Preferred variants of aprotinin according to the invention show a lower cross-reactivity to sera of aprotinin-treated patients (reduced antigenicity in a cross-reactivity score 1, see Table 4) and have a reduced immunogenicity in the T-cell epitope profiling (reduced T H epitope number , see Table 5).
  • Preferred variants of aprotinin according to the invention exhibit, upon inhibition of fibrinolysis (FIGS.
  • aprotinin BPTI-mut2 (Seq No. 3), BPTI-mut5 (Seq No. 6), BPTI-mut10 (Seq No. 11) and BPTI-mutl3 (Seq No. 14) shown in FIG ). Further variants of aprotinin according to the invention are shown in FIG.
  • This invention also relates to pharmaceutical compositions containing one or more of the variants of aprotinin according to the invention.
  • the described novel aprotinin variants are suitable for the treatment of the following disease states: loss of blood in operations with an increased risk of bleeding; Therapy of thromboembolic conditions (eg after surgery, accidents), shock, polytrauma, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), unstable angina, Heart attack, stroke, embolism, deep vein thrombosis, inflammatory diseases (eg rheumatism, asthma), invasive tumor growth and metastasis, pain and edema therapy (cerebral edema, spinal cord edema), prevention of activation of hemostasis in dialysis treatment, treatment of skin aging symptoms (elastosis, atrophy , Wrinkling, vascular changes, pigment changes, actinic keratosis, blackheads, cysts), wound healing, skin cancer, treatment of skin cancer symptoms (actinic keratosis, basal cell carcinoma, squamous cell carcinoma, malignant melanoma
  • Oligonucleotides for site directed mutagenesis experiments primers for PCR (polymerase chain reaction) and sequencing reactions were purchased from the company Operon, synthetic genes (optimized for S. cerevisiae coden-usage) from Geneart.
  • In vitro mutagenesis was performed using the Quick-change II XL site-directed mutagenesis kit according to the manufacturer (Stratagene).
  • kits from Qiagen Hot Star Mastermix
  • Stratagene PfuUltra Hotstart DNA Polymerase
  • Novagen KOD HiFi, Hot Start and XL DNA Polymerases
  • Qiagen was used PCR Purification Kit from Qiagen. All vector constructs and mutagenesis were confirmed by cycle DNA sequencing with fluorescently-labeled terminators (Big Dye Terminator, Version 1.1, Applied Biosystems) on a sequencer (3100 Avant Genetic Analyzer, Applied Biosystem).
  • Variants of BPTI bovine pancreatic trypsin inhibitor, aprotinin
  • BPTI bovine pancreatic trypsin inhibitor, aprotinin
  • BPTI-mutl 3 generated using the Stratagene PCR kit.
  • the mutagenesis primer A (down) and primer B (up) were used.
  • the mutagenesis primer A had the following sequence: mutagenesis primer A:
  • This primer generates the mutations RIA, P2E in BPTI-mut10 (coded by GCT and GAA, in bold) and can also be used to generate mutations R1A, P2E in BPTI-mutl3, the restriction site for BsaBI (GATnnnATC) at 5 '- End of the primer is underlined.
  • Primer B :
  • the PCR mixture contained approximately 100 ng of plasmid DNA (BPTI-mut2 or BPTI-mut5), 1 OpMoI mutagenesis primer A, 1 OpMoI primer B, ImM dNTPs, IxPCR reaction buffer (Stratagene), 2.5 U PfuUltra hotstart DNA polymerase (Stratagene) in a total volume of 50 ⁇ l.
  • the 'cycle' conditions were 3 min. at 94 ° C, 30 cycles of 1 min each. at 94 ° C, 1 min. at 50 ° C., 1 min. at 72 ° C and a subsequent incubation for 5 min. at 72 ° C.
  • the PCR mixture was purified with a purification kit (Qiagen), cut with the restriction enzymes BsaBI and Sphl and ligated into the yeast secretion vector pIU10.10W, also cut with BsaBI and Sphl. E.coli DH5 ⁇ cells were transformed with the ligation mixture. From the resulting clones, the successful mutagenesis was verified by DNA sequence analysis and the variants BPTI mutlO and BPTI mutl3 used for further work.
  • Yeast cells eg strain JC34.4D (MATD, ura3-52, suc2) were grown in 10 ml YEPD (2% glucose, 2% peptone, 1% Difco yeast extract) and harvested at an ODgQO of 0.6 to 0.8 , The cells were washed with 5 ml of solution A (1 M sorbitol, 3% ethylene glycol; 10 mM bicine pH 8.35), resuspended in 0.2 ml of solution A and stored at -7O 0 C.
  • solution A (1 M sorbitol, 3% ethylene glycol; 10 mM bicine pH 8.35
  • Plasmid DNA (5 ⁇ g) and carrier DNA (50 ⁇ g of herring sperm DNA) were added to the frozen cells. The cells were then thawed by shaking for 5 min at 37 ° C. After addition of 1, 5 ml of solution B (40% PEG 1000; 200 mM bicine pH 8.35) the cells were incubated for 60 min at 30 0 C, (after pelleting with 1.5 ml of solution C 0.15 M NaCl, 10 mM bicine pH 8.35) and resuspended in 100 ⁇ l of solution C. The plating took place on a selection medium with 2% agar. Were transformants after incubation for 3 days at 3O 0 C obtained Example 3
  • the contents of the SL4 solution were dissolved in demineralised water and the pH was adjusted to 3-4 with NaOH.
  • the nutrient solution was made up to 1000 ml with demineralized water and stored in aliquots at -20 ° C.
  • the starting materials of the nutrient solutions SD2 and SC5 were prepared in demineralized water and the pH was adjusted to pH 5.5. Sterilization was carried out at 121 ° C. for 20 min. Glucose was dissolved in 1/5 of the required volume in demineralized water, sterilized separately and, after cooling, added to the remaining nutrient solution. strain stocks
  • yeast transformants were created by mixing 1 ml aliquots of a preculture with 1 ml of 80% glycerol solution and stored at -14O 0 C. Precultivations The preculture fermentations were carried out in 50 ml (for main cultures in small volume) or 1 liter shake flasks (for main cultures in medium volume) filled with 10 or 100 ml SD2 nutrient solution. The inoculation took place with a trunk preserve or with a single colony from an SD2 agar plate. The cultures were incubated with constant shaking (240 rpm) for 2 - 3 days at 28 - 30 0 C. Main culture fermentations
  • the main culture fermentations on a small scale were carried out using 1 liter shake flasks filled with 100 ml SC5 nutrient solution.
  • the inoculation was usually carried out with 3 ml of the preculture described above.
  • the cultures with continuous shaking (240 rev / min) for 4 days at 28 were - incubated 30 0 C.
  • the bioreactor system of Wave Biotech (Tageiswangen, CH) was used.
  • BPTI-mutl3 Purification of BPTI-mutl3 from the supernatants of fermented yeast cells
  • the cell-free supernatants containing BPTI-mutl3 prepared in the main culture fermentation were mixed with IM NaOH until the pH was 7.8. Suspended particulates suspended in the supernatant were sedimented by centrifugation at 2,000 rpm at 4 ° C (15 minutes, Beckman-Allegra 6KR). The supernatant was applied at 1 ml / min to a 10 ml trypsin agarose column (Sigma-T1763).
  • BPTI-mutl 3 was eluted with 180 ml of 50 mM KCl / 10 mM HCl pH 2.0. The 2 ml fractions were collected in Collected tubes each containing 500 ul 200 raM Tris pH 7.6, 2 M NaCl to neutralize. Fractions containing BPTI-mutl3 were identified by the trypsin inhibition assay described below.
  • Trypsin-inhibiting fractions were pooled and dialysed twice in a dialysis tubing with a 1,000,000 dalton cut-off size (Spectra / POR6) against each 2 liters of 50 mM Tris pH 7.5.
  • the dialyzate was concentrated in an Amicon 8200 stirred cell over an ultrafiltration membrane of exclusion size 1, 000 daltons.
  • the protein concentration was determined with a Coomassie Plus test (Pierce, 23236) according to the manufacturer's instructions. The measured protein concentration was typically between 0.1 and 6 mg / ml.
  • the trypsin-inhibiting fractions were pooled after purification over trypsin agarose, mixed with the same volume of 0.1% TFA and applied to a Source 15 RPC column.
  • the column was washed with 6 ml of 0.1% TFA (buffer HPLC-A) and then BPTI-mutl 3 with a 25 ml gradient on 50% buffer HPLC-B (0.1% TFA, 60% acetonitrile) and another 5 ml Gradients to 100% buffer HPLC-B eluted.
  • the eluates containing BPTI-mutl3 were lyophilized and the lyophilizate was taken up in 250 ⁇ l of 50 mM Tris pH 7.5 per fraction.
  • the purified protein was diluted to 2 pmol / ⁇ l with a 0.1% TFA solution and acidified at the same time. After separation, this sample was analyzed by mass spectrometry using a GromSil 120 ODS-4 HE (3 ⁇ m, 250 ⁇ 0.2 mm). The molecular weights of BPTI mut 13 (6379.1 daltons) were unambiguously demonstrated with a delta of 0.7 daltons on the basis of the multiply charged substance ions obtained.
  • the protein after denaturation with guanidinium hydrochloride, reduction with dithiothreitol and derivatization with iodoacetamide, was cleaved by tryptic cleavage.
  • the resulting cleavage peptides were also analyzed by mass spectrometry and, based on the proven peptide masses and MS / MS spectra, accurate sequence coverage of the protein was established.
  • the inhibitory potency of BPTI-mutl3 against the enzymatic activities of trypsin, plasmin and plasma kallikrein were determined in biochemical assays in white 384-well microtiter plates using fluorogenic substrates.
  • the assay buffer was composed of 50 mM Tris / Cl, pH 7.4, 100 mM NaCl, 5 mM CaCl 2 , 0.08% (w / v) BSA.
  • the test conditions were as follows:
  • BPTI-mutl3 Per 10 ul of a serial dilution of BPTI-mutl3 were placed and pre-incubated with 20 ul of enzyme for 5 min at RT. Subsequently, the reaction was started by addition of 20 .mu.l of substrate. The measurement was carried out after 60-90 min in a Tecan reader at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Dose-response curves and half-maximal inhibitory constants (IC50 values) were determined using GraphPad Prism software (Version 4.02). determined.
  • trasylol aprotinin
  • Human citrated plasma was supplemented with 0.13 pM tissue factor (TF) and 164 U / ml tissue plasminogen activator (tPA) as well as BPTI mutl3 or
  • BPTI-mutl3 Inhibition of Coagulation by BPTI-mutl3
  • trasylol aprotinin
  • Human citrated plasma was spiked with 12 mM CaCl 2 to induce coagulation and with BPTI-mutl3 or aprotinin in various concentrations (0.1 ⁇ M to 10 ⁇ M).
  • physiological saline was used in place of the Kunitz domains.
  • the increase in OD at 405 nm was determined as a measure of coagulation. From this, the half-maximal coagulation time was calculated. An extension of the half-maximal coagulation time means inhibition of coagulation.
  • a competitive ELISA was established.
  • a commercially available ELISA for the detection of anti-aprotinin antibodies in human serum (CellTrend, Luckenwalde) was modified so that the competition of the variants and aprotinin for binding to anti-aprotinin antibodies could be detected with high sensitivity.
  • the anti-aprotinin antibody-containing antisera were from aprotinin-treated patients who had developed a high antibody titre to the protein.
  • the antisera in the dilution buffer together with aprotinin or the BPTI variant to be tested were preincubated at room temperature for 1 h with gentle shaking in a total volume of 300 ⁇ l each. This usually came depending on the used Antiserum dilutions of 1: 3000 to 1: 7500 are used.
  • Each protein to be tested was tested at three different concentrations (1, 10, 100 nM). An empty control without aprotinin or BPTI variant was felt as a reference. After preincubation, the batch was transferred to a well precoated with aprotinin well of the microtiter modules contained in the test kit.
  • each well was washed according to the manufacturer's instructions and then a color reaction was generated with the aid of the peroxidase-coupled secondary antibody supplied.
  • the optical density was measured in a Tecan Reader at 450 nm (reference wavelength 620 nm).
  • the measured values for each protein concentration tested were set to the reference blank value (100%) in the percentage ratio.
  • Variants with cross-reactivities reduced relative to aprotinin to the antisera used were typically> 20% residual ELISA at 100 nM protein (cross-reactivity score: 1).
  • aprotinin and variants with similar or increased cross-reactivity were characterized by residual signals of ⁇ 20% at 100 nM protein (cross-reactivity score: 0).
  • HLA class II epitopes also referred to as T H epitopes.
  • the Epibase® platform analyzes all possible 10 amino acid residue long peptide portions of a target sequence to be tested for binding to 48 HLA class II receptors (allotypes are 20x DRB1, 7x DRB3 / 4/5, 14x DQ and 7x DP).
  • the free binding energy is calculated and a dissociation constant (K d ) is determined.
  • the respective peptides are classified as strong (S), middle (M) and weak or non (N) binders. The following limits were used: S: strong binders, K d ⁇ 0.1 ⁇ M; M: middle binders, 0.1 ⁇ M ⁇ IQ ⁇ 0.8 ⁇ M; N: weak or not binder, 0.8 ⁇ M ⁇ K d .
  • the observed T H cell activation / proliferation is generally interpreted as DRBI specific.
  • participation of the DRB3 / 4/5, DQ and DP can not be ruled out. Due to the lower expression rate of these genes compared to DRBl, only strong binders for DRB3 / 4/5, DQ and DP are included in the consideration of the critical epitopes. The critical epitopes are therefore strong binders compared to the DRBl, DRB3 / 4/5, DQ and DP and additionally middle binders against DRBl counted.
  • hCAVSMC Human coronary arterial vascular smooth muscle cells
  • TEBU vascular smooth muscle cells
  • M 231 medium growth medium
  • TEBU vascular smooth muscle cells
  • the plates are previously coated with vitronectin (50 ng / cm 2) (Gibco / Invitrogen, Düsseldorf, Germany). After the incubation period, one half of the confluent cell monolayer is removed. In the cell-free area of the well about 50% of the vitronectin coating is retained.
  • the growth medium is replaced by the test medium MCDB-131 / 0.2% BSA (Molecular Cellular Developmental Biology (MCDB); Basal Medium (BSA)) (Gibco / Invitrogen, Düsseldorf, Germany) and the cells are replaced with 1OnM PDGF-BB (( R & D Systems, Wiesbaden-Nordenstadt, Germany).
  • BSA Molecular Cellular Developmental Biology
  • BSA Basal Medium
  • test substances are then added in the indicated concentrations. After 24 and 48 hours of incubation, the migration distance of the cells into the free corrugation area is determined microscopically. Each measurement point represents an average of four measured regions and at least three independent experiments were performed.
  • Neutrophils are isolated from blood by standard methods.
  • the chemotaxis of neutrophils is performed in a two chamber system.
  • a HUVEC monolayer is cultured for 24 h.
  • 1x105 Neutrophils in RPMI 1640 medium, previously loaded with a fluorescent dye, are placed in the upper chamber.
  • the lower chamber contains varying concentrations of stimulus or constant stimulus concentration (IL-8: 5nM or C5a: 1OnM) and varying concentrations of the test substance.
  • the substances to be investigated are in both chambers.
  • the assay is incubated for 45 min at 37 ° C and 5% CO2. After incubation, the cells which have migrated to the lower chamber are determined (fluorescence measurement, counting).
  • Amino acid residues 1, 2, 3, 4 may be missing 15 amino acid residues 56, 57, 58 may be missing
  • cross reactivity score 0 ⁇ 20% residual signal in ELISA at 100 nM protein
  • Cross-reactivity score 1 > 20% residual signal in ELISA at 100 nM protein
  • T H epitope number per HLA gene for aprotinin and aprotinin variants Peptides which bind to several HLAs of the same gene (DRB1, DRB3 / 4/5, DQ and DP) are counted once (for definitions, see embodiment)

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Abstract

L'objet de la présente invention est la mise au point de variants d'aprotinine ayant une efficacité comparable à celle de l'aprotinine ou améliorée, pour une antigénicité et une immunogénicité réduites. Les propriétés souhaitées sont obtenues par l'échange de restes d'acides aminés dans le centre actif et hors du centre actif contre des restes d'acides aminés correspondants d'autres domaines de Kunitz naturels et non naturels. Une analyse détaillée de l'aprotinine a révélé étonnamment des positions de restes d'acides aminés de l'aprotinine dont l'échange ciblé contre des restes d'acides aminés correspondants d'autres domaines de Kunitz naturels et non naturels donne des variantes d'aprotinine (fig. 1: séq. No 2 à séq. No 6 et séq. No 10 à séq. No 14) qui présentent une réactivité croisée plus faible avec des sérums de patients traités à l'aprotinine (antigénicité réduite) (tableau 4), présentent une immunogénicité réduite (fig. 1: séq. No 10 et séq. No 13) (tableau 5) dans des recherches de profils d'épitopes de lymphocytes T, et possèdent une efficacité comparable à l'aprotinine ou améliorée par rapport à l'inhibition enzymatique de plasmine et de kallikréine plasmatique (tableau 4), à l'inhibition de la fibrinolyse (fig. 3 et fig. 4), à l'inhibition de la coagulation (fig. 5 et fig. 6), à l'inhibition de processus inflammatoires complexes (fig. 7 et fig. 8) et à la réduction de la durée de saignement.
PCT/EP2008/001820 2007-03-13 2008-03-07 Variants d'aprotinine à propriétés améliorées WO2008110301A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102011013326A1 (de) 2011-03-08 2012-09-13 Solution Shop Ag Neue Fibrinolyse-Inhibitoren und deren mdizinische Verwendung

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DE102006060375A1 (de) * 2006-12-20 2007-08-09 Bayer Healthcare Ag Aprotininvarianten die mit einem Nicht-Protein-Polymer modifiziert sind

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EP0132732A2 (fr) * 1983-07-28 1985-02-13 Bayer Ag Les homologues d'aprotinine avec d'autres amino-acides en position 15 que la lysine, leur procédé de préparation et leur emploi comme médicaments
EP0238993A2 (fr) * 1986-03-26 1987-09-30 Bayer Ag Homologues d'aprotinine produits par génie génétique
EP0307592A2 (fr) * 1987-08-07 1989-03-22 Bayer Ag Analogues de l'inhibiteur de la trypsine pancréatique bovine, leur production et utilisation
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DE102011013326A1 (de) 2011-03-08 2012-09-13 Solution Shop Ag Neue Fibrinolyse-Inhibitoren und deren mdizinische Verwendung
WO2012119744A1 (fr) 2011-03-08 2012-09-13 Solution Shop Ag Nouveaux inhibiteurs de la fibrinolyse et leur utilisation médicale

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