Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Patent
1991-04-22
1993-12-28
Moezie, F. T.
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
514 3, 514 4, 514 8, 530399, 530324, 530303, 4351723, 4351722, 435 694, A61K 3736, C12P 2100, C12P 2102, C07K 740
Patent
active
052739660
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to glycosylated insulin-like growth factor-1.
BACKGROUND OF THE INVENTION
Insuliln-like growth factor (IGF-1) is a 70 amino acid single polypeptide chain factor which displays relatively-high homology with proinsulin. IGF-1 is known to have a mediating effect on the action of growth hormone and also to display insulin-like properties. When produced in nature by the human body, IGF-1 is unglycosylated.
IGF-1 has potential uses as a pharmaceutical agent both in the treatment of pituitary dwarfism and also diabetes. In the latter case it would be useful either as a replacement for insulin or as an adjunct to insulin. Although insulin is the traditional treatment for diabetes, type-2 diabetics have developed a resistance to insulin which means that even if very high doses of insulin are administered, the patients can still suffer from hyperglycemia. Further, excessive doses of insulin can lead to undesirable side effects such as kidney complaints, obesity, and a disturbed water balance.
Although unglycosylated IGF-1 could be used as a replacement or adjunct for insulin in an attempt to overcome some of these problems, a high proportion of unglycosylated IGF-1 tends to be sequestrated by specific binding proteins circulating in the bloodstream. Therefore, relatively high doses of the IGF-1 need to be administered for the desired pharmaceutical effect to be attained.
We have now discovered that expression of IGF-1 in yeast cells results in the production, along with the normal unglycosylated form, of an O-glycosylated analog of IGF-1, for example, an analog which carries two mannose residues on the Thr .sub.29 amino acid of the polypeptide chain. Tests have shown that O-glycosylated IGF-1 has a reduced affinity for the binding proteins and that a desired reduction in blood sugar level can be achieved by a reduced dose of O-glycosylated IGF-1 as compared with the normal unglycosylated protein. This observed affinity for the binding protein will have a profound effect on the profile and dose dependency. The other effects, although not so pronounced are of importance in the total clinical effect.
The expression "O-glycosylated IGF-1" embraces O-glycosylated molecules which comprise fragments of the whole IGF-1 polypeptide sequence, provided that those fragments display qualitatively the growth-hormone mediating effect and/or insulin-like properties of IGF-1.
SUMMARY OF THE INVENTION
According to one aspect of the invention, we therefore provide O-glycosylated IGF-1 essentially free from unglycosylated IGF-1.
According to another aspect of the invention we provide O-glycosylated IGF-1 in which the glycosylation is at the Thr .sub.29 amino acid of the polypeptide chain.
According to yet another aspect of the invention, we provide O-glycosylated IGF-1 in which the glycosylation comprises two or more mannose residues attached to the same threonine residue of the IGF-1 polypeptide chain.
According to a further aspect of the invention, we provide an O-glycosylated analog of IGF-1 in which two or more mannose residues are attached to the Thr .sub.29 amino acid of the polypeptide chain.
According to a still further aspect of the invention, we provide a method of obtaining O-glycosylated IGF-1 by the expression of IGF-1 in yeast cells, and isolating O-glycosylated IGF-1 from the medium. Preferably, the yeast is Saccharomyces cerevisiae.
The gene for IGF-1 is preferably linked to DNA coding for a secretory signal sequence, for example that of the gene for .alpha.-mating factor, so that mature authentic IGF-1 which has been O-glycosylated is secreted from the cytoplasm of the yeast cells. The .alpha.-mating factor is a 13-amino acid peptide secreted by yeast cells of the .alpha.-mating type to promote efficient conjugation of a cells to form a .alpha.-diploid cells. Sequence data of the .alpha.-mating factor structural gene shows that the .alpha.-mating factor is initially synthesized as a 165 amino acid precursor containing an 85 amino acid leader peptide and four c
REFERENCES:
Ratner, Biotechnology, vol. 7, No. 11, Nature Publishing, 1129-1133, Nov. 1989.
Hard et al., FEBS Letters, 248, No. 1,2, 111-114, May 1989.
Sknottner et al., Acta Palediatrica Scandinavia (Suppl.), 325, 107-111,1986.
Fryklund Linda
Gellerfors Par
Sknottner-Lundin Anna
Kabi Pharmacia AB
Moezie F. T.
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