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WO2009027507A2 - Aptamères se liant à une molécule cible intervenant dans l'hémostase - Google Patents

Aptamères se liant à une molécule cible intervenant dans l'hémostase Download PDF

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Publication number
WO2009027507A2
WO2009027507A2 PCT/EP2008/061395 EP2008061395W WO2009027507A2 WO 2009027507 A2 WO2009027507 A2 WO 2009027507A2 EP 2008061395 W EP2008061395 W EP 2008061395W WO 2009027507 A2 WO2009027507 A2 WO 2009027507A2
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seq
aptamer
activated protein
binding
group
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WO2009027507A3 (fr
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Günter Mayer
Jens Müller
Bernd PÖTZSCH
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Rheinische Friedrich Wilhelms Universitaet Bonn
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Rheinische Friedrich Wilhelms Universitaet Bonn
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • the invention relates to aptamers which bind to a target molecule involved in hemostasis, wherein the aptamer is an aptamer which binds to activated protein C or is a thrombin-binding fusion aptamer.
  • Activated Protein C is a serine protease and the active form of the blood-circulating zymogen protein C.
  • activated protein C inactivates the activated coagulation factors Va and Villa and thereby controls plasmatic coagulation activation.
  • Decreased production of activated protein C or disruption of activated protein C activity is associated with an increased risk of developing venous thrombosis and plays a crucial role in the pathogenesis of microcirculatory disorders, such as those associated with severe sepsis.
  • a recombinantly produced concentrate of activated protein C is used, for example, in the treatment of severe sepsis.
  • activated protein C The detection of activated protein C is hampered by its low plasma concentrations and a simultaneous 10,000-fold excess of protein C.
  • UD 40090 / SAM AL
  • amino acid sequence of activated protein C is identical to that of protein C except for a twelve amino acid propeptide.
  • US Pat. No. 6,989,241 discloses a monoclonal antibody against activated protein C for differentiating between activated protein C and protein C.
  • the affinity of this antibody for protein C is only one order of magnitude lower. Accordingly, a test procedure developed on the basis of this antibody is basically suitable for the determination of activated protein C, but requires incubation times of more than 19 hours due to the low affinity differences and shows a limited specificity. In addition, a complicated preanalytical sample processing is required.
  • the object of the invention was to provide a means which overcomes at least one of the aforementioned disadvantages of the prior art.
  • the object of the present invention was to provide a means that allows detection of activated protein C.
  • Another object of the invention was to provide thrombin-inhibiting aptamers.
  • the object is achieved by an aptamer which binds to a target molecule involved in the hemostasis, wherein the aptamer: is an aptamer that binds to activated protein C, the aptamer having a dissociation constant K D in the range of> 0.001 nM to ⁇ 80 nM of activated protein C, and wherein the aptamer has a length in the range of> 20 nucleotides to ⁇ 160 Nucleotides, or is a thrombin-binding fusion aptamer comprising at least two thrombin-binding aptamers and at least one linker, wherein the at least one linker connects the at least two thrombin-binding aptamers.
  • a further subject matter relates to a method for the determination of activated protein C in biological samples, wherein at least one aptamer binding to activated protein C is incubated with the biological sample, preferably a biological fluid, and a binding event between the aptamer and activated protein C is detected.
  • aptamer is to be understood as meaning oligonucleotides which bind specifically and with high affinity to a target molecule, in particular to a target protein involved in the hemostasis, such as activated protein C or thrombin.
  • the inventors have surprisingly been able to select aptamers capable of binding activated protein C with high affinity.
  • a particular advantage of the activated protein C binding aptamers invention is provided by the fact that these activated protein C can bind with high affinity and high selectivity.
  • the aptamers according to the invention can specifically bind to activated protein C in relation to the inactive zymogen protein C.
  • the aptamers of the invention are characterized by a high affinity in the nanomolar and sub-nanomolar range and a pronounced specificity for activated protein C over the inactive zymogen protein C.
  • the affinity and specificity of the activated protein C binding aptamers according to the invention are preferably better or at least comparable to that of antibodies.
  • a further advantage is that the aptamers provided according to the invention, in contrast to antibodies, are inexpensive to produce synthetically.
  • the selection of the aptamers according to the invention was carried out according to the known SELEX® (Systematic Evolution of Ligands by Exponential Enrichment) method, for example described in US Pat. Nos. 5,475,096, 5,270,163 and EP 0 533 838, to which reference is hereby made, in particular to the so-called counter- SELEX® process.
  • SELEX® Systematic Evolution of Ligands by Exponential Enrichment
  • the bound ssDNA was eluted, amplified, and the resulting dsDNA transferred to ssDNA before the procedure was repeated with these ssDNAs. This was repeated 11 times.
  • the sequence of individual aptamers was determined by sequencing and sequence analysis, and the binding properties were characterized by binding studies.
  • Selected aptamers of known structure can be prepared by conventional methods of nucleic acid synthesis and / or amplification. The binding ability of the selected aptamers is described by the affinity of the bond, usually expressed in terms of the dissociation constant.
  • the activated protein C binding aptamer with a dissociation constant K D in the range of> 0.002 nM to ⁇ 8 nM, preferably in the range of> 0.005 nM to ⁇ 5 nM, preferably in the range of
  • nM to ⁇ 2 nM > 0.01 nM to ⁇ 2 nM, more preferably in the range of> 0.05 nM to ⁇ 1.5 nM, very particularly preferably in the range of> 0.1 nM to ⁇ 1.3 nM, of activated protein C.
  • the dissociation constants K D were determined by non-linear regression using a 4-parameter logistics function. The determination was carried out by nonlinear regression of the data points of radioactively labeled aptamers bound to activated protein C compared to the concentration of the activated protein C used, as described in Example 3. For the calculation, the function "Logistic Dose Response in Pharmacology / Chemistry" of the software Origin 7.5 (OriginLab, Northampton, MA, USA) was used.
  • the activated protein C-binding aptamer has a number of nucleotides in the range of> 20 nucleotides to ⁇ 120 nucleotides, preferably in the range of> 40 nucleotides to ⁇ 88 nucleotides. More preferably, the activated protein C-binding aptamer has a number of nucleotides in the range of> 44 nucleotides to ⁇ 120 nucleotides, preferably in the range of> 44 nucleotides to ⁇ 88 nucleotides, preferably in the range of
  • nucleotide includes the terms “ribonucleotide” and “deoxyribonucleotide”. Accordingly, the term “2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotide”2'-fluoro,2'-methoxy and / or 2'-amino-modified ribonucleotides and deoxyribonucleotides ,
  • Aptamers according to the invention may have a DNA or an RNA sequence. It is understood that in the case that aptamers according to the invention have an RNA sequence, thymidine is replaced by uridine in the specified sequence motifs and sequences.
  • RNA sequences suitable according to the invention correspond to the DNA sequences according to the invention, T being replaced by U.
  • aptamers have a DNA sequence.
  • DNA-based aptamers advantageously have increased stability.
  • aptamers which have a sequence comprising 2'-modified nucleotides, for example 2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides.
  • aptamers which have 2'-modified ribonucleotides, for example 2'-fluoro, 2'-methoxy and / or 2'-amino-modified ribonucleotides.
  • aptamers may have deoxyribonucleotides and 2'-modified ribonucleotides, for example 2'-fluoro, 2'-methoxy and / or 2'-amino-modified ribonucleotides.
  • activating protein C-binding aptamers which could be assigned to three main groups, the sequences of the aptamers having a common sequence motif within one main group. This common sequence motif varies within a main group only in individual base positions.
  • the activated protein C binding aptamer comprises a sequence motif selected from the group comprising:
  • Ni is selected from the group comprising guanine, cytosine, adenine, thymine, uracil, 2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotide, in particular ribonucleotide, or a deletion;
  • N 2 is selected from the group comprising guanine, cytosine, adenine, thymine, uracil, 2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotide, in particular ribonucleotide, or a deletion,
  • sequence motifs selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2 and / or SEQ ID NO: 3 modified nucleotides, preferably selected from Group comprising Locked nucleic acids, 2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides.
  • C in this case corresponds to the usual one-letter code of the bases for cytosine used here, correspondingly "A” stands for adenine, "G” for guanine and “T” for thymine, "U” for uracil.
  • the positions Ni and N 2 of the sequence motifs SEQ ID NO: 1 and SEQ ID NO: 2 are highly variable.
  • the positions Ni and N 2 may comprise different bases or nucleotides or a deletion.
  • a ribonucleotide may be included in a DNA aptamer.
  • modified nucleotides in particular modified ribonucleotides may be included.
  • sequence motifs selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2 and / or SEQ ID NO: 3 of the activated protein C-binding aptamer have no modified nucleotides.
  • sequence motifs selected from the group comprising SEQ ID NOs: 1 to 3 have an essential meaning in the binding of the aptamers according to the invention to activated protein C.
  • the activated protein C-binding aptamers having a sequence motif 5'-TATCCCGNi ATGGG-3 '(SEQ ID NO: 1), four subgroups were identified, each having a common base Ni selected from the group comprising guanine, cytosine, adenine and / or thymine.
  • the activated protein C binding aptamer comprises a sequence motif selected from the group comprising:
  • sequence motifs SEQ ID NOs: 4 to 7 may have modified nucleotides, preferably selected from the group consisting of locked nucleic acids, 2'-fluoro, T-methoxy and / or 2'-amino-modified nucleotides.
  • sequence motifs selected from the group comprising SEQ ID NOs: 4 to 7 can mediate particularly good binding of the aptamers according to the invention to activated protein C.
  • the aptamer sequences SEQ ID NOs: 8 to 19 have a common consensus sequence 5'-TATCCCGTATGGG-S '(SEQ ID NO: 4) having the base thymine at position 8 within the group of SEQ ID NO: 1.
  • the activated protein C-binding aptamer comprises a sequence selected from the group comprising:
  • the activated protein C-binding aptamer may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamer modified nucleotides, preferably selected from the group comprising Locked nucleic acids, T-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides may have.
  • Portions of the aforementioned aptamer sequences include the sequence motif 5'-TATCCCGTATGGG-3 '(SEQ ID NO: 4).
  • Variants of the sequences according to the invention can be formed by modifications, such as alkylation, in particular methylation, arylation or acetylation of at least one nucleotide, incorporation of enantiomers and / or fusion of the aptamers with one or more nucleotides or a nucleic acid sequence. Mutants can be obtained, for example, by substitution, deletion, insertion, translocation, inversion and / or addition of at least one nucleotide.
  • the aptamer sequences SEQ ID NOs: 20 to 31 have a common consensus sequence 5'-TATCCCGGATGGG-S '(SEQ ID NO: 5) which has the base guanine at position 8 within the group of sequences according to SEQ ID NO: 1 ,
  • the activated protein C-binding aptamer comprises a sequence selected from the group comprising:
  • the activated protein C-binding aptamer may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamer modified nucleotides, preferably selected from the group comprising Locked nucleic acids, T-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides may have.
  • portions of the aptamer sequences include the sequence motif 5'-TATCCCGGATGGG-S '(SEQ ID NO: 5).
  • the activated protein C-binding aptamer comprises a sequence selected from the group comprising:
  • the activated protein C binding aptamer may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamer-modified nucleotides, preferably selected from the group consisting of Locked nucleic acids, T-fluoro, 2'-methoxy and / or 2'- Amino-modified nucleotides may have.
  • portions of the aptamer sequences include the sequence motif 5'-TATCCCGAATGGG-S '(SEQ ID NO: 6).
  • the aptamer sequences SEQ ID NOs: 32-34 have a common consensus sequence 5'-TATCCCGAATGGG-S '(SEQ ID NO: 6) having the base adenine at position 8 within the group of SEQ ID NO: 1.
  • the activated protein C-binding aptamer comprises a sequence selected from the group comprising:
  • the activated protein C-binding aptamer may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamer modified nucleotides, preferably selected from the group comprising Locked nucleic acids, T-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides may have.
  • Portions of the aptamer sequences include the sequence motif 5'-TATCCCGCATGGG-3 '(SEQ ID NO: 7).
  • the aptamer sequences SEQ ID NOs: 35 and 36 have a common consensus sequence 5'-TATCCCGCATGGG-3 '(SEQ ID NO: 7) having the base cytosine at position 8 within the group of SEQ ID NO: 1.
  • the activated protein C-binding aptamer comprises a sequence selected from the group comprising:
  • the activated protein C-binding aptamer may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamer modified nucleotides, preferably selected from the group comprising Locked nucleic acids, T-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides may have.
  • sequences SEQ ID NO: 103 to 121 and / or parts of these aptamer sequences include the sequence motif 5'-TATCCCGN I ATGGG-S '(SEQ ID NO: 1), in particular the sequence motifs 5'-TATCCCGTATGGG-S' (SEQ ID NO: 4), 5'-TATCCCGGATGGG-S '(SEQ ID NO: 5), 5'-TATCCCGAATGGG-3' (SEQ ID NO: 6) or 5'-TATCCCGCATGGG-S '(SEQ ID NO: 7).
  • the activated protein C-binding aptamer comprises a sequence selected from the group comprising:
  • activated protein C-binding aptamer may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamer modified nucleotides, preferably selected from the group comprising Locked nucleic acids, T-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides may have.
  • sequences SEQ ID NO: 122 and 123 and / or parts of these aptamer sequences comprise the sequence motif 5'-TATCACGGATGGGC-S '(SEQ ID NO: 124), which differs from the sequence motif SEQ ID NO: 1 in that adenine is contained at position 5.
  • the activated protein C-binding aptamer comprises a sequence selected from the group comprising:
  • the activated protein C-binding aptamer may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamer modified nucleotides, preferably selected from the group comprising Locked nucleic acids, T-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides may have.
  • sequences SEQ ID NO: 125 and 126 and / or parts of these aptamer sequences comprise the sequence motif 5'-TATCCCAAATGGGG-3 '(SEQ ID NO: 127), which differs from the sequence motif SEQ ID NO: 1 in that adenine at position 7, or the sequence motif 5'-TATCCCGTATAGGG-S '(SEQ ID NO: 128), which is different from the sequence motifs SEQ ID NO: 1 differs in that adenine is contained at the position 11.
  • activated protein C-binding aptamers having a sequence motif 5'-TCTN 2 TCGGCGG-S '(SEQ ID NO: 2)
  • subgroups have been identified, each preferably a common base N 2 selected from the group comprising guanine and / or have thymine.
  • the activated protein C-binding aptamer comprises a sequence motif selected from the group comprising:
  • the activated protein C-binding aptamer comprises a sequence selected from the group comprising:
  • the activated protein C-binding aptamer may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamer modified nucleotides, preferably selected from the group comprising Locked nucleic acids, 2'- Fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides may have.
  • portions of the aptamer sequences include the sequence motif 5'-TCTGTCGGCGG-3 '(SEQ ID NO: 37).
  • the aptamer sequences SEQ ID NOs: 39 and 40 have a common consensus sequence 5'-TCTGTCGGCGG-3 '(SEQ ID NO: 37).
  • the activated protein C-binding aptamer comprises a sequence selected from the group comprising:
  • the activated protein C-binding aptamer may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamer modified nucleotides, preferably selected from the group comprising Locked nucleic acids, T-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides may have.
  • portions of the aptamer sequences include the sequence motif 5'-TCTTTCGGCGG-3 '(SEQ ID NO: 38).
  • the aptamer sequences SEQ ID NOs: 41 to 43 have a common consensus sequence 5'-TCTTTCGGCGG-3 '(SEQ ID NO: 38).
  • the activated protein C-binding aptamer comprises the sequence motif 5'-TATCACGTATGGG-S '(SEQ ID NO: 3).
  • the activated protein C binding aptamer comprises a sequence selected from the group comprising:
  • the activated protein C-binding aptamer may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamer modified nucleotides, preferably selected from the group comprising Locked nucleic acids, T-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides may have.
  • Portions of the aptamer sequences each comprise the sequence motif 5'-TATCACGTATGGG-S '(SEQ ID NO: 3).
  • the activated protein C-binding aptamer comprises a sequence selected from the group comprising:
  • the activated protein C-binding aptamer may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamer modified nucleotides, preferably selected from the group comprising Locked nucleic acids, T-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides may have.
  • the sequences SEQ ID NO: 46, 129 and 130 and / or parts of these aptamer sequences comprise the sequence motif 5'-ATCGCTTC AGGG-3 '(SEQ ID NO: 131).
  • the activated protein C binding aptamer has a sequence
  • the activated protein C binding aptamer has a sequence
  • the activated protein C binding aptamer comprises a sequence selected from the group comprising: 5'-GCCTGTTGTGAGCCTCCTAACCATCCCGTGGCCGGTAACTTATCCCGTATGGGGGCCCTCAGGGATACTCCATGCTTATTCTTGTCTCCC-S '(SEQ ID NO: 48),
  • the activated protein C-binding aptamer may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamer modified nucleotides, preferably selected from the group comprising Locked nucleic acids, T-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides may have.
  • sequences SEQ ID NOs: 48 to 85 include the aforementioned SEQ ID NOs: 8 to 36 and 39 to 47, these in each case of two sequences 5'-GCCTGTTGTGAGCCTCCTAAC-3 '(SEQ ID NO: 86) and 5'-
  • SEQ ID NO: 86 and SEQ ID NO: 87 correspond to the primer binding sequences used in the selection of the aptamers of the present invention.
  • the activated protein C binding aptamer comprises a sequence selected from the group comprising: 5'-GCCTGTTGTGAGCCTCCTAACAGGACACATATCCCGTATGGGGGTGAAGCTTT TCCGTCGTCACCGGTGTCATGCTTATTCTTGTCTCCC-S '(SEQ ID NO: 133)
  • the activated protein C-binding aptamer may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamer modified Nucleotides, preferably selected from the group comprising Locked nucleic acids, T-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides may have.
  • sequences SEQ ID NOs: 133 to 158 include the aforementioned SEQ ID NOs: 103 to 123, 125, 126, 129, 130 and 132, each of which comprises two sequences 5'-GCCTGTTGTGAGCCTCCTAAC-3 '(SEQ ID NO: 86 ) and 5'-
  • SEQ ID NO: 86 and SEQ ID NO: 87 correspond to the primer binding sequences used in the selection of the aptamers of the present invention.
  • the activated protein C-binding aptamer can fold under defined conditions in a specific three-dimensional structure, for example, so-called hairpin structures.
  • the activated protein C-binding aptamer has at least one first hairpin structure comprising a stem and a loop.
  • hairpin structure or “hairpin” is to be understood as meaning a special form of secondary structure in the case of nucleic acid sequences, in particular aptamers, which consists of two mutually complementary sequence segments in the so-called stem and a further sequence segment in the US Pat so-called loop (loop) composed, to understand.
  • one or more bases in the trunk can form single-stranded areas called bulges.
  • the strain of the first hairpin structure in the range of> 4 base pairs to ⁇ 15 base pairs, preferably in the range of> 9 base pairs to ⁇ 13 base pairs, preferably in the range of> 11 base pairs to ⁇ 12 base pairs. It is further preferred that the loop of the first hairpin structure in the range of> 5 nucleotides to ⁇ 30 nucleotides, preferably in the range of> 6 nucleotides to ⁇ 18 nucleotides.
  • a number of paired nucleotides in the range of> 4 base pairs to ⁇ 15 base pairs, in particular in the range of> 9 base pairs to ⁇ 13 base pairs, preferably in the range of> 11 base pairs to ⁇ 12 base pairs, in the trunk of the first hairpin structure contribute to the formation of a high-affinity binding of the aptamer to activated protein C.
  • a number of paired nucleotides may contribute to enabling stable formation of the stem of the first hairpin structure.
  • the activated protein C-binding aptamer has a second hairpin structure comprising a stem and a loop.
  • the second hairpin structure is disposed in the loop of the first hairpin structure.
  • the activated protein C-binding aptamer comprises a second hairpin structure comprising a stem and a loop, wherein the second hairpin structure is disposed in the loop of the first hairpin structure.
  • the activated protein C-binding aptamer has the sequence motif SEQ ID NO: 1, wherein the sequence motif SEQ ID NO: 1 comprises the second hairpin structure.
  • the stem of the first hairpin structure has a nucleotide sequence cytidine-thymidine (CT) that forms a single-stranded region, a bulge. It has been found that in particular a nucleotide sequence cytidine-thymidine (CT) in the stem of the first hairpin structure, which forms a single-stranded region, a so-called bulge, can contribute to an improvement in the binding of the aptamer to activated protein C.
  • CT nucleotide sequence cytidine-thymidine
  • the activated protein C-binding aptamer of the invention has a sequence selected from the group comprising SEQ ID NOs: 58, 88 to 92.
  • activated protein C-binding aptamers of the invention have a sequence selected from the group comprising SEQ ID NOs: 58, 88 to 92, which have a secondary structure comprising a first hairpin structure comprising a stem and a loop and a second hairpin structure comprising a trunk and a loop is formed, wherein the second hairpin structure is arranged in the loop of the first hairpin structure.
  • inventive activated protein C-binding aptamer comprising sequences selected from the group comprising SEQ ID NOs: 58, 88 to 92 have a secondary structure comprising a first hairpin structure comprising a stem and a loop and a second hairpin.
  • a structure comprising a stem and a loop, wherein the second hairpin structure is disposed in the loop of the first hairpin structure, and wherein the sequence motif SEQ ID NO: 1 comprises the second hairpin structure.
  • inventive activated protein C binding aptamers of the sequences selected from the group comprising SEQ ID NOs: 58, 88 to 92 have a strain of the first hairpin structure with in the range of> 9 base pairs to ⁇ 13 base pairs, preferably in the range of> 11 base pairs to ⁇ 12 base pairs and / or a nucleotide sequence cytidine-thymidine (CT) in the stem of the first hairpin structure, a single-stranded region, a so-called bulge formed.
  • CT nucleotide sequence cytidine-thymidine
  • inventive activated protein C-binding aptamers comprising sequences selected from the group comprising SEQ ID NOs: 58, 88 to 92 have a loop of the first hairpin structure with a number of nucleotides in the range of> 6 nucleotides to ⁇ 18 Nucleotides on.
  • the activated protein C-binding aptamer comprises a sequence selected from the group comprising:
  • activated protein C-binding aptamer may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamer modified nucleotides, preferably selected from the group comprising Locked nucleic acids, T-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides may have.
  • the activated protein C-binding aptamer comprises a sequence selected from the group comprising:
  • the aptamers according to the invention of the sequences SEQ ID NOs: 58, 88 to 92 had a very good affinity for binding to activated protein C, the dissociation constant K D determined by filter binding experiments in the range of> 0.1 nM to ⁇ 1.3 nM.
  • Locked nucleic acids correspond to a conformationally fixed analogue of DNA.
  • the oligonucleotides of the Locked nucleic acids contain one or more bicyclic ribonucleosides in which the 2'OH group is connected to the C4 carbon atom via a methylene group.
  • locked nucleic acids show higher stability towards nucleases as compared to unmodified DNA and better hybridization properties, whereby an improvement in the affinity and / or specificity of the binding of the aptamer can be achieved.
  • a further preferred chemical modification is, for example, the addition of a so-called "3'-CAP” or “5'-CAP” structure, of a modified guanosine nucleotide (7-methyl-guanosine) to the 3'-end or 5 ' -The End. Modification of the 3'-end or 5'-end may advantageously protect the aptamer from rapid degradation by nucleases, particularly in an organism or biological sample.
  • the 5'-end of the aptamer may be pegylated.
  • a 5'-PEG modification may comprise at least one polyethylene glycol unit, preferably in the range of 1 to 900 polyethylene glycol units, preferably in the range of 1 to 450 polyethylene glycol units.
  • An advantage of a chemical modification, a change in the Phosphorzucker Weggrates or a use of enzymatically unrecognizable altered nucleotide units is that the nucleic acids used are stabilized against enzymatic degradation, especially in an organism or a biological sample.
  • the aptamers according to the invention may be present as phosphorothioate DNA or peptide nucleic acid (PNA). These modifications allow the aptamers to be stabilized against nucleic acid-cleaving enzymes. Stabilization does not affect the affinity of the modified DNA aptamers, but may retard, diminish or even prevent the degradation of the aptamers in an organism or biological sample by degrading enzymes such as nucleases or DNases.
  • the nucleotides of the sequence motifs SEQ ID NO: 1), (SEQ ID NO: 2) and / or (SEQ ID NO: 3) have no modifications.
  • nucleotides are modified in strain and / or loop of the aptamers.
  • a further subject relates to a method for the determination of activated protein C in biological samples, wherein at least one inventive activated protein C binding aptamer is incubated with the biological sample, preferably a biological fluid, and a binding event between the aptamer and activated protein C is detected ,
  • the method is a method for the quantitative determination of activated protein C in biological samples.
  • the method for determining activated protein C in biological samples is a diagnostic test procedure.
  • the aptamers of the invention which bind to activated protein C are advantageously usable in particular in clinical analysis.
  • a particular advantage of the activated protein C binding aptamers invention is provided by the fact that these activated protein C can bind with high affinity and high selectivity.
  • the aptamers according to the invention can specifically bind to activated protein C in relation to the inactive zymogen protein C. This makes it possible to use the activated protein C-binding aptamers according to the invention for the determination of activated protein C in biological samples, in particular in blood and other body fluids.
  • the aptamers of the invention which bind to activated protein C can be used in a routine-suitable test procedure.
  • a "biological sample" within the meaning of the invention is a biological material provided or obtained by sampling.
  • Bio samples are, in particular, biological materials isolated from human individuals, in particular patients, for example biological tissues and fluids.
  • Preferred biological samples are biological fluids, in particular body fluids, preferably blood or blood products, preferably selected from the group comprising blood plasma, serum and / or whole blood.
  • the samples may be pretreated, for example by mixing, addition of enzymes or markers, or purified.
  • the sample is incubated with at least one aptamer binding to activated protein C according to the invention.
  • An incubation within the meaning of the invention means contacting the sample, in particular the compounds, substances and proteins contained therein, with an aptamer.
  • At least one inventive aptamer binding to activated protein C may be added to the biological sample, preferably a biological fluid.
  • the sample may be incubated with previously immobilized aptamer, for example by adding the sample to an aptamer-coated well of an ELISA plate.
  • an aptamer arrives at its target protein activated protein C, it binds to it.
  • the binding event between aptamer and activated protein C is detected according to the invention.
  • the binding event can be detected electrochemically, optically, microgravimetrically, calorimetrically or piezoelectrically.
  • the binding event can also be detected without labeling, the aptamer being fixed on a surface, for example, and the change in the layer thickness after Attachment of activated protein C is determined, for example optically via a change in the evanescent field.
  • binding formation between aptamer and activated protein C is detected in an indicator reaction following direct or indirect coupling of a binding partner with a well-detectable label.
  • the aptamer binding to activated protein C is preferably provided with a label.
  • the label can be detected by luminescence, color reactions, enzymatic, electrochemical or radioactive.
  • labels are detectable by fluorescence, chemiluminescence or bioluminescence.
  • Preference is given to detection by means of fluorescence.
  • Preferred fluorescent dyes are bisbenzimidazoles which can be covalently coupled to aptamers.
  • Preferred fluorescent dyes are selected from the group comprising fluorescein, fluorescein-5-isothiocyanate (FITC), carbocyanine dyes, in particular indocarbocyanine dyes and indodicarbocyanine dyes, for example available under the trade name Cy3 and Cy5 from Amersham Biosciences Europe GmbH.
  • Suitable are also N-hydroxy-succinimide esters of the fluorescent dyes, such as available under the designation Alexa Fluor ® dyes at the company Invitrogen.
  • Suitable dyes for staining of DNA for example, which belongs to the Phenantrinen ethidium bromide, or belonging to the cyanine dyes SYBR (SYBR Green ®.) Made by Molecular Probes.
  • intercalating dyes such as phenantrins, for example, propidium iodide.
  • Detection of aptamer-linked activated protein C can also be detected via the amidolytic activity of the activated protein C.
  • chromogenic or fluorogenic peptide substrates can be used, which are cleaved by activated protein C.
  • detection of aptamer-linked activated protein C may be by antibodies or labeled antibodies capable of binding to activated protein C.
  • the detection of activated protein C can be carried out by the addition of the sample to immobilized aptamers and subsequent detection of the bound activated protein C by its amidolytic activity or by means of corresponding antibodies.
  • the evaluation can be carried out by means of appropriate measuring devices, for example in the fluorescence microscope, or by flow cytometry, for example in the cytofluorimeter.
  • a preferred chemical label for chemiluminescence is luminol.
  • Preferred bioluminescence involves the emission of visible light as a result of an enzyme-catalyzed redox reaction.
  • the detection can be performed with enzymes as labeling substances that convert substrates to colored products, preferably peroxidase, luciferase, beta-galactosidase or alkaline phosphatase.
  • the detection can be made radioactively via labeling by means of radioactive isotopes.
  • Evidence can also be provided by means of a so-called ELISA (enzyme-linked immunosorbent assays).
  • ELISA enzyme-linked immunosorbent assays
  • the aforementioned methods preferably include intensive washing steps to separate unbound and / or unspecifically bound aptamers and / or detection reagents.
  • the aptamer binding to activated protein C can be immobilized on a solid phase, preferably via a spacer molecule.
  • a spacer molecule This may be, for example, a linker nucleic acid.
  • Immobilization may be by means of covalent coupling or non-covalent coupling by means of suitable affinity pairs, for example biotin / streptavidin.
  • Solid phases can be selected from plates, strips, membranes, films, gels, and / or beads.
  • Suitable carrier materials are inorganic and organic polymers, in particular biodegradable Polymers or biopolymers, preferably selected from the group comprising cellulose, dextran, agar, agarose and / or Sephadex, or so-called magnetic beads.
  • coupled activated protein C-binding aptamers are useful to immobilize activated protein C (APC) present in a biological sample, for example, in plasma. Subsequently, the bound activated protein C can be quantified, for example via the hydrolysis rate of an APC-specific peptide substrate.
  • APC activated protein C
  • the advantage here is that the binding to activated protein C aptamers do not or only slightly affect the amidolytic activity of activated protein C.
  • the activated protein C present in the plasma is inactivated by the protein C inhibitor present in the plasma.
  • Aprotinin is preferably added to a biological sample, for example a plasma sample, preferably in the preanalytical phase, in particular without delay after the recovery of the sample. This may prevent inactivation of activated protein C.
  • the method according to the invention for the determination of activated protein C in biological samples comprises the following steps:
  • activated protein C-binding aptamer (s) and activated protein C for example by addition of an APC-specific peptide substrate and determination of the hydrolysis rate, preferably by determination of a dye reaction,
  • the invention furthermore relates to the use of the activated protein C-binding aptamers according to the invention for the purification of activated protein C.
  • the aptamers which bind to activated protein C are suitable, for example, for affinity purification and / or affinity chromatography.
  • the invention furthermore relates to a kit comprising at least one aptamer which binds to activated protein C according to the invention.
  • a kit comprising at least one aptamer which binds to activated protein C according to the invention.
  • it may contain signaling substances or devices, preferably fluorescent dyes, chemiluminescent cofactors, enzyme substrates or labeled antibodies.
  • activated protein C-binding aptamers according to the invention advantageously makes it possible in particular to use diagnostic methods for determining the concentration of activated protein C in hemostasis diagnostics and in extended sepsis diagnostics. Furthermore, use of the activated protein C-binding aptamers according to the invention in diagnosis makes it possible to determine a disturbed concentration of activated protein C and thus to diagnose diseases which are associated with a disruption of the protein C / activated protein C system.
  • Another object of the invention relates to the use of the inventive binding to activated protein C aptamers for the diagnosis, therapeutic and / or Prophylactic treatment of disorders associated with impaired blood clotting, bleeding disorders, preferably hemorrhagic diatheses, preferably selected from the group comprising hemophilia, in particular hemophilia A, and / or von Willebrand syndrome, and / or bleeding complications caused by an increased concentration be activated on activated protein C.
  • the invention further relates to the use of an inventive activated protein C binding aptamer, its pharmacologically acceptable salts, derivatives and / or conjugates, for the manufacture of a medicament.
  • the activated protein C binding aptamers according to the invention are suitable to bind with high affinity to activated protein C and to modulate its activity, in particular to inhibit.
  • the activated protein C-binding aptamers of the invention of SEQ ID NOs: 58, 88 to 92 are capable of blocking the activated protein C anticoagulant enzyme activity.
  • the activated protein C-binding aptamers according to the invention of the sequences SEQ ID NOs: 58, 88 to 92 are suitable for inducing an allosteric conformational change in the molecule of the activated protein C.
  • this allosteric conformational change may enhance the inactivation of activated protein C by the physiological inhibitor protein C inhibitor.
  • the allosteric conformational change of activated protein C induced by the aptamers according to the invention leads to the formation of complexes of the activated protein C with protein C inhibitor.
  • Administration of the activated protein C-binding aptamers according to the invention, their pharmacologically acceptable salts, derivatives and / or conjugates advantageously makes it possible to therapeutically influence the activity of activated protein C.
  • the invention relates to the use of an activated protein C-binding aptamer of the invention, its pharmacologically acceptable salts, derivatives and / or conjugates, for the manufacture of a medicament for the diagnosis, therapeutic and / or prophylactic treatment of disorders associated with impaired blood clotting Bleeding disorders, preferably hemorrhagic diatheses, preferably selected from the group comprising hemophilia, in particular hemophilia A, and / or von Willebrand's syndrome, and / or bleeding complications, which are triggered by an increased concentration of activated protein C.
  • Bleeding complications that are triggered by an increased concentration of activated protein C are, in particular, bleeding that occurs as a result of overactivation with activated protein C, or bleeding that occurs as a result of increased production of activated protein C as a result of secondary thrombinemia.
  • Recombinant-produced activated protein C is used, for example, in the treatment of severe sepsis.
  • aptamers according to the invention which bind to activated protein C are suitable for acting as an antidote for recombinant activated protein C in the case of absolute or relative overdosage and thereby triggered bleeding.
  • aptamers according to the invention which bind to activated protein C are particularly suitable as surrogate therapeutics in the substitution with factor VIII concentrate in patients with hemophilia A.
  • the aptamers which bind to activated protein C according to the invention may have the half-life and / or effectiveness of the factor VIII. Concentrate can be improved.
  • aptamers according to the invention which bind to activated protein C are particularly suitable for the treatment of acquired hemorrhagic diatheses, which can arise as a result of excessive formation of activated protein C as a consequence of generalized thrombin formation.
  • a constellation can be present for example in patients after cardiac surgery.
  • activated protein C-binding aptamers of the present invention are useful for the diagnostic and / or therapeutic treatment of patients treated with activated protein C concentrates, or who are prone to bleeding by the predominance of the anticoagulant activated protein C. Further, activated protein C-binding aptamers of the present invention are useful for treating patients with a congenital deficiency of blood coagulation factor VIII, and by administering the activated protein C-binding aptamers of the invention, the half-life and effectiveness of administered factor VIII concentrate can be improved.
  • the invention further relates to medicaments comprising aptamers according to the invention which bind to activated protein C.
  • the medicament comprises inventive aptamers which bind to activated protein C, preferably as a pharmacologically acceptable salt, preferably as salts of inorganic acids, for example phosphoric acid, or as salts of organic acids, for example hydrochloric acid or sulfuric acid.
  • Pharmaceuticals may further comprise pharmaceutically acceptable excipients and / or carriers.
  • the medicament is for the prophylactic or therapeutic treatment of disorders associated with impaired blood coagulation, bleeding disorders, preferably hemorrhagic diatheses, preferably selected from the group comprising hemophilia, in particular hemophilia A, and / or von Willebrand syndrome, and / or bleeding complications, which are triggered by an increased concentration of activated protein C.
  • the drug is orally, transmucosally, rectally, pulmonarily, enterally and / or parenterally administrable. Preference is given to administration by injection.
  • Another object of the invention relates to a thrombin-binding fusion aptamer comprising at least two thrombin-binding aptamers and at least one linker, wherein the at least one linker connects the at least two thrombin-binding aptamers.
  • fusion aptamer means aptamer constructs which have at least two identical or different aptamer sequences and at least one linker or spacer region connecting the aptamer sequences.
  • the thrombin-binding fusion aptamers according to the invention can show an unexpectedly strong inhibition of thrombin.
  • the thrombin-binding fusion aptamers of the invention have an unexpectedly high inhibitory effect on thrombin-mediated blood coagulation.
  • thrombin-inhibiting action of the thrombin-binding fusion aptamers according to the invention it was possible to determine, for the thrombin-inhibiting action of the thrombin-binding fusion aptamers according to the invention, that blood clotting could be inhibited even with a 30-fold lower concentration compared to individual aptamers when using thrombin-binding fusion aptamers according to the invention.
  • Thrombin is activated in the blood from an inactive precursor protein, prothrombin. This reaction is catalyzed by the prothrombinase complex.
  • the thrombin-binding fusion aptamers according to the invention can inhibit prothrombinase. This can provide a significant advantage because the formation of thrombin can already be inhibited by the thrombin-binding fusion aptamers according to the invention.
  • a significant advantage lies in particular in an improved anticoagulant effect, since a multifunctional inhibition of coagulation activation can take place. This may be particularly beneficial in the anticoagulant therapy required for use of extracorporeal circulation systems.
  • Another important advantage of the thrombin-binding fusion aptamers can be provided by the fact that the action of the aptamers can be canceled by the administration of aptamers with complementary sequence in the form of an antidote or antidote.
  • Thrombin-binding fusion aptamers according to the invention may have a DNA or an RNA sequence.
  • thrombin-binding fusion aptamers may have a DNA sequence.
  • DNA-based aptamers advantageously have increased stability.
  • aptamers which have a sequence comprising 2'-modified nucleotides, for example 2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides.
  • aptamers which have 2'-modified ribonucleotides, for example 2'-fluoro, 2'-methoxy and / or 2'-amino-modified ribonucleotides.
  • aptamers may have deoxyribonucleotides and 2'-modified ribonucleotides, for example 2'-fluoro, 2'-methoxy and / or 2'-amino-modified ribonucleotides.
  • the thrombin-binding fusion aptamer In preferred embodiments of the thrombin-binding fusion aptamer, the
  • Fusion aptamer two to four thrombin-binding aptamers; and one to three linkers, each linker linking two to thrombin
  • the thrombin-binding aptamers have a sequence selected from the group comprising:
  • N 3 , N 4 , N 5 , N 6 , N 7 , N 8 are the same or independently selected from the group comprising guanine, cytosine, adenine and / or thymine, or a deletion,
  • the thrombin-binding aptamers may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamer may modified nucleotides, preferably selected from the group comprising Locked nucleic acids, 2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides have.
  • thrombin-binding aptamers bind to exosites 1 and 2 of thrombin.
  • the linker is selected from the group comprising nucleotidic linkers, polyethylene glycol (s), peptidic linkers and / or straight or branched, saturated or unsaturated C 1 -C 50 -alkyl.
  • the nucleotide linker has a length in the range of> 1 nucleotide to ⁇ 30 nucleotides, preferably in the range of> 2 nucleotides to ⁇ 15 nucleotides, more preferably in the range of> 5 nucleotides to ⁇ 15 nucleotides, particularly preferably in the range of> 10 Nucleotides to ⁇ 15 nucleotides, wherein the nucleotides are selected from the group comprising guanosine, cytidine, adenosine and / or thymidine.
  • a preferred nucleotide is adenosine.
  • Particularly preferred linkers are so-called poly A-linkers.
  • thrombin-binding fusion aptamers according to the invention can have a particularly good affinity for thrombin, whose linkers predominantly contain the nucleotide adenosine or are formed from the nucleotide adenosine.
  • a polyethylene glycol linker has 1 to 15 polyethylene glycol units, preferably in the range of 1 to 10 polyethylene glycol units, preferably in the range of 2 to 8 polyethylene glycol units.
  • n is an integer in the range from 1 to 15, preferably in the range from 1 to 10, preferably in the range from 2 to 8 , is.
  • linker is a peptidic linker, wherein the peptidic linker preferably comprises amino acids selected from the group comprising glycine, alanine and / or ⁇ -alanine.
  • the peptidic linker comprises in the range of 2 to 4 amino acids.
  • the peptidic linker comprises ⁇ -alanine units.
  • the peptidic linker comprises in the range of 2 to 4 ⁇ -alanine units.
  • the linker is an unbranched or branched, saturated or unsaturated C 1 -C 4 -alkyl group, preferably a C 1 -C 30 -alkyl group, more preferably a C 2 -C 2 0 -alkyl group ,
  • the thrombin-binding fusion aptamer comprises a sequence selected from the group comprising: 5'-GGTTGGTGTGGTTGGAAAAAAAAAAAAAAGTCCGTGGTAGGGCAGG
  • thrombin-binding aptamers may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications , and / or the aptamers may have modified nucleotides, preferably selected from the group comprising Locked nucleic acids, T-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides.
  • a further preferred chemical modification is, for example, the addition of a so-called "3'-CAP” or “5'-CAP” structure, of a modified guanosine nucleotide (7-methyl-guanosine) to the 3'-end or 5 ' -The End. Modification of the 3'-end or 5'-end may advantageously protect the fusion aptamer from rapid degradation by nucleases, particularly in an organism or biological sample.
  • the 5 'end of the fusion aptamer may be pegylated.
  • a 5'-PEG modification may comprise at least one polyethylene glycol unit, preferably in the range of 1 to 900 polyethylene glycol units, preferably in the range of 1 to 450 polyethylene glycol units.
  • thrombin-binding fusion aptamers according to the invention, their pharmacologically acceptable salts, derivatives and / or conjugates advantageously makes it possible to therapeutically influence the activity of thrombin.
  • Another object of the invention relates to the use of the thrombin-binding fusion aptamers according to the invention for the diagnosis, therapeutic and / or prophylactic treatment of diseases that are associated with a disturbed blood coagulation or whose therapy requires an influence on the blood coagulation system.
  • the thrombin-binding fusion aptamers of the invention are useful as anticoagulants or anticoagulants.
  • the thrombin-binding fusion aptamers according to the invention are very effective anticoagulants.
  • the thrombin-binding fusion aptamer SEQ ID NO: 98 according to the invention has a significantly higher effect on thrombin-mediated blood coagulation than a combination of the single aptamers SEQ ID NO: 95 and SEQ ID NO: 97.
  • the invention furthermore relates to the use of a thrombin-binding fusion aptamer according to the invention, its pharmacologically acceptable salts, derivatives and / or conjugates, for the production of a medicament.
  • the invention relates to the use of thrombin-binding fusion aptamers of the invention, their pharmacologically acceptable salts, derivatives and / or conjugates, for the manufacture of a medicament for the diagnosis, therapeutic and / or prophylactic treatment of disorders associated with impaired blood coagulation or whose therapy requires an influence on the blood coagulation system, in particular as a blood coagulation inhibitor.
  • Diseases associated with impaired blood coagulation or whose therapy requires interference with the blood coagulation system are, in particular, selected from the group comprising arterial or venous thromboses, in particular acute myocardial and cerebral infarcts or pulmonary embolisms, and / or thromboembolic complications.
  • Thrombin-binding fusion aptamers according to the invention are particularly suitable for the therapy of arterial thromboses, in particular acute myocardial and cerebral infarction and secondary prophylaxis of thromboembolic complications after vascular reconstruction procedures.
  • Thrombin-binding fusion aptamers according to the invention are furthermore suitable for systemic anticoagulation for use in extracorporeal therapeutic methods, in particular heart-lung machines, hemodialysis and hemofiltration methods.
  • Thrombin-binding fusion aptamers of the invention are also suitable for use in the acute therapy of venous thrombosis, especially acute pulmonary embolism.
  • a further subject of the invention are medicaments comprising at least one thrombin-binding fusion aptamer according to the invention.
  • the medicament comprises thrombin-binding fusion aptamers according to the invention preferably as a pharmacologically acceptable salt, preferably as salts of inorganic acids, for example phosphoric acid, or as salts of organic acids, for example hydrochloric acid or sulfuric acid.
  • the medicament may further comprise pharmaceutically acceptable excipients and / or carriers.
  • the medicament is suitable for the prophylactic or therapeutic treatment of diseases associated with impaired blood coagulation or whose therapy requires an influence on the blood coagulation system, in particular as a blood coagulation inhibitor.
  • the drug is orally, transmucosally, rectally, pulmonarily, enterally and / or parenterally administrable. Preference is given to administration by injection.
  • FIG. 1 Sequences of DNA aptamers which bind to activated protein C.
  • the sequence of FIG. 1a corresponds to the sequence SEQ ID NO: 58
  • the sequence of FIG. 1b corresponds to the sequence SEQ ID NO: 88.
  • the sequences each have a consensus sequence SEQ ID NO. 1 on.
  • FIG. 2a corresponds to the sequence SEQ ID NO: 89
  • the sequence of FIG. 2b corresponds to the sequence SEQ ID NO: 90
  • the sequence of FIG. 2c corresponds to the sequence SEQ ID NO: 91
  • the sequence of FIG 2d corresponds to the sequence SEQ ID NO: 92.
  • the sequences each have a consensus sequence SEQ ID NO. 1 on.
  • the computation of the secondary structures was based on the folding program available on the Internet, mfold, M. Zuker, Mfold web server for nucleic acid folding and hybridization prediction, Nucleic Acid Res. 31 (13), 3406-15 (2003).
  • the drug used in severe sepsis was Drotrecogin Alpha (Xigris®, Eli Lilly, USA) as an APC source.
  • Drotrecogin Alpha Xigris®, Eli Lilly, USA
  • 100 ⁇ g of the APC were biotinylated and coupled to streptavidin-coated magnetic beads (Dynabeads® M-280 streptavidin (Dynal, Invitrogen)).
  • streptavidin-coated magnetic beads Dynabeads® M-280 streptavidin (Dynal, Invitrogen)
  • biotinylation a 3-fold molar excess of NHS-biotin was used with respect to the APC molecules used (Pierce, Rockford, USA).
  • the incubation was carried out in PBS buffer (8.0 g / l NaCl, 0.2 g / l KCl, 1.44 g / l Na 2 HPO 4 , 0.24 g / l KH 2 PO 4 , pH 7.4 ) in a total volume of 100 ⁇ l for 30 minutes on ice and then for 10 minutes at room temperature (RT). Unbound NHS-biotin (Perbio Science) was subsequently removed by column chromatography (Micro Bio-Spin 6 Chromatography columns, Biorad, Kunststoff, Germany).
  • the biotinylated APC thus obtained was incubated with 5 mg of the magnetic beads in a total volume of 600 ⁇ l of PBS containing 0.1% bovine serum albumin (BSA, bovine serum albumin) for 30 minutes at RT under constant agitation. Subsequently, the beads were Ix washed with 500 .mu.l of PBS containing 0.1% bovine serum albumin (BSA, bovine serum albumin) and finally taken up in 1 ml of PBS containing 0.1% BSA.
  • BSA bovine serum albumin
  • Dl pool with a 49-base long, flanked by two primer binding sites, variable sequence region was used (5'-GCCTGTTGTGAGCCTCCTAAC-N49-CATGCTTATTCTTGTCTCCC-3 'SEQ ID NO: 101). This was synthesized by the company Metabion (Martinsried, Germany) and obtained by HPLC purification.
  • the pool selected in this way was subsequently separated by means of polymerase chain reaction (PCR) into 5 separate 100 ⁇ l reactions (90 ⁇ l master mix and 10 ⁇ l sample each) using the primers (HPLC-pure, Metabion) 5'-GCCTGTTGTGAGCCTCCTAAC-S '(forward, SEQ ID NO: 86) and 5'-GGGAGACAAGAATAAGCATG-S '(reverse, SEQ ID NO: 102), amplified with 5'-biotin modification), followed by single-strand separation.
  • the PCR products were bound to the already described magnetic beads via the biotin molecules coupled to the 5 'end of the reverse primer.
  • the beads (500 .mu.l (5 mg) per single-strand separation) were first washed 3 times with 1000 .mu.l BW buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1.0 M NaCl) and then taken up in 500 ⁇ l 2 x BW buffer (10 mM Tris-HCl, pH 7.5, 1.0 mM EDTA, 2.0 M NaCl). After addition of the PCR products (5 ⁇ 100 ⁇ l), these mixtures were incubated for 45 minutes at RT under constant agitation.
  • 1000 .mu.l BW buffer 5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1.0 M NaCl
  • 500 ⁇ l 2 x BW buffer 10 mM Tris-HCl, pH 7.5, 1.0 mM EDTA, 2.0 M NaCl
  • the beads were each washed twice with BW buffer and once with 2x BW buffer (1000 ⁇ l each) to remove unbound components of the PCR reaction mixes. Subsequently, the beads were taken up in 50 ⁇ l of 0.1 M NaOH and incubated for 3 minutes at RT. After immobilization of the beads, the aptamers in the NaOH solution were transferred to a new reaction vessel and the process was repeated with a further 50 ⁇ l of NaOH. To neutralize the pH, a previously optimized amount of a 0.1 molar HCl solution was pipetted to the aptamer solution. After carrying out the single-strand separation described above, 50 pmol of the aptamers thus obtained were used in the next selection cycle.
  • the aptamer pool (single-stranded DNA) obtained after 10 rounds of selection was re-amplified (5 cycles with primers 5'-GCCTGTTGTGAGCCTCCTAAC-3 '(forward, SEQ ID NO: 86) and 5'-GGGAGACAAGAATAAGCATG -3 '(Reverse, SEQ ID NO: 102), in 100 ⁇ l total reaction volume using 0.75 pmol single-stranded DNA and the PCR products thus produced by TA cloning in pCR II vectors (TA Cloning® Kit, Invitrogen ) were ligated. then, transformed chemically competent E.
  • the aptamers to be tested were labeled with 32 p-ATP at the 5 'end using a polynucleotide kinase (PNK).
  • PNK polynucleotide kinase
  • 5 pmol of the aptamers were incubated with 10 .mu.Ci 32 p-ATP in the presence of 10 U PNK in a total volume of 10 .mu.l for 45 minutes at 37 0 C.
  • To remove unbound 32 p-ATP the batches were then added to 40 ul bidistilled water and purified on G25 columns (Microspin G-25 Colums, Amersham).
  • the radioactively-labeled aptamers were incubated in a final concentration of 0.4 nM in selection buffer with various concentrations of activated protein C (APC, Xigris®, Eli Lilly, USA), starting from 100 nM and subsequent, half-logarithmic dilution stages a total volume of 25 .mu.l for 30 minutes at 37 0 C incubated. After the incubation, in each case 20 ⁇ l of the mixtures were filtered by means of a vacuum system over a nitrocellulose membrane (Schleicher and Schuell, 0.45 ⁇ M).
  • APC activated protein C
  • washing buffer IxPBS 8.0 g / l NaCl, 0.2 g / l KCl, 1.44 g / l Na 2 HPO 4 , 0.24 g / l KH 2 PO 4 , pH 7.4, 1mM MgCl 2 , 1mM CaCl 2
  • the recording of the respective radiation intensities took place by means of a phosphor screen.
  • a FUJIFILM FLA-3000 with corresponding AIDA Imagequant software was used.
  • the individual K D S could be determined.
  • the function "Logistic Dose Response in Pharmacology / Chemistry" of the software Origin 7.5 (OriginLab, Northampton, MA, USA) was used.
  • Test series 1 testing of individual aptamer from that obtained after 10 rounds of selection
  • HS02-48G (SEQ ID NO: 91): 0.40 + 0.13 nM
  • APC native activated protein C
  • PC plasma-purified protein C
  • HS02-44G (SEQ ID NO: 92): 0.76 + 0.10 nM
  • HS02-52 (SEQ ID NO: 89): 0.70 + 0.17 nM
  • HS02-52G (SEQ ID NO: 90): 0.42 + 0.16 nM
  • HS02-48G (SEQ ID NO: 91): 0.36 + 0.17 nM
  • the so activated factor VIII (FVIIIa) was in a final concentration of 0.16 U / ml with 1.8 nM activated protein C (APC) (Xigris®, Eli Lilly) in assay buffer in the presence different Ap tamer- concentrations (semilogarithmic dilution series starting from 180 nM) in a total volume of 50 .mu.l incubated for 20 minutes at RT.
  • APC activated protein C
  • HS02-52G (SEQ ID NO: 90): 0.22 + 0.15 nM
  • washing buffer IxPBS 8.0 g / l NaCl, 0.2 g / l KCl, 1.44 g / l Na 2 HPO 4 , 0.24 g / l KH 2 PO 4 , pH 7 , 4, 0.05% Tween 20
  • SLT automatic microtiter plate washer
  • free binding sites with blocking buffer IxPBS 8.0 g / l NaCl, 0.2 g / l KCl, 1.44 g / l Na 2 HPO 4 , 0.24 g / l KH 2 PO 4 , pH 7.4, 2% BSA, 0.05% Tween 20) for 2 hours RT blocked.
  • Activated protein C which is now immobilized in the wells of the modules, was then followed by the addition of 100 ⁇ l / well of citrated pool plasma to which the aptamers to be tested had been added in advance in a final concentration of 100 nM. There was also a blank without aptamers carried.
  • PCI protein C inhibitor
  • the corresponding wells were incubated at different incubation times (5, 10, 20 and 30 minutes). washed by default and filled with 100 .mu.l / well dilution buffer. After completion of the series of experiments, all wells were washed again.
  • amidolytic APC activity remaining in the wells was then added to a fluorogenic peptide substrate for APC (Pefa 3342, Pentapharm) at a concentration of 200 ⁇ M in dilution buffer (100 ⁇ l / well) and the kinetics of substrate hydrolysis using a plate fluorometer (Ascent Fluoroscan, Thermo Fisher Scientific).
  • the apparent inhibition constants (k app ) as a measure of the rate of inhibition of the immobilized activated protein C (APC) were calculated from the resulting data sets by exponential interpolation.
  • HS02-52G (SEQ ID NO: 90): 0.080 + 0.002
  • HS02-48G (SEQ ID NO: 91): 0.053 + 0.000
  • HS02-44G (SEQ ID NO: 92): 0.047 + 0.000 without aptamer: 0.031 + 0.000
  • the experimental assays revealed the formation of activated protein C (APC) / protein C inhibitor (PCI) complexes as the cause of inhibition of activated protein C (APC).
  • API activated protein C
  • PCI protein C inhibitor
  • Platelet-rich plasma was recovered by centrifugation at 125 x g (Megafuge 1.0 R, Heraeus, Hanau, Germany) for 10 minutes from a healthy blood donor anticoagulated with citrate buffer (0.109 M citrate) in the ratio 1:10.
  • tissue factor reagent diluted 1: 1000 with TBS buffer (20 mM Tris-HCl, 100 mM NaCl, pH 7.6) (Recombiplastin, IL, Milan, Italy) in the presence of 15 ⁇ M phospholipids (Siemens Healthcare, Marburg, Germany) and the fibrin polymerization inhibitor H-Gly-Pro-Arg-Pro-OH.AcOH (Gly-Pro Arg-Pro, Loxo, Dossenheim, Germany) to a final concentration of 10 mM .
  • the phospholipids were presented as a 1 ⁇ g / ⁇ l (1.3 mM) stock solution of soy phospholipids prepared by dispersing dried soy phospholipids (Siemens Healthcare Diagnostics) in water.
  • Coagulation was then activated by adding 10 mM CaCl 2 to the plasma samples. This leads to an increase of thrombin which combines with the thrombomodulin and thereby can activate the endogenously present protein C of the plasma sample to activated protein C.
  • the formation of activated protein C was in the respective plasma samples after 30 seconds, 1, 2, 3, 4, 5, 6, 8 or 10 minutes by the addition of hirudin (Refludan®, Pharmion, Hamburg, Germany) to a final concentration of 100 ⁇ g / ml stopped.
  • hirudin Refludan®, Pharmion, Hamburg, Germany
  • apotrotinin (Trasylol®, Bayer, Germany) was added to a final concentration of 20 ⁇ M. Aprotinin protects the formed activated protein C from inactivation.
  • thrombin-binding fusion aptamer of sequence SEQ ID NO: 98 was determined using human platelets.
  • plasma enriched with platelets was obtained by centrifugation at 125 ⁇ g (Megafuge 1.0 R, Heraeus, Hanau, Germany) for 10 minutes from a healthy blood donor anticoagulated with citrate buffer (0.109 M citrate, pH 6.4) in a ratio of 1:10.
  • the platelet-enriched plasma was diluted in the ratio 1: 2 with a first washing buffer (140 mM NaCl, 13 mM EDTA, pH 4.2) and the mixture at 1400 ⁇ g (Megafuge 1.0 R, Heraeus, Hanau, Germany) for 10 minutes centrifuged. After removing the supernatant, the platelets were resuspended in a second wash buffer (140 mM NaCl, pH 6.5) and centrifuged again at 1400 xg for 10 minutes.
  • a first washing buffer 140 mM NaCl, 13 mM EDTA, pH 4.2
  • 1400 ⁇ g Megafuge 1.0 R, Heraeus, Hanau, Germany
  • Platelets were then transferred to HEPES-Tyrode's buffer (138mM NaCl, 2.8mM KCl, 12mM NaHCO 3 , 0.5mM NaH 2 PO 4 , 10mM glucose, 1mM CaCl 2 , 0.5mM MgCl 2 , 10mM HEPES, pH 7.4) to a final concentration of 2 x 10 6 cells / ⁇ l.
  • HEPES-Tyrode's buffer 138mM NaCl, 2.8mM KCl, 12mM NaHCO 3 , 0.5mM NaH 2 PO 4 , 10mM glucose, 1mM CaCl 2 , 0.5mM MgCl 2 , 10mM HEPES, pH 7.4
  • thrombin-induced aggregation of the platelets aliquots of in each case 250 ⁇ l of the platelet suspension were incubated with different concentrations in the range of 0 to 1 ⁇ M, namely 1 nM, 10 nM, 100 nM, 1 ⁇ M and 1 mM, the aptamers 5'- GGTTGGTGTGGTTGG-3 '(SEQ ID NO: 95), 5'-
  • the suspensions were incubated for two minutes at 37 0 C without stirring and in platelet aggregation cuvettes (lab Biomedical Technologies, Ahrensburg, Germany) transferred. Aggregation of platelets was then monitored by addition of human ⁇ -thrombin (CellSystems Biotechnologiemaschine GmbH) to a final concentration of 0.7 nM (0.1 NIH U / ml) and photo-optically measured in a 2-channel system aggregometer (APACT, LAbor Biomedical Technologies).
  • human ⁇ -thrombin CellSystems Biotechnologiemaschinemaschine GmbH
  • the aptamers and the thrombin inhibitors inhibited platelet aggregation in a concentration-dependent manner. It was found that the thrombin-binding fusion aptamer according to the invention of the sequence SEQ ID NO: 98 showed an inhibition of the platelet aggregation which was clearly higher than that of the mixture of the aptamers SEQ ID NO: 95 and SEQ ID NO: 97 and was twice or five times more effective compared to the inhibitors bivalirudin and argatroban.
  • thrombin-binding fusion aptamer according to the invention can bring about a very good inhibition of thrombin-induced platelet aggregation.
  • thrombin-forming assay the influence of the thrombin-binding fusion aptamer of the sequence SEQ ID NO: 98 on the activity of the prothrombinase was determined.
  • the pro-thrombin solutions were mixed with the aptamer of the sequence 5'- GGTTGGTGTGGTTGG-3 '(SEQ ID NO: 95) or the thrombin-binding fusion aptamer of the sequence 5'-GGTTGGTGTGGTTGGAAAAAAAAAAAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3' (SEQ ID NO: 98) to a respective final concentration of 1 ⁇ M.
  • the control was a solution of 220 nM prothrombin in assay buffer.
  • thrombin The formation of thrombin was started by adding 35 ⁇ l of prothrombinase, a solution of 1 mM factor Xa (CellSystems Biotechnologiemaschine GmbH) and 1 mM factor Va (CellSystems Biotechnologiemaschine GmbH) in assay buffer. To observe thrombin formation, 50 ⁇ l samples were taken from the batches every 2 minutes and transferred to white Fluoronunc modules (Nunc, Thermo Fisher Scientific) containing 25 ⁇ l of a 30 mM EDTA solution in TBS buffer (pH 7). 6, 1 mg / ml BSA), whereby thrombin formation was stopped.
  • the concentration of thrombin formed was calculated from a parallel series of dilutions of ⁇ -thrombin.
  • thrombin-binding fusion aptamer of the sequence SEQ ID NO: 98 showed a significantly stronger effect than the addition of the aptamer of the sequence SEQ ID NO: 95 and led to a 60% reduction in thrombin formation. This shows that the formation of thrombin can already be inhibited by the thrombin-binding fusion aptamer according to the invention.
  • the determination of the activated partial thromboplastin time is a routine test of the plasmatic coagulation analysis.
  • the analysis is basically carried out by adding a sample of the plasma with an activator. Subsequently, the coagulation reaction is started with calcium chloride. The period of time is measured until coagulation of the sample occurs.
  • Controls contained no apatamer or 2 ⁇ M of the complementary aptamer of SEQ ID NO: 159. Subsequently, the samples were incubated with 50 ⁇ l Actin FS (Siemens Healthcare Diagnostics Marburg, Germany) at 37 ° C. for 180 seconds. Thereafter, coagulation was initiated by adding 50 ⁇ l of a solution of 25 mM CaCl 2 .
  • Actin FS Siemens Healthcare Diagnostics Marburg, Germany
  • the determination of the activated partial thromboplastin time was carried out automatically using the ACL Top® coagulation device (ACL Top®, Instrumentation Laboratory).
  • the addition of the thrombin-binding fusion aptamer of SEQ ID NO: 98 markedly slowed the clotting, while the addition of the complementary aptamer of SEQ ID NO: 159 reduced the slowing down of clotting in a concentration-dependent manner.
  • the slowing down of coagulation by 1 ⁇ M of the thrombin-binding fusion aptamer of SEQ ID NO: 98 could be fully reversed by an equimolar addition of 1 ⁇ M of the complementary aptamer of SEQ ID NO: 159, so that the time to coagulate again Reached control level.

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Abstract

L'invention concerne un aptamère se liant à une molécule cible intervenant dans l'hémostase, l'aptamère étant un aptamère se liant à la protéine C activée, lequel aptamère se lie à la protéine C activée avec une constante de dissociation KD comprise entre = 0,001 nM et = 80 nM et présente une longueur comprise entre = 20 nucléotides et = 160 nucléotides, ou ledit aptamère étant un aptamère de fusion se liant à la thrombine, qui comprend au moins deux aptamères se liant à la thrombine et au moins un lieur, ledit au moins un lieur liant lesdits au moins deux aptamères se liant à la thrombine.
PCT/EP2008/061395 2007-08-31 2008-08-29 Aptamères se liant à une molécule cible intervenant dans l'hémostase Ceased WO2009027507A2 (fr)

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DE102007041476.7 2007-08-31

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WO2013042077A1 (fr) * 2011-09-23 2013-03-28 Csir Diagnostic de tuberculose
EP2980218A1 (fr) * 2014-07-30 2016-02-03 Rheinische Friedrich-Wilhelms-Universität Bonn Complexe de thrombine aptamère à utiliser comme antidote à action directe sur les inhibiteurs de la thrombine
US9545383B2 (en) 2014-04-01 2017-01-17 Massachusetts Institute Of Technology Blood clotting control
CN111440235A (zh) * 2020-04-16 2020-07-24 成都中医药大学 一种捕获水蛭素类多肽的探针及其应用
WO2021245586A3 (fr) * 2020-06-03 2022-09-09 Rhodes University Aptamères de liaison à cd4 et leurs applications

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013042077A1 (fr) * 2011-09-23 2013-03-28 Csir Diagnostic de tuberculose
US9176134B2 (en) 2011-09-23 2015-11-03 Csir Diagnosis of tuberculosis
AP3990A (en) * 2011-09-23 2017-01-07 Csir Diagnosis of tuberculosis
US9545383B2 (en) 2014-04-01 2017-01-17 Massachusetts Institute Of Technology Blood clotting control
EP2980218A1 (fr) * 2014-07-30 2016-02-03 Rheinische Friedrich-Wilhelms-Universität Bonn Complexe de thrombine aptamère à utiliser comme antidote à action directe sur les inhibiteurs de la thrombine
WO2016016259A1 (fr) * 2014-07-30 2016-02-04 Rheinische Friedrich-Wilhelms Universität Bonn Complexe aptamère-thrombine utilisable à titre d'antidote aux inhibiteurs de thrombine à action directe
CN111440235A (zh) * 2020-04-16 2020-07-24 成都中医药大学 一种捕获水蛭素类多肽的探针及其应用
WO2021245586A3 (fr) * 2020-06-03 2022-09-09 Rhodes University Aptamères de liaison à cd4 et leurs applications

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