WO2008119571A2

WO2008119571A2 - Nucleic acid myostatin antagonist aptamers obtained by (mirror- image) selex

Info

Publication number: WO2008119571A2
Application number: PCT/EP2008/050046
Authority: WO
Inventors: Jens Rosengren Daugaard; Henning THØGERSEN; Claus Bekker Jeppesen; Jesper Lau
Original assignee: Novo Nordisk A/S
Priority date: 2007-01-03
Filing date: 2008-01-03
Publication date: 2008-10-09
Also published as: WO2008119571A3

Abstract

Myostatin antagonists are used to treat muscular generative disorders, type 2 diabetes or obesity. The antanogists are nucleic acid molecules capable of binding to myostatin.

Description

MYOSTATIN ANTAGONIST

FIELD OF THE INVENTION

The invention relates to novel Myostatin antagonists, to pharmaceutical compositions comprising these compounds and to the use of the compounds for the treatment of diseases.

BACKGROUND OF THE INVENTION

Myostatin, also known as GDF8 (Growth and Differentiation Factor 8), is a single chain polypeptide of 109 amino acid residues with four disulfide bridges. It is a secreted growth factor that belongs to the transforming growth factor-β (TGF-β) superfamily of growth and differentiation factors.

Recent evidence suggests that the inhibition of Myostatin would provide an effective treatment for a number of disorders or conditions. For example, agents that block Myostatin function could be useful for slowing or even preventing the development of obesity and type 2 diabetes. Furthermore

Myostatin antagonists may be an effective therapy for disorders or conditions that cause a reduction in muscle mass and/or strength, such as muscular dystrophy, frailty, myopathy and cachexia.

For example, US 6,096,506, US 2003/0138422 and WO 2005/094446 each describe an antibody specifically reactive with the Myostatin polypeptide or an epitope thereof. US 6,468,535 describes a method for increasing animal muscle by administration of an anti-Myostatin antibody. US 6,368,597 describes the use of a Myostatin antibody for treating diabetes.

SUMMARY OF THE INVENTION

In one aspect of the invention, there is provided a Myostatin antagonist comprising one or more nucleic acid molecules which are capable of binding to Myostatin or a Myostatin analogue thereof. According to a further aspect of the invention, the invention provides a method for treating or preventing obesity, the regulation of energy balance, appetite and body weight, eating disorders, diabetes, glucose metabolism, tumour, blood pressure and cardiovascular diseases, which comprises administering to a patient in need thereof a Myostatin antagonist composition as hereinbefore defined.

There is also provided a method for treating or preventing a muscular generative disorder, which comprises administering to a patient in need thereof a Myostatin antagonist as hereinbefore defined.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1 : demonstrates a three dimensional representation of the complex of Myostatin and Activin receptor 2B fragment

DETAILED DESCRIPTION OF THE INVENTION

Myostatin is expressed primarily in developing and adult skeletal muscle, circulates in the blood and acts as a negative regulator of skeletal muscle, i.e. higher concentrations of Myostatin reduce muscle development. The precise mechanism remains unknown.

Myostatin is highly conserved across species: the amino acid sequence of the mature form of Myostatin in human, mouse, rat and cow are 100 % identical. Growth differentiation factor- 11 (GDF-11/BMP-l 1) is the member of the family that is most homologous to Myostatin (90% identity within their mature chain). U.S. 5,827,733 discloses the nucleotide and amino acid sequence of human Myostatin. The sequence of human Myostatin is shown in SEQ ID No. 1.

The term "Myostatin analogue" as used herein refers to an analogue of Myostatin, wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the peptide and or wherein one or more amino acid residues have been added to the peptide. Such addition or deletion of amino acid residues can take place at the N-terminal of the peptide and/or at the C-terminal of the peptide.

In one embodiment, the Myostatin analogue has an amino acid sequence having at least 80% identity to SEQ ID No. 1. In a further embodiment, the Myostatin analogue has an amino acid sequence having at least 85%, such at least 90%, for instance at least 95%, such as for instance at least 99% identity to SEQ ID No. 1.

The term "identity" as known in the art, refers to a relationship between the sequences of two or more peptides, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between peptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related peptides can be readily calculated by known methods.

In one embodiment, the Myostatin analogue has an amino acid sequence, which sequence is at least 80% similar to SEQ ID No. 1. In one embodiment, the Myostatin analogue has an amino acid sequence, which sequence is at least 85%, such as at least 90%, for instance at least 95%, such as for instance at least 99% similar to SEQ ID No. 1.

The term "similarity" is a concept related to identity, but in contrast to "identity", refers to a sequence relationship that includes both identical matches and conservative substitution matches. If two polypeptide sequences, each composed of 20 amino acids, have, for example, 10 identical amino acids, and the remainder are all non-conservative substitutions, then the percentage identity and similarity would both be 50%. If, in the same example, there are 5 more positions where there are conservative substitutions, then the percentage identity remains 50%, but the percentage similarity would be 75%. Therefore, in cases where there are conservative substitutions, the degree of similarity between two polypeptides will be higher than the percentage identity between those two polypeptides.

The term "nucleotide" as referred to herein means a molecule composed of a pentose sugar, a phosphate and a nitrogenous heterocyclic base, either a purine, e.g. guanine and adenine, or a pyrimidine, e.g. cytosine, thymine and uracil. The sugar may be a ribose or deoxyribose. The 'deoxy' prefix indicates that the 2'carbon atom of the sugar lacks the oxygen atom that is linked to the 2' carbon atom of the ribose. Nucleotides containing ribose are known as ribonucleotides, and those containing deoxyribose are known as deoxyribonucleotides. Normally, ribonucleotides are made up one of four bases, adenine, guanine, cytosine and uracil, whilst deoxyribonucleotides are made up from one of adenine, guanine, cytosine and thymine.

The term "L-nucleotide" as used herein refers to a nucleotide wherein the sugar moiety is either an L-ribose or an L-deoxyribose. These sugars are enantiomers of D-ribose and D-deoxyribose, respectively.

The term "D-nucleotide" as used herein refers to a nucleotide wherein the sugar moiety is either a D-ribose or a D-deoxyribose. These sugars are enantiomers of L-ribose and L-deoxyribose, respectively.

The term "nucleic acid" as referred to herein means a complex, high- molecular-weight macromolecule composed of linked nucleotides. The sugars in nucleic acids are linked to one another by phosphodiester bridges. Specifically, the 3 '-hydroxyl (3 '-OH) group of the sugar moiety of one nucleotide is esterified to a phosphate group, which is, in turn, joined to the 5 '-hydroxyl group of the adjacent sugar. The chain of sugars linked by phosphodiester bridges is referred to as the backbone of the nucleic acid. Whereas the backbone is normally constant, the bases vary from one monomer to the next.

The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). These nucleic acid types differ from each other in two respects. Firstly, they differ in the specific sugar found in their chain. The sugar in DNA is deoxyribose, whilst the sugar in RNA is ribose. The other difference is that one of the four major bases in RNA is uracil instead of thymine as found in DNA.

Artificial nucleic acids include peptide nucleic acid (PNA) and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule.

"L-nucleic acid" as used herein refers to a nucleic acid consisting of L- nucleotides, preferably consisting completely of L-nucleotides. In one embodiment, the L-nucleic acid comprises at least 50% L-nucleotides. In another embodiment, the L-nucleic acid is made up of at least 75%, such as at least 85%, for instance at least 95%, such as for instance at least 99% L- nucleotides. In a further embodiment, L-nucleic acid contains only L- nucleotides.

"D-nucleic acid" as used herein is a nucleic acid consisting of D- nucleotides, preferably consisting completely of D-nucleotides. In one embodiment, the D-nucleic acid comprises at least 50% D-nucleotides. In another embodiment, the D-nucleic acid is made up of at least 75%, such as at least 85%, for instance at least 95%, such as for instance at least 99% D- nucleotides. In a further embodiment, D-nucleic acid contains only D- nucleotides.

The term "double stranded nucleic acid" as used herein refers to two complementary nucleic acids held together by hydrogen bonding between the bases.

A "multipartite nucleic acid", as used herein, refers to a Myostatin binding nucleic acid which consists of at least two nucleic acids. The at least two nucleic acid strands may be derived from any of the Myostatin binding nucleic acids. For example, they may be formed by cleavage of a Myostatin binding nucleic acid to generate two strands. Alternatively, they may be formed by synthesising two nucleic acids, the first nucleic acid corresponding to a part of the Myostatin binding nucleic acid and the second nucleic acid corresponding to a different part of the Myostatin binding nucleic acid. It is to be acknowledged that both the cleavage and the synthesis may be applied to generate a multipartite nucleic acid where there are more than two strands as exemplified above. It should be noted that the at least two nucleic acids are typically not complementary to each other. Nevertheless, a certain extent of complementarity between the various nucleic acid parts may exist.

The term "aptamer" refers to a D-nucleic acid that binds tightly to a specific molecular target due to its three dimensional shape. In one embodiment, the specific molecular target is Myostatin.

The term "spiegelmer" refers to an L-nucleic acid that binds tightly to a specific molecular target due to its three dimensional shape. In one embodiment, the specific molecular target is Myostatin.

The term "PEG" is intended to indicate polyethylene glycol of a molecular weight between approximately 100 and approximately 1,000,000 Da, including analogues thereof, wherein for instance the terminal OH-group has been replaced by an alkoxy group, such as e.g. a methoxy group, an ethoxy group or a propoxy group. In particular, the PEG wherein the terminal -OH group has been replaced by methoxy is referred to as mPEG.

The term "equilibrium dissociation constant" is used to express the intensity of the binding between the nucleic acid and the target, which is in the present case Myostatin, and may be referred to as the "Kd value". The method for its determination is known to the one skilled in the art.

"Muscular generative disorder" refers to a disease or disorder characterised by a reduction in muscle mass and/or strength. This may be caused by any mechanism of action or combination of mechanism of action. For example, it may result from defects in muscle proteins, and/or the death of muscle cells and tissue. Muscular generative disorders include, but are not limited to, muscular dystrophy, frailty, critical care myopathy and cachexia resulting from cancer or other disorders, including but not limited to HIV infection, critical care and other myopathies.

In the present context, the term "pharmaceutically acceptable salt" is intended to indicate salts which are not harmful to the patient. Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, and ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids and the like. Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in J. Pharm. Sci. 1977, 66, 2, which is incorporated herein by reference. Examples of metal salts include lithium, sodium, potassium, magnesium salts and the like. Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts and the like.

The term "treatment" and "treating" as used herein means the management and care of a patient for the purpose of combating a condition, such as a disease or a disorder. The term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the active compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications. The patient to be treated is preferably a mammal, in particular a human being, but it may also include animals, such as dogs, cats, cows, sheep and pigs.

Composition of the Invention

The invention is based on the finding that it is possible to generate a nucleic acid that binds specifically, and with a high affinity, to Myostatin. In one aspect, there is provided a Myostatin antagonist comprising one or more nucleic acid molecules which are capable of binding to Myostatin or a Myostatin analogue thereof. It will be appreciated that references to Myostatin herein also include references to a Myostatin analogue. The nucleic acid according to the invention may be either a D-nucleic acid or an L-nucleic acid. In one embodiment, the Myostatin binding nucleic acid is an L-nucleic acid. In addition it is possible that one or several parts of the nucleic acid are present as D-nucleotides or at least one or several parts of the nucleic acids are L-nucleotide. The term "part" of the nucleic acids shall mean as little as one nucleotide. In other embodiments, the Myostatin antagonist may comprise both D-nucleic acids and L-nucleic acids.

Designing the nucleic acid as an L-nucleic acid is advantageous for several reasons. D-nucleic acids are not very stable in aqueous solutions and particularly in biological systems or biological samples, due to the widespread presence of nucleases. However naturally occurring nucleases, particularly nucleases from animal cells are not capable of degrading L- nucleic acids. Because of this the biological half-life of the L-nucleic acid is significantly increased in such a system. Furthermore due to the lack of L-nucleic acid nucleases, no nuclease degradation products are generated and thus no side effects arising therefrom are observed.

Thus, by using D or L-nucleic acid, the plasma stability and plasma clearance can be tailored to meet the requirements for once daily, once weekly administration or any other dosing regime appropriate for the specific patient population.

The Myostatin antagonist may be either a spiegelmer or an aptamer. In one embodiment, the Myostatin antagonist is a spiegelmer.

In a further embodiment, the Myostatin antagonist selectively binds to Myostatin, i.e. it provides the advantage of being significantly less reactive or non-reactive with other members of the TGF-β superfamily of proteins (e.g. GDF-I l). In one embodiment, the nucleic acid according to the invention binds to Myostatin with a Kd value of below 100 pM, suitably below lOpM, for example below IpM. The nucleic acid of the invention shall also bind to Myostatin with a Kd value of 1OnM, 3nM, InM, 10OpM, 3OpM, lOpM or below IpM.

In one embodiment, the nucleic acid may comprise deoxyribonucleotide(s), ribonucleotide(s) or combinations thereof.

In one embodiment, the nucleic acid may comprise a single stranded or a double-stranded nucleic acid. Typically, the nucleic acids are single stranded nucleic acids which exhibit defined secondary structures due to the primary sequence and may thus also form tertiary structures.

The nucleic acid according to the invention may have any length provided that it is still able to bind to the target molecule. In one embodiment, the length is between 15 and 120 nucleotides. In a further embodiment, the ranges for the length of the nucleic acid according to the invention are about 20 to 100 nucleotides, about 20 to 80 nucleotides, about 20 to 60 nucleotides, about 20 to 50 nucleotides, for example 30 to 50 nucleotides.

The Myostatin binding nucleic acid may be modified. Such modifications may be related to the single nucleotide of the nucleic acid and are well known in the art. Examples for such modifications are described in, among others, Kusser, W.(2000) J Biotechnol, 74: 27-38; Aurup, H. et al (1994) Nucleic Acids Res, 22,20-4; Cummins, L. L. et al , (1995) Nucleic Acids Res, 23, 2019-24; Eaton, RE. et al (1995) Chem Biol, 2, 633-8; Green, L. S. et al, (1995) Chem Biol, 2, 683-95; Kawasaki, A.M. et al , (1993) J Med Chem, 36, 831-41 ; Lesnik, E.A. et al , (1993) Biochemistry, 32, 7832-8; Miller, L. E. et al, (1993) J Physiol, 469, 213-43, which are hereby incorporated by reference. In a further embodiment, the nucleic acid may also comprise nucleotides that have been chemically derivatised with chemical groups. These chemical groups may serve to increase solubility, improve formulation properties, such as stability, increase in vivo stability, such as enzymatic stability, and decrease renal clearance.

Derivatisation may be achieved by attachment of a polymer such as PEG or by attachment of a chemical group that has affinity towards a plasma protein, such as e.g. albumin. The ability of a compound to bind to albumin may be determined as described in J.Med.Chem, 43, 2000, 1986-1992, which is incorporated herein by reference. In the present context, a compound is defined as binding to albumin if Ru/Da is above 0.05, such as above 0.10, such as above 0.12 or even above 0.15.

In another embodiment, the albumin binding moiety is a peptide, such as a peptide comprising less than 40 amino acid residues. A number of small peptides which are albumin binding moieties are disclosed in J. Biol Chem. 277, 38 (2002) 35035-35043, which is incorporated herein by reference.

In one embodiment, parts of the nucleic acid, that may or may not be involved in binding to Myostatin, may exhibit a function other than binding to Myostatin. In one embodiment, these additional functions may result from modification and/or chemically derivatised nucleotides. An example of an alternative function may be the interaction with other molecules, other than Myostatin, for purposes including, but not limited to, immobilization, cross-linking, detection or amplification.

Figure 1 demonstrates a three dimensional representation of the complex of Myostatin and Activin receptor 2B fragment.

The model was built based on the following structures: ILXI Refinement of BMP7 crystal structure 1M4U Crystal structure of Bone Morphogenetic Protein-7 (BMP-7) in complex with the secreted antagonist Noggin

INYS Crystal Structure of Activin A Bound to the ECD of ActRIIB P41 1 S4Y Crystal structure of the activin/actrllb extracellular domain IWAQ CRYSTAL STRUCTURE OF HUMAN GROWTH AND

DIFFERENTIATION FACTOR 5 (GDF-5)

Methods of Synthesis A nucleic acid may be synthesised from nucleotides via a variety of different routes using commercially available starting materials and/or starting materials prepared by conventional methods.

The production of nucleotides and nucleic acids is well known in the art.

The method disclosed herein for the screening of nucleic acids binding to Myostatin and in particular having the further features and characteristics as disclosed herein, is based on the so called SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process as disclosed in US patent 5,475,096, which is incorporated herein by reference. The SELEX process is known to the one skilled in the art

According to one aspect of the invention, there is provided a method of screening for one or more nucleic acid molecules which are capable of binding to Myostatin or an analogue thereof, which method comprises the following steps: a) generating a heterogeneous population of nucleic acids; b) contacting the population of step a) with Myostatin or an analogue thereof; c) separating the nucleic acid(s) not interacting with Myostatin or an analogue thereof; d) optionally separating the nucleic acid(s) interacting with Myostatin or an analogue thereof; and e) optionally sequencing the nucleic acid(s) interacting with Myostatin or an analogue thereof.

In one embodiment, the method comprises an amplification of the individual nucleic acid binding to the target molecule using a polymerase chain reaction (PCR).

A procedure has been described for efficiently mutagenizing nucleic acid sequences during PCR amplification. Thus PCR can optionally be used to change of the primary sequence of the binding nucleic acid. Because of these changes new sequences may be generated which show a binding characteristic different from the starting sequence such as, among others, increased affinity of specificity.

The method for the screening of L-nucleic acids binding to L-Myostatin, i.e. the naturally occurring form of the peptide, is based on the method as disclosed in WO 98/08856, which is incorporated herein by reference.

In one embodiment, the method comprises the following steps: a) generating a heterogeneous population of D-nucleic acids; b) contacting the population of step a) with D-Myostatin, i.e. all of the amino acids forming the peptide are D-amino acids; c) separating the D-nucleic acid not interacting with D-Myostatin; d) sequencing the D-nucleic acid interacting with D-Myostatin; and e) synthesising the L-nucleic acid sequence(s) which is/are identical to the sequence of the D-nucleic acid(s) obtained in step d).

Myostatin may be obtained from commercial sources or custom made. The production of polypeptides is well known in the art. For example, polypeptides may be produced by classical peptide synthesis, e.g. solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well established techniques, see e.g. Green and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999.

The polypeptides can also be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the polypeptide and capable of expressing the polypeptide in a suitable nutrient medium under conditions permitting the expression of the peptide. For polypeptides comprising non-natural amino acid residues, the recombinant cell should be modified such that the non-natural amino acids are incorporated into the polypeptide, for instance by use of tRNA mutants.

The polypeptides can also be produced using cell-free in vitro transcription/translation systems. The polypeptide containing novel unnatural amino acids can also be produced using frameshift or nonsense suppression systems e.g. as described in J. Am. Chem. Soc. 125 (2003): 11782-11783, Science 301 (August 2003): 964-967, Science 292 (April 2001): 498-500, Science 303 (January 2004): 371-373 and references herein.

The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The peptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration. For extra cellular products the proteinaceous components of the supernatant are isolated by filtration, column chromatography or precipitation, e.g. microfiltration, ultrafiltration, isoelectric precipitation, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, or the like, dependent on the type of polypeptide in question. For intracellular or periplasmic products the cells isolated from the culture medium are disintegrated or permeabilised and extracted to recover the product polypeptide or precursor thereof.

The DNA sequence encoding the therapeutic polypeptide may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the peptide by hybridisation using specific DNA or RNA probes in accordance with standard techniques (see, for example, Sambrook, J, Fritsch, EF and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequence encoding the polypeptide may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859- 1869, or the method described by Matthes et a , EMBO Journal 3 (1984), 801 - 805. The DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al , Science 239 (1988), 487-491.

The DNA sequence may be inserted into any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. The vector is preferably an expression vector in which the DNA sequence encoding the polypeptide is operably linked to additional segments required for transcription of the DNA, such as a promoter. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the peptide of the invention in a variety of host cells are well known in the art, cf. for instance Sambrook et al., supra.

The DNA sequence encoding the polypeptide may also, if necessary, be operably connected to a suitable terminator, polyadenylation signals, transcriptional enhancer sequences, and translational enhancer sequences. The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.

The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate. For large scale manufacture the selectable marker preferably is not antibiotic resistance, e.g. antibiotic resistance genes in the vector are preferably excised when the vector is used for large scale manufacture. Methods for eliminating antibiotic resistance genes from vectors are known in the art, see e.g. US 6,358,705 which is incorporated herein by reference.

To direct a parent peptide of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the peptide in the correct reading frame. Secretory signal sequences are commonly positioned 5 ' to the DNA sequence encoding the peptide. The secretory signal sequence may be that normally associated with the peptide or may be from a gene encoding another secreted protein.

The procedures used to ligate the DNA sequences coding for the present peptide, the promoter and optionally the terminator and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., supra).

The host cell into which the DNA sequence or the recombinant vector is introduced may be any cell which is capable of producing the present peptide and includes bacteria, yeast, fungi and higher eukaryotic cells. Examples of suitable host cells well known and used in the art are, without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.

When Myostatin is prepared in E. coli the protein may be excreted in inclusion bodies and refolded afterwards using refolding procedures described in the art.

The peptide is either synthesised as the full length peptide, or 2 or more fragments of the peptide are synthesised and ligated afterwards using native chemical ligation techniques as described in the art (see Houben-Weyl "Synthesis of Peptides and Peptidomimetics 4th edition, Volume E22a, E22b and E22c).

In the synthesis of the full-length peptide or the one or more fragments, it can be an advantage to use complementary protecting groups of the D-Cys residues to facilitate the correct folding of the peptide (see e.g. methods in Houben-Weyl "Synthesis of Peptides and Peptidomimetics 4th edition, Volume E22b). The peptide may also be synthesised as the unfolded full length peptide (e.g. using the Liberty Microwave-Enhanced Peptide Synthesis from CEM; Germany) and then folded using the procedure developed to give the active form of the natural Myostatin peptide.

The correct folding of Myostatin can be controlled by testing of the folded Myostatin in a functional assay. A model of Myostatin was prepared by sequence alignment of Myostatin with activin (see Figure 1).

In one embodiment, the D form of Myostatin may be obtained by chemical synthesis.

Uses of the Compounds

The Myostatin antagonist according to the invention may be used in therapy, and this is also an embodiment of the invention. Thus, the Myostatin antagonist may be used to treat or prevent a variety of disorders or conditions, whereby reduction of the bioavailability of Myostatin is desired.

In one aspect, the invention may be used to provide a particular beneficial therapy to increase muscle mass and muscle strength. In one embodiment, the invention provides a method of treatment or prophylaxis of a muscular generative disorder, which comprises administration of the Myostatin antagonist as hereinbefore defined.

In a specific embodiment, the Myostatin antagonist is used to treat diseases, disorders or conditions, including, but not limited to obesity, the regulation of energy balance, appetite and body weight, eating disorders, diabetes, glucose metabolism, tumour, blood pressure and cardiovascular diseases. For the purpose of the invention regulation of energy balance is regarded as a disease. The invention thus provides a method for treating these diseases or states, the method comprising administering to a patient in need thereof a therapeutically effective amount of the Myostatin antagonist according to the invention.

In one embodiment, the Myostatin antagonist is used to treat or prevent diabetes, more specifically type 1 or type 2 diabetes.

In another embodiment, the use is for the treatment or prophylaxis of any disease where the regulation of the energy balance is influenced by Myostatin, either directly or indirectly. The same applies to glucose metabolism, blood pressure and appetite and body weight.

In another embodiment, the Myostatin antagonist is used to treat or prevent tumours arising from variant tissue types, including, but not limited to, cancers of the bone, breast, respiratory tract (e.g. lung), brain, reproductive organs (e.g. cervix), digestive tract (e.g. gastro-intestinal tract and colorectal tract), urinary tract, bladder, eye, liver, skin, head, neck, thyroid, parathyroid, kidney, pancreas, blood, ovary, colon, germ/prostate, and metastatic forms thereof.

Thus, in some embodiments, there is provided a use of the Myostatin antagonist as hereinbefore defined in the manufacture of a medicament for the treatment or prevention of the diseases, disorders or conditions as hereinbefore described. In a further embodiment, there is provided a pharmaceutical composition comprising a Myostatin antagonist as hereinbefore defined for use in the treatment of the diseases, disorders or conditions as hereinbefore described.

It is to be understood that the Myostatin antagonist according to the invention may be used not only as a medicament or for the manufacture of a medicament, but also for cosmetic purposes, particularly with regard to the involvement of Myostatin in obesity. Specifically, the Myostatin antagonist may be used as a means for weight control and/or a means for appetite control.

Myostatin is highly conserved in sequence and in function across species and therefore the Myostatin antagonist may be useful for the treatment of such disorders not only in humans but also in other mammals including domestic animals, sports animals and food source animals.

Combination Therapies

Many diseases are treated using more than one medicament in the treatment, either concomitantly administered or sequentially administered. It is therefore within the scope of the invention to use the Myostatin antagonist of the invention in therapeutic methods for the treatment of one of the above mentioned diseases in combination with one another, or as an adjunct to, or in conjunction with, other established therapies normally used to in the treatment said disease. By analogy, it is also within the scope of the invention to use the Myostatin antagonist of the invention in combination with other therapeutically active compounds normally used in the treatment of one of the above mentioned diseases in the manufacture of a medicament for said disease.

Pharmaceutical Compositions

Another purpose is to provide a pharmaceutical composition comprising the Myostatin antagonist of the invention. The nucleic acids of the invention are generally utilised as the free substance or as a pharmaceutically acceptable salt thereof. The composition may further comprise a buffer system, preservative(s), tonicity agent(s), chelating agent(s), stabilizers and surfactants. The composition may further comprise one or more therapeutic agents active against the same disease state. In one embodiment of the invention the pharmaceutical composition is an aqueous composition, i.e. composition comprising water. Such composition is typically a solution or a suspension. In a further embodiment of the invention the pharmaceutical composition is an aqueous solution. The term "aqueous composition" is defined as a composition comprising at least 50 % w/w water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50 %w/w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50 %w/w water.

In another embodiment the pharmaceutical composition is a freeze-dried composition, whereto the physician or the patient adds solvents and/or diluents prior to use.

In another embodiment the pharmaceutical composition is a dried composition (e.g. freeze-dried or spray-dried) ready for use without any prior dissolution.

In a further aspect the invention relates to a pharmaceutical composition comprising an aqueous solution of the nucleic acid as hereinbefore defined, and a buffer, wherein said composition has a pH from about 2.0 to about 10.0.

In another embodiment of the invention the pH of the composition is selected from the list consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,

4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,

9.7, 9.8, 9.9, and 10.0.

In a further embodiment of the invention the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each one of these specific buffers constitutes an alternative embodiment of the invention.

In a further embodiment of the invention the composition further comprises a pharmaceutically acceptable preservative. The use of a preservative in pharmaceutical compositions is well known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20th edition, 2000.

In a further embodiment of the invention the composition further comprises an isotonic agent. The use of an isotonic agent in pharmaceutical compositions is well known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20th edition, 2000.

In a further embodiment of the invention the composition further comprises a chelating agent. The use of a chelating agent in pharmaceutical compositions is well known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20th edition, 2000.

In a further embodiment of the invention the composition further comprises a stabiliser. The use of a stabilizer in pharmaceutical compositions is well known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20th edition, 2000.

In a further embodiment of the invention the composition further comprises a stabilizer selected from the group of high molecular weight polymers or low molecular compounds. In a further embodiment of the invention the stabilizer is selected from polyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, sulphur-containing substances as monothioglycerol, thioglycolic acid and 2- methylthioethanol, and different salts (e.g. sodium chloride). Each one of these specific stabilizers constitutes an alternative embodiment of the invention.

In a further embodiment of the invention the composition further comprises a surfactant. The use of a surfactant in pharmaceutical compositions is well- known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20th edition, 2000.

It will be appreciated that following preparation, the composition may be packaged for storage, either in a liquid form, in a frozen state, or in a dried form for later reconstitution into a liquid form or other form suitable for administration to a subject.

By "dried form" is intended the liquid pharmaceutical composition or composition is dried either by freeze drying (i.e., lyophilization; see, for example, Williams and Polli (1984) J. Parenteral Sci. Technol. 38:48-59), spray drying (see Masters (1991) in Spray-Drying Hand-book (5th ed; Longman Scientific and Technical, Essez, U.K.), pp. 491-676; Broadhead et al (1992) Drug Devel. Ind. Pharm. 18: 1169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11 : 12-20), or air drying (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser (1991) Biopharm. 4:47-53).

It is possible that other ingredients may be present in the pharmaceutical composition of the invention. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (e.g., human serum albumin, gelatine or proteins) and a zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine). Such additional ingredients, of course, should not adversely affect the overall stability of the pharmaceutical composition of the invention.

Pharmaceutical compositions containing a Myostatin antagonist according to the invention may be administered to a patient in need of such treatment at several sites, for example, at topical sites, for example, skin and mucosal sites, at sites which bypass absorption, for example, administration in an artery, in a vein, in the heart, and at sites which involve absorption, for example, administration in the skin, under the skin, in a muscle or in the abdomen.

Administration of pharmaceutical compositions according to the invention may be through several routes of administration, for example, oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), transdermal, intracisternal, intraperitoneal, vaginal and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route. It will be appreciated that the preferred route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingredient chosen.

Compositions of the current invention may be administered in several dosage forms, for example, as solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules, for example, hard gelatine capsules and soft gelatine capsules, suppositories, rectal capsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops, ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal ointments, injection solution, in situ transforming solutions, for example in situ gelling, in situ setting, in situ precipitating, in situ crystallization, infusion solution, and implants. Compositions of the invention may further be compounded in, or attached to, for example through covalent, hydrophobic and electrostatic interactions, a drug carrier, drug delivery system and advanced drug delivery system in order to further enhance stability of the GH conjugate, increase bioavailability, increase solubility, decrease adverse effects, achieve chronotherapy well known to those skilled in the art, and increase patient compliance or any combination thereof. Examples of carriers, drug delivery systems and advanced drug delivery systems include, but are not limited to, polymers, for example cellulose and derivatives, polysaccharides, for example dextran and derivatives, starch and derivatives, poly(vinyl alcohol), acrylate and methacrylate polymers, polylactic and polyglycolic acid and block co-polymers thereof, polyethylene glycols, carrier proteins, for example albumin, gels, for example, thermogelling systems, for example block co-polymeric systems well known to those skilled in the art, micelles, liposomes, microspheres, nanoparticulates, liquid crystals and dispersions thereof, L2 phase and dispersions there of, well known to those skilled in the art of phase behaviour in lipid-water systems, polymeric micelles, multiple emulsions, self-emulsifying, self-microemulsifying, cyclodextrins and derivatives thereof, and dendrimers.

Compositions of the current invention are useful in the composition of solids, semi-solids, powder and solutions for pulmonary administration of the Myostatin antagonist as hereinbefore defined, using, for example a metered dose inhaler, dry powder inhaler and a nebulizer, all being devices well known to those skilled in the art.

Compositions of the current invention are specifically useful in the composition of controlled, sustained, protracting, retarded, and slow release drug delivery systems. More specifically, but not limited to, compositions are useful in composition of parenteral controlled release and sustained release systems (both systems leading to a many-fold reduction in number of administrations), well known to those skilled in the art. Even more preferably, are controlled release and sustained release systems administered subcutaneous. Without limiting the scope of the invention, examples of useful controlled release system and compositions are hydrogels, oleaginous gels, liquid crystals, polymeric micelles, microspheres, nanoparticles.

Methods to produce controlled release systems useful for compositions of the current invention include, but are not limited to, crystallization, condensation, co-crystallization, precipitation, co-precipitation, emulsification, dispersion, high pressure homogenisation, en-capsulation, spray drying, microencapsulating, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes. General reference is made to Handbook of Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99: Protein Composition and Delivery (MacNally, EJ., ed. Marcel Dekker, New York, 2000).

Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a solution or suspension for the administration of the Myostatin antagonist in the form of a nasal or pulmonal spray. As a still further option, the pharmaceutical compositions containing the Myostatin antagonist of the invention can also be adapted to transdermal administration, e.g. by needle-free injection or from a patch, optionally an iontophoretic patch, or transmucosal, e.g. buccal, administration. For the same purpose the Myostatin antagonist of the invention may be used as a food additive. Thus, in a further aspect, the invention provides a dietary composition, e.g. a drink, such as a fruit juice, sports drink, yoghurt drink, a milk drink, tea and the like or a solid foodstuff, e.g. a food snack bar, such as a fruit bar, nut bar and cereal bar, a cereal, a dessert, a chocolate (e.g. milk and dark) bar and the like, which comprises the Myostatin antagonist and/or the combinations referred to above.

When the Myostatin antagonist as described herein or a pharmaceutically acceptable salt, solvate or prodrug thereof is used in combination with a second therapeutic agent active against the same disease state, they may conveniently be administered alone or in combination, in either single or multiple doses, sequentially or simultaneously, by the same route of administration, or by a different route.

Effective Dosages

The Myostatin antagonist, or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular disease being treated. The compound(s) may be administered therapeutically to achieve therapeutic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated and/or eradication or amelioration of one or more of the systems associated with the underlying disorder. Therapeutic benefit also includes halting or slowing the progression of the disease, regardless of whether improvement is realised.

The exact dosage will depend upon the frequency and mode of administration, the sex, age, weight and general condition of the subject treated, the nature and severity of the condition treated and any concomitant diseases to be treated and other factors evident to those skilled in the art. Determination of the effective dosage is well within the capabilities of those skilled in the art. When the Myostatin antagonist or a pharmaceutically acceptable salt, solvate or prodrug thereof is used in combination with a second therapeutic agent active against the same disease state the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.

A composition comprising the Myostatin antagonist according to the invention can be used for any of the aforementioned purposes.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law).

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. EXAMPLES

The invention will be further defined by reference to the following examples. It will be apparent to those skilled in the art that many modifications, both to the materials and methods may be practiced without departing from the scope of the invention.

Analytical Methods

C2C12 skeletal muscle cell line

C2C12 skeletal muscle cell line is an in vitro bioassay system that has been used extensively in studies of the effects of muscle growth factors on muscle protein synthesis and degradation, cell replication, and apoptosis (Del Aguila et al. Am J Physiol Endocrinol Metab 276: E849-E855, 1999; Milasincic et al. MoI Cell Biol 16: 5964-5973, 1996; Semsarian C et al. Biochem J 339: 443-451, 1999).

Example 1: Inhibition of Muscle Growth by Myostatin As muscle mass represents the balance between muscle cell replication and protein synthesis and muscle protein breakdown and cell death, an assay is set-up to analyse the processes by which Myostatin inhibits muscle growth.

Cells (myoblasts and/or myotubes) are incubated with Myostatin with and/or without Myostatin inhibiting nucleic acids and subsequent cell proliferation is determined.

C2C12 cell proliferation assay

Mouse skeletal muscle cell line C2C12 is propagated as myoblasts in DMEM (DMEM containing glutamine and antibiotics) with 10% FBS, and incubated at 37°C at 70% confluence in T- 175 flasks. For studies in myoblasts, cell proliferation assays is carried out in 96 well plates (1000- 1400 myoblasts in each well). After a 16 h attachment period cells are then treated with Myostatin proteins and Myostatin antagonists in varying concentrations for 24 h, 48 h and 72 h. Cell proliferation is studied using [³H] thymidine incorporation into DNA. For differentiation into myotubes, myoblasts are plated in 96 well plates (1000-1400 myoblasts in each well). After 4 days, the medium is changed to DMEM with 2% horse serum. The myotubes begin to form in 2-4 days, and multinucleated muscle fiber cultures are used at 7-10 days. Cells are then treated with Myostatin proteins and Myostatin antagonists in varying concentrations for 24 h, 48 h and 72 h. Cell proliferation is studied using [³H] thymidine incorporation into DNA.

[³H] Thymidine incorporation into DNA

Cell cultures in 96-well plates are incubated in DMEM medium (0, 1% FBS) containing 2 μCi/ml [ H] thymidine (Amersham no. TRK296) in the absence or presence of Myostatin proteins and Myostatin antagonists. The cells are harvested at 24, 48, and 72 h, washed with NaCl, and lysed with trypsin. For analysis of 3H-labeled DNA, cell lysate are spotted onto GF/C filters and washed. Filters are dried, placed in scintillation fluid (Scinti- Safe, Fisher) and counted in a Top-counter (Tri Carb.).

Example 2: Signal Transduction Analysis of Myostatin TGF-β and related proteins initiate cellular responses by binding to two different types of serine/threonine kinase receptors, termed type I and type II. Type I receptor is activated by type II receptor upon ligand binding, and initiates specific intracellular signals by Smad proteins. Smad proteins are a group of molecules that function as intracellular signal transducers downstream of the receptors of the TGF-b superfamily.

It has been reported that ActRIIB is the type II receptor for Myostatin. The binding between Myostatin and the ActRIIB receptor is specific. An assay was set-up to analyse the signal transduction of Myostatin which requires the participation of Smad2/3 and Smad4. C2C12 cell signalling assay

Mouse skeletal muscle cell line C2C12 is propagated as myoblasts in DMEM (DMEM containing glutamine and antibiotics) with 10% FBS, and incubated at 37°C at 70% confluence in T- 175 flasks. For studies in myoblasts, cell signalling assays are carried out in 12 well plates (10000- 14000 myoblasts in each well). After a 16 h attachment period cells are then treated with Myostatin proteins and Myostatin antagonists in varying concentrations for 1-72 h. Cell signalling is studied using western blotting using specific antibodies. For differentiation into myotubes, myoblasts are plated in 12 well plates (10000-14000 myoblasts in each well). After 4 days, the medium is changed to DMEM with 2% horse serum. The myotubes begin to form in 2-4 days, and multinucleated muscle fibre cultures are used at 7-10 days. Cells are then treated with Myostatin proteins and Myostatin antagonists in varying concentrations for 1-72 h. Cell signalling is studied using western blotting using specific antibodies.

Western blotting

Cell cultures in 12-well plates are incubated in DMEM medium in the absence or presence of Myostatin proteins and or Myostatin antagonists. The cells are harvested at 1 - 72 h, washed with NaCl, and lysed. To complete cell disruption, extracts are frozen and thawed. SMAD- phosphorylation in muscle homogenates is determined using the Western blot technique. Briefly, muscle cells lysate are homogenized and briefly centrifuged. The pellet is discarded and the supernatant stored in aliquots at -80⁰C for subsequent determination of protein concentration and Western blotting. Protein concentrations are determined using Bio-Rad Protein Assay (Bio-Rad, Hercules, Ca., USA) and using Human Serum Albumin as standard.

The primary antibody is an anti-phospho-SMAD 2 and anti-phospho-SMAD 3 (for example from Santa Cruz Biotechnology, Santa Cruz, Ca., USA, and the secondary antibody is the relevant horse-radish-peroxidase-labelled secondary antibody (for example from Pierce Chemical, Rockford, II., USA, but other companies could also be relevant).

The signal is detected using the LAS-3000 imaging system (Fuji Photo Film, Tokyo, Japan) and quantified using the Image Quant software (GE Healthcare, Uppsala, Sweden).

Claims

1. A Myostatin antagonist comprising one or more nucleic acid molecules which are capable of binding to Myostatin or a Myostatin analogue thereof.

2. An antagonist according to claim 1 , wherein the nucleic acid comprises an L-nucleic acid.

3. An antagonist according to claims 1 or 2, wherein the nucleic acid comprises a D-nucleic acid.

4. An antagonist according to any of the preceding claims, wherein the antagonist selectively binds to Myostatin.

5. An antagonist according to any of the preceding claims, wherein the nucleic acid binds to Myostatin with a Kd value between IpM and lOpM.

6. An antagonist according to any of the preceding claims, wherein the nucleic acid comprises deoxyribonucleotides.

7. An antagonist according to any of the preceding claims, wherein the nucleic acid comprises ribonucleotides.

8. An antagonist according to any of the preceding claims, wherein the nucleic acid comprises a single stranded nucleic acid.

9. An antagonist according to claims 1 to 7, wherein the nucleic acid comprises a double stranded nucleic acid.

10. An antagonist according to claims 1 to 7, wherein the nucleic acid comprises a multipartite nucleic acid.

11. An antagonist according to any of the preceding claims, wherein the nucleic acid comprises between 15 and 120 nucleotides.

12. An antagonist according to any of the preceding claims, wherein the nucleic acid comprises one or more chemically derivatised nucleotides.

13. A pharmaceutical composition comprising the antagonist as defined in any of claims 1 to 12.

14. Use of the antagonist as defined in any of claims 1 to 12 in the manufacture of a medicament for the treatment or prophylaxis of a muscular generative disorder.

15. Use of the antagonist as defined in any of claims 1 to 12 in the manufacture of a medicament for the treatment or prophylaxis of Type 2 diabetes.

16. Use of the antagonist as defined in any of claims 1 to 12 in the manufacture of a medicament for the treatment or prophylaxis of obesity.

17. A method of screening for one or more nucleic acid molecules which are capable of binding to Myostatin or an analogue thereof, which method comprises the following steps: a) generating a heterogeneous population of nucleic acids; b) contacting the population of step a) with Myostatin or an analogue thereof; c) separating the nucleic acid(s) not interacting with Myostatin or an analogue thereof; d) optionally separating the nucleic acid(s) interacting with Myostatin or an analogue thereof; and e) optionally sequencing the nucleic acid(s) interacting with Myostatin or an analogue thereof.

18. A method as defined in claim 17 wherein said method comprises the following steps: a) generating a heterogeneous population of D-nucleic acids; b) contacting the population of step a) with D-Myostatin, i.e. all of the amino acids forming the peptide are D-amino acids; c) separating the D-nucleic acid not interacting with D-Myostatin; d) sequencing the D-nucleic acid interacting with D-Myostatin; and e) synthesising the L-nucleic acid sequence(s) which is/are identical to the sequence of the D-nucleic acid(s) obtained in step d).