Peptide analogs of insulin-like growth factor II

The invention provides peptide analogues of insulin-like growth factor-1 (IGF-1) or factor-2 (IGF-2). The peptide analogues of IGF-1 have at least the glutamic acid residue at position 3 replaced by another amino acid. The peptide analogues of IGF-2 are replaced at position 5 or 6 with another amino acid.

This invention relates to growth factors, related compounds and their use. 
Insulin-like growth factor-1, a somatomedin, is a small protein that has 
been shown to stimulate growth of a wide range of cells in culture. Human 
IGF-1 (hIGF-1) has been purified to homogeneity from human serum and its 
complete amino acid sequence established. The serum mediator of growth 
hormone action, somatomedin C, has been shown to have an identical 
sequence to hIGF-1 so that these two are now considered as being 
synonymous. The amino acid sequence established for hIGF-1 beginning with 
the N-terminal glycine is: 
##STR1## 
Bovine IGF-1 and porcine IGF-1 have identical sequences. 
Using the conventional numbering system of the N-terminal glycine being 
residue #1 and the C-terminal alanine residue #70, ovine and chicken IGF-1 
differ from human IGF-1 only as follows: 
##STR2## 
IGF-1 levels in serum correlate positively with growth rates in boys during 
adolescence and negatively with the degree of growth hormone deficiency in 
growth-retarded subjects, and to both growth rate and eventual size in 
mice transfected with growth hormone genes. These findings, indirectly 
linking IGF-1 concentrations with growth rates and supported by more 
direct evidence that administration of IGF-1 leads to restoration of 
growth rates in hypopituitary (growth hormone deficient) rats or mice and 
to increased growth rates in normal rats, have lead to the interpretation 
that IGF-1 might usefully be applied: (1) in humans to treat growth 
hormone deficiencies; (2) in farm animals to increase growth rates, 
increase the relative proportion of muscle and enhance food conversion 
efficiency. It is further suggested that administration of IGF-1; (3) may 
suppress the loss of body protein in severe human catabolic states such as 
following burns, infection or other trauma; (4) may improve wound healing 
in human subjects as well as in animals. IGF-1 can also be used to (5) 
support the growth of calls in culture. 
The result of the above inferences is that there is a commercial demand for 
IGF-1 for use in animal trials, clinical investigations and for cell 
culture. However, only milligram amounts of hIGF-1, for example, are 
available by purification of tonnes of human serum protein and yields from 
recombinant DNA methods remain low. 
Insulin-like growth factor-2 (IGF-2) like IGF-1, is a small protein that 
has been shown to stimulate growth of cells in culture. In most cases, 
these biological effects occur following interaction of IGF-2 with the 
same cellular receptor as is involved in IGF-1 actions. The amino acid 
sequence established for human IGF-2 (hIGF-2) beginning with the 
N-terminal alanine is shown below. Upper case letters have been used to 
indicate the amino acids equivalent to the N-terminal 5 amino acids of 
hIGF-1; 
##STR3## 
Using the conventional numbering system of the N-terminal alanine being 
residue #1 and the C-terminal glutamic acid being residue #67, bovine, 
ovine, porcine and chicken IGF-2 differ from human IGF-2 only as follows: 
##STR4## 
It has been disclosed (see PCT/AU87/00246 to applicants) that compounds 
corresponding to IGF-1 but lacking one to five, preferably three amino 
acid residues from the N-terminal of the molecule can exhibit a 
substantial increase in biological potency compared with the more complete 
compounds. 
For example, the compound destripeptide bIGF-1 but lacking the amino acid 
residues gly, pro and glu from the N-terminal, is effective in inhibiting 
protein breakdown and stimulating both protein synthesis and DNA synthesis 
in cellular systems at concentrations between 4 and 50 fold lower than 
required for entire bIGF-1. 
For IGF-1 peptides having N-terminal amino acid sequences in common with 
that of human/bovine/porcine IGF-1, the elimination of between 1 and 5 
amino acid residues from the N-terminal also results in enhanced 
biological potencies. The said N-terminal amino acid sequence is also a 
feature of the IGF-1 of rat, ovine, and chick species. 
However, a useful property of the full IGF-1 peptide but not shared by the 
IGF-1 peptides having 1 to 5 N-terminal amino acids eliminated is that 
production by recombinant DNA methods that are part of the prior art are 
facilitated by the existence of N-terminal glycine. This facilitation 
occurs because an asparagine residue can be engineered upstream from the 
glycine and the asparagine/glycine bond cleaved selectively by mild 
hydroxylamine treatment following expression of the engineered gene. 
Accordingly it is an object of the present invention to overcome, or at 
least alleviate, one or more of the difficulties related to the prior art. 
Accordingly in a first aspect of the present invention there is provided a 
peptide analogue of insulin-like growth factor-1 (IGF-1) or factor-2 
(IGF-2) where at least the glutamic acid residue is replaced at position 3 
from the N-terminal of IGF-1 by amino acid residue selected from the group 
comprising: 
______________________________________ 
Alanine (Ala) 
Asparagine (Asn) 
Phenylalanine (Phe) 
Isoleucine (Ile) 
Methionine (Met) 
Valine (Val) 
Serine (Ser) 
Proline (Pro) 
Threonine (Thr) 
Tyrosine (Tyr) 
Cysteine (Cys) 
Tryptophan (Trp) 
______________________________________ 
or at position 5 or 6 from the N-terminal of IGF-2 by an amino acid reside 
selected from the group comprising: 
______________________________________ 
Alanine (Ala) 
Asparagine (Asn) 
Phenylalanine (Phe) 
Isoleucine (Ile) 
Methionine (Met) 
Valine (Val) 
Serine (Ser) 
Proline (Pro) 
Threonine (Thr) 
Tyrosine (Tyr) 
Cysteine (Cys) 
Tryptophan (Trp) 
Leucine (Leu) 
______________________________________ 
or is absent at position 5 or 6 from the N-terminal of IGF-2 and at least 
one of the Ala.sup.1, Tyr.sup.2, Arg.sup.3, Pro.sup.4 or Ser.sup.5 
residues is also absent. 
It will be understood that in respect of chicken IGF-2 the N-terminal 
Ala-residue is absent so that the glutamic acid residue is at position 5 
from the N-terminal. 
Preferably the peptide analogue is a human, bovine, ovine, porcine or 
chicken insulin-like growth factor-1 or factor-2 analogue. 
The peptide analogue according to the present invention may be in a 
biologically pure form. 
The peptides lacking the glutamic acid residue or those having the glutamic 
acid replaced by any one of the following groups: 
______________________________________ 
Alanine (Ala) 
Asparagine (Asn) 
Phenylalanine (Phe) 
Isoleucine (Ile) 
Methionine (Met) 
Valine (Val) 
Serine (Ser) 
Proline (Pro) 
Threonine (Thr) 
Tyrosine (Tyr) 
Cysteine (Cys) 
Tryptophan (Trp) 
Leucine (Leu) 
______________________________________ 
bind poorly to the binding proteins produced by many cell types. Should a 
binding protein be present those other IGF-1 peptides that do bind have 
reduced potencies. 
The peptide analogues according to the present invention may form suitable 
replacements for IGF-1 and -2 in the following applications: (1) in humans 
to treat growth hormone deficiencies; (2) in farm animals to increae 
growth rates, increase the relative proportion of muscle or improve food 
conversion efficiency; (3) in humans to suppress the loss of body protein 
in severe catabolic states such as following burns, infection or other 
trauma; (4) in humans and animals to improve wound healing, and (5) to 
support the growth of cells in culture: 
More specifically, the present invention provides a pharmaceutical or 
veterinary composition that includes: 
(a) an effective amount of a peptide analogue of insulin-like growth 
factor-1 (IGF-1) or factor-2 (IGF-2) wherein at least the glutamic acid 
residue is replaced at position 3 from the N-terminal of IGF-1 by an amino 
acid selected from the group comprising: 
______________________________________ 
Alanine (Ala) 
Asparagine (Asn) 
Phenylalanine (Phe) 
Isoleucine (Ile) 
Methionine (Met) 
Valine (Val) 
Serine (Ser) 
Proline (Pro) 
Threonine (Thr) 
Tyrosine (Tyr) 
Cysteine (Cys) 
Tryptophan (Trp) 
______________________________________ 
or at position 5 or 6 from the N-terminal of IGF-2 by an amino acid 
selected from the group comprising: 
______________________________________ 
Alanine (Ala) 
Asparagine (Asn) 
Phenylalanine (Phe) 
Isoleucine (Ile) 
Methionine (Met) 
Valine (Val) 
Serine (Ser) 
Proline (Pro) 
Threonine (Thr) 
Tyrosine (Tyr) 
Cysteine (Cys) 
Tryptophan (Trp) 
Leucine (Leu) 
______________________________________ 
or is absent at position 5 or 6 from the N-terminal of IGF-2 and at least 
one of the Ala.sup.1, Tyr.sup.2, Arg.sup.3, Pro.sup.4 or Ser.sup.5 
residues is also absent respectively and 
(b) a pharmaceutically or veterinarily acceptable diluent, carrier or 
excipient therefor. 
The peptide analogue may be present in amounts sufficient to provide a dose 
rate of approximately 0.01 to 10, preferably 0.1 to 1 milligrams/kg body 
weight/day. The peptide analogue may be present in amounts of from 
approximately 0.02 to 2000 milligrams. For cell culture applications the 
peptide analogue may be present in concentrations from approximately 0.1 
to 100 milligrams per litre. 
In a further preferred aspect of the present invention there is provided a 
method for the treatment of protein accumulation deficiencies or protein 
loss in human subjects, which method includes 
administering to a patient to be treated an effective amount of a peptide 
analogue of insulin-like growth factor-1 (IGF-1) or factor-2 (IGF-2) as 
described above. 
The peptide analogues may be administered to human subjects as a treatment 
for disorders associated with tissue wasting including, but not limited 
to, burns, skeletal trauma, infection, cancer, cystic fibrosis, Duchenne 
muscular dystrophy, Becker dystrophy, autosomal recessive dystrophy, 
polymyositis as well as other myopathies and acquired immune deficiency 
syndrome (AIDS). The peptide analogues may be administered parenterally or 
by injection. 
In an alternative aspect there is provided a method for the treatment of 
wounds in animals including humans, which method includes 
administering to a patient to be treated an effective amount of a peptide 
analogue of mammalian insulin-like growth factor-1 (IGF-1) or factor-2 
(IGF-2) as described above For the treatment of wounds in human subjects 
or in animals the peptide analogue may be applied externally to the wound 
or it may be administered by injection. 
In a still further aspect there is provided a method for the improvement of 
growth performance in animals which method includes 
administering to an animal to be treated an effective amount of a peptide 
analogue of insulin-like growth factor-1 (IGF-1) or factor-2 (IGF-2) as 
described above. 
An implant, preferably a slow release pellet, is the preferred method of 
administration to farm animals as applied in conventional practice. 
Otherwise the peptide analogue may be administered by injection. 
The peptide analogues of the present invention may be administered to 
premature or other human infants to promote growth, improve nitrogen 
status and to treat catabolic disorders. The peptides may be administered 
as outline above for tissue wasting conditions. 
Accordingly in a still further aspect of the present invention, there is 
provided a method for the stimulation of cells in culture which method 
includes providing 
a culture medium, and 
an effective amount of a peptide analogue as described above; and 
adding the peptide analogue to the culture medium. 
Any standard culture medium may be used in accordance with this aspect of 
the present invention. For example the culture medium may include Eagle's 
Minimal Essential Medium. 
A preferred method for the preparation of a peptide analogue of IGF-1 
wherein at least the glutamic acid residue is substituted at position 3 
from the N-terminal includes 
providing a source of amino acids, and 
coupling the amino acids in sequence to form a peptide analogue. 
The peptide analogues may be produced by appropriate modifications to 
methods existing for the production of the full IGF-1 peptide. These 
modifications would be familiar to those familiar with the art. 
Specifically, the peptides related to human/bovine/porcine IGF-1 may be 
synthesized chemically using procedures developed for human IGF-1 (for 
example: Li et al., Proc. Natl. Acad. Sci, USA 80: 2216-2220, 1983) but 
with the final cycles of amino acid ligation modified. Synthetic ovine or 
chicken IGF-1 as well as related IGF-1 and IGF-2 peptides may be produced 
by techniques similar to those used for human IGF-1 using amino acid 
sequence information for these peptides. 
The peptides may also be produced following transformation of susceptible 
bacterial, yeast or tissue culture cell hosts with recombinant plasmids 
that include DNA sequences capable of directing the expression of the 
peptides. The DNA sequence may be synthetic, chromosomal, cDNA or 
combination thereof. The inserted coding sequences may incorporate 
deletion or omissions to account for differences between the sequence of 
peptide analogues and the full IGF-1 peptide.

An example to illustrate one method of synthesis of these compounds will 
now be provided. It should be understood however, that the following 
description is illustrative only and should not be taken in any way as a 
restriction on the generality of the description foregoing. 
EXAMPLE 1 
Synthesis of IGF-1 peptides 
Chemical synthesis of human/bovine/porcine IGF-1 peptides with between 1 
and 4 amino acids from the normal N-terminal modified may be effected by 
the following procedure. 
The starting material may be Boc-ala-phenylacetamido methyl resin. Coupling 
may be effected in an Applied Biosystems Inc model 430A peptide 
synthesiser with preformed symmetric anhydrides of the Bos-aminoacids in 
dichloromethane except for the derivatives of arginine, asparagine and 
glutamine which can be coupled in dimethyl formamide (DMF). In all cases a 
second coupling may be performed in DMF. Samples of resin can be removed 
after each cycle of synthesis and subjected to quantitive ninhydrin 
analysis (Sarin, V. K., Kent, S. B. H., Tam, J. P., Merifield, R. B.; 
Anal. Biochem. 17, 147-157 (1981)). Preview sequence analysis of the 
side-chain protected, resin-bound peptide may also be carried out. 
Portions of resin containing side-chain protected peptides corresponding to 
the complete sequence of hIGF-1 but with 4 to 0 amino acids not coupled at 
the N-terminal will be removed. Other portions with between 4 and 3 amino 
acids not coupled at the N-terminal may have amino acid residues coupled 
as required for specific analogues. Peptides will be cleaved and 
deprotected according to Applied Biosystems Inc procedures and recovered 
as ether precipitates. 
Peptides may be redissolved in 6M guanidine hydrochloride pH 8.5 with Tris 
containing 10 mM dithioerythritol and desalted by reverse phase HPLC and 
dried. Oxidation of the reduced peptide will be effected by dissolving in 
8M urea, 0.1M Tris (pH 8.0 with HCl) containing 13 mM oxidized 
gluthathione and incubated at 25.degree. for 15 hours. The sample can be 
purified by reverse phase HPLC using a gradient of acetonitrile in 0.1% 
trifluoroacetic acid to elute the peptides and separate the biologically 
active form of the peptide from those forms lacking the correct disulphide 
bonds and hence lacking full biological activity. The samples can be dried 
prior to resuspension. 
Biological activity may be confirmed by the ability of the peptide to 
stimulate protein synthesis in L6 myoblasts. 
It will be appreciated that various modifications and/or alterations may be 
introduced into the constructions and arrangements of parts previously 
described without departing from the spirit or ambit of the present 
invention.