U.S. patent application number 12/861511 was filed with the patent office on 2011-03-03 for glp-1 derivatives ii.
This patent application is currently assigned to NOVO NORDISK A/S. Invention is credited to PER OLAF HUUSFELDT, LISELOTTE BJERRE KNUDSEN, KJELD MADSEN, PER FRANKLIN NIELSEN.
Application Number | 20110053839 12/861511 |
Document ID | / |
Family ID | 26063667 |
Filed Date | 2011-03-03 |
United States Patent
Application |
20110053839 |
Kind Code |
A1 |
KNUDSEN; LISELOTTE BJERRE ;
et al. |
March 3, 2011 |
GLP-1 Derivatives II
Abstract
The present invention relates to a derivative of GLP-1(7-C),
wherein C is 35 or 36 which derivative has just one lipophilic
substituent which is attached to the C-terminal amino acid
residue.
Inventors: |
KNUDSEN; LISELOTTE BJERRE;
(VALBY, DK) ; HUUSFELDT; PER OLAF; (COPENHAGEN K,
DK) ; NIELSEN; PER FRANKLIN; (VAERLOSE, DK) ;
MADSEN; KJELD; (VAERLOSE, DK) |
Assignee: |
NOVO NORDISK A/S
BAGSVAERD
DK
|
Family ID: |
26063667 |
Appl. No.: |
12/861511 |
Filed: |
August 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11789953 |
Apr 26, 2007 |
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12861511 |
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09886311 |
Jun 21, 2001 |
7226990 |
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11789953 |
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09312177 |
May 14, 1999 |
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09886311 |
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PCT/DK99/00086 |
Feb 25, 1999 |
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09312177 |
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60084357 |
May 5, 1998 |
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Current U.S.
Class: |
514/7.2 ;
514/11.7; 530/308 |
Current CPC
Class: |
C07K 14/57563 20130101;
C07K 14/605 20130101; A61K 38/28 20130101; A61K 38/26 20130101;
A61K 38/28 20130101; A61K 2300/00 20130101; A61P 3/10 20180101 |
Class at
Publication: |
514/7.2 ;
530/308; 514/11.7 |
International
Class: |
A61K 38/26 20060101
A61K038/26; C07K 14/605 20060101 C07K014/605; A61P 3/10 20060101
A61P003/10 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 27, 1998 |
DK |
0274/98 |
Claims
1-91. (canceled)
92. A derivative of an analogue of exendin-4 where said analogue
has an amino acid sequence that differs from the amino acid
sequence of exendin-4 by the substitution of up to six amino acid
residues with any .alpha.-amino acid residue, and wherein said
derivative has one lipophilic substituent attached, optionally via
a spacer, to an amino acid residue of said analogue which is not
the N-terminal or C-terminal amino acid residue of said
analogue.
93. The derivative of claim 92, wherein said analogue has an amino
acid sequence that differs from the amino acid sequence of
exendin-4 by the substitution of up to four amino acid residues
with any .alpha.-amino acid residue
94. The derivative of claim 92, wherein the lipophilic substituent
has 4 to 40 carbon atoms.
95. The derivative of claim 94, wherein the lipophilic substituent
has 8 to 25 carbon atoms.
96. The derivative of claim 94, wherein the lipophilic substituent
is attached by means of a spacer.
97. The derivative of claim 96, wherein the spacer is an unbranched
alkane .alpha.,.omega.-dicarboxylic acid group having from 1 to 7
methylene groups.
98. The derivative of claim 97, wherein the spacer is an unbranched
alkane .alpha.,.omega.-dicarboxylic acid group having two methylene
groups.
99. The derivative of claim 96, wherein the spacer is an amino acid
residue except cys, or a dipeptide such as gly-lys.
100. The derivative of claim 94, wherein the lipophilic substituent
is a partially or completely hydrogenated cyclopentanophenathrene
skeleton.
101. The derivative of claim 94, wherein the lipophilic substituent
is a straight-chain or branched alkyl group.
102. The derivative of claim 94, wherein the lipophilic substituent
is a straight-chain or branched acyl group.
103. The derivative of claim 102, wherein the acyl group is of the
formula CH.sub.3(CH.sub.2).sub.nCO--, wherein n is 4 to 38.
104. The derivative of claim 103, wherein the acyl group is
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-- or
CH.sub.3(CH.sub.2).sub.22CO--.
105. The derivative of claim 94, wherein the lipophilic substituent
is an acyl group of a straight-chain or branched alkane
.alpha.,.omega.-dicarboxylic acid.
106. The derivative of claim 105, wherein the acyl group is of the
formula HOOC(CH.sub.2).sub.mCO--, wherein m is from 4 to 38.
107. The derivative of claim 106, wherein the acyl group is
HOOC(CH.sub.2).sub.14CO--, HOOC(CH.sub.2).sub.16CO--,
HOOC(CH.sub.2).sub.18CO--, HOOC(CH.sub.2).sub.20CO-- or
HOOC(CH.sub.2).sub.22CO--.
108. The derivative of claim 94, wherein the lipophilic substituent
is a group of the formula
CH.sub.3(CH.sub.2).sub.p((CH.sub.2).sub.qCOOH)CHNH--CO(CH.sub.2).sub.2CO--
-, wherein p and q are integers and p+q is an integer of from 8 to
33.
109. The derivative of claim 94, wherein the lipophilic substituent
is a group of the formula
CH.sub.3(CH.sub.2).sub.rCO--NHCH(COOH)(CH.sub.2).sub.2CO--, wherein
r is an integer of from 10 to 24.
110. The derivative of claim 94, wherein the lipophilic substituent
is a group of the formula
CH.sub.3(CH.sub.2).sub.sCO--NHCH((CH.sub.2).sub.2COOH)CO--, wherein
s is an integer of from 8 to 24.
111. The derivative of claim 94, wherein the lipophilic substituent
is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--CO(CH.sub.2).sub.uCH.sub.3, wherein
u is an integer of from 8 to 18.
112. The derivative of claim 94, wherein the lipophilic substituent
is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--COCH((CH.sub.2).sub.2COOH)NH--CO(CH.sub.2-
).sub.wCH.sub.3, wherein w is an integer of from 10 to 16.
113. The derivative of claim 94, wherein the lipophilic substituent
is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--CO(CH.sub.2).sub.2CH(COOH)NH--CO(CH.sub.2-
).sub.xCH.sub.3, wherein x is an integer of from 10 to 16.
114. The derivative of claim 94, wherein the lipophilic substituent
is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--CO(CH.sub.2).sub.2CH(COOH)NH--CO(CH.sub.2-
).sub.yCH.sub.3, wherein y is zero or an integer of from 1 to
22.
115. The derivative of claim 95, having an amino acid sequence of
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGX, wherein X.dbd.P or Y, or a
fragment or an analogue thereof.
116. The derivative of claim 95, having an amino acid sequence of
HX1X2GTFITSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 2), wherein
X1X2=SD or GE, or a fragment or an analogue thereof.
117. The derivative of claim 95, having an amino acid sequence of
DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS, or a fragment or an analogue
thereof.
118. The derivative of claim 92, which is Arg.sup.18, Leu.sup.20,
Gln.sup.34, Lys.sup.33
(N.sup..epsilon.-(.gamma.-aminobutyroyl(N.sup..alpha.-hexadecanoyl)))
Exendin-4-(7-45)-NH.sub.2.
119. The derivative of claim 92, which is Arg.sup.33, Leu.sup.20,
Gln.sup.34, Lys.sup.18
(N.sup..epsilon.-(.gamma.-aminobutyroyl(N.sup..alpha.-hexadecanoyl)))
Exendin-4-(7-45)-NH.sub.2.
120. A pharmaceutical composition comprising a derivative of claim
92 and a pharmaceutically acceptable vehicle or carrier.
121. A method of treating insulin dependent or non-insulin
dependent diabetes mellitus in a patient in need of such a
treatment, comprising administering to the patient a
therapeutically effective amount of a derivative of claim 92 and a
pharmaceutically acceptable carrier.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/789,953, filed Apr. 26, 2007, which is a
continuation of U.S. patent application Ser. No. 09/886,311, filed
Jun. 21, 2001, which is a continuation of U.S. patent application
Ser. No. 09/312,177, filed May 14, 1999, which is a continuation of
International Patent Application PCT/DK99/00086 (published as WO
1999/043708), filed Feb. 24, 1999, which claimed priority of Danish
Patent Application 0274/98, filed Feb. 27, 1998; this application
further claims priority under 35 U.S.C. .sctn.119 of U.S.
Provisional Application 60/084,357, filed May 5, 1998, the contents
of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to novel derivatives of human
glucagon-like peptide-1 (GLP-1) and fragments thereof and analogues
of such fragments which have a protracted profile of action and to
methods of making and using them. The invention furthermore relates
to novel derivatives of exendin and the uses of such
derivatives.
BACKGROUND OF THE INVENTION
[0003] Peptides are widely used in medical practice, and since they
can be produced by recombinant DNA technology it can be expected
that their importance will increase also in the years to come. When
native peptides or analogues thereof are used in therapy it is
generally found that they have a high clearance. A high clearance
of a therapeutic agent is inconvenient in cases where it is desired
to maintain a high blood level thereof over a prolonged period of
time since repeated administrations will then be necessary.
Examples of peptides which have a high clearance are: ACTH,
corticotropin-releasing factor, angiotensin, calcitonin, insulin,
glucagon, glucagon-like peptide-1, glucagon-like peptide-2,
insulin-like growth factor-1, insulin-like growth factor-2, gastric
inhibitory peptide, growth hormone-releasing factor, pituitary
adenylate cyclase activating peptide, secretin, enterogastrin,
somatostatin, somatotropin, somatomedin, parathyroid hormone,
thrombopoietin, erythropoietin, hypothalamic releasing factors,
prolactin, thyroid stimulating hormones, endorphins, enkephalins,
vasopressin, oxytocin, opiods and analogues thereof, superoxide
dismutase, interferon, asparaginase, arginase, arginine deaminase,
adenosine deaminase and ribonuclease. In some cases it is possible
to influence the release profile of peptides by applying suitable
pharmaceutical compositions, but this approach has various
shortcomings and is not generally applicable.
[0004] The hormones regulating insulin secretion belong to the
so-called enteroinsular axis, designating a group of hormones,
released from the gastrointestinal mucosa in response to the
presence and absorption of nutrients in the gut, which promote an
early and potentiated release of insulin. The enhancing effect on
insulin secretion, the so-called incretin effect, is probably
essential for a normal glucose tolerance. Many of the
gastrointestinal hormones, including gastrin and secretin
(cholecystokinin is not insulinotropic in man), are insulinotropic,
but the only physiologically important ones, those that are
responsible for the incretin effect, are the glucose-dependent
insulinotropic polypeptide, GIP, and glucagon-like peptide-1
(GLP-1). Because of its insulinotropic effect, GIP, isolated in
1973 (1) immediately attracted considerable interest among
diabetologists. However, numerous investigations carried out during
the following years clearly indicated that a defective secretion of
GIP was not involved in the pathogenesis of insulin dependent
diabetes mellitus (IDDM) or non insulin-dependent diabetes mellitus
(NIDDM) (2). Furthermore, as an insulinotropic hormone, GIP was
found to be almost ineffective in NIDDM (2). The other incretin
hormone, GLP-1 is the most potent insulinotropic substance known
(3). Unlike GIP, it is surprisingly effective in stimulating
insulin secretion in NIDDM patients. In addition, and in contrast
to the other insulinotropic hormones (perhaps with the exception of
secretin) it also potently inhibits glucagon secretion. Because of
these actions it has pronounced blood glucose lowering effects
particularly in patients with NIDDM.
[0005] GLP-1, a product of the proglucagon (4), is one of the
youngest members of the secretin-VIP family of peptides, but is
already established as an important gut hormone with regulatory
function in glucose metabolism and gastrointestinal secretion and
metabolism (5). The glucagon gene is processed differently in the
pancreas and in the intestine. In the pancreas (9), the processing
leads to the formation and parallel secretion of 1) glucagon
itself, occupying positions 33-61 of proglucagon (PG); 2) an
N-terminal peptide of 30 amino acids (PG (1-30)) often called
glicentin-related pancreatic peptide, GRPP (10, 11); 3) a
hexapeptide corresponding to PG (64-69); 4) and, finally, the
so-called major proglucagon fragment (PG (72-158)), in which the
two glucagon-like sequences are buried (9). Glucagon seems to be
the only biologically active product. In contrast, in the
intestinal mucosa, it is glucagon that is buried in a larger
molecule, while the two glucagon-like peptides are formed
separately (8). The following products are formed and secreted in
parallel: 1) glicentin, corresponding to PG (1-69), with the
glucagon sequence occupying residues Nos. 33-61 (12); 2)
GLP-1(7-36)amide (PG (78-107))amide (13), not as originally
believed PG (72-107)amide or 108, which is inactive). Small amounts
of C-terminally glycine-extended but equally bioactive GLP-1(7-37),
(PG (78-108)) are also formed (14); 3) intervening peptide-2 (PG
(111-122)amide) (15); and 4) GLP-2 (PG (126-158)) (15, 16). A
fraction of glicentin is cleaved further into GRPP (PG (1-30)) and
oxyntomodulin (PG (33-69)) (17, 18). Of these peptides, GLP-1, has
the most conspicuous biological activities.
[0006] Being secreted in parallel with glicentin/enteroglucagon, it
follows that the many studies of enteroglucagon secretion (6, 7) to
some extent also apply to GLP-1 secretion, but GLP-1 is metabolised
more quickly with a plasma half-life in humans of 2 min (19).
Carbohydrate or fat-rich meals stimulate secretion (20), presumably
as a result of direct interaction of yet unabsorbed nutrients with
the microvilli of the open-type L-cells of the gut mucosa.
Endocrine or neural mechanisms promoting GLP-1 secretion may exist
but have not yet been demonstrated in humans.
[0007] The incretin function of GLP-1(29-31) has been clearly
illustrated in experiments with the GLP-1 receptor antagonist,
exendin 9-39, which dramatically reduces the incretin effect
elicited by oral glucose in rats (21, 22). The hormone interacts
directly with the .beta.-cells via the GLP-1 receptor (23) which
belongs to the glucagon/VIP/calcitonin family of G-protein-coupled
7-transmembrane spanning receptors. The importance of the GLP-1
receptor in regulating insulin secretion was illustrated in recent
experiments in which a targeted disruption of the GLP-1 receptor
gene was carried out in mice. Animals homozygous for the disruption
had greatly deteriorated glucose tolerance and fasting
hyperglycaemia, and even heterozygous animals were glucose
intolerant (24). The signal transduction mechanism (25) primarily
involves activation of adenylate cyclase, but elevations of
intracellular Ca.sup.2+ are also essential (25, 26). The action of
the hormone is best described as a potentiation of glucose
stimulated insulin release (25), but the mechanism that couples
glucose and GLP-1 stimulation is not known. It may involve a
calcium-induced calcium release (26, 27). As already mentioned, the
insulinotropic action of GLP-1 is preserved in diabetic
.beta.-cells. The relation of the latter to its ability to convey
"glucose competence" to isolated insulin-secreting cells (26, 28),
which respond poorly to glucose or GLP-1 alone, but fully to a
combination of the two, is also not known. Equally importantly,
however, the hormone also potently inhibits glucagon secretion
(29). The mechanism is not known, but seems to be paracrine, via
neighbouring insulin or somatostatin cells (25). Also the
glucagonostatic action is glucose-dependent, so that the inhibitory
effect decreases as blood glucose decreases. Because of this dual
effect, if the plasma GLP-1 concentrations increase either by
increased secretion or by exogenous infusion the molar ratio of
insulin to glucagon in the blood that reaches the liver via the
portal circulation is greatly increased, whereby hepatic glucose
production decreases (30). As a result blood glucose concentrations
decrease. Because of the glucose dependency of the insulinotropic
and glucagonostatic actions, the glucose lowering effect is
self-limiting, and the hormone, therefore, does not cause
hypoglycaemia regardless of dose (31). The effects are preserved in
patients with diabetes mellitus (32), in whom infusions of slightly
supraphysiological doses of GLP-1 may completely normalise blood
glucose values in spite of poor metabolic control and secondary
failure to sulphonylurea (33). The importance of the
glucagonostatic effect is illustrated by the finding that GLP-1
also lowers blood glucose in type-1 diabetic patients without
residual .beta.-cell secretory capacity (34).
[0008] In addition to its effects on the pancreatic islets, GLP-1
has powerful actions on the gastrointestinal tract. Infused in
physiological amounts, GLP-1 potently inhibits pentagastrin-induced
as well as meal-induced gastric acid secretion (35, 36). It also
inhibits gastric emptying rate and pancreatic enzyme secretion
(36). Similar inhibitory effects on gastric and pancreatic
secretion and motility may be elicited in humans upon perfusion of
the ileum with carbohydrate- or lipid-containing solutions (37,
38). Concomitantly, GLP-1 secretion is greatly stimulated, and it
has been speculated that GLP-1 may be at least partly responsible
for this so-called "ileal-brake" effect (38). In fact, recent
studies suggest that, physiologically, the ileal-brake effects of
GLP-1 may be more important than its effects on the pancreatic
islets. Thus, in dose response studies GLP-1 influences gastric
emptying rate at infusion rates at least as low as those required
to influence islet secretion (39).
[0009] GLP-1 seems to have an effect on food intake.
Intraventricular administration of GLP-1 profoundly inhibits food
intake in rats (40, 42). This effect seems to be highly specific.
Thus, N-terminally extended GLP-1 (PG 72-107)amide is inactive and
appropriate doses of the GLP-1 antagonist, exendin 9-39, abolish
the effects of GLP-1 (41). Acute, peripheral administration of
GLP-1 does not inhibit food intake acutely in rats (41, 42).
However, it remains possible that GLP-1 secreted from the
intestinal L-cells may also act as a satiety signal.
[0010] Not only the insulinotropic effects but also the effects of
GLP-1 on the gastrointestinal tract are preserved in diabetic
patients (43), and may help curtailing meal-induced glucose
excursions, but, more importantly, may also influence food intake.
Administered intravenously, continuously for one week, GLP-1 at 4
ng/kg/min has been demonstrated to dramatically improve glycaemic
control in NIDDM patients without significant side effects (44).
The peptide is fully active after subcutaneous administration (45),
but is rapidly degraded mainly due to degradation by dipeptidyl
peptidase IV-like enzymes (46, 47).
[0011] The amino acid sequence of GLP-1 is given i.a. by Schmidt et
al. (Diabetologia 28 704-707 (1985). Although the interesting
pharmacological properties of GLP-1(7-37) and analogues thereof
have attracted much attention in recent years only little is known
about the structure of these molecules. The secondary structure of
GLP-1 in micelles has been described by Thorton et al.
(Biochemistry 33 3532-3539 (1994)), but in normal solution, GLP-1
is considered a very flexible molecule. Surprisingly, we found that
derivatisation of this relatively small and very flexible molecule
resulted in compounds whose plasma profile were highly protracted
and still had retained activity.
[0012] GLP-1 and analogues of GLP-1 and fragments thereof are
potentially useful i.a. in the treatment of type 1 and type 2
diabetes. However, the high clearance limits the usefulness of
these compounds, and thus there still is a need for improvements in
this field. Accordingly, it is one object of the present invention
to provide derivatives of GLP-1 and analogues thereof which have a
protracted profile of action relative to GLP-1(7-37). It is a
further object of the invention to provide derivatives of GLP-1 and
analogues thereof which have a lower clearance than GLP-1(7-37). It
is a further object of the invention to provide a pharmaceutical
composition comprising a compound according to the invention and to
use a compound of the invention to provide such a composition.
Also, it is an object of the present invention to provide a method
of treating insulin dependent and non-insulin dependent diabetes
mellitus.
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Endocrinology 1988; 123:2009-2013. [0042] 30. Hvidberg A, Toft
Nielsen M, Hilsted J, Orskov C, Holst J J. Effect of glucagon-like
peptide-1 (proglucagon 78-107amide) on hepatic glucose production
in healthy man. Metabolism 1994; 43:104-108. [0043] 31. Qualmann C,
Nauck M, Holst J J, Orskov C, Creutzfeldt W. Insulinotropic actions
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state in healthy subjects. Acta Diabetologica, 1995; 32: 13-16.
[0044] 32. Nauck M A, Heimesaat M M, Orskov C, Holst J J, Ebert R,
Creutzfeldt W. Preserved incretin activity of GLP-1(7-36amide) but
not of synthetic human GIP in patients with type 2-diabetes
mellitus. J Clin Invest 1993; 91:301-307. [0045] 33. Nauck M A,
Kleine N, Orskov C, Holst J J, Willms B, Creutzfeldt W.
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GLP-1(7-36amide) in type 2-diabetic patients. Diabetologia 1993;
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Holst J J, Nauck M A. Glucagonostatic actions and reduction of
fasting hyperglycaemia by exogenous glucagon-liem,
peptide-1(7-36amide) in type I diabetic patients. Diabetes Care
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Christiansen J, Orskov C, Holst J J. GLP-1 (glucagon-like
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inhibit gastric acid secretion in man. Dig. Dis. Sci. 1989;
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Myhre J, Christiansen J, Holst J J. Truncated GLP-1 (proglucagon
72-107amide) inhibits gastric and pancreatic functions in man. Dig
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Goebell H: Ileal release of glucagon-like peptide-1 (GLP-1):
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humoral mediator of the ileal brake in humans? Digestion 1993; 54:
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Holst J J, Schmiegel W. Inhibition of gastric emptying by
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insulin secretion. Abstract. Gut 1995; 37 (suppl. 2): A124. [0052]
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& Company Itd, 1994; pp. 363-367. [0053] 41. Tang-Christensen
M, Larsen P J, Goke R, Fink-Jensen A, Jessop D S, Moller M, Sheikh
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Ritzel R, Orskov C, Holst J J, Nauck M A. Pharmacokinetic,
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subjects. Diabetes 44: 1126-1131.
SUMMARY OF THE INVENTION
[0060] Human GLP-1 is a 37 amino acid residue peptide originating
from preproglucagon which is synthesised i.a. in the L-cells in the
distal ileum, in the pancreas and in the brain. Processing of
preproglucagon to give GLP-1(7-36)amide, GLP-1(7-37) and GLP-2
occurs mainly in the L-cells. A simple system is used to describe
fragments and analogues of this peptide. Thus, for example,
Gly.sup.8-GLP-1(7-37) designates a fragment of GLP-1 formally
derived from GLP-1 by deleting the amino acid residues Nos. 1 to 6
and substituting the naturally occurring amino acid residue in
position 8 (Ala) by Gly. Similarly,
Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-37) designates
GLP-1(7-37) wherein the .epsilon.-amino group of the Lys residue in
position 34 has been tetradecanoylated. Where reference in this
text is made to C-terminally extended GLP-1 analogues, the amino
acid residue in position 38 is Arg unless otherwise indicated, the
optional amino acid residue in position 39 is also Arg unless
otherwise indicated and the optional amino acid residue in position
40 is Asp unless otherwise indicated. Also, if a C-terminally
extended analogue extends to position 41, 42, 43, 44 or 45, the
amino acid sequence of this extension is as in the corresponding
sequence in human preproglucagon unless otherwise indicated.
[0061] In its broadest aspect, the present invention relates to
derivatives of GLP-1 and analogues thereof. The derivatives
according to the invention have interesting pharmacological
properties, in particular they have a more protracted profile of
action than the parent peptides.
[0062] In the present text, the designation "an analogue" is used
to designate a peptide wherein one or more amino acid residues of
the parent peptide have been substituted by another amino acid
residue and/or wherein one or more amino acid residues of the
parent peptide have been deleted and/or wherein one or more amino
acid residues have been added to the parent peptide. Such addition
can take place either at the N-terminal end or at the C-terminal
end of the parent peptide or both.
[0063] The term "derivative" is used in the present text to
designate a peptide in which one or more of the amino acid residues
of the parent peptide have been chemically modified, e.g. by
alkylation, acylation, ester formation or amide formation.
[0064] The term "a GLP-1 derivative" is used in the present text to
designate a derivative of GLP-1 or an analogue thereof. In the
present text, the parent peptide from which such a derivative is
formally derived is in some places referred to as the "GLP-1
moiety" of the derivative.
[0065] In a preferred embodiment, the present invention relates to
a GLP-1 derivative wherein at least one amino acid residue of the
parent peptide has a lipophilic substituent attached with the
proviso that if only one lipophilic substituent is present and this
substituent is attached to the N-terminal or to the C-terminal
amino acid residue of the parent peptide then this substituent is
an alkyl group or a group which has an .omega.-carboxylic acid
group.
[0066] In another preferred embodiment, the present invention
relates to a GLP-1 derivative having only one lipophilic
substituent.
[0067] In another preferred embodiment, the present invention
relates to a GLP-1 derivative having only one lipophilic
substituent which substituent is an alkyl group or a group which
has an .omega.-carboxylic acid group and is attached to the
N-terminal amino acid residue of the parent peptide.
[0068] In another preferred embodiment, the present invention
relates to a GLP-1 derivative having only one lipophilic
substituent which substituent is an alkyl group or a group which
has an .omega.-carboxylic acid group and is attached to the
C-terminal amino acid residue of the parent peptide.
[0069] In another preferred embodiment, the present invention
relates to a GLP-1 derivative having only one lipophilic
substituent which substituent can be attached to any one amino acid
residue which is not the N-terminal or C-terminal amino acid
residue of the parent peptide.
[0070] In another preferred embodiment, the present invention
relates to a GLP-1 derivative wherein two lipophilic substituents
are present.
[0071] In another preferred embodiment, the present invention
relates to a GLP-1 derivative wherein two lipophilic substituents
are present, one being attached to the N-terminal amino acid
residue while the other is attached to the C-terminal amino acid
residue.
[0072] In another preferred embodiment, the present invention
relates to a GLP-1 derivative wherein two lipophilic substituents
are present, one being attached to the N-terminal amino acid
residue while the other is attached to an amino acid residue which
is not N-terminal or the C-terminal amino acid residue.
[0073] In another preferred embodiment, the present invention
relates to a GLP-1 derivative wherein two lipophilic substituents
are present, one being attached to the C-terminal amino acid
residue while the other is attached to an amino acid residue which
is not the N-terminal or the C-terminal amino acid residue.
[0074] In a further preferred embodiment, the present invention
relates to a derivative of GLP-1(7-C), wherein C is selected from
the group comprising 38, 39, 40, 41, 42, 43, 44 and 45 which
derivative has just one lipophilic substituent which is attached to
the C-terminal amino acid residue of the parent peptide.
[0075] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative, being a derivative of GLP-1(7-C),
wherein C is 35 or 36 which derivative has just one lipophilic
substituent which is attached to the C-terminal amino acid
residue.
[0076] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein the lipophilic substituent
comprises from 4 to 40 carbon atoms, more preferred from 8 to 25
carbon atoms.
[0077] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein a lipophilic substituent is
attached to an amino acid residue in such a way that a carboxyl
group of the lipophilic substituent forms an amide bond with an
amino group of the amino acid residue.
[0078] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein a lipophilic substituent is
attached to an amino acid residue in such a way that an amino group
of the lipophilic substituent forms an amide bond with a carboxyl
group of the amino acid residue.
[0079] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein a lipophilic substituent is
attached to the parent peptide by means of a spacer.
[0080] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein a lipophilic
substituent--optionally via a spacer--is attached to the
.epsilon.-amino group of a Lys residue contained in the parent
peptide.
[0081] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein a lipophilic substituent is
attached to the parent peptide by means of a spacer which is an
unbranched alkane .alpha.,.omega.-dicarboxylic acid group having
from 1 to 7 methylene groups, preferably two methylene groups which
spacer forms a bridge between an amino group of the parent peptide
and an amino group of the lipophilic substituent.
[0082] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein a lipophilic substituent is
attached to the parent peptide by means of a spacer which is 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.
[0083] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein a lipophilic substituent is
attached to the parent peptide by means of a spacer which is an
amino acid residue except Cys, or is a dipeptide such as Gly-Lys
and wherein a carboxyl group of the parent peptide forms an amide
bond with an amino group of a Lys residue or a dipeptide containing
a Lys residue, and the other amino group of the Lys residue or a
dipeptide containing a Lys residue forms an amide bond with a
carboxyl group of the lipophilic substituent.
[0084] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein a lipophilic substituent is
attached to the parent peptide by means of a spacer which is an
amino acid residue except Cys, or is a dipeptide such as Gly-Lys
and wherein an amino group of the parent peptide forms an amide
bond with a carboxylic group of the amino acid residue or dipeptide
spacer, and an amino group of the amino acid residue or dipeptide
spacer forms an amide bond with a carboxyl group of the lipophilic
substituent.
[0085] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein a lipophilic substituent is
attached to the parent peptide by means of a spacer which is an
amino acid residue except Cys, or is a dipeptide such as Gly-Lys
and wherein a carboxyl group of the parent peptide forms an amide
bond with an amino group of the amino acid residue spacer or
dipeptide spacer, and the carboxyl group of the amino acid residue
spacer or dipeptide spacer forms an amide bond with an amino group
of the lipophilic substituent.
[0086] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein a lipophilic substituent is
attached to the parent peptide by means of a spacer which is an
amino acid residue except Cys, or is a dipeptide such as Gly-Lys,
and wherein a carboxyl group of the parent peptide forms an amide
bond with an amino group of a spacer which is Asp or Glu, or a
dipeptide spacer containing an Asp or Glu residue, and a carboxyl
group of the spacer forms an amide bond with an amino group of the
lipophilic substituent.
[0087] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative having a lipophilic substituent which
comprises a partially or completely hydrogenated
cyclopentanophenathrene skeleton.
[0088] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative having a lipophilic substituent which
is a straight-chain or branched alkyl group.
[0089] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative having a lipophilic substituent which
is the acyl group of a straight-chain or branched fatty acid.
[0090] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative having a lipophilic substituent which
is an acyl group selected from the group comprising
CH.sub.3(CH.sub.2).sub.nCO--, wherein n 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--.
[0091] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative having a lipophilic substituent which
is an acyl group of a straight-chain or branched alkane
.alpha.,.omega.-dicarboxylic acid.
[0092] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative having a lipophilic substituent which
is an acyl group selected from the group comprising
HOOC(CH.sub.2).sub.mCO--, wherein m is an integer from 4 to 38,
preferably an integer from 4 to 24, more preferred selected from
the group comprising HOOC(CH.sub.2).sub.14CO--,
HOOC(CH.sub.2).sub.16CO--, HOOC(CH.sub.2).sub.18CO--,
HOOC(CH.sub.2).sub.20CO-- and HOOC(CH.sub.2).sub.22CO--.
[0093] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative having a lipophilic substituent which
is a group of the formula
CH.sub.3(CH.sub.2).sub.p((CH.sub.2).sub.qCOOH)CHNH--CO(CH.sub.2).sub.2CO--
-, wherein p and q are integers and p+q is an integer of from 8 to
33, preferably from 12 to 28.
[0094] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative having a lipophilic substituent which
is a group of the formula
CH.sub.3(CH.sub.2).sub.rCO--NHCH(COOH)(CH.sub.2).sub.2CO--, wherein
r is an integer of from 10 to 24.
[0095] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative having a lipophilic substituent which
is a group of the formula
CH.sub.3(CH.sub.2).sub.sCO--NHCH((CH.sub.2).sub.2COOH)CO--, wherein
s is an integer of from 8 to 24.
[0096] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative having a lipophilic substituent which
is a group of the formula COOH(CH.sub.2).sub.tCO-- wherein t is an
integer of from 8 to 24.
[0097] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative having a lipophilic substituent which
is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--CO(CH.sub.2).sub.uCH.sub.3, wherein
u is an integer of from 8 to 18.
[0098] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative having a lipophilic substituent which
is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--COCH((CH.sub.2).sub.2COOH)NH--CO(CH.sub.2-
).sub.wCH.sub.3, wherein w is an integer of from 10 to 16.
[0099] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative having a lipophilic substituent which
is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--CO(CH.sub.2).sub.2CH(COOH)NH--CO(CH.sub.2-
).sub.xCH.sub.3, wherein x is an integer of from 10 to 16.
[0100] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative having a lipophilic substituent which
is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--CO(CH.sub.2).sub.2CH(COOH)NHCO(CH.sub.2).-
sub.yCH.sub.3, wherein y is zero or an integer of from 1 to 22.
[0101] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative having a lipophilic substituent which
can be negatively charged. Such a lipophilic substituent can for
example be a substituent which has a carboxyl group.
[0102] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative the parent peptide of which is
selected from the group comprising GLP-1(1-45) or an analogue
thereof.
[0103] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative derived from a GLP-1 fragment
selected from the group comprising GLP-1(7-35), GLP-1(7-36),
GLP-1(7-36)amide, GLP-1(7-37), GLP-1(7-38), GLP-1(7-39),
GLP-1(7-40) and GLP-1(7-41) or an analogue thereof.
[0104] In a further preferred embodiment, the present invention
relates to a GLP-1 analogue derived from a GLP-1 analogue selected
from the group comprising GLP-1(1-35), GLP-1(1-36),
GLP-1(1-36)amide, GLP-1(1-37), GLP-1(1-38), GLP-1(1-39),
GLP-1(1-40) and GLP-1(1-41) or an analogue thereof.
[0105] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein the designation analogue
comprises derivatives wherein a total of up to fifteen, preferably
up to ten amino acid residues have been exchanged with any
.alpha.-amino acid residue.
[0106] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein the designation analogue
comprises derivatives wherein a total of up to fifteen, preferably
up to ten amino acid residues have been exchanged with any
.alpha.-amino acid residue which can be coded for by the genetic
code.
[0107] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein the designation analogue
comprises derivatives wherein a total of up to six amino acid
residues have been exchanged with another .alpha.-amino acid
residue which can be coded for by the genetic code.
[0108] In a further preferred embodiment, the present invention
relates to a GLP-1(A-B) derivative wherein A is an integer from 1
to 7 and B is an integer from 38 to 45 or an analogue thereof
comprising one lipophilic substituent attached to the C-terminal
amino acid residue and, optionally, a second lipophilic substituent
attached to one of the other amino acid residues.
[0109] In a further preferred embodiment, a parent peptide for a
derivative according to the invention is selected from the group
comprising Arg.sup.26-GLP-1(7-37); Arg.sup.34-GLP-1(7-37);
Lys.sup.36-GLP-1(7-37); Arg.sup.26,34Lys.sup.36-GLP-1(7-37);
Arg.sup.26,34Lys.sup.38GLP-1(7-38);
Arg.sup.26,34Lys.sup.39-GLP-1(7-39);
Arg.sup.26,34Lys.sup.40-GLP-1(7-40);
Arg.sup.26Lys.sup.36-GLP-1(7-37); Arg.sup.34Lys.sup.36-GLP-1(7-37);
Arg.sup.26Lys.sup.39-GLP-1(7-39); Arg.sup.34Lys.sup.40-GLP-1(7-40);
Arg.sup.26,34Lys.sup.36,39-GLP-1(7-39);
Arg.sup.26,34Lys.sup.36,40-GLP-1(7-40); Gly.sup.8Arg26-GLP-1(7-37);
Gly.sup.8Arg.sup.34-GLP-1(7-37); Gly.sup.8Lys.sup.36-GLP-1(7-37);
Gly.sup.8Arg.sup.26,34Lys.sup.36-GLP-1(7-37);
Gly.sup.8Arg.sup.26,34Lys.sup.39-GLP-1(7-39);
Gly.sup.8Arg.sup.26,34Lys.sup.40-GLP-1(7-40);
Gly.sup.8Arg.sup.26Lys.sup.36-GLP-1(7-37);
Gly.sup.8Arg.sup.34Lys.sup.36-GLP-1(7-37);
Gly.sup.8Arg.sup.26Lys.sup.39-GLP-1(7-39);
Gly.sup.8Arg.sup.34Lys.sup.40-GLP-1(7-40);
Gly.sup.8Arg.sup.26,34Lys.sup.36,39-GLP-1(7-39) and
Gly.sup.8Arg.sup.26,34Lys.sup.36,40-GLP-1(7-40).
[0110] In a further preferred embodiment, a parent peptide for a
derivative according to the invention is selected from the group
comprising Arg.sup.26,34Lys.sup.38GLP-1(7-38);
Arg.sup.26,34Lys.sup.39GLP-1(7-39);
Arg.sup.26,34Lys.sup.40GLP-1(7-40);
Arg.sup.26,34Lys.sup.41GLP-1(7-41);
Arg.sup.26,34Lys.sup.42GLP-1(7-42);
Arg.sup.26,34Lys.sup.43GLP-1(7-43);
Arg.sup.26,34Lys.sup.44GLP-1(7-44);
Arg.sup.26,34Lys.sup.45GLP-1(7-45);
Arg.sup.26,34Lys.sup.38GLP-1(1-38);
Arg.sup.26,34Lys.sup.39GLP-1(1-39);
Arg.sup.26,34Lys.sup.40GLP-1(1-40);
Arg.sup.26,34Lys.sup.41GLP-1(1-41);
Arg.sup.26,34Lys.sup.42GLP-1(1-42);
Arg.sup.26,34Lys.sup.43GLP-1(1-43);
Arg.sup.26,34Lys.sup.44GLP-1(1-44);
Arg.sup.26,34Lys.sup.45GLP-1(1-45);
Arg.sup.26,34Lys.sup.38GLP-1(2-38);
Arg.sup.26,34Lys.sup.39GLP-1(2-39);
Arg.sup.26,34Lys.sup.40GLP-1(2-40);
Arg.sup.26,34Lys.sup.41GLP-1(2-41);
Arg.sup.26,34Lys.sup.42GLP-1(2-42);
Arg.sup.26,34Lys.sup.43GLP-1(2-43);
Arg.sup.26,34Lys.sup.44GLP-1(2-44);
Arg.sup.26,34Lys.sup.45GLP-1(2-45);
Arg.sup.26,34Lys.sup.38GLP-1(3-38);
Arg.sup.26,34Lys.sup.39GLP-1(3-39);
Arg.sup.26,34Lys.sup.40GLP-1(3-40);
Arg.sup.26,34Lys.sup.41GLP-1(3-41);
Arg.sup.26,34Lys.sup.42GLP-1(3-42);
Arg.sup.26,34Lys.sup.43GLP-1(3-43);
Arg.sup.26,34Lys.sup.44GLP-1(3-44);
Arg.sup.26,34Lys.sup.45GLP-1(3-45);
Arg.sup.26,34Lys.sup.38GLP-1(4-38);
Arg.sup.26,34Lys.sup.39GLP-1(4-39);
Arg.sup.26,34Lys.sup.40GLP-1(4-40);
Arg.sup.26,34Lys.sup.41GLP-1(4-41);
Arg.sup.26,34Lys.sup.42GLP-1(4-42);
Arg.sup.26,34Lys.sup.43GLP-1(4-43);
Arg.sup.26,34Lys.sup.44GLP-1(4-44);
Arg.sup.26,34Lys.sup.45GLP-1(4-45);
Arg.sup.26,34Lys.sup.38GLP-1(5-38);
Arg.sup.26,34Lys.sup.39GLP-1(5-39);
Arg.sup.26,34Lys.sup.40GLP-1(5-40);
Arg.sup.26,34Lys.sup.41GLP-1(5-41);
Arg.sup.26,34Lys.sup.42GLP-1(5-42);
Arg.sup.26,34Lys.sup.43GLP-1(5-43);
Arg.sup.26,34Lys.sup.44GLP-1(5-44);
Arg.sup.26,34Lys.sup.45GLP-1(5-45);
Arg.sup.26,34Lys.sup.38GLP-1(6-38);
Arg.sup.26,34Lys.sup.39GLP-1(6-39);
Arg.sup.26,34Lys.sup.40GLP-1(6-40);
Arg.sup.26,34Lys.sup.41GLP-1(6-41);
Arg.sup.26,34Lys.sup.42GLP-1(6-42);
Arg.sup.26,34Lys.sup.43GLP-1(6-43);
Arg.sup.26,34Lys.sup.44GLP-1(6-44);
Arg.sup.26,34Lys.sup.45GLP-1(6-45);
Arg.sup.26Lys.sup.38GLP-1(1-38); Arg.sup.34Lys.sup.38GLP-1(1-38);
Arg.sup.26,34Lys.sup.36,38GLP-1(1-38);
Arg.sup.26Lys.sup.38GLP-1(7-38); Arg.sup.34Lys.sup.38GLP-1(7-38);
Arg.sup.26,34Lys.sup.36,38GLP-1(7-38);
Arg.sup.26,34Lys.sup.38GLP-1(7-38);
Arg.sup.26Lys.sup.39GLP-1(1-39); Arg.sup.34Lys.sup.39GLP-1(1-39);
Arg.sup.26,34Lys.sup.36,39GLP-1(1-39);
Arg.sup.26Lys.sup.39GLP-1(7-39); Arg.sup.34Lys.sup.39GLP-1(7-39)
and Arg.sup.26,34Lys.sup.36,39GLP-1(7-39).
[0111] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein the parent peptide is
selected from the group comprising Arg.sup.26-GLP-1(7-37),
Arg.sup.34-GLP-1(7-37), Lys.sup.36-GLP-1(7-37),
Arg.sup.26,34Lys.sup.36-GLP-1(7-37),
Arg.sup.26Lys.sup.36-GLP-1(7-37), Arg.sup.34Lys.sup.36-GLP-1(7-37),
Gly.sup.8Arg.sup.26-GLP-1(7-37), Gly.sup.8Arg.sup.34-GLP-1(7-37),
Gly.sup.8Lys.sup.36-GLP-1(7-37),
Gly.sup.8Arg.sup.26,34Lys.sup.36-GLP-1(7-37),
Gly.sup.8Arg.sup.26Lys.sup.36-GLP-1(7-37) and
Gly.sup.8Arg.sup.34Lys.sup.36-GLP-1(7-37).
[0112] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein the parent peptide is
selected from the group comprising
Arg.sup.26Lys.sup.38-GLP-1(7-38),
Arg.sup.26,34Lys.sup.38-GLP-1(7-38),
Arg.sup.26,34Lys.sup.36,38-GLP-1(7-38),
Gly.sup.8Arg.sup.26Lys.sup.38-GLP-1(7-38) and
Gly.sup.8Arg.sup.26,34Lys.sup.36,38-GLP-1(7-38).
[0113] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein the parent peptide is
selected from the group comprising
Arg.sup.26Lys.sup.39-GLP-1(7-39),
Arg.sup.26,34Lys.sup.36,39-GLP-1(7-39),
Gly.sup.8Arg.sup.26Lys.sup.39-GLP-1(7-39) and
Gly.sup.8Arg.sup.26,34Lys.sup.36,39-GLP-1(7-39).
[0114] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative wherein the parent peptide is
selected from the group comprising
Arg.sup.34Lys.sup.40-GLP-1(7-40),
Arg.sup.26,34Lys.sup.36,40-GLP-1(7-40),
Gly.sup.8Arg.sup.34Lys.sup.40-GLP-1(7-40) and
Gly.sup.8Arg.sup.26,34Lys.sup.36,40-GLP-1(7-40).
[0115] In a further preferred embodiment, the present invention
relates to a GLP-1 derivative which is selected from the group
comprising:
TABLE-US-00001
Lys.sup.26(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-37);
Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-37);
Lys.sup.26,34-bis(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-37);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-37);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-37);
Gly.sup.8Ays.sup.26,34-bis(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-37);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-37);
Lys.sup.26(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-38);
Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-38);
Lys.sup.26,34-bis(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-38);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-38);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-38);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-38);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-38);
Lys.sup.26(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-39);
Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-39);
Lys.sup.26,34-bis(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-39);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-39);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-39);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-39);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-39);
Lys.sup.26(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-40);
Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-40);
Lys.sup.26,34-bis(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-40);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-40);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-40);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-40);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-40);
Lys.sup.26(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-36);
Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-36);
Lys.sup.26,34-bis(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-36);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-36);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-36);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-36);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-36);
Lys.sup.26(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-35);
Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-35);
Lys.sup.26,34-bis(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-35);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-35);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-35);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-35);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-35);
Lys.sup.26(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-36)amide;
Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-36)amide;
Lys.sup.26,34-bis(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-36)amide;
Gly.sup.8Lys.sup.26(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-36)amide;
Gly.sup.8Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-36)amide;
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-tetradecanoyl)-GLP-
1(7-36)amide;
Arg.sup.26Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-36)amide;
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-37);
Lys.sup.26(N.sup..epsilon.-tetradecanoyl)Arg.sup.34-GLP-1(7-37);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-tetradecanoyl)Arg.sup.34-GLP-1(7-37);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-37);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-37-
);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-38);
Lys.sup.26(N.sup..epsilon.-tetradecanoyl)Arg.sup.34-GLP-1(7-38);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-tetradecanoyl)Arg.sup.34-GLP-1(7-38);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-38);
Arg.sup.26,34Lys.sup.38(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-38);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-38-
);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-39);
Lys.sup.26(N.sup..epsilon.-tetradecanoyl)Arg.sup.34-GLP-1(7-39);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-tetradecanoyl)Arg.sup.34-GLP-1(7-39);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-39);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-39-
);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-40);
Lys.sup.26(N.sup..epsilon.-tetradecanoyl)Arg.sup.34-GLP-1(7-40);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-tetradecanoyl)Arg.sup.34-GLP-1(7-40);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-40);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-tetradecanoyl)-GLP-1(7-40-
);
Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-1(7-37);
Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-1(7-37);
Lys.sup.26,34-bis(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-37);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-37);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-37);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))--
GLP- 1(7-37);
Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-1(7-38);
Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-1(7-38);
Lys.sup.26,34-bis(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-38);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-38);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-38);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-
GLP-1(7-38);
Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-1(7-39);
Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-1(7-39);
Lys.sup.26,34-bis(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-
GLP-1(7-39);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-39);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-39);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-
GLP-1(7-39);
Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-1(7-40);
Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-1(7-40);
Lys.sup.26,34-bis(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-40);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-40);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-40);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-
GLP-1(7-40);
Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-1(7-36);
Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-1(7-36);
Lys.sup.26,34-bis(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-36);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-36);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-36);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-
GLP-1(7-36);
Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-36)amide;
Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-36)amide;
Lys.sup.26,34-bis(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-36)amide;
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-36)amide;
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-36)amide;
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-
GLP-1(7-36)amide;
Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-1(7-35);
Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-1(7-35);
Lys.sup.26,34-bis(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-35);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-35);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-35);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-
GLP-1(7-35);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-37);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl-
))- GLP-1(7-37);
Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))Arg.sup.34-GLP-
1(7-37);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))Arg.sup.-
34- GLP-1(7-37);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
- 1(7-37);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(.omega.-carboxynonadecan-
oyl))- GLP-1(7-37);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-38);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl-
))- GLP-1(7-38);
Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))Arg.sup.34-GLP-
1(7-38);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))Arg.sup.-
34-GLP- 1(7-38);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
- 1(7-38);
Arg.sup.26,34Lys.sup.38(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
- 1(7-38);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(.omega.-carboxynonadecan-
oyl))- GLP-1(7-38);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-39);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl-
))-GLP- 1(7-39);
Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))Arg.sup.34-GLP-
1(7-39);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))Arg.sup.-
34-GLP- 1(7-39);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
- 1(7-39);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(.omega.-carboxynonadecan-
oyl))- GLP-1(7-39);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
1(7-40);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(.omega.-carboxynonadecanoyl-
))-GLP- 1(7-40);
Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))Arg.sup.34-GLP-
1(7-40);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))Arg.sup.-
34-GLP- 1(7-40);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(.omega.-carboxynonadecanoyl))-GLP-
- 1(7-40);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(.omega.-carboxynonadecan-
oyl))- GLP-1(7-40);
Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-37);
Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-37);
Lys.sup.26,34-bis(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-37);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-37);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-37);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(7-deoxycholoyl))-GLP-
1(7-37);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-37);
Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-38);
Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-38);
Lys.sup.26,34-bis(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-38);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-38);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-38);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(7-deoxycholoyl))-GLP-
1(7-38);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-38);
Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-39);
Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-39);
Lys.sup.26,34-bis(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-39);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-39);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-39);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(7-deoxycholoyl))-GLP-
1(7-39);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-39);
Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-40);
Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-40);
Lys.sup.26,34-bis(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-40);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-40);
Gly.sup.8Lys.sup.34(N.sup..epsilon.s-(7-deoxycholoyl))-GLP-1(7-40);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(7-deoxycholoyl))-GLP-
1(7-40);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-40);
Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-36);
Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-36);
Lys.sup.26,34-bis(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-36);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-36);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-36);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-36);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-36);
Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-35);
Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-35);
Lys.sup.26,34-bis(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-35);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-35);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-35);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-35);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-35);
Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-36)amide;
Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-36)amide;
Lys.sup.26,34-bis(N.sup..epsilon.-(7-deoxycholoyl))-GLP-
1(7-36)amide;
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-36)amide;
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-36)amide;
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(7-deoxycholoyl))-GLP-
1(7-36)amide;
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-36)amide;
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-37-
);
Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))Arg.sup.34-GLP-1(7-37);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))Arg.sup.34-GLP-1(7-37-
);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-37);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-
-37); Lys.sup.26(N.sup..epsilon.-(choloyl))-GLP-1(7-37);
Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-37);
Lys.sup.26,34-bis(N.sup..epsilon.-(choloyl))-GLP-1(7-37);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(choloyl))-GLP-1(7-37);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-37);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(choloyl))-GLP-1(7-37);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-37);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-38-
);
Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))Arg.sup.34-GLP-1(7-38);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))Arg.sup.34-GLP-1(7-38-
);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-38);
Arg.sup.26,34Lys.sup.38(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-38);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-
-38); Lys.sup.26(N.sup..epsilon.-(choloyl))-GLP-1(7-38);
Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-38);
Lys.sup.26,34-bis(N.sup..epsilon.-(choloyl))-GLP-1(7-38);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(choloyl))-GLP-1(7-38);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-38);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(choloyl))-GLP-1(7-38);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-38);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-39-
);
Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))Arg.sup.34-GLP-1(7-39);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))Arg.sup.34-GLP-1(7-39-
);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-39);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-
-39); Lys.sup.26(N.sup..epsilon.-(choloyl))-GLP-1(7-39);
Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-39);
Lys.sup.26,34-bis(N.sup..epsilon.-(choloyl))-GLP-1(7-39);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(choloyl))-GLP-1(7-39);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-39);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(choloyl))-GLP-1(7-39);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-39);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-40-
);
Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))Arg.sup.34-GLP-1(7-40);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(7-deoxycholoyl))Arg.sup.34-GLP-1(7-40-
);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-40);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(7-deoxycholoyl))-GLP-1(7-
-40); Lys.sup.26(N.sup..epsilon.-(choloyl))-GLP-1(7-40);
Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-40);
Lys.sup.26,34-bis(N.sup..epsilon.-(choloyl))-GLP-1(7-40);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(choloyl))-GLP-1(7-40);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-40);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(choloyl))-GLP-1(7-40);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-40);
Lys.sup.26(N.sup..epsilon.-(choloyl))-GLP-1(7-36);
Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-36);
Lys.sup.26,34-bis(N.sup..epsilon.-(choloyl))-GLP-1(7-36);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(choloyl))-GLP-1(7-36);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-36);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(choloyl))-GLP-1(7-36);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-36);
Lys.sup.26(N.sup..epsilon.-(choloyl))-GLP-1(7-35);
Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-35);
Lys.sup.26,34-bis(N.sup..epsilon.-(choloyl))-GLP-1(7-35);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(choloyl))-GLP-1(7-35);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-35);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(choloyl))-GLP-1(7-35);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-35);
Lys.sup.26(N.sup..epsilon.-(choloyl))-GLP-1(7-36)amide;
Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-36)amide;
Lys.sup.26,34-bis(N.sup..epsilon.-(choloyl))-GLP-1(7-36)amide;
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(choloyl))-GLP-1(7-36)amide;
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-36)amide;
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(choloyl))-GLP-1(7-36)amide;
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-36)amide;
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-37);
Lys.sup.26(N.sup..epsilon.-(choloyl))Arg.sup.34-GLP-1(7-37);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(choloyl))Arg.sup.34-GLP-1(7-37);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(choloyl))-GLP-1(7-37);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(choloyl))-GLP-1(7-37);
Lys.sup.26(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-37);
Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-37);
Lys.sup.26,34-bis(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-37);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-37);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-37);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-37);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-37);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-38);
Lys.sup.26(N.sup..epsilon.-(choloyl))Arg.sup.34-GLP-1(7-38);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(choloyl))Arg.sup.34-GLP-1(7-38);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(choloyl))-GLP-1(7-38);
Arg.sup.26,34Lys.sup.38(N.sup..epsilon.-(choloyl))-GLP-1(7-38);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(choloyl))-GLP-1(7-38);
Lys.sup.26(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-38);
Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-38);
Lys.sup.26,34-bis(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-38);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-38);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-38);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-38);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-38);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-39);
Lys.sup.26(N.sup..epsilon.-(choloyl))Arg.sup.34-GLP-1(7-39);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(choloyl))Arg.sup.34-GLP-1(7-39);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(choloyl))-GLP-1(7-39);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(choloyl))-GLP-1(7-39);
Lys.sup.26(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-39);
Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-39);
Lys.sup.26,34-bis(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-39);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-39);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-39);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-39);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-39);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(choloyl))-GLP-1(7-40);
Lys.sup.26(N.sup..epsilon.-(choloyl))Arg.sup.34-GLP-1(7-40);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(choloyl))Arg.sup.34-GLP-1(7-40);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(choloyl))-GLP-1(7-40);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(choloyl))-GLP-1(7-40);
Lys.sup.26(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-40);
Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-40);
Lys.sup.26,34-bis(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-40);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-40);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-40);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-40);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-37);
Lys.sup.26(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-36);
Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-36);
Lys.sup.26,34-bis(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-36);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-36);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-36);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-36);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-36);
Lys.sup.26(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-35);
Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-35);
Lys.sup.26,34-bis(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-35);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-35);
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-35);
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-35);
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-35);
Lys.sup.26(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-36)amide;
Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-36)amide;
Lys.sup.26,34-bis(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-36)amide;
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-36)amide;
Gly.sup.8Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-36)amide;
Gly.sup.8Lys.sup.26,34-bis(N.sup..epsilon.-(lithocholoyl))-GLP-
1(7-36)amide;
Arg.sup.26Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-36)amide;
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-37);
Lys.sup.26(N.sup..epsilon.-(lithocholoyl))Arg.sup.34-GLP-1(7-37);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(lithocholoyl))Arg.sup.34-GLP-1(7-37);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-37);
Arg.sup.26,34Lys.sup.38(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-37);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-3-
7);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-38);
Lys.sup.26(N.sup..epsilon.-(lithocholoyl))Arg.sup.34-GLP-1(7-38);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(lithocholoyl))Arg.sup.34-GLP-1(7-38);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-38);
Arg.sup.26,34Lys.sup.38(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-38);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-3-
8);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-39);
Lys.sup.26(N.sup..epsilon.-(lithocholoyl))Arg.sup.34-GLP-1(7-39);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(lithocholoyl))Arg.sup.34-GLP-1(7-39);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-39);
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-3-
9);
Gly.sup.8Arg.sup.26Lys.sup.34(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-40);
Lys.sup.26(N.sup..epsilon.-(lithocholoyl))Arg.sup.34-GLP-1(7-40);
Gly.sup.8Lys.sup.26(N.sup..epsilon.-(lithocholoyl))Arg.sup.34-GLP-1(7-40);
Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-40)
and
Gly.sup.8Arg.sup.26,34Lys.sup.36(N.sup..epsilon.-(lithocholoyl))-GLP-1(7-4-
0).
[0116] In a further preferred embodiment, the present invention
relates to a pharmaceutical composition comprising a GLP-1
derivative and a pharmaceutically acceptable vehicle or
carrier.
[0117] In a further preferred embodiment, the present invention
relates to the use of a GLP-1 derivative according to the invention
for the preparation of a medicament which has a protracted profile
of action relative to GLP-1(7-37).
[0118] In a further preferred embodiment, the present invention
relates to the use of a GLP-1 derivative according to the invention
for the preparation of a medicament with protracted effect for the
treatment of non-insulin dependent diabetes mellitus.
[0119] In a further preferred embodiment, the present invention
relates to the use of a GLP-1 derivative according to the invention
for the preparation of a medicament with protracted effect for the
treatment of insulin dependent diabetes mellitus.
[0120] In a further preferred embodiment, the present invention
relates to the use of a GLP-1 derivative according to the invention
for the preparation of a medicament with protracted effect for the
treatment of obesity.
[0121] In a further preferred embodiment, the present invention
relates to a method of treating insulin dependent or non-insulin
dependent diabetes mellitus in a patient in need of such a
treatment, comprising administering to the patient a
therapeutically effective amount of a GLP-1 derivative of the
invention, in particular a derivative of GLP-1(7-C), wherein C is
35 or 36, together with a pharmaceutically acceptable carrier.
[0122] According to U.S. Pat. No. 5,631,224 (Novo Nordisk A/S) a
strong synergistic effect is observed in NIDDM patients by the
combined treatment with GLP-1(7-37) or GLP-1(7-36)amide and an oral
hypoglycemic agent.
[0123] Since pharmacodynamic and pharmacokinetic properties can be
changed according to patients' demand by selecting a GLP-1
derivative of the present invention, additional therapeutic
advantages can be gained by treating the NIDDM patients in a
regimen which additionally comprises treatment with another
antidiabetic agent.
[0124] Thus, the invention furthermore relates to the use of a
GLP-1 derivative according to the present invention for the
preparation of a medicament for use in the treatment of diabetes in
a regimen which additionally comprises treatment with another
antidiabetic agent.
[0125] In the present context the expression "antidiabetic agent"
includes compounds for the treatment and/or prophylaxis of insulin
resistance and diseases wherein insulin resistance is the
pathophysiological mechanism.
[0126] In one embodiment of this invention, the antidiabetic agent
is insulin or an analogue an a derivative thereof.
[0127] In another embodiment the antidiabetic agent is a
hypoglycaemic agent, preferably an oral hypoglycaemic agent.
[0128] Oral hypoglycaemic agents are preferably selected from the
group consisting of sulfonylureas, biguanides, thiazolidinediones,
glucosidase inhibitors, glucagon antagonists, GLP-1 agonists,
potasium channel openers, insulin sensitizers, hepatic enzyme
inhibitors, glucose uptake modulators, compounds modifying the
lipid metabolism, compounds lowering food intake, and agents acting
on the ATP-dependent potassium channel of the .beta.-cells.
[0129] Among the sulfonylureas, tolbutamide, glibenclamide,
glipizide and gliclazide are preferred.
[0130] Among the biguanides, metformin is preferred.
[0131] Among the thiazolidinediones, troglitazone and ciglitazone
are preferred.
[0132] Among the glucosidase inhibitors, acarbose is preferred.
[0133] Among the agents acting on the ATP-dependent potassium
channel of the .beta.-cells the following are preferred:
glibenclamide, glipizide, gliclazide, repaglinide.
[0134] U.S. Pat. No. 5,424,286 describes a method for stimulating
insulin release with exendin polypeptide(s). The exendin
polypeptides disclosed include HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGX (SEQ
ID NO:1); wherein X.dbd.P or Y, and
[0135] HX1X2GTFITSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO:2);
wherein X1X2=SD (exendin-3) or GE (exendin-4)). According to this
document the insulinotropic effect of these polypeptides is greater
than that attainable by administration of GLP-1.
[0136] The exendin-3 and -4 and fragments are useful in treatment
of diabetes mellitus (types I or II) and prevention of
hyperglycaemia. They normalise hyperglycaemia through
glucose-dependent, insulin-independent and insulin-dependent
mechanisms. These insulinotropic peptides are more active than
GLP-1. Exendin-4 is specific for exendin receptors, i.e. it does
not interact with vasoactive intestinal peptide receptors.
[0137] WO 9746584 describes truncated versions of exendin
peptide(s) for treating diabetes. The disclosed peptides increase
secretion and biosynthesis of insulin, but reduce those of
glucagon. The truncated peptides can be made more economically than
full length versions. Compared with GLP-1 and the known exendins,
they are more active (effective at lower doses), more stable to
degradation and metabolism and have a longer lasting effect.
[0138] However, the high clearance limits the usefulness of these
compounds, and thus there still is a need for improvements in this
field. Accordingly, it is one object of the present invention to
provide derivatives of exendin and analogues thereof which have a
protracted profile of action relative to native exendin.
[0139] Thus, in one aspect the invention relates to an exendin
derivative wherein at least one amino acid residue of the parent
peptide has a lipophilic substituent attached.
[0140] In a preferred embodiment only one lipophilic substituent is
present.
[0141] In another preferred embodiment, the lipophilic substituent
is attached to the N-terminal amino acid residue.
[0142] In another preferred embodiment, the lipophilic substituent
is attached to the C-terminal amino acid residue.
[0143] In another preferred embodiment, the lipophilic substituent
is attached to an amino acid residue which is not the N-terminal or
C-terminal amino acid residue.
[0144] In further preferred embodiment, two lipophilic substituents
are present.
[0145] In another preferred embodiment, one of the lipophilic
substituents is attached to the N-terminal amino acid residue while
the other is attached to the C-terminal amino acid residue.
[0146] In another preferred embodiment, one of the lipophilic
substituents is attached to the C-terminal amino acid residue while
the other is attached to an amino acid residue which is not the
N-terminal or C-terminal amino acid residue.
[0147] In another preferred embodiment, both lipophilic
substituents are attached to amino acid residues which are neither
the N-terminal nor the C-terminal amino acid residue.
[0148] In further preferred embodiment, the lipophilic substituent
comprises from 4 to 40 carbon atoms, more preferred from 8 to 25
carbon atoms, such as 12 to 18 carbon atoms.
[0149] In another preferred embodiment, a lipophilic substituent is
attached to an amino acid residue in such a way that a carboxyl
group of the lipophilic substituent forms an amide bond with an
amino group of the amino acid residue.
[0150] In another preferred embodiment, a lipophilic substituent is
attached to an amino acid residue in such a way that an amino group
of the lipophilic substituent forms an amide bond with a carboxyl
group of the amino acid residue.
[0151] In another preferred embodiment, the lipophilic substituent
is attached to the parent peptide by means of a spacer.
[0152] In another preferred embodiment, the spacer is an unbranched
alkane .alpha.,.omega.-dicarboxylic acid group having from 1 to 7
methylene groups, preferably two methylene groups, which form a
bridge between an amino group of the parent peptide and an amino
group of the lipophilic substituent.
[0153] In another preferred embodiment, the spacer is an amino acid
residue except cys, or a dipeptide such as gly-lys.
[0154] In another preferred embodiment, a carboxyl group of the
parent peptide forms an amide bond with an amino group of lys or a
dipeptide containing a lys residue, and the other amino group of
the lys spacer or a dipeptide spacer containing a lys residue forms
an amide bond with a carboxyl group of the lipophilic
substituent.
[0155] In another preferred embodiment, an amino group of the
parent peptide forms an amide bond with a carboxylic group of the
amino acid residue or dipeptide spacer, and an amino group of the
amino acid residue or dipeptide spacer forms an amide bond with a
carboxyl group of the lipophilic substituent.
[0156] In another preferred embodiment, a carboxyl group of the
parent peptide forms an amide bond with an amino group of the amino
acid residue spacer or dipeptide spacer, and a carboxyl group of
the amino acid residue spacer or dipeptide spacer forms an amide
bond with an amino group of the lipophilic substituent.
[0157] In another preferred embodiment, a carboxyl group of the
parent peptide forms an amide bond with an amino group of a spacer
which is asp or glu, or a dipeptide spacer containing an asp or glu
residue, and a carboxyl group of the spacer forms an amide bond
with an amino group of the lipophilic substituent.
[0158] In one embodiment said spacer is .gamma.-aminobutyroyl.
[0159] In a further preferred embodiment, the lipophilic
substituent comprises a partially or completely hydrogenated
cyclopentanophenathrene skeleton.
[0160] In another preferred embodiment, the lipophilic substituent
is an straight-chain or branched alkyl group.
[0161] In another preferred embodiment, the lipophilic substituent
is the acyl group of a straight-chain or branched fatty acid.
[0162] In another preferred embodiment, the acyl group is selected
from the group comprising CH.sub.3(CH.sub.2).sub.nCO--, wherein n
is 4 to 38, preferably 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--,
most preferably hexadecanoyl.
[0163] In another preferred embodiment, the lipophilic substituent
is an acyl group of a straight-chain or branched alkane
.alpha.,.omega.-dicarboxylic acid.
[0164] In another preferred embodiment, the acyl group is selected
from the group comprising HOOC(CH.sub.2).sub.mCO--, wherein m is
from 4 to 38, preferably from 4 to 24, more preferred selected from
the group comprising HOOC(CH.sub.2).sub.14CO--,
HOOC(CH.sub.2).sub.16CO--, HOOC(CH.sub.2).sub.18CO--,
HOOC(CH.sub.2).sub.20CP-- and HOOC(CH.sub.2).sub.22CO--.
[0165] In another preferred embodiment, the lipophilic substituent
is a group of the formula
CH.sub.3(CH.sub.2).sub.p((CH.sub.2).sub.qCOOH)CHNH--CO(CH.sub.2).sub.2CO--
-, wherein p and q are integers and p+q is an integer of from 8 to
33, preferably from 12 to 28.
[0166] In another preferred embodiment, the lipophilic substituent
is a group of the formula
CH.sub.3(CH.sub.2).sub.rCO--NHCH(COOH)(CH.sub.2).sub.2CO--, wherein
r is an integer of from 10 to 24.
[0167] In another preferred embodiment, the lipophilic substituent
is a group of the formula
CH.sub.3(CH.sub.2).sub.sCO--NHCH((CH.sub.2).sub.2COOH)CO--, wherein
s is an integer of from 8 to 24.
[0168] In another preferred embodiment, the lipophilic substituent
is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--CO(CH.sub.2).sub.uCH.sub.3, wherein
u is an integer of from 8 to 18.
[0169] In another preferred embodiment, the lipophilic substituent
is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--COCH((CH.sub.2).sub.2COOH)NH--CO(CH.sub.2-
).sub.wCH.sub.3, wherein w is an integer of from 10 to 16.
[0170] In another preferred embodiment, the lipophilic substituent
is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--CO(CH.sub.2).sub.2CH(COOH)NH--CO(CH.sub.2-
).sub.xCH.sub.3, wherein x is an integer of from 10 to 16.
[0171] In another preferred embodiment, the lipophilic substituent
is a group of the formula
--NHCH(COOH)(CH.sub.2).sub.4NH--CO(CH.sub.2).sub.2CH(COOH)NH--CO(CH.sub.2-
).sub.yCH.sub.3, wherein y is zero or an integer of from 1 to
22.
[0172] In another preferred embodiment, the designation analogue
comprises derivatives wherein a total of up to fifteen, preferably
up to ten amino acid residues have been exchanged with any
.alpha.-amino acid residue.
[0173] In another preferred embodiment, the designation analogue
comprises derivatives wherein a total of up to fifteen, preferably
up to ten amino acid residues have been exchanged with any
.alpha.-amino acid residue which can be coded for by the genetic
code.
[0174] In another preferred embodiment, the designation analogue
comprises derivatives wherein a total of up to six amino acid
residues have been exchanged with any .alpha.-amino acid residue
which can be coded for by the genetic code.
[0175] In another preferred embodiment, the parent peptide is
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGX (SEQ ID NO:1), wherein X.dbd.P or
Y, or a fragment or an analogue thereof.
[0176] In another preferred embodiment, the parent peptide is
HX1X2GTFITSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO:2), wherein
X1X2=SD or GE, or a fragment or an analogue thereof.
[0177] In another preferred embodiment, the parent peptide is
DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO:3), or a fragment or an
analogue thereof.
[0178] In another preferred embodiment the exendin derivative is
selected from Arg.sup.18, Leu.sup.20, Gln.sup.34, Lys.sup.33
(N.sup..epsilon.-(.gamma.-aminobutyroyl(N.sup..alpha.-hexadecanoyl)))
Exendin-4-(7-45)-NH.sub.2, Arg.sup.33, Leu.sup.20, Gln.sup.34,
Lys.sup.18
(N.sup..epsilon.-(.gamma.-aminobutyroyl(N.sup..alpha.-hexadecanoyl)))
Exendin-4-(7-45)-NH.sub.2.
[0179] The present invention furthermore relates to a
pharmaceutical composition comprising an exendin derivative
according to the present invention and a pharmaceutically
acceptable vehicle or carrier.
[0180] Moreover, the invention is concened with the use of an
exendin derivative according to the present invention for the
preparation of a medicament which has a protracted profile of
action relative to exendin.
[0181] The invention also relates to the use of an exendin
derivative according to the present invention for the preparation
of a medicament with a protracted profile of action for the
treatment of non-insulin dependent diabetes mellitus or for the
treatment of insulin dependent diabetes mellitus or for the
treatment of obesity.
[0182] The invention also relates to a method of treating insulin
dependent or non-insulin dependent diabetes mellitus in a patient
in need of such a treatment, comprising administering to the
patient a therapeutically effective amount of a exendin derivative
according to the present invention together with a pharmaceutically
acceptable carrier.
DETAILED DESCRIPTION OF THE INVENTION
[0183] To obtain a satisfactory protracted profile of action of the
GLP-1 derivative, the lipophilic substituent attached to the GLP-1
moiety preferably comprises 4-40 carbon atoms, in particular 8-25
carbon atoms. The lipophilic substituent may be attached to an
amino group of the GLP-1 moiety by means of a carboxyl group of the
lipophilic substituent which forms an amide bond with an amino
group of the amino acid residue to which it is attached.
Alternatively, the lipophilic substituent may be attached to said
amino acid residue in such a way that an amino group of the
lipophilic substituent forms an amide bond with a carboxyl group of
the amino acid residue. As a further option, the lipophilic
substituent may be linked to the GLP-1 moiety via an ester bond.
Formally, the ester can be formed either by reaction between a
carboxyl group of the GLP-1 moiety and a hydroxyl group of the
substituent-to-be or by reaction between a hydroxyl group of the
GLP-1 moiety and a carboxyl group of the substituent-to-be. As a
further alternative, the lipophilic substituent can be an alkyl
group which is introduced into a primary amino group of the GLP-1
moiety.
[0184] In one preferred embodiment of the invention, the lipophilic
substituent is attached to the GLP-1 moiety by means of a spacer in
such a way that a carboxyl group of the spacer forms an amide bond
with an amino group of the GLP-1 moiety. Examples of suitable
spacers are succinic acid, Lys, Glu or Asp, or a dipeptide such as
Gly-Lys. When the spacer is succinic acid, one carboxyl group
thereof may form an amide bond with an amino group of the amino
acid residue, and the other carboxyl group thereof may form an
amide bond with an amino group of the lipophilic substituent. When
the spacer is Lys, Glu or Asp, the carboxyl group thereof may form
an amide bond with an amino group of the amino acid residue, and
the amino group thereof may form an amide bond with a carboxyl
group of the lipophilic substituent. When Lys is used as the
spacer, a further spacer may in some instances be inserted between
the .epsilon.-amino group of Lys and the lipophilic substituent. In
one preferred embodiment, such a further spacer is succinic acid
which forms an amide bond with the .epsilon.-amino group of Lys and
with an amino group present in the lipophilic substituent. In
another preferred embodiment such a further spacer is Glu or Asp
which forms an amide bond with the .epsilon.-amino group of Lys and
another amide bond with a carboxyl group present in the lipophilic
substituent, that is, the lipophilic substituent is a
N.sup..epsilon.-acylated lysine residue.
[0185] In another preferred embodiment of the present invention,
the lipophilic substituent has a group which can be negatively
charged. One preferred group which can be negatively charged is a
carboxylic acid group.
[0186] The parent peptide 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.
[0187] 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.
[0188] The DNA sequence encoding the parent peptide 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 peptide 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 Manual, Cold Spring Harbor Laboratory Press,
New York, 1989). The DNA sequence encoding the peptide 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.
[0189] 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.
[0190] 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.
[0191] 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.
[0192] 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.
[0193] 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.
[0194] 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).
[0195] 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.
[0196] Examples of compounds which can be useful as GLP-1 moieties
according to the present invention are described in International
Patent Application No. WO 87/06941 (The General Hospital
Corporation) which relates to a peptide fragment which comprises
GLP-1(7-37) and functional derivatives thereof and to its use as an
insulinotropic agent.
[0197] Further GLP-1 analogues are described in International
Patent Application No. 90/11296 (The General Hospital Corporation)
which relates to peptide fragments which comprise GLP-1(7-36) and
functional derivatives thereof and have an insulinotropic activity
which exceeds the insulinotropic activity of GLP-1(1-36) or
GLP-1(1-37) and to their use as insulinotropic agents.
[0198] International Patent Application No. 91/11457 (Buckley et
al.) discloses analogues of the active GLP-1 peptides 7-34, 7-35,
7-36, and 7-37 which can also be useful as GLP-1 moieties according
to the present invention.
[0199] Pharmaceutical Compositions
[0200] Pharmaceutical compositions containing a GLP-1 derivative
according to the present invention may be administered parenterally
to patients in need of such a treatment. Parenteral administration
may be performed by subcutaneous, intramuscular 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 powder or a liquid for the administration of the GLP-1 derivative
in the form of a nasal or pulmonal spray. As a still further
option, the GLP-1 derivatives of the invention can also be
administered transdermally, e.g. from a patch, optionally a
iontophoretic patch, or transmucosally, e.g. bucally.
[0201] Pharmaceutical compositions containing a GLP-1 derivative of
the present invention may be prepared by conventional techniques,
e.g. as described in Remington's Pharmaceutical Sciences, 1985 or
in Remington: The Science and Practice of Pharmacy, 19.sup.th
edition, 1995.
[0202] Thus, the injectable compositions of the GLP-1 derivative of
the invention can be prepared using the conventional techniques of
the pharmaceutical industry which involves dissolving and mixing
the ingredients as appropriate to give the desired end product.
[0203] According to one procedure, the GLP-1 derivative is
dissolved in an amount of water which is somewhat less than the
final volume of the composition to be prepared. An isotonic agent,
a preservative and a buffer is added as required and the pH value
of the solution is adjusted--if necessary--using an acid, e.g.
hydrochloric acid, or a base, e.g. aqueous sodium hydroxide as
needed. Finally, the volume of the solution is adjusted with water
to give the desired concentration of the ingredients.
[0204] Examples of isotonic agents are sodium chloride, mannitol
and glycerol.
[0205] Examples of preservatives are phenol, m-cresol, methyl
p-hydroxybenzoate and benzyl alcohol.
[0206] Examples of suitable buffers are sodium acetate and sodium
phosphate.
[0207] Further to the above-mentioned components, solutions
containing a GLP-1 derivative according to the present invention
may also contain a surfactant in order to improve the solubility
and/or the stability of the GLP-1 derivative.
[0208] A composition for nasal administration of certain peptides
may, for example, be prepared as described in European Patent No.
272097 (to Novo Nordisk A/S) or in WO 93/18785.
[0209] According to one preferred embodiment of the present
invention, the GLP-1 derivative is provided in the form of a
composition suitable for administration by injection. Such a
composition can either be an injectable solution ready for use or
it can be an amount of a solid composition, e.g. a lyophilised
product, which has to be dissolved in a solvent before it can be
injected. The injectable solution preferably contains not less than
about 2 mg/ml, preferably not less than about 5 mg/ml, more
preferred not less than about 10 mg/ml of the GLP-1 derivative and,
preferably, not more than about 100 mg/ml of the GLP-1
derivative.
[0210] The GLP-1 derivatives of this invention can be used in the
treatment of various diseases. The particular GLP-1 derivative to
be used and the optimal dose level for any patient will depend on
the disease to be treated and on a variety of factors including the
efficacy of the specific peptide derivative employed, the age, body
weight, physical activity, and diet of the patient, on a possible
combination with other drugs, and on the severity of the case. It
is recommended that the dosage of the GLP-1 derivative of this
invention be determined for each individual patient by those
skilled in the art.
[0211] In particular, it is envisaged that the GLP-1 derivative
will be useful for the preparation of a medicament with a
protracted profile of action for the treatment of non-insulin
dependent diabetes mellitus and/or for the treatment of
obesity.
[0212] The present invention is further illustrated by the
following examples which, however, are not to be construed as
limiting the scope of protection. 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
[0213] The following acronyms for commercially available chemicals
are used:
[0214] DMF: N,N-Dimethylformamide.
[0215] DCC: N,N-Dicyclohexylcarbodiimide
[0216] NMP: N-Methyl-2-pyrrolidone.
[0217] EDPA: N-Ethyl-N,N-diisopropylamine.
[0218] EGTA: Ethylene glycol-bis(.beta.-aminoethyl
ether)-N,N,N',N'-tetraacetic acid.
[0219] GTP Guanosine 5'-triphosphate.
[0220] TFA: Trifluoroacetic acid.
[0221] THF: Tetrahydrofuran
[0222] H-Glu(OH)-OBu.sup.t: L-Glutamic acid .alpha.-tert-butyl
ester
[0223] Cap-ONSu: Octanoic acid 2,5-dioxopyrrolidin-1-yl ester
[0224] Lau-ONSu: Dodecanoic acid 2,5-dioxopyrrolidin-1-yl ester
[0225] Myr-ONSu: Tetradecanoic acid 2,5-dioxopyrrolidin-1-yl
ester.
[0226] Pal-ONSu: Hexadecanoic acid 2,5-dioxopyrrolidin-1-yl
ester.
[0227] Ste-ONSu Octadecanoic acid 2,5-dioxopyrrolidin-1-yl
ester.
[0228] Abbreviations:
[0229] PDMS: Plasma Desorption Mass Spectrometry
[0230] MALDI-MS: Matrix Assisted Laser Desorption/Ionisation Mass
Spectrometry
[0231] HPLC: High Performance Liquid Chromatography
[0232] amu: atomic mass units
[0233] Lit-Glu(ONSu)-OBu.sup.t: N.sup..alpha.-Lithochoyl-L-glutamic
acid .alpha.-t-butyl ester .gamma.-2,5-dioxopyrrolidin-1-yl
ester
[0234] Cap-Glu(ONSu)-OBu.sup.t: N.sup..alpha.-Octanoyl-L-glutamic
acid .alpha.-t-butyl ester .gamma.-2,5-dioxopyrrolidin-1-yl
ester
[0235] Cac-Glu(ONSu)-OBu.sup.t: N.sup..alpha.-Decanoyl-L-glutamic
acid .alpha.-t-butyl ester .gamma.-2,5-dioxopyrrolidin-1-yl
ester
[0236] Lau-Glu(ONSu)-OBu.sup.t: N.sup..alpha.-Dodecanoyl-L-glutamic
acid .alpha.-t-butyl ester .gamma.-2,5-dioxopyrrolidin-1-yl
ester
[0237] Myr-Glu(ONSu)-OBu.sup.t:
N.sup..alpha.-Tetradecanoyl-L-glutamic acid .alpha.-t-butyl ester
.gamma.-2,5-dioxopyrrolidin-1-yl ester
[0238] Pal-Glu(ONSu)-OBu.sup.t:
N.sup..alpha.-Hexadecanoyl-(L)-glutamic acid
.alpha.-t-butyl-.gamma.-2,5-dioxopyrrolidin-1-yl diester.
[0239] Ste-Glu(ONSu)-OBu.sup.t:
N.sup..alpha.-Octadecanoyl-(L)-glutamic acid
.alpha.-t-butyl-.gamma.-2,5-dioxopyrrolidin-1-yl diester
[0240] Lau-.beta.-Ala-ONSu: N.sup..beta.-Dodecanoyl-.beta.-alanine
2,5-dioxopyrrolidin-1-yl ester
[0241] Pal-.beta.-Ala-ONSu:
N.sup..beta.-Hexadecanoyl-.beta.-alanine 2,5-dioxopyrrolidin-1-yl
ester
[0242] Lau-GABA-ONSu: N.sup..gamma.-Dodecanoyl-.gamma.-aminobutyric
acid 2,5-dioxopyrrolidin-1-yl ester
[0243] Myr-GABA-ONSu:
N.sup..gamma.-Tetradecanoyl-.gamma.-aminobutyric acid
2,5-dioxopyrrolidin-1-yl ester
[0244] Pal-GABA-ONSu:
N.sup..gamma.-Hexadecanoyl-.gamma.-aminobutyric acid
2,5-dioxopyrrolidin-1-yl ester
[0245] Ste-GABA-ONSu:
N.sup..gamma.-Octadecanoyl-.gamma.-aminobutyric acid
2,5-dioxopyrrolidin-1-yl ester
[0246] Pal-Isonip-ONSu: N-Hexadecanoyl-piperidine-4-carboxylic acid
2,5-dioxopyrrolidin-1-yl ester
[0247] Pal-Glu(OBu.sup.t)-ONSu:
N.sup..alpha.-Hexadecanoyl-L-glutamic acid
.alpha.-2,5-dioxopyrrolidin-1-yl ester .gamma.-t-butyl ester
[0248] HOOC--(CH.sub.2).sub.6--COONSu: .omega.-Carboxyheptanoic
acid 2,5-dioxopyrrolidin-1-yl ester.
[0249] HOOC--(CH.sub.2).sub.10--COONSu: .omega.-Carboxyundecanoic
acid 2,5-dioxopyrrolidin-1-yl ester.
[0250] HOOC--(CH.sub.2).sub.12--COONSu: .omega.-Carboxytridecanoic
acid 2,5-dioxopyrrolidin-1-yl ester.
[0251] HOOC--(CH.sub.2).sub.14--COONSu:
.omega.-Carboxypentadecanoic acid 2,5-dioxopyrrolidin-1-yl
ester.
[0252] HOOC--(CH.sub.2).sub.16--COONSu:
.omega.-Carboxyheptadecanoic acid 2,5-dioxopyrrolidin-1-yl
ester.
[0253] HOOC--(CH.sub.2).sub.18--COONSu: .omega.-Carboxynonadecanoic
acid 2,5-dioxopyrrolidin-1-yl ester.
[0254] Analytical
[0255] Plasma Desorption Mass Spectrometry
[0256] Sample Preparation:
[0257] The sample is dissolved in 0.1% TFA/EtOH (1:1) at a
concentration of 1 .mu.g/.mu.l. The sample solution (5-10 .mu.l) is
placed on a nitrocellulose target (Bio-ion AB, Uppsala, Sweden) and
allowed to adsorb to the target surface for 2 minutes. The target
is subsequently rinsed with 2.times.25 .mu.l 0.1% TFA and
spin-dried. Finally, the nitrocellulose target is placed in a
target carrousel and introduced into the mass spectrometer.
[0258] MS Analysis:
[0259] PDMS analysis was carried out using a Bio-ion 20 time-of
flight instrument (Bio-ion Nordic AB, Uppsala, Sweden). An
acceleration voltage of 15 kV was applied and molecular ions formed
by bombardment of the nitrocellulose surface with 252-Cf fission
fragments were accelerated towards a stop detector. The resulting
time-of-flight spectrum was calibrated into a true mass spectrum
using the H.sup.+ and NO.sup.+ ions at m/z 1 and 30, respectively.
Mass spectra were generally accumulated for 1.0.times.10.sup.6
fission events corresponding to 15-20 minutes. Resulting assigned
masses all correspond to isotopically averaged molecular masses.
The accuracy of mass assignment is generally better than 0.1%.
[0260] MALDI-MS
[0261] MALDI-TOF MS analysis was carried out using a Voyager RP
instrument (PerSeptive Biosystems Inc., Framingham, Mass.) equipped
with delayed extraction and operated in linear mode.
Alpha-cyano-4-hydroxy-cinnamic acid was used as matrix, and mass
assignments were based on external calibration.
Example 1
Synthesis of N.sup..alpha.-hexadecanoyl-Glu(ONSu)-OBu.sup.t
[0262] To a suspension of H-Glu(OH)-OBu.sup.t (4.2 g, 20.6 mmol),
DMF (500 ml) and EDPA (2.65 g, 20.6 mmol) was added drop by drop a
solution of Pal-ONSu (7.3 g, 20.6 mmol) in DMF (100 ml). The
reaction mixture was stirred for 64 h at room temperature and then
concentrated in vacuo to a total volume of 20 ml. The residue was
partitioned between 10% aqueous citric acid (300 ml) and ethyl
acetate (250 ml), and the phases were separated. The organic phase
was concentrated in vacuo and the residue dissolved in DMF (50 ml).
The resulting solution was added drop by drop to a 10% aqueous
solution of citric acid (500 ml) kept at 0.degree. C. The
precipitated compound was collected and washed with iced water and
dried in a vacuum drying oven. The dried compound was dissolved in
DMF (45 ml) and HONSu (2.15 g, 18.7 mmol) was added. To the
resulting mixture was added a solution of
N,N'-dicyclohexylcarbodiimide (3.5 g, 17 mmol) in dichloromethane
(67 ml). The reaction mixture was stirred for 16 h at room
temperature, and the precipitated compound was filtered off. The
precipitate was recrystallised from n-heptane/2-propanol to give
the title compound (6.6 g, 72%).
Example 2
Synthesis of N.sup..alpha.-octadecanoyl-Glu(ONSu)-OBu.sup.t
[0263] To a suspension of H-Glu(OH)-OBu.sup.t (2.82 g, 13.9 mmol),
DMF (370 ml) and EDPA (1.79 g, 13.9 mmol) was added drop by drop a
solution of Ste-ONSu (5.3 g, 13.9 mmol) in DMF (60 ml).
Dichloromethane (35 ml) was added, and the reaction mixture was
stirred for 24 h at room temperature and then concentrated in
vacuo. The residue was partitioned between 10% aqueous citric acid
(330 ml) and ethyl acetate (200 ml), and the phases were separated.
The organic phase was concentrated in vacuo and the residue
dissolved in DMF (60 ml). The resulting solution was added drop by
drop to a 10% aqueous solution of citric acid (400 ml) kept at
0.degree. C. The precipitated compound was collected and washed
with iced water and dried in a vacuum drying oven. The dried
compound was dissolved in DMF (40 ml) and HONSu (1.63 g, 14.2 mmol)
was added. To the resulting mixture was added a solution of DCC
(2.66 g, 12.9 mmol) in dichloromethane (51 ml). The reaction
mixture was stirred for 64 h at room temperature, and the
precipitated compound was filtered off. The precipitate was
recrystallised from n-heptane/2-propanol to give the title compound
(4.96 g, 68%).
Example 3
Synthesis of Arg.sup.26,34, Lys.sup.36
(N.sup..epsilon.-(.gamma.-glutamyl(N.sup..alpha.-hexadecanoyl)))
GLP-1 (7-36)-OH
[0264] To a mixture of Arg.sup.26,34, Lys.sup.36 GLP-1 (7-36)-OH
(12.2 mg, 3.67 .mu.mol), EDPA (13.3 mg, 103 .mu.mol), NMP (1.71 ml)
and water (855 .mu.l) was added a solution of
Pal-Glu(ONSu)-OBu.sup.t (5.94 mg, 11 .mu.mol), prepared as
described above, in NMP (148 .mu.l). The reaction mixture was
gently shaken for 5 min. at room temperature, and then allowed to
stand for an additional 90 min. at room temperature. The reaction
was quenched by the addition of a solution of glycine (6 mg, 81
.mu.mol) in water (0.6 ml). A 0.5% aqueous solution of
ammonium-acetate (38 ml) was added, and the resulting mixture
eluted onto a Varian 5g C8 Mega Bond Elut.RTM., the immobilised
compound washed with 5% aqueous acetonitril (20 ml), and finally
liberated from the cartridge by elution with TFA (25 ml). The
eluate was concentrated in vacuo, and the residue purified by
column chromatography using a cyanopropyl column (Zorbax 300SB-CN)
and a standard acetonitril/TFA system. The column was heated to
65.degree. C. and the acetonitril gradient was 0-100% in 60
minutes. The title compound (3.1 mg, 23%) was isolated, and the
product was analysed by PDMS. The m/z value for the protonated
molecular ion was found to be 3695.+-.3. The resulting molecular
weight is thus 3694.+-.3 amu (theoretical value 3694 amu).
Example 4
Synthesis of Arg.sup.26,34, Lys.sup.36
(N.sup..epsilon.-(.gamma.-glutamyl(N.sup..alpha.-octadecanoyl)))
GLP-1 (7-36)-OH
[0265] To a mixture of Arg.sup.26,34, Lys.sup.36 GLP-1 (7-36)-OH
(12.2 mg, 3.7 .mu.mol), EDPA (13.3 mg, 103 .mu.mol), NMP (1.71 ml)
and water (855 .mu.l) was added a solution of
Ste-Glu(ONSu)-OBu.sup.t (6.25 mg, 11 .mu.mol), prepared as above,
in NMP (1 ml). The reaction mixture was gently shaken for 5 min. at
room temperature, and then allowed to stand for an additional 90
min. at room temperature. The reaction was quenched by the addition
of a solution of glycine (6 mg, 81 .mu.mol) in water (0.6 ml). A
0.5% aqueous solution of ammonium acetate (54 ml) was added, and
the resulting mixture eluted onto a Varian 5g C8 Mega Bond
Elut.RTM., the immobilised compound washed with 5% aqueous
acetonitril (20 ml), and finally liberated from the cartridge by
elution with TFA (25 ml). The eluate was concentrated in vacuo, and
the residue purified by column chromatography using a cyanopropyl
column (Zorbax 300SB-CN) and a standard acetonitril/TFA system. The
column was heated to 65.degree. C. and the acetonitril gradient was
0-100% in 60 minutes. The title compound (3.7 mg, 27%) was
isolated, and the product was analysed by PDMS. The m/z value for
the protonated molecular ion was found to be 3723.+-.3. The
resulting molecular weight is thus 3722.+-.3 amu (theoretical value
3722 amu).
Example 5
Synthesis of Arg.sup.18, Leu.sup.20, Gln.sup.34, Lys.sup.33
(N.sup..epsilon.-(.gamma.-aminobutyroyl(N.sup..alpha.-hexadecanoyl)))
Exendin-4-(7-45)-NH.sub.2
[0266] To a mixture of Arg.sup.18, Leu.sup.20,
Gln.sup.34-Exendin-4-NH.sub.2 (9.7 mg, 2.3 .mu.mol), EDPA (8.4 mg,
64.7 .mu.mol), NMP (1.36 ml) and water (0.68 ml) was added a
solution of Pal-GABA-ONSu (3 mg, 6.9 .mu.mol) in NMP (76 .mu.l).
The reaction mixture was gently shaken for 5 min., and then allowed
to stand for an additional 90 min. at room temperature. The
reaction was quenched by the addition of a solution of glycine (3.8
mg, 50.8 .mu.mol) in water (38 .mu.l). The resulting mixture was
purified by column chromatography using a cyanopropyl column
(Zorbax 300SB-CN) and a standard acetonitril/TFA system. The column
was heated to 65.degree. C. and the acetonitril gradient was 0-100%
in 60 minutes. The title compound (4.5 mg, 43%) was isolated, and
the product was analysed by PDMS. The m/z value for the protonated
molecular ion was found to be 4532.8.+-.3. The resulting molecular
weight is thus 4531.8.+-.3 amu (theoretical value 4534 amu).
Example 6
Synthesis of Arg.sup.33, Leu.sup.20, Gln.sup.34, Lys.sup.18
(N.sup..epsilon.-(.gamma.-aminobutyroyl(N.sup..alpha.-hexadecanoyl)))
Exendin-4-(7-45)-NH.sub.2
[0267] To a mixture of Arg.sup.33, Leu.sup.20,
Gln.sup.34-Exendin-4-NH.sub.2 (10 mg, 2.4 .mu.mol), EDPA (8.6 mg,
66.5 .mu.mol), NMP (1.4 ml) and water (0.7 ml) was added a solution
of Pal-GABA-ONSu (3.1 mg, 7.1 .mu.mol) in NMP (78 .mu.l). The
reaction mixture was gently shaken for 5 min., and then allowed to
stand for an additional 145 min. at room temperature. The reaction
was quenched by the addition of a solution of glycine (3.9 mg, 52.3
.mu.mol) in water (39 .mu.l). The resulting mixture was purified by
column chromatography using a cyanopropyl column (Zorbax 300SB-CN)
and a standard acetonitril/TFA system. The column was heated to
65.degree. C. and the acetonitril gradient was 0-100% in 60
minutes. The title compound (2.9 mg, 21%) was isolated, and the
product was analysed by PDMS. The m/z value for the protonated
molecular ion was found to be 4533.8.+-.3. The resulting molecular
weight is thus 4532.8.+-.3 amu (theoretical value 4534 amu).
[0268] Biological Findings
[0269] Protraction of GLP-1 Derivatives after s.c.
Administration
[0270] The protraction of a number GLP-1 derivatives of the
invention was determined by monitoring the concentration thereof in
plasma after sc administration to healthy pigs, using the method
described below. For comparison also the concentration in plasma of
GLP-1(7-37) after sc. administration was followed. The protraction
of other GLP-1 derivatives of the invention can be determined in
the same way.
[0271] Pigs (50% Duroc, 25% Yorkshire, 25% Danish Landrace, app 40
kg) were fasted from the beginning of the experiment. To each pig
0.5 nmol of test compound per kg body weight was 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 were drawn from a catheter in vena
jugularis. 5 ml of the blood samples were poured into chilled
glasses containing 175 .mu.l of the following solution: 0.18 M
EDTA, 1500 KIE/ml aprotinin (Novo Nordisk) and 3% bacitracin
(Sigma), pH 7.4. Within 30 min, the samples were centrifuged for 10
min at 5-6000*g. Temperature was kept at 4.degree. C. The
supernatant was pipetted into different glasses and kept at minus
20.degree. C. until use.
[0272] The plasma concentrations of the peptides were determined by
RIA using a monoclonal antibody specific for the N-terminal region
of GLP-1(7-37). The cross reactivities were less than 1% with
GLP-1(1-37) and GLP-1(8-36)amide and <0.1% with GLP-1(9-37),
GLP-1(10-36)amide and GLP-1(11-36)amide. The entire procedure was
carried out at 4.degree. C.
[0273] The assay was carried out as follows: 100 .mu.l plasma was
mixed with 271 .mu.l 96% ethanol, mixed using a vortex mixer and
centrifuged at 2600*g for 30 min. The supernatant was decanted into
Minisorp tubes and evaporated completely (Savant Speedvac AS290).
The evaporation residue was reconstituted in the assay buffer
consisting of 80 mM NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4, 0.1% HSA
(Orpha 20/21, Behring), 10 mM EDTA, 0.6 mM thiomersal (Sigma), pH
7.5. Samples were reconstituted in volumes suitable for their
expected concentrations, and were allowed to reconstitute for 30
min. To 300 .mu.l sample, 100 .mu.l antibody solution in dilution
buffer containing 40 mM NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4, 0.1%
HSA, 0.6 mM thiomersal, pH 7.5, was added. A non-specific sample
was prepared by mixing 300 .mu.l buffer with 100 .mu.l dilution
buffer. Individual standards were prepared from freeze dried
stocks, dissolved in 300 .mu.l assay buffer. All samples were
pre-incubated in Minisorp tubes with antibody as described above
for 72 h. 200 .mu.l tracer in dilution buffer containing 6-7000 CPM
was added, samples were mixed and incubated for 48 h. 1.5 ml of a
suspension of 200 ml per litre of heparin-stabilised bovine plasma
and 18 g per litre of activated carbon (Merck) in 40 mM
NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4, 0.6 mM thiomersal, pH 7.5, was
added to each tube. Before use, the suspension was mixed and
allowed to stand for 2 h at 4.degree. C. All samples were incubated
for 1 h at 4.degree. C. and then centrifuged at 3400*g for 25 min.
Immediately after the centrifugation, the supernatant was decanted
and counted in a .gamma.-counter. The concentration in the samples
was calculated from individual standard curves. Plasma
concentrations were found, calculated as % of the maximum
concentration for the individual compounds (n=2). The GLP-1
derivatives of the invention have a protracted profile of action
relative to GLP-1(7-37) and are much more persistent in plasma than
GLP-1(7-37). The time at which the peak concentration in plasma is
achieved varies within wide limits, depending on the particular
GLP-1 derivative selected.
[0274] Stimulation of cAMP Formation in a Cell Line Expressing the
Cloned Human GLP-1 Receptor
[0275] In order to demonstrate efficacy of the GLP-1 derivatives,
their ability to stimulate formation of cAMP in a cell line
expressing the cloned human GLP-1 receptor was tested. An EC.sub.50
was calculated from the dose-response curve.
[0276] Baby hamster kidney (BHK) cells expressing the human
pancreatic GLP-1 receptor were used (Knudsen and Pridal, 1996, Eur.
J. Pharm. 318, 429-435). Plasma membranes were prepared (Adelhorst
et al, 1994, J. Biol. Chem. 269, 6275) by homogenisation in buffer
(10 mmol/l Tris-HCl and 30 mmol/l NaCl pH 7.4, containing, in
addition, 1 mmol/l dithiothreitol, 5 mg/l leupeptin (Sigma, St.
Louis, Mo., USA), 5 mg/l pepstatin (Sigma, St. Louis, Mo., USA),
100 mg/l bacitracin (Sigma, St. Louis, Mo., USA), and 16 mg/l
aprotinin (Novo Nordisk A/S, Bagsvaerd, Denmark)). The homogenate
was centrifuged on top of a layer of 41 w/v % sucrose. The white
band between the two layers was diluted in buffer and centrifuged.
Plasma membranes were stored at -80.degree. C. until used.
[0277] The assay was carried out in 96-well microtiter plates in a
total volume of 140 .mu.l. The buffer used was 50 mmol/l Tris-HCl,
pH 7.4 with the addition of 1 mmol/l EGTA, 1.5 mmol/l MgSO.sub.4,
1.7 mmol/l ATP, 20 mM GTP, 2 mmol/l 3-isobutyl-1-methylxanthine,
0.01% Tween-20 and 0.1% human serum albumin (Reinst, Behringwerke
AG, Marburg, Germany). Compounds to be tested for agonist activity
were dissolved and diluted in buffer, added to the membrane
preparation and the mixture was incubated for 2 h at 37.degree. C.
The reaction was stopped by the addition of 25 .mu.l of 0.05 mol/l
HCl. Samples were diluted 10 fold before analysis for cAMP by a
scintillation proximity assay (RPA 538, Amersham, UK).
Sequence CWU 1
1
3131PRTArtificial SequenceSynthesized 1His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Xaa 20 25 30240PRTArtificial
SequenceSynthesized 2His Xaa Xaa Gly Thr Phe Ile Thr Ser Asp Leu
Ser Lys Gln Met Glu1 5 10 15Glu Glu Ala Val Arg Leu Phe Ile Glu Trp
Leu Lys Asn Gly Gly Pro 20 25 30Ser Ser Gly Ala Pro Pro Pro Ser 35
40331PRTArtificial SequenceSynthesized 3Asp Leu Ser Lys Gln Met Glu
Glu Glu Ala Val Arg Leu Phe Ile Glu1 5 10 15Trp Leu Lys Asn Gly Gly
Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30
* * * * *