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Conjugates of insulin-like growth factor-1 and poly(ethylene glycol)

a technology of insulinlike growth factor and conjugate, which is applied in the field of conjugates of insulinlike growth factor and poly(ethylene glycol) can solve the problems of unsuitability for commercial use of many nhs-pegylated proteins and complex regulation of igf-i function, and achieve the effect of superior properties in regard to therapeutic applicability

Inactive Publication Date: 2006-07-13
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is about a new type of human growth hormone (IGF-I) that can be modified to create a conjugate with poly(ethylene glycol) (PEG) for therapeutic use. The modification involves changing certain amino acids in the wild-type IGF-I sequence to create a new IGF-I variant that has improved properties for therapeutic application. Specifically, the invention describes a new IGF-I variant that has lysine at positions 27, 37, and 68, and an amino acid at position 25 that is not a lysine. This new variant can be combined with PEG to create a conjugate that has superior properties in terms of therapeutic applicability. The invention also includes methods for preparing this new type of IGF-I variant and a composition containing a mixture of different PEGylated IGF-I variants.

Problems solved by technology

The regulation of IGF-I function is quite complex.
However, a major limitation of this approach is that proteins typically contain a considerable amount of lysine residues and therefore the poly(ethylene glycol) groups are attached to the protein in a non-specific manner at all of the free ε-amino groups, resulting in a heterologous product mixture of random PEGylated proteins.
Therefore, many NHS-PEGylated proteins are unsuitable for commercial use because of low specific activity.

Method used

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  • Conjugates of insulin-like growth factor-1 and poly(ethylene glycol)
  • Conjugates of insulin-like growth factor-1 and poly(ethylene glycol)
  • Conjugates of insulin-like growth factor-1 and poly(ethylene glycol)

Examples

Experimental program
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Effect test

example 1

Chromatographical Separation of monoPEG-IGF-I Positional Isomers

[0088] IGF-I contains 4 amino groups as potential PEGylation sites and 4 possible monoPEGylated IGF-I (monoPEG-IGF-I) isomers were expected. Further derivates were expected to be oligoPEGylated depending on the reaction conditions. A strong-cation high pressure liquid chromatography method (IEC-HPLC) was developed for the separation of PEG-IGF-I or -Des(1-3)-IGF-I isomers based on their local charge differences. A preparative elution profile of 5 mg PEG-IGF-I is shown in FIG. 1. The result of this method was a separation into 5 major peaks, 3 peaks with baseline separation and 2 with partial separation. The decrease of the baseline absorption towards the end of the chromatogram suggests no additional monoPEGylated IGF-I species eluting at higher retention time. An additional peak appears between peak 3 and 4 resulting from the switch to the modified buffer B with different pH. Analytical IEC-HPLC was used to estimate t...

example 2

SDS-PAGE Analysis of monoPEG-IGF-I Isomers

[0089] SDS-PAGE was performed both under non-reducing and reducing conditions to evaluate potential unwanted cross-linking of different IGF-I molecules through intermolecular disulfide bridges. Both conditions yielded similar results indicating that no significant amount of the protein was abnormally cross-linked (FIG. 2). The SDS-PAGE analysis showed that peak 0 had an apparent molecular weight of >100 kDa whereas the major detected bands were peaks 1-3 at ˜70 kDa; additionally, peak 4 was detected at ˜7 kDa, a size expected for unPEGylated IGF-I (FIG. 2). From this running profile and the retention times obtained in the HPLC we concluded that peak 0 consists most probably of di- and oligoPEG-IGF-I. In contrast, peaks 1-3 were designated as 3 different monoPEG-IGF-I isomers. There was a discrepancy between the expected molecular weight for monoPEG-IGF-I (51.6 kDa) and the apparent size of ˜70 kDa; however, the observed higher apparent mole...

example 3

Analysis of monoPEG-IGF-I Isomers

[0092] Cleavage of rhIGF-I with Asp-N delivered 6 independent peptide fragments that were separated by HPLC between 30 and 45 minutes retention time (FIG. 3A,upper panel). After cleavage of the purified peaks 1 to 3 (FIG. 3A, lower panels), different distributions of the 6 fragments and the occurrence of an additional fragment (fragment 7) was observed eluting at 70 minutes retention time. For peak 1, specifically peptide fragment 4 was diminished with a concomitant increase of fragment 7. For peak 2, we observed a clear decrease in fragment 3 and a slight decrease in fragment 5 and the appearance of a major and a minor PEG fragment (7 and 7′). Similarly, HPLC analysis of peak 3 yielded a clear decrease in fragments 3 and 5 with the concomitant occurrence of fragments 7 and 7′. FIG. 11B shows the peptide sequences of the respective fragments as obtained by Asp-N cleavage of rhlGF-I. Using Edman N-terminal peptide degradation we analyzed the peptide...

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Abstract

A conjugate consisting of an insulin-like growth factor-1 (IGF-I) variant and one or two poly(ethylene glycol) group(s), characterized in that said IGF-I variant has an amino acid alteration at up to three amino acid positions 27, 37, 65, 68 of the wild-type IGF-I amino acid sequence so that one or two of said amino acids is / are lysine and amino acid 27 is a polar amino acid but not lysine, is conjugated via the primary amino group(s) of said lysine(s) and said poly(ethylene glycol) group(s) have an overall molecular weight of from 20 to 100 kDa is disclosed. This conjugate is useful for the treatment of neurodegenerative disorders like Alzheimer's Disease.

Description

PRIORITY TO RELATED APPLICATIONS [0001] This application claims the benefit of Eropean Application No. 04030415.6, filed Dec. 22, 2004, which is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION [0002] This invention relates to conjugates of insulin-like growth factor-I(IGF-I) with poly(ethylene glycol) (PEG), pharmaceutical compositions containing such conjugates, and methods for the production and methods of use of such conjugates. BACKGROUND OF THE INVENTION [0003] Alzheimer's disease (AD) is an increasingly prevalent form of neurodegeneration that accounts for approximately 50%-60% of the overall cases of dementia among people over 65 years of age. Death of pyramidal neurons and loss of neuronal synapses in brains regions associated with higher mental functions results in the typical symptoms, characterized by gross and progressive impairment of cognitive function. Neuropathologically, the major hallmarks of AD are the presence of two characteristic lesion...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/65A61K38/30
CPCC07K14/475A61K47/48215A61K47/60A61P25/28A61P43/00A61K47/50A61K38/30C07K14/62C07K19/00C07K14/65
Inventor AMREIN, BEATFOSER, STEFANLANG, KURTMETZGER, FRIEDRICHREGULA, JOERGSCHAUBMAR, ANDREASHESSE, FRIEDERIKEKUENKELE, KLAUS-PETERLANZENDOERFER, MARTIN
Owner F HOFFMANN LA ROCHE & CO AG
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