Site-specific preparation of polyethylene glycol-grf conjugates

Details Site-specific preparation of polyethylene glycol-grf conjugates Site-specific preparation of polyethylene glycol-grf conjugates

2000-06-05

2003-03-04

Cochrane Carlson, Karen (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S012200, C514S002600, C530S399000, C530S345000, C530S344000, C530S338000, C530S334000, C530S324000, C530S406000, C530S410000, C424S198100

Reexamination Certificate

active

06528485

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a method for the site-specific preparation of hGRF-PEG conjugates containing one or more than one PEG units (per hGRF) covalently bound to Lys
12
and/or Lys
21
and/or N
&agr;
, characterized in that the conjugation reaction between the hGRF peptide and activated PEG is carried out in solution and the desired hGRF-PEG conjugate is purified by chromatographic methods.
The conjugates prepared by this method, as well as their use in the treatment, prevention or diagnosis of growth-hormone related disorders, are also an object of the present invention.
BACKGROUND OF THE INVENTION
In the early 1980's several groups isolated and characterized growth hormone releasing factor (GRF).
GRF (also called Somatorelin) is a peptide secreted by the hypothalamus which acts on its receptor and can promote the release of growth hormone (GH) from the anterior pituitary. It exists as 44-, 40-, or 37-amino acid peptide. the 44-amino acids form may be converted physiologically into shorter forms. All three forms are reported to be active, the activity residing mainly in the first 29 amino acid residues. A synthetic peptide corresponding to the 1-29 amino acid sequence of human GRF [hGRF(1-29)], also called Sermorelin, has been prepared by recombinant DNA technology as described in European Patent EP 105 759.
Sermorelin has been used in the form of acetate for the diagnosis and treatment of growth hormone deficiency.
GRF has indeed a therapeutic value for the treatment of certain growth-hormone related disorders. The use of GRF to stimulate the release of GH is a physiological method in promoting long, bone growth or protein anabolism.
One problem associated with the use of GRF relates to its short biological half-life (about 12 to 30 minutes). The hGRF(1-29)-NH
2
is subject to enzymatic degradation and is rapidly degraded in the plasma via dipeptidylpeptidase IV (DPP-IV) cleavage between residues Ala
2
and Asp
3
.
It is therefore advantageous to develop biologically stable, long-acting GRF analogues using specific chemical modification of GRF, in order to prevent or slow down enzymatic degradation.
Polyethylene glycol (PEG) is a hydrophilic, biocompatible and non-toxic polymer of general formula H(OCH
2
CH
2
)
n
OH, wherein n≧4. Its molecular weight could vary from 200 to 20,000 daltons.
It has been demonstrated that the chemical conjugation of PEG in its mono-methoxylated form to proteins and/or peptides significantly increases their duration of biological action. Like carbohydrate moieties in a glycoprotein, PEG provides a protective coating and increases the size of the molecule, thus reducing its metabolic degradation and its renal clearance rate
PEG conjugation is an already established methodology for peptide and protein delivery pioneered by the fundamental studies of Davis and Abuchowski (Abuchowski et al., 1977a and 1977b). PEG conjugation to peptides or proteins generally resulted in non-specific chemical attachment of PEG to more than one amino acid residue. One of the key issues with this technology is therefore finding appropriate chemical methods to covalently conjugate PEG molecule(s) to specific amino acid residues.
For example, the trichlorotriazine-activated PEG, which was found to be toxic and reacted in a non-specific way, was later on replaced by various PEG reagents with chemical linkers that could react specifically to amino groups (Benchamp et al., 1983; Veronese et al., 1985; Zalipsky et al., 1983; Zalipski et al., 1990; and Delgado et al., 1990), to sulphydryl groups (Sartore et al., 1991; and Morpurgo et al., 1996) or to guanidino residues (Pande et al., 1980).
Various PEG-protein conjugates were found to be protected from proteolysis and/or to have a reduced immunogenicity (Monfardini et al., 1995; and Yamsuki et al., 1988).
Another technical difficulty in protein pegylation arises from the fact that PEG-protein conjugates usually have various number of PEG molecules attached and result in a mixture of conjugates with different PEG:protein stoichiometries. Site-specific pegylation remains a chemical challenges The conjugation of PEG to GH represents a typical example of such problem (Clark et al., 1996). It was demonstrated that Lys-residues of GH were pegylated at random positions.
To avoid or reduce the loss of enzyme activity, the active site could be protected in advance, thus allowing enzyme pegylation to occur at non-active site(s) (Caliceti et al., 1993).
Another approach was recently proposed for the site-specific conjugation of PEG to low molecular weight peptides, such as GRF. which was prepared by solid-phase peptide synthesis. In these conjugates a pegylated amino acid, prepared in advance, was introduced into the peptide sequence during the solid-phase synthesis. This procedure, however, dramatically complicates product purification that is known to be the critical step in solid phase synthesis. The presence of PEG, for its high molecular weight and its polydispersivity, is likely to yield final products with unacceptable impurities and/or products with missing amino acids, the latter being considered to occur commonly in the Merrifield procedure.
Mono-pegylation, meaning that only one PEG molecule is attached, using solid-phase synthesis to specific amino acid residues of (Ala
15
)-hGRF(1-29)-NH
2
has been recently reported in the literature (Felix et al., 1995). This study shows that (Ala
15
)-hGRF(1-29)-NH
2
pegylated at residues 21 or 25 retains the full in-vitro potency of the parent (Ala
15
)-hGRF(1-29)-NH
2
. There is however no in7-vivo data to show whether these pegylated conjugates exhibit a longer duration of action with respect to the non-pegylated counterpart.
More recently, it has been demonstrated (Campbell et al., 1997) that the attachment of PEG with different molecular weights to the C-terminus of several analogs of hGRF, again using solid-phase synthesis, had enhanced duration of action in both pig and mouse models as compared to the non-pegylated counterpart.
DESCRIPTION OF THE INVENTION
In contrast to the solid-phase preparation of mono-pegylated hGRF mentioned above, the present invention relates to site-specific pegylation of hGRF in solution phase.
hGRF was found to have a low solubility in a neutral/alkaline buffer solution, a chemical condition whereby most efficient pegylation reaction occurs. In a diluted hGRF solution, the hydrolysis of the activated PEG (such as the PEG ester) tends to decrease the yield of the pegylation reaction.
It was discovered by the Applicant that, in a suitable solvent whereby hGRF has a high solubility, it is possible to carry out a site-specific pegylation reaction in solution phase. In this way, even if the starting hGRF peptide is non-protected, the PEG chains will bind with high yields and almost exclusively to the primary amino groups (&egr;-amino groups) of Lys
12
, Lys
21
and/or N
&agr;
. depending upon the reaction conditions. The following four conjugates, which are also covered by the present invention, were obtained, the hGRF:PEG stoichiometric ratio in the conjugates mainly depending on the molar ratio of PEG to hGRF:
hGRF-PEG conjugate, in which 1 PEG molecule is covalently bound to Lys
12
,
hGRF-PEG conjugate, in which 1 PEG molecule is covalently bound to Lys
21
,
hGRF-2PEG conjugate, in which 2 PEG molecules are covalently bound to both Lys
12
and Lys
21
; and
hGRF-3PEG conjugate, in which 3 PEG molecules are covalently bound to both Lys
12
and Lys
21
and also to N
&agr;
.
“N
&agr;
” through out the present invention means the amino group at the N-terminal position of the peptide (Tyr).
Further to this step, it is possible to carry out a simple chromatographic fractionation of the conjugates obtained in the reaction either by gel filtration or by direct application to a C18 HPLC column eluted by water/acetonitrile gradient. The second method is preferred, since large scale preparation and purification of the products could be obtained.
Therefore, the main embodiment of the present invention is a method for

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