U.S. patent application number 12/676453 was filed with the patent office on 2010-11-18 for truncated glp-1 derivaties and their therapeutical use.
This patent application is currently assigned to Novo Nordisk A/S. Invention is credited to Patrick William Garibay, Janos Tibor Kodra, Jacob Kofoed, Jesper Lau, Kjeld Madsen, Igrid Pettersson, Steffen Reedtz-Runge, Lauge Schaffer, Jane Spetzler, Henning Thogersen.
Application Number | 20100292133 12/676453 |
Document ID | / |
Family ID | 40043001 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100292133 |
Kind Code |
A1 |
Spetzler; Jane ; et
al. |
November 18, 2010 |
TRUNCATED GLP-1 DERIVATIES AND THEIR THERAPEUTICAL USE
Abstract
The invention relates to truncated GLP-1 analogues, in
particular a GLP-1 analogue which is a modified GLP-1(7-35) (SEQ ID
No 1) having: i) a total of 2, 3, 4, 5 6, 7, 8, or 9 amino acid
substitutions as compared to GLP-1(7-35), including a) a Glu
residue at a position equivalent to position 22 of GLP-1(7-35), and
b) an Arg residue at a position equivalent to position 26 of
GLP-1(7-35); as well as derivatives thereof, and therapeutic uses
and compositions. These analogues and derivatives are highly
potent, have a good binding affinity to the GLP-1 receptor, also to
the extracellular domain of the GLP-1 receptor, which is of
potential relevance achieving long-acting, stable GLP-1 compounds
with a potential for once weekly administration.
Inventors: |
Spetzler; Jane; (Bronshoj,
DK) ; Schaffer; Lauge; (Lyngby, DK) ; Lau;
Jesper; (Farum, DK) ; Kodra; Janos Tibor;
(Kobenhavn, DK) ; Madsen; Kjeld; (Vaerlose,
DK) ; Garibay; Patrick William; (Holte, DK) ;
Kofoed; Jacob; (Vaerlose, DK) ; Reedtz-Runge;
Steffen; (Frederiksberg, DK) ; Thogersen;
Henning; (Farum, DK) ; Pettersson; Igrid;
(Frederiksberg, DK) |
Correspondence
Address: |
NOVO NORDISK, INC.;INTELLECTUAL PROPERTY DEPARTMENT
100 COLLEGE ROAD WEST
PRINCETON
NJ
08540
US
|
Assignee: |
Novo Nordisk A/S
Bagsvard
DK
|
Family ID: |
40043001 |
Appl. No.: |
12/676453 |
Filed: |
September 5, 2008 |
PCT Filed: |
September 5, 2008 |
PCT NO: |
PCT/EP2008/061833 |
371 Date: |
July 9, 2010 |
Current U.S.
Class: |
514/1.9 ;
514/11.7; 514/6.8; 514/7.2; 514/7.4; 530/323; 530/324; 530/326 |
Current CPC
Class: |
A61P 25/28 20180101;
A61K 38/00 20130101; A61P 1/14 20180101; A61P 1/00 20180101; A61P
9/12 20180101; A61P 3/10 20180101; A61P 3/08 20180101; A61P 3/00
20180101; A61P 3/04 20180101; C07K 14/605 20130101; A61P 3/06
20180101; A61P 9/00 20180101; A61P 1/04 20180101; A61P 9/10
20180101 |
Class at
Publication: |
514/1.9 ;
530/324; 530/323; 530/326; 514/11.7; 514/7.2; 514/6.8; 514/7.4 |
International
Class: |
C07K 14/605 20060101
C07K014/605; A61K 38/26 20060101 A61K038/26; A61P 3/10 20060101
A61P003/10; A61P 3/00 20060101 A61P003/00; A61P 3/06 20060101
A61P003/06; A61P 3/08 20060101 A61P003/08; A61P 9/10 20060101
A61P009/10; A61P 9/00 20060101 A61P009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 5, 2007 |
EP |
07115743.2 |
Jan 28, 2008 |
EP |
08101010.0 |
Claims
1. A GLP-1 analogue which is a modified GLP-1(7-35) (SEQ ID No 1)
having: i) a total of 2, 3, 4, 5 6, 7, 8, or 9 amino acid
substitutions as compared to GLP-1(7-35), including a) a Glu
residue at a position equivalent to position 22 of GLP-1(7-35), and
b) an Arg residue at a position equivalent to position 26 of
GLP-1(7-35); or a derivative thereof.
2. The GLP-1 analogue or derivative thereof according to claim 1,
wherein the amino acid(s) at a position equivalent to position 30,
31, 32, 33, 34, or 35 of GLP-1(7-35) are absent, provided that if
the amino acid at position 30, 31, 32, 33, or 34 is absent then
each amino acid residue downstream is also absent.
3. The GLP-1 analogue or derivative thereof according to claim 1,
wherein the GLP-1 analogue comprises i) a C-terminal carboxylic
acid group; or iii) a C-terminal amide group.
4. The GLP-1 analogue or derivative thereof according to claim 1,
which is derivatised with an albumin binding residue or is
pegylated.
5. The GLP-1 analogue or derivative thereof according to claim 1,
wherein the amino acid at position 35 is absent, and wherein the
total length of the GLP-1 analogue is 28 amino acids.
6. The GLP-1 analogue or derivative thereof according to claim 1,
wherein the amino acids at position 34 and 35 are absent, and
wherein the total length of the GLP-1 analogue is 27 amino
acids.
7. The GLP-1 analogue or derivative thereof according to claim 1,
wherein the amino acids at position 33, 34, and 35 are absent, and
wherein the total length of the GLP-1 analogue is 26 amino
acids.
8. The GLP-1 analogue or derivative thereof according to claim 1,
wherein the amino acids at position 32, 33, 34, and 35 are absent,
and wherein the total length of the GLP-1 analogue is 25 amino
acids.
9. The GLP-1 analogue or derivative thereof according to claim 1,
wherein the amino acids at position 31, 32, 33, 34, and 35 are
absent, and wherein the total length of the GLP-1 analogue is 24
amino acids.
10. The GLP-1 analogue or derivative thereof according to claim 1,
wherein the amino acids at position 30, 31, 32, 33, 34, and 35 are
absent, and wherein the total length of the GLP-1 analogue is 23
amino acids.
11. The GLP-1 analogue or derivative thereof according to claim 1
having a sequence according to formula (I) TABLE-US-00016 (SEQ ID
No: 2)
Xaa.sub.7-Xaa.sub.8-Xaa.sub.9-Gly-Thr-Phe-Thr-Ser-Asp-Xaa.sub.16-Ser-
Xaa.sub.18-Tyr-Xaa.sub.20-Glu-Glu-Xaa.sub.23-Xaa.sub.24-Xaa.sub.25-Arg-
Xaa.sub.27-Phe-Ile-Xaa.sub.30-Xaa.sub.31-Leu-Xaa.sub.33-Xaa.sub.34-Xaa.sub-
.35-R Formula (I)
wherein Xaa.sub.7 is L-histidine, D-histidine, desamino-histidine,
2-amino-histidine, .beta.-hydroxy-histidine, homohistidine,
N.sup..alpha.-acetyl-histidine, .alpha.-fluoromethyl-histidine,
.alpha.-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or
4-pyridylalanine; Xaa.sub.8 is Ala, Gly, Val, Leu, Ile, Lys, Aib,
(1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic
acid, (1-aminocyclopentyl)carboxylic acid,
(1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic
acid, or (1-aminocyclooctyl)carboxylic acid; Xaa.sub.9 is Glu or a
Glu derivative such as alpha, alpha dimethyl-Glu; Xaa.sub.16 is Val
or Leu; Xaa.sub.18 is Ser, Lys, Cys or Arg; Xaa.sub.20 is Leu, Lys
or Cys; Xaa.sub.23 is Gln, Glu, Lys, Cys or Arg; Xaa.sub.24 is Ala
or Asn; Xaa.sub.25 is Ala or Val; Xaa.sub.27 is Glu, Ala or Leu;
Xaa.sub.30 is Ala, Glu, Lys, Arg or absent; Xaa.sub.31 is Trp, Lys,
Cys or absent; Xaa.sub.33 is Val, Lys, Cys or absent; Xaa.sub.34 is
Lys, Glu, Asn, Arg, Cys or absent; Xaa.sub.35 is Gly, Aib or
absent; R is amide or is absent; provided that if Xaa.sub.30,
Xaa.sub.31, Xaa.sub.32, Xaa.sub.33, or Xaa.sub.34 is absent then
each amino acid residue downstream is also absent.
12. The GLP-1 analogue or derivative thereof according to claim 1
having a sequence according to formula (II) TABLE-US-00017 (SEQ ID
No: 3) Xaa.sub.7-Xaa.sub.8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-
Xaa.sub.18-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Arg-Glu-Phe-
Ile-Xaa.sub.30-Trp-Leu-Xaa.sub.33-Xaa.sub.34-Xaa.sub.35-R Formula
(II)
wherein Xaa.sub.7 is L-histidine, D-histidine, desamino-histidine,
2-amino-histidine, .beta.-hydroxy-histidine, homohistidine,
N.sup..alpha.-acetyl-histidine, .alpha.-fluoromethyl-histidine,
.alpha.-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or
4-pyridylalanine; Xaa.sub.8 is Ala, Gly, Val, Leu, Ile, Lys, Aib,
(1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic
acid, (1-aminocyclopentyl)carboxylic acid,
(1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic
acid, or (1-aminocyclooctyl)carboxylic acid; Xaa.sub.18 is Ser, Lys
or Arg; Xaa.sub.30 is Ala, Glu, Lys, Arg or is absent; Xaa.sub.33
is Val, Lys or absent; Xaa.sub.34 is Lys, Glu, Arg or is absent;
Xaa.sub.35 is Gly, Aib or is absent; R is amide or is absent.
13. The GLP-1 derivative according to claim 1, wherein at least one
amino acid residue is derivatised with A-B-C-D- wherein A- is
selected from the group consisting of ##STR00085## wherein n is
selected from the group consisting of 14, 15, 16 17, 18 and 19, p
is selected from the group consisting of 10, 11, 12, 13 and 14, and
d is selected from the group consisting of 0, 1, 2, 3, 4 and 5, -B-
is selected from the group consisting of ##STR00086## wherein x is
selected from the group consisting of 0, 1, 2, 3 and 4, and y is
selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11 and 12, -C- is selected from the group consisting of
##STR00087## wherein b and e are each independently selected from
the group consisting of 0, 1 and 2, and c and f are each
independently selected from the group consisting of 0, 1 and 2 with
the proviso that b is 1 or 2 when c is 0, or b is 0 when c is 1 or
2, and e is 1 or 2 when f is 0, or e is 0 when f is 1 or 2, and -D-
is attached to said amino acid residue and is a linker.
14. The GLP-1 derivative according to claim 13, wherein D is
selected from the group consisting of ##STR00088## and wherein k is
selected from the group consisting of 0, 1, 2, 3, 4, 5, 11 and 27,
and m is selected from the group consisting of 0, 1, 2, 3, 4, 5 and
6.
15. The GLP-1 analogue or derivative thereof according to claim 1,
which is selected from the following: [Glu22,Arg26]GLP-1(7-33)amide
N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Val25,-
Arg26,Leu27,Glu30,Lys33)GLP-1(7-33)amide; N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Arg26,-
Glu30)GLP-1(7-33)amide; [Glu22,Val25,Arg26]GLP-1(7-33)amide;
[Aib8,Lys20,Glu22,Val25,Arg26,Glu30]GLP-1(7-33)amide; N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-[Aib8,Lys20,Glu22,Val25,-
Arg26,Glu30]GLP-1(7-33)amide; [Glu22, Arg26]GLP-1(7-33)peptide;
[Glu22,Val25,Arg26]GLP-1(7-32)amide; N-epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Arg26)-
GLP-1(7-33)amide; N-epsilon31
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Glu22,Val25,Arg26,-
Lys31)GLP-1(7-33)amide; N-epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(DesaminoHis7,Lys20,Glu2-
2,Arg26)GLP-1(7-33)amide; N-epsilon31
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(DesaminoHis7,Glu22,Arg2-
6,Lys31)GLP-1(7-33)amide; N-epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Val25,-
Arg26,Leu27,Glu30,Lys31)GLP-1(7-32)amide; N-epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,
Lys20,Glu22,Val25,Arg26,Leu27,Nle30,Lys31)GLP-1(7-32)amide;
N-epsilon31-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxynona-
decanoylamino)methyl]cyclohexanecarbonyl
amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl]-[Aib8,-
Glu22,Val25,Arg26,Lys31]GLP-1-(7-33)amide; [Desamino
His7,Glu22,Arg26]-GLP-1(7-34);
[Aib8,Lys20,Glu22,Val25,Arg26,Leu27,Lys31]GLP-1(7-32)amide;
N-epsilon31-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylam-
ino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoH-
is7,Asp11,Glu18,Glu22,Val25,Arg26,Asp27,Glu30,Lys31]GLP-1(7-33)amide;
[Aib8,Glu22,Val25, Lys31]GLP-1(7-33)-amide; and
N-epsilon31-{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino-
)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-N-beta34-(2--
(bis-carboxymethylamino)acetyl)[Aib8,Glu22,Val25,Arg26,Lys31,Dap34]GLP-1(7-
-34)amide.
16. A pharmaceutical composition comprising a GLP-1 analogue or
derivative thereof according to claim 1 or a pharmaceutically
acceptable salt, amide, alkyl, or ester thereof, and a
pharmaceutically acceptable excipient.
17-19. (canceled)
20. A method of treating hyperglycemia, type 2 diabetes, impaired
glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome
X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial
infarction, coronary heart disease and other cardiovascular
disorders, stroke, inflammatory bowel syndrome, dyspepsia and
gastric ulcers in a subject in need of such treatment, the method
comprising administering to the subject a therapeutically effective
amount of a pharmaceutical composition according to claim 16.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the field of therapeutic peptides,
i.e. to new truncated Glucagon-Like Peptide-1 (GLP-1) analogues and
derivatives thereof.
BACKGROUND OF THE INVENTION
[0002] A range of different approaches have been used for modifying
the structure of glucagon-like peptide 1 (GLP-1) compounds in order
to provide a longer duration of action in vivo.
[0003] WO 2006/097538, WO 2006/097536, WO 2006/037810,
WO2006005667, WO 2005/027978. WO 98/08871 and US 2001/0011071
describes various GLP-1 analogues and derivatives thereof.
[0004] Runge et al (Journal of Biological Chemistry, vol. 283, no.
17, pp. 11340-11347), which was published after the priority dates
of the present invention, discloses the crystal structure of the
extracellular domain of the ligand-bound GLP-1 receptor.
[0005] Many diabetes patients particularly in the type 2 diabetes
segment are subject to so-called "needle-phobia", i.e. a
substantial fear of injecting themselves. In the type 2 diabetes
segment most patients are treated with oral hypoglycaemic agents,
and since GLP-1 compounds are expected to be an injectable
pharmaceutical product these patients will be administered, the
fear of injections may become a serious obstacle for the widespread
use of the clinically very promising compounds. Thus, there is a
need to develop new compounds which can be administered less than
once daily, e.g. once every second or third day preferably once
weekly, while retaining an acceptable clinical profile or
optionally via non invasive administration such as pulmonary,
nasal, sublingual, bucal or oral administration.
[0006] One object of the present invention is to provide a
chemically, physically and enzymatically stable GLP-1 analogue or
derivative thereof.
[0007] A further object of the invention is to provide a long
acting, i.e. having an administration regimen as described above,
GLP-1 analogue or derivative thereof.
[0008] Another object of this invention is to provide a GLP-1
analogue or derivative thereof with high potency (receptor
affinity) in order to reduce the therapeutic dose used for example
for once weekly s.c. dosing or alternatively for non-invasive
delivery.
[0009] Another object of this invention is to provide a GLP-1
compound with a high binding affinity to the GLP-1 receptor
(GLP-1R).
[0010] A still further object of this invention is to provide a
GLP-1 compound with a high binding affinity to the extracellular
domain of the GLP-1 receptor (nGLP-1R).
[0011] Another object of this invention is to provide a GLP-1
analogue or derivative thereof with high albumin binding affinity
which protects the peptide for proteolytic degradation and reduce
renal clearance of the peptide.
[0012] Potency, binding affinity to the GLP-1 receptor, and
possibly also to the extracellular domain of the GLP-1 receptor are
properties of potential relevance for an overall object of
achieving long-acting, stable and of course therapeutically active
GLP-1 derivatives with a potential for once weekly
administration.
SUMMARY OF THE INVENTION
[0013] The invention relates to truncated analogues of GLP-1
(7-37), and derivatives thereof.
[0014] The invention relates to a GLP-1 analogue which is a
modified GLP-1(7-35) (SEQ ID No 1) having: i) a total of 2, 3, 4, 5
6, 7, 8, or 9 amino acid substitutions as compared to GLP-1(7-35),
including a) a Glu residue at a position equivalent to position 22
of GLP-1(7-35), and b) an Arg residue at a position equivalent to
position 26 of GLP-1(7-35); or a derivative thereof.
[0015] The invention also relates to compositions and uses of these
derivatives and analogues.
[0016] Optionally the amino acid(s) at a position equivalent to
position 30, 31, 32, 33, 34, or 35 of GLP-1(7-35) can be absent
provided that if the amino acid at position 30, 31, 32, 33 or 34 is
absent then each amino acid residue downstream is also absent
[0017] Also optionally, the GLP-1 analogue of the invention
comprises a C-terminal amide group, or a C-terminal carboxylic acid
group.
[0018] In a further aspect, pharmaceutical compositions and methods
and uses of the analogues and derivatives according to the
invention, is provided.
DESCRIPTION OF THE INVENTION
Definitions and Particular Embodiments
[0019] In the present specification, the following terms have the
indicated meaning: The term "polypeptide" and "peptide" as used
herein means a compound composed of at least five constituent amino
acids connected by peptide bonds. The constituent amino acids may
be from the group of the amino acids encoded by the genetic code
and they may be natural amino acids which are not encoded by the
genetic code, as well as synthetic amino acids. Natural amino acids
which are not encoded by the genetic code are e.g.,
.gamma.-carboxyglutamate, ornithine, phosphoserine, D-alanine and
D-glutamine. Synthetic amino acids comprise amino acids
manufactured by chemical synthesis, i.e. D-isomers of the amino
acids encoded by the genetic code such as D-alanine and D-leucine,
Aib (.alpha.-aminoisobutyric acid), Abu (.alpha.-aminobutyric
acid), Tle (tert-butylglycine), .beta.-alanine, 3-aminomethyl
benzoic acid, anthranilic acid.
[0020] The 22 proteogenic amino acids are: Alanine, Arginine,
Asparagine, Aspartic acid, Cysteine, Cystine, Glutamine, Glutamic
acid, Glycine, Histidine, Hydroxyproline, Isoleucine, Leucine,
Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine,
Tryptophan, Tyrosine, Valine.
[0021] Thus a non-proteogenic (also designated non-natural) amino
acid is a moiety which can be incorporated into a peptide via
peptide bonds but is not a proteogenic amino acid. Examples are
.gamma.-carboxyglutamate, ornithine, phosphoserine, the D-amino
acids such as D-alanine and D-glutamine, Synthetic non-proteogenic
amino acids comprise amino acids manufactured by chemical
synthesis, i.e. D-isomers of the amino acids encoded by the genetic
code such as D-alanine and D-leucine, Aib (.alpha.-aminoisobutyric
acid), Abu (.alpha.-aminobutyric acid), Tle (tert-butylglycine),
3-aminomethyl benzoic acid, anthranilic acid, des-amino-Histidine,
the beta analogs of amino acids such as .beta.-alanine etc.,
D-histidine, desamino-histidine, 2-amino-histidine,
.beta.-hydroxy-histidine, homohistidine,
N.sup..alpha.-acetyl-histidine, .alpha.-fluoromethyl-histidine,
.alpha.-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or
4-pyridylalanine, (1-aminocyclopropyl)carboxylic acid,
(1-aminocyclobutyl)carboxylic acid, (1-aminocyclopentyl)carboxylic
acid, (1-aminocyclohexyl)carboxylic acid,
(1-aminocycloheptyl)carboxylic acid, or
(1-aminocyclooctyl)carboxylic acid.
[0022] The term "analogue" as used herein referring to a
polypeptide means a modified peptide wherein one or more amino acid
residues of the peptide have been substituted by other amino acid
residues and/or wherein one or more amino acid residues have been
deleted from the peptide in the C-terminal of the peptide.
[0023] The term "modified peptide" as used herein refers to a
modified peptide as defined above. For the present purposes this
term is used interchangeably with the term "modified peptide
sequence". Consistently herewith, the term "modification" when used
herein in connection with peptide sequences refers to amino acid
substitutions, additions, and/or deletions.
[0024] For the present purposes any amino acid substitution,
deletion, and/or addition refers to the sequence of human
GLP-1(7-35) which is included herein as SEQ ID No: 1. However, the
numbering of the amino acid residues in the sequence listing always
starts with no. 1, whereas for the present purpose we want,
following the established practice in the art, to start with amino
acid residue no. 7 and assign number 7 to it. Therefore, generally,
any reference herein to a position number of the GLP-1(7-35)
sequence is to the sequence starting with His at position 7 and
ending with Gly at position 35.
[0025] A simple system is often used to describe analogues: For
example [Arg.sup.34]GLP-1(7-37)Lys designates a GLP-1(7-37)
analogue wherein the naturally occurring lysine at position 34 has
been substituted with arginine and wherein a lysine has been added
to the terminal amino acid residue, i.e. to the Gly.sup.37.
[0026] The expression "a position equivalent to" when used herein
to characterize a modified GLP-1(7-35) sequence refers to the
corresponding position in the natural GLP-1(7-35) sequence (having
the sequence of SEQ ID No: 1). Corresponding positions are easily
deduced, e.g. by simple handwriting and eyeballing. In the
alternative, a standard protein or peptide alignment program may be
used, such as "align" which is a Needleman-Wunsch alignment. The
algorithm is described in Needleman, S. B. and Wunsch, C. D.,
(1970), Journal of Molecular Biology, 48: 443-453, and the align
program by Myers and W. Miller in "Optimal Alignments in Linear
Space" CABIOS (computer applications in the biosciences) (1988)
4:11-17. For the alignment, the default scoring matrix BLOSUM50 and
the default identity matrix may be used, and the penalty for the
first residue in a gap may be set at -12 and the penalties for
additional residues in a gap at -2.
[0027] All amino acids for which the optical isomer is not stated
is to be understood to mean the L-isomer.
[0028] The term "each amino acid residue downstream" as used herein
refers to each amino acid positioned towards the C-terminal
relative to a specific amino acid. As an example Lys34 and Gly35
are each amino acid residues downstream of Val33 in GLP-1
(7-35).
[0029] In embodiments of the invention a maximum of 8 amino acids
have been modified. In embodiments of the invention a maximum of 7
amino acids have been modified. In embodiments of the invention a
maximum of 6 amino acids have been modified. In embodiments of the
invention a maximum of 5 amino acids have been modified. In
embodiments of the invention a maximum of 4 amino acids have been
modified. In embodiments of the invention a maximum of 3 amino
acids have been modified. In embodiments of the invention a maximum
of 2 amino acids have been modified.
[0030] In embodiments of the invention, one or more amino acid(s)
have been deleted in the C-terminal end.
[0031] In one aspect of the invention, the C-terminal of the
analogue or derivative according to the invention may be terminated
as either an acid or amide. In a preferred aspect, the C-terminal
of the analogue or derivative of the invention is an amide.
[0032] In one aspect, the invention relates to a GLP-1 analogue or
derivative thereof, wherein the amino acids at position 35, 36 and
37 are absent, and wherein the total length of the
[0033] GLP-1 analogue is 28 amino acids. In a further aspect, the
invention relates to a GLP-1 analogue or derivative thereof,
wherein the amino acids at position 34, 35, 36 and 37 are absent,
and wherein the total length of the GLP-1 analogue is 27 amino
acids. In a further aspect, the invention relates to a GLP-1
analogue or derivative thereof, wherein the amino acids at position
33, 34, 35, 36 and 37 are absent, and wherein the total length of
the GLP-1 analogue is 26 amino acids. In a further aspect, the
invention relates to a GLP-1 analogue or derivative thereof,
wherein the amino acids at position 32, 33, 34, 35, 36 and 37 are
absent, and wherein the total length of the GLP-1 analogue is 25
amino acids. In a further aspect, the invention relates to a GLP-1
analogue or derivative thereof, wherein the amino acids at position
31, 32, 33, 34, 35, 36 and 37 are absent, and wherein the total
length of the GLP-1 analogue is 24 amino acids. In a further
aspect, the invention relates to a GLP-1 analogue or derivative
thereof, wherein the amino acids at position 30, 31, 32, 33, 34,
35, 36 and 37 are absent, and wherein the total length of the GLP-1
analogue is 23 amino acids.
[0034] In one aspect, the invention relates to a GLP-1 analogue or
derivative thereof having a C-terminal amide group.
[0035] In one aspect, the invention relates to a GLP-1 analogue or
derivative thereof having 3 amino acid substitutions compared to
the sequence 7-35 of SEQ ID NO 1 including the substitutions at
position 22 and 26. In a further aspect, the invention relates to a
GLP-1 analogue or derivative thereof, which has a substitution
selected from the group of positions 7, 8, 18, 20, 23, 24, 25, 27,
30, 31, 33 and 34 compared to the sequence 7-35 of SEQ ID NO 1. In
a further aspect, the invention relates to a GLP-1 analogue or
derivative thereof, which has a substitution selected from the
group consisting of desaminoHis7, Aib8, Lys18, Cys18, Lys20, Cys20,
Lys23, Cys23, Asn24, Val25, Ala27, Leu27, Glu30, Lys31, Cys31,
Lys33, Cys33, Lys34, Cys34 and Asn34. In a further aspect, the
invention relates to a GLP-1 analogue or derivative thereof, which
has a substitution selected from the group consisting of
desaminoHis7, Aib8, Lys18, Lys20, Lys23, Glu30, Lys31, Lys33 and
Lys34. In a further aspect, the invention relates to a GLP-1
analogue or derivative thereof, which has a substitution selected
from the group consisting of desaminoHis7 and Aib8.
[0036] In one aspect, the invention relates to a GLP-1 analogue or
derivative thereof having 4 amino acid substitutions compared to
the sequence 7-35 of SEQ ID NO 1 including the substitutions at
position 22 and 26. In a further aspect, the invention relates to a
GLP-1 analogue or derivative thereof, which has two substitutions
selected from the group of positions 7, 8, 18, 20, 23, 24, 25, 27,
30, 31, 33 and 34 compared to the sequence 7-35 of SEQ ID NO 1. In
a further aspect, the invention relates to a GLP-1 analogue or
derivative thereof, which has two substitutions selected from the
group consisting of desaminoHis7, Aib8, Lys18, Cys18, Lys20, Cys20,
Lys23, Cys23, Asn24, Val25, Ala27, Leu27, Glu30, Lys31, Cys31,
Lys33, Cys33, Lys34, Cys34 and Asn34. In a further aspect, the
invention relates to a GLP-1 analogue or derivative thereof having
an amino acid substitution selected from the group consisting of
desaminoHis7 and Aib8 and an amino acid substitution selected from
the group consisting Lys18, Lys20, Lys23, Glu30, Lys31, Lys33 and
Lys34. In a further aspect, the invention relates to a GLP-1
analogue or derivative thereof having an amino acid substitution
selected from the group consisting desaminoHis7 and Aib8, and an
amino acid substitution selected from the group consisting Lys18,
Lys20, Lys23, Glu30, Lys31, Lys33 and Lys34.
[0037] In a one aspect, the invention relates to a GLP-1 analogue
or derivative thereof having 5 amino acid substitutions compared to
the sequence 7-35 of SEQ ID NO 1 including the substitutions at
position 22 and 26. In a further aspect, the invention relates to a
GLP-1 analogue or derivative thereof, which has three amino acid
substitutions selected from the group of positions 7, 8, 18, 20,
23, 24, 25, 27, 30, 31, 33 and 34 compared to the sequence 7-35 of
SEQ ID NO 1. In a further aspect, the invention relates to a GLP-1
analogue or derivative thereof, which has three amino acid
substitutions selected from the group of desaminoHis7, Aib8, Lys18,
Cys18, Lys20, Cys20, Lys23, Cys23, Asn24, Val25, Ala27, Leu27,
Glu30, Lys31, Cys31, Lys33, Cys33, Lys34, Cys34 and Asn34. In a
further aspect, the invention relates to a GLP-1 analogue or
derivative thereof having an amino acid substitution selected from
the group consisting of desaminoHis7 and Aib8, and two amino acid
substitutions selected from the group consisting of Lys18, Lys20,
Lys23, Glu30, Lys31, Lys33 and Lys34. In a further aspect, the
invention relates to a GLP-1 analogue or derivative thereof having
an amino acid substitution selected from the group consisting of
desaminoHis7 and Aib8, and two amino acid substitutions selected
from the group consisting of Lys18, Lys20, Lys23, Glu30, Lys31,
Lys33 and Lys34.
[0038] In one aspect, the invention relates to a GLP-1 analogue or
derivative thereof having 6, 7 or 8 amino acid substitutions
compared to the sequence 7-35 of SEQ ID NO 1 including the
substitutions at position 22 and 26. In a further aspect, the
invention relates to a GLP-1 analogue or derivative thereof, which
has four, five or six amino acid substitutions selected from the
group of positions 7, 8, 18, 20, 23, 24, 25, 27, 30, 31, 33 and 34.
In a further aspect, the invention relates to a GLP-1 analogue or
derivative thereof, which has four, five or six amino acid
substitutions selected from the group consisting of desaminoHis7,
Aib8, Lys18, Cys18, Lys20, Cys20, Lys23, Cys23, Asn24, Val25,
Ala27, Leu27, Glu30, Lys31, Cys31, Lys33, Cys33, Lys34, Cys34 and
Asn34. In a further aspect, the invention relates to a GLP-1
analogue or derivative thereof having an amino acid substitution
selected from the group consisting of desaminoHis7 and Aib8 and
three, four or five amino acid substitutions selected from the
group consisting of Lys18, Lys20, Lys23, Glu30, Lys31, Lys33 and
Lys34. In a further aspect, the invention relates to a GLP-1
analogue or derivative thereof having an amino acid substitution
selected from the group consisting of desaminoHis7 and Aib8 and
three, four or five amino acid substitutions selected from the
group consisting of Lys18, Lys20, Lys23, Glu30, Lys31, Lys33 and
Lys34.
[0039] In one aspect, the invention relates to a GLP-1 analogue or
derivative thereof having the sequence of formula (I)
TABLE-US-00001 (SEQ ID No: 2)
Xaa.sub.7-Xaa.sub.8-Xaa.sub.9-Gly-Thr-Phe-Thr-Ser-Asp-Xaa.sub.16-Ser-
Xaa.sub.18-Tyr-
Xaa.sub.20-Glu-Glu-Xaa.sub.23-Xaa.sub.24-Xaa.sub.25-Arg-
Xaa.sub.27-Phe-Ile-Xaa.sub.30-Xaa.sub.31-Leu-Xaa.sub.33-Xaa.sub.34-Xaa.sub-
.35-R Formula (I)
[0040] wherein
[0041] Xaa.sub.7 is L-histidine, D-histidine, desamino-histidine,
2-amino-histidine, .beta.-hydroxy-homohistidine,
N.sup..alpha.-acetyl-histidine, .alpha.-fluoromethyl-histidine,
.alpha.-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or
4-pyridylalanine;
[0042] Xaa.sub.8 is Ala, Gly, Val, Leu, Ile, Lys, Aib,
(1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic
acid, (1-aminocyclopentyl)carboxylic acid,
(1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic
acid, or (1-aminocyclooctyl)carboxylic acid;
[0043] Xaa.sub.9 is Glu or a Glu derivative such as alpha, alpha
dimethyl-Glu;
[0044] Xaa.sub.16 is Val or Leu;
[0045] Xaa.sub.18 is Ser, Lys, Cys or Arg;
[0046] Xaa.sub.20 is Leu, Lys or Cys;
[0047] Xaa.sub.23 is Gln, Glu, Lys, Cys or Arg;
[0048] Xaa.sub.24 is Ala or Asn;
[0049] Xaa.sub.25 is Ala or Val;
[0050] Xaa.sub.27 is Glu, Ala or Leu;
[0051] Xaa.sub.30 is Ala, Glu, Lys, Arg or absent;
[0052] Xaa.sub.31 is Trp, Lys, Cys or absent;
[0053] Xaa.sub.33 is Val, Lys, Cys or absent;
[0054] Xaa.sub.34 is Lys, Glu, Asn, Arg, Cys or absent;
[0055] Xaa.sub.35 is Gly, Aib or absent;
[0056] R is amide or is absent;
[0057] provided that if Xaa.sub.30, Xaa.sub.31, Xaa.sub.32,
Xaa.sub.33, or Xaa.sub.34 is absent then each amino acid residue
downstream is also absent.
[0058] In another aspect, the invention relates to a GLP-1 analogue
or derivative thereof having the sequence of formula (II)
TABLE-US-00002 (SEQ ID No: 3)
Xaa.sub.7-Xaa.sub.8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-
Xaa.sub.18-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Arg-Glu-Phe-
Ile-Xaa.sub.30-Trp-Leu-Xaa.sub.33-Xaa.sub.34-Xaa.sub.35-R Formula
(II)
[0059] wherein
[0060] Xaa.sub.7 is L-histidine, D-histidine, desamino-histidine,
2-amino-histidine, .beta.-hydroxy-histidine, homohistidine,
N.sup..alpha.-acetyl-histidine, .alpha.-fluoromethyl-histidine,
.alpha.-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or
4-pyridylalanine;
[0061] Xaa.sub.8 is Ala, Gly, Val, Leu, Ile, Lys, Aib,
(1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic
acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl)
carboxylic acid, (1-aminocycloheptyl)carboxylic acid, or
(1-aminocyclooctyl)carboxylic acid;
[0062] Xaa.sub.18 is Ser, Lys or Arg;
[0063] Xaa.sub.30 is Ala, Glu, Lys, Arg or is absent;
[0064] Xaa.sub.33 is Val, Lys or absent;
[0065] Xaa.sub.34 is Lys, Glu, Arg or is absent;
[0066] Xaa.sub.35 is Gly, Aib or is absent;
[0067] R is amide or is absent.
[0068] In one aspect of the invention, R is absent. In a further
aspect of the invention, Xaa.sub.35 and R are absent. In a further
aspect of the invention, Xaa.sub.34, Xaa.sub.35 and R are absent.
In a further aspect of the invention, Xaa.sub.33, Xaa.sub.34,
Xaa.sub.35 and R are absent. In a further aspect of the invention,
Xaa.sub.30, Xaa.sub.33, Xaa.sub.34, Xaa.sub.35 and R are
absent.
[0069] The term "derivative" as used herein in relation to a
peptide means a chemically modified peptide or an analogue thereof,
wherein at least one substituent is not present in the unmodified
peptide or an analogue thereof, i.e. a peptide which has been
covalently modified.
[0070] Typical modifications are amides, carbohydrates, alkyl
groups, acyl groups, esters and the like. An example of a
derivative according to the invention is N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Val25,-
Arg26,Leu27,Glu30, Lys33)GLP-1(7-33)amide (structure 1) wherein the
naturally occurring Tyr at position 20 has been substituted with
lysine which has been derivatised at N-epsilon20 with epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}
##STR00001##
and wherein the naturally occurring alanine at position 8 has been
substituted with Aib and glycine in pos 22 with glutamate and
alanine at position 25 with valine and lysine at position 26 with
arginine and glutamate at position 27 with leucine and alanine at
position 30 with glutamate and valine at position 33 with
lysineamide.
[0071] In one aspect of the invention, a GLP-1 analogue or
derivative thereof, which is derivatised with an albumin binding
residue or is pegylated, is provided.
[0072] In one aspect, the invention relates to a GLP-1 analogue or
derivative, wherein the amino acid which is pegylated or
derivatised with an albumin binding residue is a Lys-residue or a
Cys-residue. In one aspect, the amino acid which is pegylated or
derivatised with an albumin binding residue is a Lys-residue. In
one aspect, the amino acid which is pegylated or derivatised with
an albumin binding residue is a Cys-residue. In one aspect, the
C-terminal amino acid is pegylated or derivatised with an albumin
binding residue. In one aspect, the invention relates to a GLP-1
analogue or derivative thereof pegylated or derivatised with an
albumin binding residue at position 18, 20, 23, 31, 33, 34 or at
the C-terminal amino acid. In a further aspect, the invention
relates to a GLP-1 analogue or derivative pegylated or derivatised
with an albumin binding residue at position 18. In further aspect,
the invention relates to a GLP-1 analogue or derivative thereof
pegylated or derivatised with an albumin binding residue at
position 20. In a further aspect, the invention relates to a GLP-1
analogue or derivative thereof pegylated or derivatised with an
albumin binding residue at position 23. In further aspect, the
invention relates to a GLP-1 analogue or derivative thereof
pegylated or derivatised with an albumin binding residue at
position 31. In a further aspect, the invention relates to a GLP-1
analogue or derivative thereof pegylated or derivatised with an
albumin binding residue at position 33. In a further aspect, the
invention relates to a GLP-1 analogue or derivative thereof
pegylated or derivatised with an albumin binding residue at
position 34.
[0073] In one aspect, the invention relates to a GLP-1 analogue or
derivative thereof, which has been derivatised with an albumin
binding residue.
[0074] The term "derivatised" as used herein means chemically
connected via a covalent bond. For example a lysine residue or
cysteine residue is linked to an albumin binding residue via a
chemical bond. Such a chemical bond can as an example be obtained
by derivatisation of an epsilon amino group of lysine by acylation
with an active ester of an albumin binding residue such as a long
fatty acid.
[0075] Other examples of connecting two chemical moieties as used
in the present invention includes but is not limited to alkylation,
ester formation, amide formation or maleimide coupling.
[0076] The term "linker" as used herein means a spacer (the two
terms spacer and linker is used interchangeably in the present
specification) that separates a peptide and an albumin binding
residue or a polyethylene glycol polymer.
[0077] In one aspect of the invention, the linker comprises one or
more alkylene glycol units, such as 1 to 5 alkylene glycol units.
The alkylene glycol units are in a further aspect ethylene glycol,
propylene glycol or butylene glycol but can also be higher alkylene
glycols.
[0078] In another aspect of the invention, the linker is a
hydrophilic linker selected from
--(CH.sub.2).sub.lD[(CH.sub.2).sub.nE].sub.m(CH.sub.2).sub.p-Q.sub.q-,
wherein
[0079] I, m and n independently are 1-20 and p is 0-10,
Q is
--Z--(CH.sub.2).sub.lD[(CH.sub.2).sub.nG].sub.m(CH.sub.2).sub.p--,
[0080] q is an integer in the range from 0 to 5,
[0081] each D, E, and G are independently selected from --O--,
--NR.sup.3--, --N(COR.sup.4)--, --PR.sup.5(O)--, and
--P(OR.sup.6)(O)--, wherein R.sup.3, R.sup.4, R.sup.5, and R.sup.6
independently represent hydrogen or C.sub.1-6-alkyl,
[0082] Z is selected from --C(O)NH--, --C(O)NHCH.sub.2--,
--OC(O)NH--, --C(O)NHCH.sub.2CH.sub.2--, --C(O)CH.sub.2--,
--C(O)CH.dbd.CH--, --(CH.sub.2).sub.s--, --C(O)--, --C(O)O-- or
--NHC(O)--, wherein s is 0 or 1.
[0083] In another aspect of the invention, the linker is a
hydrophilic linker as defined above wherein I is 1 or 2, n and m
are independently 1-10 and p is 0-10.
[0084] In another aspect of the invention, the linker is a
hydrophilic linker as defined above wherein D is --O--.
[0085] In another aspect of the invention, the linker is a
hydrophilic linker as defined above wherein E is --O--.
[0086] In yet another aspect of the invention, the hydrophilic
linker is
[0087]
--CH.sub.2O[(CH.sub.2).sub.2O].sub.m(CH.sub.2).sub.pQ.sub.q-,
wherein m is 1-10, p is 1-3, and Q is
--Z--CH.sub.2O[(CH.sub.2).sub.2].sub.m(CH.sub.2).sub.p-- wherein Z
is as defined above.
[0088] In another aspect of the invention, the linker is a
hydrophilic linker as defined above wherein q is 1.
[0089] In another aspect of the invention, the linker is a
hydrophilic linker as defined above wherein G is --O--.
[0090] In another aspect of the invention, the linker is a
hydrophilic linker as defined above wherein Z is selected from the
group consisting of --C(O)NH--, --C(O)NHCH.sub.2--, and
--OC(O)NH--.
[0091] In another aspect of the invention, the linker is a
hydrophilic linker as defined above wherein q is 0.
[0092] In another aspect of the invention, the linker is a
hydrophilic linker as defined above wherein l is 2.
[0093] In another aspect of the invention, the linker is a
hydrophilic linker as defined above wherein n is 2.
[0094] In one aspect of this invention a "hydrophilic linker" is
used that separates a peptide and an albumin binding residue with a
chemical moiety.
[0095] In one aspect of this invention, the hydrophilic linker is
--C(O)--(CH.sub.2).sub.l--O--[(CH.sub.2CH.sub.2--O].sub.m--(CH.sub.2).sub-
.p--[NHC(O)--(CH.sub.2).sub.l--O--[(CH.sub.2).sub.n--O].sub.m--(CH.sub.2).-
sub.p].sub.q--NH--, wherein l, m, n, and p independently are 1-5,
and q is 0-5.
[0096] In yet another aspect of this invention, the hydrophilic
linker is
--C(O)--CH.sub.2--O--CH.sub.2CH.sub.2--O--CH.sub.2CH.sub.2[NHC(O)--CH.sub-
.2--O--CH.sub.2CH.sub.2O--CH.sub.2CH.sub.2].sub.q--NH--, wherein q
is 0-5.
[0097] In yet another aspect of this invention, the hydrophilic
linker is
--C(O)--CH.sub.2--O--CH.sub.2CH.sub.2--O--CH.sub.2CH.sub.2--NHC(O)--CH.su-
b.2--O--CH.sub.2CH.sub.2O--CH.sub.2CH.sub.2--NH--.
[0098] In yet another aspect of the invention, the hydrophilic
linker is --[CH.sub.2CH.sub.2O].sub.m+1(CH.sub.2).sub.pQ.sub.q--
wherein m and p independently is 0-10, and Q is
--Z--(CH.sub.2).sub.lD[(CH.sub.2).sub.nG].sub.m(CH.sub.2).sub.p--
as defined above.
[0099] In yet another aspect of the invention, the hydrophilic
linker is
--(CH.sub.2).sub.l--O--[(CH.sub.2).sub.n--O].sub.m--(CH.sub.2).sub.p--[C(-
O)NH--(CH.sub.2).sub.l--O--[(CH.sub.2).sub.n--O].sub.m--(CH.sub.2).sub.p].-
sub.q--, wherein l, m, n, and p independently are 1-5, and q is
0-5.
[0100] In a further aspect of the invention, the linker comprises
an amino acid residue except Cys, or a dipeptide such as Gly-Lys.
In the present text, the expression "a dipeptide such as Gly-Lys"
is used to designate a dipeptide wherein the C-terminal amino acid
residue is Lys, His or Trp, preferably Lys, and wherein the
N-terminal amino acid residue is selected from the group comprising
Ala, Arg, Asp, Asn, Gly, Glu, Gln, Ile, Leu, Val, Phe and Pro.]
Suitable PEG polymers are typically commercially available or may
be made by techniques well-known to those skilled in the art.
[0101] In one aspect of the invention, the PEG polymer has a
molecular weight of greater than 700D, in a further aspect a
molecular weight greater than 5 kD, in yet a further aspect greater
than 10 kD, and in a even further aspect greater that 20kD. The PEG
polymer may be linear or branched. In cases where the PEG polymer
is greater than 20KDa, the PEG polymer is preferable having a
branched structure, such as for example, a 43 kD branched
PEG-peptide (Shearwater 2001 catalog #2D3XOT01, mPEG2-MAL).
[0102] The attachment of a PEG on an intact peptide can be
accomplished by attaching the PEG on the opposite side of the
peptide surface that interacts with the receptor.
[0103] There are several strategies for coupling PEG to peptides
(see, e.g. Veronese, Biomaterials 22:405-417, 2001), all of which
are incorporated herein by reference in their entirety. Those
skilled in the art, will therefore be able to utilize well-known
techniques for linking the PEG polymer to human amylin or the
amylin analogs described herein.
[0104] Briefly, cysteine PEGylation is one method for site-specific
PEGylation, and can be accomplished by introducing a unique
cysteine mutation at one of the specific positions on human amylin
or the amylin analog and then reacting the resulting peptide with a
cysteine-specific PEGylation reagent, such as PEG-maleimide. It may
be necessary to mutate the peptide in order to allow for
site-specific PEGylation. For example, if the peptide contains
cysteine residues, these will need to be substituted with
conservative amino acids in order to ensure site-specific
PEGylation. In addition, rigid linkers, including but not limited
to "GGS", "GGSGGS", and "PPPS" may be added to the C-terminus, but
before the site of PEG attachment (i.e. a unique cysteine
residue).
[0105] In one aspect of the invention, the albumin binding residue
is a lipophilic residue. In a further aspect, the lipophilic
residue is attached to a lysine residue optionally via a linker by
conjugation chemistry such as by alkylation, acylation, ester
formation, or amide formation or to a cysteine residue by maleimide
coupling.
[0106] In a further aspect of the invention, the albumin binding
residue is negatively charged at physiological pH. In another
aspect of the invention, the albumin binding residue comprises a
group which can be negatively charged. One preferred group which
can be negatively charged is a carboxylic acid group.
[0107] In yet another aspect of the invention, the albumin binding
residue is selected from the group consisting of a straight chain
alkyl group, a branched alkyl group, a group which has an
w-carboxylic acid group, and a partially or completely hydrogenated
cyclopentanophenanthrene skeleton.
[0108] In a further aspect of the invention, the albumin binding
residue is a cibacronyl residue.
[0109] In a further aspect of the invention, the albumin binding
residue has from 6 to 40 carbon atoms, from 8 to 26 carbon atoms or
from 8 to 20 carbon atoms.
[0110] In a further aspect of the invention, the albumin binding
residue is an acyl group selected from the group comprising
CH.sub.3(CH.sub.2).sub.rCO--, wherein r is an integer from 4 to 38,
preferably an integer from 4 to 24, more preferred selected from
the group comprising CH.sub.3(CH.sub.2).sub.6CO--,
CH.sub.3(CH.sub.2).sub.8CO--, CH.sub.3(CH.sub.2).sub.10CO--,
CH.sub.3(CH.sub.2).sub.12CO--, CH.sub.3(CH.sub.2).sub.14CO--,
CH.sub.3(CH.sub.2).sub.16CO--, CH.sub.3(CH.sub.2).sub.18CO--,
CH.sub.3(CH.sub.2).sub.20CO-- and
CH.sub.3(CH.sub.2).sub.22CO--.
[0111] In another aspect of the invention, the albumin binding
residue is an acyl group of a straight-chain or branched alkane
.alpha.,.omega.-dicarboxylic acid.
[0112] In one aspect, the invention relates to a GLP-1 analogue or
derivative thereof, wherein at least one amino acid residue is
derivatised with A-B-C-D-
[0113] wherein A- is selected from the group consisting of
##STR00002##
[0114] wherein n is selected from the group consisting of 14, 15,
16 17, 18 and 19, p is selected from the group consisting of 10,
11, 12, 13 and 14, and d is selected from the group consisting of
0, 1, 2, 3, 4 and 5,
[0115] -B- is selected from the group consisting of
##STR00003##
[0116] wherein x is selected from the group consisting of 0, 1, 2,
3 and 4, and .gamma. is selected from the group consisting of 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11 and 12,
[0117] -C- is selected from the group consisting of
##STR00004##
[0118] wherein b and e are each independently selected from the
group consisting of 0, 1 and 2, and c and f are each independently
selected from the group consisting of 0, 1 and 2 with the proviso
that b is 1 or 2 when c is 0, or b is 0 when c is 1 or 2, and e is
1 or 2 when f is 0, or e is 0 when f is 1 or 2, and
[0119] -D- is attached to said amino acid residue and is a
linker.
[0120] In one aspect of the invention, one amino acid residue of
the analogue according to the invention is derivatised with
A-B-C-D-.
[0121] In one aspect, the derivatised amino acid residue comprises
an amino group. In a further aspect, the derivatised amino acid
residue comprises a primary amino group in a side chain. In yet a
further aspect, the derivatised amino acid residue is lysine.
[0122] In one aspect, A- is
##STR00005##
[0123] In one aspect, n is selected from the group consisting of 15
and 17, and more is preferred 17.
[0124] In one aspect, A- is
##STR00006##
[0125] In one aspect, p is selected from the group consisting of
12, 13, and 14 and more preferred is 13. In one aspect, d is
selected from the group consisting of 0, 1, 2, 3 and 4, more
preferred 0, 1 and 2 and most preferred 1. In one aspect, d is
selected from the group consisting of 0, 1 and 2 and p is selected
from the group consisting of 12, 13 or 14, more preferred d is
selected from the group consisting of 1 and 2 and p is selected
from the group consisting of 13 and 14, and most preferred d is 1
and p is 13.
[0126] In one aspect, -B- is
##STR00007##
[0127] In one aspect, -B- is
##STR00008##
[0128] In one aspect, -B- is
##STR00009##
[0129] In one aspect, -B- is
##STR00010##
[0130] In one aspect, x is selected from the group consisting of 0,
1 and 2, more preferred x is selected from the group consisting of
0 and 1 and most preferred x is 1.
[0131] In one aspect, -B- is
##STR00011##
[0132] In one aspect, .gamma. is selected from the group consisting
of 2, 3, 4, 5, 6, 7, 8, 9 and 10 and more preferred .gamma. is
selected from the group consisting of 2, 3, 4, 5, 6, 7, and 8.
[0133] In one aspect, -C- is
##STR00012##
[0134] In one aspect, c is selected from the group consisting of 0
and 1 and b is selected from the group consisting of 1 and 2, more
preferred b is 1 and c is 0.
[0135] In one aspect, -C- is
##STR00013##
[0136] In one aspect, f is selected from the group consisting of 0
and 1 and e is selected from the group consisting of 1 and 2, more
preferred e is 1 and f is 0.
[0137] In one aspect, -C- is
##STR00014##
[0138] In one aspect, D is selected from the group consisting
of
##STR00015##
and wherein k is selected from the group consisting of 0, 1, 2, 3,
4, 5, 11 and 27, and m is selected from the group consisting of 0,
1, 2, 3, 4, 5 and 6.
[0139] In one aspect, -D- is
##STR00016##
[0140] In one aspect, k is selected from the group consisting of 1,
2, 3, 11 and 27 and more preferred k is 1. In one aspect, m is
selected from the group consisting of 0, 1, 2, 3, and 4 and more
preferred m is selected from the group consisting of 0, 1 and
2.
[0141] In one aspect, -D- is
[0142] In one aspect, -D- is
##STR00017##
[0143] In one aspect, -D- is
##STR00018##
[0144] In one aspect, -D- is
##STR00019##
[0145] In one aspect, -D- is
##STR00020##
[0146] In one aspect, m is selected from the group consisting of 0,
1, 2, 3, and 4 and more preferred m is selected from the group
consisting of 0, 1 and 2.
[0147] In one aspect, A-B-C-D- is selected and combined from
##STR00021## ##STR00022##
[0148] In one aspect, A-B-C-D- is selected and combined from
##STR00023## ##STR00024##
[0149] In one aspect, A-B-C-D- is selected from the group
consisting of
##STR00025## ##STR00026## ##STR00027## ##STR00028##
[0150] In one aspect, the invention relates to a GLP-1 analogue or
derivative thereof, wherein at least one amino acid residue is
derivatised with A-B-C-D-, and where the derivative binds to
albumin.
[0151] In one aspect, A-B-C-D is composed of an albumin binding
fragment A-B-C- and a hydrophilic linker D.
[0152] The term "GLP-1 peptide" as used herein means GLP-1(7-35)
(SEQ ID No 1) or a GLP-1(7-35) analogue thereof.
[0153] In one embodiment the the GLP-1 analogue or derivative
thereof according to the invention is an insulinotropic agent.
[0154] In the aspect of the invention, wherein the analogue is
derivatised any amino acid position in the GLP-1 analogue may be
derivatised. In one aspect of the invention, the amino acid residue
which is derivatised comprises an amino group. Examples of amino
acid residues comprising an amino group is lysine, ornithine,
Epsilon-N-alkylated lysine such as Epsilon-N methylLysine,
O-aminoethylserine, O-aminopropylserine or longer O alkylated
serines containing a primary or secondary amino group in the side
chain.
[0155] In a further aspect of the invention, the derivatised amino
acid residue comprises a primary amino group in a side chain.
Examples of amino acid residues comprising a primary amino group is
lysine ornithine, O-aminoethylserine, O-aminopropylserine or longer
O alkylated serines containing a primary amino group in the side
chain. In yet a further aspect of the invention, the derivatised
amino acid residue is lysine. In yet a further aspect of the
invention, the derivative according to the invention is only
derivatised in one position, e.g. only one amino acid residue is
derivatised. In another aspect of this invention, the amino acid
residue which is derivatised is cysteine.
[0156] Functional Properties
[0157] A number of GLP-1 compounds of the invention have been
synthesized and tested as described in the experimental part.
[0158] The GLP-1 compounds of the invention have several
advantageous and beneficial properties as explained in the
following, by reference to the Examples.
[0159] The term "insulinotropic agent" as used herein means a GLP-1
analogue or derivative thereof which is an agonist of the human
GLP-1 receptor, i.e. a GLP-1 analogue or derivative thereof which
stimulates the formation of cAMP in a suitable medium containing
the human GLP-1 receptor (one such medium disclosed below).
[0160] In a first aspect, the GLP-1 analogue or derivative of the
invention has an acceptable, preferably high potency (at the
receptor).
[0161] The potency of an insulinotropic agent such as the GLP-1
compounds of the invention may be determined by calculating the
EC.sub.50 value from the dose-response curve, e.g. as described in
Example 21.
[0162] In particular embodiments (i) baby hamster kidney (BHK)
cells expressing the cloned human GLP-1 receptor are used,
preferably BHK-467-12A, more preferably BHK-467-12A (tk-ts13); (ii)
the cells are grown in DMEM media with the addition of 100 IU/mL
penicillin, 100 .mu.g/mL streptomycin, 5% fetal calf serum and 0.5
mg/mL Geneticin G-418 (Life Technologies), preferably at 5% CO2;
(iii) the cells, preferably at approximately 80% confluence, are
washed twice in phosphate buffered saline; (iv) the cells are
harvested with an aqueous solution of tetrasodium salt of
ethylenediaminetetraacetic acid, such as Versene; (v) plasma
membranes are prepared from the cells by homogenisation, preferably
in buffer 1 ; the homogenate is centrifuged, e.g. at 48,000.times.g
for 15 min at 4.degree. C.; and/or (vii) the pellet is suspended by
homogenization in buffer 2 (.Steps (vi) and (vii) are preferably
repeated, e.g. one or two times more. The functional receptor assay
may be carried out as described in Example 21 by measuring cyclic
AMP (cAMP) as a response to stimulation by the insulinotropic
agent. cAMP formed is preferably quantified by the AlphaScreen.TM.
cAMP Kit (Perkin Elmer Life Sciences). Incubations may be carried
out in half-area 96-well microtiter plates in a total volume of 50
.mu.L buffer 3 (50 mM Tris-HCl, 5 mM HEPES, 10 mM MgCl.sub.2, pH
7.4) and with the following addiditions: 1 mM ATP, 1 .mu.M GTP, 0.5
mM 3-isobutyl-1-methylxanthine (IBMX), 0.01% Tween-20, 0.1% BSA, 6
.mu.g membrane preparation, 15 .mu.g/mL acceptor beads, 20 .mu.g/mL
donor beads preincubated with 6 nM biotinyl-cAMP. Analogues or
derivatives to be tested for agonist activity are preferably
dissolved and diluted in buffer 3. GTP is freshly prepared for each
experiment. The plate is incubated in the dark with slow agitation
for three hours at room temperature followed by counting in the
Fusion.TM. instrument (Perkin Elmer Life Sciences).
Concentration-response curves are plotted for the individual
analogues or derivatives and EC.sub.50 values estimated using a
four-parameter logistic model with Prism v. 4.0, or 5.0 (GraphPad,
Carlsbad, Calif.).
[0163] In a first particular embodiment, the GLP-1 derivative of
the invention has a potency (EC.sub.50 in nM), as determined using
the cAMP assay, below 10.00, preferably below 9.00, more preferably
below 8.00, even more preferably below 7.00, and most preferably
below 6.00 (nM).
[0164] In a second particular embodiment, the GLP-1 derivative of
the invention has a potency (EC.sub.50 in nM), as determined using
the cAMP assay, below 5.00, preferably below 4.00, more preferably
below 3.00, even more preferably below 2.00, and most preferably
below 1.00 (nM).
[0165] In a third particular embodiment, the GLP-1 derivative of
the invention has a potency (EC.sub.50 in nM), as determined using
the cAMP assay, below 0.80, preferably below 0.60, more preferably
below 0.40, even more preferably below 0.20, and most preferably
below 0.10 (nM).
[0166] In a fourth particular embodiment, the GLP-1 derivative of
the invention has a potency (EC.sub.50 in nM), as determined using
the cAMP assay, below 0.090, preferably below 0.080, more
preferably below 0.070, even more preferably below 0.060, and most
preferably below 0.050 (nM).
[0167] In a fifth particular embodiment, the GLP-1 derivative of
the invention has a potency (EC.sub.50 in nM), as determined using
the cAMP assay, below 0.040, preferably below 0.030, more
preferably below 0.020, and most preferably below 0.010 (nM).
[0168] Accordingly, exemplary ranges of potency (EC.sub.50 in nM,
as determined using the cAMP assay) of GLP-1 derivatives of the
invention are 0.010-10.0, 0.010-8.0, 0.010-6.0, 0.010-4.0,
0.010-2.0, 0.010-1.00, 0.010-0.80, 0.010-0.60, 0.010-0.40,
0.010-0.30, 0.010-0.20, 0.010-0.10, and 0.010-0.90 (nM), preferably
0.010-0.40, 0.010-0.30, 0.010-0.20, 0.010-0.10, and 0.010-0.90
(nM).
[0169] In a second aspect, the GLP-1 analogue or derivative thereof
("GLP-1 compound") has a high affinity to the GLP-1 receptor. The
affinity to the GLP-1 receptor may be determined as described in
Example 23, i.e. by way of displacement of .sup.125I-GLP-1 from the
receptor. BHK cells may be used for membrane preparation,
preferably strain tk-ts13. The membranes may be purified,
preferably as described in Example 23, A preferred binding assay
method is the SPA assay of Example 23. The IC.sub.50 value may be
read from the resulting (binding) curve as the concentration which
displaces 50% of .sup.125I-GLP-1 from the receptor.
[0170] In a first particular embodiment, the IC.sub.50 value is
below 500 nM, preferably below 400 nM, more preferably below 300
nM, even more preferably below 200 nM, and most preferably below
100 nM.
[0171] In a second particular embodiment, the IC.sub.50 value is
below 80 nM, preferably below 60 nM, more preferably below 50 nM,
even more preferably below 40 nM, and most preferably below 30
nM.
[0172] In a third particular embodiment, the IC.sub.50 value is
below 20 nM, preferably below 15 nM, more preferably below 10 nM,
even more preferably below 5.0 nM, and most preferably below 4.0
nM. In a fourth particular embodiment, the IC.sub.50 value is below
3.0 nM, preferably below 2.0 nM, more preferably below 1.0 nM, even
more preferably below 0.80 nM, and most preferably below 0.60
nM.
[0173] In a fifth particular embodiment, the IC.sub.50 value is
below 0.50 nM, preferably below 0.40 nM, more preferably below 0.30
nM, even more preferably below 0.20 nM, and most preferably below
0.10 nM.
[0174] Accordingly, exemplary ranges of IC.sub.50 are: 0.1-400,
0.2-300, 0.3-200, 0.4-100, 0.5-50, and 1-10 nM.
[0175] In a third aspect, the present invention relates to a GLP-1
analogue or derivative thereof with high affinity binding to the
isolated N-terminal extracellular domain of the GLP-1 receptor
(nGLP-1R). The affinity may be measured as the ability to displace
.sup.125I-Exendin-4(9-39) from binding to nGLP-1R, e.g. as
described in Example 22.
[0176] In this assay Exendin-4 binds nGLP-1R with an IC.sub.50
value of 5 nM, GLP-1(7-37) binds nGLP-1R with an IC.sub.50 value of
1120 nM and liraglutide binds nGLP-1R with an IC.sub.50 value of
1500 nM. In one aspect of the invention, the GLP-1 analogues or
derivatives thereof of this invention binds nGLP-1R with an
IC.sub.50 value lower than that of liraglutide. More preferable the
GLP-1 analogues or derivatives thereof of this invention binds
nGLP-1R with an IC.sub.50 value lower than 100 nM and even more
preferable below 10 nM or even below 5 nM.
[0177] The protein nGLP-1R may be prepared as described by Runge et
al 2007 (In Biochemistry, vol. 46, pp. 5830-5840). The protein is
then biotinylated and immobilized, preferably on
streptavidin-coated SPA beads. The nGLP1R in a suitable buffer such
as 0.1M NaHCO.sub.3 may be biotinylated using 75 .mu.g BNHS (Sigma
H1759) to 1 mg protein. The biotinylated nGLP1R is subsequently
preferably dialyzed against PBS. All reagents and analogues or
derivatives are preferably diluted in PBS with 0.05% v/v Tween 20.
The binding assay may e.g. be carried out in 96 well OptiPlates
(PerkinElmer 6005290) in a final volume of 200 .mu.l. Each well may
contain 2 mg streptavidin coated SPA beads (PerkinElmer RPNQ007),
0.1 pmol biotinylated nGLP1R, 50 pCi .sup.125I-Exendin (9-39) and
test peptide in suitable final concentrations, e.g. ranging from
1000 nM to 0.064 nM. The plates are incubated on a shaker,
preferably at RT for 3 hours. The SPA particles may be spun down by
centrifugation, e.g. for 10 min at 1500 rpm, and the plates are
counted, e.g. in a TopCount-NXT (PerkinElmer).
[0178] The affinity may be expressed by way of an IC.sub.50 value,
which is read from the curve as the concentration of the GLP-1
derivative which displaces 50% of .sup.125I-Exendin-4(9-39) from
binding to nGLP-1R.
[0179] In a first particular embodiment, the GLP-1 derivative of
the invention has an affinity to the extracellular domain of the
GLP-1 receptor (nGLP-1R), measured as IC.sub.50/nM in the assay of
Example 22, of below 1500, preferably below 1000, even more
preferably below 900, and most preferably below 800 (nM).
[0180] In a second particular embodiment, the GLP-1 derivative of
the invention has an affinity to the extracellular domain of the
GLP-1 receptor (nGLP-1R), measured as IC.sub.50/nM in the assay of
Example 22, of below 700, preferably below 600, even more
preferably below 500, and most preferably below 400 (nM).
[0181] In a third particular embodiment, the GLP-1 derivative of
the invention has an affinity to the extracellular domain of the
GLP-1 receptor (nGLP-1R), measured as IC.sub.50/nM in the assay of
Example 22, of below 300, preferably below 200, even more
preferably below 100, and most preferably below 80 (nM).
[0182] In a fourth particular embodiment, the GLP-1 derivative of
the invention has an affinity to the extracellular domain of the
GLP-1 receptor (nGLP-1R), measured as IC.sub.50/nM in the assay of
Example 22, of below 60, preferably below 50, even more preferably
below 40, and most preferably below 30 (nM).
[0183] In a fifth particular embodiment, the GLP-1 derivative of
the invention has an affinity to the extracellular domain of the
GLP-1 receptor (nGLP-1R), measured as IC.sub.50/nM in the assay of
Example 22, of below 20, preferably below 15, even more preferably
below 10.0, and most preferably below 5.0 (nM).
[0184] Accordingly, exemplary ranges of affinity to nGLP-1R
(IC.sub.50 in nM) of the GLP-1 derivative of the invention are:
5-1500, 5-1000, 10-500, 20-300, 50-500, 10-500, and 5-50 (nM).
[0185] The term "DPP-IV protected" as used herein referring to a
polypeptide means a polypeptide which has been chemically modified
in order to render said derivative resistant to the plasma
peptidase dipeptidyl aminopeptidase-4 (DPP-IV). The DPP-IV enzyme
in plasma is known to be involved in the degradation of several
peptide hormones, e.g. GLP-1, GLP-2, Exendin-4 etc. Thus, a
considerable effort is being made to develop analogues and
derivatives of the polypeptides susceptible to DPP-IV mediated
hydrolysis in order to reduce the rate of degradation by
DPP-IV.
[0186] In one embodiment a GLP-1 analogue or derivative thereof
according to the invention is a DPP-IV protected GLP-1 analogue or
derivative thereof.
[0187] In one embodiment a GLP-1 analogue or derivative thereof
according to the invention is a DPP-IV protected GLP-1 analogue or
derivative thereof which is more resistant to DPP-IV than
liraglutide.
[0188] Resistance of a peptide to degradation by dipeptidyl
aminopeptidase IV is determined by the following degradation
assay:
[0189] Aliquots of the peptide (5 nmol) are incubated at 37.degree.
C. with 1 .mu.L of purified dipeptidyl aminopeptidase IV
corresponding to an enzymatic activity of 5 mU for 10-180 minutes
in 100 .mu.L of 0.1 M triethylamine-HCl buffer, pH 7.4. Enzymatic
reactions are terminated by the addition of 5 .mu.L of 10%
trifluoroacetic acid, and the peptide degradation products are
separated and quantified using HPLC analysis. One method for
performing this analysis is: The mixtures are applied onto a Vydac
C18 widepore (30 nm pores, 5 .mu.m particles) 250.times.4.6 mm
column and eluted at a flow rate of 1 ml/min with linear stepwise
gradients of acetonitrile in 0.1% trifluoroacetic acid (0%
acetonitrile for 3 min, 0-24% acetonitrile for 17 min, 24-48%
acetonitrile for 1 min) according to Siegel et al., Regul. Pept.
1999;79:93-102 and Mentlein et al. Eur. J. Biochem.
1993;214:829-35. Peptides and their degradation products may be
monitored by their absorbance at 220 nm (peptide bonds) or 280 nm
(aromatic amino acids), and are quantified by integration of their
peak areas related to those of standards. The rate of hydrolysis of
a peptide by dipeptidyl aminopeptidase IV is estimated at
incubation times which result in less than 10% of the peptide being
hydrolysed.
[0190] Alternatively, the resistance of a peptide to degradation by
dipeptidyl aminopeptidase IV is determined by the following
degradation assay:
[0191] Aliquots of the peptide (4 nmol) are incubated at 37.degree.
C. with 10.9 mU of purified dipeptidyl aminopeptidase IV for 22
hours in 40 .mu.L of 0.085 M Tris-HCl buffer, pH 8.0, in presence
or absence of 1.6% human serum albumin. After 0, 4, and 22 hours
samples of 10 .mu.l are taken and enzymatic reactions are
terminated by mixing with 100 .mu.l of 1% trifluoroacetic acid. The
peptide degradation products are separated and quantified using
HPLC analysis. One method for performing this analysis is: The
mixtures are applied onto an Agilent Zorbax 300SB-C18 (5 .mu.m
particles) 150.times.2.1 mm column and eluted at a flow rate of 0.5
ml/min with a linear gradient from 0.1% trifluoroacetic acid to
100% acetonitrile with 0.07% TFA in 30 minutes. Peptides and their
degradation products are monitored by their absorbance at 214 nm,
and are quantified by integration of their peak areas. The
stability of a peptide against dipeptidyl aminopeptidase IV is
determined as the peak area of the intact peptide relative to the
sum of the peak areas of the intact peptide and the degradation
product lacking the two aminoterminal amino acids after
cleavage.
[0192] The term "pharmaceutically acceptable" as used herein means
suited for normal pharmaceutical applications, i.e. giving rise to
no serious adverse events in patients etc.
[0193] The term "excipient" as used herein means the chemical
compounds which are normally added to pharmaceutical compositions,
e.g. buffers, tonicity agents, preservatives and the like.
[0194] The term "effective amount" as used herein means a dosage
which is sufficient to be effective for the treatment of the
patient compared with no treatment.
[0195] The term "pharmaceutical composition" as used herein means a
product comprising an active GLP-1 analogue or derivative thereof
according to the invention together with pharmaceutical excipients
such as buffer, preservative, and optionally a tonicity modifier
and/or a stabilizer. Thus a pharmaceutical composition is also
known in the art as a pharmaceutical formulation.
[0196] The term "treatment of a disease" as used herein means the
management and care of a patient having developed the disease,
condition or disorder. The purpose of treatment is to combat the
disease, condition or disorder. Treatment includes the
administration of the active analogue or derivative according to
the invention to eliminate or control the disease, condition or
disorder as well as to alleviate the symptoms or complications
associated with the disease, condition or disorder.
[0197] In another aspect, the present invention relates to a GLP-1
analogue or derivative thereof that can bind to albumin and the
GLP-1 receptor simultaneously.
[0198] In another aspect the present invention relates to a GLP-1
analogue or derivative thereof that bind to the GLP-1 receptor with
an affinity below 100nM, preferable below 30 nM in the presence of
2% albumin.
[0199] In another aspect, the GLP-1 analogue or derivative thereof
("GLP-1 compound") has an affinity to the GLP-1 receptor which is
only partly decreased when comparing the affinity in the presence
of very low concentration (e.g. 0.005% to 0.2%) of human albumin to
the affinity in the presence of 2% human albumin. The shift in
binding affinity under these conditions is less than 50 fold,
preferable below 30 fold and more preferable below 10 fold.
[0200] In another aspect the present invention relates to a GLP-1
analogue or derivative thereof which is stable against the chemical
degradation normally seen with exendin-4-especially oxidation and
deamidation.
[0201] In another aspect, the present invention relates to a GLP-1
analogue or derivative thereof which has a high potency at the
receptor. For very strong albumin binding analogues with albumin
binding affinity below 100 nM, the GLP-1 potency is better than 3
micro molar and preferable the potency is better than 1 micromolar
in the cAMP assay.
[0202] For strong albumin binding analogues or derivatives with
albumin binding affinity below 500 nM, the GLP-1 potency is better
than 1 micro molar and preferable the potency is better than 0.2
micromolar in the cAMP assay.
[0203] In another aspect, the present invention relates to a GLP-1
analogue or derivative thereof which has high albumin binding
affinity. The analogues or derivatives of this invention have an
albumin binding affinity that is below 1 micromolar. More
preferable the analogues or derivatives of this invention has an
albumin binding affinity that is below 500 nM and even more
preferable below 200 nM or even below 100 nM.
[0204] The albumin binding affinity can be measured using the
following assay:
[0205] Albumin binding assay:
[0206] The affinities of the GLP-1 analogue or derivative thereof
for human serum albumin (HSA) are measured by a competition
scintillation proximity assay (SPA). Streptavidin-SPA beads (GE
Healthcare RPNQ0009) are incubated with biotinylated HSA for 5
hours. The beads are washed with buffer to remove unbound HSA. The
beads are mixed with an .sup.125I-labeled acylated GLP-1 analogue
such as
N-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoyla-
mino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][A
ib8,.sup.125I-Tyr19,Arg34]GLP-1(7-37) or
N-epsilon37-[2-(2-[2-((S)-4-((S)-4-(12-[4-(16-(1H-tetrazol-5-yl)hexadecan-
oylsulfamoyl)butyrylamino]dodecanoylamino)-4-carboxybutyrylamino)-4-carbox-
ybutyrylamino)ethoxy]ethoxy)acetyl][Aib8,.sup.125I-Tyr19,G1u22,Arg26,34,Ly-
s37] GLP-1(7-37)-NH2 in a buffer containing 100 mM Hepes 100 mM
NaCl, 10 mM MgSO.sub.4, 0.025% Tween-20, pH 7.4. The mixture is
pipetted into the wells of a Perkin Elmer Optiplate-96 6005290 (100
.mu.l per well) and 100 .mu.l of a dilution series of the GLP-1
analogue or derivative to be measured is added in the same buffer.
After 20 hours of gentle rocking at room temperature the plates are
centrifuged and counted on a TopCounter. Bound cpm are plotted as a
function of GLP-1 analogue or derivative concentration end the EC50
value of the competition curve is used as a measure of the affinity
of the analogue or derivative for HSA.
[0207] In another aspect, the present invention relates to a GLP-1
analogue or derivative thereof which has substantially improved
terminal half-life in rodent and in a non-rodent model relative to
liraglutide.
[0208] In one aspect of this invention, the terminal half-life in
rodent or in a non-rodent model is improved at least 3 fold
relative to liraglutide.
[0209] In another aspect of this invention, the terminal half-life
in a non-rodent model is improved at least 6 fold relative to
liraglutide.
[0210] In another aspect, the present invention relates to a GLP-1
analogue or derivative thereof which has an in vivo half-life of at
least 10 hrs after i.v. administration to rats.
[0211] In another aspect, the present invention relates to a GLP-1
analogue or derivative thereof which has an in vivo half-life of at
least 50 hrs after s.c. administration to mini pigs, and preferable
an in vivo half-life of at least 80 hrs after s.c. administration
to mini pigs.
[0212] In another aspect, the present invention relates to a GLP-1
analogue or derivative thereof which can be formulated into
particles suitable for pulmonary administration.
[0213] In another aspect, the present invention relates to a GLP-1
analogue or derivative thereof which is chemically and physically
stable at neutral pH, most preferably in the range 6-8.
[0214] In another aspect, the present invention relates to a GLP-1
analogue or derivative thereof which has little or no tendency to
aggregate. In one aspect the aggregation tendency is significantly
improved relatively to the aggregation tendency of liraglutide when
tested in a thioflavin assay.
[0215] In another aspect, the present invention relates to a GLP-1
analogue or derivative thereof which is suitable for pulmonal
delivery. This may be with regard to physical or chemical aspects
which are useful for a pulmonal formulation. Alternatively, the
analogues or derivatives are stable against degradation by enzymes
in the airways and lungs.
[0216] In embodiments of the invention a combination of the above
features is achieved.
[0217] The term "albumin binding moiety" as used herein means a
residue which binds non-covalently to human serum albumin. The
albumin binding residue attached to the therapeutic polypeptide
typically has an albumin binding affinity that is below 1
micromolar, preferable below 500 nM and even more preferable below
200 nM or even below 100 nM.
[0218] A range of albumin binding residues are known among linear
and branched lipohophillic moieties containing 4-40 carbon atoms
having a distal acidic group.
[0219] The term "hydrophilic linker" as used herein means a spacer
that separates a peptide and an albumin binding residue with a
chemical moiety which comprises at least 5 non-hydrogen atoms where
30-50% of these are either N or O.
[0220] In one aspect the invention relates to a GLP-1 analogue or
derivative thereof, which comprises a hydrophilic linker between
the modified GLP-1 sequence and one or more albumin binding
residue(s).
[0221] In one aspect, the hydrophilic linker is an unbranched oligo
ethylene glycol moiety with appropriate functional groups at both
terminals that forms a bridge between an amino group of the
modified GLP-1 sequence and a functional group of the albumin
binding residue.
[0222] In the formulas herein the terminal bonds from the attached
groups are to be regarded as attachment bonds and not ending in
methylene groups unless stated.
[0223] In one aspect of the invention, the GLP-1 analogue or
derivative is selected form the group consisting of
[0224] [Glu22,Arg26]GLP-1 (7-33) amide,
[0225] N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Va125,-
Arg26,Leu27,Glu30, Lys33)GLP-1(7-33)amide,
[0226] N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Arg26,-
Glu30) GLP-1(7-33) amide,
[0227] [Glu22,Va125,Arg26] GLP-1 (7-33)amide,
[0228] N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Arg26,-
Glu30) GLP-1(7-33) amide,
[0229] [Glu22, Arg26]GLP-1(7-33)peptide,
[0230] N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-[Aib8,Lys20,Glu22,Va125,-
Arg26,Glu30] GLP-1 (7-33) amide, and
[0231] [Glu22,Va125,Arg26] GLP-1 (7-32)amide.
[0232] Formulation
[0233] Another object of the present invention is to provide a
pharmaceutical formulation comprising an analogue or derivative
according to the present invention which is present in a
concentration from 0.1 mg/ml to 25 mg/ml, and wherein said
formulation has a pH from 3.0 to 9.0. The formulation may further
comprise a buffer system, preservative(s), tonicity agent(s),
chelating agent(s), stabilizers and surfactants.
[0234] In one embodiment of the invention, the pharmaceutical
formulation is an aqueous formulation, i.e. formulation comprising
water. Such formulation is typically a solution or a
suspension.
[0235] In a further embodiment of the invention, the pharmaceutical
formulation is an aqueous solution.
[0236] The term "aqueous formulation" is defined as a formulation
comprising at least 50% w/w water. Likewise, the term "aqueous
solution" is defined as a solution comprising at least 50% w/w
water, and the term "aqueous suspension" is defined as a suspension
comprising at least 50% w/w water.
[0237] In another embodiment, the pharmaceutical formulation is a
freeze-dried formulation, whereto the physician or the patient adds
solvents and/or diluents prior to use.
[0238] In another embodiment, the pharmaceutical formulation is a
dried formulation (e.g. freeze-dried or spray-dried) ready for use
without any prior dissolution.
[0239] In a further aspect, the invention relates to a
pharmaceutical formulation comprising an aqueous solution of an
analogue or derivative according to the present invention, and a
buffer, wherein said analogue or derivative is present in a
concentration from 0.1 mg/ml or above, and wherein said formulation
has a pH from about 3.0 to about 9.0.
[0240] In another embodiment of the invention, the pH of the
formulation is from about 7.0 to about 9.5. In another embodiment
of the invention, the pH of the formulation is from about 3.0 to
about 7.0. In another embodiment of the invention, the pH of the
formulation is from about 5.0 to about 7.5. In another embodiment
of the invention, the pH of the formulation is from about 7.5 to
about 9.0. In another embodiment of the invention, the pH of the
formulation is from about 7.5 to about 8.5. In another embodiment
of the invention, the pH of the formulation is from about 6.0 to
about 7.5. In another embodiment of the invention, the pH of the
formulation is from about 6.0 to about 7.0. In another embodiment,
the pharmaceutical formulation is from 8.0 to 8.5.
[0241] In an embodiment of the invention, each administered dose
contains from 0.01 mg-10 mg of active analogue or derivative
according to the invention. In an embodiment, the dose administered
contains more than 0.05 mg active analogue or derivative. In an
embodiment, the dose administered contains more than 0.1 mg active
analogue or derivative according to the invention. In an
embodiment, the dose administered contains up to 10 mg active
analogue or derivative according to the invention. In an
embodiment, the dose administered contains up to 9 mg active
analogue or derivative according to the invention. In an
embodiment, the dose administered contains up to 8 mg active
analogue or derivative according to the invention. In an
embodiment, the dose administered contains up to 7 mg active
analogue or derivative according to the invention. In an
embodiment, the dose administered contains up to 6 mg active
analogue or derivative according to the invention. In an
embodiment, the dose administered contains up to 5 mg active
analogue or derivative according to the invention. In an
embodiment, the dose administered contains from 0.2 mg to 5 mg
active analogue or derivative according to the invention.
[0242] In a further embodiment of the invention, the buffer is
selected from the group consisting of sodium acetate, sodium
carbonate, citrate, glycylglycine, histidine, glycine, lysine,
arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate,
sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine,
tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric
acid, aspartic acid or mixtures thereof. Each one of these specific
buffers constitutes an alternative embodiment of the invention.
[0243] In a further embodiment of the invention, the formulation
further comprises a pharmaceutically acceptable preservative. In a
further embodiment of the invention the preservative is selected
from the group consisting of phenol, o-cresol, m-cresol, p-cresol,
methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,
2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl
alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid,
imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol,
ethyl p-hydroxybenzoate, benzethonium chloride, chlorphenesine
(3p-chlorphenoxypropane-1,2-diol) or mixtures thereof. In an
embodiment, the preservative is phenol or m-cresol. In a further
embodiment of the invention, the preservative is present in a
concentration from 0.1 mg/ml to 20 mg/ml. In a further embodiment
of the invention, the preservative is present in a concentration
from 0.1 mg/ml to 5 mg/ml. In a further embodiment of the
invention, the preservative is present in a concentration from 5
mg/ml to 10 mg/ml. In a further embodiment of the invention, the
preservative is present in a concentration from 10 mg/ml to 20
mg/ml. Each one of these specific preservatives constitutes an
alternative embodiment of the invention. The use of a preservative
in pharmaceutical compositions is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 19.sup.th edition, 1995.
[0244] In a further embodiment of the invention, the formulation
further comprises an isotonic agent. In a further embodiment of the
invention, the isotonic agent is selected from the group consisting
of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an
amino acid (e.g. L-glycine, L-histidine, arginine, lysine,
isoleucine, aspartic acid, tryptophan, threonine), an alditol (e.g.
glycerol (glycerine), 1,2-propanediol (propyleneglycol),
1,3-propanediol, 1,3-butanediol) polyethyleneglycol (e.g. PEG400),
or mixtures thereof. In an embodiment, the isotoncity agent is
propyleneglycol. Any sugar such as mono-, di-, or polysaccharides,
or water-soluble glucans, including for example fructose, glucose,
mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose,
dextran, pullulan, dextrin, cyclodextrin, alfa and beta HPCD,
soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na
may be used. In one embodiment, the sugar additive is sucrose.
Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one
--OH group and includes, for example, mannitol, sorbitol, inositol,
galactitol, dulcitol, xylitol, and arabitol. In one embodiment, the
sugar alcohol additive is mannitol. The sugars or sugar alcohols
mentioned above may be used individually or in combination. There
is no fixed limit to the amount used, as long as the sugar or sugar
alcohol is soluble in the liquid preparation and does not adversely
effect the stabilizing effects achieved using the methods of the
invention. In one embodiment, the sugar or sugar alcohol
concentration is between about 1 mg/ml and about 150 mg/ml. In a
further embodiment of the invention, the isotonic agent is present
in a concentration from 1 mg/ml to 50 mg/ml. In a further
embodiment of the invention, the isotonic agent is present in a
concentration from 1 mg/ml to 7 mg/ml. In an embodiment of the
invention, the isotonic agent is present in a concentration from 5
mg/ml to 7 mg/ml. In a further embodiment of the invention, the
isotonic agent is present in a concentration from 8 mg/ml to 24
mg/ml. In a further embodiment of the invention, the isotonic agent
is present in a concentration from 25 mg/ml to 50 mg/ml. Each one
of these specific isotonic agents constitutes an alternative
embodiment of the invention. The use of an isotonic agent in
pharmaceutical compositions is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 19.sup.th edition, 1995.
[0245] In a further embodiment of the invention, the formulation
further comprises a chelating agent. In a further embodiment of the
invention the chelating agent is selected from salts of
ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic
acid, and mixtures thereof. In a further embodiment of the
invention the chelating agent is present in a concentration from
0.1 mg/ml to 5 mg/ml. In a further embodiment of the invention the
chelating agent is present in a concentration from 0.1 mg/ml to 2
mg/ml. In a further embodiment of the invention the chelating agent
is present in a concentration from 2 mg/ml to 5 mg/ml. Each one of
these specific chelating agents constitutes an alternative
embodiment of the invention. The use of a chelating agent in
pharmaceutical compositions is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 19.sup.th edition, 1995.
[0246] In a further embodiment of the invention, the formulation
further comprises a stabilizer. The use of a stabilizer in
pharmaceutical compositions is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 19.sup.th edition, 1995.
[0247] More particularly, compositions of the invention are
stabilized liquid pharmaceutical compositions whose therapeutically
active components include a polypeptide that possibly exhibits
aggregate formation during storage in liquid pharmaceutical
formulations.
[0248] By "aggregate formation" is intended a physical interaction
between the polypeptide molecules that results in formation of
oligomers, which may remain soluble, or large visible aggregates
that precipitate from the solution. By "during storage" is intended
a liquid pharmaceutical composition or formulation once prepared,
is not immediately administered to a subject. Rather, following
preparation, it is packaged for storage, either in a liquid form,
in a frozen state, or in a dried form for later reconstitution into
a liquid form or other form suitable for administration to a
subject. By "dried form" is intended the liquid pharmaceutical
composition or formulation is dried either by freeze drying (i.e.,
lyophilization; see, for example, Williams and Polli (1984) J.
Parenteral Sci. Technol. 38:48-59), spray drying (see Masters
(1991) in Spray-Drying Handbook (5th ed; Longman Scientific and
Technical, Essez, U.K.), pp. 491-676; Broadhead et al. (1992) Drug
Devel. Ind. Pharm. 18:1169-1206; and Mumenthaler et al. (1994)
Pharm. Res. 11:12-20), or air drying (Carpenter and Crowe (1988)
Cryobiology 25:459-470; and Roser (1991) Biopharm. 4:47-53).
Aggregate formation by a polypeptide during storage of a liquid
pharmaceutical composition can adversely affect biological activity
of that polypeptide, resulting in loss of therapeutic efficacy of
the pharmaceutical composition. Furthermore, aggregate formation
may cause other problems such as blockage of tubing, membranes, or
pumps when the polypeptide-containing pharmaceutical composition is
administered using an infusion system.
[0249] The pharmaceutical compositions of the invention may further
comprise an amount of an amino acid base sufficient to decrease
aggregate formation by the polypeptide during storage of the
composition. By "amino acid base" is intended an amino acid or a
combination of amino acids, where any given amino acid is present
either in its free base form or in its salt form. Where a
combination of amino acids is used, all of the amino acids may be
present in their free base forms, all may be present in their salt
forms, or some may be present in their free base forms while others
are present in their salt forms. In one embodiment, amino acids to
use in preparing the compositions of the invention are those
carrying a charged side chain, such as arginine, lysine, aspartic
acid, and glutamic acid. Any stereoisomer (i.e., L, D, or a mixture
thereof) of a particular amino acid (e.g. methionine, histidine,
imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan,
threonine and mixtures thereof) or combinations of these
stereoisomers, may be present in the pharmaceutical compositions of
the invention so long as the particular amino acid is present
either in its free base form or its salt form.
[0250] In one embodiment the L-stereoisomer is used. Compositions
of the invention may also be formulated with analogues of these
amino acids. By "amino acid analogue" is intended a derivative of
the naturally occurring amino acid that brings about the desired
effect of decreasing aggregate formation by the polypeptide during
storage of the liquid pharmaceutical compositions of the invention.
Suitable arginine analogues include, for example, aminoguanidine,
ornithine and N-monoethyl L-arginine, suitable methionine analogues
include ethionine and buthionine and suitable cysteine analogues
include S-methyl-L cysteine. As with the other amino acids, the
amino acid analogues are incorporated into the compositions in
either their free base form or their salt form. In a further
embodiment of the invention the amino acids or amino acid analogues
are used in a concentration, which is sufficient to prevent or
delay aggregation of the protein.
[0251] In a further embodiment of the invention, methionine (or
other sulphuric amino acids or amino acid analogous) may be added
to inhibit oxidation of methionine residues to methionine sulfoxide
when the polypeptide acting as the therapeutic agent is a
polypeptide comprising at least one methionine residue susceptible
to such oxidation. By "inhibit" is intended minimal accumulation of
methionine oxidized species over time. Inhibiting methionine
oxidation results in greater retention of the polypeptide in its
proper molecular form. Any stereoisomer of methionine (L or D) or
combinations thereof can be used. The amount to be added should be
an amount sufficient to inhibit oxidation of the methionine
residues such that the amount of methionine sulfoxide is acceptable
to regulatory agencies. Typically, this means that the composition
contains no more than about 10% to about 30% methionine sulfoxide.
Generally, this can be achieved by adding methionine such that the
ratio of methionine added to methionine residues ranges from about
1:1 to about 1000:1, such as 10:1 to about 100:1.
[0252] In a further embodiment of the invention, the formulation
further comprises a stabilizer selected from the group of high
molecular weight polymers or low molecular compounds.
[0253] In a further embodiment of the invention the stabilizer is
selected from polyethylene glycol (e.g. PEG 3350), polyvinyl
alcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycellulose or
derivates thereof (e.g. HPC, HPC-SL, HPC-L and HPMC),
cyclodextrins, sulphur-containing substances as monothioglycerol,
thioglycolic acid and 2-methylthioethanol, and different salts
(e.g. sodium chloride). Each one of these specific stabilizers
constitutes an alternative embodiment of the invention.
[0254] The pharmaceutical compositions may also comprise additional
stabilizing agents, which further enhance stability of a
therapeutically active polypeptide therein.
[0255] Stabilizing agents of particular interest to the present
invention include, but are not limited to, methionine and EDTA,
which protect the polypeptide against methionine oxidation, and a
nonionic surfactant, which protects the polypeptide against
aggregation associated with freeze-thawing or mechanical
shearing.
[0256] In a further embodiment of the invention, the formulation
further comprises a surfactant. In another embodiment of the
invention, the pharmaceutical composition comprises two different
surfactants. The term "Surfactant" as used herein refers to any
molecules or ions that are comprised of a water-soluble
(hydrophilic) part, the head, and a fat-soluble (lipophilic)
segment. Surfactants accumulate preferably at interfaces, which the
hydrophilic part is orientated towards the water (hydrophilic
phase) and the lipophilic part towards the oil- or hydrophobic
phase (i.e. glass, air, oil etc.). The concentration at which
surfactants begin to form micelles is known as the critical micelle
concentration or CMC. Furthermore, surfactants lower the surface
tension of a liquid. Surfactants are also known as amphipathic
compounds. The term "Detergent" is a synonym used for surfactants
in general.
[0257] Anionic surfactants may be selected from the group of:
Chenodeoxycholic acid, Chenodeoxycholic acid sodium salt, Cholic
acid, Dehydrocholic acid, Deoxycholic acid, Deoxycholic acid methyl
ester, Digitonin, Digitoxigenin, N,N-Dimethyldodecylamine N-oxide,
Docusate sodium, Glycochenodeoxycholic acid sodium, Glycocholic
acid hydrate, Glycodeoxycholic acid monohydrate, Glycodeoxycholic
acid sodium salt, Glycodeoxycholic acid sodium salt,
Glycolithocholic acid 3-sulfate disodium salt, Glycolithocholic
acid ethyl ester, N-Lauroylsarcosine sodium salt,
N-Lauroylsarcosine sodium salt, N-Lauroylsarcosine,
N-Lauroylsarcosine, Lithium dodecyl sulfate, Lugol,
1-Octanesulfonic acid sodium salt, 1-Octanesulfonic acid sodium
salt, Sodium 1-butanesulfonate, Sodium 1-decanesulfonate, Sodium
1-dodecanesulfonate, Sodium 1-heptanesulfonate, Sodium
1-heptanesulfonate, Sodium 1-nonanesulfonate, Sodium
1-propanesulfonate monohydrate, Sodium 2-bromoethanesulfonate,
Sodium cholate hydrate, ox or sheep bile, Sodium cholate hydrate,
Sodium choleate, Sodium deoxycholate, Sodium dodecyl sulfate,
Sodium dodecyl sulfate, Sodium hexanesulfonate, Sodium octyl
sulfate, Sodium pentanesulfonate, Sodium taurocholate,
Taurochenodeoxycholic acid sodium salt, Taurodeoxycholic acid
sodium salt monohydrate, Taurolithocholic acid 3-sulfate disodium
salt, Tauroursodeoxycholic acid sodium salt, Trizma.RTM. dodecyl
sulfate, DSS (docusate sodium, CAS registry no [577-11-7]),
docusate calcium, CAS registry no [128-49-4]), docusate potassium,
CAS registry no [7491-09-0]), SDS (sodium dodecyl sulfate or sodium
lauryl sulfate), Dodecylphosphocholine (FOS-Choline-12),
Decylphosphocholine (FOS-Choline-10), Nonylphosphocholine
(FOS-Choline-9), dipalmitoyl phosphatidic acid, sodium caprylate,
and/or Ursodeoxycholic acid.
[0258] Cationic surfactants may be selected from the group of:
Alkyltrimethylammonium bromide
[0259] Benzalkonium chloride, Benzalkonium chloride,
Benzyldimethylhexadecylammonium chloride,
Benzyldimethyltetradecylammonium chloride, Benzyltrimethylammonium
tetrachloroiodate, Dimethyldioctadecylammonium bromide,
Dodecylethyldimethylammonium bromide, Dodecyltrimethylammonium
bromide, Dodecyltrimethylammonium bromide,
Ethylhexadecyldimethylammonium bromide, Hexadecyltrimethylammonium
bromide, Hexadecyltrimethylammonium bromide,
Polyoxyethylene(10)-N-tallow-1,3-diaminopropane, Thonzonium
bromide, and/or Trimethyl(tetradecyl)ammonium bromide.
[0260] Nonionic surfactants may be selected from the group of:
BigCHAP, Bis(polyethylene glycol bis[imidazoyl carbonyl]), block
copolymers as polyethyleneoxide/polypropyleneoxide block copolymers
such as poloxamers, poloxamer 188 and poloxamer 407, Brij.RTM. 35,
Brij.RTM. 56, Brij.RTM. 72, Brij.RTM. 76, Brij.RTM. 92V, Brij.RTM.
97, Brij.RTM. 58P, Cremophor.RTM. EL, Decaethylene glycol
monododecyl ether, N-Decanoyl-N-methylglucamine,
n-Dodecanoyl-N-methylglucamide, alkyl-polyglucosides, ethoxylated
castor oil, Heptaethylene glycol monodecyl ether, Heptaethylene
glycol monododecyl ether, Heptaethylene glycol monotetradecyl
ether, Hexaethylene glycol monododecyl ether, Hexaethylene glycol
monohexadecyl ether, Hexaethylene glycol monooctadecyl ether,
Hexaethylene glycol monotetradecyl ether, Igepal CA-630, Igepal
CA-630, Methyl-6-O-(N-heptylcarbamoyl)-beta-D-glucopyranoside,
Nonaethylene glycol monododecyl ether,
N-Nonanoyl-N-methylglucamine, N-Nonanoyl-N-methylglucamine,
Octaethylene glycol monodecyl ether, Octaethylene glycol
monododecyl ether, Octaethylene glycol monohexadecyl ether,
Octaethylene glycol monooctadecyl ether, Octaethylene glycol
monotetradecyl ether, Octyl-.beta.-D-glucopyranoside, Pentaethylene
glycol monodecyl ether, Pentaethylene glycol monododecyl ether,
Pentaethylene glycol monohexadecyl ether, Pentaethylene glycol
monohexyl ether, Pentaethylene glycol monooctadecyl ether,
Pentaethylene glycol monooctyl ether, Polyethylene glycol
diglycidyl ether, Polyethylene glycol ether W-1, Polyoxyethylene 10
tridecyl ether, Polyoxyethylene 100 stearate, Polyoxyethylene 20
isohexadecyl ether, Polyoxyethylene 20 oleyl ether, Polyoxyethylene
40 stearate, Polyoxyethylene 50 stearate, Polyoxyethylene 8
stearate, Polyoxyethylene bis(imidazolyl carbonyl), Polyoxyethylene
25 propylene glycol stearate, Saponin from Quillaja bark, Span.RTM.
20, Span.RTM. 40, Span.RTM. 60, Span.RTM. 65, Span.RTM. 80,
Span.RTM. 85, Tergitol, Type 15-S-12, Tergitol, Type 15-S-30,
Tergitol, Type 15-S-5, Tergitol, Type 15-S-7, Tergitol, Type
15-S-9, Tergitol, Type NP-10, Tergitol, Type NP-4, Tergitol, Type
NP-40, Tergitol, Type NP-7, Tergitol, Type NP-9,
Tetradecyl-.beta.-D-maltoside, Tetraethylene glycol monodecyl
ether, Tetraethylene glycol monododecyl ether, Tetraethylene glycol
monotetradecyl ether, Triethylene glycol monodecyl ether,
Triethylene glycol monododecyl ether, Triethylene glycol
monohexadecyl ether, Triethylene glycol monooctyl ether,
Triethylene glycol monotetradecyl ether, Triton CF-21, Triton
CF-32, Triton DF-12, Triton DF-16, Triton GR-5M, Triton QS-15,
Triton QS-44, Triton X-100, Triton X-102, Triton X-15, Triton
X-151, Triton X-200, Triton X-207, Triton.RTM. X-100, Triton.RTM.
X-114, Triton.RTM. X-165 solution, Triton.RTM. X-305 solution,
Triton.RTM. X-405, Triton.RTM. X-45, Triton.RTM. X-705-70,
TWEEN.RTM. 20, TWEEN.RTM. 40, TWEEN.RTM. 60, TWEEN.RTM. 6,
TWEEN.RTM. 65, TWEEN.RTM. 80, TWEEN.RTM. 81, TWEEN.RTM. 85,
Tyloxapol, sphingophospholipids (sphingomyelin), and
sphingoglycolipids (ceramides, gangliosides), phospholipids, and/or
n-Undecyl .beta.-D-glucopyranoside.
[0261] Zwitterionic surfactants may be selected from the group of:
CHAPS, CHAPSO, 3-(Decyldimethylammonio)propanesulfonate inner salt,
3-(Dodecyldimethylammonio)-propanesulfonate inner salt,
3-(Dodecyldimethylammonio)propanesulfonate inner salt,
3-(N,N-Dimethylmyristylammonio)propanesulfonate,
3-(N,N-Dimethyloctadecyl-ammonio)propanesulfonate,
3-(N,N-Dimethyloctylammonio)propanesulfonate inner salt,
3-(N,N-Dimethylpalmitylammonio)propanesulfonate,
N-alkyl-N,N-dimethylammonio-1-propanesulfonates,
3-cholamido-1-propyldimethylammonio-1-propanesulfonate,
Dodecylphosphocholine, myristoyl lysophosphatidylcholine,
Zwittergent 3-12
(N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), Zwittergent
3-10 (3-(Decyldimethyl-ammonio)propanesulfonate inner salt),
Zwittergent 3-08 (3-(Octyldimethyl-ammonio)pro-panesulfonate),
glycerophospholipids (lecithins, kephalins, phosphatidyl serine),
glyceroglycolipids (galactopyranoside), alkyl, alkoxyl (alkyl
ester), alkoxy (alkyl ether)-derivatives of lysophosphatidyl and
phosphatidylcholines, e.g. lauroyl and myristoyl derivatives of
lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and
modifications of the polar head group, that is cholines,
ethanolamines, phosphatidic acid, serines, threonines, glycerol,
inositol, lysophosphatidylserine and lysophosphatidylthreonine,
acylcarnitines and derivatives, N.sup.beta-acylated derivatives of
lysine, arginine or histidine, or side-chain acylated derivatives
of lysine or arginine, N.sup.beta-acylated derivatives of
dipeptides comprising any combination of lysine, arginine or
histidine and a neutral or acidic amino acid, N.sup.beta-acylated
derivative of a tripeptide comprising any combination of a neutral
amino acid and two charged amino acids, or the surfactant may be
selected from the group of imidazoline derivatives, long-chain
fatty acids and salts thereof C.sub.6-C.sub.12 (eg. oleic acid and
caprylic acid),
N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, anionic
(alkyl-aryl-sulphonates) monovalent surfactants, palmitoyl
lysophosphatidyl-L-serine, lysophospholipids (e.g.
1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline,
serine or threonine), or mixtures thereof.
[0262] The term "alkyl-polyglucosides" as used herein in relates to
an straight or branched C.sub.5-20-alkyl, -alkenyl or -alkynyl
chain which is substituted by one or more glucoside moieties such
as maltoside, saccharide etc. Embodiments of these
alkyl-polyglucosides include C.sub.6-18-alkyl-polyglucosides.
Specific embodiments of these alkyl-polyglucosides includes the
even numbered carbon-chains such as C.sub.6, C.sub.8, C.sub.10,
C.sub.12, C.sub.14, C.sub.16, C.sub.18 and C.sub.20 alkyl chain.
Specific embodiments of the glucoside moieties include pyranoside,
glucopyranoside, maltoside, maltotrioside and sucrose. In
embodiments of the invention, less than 6 glucosid moieties are
attached to the alkyl group. In embodiments of the invention, less
than 5 glucosid moieties are attached to the alkyl group. In
embodiments of the invention, less than 4 glucosid moieties are
attached to the alkyl group. In embodiments of the invention, less
than 3 glucosid moieties are attached to the alkyl group. In
embodiments of the invention, less than 2 glucosid moieties are
attached to the alkyl group. Specific embodiments of
alkyl-polyglucosides are alkyl glucosides such n-decyl
.beta.-D-glucopyranoside, decyl .beta.-D-maltopyranoside, dodecyl
.beta.-D-glucopyranoside, n-dodecyl .beta.-D-maltoside, n-dodecyl
.beta.-D-maltoside, n-dodecyl .beta.-D-maltoside, tetradecyl
.beta.-D-glucopyranoside, decyl .beta.-D-maltoside, hexadecyl
.beta.-D-maltoside, decyl .beta.-D-maltotrioside, dodecyl
.beta.-D-maltotrioside, tetradecyl .beta.-D-maltotrioside,
hexadecyl .beta.-D-maltotrioside, n-dodecyl-sucrose,
n-decyl-sucrose, sucrose monocaprate, sucrose monolaurate, sucrose
monomyristate, and sucrose monopalmitate.
[0263] The use of a surfactant in pharmaceutical compositions is
well-known to the skilled person. For convenience reference is made
to Remington: The Science and Practice of Pharmacy, 19.sup.th
edition, 1995.
[0264] In a further embodiment of the invention, the formulation
further comprises protease inhibitors such as EDTA (ethylenediamine
tetraacetic acid) and benzamidineHCl, but other commercially
available protease inhibitors may also be used. The use of a
protease inhibitor is particular useful in pharmaceutical
compositions comprising zymogens of proteases in order to inhibit
autocatalysis.
[0265] It is possible that other ingredients may be present in the
peptide pharmaceutical formulation of the present invention. Such
additional ingredients may include wetting agents, emulsifiers,
antioxidants, bulking agents, tonicity modifiers, chelating agents,
metal ions, oleaginous vehicles, proteins (e.g., human serum
albumin, gelatine or proteins) and a zwitterion (e.g., an amino
acid such as betaine, taurine, arginine, glycine, lysine and
histidine). Such additional ingredients, of course, should not
adversely affect the overall stability of the pharmaceutical
formulation of the present invention.
[0266] Pharmaceutical compositions containing an analogue or
derivative according to the invention may be administered to a
patient in need of such treatment at several sites, for example, at
topical sites, for example, skin and mucosal sites, at sites which
bypass absorption, for example, administration in an artery, in a
vein, in the heart, and at sites which involve absorption, for
example, administration in the skin, under the skin, in a muscle or
in the abdomen.
[0267] Administration of pharmaceutical compositions according to
the invention may be through several routes of administration, for
example, lingual, sublingual, buccal, in the mouth, oral, in the
stomach and intestine, nasal, pulmonary, for example, through the
bronchioles and alveoli or a combination thereof, epidermal,
dermal, transdermal, vaginal, rectal, ocular, for examples through
the conjunctiva, uretal, and parenteral to patients in need of such
a treatment.
[0268] Compositions of the current invention may be administered in
several dosage forms, for example, as solutions, suspensions,
emulsions, microemulsions, multiple emulsion, foams, salves,
pastes, plasters, ointments, tablets, coated tablets, chewing gum,
rinses, capsules, for example, hard gelatine capsules and soft
gelatine capsules, suppositories, rectal capsules, drops, gels,
sprays, powder, aerosols, inhalants, eye drops, ophthalmic
ointments, ophthalmic rinses, vaginal pessaries, vaginal rings,
vaginal ointments, injection solution, in situ transforming
solutions, for example in situ gelling, in situ setting, in situ
precipitating, in situ crystallization, infusion solution, and
implants. Compositions of the invention may further be compounded
in, or attached to, for example through covalent, hydrophobic and
electrostatic interactions, a drug carrier, drug delivery system
and advanced drug delivery system in order to further enhance
stability of the analogue or derivative of the present invention,
increase bioavailability, increase solubility, decrease adverse
effects, achieve chronotherapy well known to those skilled in the
art, and increase patient compliance or any combination thereof.
Examples of carriers, drug delivery systems and advanced drug
delivery systems include, but are not limited to, polymers, for
example cellulose and derivatives, polysaccharides, for example
dextran and derivatives, starch and derivatives, poly(vinyl
alcohol), acrylate and methacrylate polymers, polylactic and
polyglycolic acid and block co-polymers thereof, polyethylene
glycols, carrier proteins, for example albumin, gels, for example,
thermogelling systems, for example block co-polymeric systems well
known to those skilled in the art, micelles, liposomes,
microspheres, nanoparticulates, liquid crystals and dispersions
thereof, L2 phase and dispersions there of, well known to those
skilled in the art of phase behaviour in lipid-water systems,
polymeric micelles, multiple emulsions, self-emulsifying,
self-microemulsifying, cyclodextrins and derivatives thereof, and
dendrimers.
[0269] Compositions of the current invention are useful in the
formulation of solids, semisolids, powder and solutions for
pulmonary administration of an analogue or derivative according to
the invention, using, for example a metered dose inhaler, dry
powder inhaler and a nebulizer, all being devices well known to
those skilled in the art.
[0270] Compositions of the current invention are specifically
useful in the formulation of controlled, sustained, protracting,
retarded, and slow release drug delivery systems. More
specifically, but not limited to, compositions are useful in
formulation of parenteral controlled release and sustained release
systems (both systems leading to a many-fold reduction in number of
administrations), well known to those skilled in the art. Even more
preferably, are controlled release and sustained release systems
administered subcutaneous. Without limiting the scope of the
invention, examples of useful controlled release system and
compositions are hydrogels, oleaginous gels, liquid crystals,
polymeric micelles, microspheres, nanoparticles,
[0271] Methods to produce controlled release systems useful for
compositions of the current invention include, but are not limited
to, crystallization, condensation, co-crystallization,
precipitation, co-precipitation, emulsification, dispersion, high
pressure homogenisation, encapsulation, spray drying,
microencapsulating, coacervation, phase separation, solvent
evaporation to produce microspheres, extrusion and supercritical
fluid processes. General reference is made to Handbook of
Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker,
New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99:
Protein Formulation and Delivery (MacNally, E. J., ed. Marcel
Dekker, New York, 2000).
[0272] Parenteral administration may be performed by subcutaneous,
intramuscular, intraperitoneal or intravenous injection by means of
a syringe, optionally a pen-like syringe. Alternatively, parenteral
administration can be performed by means of an infusion pump. A
further option is a composition which may be a solution or
suspension or a powder for the administration of the analogue or
derivative of the present invention in the form of a nasal or
pulmonal liquid or powder spray. As a still further option, the
pharmaceutical compositions containing the analogue or derivative
according to the invention can also be adapted to transdermal
administration, e.g. by needle-free injection or from a patch,
optionally an iontophoretic patch, or transmucosal, e.g. buccal,
administration.
[0273] The analogues or derivatives according to the present
invention can be administered via the pulmonary route in a vehicle,
as a solution, suspension or dry powder using any of known types of
devices suitable for pulmonary drug delivery. Examples of these
comprise, but are not limited to, the three general types of
aerosol-generating for pulmonary drug delivery, and may include jet
or ultrasonic nebulizers, metered-dose inhalers, or dry powder
inhalers (Cf. Yu J, Chien Y W. Pulmonary drug delivery: Physiologic
and mechanistic aspects. Crit Rev Ther Drug Carr Sys 14(4) (1997)
395-453).
[0274] Based on standardised testing methodology, the aerodynamic
diameter (d.sub.a) of a particle is defined as the geometric
equivalent diameter of a reference standard spherical particle of
unit density (1 g/cm.sup.3). In the simplest case, for spherical
particles, d.sub.a is related to a reference diameter (d) as a
function of the square root of the density ratio as described
by:
[0275] Modifications to this relationship occur for non-spherical
particles (cf. Edwards D A, Ben-Jebria A, Langer R. Recent advances
in pulmonary drug delivery using large, porous inhaled particles. J
Appl Physiol 84(2) (1998) 379-385). The terms "MMAD" and "MMEAD"
are well-described and known to the art (cf. Edwards D A,
Ben-Jebria A, Langer R and represents a measure of the median value
of an aerodynamic particle size distribution. Recent advances in
pulmonary drug delivery using large, porous inhaled particles. J
Appl Physiol 84(2) (1998) 379-385). Mass median aerodynamic
diameter (MMAD) and mass median effective aerodynamic diameter
(MMEAD) are used inter-changeably, are statistical parameters, and
empirically describe the size of aerosol particles in relation to
their potential to deposit in the lungs, independent of actual
shape, size, or density (cf. Edwards D A, Ben-Jebria A, Langer R.
Recent advances in pulmonary drug delivery using large, porous
inhaled particles. J Appl Physiol 84(2) (1998) 379-385). MMAD is
normally calculated from the measurement made with impactors, an
instrument that measures the particle inertial behaviour in air. In
a further embodiment, the formulation could be aerosolized by any
known aerosolisation technology, such as nebulisation, to achieve a
MMAD of aerosol particles less than 10 .mu.m, more preferably
between 1-5 .mu.m, and most preferably between 1-3 .mu.m. The
preferred particle size is based on the most effective size for
delivery of drug to the deep lung, where protein is optimally
absorbed (cf. Edwards D A, Ben-Jebria A, Langer A, Recent advances
in pulmonary drug delivery using large, porous inhaled particles. J
Appl Physiol 84(2) (1998) 379-385).
[0276] Deep lung deposition of the pulmonal formulations comprising
the analogue or derivative according to the invention may optional
be further optimized by using modifications of the inhalation
techniques, for example, but not limited to: slow inhalation flow
(eg. 30 L/min), breath holding and timing of actuation.
[0277] The term "stabilized formulation" refers to a formulation
with increased physical stability, increased chemical stability or
increased physical and chemical stability.
[0278] The term "physical stability" of the protein formulation as
used herein refers to the tendency of the protein to form
biologically inactive and/or insoluble aggregates of the protein as
a result of exposure of the protein to thermo-mechanical stresses
and/or interaction with interfaces and surfaces that are
destabilizing, such as hydrophobic surfaces and interfaces.
Physical stability of the aqueous protein formulations is evaluated
by means of visual inspection and/or turbidity measurements after
exposing the formulation filled in suitable containers (e.g.
cartridges or vials) to mechanical/physical stress (e.g. agitation)
at different temperatures for various time periods. Visual
inspection of the formulations is performed in a sharp focused
light with a dark background. The turbidity of the formulation is
characterized by a visual score ranking the degree of turbidity for
instance on a scale from 0 to 3 (a formulation showing no turbidity
corresponds to a visual score 0, and a formulation showing visual
turbidity in daylight corresponds to visual score 3). A formulation
is classified physical unstable with respect to protein
aggregation, when it shows visual turbidity in daylight.
Alternatively, the turbidity of the formulation can be evaluated by
simple turbidity measurements well-known to the skilled person.
Physical stability of the aqueous protein formulations can also be
evaluated by using a spectroscopic agent or probe of the
conformational status of the protein. The probe is preferably a
small molecule that preferentially binds to a non-native conformer
of the protein. One example of a small molecular spectroscopic
probe of protein structure is Thioflavin T. Thioflavin T is a
fluorescent dye that has been widely used for the detection of
amyloid fibrils. In the presence of fibrils, and perhaps other
protein configurations as well, Thioflavin T gives rise to a new
excitation maximum at about 450 nm and enhanced emission at about
482 nm when bound to a fibril protein form. Unbound Thioflavin T is
essentially non-fluorescent at the wavelengths.
[0279] Other small molecules can be used as probes of the changes
in protein structure from native to non-native states. For instance
the "hydrophobic patch" probes that bind preferentially to exposed
hydrophobic patches of a protein. The hydrophobic patches are
generally buried within the tertiary structure of a protein in its
native state, but become exposed as a protein begins to unfold or
denature. Examples of these small molecular, spectroscopic probes
are aromatic, hydrophobic dyes, such as antrhacene, acridine,
phenanthroline or the like. Other spectroscopic probes are
metal-amino acid complexes, such as cobalt metal complexes of
hydrophobic amino acids, such as phenylalanine, leucine,
isoleucine, methionine, and valine, or the like.
[0280] The term "chemical stability" of the protein formulation as
used herein refers to chemical covalent changes in the protein
structure leading to formation of chemical degradation products
with potential less biological potency and/or potential increased
immunogenic properties compared to the native protein structure.
Various chemical degradation products can be formed depending on
the type and nature of the native protein and the environment to
which the protein is exposed. Elimination of chemical degradation
can most probably not be completely avoided and increasing amounts
of chemical degradation products is often seen during storage and
use of the protein formulation as well-known by the person skilled
in the art. Most proteins are prone to deamidation, a process in
which the side chain amide group in glutaminyl or asparaginyl
residues is hydrolysed to form a free carboxylic acid. Other
degradations pathways involves formation of high molecular weight
transformation products where two or more protein molecules are
covalently bound to each other through transamidation and/or
disulfide interactions leading to formation of covalently bound
dimer, oligomer and polymer degradation products (Stability of
Protein Pharmaceuticals, Ahern. T. J. & Manning M. C., Plenum
Press, New York 1992). Oxidation (of for instance methionine
residues) can be mentioned as another variant of chemical
degradation. The chemical stability of the protein formulation can
be evaluated by measuring the amount of the chemical degradation
products at various time-points after exposure to different
environmental conditions (the formation of degradation products can
often be accelerated by for instance increasing temperature). The
amount of each individual degradation product is often determined
by separation of the degradation products depending on molecule
size and/or charge using various chromatography techniques (e.g.
SEC-HPLC and/or RP-HPLC).
[0281] Hence, as outlined above, a "stabilized formulation" refers
to a formulation with increased physical stability, increased
chemical stability or increased physical and chemical stability. In
general, a formulation must be stable during use and storage (in
compliance with recommended use and storage conditions) until the
expiration date is reached.
[0282] In one embodiment of the invention, the pharmaceutical
formulation comprising the analogue or derivative according to the
present invention is stable for more than 6 weeks of usage and for
more than 3 years of storage.
[0283] In another embodiment of the invention, the pharmaceutical
formulation comprising the analogue or derivative according to the
present invention is stable for more than 4 weeks of usage and for
more than 3 years of storage.
[0284] In a further embodiment of the invention, the pharmaceutical
formulation comprising the analogue or derivative according to the
present invention is stable for more than 4 weeks of usage and for
more than two years of storage.
[0285] In an even further embodiment of the invention, the
pharmaceutical formulation comprising the analogue or derivative
according to the present invention is stable for more than 2 weeks
of usage and for more than two years of storage.
[0286] In another aspect, the present invention relates to the use
of an analogue or derivative according to the present for the
preparation of a medicament.
[0287] In one embodiment, an analogue or derivative according to
the present is used for the preparation of a medicament for the
treatment or prevention of hyperglycemia, type 2 diabetes, impaired
glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome
X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial
infarction, stroke, coronary heart disease and other cardiovascular
disorders, inflammatory bowel syndrome, dyspepsia and gastric
ulcers.
[0288] In another embodiment, an analogue or derivative according
to the present is used for the preparation of a medicament for
delaying or preventing disease progression in type 2 diabetes.
[0289] In another embodiment an analogue or derivative according to
the present is used for the preparation of a medicament for
decreasing food intake, decreasing .beta.-cell apoptosis,
increasing .beta.-cell function and .beta.-cell mass, and/or for
restoring glucose sensitivity to .beta.-cells.
[0290] The treatment with an analogue or derivative according to
the present invention may also be combined with a second or more
pharmacologically active substances, e.g. selected from
antidiabetic agents, antiobesity agents, appetite regulating
agents, antihypertensive agents, agents for the treatment and/or
prevention of complications resulting from or associated with
diabetes and agents for the treatment and/or prevention of
complications and disorders resulting from or associated with
obesity. Examples of these pharmacologically active substances are
: Insulin, sulphonylureas, biguanides, meglitinides, glucosidase
inhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV)
inhibitors, inhibitors of hepatic enzymes involved in stimulation
of gluconeogenesis and/or glycogenolysis, glucose uptake
modulators, compounds modifying the lipid metabolism such as
antihyperlipidemic agents as HMG CoA inhibitors (statins), Gastric
Inhibitory Polypeptides (GIP analogs), compounds lowering food
intake, RXR agonists and agents acting on the ATP-dependent
potassium channel of the .beta.-cells; Cholestyramine, colestipol,
clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin,
probucol, dextrothyroxine, neteglinide, repaglinide;
.beta.-blockers such as alprenolol, atenolol, timolol, pindolol,
propranolol and metoprolol, ACE (angiotensin converting enzyme)
inhibitors such as benazepril, captopril, enalapril, fosinopril,
lisinopril, alatriopril, quinapril and ramipril, calcium channel
blockers such as nifedipine, felodipine, nicardipine, isradipine,
nimodipine, diltiazem and verapamil, and .alpha.-blockers such as
doxazosin, urapidil, prazosin and terazosin; CART (cocaine
amphetamine regulated transcript) agonists, NPY (neuropeptide Y)
antagonists, PYY agonists, Y2 receptor agonists, Y4 receptor
agonits, mixed Y2/Y4 receptor agonists, MC4 (melanocortin 4)
agonists, orexin antagonists, TNF (tumor necrosis factor) agonists,
CRF (corticotropin releasing factor) agonists, CRF BP
(corticotropin releasing factor binding protein) antagonists,
urocortin agonists, .beta.3 agonists, oxyntomodulin and analogues,
MSH (melanocyte-stimulating hormone) agonists, MCH
(melanocyte-concentrating hormone) antagonists, CCK
(cholecystokinin) agonists, serotonin re-uptake inhibitors,
serotonin and noradrenaline re-uptake inhibitors, mixed serotonin
and noradrenergic compounds, 5HT (serotonin) agonists, bombesin
agonists, galanin antagonists, growth hormone, growth hormone
releasing compounds, TRH (thyreotropin releasing hormone) agonists,
UCP 2 or 3 (uncoupling protein 2 or 3) modulators, leptin agonists,
DA agonists (bromocriptin, doprexin), lipase/amylase inhibitors,
RXR (retinoid X receptor) modulators, TR .beta. agonists; histamine
H3 antagonists, Gastric Inhibitory Polypeptide agonists or
antagonists (GIP analogs), gastrin and gastrin analogs.
[0291] The treatment with an analogue or derivative according to
the present may also be combined with surgery--a surgery that
influence the glucose levels and/or lipid homeostasis such as
gastric banding or gastric bypass.
[0292] It should be understood that any suitable combination of the
analogues or derivatives according to the present with one or more
of the above-mentioned compounds and optionally one or more further
pharmacologically active substances are considered to be within the
scope of the present invention.
[0293] Method of Manufacturing Analogues
[0294] Depending on the sequence the analogues of this invention
can be produced by a method which comprises culturing a host cell
containing a DNA sequence encoding the polypeptide and capable of
expressing the polypeptide in a suitable nutrient medium under
conditions permitting the expression of the peptide, after which
the resulting peptide is recovered from the culture.
[0295] The medium used to culture the cells may be any conventional
medium suitable for growing the host cells, such as minimal or
complex media containing appropriate supplements. Suitable media
are available from commercial suppliers or may be prepared
according to published recipes (e.g. in catalogues of the American
Type Culture Collection). The peptide produced by the cells may
then be recovered from the culture medium by conventional
procedures including separating the host cells from the medium by
centrifugation or filtration, precipitating the proteinaceous
components of the supernatant or filtrate by means of a salt, e.g.
ammonium sulphate, purification by a variety of chromatographic
procedures, e.g. ion exchange chromatography, gel filtration
chromatography, affinity chromatography, or the like, dependent on
the type of peptide in question.
[0296] The DNA sequence encoding the therapeutic polypeptide may
suitably be of genomic or cDNA origin, for instance obtained by
preparing a genomic or cDNA library and screening for DNA sequences
coding for all or part of the polypeptide by hybridisation using
synthetic oligonucleotide probes in accordance with standard
techniques (see, for example, Sambrook, J, Fritsch, E F and
Maniatis, T, Molecular Cloning: A Laboratory
[0297] Manual, Cold Spring Harbor Laboratory Press, New York,
1989). The DNA sequence encoding the polypeptide may also be
prepared synthetically by established standard methods, e.g. the
phosphoamidite method described by Beaucage and Caruthers,
Tetrahedron Letters 22 (1981), 1859-1869, or the method described
by Matthes et al., EMBO Journal 3 (1984), 801-805. The DNA sequence
may also be prepared by polymerase chain reaction using specific
primers, for instance as described in U.S. Pat. No. 4,683,202 or
Saiki et al., Science 239 (1988), 487-491.
[0298] The DNA sequence may be inserted into any vector which may
conveniently be subjected to recombinant DNA procedures, and the
choice of vector will often depend on the host cell into which it
is to be introduced. Thus, the vector may be an autonomously
replicating vector, i.e. a vector which exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g. a plasmid. Alternatively, the vector
may be one which, when introduced into a host cell, is integrated
into the host cell genome and replicated together with the
chromosome(s) into which it has been integrated.
[0299] The vector is preferably an expression vector in which the
DNA sequence encoding the peptide is operably linked to additional
segments required for transcription of the DNA, such as a promoter.
The promoter may be any DNA sequence which shows transcriptional
activity in the host cell of choice and may be derived from genes
encoding proteins either homologous or heterologous to the host
cell. Examples of suitable promoters for directing the
transcription of the DNA encoding the peptide of the invention in a
variety of host cells are well-known in the art, cf. for instance
Sambrook et al., supra.
[0300] The DNA sequence encoding the peptide may also, if
necessary, be operably connected to a suitable terminator,
polyadenylation signals, transcriptional enhancer sequences, and
translational enhancer sequences. The recombinant vector of the
invention may further comprise a DNA sequence enabling the vector
to replicate in the host cell in question.
[0301] The vector may also comprise a selectable marker, e.g. a
gene the product of which complements a defect in the host cell or
one which confers resistance to a drug, e.g. ampicillin, kanamycin,
tetracyclin, chloramphenicol, neomycin, hygromycin or
methotrexate.
[0302] To direct a parent peptide of the present invention into the
secretory pathway of the host cells, a secretory signal sequence
(also known as a leader sequence, prepro sequence or pre sequence)
may be provided in the recombinant vector. The secretory signal
sequence is joined to the DNA sequence encoding the peptide in the
correct reading frame. Secretory signal sequences are commonly
positioned 5' to the DNA sequence encoding the peptide. The
secretory signal sequence may be that normally associated with the
peptide or may be from a gene encoding another secreted protein.
The procedures used to ligate the DNA sequences coding for the
present peptide, the promoter and optionally the terminator and/or
secretory signal sequence, respectively, and to insert them into
suitable vectors containing the information necessary for
replication, are well-known to persons skilled in the art (cf., for
instance, Sambrook et al., supra).
[0303] The host cell into which the DNA sequence or the recombinant
vector is introduced may be any cell which is capable of producing
the present peptide and includes bacteria, yeast, fungi and higher
eukaryotic cells. Examples of suitable host cells well-known and
used in the art are, without limitation, E. coli, Saccharomyces
cerevisiae, or mammalian BHK or CHO cell lines.
Embodiments According to the Invention
[0304] 1. A GLP-1 analogue or derivative thereof, which comprises a
modified GLP-1 sequence 7-35 (SEQ ID No 1) having: [0305] i) a
total of 2, 3, 4, 5 6, 7 or 8 amino acid substitutions compared to
the sequence 7-35 of SEQ ID No 1, including [0306] a) a Glu residue
at a position equivalent to position 22 of SEQ ID No 1, and [0307]
b) an Arg residue at a position equivalent to position 26 of SEQ ID
No 1, [0308] ii) optionally the amino acid(s) at a position
equivalent to position 30, 31, 32, 33, 34, or 35 of SEQ ID No 1 can
be absent provided that if the amino acid at position 30, 31, 32,
33 or 34 is absent then each amino acid residue downstream is also
absent, and [0309] iii) optionally a C-terminal amide group. [0310]
2. The GLP-1 analogue or derivative thereof according to embodiment
1, which is derivatised with an albumin binding residue or is
pegylated. [0311] 3. The GLP-1 analogue or derivative thereof
according to any one of the embodiments 1-2, wherein the amino acid
at position 35 is absent, and wherein the total length of the GLP-1
analogue is 28 amino acids. [0312] 4. The GLP-1 analogue or
derivative thereof according to any one of the embodiments 1-2,
wherein the amino acids at position 34 and 35 are absent, and
wherein the total length of the GLP-1 analogue is 27 amino acids.
[0313] 5. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-2, wherein the amino acids at position 33,
34 and 35 are absent, and wherein the total length of the GLP-1
analogue is 26 amino acids. [0314] 6. The GLP-1 analogue or
derivative thereof according to any one of the embodiments 1-2,
wherein the amino acids at position 32, 33, 34 and 35 are absent,
and wherein the total length of the GLP-1 analogue is 25 amino
acids. [0315] 7. The GLP-1 analogue or derivative thereof according
to any one of the embodiments 1-2, wherein the amino acids at
position 31, 32, 33, 34 and 35 are absent, and wherein the total
length of the GLP-1 analogue is 24 amino acids. [0316] 8. The GLP-1
analogue or derivative thereof according to any one of the
embodiments 1-2, wherein the amino acids at position 30, 31, 32,
33, 34 and 35 are absent, and wherein the total length of the GLP-1
analogue is 23 amino acids. [0317] 9. The GLP-1 analogue or
derivative thereof according to any one of the embodiments 1-8
having a C-terminal amide group. [0318] 10. The GLP-1 analogue or
derivative thereof according to any one of the embodiments 1-9
having the sequence of formula (I)
TABLE-US-00003 [0318] (SEQ ID No: 2)
Xaa.sub.7-Xaa.sub.8-Xaa.sub.9-Gly-Thr-Phe-Thr-Ser-Asp-Xaa.sub.16-Ser-
Xaa.sub.18-Tyr-Xaa.sub.20-Glu-Glu-Xaa.sub.23-Xaa.sub.24-Xaa.sub.25-Arg-
Xaa.sub.27-Phe-Ile-Xaa.sub.30-Xaa.sub.31-Leu-Xaa.sub.33-Xaa.sub.34-Xaa.sub-
.35-R Formula (I)
wherein [0319] Xaa.sub.7 is L-histidine, D-histidine,
desamino-histidine, 2-amino-histidine, .beta.-hydroxy-histidine,
homohistidine, N.sup..alpha.-acetyl-histidine,
.alpha.-fluoromethyl-histidine, .alpha.-methyl-histidine,
3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine; [0320]
Xaa.sub.8 is Ala, Gly, Val, Leu, Ile, Lys, Aib,
(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl)carboxylic
acid, (1-aminocyclopentyl)carboxylic acid,
(1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic
acid, or (1-aminocyclooctyl)carboxylic acid; [0321] Xaa.sub.9 is
Glu or a Glu derivative such as alpha, alpha dimethyl-Glu; [0322]
Xaa.sub.16 is Val or Leu; [0323] Xaa.sub.18 is Ser, Lys, Cys or
Arg; [0324] Xaa.sub.20 is Leu, Lys or Cys; [0325] Xaa.sub.23 is
Gln, Glu, Lys, Cys or Arg; [0326] Xaa.sub.24 is Ala or Asn; [0327]
Xaa.sub.25 is Ala or Val; [0328] Xaa.sub.27 is Glu, Ala or Leu;
[0329] Xaa.sub.30 is Ala, Glu, Arg or absent; [0330] Xaa.sub.31 is
Trp, Lys, Cys or absent; [0331] Xaa.sub.33 is Val, Lys, Cys or
absent;
[0332] Xaa.sub.34 is Lys, Glu, Asn, Arg, Cys or absent; [0333]
Xaa.sub.35 is Gly, Aib or absent; [0334] R is amide or is absent;
[0335] provided that if Xaa.sub.30, Xaa.sub.31, Xaa.sub.32,
Xaa.sub.33, or Xaa.sub.34 is absent then each amino acid residue
downstream is also absent. [0336] 11. The GLP-1 analogue or
derivative thereof according to any one of the embodiments 1-9
having the sequence of formula (II)
TABLE-US-00004 [0336] (SEQ ID No: 3)
Xaa.sub.7-Xaa.sub.8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-
Xaa.sub.18-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Arg-Glu-Phe-
Ile-Xaa.sub.30-Trp-Leu-Xaa.sub.33-Xaa.sub.34-Xaa.sub.35-R Formula
(II)
wherein [0337] Xaa.sub.7 is L-histidine, D-histidine,
desamino-histidine, 2-amino-histidine, .beta.-hydroxy-histidine,
homohistidine, N.sup..alpha.-acetyl-histidine,
.alpha.-fluoromethyl-histidine, .alpha.-methyl-histidine,
3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine; [0338]
Xaa.sub.8 is Ala, Gly, Val, Leu, Ile, Lys, Aib,
(1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic
acid, (1-aminocyclopentyl)carboxylic acid,
(1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic
acid, or (1-aminocyclooctyl)carboxylic acid; [0339] Xaa.sub.i8 is
Ser, Lys or Arg; [0340] Xaa.sub.30 is Ala, Glu, Arg or is absent;
[0341] Xaa.sub.33 is Val, Lys or absent; [0342] Xaa.sub.34 is Lys,
Glu, Arg or is absent; [0343] Xaa.sub.35 is Gly, Aib or is absent;
[0344] R is amide or is absent. [0345] 12. The GLP-1 analogue or
derivative thereof according to any one of the embodiments 1-11,
wherein at least one amino acid residue is derivatised with
A-B-C-D- wherein A- is selected from the group consisting of
##STR00029##
[0346] wherein n is selected from the group consisting of 14, 15,
16 17, 18 and 19, p is selected from the group consisting of 10,
11, 12, 13 and 14, and d is selected from the group consisting of
0, 1, 2, 3, 4 and 5, [0347] -B- is selected from the group
consisting of
##STR00030##
[0348] wherein x is selected from the group consisting of 0, 1, 2,
3 and 4, and y is selected from the group consisting of 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11 and 12, [0349] -C- is selected from the group
consisting of
##STR00031##
[0350] wherein b and e are each independently selected from the
group consisting of 0, 1 and 2, and c and f are each independently
selected from the group consisting of 0, 1 and 2 with the proviso
that b is 1 or 2 when c is 0, or b is 0 when c is 1 or 2, and e is
1 or 2 when f is 0, or e is 0 when f is 1 or 2, and [0351] -D- is
attached to said amino acid residue and is a linker. [0352] 13. The
GLP-1 analogue or derivative thereof according to embodiment 12,
wherein D is selected from the group consisting of
##STR00032##
[0352] and wherein k is selected from the group consisting of 0, 1,
2, 3, 4, 5, 11 and 27, and m is selected from the group consisting
of 0, 1, 2, 3, 4, 5 and 6. [0353] 14. A pharmaceutical composition
comprising a GLP-1 analogue or derivative thereof according to any
one of embodiments 1-13, and a pharmaceutically acceptable
excipient. [0354] 15. A GLP-1 analogue or derivative thereof
according to any one of embodiments 1-14 for use in the treatment
or prevention of hyperglycemia, type 2 diabetes, impaired glucose
tolerance, type 1 diabetes, obesity, hypertension, syndrome X,
dyslipidemia, cognitive disorders, atheroschlerosis, myocardial
infarction, coronary heart disease and other cardiovascular
disorders, stroke, inflammatory bowel syndrome, dyspepsia and
gastric ulcers.
[0355] The amino acid sequence of human GLP-1(7-35) is included in
the Sequence Listing as SEQ ID No 1, and SEQ ID Nos 2 and 3 are
derivatives thereof according to the invention.
[0356] In the Sequence Listing, the numbering starts with amino
acid residue no. 1. Accordingly, e.g., position 1 of SEQ ID No 1 is
equivalent to position 7 of GLP-1(7-35) (His), position 16 of SEQ
ID No 1 is equivalent to position 22 of GLP-1(7-35) (Gly), and
position 20 of SEQ ID No 1 is equivalent to position 26 of
GLP-1(7-35) (Lys)--and vice versa for the other positions and the
other sequences.
[0357] Accordingly, the invention also provides, in above
embodiment 1, a GLP-1 analogue or derivative which comprises a
modified GLP-1(7-35) sequence having:
[0358] i) a total of 2, 3, 4, 5, 6, 7 or 8 amino acid substitutions
compared to the sequence of SEQ ID No 1, including
[0359] a) a Glu residue at a position equivalent to position 22 of
GLP-1(7-35) (position 16 of SEQ ID No 1), and
[0360] b) an Arg residue at a position equivalent to position 26 of
GLP-1(7-35) (position 20 of SEQ ID No 1),
[0361] ii) optionally the amino acid(s) at a position equivalent to
position 30, 31, 32, 33, 34, or 35 of GLP-1(7-35) (position 24, 25,
26, 27, 28, or 29, respectively, of SEQ ID No 1) can be absent
provided that if the amino acid at position 30, 31, 32, 33 or 34 of
GLP1(7-35) (position 24, 25, 26, 27, or 28 of SEQ ID No 1) is
absent then each amino acid residue downstream is also absent,
and
[0362] iv) optionally a C-terminal amide group.
[0363] The invention furthermore provides GLP-1 analogues or
derivatives, methods and uses thereof, and pharmaceutical
compositions with a content thereof corresponding to any of the
above embodiments and particular embodiments according to the
invention, in which corresponding position numbering amendments
have been made as explained above, and shown above for the GLP-1
analogue or derivative of above embodiment 1.
[0364] 1. A GLP-1 analogue or derivative thereof, which comprises a
modified GLP-1 sequence 7-35 (SEQ ID No 1) having:
[0365] i) a total of 2, 3, 4, 5 6, 7 or 8 amino acid substitutions
compared to the sequence 7-35 of SEQ ID No 1, including
[0366] a) a Glu residue at a position equivalent to position 22 of
SEQ ID No 1, and
[0367] b) an Arg residue at a position equivalent to position 26 of
SEQ ID No 1,
[0368] ii) optionally the amino acid(s) at a position equivalent to
position 30, 31, 32, 33, 34, or 35 of SEQ ID No 1 can be absent
provided that if the amino acid at position 30, 31, 32, 33 or 34 is
absent then each amino acid residue downstream is also absent,
and
[0369] iii) optionally a C-terminal amide group.
[0370] 2. The GLP-1 analogue or derivative thereof according to
embodiment 1, which is derivatised with an albumin binding residue
or is pegylated.
[0371] 3. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-2, wherein the amino acid at position 35
is absent, and wherein the total length of the GLP-1 analogue is 28
amino acids.
[0372] 4. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-2, wherein the amino acids at position 34
and 35 are absent, and wherein the total length of the GLP-1
analogue is 27 amino acids.
[0373] 5. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-2, wherein the amino acids at position 33,
34 and 35 are absent, and wherein the total length of the GLP-1
analogue is 26 amino acids.
[0374] 6. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-2, wherein the amino acids at position 32,
33, 34 and 35 are absent, and wherein the total length of the GLP-1
analogue is 25 amino acids.
[0375] 7. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-2, wherein the amino acids at position 31,
32, 33, 34 and 35 are absent, and wherein the total length of the
GLP-1 analogue is 24 amino acids.
[0376] 8. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-2, wherein the amino acids at position 30,
31, 32, 33, 34 and 35 are absent, and wherein the total length of
the GLP-1 analogue is 23 amino acids.
[0377] 9. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-8 having a C-terminal amide group.
[0378] 10. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-9 having the sequence of formula
(I)
TABLE-US-00005 (SEQ ID No: 2)
Xaa.sub.7-Xaa.sub.8-Xaa.sub.9-Gly-Thr-Phe-Thr-Ser-Asp-Xaa.sub.16-Ser-
Xaa.sub.18-Tyr-Xaa.sub.20-Glu-Glu-Xaa.sub.23-Xaa.sub.24-Xaa.sub.25-Arg-
Xaa.sub.27-Phe-Ile-Xaa.sub.30-Xaa.sub.31-Leu-Xaa.sub.33-Xaa.sub.34-Xaa.sub-
.35-R Formula (I)
[0379] wherein
[0380] Xaa.sub.7 is L-histidine, D-histidine, desamino-histidine,
2-amino-histidine, .beta.-hydroxy-histidine, homohistidine,
N.sup..alpha.-acetyl-histidine, .alpha.-fluoromethyl-histidine,
.alpha.-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or
4-pyridylalanine;
[0381] Xaa.sub.8 is Ala, Gly, Val, Leu, Ile, Lys, Aib,
(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl)carboxylic
acid, (1-aminocyclopentyl)carboxylic acid,
(1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic
acid, or (1-aminocyclooctyl)carboxylic acid;
[0382] Xaa.sub.9 is Glu or a Glu derivative such as alpha, alpha
dimethyl-Glu;
[0383] Xaa.sub.16 is Val or Leu;
[0384] Xaa.sub.18 is Ser, Lys, Cys or Arg;
[0385] Xaa.sub.20 is Leu, Lys or Cys;
[0386] Xaa.sub.23 is Gln, Glu, Lys, Cys or Arg;
[0387] Xaa.sub.24 is Ala or Asn;
[0388] Xaa.sub.25 is Ala or Val;
[0389] Xaa.sub.27 is Glu, Ala or Leu;
[0390] Xaa.sub.30 is Ala, Glu, Lys, Arg or absent;
[0391] Xaa.sub.31 is Trp, Lys, Cys or absent;
[0392] Xaa.sub.33 is Val, Lys, Cys or absent;
[0393] Xaa.sub.34 is Lys, Glu, Asn, Arg, Cys or absent;
[0394] Xaa.sub.35 is Gly, Aib or absent;
[0395] R is amide or is absent;
[0396] provided that if Xaa.sub.30, Xaa.sub.31, Xaa.sub.32,
Xaa.sub.33, or Xaa.sub.34 is absent then each amino acid residue
downstream is also absent.
[0397] 11. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-9 having the sequence of formula
(II)
TABLE-US-00006 (SEQ ID No: 3)
Xaa.sub.7-Xaa.sub.8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-
Xaa.sub.18-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Arg-Glu-Phe-
Ile-Xaa.sub.30-Trp-Leu-Xaa.sub.33-Xaa.sub.34-Xaa.sub.35-R Formula
(II)
[0398] wherein
[0399] Xaa.sub.7 is L-histidine, D-histidine, desamino-histidine,
2-amino-histidine, .beta.-hydroxy-histidine, homohistidine,
N.sup..alpha.-acetyl-histidine, .alpha.-fluoromethyl-histidine,
.alpha.-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or
4-pyridylalanine;
[0400] Xaa.sub.8 is Ala, Gly, Val, Leu, Ile, Lys, Aib,
(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl)carboxylic
acid, (1-aminocyclopentyl)carboxylic acid,
(1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic
acid, or (1-aminocyclooctyl)carboxylic acid;
[0401] Xaa.sub.18 is Ser, Lys or Arg;
[0402] Xaa.sub.30 is Ala, Glu, Lys, Arg or is absent;
[0403] Xaa.sub.33 is Val, Lys or absent;
[0404] Xaa.sub.34 is Lys, Glu, Arg or is absent;
[0405] Xaa.sub.35 is Gly, Aib or is absent;
[0406] R is amide or is absent.
[0407] 12. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-11, wherein at least one amino acid
residue is derivatised with A-B-C-D-
[0408] wherein A- is selected from the group consisting of
##STR00033##
[0409] wherein n is selected from the group consisting of 14, 15,
16 17, 18 and 19, p is selected from the group consisting of 10,
11, 12, 13 and 14, and d is selected from the group consisting of
0, 1, 2, 3, 4 and 5,
[0410] -B- is selected from the group consisting of
##STR00034##
[0411] wherein x is selected from the group consisting of 0, 1, 2,
3 and 4, and y is selected from the group consisting of 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11 and 12,
[0412] -C- is selected from the group consisting of
##STR00035##
[0413] wherein b and e are each independently selected from the
group consisting of 0, 1 and 2, and c and f are each independently
selected from the group consisting of 0, 1 and 2 with the proviso
that b is 1 or 2 when c is 0, or b is 0 when c is 1 or 2, and e is
1 or 2 when f is 0, or e is 0 when f is 1 or 2, and
[0414] -D- is attached to said amino acid residue and is a
linker.
[0415] 13. The GLP-1 analogue or derivative thereof according to
embodiment 12, wherein D is selected from the group consisting
of
##STR00036##
[0416] and wherein k is selected from the group consisting of 0, 1,
2, 3, 4, 5, 11 and 27, and m is selected from the group consisting
of 0, 1, 2, 3, 4, 5 and 6.
[0417] 14. A pharmaceutical composition comprising a GLP-1 analogue
or derivative thereof according to any one of embodiments 1-13, and
a pharmaceutically acceptable excipient.
[0418] 15. A GLP-1 analogue or derivative thereof according to any
one of embodiments 1-14 for use in the treatment or prevention of
hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1
diabetes, obesity, hypertension, syndrome X, dyslipidemia,
cognitive disorders, atheroschlerosis, myocardial infarction,
coronary heart disease and other cardiovascular disorders, stroke,
inflammatory bowel syndrome, dyspepsia and gastric ulcers.
[0419] 1. A GLP-1 analogue which is a modified GLP-1(7-35) (SEQ ID
No 1) having:
[0420] i) a total of 2, 3, 4, 5 6, 7, 8, or 9 amino acid
substitutions as compared to GLP-1(7-35), including
[0421] a) a Glu residue at a position equivalent to position 22 of
GLP-1(7-35), and
[0422] b) an Arg residue at a position equivalent to position 26 of
GLP-1(7-35);
[0423] or a derivative thereof.
[0424] 2. The GLP-1 analogue or derivative thereof according to
embodiment 1, wherein the amino acid(s) at a position equivalent to
position 30, 31, 32, 33, 34, or 35 of GLP-1(7-35) are absent,
provided that if the amino acid at position 30, 31, 32, 33, or 34
is absent then each amino acid residue downstream is also
absent.
[0425] 3. The GLP-1 analogue or derivative thereof according to any
one of embodiments 1-2, wherein the GLP-1 analogue comprises i) a
C-terminal carboxylic acid group; or iii) a C-terminal amide
group.
[0426] 4. The GLP-1 analogue or derivative thereof according to any
one of embodiments 1-3, which is derivatised with an albumin
binding residue or is pegylated.
[0427] 5. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-4, wherein the amino acid at position 35
is absent, and wherein the total length of the GLP-1 analogue is 28
amino acids.
[0428] 6. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-5, wherein the amino acids at position 34
and 35 are absent, and wherein the total length of the GLP-1
analogue is 27 amino acids.
[0429] 7. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-6, wherein the amino acids at position 33,
34, and 35 are absent, and wherein the total length of the GLP-1
analogue is 26 amino acids.
[0430] 8. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-7, wherein the amino acids at position 32,
33, 34, and 35 are absent, and wherein the total length of the
GLP-1 analogue is 25 amino acids.
[0431] 9. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-8, wherein the amino acids at position 31,
32, 33, 34, and 35 are absent, and wherein the total length of the
GLP-1 analogue is 24 amino acids.
[0432] 10. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-9, wherein the amino acids at position
30, 31, 32, 33, 34, and 35 are absent, and wherein the total length
of the GLP-1 analogue is 23 amino acids.
[0433] 11. A GLP-1 analogue or derivative thereof having the
sequence of formula (I)
TABLE-US-00007 (SEQ ID No: 2)
Xaa.sub.7-Xaa.sub.8-Xaa.sub.9-Gly-Thr-Phe-Thr-Ser-Asp-Xaa.sub.16-Ser-
Xaa.sub.18-Tyr-Xaa.sub.20-Glu-Glu-Xaa.sub.23-Xaa.sub.24-Xaa.sub.25-Arg-
Xaa.sub.27-Phe-Ile-Xaa.sub.30-Xaa.sub.31-Leu-Xaa.sub.33-Xaa.sub.34-Xaa.sub-
.35-R Formula (I)
[0434] wherein
[0435] Xaa.sub.7 is L-histidine, D-histidine, desamino-histidine,
2-amino-histidine, .beta.-hydroxy-histidine, homohistidine,
N.sup..alpha.-acetyl-histidine, .alpha.-fluoromethyl-histidine,
.alpha.-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or
4-pyridylalanine;
[0436] Xaa.sub.8 is Ala, Gly, Val, Leu, Ile, Lys, Aib,
(1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic
acid, (1-aminocyclopentyl)carboxylic acid,
(1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic
acid, or (1-aminocyclooctyl)carboxylic acid;
[0437] Xaa.sub.9 is Glu or a Glu derivative such as alpha, alpha
dimethyl-Glu;
[0438] Xaa.sub.16 is Val or Leu;
[0439] Xaa.sub.18 is Ser, Lys, Cys or Arg;
[0440] Xaa.sub.20 is Leu, Lys or Cys;
[0441] Xaa.sub.23 is Gln, Glu, Lys, Cys or Arg;
[0442] Xaa.sub.24 is Ala or Asn;
[0443] Xaa.sub.25 is Ala or Val;
[0444] Xaa.sub.27 is Glu, Ala or Leu;
[0445] Xaa.sub.30 is Ala, Glu, Lys, Arg or absent;
[0446] Xaa.sub.31 is Trp, Lys, Cys or absent;
[0447] Xaa.sub.33 is Val, Lys, Cys or absent;
[0448] Xaa.sub.34 is Lys, Glu, Asn, Arg, Cys or absent;
[0449] Xaa.sub.35 is Gly, Aib or absent;
[0450] R is amide or is absent;
[0451] provided that if Xaa.sub.30, Xaa.sub.31, Xaa.sub.32,
Xaa.sub.33, or Xaa.sub.34 is absent then each amino acid residue
downstream is also absent.
[0452] 12. A GLP-1 analogue or derivative thereof having the
sequence of formula (II)
TABLE-US-00008 (SEQ ID No: 3)
Xaa.sub.7-Xaa.sub.8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-
Xaa.sub.18-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Arg-Glu-Phe-
Ile-Xaa.sub.30-Trp-Leu-Xaa.sub.33-Xaa.sub.34-Xaa.sub.35-R Formula
(II)
[0453] wherein
[0454] Xaa.sub.7 is L-histidine, D-histidine, desamino-histidine,
2-amino-histidine, .beta.-hydroxy-histidine, homohistidine,
N.sup..alpha.-acetyl-histidine, .alpha.-fluoromethyl-histidine,
.alpha.-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or
4-pyridylalanine;
[0455] Xaa.sub.8 is Ala, Gly, Val, Leu, Ile, Lys, Aib,
(1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic
acid, (1-aminocyclopentyl)carboxylic acid,
(1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic
acid, or (1-aminocyclooctyl)carboxylic acid;
[0456] Xaa.sub.18 is Ser, Lys or Arg;
[0457] Xaa.sub.30 is Ala, Glu, Lys, Arg or is absent;
[0458] Xaa.sub.33 is Val, Lys or absent;
[0459] Xaa.sub.34 is Lys, Glu, Arg or is absent;
[0460] Xaa.sub.35 is Gly, Aib or is absent;
[0461] R is amide or is absent.
[0462] 13. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-12, wherein at least one amino acid
residue is derivatised with A-B-C-D-
[0463] wherein A- is selected from the group consisting of
##STR00037##
[0464] wherein n is selected from the group consisting of 14, 15,
16 17, 18 and 19, p is selected from the group consisting of 10,
11, 12, 13 and 14, and d is selected from the group consisting of
0, 1, 2, 3, 4 and 5,
[0465] -B- is selected from the group consisting of
##STR00038##
[0466] wherein x is selected from the group consisting of 0, 1, 2,
3 and 4, and y is selected from the group consisting of 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11 and 12,
[0467] -C- is selected from the group consisting of
##STR00039##
[0468] wherein b and e are each independently selected from the
group consisting of 0, 1 and 2, and c and f are each independently
selected from the group consisting of 0, 1 and 2 with the proviso
that b is 1 or 2 when c is 0, or b is 0 when c is 1 or 2, and e is
1 or 2 when f is 0, or e is 0 when f is 1 or 2, and
[0469] -D- is attached to said amino acid residue and is a
linker.
[0470] 14. The GLP-1 analogue or derivative thereof according to
embodiment 13, wherein D is selected from the group consisting
of
##STR00040##
[0471] and wherein k is selected from the group consisting of 0, 1,
2, 3, 4, 5, 11 and 27, and m is selected from the group consisting
of 0, 1, 2, 3, 4, 5 and 6.
[0472] 15. A GLP-1 analogue or derivative which is selected from
the following: [0473] [Glu22,Arg26]GLP-1(7-33)amide [0474] N
epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Val25,-
Arg26,Leu27,Glu30,Lys33)GLP-1(7-33)amide; [0475] N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Arg26,-
Glu30)GLP-1(7-33)amide; [0476] [Glu22,Val25,Arg26]GLP-1(7-33)amide;
[0477] [Aib8,Lys20,Glu22,Val25,Arg26,Glu30]GLP-1(7-33)amide; [0478]
N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-[Aib8,Lys20,Glu22,Val25,-
Arg26,Glu30]GLP-1(7-33)amide; [0479] [Glu22,
Arg26]GLP-1(7-33)peptide; [0480]
[Glu22,Val25,Arg26]GLP-1(7-32)amide; [0481] N-epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Arg26)-
GLP-1(7-33)amide; [0482] N-epsilon31
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Glu22,Val25,Arg26,-
Lys31)GLP-1(7-33)amide; [0483] N-epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(DesaminoHis7,Lys20,Glu2-
2,Arg26)GLP-1(7-33)amide; [0484] N-epsilon31
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(DesaminoHis7,Glu22,Arg2-
6,Lys31)GLP-1(7-33)amide; [0485] N-epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Val25,-
Arg26,Leu27,Glu30,Lys31)GLP-1(7-32)amide; [0486] N-epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Val25,-
Arg26,Leu27,Nle30,Lys31)GLP-1(7-32)amide; [0487]
N-epsilon31-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxynona-
decanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)-
acetylamino]ethoxy}ethoxy)acetyl]-[Aib8,Glu22,Val25,Arg26,Lys31]GLP-1-(7-3-
3)amide; [0488] [Desamino His7,Glu22,Arg26]-GLP-1(7-34); [0489]
[Aib8,Lys20,Glu22,Val25,Arg26,Leu27,Lys31]GLP-1(7-32)amide; [0490]
N-epsilon31-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylam-
ino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoH-
is7,Asp11,Glu18,Glu22,Val25,Arg26,Asp27,Glu30,Lys31]GLP-1(7-33)amide;
[0491] [Aib8,Glu22,Val25, Lys31]GLP-1(7-33)-amide; and [0492]
N-epsilon31-{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino-
)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-N-beta34-(2--
(bis-carboxymethylamino)acetyl)[Aib8,Glu22,Val25,Arg26,Lys31,Dap34]GLP-1(7-
-34)amide.
[0493] 16. A pharmaceutical composition comprising a GLP-1 analogue
or derivative thereof according to any one of embodiments 1-15 or a
pharmaceutically acceptable salt, amide, alkyl, or ester thereof,
and a pharmaceutically acceptable excipient.
[0494] 17. A GLP-1 analogue or derivative thereof according to any
one of embodiments 1-15 or a pharmaceutical composition according
to embodiment 16, for use as a medicament.
[0495] 18. A GLP-1 analogue or derivative thereof according to any
one of embodiments 1-15 or a pharmaceutical composition according
to embodiment 16, for use in the treatment or prevention of
hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1
diabetes, obesity, hypertension, syndrome X, dyslipidemia,
cognitive disorders, atheroschlerosis, myocardial infarction,
coronary heart disease and other cardiovascular disorders, stroke,
inflammatory bowel syndrome, dyspepsia and gastric ulcers.
[0496] 19. Use of a GLP-1 analogue or derivative thereof according
to any one of embodiments 1-15 or a pharmaceutical composition
according to embodiment 16, in the manufacture of a medicament for
use in the treatment or prevention of hyperglycemia, type 2
diabetes, impaired glucose tolerance, type 1 diabetes, obesity,
hypertension, syndrome X, dyslipidemia, cognitive disorders,
atheroschlerosis, myocardial infarction, coronary heart disease and
other cardiovascular disorders, stroke, inflammatory bowel
syndrome, dyspepsia and gastric ulcers.
[0497] 20. A method of treating or preventing hyperglycemia, type 2
diabetes, impaired glucose tolerance, type 1 diabetes, obesity,
hypertension, syndrome X, dyslipidemia, cognitive disorders,
atheroschlerosis, myocardial infarction, coronary heart disease and
other cardiovascular disorders, stroke, inflammatory bowel
syndrome, dyspepsia and gastric ulcers by administering a
pharmaceutically active amount of a GLP-1 analogue or derivative
thereof according to any one of embodiments 1-15 or a
pharmaceutical composition according to embodiment 16.
Embodiments
[0498] 1. A GLP-1 analogue or derivative thereof, which comprises a
modified GLP-1 sequence 7-35 (SEQ ID No 1) having:
[0499] a total of 2, 3, 4, 5 6, 7 or 8 amino acid substitutions
compared to the sequence 7-35 of SEQ ID No 1, including
[0500] a Glu residue at a position equivalent to position 22 of SEQ
ID No 1, and
[0501] an Arg residue at a position equivalent to position 26 of
SEQ ID No 1,
[0502] optionally the amino acid(s) at a position equivalent to
position 30, 31, 32, 33, 34, or 35 of SEQ ID No 1 can be absent
provided that if the amino acid at position 30, 31, 32, 33 or 34 is
absent then each amino acid residue downstream is also absent, and
optionally a C-terminal amide group.
[0503] 2. The GLP-1 analogue or derivative thereof according to
embodiment 1, which is derivatised with an albumin binding residue
or is pegylated.
[0504] 3. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-2, wherein the amino acid at position 35
is absent, and wherein the total length of the GLP-1 analogue is 28
amino acids.
[0505] 4. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-2, wherein the amino acids at position 34
and 35 are absent, and wherein the total length of the GLP-1
analogue is 27 amino acids.
[0506] 5. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-2, wherein the amino acids at position 33,
34 and 35 are absent, and wherein the total length of the GLP-1
analogue is 26 amino acids.
[0507] 6. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-2, wherein the amino acids at position 32,
33, 34 and 35 are absent, and wherein the total length of the GLP-1
analogue is 25 amino acids.
[0508] 7. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-2, wherein the amino acids at position 31,
32, 33, 34 and 35 are absent, and wherein the total length of the
GLP-1 analogue is 24 amino acids.
[0509] 8. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-2, wherein the amino acids at position 30,
31, 32, 33, 34 and 35 are absent, and wherein the total length of
the GLP-1 analogue is 23 amino acids.
[0510] 9. The GLP-1 analogue or derivative thereof according to any
one of the embodiments 1-8 having a C-terminal amide group.
[0511] 10. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-9 having 3 amino acid substitutions
compared to the sequence 7-35 of SEQ ID NO 1 including the
substitutions at position 22 and 26.
[0512] 11. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-10, which has a substitution at a
position selected from the group of position 7, 8, 18, 20, 23, 24,
25, 27, 30, 31, 33 and 34 compared to the sequence 7-35 of SEQ ID
NO 1.
[0513] 12. The GLP-1 analogue or derivative thereof according to
embodiment 11, which has a substitution selected from the group
consisting of desaminoHis7, Aib8, Lys18, Cys18, Lys20, Cys20,
Lys23, Cys23, Asn24, Val25, Ala27, Leu27, Glu30, Lys31, Cys31,
Lys33, Cys33, Lys34, Cys34 and Asn34.
[0514] 13. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 11-12, which has a substitution selected
from the group consisting of desaminoHis7, Aib8, Lys18, Lys20,
Lys23, Glu30, Lys31, Lys33 and Lys34.
[0515] 14. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 11-13, which has a substitution selected
from the group consisting of desaminoHis7 and Aib8.
[0516] 15. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-9 having 4 amino acid substitutions
compared to the sequence 7-35 of SEQ ID NO 1 including the
substitutions at position 22 and 26.
[0517] 16. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-9 and 15, which has two substitutions
at positions selected from the group of positions 7, 8, 18, 20, 23,
24, 25, 27, 30, 31, 33 and 34 compared to the sequence 7-35 of SEQ
ID NO 1.
[0518] 17. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 15-16, which has two substitutions
selected from the group consisting of desaminoHis7, Aib8, Lys18,
Cys18, Lys20, Cys20, Lys23, Cys23, Asn24, Val25, Ala27, Leu27,
Glu30, Lys31, Cys31, Lys33, Cys33, Lys34, Cys34 and Asn34.
[0519] 18. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 15-17 having an amino acid substitution
selected from the group consisting of desaminoHis7 and Aib8 and an
amino acid substitution selected from the group consisting Lys18,
Lys20, Lys23, Glu30, Lys31,Lys33 and Lys34.
[0520] 19. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 15-18 having an amino acid substitution
selected from the group consisting desaminoHis7 and Aib8, and an
amino acid substitution selected from the group consisting Lys18,
Lys20, Lys23, Glu30, Lys31, Lys33 and Lys34.
[0521] 20. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-9 having 5 amino acid substitutions
compared to the sequence 7-35 of SEQ ID NO 1 including the
substitutions at position 22 and 26.
[0522] 21. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-9 and 20, which has three amino acid
substitutions at positions selected from the group of positions 7,
8, 18, 20, 23, 24, 25, 27, 30, 31, 33 and 34 compared to the
sequence 7-35 of SEQ ID NO 1.
[0523] 22. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 20-21, which has three amino acid
substitutions selected from the group of desaminoHis7, Aib8, Lys18,
Cys18, Lys20, Cys20, Lys23, Cys23, Asn24, Val25, Ala27, Leu27,
Glu30, Lys31, Cys31, Lys33, Cys33, Lys34, Cys34 and Asn34.
[0524] 23. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 20-22 having an amino acid substitution
selected from the group consisting of desaminoHis7 and Aib8, and
two amino acid substitutions selected from the group consisting of
Lys18, Lys20, Lys23, Glu30, Lys31, Lys33 and Lys34.
[0525] 24. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 20-23 having an amino acid substitution
selected from the group consisting of desaminoHis7 and Aib8, and
two amino acid substitutions selected from the group consisting of
Lys18, Lys20, Lys23, Glu30, Lys31, Lys33 and Lys34.
[0526] 25. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-9 having 6 amino acid substitutions
compared to the sequence 7-35 of SEQ ID NO 1 including the
substitutions at position 22 and 26.
[0527] 26. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-9 and 25, which has four amino acid
substitutions at positions selected from the group of positions 7,
8, 18, 20, 23, 24, 25, 27, 30, 31, 33 and 34.
[0528] 27. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 25-26, which has four amino acid
substitutions selected from the group consisting of desaminoHis7,
Aib8, Lys18, Cys18, Lys20, Cys20, Lys23, Cys23, Asn24, Val25,
Ala27, Leu27, Glu30, Lys31, Cys31, Lys33, Cys33, Lys34, Cys34 and
Asn34.
[0529] 28. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 25-27 having an amino acid substitution
selected from the group consisting of desaminoHis7 and Aib8 and
three amino acid substitutions selected from the group consisting
of Lys18, Lys20, Lys23, Glu30, Lys31, Lys33 and Lys34.
[0530] 29. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 25-28 having an amino acid substitution
selected from the group consisting of desaminoHis7 and Aib8 and
three amino acid substitutions selected from the group consisting
of Lys18, Lys20, Lys23, Glu30, Lys31, Lys33 and Lys34.
[0531] 30. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-29 having the sequence of formula
(I)
TABLE-US-00009 (SEQ ID No: 2)
Xaa.sub.7-Xaa.sub.8-Xaa.sub.9-Gly-Thr-Phe-Thr-Ser-Asp-Xaa.sub.16-Ser-
Xaa.sub.18-Tyr-Xaa.sub.20-Glu-Glu-Xaa.sub.23-Xaa.sub.24-Xaa.sub.25-Arg-
Xaa.sub.27-Phe-Ile-Xaa.sub.30-Xaa.sub.31-Leu-Xaa.sub.33-Xaa.sub.34-Xaa.sub-
.35-R Formula (I)
[0532] wherein
[0533] Xaa.sub.7 is L-histidine, D-histidine, desamino-histidine,
2-amino-histidine, .beta.-hydroxy-histidine, homohistidine,
N.sup..alpha.-acetyl-histidine, .alpha.-fluoromethyl-histidine,
.alpha.-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or
4-pyridylalanine;
[0534] Xaa.sub.8 is Ala, Gly, Val, Leu, Ile, Lys, Aib,
(1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic
acid, (1-aminocyclopentyl)carboxylic acid,
(1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic
acid, or (1-aminocyclooctyl)carboxylic acid;
[0535] Xaa.sub.9 is Glu or a Glu derivative such as alpha, alpha
dimethyl-Glu;
[0536] Xaa.sub.16 is Val or Leu;
[0537] Xaa.sub.18 is Ser, Lys, Cys or Arg;
[0538] Xaa.sub.20 is Leu, Lys or Cys;
[0539] Xaa.sub.23 is Gln, Glu, Lys, Cys or Arg;
[0540] Xaa.sub.24 is Ala or Asn;
[0541] Xaa.sub.25 is Ala or Val;
[0542] Xaa.sub.27 is Glu, Ala or Leu;
[0543] Xaa.sub.30 is Ala, Glu, Lys, Arg or absent;
[0544] Xaa.sub.31 is Trp, Lys, Cys or absent;
[0545] Xaa.sub.33 is Val, Lys, Cys or absent;
[0546] Xaa.sub.34 is Lys, Glu, Asn, Arg, Cys or absent;
[0547] Xaa.sub.35 is Gly, Aib or absent;
[0548] R is amide or is absent;
[0549] provided that if Xaa.sub.30, Xaa.sub.31, Xaa.sub.32,
Xaa.sub.33, or Xaa.sub.34 is absent then each amino acid residue
downstream is also absent.
[0550] 31. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-29 having the sequence of formula
(II)
TABLE-US-00010 (SEQ ID No: 3)
Xaa.sub.7-Xaa.sub.8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-
Xaa.sub.18-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Arg-Glu-Phe-
Ile-Xaa.sub.30-Trp-Leu-Xaa.sub.33-Xaa.sub.34-Xaa.sub.35-R Formula
(II)
[0551] wherein
[0552] Xaa.sub.7 is L-histidine, D-histidine, desamino-histidine,
2-amino-histidine, .beta.-hydroxy-histidine, homohistidine,
N.sup..alpha.-acetyl-histidine, .alpha.-fluoromethyl-histidine,
.alpha.-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or
4-pyridylalanine;
[0553] Xaa.sub.8 is Ala, Gly, Val, Leu, Ile, Lys, Aib,
(1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic
acid, (1-aminocyclopentyl)carboxylic acid,
(1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic
acid, or (1-aminocyclooctyl)carboxylic acid;
[0554] Xaa.sub.18 is Ser, Lys or Arg;
[0555] Xaa.sub.30 is Ala, Glu, Lys, Arg or is absent;
[0556] Xaa.sub.33 is Val, Lys or absent;
[0557] Xaa.sub.34 is Lys, Glu, Arg or is absent;
[0558] Xaa.sub.35 is Gly, Aib or is absent;
[0559] R is amide or is absent.
[0560] 32. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 30-31, wherein R is absent.
[0561] 33. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 30-32, wherein Xaa.sub.35 and R are
absent.
[0562] 34. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 30-33, wherein Xaa.sub.34, Xaa.sub.35
and R are absent.
[0563] 35. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 30-34, wherein Xaa.sub.33, Xaa.sub.34,
Xaa.sub.35 and and R are absent.
[0564] 36. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 30 and 32-35 having a total of 2 amino
acids substitutions compared to the sequence 7-35 of SEQ ID NO 1,
which are a Glu residue at a position equivalent to position 22 of
SEQ ID No 2 and an Arg residue at a position equivalent to position
26 of SEQ ID No 2.
[0565] 37. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 30 and 32-35 having a total of 3 amino
acids substitutions compared to the sequence 7-35 of SEQ ID NO 1,
which are a Glu residue at a position equivalent to position 22 of
SEQ ID No 2, an Arg residue at a position equivalent to position 26
of SEQ ID No 2 and one amino acid substitution selected from the
group consisting of Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, Xaa.sub.16,
Xaa.sub.18, Xaa.sub.20, Xaa.sub.23, Xaa.sub.24, Xaa.sub.25,
Xaa.sub.27, Xaa.sub.30, Xaa.sub.31, Xaa.sub.33, Xaa.sub.34 and
Xaa.sub.35 in SEQ ID No 2 compared to the sequence 7-35 of SEQ ID
No 1.
[0566] 38. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 30 and 32-35 having a total of 4 amino
acids substitutions compared to the sequence 7-35 of SEQ ID NO 1,
which are a Glu residue at a position equivalent to position 22 of
SEQ ID No 2, an Arg residue at a position equivalent to position 26
of SEQ ID No 2 and 2 amino acid substitution selected from the
group consisting of Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, Xaa.sub.16,
Xaa.sub.18, Xaa.sub.20, Xaa.sub.23, Xaa.sub.24, Xaa.sub.25,
Xaa.sub.27, Xaa.sub.30, Xaa.sub.31, Xaa.sub.33, Xaa.sub.34 and
Xaa.sub.35 in SEQ ID No 2 compared to the sequence 7-35 of SEQ ID
No 1.
[0567] 39. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 30 and 32-35 having a total of 5 amino
acids substitutions compared to the sequence 7-35 of SEQ ID NO 1,
which are a Glu residue at a position equivalent to position 22 of
SEQ ID No 2, an Arg residue at a position equivalent to position 26
of SEQ ID No 2 and 3 amino acid substitution selected from the
group consisting of Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, Xaa.sub.16,
Xaa.sub.18, Xaa.sub.20, Xaa.sub.23, Xaa.sub.24, Xaa.sub.25,
Xaa.sub.27, Xaa.sub.30, Xaa.sub.31, Xaa.sub.33, Xaa.sub.34 and
Xaa.sub.35 in SEQ ID No 2 compared to the sequence 7-35 of SEQ ID
No 1.
[0568] 40. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 30 and 32-35 having a total of 6 amino
acids substitutions compared to the sequence 7-35 of SEQ ID NO 1,
which are a Glu residue at a position equivalent to position 22 of
SEQ ID No 2, an Arg residue at a position equivalent to position 26
of SEQ ID No 2 and 4 amino acid substitution selected from the
group consisting of Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, Xaa.sub.16,
Xaa.sub.18, Xaa.sub.20, Xaa.sub.23, Xaa.sub.24, Xaa.sub.25,
Xaa.sub.27, Xaa.sub.30, Xaa.sub.31, Xaa.sub.33, Xaa.sub.34 and
Xaa.sub.35 in SEQ ID No 2 compared to the sequence 7-35 of SEQ ID
No 1.
[0569] 41. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 30 and 32-35 having a total of 7 amino
acids substitutions compared to the sequence 7-35 of SEQ ID NO 1,
which are a Glu residue at a position equivalent to position 22 of
SEQ ID No 2, an Arg residue at a position equivalent to position 26
of SEQ ID No 2 and 5 amino acid substitution selected from the
group consisting of Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, Xaa.sub.16,
Xaa.sub.18, Xaa.sub.20, Xaa.sub.23, Xaa.sub.24, Xaa.sub.25,
Xaa.sub.27, Xaa.sub.30, Xaa.sub.31, Xaa.sub.33, Xaa.sub.34 and
Xaa.sub.35 in SEQ ID No 2 compared to the sequence 7-35 of SEQ ID
No 1.
[0570] 42. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 30 and 32-35 having a total of 8 amino
acids substitutions compared to the sequence 7-35 of SEQ ID NO 1,
which are a Glu residue at a position equivalent to position 22 of
SEQ ID No 2, an Arg residue at a position equivalent to position 26
of SEQ ID No 2 and 3 amino acid substitution selected from the
group consisting of Xaa.sub.7, Xaa.sub.8, Xaa.sub.9, Xaa.sub.16,
Xaa.sub.18, Xaa.sub.20, Xaa.sub.23, Xaa.sub.24, Xaa.sub.25,
Xaa.sub.27, Xaa.sub.30, Xaa.sub.31, Xaa.sub.33, Xaa.sub.34 and
Xaa.sub.35 in SEQ ID No 2 compared to the sequence 7-35 of SEQ ID
No 1.
[0571] 43. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 31-35 having a total of 2 amino acids
substitutions compared to the sequence 7-35 of SEQ ID NO 1, which
are a Glu residue at a position equivalent to position 22 of SEQ ID
No 3 and an Arg residue at a position equivalent to position 26 of
SEQ ID No 3.
[0572] 44. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 31-35 having a total of 3 amino acids
substitutions compared to the sequence 7-35 of SEQ ID NO 1, which
are a Glu residue at a position equivalent to position 22 of SEQ ID
No 3, an Arg residue at a position equivalent to position 26 of SEQ
ID No 3 and one amino acid substitution selected from the group
consisting of Xaa.sub.7, Xaa.sub.8, Xaa.sub.18, Xaa.sub.30,
Xaa.sub.33, Xaa.sub.34 and Xaa.sub.35 in SEQ ID No 3 compared to
the sequence 7-35 of SEQ ID No 1.
[0573] 45. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 31-35 having a total of 4 amino acids
substitutions compared to the sequence 7-35 of SEQ ID NO 1, which
are a Glu residue at a position equivalent to position 22 of SEQ ID
No 3, an Arg residue at a position equivalent to position 26 of SEQ
ID No 3 and 2 amino acid substitution selected from the group
consisting of Xaa.sub.7, Xaa.sub.8, Xaa.sub.18, Xaa.sub.30,
Xaa.sub.33, Xaa.sub.34 and Xaa.sub.35 in SEQ ID No 3 compared to
the sequence 7-35 of SEQ ID No 1.
[0574] 46. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 31-35 having a total of 5 amino acids
substitutions compared to the sequence 7-35 of SEQ ID NO 1, which
are a Glu residue at a position equivalent to position 22 of SEQ ID
No 3, an Arg residue at a position equivalent to position 26 of SEQ
ID No 3 and 3 amino acid substitution selected from the group
consisting of Xaa.sub.7, Xaa.sub.8, Xaa.sub.18, Xaa.sub.30,
Xaa.sub.33, Xaa.sub.34 and Xaa.sub.35 in SEQ ID No 3 compared to
the sequence 7-35 of SEQ ID No 1.
[0575] 47. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 31-35 having a total of 6 amino acids
substitutions compared to the sequence 7-35 of SEQ ID NO 1, which
are a Glu residue at a position equivalent to position 22 of SEQ ID
No 3, an Arg residue at a position equivalent to position 26 of SEQ
ID No 3 and 4 amino acid substitution selected from the group
consisting of Xaa.sub.7, Xaa.sub.8, Xaa.sub.18, Xaa.sub.30,
Xaa.sub.33, Xaa.sub.34 and Xaa.sub.35 in SEQ ID No 3 compared to
the sequence 7-35 of SEQ ID No 1.
[0576] 48. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 31-35 having a total of 7 amino acids
substitutions compared to the sequence 7-35 of SEQ ID NO 1, which
are a Glu residue at a position equivalent to position 22 of SEQ ID
No 3, an Arg residue at a position equivalent to position 26 of SEQ
ID No 3 and 5 amino acid substitution selected from the group
consisting of Xaa.sub.7, Xaa.sub.8, Xaa.sub.18, Xaa.sub.30,
Xaa.sub.33, Xaa.sub.34 and Xaa.sub.35 in SEQ ID No 3 compared to
the sequence 7-35 of SEQ ID No 1.
[0577] 49. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 31-35 having a total of 8 amino acids
substitutions compared to the sequence 7-35 of SEQ ID NO 1, which
are a Glu residue at a position equivalent to position 22 of SEQ ID
No 3, an Arg residue at a position equivalent to position 26 of SEQ
ID No 3 and 3 amino acid substitution selected from the group
consisting of Xaa.sub.7, Xaa.sub.8, Xaa.sub.18, Xaa.sub.30,
Xaa.sub.33, Xaa.sub.34 and Xaa.sub.35 in SEQ ID No 3 compared to
the sequence 7-35 of SEQ ID No 1.
[0578] 50. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 30-49 wherein Xaa.sub.7 is
desamino-histidine.
[0579] 51. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 30-49 wherein Xaa.sub.8 is Aib.
[0580] 52. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-51, wherein the amino acid which is
pegylated or derivatised with an albumin binding residue is a
Lys-residue or a Cys-residue.
[0581] 53. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-51, wherein the amino acid which is
pegylated or derivatised with an albumin binding residue is a
Lys-residue.
[0582] 54. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-51, wherein the C-terminal amino acid
is pegylated or derivatised with an albumin binding residue.
[0583] 55. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-54, which has been pegylated or
derivatised with an albumin binding residue at position 18, 20, 23,
31, 33, 34 or at the C-terminal amino acid.
[0584] 56. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-55, which has been pegylated or
derivatised with an albumin binding residue at position 18.
[0585] 57. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-55, which has been pegylated or
derivatised with an albumin binding residue at position 20.
[0586] 58. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-55, which has been pegylated or
derivatised with an albumin binding residue at position 23.
[0587] 59. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-55, which has been pegylated or
derivatised with an albumin binding residue at position 31.
[0588] 60. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-55, which has been pegylated or
derivatised with an albumin binding residue at position 33.
[0589] 61. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-55, which has been pegylated or
derivatised with an albumin binding residue at position 34.
[0590] 62. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-55, which has been derivatised with an
albumin binding residue.
[0591] 63. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-62, wherein at least one amino acid
residue is derivatised with A-B-C-D-
[0592] wherein A- is selected from the group consisting of
##STR00041##
[0593] wherein n is selected from the group consisting of 14, 15,
16 17, 18 and 19, p is selected from the group consisting of 10,
11, 12, 13 and 14, and d is selected from the group consisting of
0, 1, 2, 3, 4 and 5,
[0594] -B- is selected from the group consisting of
##STR00042##
[0595] wherein x is selected from the group consisting of 0, 1, 2,
3 and 4, and y is selected from the group consisting of 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11 and 12,
[0596] -C- is selected from the group consisting of
##STR00043##
[0597] wherein b and e are each independently selected from the
group consisting of 0, 1 and 2, and c and f are each independently
selected from the group consisting of 0, 1 and 2 with the proviso
that b is 1 or 2 when c is 0, or b is 0 when c is 1 or 2, and e is
1 or 2 when f is 0, or e is 0 when f is 1 or 2, and
[0598] -D- is attached to said amino acid residue and is a
linker.
[0599] 64. The GLP-1 analogue or derivative thereof according to
embodiment 61, wherein one amino acid residue is derivatised with
A-B-C-D-.
[0600] 65. The GLP-1 analogue or derivative thereof according to
any one of embodiments 63-64, wherein the derivatised amino acid
residue comprises an amino group.
[0601] 66. The GLP-1 analogue or derivative thereof according to
any one of embodiments 63-65, wherein the derivatised amino acid
residue comprises a primary amino group in a side chain.
[0602] 67. The GLP-1 analogue or derivative thereof according to
any one of embodiments 63-66, wherein the derivatised amino acid
residue is lysine.
[0603] 68. The GLP-1 analogue or derivative thereof according to
any one of embodiments 63-67, wherein only one amino acid residue
is derivatised.
[0604] 69. The GLP-1 analogue or derivative thereof according to
any one of embodiments 63-68, wherein A- is
##STR00044##
[0605] 70. The GLP-1 analogue or derivative thereof according to
any of the embodiments 63-69, wherein n is selected from the group
consisting of 15 and 17, and more is preferred 17.
[0606] 71. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 63-68, wherein A- is
##STR00045##
[0607] 72. The GLP-1 analogue or derivative thereof according to
any of the embodiments 63-68 and 69, wherein p is selected from the
group consisting of 12, 13, and 14 and more preferred is 13.
[0608] 73. The GLP-1 analogue or derivative thereof according to
any of the embodiments 61-68 and 69-70, wherein d is selected from
the group consisting of 0, 1, 2, 3 and 4, more preferred 0, 1 and 2
and most preferred 1.
[0609] 74. The GLP-1 analogue or derivative thereof according to
any of the embodiments 63-68 and 69-71, wherein d is selected from
the group consisting of 0, 1 and 2 and p is selected from the group
consisting of 12, 13 or 14, more preferred d is selected from the
group consisting of 1 and 2 and p is selected from the group
consisting of 13 and 14, and most preferred d is 1 and p is 13.
[0610] 75. The GLP-1 analogue or derivative thereof according to
any of the embodiments 63-74, wherein -B- is
##STR00046##
[0611] 76. The GLP-1 analogue or derivative thereof according to
any of the embodiments 63-74, wherein -B- is
##STR00047##
[0612] 77. The GLP-1 analogue or derivative thereof according to
any of the embodiments 63-74, wherein -B- is
##STR00048##
[0613] 78. The GLP-1 analogue or derivative thereof according to
any of the embodiments 63-74, wherein -B- is
##STR00049##
[0614] 79. The GLP-1 analogue or derivative thereof according to
embodiment 78, wherein x is selected from the group consisting of
0, 1 and 2, more preferred x is selected from the group consisting
of 0 and 1 and most preferred x is 1.
[0615] 80. The GLP-1 analogue or derivative thereof according to
any of the embodiments 63-74, wherein -B- is
##STR00050##
[0616] 81. The GLP-1 analogue or derivative thereof according to
embodiment 80, wherein y is selected from the group consisting of
2, 3, 4, 5, 6, 7, 8, 9 and 10 and more preferred y is selected from
the group consisting of 2, 3, 4, 5, 6, 7, and 8.
[0617] 82. The GLP-1 analogue or derivative thereof according to
any of the embodiments 63-81, wherein -C- is
##STR00051##
[0618] 83. The GLP-1 analogue or derivative thereof according to
embodiment 82, wherein c is selected from the group consisting of 0
and 1 and b is selected from the group consisting of 1 and 2, more
preferred b is 1 and c is 0.
[0619] 84. The GLP-1 analogue or derivative thereof according to
any of the embodiments 63-81, wherein -C- is
##STR00052##
[0620] 85. The GLP-1 analogue or derivative thereof according to
embodiment 84, wherein f is selected from the group consisting of 0
and 1 and e is selected from the group consisting of 1 and 2, more
preferred e is 1 and f is 0.
[0621] 86. The GLP-1 analogue or derivative thereof according to
any of the embodiments 63-81, wherein -C- is
##STR00053##
[0622] 87. The GLP-1 analogue or derivative thereof according to
any of the embodiments 63-86, wherein D is selected from the group
consisting of
##STR00054##
[0623] and wherein k is selected from the group consisting of 0, 1,
2, 3, 4, 5, 11 and 27, and m is selected from the group consisting
of 0, 1, 2, 3, 4, 5 and 6.
[0624] 88. The GLP-1 analogue or derivative thereof according to
any of the embodiments 63-87, wherein -D- is
##STR00055##
[0625] 89. The GLP-1 analogue or derivative thereof according to
embodiment 88, wherein k is selected from the group consisting of
1, 2, 3, 11 and 27 and more preferred k is 1.
[0626] 90. The GLP-1 analogue or derivative thereof according to
any of the embodiments 88-89, wherein m is selected from the group
consisting of 0, 1, 2, 3, and 4 and more preferred m is selected
from the group consisting of 0, 1 and 2.
[0627] 91. The GLP-1 analogue or derivative thereof according to
embodiment 91, wherein m is selected from the group consisting of
0, 1, 2, 3, and 4 and more preferred m is selected from the group
consisting of 0, 1 and 2.
[0628] 92. The GLP-1 analogue or derivative thereof according to
any of the embodiments 63-87, wherein -D- is
##STR00056##
[0629] 93. The GLP-1 analogue or derivative thereof according to
embodiment 93, wherein m is selected from the group consisting of
0, 1, 2, 3, and 4 and more preferred m is selected from the group
consisting of 0, 1 and 2.
[0630] 94. The GLP-1 analogue or derivative thereof according to
any of the embodiments 1-87, wherein -D- is
##STR00057##
[0631] 95. The GLP-1 analogue or derivative thereof according to
embodiment 95, wherein m is selected from the group consisting of
0, 1, 2, 3, and 4 and more preferred m is selected from the group
consisting of 0, 1 and 2.
[0632] 96. The GLP-1 analogue or derivative thereof according to
any of the embodiments 1-87, wherein -D- is
##STR00058##
[0633] 97. The GLP-1 analogue or derivative thereof according to
embodiment 97, wherein m is selected from the group consisting of
0, 1, 2, 3, and 4 and more preferred m is selected from the group
consisting of 0, 1 and 2.
[0634] 98. The GLP-1 analogue or derivative thereof according to
any of the embodiments 1-87, wherein -D- is
##STR00059##
[0635] 99. The GLP-1 analogue or derivative thereof according to
embodiment 99, wherein m is selected from the group consisting of
0, 1, 2, 3, and 4 and more preferred m is selected from the group
consisting of 0, 1 and 2.
[0636] 100. The GLP-1 analogue or derivative thereof according to
any of the embodiments 1-100, wherein A-B-C-D- is selected and
combined from
##STR00060## ##STR00061##
[0637] 101. The GLP-1 analogue or derivative thereof according to
any of the embodiments 1-100, wherein A-B-C-D- is selected and
combined from
##STR00062## ##STR00063##
[0638] 102. The GLP-1 analogue or derivative thereof according to
any of the embodiments 1-100, wherein A-B-C-D- is selected from the
group consisting of
##STR00064## ##STR00065## ##STR00066## ##STR00067##
[0639] 103. The GLP-1 analogue or derivative thereof according to
any one of the embodiments 1-103, which comprises a hydrophilic
linker between the modified GLP-1 sequence and one or more albumin
binding residue(s).
[0640] 104. The GLP-1 analogue or derivative thereof according to
embodiment 104, wherein the hydrophilic linker is an unbranched
oligo ethylene glycol moiety with appropriate functional groups at
both terminals that forms a bridge between an amino group of the
modified GLP-1 sequence and a functional group of the albumin
binding residue.
[0641] 105. The GLP-1 analogue or derivative thereof according to
any of the above embodiments, which is selected from the group
consisting of
[0642] [Glu22,Arg26]GLP-1(7-33)amide,
[0643] N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Val25,-
Arg26,Leu27,Glu30,Lys33)GLP-1(7-33)amide,
[0644] N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Arg26,-
Glu30)GLP-1(7-33)amide,
[0645] [Glu22,Val25,Arg26]GLP-1(7-33)amide,
[0646] N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Arg26,-
Glu30)GLP-1(7-33)amide,
[0647] [Glu22, Arg26]GLP-1(7-33)peptide,
[0648] N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-[Aib8,Lys20,Glu22,Val25,-
Arg26,Glu30]GLP-1(7-33)amide, and
[0649] [Glu22,Val25,Arg26]GLP-1(7-32)amide.
[0650] 106. A method for increasing the time of action in a patient
of a GLP-1 analogue or derivative thereof, characterised in that a
modified GLP-1 sequence 7-35 (SEQ ID No 1) is derivatised or
pegylated as disclosed in any of the preceding embodiments.
[0651] 107. A method for increasing the time of action in a patient
of a GLP-1 analogue or derivative thereof to more than about 40
hours, characterised in that a modified GLP-1 sequence 7-37 (SEQ ID
No 1) is derivatised or pegylated as disclosed in any of the
preceding embodiments.
[0652] 108. A pharmaceutical composition comprising a GLP-1
analogue or derivative thereof according to any one of embodiments
1-107, and a pharmaceutically acceptable excipient.
[0653] 109. The pharmaceutical composition according to embodiment
109, which is suited for parenteral administration.
[0654] 110. Use of a GLP-1 analogue or derivative thereof according
to any one of embodiments 1-107 for the preparation of a
medicament.
[0655] 111. Use of a GLP-1 analogue or derivative thereof according
to any one of embodiments 1-107 for the preparation of a medicament
for the treatment or prevention of hyperglycemia, type 2 diabetes,
impaired glucose tolerance, type 1 diabetes, obesity, hypertension,
syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis,
myocardial infarction, coronary heart disease and other
cardiovascular disorders, stroke, inflammatory bowel syndrome,
dyspepsia and gastric ulcers.
[0656] 112. Use of a GLP-1 analogue or derivative thereof according
to any one of embodiments 1-107 for the preparation of a medicament
for delaying or preventing disease progression in type 2
diabetes.
[0657] 113. Use of a GLP-1 analogue or derivative thereof according
to any one of embodiments 1-107 for the preparation of a medicament
for decreasing food intake, decreasing .beta.-cell apoptosis,
increasing .beta.-cell function and .beta.-cell mass, and/or for
restoring glucose sensitivity to .beta.-cells.
[0658] 114. A GLP-1 analogue or derivative thereof according to any
one of claims 1-107 for use in the treatment or prevention of
hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1
diabetes, obesity, hypertension, syndrome X, dyslipidemia,
cognitive disorders, atheroschlerosis, myocardial infarction,
coronary heart disease and other cardiovascular disorders, stroke,
inflammatory bowel syndrome, dyspepsia and gastric ulcers.
[0659] All references, including publications, patent applications
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference was individually and
specifically indicated to be incorporated by reference and was set
forth in its entirety herein.
[0660] All headings and sub-headings are used herein for
convenience only and should not be construed as limiting the
invention in any way,
[0661] Any combination of the above-described elements in all
possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by
context.
[0662] The terms "a" and "an" and "the" and similar referents as
used in the context of describing the invention are to be construed
to cover both the singular and the plural, unless otherwise
indicated herein or clearly contradicted by context.
[0663] Recitation of ranges of values herein are merely intended to
serve as a shorthand method of referring individually to each
separate value falling within the range, unless otherwise indicated
herein, and each separate value is incorporated into the
specification as if it were individually recited herein. Unless
otherwise stated, all exact values provided herein are
representative of corresponding approximate values (e.g., all exact
exemplary values provided with respect to a particular factor or
measurement can be considered to also provide a corresponding
approximate measurement, modified by "about," where
appropriate).
[0664] All methods described herein can be performed in any
suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context.
[0665] The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise indicated. No language in
the specification should be construed as indicating any element is
essential to the practice of the invention unless as much is
explicitly stated.
[0666] The citation and incorporation of patent documents herein is
done for convenience only and does not reflect any view of the
validity, patentability and/or enforceability of such patent
documents,
[0667] The description herein of any aspect or embodiment of the
invention using terms such as "comprising", "having", "including"
or "containing" with reference to an element or elements is
intended to provide support for a similar aspect or embodiment of
the invention that "consists of", "consists essentially of", or
"substantially comprises" that particular element or elements,
unless otherwise stated or clearly contradicted by context (e.g., a
formulation described herein as comprising a particular element
should be understood as also describing a formulation consisting of
that element, unless otherwise stated or clearly contradicted by
context).
[0668] This invention includes all modifications and equivalents of
the subject matter recited in the aspects or claims presented
herein to the maximum extent permitted by applicable law.
[0669] The present invention is further illustrated in the
following representative methods and examples which are, however,
not intended to limit the scope of the invention in any way.
[0670] The features disclosed in the foregoing description and in
the following examples may, both separately and in any combination
thereof, be material for realising the invention in diverse forms
thereof.
Examples
[0671] Abbreviations used: [0672] r.t: Room temperature [0673]
DIPEA: diisopropylethylamine [0674] H.sub.2O: water [0675]
CH.sub.3CN: acetonitrile [0676] DMF: NN dimethylformamide [0677]
HBTU: 2-(1H-Benzotriazol-1-yl-)-1,1,3,3 tetramethyluronium
hexafluorophosphate [0678] Fmoc: 9H-fluoren-9-ylmethoxycarbonyl
[0679] Boc: tert butyloxycarbonyl [0680] OtBu: tert butyl ester
[0681] tBu: tert butyl [0682] Trt: triphenylmethyl [0683] Pmc:
2,2,5,7,8-Pentamethyl-chroman-6-sulfonyl [0684] Dde:
1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl [0685] ivDde:
1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl [0686] Mtt:
4-methyltrityl [0687] Mmt: 4-methoxytrityl [0688] DCM:
dichloromethane [0689] TIS: triisopropylsilane) [0690] TFA:
trifluoroacetic acid [0691] Et.sub.2O: diethylether [0692] NMP:
1-Methyl-pyrrolidin-2-one [0693] DIPEA: Diisopropylethylamine
[0694] HOAt: 1-Hydroxy-7-azabenzotriazole [0695] HOBt:
1-Hydroxybenzotriazole [0696] DIC: Diisopropylcarbodiimide [0697]
DBU: 1,8-diazabicycli-[5,4,0]undecene-7 [0698] MW: Molecular
weight
[0699] A: Synthesis of Resin Bound Peptide
[0700] SPPS Method A.
[0701] The protected peptidyl resin was synthesized according to
the Fmoc strategy on an Applied Biosystems 433 peptide synthesizer
in 0.25 mmol or 1.0 mmol scale using the manufacturer supplied
FastMoc UV protocols which employ HBTU
(2-(1H-Benzotriazol-1-yl-)-1,1,3,3 tetramethyluronium
hexafluorophosphate) or HATU
(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate) mediated couplings in NMP (N-methyl
pyrrolidone), and UV monitoring of the deprotection of the Fmoc
protection group. The starting resin used for the synthesis of the
peptide amides was Rink-Amide resin and either Wang or chlorotrityl
resin was used for peptides with a carboxy C-terminal. The
protected amino acid derivatives used were standard Fmoc-amino
acids (supplied from e.g. Anaspec, or Novabiochem) supplied in
preweighed cartridges suitable for the ABI433A synthesizer with the
exception of unnatural aminoacids such as Fmoc-Aib-OH
(Fmoc-aminoisobutyric acid). The N terminal amino acid was Boc
protected at the alpha amino group (e.g. Boc-His(Boc)OH was used
for peptides with His at the N-terminal). The epsilon amino group
of lysine at position 26 was either protected with Mtt, Mmt, Dde,
ivDde, or Boc, depending on the route for attachment of the albumin
binding moiety and spacer. The synthesis of the peptides may in
some cases be improved by the use of dipeptides protected on the
dipeptide amide bond with a group that can be cleaved under acidic
conditions such but not limited to 2-Fmoc-oxy-4-methoxybenzyl or
2,4,6-trimethoxybenzyl. In cases where a serine or a threonine is
present in the peptide, the use of pseudoproline dipeptides may be
used (see e.g. catalogue from Novobiochem 2002/2003 or newer
version, or W. R. Sampson (1999), J. Pep. Sci. 5, 403.
[0702] SPPS Method B:
[0703] One alternative method (method B) of peptide synthesis was
by Fmoc chemistry on a microwave-based Liberty peptide synthesizer
(CEM Corp., North Carolina). The resin was Tentagel S RAM with a
loading of 0.24 mmol/g. The coupling chemistry was DIC/HOAt in NMP
using amino acid solutions of 0.3 M in NMP and a molar excess of
8-10 fold. Coupling conditions was 5 minutes at up to 70.degree. C.
Deprotection was with 5% piperidine in NMP at up to 70.degree. C.
When a chemical modification of a lysine side chain was desired,
the lysine was incorporated as Lys(Mtt). The Mtt group was removed
by suspending the resin in neat hexafluoroisopropanol for 20
minutes followed by washing with DCM and NMP. The chemical
modification of the lysine was performed either by manual synthesis
or by one or more automated steps on the Liberty followed by a
manual coupling. Another method of peptide synthesis was by Fmoc
chemistry on an ABI 433 with HBTU coupling. After synthesis the
resin was washed with DCM and dried, and the peptide was cleaved
from the resin by a 2 hour treatment with TFA/TIS/water
(92.5/5/2.5) followed by precipitation with diethylether. the
peptide was redissolved in 30% acetic acid or similar solvent and
purified by standard RP-HPLC on a C18 column using
acetonitrile/TFA. The identity of the peptide was confirmed by
MALDI-MS.
[0704] SPPS Method C
[0705] The protected peptidyl resin was synthesized according to
the Fmoc strategy on an Advanced ChemTech Synthesiser (APEX 348)
0.25 mmol scale using the manufacturer supplied protocols which
employ DIC (dicyclohexylcarbodiimide) and HOBt
(1-Hydroxybenzotriazole) mediated couplings in NMP (N-methyl
pyrrolidone. The starting resin used for the synthesis of the
peptide amides was Rink-Amide resin and either Wang or chlorotrityl
resin was used for peptides with a carboxy C-terminal. The
protected amino acid derivatives used were standard Fmoc-amino
acids (supplied from e.g. Anaspec, or Novabiochem. The N terminal
amino acid was Boc protected at the alpha amino group (e.g.
Boc-His(Boc)OH was used for peptides with His at the N-terminal).
The epsilon amino group of lysine at position 26 was either
protected with Mtt, Mmt, Dde, ivDde, or Boc, depending on the route
for attachment of the albumin binding moiety and spacer. The
synthesis of the peptides may in some cases be improved by the use
of dipeptides, e.g., pseudoprolines from Novabiochem,
Fmoc-Ser(tbu)-.PSI.Ser(Me,Me)-OH, see e.g. catalogue from
Novobiochem 2002/2003 or newer version, or W. R. Sampson (1999), J.
Pep. Sci. 5, 403
[0706] Procedure for Removal of ivDde or Dde-Protection.
[0707] The resin (0.25 mmol) was placed in a manual
shaker/filtration apparatus and treated with 2% hydrazine in
N-methyl pyrrolidone (20 ml, 2.times.12 min) to remove the Dde or
ivDde group and wash with N-methyl pyrrolidone (4.times.20 ml).
[0708] Procedure for Removal of Mtt or Mmt-Protection.
[0709] The resin (0.25 mmol) was placed in a manual
shaker/filtration apparatus and treated with 2% TFA and 2-3% TIS in
DCM (20 ml, 5-10 min repeated 6-12 times) to remove the Mtt or Mmt
group and wash with DCM (2.times.20 ml), 10% MeOH and 5% DIPEA in
DCM (2.times.20 ml) and N-methyl pyrrolidone (4.times.20 ml).
[0710] Alternative Procedure for Removal of Mtt-Protection:
[0711] The resin was placed in a syringe and treated with
hexafluroisopropanol for 2.times.10 min to remove the Mtt group.
The resin was then washed with DCM and NMP as described above.
[0712] Procedure for Attachment of Sidechains to Lysine
Residue.
[0713] The albumin binding residue (B--U-sidechain of formula I)
can be attached to the peptide either by acylation to resin bound
peptide or acylation in solution to the unprotected peptide using
standard acylating reagent such as but not limited to DIC,
HOBt/DIC, HOAt/DIC, or HBTU.
[0714] Attachment to Resin Bound Peptide:
[0715] Route I
[0716] Activated (active ester or symmetric anhydride) albumin
binding residue (A-B)-sidechain of formula I) such as
octadecanedioic acid mono-(2,5-dioxo-pyrrolidin-1-yl)ester (Ebashi
et al. EP511600, 4 molar equivalents relative to resin bound
peptide) was dissolved in NMP (25 mL), added to the resin and
shaken overnight at room temperature. The reaction mixture was
filtered and the resin was washed extensively with NMP,
dichloromethane, 2-propanol, methanol and diethyl ether.
[0717] Route II
[0718] The albumin binding residue (A-(B)-sidechain of formula I)
was dissolved in N-methyl pyrrolidone/methylene chloride (1:1, 10
ml). The activating reagent such as hydroxybenzotriazole (HOBt) (4
molar equivalents relative to resin) and diisopropylcarbodiimide (4
molar equivalents relative to resin) was added and the solution was
stirred for 15 min. The solution was added to the resin and
diisopropylethylamine (4 molar equivalents relative to resin) was
added. The resin was shaken 2 to 24 hours at room temperature. The
resin was washed with N-methyl pyrrolidone (2.times.20 ml),
N-methyl pyrrolidone/Methylene chloride (1:1) (2.times.20 ml) and
methylene chloride (2.times.20 ml).
[0719] Route III
[0720] Activated (active ester or symmetric anhydride) albumin
binding residue (A-B-sidechain of formula I) such as
octadecanedioic acid mono-(2,5-dioxo-pyrrolidin-1-yl)ester (Ebashi
et al. EP511600, 1-1.5 molar equivalents relative to the peptide
was dissolved in an organic solvent such as acetonitrile, THF, DMF,
DMSO or in a mixture of water/organic solvent (1-2 ml) and added to
a solution of the peptide in water (10-20 ml) together with 10
molar equivalents of DIPEA. In case of protecting groups on the
albumin binding residue such as tert.-butyl, the reaction mixture
was lyophilized O/N and the isolated crude peptide deprotected
afterwards--in case of a tert-butyl group the peptide was dissolved
in a mixture of trifluoroacetic acid, water and triisopropylsilane
(90:5:5). After for 30 min the mixture was, evaporated in vacuo and
the finale peptide purified by preparative HPLC.
[0721] Procedure for removal of Fmoc-protection: The resin (0.25
mmol) was placed in a filter flask in a manual shaking apparatus
and treated with N-methyl pyrrolidone/methylene chloride (1:1)
(2.times.20 ml) and with N-methyl pyrrolidone (1.times.20 ml), a
solution of 20% piperidine in N-methyl pyrrolidone (3.times.20 ml,
10 min each). The resin was washed with N-methyl pyrrolidone
(2.times.20 ml), N-methyl pyrrolidone/Methylene chloride (1:1)
(2.times.20 ml) and methylene chloride (2.times.20 ml).
[0722] Procedure for Cleaving the Peptide off the Resin:
[0723] The peptide was cleaved from the resin by stirring for 180
min at room temperature with a mixture of trifluoroacetic acid,
water and triisopropylsilane (95:2.5:2.5 to 92:4:4). The cleavage
mixture was filtered and the filtrate was concentrated to an oil by
a stream of nitrogen. The crude peptide was precipitated from this
oil with 45 ml diethyl ether and washed 1 to 3 times with 45 ml
diethyl ether.
[0724] Purification: The crude peptide was purified by
semipreparative HPLC on a 20 mm.times.250 mm column packed with
either 5.mu. or 7.mu. C-18 silica. Depending on the peptide one or
two purification systems were used.
[0725] TFA: After drying the crude peptide was dissolved in 5 ml
50% acetic acid H.sub.2O and diluted to 20 ml with H.sub.2O and
injected on the column which then was eluted with a gradient of
40-60% CH.sub.3CN in 0.1% TFA 10 ml/min during 50 min at 40.degree.
C. The peptide containing fractions were collected. The purified
peptide was lyophilized after dilution of the eluate with
water.
[0726] Ammonium sulphate: The column was equilibrated with 40%
CH.sub.3CN in 0.05M (NH.sub.4).sub.2SO.sub.4, which was adjusted to
pH 2.5 with concentrated H.sub.2SO.sub.4. After drying the crude
peptide was dissolved in 5 ml 50% acetic acid H.sub.2O and diluted
to 20 ml with H.sub.2O and injected on the column which then was
eluted with a gradient of 40%-60% CH.sub.3CN in 0.05M
(NH.sub.4).sub.2SO.sub.4, pH 2.5 at 10 ml/min during 50 min at
40.degree. C. The peptide containing fractions were collected and
diluted with 3 volumes of H.sub.2O and passed through a
Sep-Pak.RTM. C18 cartridge (Waters part. #:51910) which has been
equilibrated with 0.1% TFA. It was then eluted with 70% CH.sub.3CN
containing 0.1% TFA and the purified peptide was isolated by
lyophilisation after dilution of the eluate with water.
[0727] The final product obtained was characterised by analytical
RP-HPLC (retention time) and by LCMS
[0728] The RP-HPLC analysis may be performed using UV detection at
214 nm and e.g. a Vydac 218TP54 4.6 mm.times.250 mm 5.mu. C-18
silica column (The Separations Group, Hesperia, USA) and eluted at
e.g. 1 ml/min at 42.degree. C. Most often one of following specific
conditions were used:
[0729] Method 03_A1.sub.--1
[0730] HPLC (Method 03_A1.sub.--1): The RP-analysis was performed
using a Waters 2690 systems fitted with a Waters 996 diode array
detector. UV detections were collected at 214, 254, 276, and 301 nm
on a 218TP54 4.6 mm.times.250 mm 5.mu. C-18 silica column (The
Separations Group, Hesperia), which was eluted at 1 ml/min at
42.degree. C. The column was equilibrated with 10% of a 0.5 M
ammonium sulfate, which was adjusted to pH 2.5 with 4M sulfuric
acid. After injection, the sample was eluted by a gradient of 0% to
60% acetonitrile in the same aqueous buffer during 50 min.
[0731] Method 03_B1.sub.--2
[0732] HPLC (Method 03_B1.sub.--2): The RP-analysis was performed
using a Waters 2690 systems fitted with a Waters 996 diode array
detector. UV detections were collected at 214, 254, 276, and 301 nm
on a Zorbax 300SB C-18 (4.5.times.150 mm, 5.mu.), which was eluted
at 0.5 ml/min at 42.degree. C. The column was equilibrated with an
aqueous solution of TFA in water (0.1%). After injection, the
sample was eluted by a gradient of 0% to 60% acetonitrile (+0.1%
TFA) in an aqueous solution of TFA in water (0.1%) during 50
min.
[0733] Method 02_B1.sub.--1
[0734] HPLC (Method 02_B1.sub.--1): The RP-analyses was performed
using a Alliance Waters 2695 system fitted with a Waters 2487
dualband detector. UV detections at 214 nm and 254 nm were
collected using a Vydac 218TP53, C18, 300 .ANG., 5 um, 3.2
mm.times.250 mm column, 42.degree. C. Eluted with a linear gradient
of 0-60% acetonitrile, 95-35% water and 5% trifluoroacetic acid
(1.0%) in water over 50 minutes at a flow-rate of 0.50 ml/min.
[0735] Method 01_B4.sub.--2
[0736] HPLC (Method 01_B4.sub.--2): RP-analyses was performed using
a Waters 600S system fitted with a Waters 996 diode array detector.
UV detections at 214 nm and 254 nm were collected using a
Symmetry300 C18, 5 um, 3.9 mm.times.150 mm column, 42.degree. C.
Eluted with a linear gradient of 5-95% acetonitrile, 90-0% water,
and 5% trifluoroacetic acid (1.0%) in water over 15 minutes at a
flow-rate of 1.0 min/min.
[0737] Method 02_B4.sub.--4
[0738] HPLC (Method 02_B4.sub.--4): The RP-analyses was performed
using a Alliance Waters 2695 system fitted with a Waters 2487
dualband detector. UV detections at 214 nm and 254 nm were
collected using a Symmetry300 C18, 5 um, 3.9 mm.times.150 mm
column, 42.degree. C. Eluted with a linear gradient of 5-95%
acetonitrile, 90-0% water, and 5% trifluoroacetic acid (1.0%) in
water over 15 minutes at a flow-rate of 1.0 min/min.
[0739] Method 02_B6.sub.--1
[0740] HPLC (Method 02_B6.sub.--1): The RP-analyses was performed
using a Alliance Waters 2695 system fitted with a Waters 2487
dualband detector. UV detections at 214 nm and 254 nm were
collected using a Vydac 218TP53, C18, 300 .ANG., 5 um, 3.2
mm.times.250 mm column, 42.degree. C. Eluted with a linear gradient
of 0-90% acetonitrile, 95-5% water, and 5% trifluoroacetic acid
(1.0%) in water over 50 minutes at a flow-rate of 0.50 ml/min.
[0741] Method D 03_B6.sub.--1
[0742] HPLC (Method 03_B1.sub.--1): The RP-analysis was performed
using a Waters 2690 systems fitted with a Waters 996 diode array
detector. UV detections were collected at 214, 254, 276, and 301 nm
on a 218TP54 4.6 mm.times.250 mm 5.mu. C-18 silica column (The
Separations Group, Hesperia), which was eluted at 1 ml/min at
42.degree. C. The column was equilibrated with 5% acetonitrile
(+0.1% TFA) in an aqueous solution of TFA in water (0.1%). After
injection, the sample was eluted by a gradient of 0% to 90%
acetonitrile (+0.1% TFA) in an aqueous solution of TFA in water
(0.1%) during 50 min.
[0743] HPLC (Method I_BDSB2):
[0744] Buffer A: 10 mM tris, 15 mM (NH4)2SO4, pH adjusted to 7.3
with 4N H2SO4, 20% v/v acetonitrile Buffer B: 80% v/v
acetonitrile
[0745] Flow: 1.0 ml/min
[0746] Gradient: 0-20 min 10-50% B
[0747] Column: Phenomenex, Jupiter 4.6 mm.times.150 mm, C4, 5.mu.,
300 .ANG.
[0748] Column temperature: 40.degree. C.
[0749] Alternatively a preparative gradient elution can be
performed as indicated above and the percentage of acetonitrile
where the compound elutes is noted. Identity is confirmed by
MALDI.
[0750] The following instrumentation was used:
[0751] LCMS was performed on a setup consisting of Sciex API 100
Single quadropole mass spectrometer, Perkin Elmer Series 200 Quard
pump, Perkin Elmer Series 200 autosampler, Applied Biosystems 785A
UV detector, Sedex 75 evaporative light scattering detector
[0752] The instrument control and data acquisition were done by the
Sciex Sample control software running on a Windows 2000
computer.
[0753] The HPLC pump is connected to two eluent reservoirs
containing:
[0754] A: 0.05% Trifluoro acetic acid in water
[0755] B: 0.05% Trifluoro acetic acid in acetonitrile
[0756] The analysis is performed at room temperature by injecting
an appropriate volume of the sample (preferably 20 .mu.l) onto the
column which is eluted with a gradient of acetonitrile.
[0757] The HPLC conditions, detector settings and mass spectrometer
settings used are giving in the following table.
[0758] Column: Waters Xterra MS C-18.times.3 mm id 5 .mu.m
[0759] Gradient: 5%-90% acetonitrile linear during 7.5 min at 1.5
ml/min
[0760] Detection: 210 nm (analogue output from DAD)
[0761] ELS (analogue output from ELS), 40.degree. C.
[0762] MS ionisation mode API-ES
[0763] Alternatively LCMS was performed on a setup consisting of
Hewlett Packard series 1100 G1312A Bin Pump, Hewlett Packard series
1100 Column compartment, Hewlett Packard series 1100 G1315A DAD
diode array detector, Hewlett Packard series 1100 MSD and Sedere 75
Evaporative Light Scattering detector controlled by HP Chemstation
software. The HPLC pump is connected to two eluent reservoirs
containing:
[0764] A: 10 mM NH.sub.4OH in water
[0765] B: 10 mM NH.sub.4OH in 90% acetonitrile
[0766] The analysis was performed at 23.degree. C. by injecting an
appropriate volume of the sample (preferably 20 .mu.l) onto the
column which is eluted with a gradient of A and B.
[0767] The HPLC conditions, detector settings and mass spectrometer
settings used are giving in the following table.
[0768] Column Waters Xterra MS C-18.times.3 mm id 5 .mu.m
[0769] Gradient 5%-100% acetonitrile linear during 6.5 min at 1.5
ml/min
[0770] Detection 210 nm (analogue output from DAD)
[0771] ELS (analogue output from ELS)
[0772] MS ionisation mode API-ES. Scan 100-1000 amu step 0.1
amu
[0773] MALDI-MS:
[0774] Molecular weights of the peptides were determined using
matrix-assisted laser desorption time of flight mass spectroscopy
(MALDI-MS), recorded on a Microflex (Bruker). A matrix of
.alpha.-cyano-4-hydroxy cinnamic acid was used.
[0775] Analytical HPLC Conditions (Method I):
[0776] Equilibration of the column (Xterra.TM. MS C18, 5um,
4.6.times.150 mm Column, P7N 186 000490) with 0.1% TFA/H.sub.2O and
elution by a gradient of 0% CH.sub.3CN/0.1% TFA/H.sub.2O to 60%
CH.sub.3CN/0.1% TFA/H.sub.2O during 25 min followed by a gradient
from 60% to 100% over 5 min.
[0777] In the examples of this invention the nomenclature and
structurally graphics is meant as:
[0778] One letter symbols for the natural amino acids is used, e.g.
H is L-histidine, A is L-alanine ect. Three letter abbreviations
for amino acids may also be uses, e.g. His is L-histidine, Ala is
L-alanine etc. For non natural amino acids three letter
abbreviations are used, such as Aib for aminoisobutyric acid. The
position of the amino acids may either be indicated with a number
in superscript after the amino acid symbols such as Lys.sup.33, or
as Lys33. The N-terminal amino group may be symbolised either as
NH.sub.2 or as H. The C-terminal carboxylic group may be symbolised
either as --OH or as --COOH. The C-terminal amide group is
symbolised as --NH.sub.2
[0779] The sub-structures
##STR00068##
both means His-Aib-Glu-Gly-Thr-Phe.
[0780] The epsilon amino group of Lysine may be described either as
the greek symbol .epsilon. or spelled "epsilon".
[0781] The structures in the examples below are in several cases a
combination of one letter symbols for the naturally amino acids
combined with the three letter abbreviation Aib for aminoisobutyric
acid. In several cases some of the amino acids are shown in
expanded full structure. Thus lysine that has been derivatised may
be shown as the expanded full structures as in example 2 where the
lysine at position 20 is expanded. The nitrogen (with indicated H)
between tyrosine at position 19 and the expanded lysine at position
20 is thus the nitrogen of the peptide bond connecting the two
amino acids in example 2 According to the procedure above, the
following derivatives were prepared as non-limiting examples of the
invention:
Example 1
[Glu22,Arg26]GLP-1(7-33)amide
TABLE-US-00011 [0782] H--H A E G T F T S D V S S Y L E E Q A A R E
F I A W L V--NH.sub.2
[0783] Preparation method: B
[0784] The peptide was eluted at 64% acetonitrile.
[0785] Structure confirmed by MALDI-MS
[0786] Calculated MW=3056.4
Example 2
N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Val25,-
Arg26,Leu27,Glu30,Lys33)GLP-1(7-33)amide
##STR00069##
[0788] Preparation method: Method C except that the peptide was
prepared on an Apex396 from Advanced Chemtech using a molar excess
of 8-10 fold amino acid, DIC and HOAt/HOBt (1:1) and the Mtt group
was deprotected with hexaflouroisopropanol. The final product was
characterized by analytical HPLC and MALDI-MS.
[0789] HPLC (METHOD 02_B6.sub.--1):
[0790] RT=32 min
[0791] MALDI-MS=3901
[0792] Calculated MW=3900.5
Example 3
N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Arg26,-
Glu30)GLP-1(7-33)amide
##STR00070##
[0794] Preparation method: Method C except that the peptide was
prepared on an Apex396 from Advanced Chemtech using a molar excess
of 8-10 fold amino acid, DIC and HOAt/HOBt (1:1) and the Mtt group
was deprotected with hexaflouroisopropanol. The final product was
characterized by analytical HPLC and MALDI-MS.
[0795] HPLC (METHOD 02_B6.sub.--1):
[0796] RT=32.9 min
[0797] MALDI-MS=3858.7
[0798] Calculated MW=3859.3
Example 4
[Glu22,Val25,Arg26]GLP-1(7-33)amide
TABLE-US-00012 [0799] NH.sub.2--H A E G T F T S D V S S Y L E E Q A
V R E F I A W L V--NH.sub.2
[0800] Preparation method: A
[0801] HPLC (METHOD 03_A1.sub.--1)
[0802] RT=44.6 min
[0803] LCMS: m/z=1029.2 (M+3H).sup.3+
[0804] Calculated MW=3084.4
Example 5
[Aib8,Lys20,Glu22,Val25,Arg26,Glu30]GLP-1(7-33)amide
##STR00071##
[0806] Preparation method: B
[0807] The peptide was eluted at 60% acetonitrile.
[0808] Structure confirmed by MALDI-MS
[0809] Calculated MW=3171.5
Example 6
N epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-[Aib8,Lys20,Glu22,Val25,-
Arg26,Glu30]GLP-1(7-33)amide
##STR00072##
[0811] Preparation method: B
[0812] The peptide was eluted at 70% acetonitrile.
[0813] Structure confirmed by MALDI-MS
[0814] Calculated MW=3887.4
Example 7
[Glu22, Arg26]GLP-1(7-33)peptide
TABLE-US-00013 [0815] H--H A E G T F T S D V S S Y L E E Q A A R E
F I A W L V--OH
[0816] Preparation method: A
[0817] HPLC (method B6):
[0818] RT=28.09 min
[0819] LCMS: m/z=1020 (M+3H).sup.3+
[0820] Calculated MW=3057.3
Example 8
[Glu22,Val25,Arg26]GLP-1(7-32)amide
TABLE-US-00014 [0821] NH.sub.2--H A E G T F T S D V S S Y L E E Q A
V R E F I A W L--NH.sub.2
[0822] Preparation method: A
[0823] HPLC (METHOD 03_A1.sub.--1)
[0824] RT=41.9 min
[0825] LCMS: m/z=996.0 (M+3H).sup.3+
[0826] Calculated MW=2985.3
Example 9
N-epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Arg26)-
GLP-1(7-33)amide
##STR00073##
[0827] Preparation method: Method C except that the peptide was
prepared on an Apex396 from Advanced Chemtech using a molar excess
of 8-10 fold amino acid, DIC and HOAt/HOBt (1:1) and the Mtt group
was deprotected with hexaflouroisopropanol. The final product was
characterized by analytical HPLC and LC-MS.
[0828] HPLC (METHOD 02_B6.sub.--1):
[0829] RT=34.31 MIN
[0830] LCMS: m/z=1277 (M+3H).sup.3+
[0831] Calculated MW=3831
Example 10
N-epsilon31
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Glu22,Val25,Arg26,-
Lys31)GLP-1(7-33)amide
##STR00074##
[0833] Preparation method: Method C except that the peptide was
prepared on an Apex396 from Advanced Chemtech using a molar excess
of 8-10 fold amino acid, DIC and HOAt/HOBt (1:1) and the Mtt group
was deprotected with hexaflouroisopropanol. The final product was
characterized by analytical HPLC and LC-MS.
[0834] HPLC (METHOD 02_B6.sub.--1):
[0835] RT=34.02 MIN
[0836] LCMS: m/z=1253 (M+3H).sup.3+
[0837] Calculated MW=3759
Example 11
N-epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(DesaminoHis7,Lys20,Glu2-
2,Arg26)GLP-1(7-33)amide
##STR00075##
[0839] Preparation method: Method C except that the peptide was
prepared on an Apex396 from Advanced Chemtech using a molar excess
of 8-10 fold amino acid, DIC and HOAt/HOBt (1:1) and the Mtt group
was deprotected with hexaflouroisopropanol. The final product was
characterized by analytical HPLC and LC-MS.
[0840] HPLC (METHOD 02_B6.sub.--1):
[0841] RT=33.3 MIN
[0842] LCMS: m/z=1257.7 (M+3H).sup.3+
[0843] Calculated MW=3773
Example 12
N-epsilon31
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(DesaminoHis7,Glu22,Arg2-
6,Lys31)GLP-1(7-33)amide
##STR00076##
[0845] Preparation method: Method C except that the peptide was
prepared on an Apex396 from Advanced Chemtech using a molar excess
of 8-10 fold amino acid, DIC and HOAt/HOBt (1:1) and the Mtt group
was deprotected with hexaflouroisopropanol. The final product was
characterized by analytical HPLC and LC-MS.
[0846] HPLC (METHOD 02_B6.sub.--1):
[0847] RT=33.2 MIN
[0848] LCMS: m/z=1233.9 (M+3H).sup.3+
[0849] Calculated MW=3699
Example 13
N-epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Val25,-
Arg26,Leu27, Glu30,Lys31)GLP-1(7-32)amide
##STR00077##
[0851] Preparation method: Method C except that the peptide was
prepared on an Apex396 from Advanced Chemtech using a molar excess
of 8-10 fold amino acid, DIC and HOAt/HOBt (1:1) and the Mtt group
was deprotected with hexaflouroisopropanol. The final product was
characterized by analytical HPLC and MALDI-MS.
[0852] HPLC (METHOD 02_B6.sub.--1):
[0853] RT=32.6 MIN
[0854] MALDI-MS: 3715.5
[0855] Calculated MW=3714.3
Example 14
N-epsilon20
{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamin-
o]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-(Aib8,Lys20,Glu22,Val25,-
Arg26,Leu27,Nle30,Lys31)GLP-1(7-32)amide
##STR00078##
[0857] Preparation method: Method C except that the peptide was
prepared on an Apex396 from Advanced Chemtech using a molar excess
of 8-10 fold amino acid, DIC and HOAt/HOBt (1:1) and the Mtt group
was deprotected with hexaflouroisopropanol. The final product was
characterized by analytical HPLC and MALDI-MS.
[0858] HPLC (METHOD 02_B6.sub.--1):
[0859] RT=33 MIN
[0860] MALDI-MS: 3696.9
[0861] Calculated MW=3698
Example 15
N-epsilon31-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxynonad-
ecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)a-
cetylamino]ethoxy}ethoxy)acetyl]-[Aib8,Glu22,Val25,Arg26,Lys31]GLP-1-(7-33-
)amide
##STR00079##
[0863] Preparation method: Method C except that the peptide was
prepared on an Apex396 from Advanced Chemtech using a molar excess
of 8-10 fold amino acid, DIC and HOAt/HOBt (1:1) and the Mtt group
was deprotected with hexaflouroisopropanol. The final product was
characterized by analytical HPLC and MALDI-MS.
[0864] HPLC (METHOD 02_B6.sub.--1):
[0865] RT=39.3 MIN
[0866] MALDI-MS: 3696.9
[0867] Calculated MW=3698
Example 16
[Desamino His7,Glu22,Arg26]-GLP-1(7-34)
##STR00080##
[0869] HPLC (METHOD I_BDSB2)
[0870] RT=5.65 min
[0871] LCMS: m/z=1057.5 (M+3H).sup.3+
[0872] Calculated MW=3170.5
Example 17
[Aib8,Lys20,Glu22,Val25,Arg26,Leu27,Lys31]GLP-1(7-32)amide
##STR00081##
[0874] Preparation analogous to SPPS Method B.
[0875] HPLC method 02_B6.sub.--1:
[0876] RT=27.09 min
[0877] LCMS: m/z=735 (M+4H).sup.4+
[0878] Calculated (M)=2940.3
Example 18
N-epsilon31-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylami-
no)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHi-
s7,Asp11,Glu18,Glu22,Val25,Arg26,Asp27,Glu30,Lys31]GLP-1(7-33)-peptide
amide
##STR00082##
[0880] Preparation method B on ChemMatrix Rink Amide resin (0.24
mmol/g, 0.4 g).
[0881] HPLC (02_B4.sub.--4): Rt=10.649 min; 99.6% purity
[0882] HPLC (03_A3.sub.--1): Rt=8.491 min; 94.3% purity
[0883] MALDI-MS: alpha-cyano-4-hydroxycinnamic acid; m/z: 3824.543
(NEGATIVE MODE)
Example 19
[Aib8,Glu22,Val25, Lys31]GLP-1(7-33)-amide
##STR00083##
[0885] Preparation method: The peptide was prepared by SPPS Method
C and the final product was characterized by analytical HPLC and
MALDI-MS.
[0886] HPLC (02-B6-1): RT=31.5 min
[0887] HPLC (04-A3-1): RT=8.6 min
[0888] MALDI-MS: 3043.6
[0889] Calculated MW=3040.4
Example 20
N
-epsilon31-{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino-
)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-N-beta34-(2--
(bis-carboxymethylamino)acetyl)[Aib8,Glu22,Val25,Arg26,Lys31,Dap34]GLP-1(7-
-34)amide
##STR00084##
[0891] Preparation method: The peptide was prepared by SPPS Method
C and the final product was characterized by analytical HPLC and
MALDI-MS.
[0892] HPLC (02-B6-1): RT=36.7 min
[0893] HPLC (A4-A3-1): RT=8.9 min
[0894] MALDI-MS: 4015.9
[0895] Calculated MW=4015.5
[0896] Biological Findings
[0897] Protraction of GLP-1 Derivatives After i.v. or s.c.
Administration
[0898] The protraction of a number GLP-1 compounds of the invention
is determined by monitoring the concentration thereof in plasma
after sc administration to healthy pigs, using the methods
described below. For comparison also the concentration in plasma of
GLP-1(7-37) after sc. administration is followed. The protraction
of other GLP-1 compounds of the invention can be determined in the
same way.
[0899] Pharmacokinetic Testing of GLP-1 Analogues in Minipigs
[0900] The test substances are dissolved in a vehicle suitable for
subcutaneous or intravenous administration. The concentration is
adjusted so the dosing volume is approximately 1 ml.
[0901] The study is performed in 12 male Gottingen minipigs from
Ellegaard Gottingen Minipigs ApS. An acclimatisation period of
approximately 10 days is allowed before the animals enter the
study. At start of the acclimatisation period the minipigs are
about 5 months old and in the weight range of 8-10 kg.
[0902] The study is conducted in a suitable animal room with a room
temperature set at 21-23.degree. C. and the relative humidity to
.gtoreq.50%. The room is illuminated to give a cycle of 12 hours
light and 12 hours darkness. Light is from 06.00 to 18.00 h.
[0903] The animals are housed in pens with straw as bedding, six
together in each pen.
[0904] The animals have free access to domestic quality drinking
water during the study, but are fasted from approximately 4 pm the
day before dosing until approximately 12 hours after dosing.
[0905] The animals are weighed on arrival and on the days of
dosing.
[0906] The animals receive a single intravenous or subcutaneous
injection. The subcutaneous injection is given on the right side of
the neck, approximately 5-7 cm from the ear and 7-9 cm from the
middle of the neck. The injections are given with a stopper on the
needle, allowing 0.5 cm of the needle to be introduced.
[0907] Each test substance is given to three animals. Each animal
receives a dose of 2 nmol/kg body weight.
[0908] Six animals are dosed per week while the remaining six are
rested.
[0909] A full plasma concentration-time profile is obtained from
each animal. Blood samples are collected according to the following
schedule:
[0910] After Intravenous Administration:
[0911] Predose (0), 0.17 (10 minutes), 0.5, 1, 2, 4, 6, 8, 12, 24,
48, 72, 96, and 120 hours after injection.
[0912] After Subcutaneous Administration:
[0913] Predose (0), 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, and 120
hours after injection. At each sampling time, 2 ml of blood is
drawn from each animal. The blood samples are taken from a jugular
vein.
[0914] The blood samples are collected into test tubes containing a
buffer for stabilisation in order to prevent enzymatic degradation
of the GLP-1 compounds.
[0915] Plasma is immediately transferred to Micronic-tubes.
Approximately 200 .mu.l plasma is transferred to each
Micronic-tube. The plasma is stored at -20.degree. C. until
assayed. The plasma samples are assayed for the content of GLP-1
compounds using an immunoassay.
[0916] The plasma concentration-time profiles are analysed by a
non-compartmental pharmacokinetic analysis. The following
pharmacokinetic parameters are calculated at each occasion: AUC,
AUC/Dose, AUC.sub.% Extrap, C.sub.max, t.sub.max, .lamda..sub.z,
t.sub.1/2, CL, CL/f, V.sub.z, V.sub.z/f and MRT. Selected compounds
of the invention are tested in Danish Landrace pigs.
[0917] Pharmacokinetic Testing of GLP-1 Compounds in Pigs
[0918] Pigs (50% Duroc, 25% Yorkshire, 25% Danish Landrace, app 40
kg) are fasted from the beginning of the experiment. To each pig
0.5 nmol of test compound per kg body weight is administered in a
50 .mu.M isotonic solution (5 mM phosphate, pH 7.4, 0.02%
Tween.RTM. -20 (Merck), 45 mg/ml mannitol (pyrogen free, Novo
Nordisk). Blood samples are drawn from a catheter in vena
jugularis. 5 ml of the blood samples are poured into chilled
glasses containing 175 .mu.l of the following solution: 0.18 M
EDTA, 15000 KIE/ml aprotinin (Novo Nordisk) and 0.30 mM
Valine-Pyrrolidide (Novo Nordisk), pH 7.4. Within 30 min, the
samples are centrifuged for 10 min at 5-6000*g. Temperature is kept
at 4.degree. C. The supernatant is pipetted into different glasses
and kept at minus 20.degree. C. until use.
[0919] The plasma concentrations of the peptides are determined in
a sandwich ELISA or by RIA using different mono- or polyclonal
antibodies. Choice of antibodies depends of the GLP-1 compounds.
The time at which the peak concentration in plasma is achieved
varies within wide limits, depending on the particular GLP-1
compound selected.
[0920] General Assay Protocol for Sandwich ELISA in 96-Wells
Microtiter plate [0921] Coating buffer (PBS): Phosphate buffered
saline, pH7.2 [0922] Wash-buffer (PBS-wash): Phosphate buffered
saline, 0.05% v/v Tween 20, pH 7.2 [0923] Assay-buffer
(BSA-buffer): Phosphate buffered saline, 10 g/l Bovin Serum Albumin
[0924] (Fluka 05477), 0.05% v/v Tween 20, pH 7.2 [0925]
Streptavidin-buffer Phosphate buffered saline, 0.5 M NaCl, 0.05%
v/v Tween 20, pH 7.2 [0926] Standard: Individual compounds in a
plasma-matrix [0927] A-TNP: Nonsense antibody [0928] AMDEX:
Streptavin-horseradish-peroxodase (Amersham RPN4401V) [0929]
TMB-substrate: 3,3',5,5tetramethylbenzidine (<0.02%), hydrogen
peroxide
[0930] The assay is carried out as follows (volume/well):
[0931] 1.) coat with 100 .mu.l catching antibody 5 .mu.g/ml in
PBS-buffer [0932] incubate o/n, 4.degree. C. [0933] 5.times.
PBS-wash [0934] blocked with last wash in minimum 30 minute [0935]
then empty the plate
[0936] 2.) 20 .mu.l sample+100 .mu.l biotinylated detecting
antibody 1 .mu.g/ml in BSA-buffer with 10 .mu.g/ml A-TNP [0937]
incubate 2 h, room temperature, on a shaker 5.times. PBS-wash, then
empty the plate
[0938] 3.) 100 .mu.l AMDEX 1:8000 in Streptavidin-buffer [0939]
incubate 45-60 minute, room temperature, on a shaker [0940]
5.times. PBS-wash, then empty the plate
[0941] 4.) 100 .mu.l TMB-substrate [0942] incubate.times.minute at
room temperature on a shaker [0943] stop the reaction with 100
.mu.l 4 M H.sub.3PO.sub.4
[0944] Read the absorbance at 450 nm with 620 nm as reference
[0945] The concentration in the samples is calculated from standard
curves.
[0946] General Assay Protocol for RIA [0947] DB-buffer: 80 mM
phosphate buffer, 0.1% Human serum albumin, 10 mM EDTA, 0.6 mM
thiomersal, pH 7.5 [0948] FAM-buffer: 40 mM phosphate buffer, 0.1%
Human Serum Albumin, 0.6 mM thiomersal, pH 7.5 [0949] Charcoal: 40
mM phosphate buffer, 0.6 mM thiomersal, 16.7% bovine plasma, 15 g/l
activated carbon, pH 7.5 (mix the suspension minimum 1 h before use
at 4.degree. C.) [0950] Standard: Individual compounds in a
plasma-matrix
[0951] The assay is carried out in minisorp tubes 12.times.75 mm
(volumen/tube) as follows:
TABLE-US-00015 Db- FAM- buffer SAMPLE Antibody buf. Tracer Charcoal
H.sub.2O Day 1 Total 100 .mu.L NSB 330 .mu.L 100 .mu.L Sample 300
.mu.L 30 .mu.L 100 .mu.L 100 .mu.L Mix, incubate o/n at 4.degree.
C. Day 2 Total 1.5 mL NSB 1.5 mL Sample 1.5 mL
[0952] Mix--incubate 30 min at 4.degree. C.--centrifuge at 3000
rpm, 30 min--immediately after transfer supernatants to new tubes,
close with stopper and count on gamma-counter for 1 minute.
[0953] The concentration in the samples is calculated from
individual standard curves.
[0954] GLP-1 Radio Receptor Assay (RRA):
[0955] The method is a radiometric-ligand binding assay using
LEADseeker imaging particles. The assay is composed of membrane
fragments containing the GLP-1 receptor, unlabeled GLP-1 analogues,
human GLP-1 labelled with .sup.125I and PS LEADseeker particles
coated with wheat germ agglutinin (WGA). Cold and
.sup.125I-labelled GLP-1 will compete for the binding to the
receptor. When the LEADseeker particles are added they will bind to
carbohydrates residues on the membrane fragments via the
WGA-residues. The proximity between the .sup.125I-molecules and the
LEADseeker particles causes light emission from the particles. The
LEADseeker will image the emitted light and it will be reversibly
correlated to the amount of GLP-1 analogue present in the
sample.
[0956] Reagents & Materials:
[0957] Pre treatment of animal plasma: Animal plasma is heat
treated for 4 hrs at 56.degree. C. and centrifuged at 10.000 rpm
for 10 minutes. Afterwards, Val-Pyr (10 .mu.M) and aprotenin (500
KIE/mL) is added and stored at <-18.degree. C. until use.
[0958] GLP-1 analogues calibrators: GLP-1 analogues are spiked into
heat-treated plasma to produce dilution lines ranging from
approximately 1 .mu.M to 1 .mu.M.
[0959] GLP-1 RRA assay buffer: 25 mM Na-HEPES (pH=7.5), 2.5 mM
CaCl.sub.2, 1 mM MgCl.sub.2, 50 mM NaCl, 0.1% ovalbumin, 0.003%
tween 20, 0.005% bacitracin, 0.05% NaN.sub.3.
[0960] GLP-1 receptor suspension: GLP-1 receptor membrane fragments
are purified from baby hamster kidney (BHK) cells stably expressing
the human pancreatic GLP-1 receptor. Stored <-80.degree. C.
until use.
[0961] WGA-coupled polystyrene LEADseeker imaging beads (RPNQ0260,
Amersham): The beads are reconstituted with GLP-1 RRA assay buffer
to a concentration of 13.3 mg/mL. The GLP-1 receptor membrane
suspension is then added and incubated cold (2-8.degree. C.) at
end-over-end for at least 1 hr prior to use.
[0962] [.sup.125I]-GLP-1(7-36)amide (Novo Nordisk A/S). Stored
<-18.degree. C. until use.
[0963] Ethanol 99.9% vol (De Dansk Spritfabrikker A/S): Stored
<-18.degree. C. until use.
[0964] MultiScreen.RTM. Solvinert 0.45 .mu.m hydrophobic PTFE
plates (MSRPN0450, Millipore Corp.)
[0965] Poly propylene plates (cat. no. 650201, Greiner Bio-One)
[0966] White polystyrene 384-well plates (cat. no. 781075, Greiner
Bio-One)
[0967] Apparatus:
[0968] Horizontal plate mixer
[0969] Centrifuge with a standard swinging-bucket microtitre plate
rotor assembly
[0970] UltraVap--Drydown Sample Concentrator (Porvair)
[0971] LEADseeker.TM. Multimodality Imaging System (Amersham)
[0972] Assay Procedure:
[0973] Sample Preparation:
[0974] Mount the MultiScreen.RTM. Solvinert filter plate on a
chemical-comparable receiver plate (i.e. poly propylene plates) to
collect the filtrate.
[0975] Add 150 .mu.L ice-cold ethanol 99.9% into the empty wells of
the MultiScreen.RTM. Solvinert filter plate followed by 50 .mu.L
calibrator or plasma sample. Place the storage lid on the filter
plate.
[0976] Incubate 15 minutes at 18-22.degree. C. on a horizontal
plate mixer.
[0977] Place the assembled filter and receiver plate, with the lid,
into a standard swinging-bucket microtitre plate rotor assembly.
The filtrate is then collected in the empty wells of the receiver
plate at 1500 rpm for 2 minutes.
[0978] Dry down the filtrate by using the UltraVap with heated
(40.degree. C.) N.sub.2 for duration of 15 minutes. Reconstitute
the dry material by adding 100 .mu.L GLP-1 RRA assay buffer into
each well. Incubate for 5 minutes on a horizontal mixer.
[0979] GLP-1 Radio Receptor Assay:
[0980] Use the following pipetting scheme and white polystyrene
384-well plates: [0981] 35 .mu.L GLP-1 RRA assay buffer [0982] 5
.mu.L reconstituted filtrate. [0983] 10 .mu.L
[.sup.125I]GLP-1(7-36)amide. The stock solution is diluted in GLP-1
RRA assay buffer to 20.000 cpm/well prior to use. [0984] 15 .mu.L
GLP-1 receptor membrane fragments (.apprxeq.0.5 .mu.g/well)
pre-coated to WGA-polystyrene LEADseeker imaging beads (0.2
mg/well)
[0985] Seal the plates and incubate over night at 18-22.degree.
C.
[0986] The light emission from each wells is detected by using the
LEADseeker.TM. Multimodality Imaging System for duration of 10
minutes.
Example 21
Stimulation of cAMP Formation in a Cell Line Expressing the Cloned
Human GLP-1 Receptor
[0987] The potencies of a number of GLP-1 analogues and derivatives
of the invention (the compounds of Examples 1-7, 9-17, and 19-20)
were determined as described below, i.e. as the stimulation of the
formation of cyclic AMP (cAMP) in a medium containing the human
GLP-1 receptor. For comparison, the potency of truncated natural,
human GLP-1(7-33) was also determined.
[0988] Purified plasma membranes from a stable transfected cell
line, BHK467-12A (tk-ts13), expressing the human GLP-1 receptor was
stimulated with GLP-1 compound in question, and the potency of cAMP
production was measured using the AlphaScreen.TM. cAMP Assay Kit
from Perkin Elmer Life Sciences.
[0989] A stable transfected cell line has been prepared at NN A/S,
Denmark, and a high expressing clone is selected for screening. The
cells are grown at 5% CO.sub.2 in DMEM, 5% FCS, 1% Pen/Strep
(Penicillin/Streptomycin) and 0.5 mg/ml of the selection marker
G418.
[0990] Cells at approximate 80% confluence are washed 2.times. with
PBS (Phosphate Buffered Saline) and harvested with Versene (aqueous
solution of the tetrasodium salt of ethylenediaminetetraacetic
acid), centrifuged 5 min at 1000 rpm and the supernatant removed.
The additional steps are all made on ice. The cell pellet is
homogenized by the Ultrathurax for 20-30 sec. in 10 ml of Buffer 1
(20 mM Na-HEPES, 10 mM EDTA, pH=7.4), centrifuged 15 min at 20.000
rpm and the pellet resuspended in 10 ml of Buffer 2 (20 mM
Na-HEPES, 0.1 mM EDTA, pH=7.4). The suspension is homogenized for
20-30 sec and centrifuged 15 min at 20.000 rpm. Suspension in
Buffer 2, homogenization and centrifugation is repeated once and
the membranes are resuspended in Buffer 2 and ready for further
analysis or stored at -80.degree. C.
[0991] The functional receptor assay was carried out by measuring
the peptide induced cAMP production by The AlphaScreen Technology.
The basic principle of The AlphaScreen Technology is a competition
between endogenous cAMP and exogenously added biotin-cAMP. The
capture of cAMP is achieved by using a specific antibody conjugated
to acceptor beads. Formed cAMP is counted and measured at a
AlphaFusion Microplate Analyzer. The EC.sub.50 values are
calculated using the Graph-Pad Prisme software (version 5).
[0992] The truncated natural GLP-1(7-3)) peptide has a very low
potency (a high EC.sub.50 value of 11.3 nM). All tested GLP-1
compounds of the invention had a very high potency (six compounds
an EC.sub.50 below 0.10 nM, six compounds and EC.sub.50 in the
range of 0.10-0.50 nM).
[0993] Interestingly, the compounds of Examples 1, 4, 5, and 7,
none of which are derivatized, also had an EC50 below 0.30 nM,
which is strongly surprising.
Example 22
Binding to the Extracellular Domain of the GLP-1 Receptor
[0994] For a number of GLP-1 compounds of the invention (the
compounds of Examples 1-17), the affinity of the binding to the
isolated N-terminal extracellular domain of the GLP-1R receptor
(nGLP-1R) was measured as described below. For comparison, the
potency of truncated natural, human GLP-1(7-33) was also
determined.
[0995] The affinity is a measure of the ability of the GLP-1
derivative in question to displace .sup.125I-Exendin-4(9-39) from
binding to nGLP-1R, and the binding to nGLP-1R was measured in the
following assay: The protein nGLP-1R was prepared as described by
Runge et al 2007 (Biochemistry, vol. 46, pp. 5830-5840),
biotinylated and immobilized on streptavidin-coated SPA beads. The
nGLP1R in 0.1M NaHCO.sub.3 was biotinylated using 75 .mu.g BNHS
(Sigma H1759) to 1 mg protein. The biotinylated nGLP1R was
subsequently dialyzed against PBS. All reagents and derivatives
were diluted in PBS with 0.05% v/v Tween 20. The binding assay was
carried out in 96 well OptiPlates (PerkinElmer 6005290) in a final
volume of 200 .mu.l. Each well contained 2 mg streptavidin coated
SPA beads (PerkinElmer RPNQ007), 0.1 pmol biotinylated nGLP1R, 50
pCi .sup.125I-Exendin (9-39) and test peptide in final
concentrations ranging from 1000 nM to 0.064 nM. The plates were
incubated on a shaker at RT for 3 hours. The SPA particles were
spun down by centrifugation for 10 min at 1500 rpm and the plates
were counted in a TopCount-NXT (PerkinElmer).
[0996] The IC.sub.50 value is read from the respective curve as the
concentration of the GLP-1 compound in question which displaces 50%
of .sup.125I-Exendin-4(9-39) from binding to nGLP-1R.
[0997] The affinity of the truncated (natural, human) GLP-1(7-33)
to nGLP-1R (IC.sub.50) was determined to 886 nM. Eleven of the
tested GLP-1 compounds of the invention had affinities (IC.sub.50)
below 600nM (ranging from 20-556 (nM), with six compound below 100
nM. Several GLP-1 compounds of the invention accordingly exhibit a
very good binding affinity to the N-terminal GLP-1 receptor (the
lower the IC.sub.50 value, the better the binding). Compounds 1, 4,
5, and 7 which were also discussed in Example 21 had acceptable
binding affinities in the range of 200-1500 nM, three of them in
the range of 200-600 nM.
Example 23
Affinity to the GLP-1 Receptor
[0998] The binding affinity of a number of GLP-1 compounds of the
invention (compounds of Examples 1-7, and 9-18) to the human GLP-1
receptor was measured by way of their ability to displace
.sup.125I-GLP-1 from the receptor.
[0999] Conditions
[1000] Species (in vitro): Hamster
[1001] Biological End Point: Receptor Binding
[1002] Assay Method: SPA
[1003] Receptor: GLP-1 receptor
[1004] Cell Line: BHK tk-ts13
[1005] Membrane Purification:
[1006] The cells (approx. 80% confluence) were washed twice in PBS
and harvested (PBS+EDTA or Versene), following which they were
separated by centrifugation at 1000 rpm for 5 min. The cells/cell
pellet must be kept on ice to the extent possible in the subsequent
steps. The cell pellet was homogenised with Ultrathurrax for 20-30
seconds in a suitable amount of Buffer 1 (depending on the amount
of cells, but e.g. 10 ml). The homogenate as centrifuged at 20000
rpm for 15 minutes. The pellet was resuspended (homogenised) in 10
ml Buffer 2 and re-centrifuged. This step was repeated once more.
The resulting pellet was resuspended in Buffer 2, and the protein
concentration was determined. The membranes were stored at
-80.degree. C.
[1007] Buffer 1: 20 mM Na-HEPES+10 mM EDTA, pH 7.4
[1008] Buffer 2: 20 mM Na-HEPES+0.1 mM EDTA, pH 7.4
[1009] Binding Assay:
[1010] SPA:
[1011] Test compounds/peptides, membranes, SPA-particles and
[.sup.125I] are diluted in assay buffer. 25 ul (micro liter) of
test compounds/peptides are added to Optiplate. Buffer is added (50
ul). Add 5-10 ug protein/sample (50 ul) corresponding to 0.1-0.2 mg
protein/ml (to be preferably optimised for each membrane
preparation). Add SPA-particles (Wheatgerm agglutinin SPA beads)
RPNQ 0001) 0.5 mg/well (50 ul). Start the incubation with
[I.sup.125]-GLP-1 (final concentration 0.05 nM corresponding to
49.880 DPM, 25 ul). The plates are sealed with PlateSealer.
Incubate for 120 minutes at 30.degree. C. while shaking. The plates
are centrifuged (1500 rpm, 10 min) and counted in Topcounter.
[1012] Assay buffer: 50 mM HEPES
[1013] 5 mM EGTA
[1014] 5 mM MgCl.sub.2
[1015] 0.005% Tween 20
[1016] pH 7.4
[1017] The IC.sub.50 value is read from the curve as the
concentration which displaces 50% of .sup.125I-GLP-1 from the
receptor.
[1018] The IC.sub.50 value of GLP-1(7-33) (truncated natural GLP-1)
was 241 nM. Except one compound, all tested compounds of the
invention had a better binding to the GLP-1 receptor (i.e. a lower
IC.sub.50 value). Six compounds had a value below 1.00 nM, five
compounds in the range from 1-10 nM, and five compounds in the
range of 20-200 nM. Compounds 1, 4, 5, and 7 discussed in the
previous examples had IC.sub.50 values in the range of 0.24 nM to
44 nM, and three of them below 2.0 nM.
Sequence CWU 1
1
3129PRTHomo sapiensPEPTIDE(1)..(29) 1His Ala Glu Gly Thr Phe Thr
Ser Asp Val Ser Ser Tyr Leu Glu Gly1 5 10 15Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu Val Lys Gly 20 25229PRTArtificial SequenceSynthetic
2Xaa Xaa Xaa Gly Thr Phe Thr Ser Asp Xaa Ser Xaa Tyr Xaa Glu Glu1 5
10 15Xaa Xaa Xaa Arg Xaa Phe Ile Xaa Xaa Leu Xaa Xaa Xaa 20
25329PRTArtificial SequenceSynthetic 3Xaa Xaa Glu Gly Thr Phe Thr
Ser Asp Val Ser Xaa Tyr Leu Glu Glu1 5 10 15Gln Ala Ala Arg Glu Phe
Ile Xaa Trp Leu Xaa Xaa Xaa 20 25
* * * * *