WO2003068949A1

WO2003068949A1 - Treatment of muscle damage

Info

Publication number: WO2003068949A1
Application number: PCT/GB2003/000657
Authority: WO
Inventors: Nicholas Beaumont
Original assignee: Nicholas Beaumont
Priority date: 2002-02-14
Filing date: 2003-02-14
Publication date: 2003-08-21
Also published as: GB0203552D0; AU2003207315A1

Abstract

Peptides that comprise the sequence: (X1)m(X2)n(X3)G(X4)(X5)(X6)(X7)2(X8)p where X1=a basic residue; X2=any amino acid; X3=K or Q; X4=K or Q; X5=S, T, A or P; X6=I, F or L; X7=D or E; X8=any amino acid; m=2 or 3, n=0-2; and p=2-6, promote cell proliferation in muscle and nerve cells, and are useful in therapy. One example ot these peptides is the c-terminal acids of creatine kinase.

Description

TREATMENT OF MUSCLE DAMAGE

Field of the Invention

This invention relates to a peptide growth factor and its use. Background to the Invention Healthy skeletal muscle responds to exercise and training by strengthening. There are some diseases, such as muscular dystrophy, and conditions such as ageing in which the strengthening process can be weak or defective. There has been much research to identify biomolecules that are signals in the normal responses of muscle to training or stretching, in order to engineer them into therapeutic agents.

Stem cells present a unique opportunity for the repair of tissues. Therapies based upon this potential are likely to become a major focus for the biotechnology industry. The factors that control the proliferation and differentiation of these cells are not yet fully understood, and will be important in controlling stem and precursor cells for therapies.

Stem cells in muscle, and also it is thought heart, respond to damage by proliferating to form cells that differentiate to repair damaged tissue. In the case of muscle, "satellite" cells that are adjacent to muscle fibres respond to muscle fibre damage by dividing to form myoblasts which then fuse into the fibre to repair it. The chemical signals that control this process are the subject of intense study.

Yang et al, J. Muscle Res. & Cell Motility, 1996; 17: 487-495 disclosed that a splice-form of insulin-like growth factor 1 (IGF1b) is expressed by young muscle in response to stretch and exercise and that this protein (also termed mechano-growth factor or MGF) encourages the proliferation of muscle cells. MGF shares the "insulin-like" Λ/-terminal domain with IGF1, but, due to differential RNA-splicing, has a novel C-terminus. It has been shown that MGF promotes muscle growth, however, the insulin-like domain of IGF is known to cause adverse side-effects by acting as a systemic growth factor, which may promote tumour growth and cardiac defects. Summary of the Invention

The present invention is based on the surprising finding that a peptide corresponding to the C-terminus of creatine kinase can increase proliferation of a muscle cell line or nerve cell line. Antiserum raised against the peptide corresponding to the C-terminus of

MGF cross-reacts with creatine kinase (Fig. 2). Furthermore, this cross- reactivity is specific for the C-terminus of creatine kinase, which forms a peripheral region of the native protein and does not appear to be involved in the catalytic process. This has led to the surprising finding that proteins with an amino acid sequence different from that of MGF can have a similar function in the proliferation of certain cells.

According to a first aspect of the invention, an isolated peptide capable of acting as a ligand for an antibody with affinity for the C-terminus of MGF, is useful in therapy, particularly in the treatment of muscle fatigue, deterioration or damage. The isolated peptide is not MGF.

According to a second aspect of the invention, an isolated peptide useful in therapy comprises the sequence:

(X¹)_m(X²)_n(X³)G(X⁴)(X⁵)(X⁶)(X⁷)₂(X⁸)_p

where X¹ = a basic residue; X² = any amino acid; X³= K or Q; X⁴ = K or Q; X⁵ = S, T, A or P; X⁶ = I, F or L; X⁷ = D or E; X⁸ = any amino acid; m = 2 or 3; n = 0-2; and p = 2-6 (X¹ is the N-terminus).

The peptide of the invention will generally comprise the sequence defined herein as SEQ ID NO. 1 : KKLEKGQSIDDMIPAQK. Description of the Drawings

The present invention is described with reference to the accompanying drawings, wherein:

Figure 1 shows the sequence alignment of mouse IGF1 (top) with MGF (bottom), with residues encoded by variant splicing highlighted;

Figure 2 is a dot blot showing the cross-reactivity of serum raised against the C-terminus of MGF with creatine kinase, where 1 = MGF peptide, 2 = creatine kinase protein, 3 = kidney homogenate (negative control) and 4 = C- terminal peptide of creatine kinase;

Figure 3 shows the alignment of the C-termini of MGF with the C-termini from a selection of creatine kinases; Figure 4 is a graph showing the effect of modified peptides on growth/survival of C2C12 cells in 1% serum;

Figure 5 is a graph showing the effect of a PEG-modified c-terminal peptide of creatine kinase on the growth of Hep-G2 cells over 4 days; and

Figure 6 is a computer graphic representation of the structure of the creatine kinase (CK) C-terminal region and the human MGF Ec region. Description of the Invention

The peptides of the present invention may be defined on the basis of affinity to an antibody that reacts with the C-terminus of MGF. The peptide is not MGF. It has been found that suitable peptides include those derived from the C- terminus of creatine kinase (SEQ ID NO. 1). For the avoidance of doubt, suitable peptides include the full length creatine kinase protein.

The amino acid sequence of MGF does not appear to have any region in common with any creatine kinase. Therefore, computer-based sequence alignment programmes do not recognise any similarity between the two proteins.

However, there appear to be similarly charged residues in a similar order in the

C-termini of both proteins (see Fig. 3). This may be a case of "molecular mimicry" (analogy rather than hσmology) where a protein has convergently evolved to perform the same role as a pre-existing protein. In this case, it is hypothesised that there is a receptor on satellite cells (and perhaps other cells that can respond to local signals of tissue damage) that binds to both the C- termini of MGF and creatine kinase and leads to either cell division or inhibits apoptosis of precursor cells. Accordingly, peptides which have similarly charged residues in a similar order to the C-termini of MGF are within the scope of the present invention.

The co-ordinates of the crystal structure of some isoforms of creatine kinase are available in public databases. The models show the C-terminus as a separate domain that does not interact closely with the rest of the protein. This region was used as the basis for a model of the C-terminus of MGF by molecular replacement. The model of MGF that was produced has good stereochemical parameters, indicating that the model is structurally feasible. The crystal structure and the homology model possess salt bridges between basic and acidic residues. Both structures possess sharp turns that depend upon the invariant glycine residue.

The surfaces of both models have the residues, which had been identified in the alignment, featuring predominantly along one face of the peptide. This is consistent with a constrained face of both molecules binding to the same receptor, and may explain the antibody cross-reacting and binding of both peptides. The present invention makes it possible to develop "peptidomimetic" drugs based on the size, shape and charge shared by the constrained faces of these molecules. Using the known structure coordinates for the amino acids at the C- terminus of creatine kinase, it is possible to generate a 3-dimensional graphic representation of the C-terminus structure. Suitable software packages are known in the art and include Rasmol, Cerius, Insight, Quanta, Sybyl and Molcad. Various computational analyses can be used to determine whether a given molecule (for example a potential mimetic) is similar to the creatine kinase C- terminus. This analysis can be carried out using conventional software packages, including molecular similarity application of Quanta (Accelrys, San Diego, CA) version Quanta2000, or Isqkab of the CCP4 suite. The present invention therefore permits the use of molecular design techniques to identify, select and design chemical entities that share structural similarity with the creatine kinase C-terminus.

Compounds designed using computational methods can then be synthesised and tested in in vitro and in vivo models, to measure efficacy.

Suitable peptides of the invention are those of, or based on, the C- terminus of creatine kinase, or homologues thereof that are within the general definition provided above. Preferably, a peptide of the invention will have a sequence as defined above where X¹ = K; X² = L or E; X³ = K; X⁴ = Q; ⁵ = S; X⁶ = I; X⁷ = D; m = 2, n =

2; and p = 4-6. X⁸ is preferably a lysine, more preferably a lysine modified to remove the carboxyl group. The glycine residue shown in the definition provided above, is an important requirement for all the peptides of the invention. The abbreviations used herein for the amino acids are the same as those known conventionally in the art.

The peptides of the invention may be made by suitable synthetic procedures, as known in the art. Typically, the peptides will be no more than 40 amino acids in length, preferably no more than 30 amino acids in length, and most preferably no more than 20 amino acids in length. The peptides may be modified or conjugated to other molecules, e.g. PEG, to modify their pharmacological properties or to improve the stability of the peptides.

Preferably, the peptides are conjugated to a high molecular weight polysaccharide, more preferably to hyaluronic acid, or an inorganic salt thereof.

Conjugation between peptides or proteins and other molecules is known in the art.

In this aspect the peptides are part of a construct, i.e. heterologous molecules bound (covalently or non-covalently) to form a single molecule. The construct may be a fusion protein, one part of which is a peptide of the invention.

Techniques for the preparation of fusion proteins are well known in the art.

Reference to the utility of the peptides of the invention also implies utility of the constructs.

The peptides have use in therapy to treat conditions associated with muscle fatigue and injury/damage. For example, the peptides may be used during exercise, to help recovery from muscle fatigue. The peptides may also be useful to treat muscle deterioration, for example muscular dystrophy, or damage caused after a heart attack.

As the peptides can stimulate the growth of muscle and nerve cells, it is possible to use them in cell cultures to proliferate a muscle or nerve cell line. In this aspect, the peptides act as useful and cost-effective alternatives to conventional serum supplements. The peptides may also be useful in assays to identify agents that potentiate or inhibit muscle or nerve cell growth. The peptides may also be useful as a treatment to promote the growth or repair of muscle or nerve cells in vivo. The peptides are also useful to inhibit apoptosis of precursor cells. The peptides or conjugates of the invention may be administered by any suitable route, for example via injection, e.g. subcutaneous, intramuscular or intravenous injection.

Suitable formulations for administration will be apparent to the skilled person, based on conventional formulation research. Pharmaceutically acceptable carriers or diluents may be added, as will be appreciated by the skilled person. Example 1

A peptide corresponding to the C-terminus of human muscle creatine kinase (CK) was produced and modified with PEG (to improve the pharmacokinetic properties). The effect of this peptide on muscle cell growth was compared with a similarly modified MGF C-terminal peptide and a control culture (see Figs.4 and 5). The muscle cells were seeded at approximately 10% confluency and grown in only 1% of adult horse serum to reduce the cells exposure to growth factors from serum (and therefore their growth rate). The culture medium was DMEM supplemented with 2 mM glutamate, 100 units/ml penicillin and 100 μg/ml Streptomycin. The cells were incubated in 12 or 6 well plates at 37 °C.

These results suggest that the peptide derived from the analogous sequences of the C-termini of MGF and creatine kinase cause proliferation in stationary muscle cell lines. Example 2

A further study showed that at 50 μg/ml (50-fold more concentrated than in the muscle experiments) the modified peptide has no effect on the proliferation of Hep-G2 cells (a Human liver cell line that does respond to IGF1 ) (Fig.6). The peptides are therefore specific for cells that release creatine kinase, for example nerve and muscle cells (either skeletal or cardiac muscle).

Claims

I . An isolated peptide capable of acting as a ligand for an antibody with affinity for the C-terminus of mechano-growth factor, for use in therapy, with the proviso that the peptide is not MGF.

2. An isolated peptide comprising the sequence

(X¹) X²)_n(X³)G(X⁴)(X⁵)(X⁶)(X⁷)₂(X⁸)_p

where X¹ = a basic residue; X² = any amino acid; X³ = K or Q; X⁴ = K or Q; X⁵ = S, T, A or P; X⁶ = I, F or L; X⁷ = D or E; X⁸ = any amino acid; m = 2 or 3, n = 0-2; and p = 2-6, for use in therapy.

3. A peptide according to claim 2, wherein X¹ = K; X² = L or E; X³ = K; X⁴ = Q; X⁵ = S; X⁶ = I; X⁷ = D; m = 2, n = 2; and p = 4-6.

4. A peptide according to claim 1 or claim 2, having no more than 40 amino acids.

5. A construct comprising a peptide defined in any preceding claim conjugated to a polymeric molecule.

6. A construct according to claim 5, wherein the molecule is a high molecular weight polymer.

7. A construct according to claim 5 or claim 6, wherein the molecule is hyaluronic acid.

8. A construct according to any of claims 5 to 7, wherein the peptide consists of the sequence of amino acids defined as SEQ ID NO. 1.

9. Use of a peptide or construct as defined in any of claims 1 to 8, in the manufacture of a composition for the treatment of muscle damage, deterioration, or injury.

10. Use according to claim 9, wherein the treatment is for damage to skeletal muscle.

I I. Use according to claim 9 or claim 10, for the treatment of muscular dystrophy.

12. Use according to claim 9, wherein the treatment is for damage to cardiac muscle.

13. Use of a construct or peptide as defined in any of claims 1 to 8, in the manufacture of a composition for the repair of damage or loss of nerve cells.

14. Use according to any of claims 9 to 13, wherein the peptide is creatine kinase or a fragment thereof comprising the amino acids at the C-terminus.

15. Use of a peptide or construct as defined in any of claims 1 to 8, in a cell culture media to promote the growth of a muscle or nerve cell line.

16. A method for the design of a molecule that stimulates the growth of nerve and muscle cells, comprising carrying out a computational analysis to compare the structure of a candidate molecule with that of the creatine kinase C-terminus, and selecting a molecule that has the same structure.