EP1868637A1 - Mechano growth factor peptides and their use - Google Patents

Mechano growth factor peptides and their use

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Publication number
EP1868637A1
EP1868637A1 EP06726447A EP06726447A EP1868637A1 EP 1868637 A1 EP1868637 A1 EP 1868637A1 EP 06726447 A EP06726447 A EP 06726447A EP 06726447 A EP06726447 A EP 06726447A EP 1868637 A1 EP1868637 A1 EP 1868637A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
peptide
seq
extended
terminal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06726447A
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German (de)
English (en)
French (fr)
Inventor
Shi Yu Yang
Geoffrey University of Illinois at Chicago GOLDSPINK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
UCL Business Ltd
University of Illinois
Original Assignee
UCL Business Ltd
University of Illinois
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Publication date
Priority claimed from PCT/GB2006/000773 external-priority patent/WO2006097682A1/en
Application filed by UCL Business Ltd, University of Illinois filed Critical UCL Business Ltd
Publication of EP1868637A1 publication Critical patent/EP1868637A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators

Definitions

  • This invention relates to biologically active polypeptides derived from the E domain that forms the C-terminus of the insulin-like growth factor I (IGF-I) splice variant known as mechano growth factor (MGF). These peptides are modified to improve their stability compared to the naturally occurring E domain peptide.
  • IGF-I insulin-like growth factor I
  • MEF mechano growth factor
  • Mammalian IGF-I polypeptides have a number of isoforms, which arise as a result of alternative mRNA splicing. Broadly, there are two types of isoform, liver-type isoforms and non-liver-type ones. Liver-type isoforms may be expressed in the liver or elsewhere but, if expressed elsewhere, are equivalent to those expressed in the liver. They have a systemic action and are the main isoforms in mammals. Non- liver-type isoforms are less common and some are believed to have an autocrine/ paracrine action. The MGF isoform to which this invention relates is of the latter type.
  • E domains In human MGF, the C-terminus is formed by a 24 amino acid E domain, sometimes termed an Ec peptide (SEQ ID NO: 27). In rat and rabbit MGF, the corresponding E domains, sometimes termed Eb peptides, are 25 amino acids in length (SEQ ID NOS: 13/14). Liver-type IGF-I instead contains an Ea peptide at the C-termimis.
  • MGF for use against disorders of skeletal muscle, notably muscular dystrophy; for use against disorders of cardiac muscle, notably in the prevention or limitation of myocardial damage in response to ischemia or mechanical overload of the heart; for the treatment of neurological disorders in general; and for nerve repair in particular (WO97/33997; WO01/136483; WO01/85781; WO03/066082), It is becoming increasingly clear that liver-type IGF-I and MGF have different roles and functions.
  • the present inventors have found that the native human MGF C terminal Ec peptide has a short half-life in human plasma. Hence, stabilising modifications can enhance its potential for use as a pharmaceutical.
  • the inventors have also demonstrated that stabilised MGF C-terminal E peptides have neuroprotective and cardioprotective properties, as well as the ability to increase the strength of normal and dystrophic skeletal muscle.
  • the invention provides a polypeptide comprising up to 50 amino acid residues
  • polypeptide comprising a sequence of amino acids derived from the C-terminal E peptide of a Mechano Growth Factor (MGF) isoform of Insulin-like Growth Factor I (IGF-I);
  • MMF Mechano Growth Factor
  • IGF-I Insulin-like Growth Factor I
  • polypeptide incorporating one or more modifications that give it increased stability compared to the unmodified MGF E peptide;
  • the invention also provides an extended polypeptide comprising a polypeptide of the invention, extended by non-wild-type amino acid sequence N-terminal and/or C- terminal to said polypeptide.
  • the invention also provides a composition comprising a polypeptide or extended polypeptide of the invention and a carrier.
  • the invention also provides a pharmaceutical composition comprising a polypeptide or extended polypeptide of the invention and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising a polypeptide or extended polypeptide of the invention and a pharmaceutically acceptable carrier.
  • the invention also provides a polypeptide or extended polypeptide of the invention for use in a method of treatment of the human or animal body.
  • the invention also provides a method of treating a muscular disorder by administering to a patient in need thereof an effective amount of a polypeptide or extended polypeptide of the invention.
  • Said muscular disorder may be, for example, a disorder of skeletal muscle or a disorder of cardiac muscle.
  • the invention also provides a method of treating a neurological disorder by administering to a patient in need thereof an effective amount of a polypeptide or extended polypeptide of the invention.
  • the invention also provides use of a polypeptide or extended polypeptide of the invention in the manufacture of a medicament for use in a treatment as defined above.
  • polypeptide comprising up to 50 amino acid residues, said polypeptide comprising a sequence of amino acids derived from the C-terminal E peptide of a Mechano Growth Factor (MGF) isoform of Insulin-like Growth Factor I (IGF-I); or an extended polypeptide comprising said polypeptide and extended by non-wild-type amino acid sequence N-terminal and/or C-terminal to said polypeptide;
  • MMF Mechano Growth Factor
  • IGF-I Insulin-like Growth Factor I
  • the invention also provides a method of treating a disorder of cardiac muscle by administering to a patient in need thereof an effective amount of: a polypeptide comprising up to 50 amino acid residues, said polypeptide comprising a sequence of amino acids derived from the C-terminal E peptide of a Mechano Growth Factor (MGF) isoform of Insulin-like Growth Factor I (IGF-I); or an extended polypeptide comprising said polypeptide and extended by non-wild-type amino acid sequence N-terminal and/or C-terminal to said polypeptide;
  • MMF Mechano Growth Factor
  • IGF-I Insulin-like Growth Factor I
  • the invention also provides use of
  • polypeptide comprising up to 50 amino acid residues, said polypeptide comprising a sequence of amino acids derived from the C-terminal E peptide of a Mechano Growth Factor (MGF) isoform of Insulin-like Growth Factor I (IGF-I); or an extended polypeptide comprising said polypeptide and extended by non-wild-type amino acid sequence N-terminal and/or C-terminal to said polypeptide;
  • MMF Mechano Growth Factor
  • IGF-I Insulin-like Growth Factor I
  • Figure 1 Sequence alignment, showing sequences encoded by part of the sequence of each of human, rat and rabbit MGF and human, rat and rabbit liver-type IGF-I
  • Figure 2 Effect of Alanine substitution and C-terminal and N-terminal truncation on stability and biological activity - further sequence alignment, comparing modified sequences of Peptides 1-6 (SEQ ID NOS: 15-20) and Short peptides 1-4 (SEQ NOS: 21-24), and detailing impact of changes on stability as measured by incubation in human plasma and biological activity as measured by testing on muscle cell line (see Examples for details of test procedures).
  • the first two columns on the left hand side identify the peptides and give their sequences, identifying the changes made by way of substitution.
  • the third column gives the results of the tests for stability (see Example 5 for details) and the final one on the right hand side gives the results of the tests for biological activity (again, see Example 5 for details).
  • Figure 3 Increase in strength of a murine dystrophic muscle following injection of stabilised peptide after 3 weeks -
  • FIG. 4 Cardioprotection following administration of stabilised peptide - comparison of ejection fractions achieved following administration to infarcted ovine heart of stabilised peptide (third column, referred to as "Ec domain"), full length MGF (fourth column), mature IGF-I (second column) and control preparation (first column).
  • FIG. 5 Pressure/volume loop data showing preservation of function following myocardial in fraction (MI) - for normal (top left) and infarcted (MI) murine (top right) ventricle, and showing effect of stabilised peptide delivered systemically to the MI heart (bottom right, referred to as "MGF peptide") and the normal heart (bottom left).
  • AU panels show pressure (mmHg) on the Y-axis and Relative Volume Units on the X-axis.
  • FIG. 6 Neuroprotective effects in rat brain slice system - from left to right, percentage of dead cells after treatment with stabilised peptide (referred to as "MGF"), IGF-I, TBH, TBH + stabilised peptide (24 hours), TBH + IGF-I (24 hours), TBH + stabilised peptide (48 hours), TBH + IGF-I (48 hours).
  • MEF stabilised peptide
  • DMGF and CMGF Peptides C2C12 Cells were provided at 2000cell/well, in a medium containing DMEM (1000mg/L glucose), plus BSA(I OOug/ml), plus IGF-I (2ng per ml) and incubated for 36 hours. Cell proliferation was then assessed using an Alamar Blue assay.
  • the left hand group of readings shows the results for experiments with concentrations of the DMGF peptide (See Example 1.3.1 for details) of 2, 5, 50 and 100 ng/ml.
  • the middle group of readings shows the results for experiments with concentrations of the CMGF peptide (See Example 1.3.1 for details) of 2, 5, 50 and 100 ng/ml.
  • the left hand group of readings shows the results for experiments with concentrations of IGF-I alone (See Example 1.5 for details) of 2, 5, 50 and 100 ng/ml.
  • Y-axis values are fluorescence (wavelength of excitation 535nm, measurement at 590nm; mean plus standard error) in an Alamar Blue assay.
  • (B) Peptides A2, A4, A6 and A8 C2C12 muscle cells at a 500 cells/well. Cultivation was carried out for 24 hours in 10% FBS, followed by starvation for 24 hours in 0.1% BSA, stimulation for 24 hours and then treatment with BrdU for 5 hours. Concentrations of 0.1, 1, 10 and 100 ng/ml of peptides A2, A4, A6 and A8 were tested, along with 0.1 , 1 , 10 and 100 ng/ml IGF-I (See the right-hand set of results). BrdU incorporation was measured to assess the level of cell proliferation achieved. Controls containing no cells, medium only, 5% FBS and no BrdU were also provided.
  • Values on the Y-axis are for fluorescence (absorbence at 370nm; mean plus standard error across 4 wells).
  • the first column on the left relates to a control in which no cells were present.
  • the next four relate to peptide A2 at concentrations of 0.1, 1, 10 and 100 ng/ml.
  • the next four relate to peptide A4 at concentrations of 0.1, 1, 10 and 100 ng/ml.
  • the central three relate to controls containing medium only (med), 5% FBS) and no BrdU.
  • the next four relate to peptide A6 at concentrations of 0.1, 1, 10 and 100 ng/ml.
  • the next four relate to peptide A8 at concentrations of 0.1, 1 , 10 and 100 ng/ml.
  • the right-hand group of results relate to IGF-I (See Example 1.5) at concentrations of 0.1, 1, 10 and 100 ng/ml.
  • Peptide A5 HSMM cells at 500 cells/well. Cultivation was carried out for 24 hours in 10% FCS, followed by two washes in serum free medium, stimulation for 48 hours and then treatment with BrdU for 5 hours. Concentrations of 0.1, 1, 10, 100 and 500 ng/ml of peptide A5 were tested, along with 0.1, 1, lO and 100 ng/ml IGF-I. BrdU incorporation was measured to assess the level of cell proliferation achieved. Controls containing medium only, no cells (BLK), background staining (BG) and 10% FBS were also provided. Values on the Y-axis are for fluorescence (absorbence at 370nm; mean plus standard error across 4 wells).
  • the first five columns relate to peptide A5 at concentrations of 0.1, 1, 10, 100 and 500 ng/ml.
  • the next column relates to the control containing medium only.
  • the next three relate to IGF-I (See Example 1.5) alone at concentrations of 100, 10 and 0.1 ng/ml.
  • the next three relate to controls containing 10% FBS, background staining and no cells respectively. * means P ⁇ 0.05 compared to medium only control.
  • Peptide A5 HSMM cells at 500 cells/well. Cultivation was carried out for 24 hours in 10% FCS, followed by two washes in serum free medium, stimulation for 48 hours and then treatment with BrdU for 5 hours. Concentrations of 0.1, 1, 10, 100 and 500 ng/ml of peptide A5 in combination with 2 ng/ml IGF-I were tested, along with 0.1, 1, 10 and 100 ng/ml IGF-I. BrdU incorporation was measured to assess the level of cell proliferation achieved. Controls containing medium supplemented with 2 ng/ml IGF-I, no cells (BLK), and 10% FBS were also provided.
  • Values on the Y- axis are for fluorescence (absorbence at 370nm; mean plus standard error across 4 wells).
  • the first five columns relate to peptide A5 at concentrations of 0.1, 1, 10, 100 and 500 ng/ml.
  • the next three relate to IGF-I (See Example 1.5) alone at concentrations of 100, 10 and 0.1 ng/ml.
  • the next three relate to controls containing 10% FBS, medium supplemented with 2 ng/ml IGF-I and no cells respectively.
  • Peptide A5 HSMM cells at 500 cells/well. Cultivation was carried out for 24 hours in 10% FCS, followed by two washes in serum free medium, stimulation for 48 hours and then treatment with BrdU for 5 hours. Concentrations of 0.1, 1, 10, 100 and 500 ng/ml of peptide A5 were tested, along with 0.1, 1, 10 and 100 ng/ml IGF-I. BrdU incorporation was measured to assess the level of cell proliferation achieved. Controls containing medium only, no cells (BLK), background staining (BG) and 10% FBS were also provided. Values on the Y-axis are for fluorescence (absorbence at 370nm; mean plus standard error across 4 wells).
  • the first five columns relate to peptide A5 at concentrations of 0.1, 1, 10, 100 and 500 ng/ml.
  • the next column relates to the control containing medium only.
  • the next three relate to IGF-I (See Example 1.5) alone at concentrations of 100, 10 and 0.1 ng/ml.
  • the next three relate to controls containing 10% FBS, background staining and no cells respectively. * means P ⁇ 0.05 compared to medium only control.
  • Peptide A5 HSMM cells at 500 cells/well. Cultivation was carried out for 24 hours in 10% FCS, followed by two washes in serum free medium, stimulation for 48 hours and then treatment with BrdU for 5 hours. Concentrations of 0.1 , 1 , 10, 100 and 500 ng/ml of peptide A5 in combination with 2 ng/ml IGF-I were tested, along with 0.1, 1, 10 and 100 ng/ml IGF-I. BrdU incorporation was measured to assess the level of cell proliferation achieved. Controls containing medium supplemented with 2 ng/ml IGF-I, no cells (BLK), background staining (BG) and 10% FBS were also provided.
  • Values on the Y-axis are for fluorescence (absorbence at 370nm; mean plus standard error across 4 wells).
  • the first five columns relate to peptide A5 at concentrations of 0.1, 1, 10, 100 and 500 ng/ml.
  • the next column relates to the control containing medium supplemented with 2 ng/ml IGF-I only.
  • the next three relate to IGF-I (See Example 1.5) alone at concentrations of 100, 10 and 0.1 ng/ml.
  • the next three relate to controls containing 10% FBS, background staining and no cells respectively. * means P ⁇ 0.1 compared to medium control containing 2 ng/ml IGF-I.
  • Peptide A5 HSMM cells at 1000 cells/well. Cultivation was carried out for 24 hours in 10% FCS, followed by two washes in serum free medium, stimulation for 48 hours and then treatment with BrdU for 5 hours. Concentrations of 0.1, 1, 10, 100 and 500 ng/ml of peptide A5 were tested, along with 0.1, 1, 10 and 100 ng/ml IGF-I. BrdU incorporation was measured to assess the level of cell proliferation achieved. Controls containing medium only, no cells (BLk), background staining (BG) and 10% FCS were also provided. Values on the Y-axis are for fluorescence (absorbence at 370nm; mean plus Standard error across 4 wells).
  • the first five columns relate to peptide A5 at concentrations of 0.1, 1, 10, 100 and 500 ng/ml.
  • the next column relates to the control containing medium only.
  • the next three relate to IGF-I (See Example 1.5) alone at concentrations of 100, 10 and 0.1 ng/ml.
  • the next three relate to controls containing 10% FCS, background staining and no cells respectively.
  • Peptide A5 HSMM cells at 1000 cells/well. Cultivation was carried out for 24 hours in 10% FCS, followed by two washes in serum free medium, stimulation for 48 hours and then treatment with BrdU for 5 hours. Concentrations of 0.1 , 1 , 10, 100 and 500 ng/ml of peptide A5 in combination with 2 ng/ml IGF-I were tested, along with 0.1, 1, 10 and 100 ng/ml IGF-I. BrdU incorporation was measured to assess the level of cell proliferation achieved. Controls containing medium supplemented with 2 ng/ml IGF-I, no cells (BLK), background staining (BG) and 10% FBS were also provided.
  • Values on the Y-axis are for fluorescence (absorbence at 370nm; mean plus standard error across 4 wells).
  • the first five columns relate to peptide A5 at concentrations of 0.1, 1, 10, 100 and 500 ng/ml.
  • the next column relates to the control containing medium supplemented with 2 ng/ml IGF-I only.
  • the next three relate to IGF-I (See Example 1.5) alone at concentrations of 100, 10 and 0.1 ng/ml.
  • the next three relate to controls containing 10% FBS, background staining and no cells respectively. * means P ⁇ 0.1 compared to medium control containing 2 ng/ml IGF-I.
  • the DNA and amino acid sequences of human, rat and rabbit MGF DNA are given in the sequence listing as SEQ ID NOS: 1/2, 3/4 and 5/6 respectively. These are termed full-length MGF sequences in that they represent mature MGF encoded by exons 3/4/5/6 of the IGF-I gene, including the 49/52 base pair insert that changes the reading frame and creates the characteristic MGF C-terminus. Exons 1 and 2 are alternative leader sequences.
  • SEQ ID NOS: 7/8, 9/10 and 11/12 are alternative leader sequences.
  • the sequence of the native rat Eb peptide (25 amino acids; amino acids 87-111 of SEQ ID NO: 4) from the C-terminus of rat MGF is given as SEQ ID NO: 13.
  • the sequence of the native rabbit Eb peptide (25 amino acids; amino acids 87-111 of SEQ ID NO: 6) from the C-terminus of rabbit MGF is given as SEQ ID NO: 14.
  • the sequence of the native human Ec peptide (24 amino acids; amino acids 87-110 of SEQ ID NO: 2) from the C-terminus of human MGF is given as SEQ ID NO: 27.
  • SEQ ID NOS: 28 to 32 Modified sequences derived from the peptide of SEQ ID NO: 27 are given as SEQ ID NOS: 28 to 32.
  • SEQ ID NO: 28 Serine is replaced with Alanine at position 5.
  • Native human Ec peptide has Arginine in its penultimate position.
  • a variant of the native peptide with Histidine in the penultimate position has been synthesised and is shown in SEQ ID NO: 15. This peptide is also described as Peptide 1 in Figure 2.
  • SEQ ID NO: 26 represents the sequence of full-length human MGF incorporating Histidine in the penultimate position instead of Arginine.
  • SEQ ID NO: 25 is a DNA coding sequence for SEQ ID NO: 26, in which the Histidine in the penultimate position is encoded by CAC and the remaining sequence is the same as in SEQ ID NO: 1.
  • Modified sequences derived from the peptide of SEQ ID NO: 15 are given as SEQ ID NOS: 16 to 24. These are compared to peptide of SEQ ID NO: 15 and one another in Figure 2.
  • Peptide 2 (SEQ ID NO: 16), Serine is replaced with Alanine at position 5.
  • Peptide 3 (SEQ ID NO: 17), Serine is replaced with Alanine at position 12.
  • Peptide 4 (SEQ ID NO: 18), Serine is replaced with Alanine at position 18.
  • Short peptide 1 (SEQ ID NO: 21)
  • Arginine is replaced with Alanine at position 14 and the two C-terminal amino acids are removed.
  • Short peptide 2 (SEQ ID NO: 22), Arginine is replaced with Alanine at position 14 and the four C-terminal amino acids are removed.
  • Short peptide 3 (SEQ ID NO: 23), Arginine is replaced with Alanine at position 14 and the three N-terminal amino acids are removed.
  • Short peptide 4 (SEQ ID NO: 24), Arginine is replaced with Alanine at position 14 and the five N-terminal amino acids are removed.
  • SEQ ID NO: 33 is the 8 C-terminal amino acids of the variant sequence of SEQ ID NO: 15, containing Histidine in the penultimate position.
  • SEQ ID NO: 34 is the 8 C-terminal amino acids of the native human MGF C- terminus of SEQ ID NO:27, containing Arginine in the penultimate position.
  • SEQ ID NO: 35 is the sequence of SEQ ID NO: 33 with Serine in position 2 substituted with Alanine. This therefore corresponds to the 8 C-terminal amino acids of SEQ ID NO: 18 (Peptide 4).
  • SEQ ID NO: 36 is the sequence of SEQ ID NO: 34 with Serine in position 2 substituted with Alanine. This therefore corresponds to the 8 C-terminal amino acids of SEQ ID NO: 30.
  • Polypeptides of the invention are up to 50 amino acid residues in length.
  • they may be up to 10 amino acids in length, up to 30 amino acids in length, e.g. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length, or up to 35, 40, 45 or 50 amino acids in length.
  • they are from 15 to 30 amino acids in length, more preferably 20 to 28, most preferably 22, 23, 24 or 25 amino acids in length.
  • polypeptides of 5 to 10 amino acids in length i.e. 5, 6, 7, 8, 9 or 10 amino acids in length, especially those of 8 amino acids in length.
  • a polypeptide of the invention comprises a sequence of amino acids derived from the C-terminal E peptide of an MGF isoform of IGF-I.
  • An MGF isoform is, as discussed above, one in which alternative splicing introduces into the mRNA an insert which lengthens and changes the reading frame of the C-terminal E peptide found at the C- terminus of IGF-I to create an Ec or Eb peptide.
  • An MGF isoform will typically have at least 80%, preferably 85% or 90% sequence identity to one of the MGFs of SEQ ID NOS: 2, 4, or 6.
  • the insert In human MGF (SEQ ID NOS: 1 and 2), the insert is 49 base pairs and the C-terminal E peptide is known as an Ec peptide (SEQ ID NO: 27), which is 24 amino acids in length.
  • Ec peptide SEQ ID NO: 27
  • Eb peptides Eb peptides, which are 25 amino acids in length (SEQ ID NOS: 13 and 14).
  • the sequence of the invention may be derived from any of these MGF C-terminal E peptides or from any other C-terminal E peptide from the MGF of any other species.
  • the sequence comprised in the polypeptide of the invention and derived from the C- terminal E peptide of an MGF isoform may be derived from said C-terminal E peptide in any way, as long as the requirements for biological activity and stability (see below) are met.
  • the sequence may be derived from the MGF C- terminal E peptide in the sense that it has exactly the sequence of the C-terminal E peptide (e.g. SEQ ID NO: 13, 14, 27 or 34) and is merely not present within a full- length MGF molecule. It may also be derived from the MGF C-terminal E peptide in the sense that its sequence is altered (see “Modifications” below), again as long as the requirements for biological activity and stability (see below) are met.
  • the polypeptide may also comprise native MGF sequence N-terminal to the sequence derived from the C-terminal E peptide.
  • any additional sequence may be non-MGF-derived, i.e. it may be any sequence, again as long as the requirements for biological activity and stability (see below) are met.
  • the sequence derived from the C-terminal MGF E peptide may include at least 10, at least 15 or at least 20 amino acids, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 amino acids in the case of the human C-terminal MGF Ec peptide or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in the case of the rat or rabbit C-terminal MGF Eb peptide.
  • it may include up to 10 amino acids, preferably 5 to 10 amino acids, ie 5, 6, 7, 8, 9 or 10 amino acids, especially 8 amino acids.
  • Polypeptides or extended polypeptides of the invention can be assembled together to form larger structures containing two or more polypeptide of the invention, e.g. multiple copies of the same polypeptide or extended polypeptides of the invention or a mixture of different ones.
  • these structures may be made as fusion proteins, normally by recombinant expression by standard techniques from coding DNA, or assembled synthetically, or expressed as fusion proteins and then subjected to appropriate chemical modifications.
  • An extended polypeptide of the invention comprises a polypeptide of the invention, extended by non-wild-type sequence.
  • any extension sequence is non-MGF sequence in that, if the N-terminus or C-terminus of the polypeptide of the invention represents native MGF sequence, then that sequence may not simply be joined to any sequence that it adjoins in native MGF.
  • an extension may have any sequence.
  • the polypeptides of the invention may be extended at either or both of the C- and N- termini by an amino acid sequence of any length.
  • an extension may comprise up to 5, up to 10, up to 20, up to 50, or up to 100 or 200 or more amino acids.
  • any such extension will be short, e.g.
  • An extension may contain, or even consist entirely of D-form amino acids (see below), e.g. to reduce exopeptidase attack.
  • a polypeptide may be extended by 1 to 5 D-form amino acids at one or both ends.
  • an additional Cysteine residue may be incorporated at the C-terminus.
  • a polypeptide or extended polypeptide of the invention may be modified in any manner that increases its stability compared to the unmodified E peptide that they comprise a sequence derived from. Stability may be increased in various ways. For example, it is envisaged that modifications (e.g. PEGylation or other chemical modifications or L-D form amino acid conversions) to the C- and/or N-termini of the protein will protect it against exopeptidase attack, as will cyclisation, and that internal modifications (e.g. substitution, deletion, insertion and internal L-D form , conversion will protect it against cleavage by endopeptidases by disrupting their cleavage sites.
  • modifications e.g. PEGylation or other chemical modifications or L-D form amino acid conversions
  • PEGylation may be PEGylated, preferably at the N-terminus to the extent that the location of the PEGylation can be controlled, though PEGylation at other sites, such as the C-terminus and between the C- and N-termini is also contemplated.
  • PEGylation involves the covalent attachment of PEG to the polypeptide.
  • Any suitable type of PEG e.g. any suitable molecular weight, may be used as long as the resultant PEGylated polypeptide satisfies the requirements for biological activity and stability (see below).
  • polypeptide of the invention may also incorporate other chemical modifications as well as, or instead of, PEGylation. Such modifications include glycosylation, sulphation, amidation and acetylation.
  • polypeptides may be acetylated at the N-terminus are preferred or amidated at the C-terminus or both.
  • one or more hexanoic or amino-hexanoic acid moieties may be added, preferably one hexanoic or amino-hexanoic acid moiety, normally at the N-terminus.
  • polypeptide or extended polypeptide may include one or more D-form amino acids.
  • amino acids are in the L- form. Inserting D- form amino acids can improve stability.
  • a few, e.g. 1, 2, 3, 4 or 5, D-form amino acids maybe used.
  • D-form amino acids may be used at any position in the polypeptide.
  • the human MGF C-terminal E peptide of SEQ ID NO: 27 it is preferred to replace one or both of the Arginines at positions 14 and 15 with D-form amino acids.
  • Corresponding changes are also preferred in the rat and rabbit sequences of SEQ ID NOS: 13 and 14 (positions 14, 15 and 16, as the rat/rabbit sequences comprise three Arginines in succession whereas the human one has only two) and in the variant sequence of SEQ ID NO: 15.
  • Stereochemical and/or directional peptide isomers may also be used.
  • Retro (RE) peptides may be used, in which the sequence of the invention is assembled from L-amino acids but in reversed order.
  • Retro-inverso (RI) peptides may be used, in which the sequence is reversed and synthesised from D-amino acids.
  • D-form amino acids may be included at one end or the other, or both, of the polypeptide. It is envisaged that this will help to protect against exopeptidase attack. This may be achieved by converting the terminal amino acids, e.g. the terminal 1, 2, 3, 4 or 5 amino acids at one or both ends, of the sequence derived from the MGF C-terminal E peptide to D-form. Alternatively or additionally, it may be achieved by adding 1, 2, 3, 4 or 5 further D-form amino acids at one or both ends of the polypeptide. Such further amino acids may or may not correspond to those that adjoin the sequence derived from the MGF C-terminal E peptide in native MGF. Such further amino acids may be any amino acids.
  • One possible amino acid for addition in D-form in this way is Arginine. For example, a D-form Arginine residue may be added at the N-terminus, the C-terminus or both.
  • the sequence of the native human MGF C-terminal E peptide of SEQ ID NO: 27 is retained but the Arginines at positions 14 and 15 of SEQ ID NO: 27are converted to the D-form and N-terminal PEGylation is provided. C-terminal amidation may also be provided.
  • sequence of the human MGF C-terminal E peptide variant of SEQ ID NO: 15 is retained but Arginines 14 and 15 in SEQ ID NO: 15 are converted to the D-form and N-terminal PEGylation is provided.
  • sequence of the 8 C-terminal amino acids from SEQ ID NO: 15 or 27, ie the sequence of SEQ ID NO: 33 or 34, is used and N- terminal PEGylation is provided or a hexanoic or amino-hexanoic acid moiety is added at the C-terminus.
  • C-terminal amidation may also be provided.
  • polypeptides of the invention may also incorporate other modifications, for example truncation, insertion, internal deletion or substitution.
  • insertion short stretches of amino acids may be inserted into the sequence derived from that of human C-terminal MGF E peptide, as long as the resultant polypeptide satisfies the requirements for biological activity and stability (see below) and comprises less than 50 amino acids.
  • Each insertion may comprise, for example 1, 2, 3, 4 or 5 amino acids. There may be one or more, e.g. 2, 3, 4 or 5 such insertions.
  • deletion short stretches of amino acids may be deleted from the internal sequence derived from that of human C-terminal MGF E peptide, as long as the resultant polypeptide satisfies the requirements for biological activity and stability (see below).
  • One or more such deletions e.g. 1 , 2, 3, 4 or 5 deletions, may be made, up to a total of, for example, 1, 2, 3, 4, 5, 6, 1, 8, 9 or 10 amino acids.
  • substitution any amino acids in the polypeptide may in principle be substituted by any other amino acid, as, as long as the resultant polypeptide satisfies the requirements for biological activity and stability (see below).
  • substitutions may be made, e.g.
  • substitutions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 or up to 20 substitutions in total.
  • no more than 10 substitutions will be made, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.
  • at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the amino acid residues will be the same as in the native MGF C-terminal E peptide from which the sequence is derived.
  • residues at one or both ends of the polypeptide are substituted.
  • Substitutions may increase stability or biological activity.
  • the results discussed in Example 5 and Figure 2 below indicate that substitution at one or more of positions 5, 12, 14 and 18 of the peptide of SEQ ID NO: 15 can increase stability.
  • the same results show that substitutions at positions 12, 14 and 18 can also increase biological activity.
  • Substitutions in positions 5, 12, 14 and 18 of the peptides of SEQ ID NOS: 27 and 15, and in position 2 of SEQ ID NOS 33 and 34 (which corresponds to position 18 of SEQ ID NOS: a5 and 27), are therefore preferred.
  • Corresponding substitutions into positions 5, 12, 15 and 19 of rat/rabbit MGF C-terminal E peptides of SEQ ID NOS: 13 and 14 are also preferred.
  • substitution of the native amino acid with Alanine is one preferred option, as shown in Example 5 and Figure 2. However, other amino acids may equally be used.
  • the polypeptide may include substitutions that do not have a significant effect on stability or biological activity. These will typically be conservative substitutions. Conservative substitutions may be made, for example according to the following table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.
  • amino acid sequence modifications such as L-D conversions, substitutions, insertions and deletions, in the polypeptides of the invention will be found in the sequence of amino acids that is derived from the MGF C-terminal E peptide.
  • additional MGF sequence they may alternatively or additionally be found in that additional sequence.
  • modifications may be found in that sequence.
  • polypeptides of the invention include the following.
  • Histidine in the penultimate position ie a peptide having the sequence of SEQ ID NO: 27 but stabilised by converting the two Arginines at positions 14 and 15 of SEQ ID NO 27 from L-form to D-form and by N-terminal PEGylation.
  • Modifications according to the invention may confer additional advantages as well as increased stability. For example, they may confer increased therapeutic activity or be advantageous from an immunological standpoint (eg via reduced immunogenicity). This applies in particular to modifications that involve L-D conversion and/or stereochemical and/or directional isomerism (see above).
  • Polypeptides and extended polypeptides of the invention have biological activity. This activity may be selected from the following.
  • a polypeptide or extended peptide of the invention will be able to increase muscle strength (e.g. as measured by maximum attainable tetanic force) by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 50%, at least 75% or at least 100% in dystrophic and/or non-dystrophic muscle.
  • a polypeptide or extended polypeptide of the invention will have the ability to prevent or limit myocardial damage in an infarcted or mechanically overloaded heart. This can be measured by pressure/volume loops or by reference to the ability to increase ejection fraction compared to an infracted heart to which no polypeptide or extended polypeptide of the invention is administered.
  • a polypeptide or extended polypeptide of the invention will have the ability to increase ejection fraction by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9% or by at least 10% or more.
  • a polypeptide or extended polypeptide of the invention will have the ability to reduce cell death in rat organotypic hippocampal cultures and/or other similar in vitro models.
  • a polypeptide or extended polypeptide of the invention will have the ability to reduce cell death in such models by at least 20%, at least 25%, at least 30%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% or more.
  • polypeptides or extended polypeptides of the invention may have neuroprotective ability
  • polypeptides or extended polypeptides of the invention may have one or more biological properties characteristic of full-length MGF (e.g. of SEQ ID NOS: 2, 4 or 6).
  • polypeptides or extended polypeptides of the invention may have the functional properties of MGF identified in WO97/33997. hi particular, they may have the ability to induce growth of skeletal muscle tissue. Similarly, as discussed herein, they may have the ability to upregulate protein synthesis needed for skeletal muscle repair and/or to activate satellite (stem) cells in skeletal muscle.
  • one method of assessing biological activity is the Alamar Blue method as discussed in Example 5.2.2. This involves contacting a polypeptide with mononucleated myoblast cells and assessing the extent to which it causes them to proliferate. This can be scored in any suitable way, e.g. on a scale of 0 to 3 as discussed in the Examples. Activity may also be measured via cyclins, such as cyclin ID, which are early markers of cell division. Activity may also be measured via the use of bromodeoxy uridine (BrdU). BrdU will substitute itself for thymidine during DNA replication and hence can be used to identify cells whose DNA is undergoing replication and to measure how much replication and cell division is taking place.
  • bromodeoxy uridine BrdU
  • polypeptides or extended polypeptides of the invention may have the neurological properties previously identified in WO01/136483. Thus, they may have the capacity to effect motoneurone rescue. In particular, they may be able to reduce motoneurone loss following nerve avulsion by up to 20, 30, 40, 50, 60, 70, 80, 90, 95, 99 or 100% in a treated subject compared to an equivalent situation in a non-treated subject. Reduction of motoneurone loss by 70% or more, or 80% more (i.e. to 30% or less or 20% or less) is preferred. The degree of rescue may be calculated using any suitable technique, e.g. a known technique such as Stereology. As a specific test, the techniques used in WOOl/136483, which rely on measuring motoneurone rescue in response to facial nerve avulsion in rats, may be used.
  • polypeptides or extended polypeptides of the invention may have the properties identified in WO03/060882, which is to say the ability to prevent or limit myocardial damage following ischemia or mechanical overload by preventing cell death, or apoptosis, of the muscle cells of the myocardium.
  • a polypeptide or extended polypeptide of the invention will have the ability to completely prevent apoptosis in the area of cardiac muscle to which it is applied.
  • apoptosis may also be only partially prevented, i.e. limited. Damage is limited if any reduction of damage is achieved compared to that which would have taken place without a treatment of the invention, e.g.
  • ⁇ damage is reduced by 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more, as measured by the number or proportion of cells which die, or by the size of the area of muscle that loses function, or by the overall ability of the heart to pump blood.
  • reduction of damage can be estimated in vivo by determining cardiac output, ejection fraction etc using minimally invasive methods. Markers such as creatine kinase and troponin T in the serum can also be assayed. These are the parameters used in clinical situations to determine the extent damage to the cardiac muscle following injury.
  • the ability to prevent apoptosis may be measured by any suitable technique. For example, with reference to Example 4 and Figures 3 and 6, it may be measured by the ability to prevent apoptosis in a cardiac muscle cell or cardiac-like cell line, as indicated by DNA fragmentation.
  • the ability to prevent apoptosis, as indicated by DNA fragmentation may be tested by treating the cells with sorbitol or another agent that places the cells under osmotic stress for up to, e.g. 1, 2, 4, 6, 12, 24 or 48 hours, preferably 12 to 24 hours, more preferably 24 hours, and investigating whether the pattern of fragmentation associated with apoptosis can be observed.
  • An MGF polypeptide of the invention expressed in this way will typically reduce, preferably eliminate, DNA fragmentation under these conditions, as compared to an untreated cell) after 6, 12 or 24 hours' sorbitol treatment.
  • the absence of expression, or low expression, of genes that act as markers for apoptosis can also act as an indication of prevention of apoptosis.
  • One suitable marker is the Bax gene.
  • increased expression of anti-apoptotic markers in MGF-transfected cells under apoptotic conditions can be taken as a sign that the polypeptide of the invention is preventing apoptosis.
  • One suitable anti-apoptotic marker gene is Bcl2.
  • the ability to prevent apoptosis may also be measured by reference to an MGF polypeptide's ability to prevent a reduction in cell number in myocyte cells in vitro.
  • polypeptides and extended polypeptides of the invention are the ability to induce a hypertrophic phenotype in cardiac muscle cells.
  • this may be tested by assessing the ability to induce a hypertrophic phenotype in primary cardiac myocyte cultures in vitro.
  • a preferred method for determining this is to test for an increase in expression of ANF (Atrial Natriuretic Factor) and/or bMHC (Beta Myosin Heavy Chain).
  • ANF is an embryonic marker gene that is upregulated in hypertrophic conditions.
  • bMHC is an important contractile protein in muscle.
  • Polypeptides and extended polypeptides of the invention have increased stability compared to the native C-terminal MGF E peptides that they contain sequences derived from. Such comparisons are made between the polypeptide or extended polypeptide of the invention and the native C-terminal MGF E peptide in its isolated, unmodified form (e.g. an unmodified form of SEQ ID NO: 13, 14, 27, or 34, separated from the remainder of the MGF molecule and in isolated form as a 24-mer (SEQ ID NO: 27) , 25-mer (SEQ ID NOS 13/14) or 8-mer (SEQ ID NO 34)). Comparisons may also be made with the Histidine-containing sequences of SEQ ID NO: 15 and 33. Stability may be increased by any degree via the modifications discussed herein.
  • Stability may be assessed in terms of half-life in human plasma or by any other suitable technique.
  • measures of stability can be based on determining the loss of biological activity over time. This can be done by any suitable method, e.g. via an in vitro assay for any of the measures of biological activity discussed herein.
  • preferred polypeptides or extended polypeptides of the invention may have half-lives that are increased by at least 10%, at least 20%, at least 30%, at least 50%, at least 60%, at least 80%, at least 100%, at least 200% or at least 500% or more compared to the corresponding unmodified MGF C-terminal E peptide.
  • preferred polypeptides or extended polypeptides of the invention may have half-lives of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at last 12 hours, at least 24 hours or at least 48 hours or more.
  • Example 5 and Figure 2 qualitative or semi-quantitative measurements of stability may be used, as in Example 5 and Figure 2, for example by scoring the stability of polypeptides or extended polypeptides on a scale from 0 to 3. On that scale, the polypeptide of SEQ ID NO: 15 scored 1. Certain other modified polypeptides of the invention scored 2 or 3. A polypeptide or extended polypeptide of the invention will generally score more highly on such a scale than the corresponding native MGF C- terminal E peptide.
  • peptides of the invention will be stabilised, as discussed above, it may under certain circumstances be possible to make use of unstabilised polypeptides, including the native polypeptides of SEQ ID NOS: 13, 1427 and 34 or the histidine-containing variant of SEQ ID NO: 15 and 33.
  • unstabilised polypeptides including the native polypeptides of SEQ ID NOS: 13, 1427 and 34 or the histidine-containing variant of SEQ ID NO: 15 and 33.
  • Polypeptides and extended polypeptides of the invention can be used to treat a number of conditions. Broadly, these break down into three areas: disorders of skeletal muscle, disorders of cardiac muscle and neurological disorders. However, because nerve and muscle function are inter-dependent, there may be some overlap between these categories, e.g. in the area of neuromuscular disorders.
  • Neurological disorders may generally be divided into two categories, neurogenic disorders where the fault lies in the nervous system itself and myogenic or muscle- related neurological disorders. Both can be treated according to the invention.
  • Disorders of skeletal muscle that are susceptible to treatment according to the invention include: muscular dystrophy, including but not limited to Duchenne or Becker muscular dystrophy, Facioscapulohumeral Muscular Dystrophy (FSHD), congenital muscular dystrophy (CMD) and autosomal dystrophies, and related progressive skeletal muscle weakness and wasting; muscle atrophy, including but not limited to disuse atrophy, glucocorticoid-induced atrophy, muscle atrophy in ageing humans and muscle atrophy induced by spinal cord injuries or neuromuscular diseases; cachexia, for example cachexia associated with, cancers, AIDS, Chronic Obstructive Pulmonary Disease (COPD), chronic inflammatory diseases, burns injury etc; muscle weakness, especially in certain muscles such as the urinary sphincter, anal sphincter and pelvic floor muscles; sarcopeni
  • neurological (including neuromuscular) disorders include amyotrophic lateral sclerosis; spinal muscular atrophy; progressive spinal muscular atrophy; infantile or juvenile muscular atrophy, poliomyelitis or post-polio syndrome; a disorder caused by exposure to a toxin, motoneurone trauma, a motoneurone lesion or nerve damage; an injury that affects motoneurones; and motoneurone loss associated with ageing; and autosomal as well as sex-linked muscular dystrophy; Alzheimer's disease; Parkinson's disease; diabetic neuropathy; peripheral neuropathies; embolic and haemorrhagic stroke; and alcohol-related brain damage.
  • Polypeptides and extended polypeptides of the invention may also be used for maintenance of the central nervous system (CNS). The invention also finds application in nerve repair following trauma.
  • Nerve damage may also be treated according to the invention.
  • the polypeptide or extended polypeptide will typically be localised around the sites of such damage to effect repair, e.g. by means of the placement of a conduit around the two ends of a severed peripheral nerve (cf. WOO 1/85781).
  • cardiac disorders there may be mentioned diseases where promotion of cardiac muscle protein synthesis is a beneficial treatment, cardiomyopathies; acute heart failure or acute insult including myocarditis or myocardial infarction; pathological heart hypertrophy; and congestive heart failure.
  • Polypeptides and extended polypeptides of the invention may also be used for improving cardiac output by increasing heart stroke volume.
  • polypeptides and extended polypeptides of the invention may be used for prevention of myocardial damage following ischemia and/or mechanical overload.
  • the ischemia or mechanical overload in response to which the MGF polypeptide or polynucleotide is administered is a temporary condition.
  • the polypeptide or extended polypeptide of the invention is administered in response to a heart attack. Treatments of the invention will be particularly effective in helping heart attack sufferers make a good recovery; and to return to a normal, active lifestyle.
  • polypeptides and extended polypeptides of the invention may be used in combination with other pharmaceutically active agents.
  • polypeptides and extended polypeptides of the invention may be used together with IGF-I (see Examples 1.5, 7 and 8).
  • Such combined uses may involve coadministration of the polypeptides or extended polypeptides of the invention in a single pharmaceutically acceptable carrier or excipient with the other pharmaceutically active agent or agents, or they may involve separate, sequential or simultaneous injection, at the same site or at different sites.
  • Polypeptides and extended polypeptides of the invention may be produced by standard techniques. Typically, they will be obtained by standard techniques of peptide synthesis, plus appropriate chemical modifications (e.g. PEGylation) to the resulting amino acid sequence if necessary. Where there are no D-form amino acids, polypeptides and extended polypeptides may instead be obtained via recombinant expression in a host cell from the appropriate coding DNA, again by standard techniques.
  • polypeptides and extended polypeptides according to the invention will generally be isolated or purified, either completely or partially.
  • a preparation of an isolated polypeptide or extended polypeptide is any preparation that contains the polypeptide or extended polypeptide at a higher concentration than the preparation in which it was produced.
  • the polypeptide or extended polypeptide will typically have been extracted from the host cell and the major cellular components removed.
  • a polypeptide or extended polypeptide in purified form will generally form part of a preparation in which more than 90%, for example up to 95%, up to 98% or up to 99% of the polypeptide material in the preparation is that of the invention.
  • Isolated and purified preparations will often be aqueous solutions containing the polypeptide or extended polypeptide of the invention.
  • the polypeptide or extended polypeptide of the invention may be purified or isolated in other forms, e.g. as crystals or other dry preparations.
  • compositions comprising the polypeptide or extended polypeptide and a carrier.
  • a composition may be a pharmaceutical composition comprising the polypeptide or extended polypeptides and a pharmaceutically acceptable carrier or diluent. Any suitable pharmaceutical formulation may be used.
  • suitable formulations may include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, bactericidal antibiotics and solutes which render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers. For example, sealed ampoules and vials, and may be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question.
  • Sterile, pyrogen-free aqueous and non-aqueous solutions are preferred.
  • Formulations will generally be tailored, by standard formulation techniques, to the modes of administration discussed below.
  • polypeptide of the invention may be administered by any suitable route tailored to the condition to be treated, for example topical, cutaneous, parenteral, intramuscular, subcutaneous or transdermal administration; or by direct injection into the bloodstream or direct application to mucosal tissues.
  • Injection is likely to be the preferred route under many circumstances, for example subcutaneous, parenteral intramuscular or intravenous injection. Intravenous injection will often be preferred under many clinical circumstances. So-called “needle-less” injection or transcutaneous administration maybe possible under some circumstances.
  • intravenous and intramuscular injection are preferred routes.
  • Topical administration is also envisaged, e.g. via patches , to strengthen the muscles of the abdomen or for other purposes.
  • delivery will generally be intravenous.
  • direct delivery to the heart may also be possible, e.g. using a so-called “needle-less" injection system for delivery the polypeptide to the heart.
  • polypeptides and extended polypeptides of the invention may be delivered in any suitable dosage, and using any suitable dosage regime.
  • Persons of skill in the art will appreciate that the dosage amount and regime may be adapted to ensure optimal treatment of the particular condition to be treated, depending on numerous factors. Some such factors may be the age, sex and clinical condition of the subject to be treated.
  • doses in the region of 0.2 to 10 mg will be effective, for example 0.2 to 0.8 mg, preferably about 0.5 mg.
  • a solution containing the polypeptide or extended polypeptide at a concentration of 1 mg/ml may be used in an amount of 0.1 to 1 ml.
  • Single or multiple doses may be given, depending on the application in question and the clinical circumstances.
  • the peptide used in Examples 2, 3, 4 and 6 had the sequence of SEQ ID NO: 15), in which the penultimate Arginine of the native sequence (See SEQ ID NOS: 1, 2 and 27) is replaced by Histidine, stabilised by the use of the D form of Arginine instead of the naturally occurring L- form at positions 14 and 15 and the covalent attachment of the N-terminus to a polyethylene glycol (PEG) derivative (O O- bis(2aminopropyl)polyethylene glyclol 1900) (Jeffamine) via a succinic acid bridge, and amidated at the C-terminus.
  • PEG polyethylene glycol
  • Example 5 The peptides of Example 5 were obtained from Alta Biosciences, Birmingham, UK, having been synthesised via standard techniques using a peptide synthesiser. These peptides are unPEGylated and free from L-D conversion and C-terminal amidation.
  • This peptide was synthesised via standard techniques using a peptide synthesiser. The product was purified by HPLC and analyzed by MALDI-MS.
  • the peptides of Example 7.1 had the 8 amino acid sequence Gly-Ser-Thr-Phe-Glu- Glu-His-Lys (SEQ ID NO:33), plus modifications to improve stability.
  • stabilisation was achieved via N-terminal PEGylation as in 1.1 above.
  • CMGF hexanoic acid acid
  • Both DMGF and CMGF were also amidated at the C-terminal end.
  • peptides A2, A4 and A6 had the sequence Gly-Ser-Thr-Phe-Glu- Glu-Arg-Lys (SEQ ID NO:34). Peptides A2, A4 and A6 were amidated at the C- terminus. Peptide A2 was unmodified at the N-terminus. Peptide A4 had a hexanoic acid moiety attached at the N-terminus. Peptide A6 had an amino-hexanoic acid moiety attached at the N-terminus,
  • Peptide A8 had the sequence Gly-Ser-Thr-Phe-Glu-Glu-His-Lys (SEQ ID NO:33), amidated at the C-terminus and with hexanoic acid attached at the N-terminus.
  • the peptide used in Example 8 had the sequence of SEQ ID NO: 15, in which the penultimate Arginine of the native sequence (See SEQ ID NOS: 1, 2 and 27) is replaced by Histidine, stabilised by the use of the D form of Arginine instead of the naturally occurring L-form at positions 14 and 15 and amidated at the C-terminus.
  • the peptide used in Example 8 was not pegylated.
  • IGF-I peptide has been used. This is the IGF-I receptor binding domain encoded by Exons 3 and 4 that is common to all splice variants and is approximately 70 amino acids in length. In Examples 1-4, this was obtained from PeproTech, EC 3 UK. In Example 6, it was obtained from Sigma - Aldrich (ER2 IGF- I). IGF-I peptide was also used in Example 7. 2. Injection of Stabilised Peptide into Dystrophic Muscle
  • muscle strength was increased by more than 25% within a few weeks in the tibialis anterior muscle of non-dystrophic mice. This muscle is not diseased like the muscle of mdx mice (see below), although it is possible that it was physically damaged by the repeated injections.
  • MI myocardial infarction
  • the use of the stabilised peptide alone was found to markedly increase the percentage of viable myocardium and the ejection fraction as measured by echocardiography and computerised analysis of the ejection function following the MI.
  • Full-length MGF also had a significant, though smaller effect.
  • Mature liver-type IGF-I had a much smaller effect.
  • Figure 4 shows percentage change in ejection fraction on day 6 as compared to ejection fraction on day 1 before the procedure was carried out.
  • the stabilised peptide was very effective in protecting the myocardium from ischemic damage.
  • mice were produced by ligating the left anterior descending (LAD) coronary artery of the murine heart. This causes dilation of the left ventricle, the progression of which leads to heart failure.
  • Stabilised peptide administered systemically markedly improved the strength and function of the heart as measured by the pressure/volume loops ( Figure 5) that demonstrate the ability of the heart to pump blood and the dilation that results when the damaged heart can no longer cope with the venous return. This is markedly improved by the systemic administration of the stabilised peptide, through which the myocardial wall muscle is protected and increased in thickness. Therefore there is considerable potential for treatment of patients immediately following a heart attack.
  • the neuroprotective effect of the stabilised peptide was demonstrated in vitro using the well-characterised model of selective neuronal death in rat organotypic hippocampal cultures.
  • Hippocampal slices were prepared from 7-10 days old Wistar rats according to the method of Stoppini et al (1991) with minor modifications according to Sarnowska (2002). Briefly, rats were anaesthetised with Vetbutal, ice-cooled and decapitated. Brains were quickly removed to ice-cold working solution pH 7.2: 96% of
  • HBSS/HEPES- (Ca2 + and Mg2 + free) containing 2mmol/L L-glutamine, 5 mg/ml glucose, 1% amphotericine B, 0,4% penicillin-streptomycin. Hippocampi were separated and cut into 400 ⁇ m slices using Mcllwain tissue chopper.
  • Millicell-CM membranes (Millipore) in 6-well plates were pre-equilibrated with 1 ml of culture medium pH 7.2: 50% DMEM, 25% HBSS/HEPES, 25% HS, 2 mmol/L L-glutamine, 5 mg/ml glucose, 1% amphotericine B, 0.4% penicillin-streptamycine in a moist atmosphere of air and 5% CO 2 at 32 0 C for 30 minutes.
  • Four selected slices were settled on each membrane. Slices were cultivated for two weeks at 32 0 C in 5% CO 2 atmosphere of 100% humidity. The viability of the slices was checked daily under the light microscopy and evaluated additionally on the day of experiment by propidium iodide staining and observed under fluorescent microscope (Zeiss
  • Oxidative stress was induced after 14 days in culture by adding 30 mM TBH (tert- butyl peroxide) for 3 hours. After that time the slices were transferred to the fresh culture medium. Resulting cell death was assessed 24 and 48 h after the beginning of the experiment.
  • TBH tert- butyl peroxide
  • Stabilised peptide or, for the purpose of comparison, recombinant IGF-I was added to the culture medium to the final concentration of 100 ng/ml at the beginning of the experiment and was continuously present in the medium.
  • a specific anti-IGF-1 receptor (AB-I) blocking antibody (Oncogene) was included in the medium 1 hour before the slices were exposed to TBH and MGF or IGF-I peptide.
  • the concentration of the antibody 1000 ng/ml was used according to the manufacturer's recommendation.
  • a confocal laser scanning microscope (Zeiss LSM 510) was used.
  • a helium-neon laser (543 nm) was used for the excitation of propidium iodide (PI).
  • images were processed using the Zeiss LSM 510 software package v. 2.8.
  • Quantitative measurement of tissue deterioration was performed using image analyser KS 300 (Carl Zeiss Jena GmbH).
  • % of dead cells (experimental fluorescent intensity (FI)- background FI) / (maximal FI- background FI) x 100, where maximal FI was obtained by killing all cells with exposure to 100 mM glutamate.
  • Rat brain slices were isolated following induction of localized damage by TBH (tert- butyl hydroperoxide) as discussed above. The resulting cell death in treated and non- treated brain slices was determined. This is illustrated in Figure 6.
  • TBH tert- butyl hydroperoxide
  • the IGF-I receptor domain peptide (rIGF-I) which is also part of full length MGF , was also neuroprotective (as previously reported). However, this was to a lesser degree (72%) and the protective effect of IGF-I was only noticeable for up to 24 hours, whereas the stabilised peptide functioned for significantly longer as its neuroprotective effect was still clearly observed after 48 hours.
  • the peptide used in Examples 2, 3 and 4 above had the sequence of SEQ ID NO: 15 (which corresponds to that of the human Ec peptide of MGF (SEQ ID NO: 27), except that Arginine in the penultimate position is replaced by Histidine ) stabilised by the use of the D form of Arginine at positions 14 and 15 instead of the naturally occurring L-form and the covalent attachment of the N-terminus to polyethylene glycol (PEG), and amidated at the C-terminus.
  • SEQ ID NO: 15 which corresponds to that of the human Ec peptide of MGF (SEQ ID NO: 27), except that Arginine in the penultimate position is replaced by Histidine
  • PEG polyethylene glycol
  • the sequence of the native human Ec peptide from the C-terminus of human MGF is given as SEQ ID NO: 27.
  • the penultimate amino acid which is Arginine in the native peptide (See SEQ ID NOS 2 and 27) is replaced with Histidine.
  • the peptide of SEQ ID NO: 15 is described as Peptide 1 in Figure 2.
  • SEQ ID NOS: 16 to 24 Further modified sequences derived from the sequence of SEQ ID NO: 15 are given as SEQ ID NOS: 16 to 24 and compared to that of SEQ ID NO: 15 in Figure 2, where they are referred to as Peptides 2-6 and Short peptides 1-4.
  • Peptide 2 Serine is replaced with Alanine at position 5.
  • Peptide 3 Serine is replaced with Alanine at position 12.
  • hi Peptide 4 Serine is replaced with Alanine at position 18.
  • Peptide 5 SEQ ID NO: 19
  • Arginine is replaced with Alanine at position 14.
  • Peptide 6 SEQ ID NO: 20
  • Arginine is replaced with Alanine at position 14 and Arginine is also replaced with Alanine at position 15.
  • Short peptide 1 SEQ ID NO: 21
  • Arginine is replaced with Alanine at position 14 and the two C-terminal amino acids are removed.
  • Short peptide 2 (SEQ ID NO: 22), Arginine is replaced with Alanine at position 14 and the four C-terminal amino acids are removed.
  • Short peptide 3 (SEQ ID NO: 23), Arginine is replaced with Alanine at position 14 and the three N-terminal amino acids are removed.
  • Short peptide 4 (SEQ ID NO: 24), Arginine is replaced with Alanine at position 14 and the five N-terminal amino acids are removed.
  • Biological activity was determined using an in vitro system by measuring the ability of the C terminal peptides to induce mononucleated myoblasts (satellite cells) to replicate. Cell number was determined using the Alamar Blue method. This was assessed on a scale of 0 to 3 and the results are shown in Figure 2.
  • the peptide (Peptide 1) of SEQ ID NO: 15 showed little or no activity owing to its short half-life.
  • Peptide 2 (SEQ ID NO: 16) and Short Peptide 1 (SEQ ID NO: 21) scored 1 on the activity scale.
  • Peptides 4 and 5 (SEQ ID NOS: 18 and 19) scored 2 on the activity scale.
  • Peptide 3 (SEQ ID NO: 17) scored 3 on the activity scale.
  • Peptide 6 (SEQ ID NO: 20) and Short peptides 2, 3 and 4 (SEQ ID NOS: 22, 23 and
  • Stability was determined as the amount of the peptide that remained intact and bound to the specific antibody in the following way:
  • the peptide (Peptide 1) of SEQ ID NO: 15 scored 1.
  • Peptide 6 (SEQ ID NO: 20) also scored 1.
  • Peptides 3 and 4 (SEQ ID NOS: 17 and 18) scored 2.
  • Peptides 2 and 5 (SEQ ID NOS: 16 and 19) scored 3.
  • Examples 1-4 also scored 3 on this scale.
  • Short peptides 1-4 have not yet been tested, though Short peptides 2 to 4 appear to lack biological activity anyway.
  • the stabilised peptide of 1.1 above was used in these experiments. Comparisons were made with the IGF-I peptide of 1.3 above.
  • CMD congenital muscular dystrophy
  • FSHD facioscapulohumeral dystrophy
  • ALS amyotrophic lateral sclerosis
  • the stabilised peptide considerably increased stem (desmin positive) cell proliferation for normal (non-diseased) muscle (from 38.4 ⁇ 2.5% to 57.9 ⁇ 3.2% in normal (non-diseased) limb and from 49.8 ⁇ 2.4% to 68.8 ⁇ 3.9% for normal (non-diseased) craniofacial muscle biopsies). Although the initial muscle stem cell numbers were lower in patients with muscle wasting, the stabilised peptide still induced an increase (CMD 10.4 ⁇ 1.7% to 17.5 ⁇ 1.6%; FSHD 11.7 ⁇ 1.3% to 20.4 ⁇ 2.1 % and ALS 4.8 ⁇ 1.1 % to 7.2 ⁇ 0.8%). The results also confirmed that the stabilised peptide had no effect on myotube formation but that it increases myoblast progenitor cell proliferation, whilst mature IGF-I enhanced differentiation.
  • Human lower limb (vastus lateralis) muscle samples were obtained from consenting, adult healthy, FSHD and ALS patients by needle biopsy under local anaesthesia at the Royal Free Hospital, London, UK. Biopsies were pooled from several patients with the same disorder to obtain sufficient cell numbers in the primary cultures.
  • Explant cultures were incubated in serum-containing Growth Media (sGM), composed of DMEM, 20% FCS (PAA Laboratories), penicillin (100U/ml) and streptomycin (100 ⁇ g/ml) (Invitrogen), and maintained at 37°C in humidified 95% air with 5% CO 2 .
  • the first wave of migration of mononuclear cells from the explant was designated the D -wave and this population was used throughout this study.
  • Migratory human muscle cell were enzymatically harvested using trypsin-EDTA (Invitrogen) and subcultured in sGM until 70-80% confluency.
  • Passage number x (P x ), was defined as the xth sequential harvest of subconfluent cells. All experiments were performed using P 3 .. 5 cohorts. The expanded cells were then stored under cryogenic conditions until they were used in the experiments described below. At least 6 runs were made for each of the treatments used for each diseased muscle culture as well as for the two types of healthy muscle.
  • dGM serum-free, defined media
  • DMEM serum-free, defined media
  • EGF IOng/ml
  • bFGF bFGF
  • insulin 5ng/ml
  • holo-transferrin 5 ⁇ g/ml
  • sodium selenite 5ng/ml
  • dexamethasone 390ng/ml
  • vitamin C 50 ⁇ g/ml
  • vitamin H D-biotin; 250ng/ml
  • Vitamin E Terolox; 25 ⁇ g/ml) (Sigma-Aldrich
  • albumax-1 0.5mg/ml)
  • fetuin 500 ⁇ g/ml
  • streptomycin penicillin
  • lOO ⁇ g/ml penicillin
  • the stabilised peptide (1 Ong/ml) with and without rIGF-I, (1 Ong/ml) and with and without monoclonal IGF-I receptor antibody (Ab-1, 100 ⁇ g/ml, Oncogene) were added in dGM as appropriate.
  • the peptides used were (see 1.1 and 1.3 above) the stabilised peptide related to the E domain of MGF / IGF-IEc peptide [24 amino acid residues] synthesized as described previously [Dluzniewska et al, 2005] and human IGF-I peptide [70 amino acid residues] (Sigma - Aldrich ER2 IGF-I). All media were replaced every 2-3 days. The cultures were sampled at various time-points for immunocytochemical analyses.
  • cells were fixed with methanol for 10 min (-20° C), followed by detergent permeabilization with 0.5% Triton-XIOO for 10-15 min. Cells were then incubated for 60 min with an anti-desmin (1 :100; clone D33, DAKO, Glostrup, Denmark) antibody, diluted in antibody diluting solution (ADS; PBS plus 10% FCS, 0.025% sodium azide, 0.1 M lysine).
  • a class specific anti-mouse IgG antibody conjugated to FITC (1:200; Jackson ImmunoResearch Laboratories/Stratech Scientific) was used to visualize.
  • Nuclei were identified by introducing the fluorescent minor-groove DNA-binding probe, DAPI (1.0 ng/ml; Sigma-Aldrich), into the final antibody incubation step.
  • Coverslips were mounted with the glycerol-based anti-fade agent, Citifluor (Citifluor Ltd), and sealed with clear nail varnish.
  • Cell-associated fluorescence and morphology were visualized by epi-fluorescence and Leica Modulation Contrast (LMC) microscopy respectively, using an inverted Leica DMIRB microscope equipped with Leica FW4000 image processing software.
  • LMC Leica Modulation Contrast
  • For the proliferation assay all blue and green fluorescent positive cells were counted in a field. At least 30 fields in each coverslip were counted in a systematic manner; at least 100 cells were therefore counted on each coverslip. The number of cells was compared as the percentage of desmin positive cells to the total number of DAPI positive cells.
  • CPK catalyzes the reversible phosphorylation of adenosine-5-diphosphate (ADP) to form adenosine-5- triphosphate (ATP) and free creatine.
  • ADP adenosine-5-diphosphate
  • ATP adenosine-5- triphosphate
  • the reaction may be followed in either direction by measuring the formation of inorganic phosphorus, an end-product of the reaction which is proportional to CPK activity. This was measured using the colorimetric method based on the generation of inorganic phosphate [Fiske and Subbarow, 1925] procedure. This was then expressed in terms of the protein content of the culture.
  • Previously expanded primary human muscle cell cultures were re-plated at 10x10 4 cells/well in 0.2% gelatin coated 96 well plates. Cells were cultured until 70/80% confluent in sGM then the medium changed to differentiation medium (DM; DMEM, 2% FCS, penicillin (100U/ml) and streptomycin (lOO ⁇ g/ml)) containing the stabilised peptide [24 amino acid residues] synthesized as previously described [Dluzniewska et al, 2005] and/or human IGF-I peptide [70 amino acid residues] (Sigma - Aldrich IGF-I ER2).
  • DM differentiation medium
  • DMEM fetaluzniewska et al, 2005
  • human IGF-I peptide [70 amino acid residues]
  • myogenic (desmin positive) cells was determined from all of the muscles tested (See Table below). Normal (non-diseased) muscle contained a significant proportion of desmin positive cells whereas diseased muscle contained a much lower proportion of myogenic cells.
  • Muscle Type Desmin positive cells as percentage of total cells in primary culture.
  • the stabilised peptide increased proliferation (changes in the proportion of desmin- associated nuclei to total nuclei) significantly in normal craniofacial (masseter) primary cultures from 49.8 ⁇ 2.4% to 68.8 ⁇ 3.9%; pO.0001). IGF-I also induced a moderate increase (from 49.8 ⁇ 2.4% to 58.4 ⁇ 4.2%; pO.0001). Interestingly, it was found that the effect of the stabilised peptide on desmin positive cell proliferation ratio was inhibited when IGF-I was added (from 68.8 ⁇ 3.9% to 59.5 ⁇ 4.2%; p ⁇ 0.0001). The effect seen in normal lower limb (quadriceps) primary cultures was similar to that seen with craniofacial muscle.
  • the stabilised peptide increased muscle progenitor cell proliferation significantly (from 38.4 ⁇ 2.5% to 57.9 ⁇ 3.2%; p ⁇ 0.0001).
  • IGF-I had only a minor effect on proliferation (from 38.4 ⁇ 2.5% to 47.1 ⁇ 3.5%; p ⁇ 0.0001) but IGF-I completely abrogated the response to the stabilised peptide when the two peptides were added in combination (from 57.9 ⁇ 3.2% to 38.8 ⁇ 0.6%; pO.0001).
  • the stabilised peptide could reproducibly and significantly increase the number of desmin positive cells in normal muscle, the effect on disease-state muscle was investigated.
  • CMD congenital muscular dystrophy
  • the effects of the stabilised peptide on cellular proliferation of muscle cells from amyotrophic lateral sclerosis - (ALS) and FSHD produced similar results.
  • the stabilised peptide did not facilitate primary myoblast differentiation and myotube formation.
  • IGF-I at a concentration of 10 ng/ml apparently stimulates myotube formation as the numbers of cells expressing desmin is decreased by the addition of IGF-I on this stage of myogenesis.
  • the stabilised peptide acted as an agonist and, in a dose-dependent manner, prevented differentiation to the myoblast fusion competent stage.
  • the decrease of 100 ng/ml of the stabilised peptide with 10 ng/ml of systemic IGF-I was lower than 10 ng/ml of MGF with the same dose of IGF-I.
  • the stabilised peptide induced progenitor cell proliferation significantly in primary muscle culture from patients with CMD, FSHD and ALS as well as healthy individuals.
  • the stabilised peptide did not affect myotube formation, a process that IGF-I accelerates significantly.
  • This demonstrates that the biologically active MGF E domain has a distinct activity compared to mature IGF-I.
  • Our findings indicate that the different actions of IGF-I isoforms are probably mediated via different receptors.
  • the blocking of the IGF-I receptor provides evidence that MGF E domain increases satellite cell proliferation via a different signalling pathway to IGF-I, and that the initial satellite cell activation is a separate process from that which is influenced by mature IGF-I.
  • Muscle wasting is one of the main causes of death in patients with certain neuromuscular diseases. Muscle loss can be linked to the inability to express MGF, and that muscles of the mdx dystrophic mouse, a model for human Duchenne
  • Muscular Dystrophy are unable to produce MGF even during mechanical stimuli [Goldspink et al., 1996].
  • De Ban et al found that when mesenchymal stem cells were introduced into dystrophic muscles of mdx mouse, the sarcolemmal expression of dystrophin and also MGF expression was restored [De Ban et al., 2003]. Therefore, the production of MGF may depend on the compliance of the cell membrane and possibly involve some type of mechanotransduction mechanism e.g. the dystrophin complex
  • IGF-I is a neurotrophic factor, and possesses potential clinical applications for neurodegenerative disorders, particularly ALS.
  • the data presented here indicate it is the activity of MGF, not that of ordinary IGF-I, that will be most for use in the treatment of muscle wasting, because it offers an effective method of replenishing the muscle satellite (stem) cell pool that is required for muscle maintenance and repair.
  • DMGF and CMGF The 8 amino acid peptides described in 1.3.1 above and referred to in Figure 8 A as DMGF and CMGF were tested for the ability to induce proliferation of C2C 12 muscle cells at a density of 2000 cells per well in a medium containing DMEM (lOOOmg/L glucose), BSA (lOOug/ml) and IGF-I (2ng per ml). Concentrations of 2, 5, 50 and 100 ng/ml of DMGF and CMGF were tested (See the left-hand and middle sets of results in Figure 8), along with 2, 5, 50 and 100 ng/ml IGF-I alone (See the right-hand set of results in Figure 8). After 36 hours incubation, an Alamar Blue assay was used to assess the level of cell proliferation achieved. A control containing only the medium was also provided.
  • the 8 amino acid peptides described in 1.3.2 above and referred to in Figure 8B as A2, A4, A6 and A8 were tested for the ability to induce proliferation of C2C12 muscle cells at a density of 500 cells per well. Cultivation was carried out foir 24 hours in 10% FBS, followed by starvation for 24 hours in 0.1% BSA, stimulation for 24 hours and then treatment with BrdU for 5 hours. Concentrations of 0.1, 1, 10 and 100 ng/ml of peptides A2, A4, A6 and A8 were tested, along with 0.1, 1, 10 and 100 ng/ml IGF-I (See the right-hand set of results in Figure 8B). Incorporation of BrdU was measured to assess the level of cell proliferation achieved. Controls containing no cells, medium only, 5% FBS and no BrdU were also provided.
  • Peptides A2, A4, A6 and A8 induced cell proliferation.
  • the results are shown in Figure 8 in terms of fluorescence (absorbence at 370nm; mean plus standard error across 4 wells).
  • the 24 amino acid peptide described in 1.4 above and referred to in Figures 9-11 as A5 was tested for the ability to induce proliferation of human muscle progenitor cells (Cambrex). These are commercially available primary human muscle cells, ie human muscle stem (progenitor) cells. They are also sometimes known as Human Skeletal Muscle Myoblasts (HSMM). Cells were obtained from a 39 year old male subject. Cultivation was carried out for 24 hours in 200 ⁇ l of SkGM2 medium supplemented with hEGF, L-Glut, dexamethasone, antibiotics and 10% FCS. The cultivation medium was then removed and the cells were washed twice in serum free medium.
  • SkGM2 medium supplemented with hEGF, L-Glut, dexamethasone, antibiotics and 10% FCS.
  • A5 was tested for the ability to induce proliferation of Cambrex HSMM at a density of 500 ( Figures 9 and 10) or 1000 (Figure 11) cells per well in Cambrex SkGM2 medium supplemented with hEGF, L-Glut, dexamethasone and antibiotics. Concentrations of 0.1, 1, 10, 100 and 500 ng/ml of A5 were tested (See the left-hand sets of results in Figures 9A, 1 OA and 11 A), along with 0.1 , 10 and 100 ng/ml IGF-I alone (See results in Figures 9A, 1OA and HA).
  • Concentrations of 0.1, 1, 10, 100 and 500 ng/ml of A5 were also tested in the presence of 2 ng/ml IGF-I (See the left- hand set of results in Figures 9B, 1OB and 1 IB). After 48 hours incubation, the cells were treated with BrdU for 5 hours. Incorporation of BrdU was measured to assess the level of cell proliferation achieved. Controls containing no cells, medium only, 5% FBS and no BrdU were also provided. IGF-I alone had no significant effect on the proliferation of HSMM at any dose (see Figures 9-11).
  • the A5 peptide had a significant effect (P ⁇ 0.1) on the proliferation of HSMM when used in isolation at doses of lOng/ml and below ( Figures 9 A and 10A).
  • Addition of 2ng/ml IGF-I to the medium in combination with A5 resulted in a significant effect on the proliferation of HSMM at a higher confidence level (P ⁇ 0.001 ; Figures 9B 5 1OB and HB).
  • the signal would be enhanced by increasing the BrdU exposure time.

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