HK1168126B

HK1168126B - Hepcidin binding nucleic acids

Info

Publication number: HK1168126B
Application number: HK12108906.2A
Authority: HK
Inventors: S.塞尔; F．莫里希; C.马什; S．克卢斯曼; N．丁斯; K．布赫纳; F．施沃布尔
Original assignee: 诺松制药股份公司
Priority date: 2009-04-30
Filing date: 2010-04-30
Publication date: 2015-07-24

Description

Hepaciclidine-binding nucleic acids

The present invention relates to hepcidin-binding nucleic acids and their use for the preparation of medicaments (medicaments), diagnostic agents and detection agents, respectively.

The primary structure (HEPC-HUMAN, SwissProt entry P81172) of hepcidin (hepcidin) was determined in 2000 (Krause, 2000). Another team working on antimicrobial peptides found hepcidin independently (Park, 2001). The proteins are distinguished by "liver-expressed antimicrobial peptides" (abbreviation: LEAP-1) and "putative liver tumor suppressor" (abbreviation: PLTR). Hepcidin is a cysteine-rich cationic peptide and consists of 25 amino acids, resulting in a molecular weight of 2,790 daltons. The 8 cysteines form 4 disulfide bonds and give the molecule a stable and rigid structure.

The tertiary structure of hepcidin was determined by NMR analysis (Hunter, 2002). The protein consists of twisted β -pleated sheets in which unusual ortho-disulfide bridges are found at the turns of the hairpin (Hunter, 2002).

The amino acid sequences of hepcidin from different mammalian species are usually very conserved during evolution. Human hepcidin shares the following percentages of identical amino acids with hepcidin from the following species:

macaque (Macaca mulatta) (rhesus monkey) 100%

100% of Macaca fascicularis (Macaca fascicularis)

European wild boar (Sus scrofa) (pig) 84%

Mice (Mus musculus) 76%

68% of Rattus norvegicus (rat).

In addition to the biologically active hepcidin consisting of 25 amino acids (also known as hepcidin-25), two truncated inactive variants with 20 and 22 amino acids were identified: hepcidin-20 and hepcidin-22 (river, 2005). All these peptides were generated based on an 84 amino acid propeptide in humans and rats and an 83 amino acid propeptide in mice (Pigeon, 2001). The 84 amino acid hepcidin propeptide comprises a typical endoplasmic reticulum-targeting 24-amino acid signal peptide that is removed, and a common cleavage site for the prohormone converting enzyme furin (Valore, 2008). These processing steps produce the active 25 amino acid peptide hormone, which is found in blood and urine.

Hepcidin is a key signal for regulating iron homeostasis. High levels of human hepcidin lead to reduced serum iron levels, while low levels lead to increased serum iron levels, as shown in a mouse model of hepcidin deficiency and hepcidin overexpression (Nicolas, 2001; Nicolas, 2002; Nicolas, 2003). Furthermore, mutations in the hepcidin gene that lead to a lack of hepcidin activity are associated with juvenile hemochromatosis, a severe iron overload disease (Roetto, 2003). A dose-dependent and long-lasting reduction in serum iron was observed following intraperitoneal injection of hepcidin (river, 2005).

Iron is an essential element required for the growth and development of all living organisms. The iron content in mammals is regulated by controlling iron absorption, iron recycling and iron release from iron-storing cells. Iron is absorbed mainly by enterocytes in the duodenum and upper jejunum.

The feedback mechanism enhances iron absorption in individuals with iron deficiency and reduces iron absorption in individuals with iron overload. The key compound of this mechanism is the iron transporter, "ferroportin", which also acts as the hepcidin receptor (Abboud, 2000; Donovan, 2000; McKie, 2000). The membrane iron transporter is a 571 amino acid protein with 90% amino acid sequence identity between mouse, rat and human, which controls iron release (McKie, 2000). The major iron export proteins are located on the basement membrane of placental syncytiotrophoblast and intestinal cells, as well as on the cell surface of macrophages and hepatocytes.

Hepcidin inhibits iron release from these different cell types by binding to the ferroportin expressed on the above mentioned cell types and causes phosphorylation, internalization, ubiquitination (ubiquitination) and lysosomal degradation of the ferroportin, thereby reducing iron release into the blood mediated by the ferroportin (Nemeth, 2004; De Domenico, 2007). As plasma iron continues to be consumed for hemoglobin synthesis, plasma iron levels decrease and hepcidin production decreases in healthy subjects.

In the case of acute and chronic systemic inflammation, cytokines induce hepcidin production. It has been observed that hepcidin gene expression is significantly increased following inflammatory stimuli (e.g., infection) that induce an acute phase response of the vertebrate innate immune system. In mice, hepcidin gene expression was shown to be up-regulated by lipopolysaccharide (Constante, 2006), turpentine (Nemeth, 2004) and freund's complete adjuvant (Frazer, 2004), as well as by adenovirus infection. In humans, hepcidin expression is induced by the inflammatory cytokines interleukin-6 and LPS (Nemeth, 2004). A strong correlation between hepcidin expression and anemia of inflammation was also found in patients with chronic inflammatory diseases including bacterial, fungal and viral infections. In all these conditions, increased hepcidin concentrations inhibited iron efflux from macrophages, liver stores and duodenum into plasma. Under conditions of chronic inflammation, hypoferremia occurs, and erythropoiesis becomes iron-limiting and leads to anemia (Weiss, 2005; Weiss, 2008; Andrews, 2008).

The problem underlying the present invention is to provide a means for specifically interacting with hepcidin. More particularly, the problem underlying the present invention is to provide a nucleic acid based tool which specifically interacts with hepcidin.

Another problem underlying the present invention is to provide a means for the preparation of a medicament for the treatment of human or non-human diseases, wherein said diseases are characterized in that hepcidin is directly or indirectly involved in the pathogenic mechanisms of such diseases.

Another problem underlying the present invention is to provide means for the preparation of a diagnostic agent for the treatment of diseases characterized in that hepcidin is directly or indirectly involved in the pathogenic mechanisms of such diseases.

These and other problems underlying the present invention are solved by the subject matter of the appended independent claims. Preferred embodiments can be taken from the dependent claims.

Furthermore, the problem underlying the present invention is solved in a first aspect (which is also the first embodiment of the first aspect) by a nucleic acid capable of binding hepcidin.

In a second embodiment of the first aspect (which is also an embodiment of the first aspect), the nucleic acid is an antagonist of hepcidin.

In a third embodiment of the first aspect (which is also an embodiment of the first and second embodiments of the first aspect), the nucleic acid is an inhibitor of the hepcidin-ferroportin system.

In a fourth embodiment of the first aspect which is also an embodiment of the first, second and third embodiment of the first aspect, the nucleic acid comprises in the 5 '- > 3' direction a first terminal stretch of nucleotides (stretch), a central stretch of nucleotides and a second terminal stretch of nucleotides, wherein the central stretch of nucleotides comprises 32 to 40 nucleotides, preferably 32 to 35 nucleotides.

In a fifth embodiment of the first aspect (which is also an embodiment of the first and second embodiment of the first aspect), the nucleic acid comprises in the 5 '- > 3' direction a second terminal stretch of nucleotides, a central stretch of nucleotides and a first terminal stretch of nucleotides, wherein the central stretch of nucleotides comprises 32 to 40 nucleotides, preferably 32 to 35 nucleotides.

In a sixth embodiment of the first aspect (which is also an embodiment of the fourth and fifth embodiments of the first aspect), the central stretch of nucleotides is necessary for binding hepcidin.

In a seventh embodiment of the first aspect (which is also an embodiment of the fourth, fifth and sixth embodiments of the first aspect), the central nucleotide sequence segment comprises the nucleotide sequence of 5 'rkauggggakuuaaaugaggrguwggaggaar 3' or 5 'rkauggggakakagauaaauggagggrguwggaggaar 3'.

In an eighth embodiment of the first aspect (which is also an embodiment of the fourth to seventh embodiments of the first aspect), the central nucleotide sequence segment comprises the nucleotide sequence of 5 'rkauggggakuaaaauggrguggaar 3', preferably 5 'GUAUGGGAUUAAGUAAAUGAGGAGUUGGAGGAAG 3'.

In a ninth embodiment of the first aspect (which is also an embodiment of the seventh and eighth embodiments of the first aspect), the first terminal stretch of nucleotides and the second terminal stretch of nucleotides optionally hybridize to each other, wherein upon hybridization a double-stranded structure is formed; the first terminal stretch of nucleotides comprises 5 to 8 nucleotides; and the second terminal stretch of nucleotides comprises 5 to 8 nucleotides.

In a tenth embodiment of the first aspect (which is also an embodiment of the ninth embodiment of the first aspect), the double-stranded structure consists of 5 to 8 base pairs.

In an eleventh embodiment of the first aspect (which is also a particular embodiment of the seventh to tenth embodiments of the first aspect, preferably of the eighth to tenth embodiments of the first aspect), the first terminal stretch of nucleotides comprises a 5' X₁X₂X₃The nucleotide sequence of SBSBC 3 'and said second terminal nucleotide sequence segment comprises 5' GVGBVYX₄X₅X₆A nucleotide sequence of 3' of the polypeptide,

wherein

X₁Is A or absent, X₂Is G or absent, X₃Is B or absent, X₄Is S or absent, X₅Is C or absent, and X₆Is U or absent.

In a twelfth embodiment of the first aspect (which is also an embodiment of the seventh to eleventh embodiments of the first aspect), the first terminal stretch of nucleotides comprises a 5' X₁X₂X₃The nucleotide sequence of SBSBC 3 'and said second terminal stretch of nucleotides comprises 5' GVGBVBX₄X₅X₆A nucleotide sequence of 3' of the polypeptide,

wherein

a)X₁Is A, X₂Is G, X₃Is B, X₄Is S, X₅Is C, and X₆Is U, or

b)X₁Is absent, X₂Is G, X₃Is B, X₄Is S, X₅Is C, and X₆Is U, or

c)X₁Is A, X₂Is G, X₃Is B, X₄Is S, X₅Is C, and X₆Is absent.

In an embodiment 13 of the first aspect which is also an embodiment of the seventh to twelfth embodiments of the first aspect,

a) the first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'AGCGUGUC 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GGUGCGCU 3', or

b) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'AGCGUGUC 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GGCAUGCU 3', or

c) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'AGUGUGUC 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GAUGCGCU 3', or

d) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'AGUGUGUC 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GGCAUGCU 3', or

e) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'AGCGUGCC 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GGUGCGCU 3', or

f) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'AGCGCGCC 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GGCGCGCU 3'.

Embodiment 14 of the first aspect (which is also the first party)In the seventh to tenth embodiments of the above, preferably one embodiment of the eighth to tenth embodiments), the first terminal nucleotide sequence segment comprises 5' X₁X₂X₃The nucleotide sequence of SBSBC 3 'and said second terminal nucleotide sequence segment comprises 5' GVGBVYX₄X₅X₆A nucleotide sequence of 3' of the polypeptide,

wherein

a)X₁Is absent, X₂Is G, X₃Is B, X₄Is S, X₅Is C, and X₆Is absent, or

b)X₁Is absent, X₂Is absent, X₃Is B, X₄Is S, X₅Is C, and X₆Is absent, or

c)X₁Is absent, X₂Is G, X₃Is B, X₄Is S, X₅Is absent, and X₆Is absent.

In a 15 th embodiment of the first aspect (which is also a specific embodiment of the seventh to twelfth embodiments of the first aspect and 14 th embodiment), the first terminal stretch of nucleotides comprises 5' X₁X₂X₃The nucleotide sequence of SBSBC 3 'and said second terminal nucleotide sequence segment comprises 5' GVGBVYX₄X₅X₆A nucleotide sequence of 3' of the polypeptide,

wherein

X₁Is absent, X₂Is absent, X₃Is B or absent, X₄Is S or absent, X₅Is absent, and X₆Is absent.

In a 16 th embodiment of the first aspect which is also an embodiment of the 15 th embodiment of the first aspect,

a) the first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GCGCGCGC 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GCGCGC 3', or

b) The first terminal nucleotide sequence segment comprises a nucleotide sequence of 5 'GGUGUC 3' and the second terminal nucleotide sequence segment comprises a nucleotide sequence of 5 'GGCAUC 3', or

c) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GGCGUC 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GGCGCC 3', or

d) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GCGCC 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GGCGC 3', or

e) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GGCGC 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GCGCC 3'.

In a 17 th embodiment of the first aspect (which is also a specific embodiment of the seventh to 16 th embodiments of the first aspect), the nucleic acid comprises a nucleic acid sequence according to any one of SEQ ID nos. 115 to 119, SEQ ID No.121, SEQ ID No.142, SEQ ID No.144, SEQ ID No.146, SEQ ID No.148, SEQ ID No.151, SEQ ID No.152, SEQ ID No.175 or SEQ ID No. 176.

In an 18 th embodiment of the first aspect (which is also an embodiment of the fourth to sixth embodiments of the first aspect), the central nucleotide sequence segment comprises the nucleotide sequence of 5 'grcrgccggvggacaccauacagacuacakaua 3' or 5 'grcrgccggarggacacuauagacuacakaua 3'.

In a 19 th embodiment of the first aspect (which is also a specific embodiment of the fourth to sixth embodiments and 18 th embodiments of the first aspect), the central nucleotide sequence segment comprises the nucleotide sequence of 5 'grcrgccgggacaccauacagacuacakaua 3', preferably 5 'GACAGCCGGGGGACACCAUAUACAGACUACGAUA 3'.

In an embodiment 20 of the first aspect (which is also an embodiment of the 18 th and 19 th embodiments of the first aspect),

the first terminal stretch of nucleotides and the second terminal stretch of nucleotides optionally hybridize to each other, wherein upon hybridization a double-stranded structure is formed,

the first terminal stretch of nucleotides comprises 4 to 7 nucleotides, and

the second terminal stretch of nucleotides comprises 4 to 7 nucleotides.

In a 21 st embodiment of the first aspect (which is also an embodiment of the 20 th embodiment of the first aspect), the double stranded structure consists of 4 to 7 base pairs.

In a 22 nd embodiment of the first aspect (which is also a particular embodiment of the 18 th to 21 st embodiments of the first aspect), the first terminal stretch of nucleotides comprises a 5' X₁X₂X₃(ii) the nucleotide sequence of SBSN 3 'and said second terminal nucleotide sequence segment comprises 5' NSVSX₄X₅X₆A nucleotide sequence of 3' of the polypeptide,

wherein X₁Is A or absent, X₂Is G or absent, X₃Is R or absent, X₄Is Y or absent, X₅Is C or absent, and X₆Is U or absent.

In a 23 rd embodiment of the first aspect (which is also an embodiment of the 18 th to 22 th embodiments of the first aspect, preferably of the 19 th to 22 th embodiments of the first aspect), the first terminal stretch of nucleotides comprises a 5' X₁X₂X₃(ii) the nucleotide sequence of SBSN 3 'and said second terminal nucleotide sequence segment comprises 5' NSVSX₄X₅X₆A nucleotide sequence of 3' of the polypeptide,

wherein

a)X₁Is A, X₂Is G, X₃Is R, X₄Is Y, X₅Is C, and X₆Is U, or

b)X₁Is absent, X₂Is G, X₃Is R, X₄Is Y, X₅Is C, and X₆Is U, or

c)X₁Is A, X₂Is G, X₃Is R, X₄Is Y, X₅Is C, and X₆Is absent.

In an embodiment 24 of the first aspect (which is also an embodiment of the 18 th to 23 rd embodiments of the first aspect),

a) the first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'AGGCUCG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CGGGCCU 3', or

b) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'AGGCCCG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CGGGCCU 3', or

c) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'AGGCUUG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CGAGCCU 3', or

d) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'AGACUUG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CGAGUCU 3'.

In a 25 th embodiment of the first aspect (which is also an embodiment of the 18 th to 22 th embodiments of the first aspect, preferably of the 19 th to 22 th embodiments of the first aspect), the first end isThe nucleotide sequence segment comprises 5' X₁X₂X₃(ii) the nucleotide sequence of SBSN 3 'and said second terminal nucleotide sequence segment comprises 5' NSVSX₄X₅X₆A nucleotide sequence of 3' of the polypeptide,

wherein

a)X₁Is absent, X₂Is G, X₃Is R, X₄Is Y, X₅Is C, and X₆Is absent, or

b)X₁Is absent, X₂Is absent, X₃Is R, X₄Is Y, X₅Is C, and X₆Is absent, or

c)X₁Is absent, X₂Is G, X₃Is R, X₄Is Y, X₅Is absent, and X₆Is absent.

In a 26 th embodiment of the first aspect (which is also an embodiment of the 18 th to 22 th and 25 th embodiments of the first aspect), the first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GGCUCG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CGGGCC 3'.

In a 27 th embodiment of the first aspect (which is also a particular embodiment of the 18 th to 22 th embodiments of the first aspect), the first terminal stretch of nucleotides comprises a 5' X₁X₂X₃(ii) the nucleotide sequence of SBSN 3 'and said second terminal nucleotide sequence segment comprises 5' NSVSX₄X₅X₆A nucleotide sequence of 3' of the polypeptide,

wherein

X₁Is absent, X₂Is absent, X₃Is R or absent, X₄Is Y or absent, X₅Is absent, and X₆Is absent.

In a 28 th embodiment of the first aspect which is also an embodiment of the 18 th to 22 th and 27 th embodiments of the first aspect,

the first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GGCCG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CGGCC 3', or

The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GCGCG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CGCGC 3'.

In a 29 th embodiment of the first aspect (which is also a particular embodiment of the first to sixth and 18 to 28 th embodiments of the first aspect), the nucleic acid comprises a nucleic acid sequence according to any one of SEQ ID nos. 122 to 126, SEQ ID No.154, SEQ ID No.159, SEQ ID No.163 or SEQ ID No. 174.

In a 30 th embodiment of the first aspect (which is also an embodiment of the fourth to sixth embodiments of the first aspect), the central nucleotide sequence segment comprises in the 5 '- > 3' direction the following nucleotide sequence segments: box (Box) a, a linking nucleotide sequence segment and Box B; alternatively, the central nucleotide sequence segment comprises in the 5 '- > 3' direction the following nucleotide sequence segments: box B, a linking nucleotide sequence segment and box a, wherein box a comprises the nucleotide sequence of 5 'WAAAGUWGAR 3', the linking nucleotide sequence segment comprises 10 to 18 nucleotides, and box B comprises the nucleotide sequence of 5 'RGMGUGWKAGUKC 3'.

In a 31 st embodiment of the first aspect (which is also a particular embodiment of the 30 th embodiment of the first aspect), the cassette a comprises a nucleotide sequence selected from the group consisting of 5 'UAAAGUAGAG 3', 5 'AAAAGUAGAA 3', 5 'AAAAGUUGAA 3' and 5 'GGGAUAUAGUGC 3'; preferably the cartridge a comprises 5 'UAAAGUAGAG 3'.

In a 32 nd embodiment of the first aspect (which is also a particular embodiment of the 30 th to 31 th embodiments of the first aspect), the cassette B comprises a nucleotide sequence selected from the group consisting of 5 'GGCGUGAUAGUGC 3', 5 'GGAGUGUUAGUUC 3', 5 'GGCGUGAGAGUGC 3', 5 'AGCGUGAUAGUGC 3' and 5 'GGCGUGUUAGUGC 3', preferably the cassette B comprises 5 'GGCGUGAUAGUGC 3'.

In an embodiment 33 of the first aspect (which is also an embodiment of the 30 th to 32 th embodiments of the first aspect), the linking nucleotide sequence segment comprises in the 5 '- > 3' direction a first linking nucleotide subsequence segment (substretch), a second linking nucleotide subsequence segment, and a third linking nucleotide subsequence segment, wherein preferably the first linking nucleotide subsequence segment and the third linking nucleotide subsequence segment optionally hybridize to each other, wherein upon hybridization a double stranded structure is formed.

In a 34 th embodiment of the first aspect (which is also a specific embodiment of the 33 rd embodiment of the first aspect), the first and third linking nucleotide subsequences each and independently from each other comprise 3 to 6 nucleotides.

In a 35 th embodiment of the first aspect (which is also an embodiment of the 32 th to 34 th embodiments of the first aspect), the double stranded structure consists of 3 to 6 base pairs.

In a 36 th embodiment of the first aspect (which is also an embodiment of the 32 nd to 35 th embodiments of the first aspect),

a) the first linking subsequence of nucleotides comprises a nucleotide sequence selected from the group consisting of 5 'GGAC 3', 5 'GGAU 3' and 5 'GGA 3' and the third linking subsequence of nucleotides comprises a nucleotide sequence of 5 'GUCC 3', or

b) The first linking nucleotide subsequence segment comprises a nucleotide sequence of 5 'GCAG 3' and the third linking nucleotide subsequence segment comprises a nucleotide sequence of 5 'CUGC 3', or

c) The first linking subsequence of nucleotides comprises a nucleotide sequence of 5 'GGGC 3' and the third linking subsequence of nucleotides comprises a nucleotide sequence of 5 'GCCC 3', or

d) The first linking subsequence of nucleotides comprises a nucleotide sequence of 5 'GAC 3' and the third linking subsequence of nucleotides comprises a nucleotide sequence of 5 'GUC 3', or

e) The first linking subsequence of nucleotides comprises a nucleotide sequence of 5 'ACUUGU 3' and the third linking subsequence of nucleotides comprises a nucleotide sequence selected from the group consisting of 5 'GCAAGU 3' and 5 'GCAAGC 3', or

f) The first linking subsequence of nucleotides comprises a nucleotide sequence of 5 'UCCAG 3' and the third linking subsequence of nucleotides comprises a nucleotide sequence of 5 'CUGGA 3',

preferably, the first linking subsequence segment comprises a nucleotide sequence of 5 'GAC 3' and the third linking subsequence segment comprises a nucleotide sequence of 5 'GUC 3'.

In a 37 th embodiment of the first aspect (which is also a particular embodiment of the 33 rd to 36 th embodiments of the first aspect), the second linking subsequence stretch comprises 3 to 5 nucleotides.

In a 38 th embodiment of the first aspect (which is also a particular embodiment of the 33 rd to 37 th embodiments of the first aspect), the second linking subsequence of nucleotides comprises a nucleotide sequence selected from the group consisting of 5 'VBAAW 3', 5 'AAUW 3' and 5 'NBW 3'.

In a 39 th embodiment of the first aspect (which is also an embodiment of the 38 th embodiment of the first aspect), the second linking nucleotide subsequence segment comprises a nucleotide sequence of 5 'VBAAW 3', preferably a nucleotide sequence selected from the group consisting of 5 'CGAAA 3', 5 'GCAAU 3', 5 'GUAAU 3' and 5 'AUAAU 3'.

In a 40 th embodiment of the first aspect (which is also an embodiment of the 38 th embodiment of the first aspect), the second linking nucleotide subsequence segment comprises a nucleotide sequence of 5 'AAUW 3', preferably a nucleotide sequence of 5 'AAUU 3' or 5 'AAUA 3', more preferably a nucleotide sequence of 5 'AAUA 3'.

In a 41 th embodiment of the first aspect (which is also an embodiment of the 38 th embodiment), the second linking nucleotide subsequence segment comprises a nucleotide sequence of 5 'NBW 3', preferably a nucleotide sequence selected from the group consisting of 5 'CCA 3', 5 'CUA 3', 5 'UCA 3', 5 'ACA 3', 5 'GUU 3', 5 'UGA 3' and 5 'GUA 3', more preferably a nucleotide sequence selected from the group consisting of 5 'CCA 3', 5 'CUA 3', 5 'UCA 3', 5 'ACA 3' and 5 'GUU 3'.

In a 42 th embodiment of the first aspect (which is also an embodiment of the 30 th to 41 th embodiments of the first aspect), the linking nucleotide sequence segment comprises a nucleotide sequence selected from the group consisting of 5 'GGACBYAGUCC 3', 5 'GGAUACAGUCC 3', 5 'gcaggyaucugc 3', 5 'GACAAUWGUC 3', 5 'acuuguagaagcaagyu 3', 5 'UCCAGGUUCUGGA 3', 5 'GGGCUGAGCCC 3', 5 'GCAGAUAAUCUGC 3' and 5 'GGACCAGUCC 3', preferably from the group consisting of 5 'GGACCCAGUCC 3', 5 'GGACCUAGUCC 3', 5 'GGACUCAGUCC 3', 5 'GCAGGUAAUCUGC 3', 5 'GCAGGCAAUCUGC 3', 5 'GACAAUUGUC 3' and 5 'GACAAUAGUC 3'.

In a 43 rd embodiment of the first aspect (which is also an embodiment of the 30 th to 42 th embodiments of the first aspect),

the first terminal stretch of nucleotides comprises 4 to 7 nucleotides, and

the second terminal stretch of nucleotides comprises 4 to 7 nucleotides.

In a 44 th embodiment of the first aspect (which is also an embodiment of the 43 th embodiment of the first aspect), the double stranded structure consists of 4 to 7 base pairs.

In a 45 th embodiment of the first aspect (which is also a particular embodiment of the 30 th to 44 th embodiments of the first aspect), the first terminal stretch of nucleotides comprises a 5' X₁X₂X₃A nucleotide sequence of BKBKBKK 3 'and the second terminal nucleotide sequence segment comprises 5' MVVVX₄X₅X₆A nucleotide sequence of 3' of the polypeptide,

wherein X₁Is G or absent, X₂Is S or absent, X₃Is V or absent, X₄Is B or absent, X₅Is S or absent, and X₆Is C or absent.

In a 46 th embodiment of the first aspect (which is also a particular embodiment of the 30 th to 44 th embodiments of the first aspect), the first terminal stretch of nucleotides comprises a 5' X₁X₂X₃A nucleotide sequence of BKBKBKK 3 'and the second terminal nucleotide sequence segment comprises 5' MVVVX₄X₅X₆A nucleotide sequence of 3' of the polypeptide,

wherein

a)X₁Is G, X₂Is S, X₃Is V, X₄Is B, X₅Is S, and X₆Is C, or

b)X₁Is absent, X₂Is S, X₃Is V, X₄Is B, X₅Is S, and X₆Is C, or

c)X₁Is G, X₂Is S, X₃Is V, X₄Is B, X₅Is S, and X₆Is absent.

In a 47 th embodiment of the first aspect (which is also a 30 th to 46 th embodiment of the first aspect, preferably an embodiment of the 46 th embodiment of the first aspect), the first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GCACUCG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CGAGUGC 3'.

In a 48 th embodiment of the first aspect (which is also a particular embodiment of the 30 th to 45 th embodiments of the first aspect), the first terminal stretch of nucleotides comprises a 5' X₁X₂X₃A nucleotide sequence of BKBKBKK 3 'and the second terminal nucleotide sequence segment comprises 5' MVVVX₄X₅X₆A nucleotide sequence of 3' of the polypeptide,

wherein

a)X₁Is absent, X₂Is S, X₃Is V, X₄Is B, X₅Is S, and X₆Is absent, or

b)X₁Is absent, X₂Is absent, X₃Is V, X₄Is B, X₅Is S, and X₆Is absent, or

c)X₁Is absent, X₂Is S, X₃Is V, X₄Is B, X₅Is absent, and X₆Is absent.

In a 49 th embodiment of the first aspect (which is also an embodiment of the 30 th to 45 th and 48 th embodiments of the first aspect),

a) the first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GCUGUGUG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CACACACAGC 3', or

b) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CGUGUG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CACACACG 3', or

c) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CGUGCU 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'AGCACG 3', or

d) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CGCGCG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CGCGCG 3', or

e) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GCCGUG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CACGCG 3', or

f) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GCGGUG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CACCGC 3', or

g) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GCUGCG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CGCAGC 3', or

h) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GCUGGG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CCCAGC 3', or

i) The first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'GCGGCG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CGCCGC 3'.

In a 50 th embodiment of the first aspect (which is also a particular embodiment of the 30 th to 45 th embodiments of the first aspect), the first terminal stretch of nucleotides comprises a 5' X₁X₂X₃A nucleotide sequence of BKBKBKK 3' andthe two terminal nucleotide sequence segment comprises 5' MVVVX₄X₅X₆A nucleotide sequence of 3' of the polypeptide,

wherein

X₁Is absent, X₂Is absent, X₃Is V or absent, X₄Is B or absent, X₅Is absent, and X₆Is absent.

In a 51 st embodiment of the first aspect (which is also an embodiment of the 30 th to 45 th and 50 th embodiments of the first aspect), the first terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CGUG 3' and the second terminal stretch of nucleotides comprises a nucleotide sequence of 5 'CACG 3'.

In a 52 th embodiment of the first aspect (which is also a particular embodiment of the first to sixth and 30 to 11 th embodiments of the first aspect), the nucleic acid comprises a nucleic acid sequence according to any one of SEQ ID No.29, SEQ ID No.33, SEQ ID No.34, SEQ ID nos. 39 to 41, SEQ ID No.43, SEQ ID No.46, SEQ ID nos. 137 to 141 or SEQ ID No. 173.

In a 53 rd embodiment of the first aspect (which is also a particular embodiment of the first to sixth embodiments of the first aspect), the nucleic acid comprises a nucleic acid sequence according to any one of SEQ ID nos. 127 to 131.

In a 54 th embodiment of the first aspect (which is also a particular embodiment of the first to 53 th embodiments of the first aspect), the nucleic acid is capable of binding to hepcidin, wherein hepcidin is human hepcidin-25, human hepcidin-22, human hepcidin-20, simian hepcidin-25, simian hepcidin-22, simian hepcidin-20, preferably human hepcidin-25.

In a 55 th embodiment of the first aspect (which is also a specific embodiment of the first to 54 th embodiments of the first aspect, preferably of the 54 th embodiment of the first aspect), the hepcidin has an amino acid sequence according to SEQ ID No. 1.

In a 56 th embodiment of the first aspect (which is also a particular embodiment of the first to 55 th embodiments of the first aspect), the nucleic acid comprises a modification group, wherein the rate of excretion of a nucleic acid molecule comprising the modification group from an organism is reduced compared to a nucleic acid not comprising the modification group.

In a 57 th embodiment of the first aspect (which is also a particular embodiment of the first to 55 th embodiments of the first aspect), the nucleic acid comprises a modification group, wherein the nucleic acid molecule comprising the modification group has an increased residence time in an organism compared to a nucleic acid not comprising the modification group.

In a 58 th embodiment of the first aspect which is also an embodiment of the 56 th and 57 th embodiments of the first aspect, the modifying group is selected from the group comprising biodegradable and non-biodegradable modifications, preferably the modifying group is selected from the group comprising linear polyethylene glycol, branched polyethylene glycol, hydroxyethyl starch, peptides, proteins, polysaccharides, sterols, polyoxypropylene, polyoxyamidate, poly (2-hydroxyethyl) -L-glutamine and polyethylene glycol.

In a 59 th embodiment of the first aspect (which is also a particular embodiment of the 58 th embodiment of the first aspect), the modifying group is a PEG moiety consisting of a linear or branched PEG, wherein the PEG moiety preferably has a molecular weight of from about 20,000 to about 120,000Da, more preferably from about 30,000 to about 80,000Da, and most preferably about 40,000 Da.

In a 60 th embodiment of the first aspect (which is also a specific embodiment of the 58 th embodiment of the first aspect), the modifying group is a HES moiety, wherein the HES moiety preferably has a molecular weight of about 10,000 to 200,000Da, more preferably about 30,000 to 170,000Da, and most preferably about 150,000 Da.

In a 61 st embodiment of the first aspect (which is also a particular embodiment of the 56 th to 60 th embodiments of the first aspect), the modification group is coupled to the nucleic acid via a linker, wherein the linker is preferably a biodegradable linker.

In a 62 nd embodiment of the first aspect (which is also a particular embodiment of the 56 th to 61 th embodiments of the first aspect), the modifying group is coupled to the 5 '-terminal nucleotide and/or the 3' -terminal nucleotide of the nucleic acid, and/or to nucleotides of the nucleic acid between the 5 '-terminal nucleotide of the nucleic acid and the 3' -terminal nucleotide of the nucleic acid.

In a 63 rd embodiment of the first aspect (which is also an embodiment of the 56 th to 62 th embodiments of the first aspect), the organism is an animal or human body, preferably a human body.

In a 64 th embodiment of the first aspect (which is also an embodiment of the first to 63 th embodiments of the first aspect), the nucleotides of the nucleic acid or the nucleotides forming the nucleic acid are L-nucleotides.

In a 65 th embodiment of the first aspect (which is also a specific embodiment of the 1 st to 64 th embodiments of the first aspect), the nucleic acid is an L-nucleic acid.

In a 66 th embodiment of the first aspect (which is also a particular embodiment of the first to 65 th embodiments of the first aspect), the nucleic acid comprises at least one binding moiety capable of binding hepcidin, wherein such binding moiety consists of L-nucleotides.

In a 67 th embodiment of the first aspect (which is also a particular embodiment of the first to 66 th embodiments of the first aspect), the nucleic acid is for use in, or is suitable for use in, a method of treating and/or preventing a disease.

The problem underlying the present invention is solved in a second aspect (which is also the first embodiment of the second aspect) by a pharmaceutical composition comprising a nucleic acid according to any one of the embodiments of the first aspect and optionally a further component, wherein the further component is selected from the group comprising a pharmaceutically acceptable excipient, a pharmaceutically acceptable carrier and a pharmaceutically active agent.

In a second embodiment of the second aspect (which is also an embodiment of the first embodiment of the second aspect), the pharmaceutical composition comprises a nucleic acid according to any one of the embodiments of the first aspect and a pharmaceutically acceptable carrier.

The problem underlying the present invention is solved in a third aspect (which is also the first embodiment of the third aspect) by the use of a nucleic acid according to any one of the embodiments of the first aspect for the preparation of a medicament.

In a second embodiment of the third aspect (which is also an embodiment of the first embodiment of the third aspect), the medicament is for use in human medicine or for use in veterinary medicine.

The problem underlying the present invention is solved in a fourth aspect (which is also the first embodiment of the fourth aspect) by the use of a nucleic acid according to any one of the embodiments of the first aspect for the preparation of a diagnostic tool.

In a third embodiment of the third aspect (which is also an embodiment of the first and second embodiments of the third aspect), the medicament is for the treatment and/or prevention of anemia, hypoferremia, pica, a condition with elevated hepcidin levels, a condition with elevated iron levels, or a condition with iron overload.

In a fourth embodiment of the third aspect (which is also an embodiment of the third aspect), the anemia is selected from the group consisting of sideroblasts anemia, hypopigmented microcytic anemia, anemia arising from chronic diseases and/or disorders, anemia arising from inflammation, anemia arising from genetic disorders, anemia arising from acute infections, anemia arising from mutations in genes of iron metabolism and/or homeostasis, and anemia arising from cancer therapy.

In a fifth embodiment of the third aspect which is also an embodiment of the fourth embodiment of the third aspect, the chronic disease and/or disorder is selected from chronic inflammation, cancer, autoimmune disease and/or disorder, chronic infection, arteriosclerosis, atherosclerosis and cirrhosis of the liver.

In a sixth embodiment of the third aspect (which is also an embodiment of the fifth embodiment of the third aspect), the chronic inflammation is selected from chronic kidney disease, chronic obstructive pulmonary disease, multiple sclerosis, osteoarthritis, diabetes, obesity, cerebrovascular disease, congestive heart failure, myocardial infarction, coronary artery disease, peripheral occlusive arterial disease, pancreatitis and vasculitis, wherein the chronic kidney disease is preferably selected from renal disease, chronic renal failure, and wherein the chronic kidney disease is caused by renal dialysis or renal transplantation.

In a seventh embodiment of the third aspect (which is also an embodiment of the fifth embodiment of the third aspect), the autoimmune disease and/or disorder is selected from rheumatoid arthritis, irritable bowel syndrome, systemic lupus erythematosus and crohn's disease.

In an eighth embodiment of the third aspect which is also an embodiment of the fifth embodiment of the third aspect, the chronic infection is selected from the group consisting of viral infection, viral disease, bacterial infection and fungal infection, wherein preferably the viral infection comprises hepatitis and HIV infection and the bacterial infection comprises helicobacter pylori infection.

In a ninth embodiment of the third aspect (which is also an embodiment of the first to fourth embodiments of the third aspect), the anemia arising from inflammation is normocytic to microcytic and/or is characterized by a low reticulocyte production index and/or increased inflammatory markers.

In a tenth embodiment of the third aspect which is also an embodiment of the fourth embodiment of the third aspect, the genetic disorder is castleman's disease, Schnitzler's syndrome, iron refractory iron deficiency anemia (mutated matriptase-2(TMPRSS 6)), transferrin deficiency anemia, congenital erythropoietic anemia or hemoglobinopathy

In an eleventh embodiment of the third aspect which is also an embodiment of the fourth embodiment of the third aspect, the acute infection is selected from the group consisting of a viral infection, a bacterial infection and a fungal infection, preferably sepsis.

In a twelfth embodiment of the third aspect (which is also an embodiment of the fifth embodiment of the third aspect), the cancer is selected from hepatocellular carcinoma, lymphoma, multiple myeloma, head and neck cancer, breast cancer, colorectal cancer, non-myeloid cancer, renal cell carcinoma, non-small cell lung cancer, tumor, and brain tumor.

In a 13 th embodiment of the third aspect (which is also an embodiment of the third aspect), the condition is selected from the group consisting of ataxia, friedreich's ataxia, age-related macular degeneration, age-related cataracts, age-related retinal diseases and neurodegenerative diseases, wherein such neurodegenerative diseases are preferably selected from the group comprising alzheimer's disease, parkinson's disease, pantothenate kinase-associated neurodegeneration, restless leg syndrome and huntington's disease.

In a 14 th embodiment of the third aspect (which is also an embodiment of the third aspect), the medicament is for use in the treatment of iron overload, wherein hepcidin plasma levels are not elevated.

In a 15 th embodiment of the third aspect (which is also an embodiment of the 14 th embodiment of the third aspect), the iron overload is selected from the group consisting of transfusional iron overload, iron poisoning, pulmonary hemosiderosis, osteopenia, insulin resistance, african iron overload, hayasu's disease, methemoglobinemia, ceruloplasmin deficiency, neonatal hemochromatosis and erythropathy including thalassemia, alpha thalassemia, thalassemia intermedia, sickle cell disease and myelodysplastic syndrome.

In a 16 th embodiment of the third aspect (which is also an embodiment of the twelfth to 15 th embodiments of the third aspect), the agent is used in combination with an iron chelating compound.

In a 17 th embodiment of the third aspect (which is also an embodiment of the 16 th embodiment of the third aspect), the iron chelating compound is selected from curcumin, desferrioxamine, deferasirox and deferiprone.

In an 18 th embodiment of the third aspect (which is also an embodiment of the first embodiment of the third aspect), the medicament is for use in combination with or for use in combination with a further medicament or treatment method, wherein such medicament or treatment method comprises a further pharmaceutically active compound or administration of such a further pharmaceutically active compound, wherein such a further pharmaceutically active compound is selected from the group consisting of iron supplements, vitamin supplements, erythropoiesis stimulating agents, antibiotics, anti-inflammatory biologics, inhibitors of the immune system, antithrombotic lysing agents, statins, vasopressors and compounds affecting muscle contraction.

The problem underlying the present invention is solved in a fifth aspect (which is also the first embodiment of the fifth aspect) by a complex comprising a nucleic acid according to any of the embodiments of the first aspect and hepcidin, wherein the complex is preferably a crystalline complex.

In a second embodiment of the fifth aspect (which is also an embodiment of the first embodiment of the fifth aspect), the hepcidin is selected from the group comprising human hepcidin, simian hepcidin, more preferably the hepcidin is human hepcidin.

The problem underlying the present invention is solved in a sixth aspect (which is also the first embodiment of the sixth aspect) by the use of a nucleic acid according to any one of the embodiments of the first aspect for the detection of hepcidin.

In a second embodiment of the sixth aspect (which is also an embodiment of the first embodiment of the sixth aspect), the hepcidin is selected from the group comprising human hepcidin, simian hepcidin, more preferably the hepcidin is human hepcidin.

The problem underlying the present invention is solved in a seventh aspect (which is also the first embodiment of the seventh aspect) by a method for screening for an antagonist or agonist of hepcidin, the method comprising the steps of:

-providing a candidate antagonist and/or candidate agonist of hepcidin,

-providing a nucleic acid according to any one of the embodiments of the first aspect,

-providing a test system which provides a signal in the presence of an antagonist and/or agonist of hepcidin, and

-determining whether the candidate antagonist is an antagonist of hepcidin and/or whether the candidate agonist is an agonist of hepcidin.

In a second embodiment of the seventh aspect (which is also an embodiment of the first embodiment of the seventh aspect), the hepcidin is selected from the group comprising human hepcidin, simian hepcidin, more preferably the hepcidin is human hepcidin.

The problem underlying the present invention is solved in an eighth aspect (which is also the first embodiment of the eighth aspect) by a kit for the detection of hepcidin, the kit comprising a nucleic acid according to any one of the embodiments of the first aspect, wherein the hepcidin is preferably human hepcidin.

The problem underlying the present invention is solved in a ninth aspect (which is also the first embodiment of the ninth aspect) by a method for detecting a nucleic acid according to any one of the embodiments of the first aspect in a sample, wherein the method comprises the steps of:

a) providing a sample containing a nucleic acid according to the invention;

b) providing a capture probe and a detection probe, wherein the capture probe is at least partially complementary to a first portion of a nucleic acid according to any one of the embodiments of the first aspect and the detection probe is at least partially complementary to a second portion of a nucleic acid according to any one of the embodiments of the first aspect, or alternatively, the capture probe is at least partially complementary to a second portion of a nucleic acid according to any one of the embodiments of the first aspect and the detection probe is at least partially complementary to a first portion of a nucleic acid according to any one of the embodiments of the first aspect;

c) allowing the capture probe and the detection probe to react simultaneously or sequentially in any order with a nucleic acid or part thereof according to any of the embodiments of the first aspect;

d) optionally, detecting whether the capture probe hybridizes to the nucleic acid according to any one of the embodiments of the first aspect provided in step a); and

e) detecting the complex formed in step c) consisting of the nucleic acid according to any one of the embodiments of the first aspect and the capture probe and the detection probe.

In a second embodiment of the ninth aspect (which is also an embodiment of the first embodiment of the ninth aspect), the detection probe comprises detection means, and/or wherein the capture probe may be immobilised to a support, preferably a solid support.

In a third embodiment of the ninth aspect which is also a particular embodiment of the first and second embodiments of the ninth aspect, any detection probe that is not part of the complex is removed from the reaction system, such that only detection probe that is part of the complex is detected in step e).

In a fourth embodiment of the ninth aspect (which is also an embodiment of the first, second and third embodiments of the ninth aspect), step e) comprises the steps of: comparing the signals generated by said detection means when said capture probe and said detection probe hybridise in the presence of a nucleic acid or part thereof according to any one of the embodiments of the first aspect and in the absence of said nucleic acid or part thereof.

The features of the nucleic acids according to the invention described herein can be achieved in any aspect of the invention, wherein the nucleic acids are used individually or in any combination.

In connection with the present invention, preferably, the term "providing a sample" is different from and does not include a method of treatment or diagnosis of the human or animal body.

Human hepcidin-25 is a basic protein with an amino acid sequence according to SEQ ID No.1 and a pI of 8.2.

The present invention is based on the surprising finding that it is possible to generate nucleic acids which bind hepcidin specifically and with high affinity. Such nucleic acids are preferably also referred to herein as nucleic acid molecules according to the invention, nucleic acids of the invention or nucleic acid molecules of the invention.

The finding that short and high affinity binding nucleic acids to human hepcidin can be identified is surprising in this context, since the observation by Eaton et al (1997) that the generation of aptamers (aptamers) against basic proteins, i.e. D-nucleic acids binding to target molecules, is often very difficult, since this type of target gives rise to a high but non-specific signal-to-noise ratio. This high signal-to-noise ratio is due to the high non-specific affinity exhibited by nucleic acids for basic targets (e.g., human hepcidin).

As outlined in more detail in the claims and in example 1, the inventors were able to identify more surprisingly many different nucleic acid molecules binding to human hepcidin, wherein the majority of the nucleic acids can be characterized according to a nucleotide sequence segment (which is also referred to herein as a cassette). Based on the cassette and certain additional structural features and elements, various human hepcidin-binding nucleic acid molecules can be classified as hepcidin-binding nucleic acids type a, B and C, respectively.

Different types of hepcidin-binding nucleic acids comprise different nucleotide sequence segments. Thus, different types of hepcidin-binding nucleic acids show different binding behavior for different hepcidin peptides. As demonstrated in the examples, the hepcidin-binding nucleic acids according to the invention bind to human hepcidin-25, human hepcidin-22, human hepcidin-20, cynomolgus monkey hepcidin-25 and marmoset hepcidin-25.

It should be understood that whenever reference is made herein to hepcidin, such hepcidin is hepcidin-25, if not indicated to the contrary.

It is within the scope of the present invention that the nucleic acids according to the invention comprise two or more sequence segments or parts thereof which can in principle hybridize to one another. After such hybridization, a double-stranded structure is formed. One skilled in the art will appreciate that such hybridization may or may not occur, particularly under in vitro and/or in vivo conditions. Further, in the case of such hybridization, it is not necessarily the case that: the hybridization takes place over the entire length of the two sequence segments, wherein such hybridization and thus the formation of a double-stranded structure is possible in principle, at least on the basis of the base pairing rules. As preferably used herein, a double-stranded structure is a portion of a nucleic acid molecule, or a structure formed by two or more separate strands or two spatially separated stretches of a single strand of a nucleic acid molecule, whereby there is at least one, preferably two or more base pairs, which are preferably base paired according to the Watson-Crick base pairing rules. Those skilled in the art will also appreciate that other base pairing, such as Hoogsten base pairing, may be present in or form such double-stranded structures. It will also be recognized that the feature of two sequence segments hybridizing preferably indicates that such hybridization is presumed to occur due to the base complementarity of the two sequence segments.

In preferred embodiments, the term "array" as used herein denotes the order or sequence of structural or functional features or elements described herein in relation to a nucleic acid disclosed herein.

It will be appreciated by those skilled in the art that nucleic acids according to the invention are capable of binding hepcidin. Without wishing to be bound by any theory, the inventors hypothesize that the hepcidin binding is due to a combination of three-dimensional structural properties or elements of the claimed nucleic acid molecules, which result from the orientation and folding pattern of the primary sequence of nucleotides forming such properties or elements. It will be apparent that individual features or elements may be formed from a variety of different individual sequences, the degree of variation of which may vary depending on the three-dimensional structure that such elements or features must form. The overall binding characteristics of the claimed nucleic acids result from the interplay of various elements and properties, respectively, which ultimately result in the interaction of the claimed nucleic acids with their target (i.e., hepcidin). Again without wishing to be bound by any theory, the central stretch characteristic for type B and C hepcidin-binding nucleic acids and the first stretch box a and the second stretch box B characteristic for type a hepcidin-binding nucleic acids appear to be important for mediating binding of the claimed nucleic acids to hepcidin. Thus, the nucleic acid according to the invention is suitable for interacting with and detecting hepcidin. Furthermore, it will be appreciated by those skilled in the art that the nucleic acids according to the invention are antagonists of hepcidin. Thus, the nucleic acids according to the invention are suitable for the treatment and prevention of any disease or condition associated with or caused by hepcidin, respectively. Such diseases and conditions can be obtained from the prior art which identifies the involvement in or association with hepcidin, respectively, and which is incorporated herein by reference to provide the scientific rationale for the therapeutic and diagnostic use of nucleic acids according to the invention.

It is within the scope of the present invention that the nucleic acid according to the invention is a nucleic acid molecule. Thus, the terms "nucleic acid" and "nucleic acid molecule" are used synonymously herein if nothing else is indicated to the contrary. In one embodiment of the present application, the nucleic acid and thus the nucleic acid molecule comprises a nucleic acid molecule characterized in that all consecutive nucleotides forming the nucleic acid molecule are linked or linked to each other by one or more than one covalent bond. More specifically, each such nucleotide is preferably linked or linked to the other two nucleotides by a phosphodiester or other linkage, thereby forming a stretch of contiguous nucleotides. However, in such an arrangement, the two terminal nucleotides (i.e. preferably the nucleotides at the 5 'and 3' ends) are each linked to only a single nucleotide, provided that such an arrangement is a linear rather than a cyclic arrangement, and thus a linear rather than cyclic molecule.

In another embodiment of the present application, the nucleic acid and thus the nucleic acid molecule comprises at least two sets of consecutive nucleotides, wherein within each set of consecutive nucleotides each nucleotide is linked or linked to the other two nucleotides, preferably by phosphodiester or other linkages, thereby forming a stretch of consecutive nucleotides. However, in such an arrangement, the two terminal nucleotides of each of the at least two sets of consecutive nucleotides (i.e. preferably the nucleotides at the 5 'end and at the 3' end) are each linked to only a single nucleotide. However, in such embodiments, the two sets of consecutive nucleotides are not interconnected or linked by a covalent bond that links one nucleotide of one set to one nucleotide of the other or another set by a covalent bond, preferably a covalent bond formed between the sugar moiety of one of the two nucleotides and the phosphate moiety of the other of the two nucleotides or nucleosides. However, in an alternative embodiment, the two groups of consecutive nucleotides are interconnected or linked by a covalent bond linking one nucleotide of one group with one nucleotide of the other or another group by a covalent bond, preferably a covalent bond formed between the sugar moiety of one of the two nucleotides and the phosphate moiety of the other of the two nucleotides or nucleosides. Preferably, the at least two sets of consecutive nucleotides are not linked by any covalent bond. In another preferred embodiment, the at least two groups are linked by covalent bonds different from phosphodiester bonds. In another embodiment, the at least two groups are connected by a covalent bond that is a phosphodiester bond. Further, preferably, the two sets of consecutive nucleotides are connected or linked to each other by a covalent bond, wherein the covalent bond is formed between a nucleotide at the 3 'end of a first of the two sets of consecutive nucleotides and a nucleotide at the 5' end of a second of the two sets of consecutive nucleotides, or the covalent bond is formed between a nucleotide at the 5 'end of a first of the two sets of consecutive nucleotides and a nucleotide at the 3' end of a second of the two sets of consecutive nucleotides.

Nucleic acids according to the invention should also include nucleic acids that are substantially homologous to the specific sequences disclosed herein. The term "substantially homologous" should preferably be understood as meaning a homology of at least 75%, preferably 85%, more preferably 90%, and most preferably greater than 95%, 96%, 97%, 98% or 99%.

Homology between two nucleic acid molecules can be determined as known to those skilled in the art. More specifically, based on the specified program parameters, a sequence comparison algorithm can be used to calculate the percent sequence homology of a test sequence relative to a reference sequence. The test sequence is preferably a sequence or nucleic acid molecule which is said to be homologous to a different nucleic acid molecule or which is to be tested for homology (to what extent, if any) to a different nucleic acid molecule, wherein such different nucleic acid molecule is also referred to as a reference sequence. In one embodiment, the reference sequence is a nucleic acid molecule as described herein, more preferably a nucleic acid molecule having a sequence according to any one of SEQ ID nos. 29 to 43, 45 to 48, 110 to 156, 158 to 176 or 179 to 181. Optimal sequence alignment for comparison can be performed, for example, by: the algorithm of local homology of Smith & Waterman (1981)), the algorithm of homology alignment of Needleman & Wunsch (Needleman & Wunsch, 1970), the method of similarity search of Pearson & Lipman (Pearson & Lipman, 1988), the computerized implementation of these algorithms (Wisconsin Genetics software Package, Genetics Computer Group, 575 Science Dr., Madison, GAP in Wis., BESTFIT, FASTA and TFASTA), or visual inspection.

One example of an algorithm suitable for determining percent sequence identity is the algorithm used in the basic local alignment search tool (hereinafter "BLAST"), see, e.g., Altschul et al (Altschul et al, 1990; and Altschul et al, 1997). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology information (hereinafter referred to as "NCBI"). Default parameters employed in sequence identity assays using software available from NCBI, such as BLASTN (for nucleotide sequences) and BLASTP (for amino acid sequences), are described in McGinnis et al (McGinnis et al, 2004).

The term "nucleic acid of the invention" or "nucleic acid according to the invention" (wherein the two terms are used interchangeably) shall also include those nucleic acids comprising the nucleic acid sequences disclosed herein or parts thereof, preferably to the extent that said nucleic acids or said parts are involved in binding to human hepcidin. In one embodiment, such a nucleic acid is one of the nucleic acid molecules described herein, or a derivative and/or metabolite thereof, wherein such derivative and/or metabolite is preferably a truncated nucleic acid compared to the nucleic acid molecules described herein. Truncation may involve either or both termini of the nucleic acids disclosed herein. Furthermore, the truncation may relate to the internal nucleotide sequence of the nucleic acid, i.e. it may relate to the nucleotides between the 5 'and 3' terminal nucleotides, respectively. In addition, truncation shall include deletion of as few as a single nucleotide from the nucleic acid sequences disclosed herein. Truncation may also relate to more than one stretch of the nucleic acid of the invention, wherein the stretch may be only one nucleotide long. The binding of a nucleic acid according to the invention can be determined by a person skilled in the art by using routine tests or by using or employing the methods described herein, preferably the methods described herein in the examples section.

The nucleic acids according to the invention may be D-nucleic acids or L-nucleic acids. Preferably, the nucleic acid of the invention is an L-nucleic acid. Furthermore, it is also possible that one or several parts of the nucleic acid are present as D-nucleic acids or that at least one or several parts of the nucleic acid are L-nucleic acids. The term "part" of a nucleic acid shall mean as little as one nucleotide. Thus, in a particularly preferred embodiment, the nucleic acids according to the invention consist of L-nucleotides and comprise at least one D-nucleotide. Such D-nucleotides are preferably attached to a portion different from the sequence stretch defining the nucleic acid according to the invention, preferably to those portions of the nucleic acid where interaction with other portions of the nucleic acid or with the target (i.e. hepcidin) is involved. Preferably, such D-nucleotides are attached at the end of any sequence stretch according to the invention or at the end of any nucleic acid according to the invention, respectively. In a further preferred embodiment, such D-nucleotides may be used as spacers or linkers, which preferably attach modifications or modifying groups such as PEG and HES to the nucleic acid according to the invention.

It is also within the scope of embodiments of the present invention that each and any of the nucleic acid molecules described herein in their entirety in terms of their nucleic acid sequence are limited to a particular nucleotide sequence. In other words, the terms "comprising" or "including" should be understood in such embodiments as "containing" or "consisting of.

It is also within the scope of the present invention that the nucleic acid according to the invention is a portion of a longer nucleic acid, wherein the longer nucleic acid comprises several portions, wherein at least one such portion is a nucleic acid according to the invention or a portion thereof. The other part of these longer nucleic acids may be one or several D-nucleic acids or one or several L-nucleic acids. Any combination may be used in the present invention. These other portions of longer nucleic acids, alone or together, in their entirety or in a particular combination, may exhibit a function other than binding (preferably, to hepcidin). One possible function is to allow interaction with other molecules (where such other molecules are preferably different from hepcidin), e.g. for immobilization, cross-linking, detection or amplification. In a further embodiment of the invention, the nucleic acid according to the invention comprises several nucleic acids according to the invention, as individual or combined parts. Such nucleic acids comprising several nucleic acids of the invention are also encompassed by the term "longer nucleic acids".

An "L-nucleic acid" or "L-nucleic acid molecule" as used herein is a nucleic acid or nucleic acid molecule consisting of L-nucleotides, preferably a nucleic acid or nucleic acid molecule consisting entirely of L-nucleotides.

As used herein, a "D-nucleic acid" or "D-nucleic acid molecule" is a nucleic acid or nucleic acid molecule consisting of D-nucleotides, preferably consisting entirely of D-nucleotides.

Furthermore, if not indicated to the contrary, any nucleotide sequence is shown herein in the 5 '- > 3' orientation.

As preferably used herein, any nucleotide position is determined or referred to relative to the 5' end of a sequence, sequence segment or subsequence segment. Thus, the second nucleotide is the second nucleotide counted from the 5' end of the sequence, sequence segment or subsequence segment, respectively. Likewise, according to this principle, the penultimate nucleotide is the second nucleotide counted from the 3' end of the sequence, sequence segment or subsequence segment, respectively.

Whether the nucleic acid of the invention consists of D-nucleotides, of L-nucleotides or of a combination of both (the combination being, for example, a random combination or a defined sequence of stretches consisting of at least one L-nucleotide and at least one D-nucleic acid), the nucleic acid may consist of deoxyribonucleotides, ribonucleotides or a combination thereof.

The design of the nucleic acids of the invention as L-nucleic acids is advantageous for several reasons. L-nucleic acids are enantiomers of naturally occurring nucleic acids. However, due to the widespread presence of nucleases, D-nucleic acids are not very stable in aqueous solutions and in particular in biological systems or biological samples. Naturally occurring nucleases, in particular those from animal cells, are not able to degrade L-nucleic acids. Thus, the biological half-life of L-nucleic acids is significantly increased in such systems (including animals and humans). Due to the lack of degradability of the L-nucleic acids, no nuclease degradation products and thus no side effects arising therefrom are observed. This aspect defines L-nucleic acids of virtually all other compounds that are useful in the treatment of diseases and/or disorders involving the presence of hepcidin. L-nucleic acids that specifically bind to a target molecule by a mechanism other than Watson-Crick base pairing, or aptamers that are partially or completely composed of L-nucleotides, particularly those portions of the aptamer that are involved in binding of the aptamer to a target molecule, are also known as spiegelmers. As indicated, aptamers (aptamers) are known to those skilled in The art and are described, inter alia, in "The Aptamer Handbook" (eds: Klussmann, 2006).

It is also within the scope of the present invention that the nucleic acids according to the invention can be present as single-stranded or double-stranded nucleic acids, whether they are present as D-nucleic acids, L-nucleic acids or D, L-nucleic acids or whether they are DNA or RNA. Generally, the nucleic acids of the invention are single-stranded nucleic acids which exhibit a secondary structure defined by a primary sequence and thus may also form a tertiary structure. However, the nucleic acid of the invention may also be double-stranded, which means that two strands which are complementary or partially complementary to each other hybridize to each other, irrespective of whether they are two separate strands or whether they are bound (preferably covalently bound) to each other.

The nucleic acids of the invention may be modified. Such modifications may involve individual nucleotides of the nucleic acid and are well known in the art. Examples of such modifications are described in particular in Venkatesan (2003); kusser (2000); aurup (1994); cummins (1995); eaton (1995); green (1995); kawasaki (1993); lesnik (1993); and Miller (1993). Such modifications may be H atoms, F atoms or O-CH atoms at the 2' position of the individual nucleotides that are part of the nucleic acids of the invention₃Radicals or NH₂-a group. Furthermore, the nucleic acid according to the invention may comprise at least one LNA nucleotide. In one embodiment, the nucleic acid according to the invention consists of LNA nucleotides.

In one embodiment, the nucleic acid according to the invention may be a multimeric (multipartite) nucleic acid. As used herein, a "multi-variant nucleic acid" is a nucleic acid consisting of at least two separate nucleic acid strands. These at least two nucleic acid strands form a functional unit, wherein the functional unit is a ligand of a target molecule. The at least two nucleic acid strands may be derived from any of the nucleic acids of the present invention by cleaving the nucleic acid molecule to produce two strands, or by synthesizing one nucleic acid corresponding to a first portion of a (i.e., bulk) nucleic acid of the present invention and another nucleic acid corresponding to a second portion of the bulk nucleic acid. It will be appreciated that both cleavage and synthesis can be used to generate a multi-stranded nucleic acid in which more than two strands as exemplified above are present. In other words, the at least two separate nucleic acid strands are generally distinct from two strands that are complementary and hybridize to each other, although there may be a degree of complementarity between the at least two nucleic acid strands, where such complementarity may result in hybridization of the separate strands.

Finally, it is also within the scope of the present invention that a fully closed (i.e. circular) structure of the nucleic acid according to the invention is achieved, that is to say that the nucleic acid according to the invention is closed in one embodiment, preferably by covalent linkage, wherein more preferably such covalent linkage occurs between the 5 'end and the 3' end of the nucleic acid sequence disclosed herein or any derivative thereof.

One possibility for determining the binding constant of the nucleic acid molecules according to the invention is to use the surface plasmon resonance described in example 4, said example 4 confirming the finding that the nucleic acids according to the invention exhibit a favourable K_DA range of values. A suitable measure for representing the strength of binding between an individual nucleic acid molecule and a target (in this case hepcidin) is the so-called K_DValue of, wherein K_DThe values themselves and the methods for their determination are known to the person skilled in the art.

Preferably, the K displayed by the nucleic acids according to the invention_DValues below 1. mu.M. K of about 1. mu.M_DValues are considered to be characteristic of non-specific binding of nucleic acids to the target. As will be appreciated by those skilled in the art, the K of a group of compounds (e.g.nucleic acids according to the invention)_DThe value is within a certain range. K of about 1. mu.M as described above_DIs preferably K_DUpper limit of value. K binding to nucleic acids of targets_DThe lower limit of (c) may be as small as about 10pM or may be higher. Within the scope of the invention is K binding to the individual nucleic acids of hepcidin_DThe value is preferably within this range. A preferred range may be defined by the selection of any first number within the range and any second number within the range. Preferred upper limit K_DValues of 250nM and 100nM, the preferred lower limit K_DValues were 50nM, 10nM, 1nM, 100pM and 10 pM. More preferred upper limit K_DA value of 2.5nM, more preferably the lower K limit_DThe value was 400 pM.

The nucleic acid molecules according to the invention may be of any length as long as they are still capable of binding to the target molecule. It will be appreciated by those skilled in the art that there are preferred lengths of nucleic acid according to the invention. Typically, the length is between 15 and 120 nucleotides. One skilled in the art will appreciate that any integer between 15 and 120 is a possible length of a nucleic acid according to the invention. More preferred ranges of length of the nucleic acids according to the invention are lengths of about 20 to 100 nucleotides, about 20 to 80 nucleotides, about 20 to 60 nucleotides, about 20 to 50 nucleotides and about 30 to 50 nucleotides.

It is within the scope of the present invention that the nucleic acids disclosed herein comprise a moiety, preferably a high molecular weight moiety and/or preferably a moiety that enables to change the characteristics of the nucleic acids, especially with respect to the residence time in the body of an animal, preferably in a human. Particularly preferred embodiments of such modifications are pegylation and HES-ylation of nucleic acids according to the invention. As used herein, PEG represents polyethylene glycol and HES represents hydroxyethyl starch. As preferably used herein, pegylation is a modification of a nucleic acid according to the invention, wherein such modification consists of a PEG moiety attached to a nucleic acid according to the invention. As preferably used herein, HES-functionalization is a modification of a nucleic acid according to the invention, wherein such modification consists of a HES part attached to a nucleic acid according to the invention. Such modifications as linear polyethylene glycol, branched polyethylene glycol, hydroxyethyl starch, peptides, proteins, polysaccharides, sterols, polyoxypropylene, polyoxyamidates, poly (2-hydroxyethyl) -L-glutamine and polyethylene glycol, and methods of modifying nucleic acids with such modifications are described in european patent application EP 1306382, the disclosure of which is hereby incorporated by reference in its entirety.

Preferably, the molecular weight of the modification consisting of or comprising the high molecular weight moiety is from about 2,000 to 250,000Da, preferably from 20,000 to 200,000 Da. Where PEG is such a high molecular weight moiety, the molecular weight is preferably from 20,000 to 120,000Da, more preferably from 40,000 to 80,000 Da. Where HES is such a high molecular weight fraction, the molecular weight is preferably from 20,000 to 200,000Da, more preferably from 40,000 to 150,000 Da. The HES modification method is described, for example, in German patent application DE 12004006249.8, the disclosure of which is hereby incorporated by reference in its entirety.

It is within the scope of the present invention that either of PEG and HES may be used in linear or branched form, as further described in patent applications WO2005/074993, WO2003/035665 and EP 1496076. In principle, such modifications can be made at any position of the nucleic acid molecule of the invention. Preferably, such modifications are made on the 5 '-terminal nucleotide of the nucleic acid molecule according to the invention, on the 3' -terminal nucleotide of the nucleic acid molecule according to the invention and/or on any nucleotide between the 5 'nucleotide and the 3' nucleotide of the nucleic acid molecule according to the invention.

The modification, preferably PEG and/or HES moieties, may be attached directly or indirectly (preferably via a linker) to the nucleic acid molecule of the invention. It is also within the scope of the present invention that the nucleic acid molecule according to the invention comprises one or more modifications, preferably one or more PEG and/or HES moieties. In one embodiment, a single linker molecule attaches more than one PEG moiety or HES moiety to a nucleic acid molecule according to the invention. The linker used in the present invention may itself be linear or branched. Linkers of this type are known to the person skilled in the art and are further described in patent applications WO2005/074993, WO2003/035665 and EP 1496076.

In a preferred embodiment, the linker is a biodegradable linker. The biodegradable linker enables to change the characteristics of the nucleic acid according to the invention, in particular with respect to the residence time in the animal body, preferably in the human body, as a result of the release of the modification from the nucleic acid according to the invention. The use of a biodegradable linker may enable better control of the residence time of the nucleic acid according to the invention. Preferred embodiments of such biodegradable linkers are the biodegradable linkers described in international patent applications WO2006/052790, WO2008/034122, WO2004/092191 and WO2005/099768, but are not limited thereto.

It is within the scope of the present invention that the modification or modification group is a biodegradable modification, wherein the biodegradable modification may be attached directly or indirectly (preferably via a linker) to the nucleic acid molecule of the invention. The biodegradable modification makes it possible to modify the characteristics of the nucleic acid according to the invention, in particular with respect to the residence time in the animal body, preferably in the human body, as a result of the release or degradation of the modification from the nucleic acid according to the invention. The use of biodegradable modifications may enable better control of the residence time of the nucleic acid according to the invention. Preferred embodiments of such biodegradable modifications are, but not limited to, the biodegradable modifications described in international patent applications WO2002/065963, WO2003/070823, WO2004/113394 and WO2000/41647 (preferably, page 18, lines 4 to 24 of WO 2000/41647).

In addition to the modifications described above, other modifications may be used to alter the characteristics of the nucleic acid according to the invention, wherein such other modifications may be selected from the group consisting of proteins, lipids (e.g. cholesterol) and sugar chains (e.g. amylase, dextran, etc.).

Without wishing to be bound by any theory, it appears that the excretion kinetics is altered by modifying the nucleic acid according to the invention with a high molecular weight moiety, such as a polymer, more particularly one or several of the polymers disclosed herein (which are preferably physiologically acceptable). More specifically, it appears that due to the increased molecular weight of such modified nucleic acids of the invention and due to the fact that the nucleic acids of the invention do not undergo metabolism (particularly in the L form), the excretion from the animal body, preferably from the mammalian body, and more preferably from the human body, is reduced. Since excretion usually occurs through the kidneys, the inventors speculate that: the glomerular filtration rate of the thus modified nucleic acids is significantly reduced compared to nucleic acids without this type of high molecular weight modification, which leads to an increased residence time in the animal. In this connection, it is particularly noteworthy that, despite this high molecular weight modification, the specificity of the nucleic acids according to the invention is not adversely affected. In this case, the nucleic acids according to the invention have, inter alia, the surprising feature that cannot generally be expected from pharmaceutically active compounds, so that pharmaceutical preparations having sustained-release properties are not necessarily required in order to provide sustained release of the nucleic acids according to the invention. Rather, in their modified form comprising high molecular weight moieties, the nucleic acids according to the invention can themselves be used as sustained release formulations, since, owing to their modification, they already behave as if they were released from sustained release formulations. In this case, the modification of the nucleic acid molecule according to the invention disclosed herein and the thus modified nucleic acid molecule according to the invention as well as any composition comprising the same may provide different, preferably controlled pharmacokinetics and biodistribution thereof. This also includes residence time in the cycle and distribution into the tissue. Such modifications are further described in patent application WO 2003/035665.

However, it is also within the scope of the present invention that the nucleic acid according to the invention does not comprise any modification, and in particular does not comprise high molecular weight modifications such as pegylation or HES ylation. Such embodiments are particularly preferred when the nucleic acid according to the invention shows preferential distribution to any target organ or tissue in the body or when it is desired to rapidly eliminate the nucleic acid according to the invention from the body after administration. Nucleic acids according to the invention disclosed herein having preferential distribution characteristics to any target organ or tissue in the body will enable the establishment of effective local concentrations in the target tissue while maintaining low systemic nucleic acid concentrations. This will enable the use of low doses, which is not only advantageous from an economic point of view, but also reduces the unnecessary exposure of other tissues to the nucleic acid agent, thereby reducing the potential risk of side effects. Rapid clearance of a nucleic acid according to the invention from the body after administration may be desirable, especially in the case of in vivo imaging or particular therapeutic dosing requirements using a nucleic acid according to the invention or a medicament comprising the same.

The nucleic acids according to the invention and/or the antagonists according to the invention can be used for producing or producing a medicament or a pharmaceutical composition or for use in producing or producing a medicament or a pharmaceutical composition. Such medicament or pharmaceutical composition according to the invention comprises at least one nucleic acid according to the invention, optionally together with at least one further pharmaceutically active compound, wherein the nucleic acid according to the invention is preferably used as a pharmaceutically active compound per se. In a preferred embodiment, such an agent or pharmaceutical composition comprises at least a pharmaceutically acceptable carrier. Such carriers may be, for example, water, buffer, PBS, glucose solution (preferably 5% glucose in salt equilibrium), starch, sugar, gelatin or any other acceptable carrier substance. Such vectors are generally known to those skilled in the art. It will be appreciated by the skilled person that any embodiment, use or aspect of the medicament of the invention or any embodiment, use or aspect associated therewith may also be applied to the pharmaceutical composition of the invention and vice versa.

The indications, diseases and conditions for which the use or the intended use of the nucleic acids, pharmaceutical compositions and medicaments, respectively according to the invention or prepared according to the invention, for the treatment and/or prophylaxis is due to the direct or indirect involvement of hepcidin in the respective pathogenic mechanism.

As mentioned in the introductory part, hepcidin is a key signal in the regulation of iron homeostasis, with high levels of human hepcidin resulting in reduced serum iron levels and low levels resulting in increased serum iron levels, as shown in a mouse model of hepcidin deficiency and hepcidin overexpression (Nicolas, 2001; Nicolas, 2002; Nicolas, 2003).

As also mentioned herein, the binding of hepcidin to ferroportin leads to immediate internalization of ferroportin and subsequent long-lasting reduction in serum iron (river, 2005), where the reduction in serum iron is responsible for anemia. Anemia is defined as the absolute decrease in the amount of hemoglobin in the circulating blood and is often a symptom of a disease that manifests as low hemoglobin, and is not a diagnosis isolated per se. Anemia is caused by a medical condition that negatively impairs the production and/or life of red blood cells. Additionally, anemia can be the result of blood loss.

Therefore, to understand the development of anemia, anemia is divided into three etiological classes based on the underlying mechanisms:

a) the reduction in the production of red blood cells,

b) increased destruction of red blood cells, and

c) blood loss.

However, these three categories (reduced red blood cell production, increased red blood cell destruction and blood loss) are not strictly distinguished from each other, but can occur concomitantly or independently of each other.

In many diseases, the combination of mechanisms can lead to anemia. Thus, neutralization of hepcidin may be beneficial in many anemic conditions.

Since the hepcidin-binding nucleic acids according to the invention interact with or bind to human hepcidin, the skilled person will appreciate that hepcidin-binding nucleic acids according to the invention can be used for the treatment, prevention and/or diagnosis of any disease in humans and animals as described herein. In this regard, it is to be understood that the nucleic acid molecules according to the invention may be used for the treatment and prevention of any of the diseases, disorders or conditions described herein.

In the following, and without wishing to be bound by any theory, the principle of using the nucleic acid molecules according to the invention is provided for various diseases, disorders and conditions, thus making the claimed therapeutic, prophylactic and diagnostic applicability of the nucleic acid molecules according to the invention seem plausible. In order to avoid any unnecessary repetition, it is understood that, as it relates to hepcidin-ferroportin interactions known to the person skilled in the art and also outlined herein, said interactions can be carried out by the nucleic acid molecules according to the invention, so as to achieve the claimed therapeutic and/or prophylactic effect.

Thus, diseases and/or disorders and/or diseased conditions that may be treated and/or prevented using agents according to the present invention include, but are not limited to, anemia, hypoferremia, pica, conditions with elevated hepcidin levels, conditions with elevated iron levels, and/or conditions with iron overload.

Preferably, the anemia is selected from sideroblasts anemia, hypopigmented microcytic anemia, anemia arising from chronic diseases and/or disorders, anemia arising from inflammation, anemia arising from genetic disorders, anemia arising from acute infections, and/or anemia arising from mutations in genes of iron metabolism and/or homeostasis.

The various chronic diseases and/or disorders that can cause anemia are selected from chronic inflammation, cancer, autoimmune diseases and/or autoimmune disorders, chronic infection, arteriosclerosis, atherosclerosis and cirrhosis of the liver. In such cases, the anemia that can be treated by the nucleic acids of the invention is anemia that is caused by or associated with any of the various chronic diseases and/or disorders described. In addition, the anemia may be anemia arising from a cancer treatment (preferably chemotherapy).

Subpopulations of chronic inflammation are chronic kidney disease, chronic obstructive pulmonary disease, multiple sclerosis, osteoarthritis, diabetes, obesity, cerebrovascular disease, congestive heart failure, myocardial infarction, coronary artery disease, peripheral occlusive artery disease, pancreatitis, vasculitis, wherein such chronic kidney disease includes kidney disease, chronic renal failure, and/or is caused by kidney dialysis or kidney transplantation.

The cancer subpopulations are hepatocellular carcinoma, lymphoma, multiple myeloma, head and neck cancer, breast cancer, colorectal cancer, non-myeloid cancer, renal cell carcinoma, non-small cell lung cancer, tumors, and brain tumors.

Subpopulations of autoimmune diseases and/or disorders are rheumatoid arthritis, irritable bowel syndrome, systemic lupus erythematosus and crohn's disease.

Subgroups of chronic infections are viral infections, viral diseases, bacterial infections and fungal infections, wherein said viral infections include, but are not limited to, hepatitis and HIV infections, and said bacterial infections include, but are not limited to, helicobacter pylori infections.

Anemia arising from inflammation is normocytic to microcytic and is characterized by a low reticulocyte formation index, low or normal Total Iron Binding Capacity (TIBC). In the case of anemia arising from inflammation, hepcidin, acute phase proteins and other inflammatory markers (e.g. C-reactive protein) are increased. Anemia arising from inflammation is also known as inflammatory anemia.

Various genetic disorders that can cause anemia are selected from the group consisting of castleman's disease, zenitler's syndrome, iron refractory iron deficiency anemia (mutations in matriptase-2(TMPRSS 6)), transferrin deficiency anemia, congenital erythropoietic anemia, and hemoglobinopathies.

The various acute infections that can cause anemia are selected from viral infections, bacterial infections, and fungal infections, wherein viral infections, bacterial infections, and fungal infections alone or in combination with each other can cause sepsis.

The term "condition with elevated hepcidin levels" refers to a condition in a mammal (preferably, a human) wherein the hepcidin levels in the body are elevated compared to the normal hepcidin levels of such mammal, e.g. elevated hepcidin serum levels compared to the normal hepcidin serum levels of the mammal (in the case of a human, about 120 ng/mL). Elevated serum hepcidin levels can be determined, inter alia, by enzyme-linked immunoassays (commercially available kits from DRG diagnostics, Marburg, Germany).

Thus, patients to whom the medicament according to the present invention may preferably be applied include, but are not limited to, patients treated with erythropoietin and other red blood cell stimulation therapies and preferably show a low responsiveness to erythropoietin, wherein more preferably the patients have chronic kidney disease or have cancer, wherein the cancer is selected from hepatocellular carcinoma, lymphoma, multiple myeloma, head and neck cancer, breast cancer, colorectal cancer, non-myeloid cancer, renal cell carcinoma, non-small cell lung cancer, tumors, and brain tumors.

In a further embodiment, the medicament according to the invention comprises an additional pharmaceutically active compound. Such additional pharmaceutically active compounds are preferably compounds that can modulate the activity, concentration or expression of hepcidin or ferroportin. Such compounds are preferably inhibitors of pro-hepcidin cleaving proteases, antibodies to pro-hepcidin, antagonists of ferroportin such as antibodies to ferroportin, JAK2 inhibitors, GDF15, BMP modulators, soluble hemojuvelin or TGF-beta inhibitors.

Other additional pharmaceutically active compounds which can be used with or in the medicament comprising a nucleic acid according to the invention are those compounds which are known and/or are used for the treatment of anemia and/or inflammatory conditions, wherein the treatment of said inflammatory conditions has a positive effect on anemia. Such pharmaceutically active compounds are selected from the group comprising iron supplements, vitamin supplements, erythropoiesis-stimulating agents, antibiotics, anti-inflammatory biologicals, inhibitors of the immune system, anti-thrombolytics, statins, vasopressors and compounds affecting muscle contraction.

Non-limiting examples of iron supplements are ferrous sulfate, ferrous gluconate, ferric dextran, ferric sodium gluconate, ferric carboxymaltose, ferric polymaltose hydroxide, ferric fumarate, ferric sucrose, and ferric sucrose hydroxide.

Non-limiting examples of vitamin supplements are vitamin C, folic acid, vitamin B12, vitamin B6, and vitamin D.

Non-limiting examples of erythropoiesis-stimulating agents are erythropoietin, epoetin, darbepotine, CERA, HI F prolyl hydroxylase inhibitors (e.g., FG-2216 and FG-4592), and other agents that stimulate erythropoiesis.

Non-limiting examples of antibiotics are aminoglycosides, β -lactam antibiotics, peptide antibiotics, gyrase inhibitors, lincosamides, macrolide antibiotics, nitroimidazole derivatives, polypeptide antibiotics, sulfonamides, tetracycline and trimethoprim.

Non-limiting examples of anti-inflammatory biologies are:

a) IL-6 receptor antagonists, e.g. Tolizumab or Attlizumab (Atlizumab)

b) TNF antagonists, e.g. etanercept, infliximab, adalimumab, Certolizumab (Certolzumab)

c) IL-1 receptor antagonists, e.g. anakinra, and

d) CD20 binding molecules, such as rituximab and ibermumab (Ibritumab).

Non-limiting examples of inhibitors of the immune system are azathioprine, brequinar, calcineurin inhibitors, chlorambucil, cyclosporin A, deoxyspergualin, leflunomide, methotrexate, mizoribine, mycophenolate mofetil, rapamycin, tacrolimus, and thalidomide.

Non-limiting examples of anti-inflammatory agents are PDE4 inhibitors such as roflumilast, and corticosteroids such as prednisolone, methylprednisolone, hydrocortisone, dexamethasone, triamcinolone, betamethasone, effervescent (effervescet), budesonide, ciclesonide, and fluticasone.

A non-limiting example of an anti-thrombolytic agent is activated human protein C, such as curcurdlan alpha.

Non-limiting examples of statins are atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.

Non-limiting examples of vasopressors and/or compounds that affect muscle contraction (inotropic compounds) are norepinephrine, vasopressin and dobutamine.

In addition to situations with elevated hepcidin plasma levels, the nucleic acid molecules according to the invention may also be used to antagonize hepcidin in patients with elevated iron levels and/or in conditions with iron overload and non-elevated hepcidin plasma levels. Treatment of such patients with a nucleic acid molecule according to the invention is preferably carried out to reduce the cellular iron concentration, wherein the treatment is preferably in combination with an iron chelating compound. The neutralization of physiological hepcidin by the nucleic acid according to the invention protects the expression of ferroportin and thus supports further iron release from intracellular stores. Ferroportin protection in combination with iron chelating compounds removes iron via urine and reduces the total body iron content.

In the medical field, iron overload means the accumulation of iron in the body due to any cause. In the human case, iron overload is characterized by a systemic iron content of > 5 mg. Iron overload is also known as hemochromatosis.

The term "condition with iron overload" refers to a condition in a mammal (preferably, a human) wherein the level of iron in the mammal is elevated compared to the normal iron level of such mammal, for example an elevated iron serum level compared to the normal iron serum level of the mammal (in the case of a human, about 20 μmol/L), or an increased iron level in the liver of the mammal compared to the normal iron level in the liver of the mammal. The elevated serum iron levels are determined by direct measurement of serum iron (among other things using colorimetric assays), by standard transferrin saturation assays (which reveal how much iron is bound to proteins carrying iron in the Blood), or by standard serum Ferritin assays (e.g.: Ferritin Blood Test ELISA kit from Calbiotech, USA). For example, transferrin saturation levels of 45% or higher often indicate abnormally high levels of iron in serum. In particular, elevated iron levels in the liver can be determined by measuring the iron content of the liver from tissue obtained by liver biopsy, or by imaging techniques such as MRI and/or SQUID. The extent of iron levels in other tissues (e.g., brain, heart) can also be assessed using these and other imaging techniques.

Subpopulations of iron overload are transfusional iron overload, iron poisoning, pulmonary hemosiderosis, osteopenia, insulin resistance, african iron overload, hayashi disease, methemoglobinemia, ceruloplasmin deficiency, neonatal hemochromatosis and erythropathies including thalassemia, alpha thalassemia, thalassemia intermedia, sickle cell disease and myelodysplastic syndrome.

Patients suffering from other disorders/diseases associated with elevated iron levels should also benefit from therapy with a nucleic acid molecule according to the invention, preferably in combination with an iron chelating compound. Thus, diseases and/or disorders and/or diseased conditions that may be treated and/or prevented using the agents according to the invention include, but are not limited to, diseases with elevated iron levels, including ataxia, friedreich's ataxia, age-related macular degeneration, age-related cataracts, age-related retinal diseases and neurodegenerative diseases, wherein such neurodegenerative diseases include alzheimer's disease, parkinson's disease, pantothenate kinase-associated neurodegeneration, restless leg syndrome and huntington's chorea.

In a further embodiment, the medicament according to the invention comprises a further pharmaceutically active compound, preferably a compound that can bind iron and remove iron from the tissues of the mammalian body (in particular the human body) or from the circulation. Such pharmaceutically active compounds are preferably selected from iron chelating compounds. The combination of such compounds with the nucleic acid molecule according to the invention will further reduce physiological hepcidin concentrations and thereby reduce the iron burden on the cells.

Non-limiting examples of iron chelating compounds are curcumin, desferrioxamine, deferasirox and deferiprone.

Finally, the additional pharmaceutically active agent may be a modulator of iron metabolism and/or iron homeostasis. Alternatively or additionally, such further pharmaceutically active agent is a further, preferably a second, species of nucleic acid according to the invention. Alternatively, the agent comprises at least one further nucleic acid which binds to a target molecule different from hepcidin or which exhibits a different function than one of the nucleic acids according to the invention. Preferably, such at least one further nucleic acid exhibits a function similar or identical to one of said one or several further pharmaceutically active compounds disclosed herein.

Within the scope of the present invention, an agent comprising a nucleic acid according to the invention (also referred to herein as an agent of the (invention)) is in principle alternatively or additionally used for the prevention of any of the diseases disclosed in connection with the use of said agent for the treatment of said disease. Thus, the respective markers, i.e. the respective markers of the respective diseases, are known to the person skilled in the art. Preferably, the respective marker is hepcidin.

In one embodiment of the agents of the invention, such agents are used in combination with other therapies for any of the diseases disclosed herein (particularly, those to be treated with the agents of the invention).

As preferably used herein, "combination therapy" or "co-therapy" includes the administration of an agent of the invention and at least a second agent as part of a treatment regimen in an attempt to provide a beneficial effect from the combined action of these therapeutic agents, i.e., the agent of the invention and the second agent. Administration of these therapeutic agents as a combination or in a combined manner is typically carried out over a defined period of time (typically minutes, hours, days or weeks depending on the combination selected).

"combination therapy" is intended to include the administration of two or more therapeutic agents as part of separate monotherapy regimens that occasionally and variably result in the combination of the invention. "combination therapy" is intended to include the administration of these therapeutic agents in a sequential manner, that is, where each therapeutic agent is administered at a different time, as well as the administration of these therapeutic agents or at least two of the therapeutic agents in a substantially simultaneous manner. Substantially simultaneous administration may be accomplished, for example, by: a single capsule having a fixed ratio of each therapeutic agent or multiple single capsules for each therapeutic agent is administered to the subject.

Sequential administration or substantially simultaneous administration of the therapeutic agents may be achieved by any suitable route, including but not limited to topical, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents may be administered by the same route or by different routes. For example, a first therapeutic agent of a particular combination of therapeutically effective agents may be administered by injection, while another or other therapeutic agents of the combination may be administered topically.

Alternatively, for example, all therapeutic agents may be administered topically or all therapeutic agents may be administered by injection. The order of administration of the therapeutic agents is not critical unless otherwise indicated. If the combination therapy also includes a non-drug treatment, the non-drug treatment can be carried out at any suitable time so long as the beneficial effect obtained from the combination of the therapeutic agent and the non-drug treatment is achieved. For example, where appropriate, beneficial effects may still be achieved when the non-drug treatment is delayed in time, perhaps for days or even weeks, while the therapeutic agent is still being administered.

As discussed in the above general terms, the medicament according to the invention may in principle be administered in any form known to the person skilled in the art. The preferred route of administration is systemic, more preferably it is by parenteral administration, preferably by injection. Alternatively, the agent may be administered topically. Other routes of administration include intramuscular, intraperitoneal, subcutaneous, oral, intranasal, intratracheal and pulmonary administration, with the preferred route of administration being the least invasive and ensuring efficacy.

Parenteral administration is commonly used for subcutaneous, intramuscular or intravenous injection and infusion. In addition, one method for parenteral administration employs implantation of a sustained release or sustained release system, which ensures that a constant dosage level is maintained, and is well known to those skilled in the art.

In addition, preferred agents of the invention may be administered by the intranasal route by topical use of a suitable intranasal vehicle (vehicle), inhalant, or may be administered by the transdermal route in the form of transdermal patches well known to those skilled in the art. For administration in the form of a transdermal delivery system, the dosage will generally be administered continuously rather than intermittently throughout the dosage regimen. Other preferred topical formulations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of the active ingredient will typically range from 0.01% to 15% (w/w or w/v).

The agents of the invention will generally comprise an active ingredient in an amount effective for the therapy, including but not limited to the nucleic acid molecules of the invention, preferably dissolved or dispersed in a pharmaceutically acceptable medium. Pharmaceutically acceptable media or carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients may also be incorporated into the agents of the present invention.

In a further aspect, the present invention relates to a pharmaceutical composition. Such pharmaceutical compositions comprise at least one nucleic acid according to the invention, and preferably a pharmaceutically acceptable vehicle. Such a vehicle may be any vehicle or any binder used and/or known in the art. More particularly, such binders or vehicles are any of those discussed in relation to the preparation of the agents disclosed herein. In a further embodiment, the pharmaceutical composition comprises an additional pharmaceutically active agent.

The preparation of pharmaceutical agents and pharmaceutical compositions, respectively, is known to those skilled in the art in light of the present disclosure. Typically, such compositions may be prepared as injectables (as liquid solutions or suspensions); prepared in solid form suitable for dissolution or suspension in a liquid prior to injection; tablets or other solid dosage forms prepared for oral administration; preparing into timed release capsule; or in any other form currently used, including eye drops, creams, lotions, ointments, inhalants, and the like. It may also be particularly useful for a surgeon, physician or health care worker to treat a particular area in the surgical field with a sterile formulation (e.g., a saline-based wash solution). The composition may also be delivered by a microdevice, microparticle or sponge.

In this case, the amount of active ingredient to be administered and the volume of the composition depend on the individual or subject to be treated. The specific amount of active compound necessary for administration depends on the judgment of the practitioner and is specific to each individual.

It is common to utilize the minimum volume of medicament necessary to disperse the active compound. Suitable administration regimens are also variable, but are typically: the compound is administered initially and the results monitored, then further controlled doses are provided at further intervals.

For example, for oral administration in the form of a tablet or capsule (e.g., a gelatin capsule), the active pharmaceutical ingredient, i.e., the nucleic acid molecule according to the invention and/or any additional pharmaceutically active agent (which will also be collectively referred to herein as a therapeutic agent or active compound), can be combined with an oral, non-toxic, pharmaceutically acceptable and preferably inert carrier, e.g., ethanol, glycerol, water, and the like. Moreover, suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture, as desired or needed. Suitable binders include starch, magnesium aluminum silicate, starch slurry, gelatin, methyl cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talc, stearic acid, its magnesium or calcium salt, and/or polyethylene glycol and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum starch, agar, alginic acid or its sodium salt, effervescent mixtures, and the like. Diluents include, for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and/or glycine.

The medicament according to the present invention may also be administered in oral dosage forms such as timed release and sustained release tablets or capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions. Advantageously, suppositories are prepared from fat emulsions or suspensions.

The pharmaceutical compositions or medicaments according to the invention can be sterilized and/or contain adjuvants, such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters (solvothermotors), salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods and typically contain from about 0.1% to 75%, preferably from about 1% to 50% of the active ingredient.

Liquid, in particular injectable, compositions may be prepared, for example, by dissolution, dispersion, or the like. The active compound is dissolved in or mixed with a pharmaceutically pure solvent (e.g., water, saline, aqueous dextrose, glycerol, ethanol, etc.) to form an injectable solution or suspension. In addition, solid forms suitable for dissolution in liquid prior to injection may be formulated.

For solid compositions, excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. The active compounds defined above may also be formulated as suppositories with, for example, a polyalkylene glycol such as propylene glycol as the carrier. In some embodiments, suppositories are advantageously prepared from fat emulsions or suspensions.

The agents and nucleic acid molecules of the invention may also be administered in the form of liposome delivery systems such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles, respectively. Liposomes can be formed from a variety of phospholipids, including cholesterol, stearylamine or phosphatidylcholines. In some embodiments, the film of lipid component is hydrated with an aqueous solution of the drug to form a lipid layer encapsulating the drug, as is well known to those skilled in the art. For example, the nucleic acid molecules according to the invention may be provided as complexes with lipophilic compounds or non-immunogenic high molecular weight compounds constructed using methods known in the art. Additionally, liposomes may carry such nucleic acid molecules on their surface for targeting and carrying cytotoxic agents internally to mediate cell killing. Examples of nucleic acid-related complexes are provided in U.S. patent No. 6,011,020.

The agents and nucleic acid molecules of the invention may also be coupled to soluble polymers as targetable drug carriers, respectively. Such polymers may include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxide polylysine, substituted with palmitoyl residues. Furthermore, the agents and nucleic acid molecules of the present invention may be coupled to a class of biodegradable polymers useful for achieving controlled release of drugs, such as polylactic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and crosslinked or amphipathic block copolymers of hydrogels, respectively.

The pharmaceutical compositions and medicaments to be administered may also contain, respectively, if desired, some, usually minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents and other substances such as sodium acetate and triethanolamine oleate.

The dosage regimen for each of the use of the nucleic acid molecules and agents of the invention may be selected in accordance with a variety of factors, including the type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; kidney and liver function in the patient; and the particular nucleic acid or salt thereof employed according to the invention. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.

In the treatment of any of the diseases disclosed herein, the effective plasma level of the nucleic acid according to the invention preferably ranges from 500fM to 500. mu.M.

Individually, the nucleic acid molecules and agents of the invention may preferably be administered in a single daily dose, every second or third day, weekly, biweekly, in a single monthly dose, or every third month.

It is within the scope of the present invention that the agents described herein constitute the pharmaceutical compositions disclosed herein.

In a further aspect, the present invention relates to a method for the treatment of a subject in need of such treatment, wherein the method comprises administering a pharmaceutically effective amount of at least one nucleic acid according to the invention. In one embodiment, the subject has a disease or is at risk of developing such a disease, wherein the disease is any of those disclosed herein, in particular any of those disclosed in relation to the use of any nucleic acid according to the invention for the preparation of a medicament.

It will be appreciated that the nucleic acids and antagonists according to the invention may be used not only as medicaments or for the preparation of medicaments, but also for cosmetic purposes, in particular in connection with regional skin lesions where hepcidin is involved in inflammation. Thus, other conditions or diseases that can be treated or prevented using the nucleic acids, agents and/or pharmaceutical compositions according to the invention are inflamed regional skin lesions.

As preferably used herein, the diagnostic or diagnostic agent or diagnostic tool is suitable for the direct or indirect detection of hepcidin, preferably hepcidin as described herein, and more preferably hepcidin as described herein in relation to the various conditions and diseases described herein. The diagnosis is suitable for detecting and/or following any of the conditions and diseases each described herein. Such detection is made possible by the binding of the nucleic acid according to the invention to hepcidin. This binding can be detected directly or indirectly. Corresponding methods and tools are known to the person skilled in the art. In particular, the nucleic acid according to the invention may comprise a label enabling the detection of the nucleic acid according to the invention, preferably the nucleic acid bound to hepcidin. Such labels are preferably selected from the group comprising radioactive labels, enzymatic labels and fluorescent labels. In principle, all known assays developed for antibodies can be employed and adapted for use with the nucleic acids according to the invention, wherein the target-binding antibody is replaced by the target-binding nucleic acid of the invention. In antibody-assays using unlabeled target-binding antibodies, the detection is preferably performed by a secondary antibody that is modified with radiolabel, enzyme label and fluorescent label and binds the target-binding antibody at the Fc-fragment of the target-binding antibody. In the case of nucleic acids, preferably according to the invention, the nucleic acids are modified with such labels, wherein preferably such labels are selected from the group comprising biotin, Cy-3 and Cy-5, and such labels are detected by antibodies against such labels, such as avidin, anti-Cy 3 or anti-Cy 5 antibodies, or in the case of the label being biotin, by streptavidin or avidin which naturally binds biotin. Such antibodies, streptavidin or avidin are then preferably modified with a corresponding label, such as a radiolabel, an enzymatic label or a fluorescent label, as are secondary antibodies that allow their detection.

In a further embodiment, the nucleic acid molecule according to the invention is detected or analyzed by a second detection means, wherein the second detection means is a molecular beacon. The methodology of molecular beacons is known to those skilled in the art.

Briefly, a nucleic acid probe (which is also referred to as a molecular beacon) is the reverse complement of a nucleic acid to be detected and thus hybridizes to part or all of the nucleic acid to be detected. Upon binding to the nucleic acid to be detected, the fluorophores of the molecular beacon separate, which results in a change in the fluorescent signal, preferably in the intensity. This change is related to the amount of nucleic acid to be detected.

It will be appreciated that the detection of hepcidin with a nucleic acid according to the invention will in particular enable the detection of hepcidin as defined herein.

With respect to the detection of hepcidin, a preferred method comprises the following steps:

(a) providing a sample to be tested for the presence of hepcidin,

(b) providing a nucleic acid according to the invention,

(c) reacting said sample with said nucleic acid, preferably in a reaction vessel,

wherein step (a) may be performed before step (b), or step (b) may be performed before step (a).

In a preferred embodiment, an additional step d) is provided, which consists in detecting whether the nucleic acid has reacted with hepcidin. Preferably, the nucleic acid of step b) is immobilized to a surface. The surface may be the surface of a reaction vessel, such as a reaction tube, a well on a plate, or may be the surface of a device (e.g., a bead) contained in such a reaction vessel. Immobilization of the nucleic acid to the surface may be accomplished by any means known to those skilled in the art, including but not limited to non-covalent or covalent attachment. Preferably, the linkage is established by a covalent chemical bond between the surface and the nucleic acid. However, it is also within the scope of the present invention to indirectly immobilize the nucleic acids to the surface, wherein such indirect immobilization involves the use of additional components or a pair of interaction partners. Such further components are preferably compounds which specifically interact with the nucleic acid to be immobilized (which are also referred to as interaction partners) and thus mediate the attachment of the nucleic acid to the surface. The interaction partner is preferably selected from the group comprising nucleic acids, polypeptides, proteins and antibodies. Preferably, the interaction partner is an antibody, more preferably a monoclonal antibody. Alternatively, the interaction partner is a nucleic acid, preferably a functional nucleic acid. More preferably, such functional nucleic acids are selected from aptamers, spiegelmers and nucleic acids at least partially complementary to the nucleic acids. In a further alternative embodiment, the binding of the nucleic acid to the surface is mediated by a multi-partition interaction partner (multi-partition interaction partner). Such a multi-variant interaction partner is preferably a pair of interaction partners or an interaction partner consisting of a first member and a second member, wherein the first member is comprised in or attached to the nucleic acid and the second member is attached to or comprised within the surface. The multi-part interaction partner is preferably selected from the group consisting of pairs of interaction partners comprising biotin and avidin, biotin and streptavidin, and biotin and neutravidin. Preferably, the first member of the interaction partner pair is biotin.

A preferred result of such a method for detecting hepcidin is that an immobilized complex of hepcidin and the nucleic acid is formed, wherein more preferably the complex is detected. Within the scope of embodiments, hepcidin contained in or released from the complex is detected.

The corresponding detection means suitable for detecting said hepcidin are any detection means specific for hepcidin. Particularly preferred detection means are detection means selected from the group comprising nucleic acids, polypeptides, proteins and antibodies.

The method for detecting hepcidin according to the invention further comprises removing the sample from the reaction vessel that has preferably been used for carrying out step c).

In a further embodiment, the method of the invention further comprises the step of immobilizing an interaction partner of hepcidin on a surface (preferably a surface as defined above), wherein the interaction partner is as defined herein, and preferably as defined above in relation to the respective method, and more preferably comprises in their various embodiments nucleic acids, polypeptides, proteins and antibodies. In this embodiment, a particularly preferred detection means is a nucleic acid according to the invention, wherein such nucleic acid may be labeled or unlabeled. Where such nucleic acids are labeled, the nucleic acid comprises a detection label. Such detection labels may be detected directly or indirectly. In various embodiments described herein, such detection may also involve the use of a second detection means, which is also preferably selected from the group comprising nucleic acids, polypeptides, proteins and antibodies. Such second detection means are preferably specific for the nucleic acid according to the invention and, in case the nucleic acid according to the invention comprises a detection label, such second detection means are specific for said detection label. In a more preferred embodiment, the second detection means is a molecular beacon. It is also within the scope of the present invention that the second detection means in a preferred embodiment comprises a detection label. Whether or not comprised by a nucleic acid according to the invention or by the second detection means, the detection label is preferably selected from the group comprising biotin, bromodeoxyuridine labels, digoxigenin labels, fluorescent labels, UV-labels, radioactive labels and chelator molecules. Particularly preferred combinations are as follows:

the detection label attached to the nucleic acid according to the invention is biotin and the second detection means is an antibody directed against biotin, or wherein

The detection label attached to the nucleic acid according to the invention is biotin and the second detection means is avidin or a molecule carrying avidin, or wherein

The detection label attached to the nucleic acid according to the invention is biotin and the second detection means is streptavidin or a molecule carrying streptavidin, or wherein

The detection label attached to the nucleic acid according to the invention is biotin and the second detection means is neutravidin or a molecule carrying neutravidin, or wherein

The detection label attached to the nucleic acid according to the invention is bromodeoxyuridine and the second detection means is an antibody directed against bromodeoxyuridine, or wherein

The detection label attached to the nucleic acid according to the invention is digoxigenin and the second detection means is an antibody directed against digoxigenin, or wherein

The detection label attached to the nucleic acid according to the invention is a chelator and the second detection means is a radionuclide,

wherein preferably the detection label is attached to a nucleic acid according to the invention.

It will be appreciated that these types of combinations may also be applied to embodiments in which the nucleic acid according to the invention is attached to a surface. In such embodiments, it is preferred that the detection label is attached to the second detection means, i.e. preferably the interaction partner.

Finally, it is also within the scope of the invention to use a third detection means for detecting the second detection means, preferably the third detection means is an enzyme, more preferably an enzyme showing an enzymatic reaction after reaction with the second detection means. Alternatively, the third detection means is a means for detecting radiation, more preferably radiation emitted by a radionuclide attached to the nucleic acid according to the invention or the second detection means (preferably the second detection means). Preferably, the third detection means specifically detects and/or interacts with the second detection means.

Furthermore, in embodiments wherein the interaction partner of hepcidin is immobilized on a surface and the nucleic acid according to the invention is preferably added to the complex formed between the interaction partner and the hepcidin, the sample may be removed from the reaction system, more preferably from the reaction vessel in which step c) and/or d) is performed.

In one embodiment, the nucleic acid according to the invention comprises a fluorescent moiety, and wherein the fluorescence of the fluorescent moiety is different when forming a complex between the nucleic acid and hepcidin on the one hand and in the case of the nucleic acid and free hepcidin on the other hand.

In a further embodiment, the nucleic acid used in the method for detecting hepcidin according to the invention is a derivative of a nucleic acid according to the invention, wherein the derivative of the nucleic acid comprises at least one fluorescent derivative of adenosine in place of adenosine. In a preferred embodiment, the fluorescent derivative of adenosine is vinylidene adenosine.

In a further embodiment, fluorescence is used to detect a complex consisting of a derivative of a nucleic acid according to the invention and hepcidin.

In one embodiment of the method, a signal is generated in step (c) or step (d), and preferably, the signal is correlated with the concentration of hepcidin in the sample.

In a preferred embodiment, the method may be performed in a 96-well plate, wherein the components are immobilized in a reaction vessel as described above and the wells are used as reaction vessels.

The nucleic acids of the invention can further be used as starting materials for drug design. There are mainly two possible approaches. One approach is to screen compound libraries, wherein such compound libraries are preferably low molecular weight compound libraries. In one embodiment, the screening is a high throughput screening. Preferably, high throughput screening is a rapid and efficient trial and error (trial-and-error) assessment of compounds in a target-based assay. In the best case, the assay format of the target-based assay is based on colorimetric measurements. Libraries used in connection therewith are known to the person skilled in the art.

Alternatively, the nucleic acids according to the invention can be used for rational design of medicaments. Preferably, the rational drug design is the design of a pharmacological lead structure. Starting from the three-dimensional structure of the target, a database containing the structures of many different compounds is searched once with a computer program, which is usually determined by methods such as X-ray crystallography or nuclear magnetic resonance spectroscopy. This selection is done by computer and the identified compounds can then be tested in the laboratory.

Rational drug design may start from any nucleic acid according to the invention and relate to a structure, preferably a three-dimensional structure, similar to or identical to that part of the structure of the nucleic acid of the invention which mediates the binding of the nucleic acid to the target, i.e. hepcidin. In a further step or as an alternative step in rational drug design, the (preferably) three-dimensional structure of those parts of the nucleic acid that bind to hepcidin is mimicked with a chemical group different from nucleotides and nucleic acids. By means of this simulation, compounds different from the nucleic acids according to the invention can be designed. Such compounds are preferably small molecules or peptides.

In case the compound library is screened, for example by using competitive assays known to the person skilled in the art, suitable hepcidin analogues, hepcidin agonists or hepcidin antagonists can be found. Such a competitive assay can be established as follows. The nucleic acid of the invention (preferably, a spiegelmer aptamer of an L-nucleic acid as a binding target) is coupled to a solid phase. To identify hepcidin analogs, labeled hepcidin may be added to the assay. A potential analogue will compete with the hepcidin molecule for binding to the spiegelmer, which will be accompanied by a reduction in the signal obtained by the corresponding label. Screening for agonists or antagonists may involve the use of cell culture assays known to those skilled in the art.

The kit according to the invention may comprise at least one or several nucleic acids according to the invention. In addition, the kit may comprise at least one or several positive or negative controls. The positive control may for example be hepcidin, in particular hepcidin bound by the nucleic acid of the invention, wherein preferably the positive control is present in liquid or lyophilized form. The negative control may, for example, be a peptide defined on the basis of biophysical properties similar to hepcidin, but which is not recognized by the nucleic acid of the invention. In addition, the kit may further comprise one or several buffers. The various components may be included in the kit in dry or lyophilized form, or may be dissolved or dispersed in a liquid. The kit may further comprise one or more containers which, in turn, may contain one or more of the components of the kit. In still further embodiments, the kit further comprises instructions or instruction leaflet providing information to the user about how to use the kit and its various components.

The quantification of the nucleic acids according to the invention in several body fluids, tissues and organs of the human or non-human body is essential for the evaluation of the pharmacokinetic and pharmacodynamic properties of the nucleic acids according to the invention. For this purpose, any detection method disclosed herein or known to the person skilled in the art using nucleic acids according to the invention can be employed. In a further aspect of the invention, a sandwich hybridization assay for detecting a nucleic acid according to the invention is provided. For such sandwich hybridization assays, capture probes and detection probes are used. The capture probe is substantially complementary to a first part of the nucleic acid according to the invention and the detection probe is substantially complementary to a second part of the nucleic acid according to the invention. Both the capture probes and the detection probes may be formed from DNA nucleotides, modified RNA nucleotides, LNA nucleotides and/or PNA nucleotides.

Thus, the capture probe comprises a stretch of nucleotide sequence substantially complementary to the 5 '-end of the nucleic acid according to the invention, whereas the detection probe comprises a stretch of nucleotide sequence substantially complementary to the 3' -end of the nucleic acid according to the invention. In this case, the capture probe is immobilized via its 5 ' -end to a surface or a substrate, wherein the capture probe may be immobilized directly at its 5 ' -end or via a linker between its 5 ' -end and the surface or substrate. In principle, however, the linker may be attached to each nucleotide of the capture probe. The linker may be formed from a hydrophilic linker, or from a D-DNA nucleotide, a modified D-DNA nucleotide, a D-RNA nucleotide, a modified D-RNA nucleotide, a D-LNA nucleotide, a PNA nucleotide, an L-RNA nucleotide, an L-DNA nucleotide, a modified L-RNA nucleotide, a modified L-DNA nucleotide and/or an L-LNA nucleotide, as known to the person skilled in the art.

Alternatively, the capture probe may comprise a stretch of nucleotide sequence substantially complementary to the 3 '-end of the nucleic acid according to the invention, while the detection probe comprises a stretch of nucleotide sequence substantially complementary to the 5' -end of the nucleic acid according to the invention. In this case, the capture probe is immobilized via its 3 ' -end to a surface or a substrate, wherein the capture probe may be immobilized directly at its 3 ' -end or via a linker between its 3 ' -end and the surface or substrate. In principle, however, the linker may be attached to each nucleotide of a stretch of nucleotide sequences which is substantially complementary to the nucleic acid according to the invention. The linker may be formed from a hydrophilic linker, or from a D-DNA nucleotide, a modified D-DNA nucleotide, a D-RNA nucleotide, a modified D-RNA nucleotide, a D-LNA nucleotide, a PNA nucleotide, an L-RNA nucleotide, an L-DNA nucleotide, a modified L-RNA nucleotide, a modified L-DNA nucleotide and/or an L-LNA nucleotide, as known to the person skilled in the art.

The number of nucleotides of the capture probe and the detection probe, respectively, which can hybridize with the nucleic acid according to the invention is variable and can depend on the number of nucleotides of the capture probe and/or the detection probe and/or the nucleic acid according to the invention itself. The maximum value for the total number of nucleotides of the capture probe and the detection probe that can hybridize to a nucleic acid according to the invention should be the number of nucleotides comprised by the nucleic acid according to the invention. The minimum number of individual nucleotides of the detection probe and the capture probe (typically 2 to 10 nucleotides) should be such as to enable hybridization to the 5 '-end or the 3' -end, respectively, of the nucleic acid according to the invention.

Furthermore, the detection probe preferably carries a detectable marker molecule or label as described previously herein. In principle, the label or marker molecule may be attached to each nucleotide of the detection probe or to each part of the detection probe. Preferably, the label or marker is located at the 5 '-end or at the 3' -end of the detection probe, wherein a linker may be inserted between the nucleotide in the detection probe that is complementary to the nucleic acid according to the invention and the label. The linker may be formed from a hydrophilic linker, or from a D-DNA nucleotide, a modified D-DNA nucleotide, a D-RNA nucleotide, a modified D-RNA nucleotide, a D-LNA nucleotide, a PNA nucleotide, an L-RNA nucleotide, an L-DNA nucleotide, a modified L-RNA nucleotide, a modified L-DNA nucleotide and/or an L-LNA nucleotide, as known to the person skilled in the art.

In one embodiment of the method for detecting hepcidin, the detection of the nucleic acid according to the invention can be carried out as follows:

the nucleic acid according to the invention is hybridized with a capture probe at one end and a detection probe at the other end. Unbound detection probes, i.e. detection probes which are not bound to the nucleic acid according to the invention, are then removed, for example by one or more washing steps. Subsequently, the amount of bound detection probe, which preferably carries a label or a marker molecule, may be measured, e.g. as outlined in more detail in WO/2008/052774 (which is incorporated herein by reference).

As preferably used herein, the term "treatment" additionally or alternatively includes prophylaxis and/or follow-up in a preferred embodiment.

As preferably used herein, the terms "disease" and "condition" should be used interchangeably if not indicated to the contrary.

As used herein, the term "comprising" is preferably not intended to limit subject matter preceding or described by the term. However, in an alternative embodiment, the term "comprising" should be interpreted in an inclusive sense and thus should be interpreted as limiting the subject matter preceding the term or the subject matter described by the term.

The individual SEQ ID No. s, chemical properties, their actual sequences, and internal reference numbers of the nucleic acid molecules and target molecules hepcidin according to the invention used herein are summarized in the following table.

The invention is further illustrated by the accompanying drawings, examples and sequence listing from which further features, embodiments and advantages can be obtained

FIGS. 1 and 2 show an alignment of the sequences of type A hepcidin binding nucleic acids;

FIG. 3 shows derivatives of type A hepcidin binding nucleic acids 223-C5-001;

FIG. 4 shows a derivative of type A hepcidin binding nucleic acid 229-B1-001;

FIG. 5 shows an alignment of the sequences of type B hepcidin binding nucleic acids;

FIG. 6 shows derivatives of type B hepcidin-binding nucleic acids 238-D4-001;

FIG. 7 shows an alignment of the sequences of type C hepcidin binding nucleic acids;

FIG. 8 shows derivatives of hepcidin-binding nucleic acids 238-C4-001 in form C;

FIG. 9 shows an alignment of the sequences of other hepcidin binding nucleic acids;

FIG. 10 shows data on the binding of hepcidin-binding nucleic acids 223-C5-001, 229-B1-002, 238-C4-006, 238-D4-001 and 238-D4-008 to human hepcidin-25, cynomolgus monkey hepcidin-25, marmoset hepcidin-25, mouse hepcidin-25 and rat hepcidin-25;

FIG. 11 shows data on the binding of hepcidin-binding nucleic acids 223-C5-001, 229-B1-002, 238-C4-006, 238-D4-001 and 238-D4-008 to human hepcidin-25, hepcidin-22 and hepcidin-20;

FIG. 12 shows data on the binding of hepcidin-binding nucleic acids 223-C5-001-5 ' -PEG, 229-B1-002-5 ' -PEG, 238-C4-006-5 ' -PEG, 238-D4-002-5 ' -PEG and 238-D4-008-5 ' -PEG to human hepcidin-25;

FIG. 13 shows a Biacore 2000 sensorgram (sensorgram) indicating K binding of an aptamer of type A hepcidin-binding nucleic acid 223-C5-001 to biotinylated human D-hepcidin-25 at 37 ℃_DValues, wherein the biotinylated human D-hepcidin is immobilized on a streptavidin-conjugated sensor chip by a streptavidin coupling process at 37 ℃, expressed as a time-varying Response (RU);

FIGS. 14 and 15 show the results of a fractionation test for comparing hepcidin-binding nucleic acids with each other and identifying the best hepcidin-binding nucleic acids, wherein the type A hepcidin-binding nucleic acid 223-C5-001 is labeled and binding of the nucleic acid 223-C5-001 to biotinylated human D-hepcidin-25 is performed in the presence of 10, 50 or 250nM unlabeled competitor RNA (different types of hepcidin-binding nucleic acids), respectively, at 37 ℃ as binding of 223-C5-001 as a function of the concentration of biotinylated human D-hepcidin-25 ("competitive pull-down assay");

FIG. 16 shows a Biacore 2000 sensorgram, whichIndicating that an aptamer of type A hepcidin-binding nucleic acid 229-B1-001 binds K to biotinylated human D-hepcidin-25 at 37 ℃_DValues, wherein the biotinylated human D-hepcidin-25 was immobilized on a streptavidin-conjugated sensor chip by a streptavidin coupling process at 37 ℃, expressed as a time-varying Response (RU);

FIGS. 17A/17B show the effect of the spiegelmers 223-C5-001-5 ' -PEG, 238-D4-008-5 ' -amino, and 238-D4-008-5 ' -PEG (═ NOX-H94) on the effect of human hepcidin-25 on iron-induced ferroportin upregulation, wherein lysates obtained from J774.1 cells following stimulation with either human hepcidin-25 or hepcidin-25 + spiegelmers were separated by SDS-gel electrophoresis and analyzed by Western blotting using antibodies against mouse ferroportin;

FIG. 18 shows the effect of the spiegelmer 223-C5-001-5 '-PEG on hepcidin activity in vivo, where the reduction in serum iron by human hepcidin was completely blocked by administration of the spiegelmer 223-C5-001-5' -PEG prior to injection of human hepcidin;

FIG. 19 shows a Biacore 2000 sensorgram indicating the K-linked spisomer of the hepcidin-binding nucleic acid NOX-H94(═ 238-D4-008-5' -PEG) and biotinylated human L-hepcidin at 37 deg.C_DValues, wherein the biotinylated human L-hepcidin is immobilized on a streptavidin-conjugated sensor chip by a streptavidin coupling process at 37 ℃, expressed as a time-varying Response (RU);

figure 20 shows the effect of the spiegelmer aptamer NOX-H94(═ 238-D4-008-5 '-PEG) on hepcidin activity in vivo, where the reduction in serum iron by human hepcidin was completely blocked by administration of the spiegelmer aptamer NOX-H94(═ 238-D4-008-5' -PEG) prior to injection of human hepcidin;

figure 21 shows the effect of the spiegelmer NOX-H94(═ 238-D4-008-5' -PEG) in an animal model of anemia of inflammation (cynomolgus monkey), where IL-6 induces hepcidin secretion, which subsequently leads to anemia in non-human primates; in this experiment, human IL-6 caused a reduction in serum iron concentration to 27% of the pre-dose (predose) value of vehicle/IL-6 treated monkeys, which was completely blocked by the administration of the spiegelmer aptamer 238-D4-008-5' -PEG prior to injection of human IL-6.

Example 1: nucleic acids binding to human hepcidin

By using biotinylated human D-hepcidin-25 as a target, several nucleic acids binding to human hepcidin (in particular, human hepcidin-25, human hepcidin-22 and human hepcidin-20) can be generated: their nucleotide sequences are depicted in fig. 1 to 9. These nucleic acids were characterized as follows: characterization was performed at the aptamer (i.e., D-nucleic acid) level using a direct pull-down assay using biotinylated human D-hepcidin-25 (example 3), a competitive pull-down assay (example 3) and/or surface plasmon resonance measurement (example 4), or at the spiegelmer (i.e., L-nucleic acid) level using the natural configuration of human hepcidin-25 (human L-hepcidin-25) in competitive pull-down assay (example 3), surface plasmon resonance measurement (example 4), in vitro assay (example 5) and/or in vivo assay (examples 6 and 7). The spiegelmers and aptamers were synthesized as described in example 2.

The nucleic acid molecules so produced show different sequence motifs, of which three main types were identified and defined as type a, type B and type C hepcidin binding nucleic acids and are depicted in fig. 1 to 8.

For the definition of nucleotide sequence motifs, IUPAC abbreviations are used for undefined nucleotides:

s is strong G or C;

w is weak A or U;

r purine G or A;

y pyrimidine C or U;

k is keto G or U;

m imino A or C;

b is not A C or U or G;

d is not C A or G or U;

h is not G A or C or U;

v is not U A or C or G;

n is all A or G or C or U.

If this is not indicated to the contrary, any nucleic acid sequences or sequence segments and the sequences of the cassettes are shown in the 5 '- > 3' orientation, respectively.

1.1 type A hepcidin-binding nucleic acids

As depicted in fig. 1, fig. 2, fig. 3 and fig. 4, a type a hepcidin-binding nucleic acid comprises one central nucleotide sequence segment, wherein the central nucleotide sequence segment comprises at least two nucleotide sequence segments (also referred to herein as nucleotide cassettes) defining a potential hepcidin-binding motif: a first nucleotide sequence segment cassette a and a second nucleotide sequence segment cassette B.

The first and second nucleotide sequence segment cassettes a and B are linked to each other by a linking nucleotide sequence segment.

In the linking nucleotide sequence segment, some nucleotides may hybridize to each other, wherein upon hybridization a double-stranded structure is formed. However, such hybridization is not necessarily given in the molecule.

Typically, a type a hepcidin-binding nucleic acid comprises terminal nucleotide sequence segments at its 5 '-end and 3' -end: a first terminal stretch of nucleotides and a second terminal stretch of nucleotides. The first terminal stretch of nucleotides and the second terminal stretch of nucleotides may hybridize to each other, wherein upon hybridization a double-stranded structure is formed. However, such hybridization is not necessarily given in the molecule.

The five nucleotide sequence segments (box a, box B, linking nucleotide sequence segment, first terminal nucleotide sequence segment and second terminal nucleotide sequence segment) of the type a hepcidin-binding nucleic acid may be arranged differently from each other: a first terminal nucleotide sequence segment-box a-linking nucleotide sequence segment-box B-second terminal nucleotide sequence segment, or a first terminal nucleotide sequence segment-box B-linking nucleotide sequence segment-box a-second terminal nucleotide sequence segment.

However, the five nucleotide sequence segments (box a, box B, linker nucleotide sequence segment, first terminal nucleotide sequence segment and second terminal nucleotide sequence segment) of the type a hepcidin-binding nucleic acids can also be arranged relative to each other as follows: a second terminal nucleotide sequence segment-box a-linking nucleotide sequence segment-box B-first terminal nucleotide sequence segment, or a second terminal nucleotide sequence segment-box B-linking nucleotide sequence segment-box a-first terminal nucleotide sequence segment.

The sequence of the defined cassette or nucleotide sequence segments may differ between type a hepcidin-binding nucleic acids, which affects the binding affinity for human hepcidin (in particular, human hepcidin-25). Based on the binding analysis of the different type a hepcidin binding nucleic acids, cassette a and cassette B described hereinafter and their nucleotide sequences are necessary for binding human hepcidin (in particular human hepcidin-25) individually, more preferably in their entirety.

Type a hepcidin-binding nucleic acids according to the invention are shown in figures 1 to 4. They were all tested as aptamers and/or spiegelmers for their ability to bind to human hepcidin-25 (more precisely, biotinylated human D-hepcidin-25 and biotinylated human L-hepcidin-24, respectively). Characterized in that itBinding affinity to human hepcidin-25 the first type a hepcidin-binding nucleic acid is hepcidin-binding nucleic acid 223-C5-001. Determination of the equilibrium binding constant K for human hepcidin-25 by surface plasmon resonance measurement_D(K determined by aptamer sequence)_D1.2nM, fig. 13; k determined by sequence of spiegelmers_D2.7nM, fig. 11). In addition to human hepcidin-25, hepcidin-binding nucleic acid 223-C5-001 bound human hepcidin-20 with nearly the same binding affinity (FIG. 11).

Derivatives 223-C5-002, 223-C5-007 and 223-C5-008 of type A hepcidin-binding nucleic acid 223-C5-001 showed reduced binding affinity in a competitive pull-down assay compared to type A hepcidin-binding nucleic acid 223-C5-001 (FIG. 3). Indeed, hepcidin-binding nucleic acid 223-C5-006 showed similar binding to human hepcidin-25 in the same assay format as 223-C5-001 (FIG. 3).

Type A hepcidin-binding nucleic acids 223-B5-001, 223-A5-001, 223-A3-001, 223-F5-001, 223-G4-001, 223-A4-001, 229-C2-001, 229-B4-001, 229-E2-001, 229-B1-001, 229-G1-001, 229-C4-001, 238-A1-001, 238-E2-001, 237-A7-001, 236-G2-001, 236-D1-001, 229-D1-001 and 229-E1-as aptamers are tested against type A hepcidin-binding nucleic acids 223-C5-001 in a competitive pulldown assays, wherein the binding of a radiolabeled aptamer 223-C5-C67ll 26-001 to hepcidin-a-is first determined by using a direct pulldown assay Affinity. No competition could be observed for binding of nucleic acid 229-E1-001 to hepcidin type A binding nucleic acid 223-C5-001 (FIGS. 2 and 15). This observation led to the hypothesis that nucleic acid 229-E1-001 has no or very low binding affinity for human hepcidin-25. Type A hepcidin-binding nucleic acids 223-B5-001, 223-A5-001, 223-A3-001, 223-A4-001, 229-C2-001, 229-B4-001, 229-E2-001, 229-C4-001, 238-A1-001, 238-E2-001, 237-A7-001, 236-G2-001 and 236-D1-001 showed reduced binding affinity in a competitive pull-down assay compared to type A hepcidin-binding nucleic acids 223-C5-001 (FIGS. 1, 14 and 15). Type AHepcidin-binding nucleic acids 223-F5-001, 223-G4-001, 229-G1-001, and 229-D1-001 showed similar binding affinities to 223-C5-001 (FIGS. 1, 2, 14, and 15). For type a hepcidin-binding nucleic acid 229-B1-001, a better binding affinity for biotinylated human D-hepcidin-25 was observed (fig. 1 and 15). Thus, type A hepcidin-binding nucleic acid 229-B1-001 was further characterized. Determination of the equilibrium binding constant K of the type A hepcidin-binding nucleic acid 229-B1-001 by surface plasmon resonance measurement_D(K determined by aptamer sequence)_D0.5nM, fig. 16; k determined by sequence of spiegelmers_DData not shown) at 1.25 nM.

Derivatives 229-B1-003, 229-B1-004, 229-B1-005 and 229-B1-006 of type A hepcidin-binding nucleic acid 229-B1-001 showed reduced binding affinity in a competitive pull-down assay compared to type A hepcidin-binding nucleic acid 229-B1-001 (FIG. 4). Indeed, type A hepcidin-binding nucleic acids 229-B1-002, 229-B1-007, 229-B1-008, 229-B1-009, 229-B1-010 and 229-B1-011 show binding to human hepcidin-25 similar to 229-B1-001 or slightly improved compared to 229-B1-001 in the same assay format (FIG. 4).

Type A hepcidin-binding nucleic acids 229-B1-002 were further characterized. Determination of the equilibrium binding constant K of the type A hepcidin-binding nucleic acid 229-B1-002 by surface plasmon resonance measurement_D(K determined by sequence of spiegelmers)_D1.47nM, fig. 10 and 11).

In addition, the binding specificity/selectivity of type a hepcidin binding nucleic acids 229-B1-002 was tested with the following hepcidin molecules: human hepcidin-25, cynomolgus monkey hepcidin-25, mouse hepcidin-25, rat hepcidin-25, human hepcidin-22, and human hepcidin-20 (FIGS. 10 and 11). Type A hepcidin-binding nucleic acid 229-B1-002 showed similar binding to human hepcidin-25, cynomolgus monkey hepcidin-25, human hepcidin-22 and human hepcidin-20, but not to mouse hepcidin-25 and rat hepcidin-25 (FIGS. 10 and 11).

All type A hepcidin binding nucleic acids according to the invention, except the type A nucleic acid 229-E1-001, comprise a first stretch box A. In type A hepcidin-binding nucleic acid 229-D1-001, cassette A is linked with its 3 '-end to the 5' -end of the second terminal stretch (FIG. 2). In all other hepcidin binding type a nucleic acids, cassette a is linked with its 5 '-end to the 3' -end of the first terminal stretch (fig. 1 to 4). The type a hepcidin-binding nucleic acid comprising cassette a shares the sequence 5 'WAAAGUWGAR 3' with cassette a. The sequence of cassette A of all other type A hepcidin binding nucleic acids is 5 'UAAAGUAGAG 3', except for type A hepcidin binding nucleic acids 229-C4-001/236-G2-001 and 236-D1-001 which comprise sequences 5 'AAAAGUAGAA 3' and 5 'AAAAGUUGAA 3' with respect to cassette A, respectively.

All hepcidin-binding nucleic acids of type a contain a cassette B with the sequence 5 'RGMGUGWKAGUKC 3', except for hepcidin-binding nucleic acid type a 236-D1-001 (see fig. 2). Type a hepcidin-binding nucleic acid 236-D1-001 comprises cassette B which differs from the consensus sequence of the cassettes of the other type a hepcidin-binding nucleic acids: 5 'GGGAUAUAGUGC 3'. Since nucleic acid 229-E1-001, which does not comprise cassette a, does not bind to human hepcidin-25 or binds weakly to human hepcidin-25 as described above, it was hypothesized that, in addition to cassette B, cassette a is also necessary for binding to human hepcidin-25, in particular for binding with high affinity to human hepcidin-25. In type A hepcidin-binding nucleic acid 229-D1-001, cassette B is linked with its 5 '-end to the 3' -end of the first terminal stretch (FIG. 2). In all other hepcidin-binding nucleic acids of type a, cassette B (except hepcidin-binding nucleic acid 229-E1-001) was linked with its 3 '-end to the 5' -end of the second terminal stretch (fig. 1, 3 and 4). Hepcidin-binding nucleic acids with different cassette B sequences show high binding affinity for human hepcidin-25:

a)229-B1-001 and derivatives, 223-C5-001 and derivatives, 223-B5-001, 223-A5-001, 223-A3-001, 223-F5-001, 223-G4-001, 223-A4-001, 238-E2: 5 'GGCGUGAUAGUGC 3';

b)229-B4-001，229-C2-001，229-E2-001：5’GGAGUGUUAGUUC 3’；

c)229-G1-001：5’GGCGUGAGAGUGC 3’；

d)229-C4-001，236-G2-001：5’AGCGUGAUAGUGC 3’；

e)238-A1-001：5’GGCGUGUUAGUGC 3’；

f)236-D1-001：5’GGGAUAUAGUGC 3’。

the hepcidin-binding nucleic acids comprising cassette a and cassette B are linked to each other by a 10 to 18 nucleotide linking nucleotide sequence stretch. The linking nucleotide sequence segment comprises in the 5 '- > 3' direction a first linking nucleotide subsequence segment, a second linking nucleotide subsequence segment, and a third linking nucleotide subsequence segment, wherein preferably the first linking nucleotide subsequence segment and the third linking nucleotide subsequence segment optionally hybridize to each other, wherein upon hybridization a double-stranded structure is formed. However, such hybridization is not necessarily given in the molecule. If the nucleotides of the first linking subsequence segment and the nucleotides of the third linking subsequence segment hybridize to each other, they form a loop of nucleotides in between that do not hybridize to each other (i.e., the second subsequence segment). The first and third nucleotide subsequences of the linking nucleotide sequence segment of a hepcidin-binding nucleic acid comprise 3 (see 229-B1-001 and derivatives, 229-G1-001), 4 (see 223-C5-001 and derivatives, 223-B5-001, 223-A5-001, 223-A3-001, 223-F5-001, 223-G4-001, 223-A4-001, 229-C2-001, 229-B4-001, 229-E2-001, 238-A1-001, 238-E2-001, 237-A7-001), 5 (229-D1-001), or 6 (229-C4-001, 236-G2-001) nucleotides. The type a binding nucleic acid 236-D1-001 comprises a 18 nucleotide linking stretch of nucleotides that cannot be categorized into a first linking stretch of nucleotides, a second linking stretch of nucleotides, and a third linking stretch of nucleotides due to the particular sequence of the linking stretch of nucleotides.

As shown for the hepcidin binding nucleic acids 223-C5-001 and derivatives thereof, 223-B5-001, 223-A5-001, 223-A3-001, 223-F5-001, 223-G4-001, 238-E2-001 and 223-A4-001, the first subsequence of the linking nucleotide sequence segment comprises the sequence 5 'GGAC 3' or 5 'GGAU 3' or 5 'GGA 3', and the third subsequence of the linking nucleotide sequence segment comprises the nucleotide sequence 5 'GUCC 3'. Other combinations of the first and third subsequences of the linking nucleotide sequence segment are:

a)5 'GCAG 3' and 5 'CUGC 3' (229-C2-001, 229-B4-001, 229-E2-001, 237-A7-001), or

b)5 'GAC 3' and 5 'GUC 3' (229-B1-001 and derivatives thereof, 229-G1-001), or

c)5 'ACUUGU 3' and 5 'GCAAGU 3' (229-C4-001), or

d)5 'ACUUGU 3' and 5 'GCAAGC 3' (236-G2-001), or

e)5 'UCCAG 3' and 5 'CUGGA 3' (229-D1-001), or

f)5 'GGGC 3' and 5 'GCCC 3' (238-A1-001).

As shown in figures 1, 2, 3 and 4, the second subsequence segment of the linking nucleotide sequence segment comprises 3 to 5 nucleotides, wherein the different sequences are very heterologous: 5 'CGAAA 3', 5 'GCAAU 3', 5 'GUAAU 3', 5 'AAUU 3', 5 'AUAAU 3', 5 'AAUA 3', 5 'CCA 3', 5 'CUA 3', 5 'UCA 3', 5 'ACA 3', 5 'GUU 3', 5 'UGA 3' and 5 'GUA 3'. The second subsequence of said contiguous nucleotide sequence segment of a hepcidin-binding nucleic acid may be summarized as the following general sequence: 5 'VBAAW 3', 5 'AAUW 3' or 5 'NBW 3'.

However, the hepcidin-binding nucleic acid with the best binding affinity comprises the following sequence for the second subsequence section of the linking nucleotide sequence section:

a)5 'AAUU 3' (229-B1 and its derivatives)

b)5 'CCA 3' (223-C5 and derivatives thereof)

c)5’CUA 3’(223-F5-001)

d)5’UCA 3’(223-G4-001)

e)5’AAUA 3’(229-G1-001)。

As described above, the nucleotide sequences of the first and third subsequences of the connecting sequence segment are related to each other. In addition, the nucleotide sequence of the second subsequence of said linking nucleotide sequence segment is related to a specific pair of first and third nucleotide subsequence segments, which results in the following sequence or generic sequence of said linking nucleotide sequence segment of the hepcidin-binding nucleic acid:

a)5 'GGACBYAGUCC 3' (223-C5-001, 223-C5-002, 223-C5-006, 223-C5-007, 223-B5-001, 223-a5-001, 223-A3-001, 223-F5-001, 223-G4-001, 238-E2-001), preferably 5 'GGACCCAGUCC 3', 5 'GGACCUAGUCC 3' or 5 'GGACUCAGUCC 3' or 5 'GGACGUAGUCC 3', more preferably 5 'GGACCCAGUCC 3', 5 'GGACCUAGUCC 3' or 5 'GGACUCAGUCC 3'; or

b)5 'GGAUACAGUCC 3' (223-a 4-001); or

c)5 'gcaggyaaucgc 3' (229-C2-001, 229-B4-001, 229-E2-001), preferably 5 'GCAGGUAAUCUGC 3' or 5 'GCAGGCAAUCUGC 3', more preferably 5 'GCAGGUAAUCUGC 3'; or

d)5 'GACAAUWGUC 3' (229-B1-001 and derivatives, 229-G1-001), preferably 5 'GACAAUUGUC 3' or 5 'GACAAUAGUC 3'; or

e)5 'ACUUGUCGAAAGCAAGy 3' (229-C4-001, 236-G2-001); or

f)5 'UCCAGGUUCUGGA 3' (229-D1-001); or

g)5 'GGGCUGAGCCC 3' (238-a 1-001); or

h)5 'GCAGAUAAUCUGC 3' (237-a 7-001); or

i)5’GGACCAGUCC 3’(223-C5-008)。

As previously mentioned, the linking stretch of nucleotides of the type A binding nucleic acid 236-D1-001 cannot be classified into a first linking stretch of nucleotides, a second linking stretch of nucleotides, and a third linking stretch of nucleotides. However, the sequence of the linking nucleotide sequence segment of the type A binding nucleic acid 236-D1-001 is 5 'AUUUGUUGGAAUCAAGCA 3'.

The first and second terminal nucleotide stretches of the hepcidin-A-binding nucleic acid comprise 4 (e.g., 229-C4-001), 5 (e.g., 223-C5-007), 6 (e.g., 229-B1-001), or 7 (e.g., 223-C5-001) nucleotides, wherein the stretches optionally hybridize to each other, wherein upon hybridization a double-stranded structure is formed. The double-stranded structure may consist of 4 to 7 base pairs. However, such hybridization is not necessarily given in the molecule.

By combining the first and second terminal nucleotide sequence segments of all tested hepcidin-binding nucleic acids, the general formula for the first terminal nucleotide sequence segment and for the second terminal nucleotide sequence segment is 5' X₁X₂X₃BKBKK 3 '(first terminal stretch of nucleotides) and 5' MVVVX₄X₅X₆3' (second terminal nucleotide sequence segment), wherein

X₁Is G or absent, X₂Is S or absent, X₃Is V or absent, X₄Is B or absent, X₅Is S or absent, and X₆Is either C or is absent,

preferably

a)X₁Is G, X₂Is S, X₃Is V, X₄Is B, X₅Is S, and X₆Is C, or

b)X₁Is absent, X₂Is S, X₃Is V, X₄Is B, X₅Is S, and X₆Is C, or

c)X₁Is G, X₂Is S, X₃Is V, X₄Is B, X₅Is S, and X₆Is absent, or

d)X₁Is absent, X₂Is S, X₃Is V, X₄Is B, X₅Is S, and X₆Is absent, or

e)X₁Is absent, X₂Is absent, X₃Is V, X₄Is B, X₅Is S, and X₆Is absent, or

f)X₁Is absent, X₂Is S, X₃Is V, X₄Is B, X₅Is absent, and X₆Is absent, or

g)X₁Is absent, X₂Is absent, X₃Is V or absent, X₄Is B or absent, X₅Is absent, and X₆Is absent.

However, the hepcidin-binding nucleic acid with the best binding affinity comprises the following combination of the first and second terminal nucleotide sequence segments:

a)223-C5-001, 223-F5-001, 223-G4-001: 5 'GCACUCG 3' (first terminal nucleotide sequence segment) and 5 'CGAGUGC 3' (second terminal nucleotide sequence segment);

b) 229-B1-002: 5 'GCUGUG 3' (first terminal nucleotide sequence segment) and 5 'CACAGC 3' (second terminal nucleotide sequence segment);

c)229-B1-001, 229-G1-001: 5 'CGUGUG 3' (first terminal nucleotide sequence segment) and 5 'CACACG 3' (second terminal nucleotide sequence segment);

d) 229-D1-001: 5 'CGUGCU 3' (first terminal nucleotide sequence segment) and 5 'AGCACG 3' (second terminal nucleotide sequence segment);

e) 223-C5-006: 5 'CGCGCG 3' (first terminal nucleotide sequence segment) and 5 'CGCGCG 3' (second terminal nucleotide sequence segment);

f) 229-B1-007: 5 'GCCGUG 3' (first terminal stretch of nucleotides) and 5 'CACGGC 3' (second terminal stretch of nucleotides);

g) 229-B1-008: 5 'GCGGUG 3' (first terminal stretch of nucleotides) and 5 'CACCGC 3' (second terminal stretch of nucleotides);

h) 229-B1-009: 5 'GCUGCG 3' (first terminal nucleotide sequence segment) and 5 'CGCAGC 3' (second terminal nucleotide sequence segment);

i) 229-B1-010: 5 'GCUGGG 3' (first terminal stretch of nucleotides) and 5 'CCCAGC 3' (second terminal stretch of nucleotides);

j) 229-B1-011: 5 'GCGGCG 3' (first terminal nucleotide sequence segment) and 5 'CGCCGC 3' (second terminal nucleotide sequence segment).

To confirm the functionality of hepcidin-binding nucleic acids as spiegelmers, hepcidin-binding nucleic acids type a 223-C5-001 and 229-B1-002 were synthesized as spiegelmers comprising an amino group at their 5' -end. The amino-modified spiegelmers 223-C5-001-5 '-amino and 229-B1-002-5' -amino were coupled with a 40kDa PEG-moiety, resulting in type A hepcidin-binding nucleic acids 223-C5-001-5 '-PEG and 229-B1-002-5' -PEG. The synthesis and pegylation of the spiegelmers are described in example 2.

Determination of equilibrium binding constant K for the spiegelmers 223-C5-001-5 '-PEG and 229-B1-002-5' -PEG by surface plasmon resonance measurement_D(FIG. 12):

223-C5-001-5’-PEG：K_D＝4.44nM；

229-B1-002-5’-PEG：K_D＝1.92nM。

the spiegelmer 223-C5-001-5' -PEG was tested for inhibition/antagonism of hepcidin function in vitro and in vivo. As shown in example 5, the spiegelmer aptamer 223-C5-001-5' -PEG inhibited hepcidin-induced down-regulation of ferroportin in vitro. The applicability of the in vivo use of the spiegelmer 223-C5-001-5' -PEG was tested in animal models of inflammatory anemia, where the known property of inducing serum iron decline with human hepcidin-25 was exploited (example 5).

Type 1.2B hepcidin-binding nucleic acids

As depicted in fig. 5 and 6, type B hepcidin-binding nucleic acids comprise a central nucleotide sequence segment that defines a potential hepcidin-binding motif.

Generally, type B hepcidin-binding nucleic acids comprise terminal nucleotide sequence segments at their 5 '-end and 3' -end: a first terminal stretch of nucleotides and a second terminal stretch of nucleotides. The first terminal stretch of nucleotides and the second terminal stretch of nucleotides may hybridize to each other, wherein upon hybridization a double-stranded structure is formed. However, such hybridization is not necessarily given in the molecule.

The three stretches of type B hepcidin-binding nucleic acid (first terminal stretch of nucleotides, central stretch of nucleotides and second terminal stretch of nucleotides) may be arranged differently from each other: a first terminal stretch of nucleotides-a central stretch of nucleotides-a second terminal stretch of nucleotides, or a second terminal stretch of nucleotides-a central stretch of nucleotides-a first terminal stretch of nucleotides.

The sequence of the defined sequence segments may differ between type B hepcidin-binding nucleic acids, which affects the binding affinity for human hepcidin (in particular, human hepcidin-25). The central nucleotide sequence stretch and its nucleotide sequence described hereinafter are required for binding to human hepcidin-25 individually, more preferably in its entirety, based on binding analysis of different hepcidin-binding nucleic acids.

Type B hepcidin-binding nucleic acids according to the invention are shown in figures 5 and 6. They were all tested as aptamers or spiegelmers for their ability to bind to human hepcidin-25 (more precisely, biotinylated human D-hepcidin-25 and biotinylated human L-hepcidin-25, respectively).

Type B hepcidin-binding nucleic acids 238-D2-001, 238-D4-001, 238-H1-001, 238-A2-001, 238-G2-001, 238-G4-001, 238-G3-001 were tested as aptamers in a competitive pull-down assay against type A hepcidin-binding nucleic acids 229-B1-001. Only type B hepcidin-binding nucleic acid 238-G4-001 showed reduced binding affinity in a competitive pull-down assay compared to type a hepcidin-binding nucleic acid 229-B1-001 (fig. 5). Type B hepcidin-binding nucleic acids 238-D2-001, 238-D4-001, 238-H1-001, 238-A2-001, 238-G2-001 and 238-G3-001 showed improved binding affinity compared to type A hepcidin-binding nucleic acids 229-B1-001 (FIG. 5). Type B hepcidin-binding nucleic acids 238-D4-001 were further characterized. Determination of the equilibrium binding constant K of the spiegelmer aptamer 238-D4-001 by surface plasmon resonance measurement_D(K_D0.51 nM; fig. 5).

Derivatives 238-D4-003, 238-D4-005, 238-D4-007, 238-D4-009, 238-D4-010, 238-D4-011 and 238-D4-013 of hepcidin B-binding nucleic acid 238-D4-001 showed reduced binding affinity in a competitive pull-down assay (or as shown by surface plasmon resonance measurements) compared to hepcidin B-binding nucleic acid 238-D4-001 (FIG. 6). Indeed, hepcidin-binding nucleic acids 238-D4-002, 238-D4-004, 238-D4-006, 238-D4-008 and 238-D4-012 showed similar binding to human hepcidin in the same assay format as 238-D4-001 (FIG. 6). Determination of the equilibrium binding constant K of the spiegelmers 238-D4-002, 238-D4-006 and 238-D4-008 by surface plasmon resonance measurement_D. The calculated equilibrium binding constant of the 238-D4-001 derivative lies with respect to 238-D4-001 itselfThe same range is shown (fig. 6).

In addition, the binding selectivity of type B hepcidin-binding nucleic acids 238-D4-001 and 238-D4-008 was tested using the following hepcidin molecules: human hepcidin-25, cynomolgus monkey hepcidin-25, marmoset hepcidin-25 (for 238-D4-008 only), mouse hepcidin-25, rat hepcidin-25, human hepcidin-22 (not for 238-D4-008) and human hepcidin-20 (fig. 10 and 11). Type B hepcidin-binding nucleic acids 238-D4-001 and 238-D4-008 showed similar binding to human hepcidin-25, human hepcidin-22, human hepcidin-20 and cynomolgus hepcidin-25, weaker binding to marmoset hepcidin-25 and no binding to mouse hepcidin-25 and rat hepcidin-25 (FIGS. 10 and 11).

The hepcidin B-binding nucleic acids according to the invention share the sequence 5 'rkauggggakuuaaauggggrguwggaggaar 3' or 5 'rkauggggakakauaaaugaggrguwggaggaar 3' with respect to the central nucleotide sequence segment. Type B hepcidin-binding nucleic acids 238-D4-001 and derivatives thereof that exhibit the same binding affinity for human hepcidin-25 share a consensus sequence comprising the sequence 5 'GUAUGGGAUUAAGUAAAUGAGGAGUUGGAGGAAG 3' with respect to the central nucleotide sequence segment.

The first and second terminal nucleotide sequence segments of the type B hepcidin-binding nucleic acid comprise 5 (238-D4-004, 238-D4-005, 238-D4-008, 238-D4-009), 6 (238-D4-002, 238-D4-003, 238-D4-006, 238-D4-007, 238-D4-010, 238-D4-011, 238-D4-012, 238-D4-013) or 8 (238-D2-001, 238-D4-001, 238-H1-001, 238-A2-001, 238-G2-001, 238-G4-001, 238-G3-001) nucleotides, wherein the sequence segments optionally hybridize with one another, wherein a double-stranded structure is formed after hybridization. The double-stranded structure may consist of 5 to 8 base pairs. However, such hybridization is not necessarily given in the molecule.

By combining the first and second terminal nucleotide sequence segments of all tested type B hepcidin-binding nucleic acids, with respect to the first terminal nucleotide sequence segment and with respect to the second terminal nucleotideThe general formula of the sequence segment is 5' X₁X₂X₃SBSBC 3 '(first terminal nucleotide sequence segment) and 5' GVGBVBX₄X₅X₆3' (second terminal nucleotide sequence segment), wherein

X₁Is A or absent, X₂Is G or absent, X₃Is B or absent, X₄Is S or absent, X₅Is C or absent, and X₆Is a group of U or is absent,

preferably

a)X₁Is A, X₂Is G, X₃Is B, X₄Is S, X₅Is C, and X₆Is U, or

b)X₁Is absent, X₂Is G, X₃Is B, X₄Is S, X₅Is C, and X₆Is U, or

c)X₁Is A, X₂Is G, X₃Is B, X₄Is S, X₅Is C, and X₆Is absent, or

d)X₁Is absent, X₂Is G, X₃Is B, X₄Is S, X₅Is C, and X₆Is absent, or

e)X₁Is absent, X₂Is absent, X₃Is B, X₄Is S, X₅Is C, and X₆Is absent, or

f)X₁Is absent, X₂Is G, X₃Is B, X₄Is S, X₅Is absent, and X₆Is absent, or

g)X₁Is absent, X₂Is absent, X₃Is B or absent, X₄Is S or absent, X₅Is absent, and X₆Is absent.

However, the most binding type B hepcidin binding nucleic acid comprises the following combination of first and second terminal nucleotide sequence segments:

a) 238-D2-001: 5 'AGCGUGUC 3' (first terminal nucleotide sequence segment) and 5 'GGUGCGCU 3' (second terminal nucleotide sequence segment);

b) 238-D4-001: 5 'AGCGUGUC 3' (first terminal stretch of nucleotides) and 5 'GGCAUGCU 3' (second terminal stretch of nucleotides);

c) 238-H1-001: 5 'AGUGUGUC 3' (first terminal nucleotide sequence segment) and 5 'GAUGCGCU 3' (second terminal nucleotide sequence segment);

d) 238-A2-001: 5 'AGUGUGUC 3' (first terminal stretch of nucleotides) and 5 'GGCAUGCU 3' (second terminal stretch of nucleotides);

e) 238-G2-001: 5 'AGCGUGCC 3' (first terminal nucleotide sequence segment) and 5 'GGUGCGCU 3' (second terminal nucleotide sequence segment);

f) 238-G3-001: 5 'AGCGCGCC 3' (first terminal nucleotide sequence segment) and 5 'GGCGCGCU 3' (second terminal nucleotide sequence segment);

g) 238-D4-002: 5 'GCGCGC 3' (first terminal nucleotide sequence segment) and 5 'GCGCGC 3' (second terminal nucleotide sequence segment);

h) 238-D4-006: 5 'GGUGUC 3' (first terminal stretch of nucleotides) and 5 'GGCAUC 3' (second terminal stretch of nucleotides);

i) 238-D4-012: 5 ' GGCGUC 3 ' (first terminal nucleotide sequence segment) and 5 ' GGCGCC 3 ' (3 ' -terminal nucleotide sequence segment);

j) 238-D4-008: 5 'GCGCGCC 3' (first terminal nucleotide sequence segment) and 5 'GGCGC 3' (second terminal nucleotide sequence segment);

k) 238-D4-004: 5 'GGCGC 3' (first terminal nucleotide sequence segment) and 5 'GCGCC 3' (second terminal nucleotide sequence segment).

To confirm the functionality of type B hepcidin-binding nucleic acids as spiegelmers, hepcidin-binding nucleic acids 238-D4-002 and 238-D4-008 were synthesized as spiegelmers comprising an amino group at their 5' -end. The amino-modified spiegelmers 238-D4-002-5 '-amino and 238-D4-008-5' -amino were coupled with a 40kDa PEG-moiety, resulting in hepcidin-binding nucleic acids 238-D4-002-5 '-PEG and 238-D4-008-5' -PEG. The synthesis and pegylation of the spiegelmers are described in example 2.

Determination of equilibrium binding constant K for the spiegelmers 238-D4-002-5 '-PEG and 238-D4-008-5' -PEG by surface plasmon resonance measurement_D(FIG. 12):

238-D4-002-5’-PEG：0.53nM；

238-D4-008-5’-PEG：0.64nM。

the spiegelmer aptamer 238-D4-008-5' -PEG was tested for inhibition/antagonism of hepcidin function in vitro and in vivo. As shown in example 5, the spiegelmer aptamer 238-D4-008-5' -PEG inhibited hepcidin-induced down-regulation of ferroportin in vitro. The applicability of the in vivo use of the spiegelmer aptamer 238-D4-008-5' -PEG was tested in animal models of inflammatory anemia, where the known property of inducing serum iron decline with human hepcidin-25 was exploited (example 5, FIG. 20). In addition, the spiegelmer aptamer 238-D4-008-5' -PEG was tested in another animal model for anemia of inflammation (cynomolgus monkey), where IL-6 induces hepcidin secretion, which subsequently leads to anemia in non-human primates. In this assay, human IL-6 caused a decrease in serum iron concentration (example 6, FIG. 21).

1.3 Hepacidin binding nucleic acids of type C

As depicted in fig. 7 and 8, type C hepcidin-binding nucleic acids comprise a central nucleotide sequence segment defining a potential hepcidin-binding motif.

Typically, a type C hepcidin-binding nucleic acid comprises at its 5 '-end and 3' -end terminal sequence segments: a first terminal stretch of nucleotides and a second terminal stretch of nucleotides. The first terminal stretch of nucleotides and the second terminal stretch of nucleotides may hybridize to each other, wherein upon hybridization a double-stranded structure is formed. However, such hybridization is not necessarily given in the molecule.

The three nucleotide sequence segments (first terminal nucleotide sequence segment, central nucleotide sequence segment and second terminal nucleotide sequence segment) of the type C hepcidin-binding nucleic acid may be arranged differently from each other: a first terminal stretch of nucleotides-a central stretch of nucleotides-a second terminal stretch of nucleotides, or a second terminal stretch of nucleotides-a central stretch of nucleotides-a first terminal stretch of nucleotides.

The sequence of the defined sequence segments may differ between type C hepcidin-binding nucleic acids, which affects the binding affinity for human hepcidin (in particular, human hepcidin-25). The central nucleotide sequence stretch and its nucleotide sequence described hereinafter are required for binding human hepcidin, individually and more preferably in its entirety, based on binding analysis of different type C hepcidin-binding nucleic acids.

Type C hepcidin-binding nucleic acids according to the invention are shown in figures 7 and 8. They were all tested as aptamers or spiegelmers for their ability to bind to human hepcidin-25 (more precisely, biotinylated human D-hepcidin-25 and biotinylated human L-hepcidin-25).

Type C hepcidin-binding nucleic acids 238-C4-001, 238-E3-001, 238-F2-001, 238-A4-001 and 238-E1-001 were tested as aptamers in a competitive pull-down assay against type A hepcidin-binding nucleic acid 229-B1-001. Type C hepcidin-binding nucleic acids showed improved binding affinity compared to type a hepcidin-binding nucleic acids 229-B1-001 (fig. 7). The hepcidin-binding type C nucleic acid 238-C4-001 was further characterized. Determination of the equilibrium binding constant K of the spiegelmer aptamer 238-C4-001 by surface plasmon resonance measurement_D(K_D0.9 nM; fig. 7).

Derivatives 238-C4-003, 238-C4-004, 238-C4-005, 238-C4-007, 238-C4-008, 238-C4-009, 238-C4-011, 238-C4-012, 238-C4-013, 238-C4-014, 238-C4-024, 238-C4-025 and 238-C4-062 of type C hepcidin-binding nucleic acids 238-C4-001 showed reduced binding affinity compared to hepcidin-binding nucleic acids 238-C4-001 or 238-C4-006 in a competitive pull-down assay or by plasmon resonance measurements (FIG. 8). Nucleic acid 238-C4-063 was shown not to bind to hepcidin. Indeed, hepcidin-binding nucleic acids 238-C4-002, 238-C4-006 and 238-C4-010 showed similar binding to human hepcidin-25 in the same assay as 238-C4-001 (FIG. 8). Determination of equilibrium binding constant K for the spiegelmers 238-C4-002 and 238-C4-006 by surface plasmon resonance measurement_D. The equilibrium binding constant for the derivative of 238-C4-001 was calculated to lie within the same range as shown for 238-C4-001 itself (FIG. 8).

In addition, the binding specificity/selectivity of the type C hepcidin-binding nucleic acid 238-C4-006 was tested using the following hepcidin molecules: human hepcidin-25, cynomolgus monkey hepcidin-25, marmoset hepcidin-25, mouse hepcidin-25, rat hepcidin-25, human hepcidin-22 and human hepcidin-20 (figures 10 and 11). Type C hepcidin-binding nucleic acid 238-C4-006 showed similar binding to human hepcidin-25, human hepcidin-22, human hepcidin-20 and cynomolgus monkey hepcidin-25 and not to marmoset hepcidin-25, mouse hepcidin-25 and rat hepcidin-25 (figures 10 and 11).

In addition to the nucleic acids 238-C4-063 which show no binding to hepcidin-25, the hepcidin-binding nucleic acids of type C according to the invention share the sequence 5 'GRCRGCCGGVGGGACACCAUACAGACUACUACKAUA 3' or 5 'GRCRGCCGGGAGACACUAUAUAUACAGACUACKAUA 3' with respect to the central nucleotide sequence segment. The hepcidin-binding nucleic acids 238-C4-001 and derivatives 238-C4-002, 238-C4-005, 238-C4-010 of type C and hepcidin-binding nucleic acids 238-E3-001, 238-F2-001, 238-A4-001, 238-E1-001 (which all show the same binding affinity) share the consensus sequence 5 'GRCRGCCGACAGGGACACCAUGACUACKAUA 3', preferably the sequence 5 'GACAGCCGGGGGACACCAUAUACAGACUACGAUA 3'.

The first and second terminal nucleotide sequence segments of a hepcidin-binding nucleic acid of type C comprise 4 (238-C4-004, 238-C4-011, 238-C4-012, 238-C4-013, 238-C4-014), 5 (238-C4-003, 238-C4-005, 238-C4-006, 238-C4-007, 238-C4-008, 238-C4-009, 238-C4-010, 238-C4-024, 238-C4-025, 238-C4-062), 6 (238-C4-002) or 7 (238-C4-001, E3-001, 238-F2-001, 238-A4-001, 238-E1-001) nucleotides, wherein these sequence segments optionally hybridize with one another, wherein upon hybridization a double stranded structure is formed. The double-stranded structure may consist of 4 to 7 base pairs. However, such hybridization is not necessarily given in the molecule.

By combining the first and second terminal nucleotide sequence segments of all tested C-type hepcidin-binding nucleic acids, the general formula for the first terminal nucleotide sequence segment and for the second terminal nucleotide sequence segment is 5' X₁X₂X₃SBSN 3 '(first terminal nucleotide sequence stretch) and 5' NSVSX₄X₅X₆3' (second terminal nucleotide sequence segment), wherein

X₁Is A or absent, X₂Is G or absent, X₃Is R or absent, X₄Is Y or absent, X₅Is C or absent, and X₆Is a group of U or is absent,

preferably

a)X₁Is A, X₂Is G, X₃Is R, X₄Is Y, X₅Is C, and X₆Is U, or

b)X₁Is absent, X₂Is G, X₃Is R, X₄Is Y, X₅Is C, and X₆Is U, or

c)X₁Is A, X₂Is G, X₃Is R, X₄Is Y, X₅Is C, and X₆Is absent, or

d)X₁Is absent, X₂Is G, X₃Is R, X₄Is Y, X₅Is C, and X₆Is absent, or

e)X₁Is absent, X₂Is absent, X₃Is R, X₄Is Y, X₅Is C, and X₆Is absent, or

f)X₁Is absent, X₂Is G, X₃Is R, X₄Is Y, X₅Is absent, and X₆Is absent, or

g)X₁Is absent, X₂Is absent, X₃Is R or absent, X₄Is Y or absent, X₅Is absent, and X₆Is absent.

However, the most binding C-type hepcidin-binding nucleic acid comprises the following combination of the first and 3' -terminal nucleotide sequence segments:

a)238-C4-001, 238-E3-001: 5 'AGGCUCG 3' (first terminal nucleotide sequence segment) and 5 'CGGGCCU 3' (second terminal nucleotide sequence segment);

b) 238-F2-001: 5 'AGGCCCG 3' (first terminal nucleotide sequence segment) and 5 'CGGGCCU 3' (second terminal nucleotide sequence segment);

c) 238-A4-001: 5 'AGGCUUG 3' (first terminal nucleotide sequence segment) and 5 'CGAGCCU 3' (second terminal nucleotide sequence segment);

d) 238-E1-001: 5 'AGACUUG 3' (first terminal nucleotide sequence segment) and 5 'CGAGUCU 3' (second terminal nucleotide sequence segment);

e) 238-C4-002: 5 'GGCUCG 3' (first terminal nucleotide sequence segment) and 5 'CGGGCC 3' (second terminal nucleotide sequence segment);

f) 238-C4-006: 5 'GGCCG 3' (first terminal nucleotide sequence segment) and 5 'CGGCC 3' (second terminal nucleotide sequence segment);

g) 238-C4-010: 5 'GCGCGCG 3' (first terminal nucleotide sequence segment) and 5 'CGCGC 3' (second terminal nucleotide sequence segment).

To confirm the functionality of a hepcidin-binding nucleic acid as an spiegelmer, a hepcidin-binding nucleic acid 238-C4-006 was synthesized as a spiegelmer comprising an amino group at its 5' -end. Coupling of a 40kda PEG-moiety to an amino-modified spiegelmer aptamer 238-C4-006-5 '-amino group resulted in a type C hepcidin binding nucleic acid 238-C4-006-5' -PEG. The synthesis and pegylation of the spiegelmers are described in example 2.

Determination of the equilibrium binding constant K of the spiegelmer aptamer 238-C4-006-5' -PEG by surface plasmon resonance measurement_D(FIG. 12): 0.76 nM.

1.4 other hepcidin-binding nucleic acids

As depicted in fig. 9, other hepcidin-binding nucleic acids are shown unrelated to hepcidin-binding nucleic acids of type a, type B and type C. The binding affinity of these hepcidin nucleic acids was determined by plasmon resonance measurements as well as by competitive binding assays against type a hepcidin-binding nucleic acids 229-G1-001. All nucleic acids showed weaker binding affinity than type a hepcidin-binding nucleic acid 229-G1-001 (fig. 9).

Example 2: synthesis and derivatization of aptamers and spiegelmers

Small scale synthesis

Aptamers (D-RNA nucleic acids) and spiegelmers (L-RNA nucleic acids) were prepared by solid phase synthesis using 2' TBDMS RNA phosphoramidite chemistry (Damha and Ogilvie, 1993) using an ABI 394 synthesizer (Applied Biosystems, Foster City, Calif., USA). D-and L-configuration rA (N-Bz) -, rC (Ac) -, rG (N-ibu) -and rU-phosphoramidites were purchased from ChemGenes, Wilmington, MA. Aptamers and spiegelmers were purified by gel electrophoresis.

Large Scale Synthesis plus modification

Using 2' TBDMS RNA phosphoramidite chemistry (Damha and Ogilvie, 1993)The spiegelmers were prepared by solid phase synthesis using a synthesizer (Amersham Biosciences; General electric healthcare, Freiburg). L-rA (N-Bz) -, L-rC (Ac) -, L-rG (N-ibu) -and L-rU-phosphoramidites were purchased from ChemGenes, Wilmington, MA. 5' -amino-modifying agents were purchased from American International Chemicals Inc. (Framingham, MA, USA). Synthesis of unmodified or 5' -amino-modified spiegelmers starting from L-riboG, L-riboC, L-riboA or L-riboU modified CPG with a pore size of(Linktechnology, Glasgow, UK). For the coupling (15 min for each cycle), 0.3M benzylthiotetrazole in acetonitrile (CMS-Chemicals, Abingdon, UK) and 3.5 equivalents of each 0.1M phosphoramidite solution in acetonitrile were used. An oxidative capping cycle is used. Other standard solvents and reagents for oligonucleotide synthesis were purchased from Biosolve (Valkenswaard, NL). Synthesizing a spiegelmer aptamer, DMT-ON; after deprotection, purification was performed by preparative RP-HPLC (Wincott et al, 1995) using Source15RPC medium (Amersham). The 5' DMT-group was removed with 80% acetic acid (30 min at room temperature). Subsequently, 2M NaOAc in water was added and the spiegelmers were desalted by tangential flow filtration using 5K regenerated cellulose membranes (Millipore, Bedford, MA).

PEGylation of spiegelmers

To extend the plasma residence time of the spiegelmers in vivo, the spiegelmers were covalently coupled at the 5' -end to a 40kDa polyethylene glycol (PEG) moiety.

5' -PEGylation of spiegelmers

For PEGylation (for technical details of the PEGylation method, see European patent application EP 1306382), the purified 5' -amino-modified spiegelmers are dissolved in H₂O (2.5ml), DMF (5ml) and buffer A (5 ml; prepared by mixing citric acid. H₂O[7g]Boric acid [3.54g ]]2.26ml of phosphoric acid]And 1M NaOH [343ml]And adding water to reach a final volume of 1L; pH 8.4 with 1M HCl).

The pH of the spiegelmer solution was brought to 8.4 with 1M NaOH. Then, 40kDa PEG-NHS ester (JenkemTtechnology, Allen, TX, USA) was added in 6 parts (0.25 equivalents per part) every 30 minutes at 37 ℃ until a maximum yield of 75 to 85% was reached. During the addition of PEG-NHS ester, the pH of the reaction mixture was maintained at 8-8.5 with 1M NaOH.

The reaction mixture was mixed with 4ml of urea solution (8M) and 4ml of buffer B (0.1M in H)₂Triethylammonium acetate in O) and heated to 95 ℃ for 15 minutes. The PEGylated spiegelmers were then purified by RP-HPLC using an acetonitrile gradient (buffer B; buffer C: 0.1M triethylammonium acetate in acetonitrile) using Source15RPC medium (Amersham). Excess PEG was eluted at 5% buffer C and PEGylated spiegelmers were eluted at 10-15% buffer C. Product fractions with a purity > 95% (as assessed by HPLC) were combined and mixed with 40ml 3M NaOAC. The pegylated spiegelmers were desalted by tangential flow filtration (5K regenerated cellulose membrane, Millipore, Bedford MA).

Example 3: determination of the binding constant with hepcidin (Pull-Down assay) direct Pull-Down assay

The affinity of hepcidin-binding nucleic acids as aptamers (D-RNA nucleic acids) for biotinylated human D-hepcidin-25 (SEQ ID No.7) was measured in a pull down assay format at 37 ℃. Using [ gamma-³²P]Labeled ATP (Hartmann analytical, Braunschweig, Germany), 5' -phosphate labeling of aptamers by T4 polynucleotide kinase (Invitrogen, Karlsruhe, Germany). The specific radioactivity of the labeled aptamer was 200,000-800,000 cpm/pmol. Aptamers were mixed at a concentration of 20pM with varying amounts of biotinylated human D-hepcidin at 37 ℃ in selection buffer (20mM Tris-HCl pH 7.4; 137mM NaCl; 5mM KCl; 1mM MgCl) after denaturation and renaturation₂；1mM CaCl₂；0.1％[w/vol]Tween-20) for 2-12 hours in order to reach equilibrium at low concentrations. The selection buffer was supplemented with 10. mu.g/ml human serum albumin (Sigma-Aldrich, Steinheim, Germany) and 10. mu.g/ml yeast RNA (Ambion, Austin, USA) in order to prevent adsorption of the binding partner to the surface of the plastic ware or immobilization matrix used. The concentration range of biotinylated human D-hepcidin was set to 32pM to 500 nM; the total reaction volume was 1 ml. Biotinylated human D-hepcidin and complexes of aptamer and biotinylated human D-hepcidin were immobilized on 6. mu.l NeutrAvidin or Streptavidin Ultralink Plus particles (Thermo Scientific, Rockford, USA) that had been pre-equilibrated with selection buffer and resuspended in a total volume of 12. mu.l. The particles were kept suspended in the hot homogenizer for 30 minutes at the corresponding temperature. After separation of the supernatant and appropriate washing, the fixed radioactivity was quantified in a scintillation counter. The percent binding was plotted against the concentration of biotinylated human D-hepcidin and the dissociation constants were obtained by using a Software algorithm (GRAFIT; Erithacus Software; Surrey u.k.) and assuming a 1: 1 stoichiometry.

Aptamer competitive pull-down assay

To compare different aptamers binding to hepcidin nucleic acids, a competitive fractionation assay was performed. For this purpose, the most affinity aptamers available were radiolabeled (see above) and used as reference. After denaturation and renaturation, it was incubated with biotinylated human D-hepcidin in 0.8ml of selection buffer at 37 ℃ under conditions that resulted in approximately 5-10% binding (no competition) to the biotinylated human D-hepcidin-25 after immobilization and washing on NeutrAvidin agarose or Streptavidin Ultralink Plus (both from Thermo Scientific). Excess denatured and renatured unlabeled D-RNA aptamer variants were added at different concentrations (e.g., 10, 50, and 250nM) with labeled reference aptamers to allow parallel binding reactions. The aptamer to be tested competes with the reference aptamer for binding to the target, thereby reducing the binding signal, which is dependent on their binding characteristics. The most active aptamer found in this assay can then be used as a new reference for comparative analysis of other aptamer variants.

Competitive pull-down assay for spiegelmers

In addition, a competitive pull-down assay was performed to analyze the affinity of the spiegelmers that bind hepcidin. For this purpose, spiegelmers which bind biotinylated human L-hepcidin-25 were used. The addition of two additional guanosine residues in the D-configuration at the 5' -terminus of the spiegelmers enabled radiolabeling of spiegelmers by T4 polynucleotide kinase (see above). After denaturation and renaturation, 5-fold dilutions of the labeled spiegelmers and a set of 0.032 to 500nM competitor molecules (e.g.different species of hepcidin, truncated forms of hepcidin or spiegelmers; see below) were incubated with a constant amount of biotinylated human L-hepcidin at 37 ℃ for 2-4 hours in 0.8ml selection buffer. The peptide concentration chosen should result in a final binding of approximately 5-10% of the radiolabeled spiegelmer aptamer at the lowest competitor concentration. In one version of the competitive pull-down assay, excess denatured and renatured unlabeled L-RNA spiegelmer variants were used as competitors, while unmodified as well as PEGylated versions were tested. In another assay method, non-biotinylated L-hepcidin-25 from various species (e.g., human L-hepcidin-25, cynomolgus monkey L-hepcidin-25, marmoset L-hepcidin-25 or rat L-hepcidin-25) or non-biotinylated N-terminally truncated L-hepcidin-20 and L-hepcidin-22 competed with biotinylated L-hepcidin for binding to spiegelmers. After immobilization of biotinylated L-hepcidin-25 and bound spiegelmers on 1.5-3 μ L Streptavidin Ultralink Plus substrate (Thermo Scientific, Rockford, USA), washing and scintillation counting (see above), normalized percentages of bound radiolabeled spiegelmers were plotted against the corresponding competitor molecule concentrations. The resulting dissociation constants were calculated by using grafit software.

Example 4: binding analysis by surface plasmon resonance measurement

The binding of aptamers binding to hepcidin nucleic acid to biotinylated human D-hepcidin-25 and the binding of spiegelmers binding to hepcidin nucleic acid to biotinylated human L-hepcidin-20 and human, rat and mouse L-hepcidin 25 was analyzed using a Biacore 2000 instrument (Biacore AB, Uppsala, Sweden).

The instrument was set to a permanent temperature of 37 ℃. Biacore was cleaned using the DESORB method according to the manufacturer's instructions before the start of each experiment. After the maintenance chip was installed, the instrument was primed (prime) with DESORB solution 1 (0.5% sodium dodecyl sulfate, SDS), DESORB solution 2(50mM glycine, pH 9.5), and finally degassed MilliQ water in succession. Subsequently, the SANATIZE method was run with 0.1M NaOCl, and the system was then primed with MilliQ water.

Biotinylated human D-hepcidin 25, human L-hepcidin 20 and human, rat and mouse L-hepcidin 25 (all peptides from BACHEM, committed synthesis) were dissolved in 1mM concentration in screw-lock vials (screen lock visual) in water with 1mg/ml fatty acid free BSA and stored at 4 ℃ until use.

After mounting the Sensor chip with carboxymethylated dextran matrix (Sensor ChipCM5, GE, BR-1000-14), the Biacore instrument was prepared with MilliQ water and then with HBS-EP buffer (0.01M HEPES buffer [ pH 7.4 ], 0.15M NaCl with 0.005% surfactant P20; GE, BR-1001-88) and equilibrated until a stable baseline was observed. Flow Cell (FC) was immobilized starting from flow cell 4 to flow cell 1 to avoid peptides being entrained to other flow cells.

Mu.l of a 1: 1 mixture of 0.4M EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide in water; GE, BR-1000-50) and 0.1M NHS (N-hydroxysuccinimide in water; GE, BR-1000-50) were injected at a flow rate of 10. mu.l/min using the QUICKINJECT command. Activation of the flow cell was monitored by RU increase after NHS/EDC injection (typically 500-600RU for CM5 chips).

Soluble neutravidin (neutravidin) was dissolved in water to a concentration of 1mg/ml, diluted to 50 μ g/ml in HBS-EP, and subsequently injected at a flow rate of 10 μ l/min using the MANUALINJECT command. The maximum amount observed for covalently immobilized neutravidin is approximately 10,000-15,000 RU. The flow cell was closed by injecting 70. mu.l of 1M ethanolamine hydrochloride (GE, BR-1000-50) at a flow rate of 10. mu.l/min; often the non-covalently bound peptides/proteins are removed by this procedure. The non-covalently coupled neutravidin was removed by injection of 10-30. mu.l of 50mM NaOH solution. Biotinylated human D-hepcidin 25, human L-hepcidin 20, and human, rat, and mouse L-hepcidin 25 were directly diluted to a final concentration of 10-20nM in HBS-EP buffer and immediately vortexed. Transfer 1000. mu.l of this sample toGlass Vials (Glass glasses,GE, BR-1002-07) and injected at a flow rate of 10. mu.l/min using the MANUALINJECT command. For binding assays, up to 5000 Response Units (RU) of biotinylated human D-hepcidin 25, human L-hepcidin 20, and human, rat and mouse L-hepcidin 25 were immobilizedOn the flow cell; and for kinetic evaluation, 500-. Subsequently, the flow cell was washed with 1M NaCl (Ambion, cat. No. am9759) to avoid entrainment of biotinylated human D-hepcidin 25, human L-hepcidin 20, and human, rat, and mouse L-hepcidin 25 due to non-specific interactions of biotinylated human D-hepcidin 25, human L-hepcidin 20, and human, rat, and mouse L-hepcidin 25 with Biacore tubing and other surfaces. FC1 was used as a closed control flow cell.

Finally, all sensor flow cells (from FC1 to FC4) were blocked by injecting 20. mu.l/min of a saturated Biotin solution (Biotin, Sigma-Aldrich B-4501Lot 68H1373) at a flow rate of 20. mu.l/min diluted 1: 10 in HBS-EP buffer. Degassed running buffer (20mM Tris pH 7.4; 150mM NaCl; 5mM KCl, 1mM MgCl) for sensor chips₂，1mM CaCl₂And 0.1% Tween20) were primed twice and equilibrated at 30 μ l/min until baseline appeared stable.

Typically, for analytical purposes, the aptamer/spiegelmer of hepcidin-binding nucleic acids was diluted in water to a stock concentration of 100 μ M (quantified by UV measurement), heated to 95 ℃ for 30 seconds in a water bath or hot blender, and snap-cooled on ice to ensure a homogeneous dissolution solution.

Kinetic parameters and dissociation constants were evaluated by a series of aptamer injections performed at concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.8, 3.9 and 0nM diluted in running buffer. In all experiments, the analysis was performed using Kinject commands (which define a 360 second binding time and 360 seconds dissociation time, at a flow rate of 30. mu.l/min) at 37 ℃. The assay was double-referenced, while FC1 served as a (blocked) surface control (overall contribution per aptamer concentration), and a series of buffer injections without analyte determined the overall contribution of the buffer itself. Data analysis and calculation of dissociation constants (KD) were performed using the Langmuir 1: 1 chemometric fitting algorithm with BIAevaluation 3.0 software (BIACORE AB, Uppsala, Sweden).

Example 5: inhibition of ferroportin down-regulation induced by human and mouse hepcidins by binding to hepcidin spiegelmers

Method of producing a composite material

Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 100 units/ml penicillin and 100. mu.g/ml streptomycin with Glutamax (Invitrogen, Karlsruhe, Germany) at 37 ℃ and 5% CO₂J774.1 cells (mouse monocyte-macrophages from DSMZ, Braunschweig) were cultured. For these assays, cells were plated at 7.3X 10 in 12-well plates⁵One cell/well (2X 10)⁵Individual cell/cm²) Is inoculated in 2ml of medium and at 37 ℃ and 5% CO₂The cells were incubated for several hours. After cell adherence, the cells were loaded with iron by adding 20. mu.l of Fe-NTA-solution by mixing 1 part of 0.3M FeCl in water₃And 2 parts NTA (nitrilotriacetate) in water at 0.3M followed by 1: 10 dilution with DMEM. Cells were cultured overnight as described. The next day, a stimulation solution containing human hepcidin and (when indicated) the spiegelmer aptamer (see below, added spiegelmer aptamer) each at the desired final concentration of 5X was prepared in DMEM and preincubated at 37 ℃ for 30 minutes. 0.5ml of the solution was added to each well of a 12-well plate. After 3 hours of stimulation, the medium was removed and the cells were washed rapidly once with 1ml ice-cold Phosphate Buffered Saline (PBS). Cells were then scraped from the wells in 1ml of cold PBS and collected in pre-cooled Eppendorf tubes. After centrifugation at 500g for 5 min at 4 ℃ the supernatant was removed and the pellet resuspended in 75. mu.l of lysis buffer (Tris/HCl, pH 7.5, 150mM NaCl, 1mM EDTA, 1% Triton X-100 and protease inhibitor (protease inhibitor cocktail tablet, Roche # 11873580001)). The cell suspension was frozen on dry ice, thawed, vortexed thoroughly, and centrifuged at 1000g for 10 min at 4 ℃. The lysate supernatant was collected and stored at-80 ℃ until further analysis.

Protein assays were performed using the bicinchoninic acid method. The amount of lysate containing 20. mu.g of protein was mixed with 2 Xsample buffer (125mM Tris/HCl, pH 6.8; 20% glycerol; 4% SDS; 0.02% bromophenol blue) and incubated at 37 ℃ for 10 minutes. Proteins were separated on a 10% SDS-polyacrylamide gel and transferred by electroblotting onto Hybond ECL nitrocellulose or Hybond-P PVDF membrane (GE Healthcare, Munich, Germany). After blotting, the membrane was stained with ponceau (0.2% in 3% trichloroacetic acid) to check protein loading and transfer. Membrane iron transporters were detected with rabbit anti-mouse membrane iron transporter antibody (Alpha Diagnostics, # MTP11-A) and anti-rabbit IgG-HRP conjugate (New England Biolabs, Frankfurt a.M., Germany), using LumiGlo^RChemiluminescent reagent (CellSignaling Technology, Frankfurt a.M., Germany) and Hyperfilm^TMECL chemiluminescent films (GEHealthcare, Munich, Germany).

Results

Lysates obtained from J774.1 cells after stimulation with human hepcidin or hepcidin + respective spiegelmers were separated by SDS-gel electrophoresis and analyzed by Western blotting using antibodies against mouse ferroportin.

Treatment of J774.1 cells with Fe/NTA resulted in a significant upregulation of ferroportin expression. This effect was significantly reversed by stimulating the cells with 100nM human hepcidin-25 for 3 hours. The hepcidin effect was blocked when pre-incubated with the spiegelmer aptamer 226-C5-001-5 ' -PEG, 238-D4-008-5 ' -amino and 238-D4-008-5 ' -PEG (═ NOX-H94).

FIG. 17A: the ferroportin (arrow) which was barely detectable in untreated cells (lane 1) was up-regulated by treatment with Fe/NTA (lanes 2, 3). 100nM human hepcidin-25 (HEP) resulted in down-regulation of ferroportin (lanes 4, 5) and this effect could be strongly inhibited by the spiegelmer 226-C5-001(C5-PEG) (lanes 6, 7).

FIG. 17B: human hepcidin caused ferroportin down-regulation in Fe/NTA treated J774.1 cells (lanes 6, 7). This effect of human hepcidin-25 could be strongly inhibited by the spiegelmer aptamer NOX-H94 (lanes 12-15) and the spiegelmer HEP-238-D4-008a, which is an amino-modified oligonucleotide intermediate of 238-D4-008-5' -PEG (═ NOX-H94) (lanes 8-11).

Example 6: activity of hepcidin-binding spiegelmers in vivo

The current concept of anemia of chronic disease is that proinflammatory cytokines (especially IL-6) stimulate the synthesis and release of hepcidin in hepatocytes. Hepcidin then binds to different cell types expressing the ferroportin, a ferroportin. This interaction induces internalization and degradation of the hepcidin-ferroportin complex, followed by a decrease in serum iron. Long-term reduction of serum iron negatively affects erythropoiesis and eventually manifests as anemia. The known property of human hepcidin-25 to induce serum iron decline in mice (river, 2005) was used as a model for inflammatory anemia. To test the in vivo activity of the spiegelmers, hypoferremic status was induced in C57BL/6 mice with human hepcidin-25. To characterize the spiegelmers in this model, animals received prophylactic treatment with spiegelmers to block the effect of human hepcidin.

Method of producing a composite material

Female C57B1/6 mice (elevoge Janvier, France, 6 weeks old, n 6-7 per group) received a single intravenous injection of either anti-hepcidin spiegelmer (10-20ml/kg body weight) or vehicle (5% glucose, 10-20ml/kg body weight). After 30 minutes, synthetic human hepcidin-25 (Bachem, Weil am Rhein, Germany, Cat No. H-5926) (10ml/kg body weight) was injected intraperitoneally at a dose of 1-2mg/kg body weight. Blood was collected 2 hours after hepcidin injection. Serum and plasma samples were obtained for iron determination and whole blood cell count, respectively. For each animal, serum iron, hemoglobin, hematocrit, white blood cell count, red blood cell count, platelet count, mean red blood cell volume, and mean red blood cell hemoglobin values were determined.

Results

Injection of synthetic human hepcidin-25 resulted in a rapid decrease in serum iron. At 2 hours post injection, serum iron concentrations were reduced to 56% of the values of vehicle-treated mice. These in vivo findings are consistent with the data published by river et al (Ribera et al), which reported a reduction to about 25% in a very similar trial with higher hepcidin doses. As depicted in figure 9, the reduction in serum iron was completely blocked by the administration of the spiegelmer aptamer 223-C5-001-5' -PEG prior to injection of human hepcidin (98% of control). The same effect was observed with 239-D4-008-5' -PEG, as depicted in FIG. 20.

Example 7: activity of spiegelmers binding to hepcidin in cynomolgus monkeys stimulated with human interleukin-6

The major role of interleukin-6 (IL-6) in anemia of chronic disease was demonstrated with the IL-6 receptor antibody tollizumab. Treatment with this antibody showed utility in patients with castleman's disease (Nishimoto, 2008) and also in cynomolgus monkey arthritis models (Hashizume, 2009). The known property of IL-6 to induce hepcidin secretion, which subsequently leads to anemia in non-human primates, is used as another model for anemia of inflammation (Asano, 1990; Klug 1994). Instead of using hemoglobin as a parameter, serum iron levels were chosen as endpoints to show utility against hepcidin spiegelmers. A condition of hypoferremia was induced in cynomolgus monkeys using human recombinant IL-6. This model is important to show that anti-hepcidin spiegelmers also bind endogenous hepcidin, just as synthetic human hepcidin was used in all other experiments. To test the in vivo activity of the spiegelmers, hypoferremic status was induced in cynomolgus monkeys with human recombinant IL-6. To characterize the spiegelmers in this model, animals received prophylactic treatment with spiegelmers to block the effect of cynomolgus monkey hepcidin.

Method of producing a composite material

Male cynomolgus monkeys (Roberto c. hartelust, Tilburg, The Netherlands, 34 to 38 months of age, n 3 per group) received a single intravenous injection of either anti-hepcidin spiegelmer (1ml/kg body weight) or vehicle (5% glucose, 1ml/kg body weight). After 30 minutes, recombinant human IL-6(Miltenyi Biotech, Bergisch Gladbach, Germany) was injected subcutaneously at a dose of 10. mu.g/kg body weight (1ml/kg body weight). Blood was collected 8 hours after IL-6 injection. Serum and plasma samples were obtained for iron determination and whole blood cell count, respectively. For each animal, serum iron, hemoglobin, hematocrit, white blood cell count, red blood cell count, platelet count, mean red blood cell volume, and mean red blood cell hemoglobin values were determined.

Results

Injection of recombinant human IL-6 results in a decrease in serum iron. At 8 hours post-injection, serum iron concentrations were reduced to 27% of the pre-dose values of vehicle/IL-6 treated monkeys. As depicted in FIG. 21, the reduction in serum iron was completely blocked by administration of the spiegelmer aptamer 238-D4-008-5' -PEG prior to injection of human IL-6.

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The features of the present invention disclosed in the specification, the claims, the sequence listing and/or the drawings (separately and in any combination thereof) may be material for realising the invention in diverse forms thereof.

Claims

1. An L-nucleic acid capable of binding hepcidin, wherein the nucleic acid consists of a nucleic acid sequence according to any one of SEQ ID No.116, SEQ ID No.142, SEQ ID No.144, SEQ ID No.146, SEQ ID No.148 and SEQ ID No. 152.

2. The nucleic acid according to claim 1, wherein the nucleic acid comprises a modification group, wherein the modification group is a polyethylene glycol moiety consisting of linear or branched polyethylene glycol, wherein the polyethylene glycol moiety has a molecular weight of 40,000 Da.

3. The nucleic acid according to claim 2, wherein the modifying group is coupled to the nucleic acid via a linker.

4. The nucleic acid according to claim 2, wherein the modification group is coupled to the 5 '-terminal nucleotide or the 3' -terminal nucleotide of the nucleic acid.

5. The nucleic acid according to claim 2, wherein said nucleic acid is the nucleic acid shown as SEQ ID No.175 or SEQ ID No. 176.