Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
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NOTE POUR LE TOME / VOLUME NOTE:
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MECHANO GROWTH FACTOR PEPTIDES AND THEIR USE
FIELD OF THE INVENTION
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.
BACKGROUND TO THE INVENTION
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.
In MGF (Yang et al, 1996; McKoy et al, 1999), alternative splicing introduces
an
insert which changes the reading frame of the C-terminal portion of the
inolecule.
This insert is 49 base pairs long in human MGF. A 52 base pair insert has a
similar
effect in rat and rabbit MGF. The result is that MGF is sliglltly longer than
liver-type
IGF-I (because the terminator codon appears later owing to the reading frame
shift)
and that the C-terminal E domain has a different sequence. It is also smaller
overall
because it lacks glycosylation.
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:
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13/14). Liver-type IGF-I instead contains an Ea peptide at the C-terminus. The
sequences of the Ea and Ec/Eb peptides are unrelated to one another because of
the
reading frame shift discussed above.The presence of a splice variant with what
can
now be seen to be the MGF C-terminal was first noted by Chew et al (1995), who
identified it in liver tissue during studies on patients suffering from liver
cancer, but
did not investigate it at all in ternns of potential function or therapeutic
significance.
Goldspink and co-workers have already identified 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 (W097/3 3997;
WO01/136483;
WO01/85781; W003/066082). It is becoming increasingly clear that liver-type
IGF-I
and MGF have different roles and functions. Thus, Hill and Goldspink (2003)
have
shown that, in the rat anterior tibialis muscle, MGF is expressed rapidly in
response
to mechanical damage caused by electrical stimulation or resulting from
bupivacaine
injection, but that its expression then declines within a few days.
Conversely, liver-
type IGF-I is more slowly upregulated and its increase is commensurate with
the
decline in MGF expression. In addition, Yang and Goldspink (2002) have shown,
using the mouse C2C 12 muscle cell line as an in vitro model, that a 24 amino
acid
peptide related to the Ec peptide from the C-terminus of human MGF, but with
Histidine in the penultimate position rather than the native Arginine, and an
additional C-tenninal cysteine, has a distinct activity compared to that of
mature
IGF-I in that it increases myoblast proliferation but inhibits myotube
formation.
Dluzniewska et al (September 2005) have also demonstrated a strong
neuroprotective effect of the a related peptide, again with with Histidine in
the
penultimate position rather than the native Arginine and some modifications by
way
of conversion of L-Arginine to D-Arginine at positions 14 and 15, plus C-
terminal
amidation and PEGylation.
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SUMMARY OF THE INVENTION
However, 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.
Accordingly, the invention provides 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);
said polypeptide incorporating one or more modifications that give it
increased
stability compared to the unmodified MGF E peptide;
and said polypeptide possessing biological activity.
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.
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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 metliod 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.
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 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;
and said polypeptide or extended polypeptide possessing biological activity.
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:
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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;
and said polypeptide possessing biological activity.
The invention also provides use 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;
and said polypeptide possessing biological activity
in the manufacture of a medicament for use in the treatment of a neurological
disorder or a disorder of cardiac muscle.
BRIEF DESCRIPTION OF THE DRAWINGS
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
(Amino acids 26 to 110 of SEQ ID NO: 2 and to 26 to 111 of SEQ ID NOS: 4 and
6:
see below), and highlighting differences between MGF and liver-type IGF-I at C-
terminus; created by 49 base pair insert in human MGF and 52 base pair insert
in
rat/rabbit MGF, leading to reading frame shift and divergence at C-terminus.
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Figure 2: Effect of Alanine substitution and C-terniinal 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).
In the Figure, 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 -
(A) percentage change in tetanic force in dystrophic muscle of mdx mice
following
injection of stabilised peptide (left hand column) and IGF (right hand
column).
(B) percentage change in tetanic force in dystrophic muscle of mdx mice
following
injection of stabilised peptide (left hand column) and PBS vehicle control
(right hand
column).
Figure 4: Cardioprotection following adniinistration 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).
Figure 5: Pressure/volume loop data showing preservation of function following
myocardial in fraction (MI) - for normal (top left) and infarcted (MI) murine
(top
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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). All panels show pressure (mmHg) on the Y-axis and Relative Volume Units
on
the X-axis.
Figure 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).
Figure 7: Western blots demonstrating the greater stability of the stabilised
peptide that incorporates conversion of Arginine from L to D form and N-
terminal PEGylation - the stability of the stabilised peptide compared to a
corresponding one lacking the L to D form conversions and N-terminal
PEGylation
was investigated by incubation in fresh liuman plasma for a range of different
time
intervals. Western blotting was then used to assess the survival of each
peptide over
those time intervals: A = 0 minutes; B= 30 minutes; C = 2 hours; D = 24 hours.
The
results for the peptide with L-D conversion and N-terminal PEGylation are
shown on
the right; those for the peptide lacking the L to D form conversions and N-
terminal
PEGylation are on the left.
Figure 8: Effect of.8 amino acid C-terminal peptides on proliferation of C2C12
muscle cells:
(A) DMGF and CMGF Peptides : C2C12 Cells were provided at 2000cell/well, in
a medium containing DMEM (1000mg/L glucose), plus BSA(100ug/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
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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.
Figure 9 : Effect on proliferation on HSMM cells
(A) 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
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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.
(B) 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. *
means P < 0.01 and ** means P < 0.001 compared to medium control containing 2
ng/ml IGF-I.
Figure 10 : Effect on proliferation on HSMM cells
(A) 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
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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.
(B) 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.
Figure 11: Effect on proliferation on HSMM cells
(A) 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
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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.
(B) 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.
SEOUENCE INFORMATION
The DNA and amino acid sequences of human, rat and rabbit MGF DNA and 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
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alternative leader sequences. For comparison, the corresponding DNA and amino
acid sequences from human, rat and rabbit liver-type IGF-I are given as SEQ ID
NOS: 7/8, 9/10 and 11/12 respectively. A comparison of the six amino acid
sequences, from the beginning of the sequence encoded by exon 4 onwards, is
made
in Figure 1.
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.
Modified sequences derived from the peptide of SEQ ID NO: 27 are given as SEQ
ID NOS: 28 to 32.
In SEQ ID NO: 28, Serine is replaced with Alanine at position 5.
In SEQ ID NO: 29, Serine is replaced with Alanine at position 12.
In SEQ ID NO: 30, Serine is replaced with Alanine at position 18.
In SEQ ID NO: 31, Arginine is replaced with Alanine at position 14.
In SEQ ID NO: 32, Arginine is replaced with Alanine at position 14 and
Arginine is
also replaced with Alanine at position 15.
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
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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.
In Peptide 2 (SEQ ID NO: 16), Serine is replaced with Alanine at position 5.
In Peptide 3 (SEQ ID NO: 17), Serine is replaced with Alanine at position 12.
In Peptide 4 (SEQ ID NO: 18), Serine is replaced with Alanine at position 18.
In Peptide 5 (SEQ ID NO: 19), Arginine is replaced with Alanine at position
14.
In Peptide 6 (SEQ ID NO: 20), Arginine is replaced with Alanine at position 14
and
Arginine is also replaced with Alanine at position 15.
In Short peptide 1(SEQ ID NO: 21), Arginine is replaced with Alanine at
position
14 and the two C-terminal amino acids are removed.
In Short peptide 2 (SEQ ID NO: 22), Arginine is replaced with Alanine at
position
14 and the four C-terminal amino acids are removed.
In Short peptide 3 (SEQ ID NO: 23), Arginine is replaced with Alanine at
position
14 and the three N-terminal amino acids are removed.
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In Short peptide 4 (SEQ ID NO: 24), Arginine is replaced with Alanine at
position
14 and the five N-terminal amino acids are removed.
Four 8 amino acid peptide sequences are also included in the Sequence Listing.
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.
For ease of reference, these sequences are also described in the following
Table.
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SEQ ID Description
NO: ("aa" denotes "amino acid")
1 Full length human IGF-1-Ec (= MGF) (Nucleotide and amino acid)
2 Full length human IGF-1-Ec (= MGF) Amino acid only)
3 Full length rat IGF-1-Eb (= rat MGF) (Nucleotide and amino acid)
4 Full length rat IGF-1-Eb (= rat MGF) (Amino acid only)
Full length rabbit IGF-1-Eb (= rabbit MGF) (Nucleotide and amino acid)
6 Full length rabbit IGF-1-Eb rabbitMGF) (Amino acid only)
7 Full length human liver-type IGF-1 (Nucleotide and amino acid)
8 Full length human liver-type IGF-1 (Amino acid only)
9 Full length rat liver-type IGF-1 (Nucleotide and amino acid)
Full length rat liver-type IGF-1 (Amino acid only)
11 Full length rabbit liver-type IGF-1 ( Nucleotide and amino acid)
12 Full length rabbit liver-type IGF-1 (Amino acid only)
13 Synthetic peptide corresponding to aa 87 - 111 of SEQ ID NO: 4
14 Synthetic peptide corresponding to aa 87 - 111 of SEQ ID NO: 6
Synthetic peptide corresponding to aa 87 - 110 of SEQ ID NO: 2 with
Arg109->His (= Ar 23->His using SEQ ID NO: 15 numbering)
16 Peptide of SEQ ID NO: 15 with Ser5->Ala
17 Peptide of SEQ ID NO: 15 with Ser12--*Ala
18 Peptide of SEQ ID NO: 15 with Ser18-Ala
19 Peptide of SEQ ID NO: 15 with Arg14--+Ala
Peptide of SEQ ID NO: 15 with Ar l4--->Ala , Ar 15-Ala
21 Synthetic peptide corresponding to aa 1-22 of SEQ ID NO: 15 with Arg14->Ala
22 Synthetic peptide corresponding to aa 1-20 of SEQ ID NO: 15 with Ar l4--
>Ala
23 Synthetic peptide corresponding to aa 4- 24 of SEQ ID NO: 15 with
Ar 14->Ala and Ar 23->His
24 Synthetic peptide corresponding to aa 6- 24 of SEQ ID NO: 2 with
Ar 14-+Ala and Ar 23-aHis
Se uence of SEQ ID NO: 1 with Ar 109-->His (Nucleotide and amino acid)
26 Sequence of SEQ ID NO: 2 with Arg109->His (Amino acid only)
27 Synthetic peptide corresponding to aa 87 - 110 of SEQ ID NO: 2
28 Peptide of SEQ ID NO: 27 with Ser5->Ala
29 Peptide of SEQ ID NO: 27 with Ser12--->Ala
Peptide of SEQ ID NO: 27 with Serl 8->Ala
31 Peptide of SEQ ID NO: 27 with Ar 14-iAla
32 Peptide of SEQ ID NO: 27 with Arg14---- >Ala , Ar 15-->Ala
33 Peptide corresponding to the 8 C-terminal amino acids of SEQ ID NO: 15
34 Peptide corresponding to the 8 C-terminal amino acids of SEQ ID NO: 27
Peptide corres ondin to the 8 C-terminal amino acids of SEQ ID NO: 18
36 Peptide corresponding to the 8 C-terminal amino acids of SEQ ID NO: 30
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DETAILED DESCRIPTION OF THE INVENTION
Polypeptides and Extended Polypeptides of the invention
Polypeptides of the Invention
Polypeptides of the invention are up to 50 amino acid residues in length. For
example, 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.
Preferably,
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. Also preferred are
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. 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. In rat and rabbit MGF (SEQ ID NOS: 3-6),
the
insert is 49 base pairs and the C-terminal E peptides are known as 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
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peptide in any way, as long as the requirements for biological activity and
stability
(see below) are met. In particular, 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.
Up to the maximum length of 50 amino acids, the polypeptide may also comprise
native MGF sequence N-terminal to the sequence derived from the C-terminal E
peptide. Alternatively, 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. Alternatively, it may include up to 10 ainino 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. Depending on the nature of the polypeptides and
in
particular whether they contain any L-D conversions (see below), 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.
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Extended Polypeptides of the Invention
An extended polypeptide of the invention comprises a polypeptide of the
invention,
extended by non-wild-type sequence. By this is meant that 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. Apart from that, an
extension
may have any sequence. Thus, 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. For
example, 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. Typically, any such extension will be short,
e.g. 1,
2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length. An extension may contain,
or even
consist entirely of D-form amino acids (see below), e.g. to reduce
exopeptidase
attack. For example, a polypeptide may be extended by 1 to 5 D-form amino
acids at
one or both ends. For example, in some embodiments an additional Cysteine
residue
may be incorporated at the C-terminus.
Modifications
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 ainino 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.
For example, it 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
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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).
Whether to achieve stabilisation or otherwise, 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. In
particular, polypeptides may be acetylated at the N-terminus are preferred or
amidated at the C-terminus or both. Alternatively or additionally, 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.
In addition or alternatively, the polypeptide or extended polypeptide may
include one
or more D-form amino acids. In nature, amino acids are in the L-form.
Inserting D-
form amino acids can improve stability. Typically, a few, e.g. 1, 2, 3, 4 or
5, D-form
amino acids may be used. However, more can also be used, e.g. 5 to 10, 10 to
15, 15
to 20 or 20 or more as long as the resultant PEGylated polypeptide satisfies
the
requirements for biological activity and stability (see below). If those
requirements
are satisfied, the entire polypeptide may even be synthesised using D-form
amino
acids.
D-form amino acids may be used at any position in the polypeptide. In 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.
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Stereochemical and/or directional peptide isomers may also be used. For
example,
Retro (RE) peptides may be used, in which the sequence of the invention is
assembled from L-amino acids but in reversed order. Alternatively, Retro-
inverso
(RI) peptides may be used, in which the sequence is reversed and synthesised
from
D-amino acids.
Additionally or alternatively, 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.
In one embodiment, 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.
In another embodiment, the 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.
In some further embodiments, the 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.
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Alternatively or additionally, polypeptides of the invention may also
incorporate
other modifications, for example truncation, insertion, internal deletion or
substitution.
As to truncation, it has has also been found that shorter peptides, based on
the C-
terminal eight amino acids of SEQ ID NO: 15 are active. However, the results
in
Example 5 below suggest that the activity of longer peptides related to the
MGF C-
terminus can be quite sensitive to truncation, particularly of the N-terminus
of the
peptides. At the N-terminus of the peptide of SEQ ID NO: 15, truncation by 3
amino
acids led to loss of activity in the muscle cell model used in Example 5. At
the C-
terminus of the peptide of SEQ ID NO: 15, truncation by four amino acids led
to loss
of activity in the muscle cell model, though truncation by two did not. In the
case of
the native human, rat and rabbit E peptide sequences, and in the variant one
of SEQ
ID NO: 15 and other peptides of the invention that have lengths comparable to
those
of the native peptides (eg 18 or more amino acids), it is therefore envisaged
that it
will be possible to truncate by 1, 2 or 3 amino acids at the C-terminus
without loss of
activity. It is also envisaged that it will be possible to truncate by 1 or 2
amino acids
at the N-terminus without loss of activity.
As to 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.
As to internal 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, 7, 8, 9 or 10 amino
acids.
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As to 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). One or more
such
substitutions may be made, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 or up
to 20
substitutions in total. Preferably, in the sequence derived from the MGF C-
terminal E
peptide, no more than 10 substitutions will be made, e.g. 1, 2, 3, 4, 5, 6, 7,
8, 9 or 10
substitutions. Preferably, in the in the sequence derived from the MGF C-
terminal E
peptide, 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. In one preferred approach, residues at one or
both
ends of the polypeptide (terminal residues) are substituted. It is envisaged
that this
will protect against exopeptidase attack. Thus, for example, it may be
preferred to
substitute residues in the N-terminal and for C-terminal positions, or in the
positions
immediated adjacent to the terminal ones, or up to 3, 4 or 5 positions from
one or
both ends.
Substitutions may increase stability or biological activity. For example, 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.
Whether in positions 5, 12, 14 or 18 of SEQ ID NOS: 27 or 15, position 2 of
SEQ ID
NOS: 33 and 34, positions 5, 12, 15 and 19 of SEQ ID NOS: 13 and 14, or
elsewhere, substitution of the native amino acid with Alanine is one preferred
option,
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as shown in Example 5 and Figure 2. However, other amino acids may equally be
used.
Alternatively or additionally, 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.
ALIPHATIC Non-polar G A P
ILV
Polar-uncharged C S T M
NQ
Polar-charged D E
KR
AROMATIC H F W Y
Typically, 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. However, where the polypeptide contains additional MGF sequence, they
may alternatively or additionally be found in that additional sequence. For
example,
if a polypeptide of the invention contains further MGF sequence that is N-
terminal to
the sequence of the E peptide (e.g. SEQ ID NO: 13, 14 or 27) in native MGF,
modifications may be found in that sequence.
Alternatively or additionally, stability can also be increased by cyclisation
of the
polypeptides or extended polypeptides of the invention. It is envisaged that
this will
protect against exopeptidase attack.
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Preferred polypeptides of the invention include the following.
(i) A peptide which is 24 amino acids in length and has the sequence of SEQ ID
NO: 15 but is stabilised by converting the two Arginines of SEQ ID NO 15
(positions 14 and 15) from L-form to D-form and by N-terminal PEGylation.
(ii) A peptide as in (i) above but lacking PEGylation, ie having the sequence
of
SEQ ID NO: 15 but stabilised by converting the two Arginines of SEQ ID
NO 15 (positions 14 and 15) from L-form to D-form.
(iii) The peptides described in Example 5 and Figure 2 as Peptides 2, 3, 4 and
5
(SEQ ID NOS: 16 to 19).
(iv) The peptide described in Example 5 and Figure 2 as Short peptide 1(SEQ ID
NO: 21), which has the sequence of SEQ ID NO: 19 (in which Arginine at
position 14 is replaced by Alanine) but is truncated by 2 amino acids at the C-
terminus.
(v) A peptide corresponding to that of (i) above but based on the native
huinan
C-terminal peptide of SEQ ID NO: 27, which contains Arginine rather than
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.
(vi) A peptide as in (v) above but lacking PEGylation, ie having the sequence
of
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.
(vii) Peptides corresponding to those of (iii) above but based on the native
human
C-terminal peptide of SEQ ID NO: 27, which contains Arginine rather than
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Histidine in the penultimate position; shown herein as SEQ ID NOS: 28 to
31.
(viii) Peptides of any of SEQ ID NOS 33-36 with N-terminal PEGylation or the
attachment of an N-terminal hexanoic or amino-hexanoic acid moiety.
(ix) Any of the peptides of (i), (ii), (iii), (iv), (v), (vi), (vii), or (vii)
above with C-
terminal amidation, notably the peptides of (ii) and (vi) above with C-
terminal amidation, i.e. peptides having the sequences of SEQ ID NOS: 15
and 27, with conversion of L-Arginine to D-Arginine at positions 14 and 15
and C-terminal amidation, but lacking PEGylation.
(x) Any of the peptides of (i), (ii), (iii), (iv), (v), (vi), (vii), (viii) or
(ix) above
with an additional Cysteine residue at the C-terminus.
(xi) Any of the peptides of (i), (ii), (iii), (iv), (v), (vi), (vii), (viii)
or (ix) above
with an additional D-form Arginine residue at the N-terminus.
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).
Biological Activity
Polypeptides and extended polypeptides of the invention have biological
activity.
This activity may be selected from the following.
The ability to increase muscle strength in dystrophic and/or non-dystrophic
skeletal
muscle in mice, humans or other mammals (cf. Example 2 below). Preferably, a
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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.
Cardioprotective ability in sheep, mice, humans or other mammals (cf. Example
3
below). Preferably, 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.
Preferably 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.
Neuroprotective ability in vitro or in vivo in mice, gerbils, humans or other
mammals
(cf. Example 4 below). Preferably, 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. Preferably, following exposure
to TBH
or other another agent that induces oxidative stress or causes damage in other
ways, 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. Alternatively or additionally, polypeptides or extended polypeptides of
the
invention may have neuroprotective ability
Furthermore, the 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). For example, polypeptides or extended polypeptides of the
invention
may have the functional properties of MGF identified in W097/33997. In
particular,
they may have the ability to induce growth of skeletal muscle tissue.
Similarly, as
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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.
In this regard, 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 1D, 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.
Alternatively or additionally, 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 WO01/136483, which rely on
measuring
motoneurone rescue in response to facial nerve avulsion in rats, may be used.
Alternatively or additionally, polypeptides or extended polypeptides of the
invention
may have the properties identified in W003/060882, 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.
Preferably, 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
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applied. However, 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. if damage is
reduced by 1% or more, 5% or more, 10% or more, 20% or more, 3 0% 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.
In particular, 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. Similarly, increased expression of anti-apoptotic
markers in
MGF-transfected cells under apoptotic conditions can be taken as a sign that
the
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polypeptide of the invention is preventing apoptosis. One suitable anti-
apoptotic
marker gene is Bc12. 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.
Another preferred property of polypeptides and extended polypeptides of the
invention is the ability to induce a hypertrophic phenotype in cardiac muscle
cells.
In particular, 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.
Stability of Polypeptides and Extended Polypeptides of the Invention
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. In particular, stability can be measured by assessing
peptides'
susceptibility to proteolytic cleavage in fresh human plasma according to the
technique of Example 5.1 below, in which the plasma was stored until used at -
70 C,
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l0 g of peptide was added to 2m1 of plasma, plus 7 ml of PBS and the mixture
was
incubated at 37 C for different time intervals. Western blotting was then used
to
detect each peptide over those time intervals. (In Figure 7: A= 0 minutes; B=
30
minutes; C = 2 hours; D = 24 hours. The results for the peptide with L-D
conversion
and N-terminal PEGylation are shown on the right; those for the peptide
lacking the
L to D form conversions and N-terminal PEGylation are on the left.) Relatively
little
of the peptide lacking L-D conversion and PEGylation could be detected after
30
minutes, very little after 2 hours and none or almost none after 24 hours. In
contrast,
the peptide with L-D conversion and PEGylation could be detected in much
greater
abundance and 2 hours and 24 hours.
Other 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.
Quantitatively, in relative terms, 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.
Quantitatively, in absolute terms, 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.
Alternatively, 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
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generally score more highly on such a scale than the corresponding native MGF
C-
terminal E peptide.
Further Peptides of the Invention
Whilst many of the 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, 14 27 and
34 or
the histidine-containing variant of SEQ ID NO: 15 and 33. In the treatment of
neurological and cardiac disorders according to the invention, it may be
desirable for
the polypeptide or extended polypeptide of the invention to be degraded
relatively
rapidly, i.e. to exert its effect for a relatively short period of time.
Therefore,
stabilisation will not necessarily be required in the context of such
treatments.
Where stabilisation is not required, it is preferred to use the native
polypeptides of
SEQ ID NOS: 13, 14, 27 and 34 or the Histidine-containing variant of SEQ ID
NO:
15 or 33 without stabilising modifications. However, modified polypeptides may
also be used. Any of the modifications discussed herein may be applied except
that,
in this aspect, it is not required that those modifications result in
increased stability.
Treatments According to the Invention
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.
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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; sarcopenia and frailty in
the
elderly. The invention also fmds application in muscle repair following
trauma.
So far as neurological disorders are concerned, treatment of neurodegenerative
disorders is one possibility. Treatment of motoneurone disorders, especially
neurodegenerative disorders of motoneurones is also a possibility.
Examples of 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
dystroplzy;
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. In this
embodiment,
the polypeptide or extended polypeptide will typically be localised around the
sites
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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. WO01/85781).
As to 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. In particular, polypeptides and extended
polypeptides
of the invention may be used for prevention of myocardial damage following
ischemia and/or mechanical overload.
In this case, they will generally be administered as rapidly as possible after
the onset
of the ischemia or mechanical overload to the heart, for example as soon as a
heart
attack resulting from ischemia has been diagnosed. Preferably, they will be
administered within 5, 10, 15, 30 or 60 minutes, or within 2 or 5 hours.
Preferably,
the ischemia or mechanical overload in response to which the MGF polypeptide
or
polynucleotide is administered is a temporary condition. In a particularly
preferred
embodiment, 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.
Under somce circumstances, it may be desirable to use polypeptides and
extended
polypeptides of the invention in combination with other pharmaceutically
active
agents. For example, 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.
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Production of Polypeptides and Extended Polypeptides of the Invention
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.
Isolation and purification to any desired degree may also be carried out 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. In particular, where the polypeptide or extended
polypeptide
is obtained recombinantly, 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. However, 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, Formulations, Administration and Dosages
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The polypeptides and extended polypeptides of the invention are preferably
provided
in the form of compositions comprising the polypeptide or extended polypeptide
and
a carrier. In particular, such a composition may be a pharmaceutical
composition
comprising the polypeptide or extended polypeptides and a pharmaceutically
acceptable carrier or diluent. Any suitable phannaceutical formulation may be
used.
For example, 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.
It should be understood that in addition to the ingredients particularly
mentioned
above the 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.
The 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
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injection will often be preferred under many clinical circumstances. So-called
"needle-less" injection or transcutaneous administration may be possible under
some
circumstances.
In the treatment of skeletal muscle disorders, 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.
In the treatment of cardiac muscle disorders, delivery will generally be
intravenous.
Under appropriate clinical circumstances (e.g. in specialist cardiac units)
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.
The 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.
Based on the Inventors' experience, it is envisaged that 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.
For
example, 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 following Examples illustrate the invention.
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EXAMPLES
1. Peptides
1.1 Peptides of Examples 2, 3, 4 and 6
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 (0'O-
bis(2aminopropyl)polyethylene glyclol 1900) (Jeffamine) via a succinic acid
bridge,
and amidated at the C-terminus.
1.2 Peptides of Exatnple 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.
Also, a peptide corresponding to that of 1.1 above, with the same L-D
conversions,
but without PEGylation, has also been tested for stability (see 5.2.3 below).
This
peptide was synthesised via standard techniques using a peptide synthesiser.
The
product was purified by HPLC and analyzed by MALDI-MS.
1.3 Peptides of Example 7
1.3.1 Peptides of Exaynple 7.1
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. In the
peptide
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referred to as DMGF in Figure 8, stabilisation was achieved via N-terminal
PEGylation as in 1.1 above. In the peptide referred to as CMGF in Figure 8,
stabilisation was achieved via N-terminal attachment of hexanoic acid
acid. Both DMGF and CMGF were also amidated at the C-terminal end.
1. 3.2 Peptides of Example 7.2
In Example 7.2, 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.
1. 4 Peptide of Example 8
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.
1.5 IGF-I Peptide
For comparison, 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, UK. In Example 6, it was obtained from Sigma - Aldrich (ER2 IGF-
I). IGF-I peptide was also used in Example 7.
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2. Injection of Stabilised Peptide into Dystrophic Muscle
Using intramuscular injections (twice weekly injections of 25 1 containing 17
g of
the chemically stabilised peptide), muscle strength was increased by more than
25%
within a few weeks in the tibialis anterior muscle of non-dystrophic miceThis
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.
Greater increases, of up to around 35% (Figures 3A, 3B), were recorded for
intramuscular injections (two per week for three weeks) in the dystrophic
muscles of
the mdx mouse, which has the same type of mutation as that in human Duchenne
muscular dystrophy. Injections of IGF-I led only to an increase of around 5%,
as
shown in Figure 3A On the same basis, the results of a comparison between the
stabilised peptide and a PBS vehicle-only control are shown in Figure 3B.
These data relating to muscle protection and repair show that the stabilised
peptide is
effective in increasing the strength of dystrophic and non-dystrophic muscle.
3. Cardioprotection and Myocardial Repair by Stabilised Peptide
A myocardial infarction (MI) was induced in ovine hearts by catheterising a
marginal
branch of the circumflex coronary artery and injecting a small bolus of
microspheres
to induce localised ischemia. Full-length MGF (native C-terminal peptide plus
sequence encoded by exons 3 and 4 and common to MGF and liver-type IGF-I) or
stabilised peptide was injected (200nm, intracoronary) 15 minutes later using
the
same catheter whilst the animal was still under the anaesthetic. As a control,
mature
liver-type IGF-I was used. 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.
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Mature liver-type IGF-I had a much smaller effect. The results are given in
Figure 4,
which shows percentage change in ejection fraction on day 6 as compared to
ejection
fraction on day 1 before the procedure was carried out. Thus, the stabilised
peptide
was very effective in protecting the myocardium from ischemic damage.
Additional experiments were carried out on mice. In these studies the MI was
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.
4. Neuroprotection by Stabilised Peptide Following Ischemia and General
Damage
4.1 Neuroprotective Effect In Vitro
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
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glucose, 1% amphotericine B, 0,4% penicillin-streptomycin. Hippocampi were
separated and cut into 400 m slices using McIlwain 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% CO2 at 32 C for 30 minutes. Four selected slices were
settled on each membrane. Slices were cultivated for two weeks at 32 C in 5%
CO2
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
Axiovert 25) with MC-10095 camera (Carl Zeiss Jena GmbH) in order to record
initial PI uptake (Sarnowska, 2002).
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.
Stabilised peptide or, for the purpose of comparison, recombinant IGF-1 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.
In order to investigate a pathway in which the MGF acts, a specific anti-IGF-1
receptor (AB-1) blocking antibody (Oncogene) was included in the medium 1 hour
before the slices were exposed to TBH and MGF or IGF-1 peptide. The
concentration of the antibody (1000 ng/ml) was used according to the
manufacturer's
recommendation.
To obtain detailed images of the slices, 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). Following acquisition, images were processed using the
Zeiss
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LSM 510 software package v. 2.8. Quantitative measurement of tissue
deterioration
was performed using image analyser KS 300 (Carl Zeiss Jena GmbH).
Cell damage was quantified on fluorescence images of PI-stained cultures 24
and 48
hours after TBH challenge. The relative extent of cell death was calculated
from each
standardized CAl region as follows: % of dead cells = (experimental
fluorescent
intensity (FI)- background FI) / (maximal Fl- background FI) x 100, where
maximal
FI was obtained by killing all cells with exposure to 100 mM glutamate.
All the measureinents were repeated for 5 independent culture preparations and
8
slices were used for each experimental condition. Statistical significance of
the
differences between the results was calculated using one-way Anova followed by
Dunnet's test, (GraphPad Prism 3.02).
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. In the
absence of
treatment peptide, TBH caused about 60% of the cells to die within 24 hours
but,
following treatment with the stabilised peptide (100ng/ml), 85% protection was
observed. 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-1 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.
4.2 Neuroprotective Effect in Gerbil Model
Other experiments were carried out using a Gerbil model of brain ischemia. To
assess neuroprotection, confocal microscopy was carried out on the brain after
administration of the stabilised peptide or the IGF-I receptor binding domain.
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In the gerbil brain, bilateral ligation of the common carotid arteries
invariably
produces specific hippocampal lesions: in the CAl region, pyramidal neurones
start
to die 3-4 days after ischemia.
Male Mongolian gerbils weighing 50-60 g were used. The ischemic insult was
performed by 5 min. ligation of the common carotid arteries under halotane in
N20:02 (70:30) anaesthesia in strictly controlled normothermic conditions as
previously described (Domanska-Janik et al., 2004). The cerebral blood flow
was
continuously monitored by laser Doppler flowmetry (Muro, Inc.). A group of
animals received stabilised peptide or IGF-1 (1 g/ l in PBS) by injection at
a dose
of 25 g directly to the left carotid artery immediately upon the reperfusion.
Sham
operated animals were injected with the same dose of the peptide.
Usually, 10 - 15 minutes after the procedure, treated animals were standing up
on
their legs and behaving as untreated ones. The animals were allowed a recovery
period of one week, then were perfused with ice-cold 4% paraformaldehyde in
PBS
under pentobarbital anaesthesia. The histological evaluation was performed on
paraffin-embedded and fixed, 10 mm-thick sections stained by
hematoxylline/eosine.
The extent of cell damage in the CA1 hippocampal region was quantified, under
a
Zeiss Axioscop 2, as the mean number of the persisted, intact neurons in the
coronal
sections. At least three defined 300 m fields of the CAl region were captured
using
a MC 10095 camera (Carl Zeiss Jena GmbH) and counted in a computer-assisted
image analysis system (KS 300, Carl Zeiss Jena GmbH).
In control animals, the mean number of morphologically intact neurones per 300
m
length scored in the CAl region was 121.25 + 12.5 (mean + SD, n=5). In
contrast to
the untreated animals, where only about 12% (15.2 5, n=7) of neurones
survived
the ischemic episode, injection (single bolus of 25 g) of the stabilised MGF
C-
terminal peptide into the left carotid artery, immediately after re-perfusion,
provided
a very significant neuroprotection. 83.2 25 (n=10) neurones were scored on
the
injected side (74.5 % of non-operated control value) and 65.8 30 (n=10) on
the
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contralateral side (54 % of non-operated control value). Thus, treatment with
the
stabilised MGF C-terminal peptide enabled a high proportion of the CA1
hippocampal neurones to survive the ischemic insult. In most animals, the
protective
effect was noticeable bilaterally while in a minority it was mostly evident on
the
injected (left) side.
In contrast, similar injection of 25 g of IGF-1 peptide had little influence
on the
postischemic survival of CAl neurones; 7 days after the insult there were 19.2
7.3
neurones (n=5) left, which is only 15.8 % of the control neuronal cell number
and not
significantly different from the untreated postischemic group.
5. Biological Activity and Stability of Modified Peptides
5.1 Peptide Stabilised by L-D Conversion and N-terminal PEGylation
The peptide used in Examples 2, 3 and 4 above had the sequence of SEQ ID NO:
15
(which corresponds to that of the the lluman 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.
The biological activity of this peptide is confirmed in Examples 2, 3 and 4.
Its stability is demonstrated by Figure 7. Stability of the peptides with and
without
PEGylation and L-D conversion of Arginine at positions 14 and 15 was
investigated
by assessing the peptides' susceptibility to proteolytic cleavage in fresh
human
plasma.
The plasma was stored until used at -70 C. 10 g of peptide were added to 2m1
of
plasma, plus 7 ml of PBS. This mixture was incubated at 37 C for different
time
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intervals. Western blotting with a polyclonal antibody having specificity to
peptides
with the amino acid sequence of SEQ ID NO : 15 was then used to detect each
peptide over those time intervals. (In Figure 7: A = 0 minutes; B = 30
minutes; C = 2
hours; D = 24 hours. The results for the peptide with L-D conversion and N-
terminal
PEGylation are shown on the right; those for the peptide lacking the L to D
form
conversions and N-terminal PEGylation are on the left.). Relatively little of
the
peptide lacking L-D conversion and PEGylation could be detected after 30
minutes,
very little after 2 hours and none or almost none after 24 hours. In contrast,
the
peptide with L-D conversion and PEGylation could be detected in abundance even
after 24 hours.
5.2 Further Peptides - Replacement of Serine or Arginine with Alanine and C-
ternzinal and N-terminal Truncation
5.2.1 Further Peptides
Herein, the sequence of the native human Ec peptide from the C-terminus of
human
MGF is given as SEQ ID NO: 27. In the peptide of SEQ ID NO: 15, 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.
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.
In Peptide 2 (SEQ ID NO: 16), Serine is replaced with Alanine at position 5.
In
Peptide 3 (SEQ ID NO: 17), Serine is replaced with Alanine at position 12. In
Peptide 4 (SEQ ID NO: 18), Serine is replaced with Alanine at position 18. In
Peptide 5 (SEQ ID NO: 19), Arginine is replaced with Alanine at position 14.
In
Peptide 6 (SEQ ID NO: 20), Arginine is replaced with Alanine at position 14
and
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Arginine is also replaced with Alanine at position 15. In Short peptide 1(SEQ
ID
NO: 21), Arginine is replaced with Alanine at position 14 and the two C-
terminal
amino acids are removed. In Short peptide 2 (SEQ ID NO: 22), Arginine is
replaced
with Alanine at position 14 and the four C-terminal amino acids are removed.
In
Short peptide 3 (SEQ ID NO: 23), Arginine is replaced with Alanine at position
14
and the three N-terminal amino acids are removed. In Short peptide 4 (SEQ ID
NO:
24), Arginine is replaced with Alanine at position 14 and the five N-terminal
amino
acids are removed.
5.2.2 Biological Activity of Further Peptides
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 metliod. This was
assessed on a scale of 0 to 3 and the results are shown in Figure 2.
0= no measureable increase in cell number at 6h.
1= significant increase in cell number at 4h.
2= significant increase in cell number at 2h.
3 = significant increase in cell number at 1 h.
Significance was at the level of P>0.05 using the T test.
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
24) exhibited no measurable activity (zero score).
5.2.3 Stability of Furtlaef= Peptides
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The stability of each peptide was determined by introducing it into fresh
human
plasma and using Western blotting as discussed in Example 5.1 above. Like
biological activity, stability was scored on a scale of 0 to 3. The results
are shown in
Figure 2.
Stability was determined as the amount of the peptide that remained intact and
bound
to the specific antibody in the following way:
1= marked loss of detectable antibody binding by %Z hours.
2 = marked loss of detectable binding by 2 hours.
3 = no marked loss of antibody binding by 24 hours.
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.
The peptide of Examples 1-4 also scored 3 on this scale. The same peptide, but
lacking PEGylation, 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.
6. Effects of Stabilised Peptide on Muscle Satellite Cell Proliferation in
Dystrophic, ALS and Healthy Human Muscle
The stabilised peptide of 1.1 above was used in these experiments. Comparisons
were made with the IGF-I peptide of 1.3 above.
6.1 Sufninary
Primary human muscle cell cultures were derived from biopsies of congenital
muscular dystrophy (CMD), facioscapulohumeral dystrophy (FSHD) and motor
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neurone disease or amyotrophic lateral sclerosis (ALS) patients as well as
from
healthy muscle using proliferation/differentiation assays. Cell cultures were
treated
with the two peptides and immunocytochemistry techniques were used to detect
cells
expressing the differentiation marker desmin, and total number of nuclei using
DAPI.
Creatine phosphokinase (CPK) and protein assays were used to determine
myogenic
differentiation following peptide treatment. The stabilised peptide
considerably
increased stem (desmin positive) cell proliferation for normal (non-diseased)
muscle
(from 38.4zL2.5% 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 10.4d:1.7% to 17.5 1.6%;
FSHD
11.7 1.3% to 20.4- 2.1% and ALS 4.811.1% 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.
6.2 Isolation of Human Muscle-Derived Cells
Human primary muscle cell cultures were isolated as previously described
[Lewis et
al., 2000; Sinanan et al., 2004]. Briefly, following informed consent,
craniofacial
(masseter) muscle biopsies were obtained from healthy adult and CMD patients
during elective surgery at the Eastman and Middlesex Hospitals, London, UK.
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.
These were washed with antibiotic (penicillin, 100U/ml; streptomycin, 100
g/ml;
fungizone, 2.5 g/ml; Invitrogen) supplemented DMEM (high glucose; Invitrogen),
scissor-minced and tissue fragments plated into 0.2% gelatin-coated (Sigma-
Aldrich)
T150cm2 culture flasks (Helena Biosciences). Explant cultures were incubated
in
serum-containing Growth Media (sGM), composed of DMEM, 20% FCS (PAA
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Laboratories), penicillin (100U/ml) and streptomycin (100 g/ml) (Invitrogen),
and
maintained at 37 C in humidified 95% air with 5% CO2. The first wave of
migration
of mononuclear cells from the explant was designated the ~-wave and this
population was used throughout this study. Migratory human muscle cell were
enzymatically harvested using trypsin-EDTA (Invitrogen) and subcultured in sGM
unti170-80% confluency. Passage number x(PX), was defined as the xth
sequential
harvest of subconfluent cells. All experiments were performed using P3_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.
6.3 Determination of the myogenic progenitor (stent) cell population in vitro
Assessment of the number of myogenic precursors was performed as described
previously (Sinanan et al., 2004). Cells were re-plated on gelatin-coated
(0.2%)
13mm coverslips at an initial density of 4.5x 103 cells cm 2. To avoid
confounding
effects of IGF and related protein in FCS, cells were cultured in a serum-
free,
defined media (dGM); DMEM supplemented with EGF (10ng/ml), bFGF (2ng/ml),
insulin (5ng/ml), holo-transferrin (5 g/ml), sodium selenite (5ng/ml),
dexamethasone
(390ng/ml), vitamin C (50 ghnl), vitamin H (D-biotin; 250ng/ml), Vitamin E
(Trolox; 25 g/ml) (Sigma-Aldrich), albumax-1 (0.5mg/ml) (Invitrogen), fetuin
(500gg/ml) (Clonetics/BioWhittaker), penicillin (100U/ml) and streptomycin
(100 g/ml) (Invitrogen). After allowing 24 hours for adherence, the stabilised
peptide (10ng/ml) with and without rIGF-I, (10ng/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
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replaced every 2-3 days. The cultures were sampled at various time-points for
immunocytochemical analyses.
6.4 Ina mu n o cyto ch ena istry
At the appropriate time-points, cells were fixed with methanol for 10 min (-20
C),
followed by detergent permeabilization with 0.5% Triton-X100 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
p1us10% FCS, 0.025% sodium azide, 0.1M 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. 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.
6.5 Creatine phosphokitaase (CPK) assay
This assay was performed using previously published protocols [Auluck et al.,
2005].
Measurement of CPK allows for the quantitative comparison of myogenesis [Goto
et
al., 1999], as it is a marker of myotube formation. The enzyme CPK catalyzes
the
reversible phosphorylation of adenosine-5-diphosphate (ADP) to form adenosine-
5-
triphosphate (ATP) and free creatine. The reaction may be followed in either
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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
10x104
cells/well in 0.2% gelatin coated 96 well plates. Cells were cultured
unti170/80%
confluent in sGM then the medium changed to differentiation medium (DM; DMEM,
2% FCS, penicillin (100U/ml) and streptomycin (100 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). After 48 hours, cells were washed twice with ice
cold
PBS and then stored frozen in 0.5 mM glycine buffer (pH 6.75) at -70 C. Fixed
cells
were lysed by rapid thawing and CPK assay kit used according to manufacturers
instructions (Sigma-Aldrich). The protein concentration of each sample was
determined against an albumin standard curve using the Pierce Micro BCA Kit
(PerBio Science, UK Ltd., Northumberland, UK).
6.6 Statistical analysis
1-way ANOVA test was applied using StatView 4.51 (SAS Institute Inc., Cherwell
Scientific Publishing Ltd, Oxford, UK) followed by the Fisher's PLSD post hoc
test.
p<0.05 was considered significant. Data were pooled for all runs (minimum of
6) for
the 4 types of experiments for each condition including the two types of
healthy
muscle and presented as mean s.d.
6.7 The proportion of tnyogenic precuf-sors in hunaan inuscle primary cultures
The percentage of myogenic (desmin positive) cells was determined from all of
the
muscles tested (See Table below). Normal (non-diseased) muscle contained a
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significant proportion of desmin positive cells whereas diseased muscle
contained a
much lower proportion of myogenic cells.
Table - Human primary muscle cultures derived from different nzuscle sources
that contain differing proportions of nzyogenic (desmin positive) cells before
addition ofpeptides
Muscle Type Desmin positive cells as percentage
of total cells in primary culture.
Normal (non-diseased) 49.8 2.4%
Craniofacial
Normal (non-diseased) Limb 38.4L2.5%
CMD Limb 10.4~:1.7%
ALS Limb 4.8 1.1 %
FSHD Limb 11.7 1.3%
6.8 Effect on normal (non-diseased) human primary muscle progenitor cells
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%; p<0.0001). IGF-I also induced a
moderate increase (from 49.8 2.4% to 58.4 4.2%; p<0.0001). 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 59.5f4.2%;
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-L-2.5% to 47.1 3.5%;
p<0.0001) but IGF-I completely abrogated the response to the stabilised
peptide
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when the two peptides were added in combination (from 57.9 3.2% to 38.8 0.6%;
p<0.0001).
6.9 Effect on disease-state human primary muscle derived cell proliferation
Following the observation that 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. In primary cultures derived
from
congenital muscular dystrophy (CMD), the stabilised peptide significantly
increased
muscle progenitor cell proliferation (from 10.4 1.7% to 17.5 1.6%; p<0.0001),
whilst IGF-I had a small effect (10.4 1.7% to 13.2 1.7%; p=0.005). When
combining both peptides the inhibiting effect was again as observed as for
normal
inuscle, with effect of the stabilised peptide being reduced to control levels
(from
17.5 1.6% to 13.1+1.2%; p=0.0001). 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 increased the numbers of
desmin
expressing cells markedly in these disorders (ALS from 4.8 1.1% to 7.2 0.8%;
p=0.0002, FSHD from 11.7 1.3% to 20.412.1%; p<0.0001). As was the case for
normal muscle, IGF-I again had a negligible effect (ALS from 4.8 1.1 % to
4.7 1.4%; p=0.7719, FSHD from 11.7 1.3% to 14.1 1.6%; p=0.0107)). When both
isoforms were used together, MGF-induced desmin expressing increase was again
inhibited (ALS from 7.2 0.8% to 5.3 1.0%; p=0.0024, FSHD from 20.412.1% to
14.5+1.4%; p<0.0001).
6.10 Increased progenitor cell pf=olifet=ation by MGF E domain in relation to
the
IGF-I receptor
In normal muscle, the increase in proliferation induced by the stabilised
peptide was
not inhibited by the presence of an anti-IGFIR antibody (68.8 3.9% in MGF
treated
and 71.1 6.2% in MGF plus Ab-I treated cells; p=0.2472). The same effect was
also
observed for both CMD and ALS muscle (17.5 1.6% vs. 16.7--I= 1.8% p=0.4589 for
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CMD; 7.2 0.8% vs. 6.5 0.8%,p=0.2933 for ALS). This indicates that the action
of
the MGF E domain does not involve the IGF-I receptor.
6.11 Effects of MGF E domain on preventitag terminal differentiation.
In the CPK assays of 6.4 above, the stabilised peptide did not facilitate
primary
myoblast differentiation and myotube formation. In contrast, 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. Indeed, in the presence of l Ong/ml IGF-I, 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.
6.12 Conclusions
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.
It has been proposed that muscle wasting in neurological conditions and ageing
is
due to a loss of satellite cells. We have demonstrated that the ratio of
progenitor
(desmin positive) cells to total myoblasts from the patients with CMD, FSHD
and
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ALS is low compared to the ratio of myoblasts from healthy individuals. Thus
it is
debatable whether muscles degenerate because of lack of satellite cells or
because of
inability to express some factor for satellite cell activation. We have
previously
demonstrated that elderly people are unable to express MGF at the levels
required to
maintain muscle [Hameed et al., 2004], with similar findings for FSHD and ALS
patients (unpublished findings).
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 Bari et al found that when mesenchyrnal stem
cells
were introduced into dystrophic muscles of mdx mouse, the sarcolemmal
expression
of dystrophin and also MGF expression was restored [De Bari 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
It has been known for some time that IGF-I is a neurotrophic factor, and
possesses
potential clinical applications for neurodegenerative disorders, particularly
ALS.
Using animal models, systemic delivery of human recombinant IGF-I (mature IGF-
I)
has been used in animal models and to treat ALS patients. Most recently, it
was
reported that exercise, when combined with IGF-I gene therapy by AAV2 vector,
has
some synergistic effects in treatment of an animal model of ALS [Kaspar et
al.,
2005].
However, 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. This supports the use of
peptides of
the invention as therapeutic agents for muscle degeneration in disorders such
as
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CMD, FSHD and ALS in which there is an apparent impairment in expressing the
MGF splice variant. There is also the potential for using peptides of the
invention to
multiply the muscle satellite cells in culture for cell therapy purposes.
7. Cell Proliferation Assays with 8 Amino Acid Peptides
7.1 DMGF and CMGFpeptides
The 8 amino acid peptides described in 1.3.1 above and referred to in Figure
8A as
DMGF and CMGF were tested for the ability to induce proliferation of C2C12
muscle cells at a density of 2000 cells per well in a medium containing DMEM
(1000mg/L glucose), BSA (100ug/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.
Both DMGF and CMGF induced cell proliferation. The results are shown in Figure
8A in terms of fluorescence in the Alamar Blue Assay. All values for DMGF and
CMGF, and those for IGF-I alone, were statistically different to the control
value for
the medium only. Increasing levels of proliferation were observed with
increasing
concentration of DMGF/CMGF.
7.2 Peptides A2, A4, A6 a:ad A8
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 C2C
12
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
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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).
8. Cell Proliferation Assays with Human primary cells (HSMM)
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 1 of SkGM2 medium supplemented
with hEGF, L-Glut, dexametllasone, antibiotics and 10% FCS. The cultivation
medium was then removed and the cells were washed twice in serum free medium.
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, 10A and 11A), along with 0.1, 10 and 100 ng/ml
IGF-I
alone (See results in Figures 9A, 10A and 11 A). 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, 10B and 11B). 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.
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IGF-I alone had no significant effect on the proliferation of HSMM at any dose
(see
Figures 9-11). After 48 hours, the A5 peptide had a significant effect (P <
0.1) on
the proliferation of HSMM when used in isolation at doses of l Ong/ml and
below
(Figures 9A 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, 10B and 11 B). As the cells are
comparatively slow growing, it is recommended to increase incubation period
with
peptide to 72 hours. Secondly, the signal would be enhanced by increasing the
BrdU
exposure time.
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