U.S. patent application number 14/095667 was filed with the patent office on 2014-11-13 for peptide agonists of glp-1 activity.
This patent application is currently assigned to Zealand Pharma A/S. The applicant listed for this patent is Zealand Pharma A/S. Invention is credited to Bjarne D. LARSEN, Jens D. Mikkelsen, Soren Neve.
Application Number | 20140336356 14/095667 |
Document ID | / |
Family ID | 29709523 |
Filed Date | 2014-11-13 |
United States Patent
Application |
20140336356 |
Kind Code |
A1 |
LARSEN; Bjarne D. ; et
al. |
November 13, 2014 |
PEPTIDE AGONISTS OF GLP-1 ACTIVITY
Abstract
Novel peptide agonists of GLP-1 activity useful for lowering
blood glucose levels. The novel peptides comprise variants of the
GLP-1 or the exendin-4 polypeptide sequence and are
pharmacologically active and stable. These peptides are useful in
the treatment of diseases that benefit from regulation of excess
levels of blood glucose and/or regulation of gastric emptying, such
as diabetes and eating disorders.
Inventors: |
LARSEN; Bjarne D.;
(Roskilde, DK) ; Mikkelsen; Jens D.; (Kgs. Lyngby,
DK) ; Neve; Soren; (Kgs. Lyngby, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zealand Pharma A/S |
Glostrup |
|
DK |
|
|
Assignee: |
Zealand Pharma A/S
Glostrup
DK
|
Family ID: |
29709523 |
Appl. No.: |
14/095667 |
Filed: |
December 3, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13850762 |
Mar 26, 2013 |
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14095667 |
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12770667 |
Apr 29, 2010 |
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13850762 |
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11515812 |
Sep 5, 2006 |
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12770667 |
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10291226 |
Nov 8, 2002 |
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11515812 |
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09614847 |
Jul 12, 2000 |
6528486 |
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10291226 |
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60143591 |
Jul 12, 1999 |
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Current U.S.
Class: |
530/308 |
Current CPC
Class: |
C07K 14/57563 20130101;
A61K 47/62 20170801; A61P 3/04 20180101; A61P 3/10 20180101; A61K
38/26 20130101; A61K 38/00 20130101; C07K 14/605 20130101 |
Class at
Publication: |
530/308 |
International
Class: |
C07K 14/605 20060101
C07K014/605 |
Claims
1. A peptide conjugate comprising a peptide X selected from the
group consisting of (a) an exendin having at least 90% homology to
exendin-4; (b) a variant of said exendin wherein said variant
comprises a modification selected from the group consisting of
between one and five deletions at positions 34-39 and contains a
Lys at position 40 having a lipophilic substituent; or (c) GLP-1
(7-36) (SEQ ID NO: 114) or GLP-1 (7-37) (SEQ ID NO: 123) having at
least one modification selected from the group consisting of: (i)
substitution of D-alanine, glycine or alpha-amino isobutyric acid
for alanine at position 8 and (ii) a lipophilic substituent; and Z,
a peptide sequence of 4-20.amino acid units covalently bound to
said variant, wherein each amino acid unit in said peptide
sequence, Z is selected from the group consisting of Ala, Leu, Ser,
Thr, Tyr, Asn, Gin, Asp, Glu, Lys, Arg, His, Met, Orn, and amino
acid units of the general formula I
--NH--C(R.sup.1)(R.sup.2)--C(.dbd.O)-(1) wherein R.sup.1 and
R.sup.2 are selected from the group consisting of hydrogen,
C.sub.1-6-alkyl, phenyl, and phenyl-methyl, wherein C.sub.1-6-alkyl
is optionally substituted with from one to three substituents
selected from halogen, hydroxy, amino, cyano, nitro, sulfono, and
carboxy, and phenyl and phenyl-methyl is optionally substituted
with from one to three substituents selected from C.sub.1-6-alkyl,
C.sub.2-6-alkenyl, halogen, hydroxy, amino, cyano, nitro, sulfono,
and carboxy, or R.sup.1 and R.sup.2 together with the carbon atom
to which they are bound form a cyclopentyl, cyclohexyl, or
cycloheptyl ring, e.g. 2,4-diaminobutanoic acid and
2,3-diaminopropanoic acid; and a pharmaceutically acceptable salt
or the c-terminal amide of said peptide conjugate, with the proviso
that x is not exendin-4 or exendin-3.
2-48. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel peptide agonists of
GLP-1 activity. More specifically the invention relates to novel
peptides that lower blood glucose levels comprising variants of the
exendin-4 polypeptide sequence and peptide conjugates comprising
variants of the GLP-1 or the exendin-4 polypeptide sequences which
are pharmacologically active and stable, and as agonists of GLP-1
activity are useful in the treatment of diseases that benefit from
regulation of excess levels of blood glucose and/or regulation of
gastric emptying, such as diabetes and eating disorders. The
present invention also relates to methods of preparing said novel
peptides, a composition, e.g., a pharmaceutical composition,
comprising a peptide of the invention and a physiologically
acceptable carrier, to said peptide for use in therapy, a method of
treating a disorder and to the use of said peptide for the
manufacture of a pharmaceutical composition for use in therapy.
BACKGROUND OF THE INVENTION
[0002] A number of hormones that lower blood glucose levels are
released from the gastrointestinal mucosa in response to the
presence and absorption of nutrients in the gut. These include
gastrin, secretin, glucose-dependent insulinotropic polypeptide
(GIP) and glucagon-like peptide-1 (GLP-1). The most potent
substance known is GLP-1 (Orskov, 1992, Diabetologia 35:701-711).
Glucagon-like peptide 1 (GLP-1) is a product of proglucagon, a 180
amino acid peptide (Drucker, 1998, Diabetes 47:159-169). The
overall sequence of proglucagon contains the 29-amino acid sequence
of glucagon, the 36 or 37 amino acid sequence of GLP-1 and the 34
amino acid sequence of glucagon-like peptide-2 (GLP-2), an
intestinotrophic peptide. GLP-1 has a number of functions. It is a
physiological hormone that enhances the effect on insulin secretion
in normal humans and is therefore an incretin hormone. In addition,
GLP-1 also lowers glucagon concentrations, slows gastric emptying,
stimulates (pro)insulin biosynthesis, and enhances insulin
sensitivity (Hauck, 1997, Horm. Metab. Res. 47:1253-1258). The
peptide also enhances the ability for the .beta.-cells to sense and
respond to glucose in subjects with impaired glucose tolerance
(Byrne, 1998, Eur. J. Clin. Invest. 28:72-78). The insulinotropic
effect of GLP-1 in humans increases the rate of glucose
disappearance partly because of increased insulin levels and partly
because of enhanced insulin sensitivity (D'Alessio, 1994, Eur. J.
Clin. Invest. 28:72-78). This has placed GLP-1 as a promising agent
for treatment for type II diabetes. Active fragments of GLP-1 have
been found to be GLP-1(7-36) (SEQ ID NO: 114) and GLP-1(7-37) (SEQ
ID NO: 124).
[0003] However, a major pharmacological problem with native GLP-1
is its short half-life. In humans and rats, GLP-1 is rapidly
degraded by dipeptidyl peptidase-IV (DPP-IV) into GLP-1(9-36)amide
(SEQ ID NO: 125), acting as an endogenous GLP-1 receptor antagonist
(Deacon, 1998, Diabetologia 41:271-278). Several strategies
circumventing this problem have been proposed, some using
inhibitors of DPP-1V and others DPP-IV resistant analogues of GLP-1
(7-36)amide (SEQ ID NO: 114) (Deacon, 1998, Diabetologia
41:271-278; Deacon et al., 1998, Diabetes 47:764-769; Ritzel, 1998,
J. Endocrinol. 159:93-102; U.S. Pat. No. 5,545,618; Pederson, 1998,
Diabetes 47:1253-1258).
[0004] Exendins, another group of peptides that lower blood glucose
levels have some sequence similarity (53%) to GLP-1[7-36]NH.sub.2
(SEQ ID NO: 114) (Goke et al., 1993, J. Biol. Chem. 268:19650-55).
The exendins are found in the venom of Helodermatidae or beaded
lizards (Raufman, 1996, Reg. Peptides 61:1-18). Exendin-3 is
present in the venom of Heloderma horridum, the Mexican beaded
lizard and exendin-4 is present in the venom of Heloderma
suspectum, the Gila monster. Exendin-4 differs from exendin-3 at
just positions two and three. The cDNA encoding the exendin-4
precursor protein, a 47 amino acid peptide fused to the amino
terminus of exendin-4 has been cloned and sequenced (Pohl et al.,
1998, J. Biol. Chem. 273:9778-9784 and WO98/35033). Both exendin-3
and exendin-4 stimulate an increase in cellular cAMP production in
guinea pig pancreatic acinar cells by interacting with exendin
receptors (Raufman, 1996, Reg. Peptides 61:1-18). Exendin-3 causes
a biphasic increase in cellular cAMP production, but a monophasic
increase in amylase release in pancreatic acinar cells. In
contrast, exendin-4 causes a monophasic increase in cAMP production
and does not alter amylase release.
[0005] Exendin-4 is a strong GLP-1 receptor agonist on isolated rat
insulinoma cells (Goke et al., 1993, J. Biol. Chem. 268:19650-55).
This is expected as the (His Ala) domain of GLP-1 recognised by
DPP-IV is not present in exendin-4 (Goke et al., 1993, J. Biol.
Chem. 268:19650-55). Binding of [.sup.125I]GLP-1 to the nucleus of
the solitary tract was inhibited concentration-dependently by
unlabelled GLP-1 and [Tyr39]exendin-4 (SEQ ID NO: 126) with Ki
values of 3.5 and 9.4 nM respectively, and similar values are found
in cell lines (Goke et al., 1995, Eur. J. Neurosci. 7:2294-2300 and
Goke et al., 1993, J. Biol. Chem. 268:19650-55). Further, exendin-4
given systemically lowers blood glucose levels by 40% in diabetic
db/db mice (WO99/07404). Recently, Grieg et al. (1999, Diabetologia
42:45-50) has shown a long lasting blood glucose lowering effect of
once daily intraperitoneal injection of exendin-4 to diabetic ob/ob
mice). U.S. Pat. No. 5,424,286 discloses that a considerable
portion of the N-terminal sequence is essential in order to
preserve insulinotropic activity (exendin-4(1-31) (SEQ ID NO: 127)
and Y.sup.31-exendin-4(1-31)) (SEQ ID NO: 148) whereas an
N-terminally truncated exendin (exendin-4(9-39) (SEQ ID NO: 128)
has inhibitory properties.
[0006] The use of exendin-3, exendin-4 and exendin agonists has
been proposed for the treatment of diabetes mellitus, reducing
gastric motility and delaying gastric emptying and the prevention
of hyperglycemia (U.S. Pat. No. 5,424,286, WO98/05351) as well as
for the reduction of food intake (WO98/30231). There has been
proposed ways of obtaining novel compounds by modifying the native
exendin sequences. One way is to attach lipophilic substituents to
the molecule, e.g. as described in WO 99/43708 which discloses
derivatives of exendin with just one lipophilic substituent
attached to the C-terminal amino acid residue.
[0007] A major approach has been to devise exendin analogues
characterised by amino acid substitutions and/or C-terminal
truncation of the native exendin-4 sequence. This approach is
represented by the compounds of WO99/07404, WO 99/25727 and WO
99/25728. WO99/07404 discloses exendin agonists having a general
formula I that defines a peptide sequence of 39 amino acid residues
with Gly Thr in positions 4-5, Ser Lys Gln in positions 11-13, Glu
Glu Glu Ala Val Arg Leu in positions 15-21, Leu Lys Asn Gly Gly in
positions 26-30, Ser Ser Gly Ala in positions 32-35, and wherein
the remaining positions may be occupied by wild-type exendin amino
acid residues or may be occupied by specified amino acid
substitutions. The formula I does not cover any exendin agonists or
analogues having specific amino acid deletions and/or being
conjugates as described herein, such as the novel compounds
desPro.sup.36-exendin-4(1-39) (SEQ ID NO: 101),
exendin-4(1-39)-K.sub.6 (SEQ ID NO: 92) or
desPro.sup.36-exendin-4(1-39)-K.sub.6(SEQ ID NO: 93).
[0008] WO 99/25727 discloses exendin agonists having a general
formula I that defines a peptide sequence of from 28 to 38 amino
acid residues with Gly in position 4 and Ala in position 18, and
wherein the remaining positions may be occupied by wild-type
exendin amino acid residues or may be occupied by specified amino
acid substitutions. Formula I does not comprise a peptide sequence
having Ser as the C-terminal amino acid and exendin agonists or
analogues having specific amino acid deletions and/or being
conjugates as described herein, such as the novel compounds
desPro.sup.36-exendin-4(1-39) (SEQ ID NO: 101),
exendin-4(1-39)-K.sub.6 (SEQ ID NO: 92) or
desPro.sup.36-exendin-4(1-39)-K.sub.6(SEQ ID NO: 93). Further,
formula II of WO 99/25727 defines a peptide sequence similar to
formula I, but including exendin derivatives having a
C(1-10)alkanoyl or cycloalkylalkanoyl substituent on lysine in
position 27 or 28.
[0009] When treating inappropriate post-prandial blood glucose
levels the compounds are administered frequently, for example one,
two or three times a day.
[0010] WO 99/25728 discloses exendin agonists having a general
formula I that defines a peptide sequence of from 28 to 39 amino
acid residues with fixed Ala in position 18, and wherein the
remaining positions may be occupied by wild-type exendin amino acid
residues or may be occupied by specified amino acid substitutions.
Said exendin agonists all correspond to a truncated exendin
analogue having a varying degree of amino acid substitutions.
Peptide sequences of from 34 to 38 amino acid residues do not have
Ser C-terminally. A peptide sequence of 39 amino acid residues may
have either Ser or Tyr C-terminally, but no further residues.
Exendin agonists or analogues having specific amino acid deletions
and/or being conjugates according to the invention described herein
are not comprised by formula I. Further, formula II defines a
peptide sequence similar to formula I, but including exendin
derivatives having a C(1-10)alkanoyl or cycloalkylalkanoyl
substituent on lysine in position 27 or 28.
[0011] WO 99/46283 (published 16.09.99) discloses peptide
conjugates comprising a pharmacologically active peptide X and a
stabilising peptide sequence Z of 4-20 amino acid residues
covalently bound to X, where said conjugates are characterised in
having an increased half-life compared to the half-life of X. X may
be exendin-4 or exendin-3.
OBJECTIVE OF THE INVENTION
[0012] There is a need for compounds that lower blood glucose
levels in mammals, and are stable and effective. Therefore, it is
an objective of the invention to provide novel compounds that lower
blood glucose levels in mammals. Ideally, these should be effective
when administered orally. It is a further object of the invention
to provide novel peptide agonists of GLP-1 activity and/or
exendin-4 activity. It is a still further purpose of the invention
to provide peptide agonists of GLP-1 activity and/or exendin-4
activity having an increased half-life and/or a decreased
clearance.
SUMMARY OF THE INVENTION
[0013] The invention is directed to a peptide conjugate comprising
a peptide X selected from the group consisting of
(a) an exendin having at least 90% homology to exendin-4; (b) a
variant of said exendin wherein said variant comprises a
modification selected from the group consisting of between one and
five deletions at positions 34-39 and contains a Lys at position 40
having a lipophilic substituent; or (c) GLP-1 (7-36) (SEQ ID NO:
114) or GLP-1 (7-37) (SEQ ID NO: 124) having at least one
modification selected from the group consisting of: [0014] (i)
substitution of D-alanine, glycine or alpha-amino isobutyric acid
for alanine at position 8 and [0015] (ii) a lipophilic substituent,
and Z, a peptide sequence of 4-20 amino acid units covalently bound
to said variant, wherein each amino acid unit in said peptide
sequence, Z is selected from the group consisting of Ala, Leu, Ser,
Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn, and amino
acid units of the general formula I
[0015] --NH--C(R.sup.1)(R.sup.2)--C(.dbd.O)-- (I)
wherein R.sup.1 and R.sup.2 are selected from the group consisting
of hydrogen, C.sub.1-6-alkyl, phenyl, and phenyl-methyl, wherein
C.sub.1-6-alkyl is optionally substituted with from one to three
substituents selected from halogen, hydroxy, amino, cyano, nitro,
sulfono, and carboxy, and phenyl and phenyl-methyl is optionally
substituted with from one to three substituents selected from
C.sub.1-6-alkyl, C.sub.2-6-alkenyl, halogen, hydroxy, amino, cyano,
nitro, sulfono, and carboxy, or R.sup.1 and R.sup.2 together with
the carbon atom to which they are bound form a cyclopentyl,
cyclohexyl, or cycloheptyl ring, e.g. 2,4-diaminobutanoic acid and
2,3-diaminopropanoic acid, with the proviso that X is not exendin-4
or exendin-3.
[0016] The peptide X is further characterised in being effective in
improving glucose tolerance in a diabetic mammal.
[0017] Furthermore, the invention is directed to a novel variant of
a parent exendin, wherein said parent exendin has an amino acid
sequence having at least an 90% homology to exendin-4 and wherein
said variant lowers the blood glucose level in a mammal, binds to a
GLP-1 receptor and has at least one modification selected from the
group consisting of (a) between one and five deletions at positions
34-38, and (b) contains a Lys at position 40 having a lipophilic
substituent attached to the epsilon amino group of said lysine.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 shows the effect of Compound 1 (SEQ ID NO:101) (des
Pro.sup.36-exendin-4(1-39)-NH.sub.2) on blood glucose levels of
mice, cf. Example 25.
[0019] FIG. 2 shows the effect of Compound 2 (SEQ ID NO:93) (des
Pro.sup.36-exendin-4(1-39)-Lys.sub.6-NH.sub.2 on the blood glucose
levels of mice, cf. Example 25.
[0020] FIG. 3 shows the effect of Compound 5 (SEQ ID NO:89)
(Gly.sup.8, Lys.sup.37
(palmitoyl)-GLP1-(7-36)(Human)-(Lys).sub.7-NH.sub.2 on the blood
glucose levels of mice, cf. Example 25.
[0021] FIG. 4 shows in vivo degradation kinetics in rabbits after
i.v. injection of 1 .mu.mol/kg of Compound 4 and Compound (iii),
cf. Example 27.
[0022] FIG. 5 is a plot of AUC (area under the curve) values
(mean.+-.SEM) for Compounds 2, 14-16, 18 and 19 in an oral glucose
tolerance test (OGTT), cf. Example 28.
[0023] FIG. 6 shows a synthetic cDNA constructed for heterolog
expression of Compound 2 in yeast. The new construct was designated
pYES0010, cf. Example 20.
[0024] FIG. 7 is a plot of dose-response on GTT in db/db mice based
on relative AUC.sub.0-240 min values (mean.+-.SEM) for Compound 2
and Compound (i), cf. Example 29.
[0025] FIG. 8 shows the effects of a maximal dose of Compound 2,
i.e. 100 nmol/kg i.p., on the oral glucose tolerance test (OGTT)
when administered up to 24 hours before the OGTT.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The compounds of the present invention include hitherto
unknown deletion variants of a parent exendin. In contrast to known
substitution and/or truncation variants of exendin-4(1-39) the
novel compounds are believed to exhibit a stabilised alpha-helix
structure with superior stability properties and unreduced or
enhanced binding properties. Moreover, conjugation of the novel
variants, modified GLP-1(7-36)-NH.sub.2(SEQ ID NO: 114), and
modified GLP-1(7-37) (SEQ ID NO: 124) to specific short peptide
sequences (Z) render stability to these compounds without
compromising the pharmacological properties. These conjugations
confer in vivo stability and hydrophilicity to the peptide
molecule. The Z is composed of amino-acid residues, and has alone
no structural characteristics in terms of .alpha.-helix
conformation. However, from studies using both circular dichroism
and nuclear magnetic resonance (NMR) spectroscopy, addition of Z
dramatically alters the structural characteristics of some peptides
as evidenced by the increased amount of .alpha.-helix conformation
in the peptide. For example, circular dichroism demonstrated that a
Z-modified (Gly.sup.8)-GLP-1 (SEQ ID NO: 87) had much more
.alpha.-helix conformation than (Gly.sup.8)-GLP-1(SEQ ID NO: 87).
Together with the pharmacological results, the structural analyses
suggest that Z is modifying the conformation of the peptide leading
to higher enzyme-stability, but without losing its potency. Also
the physical and chemical properties of peptides may be altered
considerably by Z-modification with resulting impact on
pharmacological formulation strategy.
Exendin Variants
[0027] The exendin variant of the present invention is a variant of
a parent exendin peptide having at least about 90% homology and
most preferably at least about 95% to exendin-4, which have exendin
activity, e.g., lowers the blood glucose level in a mammal and
binds to a GLP-1 receptor. In a preferred embodiment, the parent
exendin peptide has an amino acid sequence which differs by five
amino acids, preferably by four amino acids, more preferably by
three amino acids, even more preferably by two amino acids, and
still more preferably by one amino acid residue from the amino acid
sequence of exendin-4(1-39) (SEQ ID NO: 102).
[0028] In one embodiment, the exendin variant comprises between one
and five deletions at positions 34-38. Preferably the variant
comprises between 1 and 4 deletions at positions 34-38, more
preferably between 1 and 3 deletions at positions 36-38. Preferably
the parent exendin is exendin-4, and a preferred variant included
as peptide X in the peptide conjugates herein has an amino acid
sequence wherein 1, 2 or 3 of the Pro residues in positions 36, 37
and 38 have been deleted from the amino acid sequence of exendin-4
and preferably from the amino acid sequence of exendin-4(1-39) (SEQ
ID NO: 102).
[0029] Coupling of a Z sequence to the X peptide herein is believed
to increase the stability of these compounds. Proline is a rigid
amino acid that may interfere with the effect of Z to stabilise the
structure of the X peptide. Deletion of one, two or all of the
proline amino acids in positions 36, 37 and 38 of the exendin
backbone is therefore preferred in the peptide conjugates
comprising a variant of a parent exendin according to the
invention, as long as the efficacy of said conjugates as measured
in, e.g. an oral glucose tolerance test (OGTT) in diabetic db/db
mice, is not negatively affected.
[0030] In another embodiment, the variant comprises an additional
residue at position 40, a lysine residue which comprises a
lipophilic substituent bound to the epsilon amino group of lysine
via an amide bond. The lipophilic substituent may be the acyl group
of a straight-chain or branched fatty acid or a straight-chain or
branched alkane .alpha.,.omega.-dicarboxylic acid. The acyl group
may have the formula CH.sub.3(CH.sub.2).sub.nCO--, wherein n is an
integer from 4-38 and preferably from 4-24. In a specific
embodiment, the acyl group is selected from the group consisting of
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--. The acyl group may have the formula
HOOC(CH.sub.2).sub.mCO--, wherein n is an integer from 4-38 and
preferably from 4-24. In a specific embodiment, the acyl group is
selected from the group consisting of 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--. In a more
specific embodiment, the lipophilic substituent is selected from
the group consisting of tetradecanoyl, .omega.-carboxynonadecanoyl,
7-deoxycholoyl, choloyl, palmitoyl and lithocholyl. In a most
specific embodiment, the lipophilic substituent is palmitoyl.
[0031] Alternatively, the liphophilic substituent may have an NH
group. Specific embodiments include but are not limited to the
formulae
CH.sub.3(CH.sub.2).sub.a((CH.sub.2).sub.bCOOH)CHNHCO(CH.sub.2).sub.2CO--
wherein a and b are integers and a+b is an integer of from 8 to 33,
preferably from 12 to 28;
CH.sub.3(CH.sub.2).sub.cCONHCH(COOH)(CH.sub.2).sub.2CO-- wherein c
is an integer of from 10 to 24;
CH.sub.3(CH.sub.2).sub.dCONHCH(CH.sub.2).sub.2(COOH)CO-- wherein d
is an integer of from 8 to 24; COOH(CH.sub.2).sub.eCO-- wherein e
is an integer of from 8 to 24;
--NHCH(COOH)(CH.sub.2).sub.4NHCO(CH.sub.2).sub.fCH.sub.3 wherein f
is an integer of from 8 to 18;
--NHCH(COOH)(CH.sub.2).sub.4NHCOCH(CH.sub.2).sub.2COOH)NHCO(CH.sub.2).sub-
.gCH.sub.3 wherein g is an integer of from 10 to 16; and
--NHCH(COOH)(CH.sub.2).sub.4NHCO(CH.sub.2).sub.2CH(COOH)NHCO(CH.sub.2).su-
b.hCH.sub.3 wherein h is an integer of 0 or from 1 to 22 and
preferably from 10 to 16.
[0032] The exendin variants having a lysine residue at position 40
carrying a lipophilic substituent optionally further comprise
between one and five deletions, preferably between one and three
deletions, at positions 34 to 39, preferably at positions 34-38,
such as [des Ser.sup.39, Lys.sup.40 (palmitoyl)]exendin-4(1-39)
(SEQ ID NO: 107), [des Pro.sup.36, Lys.sup.40
(palmitoyl)]exendin-4(1-39) (SEQ ID NO: 110) and [des Pro.sup.36,
Lys.sup.40 (palmitoyl)]exendin-4(1-40) (SEQ ID NO: 152).
[0033] The variant may be in a most specific embodiment selected
from the group consisting of:
Compound 1: des Pro.sup.36-exendin-4(1-39)-NH.sub.2 (SEQ ID
NO:101), des Pro.sup.36-exendin-4(1-40)-NH.sub.2 (SEQ ID NO: 139),
Compound 14: des Pro.sup.36, Pro.sup.37,
Pro.sup.38-exendin-4(1-39)-NH.sub.2(SEQ ID NO: 132), des
Pro.sup.36, Pro.sup.37, Pro.sup.38-exendin-4(1-40)-NH.sub.2(SEQ ID
NO: 140), des Pro.sup.36, Pro.sup.37-exendin-4(1-39)-NH.sub.2(SEQ
ID NO: 130), des Ala.sup.35-exendin-4(1-39)-NH.sub.2 (SEQ ID
NO:105), des Gly.sup.34-exendin-4(1-39)-NH.sub.2 (SEQ ID NO:106),
des Ser.sup.39-(Lys.sup.40 (palmitoyl))exendin-4(1-39)-NH.sub.2
(SEQ ID NO:107), des Gly.sup.34-(Lys.sup.40
(palmitoyl))exendin-4(1-39)-NH.sub.2 (SEQ ID NO:108), des
Ala.sup.35-(Lys.sup.40 (palmitoyl))exendin-4(1-39):NH.sub.2 (SEQ ID
NO:109), des Pro.sup.36-(Lys.sup.40
(palmitoyl))exendin-4(1-39)-NH.sub.2 (SEQ ID NO:110), and the free
acid thereof and a pharmaceutically acceptable salt thereof.
Modified GLP-1
[0034] A preferred modified GLP-1 included as peptide X in the
peptide conjugates herein has an amino acid sequence of GLP-1
(7-36)-NH.sub.2 (SEQ ID NO: 114) or GLP-1 (7-37) (SEQ ID NO: 124)
having a substitution of glycine for alanine at position 8.
Alternatively, a preferred modified GLP-1 has an amino acid
sequence of GLP-1 (7-36) (SEQ ID NO: 114) or GLP-1 (7-37) (SEQ ID
NO: 124) having a substitution of glycine for alanine at position 8
and a lipophilic substituent, preferably palmitoyl, on one lysine
residue at position 26, 34 or 37. The lipophilic substituent is
preferably attached to the epsilon amino group of said lysine and
includes the specific embodiments described above for the exendin
variants. The modified GLP-1(7-36) (SEQ ID NO: 114) or GLP-1(7-37)
(SEQ ID NO: 124) used as X in the conjugates of the invention may
be those cited in WO 99/43707 and WO 98/08871 comprising a
lipophilic substituent or, more preferably those GLP-1 analogues
having a glycine substitution at position 8. Preferred peptides X
are
Gly.sup.8-GLP-1(7-36) (SEQ ID NO: 87);
Gly.sup.8-GLP-1(7-37) (SEQ ID NO: 123), and
[0035] Gly.sup.8-GLP-1(7-36)-Lys.sup.37(palmitoyl) (SEQ ID NO:
147).
[0036] The compounds of the invention having a lipophilic
substituent would have a more protracted profile of action than the
parent peptides as demonstrated for GLP-1 derivatives in WO
98/08871.
Peptide Conjugates
[0037] The peptide sequence Z may be bound to the C-terminal or the
N-terminal of the peptide sequence, X, or two peptide sequences may
be bound individually to both the C- and N-terminal of X. In case
the native peptide X possesses a free C-terminal carboxylic acid,
the peptide sequence Z may be attached to either the C-terminal of
the peptide X or to the N-terminal of the peptide X, or the C- and
N-terminal of X may both be bound to each individual peptide
sequence Z. Alternatively, Z may be bound to the nitrogen atom on
the side chain of lysine, histidine or arginine or a carbonyl
function on the side chain of glutamic acid or aspartic acid
anywhere within the peptide sequence X. In one embodiment, Z may be
attached to X within the sequence and to the N- and/or C-terminal
of X. Whether the sequence should be attached to the peptide
sequence X at its C-terminal, at its N-terminal, or both, or within
the peptide sequence X depends on the specific peptide X and can be
easily determined by the person skilled in the art. Preferably, X
is bound to Z via a peptide bond and preferably at the C-terminal
of X.
[0038] One aspect of the invention is directed to a peptide
conjugate comprising a peptide X which reduces the blood glucose
level in a mammal, wherein X is (a) an exendin having at least 90%
homology to exendin-4; (b) a variant of said exendin wherein said
variant comprises a modification selected from the group consisting
between One and five deletions at positions 34-39 and contains a
Lys at position 40 having a lipophilic substituent; or (c) GLP-1
(7-36) (SEQ ID NO: 114) or GLP-1 (7-37) (SEQ ID NO: 124) having at
least one modification selected from the group consisting of: (i)
substitution of D-alanine, glycine or alpha-amino isobutyric acid
(Aib) for alanine at position 8 and (ii) a lipophilic substituent;
and Z, a peptide sequence of 4-20 amino acid units covalently bound
to X, wherein each amino acid unit in said peptide sequence Z is
selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn,
Gln, Asp, Glu, Lys, Arg, His, Met, Orn, and amino acid units of the
general formula
--NH--C(R.sup.1)R.sup.2)--C(.dbd.O)-- (I)
wherein R.sup.1 and R.sup.2 are selected from the group consisting
of hydrogen, C.sub.1-6-alkyl, phenyl, and phenyl-methyl, wherein
C.sub.1-6-alkyl is optionally substituted with from one to three
substituents selected from halogen, hydroxy, amino, cyano, nitro,
sulfono, and carboxy, and phenyl and phenyl-methyl is optionally
substituted with from one to three substituents selected from
C.sub.1-6-alkyl, C.sub.2-6-alkenyl, halogen, hydroxy, amino, cyano,
nitro, sulfono, and carboxy, or R.sup.1 and R.sup.2 together with
the carbon atom to which they are bound form a cyclopentyl,
cyclohexyl, or cycloheptyl ring, e.g. 2,4-diaminobutanoic acid and
2,3-diaminopropanoic acid. Preferably, X binds to a GLP-1 receptor
and does not include exendin-4 or exendin-3.
[0039] Z is typically a peptide sequence of 4-20 amino acid
residues, e.g., in the range of 4-15, more preferably in the range
of 4-10 in particular in the range of 4-7 amino acid residues,
e.g., of 4, 5, 6, 7, 8 or 10 amino acid residues, where 6 amino
acid residues are preferred. Preferably, Z contains at least one
Lys residue. In a preferred embodiment of the invention each of the
amino acid residues in the peptide sequence Z are independently
selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn,
Gln, Asp, Glu, Lys, Arg, His, Met, Orn, diaminobutanoic acid and
diaminopropanoic acid. Preferably, the amino acid residues are
selected from Glu, Lys, and Met, especially Lys, or the amino acid
residues are selected from the group consisting of Asn, Glu and
Lys. The above-mentioned amino acids may have either D- or
L-configuration, but preferably the above-mentioned amino acids
have an L-configuration. In a preferred embodiment of the invention
Z contains at least 1 lysine residue or when Z is attached via a
peptide bond to the N-terminal of said peptide X then Z has an
amino acid sequence selected from the group consisting of
Asn-(Glu)n wherein n is an integer from 3 to 7.
[0040] Thus, illustrative examples of the peptide sequence Z
are:
Lys-Lys-Lys-Lys (SEQ ID NO:1), Xaa-Lys-Lys-Lys, Lys-Xaa-Lys-Lys,
Lys-Lys-Xaa-Lys, Lys-Lys-Lys-Xaa, Xaa-Xaa-Lys-Lys, Xaa-Lys-Xaa-Lys,
Xaa-Lys-Lys-Xaa, Lys-Xaa-Xaa-Lys, Lys-Xaa-Lys-Xaa, Lys-Lys-Xaa-Xaa,
Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Xaa, Xaa-Lys-Xaa-Xaa, Lys-Xaa-Xaa-Xaa,
Xaa-Xaa-Xaa-Xaa (SEQ ID NO:2), Lys-Lys-Lys-Lys-Lys (SEQ ID NO:3),
Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:4), Lys-Xaa-Lys-Lys-Lys (SEQ ID
NO:5), Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:6), Lys-Lys-Lys-Xaa-Lys (SEQ
ID NO:7), Lys-Lys-Lys-Lys-Xaa, Xaa-Xaa-Lys-Lys-Lys,
Xaa-Lys-Xaa-Lys-Lys, Xaa-Lys-Lys-Xaa-Lys, Xaa-Lys-Lys-Lys-Xaa,
Lys-Xaa-Xaa-Lys-Lys, Lys-Xaa-Lys-Xaa-Lys, Lys-Xaa-Lys-Lys-Xaa,
Lys-Lys-Xaa-Xaa-Lys, Lys-Lys-Xaa-Lys-Xaa, Lys-Lys-Lys-Xaa-Xaa,
Lys-Lys-Xaa-Xaa-Xaa, Lys-Xaa-Lys-Xaa-Xaa, Lys-Xaa-Xaa-Lys-Xaa,
Lys-Xaa-Xaa-Xaa-Lys, Xaa-Lys-Lys-Xaa-Xaa, Xaa-Lys-Xaa-Xaa-Lys,
Xaa-Xaa-Lys-Lys-Xaa, Xaa-Xaa-Lys-Xaa-Lys, Xaa-Xaa-Xaa-Lys-Lys,
Lys-Xaa-Xaa-Xaa-Xaa, Xaa-Lys-Xaa-Xaa-Xaa, Xaa-Xaa-Lys-Xaa-Xaa,
Xaa-Xaa-Xaa-Lys-Xaa, Xaa-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Xaa-Xaa-Xaa (SEQ
ID NO:8), Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO:9),
Xaa-Lys-Lys-Lys-Lys-Lys (SEQ ID NO:10), Lys-Xaa-Lys-Lys-Lys-Lys
(SEQ ID NO:11), Lys-Lys-Xaa-Lys-Lys-Lys (SEQ ID NO:12),
Lys-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:13), Lys-Lys-Lys-Lys-Xaa-Lys
(SEQ ID NO:14), Lys-Lys-Lys-Lys-Lys-Xaa (SEQ ID NO:15),
Xaa-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:16), Xaa-Lys-Xaa-Lys-Lys-Lys
(SEQ ID NO:17), Xaa-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:18),
Xaa-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:19), Xaa-Lys-Lys-Lys-Lys-Xaa
(SEQ ID NO:20), Lys-Xaa-Xaa-Lys-Lys-Lys (SEQ ID NO:21),
Lys-Xaa-Lys-Xaa-Lys-Lys (SEQ ID NO:22), Lys-Xaa-Lys-Lys-Xaa-Lys
(SEQ ID NO:23), Lys-Xaa-Lys-Lys-Lys-Xaa (SEQ ID NO:24),
Lys-Lys-Xaa-Xaa-Lys-Lys (SEQ ID NO:25), Lys-Lys-Xaa-Lys-Xaa-Lys
(SEQ ID NO:26), Lys-Lys-Xaa-Lys-Lys-Xaa (SEQ ID NO:27),
Lys-Lys-Lys-Xaa-Xaa-Lys (SEQ ID NO:28), Lys-Lys-Lys-Xaa-Lys-Xaa
(SEQ ID NO:29), Lys-Lys-Lys-Lys-Xaa-Xaa, Xaa-Xaa-Xaa-Lys-Lys-Lys,
Xaa-Xaa-Lys-Xaa-Lys-Lys, Xaa-Xaa-Lys-Lys-Xaa-Lys,
Xaa-Xaa-Lys-Lys-Lys-Xaa, Xaa-Lys-Xaa-Xaa-Lys-Lys,
Xaa-Lys-Xaa-Lys-Xaa-Lys, Xaa-Lys-Xaa-Lys-Lys-Xaa,
Xaa-Lys-Lys-Xaa-Xaa-Lys, Xaa-Lys-Lys-Xaa-Lys-Xaa,
Xaa-Lys-Lys-Lys-Xaa-Xaa, Lys-Lys-Lys-Xaa-Xaa-Xaa,
Lys-Lys-Xaa-Lys-Xaa-Xaa, Lys-Lys-Xaa-Xaa-Lys-Xaa,
Lys-Lys-Xaa-Xaa-Xaa-Lys, Lys-Xaa-Lys-Lys-Xaa-Xaa,
Lys-Xaa-Lys-Xaa-Lys-Xaa, Lys-Xaa-Lys-Xaa-Xaa-Lys,
Lys-Xaa-Xaa-Lys-Lys-Xaa, Lys-Xaa-Xaa-Lys-Xaa-Lys,
Lys-Xaa-Xaa-Xaa-Lys-Lys, Lys-Lys-Xaa-Xaa-Xaa-Xaa,
Lys-Xaa-Lys-Xaa-Xaa-Xaa, Lys-Xaa-Xaa-Lys-Xaa-Xaa-Lys,
Lys-Xaa-Xaa-Xaa-Lys-Xaa-Lys, Lys-Xaa-Xaa-Xaa-Xaa-Lys-Lys,
Xaa-Lys-Lys-Xaa-Xaa-Xaa, Xaa-Lys-Xaa-Lys-Xaa-Xaa,
Xaa-Lys-Xaa-Xaa-Lys-Xaa, Xaa-Lys-Xaa-Xaa-Xaa-Lys,
Xaa-Xaa-Lys-Lys-Xaa-Xaa, Xaa-Xaa-Lys-Xaa-Lys-Xaa,
Xaa-Xaa-Lys-Xaa-Xaa-Lys, Xaa-Xaa-Xaa-Lys-Lys-Xaa,
Xaa-Xaa-Xaa-Lys-Xaa-Lys, Xaa-Xaa-Xaa-Xaa-Lys-Lys,
Lys-Xaa-Xaa-Xaa-Xaa-Xaa, Xaa-Lys-Xaa-Xaa-Xaa-Xaa,
Xaa-Xaa-Lys-Xaa-Xaa-Xaa, Xaa-Xaa-Xaa-Lys-Xaa-Xaa,
Xaa-Xaa-Xaa-Xaa-Lys-Xaa, Xaa-Xaa-Xaa-Xaa-Xaa-Lys,
Xaa-Xaa-Xaa-Xaa-Xaa-Xaa, Lys-Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID
NO:30), Xaa-Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO:31),
Lys-Xaa-Lys-Lys-Lys-Lys-Lys (SEQ ID NO:32),
Lys-Lys-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:33),
Lys-Lys-Lys-Xaa-Lys-Lys-Lys (SEQ ID NO:34),
Lys-Lys-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:35),
Lys-Lys-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:36),
Lys-Lys-Lys-Lys-Lys-Lys-Xaa (SEQ ID NO:37),
Xaa-Xaa-Lys-Lys-Lys-Lys-Lys (SEQ ID NO:38),
Xaa-Lys-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:39),
Xaa-Lys-Lys-Xaa-Lys-Lys-Lys (SEQ ID NO:40),
Xaa-Lys-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:41),
Xaa-Lys-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:42),
Lys-Xaa-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:43),
Lys-Xaa-Lys-Xaa-Lys-Lys-Lys (SEQ ID NO:44),
Lys-Xaa-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:45),
Lys-Xaa-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:46),
Lys-Lys-Xaa-Xaa-Lys-Lys-Lys (SEQ ID NO:47),
Lys-Lys-Xaa-Lys-Xaa-Lys-Lys (SEQ ID NO:48),
Lys-Lys-Xaa-Lys-Lys-Xaa-Lys (SEQ ID NO:49),
Lys-Lys-Lys-Xaa-Xaa-Lys-Lys (SEQ ID NO:50),
Lys-Lys-Lys-Xaa-Lys-Xaa-Lys (SEQ ID NO:51),
Lys-Lys-Lys-Lys-Xaa-Xaa-Lys (SEQ ID NO:52),
Xaa-Xaa-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:53),
Xaa-Xaa-Lys-Xaa-Lys-Lys-Lys (SEQ ID NO:54),
Xaa-Xaa-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:55),
Xaa-Xaa-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:56),
Xaa-Lys-Xaa-Xaa-Lys-Lys-Lys (SEQ ID NO:57),
Xaa-Lys-Xaa-Lys-Xaa-Lys-Lys (SEQ ID NO:58),
Xaa-Lys-Xaa-Lys-Lys-Xaa-Lys (SEQ ID NO:59),
Xaa-Lys-Lys-Xaa-Xaa-Lys-Lys (SEQ ID NO:60),
Xaa-Lys-Lys-Xaa-Lys-Xaa-Lys (SEQ ID NO:61),
Xaa-Lys-Lys-Lys-Xaa-Lys-Xaa (SEQ ID NO:62),
Xaa-Lys-Lys-Xaa-Lys-Lys-Xaa (SEQ ID NO:63),
Xaa-Lys-Xaa-Lys-Lys-Lys-Xaa (SEQ ID NO:64),
Xaa-Lys-Lys-Lys-Xaa-Xaa-Lys (SEQ ID NO:65),
Lys-Xaa-Lys-Lys-Lys-Xaa-Xaa (SEQ ID NO:66),
Xaa-Lys-Lys-Lys-Lys-Xaa-Xaa (SEQ ID NO:67),
Xaa-Lys-Lys-Lys-Xaa-Lys-Xaa (SEQ ID NO:68),
Xaa-Lys-Lys-Lys-Xaa-Xaa-Lys (SEQ ID NO:69),
Lys-Lys-Lys-Lys-Xaa-Xaa-Xaa (SEQ ID NO:70),
Lys-Lys-Lys-Xaa-Xaa-Xaa-Lys (SEQ ID NO:71),
Lys-Lys-Lys-Xaa-Lys-Xaa-Xaa (SEQ ID NO:72),
Lys-Lys-Xaa-Lys-Lys-Xaa-Xaa (SEQ ID NO:73),
Lys-Lys-Xaa-Xaa-Lys-Xaa-Lys (SEQ ID NO:74),
Lys-Lys-Xaa-Xaa-Xaa-Lys-Lys (SEQ ID NO:75),
Lys-Lys-Xaa-Lys-Lys-Xaa-Xaa (SEQ ID NO:76),
Lys-Xaa-Lys-Lys-Xaa-Xaa-Lys (SEQ ID NO:77),
Lys-Xaa-Lys-Xaa-Lys-Xaa-Lys (SEQ ID NO:78),
Lys-Xaa-Lys-Xaa-Xaa-Lys-Lys (SEQ ID NO:79),
Lys-Xaa-Xaa-Lys-Lys-Xaa-Lys (SEQ ID NO:80),
Lys-Xaa-Xaa-Lys-Xaa-Lys-Lys (SEQ ID NO:81),
Lys-Xaa-Xaa-Xaa-Lys-Lys-Lys (SEQ ID NO:82),
Lys-Lys-Xaa-Xaa-Xaa-Xaa-Lys, Lys-Xaa-Lys-Xaa-Xaa-Xaa-Lys,
Lys-Xaa-Xaa-Lys-Xaa-Xaa-Lys, Lys-Xaa-Xaa-Xaa-Lys-Xaa-Lys,
Lys-Xaa-Xaa-Xaa-Xaa-Lys-Lys, Xaa-Lys-Lys-Xaa-Xaa-Xaa-Lys,
Xaa-Lys-Xaa-Lys-Xaa-Xaa-Lys, Xaa-Lys-Xaa-Xaa-Lys-Xaa-Lys,
Xaa-Lys-Xaa-Xaa-Xaa-Lys-Lys, Xaa-Xaa-Lys-Lys-Xaa-Xaa-Lys,
Xaa-Xaa-Lys-Xaa-Lys-Xaa-Lys, Xaa-Xaa-Lys-Xaa-Xaa-Lys-Lys,
Xaa-Xaa-Xaa-Lys-Lys-Xaa-Lys, Xaa-Xaa-Xaa-Lys-Xaa-Lys-Lys,
Xaa-Xaa-Xaa-Xaa-Lys-Lys-Lys, Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Lys,
Xaa-Lys-Xaa-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Xaa-Xaa-Xaa-Lys,
Xaa-Xaa-Xaa-Lys-Xaa-Xaa-Lys, Xaa-Xaa-Xaa-Xaa-Lys-Xaa-Lys,
Xaa-Xaa-Xaa-Xaa-Xaa-Lys-Lys, Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Lys,
Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa, Xaa-Xaa-Xaa-Xaa-Xaa-Lys-Xaa,
Xaa-Lys-Xaa-Xaa-Xaa-Xaa-Xaa, Xaa-Xaa-Lys-Xaa-Xaa-Xaa,
Xaa-Xaa-Xaa-Xaa-Lys-Xaa, Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa, wherein each
Xaa is independently selected from the group consisting of Ala,
Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Arg, His, Met, Orn, and
amino acids of the formula I as defined herein, e.g., Dbu or
Dpr.
[0041] As indicated above, the amino acid residues of Z may of
course all be different or all be identical. However, in
interesting embodiments of the present invention, the amino acid
residues in Z are selected from two or three different amino acids,
or are identical amino acids. Examples of suitable peptide
sequences, wherein the amino acid residues in Z are identical are
e.g., (Lys).sub.n, wherein n is an integer in the range from 4 to
15, preferably in the range from 4 to 10, such as in the range from
4 to 8, e.g., in the range from about 4 to 7, e.g., Lys.sub.4 (SEQ
ID NO:1), Lys.sub.5 (SEQ ID NO:2); Lys.sub.6 (SEQ ID NO:8),
Lys.sub.7 (SEQ ID NO:30). Preferred is (Lys).sub.6 bound via a
peptide bond to the C-terminal of X.
[0042] Examples of suitable peptide sequences, wherein the amino
acid residues in Z are selected from about two different amino
acids are e.g., (Lys-Xaa).sub.m or (Xaa-Lys).sub.m, wherein m is an
integer in the range from about 2 to 7, preferably in the range
from 2 to 5, such as in the range from 2 to 4, e.g., 3, and Xaa is
independently selected from the group consisting of Ser, Thr, Tyr,
Asn, Gln, Asp, Glu, Arg, His, Orn, 2,4-diaminobutanoic acid,
2,3-diaminopropanoic acid and Met. More preferably such peptide
sequences are e.g., (Lys-Xaa).sub.3 or (Xaa-Lys).sub.3, wherein Xaa
is as defined above, such as (Lys-Glu).sub.3 (SEQ ID NO:83) or
(Glu-Lys).sub.3 (SEQ ID NO:84). Other examples of suitable peptide
sequences, wherein the amino acid residues in Z are selected from
about two amino acid residues are e.g., Lys.sub.p-Xaa.sub.q or
Xaa.sub.p-Lys.sub.q, wherein p and q are integers in the range from
1 to 14, with the proviso that p+q is in the range from 4 to 15,
preferably in the range from 4 to 10, such as in the range from 4
to 8, e.g., in the range from 4 to 6, e.g., 4, 5 or 6, and Xaa is
independently selected from the group consisting of Ser, Thr, Tyr,
Asn, Gln, Asp, Glu, Arg, His and Met. More preferably such peptide
sequences are e.g., Lys.sub.3-Xaa.sub.3 or Xaa.sub.3-Lys.sub.3,
wherein Xaa is as defined above, such as Lys.sub.3-Glu.sub.3 (SEQ
ID NO:85) or Glu.sub.3-Lys.sub.3 (SEQ ID NO:86). More preferred Z
sequences consists of a sequence of amino acid residues selected
from Asn and Gln together with 4-7 amino acid residues selected
from Glu and Asp, such as Asn-(Glu).sub.5(SEQ ID NO: 141),
Asn-(Glu).sub.6(SEQ ID NO: 142), Gln-(Glu).sub.5(SEQ ID NO: 143),
Asn-(Asp).sub.5(SEQ ID NO: 144), and Gln-(Asp).sub.5(SEQ ID NO:
145), which is the N-terminal part of the peptide conjugate of the
invention.
[0043] Examples of suitable peptide sequences, wherein the amino
acid residues in Z are selected from three different amino acids
are e.g., Xaa.sup.1-(Lys).sub.x-(Xaa.sup.2).sub.y,
Xaa.sup.1-(Xaa.sup.1).sub.x-(Lys).sub.y,
(Xaa.sup.2).sub.y-Xaa.sup.1,
(Xaa.sup.1).sub.x(Lys).sub.y-Xaa.sup.2,
(Lys).sub.x-Xaa.sup.1-(Xaa.sup.2).sub.y,
(Xaa.sup.1).sub.x-Xaa.sup.2-(Lys).sub.y,
Xaa.sup.1-Lys-Xaa.sup.2-Lys, Xaa.sup.1-Lys-Xaa.sup.2-Lys-Xaa.sup.2,
Xaa.sup.1Lys-Xaa.sup.2-Lys-Xaa.sup.2-Lys,
Xaa.sup.1-Xaa.sup.2-Lys-Xaa.sup.2,
Xaa.sup.1-Xaa.sup.2-Lys-Xaa.sup.2-Lys,
Xaa.sup.1-Xaa.sup.1-Lys-Xaa.sup.2-Lys-Xaa.sup.2,
Lys-Xaa.sup.2-Lys-Xaa.sup.1, Lys-Xaa.sup.2-Lys-Xaa.sup.2-Xaa.sup.1,
Lys-Xaa.sup.2-Lys-Xaa.sup.2-Lys-Xaa.sup.1,
Xaa.sup.2-Lys-Xaa.sup.2-Xaa.sup.1,
Xaa.sup.2-Lys-Xaa.sup.2-Lys-Xaa.sup.1,
Xaa.sup.2-Lys-Xaa.sup.1-Lys-Xaa.sup.2-Xaa.sup.1, etc., wherein x
and y are integers in the range from about 1 to 5 with the proviso
that x+y is at the most 6, and Xaa.sup.1 and Xaa.sup.2 is
independently selected from about the group consisting of Ala, Leu,
Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Arg, His, Met, Orn,
2,3-diaminopropanoic acid, 2,4-diaminobutanoic acid and amino acids
of the formula I as defined herein.
[0044] In preferred embodiments of the invention the ratio between
the minimum effective oral dose of said peptide conjugate and the
minimum effective dose of the peptide, X is at least 1:5.
[0045] A most preferred embodiment of the invention is directed to
a novel peptide conjugate comprising a peptide X being an agonist
of GLP-1 and/or exendin-4 activity selected from the group
consisting of
des Pro.sup.36-exendin-4(1-39)-NH.sub.2 (SEQ ID NO:101), des
Pro.sup.36-exendin-4(1-40)-NH.sub.2(SEQ ID NO: 139), des
Pro.sup.36-des Pro.sup.37-exendin-4(1-39)-N1-1(SEQ ID NO: 130), des
Pro.sup.36-des Pro.sup.37-des
Pro.sup.38-exendin-4(1-39)-NH.sub.2(SEQ ID NO: 132), des
Pro.sup.36-des Pro.sup.37-des
Pro.sup.33-exendin-4(1-40)-NH.sub.2(SEQ ID NO: 140), des
Ala.sup.35-exendin-4(1-39)-NH.sub.2 (SEQ ID NO:105), des
Gly.sup.34-exendin-4(1-39)-NH.sub.2 (SEQ ID NO:106), des
Gly.sup.34-(Lys.sup.40 (palmitoyl))exendin-4(1-39)-NH.sub.2 (SEQ ID
NO:108), des Ala.sup.35-(Lys (palmitoyl))exendin-4(1-39)-NH.sub.2
(SEQ ID NO:109), des Pro.sup.36-(Lys.sup.40
(palmitoyl))exendin-4(1-39)-NH.sub.2 (SEQ ID NO:110),
[0046] Compound (iii) Gly.sup.8-GLP-1 (7-36)-NH.sub.2(SEQ ID NO:
87), Gly.sup.8-GLP-1(7-37) (SEQ ID NO: 123), and
Gly.sup.8-GLP-1(7-36)-Lys.sup.37 (palmitoyl)-NH.sub.2(SEQ ID NO:
147), and being C-terminally bound via a peptide bond to a peptide
sequence Z selected from the group consisting of (Lys)n where n is
an integer from 4 to 8, preferably n is 6.
[0047] It should be understood that the peptide conjugates of the
invention might also be in the preferred amide (NH.sub.2) or in the
free acid (OH) form or in the form of a salt thereof. Exemplary
peptide conjugates of the invention are
Gly.sup.8-GLP-1 (7-36)-Lys.sub.6-NH.sub.2 (SEQ ID NO:88),
[0048] (Gly.sup.8,Lys.sup.37
(palmitoyl)-GLP-1(7-36)(Human)-Lys.sub.7-NH.sub.2 (SEQ ID NO:89),
des Ser.sup.39-exendin-4(1-39)-(Lys).sub.6-NH.sub.2 (SEQ ID NO:91),
exendin-4(1-39)-Lys.sub.6-NH.sub.2 (SEQ ID NO:92), des
Pro.sup.36-exendin-4(1-39)-Lys.sub.6-NH.sub.2 (SEQ ID NO:93), des
Ala.sup.35-exendin-4(1-39)-Lys.sub.6-NH.sub.2 (SEQ ID NO:94), des
Gly.sup.34-exendin-4(1-39)-Lys.sub.6-NH.sub.2 (SEQ ID NO:95), des
Ser.sup.39-(Lys.sup.40
(palmitoyl))exendin-4(1-39)-Lys.sub.7-NH.sub.2 (SEQ ID NO:96), des
Gly.sup.34-(Lys.sup.40
(palmitoyl))exendin-4(1-39)-Lys.sub.7-NH.sub.2 (SEQ ID NO:97), des
Ala.sup.35-(Lys.sup.40
(palmitoyl))exendin-4(1-39)-Lys.sub.7-NH.sub.2 (SEQ ID NO:98), des
Pro.sup.36-(Lys.sup.40
(palmitoyl))exendin-4(1-39)-Lys.sub.7-NH.sub.2 (SEQ ID NO:99),
Lys.sup.40 (palmitoyl)exendin-4(1-39)-Lys.sub.7-NH.sub.2 (SEQ ID
NO:100), des Pro.sup.36,
Pro.sup.37-exendin-4(1-39)-Lys.sub.6-NH.sub.2(SEQ ID NO: 131),
Lys.sub.6-des Pro.sup.36, Pro.sup.37,
Pro.sup.3.sup.8-exendin-4(1-39)-NH.sub.2(SEQ ID NO: 134)
Asn(Glu).sub.5-des Pro.sup.36, Pro.sup.37,
Pro.sup.3.sup.8-exendin-4(1-39)-NH.sub.2(SEQ ID NO: 137),
Lys.sub.6-des Pro.sup.36, Pro.sup.37,
Pro.sup.3.sup.8-exendin-4(1-39)-Lys.sub.6-NH.sub.2(SEQ ID NO: 135),
Asn(Glu).sub.5-des Pro.sup.36, Pro.sup.37,
Pro.sup.3.sup.8-exendin-4(1-39)-Lys.sub.6-NH.sub.2(SEQ ID NO: 136),
des Pro.sup.36, Pro.sup.37,
Pro.sup.3.sup.8-exendin-4(1-39)-Lys.sub.6-NH.sub.2(SEQ ID NO:
133),
Ser.sup.8-GLP-1 (7-36)-Lys.sub.6-NH.sub.2 (SEQ ID NO: 115),
Aib.sup.8-GLP-1 (7-36)-Lys.sub.6-NH.sub.2 (SEQ ID NO: 116),
Lys.sub.6-Gly.sup.8-GLP-1 (7-36)-Lys.sub.6-NH.sub.2 (SEQ ID NO:
118),
Lys.sub.6-Gly.sup.8-GLP-1 (7-36)-NH.sub.2 (SEQ ID NO: 119),
[0049]
(Gly.sup.8,Lys.sup.2.sup.6(palmitoyl)-GLP-1(7-36)(Human)-Lys.sub.6--
NH.sub.2 (SEQ ID NO: 103), (Gly.sup.8,Lys.sup.34
(palmitoyl)-GLP-1(7-36)(Human)-Lys.sub.6-NH.sub.2 (SEQ ID NO:
90),
Gly.sup.8-GLP-1 (7-36)-Lys.sub.8-NH.sub.2(SEQ ID NO: 120),
Gly.sup.8-GLP-1 (7-36)-Lys.sub.10-NH.sub.2(SEQ ID NO: 121),
Gly.sup.8-GLP-1 (7-37)-Lys.sub.6-NH.sub.2(SEQ ID NO: 122),
[0050] and the free acid thereof and a pharmaceutically acceptable
salt thereof.
[0051] Among the Preferred conjugates are
des Pro.sup.36-exendin-4(1-39)-Lys.sub.6-NH.sub.2 (SEQ ID
NO:93),
Gly.sup.8-GLP-1 (7-36)-Lys.sub.6-NH.sub.2 (SEQ ID NO:88),
[0052] des
Pro.sup.36,Pro.sup.37,Pro.sup.38-exendin-4(1-39)-Lys.sub.6-NH.s-
ub.2(SEQ ID NO: 133), and their salts as defined herein.
[0053] In a most specific embodiment, the conjugates are selected
from the group consisting of
Gly.sup.8-GLP-1-(7-36)(Human)-NH.sub.2(SEQ ID NO: 87),
Gly.sup.8-GLP-1-(7-36)(Human)-Lys.sub.6-NH.sub.2(SEQ ID NO: 88),
Gly.sup.8Lys.sup.37(palmitoyl)-GLP-1-(7-36)(Human)-Lys.sub.7-NH.sub.2(SEQ
ID NO: 89),
Gly.sup.8Lys.sup.34(palmitoyl)-GLP-1-(7-36)(Human)-Lys.sub.6-NH.sub.2(SEQ
ID NO: 90), des Ser.sup.39-exendin-4(1-39)-Lys.sub.6-NH.sub.2(SEQ
ID NO: 91), exendin-4(1-39)-Lys.sub.6-NH.sub.2(SEQ ID NO: 92), des
Pro.sup.36-exendin-4(1-39)-Lys.sub.6-NH.sub.2(SEQ ID NO: 93), des
Ala.sup.35-exendin-4(1-39)-Lys.sub.6-NH.sub.2(SEQ ID NO: 94), des
Gly.sup.34-exendin-4(1-39)-Lys.sub.6-NH.sub.2(SEQ ID NO: 95), des
Ser.sup.39-(Lys.sup.40
(palmitoyl))exendin-4(1-39)-Lys.sub.7-NH.sub.2(SEQ ID NO: 96), des
Gly.sup.34-(Lys.sup.40(palmitoyl))exendin-4(1-39)-Lys.sub.7-NH.sub.2(SEQ
ID NO: 97), des
Ala.sup.35-(Lys.sup.40(palmitoyl))exendin-4(1-39)-Lys.sub.7-NH.sub.2(SEQ
ID NO: 98), des
Pro.sup.36-(Lys.sup.40(palmitoyl))exendin-4(1-39)-Lys.sub.7-NH.sub.2
(SEQ ID NO: 99) and Lys.sup.40
(palmitoyl))exendin-4(1-39)-Lys.sub.7-NH.sub.2(SEQ ID NO: 100).
[0054] The provision of the peptide conjugates of the present
invention enables blood glucose lowering peptides, such as GLP-1
and exendins and their active analogues to be administered orally.
The herein preferred terminal peptide fragments Z are chosen so as
to induce an alpha-helical structure to the peptide X without
significantly affecting the desired activity of X. Said helical
structure stabilises the peptide chain, e.g. against degradation,
as evidenced by the increased half life of from 2 to 3 times of the
conjugated peptide compared to the unconjugated peptide, cf. table
5 below. The peptide sequence Z is the part of the peptide
conjugate responsible for introducing of a certain structure into
the molecule so that the minimum effective dose is lowered at least
five fold. Preferably the minimum effective dose is lowered at
least ten fold, more preferably 25 fold, even more preferably 40
fold, and most preferably 50 fold. Therefore, the present invention
also relates to the use of a peptide sequence (Z) as defined above
for the preparation of a said peptide conjugate as defined
above.
[0055] Thus, the invention also relates to a novel peptide
conjugate comprising a peptide X as defined herein and wherein X
reduces the blood glucose level in a mammal where the ratio between
the minimum effective oral dose of said peptide conjugate and the
minimum effective oral dose of the peptide X is at least 1:5.
[0056] Specifically, the invention is directed to a method for
stimulating insulin release in a mammal comprising administering an
effective insulinotropic amount of the peptide conjugate of the
present invention, a method of lowering blood glucose level in a
mammal comprising administering an amount of the peptide conjugate
of the present invention effective to lower blood glucose level in
said mammal, a method of reducing gastric motility in a mammal in
an amount of the peptide conjugate of the present invention
effective to reduce gastric motility, a method of delaying gastric
emptying in a mammal in an amount of the peptide conjugate of the
present invention effective to delay gastric emptying, a method of
inhibiting food uptake in a mammal in an amount of the peptide
conjugate of the present invention effective to inhibit food uptake
and a method of lowering plasma lipid level in a mammal comprising
administering an amount of peptide conjugate of the present
invention effective to lower plasma lipid level in said mammal.
Specifically, the peptide conjugate of the present invention may be
used in treatment of diabetes type 1 or type 2, obesity, eating
disorders, hyperglycemia, metabolic disorders, gastric disease and
insulin resistance syndrome.
[0057] The present invention also relates to methods for the
preparation of said peptide conjugate, by means of recombinant DNA
technology comprising the steps of (a) introducing a nucleic acid
sequence encoding said conjugate into a host cell and (b) culturing
said host cell and (c) isolating said conjugate from the culture or
(a) culturing a recombinant host cell comprising a nucleic acid
sequence encoding said conjugate under conditions permitting the
production of said conjugate and (b) isolating said conjugate from
the culture.
[0058] The method also relates to methods for the preparation of
said peptide conjugate in which peptide X is obtained via
recombinant DNA methods by isolating said peptide. X is then
conjugated to Z which is attached to a solid support or has been
prepared by solid phase synthetic methods. Furthermore, the
invention relates to the preparation of the peptide conjugate of
the present invention by peptide synthetic methods. Furthermore,
the invention relates to the preparation of the peptide conjugate
of the present invention by peptide synthetic methods.
[0059] The conjugates of the invention comprising an N-terminal
sequence of from 33 to 39, preferably from 36 to 38, amino acid
residues having a substantial homology to the native exendin-4
N-terminal sequence thought to be essential for receptor binding
(insulinotropic activity) and a C-terminal sequence Z possess as a
further advantage improved stability compared to native exendins
and C-terminally truncated forms of exendin. Likewise, the GLP-1
peptide conjugate Compound 4 shows improved stability compared to
the unconjugated Compound (iii).
Compositions
[0060] The invention also concerns a composition comprising the
exendin variant or the peptide conjugate of the present invention
in combination with a physiologically acceptable carrier. Such
compositions may be in a form adapted to oral, parenteral
(including subcutaneous (s.c.), intravenous (i.v.), intramuscular
(i.m.), epidural, direct brain and intraperitoneal (i.p.)), rectal,
intratracheal, intranasal, dermal, vaginal, buccal, ocularly, or
pulmonary administration, preferably in a form adapted to
subcutaneous or oral administration, and such compositions may be
prepared in a manner well-known to the person skilled in the art,
e.g., as generally described in "Remington's Pharmaceutical
Sciences", 17. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing
Company, Easton, Pa., U.S.A., 1985 and more recent editions and in
the monographs in the "Drugs and the Pharmaceutical Sciences"
series, Marcel Dekker. The compositions may appear in conventional
forms, for example, capsules, tablets, aerosols, topical
application forms, liquid or semiliquid forms, such as solutions,
suspensions, dispersions, emulsions, micelles or liposomes.
Preferred are liquid compositions suitable for s.c. administration.
In a preferred embodiment, the compositions of the present
invention are administered subcutaneously. In an alternative
preferred embodiment, the compositions of the present invention are
administered orally, and in such cases one preferred administration
form is a tablet or capsule.
[0061] The pharmaceutical carrier or diluent employed may be a
conventional solid or liquid carrier. Examples of solid carriers
are lactose, terra alba, sucrose, cyclodextrin, talc, gelatin,
agar, pectin, acacia, magnesium stearate, stearic acid to lower
alkyl ethers of cellulose. Examples of liquid carriers are syrup,
peanut oil, olive oil, phospholipids, sterols, fatty acids, fatty
acid amines, polyoxyethylene, isotonic buffer solutions and water.
Similarly, the carrier or diluent may include any sustained release
material known in the art, such as glyceryl monostearate or
glyceryl distearate, alone or mixed with a wax. If a solid carrier
is used for oral administration, the preparation may be tabletted,
placed in a hard gelatin capsule in powder or pellet form or it can
be in the form of a troche or lozenge. The amount of solid carrier
will vary widely but will usually be from about about 25 mg to
about 1 g.
[0062] A typical tablet which may be prepared by conventional
tabletting techniques may contain: [0063] Core: active compound (as
free compound of the invention or salt thereof) 100 mg; colloidal
silicon dioxide (Aerosil) 1.5 mg; cellulose, microcryst. (Avicel)
70 mg; modified cellulose gum (Ac-Di-Sol) 7.5 mg; magnesium
stearate. [0064] Coating: HPMC approx. 9 mg; *Mywacett 9-40T
approx. 0.9 mg; *acylated monoglyceride used as plasticizer for
film coating.
[0065] If a liquid carrier is used, the preparation may be in the
form of a syrup, emulsion, soft gelatin capsule or sterile
injectable liquid such as an aqueous or non-aqueous liquid
suspension or solution.
[0066] For nasal administration, the preparation may contain a
compound of the present invention, preferably a conjugate,
dissolved or suspended in a liquid carrier, in particular, an
aqueous carrier, for aerosol application. The carrier may contain
additives such as solubilizing agents, e.g., propylene glycol,
surfactants such as bile acid salts or polyoxyethylene higher
alcohol ethers, absorption enhancers such as lecithin
(phosphatidylcholine) or cyclodextrin, or preservatives such as
parabines.
[0067] The composition may also be in a form suited for local or
systemic injection or infusion and may, as such, be formulated with
sterile water or an isotonic saline or glucose solution. The
compositions may be sterilized by conventional sterilization
techniques which are well known in the art. The resulting aqueous
solutions may be packaged for use or filtered under aseptic
conditions and lyophilized, the lyophilized preparation being
combined with the sterile aqueous solution prior to administration.
Preferably, the formulation to be used for intravenous,
subcutaneous and oral dosing will be a solution of the active
compound in buffer. The preparation may be produced immediately
before use from active drug substance and sterile buffer solution.
One preferred method of sterilization may be by sterile filtration
of a solution made immediately prior to use. The composition may
contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions, such as buffering
agents, tonicity adjusting agents and the like, for instance sodium
acetate, sodium lactate, sodium chloride, potassium chloride,
calcium chloride, etc.
[0068] The compounds of the invention possess valuable
pharmacological properties, e.g. stability towards proteolytic
enzymes. In vitro stability studies with the present peptides and
peptide conjugates in the presence of selected proteolytic enzymes
show increased half lives of the novel peptides compared to prior
art peptides. Thus, the compounds of the invention exhibit
considerably extended duration of action in vivo compared to GLP-1
and other GLP-1 agonists. Furthermore, the compounds of the
invention stimulate cAMP formation. This effect may be demonstrated
in a cAMP assay, e.g. as described in WO 98/08871.
[0069] The peptide compounds of the present invention are agonists
of GLP-1 activity and/or exendin-4 activity and improves blood
glucose tolerance in diabetic mammals as determined by assays known
in the art for a particular peptide. Examples of such an assay are
described herein. Thus, the invention also concerns the exendin
variants and peptide conjugates as defined above for use in
therapy, and the use of the peptide conjugates as defined above for
the manufacture of a pharmaceutical composition for use in therapy,
e.g., in the treatment of diabetes type 1 or type 2, obesity,
eating disorders and insulin resistance syndrome.
[0070] In specific embodiments, the exendin variants and peptide
conjugates of the invention may be used to stimulate insulin
release, lower blood glucose level, reduce gastric motility, delay
gastric emptying, inhibit food uptake, e.g. by suppression of
appetite, or lower the plasma lipid level in a vertebrate or a
mammal. The novel compounds of the invention may also be used
generally in the treatment of diabetes mellitus associated with a
risk for hyperglycemia, i.e. where insulin sensitivity is decreased
with stress, myocardia infection, stroke and infections, or in
cases of insulin resistance during pregnancy. The novel compounds
may also be used in the treatment of other types of diabetes, such
as cases where diabetes may be secondary to other endocrine
diseases such as acromegaly, Cushing's syndrome, pheochromocytoma,
glucagonoma, somatostatinoma, primary aldosteronism, or secondary
to administration of certain hormones causing hyperglycemia, or
secondary to certain drugs (antihypertensive drugs, thiazide
diuretics, preparations containing estrogen, psychoactive drugs,
sympathomimetic agents. Furthermore, the novel compounds of the
invention may be used generally in the treatment of diseases and
conditions associated with a risk for hypoglycemia, i.e. where
endogenous glucose production is decreased, as following alcohol
ingestion, or in cases where the sensitivity to insulin is
increased in patients with hypopituitarism or primary
adrenocortical insufficiency, or where insulin clearance is
decreased as with progressive renal insufficiency.
[0071] Other specific therapeutic uses are described in WO 99/40788
(relating to the inotropic and diuretic effects of exendin and
GLP-1) WO 98/39022 (relating to a method of sedating a mammalian
subject having increased activation of the central or peripheral
nervous system comprising administering exendin or GLP-1 or an
agonist of exendin or GLP-1 to the subject to produce a sedative or
anxiolytic effect on the subject), WO 93/18786 (relating to the
treatment of diabetes using GLP-1(7-37) (SEQ ID NO: 124) or
GLP-1(7-36)amide (SEQ ID NO: 114) in a regimen which additionally
comprises treatment with an oral hypoglycemic agent, such as
sulfonylurea, producing a strong synergistic effect), WO 98/19698
(relating to the use of GLP-1 analogs for the regulation of
obesity), WO 98/08531 (relating to the use of GLP-1 or analogs in a
method of reducing mortality and morbidity after myocardial
infarction), WO 98/08873 (relating to the use of GLP-1 or analogs
in a method of attenuating post-surgical catabolic changes and
hormonal responses to stress). Besides, the compounds of the
invention are suitable in a combination therapy with other
antidiabetic agents, such as insulin, metformin, sulfonyl ureas and
thiazolidinediones, or in combination therapy with other
antiobesity agents, such as leptin, dexphenfluramine, amphetamin
etc.
DEFINITIONS
[0072] A "peptide" as used herein is any compound produced by amide
formation between a carboxyl group of one amino acid and an amino
group of another. The amide bonds in peptides may be called peptide
bonds. The word peptide usually applies to compounds whose amide
bonds are formed between C-1 of one amino acid and N-2 of another
(sometimes called eupeptide bonds), but it includes compounds with
residues linked by other amide bonds (sometimes called isopeptide
bonds). Peptides with fewer than about 10-20 residues may also be
called oligopeptides; those with more, polypeptides. Polypeptides
of specific sequence of more than about 50 residues are usually
known as proteins. A "natural polypeptide sequence" as used herein
refers to a polypeptide sequence consisting of natural L-amino acid
residues and which is capable of being expressed by a recombinant
host cell. The X compounds herein are all peptide sequences of 40
amino acid residues or less.
[0073] "GLP-1" as used herein includes GLP-1(7-37)-OH(SEQ ID NO:
124), GLP-1(7-37)-NH.sub.2(SEQ ID NO: 124), GLP-1(7-36)-OH(SEQ ID
NO: 114), and GLP-1(7-36)-NH.sub.2(SEQ ID NO: 114).
[0074] "Agonist" refers to an endogenous substance or a drug that
can interact with a receptor and initiate a physiological or a
pharmacological response characteristic of that receptor
(contraction, relaxation, secretion, enzyme activation, etc.).
[0075] "Antagonist" refers to a drug or a compound that opposes the
physiological effects of another. At the receptor level, it is a
chemical entity that opposes the receptor-associated responses
normally induced by another bioactive agent.
[0076] "Partial agonist" refers to an agonist which is unable to
induce maximal activation of a receptor population, regardless of
the amount of drug applied. A "partial agonist" may be termed
"agonist with intermediate intrinsic efficacy" in a given tissue.
Moreover, a partial agonist may antagonize the effect of a full
agonist that acts on the same receptor.
[0077] "Receptor" refers to a molecule or a polymeric structure in
or on a cell that specifically recognizes and binds a compound
acting as a molecular messenger (neurotransmitter, hormone,
lymphokine, lectin, drug, etc.).
[0078] By "exendin variant" of the present invention is to be
understood a variant of a parent exendin peptide having at least
about 90% homology to exendin-4 and most preferably having at least
about 95% homology to exendin-4(1-39) (SEQ ID NO: 102), which has
exendin activity, e.g., lowers the blood glucose level in a mammal
and binds to a GLP-1 receptor. "Exendin-4" as used herein refers to
exendin-4(1-39) (SEQ ID NO: 102) the amino acid sequence of which
is disclosed in U.S. Pat. No. 5,424,286, SEQ ID NO:2, and
exendin-4(1-40) (SEQ ID NO: 138) as disclosed by Chen & Drucker
in The Journal of Biological Chemistry, Vol. 272, No. 7, pp.
4108-15 which differs only in having glycine in position 40 as
C-terminal amino acid residue. The homology of the parent exendin
is determined as the degree of identity between two protein
sequences indicating a derivation of the first sequence from the
second. The homology may suitably be determined by means of
computer programs known in the art such as GAP provided in the GCG
program package (Program Manual for the Wisconsin Package, Version
8, August 1994, Genetics Computer Group, 575 Science Drive,
Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D.,
(1970), J. Mol. Biol. 48:443-453). The following settings for
polypeptide sequence comparison may be used: GAP creation penalty
of 3.0 and GAP extension penalty of 0.1.
[0079] "Salts" include pharmaceutically acceptable salts, such as
acid addition salts and basic salts. Examples of acid addition
salts are hydrochloride salts, sodium salts, hydrobromide salts,
etc. Examples of basic salts are salts where the cation is selected
from alkali metals, such as sodium and potassium, alkaline earth
metals, such as calcium, and ammonium ions
.sup.+N(R.sup.3).sub.3(R.sup.4), where R.sup.3 and R.sup.4
independently designates optionally substituted C.sub.1-6-alkyl,
optionally substituted C.sub.2-6-alkenyl, optionally substituted
aryl, or optionally substituted heteroaryl. Other examples of
pharmaceutically acceptable salts are; e.g., those described in
"Remington's Pharmaceutical Sciences" 17. Ed. Alfonso R. Gennaro
(Ed.), Mark Publishing Company, Easton, Pa., U.S.A., 1985 and more
recent editions, and in Encyclopedia of Pharmaceutical
Technology.
Preparation of Variants and Conjugates
[0080] The exendin variants and the peptide conjugates of the
invention may be prepared by methods known per se in the art. Thus,
the variants and the peptide sequences X and Z may be prepared by
standard peptide-preparation techniques such as solution synthesis
or Merrifield-type solid phase synthesis. It is believed that the
Boc (tert.butyloxycarbonyl) as well as the Fmoc
(9-fluorenylmethyloxycarbonyl) strategies are applicable.
[0081] In one possible synthesis strategy, the peptide conjugates
of the invention may be prepared by solid phase synthesis by first
constructing the peptide sequence Z using well-known standard
protection, coupling and deprotection procedures, thereafter
sequentially coupling the peptide sequence X on Z in a manner
similar to the construction of Z, and finally cleaving off the
entire peptide conjugate from the carrier. This strategy yields a
peptide conjugate, wherein the peptide sequence Z is covalently
bound to the peptide X at the C-terminal carbonyl function of X. If
the desired peptide conjugate, however, is a peptide conjugate,
wherein two stabilising sequences Z are covalently and
independently bound to both the C- and the N-terminal of the
peptide X, the above strategy is also applicable but, as will be
understood by the person skilled in the art, before cleaving the
off the C-terminal bound peptide conjugate from the solid support,
it is necessary to sequentially couple the second peptide sequence
Z to the N-terminal of X in a manner similar to the procedure
described above. This strategy may also be used to attach Z to the
carbonyl function on the side chain of Glu or Asp. A possible
strategy for the preparation of peptide conjugates, wherein the
peptide sequence Z is covalently bound to the N-terminal nitrogen
atom or covalently bound to the nitrogen atom on the side chain of
Lys, Arg or His of X is analogous with the method described above,
i.e. said peptide conjugates may be prepared by solid phase
synthesis by first constructing the peptide sequence X using
well-known standard protection, coupling and deprotection
procedures, thereafter sequentially coupling the peptide sequence Z
on X in a manner similar to the construction of X, and finally
cleaving off the entire peptide conjugate from the carrier. Another
possible strategy is to prepare one or both of the two sequences X
and Z (or parts thereof) separately by solution synthesis, solid
phase synthesis, recombinant techniques, or enzymatic synthesis,
followed by coupling of the two sequences by well-known segment
condensation procedures, either in solution or using solid phase
techniques or a combination thereof. In one embodiment, X may be
prepared by recombinant DNA methods and Z may be prepared by solid
phase synthesis. The conjugation of X and Z may be carried out by
using chemical ligation. This technique allows for the assembling
of totally unprotected peptide segments in a highly specific manner
(Liu et al., 1996, J. Am. Chem. Soc. 118:307-312 and Dawson et al.,
1996, 226:776). The conjugation can also be performed by
protease-catalysed peptide bond formation, which offers a highly
specific technique to combine totally unprotected peptide segments
via a peptide bond (W. Kullmann, 1987, Enzymatic Peptide Synthesis,
CRC Press, Boca Raton, Fla., pp. 41-59).
[0082] Side chain derivatization of Lys, Arg, His, Trp, Ser, Thr,
Cys, Tyr, Asp and Glu with the peptide sequence, Z, can be carried
out by traditional convergent peptide synthesis using suitable
orthogonal protecting schemes as known in the art, or by using the
equally well known general solid phase method with suitable
orthogonal removable chain protection.
[0083] Furthermore, it is envisaged that a combination of the
above-mentioned strategies may be especially applicable where a
modified peptide sequence, e.g., from a peptide X comprising
isosteric bonds such as reduced peptide bonds, is to be coupled to
a peptide sequence Z. In this case, it may be advantageous to
prepare the immobilised fragment of Z by successive coupling of
amino acids, and then couple a complete peptide sequence X
(prepared in solution or fully or partially using solid phase
techniques or by means of recombinant techniques) to the
fragment.
[0084] Examples of suitable solid support materials (SSM) are e.g.,
functionalised resins such as polystyrene, polyacrylamide,
polydimethylacrylamide, polyethyleneglycol, cellulose,
polyethylene, polyethyleneglycol grafted on polystyrene, latex,
dynabeads, etc. It should be understood that it may be necessary or
desirable that the C-terminal amino acid of the peptide sequence Z
or the C-terminal amino acid of the peptide X is attached to the
solid support material by means of a common linker such as
2,4-dimethoxy-4'-hydroxy-benzophenone,
4-(4-hydroxy-methyl-3-methoxyphenoxy)-butyric acid,
4-hydroxy-methyl-benzoic acid, 4-hydroxymethyl-phenoxyacetic acid,
3-(4-hydroxymethylphenoxy)propionic acid, and
p-[(R,S)-a[1-(9H-fluoren-9-yl)methoxyformamido]-2,4-dimethoxybenzyl]-phen-
oxy-acetic acid.
[0085] The variants and the peptide conjugates of the invention may
be cleaved from the solid support material by means of an acid such
as trifluoracetic acid, trifluoromethanesulfonic acid, hydrogen
bromide, hydrogen chloride, hydrogen fluoride, etc. optionally in
combination with one or more "scavengers" suitable for the purpose,
e.g., ethanedithiol, triisopropylsilane, phenol, thioanisole, etc.,
or the peptide conjugate of the invention may be cleaved from the
solid support by means of a base such as ammonia, hydrazine, an
alkoxide, such as sodium ethoxide, an hydroxide, such as sodium
hydroxide, etc.
[0086] Thus, the present invention also relates to a method for the
preparation of a pharmacologically active peptide conjugate,
wherein Z is covalently bound to X, preferably via a peptide bond.
A method for the preparation of a peptide conjugate of formula I
(X--Z), comprises the steps of:
a) coupling an amino acid or dipeptide having suitable protecting
groups, including an N-.alpha.-protecting group, in the activated
form to an immobilised peptide sequence H-Z-SSM, thereby forming an
immobilised N-.alpha.-protected peptide fragment, b) removing said
N-.alpha.-protecting group, thereby forming an immobilised
protected peptide fragment having an unprotected N-terminal, c)
coupling an additional amino acid or dipeptide having suitable
protecting groups including an N-.alpha.-protecting group in the
carboxyl activated form to the N-terminal of the immobilised
peptide fragment, and repeating the removal/coupling step procedure
in step b) and c) until the desired peptide sequence X is obtained,
and then d) cleaving off the peptide conjugate from the solid
support material.
[0087] A method for the preparation of a peptide conjugate of
formula II (Z--X), comprises the steps of:
a) coupling an amino acid or dipeptide having suitable protecting
groups, including an N-.alpha.-protecting group, in the activated
form to a solid support material (SSM), thereby forming an
immobilised protected amino acid or a protected dipeptide, b)
removing said N-.alpha.-protecting group, thereby forming an
immobilised amino acid or peptide fragment having an unprotected
N-terminal, c) coupling an additional amino acid or dipeptide
having suitable protecting groups, including an
N-.alpha.-protecting group, in the carboxyl activated form to the
N-terminal of the immobilised amino acid or peptide fragment, and
repeating the removal/coupling step procedure in step b) and c)
until the desired peptide sequence X is obtained, d) coupling an
additional amino acid or dipeptide having suitable protecting
groups, including an N-.alpha.-protecting group, in the carboxyl
activated form to the N-terminal of the immobilised peptide
fragment, and repeating the removal/coupling step procedure in step
b) and d) until the desired peptide sequence Z is obtained, and
then e) cleaving off the peptide conjugate from the solid support
material.
[0088] Furthermore, a method for the preparation of a peptide
conjugate of formula III (Z--X--Z), comprises the steps of:
a) coupling an amino acid or dipeptide having suitable protecting
groups, including an N-.alpha.-protecting group, in the carboxyl
activated form to an immobilised peptide sequence H-Z-SSM, thereby
forming an immobilised N-.alpha.-protected peptide fragment, b)
removing said N-.alpha.-protecting group, thereby forming an
immobilised peptide fragment having an unprotected N-terminal, c)
coupling an additional amino acid or dipeptide having suitable
protecting groups, including an N-.alpha.-protecting group, in the
carboxyl activated form to the N-terminal of the immobilised
peptide fragment, and repeating the removal/coupling step procedure
in step b) and c) until the desired peptide sequence X is obtained,
and then d) coupling an additional amino acid or dipeptide having
suitable protecting groups, including an N-.alpha.-protecting
group, in the carboxyl activated form to the N-terminal of the
immobilised peptide fragment, and repeating the removal/coupling
step procedure in step b) and d) until the desired peptide sequence
Z is obtained, and then e) cleaving off the peptide conjugate from
the solid support material.
[0089] The coupling, removal and cleavage steps are performed by
methods known to the person skilled in the art taking into
consideration the protection strategy and the selected solid phase
material. In general, however, it is believed that the Boc
(tert.butyloxycarbonyl) as well as the Fmoc
(9-fluorenylmethyloxycarbonyl) protection strategies are applicable
and that peptide bonds may be formed using the various activation
procedures known to the person skilled in the art, e.g., by
reacting a C-terminal activated derivative (acid halide, acid
anhydride, activated ester e.g., HObt-ester, etc.) of the
appropriate amino acid or peptide with the amino group of the
relevant amino acid or peptide as known to a person skilled in
peptide chemistry. Furthermore, it may be necessary or desirable to
include side-chain protection groups when using amino acid residues
carrying functional groups which are reactive under the prevailing
conditions. The necessary protection scheme will be known to the
person skilled in the art (cf., e.g., M. Bodanszky and A.
Bodanszky, "The Practice of Peptide Synthesis", 2. Ed,
Springer-Verlag, 1994, J. Jones, "The Chemical Synthesis of
Peptides", Clarendon Press, 1991, and Dryland et al., 1986, J.
Chem. Soc., Perkin Trans. 1:125-137).
[0090] The peptides and peptide conjugates of the invention may
also be prepared by means of recombinant DNA technology using
general methods and principles known to the person skilled in the
art. A nucleic acid sequence encoding the peptides and peptide
conjugates may be prepared synthetically by established standard
methods, e.g., the phosphoamidite method described by S. L.
Beaucage and M. H. Caruthers, Tetrahedron Letters 22, 1981, pp.
1859-1869, or the method described by Mathes et al., EMBO Journal
3, 1984, pp. 801-805. According to the phosphoamidite method,
oligonucleotides are synthesized, e.g., in an automatic DNA
synthesizer, purified, annealed, ligated and cloned in suitable
vectors. The techniques used to isolate or clone a nucleic acid
sequence encoding peptide X are known in the art and include
isolation from genomic DNA, preparation from cDNA, or a combination
thereof. The cloning of the nucleic acid sequences of the present
invention from such genomic DNA can be effected, e.g., by using the
well known polymerase chain reaction (PCR) or antibody screening of
expression libraries to detect cloned DNA fragments with shared
structural features. See, e.g., Innis et al., 1990, A Guide to
Methods and Application, Academic Press, New York. Other nucleic
acid amplification procedures such as ligase chain reaction (LCR),
ligated activated transcription (LAT) and nucleic acid
sequence-based amplification (NASBA) may be used. It can then be
ligated to a nucleic acid sequence encoding Z.
[0091] The nucleic acid sequence encoding the peptides and peptide
conjugates is then inserted into a recombinant expression vector
which may be any vector which may conveniently be subjected to
recombinant DNA procedures. 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.
[0092] In the vector, the nucleic acid sequence encoding the
peptides and peptide conjugates of the present invention should be
operably connected to a suitable promoter sequence. The promoter
may be any nucleic acid 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 nucleic acid sequence encoding said peptides
and peptide conjugates in mammalian cells are the SV 40 promoter
(Subramani et al., Mot. Cell Biol. 1, 1981, pp. 854-864), the MT-1
(metallothionein gene) promoter (Palmiter et al., Science 222,
1983, pp. 809-814) or the adenovirus 2 major late promoter, a Rous
sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter
(Boshart et al., 1981, Cell 41:521-530) and a bovine papilloma
virus promoter (BPV). A suitable promoter for use in insect cells
is the polyhedrin promoter (Vasuvedan et al., FEBS Lett. 311, 1992,
pp. 7-11).
[0093] Examples of suitable promoters for directing the
transcription of the nucleic acid sequence encoding the peptides
and peptide conjugates, especially in a bacterial host cell, are
the promoters obtained from the E. coli lac operon, the
Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis
levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase
gene (amyL), the Bacillus stearothermophilus maltogenic amylase
gene (amyM), the Bacillus amyloliquefaciens alpha amylase gene
(amyQ), the Bacillus licheniformis penicillinase gene (penP), the
Bacillus subtilis xylA and xylB genes, and the prokaryotic
beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of
the National Academy of Sciences USA 75:3727-3731), as well as the
tac promoter (DeBoer et al., 1983, Proceedings of the National
Academy of Sciences USA 80:21 25). Further promoters are described
in "Useful proteins from recombinant bacteria" in Scientific
American, 1980, 242:74-94; and in Sambrook et al., 1989, supra.
Examples of suitable promoters for directing the transcription of
the nucleic acid sequence encoding the peptides and peptide
conjugates in a filamentous fungal host cell are promoters obtained
from the genes encoding Aspergillus orysae TAKA amylase, Rhizomucor
miehei aspartic proteinase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase, Fusarium oxysporum trypsin-like protease (as described
in U.S. Pat. No. 4,288,627, which is incorporated herein by
reference), and hybrids thereof. Particularly preferred promoters
for use in filamentous fungal host cells are the TAKA amylase,
NA2-tpi (a hybrid of the promoters from the genes encoding
Aspergillus niger neutral a amylase and Aspergillus oryzae triose
phosphate isomerase), and glaA promoters. In a yeast host, useful
promoters are obtained from the Saccharomyces cerevisiae enolase
(ENO-1) gene, the Saccharomyces cerevisiae galactokinase gene
(GAL1), the Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes
(ADH2/GAP), and the Saccharomyces cerevisiae 3-phosphoglycerate
kinase gene. Other useful promoters for yeast host cells are
described by Romanos et al., 1992, Yeast 8:423-488.
[0094] The nucleic acid sequence encoding said peptides and peptide
conjugates may also be operably connected to a suitable terminator,
such as the human growth hormone terminator (Palmiter et al., op.
cit.) Preferred terminators for filamentous fungal host cells are
obtained from the genes encoding Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase, and Fusarium
oxysporum trypsin-like protease. Preferred terminators for yeast
host cells are obtained from the genes encoding Saccharomyces
cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1),
or Saccharomyces cerevisiae glyceraldehyde-3-phosphate
dehydrogenase. Other useful terminators for yeast host cells are
described by Romanos et al., 1992, supra.
[0095] The vector may further comprise elements such as
polyadenylation signals (e.g., from SV 40 or the adenovirus 5 Elb
region), transcriptional enhancer sequences (e.g., the SV 40
enhancer) and translational enhancer sequences (e.g., the ones
encoding adenovirus VA RNAs). Furthermore, preferred
polyadenylation sequences for filamentous fungal host cells are
obtained from the genes encoding Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, and Aspergillus niger alpha-glucosidase. Useful
polyadenylation sequences for yeast host cells are described by Guo
and Sherman, 1995, Molecular Cellular Biology 15:5983-5990.
[0096] The recombinant expression vector may further comprise a DNA
sequence enabling the vector to replicate in the host cell in
question. Examples of such a sequence (when the host cell is a
mammalian cell) is the SV 40 or polyoma origin of replication.
Examples of bacterial origins of replication are the origins of
replication of plasmids pBR322, pUC19, pACYC177, pACYC184, pUB110,
pE194, pTA1060, and pAM.beta.1. Examples of origin of replications
for use in a yeast host cell are the 2 micron origin of
replication, the combination of CEN6 and ARS4, and the combination
of CEN3 and ARS1. The origin of replication may be one having a
mutation to make its function temperature-sensitive in the host
cell (see, e.g., Ehrlich, 1978, Proc. Natl. Acad. Sci. USA
75:1433).
[0097] The vector may also comprise a selectable marker, e.g., a
gene the product of which complements a defect in the host cell,
such as the gene coding for dihydrofolate reductase (DHFR) or one
which confers resistance to a drug, e.g., neomycin, geneticin,
ampicillin, or hygromycin. Suitable markers for yeast host cells
are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. A selectable
marker for use in a filamentous fungal host cell may be selected
from the group including, but not limited to, amdS (acetamidase),
argB (ornithine carbamoyltransferase), bar (phosphinothricin
acetyltransferase), hygB (hygromycin phosphotransferase), niaD
(nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase),
sC (sulfate adenyltransferase), trpC (anthranilate synthase), and
glufosinate resistance markers, as well as equivalents from other
species. Preferred for use in an Aspergillus cell are the amdS and
pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the
bar marker of Streptomyces hygroscopicus. Furthermore, selection
may be accomplished by cotransformation, e.g., as described in WO
91/17243, where the selectable marker is on a separate vector.
[0098] The procedures used to ligate the nucleic acid sequences
coding for the peptides and peptide conjugates, the promoter and
the terminator, 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., op.cit.).
[0099] The host cell into which the expression vector is introduced
may be any cell which is capable of producing the peptides and
peptide conjugates and is may be a eukaryotic cell, such as
invertebrate (insect) cells or vertebrate cells, e.g., Xenopus
laevis oocytes or mammalian cells, in particular insect and
mammalian cells. Examples of suitable mammalian cell lines are the
COS (e.g., ATCC CRL 1650), KIK (e.g., ATCC CRL 1632, ATCC CCL 10)
or CHO (e.g., ATCC CCL 61) cell lines. Methods for transfecting
mammalian cells and expressing DNA sequences introduced in the
cells are described in e.g., Kaufman and Sharp, 1982, J. Mol. Biol.
159:601-621; Southern and Berg, 1982, J. Mol. Appl. Genet.
1:327-341; Loyter et al., 1982, Proc. Natl. Acad. Sci. USA
79:422-426; Wigler et al., 1978, Cell 14:725; Corsaro and Pearson,
1981, Somatic Cell Genetics 7:603, Graham and van der Eb, 1973,
Virology 52:456; Fraley et al., 1980, JBC 225:10431; Capecchi,
1980, Cell 22:479; Wiberg et al., 1983, NAR 11:7287; and Neumann et
al., 1982, EMBO J. 1:841-845. The host cell may also be a
unicellular pathogen, e.g., a prokaryote, or a non-unicellular
pathogen, e.g., a eukaryote. Useful unicellular cells are bacterial
cells such as gram positive bacteria including, but not limited to,
a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus
coagulans, Bacillus lautus, Bacillus lentus, Bacillus
licheniformis, Bacillus megaterium, Bacillus stearothermophilus,
Bacillus subtilis, and Bacillus, thuringiensis; or a Streptomyces
cell, e.g., Streptomyces lividans or Streptomyces murinus, or gram
negative bacteria such as E. coli and Pseudomonas sp. In a
preferred embodiment, the bacterial host cell is a Bacillus lentus,
Bacillus licheniformis, Bacillus stearothermophilus or Bacillus
subtilis cell. The transformation of a bacterial host cell may, for
instance, be effected by protoplast transformation (see, e.g.,
Chang and Cohen, 1979, Molecular General Genetics 168:111-115), by
using competent cells (see, e.g., Young and Spizizin, 1961, Journal
of Bacteriology 81:823-829, or Dubnar and Davidoff Abelson, 1971,
Journal of Molecular Biology 56:209-221), by electroporation (see,
e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or by
conjugation (see, e.g., Koehler and Thorne, 1987, Journal of
Bacteriology 169:5771-5278). The host cell may be a fungal cell.
The fungal host cell may also be a yeast cell. "Yeast" as used
herein includes ascosporogenous yeast (Endomycetales),
basidiosporogenous yeast, and yeast belonging to the Fungi
Imperfecti (Blastomycetes).
[0100] The medium used to culture the cells may be any conventional
medium suitable for growing mammalian cells, such as a
serum-containing or serum-free medium containing appropriate
supplements, or a suitable medium for growing insect, yeast or
fungal cells. 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).
[0101] Thus, the invention also relates to a method for producing
the exendin variants and peptide conjugates of the invention having
a natural polypeptide sequence, comprising [0102] a) introducing a
nucleic acid sequence encoding a polypeptide sequence comprising
the peptide sequence of the exendin variant or the peptide
conjugate of the invention and a selectable marker contained within
a nucleic acid construct or a vector into a host cell to obtain a
recombinant host cell; [0103] b) selecting said recombinant host
cell; [0104] c) culturing said recombinant host cells under
conditions permitting the production of said polypeptide sequence;
[0105] d) isolating said polypeptide sequence from the culture; and
[0106] e) optionally cleaving said polypeptide sequence using an
appropriate protease to obtain said peptide conjugate.
[0107] The variants and peptide conjugates of the invention having
a natural polypeptide sequence thus 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, affinity
chromatography, or the like. The lipophilic substituent(s) may be
attached to the peptide of the present invention using procedures
known in the art. In one embodiment, the lipophilic substituent may
be attached by incorporating an amino acid with the lipophilic
substituent already attached in the standard synthesis method (see,
for example, synthesis of compound 7 in the Examples section).
Alternatively, the substituent may be attached after the peptide
has been synthesized and isolated as, for example, described in
WO98/08871.
[0108] The invention is further illustrated by the following
examples.
EXAMPLES
Peptide Synthesis, General Procedures
[0109] Apparatus and Synthetic Strategy
[0110] Peptides are synthesized batchwise in a polyethylene vessel
equipped with a polypropylene filter for filtration using
9-fluorenylmethyloxycarbonyl (Fmoc) as the N-.alpha.-amino
protecting group and suitable common protection groups for
side-chain functionalities (Dryland et al., 1986, J. Chem. Soc.,
Perkin Trans. 1:125-137).
[0111] Solvents
[0112] Solvent DMF (N,N-dimethylformamide, Riedel de-Haen, Germany)
is purified by passing it through a column packed with a strong
cation exchange resin (Lewatit S 100 MB/H strong acid, Bayer AG
Leverkusen, Germany) and analysed for free amines prior to use by
addition of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine
(Dhbt-OH) giving rise to a yellow color (Dhbt-O-anion) if free
amines are present. Solvent DCM (dichloromethane, analytical grade,
Riedel de-Haen, Germany) is used directly without purification. THF
(tetrahydrofuran, analytical grade, Riedel de-Haen, Germany) is
used directly without further purification.
[0113] Amino Acids
[0114] Fmoc-protected amino acids are purchased from MilliGen (UK)
and from PerSeptive Biosystems GmbH Hamburg, Germany in suitable
side-chain protected forms. FmocLys(palmitoyl)-OH is purchased from
Bachem (Switzerland).
[0115] Linker
[0116] (4-hydroxymethylphenoxy)acetic acid (HMPA), Novabiochem,
Switzerland is coupled to the resin either as a preformed or in
situ generated 1-hydroxybenzotriazole (HObt) ester by means of
DIC.
[0117] Coupling Reagents
[0118] Coupling reagent diisopropylcarbodiimide (DIC) is purchased
from (Riedel de-Haen, Germany) and distilled prior to use,
dicyclohexylcarbodiimide (DCC) is purchased from Merck-Schuchardt,
Munchen, Germany, and purified by distillation.
[0119] Solid Supports
[0120] Peptides synthesized according to the Fmoc-strategy are
synthesized on the following types of solid support using 0.05 M or
higher concentrations of Fmoc-protected activated amino acid in
DMF. TentaGel S resins 0.22-0.31 mmol/g (TentaGel S-Ram, TentaGel S
RAM-Lys(Boc)Fmoc; Rapp polymere, Germany).
[0121] Catalysts and Other Reagents
[0122] Diisopropylethylamine (DIEA) is purchased from Aldrich,
Germany, and ethylenediamine from Fluka, piperidine and pyridine
from Riedel-de Haen, Frankfurt, Germany.
4-(N,N-di-methylamino)pyridine (DMAP) is purchased from Fluka,
Switzerland and used as a catalyst in coupling reactions involving
symmetrical anhydrides. Ethanedithiol is purchased from Riedel-de
Haen, Frankfurt, Germany.
3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH) and
1-hydroxybenzotriazole (HObt) are obtained from Fluka,
Switzerland.
[0123] Coupling Procedures
[0124] The first amino acid is coupled as a symmetrical anhydride
in DMF generated from the appropriate N-.alpha.-protected amino
acid by means of DIC or DCC. The following amino acids are coupled
as preformed HObt esters made from appropriate N-.alpha.-protected
amino acids and HObt by means of DIC in DMF. Acylations are checked
by the ninhydrin test performed at 80.degree. C. in order to
prevent Fmoc deprotection during the test (Larsen, B. D. and Holm,
A., 1994, Int. J. Peptide Protein Res. 43:1-9).
[0125] Coupling as HObt-Ester
[0126] Method a. 3 eq. N-.alpha.-amino protected amino acid is
dissolved in DMF together with 3 eq. HObt and 3 eq DIC. The
solution is left at r.t. for 10 minutes and then added to the
resin, which had been washed with a solution of 0.2% Dhbt-OH in DMF
prior to the addition of the preactivated amino acid.
[0127] Method b. 3 eq. N-.alpha.-amino protected amino acid is
dissolved in DMF together with 3 eq. HObt. 3 eq DIC are added just
prior to use. The final solution is added to the resin.
[0128] Preformed Symmetrical Anhydride
[0129] 6 eq. N-.alpha.-amino protected amino acid is dissolved in
DCM and cooled to 0.degree. C. DCC or DIC (3 eq.) is added and the
reaction continued for 10 min. The solvent is removed in vacuo and
the residue dissolved in DMF. The DMF-solution is filtered in case
of using DCC and immediately added to the resin followed by 0.1 eq.
of DMAP.
[0130] Deprotection of the N-.alpha.-Amino Fmoc Protecting
Group
[0131] Deprotection of the Fmoc group is performed by treatment
with 20% piperidine in DMF (1.times.5 and 1.times.10 min.),
followed by wash with DMF until no yellow colour (Dhbt-O-) could be
detected after addition of Dhbt-OH to the drained DMF.
[0132] Cleavage of Peptide from Resin with Acid
[0133] Method a. Peptides are cleaved from the resins by treatment
with 95% trifluoroacetic acid (TFA, Riedel-de Haen, Frankfurt,
Germany)-water v/v or with 95% TFA and 5% ethanedithiol v/v at r.t.
for 2 h. The filtered resins are washed with 95% TFA-water and
filtrates and washings are diluted by adding 10% acetic acid. The
resulting mixture is extracted 3 times with ether and finally
freeze dried. The crude freeze dried product is analysed by
high-performance liquid chromatography (HPLC) and identified by
mass spectrometry (MS).
[0134] Batchwise Peptide Synthesis on TentaGel S-RAM
[0135] TentaGel S-RAM resin (100-1000 mg, 0.22-0.31 mmol/g) is
placed in a polyethylene vessel equipped with a polypropylene
filter for filtration. The resin is swelled in DMF (5-10 ml), and
the Fmoc group is removed according to the procedure described
above. The following amino acids according to the sequence are
coupled as Fmoc-protected HObt esters (3 eq.) generated in situ by
means of DIC as described above. The couplings are continued for 3
h, unless otherwise specified. The resin is drained and washed with
DMF (4.times.5-10 ml, 2 min each) in order to remove excess
reagent. All acylations are checked by the ninhydrin test performed
at 80.degree. C. After completion of the synthesis, the
peptide-resin is washed with DMF (3.times.5-10 ml, 5 min each), DCM
(3.times.5-10 ml, 1 min each) and finally diethyl ether
(3.times.5-10 ml, 1 min each) and dried in vacuo.
[0136] HPLC Conditions
[0137] Isocratic HPLC analysis is preformed on a Shimadzu system
consisting of an LC-6A pump, an MERCK HITACHI L-4000 UV detector
operated at 215 nm and a Rheodyne 7125 injection valve with a 20
.mu.l loop. The column used for isocratic analysis is a Spherisorb
ODS-2 (100.times.3 mm; 5-.mu.m particles) (MicroLab, Aarhus,
Denmark). HPLC analysis using gradients is performed on a
MERCK-HITACHI L-6200 Intelligent pump, an MERCK HITACHI L-4000 UV
detector operated at 215 nm and a Rheodyne 7125 injection valve
with a 20 .mu.l loop, or on a Waters 600 E instrument equipped with
a Waters 996 photodiode array detector. The columns used are a
Rescorce.TM. RPC 1 ml (Waters) or a LiChroCART 125-4, LiChrospher
100 RP-18 (5 .mu.m) (Merck).
[0138] Buffer A is 0.1 vol % TFA in water and buffer B 90 vol %
acetonitrile, 9.9 vol % water and 0.1 vol % TFA. The buffers are
pumped through the columns at a flow rate of 13-1.5 ml/min using
either of the following gradients for peptide analysis 1) Linear
gradient from 0%-100% B (30 min) or 2) 0% B (2 min) linear gradient
from 0-50% B (23 min) 50-100% B (5 min).
[0139] For Preparative HPLC, purification is performed on a Waters
600 E instrument equipped with a Waters 996 photodiode array
detector. The column used is a Waters Delta-Pak C-18 15 .mu.m, 100
.ANG., 25.times.100 mm. Gradient "2)" is used with a flow rate of 9
ml/min.
[0140] Mass Spectroscopy
[0141] Mass spectra are obtained on a Finnigan Mat LCQ instrument
equipped with an electrospray (ESI) probe (ES-MS) and on a TofSpec
E, Fisons Instrument (MALDI-TOF) using
.beta.-cyano-p-hydroxycinnamic acid as matrix. Alternatively,
spectra may be obtained by a Micromass LCT instrument.
Peptide Synthesis of Prior Art Peptides
(i) Peptide Synthesis of Compound (i),
[0142]
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-G-
lu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-
-Pro-Pro-Pro-Ser-NH.sub.2
[0143] (exendin-4(1-39)-NH.sub.2) (SEQ ID NO:102) on TentaGel
S-RAM.
[0144] Dry TentaGel S-RAM resin (0.25 mmol/g, 1000 mg) is placed in
a polyethylene vessel equipped with a polypropylene filter for
filtration and swelled for two hours in DMF (5 ml). The Fmoc group
is removed according to the procedure described above, and the
peptide according to the sequence is assembled as described under
"Batchwise peptide synthesis on TentaGel S-RAM resins". After
completion of the synthesis, the peptide-resin is washed with DMF
(3.times.5 ml, 1 min each), DCM (3.times.5 ml, 1 min each), diethyl
ether (3.times.5 ml, 1 min each) and dried in vacuo. The peptide is
cleaved from the resin according to method a as described above and
freeze dried from acetic acid. The crude peptide is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 90%. The identity of the peptide is confirmed by ES-MS.
Yield 17%.
(ii) Peptide Synthesis of Compound (ii),
[0145]
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-G-
lu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-
-Pro-Pro-Pro-NH.sub.2(SEQ ID NO: 129)
[0146] (des Ser.sup.39 exendin-4(1-39)-NH.sub.2) (SEQ ID NO: 129)
on TentaGel S-RAM.
[0147] Dry TentaGel S-RAM resin (0.25 mmol/g, 1000 mg) is placed in
a polyethylene vessel equipped with a polypropylene filter for
filtration and swelled for two hours in DMF (5 ml). The Fmoc group
is removed according to the procedure described above, and the
peptide according to the sequence is assembled as described under
"Batchwise peptide synthesis on TentaGel S-RAM resins". After
completion of the synthesis, the peptide-resin is washed with DMF
(3.times.5 ml, 1 min each), DCM (3.times.5 ml, 1 min each), diethyl
ether (3.times.5 ml, 1 min each) and dried in vacuo. The peptide is
cleaved from the resin according to method a as described above and
freeze-dried from acetic acid. The crude peptide is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 97%. The identity of the peptide is confirmed by ES-MS.
Yield 22%.
(iii) Peptide Synthesis of Compound (iii),
[0148]
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser:-Ser-Tyr-Leu-Glu-Gly--
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH.sub.2
(SEQ ID NO: 87)
[0149] (Gly.sup.8-GLP1-(7-36)(Human)-NH.sub.2) (SEQ ID NO:87) on
TentaGel S-RAM.
[0150] Dry TentaGel S-RAM resin (0.25 mmol/g, 1000 mg) is placed in
a polyethylene vessel equipped with a polypropylene filter for
filtration and swelled for two hours in DMF (5 ml). The Fmoc group
is removed according to the procedure described above, and the
peptide according to the sequence is assembled as described under
"Batchwise peptide synthesis on TentaGel S-RAM resins". After
completion of the synthesis, the peptide-resin is washed with DMF
(3.times.5 ml, 1 min each), DCM (3.times.5 ml, 1 min each), diethyl
ether (3.times.5 ml, 1 min each) and dried in vacuo. The peptide is
cleaved from the resin according to method a as described above and
freeze dried from acetic acid. The crude peptide is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 95%. The identity of the peptide is confirmed by ES-MS.
Yield 9%.
Synthesis of Peptide Sequences of the Invention
1. Peptide Synthesis of Compound 1,
[0151]
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-G-
lu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Fro-Ser-Ser-Gly-Ala-
-Pro-Pro-Ser-NH.sub.2 (SEQ ID NO: 101)
[0152] (des Pro.sup.36-exendin-4(1-39)-NH.sub.2) (SEQ ID NO:101) on
TentaGel S-RAM.
[0153] Dry TentaGel S-RAM resin (0.25 mmol/g, 1500 mg) is placed in
a polyethylene vessel equipped with a polypropylene filter for
filtration and swelled for two hours in DMF (5 ml). The Fmoc group
is removed according to the procedure described above, and the
peptide according to the sequence is assembled as described under
"Batchwise peptide synthesis on TentaGel S-RAM resins". After
completion of the synthesis, the peptide-resin is washed with DMF
(3.times.5 ml, 1 min each), DCM (3.times.5 ml, 1 min each), diethyl
ether (3.times.5 ml, 1 min each) and dried in vacuo. The peptide is
cleaved from the resin according to method a as described above and
freeze dried from acetic acid. The crude peptide is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 95%. The identity of the peptide is confirmed by ES-MS.
Yield 18.3%.
2. Peptide Synthesis of Compound 2,
[0154]
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-G-
lu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-
-Pro-Pro-Ser-(Lys).sub.6-NH.sub.2 (SEQ ID NO: 93)
[0155] (des-Pro.sup.36-exendin-4(1-39)-Lys.sub.6-NH.sub.2) (SEQ ID
NO:93) on TentaGel S-RAM-Lys(Boc)Fmoc.
[0156] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1500 mg)
is placed in a poly-ethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the first lysine is removed as described above and
the synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Lys(Boc)Fmoc". After completion of the synthesis, the
peptide-resin is washed with DMF (3.times.5 ml, 1 min each), DCM
(3.times.5 ml, 1 min each), diethyl ether (3.times.5 ml, 1 min
each) and dried in vacuo. The peptide is cleaved from the resin
according to method a as described above and freeze dried from
acetic acid. The crude freeze dried product is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 95%. The identity of the peptide is confirmed by ES-MS.
Yield 22.1%.
3. Peptide Synthesis of Compound 3,
[0157]
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-G-
lu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-
-Pro-Pro-Pro-Ser-(Lys).sub.6-NH.sub.2 (SEQ ID NO: 92)
[0158] (exendin-4(1-39)-Lys.sub.6-NH.sub.2) (SEQ ID NO:92) on
TentaGel S-RAM-Lys(Boc)Fmoc.
[0159] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg)
is placed in a poly-ethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the first lysine is removed as described above and
the synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Lys(Boc)Fmoc". After completion of the synthesis, the
peptide-resin is washed with DMF (3.times.5 ml, 1 min each), DCM
(3.times.5 ml, 1 min each), diethyl ether (3.times.5 ml, 1 min
each) and dried in vacuo. The peptide is cleaved from the resin
according to method a as described above and freeze dried from
acetic acid. The crude freeze dried product is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 90%. The identity of the peptide is confirmed by ES-MS.
Yield 20.5%.
4. Peptide synthesis of Compound 4,
[0160]
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-G-
ln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-(Lys).sub.6-NH.sub.-
2 (SEQ ID NO: 88) (Gly.sup.8-GLP1-(7-36)(Human)-Lys.sub.6-NH.sub.2)
(SEQ ID NO:88) on TentaGel S-RAM-Lys(Boc)Fmoc.
[0161] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg)
is placed in a polyethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the first lysine is removed as described above and
the synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Lys(Boc)Fmoc". After completion of the synthesis, the
peptide-resin is washed with DMF (3.times.5 ml, 1 min each), DCM
(3.times.5 ml, 1 min each), diethyl ether (3.times.5 ml, 1 min
each) and dried in vacuo. The peptide is cleaved from the resin
according to method a as described above and freeze dried from
acetic acid. The crude freeze dried product is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 95%. The identity of the peptide is confirmed by ES-MS.
Yield 11.7%.
4a. Peptide Synthesis of Compound 4,
[0162]
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-G-
ln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys-Lys-Lys-Lys-Lys-
-Lys-NH.sub.2 (SEQ ID NO: 88)
[0163] ([Gly.sup.8]hGLP-1(7-36)-(Lys).sub.6-NH.sub.2) (SEQ ID
NO:88) on TentaGel S-RAM-Lys(Boc)Fmoc.
[0164] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (022 mmol/g, 2013 mg)
is placed in a glass vessel equipped with a polypropylene filter
for filtration and swelled for two hours in DMF (5 ml). The Fmoc
group on the first lysine is removed as described above and the
synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Lys(Boc)Fmoc". After completion of the synthesis, the
peptide-resin is washed with DMF (3.times.5 ml, 1 min each), DCM
(3.times.5 ml, 1 min each), diethyl ether (3.times.5 ml, 1 min
each) and dried in vacuo. The peptide is cleaved from the resin
according to method a as described above and freeze dried from
acetic acid. The crude freeze dried product is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 90%. The identity of the peptide is confirmed by ES-MS.
Yield 13%.
5. Peptide Synthesis of Compound 5,
[0165]
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-G-
ln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(palmitoyl)-(Lys-
).sub.6-NH.sub.2 (SEQ ID NO: 89)
([Gly.sup.8,Lys.sup.37(palmitoyl)]GLP1-(7-36)(Human)-(Lys).sub.7-NH.sub.2-
) (SEQ ID NO:89) on TentaGel S-RAM-Lys(Boc)Fmoc.
[0166] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg)
is placed in a polyethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the first lysine is removed as described above and
the synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Lys(Boc)Fmoc". The reagent Fmoc-Lys(palmitoyl)-OH is coupled
in a slightly modified manner due to its poor solubility in DMF.
Approximately 400 mg of Fmoc-Lys(palmitoyl)-OH is dissolved in
approximately 6 ml THF rather than DMF. After completion of the
synthesis, the peptide-resin is washed with DMF (3.times.5 ml, 1
min each), DCM (3.times.5 ml, 1 min each), diethyl ether (3.times.5
ml, 1 min each) and dried in vacuo. The peptide is cleaved from the
resin according to method b as described above and freeze dried
from acetic acid. The crude freeze dried product is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 95%. The identity of the peptide is confirmed by ES-MS.
Yield 93%.
6. Peptide Synthesis of Compound 6,
[0167]
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-G-
ln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys(palmitoyl)-Gly-Arg-(Lys).su-
b.6-NH.sub.2 (SEQ ID NO: 90)
[0168]
([Gly.sup.8,Lys.sup.34(palmitoyl)]GLP1-(7-36)(Human)-(Lys).sub.6-NH-
.sub.2) (SEQ ID NO:90) on TentaGel S-RAM-Lys(Boc)Fmoc.
[0169] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (022 mmol/g, 1000 mg)
is placed in a polyethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the first lysine is removed as described above and
the synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Lys(Boc)Fmoc". The reagent Fmoc-Lys(palmitoyl)-OH is coupled
in a slightly modified manner due to its poor solubility in DMF.
Approximately 400 mg of Fmoc-Lys(palmitoyl)-OH is dissolved in
approximately 6 ml THF rather than DMF. After completion of the
synthesis, the peptide-resin is washed with DMF (3.times.5 ml, 1
min each), DCM (3.times.5 ml, 1 min each), diethyl ether (3.times.5
ml, 1 min each) and dried in vacuo. The peptide is cleaved from the
resin according to method a as described above and freeze dried
from acetic acid. The crude freeze dried product is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 90%. The identity of the peptide is confirmed by ES-MS.
Yield 42%.
7. Peptide Synthesis of Compound 7,
[0170]
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-G-
ln-Ala-Ala-Lys(palmitoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-(Lys)6-N-
H.sub.2 (SEQ ID NO: 103)
[0171] ([Gly.sup.8,
Lys.sup.2.sup.6(palmitoyl)]GLP1-(7-36)(Human)-(Lys).sub.6-NH.sub.2)
(SEQ ID NO:103) on TentaGel S-RAM-Lys(Boc)Fmoc.
[0172] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (022 mmol/g, 1000 mg)
is placed in a polyethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the first lysine is removed as described above and
the synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Lys(Boc)Fmoc". The reagent Fmoc-Lys(palmitoyl)-OH is coupled
in a slightly modified manner due to its poor solubility in DMF.
Approximately 400 mg of Fmoc-Lys(palmitoyl)-OH is dissolved in
approximately 6 ml THF rather than DMF. After completion of the
synthesis, the peptide-resin is washed with DMF (3.times.5 ml, 1
min each), DCM (3.times.5 ml, 1 min each), diethyl ether (3.times.5
ml, 1 min each) and dried in vacuo. The peptide is cleaved from the
resin according to method a as described above and freeze dried
from acetic acid. The crude freeze dried product is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 90%. The identity of the peptide is confirmed by ES-MS.
Yield 22%.
8. Peptide Synthesis of Compound 8,
[0173]
H-Lys-Lys-Lys-Lys-Lys-Lys-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-S-
er-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-
-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-NH.sub.2 (SEQ ID NO: 149)
[0174] (H-(Lys).sub.6-des Pro.sup.36exendin-4(1-39)-NH.sub.2) (SEQ
ID NO: 149) on TentaGel S-RAM-Fmoc.
[0175] Dry TentaGel S-RAM-Fmoc resin (0.23 mmol/g, 1000 mg) is
placed in a poly-ethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the resin is removed as described above and the
synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Fmoc". After completion of the synthesis, the peptide-resin
is washed with DMF (3.times.5 ml, 1 min each), DCM (3.times.5 ml, 1
min each), diethyl ether (3.times.5 ml, 1 min each) and dried in
vacuo. The peptide is cleaved from the resin according to method a
as described above and freeze dried from acetic acid. The crude
freeze dried product is purified by preparative HPLC using the
procedure described above. The purified product is found to be
homogeneous and the purity is found to be better than 95%. The
identity of the peptide is confirmed by ES-MS. Yield 26%.
9. Peptide Synthesis of Compound 9,
[0176]
H-Lys.sub.6-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-
-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-S-
er-Gly-Ala-Pro-Pro-Ser-(Lys).sub.6-NH.sub.2 (SEQ ID NO: 150)
[0177] (H-Lys.sub.6-des
Pro.sup.36exendin-4(1-39)-Lys.sub.6-NH.sub.2) (SEQ ID NO: 150) on
TentaGel S-RAM-Lys(Boc)Fmoc.
[0178] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg)
is placed in a poly-ethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the first lysine is removed as described above and
the synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Lys(Boc)Fmoc". After completion of the synthesis, the
peptide-resin is washed with DMF (3.times.5 ml, 1 min each), DCM
(3.times.5 ml, 1 min each), diethyl ether (3.times.5 ml, 1 min
each) and dried in vacuo. The peptide is cleaved from the resin
according to method a as described above and freeze dried from
acetic acid. The crude freeze dried product is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 90%. The identity of the peptide is confirmed by ES-MS.
Yield 32%.
10. Peptide Synthesis of Compound 10,
[0179]
H-Lys-Lys-Lys-Lys-Lys-Lys-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-S-
er-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-
-Arg-Lys-Lys-Lys-Lys-Lys-Lys-NH.sub.2 (SEQ ID NO: 118)
(H-(Lys).sub.6-([Gly.sup.5]hGLP-1(7-36)-(Lys).sub.6-NH.sub.2) (SEQ
ID NO: 118) on TentaGel S-RAM-Lys(Boc)Fmoc.
[0180] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (022 mmol/g, 1000 mg)
is placed in a poly-ethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the first lysine is removed as described above and
the synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Lys(Boc)Fmoc". After completion of the synthesis, the
peptide-resin is washed with DMF (3.times.5 ml, 1 min each), DCM
(3.times.5 ml, 1 min each), diethyl ether (3.times.5 ml, 1 min
each) and dried in vacuo. The peptide is cleaved from the resin
according to method a as described above and freeze dried from
acetic acid. The crude freeze dried product is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 90%. The identity of the peptide is confirmed by ES-MS.
Yield 18%.
11. Peptide Synthesis of Compound 11,
[0181]
H-Lys-Lys-Lys-Lys-Lys-Lys-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-S-
er-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-
-Arg-NH.sub.2 (SEQ ID NO: 119)
[0182] (H-(Lys).sub.6-[Gly.sup.8]hGLP-1(7-36)-NH.sub.2) (SEQ ID NO:
119) on TentaGel S-RAM-Lys(Boc)Fmoc.
[0183] Dry TentaGel S-RAM-Fmoc resin (0.23 mmol/g, 1000 mg) is
placed in a poly-ethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the resin is removed as described above and the
synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Fmoc". After completion of the synthesis, the peptide-resin
is washed with DMF (3.times.5 ml, 1 min each), DCM (3.times.5 ml, 1
min each), diethyl ether (3.times.5 ml, 1 min each) and dried in
vacuo. The peptide is cleaved from the resin according to method a
as described above and freeze dried from acetic acid. The crude
freeze dried product is purified by preparative HPLC using the
procedure described above. The purified product is found to be
homogeneous and the purity is found to be better than 98%. The
identity of the peptide is confirmed by ES-MS. Yield 15%.
12. Peptide Synthesis of Compound 12,
[0184]
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-G-
ln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys-Lys-Lys-Lys-Lys-
-Lys-Lys-Lys-NH.sub.2 (SEQ ID NO: 120)
[0185] ([Gly.sup.8]hGLP-1(7-36)-(Lys).sub.8-NH.sub.2) (SEQ ID NO:
120) on TentaGel S-RAM-Lys(Boc)Fmoc.
[0186] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg)
is placed in a poly-ethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the first lysine is removed as described above and
the synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Lys(Boc)Fmoc". After completion of the synthesis, the
peptide-resin is washed with DMF (3.times.5 ml, 1 min each), DCM
(3.times.5 ml, 1 min each), diethyl ether (3.times.5 ml, 1 min
each) and dried in vacuo. The peptide is cleaved from the resin
according to method a as described above and freeze dried from
acetic acid. The crude freeze dried product is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 98%. The identity of the peptide is confirmed by ES-MS.
Yield 4.2%.
13. Peptide Synthesis of Compound 13,
[0187]
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-G-
ln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys-Lys-Lys-Lys-Lys-
-Lys-Lys-Lys-Lys-Lys-NH.sub.2 (SEQ ID NO: 121)
[0188] ([Gly.sup.8]hGLP-1(7-36)-(Lys).sub.10-NH.sub.2) (SEQ ID NO:
121) on TentaGel S-RAM-Lys(Boc)Fmoc.
[0189] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg)
is placed in a poly-ethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the first lysine is removed as described above and
the synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Lys(Boc)Fmoc". After completion of the synthesis, the
peptide-resin is washed with DMF (3.times.5 ml, 1 min each), DCM
(3.times.5 ml, 1 min each), diethyl ether (3.times.5 ml, 1 min
each) and dried in vacuo. The peptide is cleaved from the resin
according to method a as described above and freeze dried from
acetic acid. The crude freeze dried product is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 95%. The identity of the peptide is confirmed by ES-MS.
Yield 2%.
14. Peptide Synthesis of Compound 14,
[0190]
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-G-
lu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-
-Ser-NH.sub.2 (SEQ ID NO: 132)
[0191] (H-des Pro.sup.36, Pro.sup.37,
Pro.sup.38exendin-4(1-39)-NH.sub.2) (SEQ ID NO: 132) on TentaGel
S-RAM-Fmoc.
[0192] Dry TentaGel S-RAM-Fmoc resin (0.23 mmol/g, 1000 mg) is
placed in a poly-ethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the resin is removed as described above and the
synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Fmoc". After completion of the synthesis, the peptide-resin
is washed with DMF (3.times.5 ml, 1 min each), DCM (3.times.5 ml, 1
min each), diethyl ether (3.times.5 ml, 1 min each) and dried in
vacuo. The peptide is cleaved from the resin according to method a
as described above and freeze dried from acetic acid. The crude
freeze dried product is purified by preparative HPLC using the
procedure described above. The purified product is found to be
homogeneous and the purity is found to be better than 95%. The
identity of the peptide is confirmed by ES-MS. Yield 11%.
15. Peptide Synthesis of Compound 15,
[0193]
H-Lys-Lys-Lys-Lys-Lys-Lys-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-S-
er-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-
-Gly-Pro-Ser-Ser-Gly-Ala-Ser-NH.sub.2 (SEQ ID NO: 134)
[0194] (H-(Lys).sub.6-des Pro.sup.36, Pro.sup.37,
Pro.sup.38exendin-4(1-39)-NH.sub.2) (SEQ ID NO: 134) on TentaGel
S-RAM-Fmoc.
[0195] Dry TentaGel S-RAM-Fmoc resin (0.23 mmol/g, 1000 mg) is
placed in a poly-ethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the resin is removed as described above and the
synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Fmoc". After completion of the synthesis, the peptide-resin
is washed with DMF (3.times.5 ml, 1 min each), DCM (3.times.5 ml, 1
min each), diethyl ether (3.times.5 ml, 1 min each) and dried in
vacuo. The peptide is cleaved from the resin according to method a
as described above and freeze dried from acetic acid. The crude
freeze dried product is purified by preparative HPLC using the
procedure described above. The purified product is found to be
homogeneous and the purity is found to be better than 94%. The
identity of the peptide is confirmed by ES-MS. Yield 17%.
16. Peptide Synthesis of Compound 16,
[0196]
H-Asn-Glu-Glu-Glu-Glu-Glu-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-S-
er-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-
-Gly-Pro-Ser-Ser-Gly-Ala-Ser-NH.sub.2 (SEQ ID NO: 137)
[0197] (H-Asn-(Glu).sub.5-des Pro.sup.36, Pro.sup.37,
Pro.sup.38exendin-4(1-39)-NW) (SEQ ID NO: 137) on TentaGel
S-RAM-Fmoc.
[0198] Dry TentaGel S-RAM-Fmoc resin (0.23 mmol/g, 1000 mg) is
placed in a poly-ethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the resin is removed as described above and the
synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Fmoc". After completion of the synthesis, the peptide-resin
is washed with DMF (3.times.5 ml, 1 min each), DCM (3.times.5 ml, 1
min each), diethyl ether (3.times.5 ml, 1 min each) and dried in
vacuo. The peptide is cleaved from the resin according to method a
as described above and freeze dried from acetic acid. The crude
freeze dried product is purified by preparative HPLC using the
procedure described above. The purified product is found to be
homogeneous and the purity is found to be better than 90%. The
identity of the peptide is confirmed by ES-MS. Yield 9%.
17. Peptide Synthesis of Compound 17,
[0199] Compound 3,
H-(Lys).sub.6-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-
-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-G-
ly-Ala-Ser-(Lys).sub.6-NH.sub.2 (SEQ ID NO: 135)
[0200] (H-(Lys).sub.6-des Pro.sup.36, Pro.sup.37,
Pro.sup.38exendin-4(1-39)-(Lys).sub.6-NH.sub.2) (SEQ ID NO: 135) on
TentaGel S-RAM-Lys(Boc)Fmoc.
[0201] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg)
is placed in a poly-ethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the first lysine is removed as described above and
the synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Lys(Boc)Fmoc". After completion of the synthesis, the
peptide-resin is washed with DMF (3.times.5 ml, 1 min each), DCM
(3.times.5 ml, 1 min each), diethyl ether (3.times.5 ml, 1 min
each) and dried in vacuo. The peptide is cleaved from the resin
according to method a as described above and freeze dried from
acetic acid. The crude freeze dried product is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 90%. The identity of the peptide is confirmed by ES-MS.
Yield 10%.
18. Peptide Synthesis of Compound 18,
[0202]
H-Asn-Glu-Glu-Glu-Glu-Glu-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-S-
er-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-
-Gly-Pro-Ser-Ser-Gly-Ala-Ser-(Lys).sub.6-NH.sub.2 (SEQ ID NO:
136)
[0203] (H-Asn-(Glu); des Pro.sup.36, Pro.sup.37,
Pro.sup.38exendin-4(1-39)-(Lys).sub.6-NH.sub.2) (SEQ ID NO: 136) on
TentaGel S-RAM-Lys(Boc)Fmoc.
[0204] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg)
is placed in a poly-ethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the first lysine is removed as described above and
the synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Lys(Boc)Fmoc". After completion of the synthesis, the
peptide-resin is washed with DMF (3.times.5 ml, 1 min each), DCM
(3.times.5 ml, 1 min each), diethyl ether (3.times.5 ml, 1 min
each) and dried in vacuo. The peptide is cleaved from the resin
according to method a as described above and freeze dried from
acetic acid. The crude freeze dried product is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 92%. The identity of the peptide is confirmed by ES-MS.
Yield 14%.
19. Peptide Synthesis of Compound 19,
[0205]
H-His-Gly-Glu-GIy-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-G-
lu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-
-Ser-(Lys).sub.6-NH.sub.2 (SEQ ID NO: 133)
[0206] (des Pro.sup.36, Pro.sup.37,
Pro.sup.38exendin-4(1-39)-(Lys).sub.6-NH.sub.2) (SEQ ID NO: 133) on
TentaGel S-RAM-Lys(Boc)Fmoc.
[0207] Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg)
is placed in a poly-ethylene vessel equipped with a polypropylene
filter for filtration and swelled for two hours in DMF (5 ml). The
Fmoc group on the first lysine is removed as described above and
the synthesis is continued until finishing the peptide sequence as
described under "Batchwise peptide synthesis on TentaGel
S-Ram-Lys(Boc)Fmoc". After completion of the synthesis, the
peptide-resin is washed with DMF (3.times.5 ml, 1 min each), DCM
(3.times.5 ml, 1 min each), diethyl ether (3.times.5 ml, 1 min
each) and dried in vacuo. The peptide is cleaved from the resin
according to method a as described above and freeze dried from
acetic acid. The crude freeze dried product is purified by
preparative HPLC using the procedure described above. The purified
product is found to be homogeneous and the purity is found to be
better than 97%. The identity of the peptide is confirmed by ES-MS.
Yield 19%.
20. Recombinant Preparation of Compound 2
[0208] Construction of the pYES0010 Expression Vector
[0209] A synthetic cDNA was constructed for heterolog expression in
yeast. The protein sequence encoding Compound 2 was back translated
using a Saccharomyces cerevisiae codon usage table (Saccharomyces
Genome Database). To enable translation of the synthetic cDNA an
additional ATG start codon was added to the 5' end and a TAA stop
codon was added to the 3' end. The construct was inserted into
HindIII and The EcoRI site of the pYES2 shuttle vector comprising
an ampicilline resistance gene, and the new construct was
designated pYES0010, cf. FIG. 6. pYES0010 was subsequently
transformed into E. coli and subjected to ampicillin selection
pressure. Positive clones were selected and sequenced.
Transformation into Yeast.
[0210] In order to make transform the pYES0010 into the yeast
haploid INVSc1: MATa his3delta1 leu2 trp1-289 ura3-52. Yeast were
grown in YPD medium (1% yeast extract, 2% peptone, 2% glucose, and
0.004% adenine sulfate) at 30 C to saturation. 1 ml of culture was
harvested for transformations. 2 .mu.l of 10 mend carrier DNA was
added and 1 .mu.g of pYES0010 was added and mixed. 0.5 ml (45% PEG
4000, 1 M Li OAc, 0.5M EDTA and 1M Tris-HCl (pH 7.5) was added and
mixed. Finally 20 .mu.l M DTT was added and the mixture was
incubated for 16 h at room temperature. After incubation the cells
were heat shocked at 42 C for 10 min and plated selective plates
(6.7% yeast nitrogen base, 2% glucose, 20 .mu.g/ml adenine, 20
.mu.g/ml arginine, 29 .mu.g/ml isoleucine, 20 .mu.g/ml histidine,
60 .mu.g/ml leucine, 20 .mu.g/ml lysine, 20 .mu.g/ml tryptophan, 20
.mu.g/ml methionine 50 .mu.g/ml phenylalanine 150 .mu.g/ml valine,
30 .mu.g/ml Tyrosine and 2.5% agar. Plates were incubated at 30 C
for 3 to 5 days until transformants appear.
Expression and Purification of Compound 2.
[0211] Transformants were cultivated in selective media (6.7% Yeast
nitrogen base, 2% glucose, 20 .mu.g/ml adenine, 20 .mu.g/ml
arginine, 29 .mu.g/ml isoleucine, 20 .mu.g/ml histidine, 60
.mu.g/ml leucine, 20 .mu.g/ml lysine, 20 .mu.g/ml Tryptophan, 20
.mu.g/ml methionine 50 .mu.g/ml phenylalanine 150 .mu.g/ml valine,
30 .mu.g/ml Tyrosine) for 1.5 days. The cells were harvested and
resuspended in galactose induction medium (6.7% Yeast nitrogen
base, 4% galactose, 20 .mu.g/ml adenine, 20 .mu.g/ml arginine, 29
.mu.g/ml isoleucine, 20 .mu.g/ml histidine, 60 .mu.g/ml leucine, 20
.mu.g/ml lysine, 20 .mu.g/ml Tryptophan, 20 .mu.g/ml methionine 50
.mu.g/ml phenylalanine 150 .mu.g/ml valine, 30 .mu.g/ml Tyrosine
for 1 day. The cells were harvested and homogenized in 10 mM
Tris-HCl pH 7.5 containing protease inhibitors (Roche). The lysate
was clarified centrifugation at 20.000.times.g for 30 min. The
supernatant was loaded onto a Superdex 12 HR 10/30 column (Amersham
Pharmacia Biotech) equilibrated with 10 mM Tris-HCl pH 7.5. The
column was eluted in 50 Mm ammonia bicarbonate buffer pH 8.0.
Samples containing recombinant Compound 2 were pooled. The
N-terminal methionine was removed by methionine aminopeptidase and
the samples were further purified on a HPLC Column.
HPLC Settings for Compound 2 Purification.
[0212] HPLC column Kromasil RP CS; K 100-10-C8 nr. CER 2230,
compound
[0213] Temp: 22 C
[0214] Flow rate: 35 ml/min
[0215] HPLC solvents:
[0216] A: 0.10% trifluoroacetic acid in water
[0217] B: 0.10% trifluoroacetic acid in acetonitrile: water
90:10.
[0218] Compound 2 was eluted from the HPLC column with 0.10%
trifluoroacetic acid in 20% to 80% Acetonitrile in 40 min.
21. Injection Formulations of Peptide
[0219] Fixed dose formulations of peptide for intra venous
injection are prepared by dissolving the peptide in sterile,
isotonic saline, and storing the resulting solution in glass
ampoules filled with inert gas under sterile conditions. Each dose
of the peptide is stored dry in ampoules or capped vials filled
with inert gas. Multi-dose formulations of peptide for intra venous
injection are prepared by dissolving the peptide in sterile,
isotonic saline, storing the resulting solution in capped vials, if
necessary adding preservative (for instance 0.1%
parahydroxybenzoate, 1% benzyl alcohol or 0.1% chlorocresole).
Example of Multi-Dose Peptide Formulation:
TABLE-US-00001 [0220] Compound 2 12.25 mg Sodiumdihydrogenphosphate
1.380 g Parahydroxybenzoate 0.1 g Aqua ad injectabile 100 ml
22. Stability Experiments
[0221] In vitro stability studies with the present peptides and
peptide conjugates in the presence of selected proteolytic enzymes
are applied as a tool for evaluating the protection of said
peptides against proteolysis in vivo. The aim of the experiments
performed was to measure and compare the in vitro stability of
Compounds 4, 5, 6 and 7 to that of the prior art compounds Compound
(iii) H-(Gly.sup.8)-hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 87) and
hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 114) in solutions of one or more
of the enzymes leucine aminopeptidase, carboxypeptidase A and
dipeptidyl aminopeptidase IV at 37.degree. C.
Materials and Apparatus for In Vitro Stability
[0222] Water used was of highest quality obtained from a Milli-Q
water treatment system (Millipore, Bedford, Mass., USA).
Acetonitrile (ACN) was of super gradient quality obtained from
Labscan Ltd. (Dublin, Ireland). Trifluoracetic acid (TFA) 99.9%,
dihydrogen phosphate (NaH.sub.2PO.sub.4), sodium hydroxide (NaOH)
and all other chemicals used were of analytical grade. Leucine
aminopeptidase (EC 3.4.11.1), Carboxypeptidase A (EC 3.4.17.1) and
Dipeptidyl peptidase (Dipeptidyl aminopeptidase IV, EC 3.4.14.5)
were all obtained from Sigma (St. Louis, Mo., USA). Gradient HPLC
analysis was done using a Hewlett Packard HP 1100 HPLC system
consisting of a HP 1100 Binary Pump, a HP 1100 Autosampler, A HP
1100 Column Thermostat and a HP 1100 Variable Wavelength Detector.
Hewlett Packard Chemstation for LC software (Rev. A.06.01) was used
for instrument control and data acquisition. A Vydac 238TP54
(150.times.4.6 mm I.D.) column packed with 5 m, C18, 300 particles
was used with the instrument. A SHT200D block heater from Stuart
Scientific was used for heating of the peptide/enzyme solutions
during the stability experiments. The degradation of the test
compounds was studied at 37.degree. C. in 50 mM phosphate buffer
solutions of pH 7.4 containing leucine aminopeptidase (25 U/ml) or
carboxypeptidase A (1 U/ml) or 100 mM ammoniumbicarbonate buffer of
pH 8.0 containing dipeptidyl aminopeptidase IV (0.5 U/ml).
Experiments were initiated by addition of an aliquot (100 l) of a
stock solution (1 mg/ml) of the peptide in water to 900 l preheated
enzyme solution in an Eppendorf microvial giving an initial
concentration of 0.1 mg/ml (.about.1.710-5-1.810-5 M) of the
peptide. The peptide/enzyme solution was kept at 37.degree. C. and
at appropriate time intervals samples of 100 l were withdrawn from
the peptide/enzyme solution and mixed thoroughly with 20 l 25% TFA
in acetonitrile in order to stop the enzymatic degradation process.
The inactivated samples were transferred to autosampler vials and
analysed for content of intact test compound by HPLC as described
below. Half-lives (VA) for the test compounds in enzyme solutions
were calculated from plots of natural logarithm to the residual
concentration (i.e. HPLC peak heights) against time using the
formula:
t.sub.1/2=1/k.sub.obs ln(2), where k.sub.obs is the apparent
first-order rate constant for the observed degradation.
HPLC Analysis
[0223] Samples from the stability experiments performed as
described above were analysed by gradient HPLC analysis using the
instrumentation described above and the following experimental
conditions.
[0224] Column temperature: 30.degree. C.
[0225] Injection volume: 10 l
[0226] Mobile phase A: 0.1% TFA in water
[0227] Mobile phase B: 0.085% TFA in acetonitrile (ACN)
[0228] Gradient: 32-52% B in 21 min
[0229] Detection: UV at 215 nm
[0230] The experimental results obtained from the individual
stability experiments are shown in Table 1 below. It appears from
the table that the half life of the compounds of the invention is
considerably extended in solution with all enzymes tested.
TABLE-US-00002 TABLE 1 Test Compound Compound Enzyme Solution
Half-life No. Name Enzyme Conc. (t.sub.1/2) Compound 5
H-(Gly.sup.8)-hGLP- LAP 25 U/ml >3 days 1(7-36)-Lys(Palm)- CPA 1
U/ml >2 days Lys.sub.6-NH.sub.2 DPP IV 0.5 U/ml 440 min (SEQ ID
NO: 153) Compound 7 H-(Gly.sup.8, Lys.sup.2.sup.6(Palm))- LAP 25
U/ml 1150 min hGLP-1(7-36)- CPA 1 U/ml 1058 min Lys.sub.6-NH.sub.2
DPP IV 0.5 U/ml 526 min (SEQ ID NO: 103) Compound 6 H-(Gly.sup.8,
Lys.sup.34(Palm))- LAP 25 U/ml ~1.5 day hGLP-1(7-36)- CPA 1 U/ml
>1 day Lys.sub.6-NH.sub.2 DPP IV 0.5 U/ml 177 min (SEQ ID NO:
90) GLP-1 H-hGLP-1(7-36)-NH.sub.2 LAP 25 U/ml 152 min (SEQ ID NO:
114) CPA 1 U/ml 48 min DPP IV 0.5 U/ml 2.0 min Compound 4
H-(Gly.sup.8)-hGLP- LAP 25 U/ml ~1.5 day 1(7-36)-Lys.sub.6-NH.sub.2
CPA 1 U/ml 145 min (SEQ ID NO: 88) DPP IV 0.5 U/ml 292 min
Compound(iii) H-(Gly.sup.8)-hGLP- LAP 25 U/ml 693 min
1(7-36)-NH.sub.2 CPA 1 U/ml 127 min (SEQ ID NO: 87) DPP IV 0.5 U/ml
56 min LAP: Leucine aminopeptidase, CPA: Carboxypeptidase A, DPP
IV: Dipeptidyl aminopeptidase IV
23. In Vitro Stability Studies of Compound (iii) and Compound 4 in
Rat Plasma
[0231] The degradation of the two test compounds and in heparin
stabilised rat (Sprague-Dawley) plasma was followed by the
combination of solid phase extraction and LC-MS. The degradation
was followed for 720 minutes in plasma. The half-life of Compound
(iii) was found to be 238 min. in rat plasma. This finding was
compared with the half-life of Compound 4, which was found to be
466 min. in rat plasma.
Materials and Methods
[0232] Blank rat plasma in sodium heparin (5000 units/mL) were
obtained from Harlan Sera Lab Ltd. (Loughborough, UK). Test
Substances and Solutions The test substances used in the study are
listed in the table below. For the in vitro experiments a stock
solution of 100 .mu.g/ml milli-Q water was used (corresponding to
26.0 .mu.M Compound (iii) H-(Gly.sup.8)-GLP-1(7-36) (SEQ ID NO:
87)-NH.sub.2 or 17.8 .mu.M Compound 4).
TABLE-US-00003 Substance Name Batch No. Average Mw. Peptide Content
Compound (iii) ZP 7.73-1F 3284 g/mol 85% Compound 4 ZP 7.69-1C 4053
g/mol 72%
[0233] The LC-MS analysis was performed on an HP 1100 instrument
consisting of an on-line degasser, a quaternary gradient pump, an
auto sampler, a column oven, Hewlett Packard (Wilmington, Del.,
USA) in combination with a Quattro Ultima mass spectrometer from
Micromass (Altrincham, UK). Both the LC and MS were controlled by
MassLynx 33 software. The LC separations prior to MS detection were
performed on a Vydac 218MS52 (2.1.times.250 mm) column (Hesperia,
Calif., USA).
[0234] The initial plasma volume was 1000 .mu.l (37.degree. C.).
From the initial plasma volume, 100 .mu.l was transferred to a 0.75
ml HPLC vial (used as blank), mixed with 560 .mu.l extraction
solution (MeCN:0.18 M ammonium carbonate pH 9.5 (6:94 v/v), 4 C)
and extracted by Solid Phase Extraction using ASPEC XL4 Robot. A
volume of 100 .mu.l stock solution was added to the remaining 900
.mu.l plasma, mixed thoroughly and incubated at 37 C (corresponding
to an initial concentration of 10 .mu.g of the test compounds/nil).
At each time point (0.2, 60, 120, 180, 240, 360, 480, 662 and 720
min., respectively) 100 .mu.l of the drug containing plasma was
collected, mixed with 560 .mu.l ice cold extraction solution and
immediately extracted by SPE as described above. The extracted
plasma samples were analysed by LC-MS.
[0235] The LC-MS analysis were performed on an HP 1100 series LC in
combination with a Quattro Ultima II triple quadrupole MS
instrument.
[0236] The samples were kept at 18.degree. C. in the autosampler
tray prior to injection of 10 .mu.l. The separations were performed
at 30.degree. C. on a Vydac 218MS52 (2.1.times.250 mm) LC column
using a linear gradient from 15 to 50% B within 14 min. at a flow
rate of 250 .mu.l/min. 0.1% formic acid in water was used as mobile
phase A and 0.1% formic acid in MeCN as mobile phase B. Compound 4
and Compound (iii) were detected by single ion recording (SIR)
using the 6 H+ (m/z=676.7) and 4 H+ (m/z=822.1) ion species,
respectively. The cone voltage for the analysis of compound (iii)
and Compound 4 was set to 100 and 70 V, respectively.
[0237] The in vitro stability of Compound (iii) and Compound 4 have
been investigated in rat plasma by LC-MS. The degradation of the
two compounds were followed for 720 min. and the results were
plotted as the natural logarithm of the peak area vs. time. The
degradation rates (k.sub.obs) of the compounds were found as the
slope after linear regression, and the half-life (T1/2) was found
as ln 2/k.sub.obs. The results from the experiment are listed
below.
TABLE-US-00004 Degradation Study over 720 minutes in Rat Plasma
Compound T1/2 (min) k.sub.obs (min.sup.-1) r.sup.2 Compound (iii)
238.4 0.0029 0.9785 Compound 4 466.1 0.0015 0.8596
[0238] The conclusion of the experiment is therefore that the
provision of a C-terminal Lys (SEQ ID NO: 9) peptide conjugation to
the (Gly.sup.8)hGLP-1(7-36) (SEQ ID NO: 87) sequence results in a
two fold increased stability in rat plasma.
24. Single Dose Effect of Oral and Parenteral Administration of
Compound 5 on Blood Glucose Levels in Diabetic ob/ob Mice.
[0239] The compounds of the invention possess blood glucose
lowering properties. This was examined using Compound 5 to test the
effect on blood glucose (BG) levels in the ob/ob mutant mice after
intraperitoneal (i.p.) and peroral (p.o.) administration. Compound
5 reduced BO levels in diabetic mice in a dose of 110 .mu.g/mouse
when administered i.p. Likewise p.o. administration of Compound 5
elicited a similar decrease in BG levels in a dose of 1100
.mu.g/mouse, but not at lower doses.
Experimental
[0240] Forty female diabetic ob/ob mice (Ume{dot over (a)} strain,
Bomholtgaard), which are obese due to a dominant mutant leptin
(Tomita, T., Doull, V., Pollock, H. G, and Krizsan, D. 1992.
Pancreatic islets of obese hyperglycemic mice (ob/ob). Pancreas 7:
367-75) were housed (3 mice/cage) under controlled ambient
conditions following a 12:12-h light:dark cycle and fed standard
Altromin no 1324 diet with free access to tap water. At arrival the
animals were 8 weeks of age. The mice were allowed 2 weeks of
acclimatization before experiments were initiated. At the time of
experiment the mice were 13 weeks old with a body weight of
41.8.+-.3.2 g (mean.+-.SD; n=42). Handling of the mice one and
three days before the experiment was performed in order to reduce
stress-induced BG excursions. On the day of the experiment, blood
was taken from the tip of the tail 2-3 hours after the light was
turned on. A single drop of blood (<5 .mu.l) was dropped on the
glucose strip for analysis and measured by an Elite Autoanalyser,
Bayer, Denmark. Whole blood glucose (BG) concentration was analysed
by the immobilised glucose oxidase method. Blood glucose levels
varied between normoglycaemia and severe hyperglycaemia (range:
3.6-15.6 mM; mean.+-.SD: 9.4.+-.3.3 mM; n=42). Six animals with
BG<5.8 mM were excluded from the study (total n=36). The
remaining animals were stratified based on their BG levels in order
to ensure that the mean BG was similar among groups. One hour after
the initial control blood sampling, drugs were administered and BG
was measured at t=60 min, t=120 min, t=240 min, t=480 min.
Peptides and Other Materials
[0241] Compound 5 (batch nr. ZP 3.12 fraction 1-2. Purification)
was synthesised by the Department of Chemistry, Zealand
Pharmaceuticals. The peptide was dissolved in sterile isotonic NaCl
shortly before dosing and given in a volume of 0.2 ml. The same
solutions were used for both p.o. and i.p. administration. For each
animal, a data log sheet was filled out at the time of each blood
sampling.
Drug Administration
[0242] Animals were administered with Compound 5, and the maximum
dose was 1100 .mu.g/mouse and the lowest dose was 1.1 .mu.g/mouse.
As a negative control, saline was administered p.o. and as positive
control the test compound was given i.p. in a dose of 110
.mu.g/mouse.
[0243] During control conditions, BG levels in non-fasted ob/ob
mice were similar in all groups (individual group data not shown),
but within groups, there was a great scatter on BG levels (BG range
for all animals: 5.8-15.6 mM). Therefore, to correct for the
varying degree of hyperglycemia, results are expressed as the
relative difference from baseline (% control). Intraperitoneal
administration of 110 .mu.g Compound 5 produced a sustained
decrease in BG that reached nadir at 1-2 hrs after administration
of the compound. No changes were observed in saline treated
animals. In most groups (5/6), BG increased between 4 and 8 hrs
after drug administration. Compound 5 reduced the BG levels in a
dose of 110 .mu.g/mouse when administered i.p. in diabetic ob/ob
mice (data not shown). The antidiabetic effect was observed after
60 minutes and was maximal 2-4 h after administration of the
compound. Furthermore, a long-lasting effect (>8 hours) suggests
that Compound 5 has a longer duration of action than the
notoriously short-acting native GLP-1 (Bailey, C. J. & Flatt,
P. R. 1987. Glucagon-like peptide-1 and the entero-insular axis in
obese hyperglycaemic (ob/ob) mice. Life Sci, 40, 521-5). The dose
1100 .mu.g/mouse p.o. elicited a similar decrease in BG as observed
in animals treated with 110 .mu.g i.p.
[0244] We have shown that Compound 5 effectively lowers BG levels
in diabetic ob/ob mice following i.p. administration of 110
.mu.g/mouse the compound. A similar effect is seen after 1100
.mu.g/mouse of Compound 5 when given by the oral route. This
suggests that the compound is absorbed from the gastrointestinal
tract.
25. In Vivo Studies with
TABLE-US-00005 Compound 1 (des Pro.sup.36-exendin-4(1-39)-NH.sub.2
(SEQ ID NO: 101)), Compound 2 (des
Pro.sup.36-exendin-4(1-39)-Lys6-NH.sub.2 (SEQ ID NO: 93)), Compound
(iii) (Gly.sup.8-GLP1-(7-36)(Human)-NH.sub.2 (SEQ ID NO: 87)),
Compound 4 (Gly.sup.8-GLP1-(7-36)(Human)-Lys.sub.6-NH.sub.2 (SEQ ID
NO: 88)) and Compound 5
(Gly.sup.8Lys.sup.37(palmitoyl)-GLP1-(7-36)(Human)-
Lys.sub.7-NH.sub.2 (SEQ ID NO: 89))
[0245] Various concentrations of each peptide are administered
orally and intraperitoneally to ob/ob mice to determine if these
compounds affect blood glucose levels. The experimental conditions
used were the same as described in Example 24.
Peptides and Other Materials
[0246] Des Pro.sup.36-exendin-4(1-39)-NH.sub.2 (Compound 1, SEQ ID
NO:101) and the same peptide, but with an additional sequence,
Lys.sub.6, attached at the C-terminal, des
Pro.sup.36-exendin-4(1-39)-Lys6-NH.sub.2 (Compound 2, SEQ ID
NO:93), Gly.sup.8-GLP1-(7-36)(Human)-NH.sub.2 (Compound (ii), SEQ
ID NO:87) and the same peptide, but with an additional sequence,
Lys.sub.6, attached at the C-terminal,
Gly.sup.8-GLP1-(7-36)(Human)-Lys.sub.6-NH.sub.2 (Compound 4, SEQ ID
NO:88) and
Gly.sup.8Lys.sup.37(palmitoyl)-GLP1-(7-36)(Human)-Lys.sub.7-NH.sub.2
(Compound 5, SEQ ID NO:89) are synthesized using methods described
above. Solutions are prepared on the morning of dosing, immediately
before the animals are administered. The same solutions are used
for both peroral and interperitoneal administration. All peptides
are dissolved in sterile isotonic NaCl and given in a volume of 0.2
ml. All experiments are carried out in the same mice to compare the
active doses of the peptides shown in Table 2. Blood sampling is
performed as described above and the animals are administered with
the doses shown in Table 3. As negative control, saline is
administered perorally. Results are shown in Table 4.
TABLE-US-00006 TABLE 2 Number Compound Compound 1 des
Pro.sup.36-exendin-4(1-39)-NH.sub.2 (SEQ ID NO: 101) Compound 2 des
Pro.sup.36-exendin-4(1-39)-Lys.sub.6-NH2 (SEQ ID NO: 93) Compound
(ii) G1y.sup.8-GLP1-(7-36)(Human)-NH2 (SEQ ID NO: 87) Compound 4
Gly.sup.8-GLP1-(7-36)(Human)-Lys.sub.6-NH2 (SEQ ID NO: 88) Compound
5 Gly.sup.8Lys.sup.37(palmitoyl)-GLP1-
(7-36)(Human)-Lys.sub.7-NH.sub.2 (SEQ ID NO: 89)
TABLE-US-00007 TABLE 3 Group Group Group Group Group 5 Group 1 2 3
4 Dose 6 Dose Dose Dose Dose peroral Dose peroral peroral peroral
peroral .mu.l/mouse i.p .mu.g/ .mu.g/ .mu.g/ .mu.g/ Isotonic .mu.g/
Compound mouse mouse mouse mouse saline mouse Compound 1 400 40 4
0.4 200 .mu.l 40 Compound 2 1000 100 10 1 200 .mu.l 100 Compound
(ii) 1000 100 10 1 200 .mu.l 100 Compound 4 1000 100 10 1 200 .mu.l
100 Compound 5 1000 100 10 1 200 .mu.l 100
[0247] Group data were summarised as the mean.+-.SEM of the
individual results in each treatment group. In order to analyse the
effects of the compounds, the absolute and the relative (% of t=0)
difference from baseline was calculated for each time point.
TABLE-US-00008 TABLE 4 0 1 hour 2 hours 4 hours Compound 1-saline
100 103 107 92 Compound 1-400 .mu.g po 100 93 88 93 Compound 1-40
.mu.g po 100 89 89 91 Compound 1-4 .mu.g po 100 105 88 91 Compound
1-0.4 .mu.g po 100 106 103 100 Compound 1-40 .mu.g ip 100 68 69 74
Compound 2-saline 100 100 112 114 Compound 2-1000 .mu.g po 100 67
69 64 Compound 2 100 .mu.g po 100 78 71 72 Compound 2-10 .mu.g po
100 86 72 72 Compound 2 1 .mu.g po 100 112 101 96 Compound 2 100
.mu.g ip 100 75 67 63 Compound (ii)-saline 100 95 87 100 Compound
(ii)-1000 .mu.g po 100 87 105 94 Compound (ii)-100 .mu.g po 100 118
111 92 Compound (ii)-10 .mu.g po 100 101 94 104 Compound (ii)-1
.mu.g po 100 94 89 96 Compound (ii)-100 .mu.g ip 100 70 60 81
Compound 4-saline 100 102 94 79 Compound 4-1000 .mu.g po 100 128 72
78 Compound 4-100 .mu.g po 100 72 70 58 Compound 4-10 .mu.g po 100
98 95 81 Compound 4-1 .mu.g po 100 99 89 84 Compound 4 100 .mu.g ip
100 83 58 56 Compound 5-saline 100 90 86 103 Compound 5-1000 .mu.g
po 100 73 75 67 Compound 5-100 .mu.g po 100 97 140 107 Compound
5-10 .mu.g po 100 90 120 126 Compound 5-1 .mu.g po 100 111 133 114
Compound 5-100 .mu.g ip 100 63 50 52
[0248] The results obtained are shown in Table 4 and in FIGS.
1-3.
[0249] These results show that all tested compounds have an effect
in lowering blood glucose levels.
[0250] The effect is most pronounced when Compound 1 is given
intraperitoneally whereas the effect of 1000 .mu.g po of Compound 2
is comparable to the effect of 100 .mu.g ip of Compound 2. The
potency of Compound 1 (des Pro.sup.36-exendin-4(1-39)-NH.sub.2, SEQ
ID NO:101) and Compound 2 (des
Pro.sup.36-exendin-4(1-39)-Lys.sub.6-NH.sub.2, SEQ ID NO:93) when
given intraperitoneally is shown to be very similar to
exendin-4(1-39)-NH.sub.2 (SEQ ID NO: 102) (Compound (i)) itself
(data not given) administered in the same way.
[0251] For Compound 1, des Pro.sup.36-exendin-4(1-39)-NH.sub.2 (SEQ
ID NO:101), there is no effect in lowering blood glucose levels up
to a dose of 400 .mu.g/mouse when the compound is administered
perorally, whereas for the same compound with the addition of the
Lys6 fragment there is activity seen at a dose of 10 .mu.g/mouse.
This indicates that the minimum effective oral dose of the des
Pro.sup.36-exendin-4(1-39)-Lys.sub.6-NH2 (SEQ ID NO:93) is at least
40 times lower than for des Pro.sup.36-exendin-4(1-39)-NH.sub.2
(SEQ ID NO:101).
[0252] These results show that the attachment of the sequence Z has
no significant effect on the potency of the various peptides when
administered interperitoneally while significantly enhancing the
potency of the compound when administered perorally.
26. Bioavailability of Compound 4 and Compound (iii) after
Gastro-Intestinal Delivery in Duodenum in Conscious Rats.
[0253] Various peptide based GLP-1 analogues have been developed
for parenteral use, but none of these substances has been
pharmacologically effective after oral administration [Hoist, J.
J.: Enteroglucagon. Annu Rev Physiol, 59:257-271, 1997]. It was
decided to examine the absorption of the test compound from the
duodenum in conscious rats. Compound (iii)
(Gly.sup.8)hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 87) was used as
reference.
Chemicals and Reagents
[0254] Blank rat plasma in sodium heparin (5000 units/mL) were
obtained from Harlan Sera Lab Ltd. (Loughborough, UK). OASIS.TM.
HLB solid phase extraction columns, 1 cc, 30 mg sorbent, were
obtained from Waters (Milford, Mass., USA) and ISOLUTE C18 (EC), 1
cc, SPE columns were obtained from IST (Mid Glamorgan, U.K.). The
LC/MS analysis was performed on a HP 1100 instrument consisting of
an on-line degasser, a binary gradient pump, an auto sampler, a
column oven, Hewlett Packard (Wilmington, Del., USA) in combination
with a Quattro Ultima mass spectrometer from Micromass (Altrincham,
UK) both the LC and MS were controlled by MassLynx 3.3 software.
The LC separations prior to MS detection were performed on a Vydac
218MS52 (2.1.times.250 mm) column (Hesperia, Calif., USA).
Drugs and Dose Levels:
[0255] Compound 4 (batch No. ZP 7.97-5-F, 4053 g/mol) and Compound
(iii) (batch No. ZP 7.73-2-G, 3854 g/mol) were synthesised in-house
using the Fmoc strategy. The identification was performed by mass
spectrometry and the purity of both batches was determined by
RP-HPLC to 97 and 99.7% for the test compounds, respectively. The
peptide content of the batches were 72% and 80% for ZP 7.97-5-F and
ZP 7.73-2-G, respectively. The peptides were dissolved in pyrogen
free isotonic saline and doses of 1.000 or 10.000 nmol/kg
administered through the intra duodenal catheter in a volume of 100
.mu.l.
Animals:
[0256] Fourteen Sprague-Dawley rats weighing 250 to 350 g. were
used for the experiment. The rats were anaesthetised with
Hypnorm.RTM.-Dormicum.RTM. s.c. and a catheter was inserted into
the femoral artery for arterial blood sampling. An additional
catheter was inserted into the duodenum via an incision in the
ventricle. Before the experiment was started, the rats were allowed
to recover for one week after the operation. The operated rats were
conscious at the day of the experiment. In order to establish
whether the intra duodenal catheters were situated in the duodenum,
an autopsy was performed on the rats immediately after the
experiment.
Sample Treatment:
[0257] Blood samples were collected at t=-5, 5, 10, 15, 20, 40, and
60 min. The blood was collected in EDTA containing ice-chilled
tubes and immediately centrifuged at 4.degree. C. for 5 min
(4.000.times.g). Plasma (250 .mu.l) was transferred to ice-chilled
0.75 ml PLC vials containing 250 .mu.l extraction solution (MeCN:
0.18 M Ammonium Carbonate pH 9.5, 10:90 v/v). The plasma samples
were stored at -20?C until SPE and LC/MS analysis.
Solid Phase Extraction:
[0258] The drug containing plasma samples (400 .mu.l) were loaded
onto solid phase extraction columns preconditioned with 950 .mu.l
MeCN followed by 950 .mu.l water. The columns were washed with 950
.mu.l 2% TFA in water followed by an equal volume of 2% TFA in
MeCN:water (20:78 v/v). The analytes were eluted with 500 .mu.l 2%
TFA in MeCN:water (60:38 v/v) and analysed by LC/MS.
LC/MS
[0259] The samples were kept at 18.degree. C. in the auto sampler
tray prior to injection of 20 to 50 .mu.l onto the LC column (Vydac
218MS52 (2.1.times.250 mm). The separations were performed at
30.degree. C. using a flow rate of 250 .mu.l/min and a gradient
according to Table 1. Both the test compound and the reference drug
were detected by single ion recording (SIR) using the m/z=676.7 and
the m/z=1095.2 and 821.8 ion species, respectively. All instrument
conditions were controlled by MassLynx software ver. 3.3
software.
TABLE-US-00009 Compound Gradient Compound 4 Initial: 15% B, 0-14
min; 15-50% B, 14-15 min; 50-15% B and 15-20 min 15% B. Compound
(iii) Initial: 25% B, 1-1.5 min; 25-30% B, 1.5-10 min; 30-40% B,
10-10.5 min; 40-90% B, 11.5-12 min; 90-25% B, and 12-17 min 25% B.
The gradient used for the analysis of the test compounds using 0.1%
formic acid in water or MeCN as Mobile phase A or B,
respectively.
[0260] The plasma samples were analysed as described under
materials and methods. The bioavailability of Compound 4 was
examined in doses of 1.000 (n=4) and 10.000 (n=5) nmol/k, whereas
Compound (iii) was only studied in a dose of 10.000 (n=5) nmol/kg.
At all the investigated time points the concentration of Compound
(iii) was below the detection limit (approx. 0.5 nM), the exact
bioavailability could therefore not be estimated. In contrast,
Compound 4 was detected in the plasma samples from two out of four
rats after intra duodenal administration of 1.000 nmol/kg and in
four out of five rats following administration of 10.000
nmol/kg.
27. In Vivo Pharmacokinetics of Compound 1, Compound 2, Compound 4,
and Compound (iii) After i.v. Administration to Rabbits and
Pigs
[0261] We have shown an increased in vitro stability of the GLP-1
agonist Compound 4 when compared to the reference drug Compound
(iii) in rat plasma. In order to establish whether this effect is
sustained in vivo, the pharmacokinetic parameters of the two
compounds are examined in rabbits. Using the same experimental
conditions these parameters were also measured for Compounds 1 and
2 in rabbits and using similar conditions in pigs.
Chemicals and Reagents
[0262] Blank rabbit plasma in sodium heparin (5000 units/mL) were
obtained from Harlan Sera Lab Ltd. (Loughborough, UK). OASIS.TM.
HLB solid phase extraction columns, 1 cc, 30 mg sorbent, were
obtained from Waters (Milford, Mass., USA) and ISOLUTE C18 (EC), 1
cc, SPE columns were obtained from IST (Mid Glamorgan, U.K.). The
LC/MS analysis was performed on a HP 1100 instrument consisting of
an on-line degasser, a binary gradient pump, an auto sampler, a
column oven, Hewlett Packard (Wilmington, Del., USA) in combination
with a Quattro Ultima mass spectrometer from Micromass (Altrincham,
UK) both the LC and MS were controlled by MassLynx 3.3 software.
The LC separations prior to MS detection were performed on a Vydac
218MS52 (2.1.times.250 mm) column (Hesperia, Calif., USA).
Drugs and Dose Levels:
[0263] Compound 4 (batch No. ZP 7.97-5-F, 4053 g/mol) and Compound
(iii) (batch No. ZP 7.73-2-G, 3854 g/mol) were synthesised in-house
using the Fmoc strategy. The identification was performed by mass
spectrometry and the purity of both batches were determined by
RP-HPLC to 97 and 99.7% for the test compounds, respectively. The
peptide content of the batches were 72% and 80% for ZP 7.97-5-F and
ZP 7.73-2-G, respectively. The peptides were dissolved in pyrogen
free isotonic saline and both peptides were administered i.v. to
rabbits and rats using a dose of 1000 nmol/kg.
Rabbits:
[0264] Fifteen New Zealand White rabbits weighing 2.5 to 3.0 kg
were used for the experiment. On the day of the experiment, the
rabbits were anaesthetised with Hypnorm.RTM. i.m. followed by
Dormicum.RTM. i.v. Catheters were inserted into the femoral vein
and artery for i.v. administration of drugs and arterial blood
sampling. The rabbits stayed unconscious throughout the
experiment.
Sample Treatment:
[0265] Blood Samples were collected at t=1, 3, 5, 10, 15, 20, 30,
40, 60, 90, 120, 150, 180 and 240 min. The blood was collected in
EDTA containing ice-chilled tubes and immediately centrifuged at 4
C for 5 min (20.000.times.g). Plasma (250 .mu.l) was transferred to
ice-chilled 0.75 ml PLC vials containing 250 .mu.l extraction
solution (MeCN: 0.18 M Ammonium Carbonate pH 9.5, 10:90 v/v). The
plasma samples were stored at -20 C until SPE and LC/MS
analysis.
Solid Phase Extraction:
[0266] The drug containing plasma samples (400 .mu.l) are loaded
onto OASIS.TM. HLB (Compound 4) or ISOLUTE.TM. (Compound (iii))
solid phase extraction columns preconditioned with 950 .mu.l MeCN
followed by 950 .mu.l water. The columns are washed with 950 .mu.l
2% TFA in water followed by an equal volume of 2% TFA in MeCN:water
(20:78 v/v). The analytes are eluted with 500 .mu.l 2% TFA in
MeCN:water (60:38 v/v) and analysed by LC/MS.
LC/MS
[0267] The samples were kept at 18 C in the auto sampler tray prior
to injection of 20 to 50 .mu.l onto the LC column (Vydac 218MS52
(2.1.times.250 mm). The separations were performed at 30.degree. C.
using a flow rate of 250 .mu.l/min and a gradient according to the
table below. Both the test compound and the reference drug are
detected by single ion recording (SIR) using the m/z=676.7 and the
m/z=1095.2 and 821.8 ion species, respectively. All instrument
conditions were controlled by MassLynx software ver. 3.3
software.
TABLE-US-00010 Compound Gradient Compound 4 Initial: 15% B, 0-14
min; 15-50% B, 14-15 min; 50-15% B and 15-20 min 15% B. Compound
(iii) Initial: 25% B, 1-1.5 min; 25-30% B, 1.5-10 min; 30-40% B,
10-10.5 min; 40-90% B, 11.5-12 min; 90-25% B, and 12-17 min 25% B.
The gradient used for the analysis of the test compounds using 0.1%
formic acid in water or MeCN as Mobile phase A or B,
respectively.
[0268] The plasma samples were analysed as described under
materials and methods and the plasma concentration (C.sub.pl)
plotted versus time in a semi log diagram. The plasma concentration
were followed for three hours in rabbits, whereas the limited blood
volume of rats restricted the blood sampling in this specie to one
hour. The C.sub.pl vs. time curves from the individual rabbits were
fitted to a two-compartment open model (figure not shown) using
1/y.sup.2 weighted least squares in WinNonlin 3.1 (Pharsight Corp.
(Mountain View, Calif.)). The pharmacokinetic constants obtained
from the data analysis are listed in Table 5 and the degradation
kinetics in rabbit after i.v. injection of 1 .mu.mol/kg of Compound
4 and Compound (iii), respectively, is shown in FIG. 4.
TABLE-US-00011 TABLE 5 In vivo kinetics in rabbits and pigs **
Comp. (iii) Comp. 4 Comp. 1 Comp. 2 Comp. 2 ** (n = 7) (n = 8) (n =
5) (n = 5) (n = 2) Parameter Mean Mean Mean Mean Mean T.sub.1/2,
.alpha. min 23 6.8 4.4 11 16 T.sub.1/2, .beta. min 10.8 28.0 23 69
252 Table 5: The pharmacokinetic constants were obtained from
rabbits when the C.sub.pl vs. time curves was fitted
mathematically. The compounds were administered iv in a
concentration of 1000 nmol/kg. T.sub.1/2 values are given in
minutes (min) for the .alpha. and .beta. phase. Statistics:
two-tailed t-test assuming samples with unequal variances showed p
< 0.001 for all measured parameters. In conclusion the T1/2
value for Compound 4 is approximately three times the value for the
reference Compound (iii) and, likewise, the T1/2 value for Compound
2 is approximately three times the value calculated for Compound 1
which represents the unconjugated equivalent.
28. Glucose Tolerance Test of Compounds 2, 14-16, 18 and 19
Compared to Compound (i)
[0269] Male diabetic db/db mice (M&B, Bomholdtgaard, LI.
Skensved, Denmark) are used. This well-described mouse model has
inherited malfunctions of the glucose metabolism due to a mutation
in the leptin receptor. Like human patients with uncontrolled
non-insulin demanding diabetes mellitus (NIDDM), homozygous db/db
mice experience polydipsia, polyuria and glycosuria and gain weight
during their first 3 months of life despite their hyperglycaemic
stage. However, in this model the hyperglycaemia is associated with
progressive pancreatic islet atrophy with possible ketosis and
death at 6-8 months of age. Thus, attention should be paid to the
progression and status of their disease state. Therefore,
preferably only db/db mice less than 16 weeks old should be used
for drug testing og GLP-1 analogues.
[0270] All animals are acclimatised for at least one week and
handled daily for two days prior to the first oral glucose
tolerance test (OGTT). Furthermore, to reduce stress-induced
glucose excursions, the animals should be subjected to at least one
OGTT without compound as described below prior to the experiment.
Due to the great scatter of glucose tolerance among diabetic mice,
the animals are stratified by an OGTT prior to their first use.
Peptides
[0271] Peptides are dissolved in 0.1 M phosphate-buffered saline
(PBS) with 0.1% bovine albumin where pH is adjusted to 7.4 by
adding 5 M NaOH. All solutions are prepared fresh on the morning
immediately before the experiment. Vehicle treated animals are
given PBS with 0.1% albumin alone.
Glucose Tolerance Test and Dosing
[0272] Before the oral glucose tolerance test, the animals are
fasted for 17 hours (from 4 p.m. until 9 a.m. the following
morning). Beginning at 9.00 a.m. blood is taken from the tail tip
(t=-15 min) and blood glucose is measured. The whole blood glucose
(mM) concentration is analysed by the immobilised glucose oxidase
method using a drop of blood (<5 .mu.l, Elite Autoanalyser,
Bayer, Denmark) following the manufacturer's manual. Animals with
severe diabetes (>10 mM) are excluded. Immediately after the
initial blood sample, the animals receive an intraperitoneal (i.p.)
injection of vehicle or a dose of antidiabetic compound. Injection
volume is 200 .mu.l/50 g body weight in all groups. Fifteen minutes
after i.p. administration of the substance an oral dose of 1 g/kg
glucose (Sigma, St. Louis) dissolved in water (200 .mu.l/50 g body
weight) is given, and the animals are returned to their home cages
(t=0). Blood glucose levels are measured at t=30 mM, t=60 min,
t=120 mM and t=240 min. The animals are fasted during the
observation period. For each animal a data log sheet was filled in
at the time of each blood sampling.
Calculations and Statistics
[0273] In order to analyse the effects of the compounds, the
absolute and the relative difference from baseline (t=0) are
calculated for each time point. The area under the curve for the
whole experiment (AUC 0-240 min) is determined using the trapezoid
method. On the day of stratification, the mice are distributed in
order to ensure that the glucose tolerances are similar in all
groups. However, to correct for the progression of the diabetes
with time, a vehicle treated control group is tested on each day of
experiment and the response to drugs are expressed relative to
response observed in vehicle-treated time-control animals.
Dose-response curves for each substance are plotted, cf. FIG. 5,
and the effect of drug relative to responses obtained during
treatment with vehicle are analysed using an ANCOVA analysis
(analysis of covariance). Treatment (drug or vehicle) is considered
the independent variable, AUC 0-240 min expressed as percent
response in vehicle-treated time-control mice is the dependent
variable, and drug dose is defined as covariate. Post-hoc analysis
is performed using Fisher's Least Significant test. Differences are
considered significant at the 0.05 level. Statistical analyses were
performed using Statistica version 5.5 for Windows NT, StatSoft,
Tulsa, Okla., U.S.A. The dose response curves shown in FIG. 5
clearly shows that all tested compounds exhibit a glucose lowering
effect comparable to that of the reference drug.
29. Effects of Compound 2 and Compound (i) on OGGT in db/db
Mice
[0274] FIG. 7 is a plot of AUC for Compound 2 and Compound (i) in
an OGTT performed using the same experimental conditions as
described in Example 28. The figure shows that the blood glucose
lowering effect of Compound 2 is the same as the effect of the
prior art compound (iii).
30. Long Term Effects of Compound 2, 100 Nmol/Kg i.p. On the Oral
Glucose Tolerance Test (OGTT) when Administered Up to 24 Hours
Before the OGTT
[0275] This experiment uses the maximal dose of 100 nmol/kg i.p. in
db/db mice and otherwise, the same experimental conditions as
described in Example 28 are used. Results are shown in FIG. 8 and
the conclusion of the experiment is that the duration of action of
Compound 2 is up to 18 hours in db/db mice.
[0276] The invention described and claimed herein is not to be
limited in scope by the specific embodiments herein disclosed,
since these embodiments are intended as illustrations of several
aspects of the invention. Any equivalent embodiments are intended
to be within the scope of this invention. Indeed, various
modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art
from the fore-going description. Such modifications are also
intended to fall within the scope of the appended claims. Various
references are cited herein, the disclosure of which are
incorporated by reference in their entireties.
Sequence CWU 1
1
15314PRTArtificial Sequencesynthetic peptide sequence 1Lys Lys Lys
Lys 1 25PRTArtificial Sequencesynthetic peptide sequence 2Lys Lys
Lys Lys Lys 1 5 35PRTArtificial Sequencesynthetic peptide sequence
3Xaa Lys Lys Lys Lys 1 5 45PRTArtificial Sequencesynthetic peptide
sequence 4Lys Xaa Lys Lys Lys 1 5 55PRTArtificial Sequencesynthetic
peptide sequence 5Lys Lys Xaa Lys Lys 1 5 65PRTArtificial
Sequencesynthetic peptide sequence 6Lys Lys Lys Xaa Lys 1 5
75PRTArtificial Sequencesynthetic peptide sequence 7Lys Lys Lys Lys
Xaa 1 5 86PRTArtificial Sequencesynthetic peptide sequence 8Lys Lys
Lys Lys Lys Lys 1 5 96PRTArtificial Sequencesynthetic peptide
sequence 9Xaa Lys Lys Lys Lys Lys 1 5 106PRTArtificial
Sequencesynthetic peptide sequence 10Lys Xaa Lys Lys Lys Lys 1 5
116PRTArtificial Sequencesynthetic peptide sequence 11Lys Lys Xaa
Lys Lys Lys 1 5 126PRTArtificial Sequencesynthetic peptide sequence
12Lys Lys Lys Xaa Lys Lys 1 5 136PRTArtificial Sequencesynthetic
peptide sequence 13Lys Lys Lys Lys Xaa Lys 1 5 146PRTArtificial
Sequencesynthetic peptide sequence 14Lys Lys Lys Lys Lys Xaa 1 5
156PRTArtificial Sequencesynthetic peptide sequence 15Xaa Xaa Lys
Lys Lys Lys 1 5 166PRTArtificial Sequencesynthetic peptide sequence
16Xaa Lys Xaa Lys Lys Lys 1 5 176PRTArtificial Sequencesynthetic
peptide sequence 17Xaa Lys Lys Xaa Lys Lys 1 5 186PRTArtificial
Sequencesynthetic peptide sequence 18Xaa Lys Lys Lys Xaa Lys 1 5
196PRTArtificial Sequencesynthetic peptide sequence 19Xaa Lys Lys
Lys Lys Xaa 1 5 206PRTArtificial Sequencesynthetic peptide sequence
20Lys Xaa Xaa Lys Lys Lys 1 5 216PRTArtificial Sequencesynthetic
peptide sequence 21Lys Xaa Lys Xaa Lys Lys 1 5 226PRTArtificial
Sequencesynthetic peptide sequence 22Lys Xaa Lys Lys Xaa Lys 1 5
236PRTArtificial Sequencesynthetic peptide sequence 23Lys Xaa Lys
Lys Lys Xaa 1 5 246PRTArtificial Sequencesynthetic peptide sequence
24Lys Lys Xaa Xaa Lys Lys 1 5 256PRTArtificial Sequencesynthetic
peptide sequence 25Lys Lys Xaa Lys Xaa Lys 1 5 266PRTArtificial
Sequencesynthetic peptide sequence 26Lys Lys Xaa Lys Lys Xaa 1 5
276PRTArtificial Sequencesynthetic peptide sequence 27Lys Lys Lys
Xaa Xaa Lys 1 5 286PRTArtificial Sequencesynthetic peptide sequence
28Lys Lys Lys Xaa Lys Xaa 1 5 296PRTArtificial Sequencesynthetic
peptide sequence 29Lys Lys Lys Lys Xaa Xaa 1 5 307PRTArtificial
Sequencesynthetic peptide sequence 30Lys Lys Lys Lys Lys Lys Lys 1
5 317PRTArtificial Sequencesynthetic peptide sequence 31Xaa Lys Lys
Lys Lys Lys Lys 1 5 327PRTArtificial Sequencesynthetic peptide
sequence 32Lys Xaa Lys Lys Lys Lys Lys 1 5 337PRTArtificial
Sequencesynthetic peptide sequence 33Lys Lys Xaa Lys Lys Lys Lys 1
5 347PRTArtificial Sequencesynthetic peptide sequence 34Lys Lys Lys
Xaa Lys Lys Lys 1 5 357PRTArtificial Sequencesynthetic peptide
sequence 35Lys Lys Lys Lys Xaa Lys Lys 1 5 367PRTArtificial
Sequencesynthetic peptide sequence 36Lys Lys Lys Lys Lys Xaa Lys 1
5 377PRTArtificial Sequencesynthetic peptide sequence 37Lys Lys Lys
Lys Lys Lys Xaa 1 5 387PRTArtificial Sequencesynthetic peptide
sequence 38Xaa Xaa Lys Lys Lys Lys Lys 1 5 397PRTArtificial
Sequencesynthetic peptide sequence 39Xaa Lys Xaa Lys Lys Lys Lys 1
5 407PRTArtificial Sequencesynthetic peptide sequence 40Xaa Lys Lys
Xaa Lys Lys Lys 1 5 417PRTArtificial Sequencesynthetic peptide
sequence 41Xaa Lys Lys Lys Xaa Lys Lys 1 5 427PRTArtificial
Sequencesynthetic peptide sequence 42Xaa Lys Lys Lys Lys Xaa Lys 1
5 437PRTArtificial Sequencesynthetic peptide sequence 43Lys Xaa Xaa
Lys Lys Lys Lys 1 5 447PRTArtificial Sequencesynthetic peptide
sequence 44Lys Xaa Lys Xaa Lys Lys Lys 1 5 457PRTArtificial
Sequencesynthetic peptide sequence 45Lys Xaa Lys Lys Xaa Lys Lys 1
5 467PRTArtificial Sequencesynthetic peptide sequence 46Lys Xaa Lys
Lys Lys Xaa Lys 1 5 477PRTArtificial Sequencesynthetic peptide
sequence 47Lys Lys Xaa Xaa Lys Lys Lys 1 5 487PRTArtificial
Sequencesynthetic peptide sequence 48Lys Lys Xaa Lys Xaa Lys Lys 1
5 497PRTArtificial Sequencesynthetic peptide sequence 49Lys Lys Xaa
Lys Lys Xaa Lys 1 5 507PRTArtificial Sequencesynthetic peptide
sequence 50Lys Lys Lys Xaa Xaa Lys Lys 1 5 517PRTArtificial
Sequencesynthetic peptide sequence 51Lys Lys Lys Xaa Lys Xaa Lys 1
5 527PRTArtificial Sequencesynthetic peptide sequence 52Lys Lys Lys
Lys Xaa Xaa Lys 1 5 537PRTArtificial Sequencesynthetic peptide
sequence 53Xaa Xaa Xaa Lys Lys Lys Lys 1 5 547PRTArtificial
Sequencesynthetic peptide sequence 54Xaa Xaa Lys Xaa Lys Lys Lys 1
5 557PRTArtificial Sequencesynthetic peptide sequence 55Xaa Xaa Lys
Lys Xaa Lys Lys 1 5 567PRTArtificial Sequencesynthetic peptide
sequence 56Xaa Xaa Lys Lys Lys Xaa Lys 1 5 577PRTArtificial
Sequencesynthetic peptide sequence 57Xaa Lys Xaa Xaa Lys Lys Lys 1
5 587PRTArtificial Sequencesynthetic peptide sequence 58Xaa Lys Xaa
Lys Xaa Lys Lys 1 5 597PRTArtificial Sequencesynthetic peptide
sequence 59Xaa Lys Xaa Lys Lys Xaa Lys 1 5 607PRTArtificial
Sequencesynthetic peptide sequence 60Xaa Lys Lys Xaa Xaa Lys Lys 1
5 617PRTArtificial Sequencesynthetic peptide sequence 61Xaa Lys Lys
Xaa Lys Xaa Lys 1 5 627PRTArtificial Sequencesynthetic peptide
sequence 62Xaa Lys Lys Lys Xaa Lys Xaa 1 5 637PRTArtificial
Sequencesynthetic peptide sequence 63Xaa Lys Lys Xaa Lys Lys Xaa 1
5 647PRTArtificial Sequencesynthetic peptide sequence 64Xaa Lys Xaa
Lys Lys Lys Xaa 1 5 657PRTArtificial Sequencesynthetic peptide
sequence 65Xaa Lys Lys Lys Xaa Xaa Lys 1 5 667PRTArtificial
Sequencesynthetic peptide sequence 66Lys Xaa Lys Lys Lys Xaa Xaa 1
5 677PRTArtificial Sequencesynthetic peptide sequence 67Xaa Lys Lys
Lys Lys Xaa Xaa 1 5 687PRTArtificial Sequencesynthetic peptide
sequence 68Xaa Lys Lys Lys Xaa Lys Xaa 1 5 697PRTArtificial
Sequencesynthetic peptide sequence 69Xaa Lys Lys Lys Xaa Xaa Lys 1
5 707PRTArtificial Sequencesynthetic peptide sequence 70Lys Lys Lys
Lys Xaa Xaa Xaa 1 5 717PRTArtificial Sequencesynthetic peptide
sequence 71Lys Lys Lys Xaa Xaa Xaa Lys 1 5 727PRTArtificial
Sequencesynthetic peptide sequence 72Lys Lys Lys Xaa Lys Xaa Xaa 1
5 737PRTArtificial Sequencesynthetic peptide sequence 73Lys Lys Xaa
Lys Lys Xaa Xaa 1 5 747PRTArtificial Sequencesynthetic peptide
sequence 74Lys Lys Xaa Xaa Lys Xaa Lys 1 5 757PRTArtificial
Sequencesynthetic peptide sequence 75Lys Lys Xaa Xaa Xaa Lys Lys 1
5 767PRTArtificial Sequencesynthetic peptide sequence 76Lys Lys Xaa
Lys Lys Xaa Xaa 1 5 777PRTArtificial Sequencesynthetic peptide
sequence 77Lys Xaa Lys Lys Xaa Xaa Lys 1 5 787PRTArtificial
Sequencesynthetic peptide sequence 78Lys Xaa Lys Xaa Lys Xaa Lys 1
5 797PRTArtificial Sequencesynthetic peptide sequence 79Lys Xaa Lys
Xaa Xaa Lys Lys 1 5 807PRTArtificial Sequencesynthetic peptide
sequence 80Lys Xaa Xaa Lys Lys Xaa Lys 1 5 817PRTArtificial
Sequencesynthetic peptide sequence 81Lys Xaa Xaa Lys Xaa Lys Lys 1
5 827PRTArtificial Sequencesynthetic peptide sequence 82Lys Xaa Xaa
Xaa Lys Lys Lys 1 5 836PRTArtificial Sequencesynthetic peptide
sequence 83Lys Glu Lys Glu Lys Glu 1 5 846PRTArtificial
Sequencesynthetic peptide sequence 84Glu Lys Glu Lys Glu Lys 1 5
856PRTArtificial Sequencesynthetic peptide sequence 85Lys Lys Lys
Glu Glu Glu 1 5 866PRTArtificial Sequencesynthetic peptide sequence
86Glu Glu Glu Lys Lys Lys 1 5 8730PRTArtificial
SequenceGly8-GLP-1-(7-36)(Human)-NH2 87His Gly Glu Gly Thr Phe Thr
Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu
Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 8836PRTArtificial
SequenceGly8-GLP-1-(7-36)(Human)-Lys6-NH2 88His Gly Glu Gly Thr Phe
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys
Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys Lys 20 25 30 Lys Lys
Lys Lys 35 8938PRTArtificial Sequence
Gly8Lys37(palmitoyl)-GLP-1-(7-36)(Human)-Lys7-NH2 89His Gly Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala
Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys Lys 20 25 30
Lys Lys Lys Lys Lys Lys 35 9036PRTArtificial Sequence
Gly8Lys34(palmitoyl)-GLP-1-(7-36)(Human)-Lys6-NH2 90His Gly Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala
Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys Lys 20 25 30
Lys Lys Lys Lys 35 9144PRTArtificial Sequencedes
Ser39-exendin-4-Lys6-NH2 91His Gly Glu Gly Thr Phe Thr Ser Asp Leu
Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro
Lys Lys Lys Lys Lys Lys 35 40 9245PRTArtificial
Sequenceexendin-4-Lys6-NH2 92His Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile
Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro
Pro Ser Lys Lys Lys Lys Lys Lys 35 40 45 9344PRTArtificial
Sequencedes Pro36-exendin-4-Lys6-NH2 93His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu
Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala
Pro Pro Ser Lys Lys Lys Lys Lys Lys 35 40 9444PRTArtificial
Sequencedes Ala35-exendin-4-Lys6-NH2 94His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu
Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Pro
Pro Pro Ser Lys Lys Lys Lys Lys Lys 35 40 9544PRTArtificial
Sequencedes Gly34-exendin-4-Lys6-NH2 95His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu
Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Ala Pro
Pro Pro Ser Lys Lys Lys Lys Lys Lys 35 40 9646PRTArtificial
Sequencedes Ser39-(Lys40(palmitoyl))exendin-4-Lys7-NH2 96His Gly
Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20
25 30 Ser Gly Ala Pro Pro Pro Lys Lys Lys Lys Lys Lys Lys Lys 35 40
45 9746PRTArtificial Sequencedes
Gly34-(Lys40(palmitoyl))exendin-4-Lys7-NH2 97His Gly Glu Gly Thr
Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val
Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser
Ala Pro Pro Pro Ser Lys Lys Lys Lys Lys Lys Lys Lys 35 40 45
9846PRTArtificial Sequencedes
Ala35-(Lys40(palmitoyl))exendin-4-Lys7-NH2 98His Gly Glu Gly Thr
Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val
Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser
Gly Pro Pro Pro Ser Lys Lys Lys Lys Lys Lys Lys Lys 35 40 45
9946PRTArtificial Sequencedes
Pro36-(Lys40(palmitoyl))exendin-4-Lys7 99His Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly
Ala Pro Pro Ser Lys Lys Lys Lys Lys Lys Lys Lys 35 40 45
10047PRTArtificial SequenceLys40(palmitoyal)exendin-4-Lys7-NH2
100His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly
Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser Lys Lys Lys Lys Lys
Lys Lys Lys 35 40 45 10138PRTArtificial Sequencedes
Pro36-exendin-4-NH2 101His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp
Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Ser 35
10239PRTHeloderma suspectum 102His Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile
Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro
Pro Ser 35 10336PRTArtificial Sequence
Gly8Lys26(palmitoyal)-GLP-1-(7-36)(Human)-Lys6-NH2 103His Gly Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys Lys 20 25
30 Lys Lys Lys Lys 35 10438PRTArtificial Sequencedes
Ser39-exendin-4-NH2 104His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp
Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro 35
10538PRTArtificial Sequencedes Ala35-exendin-4-NH2 105His Gly Glu
Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25
30 Ser Gly Pro Pro Pro Ser 35 10638PRTArtificial Sequencedes
Gly34-exendin-4-NH2 106His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp
Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Ala Pro Pro Pro Ser 35
10739PRTArtificial Sequencedes Ser39-(Lys40
(palmitoyal))exendin-4-NH2 107His Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile
Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro
Pro Lys 35 10839PRTArtificial Sequencedes Gly34-(lys40
(palmitoyal))exendin-4-NH2 108His Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile
Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30 Ser Ala Pro Pro Pro Ser Lys 35 10939PRTArtificial
Sequencedes Ala35-(Lys40(palmitoyal))exendin-4-NH2 109His Gly Glu
Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25
30 Ser Gly Pro Pro Pro Ser Lys 35 11039PRTArtificial Sequencedes
Pro36-(Lys40 (palmitoyal))exendin-4-NH2 110His Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly
Ala Pro Pro Ser Lys 35 11131PRTArtificial
SequenceGly8Lys37N-palmitoyal-GLP-1 (7-36) 111His Gly Glu Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys 20 25 30
11230PRTArtificial SequenceGly8Lys34N-palmitoyal-GLP-1 (7-36)
112His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30 11330PRTArtificial SequenceGly8Lys26N-palmitoyal-GLP-1
(7-36) 113His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 30 11430PRTHomo sapiens 114His Ala Glu Gly Thr Phe
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys
Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 11536PRTArtificial
SequenceSer8-GLP-1(7-36)-Lys6 115His Ser Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu Val Lys Gly Arg Lys Lys 20 25 30 Lys Lys Lys Lys 35
11636PRTArtificial SequenceAib8-GLP-1(7-36)-Lys6 116His Xaa Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala
Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys Lys 20 25 30
Lys Lys Lys Lys 35 11737PRTArtificial SequenceGly8-GLP-1
(7-36)-Lys7 117His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val
Lys Gly Arg Lys Lys 20 25 30 Lys Lys Lys Lys Lys 35
11842PRTArtificial SequenceLys6-Gly8-GLP-1(7-36)-Lys6 118Lys Lys
Lys Lys Lys Lys His Gly Glu Gly Thr Phe Thr Ser Asp Val 1 5 10 15
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu 20
25 30 Val Lys Gly Arg Lys Lys Lys Lys Lys Lys 35 40
11936PRTArtificial SequenceLys6-Gly8-GLP-1(7-36) 119Lys Lys Lys Lys
Lys Lys His Gly Glu Gly Thr Phe Thr Ser Asp Val 1 5 10 15 Ser Ser
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu 20 25 30
Val Lys Gly Arg 35 12038PRTArtificial SequenceGly8-GLP-1(7-36)-Lys8
120His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
Lys Lys 20 25 30 Lys Lys Lys Lys Lys Lys 35 12140PRTArtificial
SequenceGly8-GLP-1(7-36)-Lys10 121His Gly Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu Val Lys Gly Arg Lys Lys 20 25 30 Lys Lys Lys Lys
Lys Lys Lys Lys 35 40 12237PRTArtificial
SequenceGly8-GLP-1(7-37)-Lys6 122His Gly Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu Val Lys Gly Arg Gly Lys 20 25 30 Lys Lys Lys Lys
Lys 35 12331PRTArtificial SequenceGly8-GLP-1(7-37) 123His Gly Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30
12431PRTHomo sapiens 124His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser
Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp
Leu Val Lys Gly Arg Gly 20 25 30 12528PRTArtificial
SequenceGLP-1(9-36)(Human) 125Glu Gly Thr Phe Thr Ser Asp Val Ser
Ser Tyr Leu Glu Gly Gln Ala 1 5 10 15 Ala Lys Glu Phe Ile Ala Trp
Leu Val Lys Gly Arg 20 25 12639PRTArtificial
Sequence[Tyr39]exendin-4 126His Gly Glu Gly Thr Phe Thr Ser Asp Leu
Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro
Tyr 35 12730PRTHeloderma suspectum 127His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu
Phe Ile Glu Trp Leu Lys Asn Gly Gly 20 25 30 12830PRTHeloderma
suspectum 128Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe
Ile Glu Trp 1 5 10 15 Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro
Pro Pro Ser 20 25 30 12938PRTArtificial Sequencedes Ser39-exendin-4
129His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly
Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro 35 13037PRTArtificial
Sequencedes-Pro36,Pro37-exendin-4 130His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu
Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala
Pro Ser 35 13143PRTArtificial
Sequencedes-Pro36,Pro37-exendin-4-Lys6 131His Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly
Ala Pro Ser Lys Lys Lys Lys Lys Lys 35 40 13236PRTArtificial
Sequencedes-Pro36,Pro37, Pro38-exendin-4 132His Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly
Ala Ser 35 13342PRTArtificial Sequencedes-Pro36,Pro37,
Pro38-exendin-4-Lys6 133His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp
Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Ser Lys Lys Lys
Lys Lys Lys 35 40 13442PRTArtificial SequenceLys6-des-Pro36, pro37,
Pro38-exendin-4 134Lys Lys Lys Lys Lys Lys His Gly Glu Gly Thr Phe
Thr Ser Asp Leu 1 5 10 15 Ser Lys Gln Met Glu Glu Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu 20 25 30 Lys Asn Gly Gly Pro Ser Ser Gly
Ala Ser 35 40 13548PRTArtificial SequenceLys6-des-Pro36, Pro37,
Pro38-exendin-4-Lys6 135Lys Lys Lys Lys Lys Lys His Gly Glu Gly Thr
Phe Thr Ser Asp Leu 1 5 10 15 Ser Lys Gln Met Glu Glu Glu Ala Val
Arg Leu Phe Ile Glu Trp Leu 20 25 30 Lys Asn Gly Gly Pro Ser Ser
Gly Ala Ser Lys Lys Lys Lys Lys Lys 35 40 45 13648PRTArtificial
SequenceAsn(Glu)5-des-pro36,Pro37, Pro38-exendin-4-Lys6 136Asn Glu
Glu Glu Glu Glu His Gly Glu Gly Thr Phe Thr Ser Asp Leu 1 5 10 15
Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu 20
25 30 Lys Asn Gly Gly Pro Ser Ser Gly Ala Ser Lys Lys Lys Lys Lys
Lys 35 40 45 13742PRTArtificial SequenceAsn(Glu)5-des-Pro36, Pro37,
Pro38-exendin-4 137Asn Glu Glu Glu Glu Glu His Gly Glu Gly Thr Phe
Thr Ser Asp Leu 1 5 10 15 Ser Lys Gln Met Glu Glu Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu 20 25 30 Lys Asn Gly Gly Pro Ser Ser Gly
Ala Ser 35 40 13840PRTHeloderma suspectum 138His Gly Glu Gly Thr
Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val
Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser
Gly Ala Pro Pro Pro Ser Gly 35 40 13939PRTArtificial
Sequencedes-Pro36-exendin-4 (1-40) 139His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu
Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala
Pro Pro Ser Gly 35 14037PRTArtificial Sequencedes-Pro36,Pro37,
Pro38-exendin-4 (1-40) 140His Gly Glu Gly Thr Phe Thr Ser Asp Leu
Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Ser Gly 35
1416PRTArtificial Sequencesynthetic peptide sequence 141Asn Glu Glu
Glu Glu Glu 1 5 1427PRTArtificial Sequencesynthetic peptide
sequence 142Asn Glu Glu Glu Glu Glu Glu 1 5 1436PRTArtificial
Sequencesynthetic peptide sequence 143Gln Glu Glu Glu Glu Glu 1 5
1446PRTArtificial Sequencesynthetic peptide sequence 144Asn Asp Asp
Asp Asp Asp 1 5 1456PRTArtificial Sequencesynthetic peptide
sequence 145Gln Asp Asp Asp Asp Asp 1 5 146138DNAArtificial
SequenceSynthetic cDNA 146atgcatggtg agggtacatt cacatctgat
ttgtctaagc aaatggagga ggaggctgtt 60cgtttgttca ttgagtggtt gaagaatggt
ggtccatctt ctggtgctcc accatctaag 120aagaagaaga agaagtaa
13814732PRTArtificial SequenceGly8-Glp-1(7-36)-Lys37(palmitoyal)
(Human) 147His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg Gly Lys 20 25 30 14831PRTArtificial SequenceY31-exendin-4
(1-31) Human) 148His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys
Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu
Lys Asn Gly Gly Lys 20 25 30 14944PRTArtificial
SequenceLys6-des-Pro36-exendin-4 149Lys Lys Lys Lys Lys Lys His Gly
Glu Gly Thr Phe Thr Ser Asp Leu 1 5 10 15 Ser Lys Gln Met Glu Glu
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu 20 25 30 Lys Asn Gly Gly
Pro Ser Ser Gly Ala Pro Pro Ser 35 40 15050PRTArtificial
SequenceLys6-des-Pro36-exendin-4-Lys6 150Lys Lys Lys Lys Lys Lys
His Gly Glu Gly Thr Phe Thr Ser Asp Leu 1 5 10 15 Ser Lys Gln Met
Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu 20 25 30 Lys Asn
Gly Gly Pro Ser Ser Gly Ala Pro Pro Ser Lys Lys Lys Lys 35 40 45
Lys Lys 50 15140PRTArtificial Sequence(Lys40(palmitoyal)exendin-4
(1-39) 151His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met
Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser Lys 35 40
15239PRTArtificial Sequence[Des Pro36,
Lys40(palmitoyal)]exendin-4(1-40)(Human) 152His Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly
Ala Pro Pro Ser Lys 35 15337PRTArtificial
SequenceGly8Lys37(palmitoyal)-Glp-11(7-36)(Human)-Lys6 153His Gly
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys Lys 20
25 30 Lys Lys Lys Lys Lys 35
* * * * *