Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Chemical aftertreatment – e.g. – acylation – methylation – etc.
Reexamination Certificate
2000-03-13
2002-09-17
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Chemical aftertreatment, e.g., acylation, methylation, etc.
C530S308000, C530S333000, C530S402000, C436S086000, C436S090000
Reexamination Certificate
active
06451974
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of introducing one or more acyl groups into a peptide or a protein. More particularly, the invention relates to an improved method of acylating the &egr;-amino group of a lysine residue contained in a naturally occurring GLP-1 or an analogue thereof. Furthermore, the present invention relates to compounds useful as acylating agents in the method.
2. Description of the Related Art
Peptides are widely used in medical practice, and since they can be produced by recombinant DNA technology, it can be expected that their importance will increase also in the years to come. When native peptides or analogues thereof are used in therapy, it is generally found that they have a high clearance. A high clearance of a therapeutic agent is inconvenient in cases where it is desired to maintain a high blood level thereof over a prolonged period of time since repeated administrations will then be necessary. Examples of peptides which in their native form have a high clearance are: ACTH, corticotropin-releasing factor, angiotensin, calcitonin, exendin, exendin-3, exendin4, insulin, glucagon, glucagon-like peptide-1, glucagon-like peptide-2, insulin-like growth factor-1, insulin-like growth factor-2, gastric inhibitory peptide, growth hormone-releasing factor, pituitary adenylate cyclase activating peptide, secretin, enterogastrin, somatostatin, somatotropin, somatomedin, parathyroid hormone, thrombopoietin, erythropoietin, hypothalamic releasing factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, vasopressin, oxytocin, opiods and analogues thereof, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase and ribonuclease.
Introduction of lipophilic acyl groups into naturally occurring peptides or analogues thereof leads to acylated peptides which have a protracted profile of action relative to the native peptide (or unmodified analogue). This phenomenon has been thoroughly described and demonstrated in the present applicant's previous applications, WO98/08871, which i.a. discloses acylation of GLP-1 and analogues, and WO98/08872, which i.a. discloses acylation of GLP-2 and analogues, and WO99/43708, which i.a. discloses acylation of exendin and analogues. Furthermore, it has been suggested that the inclusion of a group which can be negatively charged, e.g., a carboxylic acid group, adjacent to the lipophilic group may be advantageous.
European patent application No. 92107035.5 (Kuraray Co.) describes reactive monoesters of long chain dicarboxylic acids for the introduction of long chain carboxylic acids into proteins. Introduction of lipophilic acyl groups into GLP-1 via mono- or dipeptide spacers may be especially interesting and has been suggested and exemplified in WO98/08871. Asparatic acid and glutamic acid were mentioned as suitable linkers. However, as such mono- and dipeptide spacers include a supplementary carboxylic acid group, protection and subsequent deprotection steps were considered necessary. Deprotection was performed under acidic conditions which to a certain degree led to destruction of the peptide (GLP-1). Thus, alternative methods for the preparation of these variants are desirable.
Thus, it has been the aim of the present invention to provide an alternative method for the introduction of lipophilic groups into peptides via &agr;-amino-&agr;, &ohgr;-dicarboxylic acid spacers. Such a method will facilitate the preparation of modified peptides where chargeable carboxylic acid groups are introduced in the proximity of lipophilic groups, but without direct influence on the lipophilic group.
SUMMARY OF THE INVENTION
The present invention provides a method for acylating one or more amino groups of a peptide (or protein), the method comprising:
(a) reacting a peptide (or protein) having at least one free amino group with an acylating agent of formula I
wherein
n is 0-8;
R
1
is COOR
4
;
R
2
is a lipophilic moiety, e.g., selected from C
3-39
-alkyl, C
3-39
-alkenyl, C
3-39
-alkadienyl and steroidal residues;
R
3
together with the carboxyl group to which R
3
is attached designate a reactive ester or a reactive N-hydroxy imide ester; and
R
4
is selected from hydrogen, C
1-12
-alkyl and benzyl, under basic conditions in a mixture of an aprotic polar solvent and water; and
(b) if R
4
is not hydrogen, saponifying the acylated peptide ester group (COOR
4
) under basic conditions;
in order to obtain an N-acylated peptide (or an N-acylated protein).
It has been found that saponification of the acylated peptide ester (where R
4
is an alkyl or benzyl group) under basic conditions is possible with only minor or no racemization of the various &agr;-amino acid fragments of the peptide and the spacer. The present invention has been found to have certain advantages over the previously used acidic hydrolysis with respect to purity and suppression of side products, e.g. degradation products.
It has also been found that acylation using the acylating agent as the free acid (where R
4
is hydrogen) under basic conditions does essentially lead directly to the desired product, the acylated peptide, without side products and without the need for a deprotection step.
The present invention also provides novel compounds useful as acylating agents in the above-mentioned method, such novel compounds having formula I
wherein
n is 0-8;
R
1
is COOH;
R
2
is a lipophilic moiety, e.g., selected from C
3-39
-alkyl, C
3-39
-alkenyl, C
3-39
-alkadienyl and steroidal residues; and
R
3
together with the carboxyl group to which R
3
is attached designate a reactive ester or a reactive N-hydroxy imide ester.
DETAILED DESCRIPTION OF THE INVENTION
Peptides and Proteins
It is generally believed that the present invention is useful for the introduction of lipophilic acyl groups into any peptide (or protein) in order to reduce the in vivo clearance rate. Examples of such peptides and proteins are ACTH, corticotropin-releasing factor, angiotensin, calcitonin, exendin and analogues thereof, insulin and analogues thereof, glucagon and analogues thereof, glucagon-like peptide-1 and analogues thereof, glucagon-like peptide-2 and analogues thereof, insulin-like growth factor-1, insulin-like growth factor-2, gastric inhibitory peptide, growth hormone-releasing factor, pituitary adenylate cyclase activating peptide, secretin, enterogastrin, somatostatin, somatotropin, somatomedin, parathyroid hormone, thrombopoietin, erythropoietin, hypothalamic releasing factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, vasopressin, oxytocin, opiods and analogues thereof, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase and ribonuclease.
It should be understood that the peptide (or protein) should carry at least one free amino group, such an amino group being the N-terminal amino group or a side chain amino group.
Particularly interesting are amino groups of lysine and ornithine amino acid residues. The method is particularly relevant for the N-acylation of the &egr;-amino group of lysine residues. It should also be understood that the peptide or protein in question may comprise two or more pendant amino groups which all may be N-acylated according to the present invention.
It is presently believed that the present invention is especially suitable for the modification of GLP-1 and analogues thereof. Examples of GLP-1 and analogues which can be N-acylated according to the present invention are GLP-1 and truncated analogues, such as Arg
26
-GLP-1(7-37); Arg
34
-GLP-1(7-37); Lys
36
-GLP-1(7-37); Arg
26,34
Lys
36
-GLP-1(7-37); Arg
26,34
Lys
38
GLP-1(7-38); Arg
28,34
Lys
39
-GLP-1(7-39); Arg
26,34
Lys
40
-GLP-1(7-40); Arg
26
Lys
36
-GLP-1(7-37); Arg
34
Lys
36
-GLP-1(7-37); Arg
26
Lys
39
-GLP-1(7-39); Arg
34
Lys
40
-GLP-1(7-40); Arg
26,34
Lys
36,39
-GLP-1(7-39); Arg
26,34
Lys
36,40
-GLP-1(7-40); Gly
8
Arg
26
-GLP-1(7-37); Gly
8
Arg
34
-GLP-1(7-37); Gly
8
Lys
36
-GLP-1(7-37); Gly
8
Arg
26,34
Lys
36
-GLP-1(7
Bork, Esq. Richard W.
Green, Esq. Rezo
Low Christopher S. F.
Mohamed Abdel A.
Novo Nordisk A S
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