DE102007041476A1 - Aptamers that bind to a target molecule involved in hemostasis - Google Patents

Abstract

The invention relates to an aptamer which binds to a target molecule involved in hemostasis, the aptamer being an aptamer which binds to activated protein C, the aptamer having a dissociation constant KD in the range of> = 0.001 nM to <= 80 nM activated protein C and wherein the aptamer has a length in the range of> = 20 nucleotides to <= 160 nucleotides, or wherein the aptamer is a thrombin-binding fusion aptameter comprising at least two thrombin-binding aptamers and at least one linker, said at least one linker linking at least two thrombin-binding aptamers.

Description

The The invention relates to aptamers which are linked to hemostasis bind involved target molecule, the aptamer an aptamer which binds to activated protein C, or a thrombin-binding one Fusion aptamer is.

activated Protein C (APC) is a serine protease and the active form of im Blood circulating zymogens protein C. As so-called "endogenous Inhibitor "activates activated protein C the activated coagulation factors Va and VIIIa and thereby controls the plasmatic coagulation activation. A reduced formation of activated protein C or a disorder The activity of activated protein C is elevated Risk linked to the development of venous thrombosis and plays in the pathogenesis of microcirculatory disorders, as they occur, for example, in the context of severe sepsis, a decisive role. A recombinantly produced concentrate of Activated protein C is used, for example, in the treatment of heavy sepsis used.

In spite of the central role, the activated protein C in the pathogenesis, Pathophysiology and therapy of various vascular and inflammatory diseases is not a routine diagnostic test method available, with which the concentration of activated protein C in plasma or whole blood can.

Of the Detection of activated protein C is due to its low plasma concentrations and a concurrent 10,000-fold excess of protein C is difficult. On top of that, the amino acid sequence of activated protein C up to a twelve amino acids long propeptide is identical to that of protein C.

method for determining the activity of activated protein C, for example by determining the protein C-propeptide concentration, are known, but due to multiple technical problems can not be used in the diagnosis.

Furthermore, from the Scriptures US 6989241 a monoclonal antibody against activated protein C known for differentiation between activated protein C and protein C. However, 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.

Of the Invention was the object of a means available to provide at least one of the aforementioned disadvantages of The state of the art overcomes. In particular, the Object of the present invention therein, a means available to provide evidence of activated protein C.

A Another object of the invention was thrombin-inhibiting aptamers to provide.

• – ein Aptamer ist, das an aktiviertes Protein C bindet, wobei das Aptamer mit einer Dissoziationskonstante K_D im Bereich von ≥ 0,001 nM bis ≤ 80 nM an aktiviertes Protein C bindet, und wobei das Aptamer eine Länge im Bereich von ≥ 20 Nukleotide bis ≤ 160 Nukleotide aufweist, oder
• – ein Thrombin-bindendes Fusions-Aptamer ist, umfassend wenigstens zwei an Thrombin bindende Aptamere und wenigstens einen Linker, wobei der wenigstens eine Linker die wenigstens zwei an Thrombin bindenden Aptamere verbindet.

According to the invention, the object is achieved by an aptamer which binds to a target molecule involved in the hemostasis, wherein the aptamer:

• An aptamer that binds to activated protein C, the aptamer binding to activated protein C with a dissociation constant K _D in the range of ≥ 0.001 nM to ≤ 80 nM, and wherein the aptamer has a length in the range of ≥ 20 nucleotides to ≤ Has 160 nucleotides, or
• A thrombin-binding fusion aptamer comprising at least two thrombin-binding aptamers and at least one linker, said at least one linker linking the at least two aptamers binding to thrombin.

One Another object relates to a method for the determination of activated Protein C in biological samples, wherein at least one activated Protein C binding aptamer with the biological sample, preferably a biological fluid, and incubated Binding event between the aptamer and activated protein C is detected.

Further advantageous embodiments of the invention will become apparent from the Dependent claims.

Under The term "aptamer" in the context of this invention are oligonucleotides to understand the specific and high affinity a target molecule, especially one at the hemostasis involved target protein such as activated protein C or thrombin, tie.

The Inventors were able to surprisingly select aptamers, can bind the activated protein C with high affinity. A particular advantage of the invention activated protein C binding aptamer is thereby made available provided that this activated protein C with high affinity and high selectivity. Especially can the aptamers of the invention specifically to activated protein C over the inactive one Zymogen protein C bind.

Especially the aptamers of the invention are distinguished a high affinity in nanomolar and sub-nanomolar Range and a pronounced specificity for activated protein C over the inactive zymogen protein C off. These features enable a reliable Determination of activated protein C even in the presence of a large excess of inactive zymogen protein C, and a reproducible determination of activated protein C in biological samples. The affinity and specificity of according to the invention binding to activated protein C. Aptamers are preferably better or at least comparable to of antibodies. Another advantage is that the inventions provided Aptamers are inexpensive compared to antibodies are synthetically produced.

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 5,475,096 . US 5,270,163 and EP 0 533 838 , which is hereby incorporated by reference, in particular the so-called Counter- ^SELEX® method. First, the synthesis of a randomized DNA pool was carried out. Aptamers were generated schematically by the following steps. Aptamers from the DNA pool were enriched by binding the pool with activated protein C and separating the binding complex from unbound ssDNA. In order to suppress an accumulation of matrix binders, a pre-selection against streptavidin beads took place before the selected selection step. After incubation with activated protein C, 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. After cloning the selected aptamer pool, 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 through the affinity of the bond is usually expressed via described the dissociation constant.

In a preferred embodiment of the present invention, 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 ≥ 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 non-linear regression of the data points of radioactively-labeled aptamers bound to activated protein C in relation 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.

In a further preferred embodiment of the present invention Invention has the activated protein C binding aptamer one Number of nucleotides in the range of ≥ 20 nucleotides to ≤ 120 Nucleotides, preferably in the range of ≥ 40 nucleotides up to ≤ 88 nucleotides, up. This is especially preferred activated protein C binding aptamer 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 ≥ 44 to ≤ 52 Nucleotides on.

Unless otherwise stated below, the term "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 Deso includes xyribonukleotide.

invention Aptamers may have a DNA or an RNA sequence. It is understood that in the event that inventive Aptamers have an RNA sequence in the specified sequence motifs and sequences thymidine is replaced by uridine.

The according to the invention suitable RNA sequences the DNA sequences of the invention, wherein T by U is replaced.

Preferably Aptamers have a DNA sequence. Own aptamers based on DNA advantageously an increased stability.

Farther suitable are aptamers having a sequence comprising 2'-modified Nucleotides, for example 2'-fluoro, 2'-methoxy and / or 2'-amino-modified Nucleotides. Also suitable are aptamers which are 2'-modified Ribonucleotides, for example, 2'-fluoro, 2'-methoxy and / or 2'-amino-modified ribonucleotides. Furthermore you can Aptamers deoxyribonucleotides and 2'-modified ribonucleotides, For example, 2'-fluoro, 2'-methoxy and / or 2'-amino-modified Have ribonucleotides.

in the Within the scope of the present invention, activated protein C binding aptamers are selected, which are assigned to three main groups could be, with the sequences of aptamers within a Main group have a common sequence motif. This common Sequence motif varies within a main group only in individual Base positions.

worin:
N₁ ist ausgewählt aus der Gruppe umfassend Guanin, Cytosin, Adenin, Thymin, Uracil, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifiziertes Nukleotid, insbesondere Ribonukleotid, oder eine Deletion;

worin:
N₂ ist ausgewählt aus der Gruppe umfassend Guanin, Cytosin, Adenin, Thymin, Uracil, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifiziertes Nukleotid, insbesondere Ribonukleotid, oder eine Deletion,
und/oder

wobei die Sequenzmotive ausgewählt aus der Gruppe umfassend SEQ ID NO: 1, SEQ ID NO: 2 und/oder SEQ ID NO: 3 modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen können.In a preferred embodiment of the present invention, the activated protein C binding aptamer comprises a sequence motif selected from the group comprising:

wherein:
N ₁ 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;

wherein:
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,
and or

wherein the 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 the group comprising Locked nucleic acids, 2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides.

"C" is here according to the usual used here One-letter code of the bases for cytosine, accordingly "A" stands for adenine, "G" for guanine and "T" for thymine, "U" for uracil.

In particular, the positions N ₁ and N _{2 of} the sequence motifs SEQ ID NO: 1 and SEQ ID NO: 2 are highly variable. Thus, positions N ₁ and N _{2 may} comprise different bases or nucleotides or a deletion. For example, at positions N ₁ and N _2, a ribonucleotide may be included in a DNA aptamer. Furthermore, in particular at the positions N ₁ and N ₂ modified nucleotides, in particular modified ribonucleotides may be included.

In preferred embodiments have the 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 Aptamers do not have modified nucleotides.

Without being committed to a particular theory is believed that the sequence motifs are selected from the group comprising SEQ ID NOs: 1 to 3 an essential role in the binding of Aptamers according to the invention of activated protein C have.

Among the activated protein C-binding aptamers having a sequence motif 5'-TATCCCGN ₁ ATGGG-3 '(SEQ ID NO: 1), four subgroups were identified, each having a common base N ₁ selected from the group comprising guanine, cytosine , Adenine and / or thymine.

In a preferred embodiment of the present invention, the activated protein C binding aptamer comprises a sequence motif selected from the group comprising:

in this connection may modify the sequence motifs SEQ ID NOs: 4 to 7 Nucleotides, preferably selected from the group comprising Locked nucleic acids, 2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides.

Without being committed to a particular theory is believed that the sequence motifs are selected from the group comprising SEQ ID NOs: 4 to 7 a particularly good binding of the invention Aptamers to activated protein C can mediate.

The Aptamer Sequences SEQ ID NOs: 8 to 19 have a common consensus sequence 5'-TATCCCGTATGGG-3 ' (SEQ ID NO: 4) containing the base thymine at position 8 within the group of the sequences SEQ ID NO: 1.

und/oder deren Varianten, Mutanten und/oder Teile, wobei das an aktiviertes Protein C bindende Aptamer Modifikationen aufweisen kann, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder das Aptamer modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen kann.In preferred embodiments of the present invention, the activated protein C-binding aptamer comprises a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein 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.

parts the aforementioned aptamer sequences comprise the sequence motif 5'-TATCCCGTATGGG-3 '(SEQ ID NO: 4).

Variants of the sequences according to the invention can be obtained by modifications, such as alkylation, in particular methylation, arylation or acetylation of at least one nucleotide, incorporation of enantiomers and / or by 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-3 ' (SEQ ID NO: 5) containing the base guanine at position 8 within the group of sequences according to SEQ ID NO: 1.

und/oder deren Varianten, Mutanten und/oder Teile, wobei das an aktiviertes Protein C bindende Aptamer Modifikationen aufweisen kann, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder das Aptamer modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen kann. Teile der Aptamer-Sequenzen umfassen das Sequenzmotiv 5'-TATCCCGGATGGG-3' (SEQ ID NO: 5).In preferred embodiments of the present invention, the activated protein C-binding aptamer comprises a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein 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'-TATCCCGGATGGG-3 '(SEQ ID NO: 5).

und/oder deren Varianten, Mutanten und/oder Teile, wobei das an aktiviertes Protein C bindende Aptamer Modifikationen aufweisen kann, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder das Aptamer modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen kann. Teile der Aptamer-Sequenzen umfassen das Sequenzmotiv 5'-TATCCCGAATGGG-3' (SEQ ID NO: 6).In preferred embodiments of the present invention, the activated protein C-binding aptamer comprises a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein 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'-TATCCCGAATGGG-3 '(SEQ ID NO: 6).

The Aptamer Sequences SEQ ID NOs: 32 to 34 have a common consensus sequence 5'-TATCCCGAATGGG-3 ' (SEQ ID NO: 6) containing the base adenine at position 8 within the group of the sequences SEQ ID NO: 1.

und/oder deren Varianten, Mutanten und/oder Teile, wobei das an aktiviertes Protein C bindende Aptamer Modifikationen aufweisen kann, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder das Aptamer modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen kann. Teile der Aptamer-Sequenzen umfassen das Sequenzmotiv 5'-TATCCCGCATGGG-3' (SEQ ID NO: 7).In preferred embodiments of the present invention, the activated protein C-binding aptamer comprises a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein 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'-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) containing the base cytosine at position 8 within the group of the sequences SEQ ID NO: 1.

und/oder deren Varianten, Mutanten und/oder Teile, wobei das an aktiviertes Protein C bindende Aptamer Modifikationen aufweisen kann, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder das Aptamer modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen kann. Die Sequenzen SEQ ID NO: 103 bis 121 und/oder Teile dieser Aptamer-Sequenzen umfassen das Sequenzmotiv 5'-TATCCCGN₁ATGGG-3' (SEQ ID NO: 1), insbesondere die Sequenzmotive 5'-TATCCCGTATGGG-3' (SEQ ID NO: 4), 5'-TATCCCGGATGGG-3' (SEQ ID NO: 5), 5'-TATCCCGAATGGG-3' (SEQ ID NO: 6) oder 5'-TATCCCGCATGGG-3' (SEQ ID NO: 7).In further preferred embodiments of the present invention, the activated protein C-binding aptamer comprises a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein 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. The sequences SEQ ID NO: 103 to 121 and / or parts of these aptamer sequences include the sequence motif 5'-TATCCCGN ₁ ATGGG-3 '(SEQ ID NO: 1), in particular the sequence motifs 5'-TATCCCGTATGGG-3' (SEQ ID NO: 4), 5'-TATCCCGGATGGG-3 '(SEQ ID NO: 5), 5'-TATCCCGAATGGG-3' (SEQ ID NO: 6) or 5'-TATCCCGCATGGG-3 '(SEQ ID NO: 7).

und/oder deren Varianten, Mutanten und/oder Teile, wobei das an aktiviertes Protein C bindende Aptamer Modifikationen aufweisen kann, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder das Aptamer modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen kann. Die Sequenzen SEQ ID NO: 122 und 123 und/oder Teile dieser Aptamer-Sequenzen umfassen das Sequenzmotiv 5'-TATCACGGATGGGC-3' (SEQ ID NO: 124), das sich von dem Sequenzmotive SEQ ID NO: 1 dadurch unterscheidet, dass Adenin an der Position 5 enthalten ist.In also preferred embodiments of the present invention, the activated protein C-binding aptamer comprises a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein 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. The sequences SEQ ID NO: 122 and 123 and / or parts of these aptamer sequences comprise the sequence motif 5'-TATCACGGATGGGC-3 '(SEQ ID NO: 124), which differs from the sequence motif SEQ ID NO: 1 in that adenine is contained at position 5.

und/oder deren Varianten, Mutanten und/oder Teile, wobei das an aktiviertes Protein C bindende Aptamer Modifikationen aufweisen kann, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder das Aptamer modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen kann. Die Sequenzen SEQ ID NO: 125 und 126 und/oder Teile dieser Aptamer-Sequenzen umfassen das Sequenzmotiv 5'-TATCCCAAATGGGG-3' (SEQ ID NO: 127), das sich von dem Sequenzmotive SEQ ID NO: 1 dadurch unterscheidet, dass Adenin an der Position 7 enthalten ist, oder das Sequenzmotiv 5'-TATCCCGTATAGGG-3' (SEQ ID NO: 128), das sich von dem Sequenzmotive SEQ ID NO: 1 dadurch unterscheidet, dass Adenin an der Position 11 enthalten ist.In still preferred embodiments of the present invention, the activated protein C-binding aptamer comprises a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein 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. The 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-3 '(SEQ ID NO: 128), which is different from the sequence motif SEQ ID NO: 1 in that adenine is contained at position 11.

Further, among the activated protein C-binding aptamers having a sequence motif 5'-TCTN ₂ TCGGCGG-3 '(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.

In also preferred embodiments of the present invention, the activated protein C-binding aptamer comprises a sequence motif selected from the group comprising:

und/oder deren Varianten, Mutanten und/oder Teile, wobei das an aktiviertes Protein C bindende Aptamer Modifikationen aufweisen kann, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder das Aptamer modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'- Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen kann. Teile der Aptamer-Sequenzen umfassen das Sequenzmotiv 5'-TCTGTCGGCGG-3' (SEQ ID NO: 37).In preferred embodiments of the present invention, the activated protein C-binding aptamer comprises a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein 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).

und/oder deren Varianten, Mutanten und/oder Teile, wobei das an aktiviertes Protein C bindende Aptamer Modifikationen aufweisen kann, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder das Aptamer modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen kann. Teile der Aptamer-Sequenzen umfassen das Sequenzmotiv 5'-TCTTTCGGCGG-3' (SEQ ID NO: 38).In still preferred embodiments of the present invention, the activated protein C-binding aptamer comprises a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein 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'-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).

In also preferred embodiments of the present invention the activated protein C binding aptamer comprises the sequence motif 5'-TATCACGTATGGG-3 '(SEQ ID NO: 3).

und/oder deren Varianten, Mutanten und/oder Teile, wobei das an aktiviertes Protein C bindende Aptamer Modifikationen aufweisen kann, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder das Aptamer modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen kann. Teile der Aptamer-Sequenzen umfassen jeweils das Sequenzmotiv 5'-TATCACGTATGGG-3' (SEQ ID NO: 3).In further embodiments of the present invention, the activated protein C binding aptamer comprises a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein 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 each comprise the sequence motif 5'-TATCACGTATGGG-3 '(SEQ ID NO: 3).

und/oder deren Varianten, Mutanten und/oder Teile, wobei das an aktiviertes Protein C bindende Aptamer Modifikationen aufweisen kann, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder das Aptamer modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen kann. Die Sequenzen SEQ ID NO: 46, 129 und 130 und/oder Teile dieser Aptamer-Sequenzen umfassen das Sequenzmotiv 5'-ATCGCTTCAGGG-3' (SEQ ID NO: 131).In another embodiment of the present invention, the activated protein C-binding aptamer comprises a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein 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. The sequences SEQ ID NO: 46, 129 and 130 and / or parts of these aptamer sequences comprise the sequence motif 5'-ATCGCTTCAGGG-3 '(SEQ ID NO: 131).

In another embodiment of the present invention For example, the activated protein C-binding aptamer has a sequence 5'-ACGTGAATGTCTGGCTGGTGTAGGTCTGGGCTGGACGTGGGGCTAGTGA-3 '(SEQ ID NO: 47).

In another embodiment of the present invention For example, the activated protein C-binding aptamer has a sequence 5'-ACGTGAATGTCTGGCTGGTGTAGGTCTGGGCTGGACGTGGGGCTAGTGA-3 '(SEQ ID NO: 132).

und/oder deren Varianten, Mutanten und/oder Teile, wobei das an aktiviertes Protein C bindende Aptamer Modifikationen aufweisen kann, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder das Aptamer modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen kann.In further embodiments of the present invention, the activated protein C binding aptamer comprises a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein 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.

in this connection the sequences SEQ ID NOs: 48 to 85 include the aforementioned SEQ ID NOs: 8 to 36 and 39 to 47, these being each of two sequences 5'-GCCTGTTGTGAGCCTCCTAAC-3 '(SEQ ID NO: 86) and 5'-CATGCTTATTCTTGTCTCCC-3' (SEQ ID NO: 87) flanking. These sequences SEQ ID NO: 86 and SEQ ID NO: 87 correspond to the primer binding sequences used in the Selection of the aptamers of the invention used were.

Surprisingly, it was found that in particular the aptamers according to the invention of the sequences SEQ ID NOs: 49, 58, 70 and 83 had a very good affinity for binding to activated protein C. In particular in filter binding experiments it could be determined that the determined dissociation constant K _{D was} in the range of ≥ 0.1 nM to ≤ 0.4 nM.

und/oder deren Varianten, Mutanten und/oder Teile, wobei das an aktiviertes Protein C bindende Aptamer Modifikationen aufweisen kann, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder das Aptamer modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen kann.In further embodiments of the present invention, the activated protein C binding aptamer comprises a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein 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.

in this connection For example, the sequences SEQ ID NOs: 133 to 158 include the foregoing SEQ ID NOs: 103 to 123, 125, 126, 129, 130 and 132, these being each of two sequences 5'-GCCTGTTGTGAGCCTCCTAAC-3 '(SEQ ID NO: 86) and 5'-CATGCTTATTCTTGTCTCCC-3 '(SEQ ID NO: 87). These Sequences SEQ ID NO: 86 and SEQ ID NO: 87 correspond to the primer binding sequences, in the selection of the aptamers of the invention were used.

aptamers can change into a specific one under defined conditions three-dimensional structure, for example, so-called hairpin structures wrinkles. Preferably, the binding to activated protein C has Aptamer at least a first hairpin structure comprising a Trunk and a loop on.

For the purposes of the present invention, the term "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. Here you can form in the trunk one or more bases single-stranded areas so-called bulges.

It It is preferred that the stem of the first hairpin structure in the area from ≥ 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 Has base pairs. It is further preferred that the loop the first hairpin structure in the range of ≥ 5 nucleotides to ≤ 30 nucleotides, preferably in the range of ≥ 6 Nucleotides to ≤ 18 nucleotides.

In Advantageously, a number of paired nucleotides in the range from ≥ 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 that a high-affinity binding of the aptamer to activated protein C is formable.

Without Being committed to a particular theory is believed to be a number of paired nucleotides, in particular in the range of ≥ 9 Base pairs up to ≤ 13 base pairs can contribute to that a stable formation of the trunk of the first hairpin structure is possible.

In preferred embodiments, the activated on Protein C-binding aptamer comprising a second hairpin structure a trunk and a bow on. Preferably, the second hairpin structure arranged in the loop of the first hairpin structure.

In further preferred embodiments, the activated on Protein C-binding aptamer comprising a second hairpin structure a trunk and a loop on, with the second hairpin structure arranged in the loop of the first hairpin structure.

In Particularly preferred embodiments, the activated on 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. In further preferred embodiments, the strain the first hairpin structure has a nucleotide sequence cytidine-thymidine (CT), which has a single-stranded region, a bulge, formed.

It could be found that in particular a nucleotide sequence Cytidine thymidine (CT) in the trunk of the first hairpin structure, the a single-stranded area, called a bulge forms to improve the binding of the aptamer to activated Protein C can contribute.

In preferred embodiments, the inventive Activated protein C binding aptamer selected a sequence from the group comprising SEQ ID NOs: 58, 88 to 92. Especially preferred embodiments according to the invention Activated protein C binding aptamer selected a sequence from the group comprising SEQ ID NOs: 58, 88 to 92, these being have a secondary structure that has a first hairpin structure comprising a trunk and a loop and a second hairpin structure comprising a trunk and a loop, wherein the second Hairpin structure in the loop of the first hairpin structure is arranged.

In very particularly preferred embodiments have inventive activated protein C-binding aptamer having selected sequences from the group comprising SEQ ID NOs: 58, 88 to 92 a secondary structure on which a first hairpin structure comprising a trunk and a loop and a second hairpin structure comprising one Trunk and a loop includes, with the second hairpin structure arranged in the loop of the first hairpin structure, and wherein the sequence motif SEQ ID NO: 1 is the second hairpin structure includes.

In further particularly preferred embodiments exhibit according to the invention binding to activated protein C. Aptamers of the sequences selected from the group comprising SEQ ID NOs: 58, 88 to 92 a strain of the first hairpin structure on 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 trunk the first hairpin structure, which is a single-stranded Area, forming a so-called bulge. In very special, too preferred embodiments according to the invention activated protein C-binding aptamers having sequences selected from the group comprising SEQ ID NOs: 58, 88 to 92 a loop of the first hairpin structure with a number Nucleotides in the range of ≥6 nucleotides to ≤18 Nucleotides on.

und/oder deren Varianten, Mutanten und/oder Teile, wobei das an aktiviertes Protein C bindende Aptamer Modifikationen aufweisen kann, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder das Aptamer modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen kann.In particularly preferred embodiments, the activated protein C binding aptamer a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein 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.

In other embodiments, the activated protein C-binding aptamer comprises a sequence selected from the group comprising:

Surprisingly, it was found that in particular 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 ranging from ≥ 0.1 nM to ≤ 1.3 nM.

Locked nucleic acids (LNA, Locked Nucleic Acid) correspond to a conformationally fixed one Analog of DNA. The oligonucleotides of the locked nucleic acids contain one or more bicyclic ribonucleosides in which the 2'OH group with the C4 carbon atom via a methylene group connected is. Advantageously, Locked nucleic acids compared to unmodified DNA compared to a higher stability Nucleases and better hybridization properties, creating a Improvement of affinity and / or specificity the binding of the aptamer can be achieved.

A Another 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'-end. The modification of the 3'-end or 5'-end The aptamer can advantageously prevent rapid degradation by nucleases, in particular in an organism or a biological Protect the sample.

In other embodiments, in particular, the 5'-end of the aptamer may be pegylated. For example, 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. Preference is given to using linear polyethylene glycol (PEG) units HO- (CH ₂ CH ₂ O) _n -H, where n is an integer in the range from 1 to 900, preferably in the range from 1 to 450.

From Advantage of a chemical modification, a change the phosphoric sugar backbone or a use of enzymatically unrecognizable altered nucleotide units is that the nucleic acids used against an enzymatic Degradation especially in an organism or a biological sample are stabilized.

In another embodiment of the present invention can the aptamers of the invention as phosphorothioate DNA or peptide nucleic acid (PNA) are present. Through these modifications, the aptamers can counteract nucleic acid-cleaving Enzymes are stabilized. The stabilization touched the affinity of the modified DNA aptamers can not but the decomposition of aptamers in an organism or a biological Delay sample by degrading enzymes such as nucleases or DNases, reduce or even prevent.

Preferably have the nucleotides of the sequence motifs (SEQ ID NO: 1), (SEQ ID NO: 2) and / or (SEQ ID NO: 3) have no modifications. Prefers nucleotides are modified in the stem and / or loop of the aptamers.

One Another object relates to a method for the determination of activated Protein C in biological samples, wherein at least one inventive activated protein C binding aptamer with the biological Sample, preferably a biological fluid, incubated and a binding event between the aptamer and activated Protein C is detected.

In In preferred embodiments, the method is a method for the quantitative determination of activated protein C in biological Rehearse. In particularly preferred embodiments the method for the determination of activated protein C in biological Samples a diagnostic test procedure. The invention Activated protein C binding aptamers are particularly useful in advantageous for clinical analysis.

One particular advantage of the invention activated on Protein C-binding aptamer is thereby made available provided that this activated protein C with high affinity and high selectivity. Especially can the aptamers of the invention specifically to activated protein C over the inactive one Zymogen protein C bind. This allows the inventive Activated protein C-binding aptamers for the determination of activated Protein C in biological samples, especially in blood and others To use body fluids. Especially are the activated protein of the invention C binding aptamers in a routine suitable test method usable.

A "biological sample" in the sense of the invention is a provided or biological material obtained by sampling. biological Specimens are in particular biological materials derived from human Individuals in particular patients are isolated, for example biological tissues and fluids. Preferred biological Samples are biological fluids, especially 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 be purified.

The Sample is at least one inventive incubated with activated protein C binding aptamer. Under one Incubation in the sense of the invention is a contacting of the sample, in particular the compounds, substances and proteins contained therein, to understand with an aptamer.

For example, at least one inventive aptamer binding to activated protein C may be used the biological sample, preferably a biological fluid, are added. Furthermore, the sample may be incubated with previously immobilized aptamer, for example by adding the sample to an aptamer-coated well of an ELISA plate.

reaches an aptamer binds to its target protein-activated protein C. it to this. The binding event between aptamer and activated Protein C is detected according to the invention. The Binding event can be electrochemical, optical, microgravimetric, calorimetrically or piezoelectrically detected.

The Binding event can also be detected without labeling, for example, the aptamer is fixed on a surface and the change in layer thickness activated after annealing Protein C is determined, for example optically via a change of the evanescent field.

Preferably becomes a bond formation between aptamer and activated protein C in an indicator reaction after a direct or indirect Coupling of a binding partner with a well-detectable labeling substance demonstrated. Preference is given to this activated Labeling protein C-binding aptamer.

The label can be detected by luminescence, color reactions, enzymatic, electrochemical or radioactive. Preferably, 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. Also suitable for labeling double-stranded DNA or RNA are intercalating dyes, such as phenantrins, for example, propidium iodide.

One Detection of aptamer-linked activated protein C can also be over detects the amidolytic activity of the activated protein C. become. Chromogenic or fluorogenic peptide substrates can be used for this purpose which are cleaved by activated protein C.

Of Further, the detection of aptamer-bound activated protein C by antibodies or labeled antibodies, which are able to bind to activated protein C. In particularly preferred Embodiments may include the detection of activated protein C by adding the sample to immobilized aptamers and then Detection of bound activated protein C by its amidolytic Activity or via appropriate antibodies respectively.

The Evaluation can 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. A preferred bioluminescence involves the emission of visible light as a result of an enzyme catalyzed redox reaction. Furthermore, the detection with enzymes as Labeling substances are added to the substrates implement colored products, preferably peroxidase, luciferase, beta-galactosidase or alkaline phosphatase. Furthermore, the Detection radioactively via radioactive labeling Isotopes take place. A proof can continue by means of a so-called ELISA (enzyme-linked immunosorbent assays). The aforementioned Methods preferably include intensive washing steps to unbound and / or nonspecifically bound aptamers and / or detection reagents separate.

In other embodiments of the method according to the invention, the aptamer binding to activated protein C can be immobilized on a solid phase, preferably via 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.

The The invention further relates to the use of the invention activated protein C binding aptamers for the purification of activated protein C. The aptamers that bind to activated protein C are for example for affinity purification and / or affinity chromatography suitable.

The The invention further relates to a kit comprising at least one according to the invention binding to activated protein C. Aptamer. In a preferred embodiment of the kit can this contain signaling substances or devices, preferably Fluorescent dyes, chemiluminescent cofactors, enzyme substrates or labeled antibodies.

A Use of the invention activated at Protein C-binding aptamers allows in advantageous In particular, diagnostic method for determining the concentration on activated protein C in hemostasis diagnostics and advanced sepsis diagnostics. Furthermore possible a use of the inventive activated Protein C-binding aptamers in diagnostics make a determination of a disturbed Concentration of activated protein C and thus a diagnosis of disorders associated with a system disorder protein C / activated protein C go along.

One Another object of the invention relates to the use of the invention to activated protein C binding aptamers for the diagnosis, therapeutic and / or prophylactic treatment of disorders associated with a 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, due to an increased concentration of activated protein C are triggered.

The The invention further relates to the use of an inventive activated protein C binding aptamers, its pharmacologically acceptable salts, derivatives and / or conjugates, for the preparation of a medicine.

The according to the invention binding to activated protein C. Aptamers are suitable with high affinity for activated Bind protein C and modulate its activity, in particular to inhibit. In particular, the invention Activated protein C-binding aptamers of the sequences SEQ ID NOs: 58, 88 to 92, suitable, the anticoagulant enzyme activity of activated protein C.

About that In addition, it was found that in particular the invention Activated protein C-binding aptamers of the sequences SEQ ID NOs: 58, 88 to 92 are suitable, an allosteric conformational change in the molecule of the activated protein C to induce. Without being committed to a particular theory is believed that this allosteric conformational change is the inactivation of activated protein C by the physiological inhibitor protein C-inhibitor can amplify. Furthermore, without on one certain theory is supposed to be set by that the aptamers according to the invention induced allosteric Conformational change of activated protein C to a Formation of complexes of activated protein C with protein C inhibitor leads.

A Administration of the inventive activated Protein C-binding aptamers, their pharmacologically acceptable Salts, derivatives and / or conjugates allows in advantageous Way of therapeutically influencing the activity of activated protein C.

The The invention relates in particular to the use of an inventive activated protein C binding aptamers, its pharmacologically acceptable salts, derivatives and / or conjugates, for the preparation a drug for diagnosis, therapeutic and / or prophylactic Treatment of disorders associated with impaired blood clotting related, bleeding disorders, preferably hemorrhagic Diathesis, preferably selected from the group comprising Hemophilia, especially hemophilia A, and / or von Willebrand syndrome, and / or bleeding complications caused by triggered an increased concentration of activated protein C. become.

Bleeding complications, due to an increased concentration of activated protein C are triggered, in particular, bleeding, as Result of an overdose with activated protein C occur or bleeding resulting from an increased formation of activated Protein C as a result of secondary thrombinemia occur.

recombinantly Activated protein C produced, for example, in the treatment used the severe sepsis. In particular, inventive suitable for activated protein C aptamers, as an antidote for recombinated activated protein C in the case of absolute or relative overdose and thereby triggered Bleeding, to work. Furthermore, inventive particularly suitable for activated protein C-binding aptamers, as a surrogate in the substitution with factor VIII concentrate in patients with hemophilia A. In particular, by the inventive activated protein C-binding aptamers the half-life and / or Effectiveness of the factor VIII concentrate can be improved. Farther are activated protein C according to the invention binding aptamers especially for the treatment of acquired haemorrhagic Diathesis caused by an excessive education of activated protein C as a result of generalized thrombin formation can arise, suitable. Such a constellation For example, in patients after cardiac surgery. available.

Especially are activated protein C according to the invention binding aptamers for diagnostic and / or therapeutic treatment suitable for patients with concentrates of activated protein C, or by the preponderance of the anticoagulant acting activated protein C have a bleeding tendency. Furthermore, the invention are activated at Protein C-binding aptamers suitable for the treatment of patients with a congenital deficiency of blood coagulation factor VIII, where by administering the inventive activated Protein C-binding aptamers the half-life and effectiveness can be improved by administered factor VIII concentrate.

One Another object of the invention are pharmaceutical compositions comprising the invention activated protein C-binding aptamers. The medicine includes Inventive activated protein C binding aptamers 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. Medicines can continue pharmaceutically acceptable excipients and / or carriers include.

In Advantageously, the drug for prophylactic or therapeutic treatment of disorders associated with a disturbed Blood clotting, bleeding disorders, preferably hemorrhagic diatheses are preferably selected from the group comprising hemophilia, in particular hemophilia A, and / or von Willebrand syndrome, and / or bleeding complications, due to an increased concentration of activated protein C are triggered, suitable.

The Medicines are oral, transmucosal, rectal, pulmonary, enteral and / or parenterally administrable. Preference is given to administration by Injection.

One Another object of the invention relates to a thrombin-binding A fusion aptamer comprising at least two thrombin-binding Aptamers and at least one linker, wherein the at least one linker which connects at least two thrombin-binding aptamers.

Under The term "fusion aptamer" in the context of this invention are aptamer constructs to understand the at least two same or different Aptamersequenzen and at least one connecting the Aptamersequenzen Have left or spacer region.

surprisingly Way could be found that the inventive Thrombin-binding fusion aptamers an unexpectedly strong inhibition of thrombin. In particular, it was found be that thrombin-binding the invention Fusion aptamers an unexpectedly high inhibitory effect on the have thrombin-mediated blood coagulation.

For example could thrombin-inhibiting effect of the invention Thrombin-binding fusion aptamers are found to be one Blood coagulation when using inventive Thrombin-binding fusion aptamers already at a 30-fold lower level Concentrations against individual aptamers are inhibited could.

invention Thrombin-binding fusion aptamers can be a DNA or have an RNA sequence. Preferably, thrombin-binding Fusion aptamers to a DNA sequence. Own aptamers based on DNA advantageously an increased stability.

Also suitable are aptamers which have a sequence comprising 2'-modified nucleotides, for example 2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides. Also suitable are aptamers which are 2'-modified ribonucleotides, for example 2'-fluoro, 2'-methoxy and / or 2'-amino-modified Ribonucleotides. Furthermore, aptamers may have deoxyribonucleotides and 2'-modified ribonucleotides, for example 2'-fluoro, 2'-methoxy and / or 2'-amino-modified ribonucleotides.

• – zwei bis vier an Thrombin bindende Aptamere; und
• – ein bis drei Linker, wobei die Linker jeweils zwei an Thrombin bindende Aptamere verbinden.

In preferred embodiments of the thrombin-binding fusion aptamer, the fusion aptamer comprises:

• - two to four thrombin-binding aptamers; and
• One to three linkers, each linker linking two thrombin-binding aptamers.

worin
N₃, N₄, N₅, N₆, N₇, N₈ sind gleich oder unabhängig voneinander ausgewählt aus der Gruppe umfassend Guanin, Cytosin, Adenin und/oder Thymin, oder eine Deletion,
und/oder

und/oder deren Varianten, Mutanten und/oder Teile. Hierbei können die an Thrombin bindenden Aptamere Modifikationen aufweisen, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder das Aptamer kann modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide, aufweisen.Preferably, the thrombin-binding aptamers have a sequence selected from the group comprising:

wherein
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,
and or

and / or their variants, mutants and / or parts. In this case, 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 be modified nucleotides, preferably selected from the group comprising locked nucleic acids, 2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides.

Prefers usable thrombin-binding aptamers bind to the exosites 1 and 2 of thrombin.

Other useful aptamers which bind to thrombin are disclosed, for example, in the document WO 03/002592 , to which reference is made in full.

In preferred embodiments of the thrombin-binding fusion aptamer, 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.

Preferably The nucleotide linker has a length in the range from ≥ 1 nucleotide to ≤ 30 nucleotides, preferred in the range of ≥ 2 nucleotides to ≤ 15 nucleotides, more preferably in the range of ≥ 5 nucleotides to ≦ 15 Nucleotides, more preferably in the range of ≥ 10 nucleotides to ≤ 15 nucleotides, with the nucleotides selected are from the group comprising guanosine, cytidine, adenosine and / or Thymidine. A preferred nucleotide is adenosine. Especially preferred Linkers are so-called poly A linkers.

It could surprisingly be found that the inventive Thrombin-binding fusion aptamers have a particularly good affinity may have thrombin, the linker predominantly contain the nucleotide adenosine or from the nucleotide adenosine are formed.

Preferably For example, a polyethylene glycol linker has 1 to 15 polyethylene glycol units, preferably in the range of 1 to 10 polyethylene glycol units, preferably ranging from 2 to 8 polyethylene glycol units.

Preference is given to using linear polyethylene glycol (PEG) units HO- (CH ₂ CH ₂ O) _n -H, where 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.

In preferred embodiments of the thrombin-binding fusion aptamer Linker is a peptidic linker, where the peptide linker preferably amino acids selected from the group comprising glycine, alanine and / or β-alanine. Preferably includes the peptidic linker in the range of 2 to 4 amino acids. Preferably, the peptidic linker comprises β-alanine units. Preferably, the peptidic linker comprises in the range of 2 to 4 β-alanine units.

In further preferred embodiments of the thrombin-binding fusion aptamer, the linker is an unbranched or branched, saturated or unsaturated C ₁ -C ₄₀ -alkyl group, preferably a C ₁ -C ₃₀ -alkyl group, more preferably a C ₂ -C ₂₀ -alkyl group , Preferably, the linker is an alkl group - (CH ₂ ) _n - wherein n is an integer in the range of 1 to 40, preferably in the range of 1 to 30, also preferably in the range of 2 to 20, more preferably n is in the range of 2 to 18, very particularly preferably n = 2, 6, 12 or 18.

und/oder deren Varianten, Mutanten und/oder Teile, wobei die an Thrombin bindenden Aptamere Modifikationen aufweisen können, vorzugsweise ausgewählt aus der Gruppe umfassend 5'-PEG- und/oder 3'-CAP-Modifikationen, und/oder die Aptamere modifizierte Nukleotide, vorzugsweise ausgewählt aus der Gruppe umfassend Locked-Nukleinsäuren, 2'-Fluoro-, 2'-Methoxy- und/oder 2'-Amino-modifizierte Nukleotide aufweisen können.In particularly preferred embodiments of the thrombin-binding fusion aptamer, the thrombin-binding fusion aptamer comprises a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein the thrombin-binding aptamers may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamers modified nucleotides , preferably selected from the group comprising Locked nucleic acids, 2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides.

A Another 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'-end. The modification of the 3'-end or 5'-end The fusion aptamer may advantageously be faster than one Degradation by nucleases, in particular in an organism or a protect biological sample.

In other embodiments, in particular, the 5 'end of the fusion aptamer may be pegylated. For example, 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. Preference is given to using linear polyethylene glycol (PEG) units HO- (CH ₂ CH ₂ O) _n -H, where n is an integer in the range from 1 to 900, preferably in the range from 1 to 450.

A Administration of the thrombin-binding according to the invention Fusion aptamers, their pharmacologically acceptable salts, derivatives and / or conjugates advantageously allows one therapeutically influencing the activity of thrombin.

One Another object of the invention relates to the use of the invention Thrombin-binding fusion aptamers for diagnosis, therapeutic and / or prophylactic treatment of disorders associated with a disturbed blood coagulation or related Therapy requires an influence on the blood coagulation system.

Especially are the thrombin-binding according to the invention Fusion aptamers useful as anticoagulants or anticoagulants.

Surprised could be found that the invention Thrombin-binding fusion aptamers are very effective anticoagulants are. In particular, it was found that the invention Thrombin-binding fusion aptamer SEQ ID NO: 98 a significantly higher Have an effect on thrombin-mediated blood coagulation, as a combination of the single aptamers SEQ ID NO: 95 and SEQ ID NO: 97.

The The invention further relates to the use of an inventive Thrombin-binding fusion aptamer, its pharmacologically acceptable Salts, derivatives and / or conjugates for the manufacture of a medicament.

The The invention particularly relates to the use of the invention Thrombin-binding fusion aptamers, their pharmacologically acceptable Salts, derivatives and / or conjugates for the manufacture of a medicament for diagnosis, therapeutic and / or prophylactic treatment of disorders with a disturbed blood clotting or their therapy influence the Blood coagulation system requires, especially as anticoagulant.

diseases which are associated with a disturbed blood clotting or their therapy influencing the blood clotting system in particular are selected from the group comprising arterial or venous thrombosis, especially acute Myocardial and cerebral infarction or pulmonary embolism, and / or thromboembolic Complications.

invention Thrombin-binding fusion aptamers are particularly suitable for Treatment of arterial thrombosis, especially acute myocardial and cerebral infarction and secondary prophylaxis of thromboembolic Complications after vascular reconstruction procedures. Thrombin-binding fusion aptamers according to the invention are also suitable for systemic anticoagulation for use in extracorporeal therapy, in particular cardiopulmonary bypass, Hemodialysis and hemofiltration. invention Thrombin-binding fusion aptamers are also suitable for use in the acute therapy of venous thrombosis, in particular acute pulmonary embolism.

One Another object of the invention are medicaments comprising at least a 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 drug may continue to be pharmaceutical compatible excipients and / or carriers.

In Advantageously, the drug for prophylactic or therapeutic treatment of disorders associated with a disturbed Blood clotting or their therapy influence of the blood coagulation system, in particular as a anticoagulant, suitable.

The Medicines are oral, transmucosal, rectal, pulmonary, enteral and / or parenterally administrable. Preference is given to administration by Injection.

If not otherwise stated, have the technical used and scientific terms the meaning of how commonly owned by one of ordinary skill in the art to which this invention belongs is understood.

All Publications, patent applications, patents and others References herein are fully incorporated by reference added.

Examples and Figures illustrating the present invention serve are given below.

In the figures shows:

1 Sequences of DNA aptamers that bind to activated protein C. The sequence of 1a corresponds to the sequence SEQ ID NO: 58, the sequence of 1b corresponds to the sequence SEQ ID NO: 88. The sequences each have a consensus SEQ ID NO. 1 on.

2 Sequences of DNA aptamers that bind to activated protein C. The sequence of 2a corresponds to the sequence SEQ ID NO: 89, the sequence of 2 B corresponds to the sequence SEQ ID NO: 90, the sequence of 2c corresponds to the sequence SEQ ID NO: 91, and the sequence of 2d corresponds to the sequence SEQ ID NO: 92. The sequences each have a consensus SEQ ID NO. 1 on.

The calculation of the secondary structures was based on the folding program mfold available on the internet, M. Zuker, Mfold web server for nucleic acid folding and hybridization prediction, Nucleic Acid Res. 31 (13), 3406-15 (2003) ,

example 1

In vitro selection (SELEX ^® / Counter-SELEX ^® )

For the selection of DNA aptamers to activated protein C (APC) the drotrecogin alpha (Xigris ^®) used as a medicament in severe sepsis was used as APC source. For the immobilization of the target proteins necessary for the selection, 100 μg of the APC were biotinylated and coupled to streptavidin-coated magnetic beads (Dynabeads M-280 streptavidin [Dynal, Invitrogen]). For 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 (1 × PBS, pH 7.4) in a total volume of 100 μl for 30 min on ice and then for 10 min at room temperature (RT). Unbound NHS-biotin was subsequently removed by column chromatography (Micro Bio-Spin 6 Chromatography columns, Biorad, Munich, Germany). The biotinylated APC thus obtained was incubated with 5 mg of the magnetic beads in a total volume of 600 μl PBS (0.1% BSA) for 30 min at RT under constant agitation. Subsequently, the beads were washed 1 × with 500 μl PBS (0.1% BSA) and finally taken up in 1 ml PBS (0.1% BSA). The DNA aptamer start library used was the so-called D1 pool with a 49-base-long variable sequence region flanked by two primer binding sites (5'-GCCTGTTGTGAGCCTCCTAAC-N ₄₉ -CATGCTTATTCTTGTCTCCC-3 'SEQ ID NO: 101). This was synthesized by the company Metabion (Martinsried, Germany) and obtained by HPLC purification.

Initially, 500 pmol of the D1 pool was incubated with 80 μl (8 μg) of immobilized APC in a final final volume of 160 μl in selection buffer (1X PBS, pH 7.4, 0.1% BSA, 1mM MgCl ₂ , 1mM CaCl ₂ ) incubated. After incubation, the beads were washed 1 × with 150 μl of selection buffer and bound DNA was eluted at 80 ° C. in 55 μl of aqua. The pool selected in this way was subsequently separated by means of polymerase chain reaction (PCR) into 5 separate 100 μl reactions (90 μl master mix + 10 μl sample each) using the primers (HPLC-pure, Metabion) 5'-GCCTGTTGTGAGCCTCCTAAC-3 '(forward, SEQ ID NO: 86) and 5'-GGGAGACAAGAATAAGCATG-3 '(reverse, SEQ ID NO: 102), amplified with 5'-biotin modification), followed by single-strand separation. For this purpose, the PCR products were bound to the already described magnetic beads via the biotin molecules coupled to the 5 'end of the reverse primer. Here, 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 μl 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 μl), these mixtures were incubated for 45 minutes at RT under constant agitation. After incubation, the beads were washed 2X each with BW buffer and 1X with 2X BW buffer (1000μl each) to remove unbound components of the PCR reaction mixes. Subsequently, the beads were taken up in 50 μl 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 μl of NaOH. To neutralize the pH, a pre-optimized amount of a 0.1 molar HCl solution was pipetted to the aptamer solution.

To Carrying out the single-strand separation described above 50 pmol of the aptamer thus obtained was added to the next Selection cycle used.

To increase the specificity, a counter-selex was carried out against the streptavidin beads used from the third round and the number of washing steps after binding of the aptamers to the immobilized APC was increased stepwise. Because of the selection-related enrichment of APC-binding aptamer sequences, the number of PCR cycles performed for amplification was readjusted after each selection cycle. In total, 12 selection cycles were performed. An overview of the parameters varied during the selection can be found in Table 1. Table 1. Overview of the selected selection parameters round Use ssDNA Counter-SELEX washes PCR cycles [#] [Pmol] [Yes No] [Number] [Number] 1 500 No 1 18 2 50 No 2 18 3 50 Yes 4 18 4 50 Yes 8th 18 5 50 Yes 8th 12 6 50 Yes 8th 9 7 50 Yes 8th 12 8th 50 Yes 8th 12 9 50 Yes 8th 11 10 50 Yes 8th 10 11 50 Yes 8th 9 12 50 Yes 8th 10

Example 2

Cloning and sequencing

To analyze the sequences of the selected aptamers, 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 .mu.l total reaction volume with the use of 0.75 pmol single-stranded DNA) and the PCR products thus produced by TA cloning into pCR II vector (TA Cloning ^® Kit, Invitrogen). Subsequently, chemically competent E. coli cells (XL-10 Gold, Stratagene) were transformed with the vectors and plated on LB agar with 100 μg / ml ampicillin. Of the colony-forming units (CFU) produced overnight at 37 ° C., a selection in 5 ml suspension cultures (LB containing 100 μg / ml ampicillin) was further propagated with constant movement overnight at 37 ° C. and the vectors after centrifugation of the Purify cells at 5,000 rpm for 15 min using the QIAprep Spin Mini Kit (Qiagen). Sequencing of the vector inserts (aptamer sequences) was performed using M13 sequencing primers using an ABI 3130x1 Genetic Analyzer.

Example 3

Determination of the dissociation constant K _{D of} individual activated protein C binding aptamers (filter binding tests)

To test the APC binding properties, the aptamers to be tested were labeled with ³² p-ATP at the 5 'end using a polynucleotide kinase (PNK). For this purpose, 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 min at 37.degree. To remove unbound ³² P-ATP, the batches were then treated with 40 ul of Aqua and purified on G25 columns (Microspin G-25 Colums, Amersham).

The thus radiolabelled aptamers were incubated at a final concentration of 0.4 nM in selection buffer with various concentrations of APC starting at 100 nM and subsequent half-log dilution steps in a total volume of 25 μl for 30 minutes at 37 ° C , After the incubation, in each case 20 μl of the mixtures were filtered by means of a vacuum system over a nitrocellulose membrane (Schleicher and Schuell, 0.45 μM). Subsequently, several washes (4 x 200 μl) with wash buffer (1 x PBS, pH 7.4, 1 mM MgCl ₂ , 1 mM CaCl ₂ ) to remove excess non-protein bound radioactivity from the membrane. The recording of the respective radiation intensities took place by means of a phosphor screen. To evaluate the data, a FUJIFILM FLA-3000 with corresponding AIDA Imagequant software was used. By non-linear regression of the obtained data points, the bound radioactivity on APC concentration, the individual Kps could be determined. For calculation wur en uses the Logistic Dose Response in Pharmacology / Chemistry feature of Origin 7.5 software (OriginLab, Northampton, MA, USA).

With regard to the binding of individual aptamers to the used for selection Xigris ^® following K _D s could be determined using the procedure described above: Test series 1: screening of individual aptamer from the obtained after 10 rounds of selection aptamer pool aptamer SEQ ID NO: HS02 (SEQ ID NO: 58): 0.12 ± 0.02 nM HS03 (SEQ ID NO: 70): 0.19 ± 0.03 nM HS04 (SEQ ID NO: 49): 0.21 ± 0.02 nM HS08 (SEQ ID NO: 83): 0.30 ± 0.09 nM Test Series 2: Testing of aptamer variants based on (SEQ ID NO: 58) aptamer SEQ ID NO: HS02-88 (SEQ ID NO: 58): 0.43 ± 0.08 nM HS02-62 (SEQ ID NO: 88): 0.56 ± 0.13 nM HS02-52 (SEQ ID NO: 89): 0.35 ± 0.09 nM HS02-52G (SEQ ID NO: 90): 0.47 ± 0.11 nM HS02-48G (SEQ ID NO: 91): 0.40 ± 0.13 nM HS02-44G (SEQ ID NO: 92): 0.17 ± 0.06 nM

It showed that the activated protein C binding aptamers very good dissociation constants for binding to activated Had protein C.

Example 4

Determination of specificity of individual activated protein C-binding aptamers

The determination of the APC specificity of the variants SEQ ID NO: 58 and 88 to 92 was carried out by incubation of different concentrations of native APC (plasma purified PC [Ceprotin ^® , Baxter], which with human alpha-thrombin [Cellsystems, St. Katharinen , Germany] was activated), PC (Ceprotin ^®), alpha-thrombin (Cell Systems), factor VIIa (NovoSeven ^®, Novo Nordisk), factor Xa (Cell Systems) and FIXa (Cell Systems) with radioactively-labeled aptamers of the sequences SEQ ID NO : 58, 88 to 92 and subsequent filter binding experiment as described in Example 3. Here, the native APC was used in a semilogarithmic dilution series starting from 100 nM and the zymogen PC also in a semilogarithmic dilution series starting from 1000 nM. Alpha-thrombin, Factor VIIa, Factor Xa and FIXa were each tested at concentrations of 320 nM, 32 nM and 3.2 nM.

Up to a concentration of 320 nM, no binding of the aptamers was observed with respect to alpha-thrombin, factor VIIa, factor Xa and FIXa. Although binding of all aptamers to PC was observed, the K _D s were not quantifiable, but were> 320 nM in all cases. Studies using a fluorogenic peptide substrate suggest that at least part of the observed binding of aptamers to a contamination of the PC used (Ceprotin ^®) is due to APC.

58 and 88 to 92 also effective binding to native APC (activated Ceprotin ^®) be confirmed: In addition to binding to Xigris ^® was for the high-affinity binding HS02 variants SEQ ID NO. The determined K _D s were included in this case: aptamer SEQ ID NO: HS02-88 (SEQ ID NO: 58): 0.47 ± 0.06 nM HS02-62 (SEQ ID NO: 88): 1.06 ± 0.23 nM HS02-52 (SEQ ID NO: 89): 0.97 ± 0.18 nM HS02-52G (SEQ ID NO: 90): 0.74 ± 0.26 nM HS02-48G (SEQ ID NO: 91): 0.85 ± 0.06 nM HS02-44G: (SEQ ID NO: 92): 0.76 ± 0.10 nM

It showed that the activated protein C binding aptamers very good dissociation constants for binding to native had activated protein C.

Example 5

Attempts to inhibit the APC-mediated Factor Va inactivation

For checking inhibition of anticoagulant APC activity the various HS02 variants SEQ ID NO: 58 and 88 to 92 were each in ascending concentrations in a purified System for determining APC-mediated inhibition of FVα cofactor activity used.

Here, (Xigris ^®) was 625 pM FVa (Cell Systems) at 18.3 pM APC in assay buffer (20 mM Tris-HCl, pH 7.4, 0.1% BSA, 137 mM NaCl, 5 mM CaCl2, 10 ug / ml phospholipids) in the presence of different aptamer concentrations (semilogarithmic dilution series starting from 180 nM) in a total volume of 50 .mu.l incubated for 20 min at RT.

The Determination of remaining FVa cofactor activity was with the aid of a plate fluorometer (Ascent Fluoroscan, Thermo Fisher Scientific). Here were 75 μl each of a 42 pM human FXa solution (Cellsystems) and 667 μM of a fluorogenic peptide sub-start (Z-Gly-Gly-Arg-AMC, Bachem, Weil am Rhein, Germany) into the pits of white C8 Fluoronunc modules (Nunc, Thermo Fisher Scientific) and 25 μl of the test mixture described above. Immediately before the beginning of the kinetic measurement of the substrate turnover (one measurement per minute for 20 minutes) the addition was made of 50 μl of a 25 nM prothrombin solution in assay buffer (Cell Systems).

By nonlinear regression of the data points obtained (cofactor residual activity via aptamer concentration), the following IC ₅₀ values were obtained: aptamer SEQ ID NO: HS02-88 (SEQ ID NO: 58): 0.58 ± 0.04 nM HS02-62 (SEQ ID NO: 88): 1.20 ± 0.09 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 HS02-44G (SEQ ID NO: 92): 0.28 ± 0.11 nM

In the experiments described above it was possible to show that the investigated aptamers were able to inhibit the functional activity of APC with respect to the inactivation of FVa with low IC ₅₀ values.

Example 6

Attempts to inhibit the APC-mediated Factor VIIIa inactivation

To further test the inhibition of anticoagulant APC activity by the various HS02 variants SEQ ID NO: 58 and 88 to 92, these were also each used in ascending concentrations in a purified system for the determination of APC-mediated inhibition of FVIIIa cofactor activity. For this purpose, 1 U recombinant FVIII (Recombinate ^®, Baxter) by incubation with 0.025 NIH units of human alpha-thrombin (Cell Systems) for 2 (0.1% BSA) activated min in a total volume of 100 ul in PBS buffer. To stop the activation reaction the batch 5 ul of a 1.4 nM hirudin (Refludan ^®, Bayer Healthcare) was added. Subsequently, the so-activated FVIII (FVIIIa) was in a final concentration of 0.16 U / ml with 1.8 nM APC (Xigris ^® ) in assay buffer in the presence of different aptamer concentrations (semilogarithmic dilution series starting from 180 nM) in one Total volume of 50 .mu.l incubated for 20 min at RT. Determination of residual FVIIIa cofactor activity was also performed using a plate fluorometer (FLx-800, Bio-Tek). In each case 75 μl of a solution of 3 nM human FIXa (Cellsystems) and 333 μM of a fluorogenic peptide sub-start (Boc-Ile-Glu-Gly-Arg-AMC, Bachem) were introduced into the wells of black F16 Fluoronunc modules (Nunc, Thermo Fisher Scientific) and added 25 .mu.l of the test mixture described above. Immediately prior to initiating the kinetic measurement of substrate turnover (one measurement per minute for 20 minutes), 50 μl of a 25 nM FXa solution was added to assay buffer (Cellsystems).

By nonlinear regression of the data points obtained (cofactor residual activity via aptamer concentration), the following IC ₅₀ values were obtained: aptamer SEQ ID NO: HS02-88 (SEQ ID NO: 58): 1.27 ± 0.41 nM HS02-62 (SEQ ID NO: 88): 0.88 ± 0.46 nM HS02-52 (SEQ ID NO: 89): 2.46 ± 0.08 nM HS02-52G (SEQ ID NO: 90): 0.22 ± 0.15 nM HS02-48G (SEQ ID NO: 91): 1.06 ± 0.43 nM HS02-44G (SEQ ID NO: 92): 0.88 ± 0.11 nM

In the experiments described above it was possible to show that the investigated aptamers are able to inhibit the functional activity of APC with respect to the inactivation of FVIIIa with low IC ₅₀ values.

Example 7

Experiments to determine the influence the APC aptamer on the inhibition of APC by the protein C inhibitor (PCI)

To test the influence of APC aptamers on the kinetics of inhibition of APC by PCI, a test system in microtiter plate format was used. The wells of white C8 Fluoronunc modules (Nunc, Thermo Fisher Scientific) were incubated with a polyclonal rabbit anti human PC antibody (Dako) in coating buffer (30 mM Na ₂ CO ₂ , 200 mM NaHCO ₃ , pH 9.0) at 4 ° C overnight (100 μl / well). Subsequently, the wells were washed 3x with wash buffer (1 x PBS, pH 7.4, 0.05% Tween 20) (this is the standard washing sequence used in this test procedure, performed with an automatic microtiter plate washer [SLT, Tecan, Germany]) and free Binding sites blocked with blocking buffer (1 × PBS, pH 7.4, 2% BSA, 0.05% Tween 20) for 2 h at RT. After washing the wells, the addition was made of 100 ul / well of a 360 pM APC (Xigris ^®) solution in dilution buffer (1 x PBS, pH 7.4, 0.1% BSA, 0.05% Tween 20) for 1 h at RT was incubated and then removed by washing. The APC, which was now immobilized in the wells of the modules, was then followed by the addition of 100 μl / 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. In order to stop binding of the PCI contained in the pool plasma to the APC immobilized in the wells at defined times, the corresponding wells were washed by default after different incubation times (5, 10, 20 and 30 min) and filled with 100 μl / well of dilution buffer. After completion of the series of experiments, all wells were washed again. To determine the amidolytic APC activity remaining in the wells, a fluorogenic peptide substrate for APC (Pefa 3342, Pentapharm) in a concentration of 200 μM in dilution buffer was then added (100 μl / well) and the kinetics of the substrate hydrolysis using a plate fluorometer (Ascent Fluoroscan , Thermo Fisher Scientific). The apparent inhibition constants (kapp) as a measure of the rate of inhibition of the immobilized APC were calculated from the resulting data sets by exponential interpolation. To demonstrate the PCI specificity of the effect found, the wells were rinsed after inhibition kinetics were recorded and then 100 μl / well of a POD-labeled polyclonal anti-PCI antibody (Affinity Biologicals) in a 1: 2000 dilution in dilution buffer was added. After an incubation period of 1 h, the well was washed again and 100 μl / well of a chemiluminescent substrate (Roche) added. The intensity of the released chemiluminescence is determined after an incubation period of 3 min with the Ascent Fluoroscan.

The apparent inhibition constants (k _app ) of immobilized APC determined in the above-described immunoassay in the presence of the various aptamers of the sequences SEQ ID NO: 58 and 88 to 92 (100 nM) in the plasma matrix are shown below: aptamer SEQ ID NO: HS02-88 (SEQ ID NO: 58): 0.143 ± 0.012 HS02-62 (SEQ ID NO: 88): 0.095 ± 0.002 HS02-52 (SEQ ID NO: 89): 0.085 ± 0.006 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

In The experimental approaches could be the formation of APC / PCI complexes be proven as the cause of the inhibition of APC. Here, the determined number of complexes formed was proportional to the found inhibition constants.

Example 8

Experiments to anticoagulant by the thrombin-binding fusion aptamer of the sequence SEQ ID NO: 98

The Thrombin-inhibiting action of the thrombin-binding fusion aptamer of the sequence 5'-GGTTGGTGTGGTTGGAAAAAAAAAAAAAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3 ' (SEQ ID NO: 98) was in a single-step thrombin-based Coagulation test using Amelung Coagulometer AMAX CS 190 (Amelung, Lemgo) determined.

For this purpose, human α-thrombin (CellSystems, St. Katharinen) in PBS (pH 7.4, 3 mM MgCl ₂ , 0.1% BSA) to a concentration of 2.5 NIH units / ml (NIH = National Institute of Health , Defined biological units) diluted and mixed with different concentrations of the thrombin-binding fusion aptamer of the sequence SEQ ID NO: 98 and the aptamers of the sequences SEQ ID NO: 95 and SEQ ID NO: 97, starting a semilogarithmic dilution series starting from 1500 nM was used.

To incubation time of 10 minutes at 37 ° C 25 μl of the reaction mixture to 50 μl of human citrated pool plasma (own production) given and the time until the coagulation of the Assays determined.

It could be found to coagulate upon addition of thrombin-binding Fusion aptamers of the sequence SEQ ID NO: 98 already at a 30-fold lower concentrations compared to the aptamer of the sequence SEQ ID NO: 95 was inhibited.

It follows Sequence listing according to WIPO St. 25. This can of the official publication platform of the DPMA become.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• - US 6989241 [0006]
• US 5475096 [0015]
• US 5270163 [0015]
• - EP 0533838 [0015]
• WO 03/002592 [0123]

Cited non-patent literature

• M. Zuker, Mfold web server for nucleic acid folding and hybridization prediction, Nucleic Acid Res. 31 (13), 3406-15 (2003) [0151]

Claims (28)

Aptamer that binds to a target molecule involved in hemostasis, the aptamer being: - 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, said at least one linker containing at least connects two thrombin-binding aptamers. Aptamer which binds to activated protein C according to claim 1, characterized in that the activated protein C binding aptamer having 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 ≥ 0.01 nM to ≤ 2 nM, particularly 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 , binds to activated protein C. Aptamer, which binds to activated protein C, after Claim 1 or 2, characterized in that the activated Protein C-binding aptamer has a number of nucleotides in the range from ≥ 20 nucleotides to ≤ 120 nucleotides, preferably in the range of ≥ 40 nucleotides to ≤ 88 nucleotides, having. Aptamer which binds to activated protein C according to any one of the preceding claims, characterized in that the activated protein C binding aptamer comprises a sequence motif selected from the group comprising:

wherein: N ₁ 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;

wherein: 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, and /or

wherein the 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 the group comprising Locked nucleic acids, 2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides. Aptamer, which binds to activated protein C, after one of the preceding claims, characterized that the activated protein C binding aptamer at least one first hairpin structure comprising a trunk and a loop having. Aptamer, which binds to activated protein C, after one of the preceding claims, characterized the activated protein C-binding aptamer has a second hairpin structure comprising a trunk and a loop, wherein the second Hairpin structure in the loop of the first hairpin structure is arranged. Aptamer, which binds to activated protein C, after one of the preceding claims, characterized the sequence SEQ ID NO: 1 comprises the second hairpin structure. Aptamer, which binds to activated protein C, after one of the preceding claims, characterized that the stem of the first hairpin structure is a nucleotide sequence Cytidine-thymidine, which forms a bulge. Aptamer which binds to activated protein C according to any one of the preceding claims, characterized in that the strain of the first hairpin structure in the range of ≥ 4 base pairs to 5 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. Aptamer, which binds to activated protein C, after one of the preceding claims, characterized that the loop of the first hairpin structure is in the range of ≥ 5 Nucleotides up to ≤ 30 nucleotides, preferably in the range from ≥ 6 nucleotides to ≤ 18 nucleotides. Aptamer which binds to activated protein C according to any one of the preceding claims, characterized in that the activated protein C binding aptamer has a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein the activated protein C-binding aptamer may have modifications, preferably selected from the group comprising 5'-PEG and / or Y-CAP modifications, and / or modified the aptamer Nucleotides, preferably selected from the group comprising Locked nucleic acids, 2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides may have. Method for the determination of activated protein C in biological samples, characterized in that at least an activated protein C binding aptamer after one of the previous Claims with the biological sample, preferably one biological fluid, incubated and a binding event between the aptamer and activated protein C is detected. Method according to claim 12, characterized in that that the biological fluid is a body fluid is, preferably blood or blood products, preferably selected from the group comprising blood plasma, serum and / or whole blood. Kit comprising at least one activated protein C-binding aptamer according to one of the preceding claims. Use of an activated protein C binding Aptamers according to one of the preceding claims, the pharmacologically acceptable salts, derivatives and / or conjugates, for the preparation of a medicine. Use of an activated protein C binding Aptamers according to one of the preceding claims, the pharmacologically acceptable salts, derivatives and / or conjugates, for the preparation a drug for diagnosis, therapeutic and / or prophylactic Treatment of disorders associated with impaired blood clotting related, bleeding disorders, preferably hemorrhagic Diathesis preferably selected from the group comprising Hemophilia, especially hemophilia A, and / or von Willebrand syndrome, and / or bleeding complications caused by triggered an increased concentration of activated protein C. become. Medicaments comprising activated protein C binding aptamers according to one of the preceding claims. Thrombin-binding fusion aptamer according to claim 1, characterized in that the fusion aptamer comprises: - two to four thrombin-binding aptamers; and - one to three linkers, where the linkers each have two thrombin-binding Connect aptamers. Thrombin-binding fusion aptamer according to claim 1 or 18, characterized in that the thrombin-binding aptamers have a sequence selected from the group comprising:

wherein 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, and / or

and / or their variants, mutants and / or parts, wherein the thrombin-binding aptamers may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamers modified nucleotides , preferably selected from the group comprising Locked nucleic acids, 2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides. Thrombin-binding fusion aptamer according to one of the preceding claims, characterized in that the linker is selected from the group comprising nucleotidic linkers, polyethylene glycol (s), peptidic linkers and / or unbranched or branched, saturated or unsaturated C ₁ -C ₅₀ - alkyl. Thrombin-binding fusion aptamer after one of previous claims, characterized in that the nucleotidic linkers have a length in the range of ≥ 1 Nucleotides up to ≤ 30 nucleotides, preferably in the range from ≥ 2 nucleotides to ≤ 15 nucleotides, preferred in the range of ≥ 10 nucleotides to ≤ 15 nucleotides selected from the group comprising guanosine, cytidine, Adenosine and / or thymidine, preferably adenosine. Thrombin-binding fusion aptamer according to one of the preceding claims, characterized in that the linker linear polyethylene glycol (PEG) units HO- (CH ₂ CH ₂ O) _n -H, wherein n is an integer in the range of 1 to 15 , preferably in the range of 1 to 10, preferably in the range of 2 to 8, is. Thrombin-binding fusion aptamer after one of previous claims, characterized in that the peptidic linker amino acids selected from the group comprising glycine, alanine and / or β-alanine, Preferably, the peptidic linker comprises in the range of 2 to 4 amino acids. Thrombin-binding fusion aptamer according to one of the preceding claims, characterized in that the linker is a straight or branched, saturated or unsaturated C ₁ -C ₄₀ alkyl group, preferably C ₁ -C ₃₀ alkyl, more preferably C ₂ -C _20- alkyl. Thrombin-binding fusion aptamer according to one of the preceding claims, characterized in that the thrombin-binding fusion aptamers have a sequence selected from the group comprising:

and / or their variants, mutants and / or parts, wherein the thrombin-binding aptamers may have modifications, preferably selected from the group comprising 5'-PEG and / or 3'-CAP modifications, and / or the aptamers modified nucleotides , preferably selected from the group comprising Locked nucleic acids, 2'-fluoro, 2'-methoxy and / or 2'-amino-modified nucleotides. Use of thrombin-binding fusion aptamer according to one of the preceding claims, their pharmacological acceptable salts, derivatives and / or conjugates, for the preparation of a medicine. Use of thrombin-binding fusion aptamer according to one of the preceding claims, their pharmacological acceptable salts, derivatives and / or conjugates, for the preparation a drug for diagnosis, therapeutic and / or prophylactic Treatment of disorders associated with impaired blood clotting or their therapy influence the Blood coagulation system requires selected in particular from the group comprising arterial or venous thromboses, especially acute myocardial and cerebral infarcts or pulmonary embolisms and / or thromboembolic complications, especially as anticoagulants. Medicament comprising at least one thrombin-binding Fusion aptamer according to one of the preceding claims.