U.S. patent application number 14/608284 was filed with the patent office on 2015-07-30 for double-acylated glp-1 derivatives with a linker.
The applicant listed for this patent is Novo Nordisk A/S. Invention is credited to Patrick William Garibay, Jacob Kofoed, Thomas Kruse, Jesper Lau, Lars Linderoth.
Application Number | 20150210745 14/608284 |
Document ID | / |
Family ID | 46050422 |
Filed Date | 2015-07-30 |
United States Patent
Application |
20150210745 |
Kind Code |
A1 |
Kofoed; Jacob ; et
al. |
July 30, 2015 |
Double-Acylated GLP-1 Derivatives with a Linker
Abstract
The invention relates to a derivative of a GLP-1 analogue, which
analogue comprises a first K residue at a position corresponding to
position 18 of GLP-1(7-37) (SEQ ID NO: 1), a second K residue at
another position, and a maximum of twelve amino acid changes as
compared to GLP-1(7-37); which derivative comprises two protracting
moieties attached to said first and second K residue, respectively,
via a linker, wherein the protracting moiety is selected from
HOOC--(CH2)x-CO--*, and Chem. 1: HOOC--C6H4-0-(CH2)y-CO--*, Chem.
2: in which x is an integer in the range of 6-18, and y is an
integer in the range of 3-17; and the linker comprises
*--NH--(CH2)q-CH[(CH2)w-NH2]-CO--*, Chem. 3: wherein q is an
integer in the range of 0-5, and w is an integer in the range of
0-5; or a pharmaceutically acceptable salt, amide, or ester
thereof. The invention also relates to the pharmaceutical use
thereof, for example in the treatment and/or prevention of all
forms of diabetes and related diseases, as well as to corresponding
novel peptides and side chain intermediates. The derivatives are
potent, protracted, and suitable for oral administration.
Inventors: |
Kofoed; Jacob; (Vaerloese,
DK) ; Lau; Jesper; (Farum, DK) ; Linderoth;
Lars; (Alleroed, DK) ; Garibay; Patrick William;
(Holte, DK) ; Kruse; Thomas; (Herlev, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novo Nordisk A/S |
Bagsvaerd |
|
DK |
|
|
Family ID: |
46050422 |
Appl. No.: |
14/608284 |
Filed: |
January 29, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13883946 |
Jul 17, 2013 |
9006178 |
|
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PCT/EP11/69743 |
Nov 9, 2011 |
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14608284 |
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61414221 |
Nov 16, 2010 |
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61497123 |
Jun 15, 2011 |
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Current U.S.
Class: |
530/308 |
Current CPC
Class: |
A61P 1/00 20180101; A61P
15/08 20180101; A61P 3/04 20180101; A61P 3/08 20180101; A61P 1/14
20180101; A61P 7/12 20180101; A61P 9/00 20180101; A61P 3/06
20180101; A61P 15/00 20180101; A61P 43/00 20180101; A61K 47/542
20170801; A61P 3/10 20180101; C07K 14/605 20130101 |
International
Class: |
C07K 14/605 20060101
C07K014/605 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 9, 2010 |
EP |
10190515.6 |
Jun 9, 2011 |
EP |
11169276.0 |
Claims
1. A GLP-1 analogue which comprises the following changes as
compared to GLP-1(7-37) (SEQ ID NO: 1): (a) 7Imp, 8Aib, 18K, 22E,
34Q; (b) 7Imp, 18K, 22E, 25V, 26R, 31K, 34R; or (c) 8Aib, 18K, 19Q,
22E, 34Q; or a pharmaceutically acceptable salt, amide, or ester of
any of the analogues of (a)-(c).
2. A GLP-1 analogue selected from the following analogues of
GLP-1(7-37) (SEQ ID NO: 1): (a) 7Imp, 8Aib, 18K, 22E, 34Q (SEQ ID
NO: 5); (b) 7Imp, 18K, 22E 25V, 26R, 31K, 34R (SEQ ID NO: 6); or
(c) 8Aib, 18K, 19Q, 22E, 34Q (SEQ ID NO: 7); or a pharmaceutically
acceptable salt, amide, or ester of any of the analogues of
(a)-(c).
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/883,946, filed Jul. 17, 2013, which is a 35 U.S.C. .sctn.371
National Stage application of International Application
PCT/EP2011/069743 (WO 2012/062804 A1), filed Nov. 9, 2011, which
claims priority from EP10190515.6 filed 9 Nov. 2010 and EP
11169276.0 filed 9 Jun. 2011. It also claims priority under 35
U.S.C. .sctn.119 of U.S. Provisional Patent Application Ser. No.
61/414,221 filed on 16 Nov. 2010, and U.S. Provisional Patent
Application Ser. No. 61/497,123 filed on 15 Jun. 2011 under 35
U.S.C. Each of these is incorporated herein by reference in its
entirety.
TECHNICAL FIELD
[0002] The present invention relates to derivatives of analogues of
Glucagon-Like Peptide 1 (GLP-1), more in particular to GLP-1
derivatives that are double-acylated at K.sup.18 and at another K
residue of the peptide, via a novel linker, and their
pharmaceutical use.
INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING
[0003] The Sequence Listing, entitled "SEQUENCE LISTING", is 10,605
bytes, was created on Sep. 23, 2014 and is incorporated herein by
reference.
BACKGROUND
[0004] WO 99/43706 discloses a number of mono- and double-acylated
GLP-1 derivatives including some K.sup.18,26 and K.sup.18,34
derivatives.
[0005] WO 2011/080103 which published after the priority dates of
the present application discloses a number of GLP-1 derivatives
that are double-acylated at K.sup.26,37.
[0006] WO 06/097537 discloses a number of GLP-1 derivatives
including semaglutide (Example 4), a mono-acylated GLP-1 derivative
for once weekly administration which is under development by Novo
Nordisk A/S.
[0007] Angewandte Chemie International Edition 2008, vol. 47, p.
3196-3201 reports the discovery and characterisation of a class of
4-(p-iodophenyl)butyric acid derivatives which purportedly display
a stable noncovalent binding interaction with both mouse serum
albumin (MSA) and human serum albumin (HSA).
SUMMARY
[0008] The invention relates to derivatives of GLP-1 peptides.
[0009] The derivatives are acylated at a lysine substituted for the
native serine at position 18, as well as at another lysine residue.
The other lysine residue may be a native lysine, or a lysine
substituted for another amino acid residue. The side chains are
albumin binding moieties. They comprise a protracting moiety,
preferably selected from fatty diacids, and fatty acids with a
terminal, or distal, phenyl or phenoxy group, both optionally
substituted. A carboxy group of the fatty acid or fatty diacid,
optionally via a linker, is acylated to a lysine residue of the
GLP-1 peptide, preferably at the epsilon-amino group thereof. The
GLP-1 peptide may be an analogue of GLP-1 (7-37) (SEQ ID NO: 1)
having a total of up to twelve amino acid differences as compared
to GLP-1 (7-37), for example one or more additions, one or more
deletions, and/or one or more substitutions. The protracting moiety
is attached to the peptide via a linker. The linker comprises a
free amino group (--NH.sub.2 substituent), and is a diradical with
a *--NH end and a CO--* end.
[0010] More in particular, the invention relates to a derivative of
a GLP-1 analogue, which analogue comprises a first K residue at a
position corresponding to position 18 of GLP-1(7-37) (SEQ ID NO:
1), a second K residue at another position, and a maximum of twelve
amino acid changes as compared to GLP-1 (7-37), which derivative
comprises two protracting moieties attached to said first and
second K residue, respectively, each via a linker, wherein the
protracting moiety is selected from Chem. 1, and Chem. 2:
Chem. 1: HOOC--(CH.sub.2).sub.x--CO--*; and Chem. 2:
HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--*, in which x is an
integer in the range of 6-18, and y is an integer in the range of
3-17; and the linker comprises Chem. 3:
*--NH--(CH.sub.2).sub.q--CH[(CH.sub.2).sub.w--NH.sub.2]-CO--*,
wherein q is an integer in the range of 0-5, and w is an integer in
the range of 0-5; or a pharmaceutically acceptable salt, amide, or
ester thereof.
[0011] The invention also relates to such derivative for use as a
medicament, in particular for use in the treatment and/or
prevention of all forms of diabetes and related diseases, such as
eating disorders, cardiovascular diseases, gastrointestinal
diseases, diabetic complications, critical illness, and/or
polycystic ovary syndrome; and/or for improving lipid parameters,
improving .beta.-cell function, and/or for delaying or preventing
diabetic disease progression.
[0012] The invention furthermore relates to intermediate products
in the form of the GLP-1 peptides of the GLP-1 derivatives of the
invention.
[0013] The derivatives of the invention are biologically active.
Also, or alternatively, they have a protracted pharmacokinetic
profile. Also, or alternatively, they have a high oral
bioavailability. These properties are of importance in the
development of next generation GLP-1 compounds for subcutaneous,
intravenous, and/or in particular oral administration.
DESCRIPTION
[0014] In what follows, Greek letters may be represented by their
symbol or the corresponding written name, for example:
.alpha.=alpha; .beta.=beta; .gamma.=gamma; .delta.=delta;
.epsilon.=epsilon; .zeta.=zeta; .omega.=omega; etc. Also, the Greek
letter of p may be represented by "u", e.g. in .mu.l=ul, or in
.mu.M=uM.
[0015] An asterisk (*) in a chemical formula designates i) a point
of attachment, ii) a radical, and/or iii) an unshared electron.
[0016] The invention relates to a derivative of a GLP-1 analogue,
which analogue comprises a first K residue at a position
corresponding to position 18 of GLP-1(7-37) (SEQ ID NO: 1), a
second K residue at another position, and a maximum of twelve amino
acid changes as compared to GLP-1(7-37), which derivative comprises
two protracting moieties attached to said first and second K
residue, respectively, via a linker, wherein the protracting moiety
is selected from Chem. 1: HOOC--(CH.sub.2).sub.x--CO--*, and Chem.
2: HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--*, in which x is
an integer in the range of 6-18, and y is an integer in the range
of 3-17; and the linker comprises Chem. 3:
*--NH--(CH.sub.2).sub.q--CH[(CH.sub.2).sub.w--NH.sub.2]-CO--*,
wherein q is an integer in the range of 0-5, and w is an integer in
the range of 0-5; or a pharmaceutically acceptable salt, amide, or
ester thereof.
GLP-1 Analogues
[0017] The term "GLP-1 analogue" or "analogue of GLP-1" as used
herein refers to a peptide, or a compound, which is a variant of
the human Glucagon-Like Peptide-1 (GLP-1(7-37)), the sequence of
which is included in the sequence listing as SEQ ID NO: 1. The
peptide having the sequence of SEQ ID NO: 1 may also be designated
native GLP-1.
[0018] In the sequence listing, the first amino acid residue of SEQ
ID NO: 1 (histidine) is assigned no. 1. However, in what
follows--according to established practice in the art--this
histidine residue is referred to as no. 7, and subsequent amino
acid residues are numbered accordingly, ending with glycine no. 37.
Therefore, generally, any reference herein to an amino acid residue
number or a position number of the GLP-1 (7-37) sequence is to the
sequence starting with His at position 7 and ending with Gly at
position 37.
[0019] GLP-1 analogues of the derivatives of the invention may be
described by reference to i) the number of the amino acid residue
in native GLP-1(7-37) which corresponds to the amino acid residue
which is changed (i.e., the corresponding position in native
GLP-1), and to ii) the actual change.
[0020] The GLP-1 analogue of the derivative of the invention
comprises a first lysine residue at a position corresponding to
position 18 of GLP-1(7-37). If the amino acid sequence of this
analogue is otherwise identical to that of native GLP-1, such
analogue may be designated K.sup.18-GLP-1(7-37). This designation
accordingly represents the amino acid sequence of native GLP-1
where serine at position 18 has been substituted with lysine. As an
added remark, this analogue comprises a second Lys residue at
position 26, and a third Lys residue at position 34 (viz. the
native lysines of GLP-1(7-37)).
[0021] The GLP-1 analogue of the derivative of the invention
furthermore comprises a second lysine residue at another position,
which position may be designated "T". T accordingly represents any
other position than position 18.
[0022] For example, T may represent 26, in which case the analogue,
in addition to the lysine at position 18, comprises a lysine at a
position corresponding to position 26 in native GLP-1. Such
analogue would still be designated K.sup.18-GLP-1(7-37), provided
that, except for the K.sup.18-substitution, its amino acid sequence
would be identical to that of native GLP-1.
[0023] As another example, T may represent 34, in which case the
analogue, in addition to the lysine at position 18, comprises a
lysine at a position corresponding to position 34 in native GLP-1.
Such analogue would also still be designated K.sup.18-GLP-1(7-37),
provided that, except for the K.sup.18-substitution, its amino acid
sequence would be identical to that of native GLP-1.
[0024] But T may also represent a number in the range of 7-37 other
than 18, 26, or 34. Such analogue would be designated
K.sup.18,K.sup.T-GLP-1(7-37), provided that, except for the
K.sup.18- and the K.sup.T-substitutions, its amino acid sequence is
identical to that of native GLP-1.
[0025] The GLP-1 analogue forming part of the derivative of the
invention comprises, preferably has, a maximum of twelve amino acid
changes when compared with native GLP-1 (SEQ ID NO: 1)--in other
words, it is a GLP-1 peptide in which a number of amino acid
residues have been changed when compared to native GLP-1(7-37) (SEQ
ID NO: 1). These changes may represent, independently, one or more
amino acid substitutions, additions, and/or deletions.
[0026] The following are non-limiting examples of appropriate
analogue nomenclature.
[0027] For example, the analogue
[Aib8,Lys18,Glu22,Val25,Arg26,Lys31,Arg34]-GLP-1-(7-37) (SEQ ID
NO:3) designates a GLP-1 (7-37) peptide which, when compared to
native GLP-1, is changed by the following substitutions:
Substitution of alanine at position 8 with Aib
(.alpha.-aminoisobutyric acid), of serine at position 18 with
lysine, of glycine at position 22 with glutamic acid, of alanine at
position 25 with valine, of lysine at position 26 with arginine, of
tryptophan at position 31 with lysine, and of lysine at position 34
with arginine. This analogue may also be briefly designated (8Aib,
18K, 22E, 25V, 26R, 31K, 34R)(SEQ ID NO:3).
[0028] As another example, the analogue
[Lys18,Glu22,Arg26,Lys27,His31,Gly34]-GLP-1-(7-34) (SEQ ID NO:2)
designates a GLP-1(7-37) peptide, which, when compared to native
GLP-1, is changed by substitution of serine at position 18 with
lysine, substitution of glycine at position 22 with glutamic acid,
substitution of lysine at position 26 with arginine, substitution
of glutamic acid at position 27 with lysine, substitution of
tryptophan at position 31 with histidine, substitution of lysine at
position 34 with glycine, and by deletion of the C-terminus of
glycine-arginine-glycine at position 35-36-37. This analogue may
also be briefly designated (18K, 22E, 26R, 27K, 31H, 34G, des35-37)
(SEQ ID NO:2), where reference to GLP-1(7-37) is implied, and "des"
represents a deletion.
[0029] As a still further example, an analogue comprising
Imp.sup.7, and/or (Aib.sup.8 or S.sup.8) refers to a GLP-1(7-37)
peptide, which, when compared to native GLP-1, comprises a
substitution of histidine at position 7 with imidazopropionic acid
(Imp); and/or a substitution of alanine at position 8 with
.alpha.-aminoisobutyric acid (Aib), or with serine. This analogue
may comprise further changes as compared to SEQ ID NO: 1.
[0030] As is apparent from the above examples, amino acid residues
may be identified by their full name, their one-letter code, and/or
their three-letter code. These three ways are fully equivalent.
[0031] The expressions "a position equivalent to" or "corresponding
position" may be used to characterise the site of change in a GLP-1
sequence by reference to native GLP-1(7-37) (SEQ ID NO: 1).
Equivalent or corresponding positions are easily deduced, e.g. by
simple handwriting and eyeballing; and/or a standard protein or
peptide alignment program may be used, such as "align" which is a
Needleman-Wunsch alignment. The algorithm is described in
Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular
Biology, 48: 443-453, and the align program by Myers and W. Miller
in "Optimal Alignments in Linear Space" CABIOS (computer
applications in the biosciences) (1988) 4:11-17. For the alignment,
the default scoring matrix BLOSUM62 and the default identity matrix
may be used, and the penalty for the first residue in a gap may be
set at -10 (minus 10) and the penalties for additional residues in
a gap at -0.5 (minus 0.5).
[0032] An example of such alignment is inserted hereinbelow, in
which sequence no. 1 is SEQ ID NO: 1, and sequence no. 2 is SEQ ID
NO:2; the analogue (18K, 22E, 26R, 27K, 31H, 34G, des35-37)
thereof:
##STR00001##
[0033] In case of non-natural amino acids such as Imp and/or Aib
being included in the sequence, they may, for alignment purposes,
be replaced with X. If desired, X can later be manually
corrected.
[0034] The term "peptide", as e.g. used in the context of the GLP-1
analogues of the derivatives of the invention, refers to a compound
which comprises a series of amino acids interconnected by amide (or
peptide) bonds.
[0035] The peptides of the invention comprise at least five
constituent amino acids connected by peptide bonds. In particular
embodiments the peptide comprises at least 10, preferably at least
15, more preferably at least 20, even more preferably at least 25,
or most preferably at least 27 amino acids.
[0036] In particular embodiments, the peptide is composed of at
least five constituent amino acids, preferably composed of at least
10, at least 15, at least 20, at least 25, or most preferably
composed of at least 27 amino acids. In still further particular
embodiments the peptide is composed of at least 28, at least 29, at
least 30, at least 31, or at least 32 amino acids.
[0037] In a still further particular embodiment the peptide
consists of amino acids interconnected by peptide bonds.
[0038] Amino acids are molecules containing an amine group and a
carboxylic acid group, and, optionally, one or more additional
groups, often referred to as the amino acid side chain.
[0039] The term "amino acid" includes proteogenic amino acids
(encoded by the genetic code, including natural amino acids, and
standard amino acids), as well as non-proteogenic (not found in
proteins, and/or not coded for in the standard genetic code), and
synthetic amino acids. Thus, the amino acids may be selected from
the group of proteinogenic amino acids, non-proteinogenic amino
acids, and/or synthetic amino acids.
[0040] Non-limiting examples of amino acids which are not encoded
by the genetic code are gamma-carboxyglutamate, ornithine, and
phosphoserine. Non-limiting examples of synthetic amino acids are
the D-isomers of the amino acids such as D-alanine and D-leucine,
Aib (.alpha.-aminoisobutyric acid), .beta.-alanine, and
des-amino-histidine (desH, alternative name imidazopropionic acid,
abbreviated Imp).
[0041] In what follows, all amino acids for which the optical
isomer is not stated is to be understood to mean the L-isomer
(unless otherwise specified).
[0042] The GLP-1 derivatives and analogues of the invention have
GLP-1 activity. This term refers to the ability to bind to the
GLP-1 receptor and initiate a signal transduction pathway resulting
in insulinotropic action or other physiological effects as is known
in the art. For example, the analogues and derivatives of the
invention may suitably be tested for GLP-1 activity using the in
vitro potency assay described in Example 59 herein.
GLP-1 Derivatives
[0043] The term "derivative" as used herein in the context of a
GLP-1 peptide or analogue means a chemically modified GLP-1 peptide
or analogue, in which one or more substituents have been covalently
attached to the peptide. The substituent may also be referred to as
a side chain.
[0044] In a particular embodiment, the side chain is capable of
forming non-covalent aggregates with albumin, thereby promoting the
circulation of the derivative with the blood stream, and also
having the effect of protracting the time of action of the
derivative, due to the fact that the aggregate of the
GLP-1-derivative and albumin is only slowly disintegrated to
release the active pharmaceutical ingredient. Thus, the
substituent, or side chain, as a whole may be referred to as an
albumin binding moiety.
[0045] In another particular embodiment the albumin binding moiety
comprises a portion which is particularly relevant for the albumin
binding and thereby the protraction, which portion may accordingly
be referred to as a protracting moiety. The protracting moiety may
be at, or near, the opposite end of the albumin binding moiety,
relative to its point of attachment to the peptide.
[0046] In a still further particular embodiment the albumin binding
moiety comprises a portion inbetween the protracting moiety and the
point of attachment to the peptide, which portion may be referred
to as a linker, a linker moiety, a spacer, or the like.
[0047] In particular embodiments, the protracting moiety is
lipophilic, and/or negatively charged at physiological pH
(7.4).
[0048] The albumin binding moiety, the protracting moiety, or the
linker may be covalently attached to a lysine residue of the GLP-1
peptide by acylation.
[0049] In a preferred embodiment, an active ester of the albumin
binding moiety, preferably comprising a protracting moiety and a
linker, is covalently linked to an amino group of a lysine residue,
preferably the epsilon amino group thereof, under formation of an
amide bond (this process being referred to as acylation).
[0050] Unless otherwise stated, when reference is made to an
acylation of a lysine residue, it is understood to be to the
epsilon-amino group thereof.
[0051] A derivative comprising two protracting moieties attached to
a first and a second K residue (e.g., to K.sup.18 and K.sup.T) via
a linker may be referred to as a derivative which has been acylated
twice, double-acylated, or dual acylated at the epsilon-amino
groups of the first and second lysine residues, e.g. at position 18
and T, respectively, of the GLP-1 peptide.
[0052] For the present purposes, the terms "albumin binding
moiety", "protracting moiety", and "linker" include the molecule
itself as well as radicals thereof. Whether or not one or the other
form is meant is clear from the context in which the term is used.
In a preferred embodiment, these terms refer to radicals. The
radicals are preferably suitable for forming one or more amide
bonds, i.e. with one or two unshared electrons (*) in connection
with a carbonyl group and/or an amino group. Examples of such
radicals are Chem. 1, Chem. 2, and Chem. 3, the structures of which
are shown in the following.
[0053] In one aspect, each protracting moiety comprises, or
consists of, a protracting moiety, independently selected from
Chem. 1: HOOC--(CH.sub.2).sub.x--CO--*, and Chem. 2:
HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--*, in which x is an
integer in the range of 6-18, and y is an integer in the range of
3-17.
[0054] In one embodiment, *--(CH.sub.2).sub.x--* refers to straight
or branched, preferably straight, alkylene in which x is an integer
in the range of 6-18.
[0055] In another embodiment, *--(CH.sub.2).sub.y--* refers to
straight or branched, preferably straight, alkylene in which y is
an integer in the range of 3-17.
[0056] The nomenclature is as is usual in the art, for example in
the above formulas *--COOH refers to carboxy, *--C.sub.6H.sub.4--*
to phenylene, and *--CO--* to carbonyl (O.dbd.C<**). In
particular embodiments, the phenylene radical is ortho, meta, or
para, respectively.
[0057] In a particular embodiment, the derivative of the invention
has a first protracting moiety attached to a first K residue at a
position corresponding to position 18 of GLP-1(7-37) (SEQ ID NO:
1), and a second protracting moiety attached to a second K residue
at another position.
[0058] In other particular embodiments, the two albumin binding
moieties (i.e. the entire side chains) are similar, preferably
substantially identical, or, most preferably, identical.
[0059] In still further particular embodiments, the two protracting
moieties (or the first and the second protracting moiety), are
similar, preferably substantially identical, or, most preferably,
identical.
[0060] In still further particular embodiments, the two linkers are
similar, preferably substantially identical, or, most preferably
identical.
[0061] The term "substantially identical" includes differences from
identity which are due to formation of one or more salts, esters,
and/or amides; preferably formation of one or more salts, methyl
esters, and simple amides; more preferably formation of no more
than two salts, methyl esters, and/or simple amides; even more
preferably formation of no more than one salt, methyl ester, and/or
simple amide; or most preferably formation of no more than one
salt.
[0062] In the context of chemical compounds such as albumin binding
moieties, protracting moieties, and linkers, similarity and/or
identity may be determined using any suitable computer program
and/or algorithm known in the art.
[0063] For example, the similarity of two protracting moieties, two
linkers, and/or two entire side chains may suitably be determined
using molecular fingerprints. Fingerprints is a mathematical method
of representing a chemical structure (see e.g. Chemoinformatics: A
textbook, Johann Gasteiger and Thomas Engel (Eds), Wiley-VCH
Verlag, 2003).
[0064] Examples of suitable fingerprints include, without
limitation, UNITY fingerprints, MDL fingerprints, and/or ECFP
fingerprints, such as ECFP.sub.--6 fingerprints (ECFP stands for
extended-connectivity fingerprints).
[0065] In particular embodiments, the two protracting moieties, the
two linkers, and/or the two entire side chains are represented as
a) ECFP.sub.--6 fingerprints; b) UNITY fingerprints; and/or c) MDL
fingerprints.
[0066] The Tanimoto coefficient is preferably used for calculating
the similarity of the two fingerprints, whether a), b), or c) is
used.
[0067] In particular embodiments, whether a), b) or c) is used, the
two protracting moieties, the two linkers, and/or the two entire
side chains, respectively, have a similarity of at least 0.5 (50%);
preferably at least 0.6 (60%); more preferably at least 0.7 (70%),
or at least 0.8 (80%); even more preferably at least 0.9 (90%); or
most preferably at least 0.99 (99%), such as a similarity of 1.0
(100%).
[0068] UNITY fingerprints may be calculated using the programme
SYBYL (available from Tripos, 1699 South Hanley Road, St. Louis,
Mo. 63144-2319 USA). ECFP.sub.--6 and MDL fingerprints may be
calculated using the programme Pipeline Pilot (available from
Accelrys Inc., 10188 Telesis Court, Suite 100, San Diego, Calif.
92121, USA). For more details, see for example J. Chem. Inf. Model.
2008, 48, 542-549; J. Chem. Inf. Comput. Sci. 2004, 44, 170-178; J.
Med. Chem. 2004, 47, 2743-2749; J. Chem. Inf. Model. 2010, 50,
742-754; as well as SciTegic Pipeline Pilot Chemistry Collection:
Basic Chemistry User Guide, March 2008, SciTegic Pipeline Pilot
Data Modeling Collection, 2008--both from Accelrys Software Inc.,
San Diego, US, and the guides
http://www.tripos.com/tripos_resources/fileroot/pdfs/Unity.sub.--111408.p-
df, and
http://www.tripos.com/data/SYBYL/SYBYL.sub.--072505.pdf.
[0069] An example of a similarity calculation is inserted
hereinbelow, in which a known entire side chain of a known GLP-1
derivative was compared with a methyl ester thereof, the two side
chains being shown hereinbelow:
##STR00002##
[0070] Using a) ECFP.sub.--6 fingerprints the similarity is 0.798,
using b) UNITY fingerprints the similarity is 0.957; and using MDL
fingerprints the similarity is 0.905.
[0071] In case of two identical side chains (albumin binding
moieties) the derivative may be designated symmetrical.
[0072] Each of the two linkers of the derivative of the invention
comprises the following first linker element (A): Chem. 3:
*--NH--(CH.sub.2).sub.q--CH[(CH.sub.2).sub.w--NH.sub.2]-CO--*,
wherein q is an integer in the range of 0-5, and w is an integer in
the range of 0-5.
[0073] In a particular embodiment, the first protracting moiety is
attached to the first K residue at a position corresponding to
position 18 of GLP-1(7-37) (SEQ ID NO: 1) via a first linker
comprising the above first linker element, and the second
protracting moiety is attached to the second K residue at another
position, via a second linker comprising the above first linker
element.
[0074] Non-limiting examples of linkers comprising this first
linker element of Chem. 3 include diradicals of ornithine, lysine,
and homolysine; each in an alpha-version or in an omega-version.
Ornithine refers to 2,5-diaminopentanoic acid, lysine refers to
2,6-diaminohexanoic acid, and homolysine refers to
2,7-diaminoheptanoic acid.
[0075] For the alpha-versions, q=0. In other words, the alpha
refers to the fact that it is the amino group in the alpha position
(to the --CO--* radical) that is radicalised (to *--NH). When w=3,
4, and 5, the formula Chem. 3 refers to alpha-ornithine (alpha-Orn;
Chem. 9), alpha-lysine (alpha-Lys; Chem. 7), and alpha-homolysine
(alpha-Homolys; Chem. 11), respectively.
[0076] For the omega versions, w=0. In other words, the omega
refers to the fact that it is the amino group at the distal C-atom
of the alkyl substituent chain that is radicalised (to *--NH). When
q=3, 4, and 5, the formula Chem. 3 refers to delta-ornithine
(delta-Orn; Chem. 8), epsilon-lysine (eps-Lys; Chem. 6), and
zeta-homolysine (zeta-Homolys; Chem. 10), respectively.
[0077] In a preferred embodiment, these linkers are in their
L-form. The linker may comprise 1 or 2 times Chem. 3. When z is 2
the Chem. 3 elements are preferably interconnected via an amide
bond. For example, the linker may comprise two times epsilon-Lys
(2.times.eps-Lys; 2.times.Chem. 6).
[0078] The linker (each of the first and second linker) may further
(i.e., in addition to one or two times the first linker element
(A)) comprise one or more additional linker elements, independently
selected from the second (B), third (C), and/or fourth (D) linker
elements, as defined in the following:
[0079] A second linker element (B):
##STR00003##
wherein k is an integer in the range of 1-5, and n is an integer in
the range of 1-5.
[0080] In a particular embodiment, when k=1 and n=1, this linker
element may be designated OEG, or 8-amino-3,6-dioxaoctanic acid,
and/or it may be represented by the following formula:
*--NH--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--CH.sub.2--CO--*.
Chem. 12a:
[0081] A third linker element (C), glutamic acid (Glu), in either
of the following two versions:
##STR00004##
[0082] In the Chem. 14 version, the third linker element may also
be referred to as gamma-Glu, or briefly gGlu, due to the fact that
it is the gamma carboxy group of the amino acid glutamic acid which
is here used for connection to another linker element, or to the
epsilon-amino group of lysine. In the Chem. 15 version, it may also
be referred to as alpha-Glu, or briefly aGlu, due to the fact that
it is the alpha carboxy group which is used for the connection.
[0083] In particular embodiments, Chem. 14 is a) in the L-form, or
b) in the D-form.
[0084] A fourth linker element (D):
*--NH--(CH.sub.2).sub.s--CO--*, Chem. 16:
in which s is an integer in the range of 3-13.
[0085] In Chem. 16, the group *--(CH.sub.2).sub.s--* may represent
straight or branched, preferably straight, alkylene.
[0086] In still further particular embodiments the linker has a)
from 6 to 41 C-atoms; and/or b) from 4 to 28 hetero atoms.
Particular and non-limiting examples of hetero atoms are N--, and
O-atoms. H-atoms are not hetero atoms.
[0087] In a particular embodiment, each linker consists of Chem. 14
and two times Chem. 6, interconnected via amide bonds and in the
sequence indicated, connected at its *--NH end to the *--CO end of
the protracting moiety, and at its *--CO end to the epsilon amino
group of the first or the second K residue of the GLP-1
analogue.
[0088] For example, the first linker consists of Chem. 14 and two
times Chem. 6, interconnected via amide bonds and in the sequence
indicated, connected at its *--NH end to the *--CO end of the first
protracting moiety, and at its *--CO end to the epsilon amino group
of the first K residue of the GLP-1 analogue; and the second linker
consists of Chem. 14 and two times Chem. 6, interconnected via
amide bonds and in the sequence indicated, connected at its *--NH
end to the *--CO end of the second protracting moiety, and at its
*--CO end to the epsilon amino group of the second K residue of the
GLP-1 analogue.
[0089] Needless to say, just for the sake of good order: Here and
in the following the phrase "in the sequence indicated" means, that
the *--NH end of the first-mentioned linker element (here Chem. 14)
is connected to the *--CO end of the protractor, and the *--CO end
of the last-mentioned linker element (here the last one of the two
times Chem. 6) is connected to the epsilon amino group of the K
residue in question of the GLP-1 analogue.
[0090] In another particular embodiment, each linker (the first and
the second linker) consists of Chem. 14, two times Chem. 13, and
Chem. 6, interconnected via amide bonds and in the sequence
indicated, connected at its *--NH end to the *--CO end of the
protracting moiety, and at its *--CO end to the epsilon amino group
of the first or the second K residue of the GLP-1 analogue.
[0091] For example, the first linker consists of Chem. 14, two
times Chem. 13, and Chem. 6, interconnected via amide bonds and in
the sequence indicated, connected at its *--NH end to the *--CO end
of the first protracting moiety, and at its *--CO end to the
epsilon amino group of the first K residue of the GLP-1 analogue;
and the second linker consists of Chem. 14, two times Chem. 13, and
Chem. 6, interconnected via amide bonds and in the sequence
indicated, connected at its *--NH end to the *--CO end of the
second protracting moiety, and at its *--CO end to the epsilon
amino group of the second K residue of the GLP-1 analogue.
[0092] Additional PARTICULAR EMBODIMENTS (numbered 58-66) are
listed further below, and additional embodiments in which these
PARTICULAR EMBODIMENTS have been reformulated in a corresponding
way as explained above for the linkers (Chem. 14, 2.times.Chem. 6)
and (Chem. 14, 2.times.Chem. 13, and Chem. 6), in the sentences
starting "For example, - - - ", are specifically herein
incorporated by reference.
[0093] In still further particular embodiments, the invention
relates to:
(a) A derivative of a GLP-1 analogue, which analogue comprises a
first K residue at a position corresponding to position 18 of
GLP-1(7-37) (SEQ ID NO: 1), a second K residue at position 26 of
GLP-1(7-37) (SEQ ID NO: 1), and a maximum of four amino acid
changes as compared to GLP-1(7-37), which derivative comprises two
protracting moieties attached to said first and second K residue,
respectively, via a linker, wherein the protracting moiety is Chem.
1: HOOC--(CH.sub.2).sub.x--CO--*, or Chem. 2:
HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--*, in which x is 12,
and y is 9 or 11; and the linker comprises Chem. 3:
*--NH--(CH.sub.2).sub.q--CH[(CH.sub.2).sub.w--NH.sub.2]-CO--*,
wherein q is 4, and w is 0; or a pharmaceutically acceptable salt,
amide, or ester thereof. (b) The derivative of (a), wherein the
linker consists of Chem. 14 and two times Chem. 6, interconnected
via amide bonds and in the sequence indicated, connected at its
*--NH end to the *--CO end of the protracting moiety, and at its
*--CO end to the epsilon amino group of the first or the second K
residue of the GLP-1 analogue. (c) The derivative of (a), wherein
the linker consists of Chem. 14, two times Chem. 13, and Chem. 6,
interconnected via amide bonds and in the sequence indicated,
connected at its *--NH end to the *--CO end of the protracting
moiety, and at its *--CO end to the epsilon amino group of the
first or the second K residue of the GLP-1 analogue. (d) The
derivative of any of (a), (b), or (c), wherein the analogue, in
addition to the change K.sup.18, further comprises Q.sup.34. (e)
The derivative of any of (a), (b), (c), or (d), wherein the
analogue comprises Aib.sup.8. (f) The derivative of any of (a),
(b), (c), (d), or (e), wherein the analogue comprises E.sup.22. (g)
The derivative of any of (a), (b), (c), (d), (e), or (f), wherein
the analogue comprises, preferably has, Formula I:
[0094]
Xaa.sub.7-Xaa.sub.8-Glu-Gly-Thr-Xaa.sub.12-Thr-Ser-Asp-Xaa.sub.16-S-
er-Lys-Xaa.sub.19-Xaa.sub.20-Glu-Xaa.sub.22-Xaa.sub.23-Ala-Xaa.sub.25-Xaa.-
sub.26-Xaa.sub.27-Phe-Ile-Xaa.sub.30-Xaa.sub.31-Leu-Xaa.sub.33-Xaa.sub.34--
Xaa.sub.35-Xaa.sub.36-Xaa.sub.37-Xaa.sub.38 (SEQ ID NO: 4), wherein
Xaa.sub.7 is His or desamino-histidine (imidazopropionyl);
Xaa.sub.8 is Aib; Xaa.sub.12 is Phe; Xaa.sub.16 is Val; Xaa.sub.19
is Tyr; Xaa.sub.20 is Leu; Xaa.sub.22 is Gly or Glu; Xaa.sub.23 is
Gln; Xaa.sub.25 is Ala or Val; Xaa.sub.26 is Lys; Xaa.sub.27 is
Glu; Xaa.sub.30 is Ala; Xaa.sub.31 is Trp; Xaa.sub.33 is Val;
Xaa.sub.34 is Gln; Xaa.sub.35 is Gly; Xaa.sub.36 is Arg; Xaa.sub.37
is Gly; and Xaa.sub.38 is absent.
(h) A compound selected from Chem. 24, Chem. 25, Chem. 30, Chem.
38, Chem. 37, and Chem. 39; or a pharmaceutically acceptable salt,
amide, or ester thereof. (i) The derivative of any of embodiments
(a), (b), (c), (d), (e), (f), (g), or (h), which has a potency
corresponding to an EC.sub.50 below 500 pM, preferably below 400
pM, more preferably below 300 pM, even more preferably below 200
pM, or most preferably below 100 pM; wherein the potency is
determined as EC.sub.50 for stimulation of the formation of cAMP in
a medium containing the human GLP-1 receptor, using a stable
transfected cell-line such as BHK467-12A (tk-ts13); and wherein
cAMP is determined using a functional receptor assay, e.g. based on
competition between endogenously formed cAMP and exogenously added
biotin-labelled cAMP, and e.g. capturing cAMP using a specific
antibody, such as the AlphaScreen cAMP Assay, e.g. as described in
Example 59. (j) The derivative of any of embodiments (a), (b), (c),
(d), (e), (f), (g), (h), or (i), for which the GLP-1 receptor
binding affinity (IC.sub.50) in the presence of 0.005% HSA (low
albumin) is below 10 nM, preferably below 8.0 nM, still more
preferably below 6.0 nM, even more preferably below 4.0 nM, or most
preferably below 2.00 nM; wherein the binding affinity to the GLP-1
receptor is measured by way of displacement of .sup.125I-GLP-1 from
the receptor, for example using a SPA binding assay; and wherein
the GLP-1 receptor is prepared using a stable, transfected baby
hamster kidney cell line, such as BHK tk-ts13; and wherein the
IC.sub.50 value is determined as the concentration which displaces
50% of .sup.125I-GLP-1 from the receptor. (k) The derivative of any
of embodiments (a), (b), (c), (d), (e), (f), (g), (h), (i), or (j),
wherein the terminal half-life (T.sub.1/2) after i.v.
administration in rat is at least three times the terminal
half-life of semaglutide; wherein the half-life is determined in in
vivo pharmacokinetic studies in rat, for example as described in
Example 65. (l) The derivative of any of embodiments (a), (b), (c),
(d), (e), (f), (g), (h), (i), (j), or (k), wherein in an oral
gavage experiment in rats in vivo the AUC of the dose-corrected
plasma exposure from time 30 to 180 min (in the unit of
(min=pM/pmol), is at least 20, preferably at least 40, more
preferably at least 60, or most preferably at least 75; wherein the
AUC may be determined as described in Example 62.
[0095] The derivatives of the invention may exist in different
stereoisomeric forms having the same molecular formula and sequence
of bonded atoms, but differing only in the three-dimensional
orientation of their atoms in space. The stereoisomerism of the
exemplified derivatives of the invention is indicated in the
experimental section, in the names as well as the structures, using
standard nomenclature. Unless otherwise stated the invention
relates to all stereoisomeric forms of the claimed derivative.
[0096] The concentration in plasma of the GLP-1 derivatives of the
invention may be determined using any suitable method. For example,
LC-MS (Liquid Chromatography Mass Spectroscopy) may be used, or
immunoassays such as RIA (Radio Immuno Assay), ELISA (Enzyme-Linked
Immuno Sorbent Assay), and LOCI (Luminescence Oxygen Channeling
Immunoasssay). General protocols for suitable RIA and ELISA assays
are found in, e.g., WO09/030738 on p. 116-118. A preferred assay is
the LOCI assay described in 150 herein.
Intermediate Products
[0097] The invention also relates to an intermediate product in the
form of a GLP-1 analogue which comprises the following changes as
compared to GLP-1(7-37) (SEQ ID NO: 1): (a) 7Imp, 8Aib, 18K, 22E,
34Q (SEQ ID NO: 5); (b) 7Imp, 18K, 22E 25V, 26R, 31K, 34R(SEQ ID
NO: 6); or (c) 8Aib, 18K, 19Q, 22E, 34Q (SEQ ID NO: 7); or a
pharmaceutically acceptable salt, amide, or ester thereof.
Pharmaceutically Acceptable Salt, Amide, or Ester
[0098] The analogues and derivatives of the invention may be in the
form of a pharmaceutically acceptable salt, amide, or ester.
[0099] Salts are e.g. formed by a chemical reaction between a base
and an acid, e.g.:
2NH.sub.3+H.sub.2SO.sub.4.fwdarw.(NH.sub.4).sub.2SO.sub.4.
[0100] The salt may be a basic salt, an acid salt, or it may be
neither nor (i.e. a neutral salt). Basic salts produce hydroxide
ions and acid salts hydronium ions in water.
[0101] The salts of the derivatives of the invention may be formed
with added cations or anions that react with anionic or cationic
groups, respectively. These groups may be situated in the peptide
moiety, and/or in the side chain of the derivatives of the
invention.
[0102] Non-limiting examples of anionic groups of the derivatives
of the invention include free carboxylic groups in the side chain,
if any, as well as in the peptide moiety. The peptide moiety often
includes a free carboxylic acid group at the C-terminus, and it may
also include free carboxylic groups at internal acid amino acid
residues such as Asp and Glu.
[0103] Non-limiting examples of cationic groups in the peptide
moiety include the free amino group at the N-terminus, if present,
as well as any free amino group of internal basic amino acid
residues such as His, Arg, and Lys.
[0104] The ester of the derivatives of the invention may, e.g., be
formed by the reaction of a free carboxylic acid group with an
alcohol or a phenol, which leads to replacement of at least one
hydroxyl group by an alkoxy or aryloxy group
[0105] The ester formation may involve the free carboxylic group at
the C-terminus of the peptide, and/or any free carboxylic group in
the side chain.
[0106] The amide of the derivatives of the invention may, e.g., be
formed by the reaction of an activated form of a free carboxylic
acid group with an amine or a substituted amine, or by reaction of
a free or substituted amino group with an activated form of a
carboxylic acid.
[0107] The amide formation may involve the free carboxylic group at
the C-terminus of the peptide, any free carboxylic group in the
side chain, the free amino group at the N-terminus of the peptide,
and/or any free or substituted amino group of the peptide in the
peptide and/or the side chain.
[0108] In a particular embodiment, the peptide or derivative is in
the form of a pharmaceutically acceptable salt. In another
particular embodiment, the derivative is in the form of a
pharmaceutically acceptable amide, preferably with an amide group
at the C-terminus of the peptide. In a still further particular
embodiment, the peptide or derivative is in the form a
pharmaceutically acceptable ester.
Functional Properties
[0109] In a first aspect, the derivatives of the invention have a
good potency. Also, or alternatively, in a second aspect, they have
a protracted pharmacokinetic profile. Also, or alternatively, in a
third aspect, they have a high oral bioavailability. Also, or
alternatively, in a fourth aspect, they have good biophysical
properties.
Biological Activity (Potency)
[0110] According to the first aspect, the derivatives of the
invention, as well as the constituent GLP-1 peptides as such (such
as K.sup.18-GLP-1(7-37) or analogues thereof), are biologically
active, or potent. In fact, the derivatives of the invention have a
surprisingly good potency. This may appear to be so in particular
when the second acylation position is at a position corresponding
to around position 26 of GLP-1(7-37) (SEQ ID NO: 1). Without
wishing to be bound by this theory it is contemplated that this may
have to do with the free amino group of the specific linker.
[0111] In a particular embodiment, potency and/or activity refers
to in vitro potency, i.e. performance in a functional GLP-1
receptor assay, more in particular to the capability of stimulating
cAMP formation in a cell line expressing the cloned human GLP-1
receptor.
[0112] The stimulation of the formation of cAMP in a medium
containing the human GLP-1 receptor may preferably be determined
using a stable transfected cell-line such as BHK467-12A (tk-ts13),
and/or using for the determination of cAMP a functional receptor
assay, e.g. based on competition between endogenously formed cAMP
and exogenously added biotin-labelled cAMP, in which assay cAMP is
more preferably captured using a specific antibody, and/or wherein
an even more preferred assay is the AlphaScreen cAMP Assay, most
preferably the one described in Example 59.
[0113] The term half maximal effective concentration (EC.sub.50)
generally refers to the concentration which induces a response
halfway between the baseline and maximum, by reference to the dose
response curve. EC.sub.50 is used as a measure of the potency of a
compound and represents the concentration where 50% of its maximal
effect is observed.
[0114] The in vitro potency of the derivatives of the invention may
be determined as described above, and the EC.sub.50 of the
derivative in question determined. The lower the EC.sub.50, the
better the potency.
[0115] In a particular embodiment, the medium has the following
composition (final in-assay concentrations): 50 mM TRIS-HCl; 5 mM
HEPES; 10 mM MgCl.sub.2, 6H.sub.2O; 150 mM NaCl; 0.01% Tween; 0.1%
BSA; 0.5 mM IBMX; 1 mM ATP; 1 uM GTP. A first alternative medium
is: 50 mM TRIS-HCl; 5 mM HEPES; 10 mM MgCl.sub.2, 6H.sub.2O; 150 mM
NaCl; 0.01% Tween. A second alternative medium is: 50 mM Tris-HCl,
1 mM EGTA, 1.5 mM MgSO.sub.4, 1.7 mM ATP, 20 mM GTP, 2 mM
3-isobutyl-1-methylxanthine (IBMX), 0.01% Tween-20, pH 7.4.
[0116] In a further particular embodiment, the derivative of the
invention has an in vitro potency corresponding to an EC.sub.50 at
or below 10000 pM, more preferably below 5000 pM, even more
preferably below 1000 pM, or most preferably below 500 pM.
[0117] The ability of the derivatives of the invention to bind to
the GLP-1 receptor may also be used as a measure of the GLP-1
activity (receptor affinity). This ability may be determined as
described in Example 60. Generally, the binding to the GLP-1
receptor at low albumin concentration should be as good as
possible, corresponding to a low IC.sub.50 value. In particular
embodiments, the IC.sub.50 value of a derivative of the invention,
in the presence of 0.005% HSA (low albumin), is below the
corresponding IC.sub.50 value for semaglutide, preferably below 90%
thereof, more preferably below 80% thereof, even more preferably
below 70% thereof, or most preferably below 50% thereof.
[0118] In another particular embodiment the derivatives of the
invention are potent in vivo, which may be determined as is known
in the art in any suitable animal model, as well as in clinical
trials.
[0119] The diabetic db/db mouse is one example of a suitable animal
model, and the blood glucose lowering effect, and/or the body
weight lowering effect may be determined in such mice in vivo, e.g.
as described in Example 63.
[0120] The LYD pig is another example of a suitable animal model,
and the reduction in food intake may be determined in such pigs in
vivo, e.g. as described in Example 64.
Protraction--Receptor Binding/Low and High Albumin
[0121] According to the second aspect, the derivatives of the
invention are protracted.
[0122] The ability of the derivatives of the invention to bind to
the GLP-1 receptor in the presence of a low and a high
concentration of albumin, respectively, may be determined as
described in Example 60.
[0123] Generally, the binding to the GLP-1 receptor at low albumin
concentration should be as good as possible, corresponding to a low
IC.sub.50 value.
[0124] The IC.sub.50 value at high albumin concentration is a
measure of the influence of albumin on the binding of the
derivative to the GLP-1 receptor. As is known, the GLP-1
derivatives also bind to albumin. This is a generally desirable
effect, which extends their lifetime in plasma. Therefore, the
IC.sub.50 value at high albumin will generally be higher than the
IC.sub.50 value at low albumin, corresponding to a reduced binding
to the GLP-1 receptor, caused by albumin binding competing with the
binding to the GLP-1 receptor.
[0125] A high ratio (IC.sub.50 value (high albumin)/IC.sub.50 value
(low albumin)) may therefore be taken as an indication that the
derivative in question binds well to albumin (may have a long
half-life), and also per se binds well to the GLP-1 receptor (the
IC.sub.50 value (high albumin) is high, and the IC.sub.50 value
(low albumin) is low). On the other hand, albumin binding may not
always be desirable, or the binding to albumin may become too
strong. Therefore, the desirable ranges for IC.sub.50 (low
albumin), IC.sub.50 (high albumin)/, and the ratio high/low may
vary from compound to compound, depending on the intended use and
the circumstances surrounding such use, and on other compound
properties of potential interest.
[0126] As an example, in one particular embodiment, the ratio
(hi/lo), vis. [GLP-1 receptor binding affinity (IC.sub.50) in the
presence of 2.0% human serum albumin (HSA), divided by GLP-1
receptor binding affinity (IC.sub.50) in the presence of 0.005%
HSA], is at least 1, preferably at least 10, more preferably at
least 20, even more preferably at least 30, or most preferably at
least 50.
Protraction--Half Life In Vivo
[0127] According to the second aspect, the derivatives of the
invention are protracted.
[0128] Protraction may be determined as terminal half-life
(T.sub.1/2) in vivo in rats after i.v. administration, as described
in Example 65. In particular embodiments, the half-life in rat is
at least 7 hours, preferably at least 10 hours, even more
preferably at least 20 hours, or most preferably at least 30
hours.
[0129] Or, protraction may be determined in another animal species,
for example as terminal half-life (T.sub.1/2) in vivo in minipigs
after i.v. administration, as described in Example 66. In
particular embodiments, the terminal half-life in minipigs is at
least 8 hours, preferably at least 24 hours, even more preferably
at least 40 hours, or most preferably at least 60 hours.
[0130] Surprisingly, the present inventors identified a novel class
of GLP-1 derivatives, object of the present invention, which have a
high potency, and at the same time preferably a good half-life.
Oral Bioavailability
[0131] According to the third aspect, the derivatives of the
invention have a high oral bioavailability.
[0132] The oral bioavailability of commercial GLP-1 derivatives is
very low. The oral bioavailability of GLP-1 derivatives under
development for i.v. or s.c. administration is also low.
[0133] Accordingly, there is a need in the art for GLP-1
derivatives of an improved oral bioavailability. Such derivatives
could be suitable candidates for oral administration, as long as
mainly their potency is generally satisfactory, and/or as long as
their half-life is also generally satisfactory.
[0134] Generally, the term bioavailability refers to the fraction
of an administered dose of an active pharmaceutical ingredient
(API), such as a derivative of the invention that reaches the
systemic circulation unchanged. By definition, when an API is
administered intravenously, its bioavailability is 100%. However,
when it is administered via other routes (such as orally), its
bioavailability decreases (due to incomplete absorption and
first-pass metabolism). Knowledge about bioavailability is
important when calculating dosages for non-intravenous routes of
administration.
[0135] Absolute oral bioavailability compares the bioavailability
(estimated as the area under the curve, or AUC) of the API in
systemic circulation following oral administration, with the
bioavailability of the same API following intravenous
administration. It is the fraction of the API absorbed through
non-intravenous administration compared with the corresponding
intravenous administration of the same API. The comparison must be
dose normalised if different doses are used; consequently, each AUC
is corrected by dividing by the corresponding dose
administered.
[0136] A plasma API concentration vs time plot is made after both
oral and intravenous administration. The absolute bioavailability
(F) is the dose-corrected AUC-oral divided by AUC-intravenous.
[0137] In a particular embodiment, the derivative of the invention
has an absolute oral bioavailability which is higher than that of
semaglutide, preferably at least 10% higher, more preferably at
least 20% higher, even more preferably at least 30% higher, or most
preferably at least 40% higher. In additional particular
embodiments, it has an absolute oral bioavailability which is at
least 1.5 times that of semaglutide, preferably at least 2.0 times,
more preferably at least 3.0 times, even more preferably at least
4.0 times, or most preferably at least 5.0 times that of
semaglutide.
[0138] Before testing oral bioavailability the derivatives of the
invention may suitably be formulated as is known in the art of oral
formulations of insulinotropic compounds, e.g. using any one or
more of the formulations described in WO 2008/145728.
[0139] A couple of tests have been developed, described in Examples
61 and 62, which were found to give an acceptable prediction of
oral bioavailability. According to these tests, after direct
injection of the GLP-1 derivative into the intestinal lumen of
rats, or after oral gavage of rats, the concentration (exposure)
thereof in plasma is determined, and the ratio of plasma
concentration (pmol/l) divided by the concentration of the dosing
solution (umol/l) is calculated for t=30 min; or the AUC of the
dose-corrected plasma exposure from time 30 to 180 min is
calculated (min.times.pM/pmol). These figures are measures of
oral/intestinal bioavailability, and they are expected to correlate
with actual oral bioavailability data.
Biophysical Properties
[0140] According to the fourth aspect, the peptides/derivatives of
the invention have good biophysical properties. These properties
include but are not limited to physical stability and/or
solubility. These and other biophysical properties may be measured
using standard methods known in the art of protein chemistry. In a
particular embodiment, these properties are improved as compared to
native GLP-1 (SEQ ID NO: 1). Changed oligomeric properties of the
peptides/derivatives may be at least partly responsible for the
improved biophysical properties.
[0141] Additional particular embodiments of the derivatives of the
invention are described in the sections headed "PARTICULAR
EMBODIMENTS", "Additional particular embodiments", and "Still
further additional particular embodiments", before the experimental
section.
Production Processes
[0142] The production of peptides like GLP-1(7-37) and GLP-1
analogues is well known in the art.
[0143] The GLP-1 moiety of the derivatives of the invention, viz.
K.sup.18-GLP-1(7-37) or an analogue thereof, may for instance be
produced by classical peptide synthesis, e.g., solid phase peptide
synthesis using t-Boc or Fmoc chemistry or other well established
techniques, see, e.g., Greene and Wuts, "Protective Groups in
Organic Synthesis", John Wiley & Sons, 1999, Florencio Zaragoza
Dorwald, "Organic Synthesis on solid Phase", Wiley-VCH Verlag GmbH,
2000, and "Fmoc Solid Phase Peptide Synthesis", Edited by W. C.
Chan and P. D. White, Oxford University Press, 2000.
[0144] Also, or alternatively, they may be produced by recombinant
methods, viz. by culturing a host cell containing a DNA sequence
encoding the analogue and capable of expressing the peptide in a
suitable nutrient medium under conditions permitting the expression
of the peptide. Non-limiting examples of host cells suitable for
expression of these peptides are: Escherichia coli, Saccharomyces
cerevisiae, as well as mammalian BHK or CHO cell lines.
[0145] Those derivatives of the invention which include non-natural
amino acids and/or a covalently attached N-terminal mono- or
dipeptide mimetic may e.g. be produced as described in the
experimental part. Or see e.g., Hodgson et al: "The synthesis of
peptides and proteins containing non-natural amino acids", Chemical
Society Reviews, vol. 33, no. 7 (2004), p. 422-430; and WO
2009/083549 A1 entitled "Semi-recombinant preparation of GLP-1
analogues".
[0146] Specific examples of methods of preparing a number of the
derivatives of the invention are included in the experimental
part.
Pharmaceutical Compositions
[0147] Pharmaceutical composition comprising a derivative of the
invention or a pharmaceutically acceptable salt, amide, or ester
thereof, and a pharmaceutically acceptable excipient may be
prepared as is known in the art.
[0148] The term "excipient" broadly refers to any component other
than the active therapeutic ingredient(s). The excipient may be an
inert substance, an inactive substance, and/or a not medicinally
active substance.
[0149] The excipient may serve various purposes, e.g. as a carrier,
vehicle, diluent, tablet aid, and/or to improve administration,
and/or absorption of the active substance.
[0150] The formulation of pharmaceutically active ingredients with
various excipients is known in the art, see e.g. Remington: The
Science and Practice of Pharmacy (e.g. 19.sup.th edition (1995),
and any later editions).
[0151] Non-limiting examples of excipients are: Solvents, diluents,
buffers, preservatives, tonicity regulating agents, chelating
agents, and stabilisers.
[0152] Examples of formulations include liquid formulations, i.e.
aqueous formulations, i.e. formulations comprising water. A liquid
formulation may be a solution, or a suspension. An aqueous
formulation typically comprises at least 50% w/w water, or at least
60%, 70%, 80%, or even at least 90% w/w of water.
[0153] Alternatively a pharmaceutical composition may be a solid
formulation, e.g. a freeze-dried or spray-dried composition, which
may be used as is, or whereto the physician or the patient adds
solvents, and/or diluents prior to use.
[0154] The pH in an aqueous formulation may be anything between pH
3 and pH 10, for example from about 7.0 to about 9.5; or from about
3.0 to about 7.0.
[0155] A pharmaceutical composition may comprise a buffer. The
buffer may e.g. be selected from the group consisting of sodium
acetate, sodium carbonate, citrate, glycylglycine, histidine,
glycine, lysine, arginine, sodium dihydrogen phosphate, disodium
hydrogen phosphate, sodium phosphate, and
tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid,
succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid,
and mixtures thereof.
[0156] A pharmaceutical composition may comprise a preservative.
The preservative may e.g. be selected from the group consisting of
phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate,
propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl
p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol,
and thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine,
sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate,
benzethonium chloride, chlorphenesine
(3p-chlorphenoxypropane-1,2-diol), and mixtures thereof. The
preservative may be present in a concentration from 0.1 mg/ml to 20
mg/ml.
[0157] A pharmaceutical composition may comprise an isotonic agent.
The isotonic agent may e.g. be selected from the group consisting
of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an
amino acid (e.g. glycine, histidine, arginine, lysine, isoleucine,
aspartic acid, tryptophan, threonine), an alditol (e.g. glycerol
(glycerine), 1,2-propanediol (propyleneglycol), 1,3-propanediol,
1,3-butanediol) polyethyleneglycol (e.g. PEG400), and mixtures
thereof. Any sugar such as mono-, di-, or polysaccharides, or
water-soluble glucans, including for example fructose, glucose,
mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose,
dextran, pullulan, dextrin, cyclodextrin, alfa and beta HPCD,
soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na
may be used. Sugar alcohol is defined as a C4-C8 hydrocarbon having
at least one --OH group and includes, for example, mannitol,
sorbitol, inositol, galactitol, dulcitol, xylitol, and
arabitol.
[0158] A pharmaceutical composition may comprise a chelating agent.
The chelating agent may e.g. be selected from salts of
ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic
acid, and mixtures thereof.
[0159] A pharmaceutical composition may comprise a stabiliser. The
stabiliser may e.g. be one or more oxidation inhibitors,
aggregation inhibitors, surfactants, and/or one or more protease
inhibitors.
[0160] The term "aggregate formation" refers to a physical
interaction between the polypeptide molecules resulting in
formation of oligomers, which may remain soluble, or large visible
aggregates that precipitate from the solution. Aggregate formation
by a polypeptide during storage of a liquid pharmaceutical
composition can adversely affect biological activity of that
polypeptide, resulting in loss of therapeutic efficacy of the
pharmaceutical composition. Furthermore, aggregate formation may
cause other problems such as blockage of tubing, membranes, or
pumps when the polypeptide-containing pharmaceutical composition is
administered using an infusion system.
[0161] A pharmaceutical composition may comprise an amount of an
amino acid base sufficient to decrease aggregate formation of the
peptide during storage of the composition. The term "amino acid
base" refers to one or more amino acids (such as methionine,
histidine, imidazole, arginine, lysine, isoleucine, aspartic acid,
tryptophan, threonine), or analogues thereof. Any amino acid may be
present either in its free base form or in its salt form. Any
stereoisomer (i.e., L, D, or a mixture thereof) of the amino acid
base may be present. Methionine (or other sulphuric amino acids or
amino acid analogous) may be added to inhibit oxidation of
methionine residues to methionine sulfoxide when the peptide is a
polypeptide comprising at least one methionine residue susceptible
to such oxidation. Any stereoisomer of methionine (L or D) or
combinations thereof can be used.
[0162] A pharmaceutical composition may comprise a stabiliser
selected from the group of high molecular weight polymers or low
molecular compounds. The stabiliser may e.g. be selected from
polyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PVA),
polyvinylpyrrolidone, carboxy-/hydroxycellulose or derivates
thereof (e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins,
sulphur-containing substances as monothioglycerol, thioglycolic
acid and 2-methylthioethanol, and different salts (e.g. sodium
chloride).
[0163] A pharmaceutical composition may comprise additional
stabilising agents such as, but not limited to, methionine and
EDTA, which protect the polypeptide against methionine oxidation,
and a nonionic surfactant, which protects the polypeptide against
aggregation associated with freeze-thawing or mechanical
shearing.
[0164] A pharmaceutical composition may comprise one or more
surfactants, for example a surfactant, at least one surfactant, or
two different surfactants. The term "surfactant" refers to any
molecules or ions that are comprised of a water-soluble
(hydrophilic) part, and a fat-soluble (lipophilic) part. The
surfactant may e.g. be selected from the group consisting of
anionic surfactants, cationic surfactants, nonionic surfactants,
and/or zwitterionic surfactants.
[0165] A pharmaceutical composition may comprise one or more
protease inhibitors, such as, e.g., EDTA (ethylenediamine
tetraacetic acid), and/or benzamidineHCl. Additional, optional,
ingredients of a pharmaceutical composition include, e.g., wetting
agents, emulsifiers, antioxidants, bulking agents, metal ions, oily
vehicles, proteins (e.g., human serum albumin, gelatine), and/or a
zwitterion (e.g., an amino acid such as betaine, taurine, arginine,
glycine, lysine and histidine).
[0166] Still further, a pharmaceutical composition may be
formulated as is known in the art of oral formulations of
insulinotropic compounds, e.g. using any one or more of the
formulations described in WO 2008/145728.
[0167] An administered dose may contain from 0.01 mg-100 mg of the
derivative, or from 0.01-50 mg, or from 0.01-20 mg, or from 0.01
mg-10 mg of the derivative.
[0168] The derivative may be administered in the form of a
pharmaceutical composition. It may be administered to a patient in
need thereof at several sites, for example, at topical sites such
as skin or mucosal sites; at sites which bypass absorption such as
in an artery, in a vein, or in the heart; and at sites which
involve absorption, such as in the skin, under the skin, in a
muscle, or in the abdomen.
[0169] The route of administration may be, for example, lingual;
sublingual; buccal; in the mouth; oral; in the stomach; in the
intestine; nasal; pulmonary, such as through the bronchioles, the
alveoli, or a combination thereof; parenteral, epidermal; dermal;
transdermal; conjunctival; uretal; vaginal; rectal; and/or ocular.
In a particular embodiment the route of administration is per
oral.
[0170] A composition may be administered in several dosage forms,
for example as a solution; a suspension; an emulsion; a
microemulsion; multiple emulsions; a foam; a salve; a paste; a
plaster; an ointment; a tablet; a coated tablet; a chewing gum; a
rinse; a capsule such as hard or soft gelatine capsules; a
suppositorium; a rectal capsule; drops; a gel; a spray; a powder;
an aerosol; an inhalant; eye drops; an ophthalmic ointment; an
ophthalmic rinse; a vaginal pessary; a vaginal ring; a vaginal
ointment; an injection solution; an in situ transforming solution
such as in situ gelling, setting, precipitating, and in situ
crystallisation; an infusion solution; or as an implant. A
composition may further be compounded in a drug carrier or drug
delivery system, e.g. in order to improve stability,
bioavailability, and/or solubility. A composition may be attached
to such system through covalent, hydrophobic, and/or electrostatic
interactions. The purpose of such compounding may be, e.g., to
decrease adverse effects, achieve chronotherapy, and/or increase
patient compliance.
[0171] A composition may also be used in the formulation of
controlled, sustained, protracting, retarded, and/or slow release
drug delivery systems.
[0172] Parenteral administration may be performed by subcutaneous,
intramuscular, intraperitoneal, or intravenous injection by means
of a syringe, optionally a pen-like syringe, or by means of an
infusion pump.
[0173] A composition may be administered nasally in the form of a
solution, a suspension, or a powder; or it may be administered
pulmonally in the form of a liquid or powder spray.
[0174] Transdermal administration is a still further option, e.g.
by needle-free injection, from a patch such as an iontophoretic
patch, or via a transmucosal route, e.g. buccally.
[0175] A composition may be a stabilised formulation. The term
"stabilised formulation" refers to a formulation with increased
physical and/or chemical stability, preferably both. In general, a
formulation must be stable during use and storage (in compliance
with recommended use and storage conditions) until the expiration
date is reached.
[0176] The term "physical stability" refers to the tendency of the
polypeptide to form biologically inactive and/or insoluble
aggregates as a result of exposure to thermo-mechanical stress,
and/or interaction with destabilising interfaces and surfaces (such
as hydrophobic surfaces). The physical stability of an aqueous
polypeptide formulation may be evaluated by means of visual
inspection, and/or by turbidity measurements after exposure to
mechanical/physical stress (e.g. agitation) at different
temperatures for various time periods. Alternatively, the physical
stability may be evaluated using a spectroscopic agent or probe of
the conformational status of the polypeptide such as e.g.
Thioflavin T or "hydrophobic patch" probes.
[0177] The term "chemical stability" refers to chemical (in
particular covalent) changes in the polypeptide structure leading
to formation of chemical degradation products potentially having a
reduced biological potency, and/or increased immunogenic effect as
compared to the intact polypeptide. The chemical stability can be
evaluated by measuring the amount of chemical degradation products
at various time-points after exposure to different environmental
conditions, e.g. by SEC-HPLC, and/or RP-HPLC.
[0178] The treatment with a derivative according to the present
invention may also be combined with one or more additional
pharmacologically active substances, e.g. selected from
antidiabetic agents, antiobesity agents, appetite regulating
agents, antihypertensive agents, agents for the treatment and/or
prevention of complications resulting from or associated with
diabetes and agents for the treatment and/or prevention of
complications and disorders resulting from or associated with
obesity. Examples of these pharmacologically active substances are:
Insulin, sulphonylureas, biguanides, meglitinides, glucosidase
inhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV)
inhibitors, inhibitors of hepatic enzymes involved in stimulation
of gluconeogenesis and/or glycogenolysis, glucose uptake
modulators, compounds modifying the lipid metabolism such as
antihyperlipidemic agents as HMG CoA inhibitors (statins), Gastric
Inhibitory Polypeptides (GIP analogs), compounds lowering food
intake, RXR agonists and agents acting on the ATP-dependent
potassium channel of the .beta.-cells; Cholestyramine, colestipol,
clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin,
probucol, dextrothyroxine, neteglinide, repaglinide;
.beta.-blockers such as alprenolol, atenolol, timolol, pindolol,
propranolol and metoprolol, ACE (angiotensin converting enzyme)
inhibitors such as benazepril, captopril, enalapril, fosinopril,
lisinopril, alatriopril, quinapril and ramipril, calcium channel
blockers such as nifedipine, felodipine, nicardipine, isradipine,
nimodipine, diltiazem and verapamil, and .alpha.-blockers such as
doxazosin, urapidil, prazosin and terazosin; CART (cocaine
amphetamine regulated transcript) agonists, NPY (neuropeptide Y)
antagonists, PYY agonists, Y2 receptor agonists, Y4 receptor
agonits, mixed Y2/Y4 receptor agonists, MC4 (melanocortin 4)
agonists, orexin antagonists, TNF (tumor necrosis factor) agonists,
CRF (corticotropin releasing factor) agonists, CRF BP
(corticotropin releasing factor binding protein) antagonists,
urocortin agonists, .beta.3 agonists, oxyntomodulin and analogues,
MSH (melanocyte-stimulating hormone) agonists, MCH
(melanocyte-concentrating hormone) antagonists, CCK
(cholecystokinin) agonists, serotonin re-uptake inhibitors,
serotonin and noradrenaline re-uptake inhibitors, mixed serotonin
and noradrenergic compounds, 5HT (serotonin) agonists, bombesin
agonists, galanin antagonists, growth hormone, growth hormone
releasing compounds, TRH (thyreotropin releasing hormone) agonists,
UCP 2 or 3 (uncoupling protein 2 or 3) modulators, leptin agonists,
DA agonists (bromocriptin, doprexin), lipase/amylase inhibitors,
RXR (retinoid X receptor) modulators, TR .beta. agonists; histamine
H3 antagonists, Gastric Inhibitory Polypeptide agonists or
antagonists (GIP analogs), gastrin and gastrin analogs.
[0179] The treatment with a derivative according to this invention
may also be combined with a surgery that influences the glucose
levels, and/or lipid homeostasis such as gastric banding or gastric
bypass.
Pharmaceutical Indications
[0180] The present invention also relates to a derivative of the
invention for use as a medicament.
[0181] In particular embodiments, the derivative of the invention
may be used for the following medical treatments, all preferably
relating one way or the other to diabetes:
(i) prevention and/or treatment of all forms of diabetes, such as
hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1
diabetes, non-insulin dependent diabetes, MODY (maturity onset
diabetes of the young), gestational diabetes, and/or for reduction
of HbA1C; (ii) delaying or preventing diabetic disease progression,
such as progression in type 2 diabetes, delaying the progression of
impaired glucose tolerance (IGT) to insulin requiring type 2
diabetes, and/or delaying the progression of non-insulin requiring
type 2 diabetes to insulin requiring type 2 diabetes; (iii)
improving .beta.-cell function, such as decreasing .beta.-cell
apoptosis, increasing .beta.-cell function and/or .beta.-cell mass,
and/or for restoring glucose sensitivity to .beta.-cells; (iv)
prevention and/or treatment of cognitive disorders; (v) prevention
and/or treatment of eating disorders, such as obesity, e.g. by
decreasing food intake, reducing body weight, suppressing appetite,
inducing satiety; treating or preventing binge eating disorder,
bulimia nervosa, and/or obesity induced by administration of an
antipsychotic or a steroid; reduction of gastric motility; and/or
delaying gastric emptying; (vi) prevention and/or treatment of
diabetic complications, such as neuropathy, including peripheral
neuropathy; nephropathy; or retinopathy; (vii) improving lipid
parameters, such as prevention and/or treatment of dyslipidemia,
lowering total serum lipids; lowering HDL; lowering small, dense
LDL; lowering VLDL: lowering triglycerides; lowering cholesterol;
increasing HDL; lowering plasma levels of lipoprotein a (Lp(a)) in
a human; inhibiting generation of apolipoprotein a (apo(a)) in
vitro and/or in vivo; (iix) prevention and/or treatment of
cardiovascular diseases, such as syndrome X; atherosclerosis;
myocardial infarction; coronary heart disease; stroke, cerebral
ischemia; an early cardiac or early cardiovascular disease, such as
left ventricular hypertrophy; coronary artery disease; essential
hypertension; acute hypertensive emergency; cardiomyopathy; heart
insufficiency; exercise tolerance; chronic heart failure;
arrhythmia; cardiac dysrhythmia; syncopy; atheroschlerosis; mild
chronic heart failure; angina pectoris; cardiac bypass reocclusion;
intermittent claudication (atheroschlerosis oblitterens); diastolic
dysfunction; and/or systolic dysfunction; (ix) prevention and/or
treatment of gastrointestinal diseases, such as inflammatory bowel
syndrome; small bowel syndrome, or Crohn's disease; dyspepsia;
and/or gastric ulcers; (x) prevention and/or treatment of critical
illness, such as treatment of a critically ill patient, a critical
illness poly-nephropathy (CIPNP) patient, and/or a potential CIPNP
patient; prevention of critical illness or development of CIPNP;
prevention, treatment and/or cure of systemic inflammatory response
syndrome (SIRS) in a patient; and/or for the prevention or
reduction of the likelihood of a patient suffering from
bacteraemia, septicaemia, and/or septic shock during
hospitalisation; and/or (xi) prevention and/or treatment of
polycystic ovary syndrome (PCOS). In a particular embodiment, the
indication is selected from the group consisting of (i)-(iii) and
(v)-(iix), such as indications (i), (ii), and/or (iii); or
indication (v), indication (vi), indication (vii), and/or
indication (iix).
[0182] In another particular embodiment, the indication is (i). In
a further particular embodiment the indication is (v). In a still
further particular embodiment the indication is (iix).
[0183] The following indications are particularly preferred: Type 2
diabetes, and/or obesity.
Particular Embodiments
[0184] The following are particular embodiments of the
invention:
1. A derivative of a GLP-1 analogue,
[0185] which analogue comprises a first K residue at a position
corresponding to position 18 of GLP-1(7-37) (SEQ ID NO: 1), a
second K residue at another position, and a maximum of twelve amino
acid changes as compared to GLP-1(7-37),
[0186] which derivative comprises two protracting moieties attached
to said first and second K residue, respectively, via a linker,
wherein
[0187] the protracting moiety is selected from Chem. 1, and Chem.
2:
HOOC--(CH.sub.2).sub.x--CO--* Chem. 1:
HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--*, Chem. 2:
[0188] in which x is an integer in the range of 6-18, and y is an
integer in the range of 3-17; and
[0189] the linker comprises
*--NH--(CH.sub.2).sub.q--CH[(CH.sub.2).sub.w--NH.sub.2]-CO--*,
Chem. 3:
[0190] wherein q is an integer in the range of 0-5, and w is an
integer in the range of 0-5;
[0191] or a pharmaceutically acceptable salt, amide, or ester
thereof.
2. The derivative of embodiment 1, wherein the linker comprises z
times Chem. 3, wherein z is an integer in the range of 1-2. 3. The
derivative of embodiment 2, wherein z is 1. 4. The derivative of
embodiment 2, wherein z is 2. 5. The derivative of any of
embodiments 2 and 4, wherein when z is 2 the Chem. 3 elements are
interconnected via an amide bond. 6. The derivative of any of
embodiments 1-5, wherein w is 0. 7. The derivative of any of
embodiments 1-6, wherein q is an integer in the range of 3-5. 8.
The derivative of any of embodiments 1-7, wherein the linker
comprises Chem. 4: *--NH--(CH.sub.2).sub.q--CH(NH.sub.2)--CO--*,
wherein q is an integer in the range of 3-5. 9. The derivative of
any of embodiments 1-8, wherein q is 3. 10. The derivative of any
of embodiments 1-8, wherein q is 4. 11. The derivative of any of
embodiments 1-8, wherein q is 5. 12. The derivative of any of
embodiments 1-5, wherein q is 0. 13. The derivative of any of
embodiments 1-5, and 12, wherein w is an integer in the range of
3-5. 14. The derivative of any of embodiments 1-5, and 12-13,
wherein the linker comprises Chem. 5:
*--NH--CH[(CH.sub.2).sub.w--NH.sub.2]-CO--*, wherein w is an
integer in the range of 3-5. 15. The derivative of any of
embodiments 1-5, and 12-14, wherein w is 3. 16. The derivative of
any of embodiments 1-5, and 12-14, wherein w is 4. 17. The
derivative of any of embodiments 1-5, and 12-14, wherein w is 5.
18. The derivative of any of embodiments 1-8, 10, 12-14, and 16,
wherein Chem. 3, Chem. 4, or Chem. 5, respectively, is a di-radical
of lysine. 19. The derivative of any of embodiments 1-8, 10, and
18, wherein the linker comprises
*--NH--(CH.sub.2).sub.4--CH(NH.sub.2)--CO--*. Chem. 6:
20. The derivative of any of embodiments 1-2, 4-8, 10, and 18,
wherein the linker comprises
*--NH--(CH.sub.2).sub.4--CH(NH.sub.2)--CO--NH--(CH.sub.2).sub.4--CH(NH.s-
ub.2)--CO--*. 2.times.Chem. 6:
21. The derivative of any of embodiments 1-5, 12-14, 16, and 18,
wherein the linker comprises
*--NH--CH[(CH.sub.2).sub.4--NH.sub.2]-CO--*. Chem. 7:
22. The derivative of any of embodiments 18-21, wherein lysine is
L-lysine. 23. The derivative of any of embodiments 1-9, and 12-15,
wherein Chem. 3, Chem. 4, or Chem. 5, respectively, is a di-radical
of ornithine. 24. The derivative of any of embodiments 1-9, and 23,
wherein the linker comprises Chem. 8:
*--NH--(CH.sub.2).sub.3--CH(NH.sub.2)--CO--*. 25. The derivative of
any of embodiments 1-5, 12-15, and 23, wherein the linker
comprises
*--NH--CH[(CH.sub.2).sub.3--NH.sub.2]-CO--*. Chem. 9:
26. The derivative of any of embodiments 23-25, wherein the linker
is L-ornithine. 27. The derivative of any of embodiments 1-8,
11-14, and 17, wherein Chem. 3, Chem. 4, or Chem. 5, respectively,
is a di-radical of homolysine. 28. The derivative of any of
embodiments 1-8, 11, and 27, wherein the linker comprises
*--NH--(CH.sub.2).sub.5--CH(NH.sub.2)--CO--*. Chem. 10:
29. The derivative of any of embodiments 1-5, 12-14, 17, and 27,
wherein the linker comprises
*--NH--CH[(CH.sub.2).sub.5--NH.sub.2]-CO--*. Chem. 11:
30. The derivative of any of embodiments 27-29, wherein the linker
comprises L-homolysine. 31. The derivative of any of embodiments
1-30, wherein Chem. 3, Chem. 4, Chem. 5, Chem. 6, 2.times.Chem. 6,
Chem. 7, Chem. 8, Chem. 9, Chem. 10, or Chem. 11, respectively, is
a first linker element. 32. The derivative of any of embodiments
1-31, wherein the linker comprises a second linker element, Chem.
12:
##STR00005##
wherein k is an integer in the range of 1-5, and n is an integer in
the range of 1-5. 33. The derivative of embodiment 32, wherein k is
1. 34. The derivative of any of embodiments 32-33, wherein n is 1.
35. The derivative of any of embodiments 32-34, wherein the second
linker element is
##STR00006##
36. The derivative of any of embodiments 32-35, wherein Chem. 13 is
included m times, wherein m is 0, or an integer in the range of
1-10. 37. The derivative of embodiment 36, wherein m is 0, 1, or 2.
38. The derivative of any of embodiments 36-37, wherein m is 0. 39.
The derivative of any of embodiments 36-37, wherein m is 1. 40. The
derivative of any of embodiments 36-37, wherein m is 2. 41. The
derivative of any of embodiments 36-37, and 40, wherein, when m is
different from 1, the Chem. 13 elements are interconnected via
amide bond(s). 42. The derivative of any of embodiments 1-41,
wherein the linker comprises a third linker element selected from
Chem. 14 and Chem. 15:
##STR00007##
43. The derivative of embodiment 42, wherein the third linker
element is Chem. 14. 44. The derivative of embodiment 43, wherein
Chem. 14 is included p times, wherein p is 0, or an integer in the
range of 1-3. 45. The derivative of embodiment 44, wherein p is 0.
46. The derivative of embodiment 44, wherein p is 1. 47. The
derivative of embodiment 44, wherein p is 2. 48. The derivative of
embodiment 44, wherein p is 3. 49. The derivative any of
embodiments 42-48, wherein Chem. 14 is a di-radical of L-Glu. 50.
The derivative of any of embodiments 42-44, and 47-49, wherein,
when p is different from 0 and different from 1, the Chem. 14
elements are interconnected via amide bond(s). 51. The derivative
of any of embodiments 1-50, wherein the linker comprises a fourth
linker element:
*--NH--(CH.sub.2).sub.s-CO--*, Chem. 16:
in which s is an integer in the range of 3-13. 52. The derivative
of embodiment 51, wherein s is 5, 7, or 11; preferably 7. 53. The
derivative of embodiment 52, wherein the fourth linker element is
Chem. 17:
*--NH--(CH.sub.2).sub.7-CO--*.
54. The derivative of any of embodiments 51-53, wherein Chem. 16 is
a di-radical of amino octanoic acid. 55. The derivative of any of
embodiments 1-54, wherein the linker and the protracting moiety are
interconnected via an amide bond. 56. The derivative of any of
embodiments 1-55, wherein the linker and the GLP-1 analogue are
interconnected via an amide bond. 57. The derivative of any of
embodiments 1-55, wherein the linker is attached to the
epsilon-amino group of the first or the second K residue. 58. The
derivative of any of embodiments 1-3, 6-8, 10, 18-19, 22, 32-38,
42-44, 46, and 49, wherein the linker consists of Chem. 14 and
Chem. 6, interconnected via an amide bond and in the sequence
indicated, connected at its *--NH end to the *--CO end of the
protracting moiety, and at its *--CO end to the epsilon amino group
of the first or the second K residue of the GLP-1 analogue. 59. The
derivative of any of embodiments 1-2, 4-5, 6-8, 10, 18-20, 22,
32-38, 42-44, 46, and 49, wherein the linker consists of Chem. 14
and two times Chem. 6, interconnected via amide bonds and in the
sequence indicated, connected at its *--NH end to the *--CO end of
the protracting moiety, and at its *--CO end to the epsilon amino
group of the first or the second K residue of the GLP-1 analogue.
60. The derivative of any of embodiments 1-3, 6-8, 10, 18-19, 22,
32-38, 42-44, 47, and 49, wherein the linker consists of two times
Chem. 14 and Chem. 6, interconnected via amide bonds and in the
sequence indicated, connected at its *--NH end to the *--CO end of
the protracting moiety, and at its *--CO end to the epsilon amino
group of the first or the second K residue of the GLP-1 analogue.
61. The derivative of any of embodiments 1-3, 6-8, 10, 18-19, 22,
32-38, 42-44, 48, and 49, wherein the linker consists of three
times Chem. 14 and Chem. 6, interconnected via amide bonds and in
the sequence indicated, connected at its *--NH end to the *--CO end
of the protracting moiety, and at its *--CO end to the epsilon
amino group of the first or the second K residue of the GLP-1
analogue. 62. The derivative of any of embodiments 1-3, 6-8, 10,
18-19, 22, 32-37, 39, and 42-45, wherein the linker consists of
Chem. 13 and Chem. 6, interconnected via amide bonds and in the
sequence indicated, connected at its *--NH end to the *--CO end of
the protracting moiety, and at its *--CO end to the epsilon amino
group of the first or the second K residue of the GLP-1 analogue.
63. The derivative of any of embodiments 1-3, 6-8, 10, 18-19, 22,
32-37, 39, 42-44, 46, and 49, wherein the linker consists of Chem.
14, Chem. 13, and Chem. 6, interconnected via amide bonds and in
the sequence indicated, connected at its *--NH end to the *--CO end
of the protracting moiety, and at its *--CO end to the epsilon
amino group of the first or the second K residue of the GLP-1
analogue. 64. The derivative of any of embodiments 1-3, 6-8, 10,
18-19, 22, 32-37, 40-44, 46, and 49, wherein the linker consists of
Chem. 14, two times Chem. 13, and Chem. 6, interconnected via amide
bonds and in the sequence indicated, connected at its *--NH end to
the *--CO end of the protracting moiety, and at its *--CO end to
the epsilon amino group of the first or the second K residue of the
GLP-1 analogue. 65. The derivative of any of embodiments 1-3, 6-8,
10, 18-19, 22, 32-37, 40, 42-44, 46, and 49, wherein the linker
consists of Chem. 13, Chem. 14, Chem. 13, and Chem. 6,
interconnected via amide bonds and in the sequence indicated,
connected at its *--NH end to the *--CO end of the protracting
moiety, and at its *--CO end to the epsilon amino group of the
first or the second K residue of the GLP-1 analogue. 66. The
derivative of any of embodiments 1-2, 4, 6-8, 10, 18-20, 22, 32-38,
42-44, 46, and 49, wherein the linker consists of Chem. 17, Chem.
14, and two times Chem. 6, interconnected via amide bonds and in
the sequence indicated, connected at its *--NH end to the *--CO end
of the protracting moiety, and at its *--CO end to the epsilon
amino group of the first or the second K residue of the GLP-1
analogue. 67. The derivative of any of embodiments 1-66, wherein
the protracting moiety is Chem. 1. 68. The derivative of embodiment
67, wherein x is an even number. 69. The derivative of any of
embodiments 67-68, wherein x is an integer in the range of 10-18.
70. The derivative of any of embodiments 67-69, wherein x is 12.
71. The derivative of any of embodiments 67-69, wherein x is 14.
72. The derivative of any of embodiments 67-69, wherein x is 16.
73. The derivative of any of embodiments 67-69, wherein x is 18.
74. The derivative of any of embodiments 1-66, wherein the
protracting moiety is Chem. 2. 75. The derivative of embodiment 74,
wherein y is an odd number. 76. The derivative of any of
embodiments 74-75, wherein y is an integer in the range of 7-11.
77. The derivative of any of embodiments 74-76, wherein y is 9. 78.
The derivative of any of embodiments 74-76, wherein y is 11. 79.
The derivative of any of embodiments 1-73, wherein Chem. 1 is
represented by
##STR00008##
80. The derivative of any of embodiments 1-66 and 74-78, wherein
Chem. 2 is represented by
##STR00009##
81. The derivative of any of embodiments 1-80, wherein the two
protracting moieties are substantially identical. 82. The
derivative of any of embodiments 1-81, wherein the two protracting
moieties have a similarity of at least 0.5; preferably at least
0.6; more preferably at least 0.7, or at least 0.8; even more
preferably at least 0.9; or most preferably at least 0.99, such as
a similarity of 1.0. 83. The derivative of any of embodiments 1-82,
wherein the two linkers are substantially identical. 84. The
derivative of any of embodiments 1-83, wherein the two linkers have
a similarity of at least 0.5; preferably at least 0.6; more
preferably at least 0.7, or at least 0.8; even more preferably at
least 0.9; or most preferably at least 0.99, such as a similarity
of 1.0. 85. The derivative of any of embodiments 1-84, wherein the
two side chains consisting of protracting moiety and linker are
substantially identical. 86. The derivative of any of embodiments
1-85, wherein the two side chains consisting of protracting moiety
and linker have a similarity of at least 0.5; preferably at least
0.6; more preferably at least 0.7, or at least 0.8; even more
preferably at least 0.9; or most preferably at least 0.99, such as
a similarity of 1.0. 87. The derivative of any of embodiments
81-86, wherein the two chemical structures to be compared are
represented as fingerprints, such as a) ECFP.sub.--6 fingerprints;
b) UNITY fingerprints; and/or c) MDL fingerprints; and wherein for
each of a), b) and c) the Tanimoto coefficient is preferably used
for calculating the similarity of the two fingerprints. 88. The
derivative of any of embodiments 1-87, wherein the first K residue
is designated K.sup.18. 89. The derivative of any of embodiments
1-88, wherein the second K residue is at a position corresponding
to position T of GLP-1 (7-37) (SEQ ID NO: 1). 90. The derivative of
any of embodiments 1-89, wherein the second K residue is designated
K.sup.T. 91. The derivative of any of embodiments 89-90, wherein T
is an integer selected from the range of 7-17 or from the range of
19-37. 92. The derivative of any of embodiments 89-91, wherein T is
an integer selected from the range of 12-17. 93. The derivative of
any of embodiments 89-92, wherein T is selected from the range of
19-37. 94. The derivative of any of 89-93, wherein T is selected
from the group consisting of 22, 26, 27, 30, 31, 34, and 37 95. The
derivative of any of embodiments 89-93, wherein T=22. 96. The
derivative of any of embodiments 89-93, wherein T=26. 97. The
derivative of any of embodiments 89-93, wherein T=27. 98. The
derivative of any of embodiments 89-93, wherein T=30. 99. The
derivative of any of embodiments 89-93, wherein T=31. 100. The
derivative of any of embodiments 89-93, wherein T=34. 101. The
derivative of any of embodiments 89-93, wherein T=37. 102. The
derivative of any of embodiments 89-93, wherein T is 26 or 31. 103.
The derivative of any of embodiments 1-102, wherein the position
corresponding to position 18 of GLP-1 (7-37) (SEQ ID NO: 1) is
identified by handwriting and eyeballing. 104. The derivative of
any of embodiments 89-103, wherein the position corresponding to
position T of GLP-1 (7-37) (SEQ ID NO: 1) is identified by
handwriting and eyeballing. 105. The derivative of any of
embodiments 1-104, wherein the number of amino acid changes as
compared to GLP-1(7-37) (SEQ ID NO: 1) are identified by
handwriting and eyeballing. 106. The derivative of any of
embodiments 1-105, wherein the position corresponding to position
18 of GLP-1(7-37) (SEQ ID NO: 1) is identified by use of a standard
protein or peptide alignment program. 107. The derivative of any of
embodiments 89-106, wherein the position corresponding to position
T of GLP-1 (7-37) (SEQ ID NO: 1) is identified by use of a standard
protein or peptide alignment program. 108. The derivative of any of
embodiments 1-107, wherein the number of amino acid changes as
compared to GLP-1(7-37) (SEQ ID NO: 1) are identified by use of a
standard protein or peptide alignment program. 109. The derivative
of any of embodiments 107-108, wherein the alignment program is a
Needleman-Wunsch alignment. 110. The derivative of any of
embodiments 107-109, wherein the default scoring matrix and the
default identity matrix is used. 111. The derivative of any of
embodiments 107-110, wherein the scoring matrix is BLOSUM62. 112.
The derivative of any of embodiments 107-111, wherein the penalty
for the first residue in a gap is -10 (minus ten). 113. The
derivative of any of embodiments 107-112, wherein the penalties for
additional residues in a gap is -0.5 (minus point five). 114. The
derivative of any of embodiments 1-113, wherein the analogue
comprises no K residues other than the first and the second K
residue. 115. The derivative of any of embodiments 1-114, wherein
the maximum twelve amino acid change(s) is (are) at one or more
positions corresponding to the following positions in GLP-1(7-37)
(SEQ ID NO: 1): 7, 8, 12, 18, 19, 22, 23, 25, 26, 27, 30, 31, 34,
35, 36, and 37. 116. The derivative of any of embodiments 1-115,
wherein the maximum twelve amino acid change(s) is (are) at one or
more positions corresponding to the following positions in
GLP-1(7-37) (SEQ ID NO: 1): 7, 8, 18, 19, 22, 23, 25, 26, 27, 30,
31, 34, 35, 36, and 37. 117. The derivative of any of embodiments
1-116, wherein the analogue comprises K.sup.18. 118. The derivative
of any of embodiments 1-117, wherein the analogue comprises at
least one of the following changes: Imp.sup.7, Aib.sup.8 or
S.sup.8, L.sup.12, Q.sup.19, K.sup.22 or E.sup.22, R.sup.23 or
E.sup.23, V.sup.25, R.sup.26 or H.sup.26 or V.sup.26, K.sup.27 or
L.sup.27 or H.sup.27, K.sup.30 or E.sup.30, K.sup.31 or H.sup.31,
G.sup.34 or R.sup.34 or Q34 or Des.sup.34 or H.sup.34, Des.sup.35,
Des.sup.36, K.sup.37 or Des.sup.37. 119. The derivative of any of
embodiments 1-118, wherein the second K residue is K.sup.22, and
wherein the analogue, in addition to the change K.sup.18, further
comprises i) a change selected from Des.sup.34, G.sup.34, R.sup.34,
and Q.sup.34, and ii) a change selected from R.sup.26, H.sup.26,
and V.sup.26. 120. The derivative of any of embodiments 1-119,
wherein the second K residue is K.sup.22, and wherein the analogue,
in addition to the change K.sup.18, further comprises Q.sup.34, and
R.sup.26. 121. The derivative of any of embodiments 1-118, wherein
the second K residue is K.sup.26, and wherein the analogue, in
addition to the change K.sup.18, further comprises a change
selected from G.sup.34, R.sup.34, H.sup.34, and Q.sup.34;
preferably selected from R.sup.34, and Q.sup.34. 122. The
derivative of any of embodiments 1-118, and 121, wherein the second
K residue is K.sup.26, and wherein the analogue, in addition to the
change K.sup.18, further comprises R.sup.34. 123. The derivative of
any of embodiments 1-118, and 121, wherein the second K residue is
K.sup.26, and wherein the analogue, in addition to the change
K.sup.18, further comprises Q.sup.34. 124. The derivative of any of
embodiments 1-118, and 121, wherein the second K residue is
K.sup.26, and wherein the analogue, in addition to the change
K.sup.18, further comprises H.sup.34. 125. The derivative of any of
embodiments 1-118, wherein the second K residue is K.sup.27, and
wherein the analogue, in addition to the change K.sup.18, further
comprises i) a change selected from Des.sup.34, G.sup.34, R.sup.34,
and Q.sup.34, and ii) a change selected from R.sup.26, H.sup.26,
and V.sup.26. 126. The derivative of any of embodiments 1-118, and
125, wherein the second K residue is K.sup.27, and wherein the
analogue, in addition to the change K.sup.18, further comprises
Q.sup.34 and R.sup.26. 127. The derivative of any of embodiments
1-118, and 125, wherein the second K residue is K.sup.27, and
wherein the analogue, in addition to the change K.sup.18, further
comprises R.sup.34 and R.sup.26. 128. The derivative of any of
embodiments 1-118, and 125, wherein the second K residue is
K.sup.27, and wherein the analogue, in addition to the change
K.sup.18, further comprises G.sup.34 and R.sup.26. 129. The
derivative of any of embodiments 1-118, and 125, wherein the second
K residue is K.sup.27, and wherein the analogue, in addition to the
change K.sup.18, further comprises Q.sup.34 and H.sup.26. 130. The
derivative of any of embodiments 1-118, and 125, wherein the second
K residue is K.sup.27, and wherein the analogue, in addition to the
change K.sup.18, further comprises R.sup.34 and H.sup.26. 131. The
derivative of any of embodiments 1-118, and 125, wherein the second
K residue is K.sup.27, and wherein the analogue, in addition to the
change K.sup.18, further comprises R.sup.34 and V.sup.26. 132. The
derivative of any of embodiments 1-118, wherein the second K
residue is K.sup.30 and wherein the analogue, in addition to the
change K.sup.18, further comprises i) a change selected from
Des.sup.34, G.sup.34, R.sup.34, and Q.sup.34, and ii) a change
selected from R.sup.26, H.sup.26, and V.sup.26. 133. The derivative
of any of embodiments 1-118, and 132, wherein the second K residue
is K.sup.30, and wherein the analogue, in addition to the change
K.sup.18, further comprises R.sup.34 and R.sup.26. 134. The
derivative of any of embodiments 1-118, and 132, wherein the second
K residue is K.sup.30, and wherein the analogue, in addition to the
change K.sup.18, further comprises G.sup.34 and R.sup.26. 135. The
derivative of any of embodiments 1-118, and 132, wherein the second
K residue is K.sup.30, and wherein the analogue, in addition to the
change K.sup.18, further comprises R.sup.34 and H.sup.26. 136. The
derivative of any of embodiments 1-118, and 132, wherein the second
K residue is K.sup.31, and wherein the analogue, in addition to the
change K.sup.18, further comprises i) a change selected from
Des.sup.34, G.sup.34, R.sup.34, and Q.sup.34, and ii) a change
selected from R.sup.26, H.sup.26, and V.sup.26. 137. The derivative
of any of embodiments 1-118, wherein the second K residue is
K.sup.31 and wherein the analogue, in addition to the change
K.sup.18, further comprises Des.sup.34 and R.sup.26. 138. The
derivative of any of embodiments 1-118, and 137, wherein the second
K residue is K.sup.31, and wherein the analogue, in addition to the
change K.sup.18, further comprises Q.sup.34 and R.sup.26. 139. The
derivative of any of embodiments 1-118, and 137, wherein the second
K residue is K.sup.31, and wherein the analogue, in addition to the
change K.sup.18, further comprises R.sup.34 and R.sup.26. 140. The
derivative of any of embodiments 1-118, and 137, wherein the second
K residue is K.sup.31, and wherein the analogue, in addition to the
change K.sup.18, further comprises G.sup.34 and R.sup.26. 141. The
derivative of any of embodiments 1-118, and 137, wherein the second
K residue is K.sup.31, and wherein the analogue, in addition to the
change K.sup.18, further comprises R.sup.34 and H.sup.26. 142. The
derivative of any of embodiments 1-118, and 137, wherein the second
K residue is K.sup.31, and wherein the analogue, in addition to the
change K.sup.18, further comprises G.sup.34 and H.sup.26. 143. The
derivative of any of embodiments 1-118, wherein the second K
residue is K.sup.34 and wherein the analogue, in addition to the
change K.sup.18, further comprises a change selected from R.sup.26,
H.sup.26, and V.sup.26. 144. The derivative of any of embodiments
1-118, and 143, wherein the second K residue is K.sup.34, and
wherein the analogue, in addition to the change K.sup.18, further
comprises R.sup.26. 145. The derivative of any of embodiments
1-118, wherein the second K residue is K.sup.37, and wherein the
analogue, in addition to the change K.sup.18, further comprises i)
a change selected from Des.sup.34, G.sup.34, R.sup.34, and
Q.sup.34, and ii) a change selected from R.sup.26, H.sup.26, and
V.sup.26. 146. The derivative of any of embodiments 1-118, and 145,
wherein the second K residue is K.sup.37, and wherein the analogue,
in addition to the change K.sup.18, further comprises R.sup.34 and
R.sup.26. 147. The derivative of any of embodiments 1-146, wherein
the analogue comprises at least one of the following changes:
Imp.sup.7, Aib.sup.8 or S.sup.8, L.sup.12, Q.sup.19, E.sup.22,
R.sup.23 or E.sup.23, V.sup.25, L.sup.27 or H.sup.27, E.sup.30,
H.sup.31, Des.sup.34, Des.sup.35, Des.sup.36, or Des.sup.37. 148.
The derivative of any of embodiments 1-147, wherein the analogue
comprises at least one of the following changes: Imp.sup.7,
Aib.sup.8, Q.sup.19, E.sup.22, R.sup.23 or E.sup.23, V.sup.25,
L.sup.27 or H.sup.27, E.sup.30, H.sup.31, Des.sup.34, Des.sup.35,
Des.sup.36, or Des.sup.37; preferably at least one of the following
changes: Imp.sup.7, Aib.sup.8, Q.sup.19, E.sup.22, Or V.sup.25.
149. The derivative of any of embodiments 115-148, wherein if the
amino acid residue at the position corresponding to position 34 is
deleted (Des.sup.34), then the amino acid residues at the positions
corresponding to positions 35-37 are also deleted (Des.sup.35,
Des.sup.36, and Des.sup.37). 150. The derivative of any of
embodiments 115-148, wherein if the amino acid residue at the
position corresponding to position 35 is deleted (Des.sup.35), then
the amino acid residues at the positions corresponding to positions
36-37 are also deleted (Des.sup.36 and Des.sup.37). 151. The
derivative of any of embodiments 115-148, wherein if the amino acid
residue at the position corresponding to position 36 is deleted
(Des.sup.36), then the amino acid residue at the position
corresponding to position 37 is also deleted (Des.sup.37). 152. The
derivative of any of embodiments 1-151, wherein the analogue
comprises Imp.sup.7. 153. The derivative of any of embodiments
1-152, wherein the analogue comprises Aib.sup.8. 154. The
derivative of any of embodiments 1-152, wherein the analogue
comprises S.sup.8. 155. The derivative of any of embodiments 1-154,
wherein the analogue comprises L.sup.12. 156. The derivative of any
of embodiments 1-155, wherein the analogue comprises Q.sup.19. 157.
The derivative of any of embodiments 1-156, wherein the analogue
comprises E.sup.22. 158. The derivative of any of embodiments
1-157, wherein the analogue comprises R.sup.23. 159. The derivative
of any of embodiments 1-156, wherein the analogue comprises
E.sup.23. 160. The derivative of any of embodiments 1-159, wherein
the analogue comprises V.sup.25. 161. The derivative of any of
embodiments 1-160, wherein the analogue comprises L.sup.27. 162.
The derivative of any of embodiments 1-161, wherein the analogue
comprises H.sup.27. 163. The derivative of any of embodiments
1-162, wherein the analogue comprises E.sup.30. 164. The derivative
of any of embodiments 1-163, wherein the analogue comprises
H.sup.31. 165. The derivative of any of embodiments 1-164, wherein,
for determination of the changes in the analogue, the amino acid
sequence of the analogue is compared to the amino acid sequence of
native GLP-1(7-37) (SEQ ID NO: 1). 166. The derivative of any of
embodiments 1-165, wherein, for determination of a position in an
analogue which corresponds to a specified position in native GLP-1
(7-37) (SEQ ID NO: 1), the amino acid sequence of the analogue is
compared to the amino acid sequence of native GLP-1(7-37) (SEQ ID
NO: 1). 167. The derivative of any of embodiments 1-166, wherein
the comparison of the amino acid sequence of the analogue with that
of GLP-1 (7-37) (SEQ ID NO: 1) is done by handwriting and
eyeballing. 168. The derivative of any of embodiments 1-167,
wherein the comparison of the amino acid sequence of the analogue
with that of GLP-1 (7-37) (SEQ ID NO: 1) is done by use of a
standard protein or peptide alignment program. 169. The derivative
of embodiment 168, wherein the alignment program is a
Needleman-Wunsch alignment. 170. The derivative of any of
embodiments 168-169, wherein the default scoring matrix and the
default identity matrix is used. 171. The derivative of any of
embodiments 168-170, wherein the scoring matrix is BLOSUM62. 172.
The derivative of any of embodiments 168-171, wherein the penalty
for the first residue in a gap is -10 (minus ten). 173. The
derivative of any of embodiments 168-172, wherein the penalties for
additional residues in a gap is -0.5 (minus point five). 174. The
derivative of any of embodiments 168-173, wherein the position
corresponding to any of the indicated positions of GLP-1 (7-37)
(SEQ ID NO: 1) is identified by handwriting and eyeballing. 175.
The derivative of any of embodiments 168-173, wherein the position
corresponding to any of the indicated positions of GLP-1 (7-37)
(SEQ ID NO: 1) is identified as described for position 18 and
position T in any of embodiments 103-113. 176. The derivative of
any of embodiments 1-175, which is a derivative of GLP-1(7-33)
(amino acids 1-27 of SEQ ID NO: 1). 177. The derivative of any of
embodiments 1-175, which is a derivative of GLP-1(7-34) (amino
acids 1-28 of SEQ ID NO: 1). 178. The derivative of any of
embodiments 1-175, which is a derivative of GLP-1(7-35) (amino
acids 1-29 of SEQ ID NO: 1). 179. The derivative of any of
embodiments 1-178, wherein the analogue has a maximum of eleven
amino acid changes. 180. The derivative of any of embodiments
1-178, wherein the analogue has a maximum of ten amino acid
changes. 181. The derivative of any of embodiments 1-178, wherein
the analogue has a maximum of nine amino acid changes. 182. The
derivative of any of embodiments 1-178, wherein the analogue has a
maximum of eight amino acid changes. 183. The derivative of any of
embodiments 1-178, wherein the analogue has a maximum of seven
amino acid changes. 184. The derivative
of any of embodiments 1-178, wherein the analogue has a maximum of
six amino acid changes. 185. The derivative of any of embodiments
1-178, wherein the analogue has a maximum of five amino acid
changes. 186. The derivative of any of embodiments 1-178, wherein
the analogue has a maximum of four amino acid changes. 187. The
derivative of any of embodiments 1-178, wherein the analogue has a
maximum of three amino acid changes. 188. The derivative of any of
embodiments 1-178, wherein the analogue has a maximum of two amino
acid changes. 189. The derivative of any of embodiments 1-178,
wherein the analogue has a minimum of one amino acid modification.
190. The derivative of any of embodiments 1-178, wherein the
analogue has a minimum of two amino acid changes. 191. The
derivative of any of embodiments 1-178, wherein the analogue has a
minimum of three amino acid changes. 192. The derivative of any of
embodiments 1-178, wherein the analogue has a minimum of four amino
acid changes. 193. The derivative of any of embodiments 1-178,
wherein the analogue has a minimum of five amino acid changes. 194.
The derivative of any of embodiments 1-178, wherein the analogue
has a minimum of six amino acid changes. 195. The derivative of any
of embodiments 1-178, wherein the analogue has a minimum of seven
amino acid changes. 196. The derivative of any of embodiments
1-178, wherein the analogue has a minimum of eight amino acid
changes. 197. The derivative of any of embodiments 1-178, wherein
the analogue has a minimum of nine amino acid changes. 198. The
derivative of any of embodiments 1-178, wherein the analogue has a
minimum of ten amino acid changes. 199. The derivative of any of
embodiments 1-178, wherein the analogue has a minimum of eleven
amino acid changes. 200. The derivative of any of embodiments
1-178, wherein the analogue has one amino acid changes. 201. The
derivative of any of embodiments 1-178, wherein the analogue has
two amino acid changes. 202. The derivative of any of embodiments
1-178, wherein the analogue has three amino acid changes. 203. The
derivative of any of embodiments 1-178, wherein the analogue has
four amino acid changes. 204. The derivative of any of embodiments
1-178, wherein the analogue has five amino acid changes. 205. The
derivative of any of embodiments 1-178, wherein the analogue has
six amino acid changes. 206. The derivative of any of embodiments
1-178, wherein the analogue has seven amino acid changes. 207. The
derivative of any of embodiments 1-178, wherein the analogue has
eight amino acid changes. 208. The derivative of any of embodiments
1-178, wherein the analogue has nine amino acid changes. 209. The
derivative of any of embodiments 1-178, wherein the analogue has
ten amino acid changes. 210. The derivative of any of embodiments
1-178, wherein the analogue has eleven amino acid changes. 211. The
derivative of any of embodiments 1-210, wherein the changes are,
independently, substitutions, additions, and/or deletions. 212. The
derivative of any of embodiments 1-210, wherein the changes are,
independently, substitutions, and/or deletions. 213. The derivative
of any of embodiments 1-212, wherein the changes are substitutions.
214. The derivative of any of embodiments 1-212, wherein the
changes are deletions. 215. The derivative of any of embodiments
1-214, wherein the analogue a) comprises a GLP-1 analogue of
Formula I; and/or b) is a GLP-1 analogue of Formula I:
Xaa.sub.7-Xaa.sub.8-Glu-Gly-Thr-Xaa.sub.12-Thr-Ser-Asp-Xaa.sub.6-Ser-Lys-
-Xaa.sub.19-Xaa.sub.20-Glu-Xaa.sub.22-Xaa.sub.23-Ala-Xaa.sub.25-Xaa.sub.26-
-Xaa.sub.27-Phe-Ile-Xaa.sub.30-Xaa.sub.31-Leu-Xaa.sub.33-Xaa.sub.34-Xaa.su-
b.35-Xaa.sub.36-Xaa.sub.37-Xaa.sub.38(SEQ ID NO: 8), Formula I:
wherein
[0192] Xaa.sub.7 is L-histidine, imidazopropionyl,
.alpha.-hydroxy-histidine, D-histidine, desamino-histidine,
2-amino-histidine, .beta.-hydroxy-histidine, homohistidine,
N.sup..alpha.-acetyl-histidine, N.sup..alpha.-formyl-histidine,
.alpha.-fluoromethyl-histidine, .alpha.-methyl-histidine,
3-pyridylalanine, 2-pyridylalanine, or 4-pyridylalanine;
[0193] Xaa.sub.8 is Ala, Gly, Val, Leu, Ile, Thr, Ser, Lys, Aib,
(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl)
carboxylic acid, (1-aminocyclopentyl) carboxylic acid,
(1-aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl)
carboxylic acid, or (1-aminocyclooctyl) carboxylic acid;
[0194] Xaa.sub.12 is Phe or Leu;
[0195] Xaa.sub.16 is Val or Leu;
[0196] Xaa.sub.19 is Tyr or Gln;
[0197] Xaa.sub.20 is Leu or Met;
[0198] Xaa.sub.22 is Gly, Glu, Lys, or Aib;
[0199] Xaa.sub.23 is Gln, Glu, or Arg;
[0200] Xaa.sub.25 is Ala or Val;
[0201] Xaa.sub.26 is Val, His, Lys, or Arg;
[0202] Xaa.sub.27 is Glu, Leu, or Lys;
[0203] Xaa.sub.30 is Ala, Glu, Lys, or Arg;
[0204] Xaa.sub.31 is Trp, Lys, or His
[0205] Xaa.sub.33 is Val or Lys;
[0206] Xaa.sub.34 is Lys, Glu, Asn, Gly, Gln, Arg, His, or
absent;
[0207] Xaa.sub.35 is Gly, Aib, or absent;
[0208] Xaa.sub.36 is Arg, Gly, Lys, or absent;
[0209] Xaa.sub.37 is Gly, Ala, Glu, Pro, Lys, Arg, or absent;
and
[0210] Xaa.sub.38 is Ser, Gly, Ala, Glu, Pro, Lys, Arg, or
absent.
216. The derivative of embodiment 215, wherein the peptide of
Formula I is an analogue of GLP-1(7-37) (SEQ ID NO: 1). 217. The
derivative of any of embodiments 215-216, wherein if Xaa.sub.37 is
absent, then Xaa.sub.38 is also absent. 218. The derivative of any
of embodiments 215-217, wherein if Xaa.sub.36 is absent, then
Xaa.sub.37, and Xaa.sub.38 are also absent. 219. The derivative of
any of embodiments 215-218, wherein if Xaa.sub.35 is absent, then
Xaa.sub.36, Xaa.sub.37, and Xaa.sub.38 are also absent. 220. The
derivative of any of embodiments 215-219, wherein if Xaa.sub.34 is
absent, then Xaa.sub.35, Xaa.sub.36, Xaa.sub.37, and Xaa.sub.38 are
also absent. 221. The derivative of any of embodiments 215-220,
wherein Xaa.sub.7 is His or desamino-histidine (imidazopropionyl);
Xaa.sub.8 is Ala, Ser, or Aib; Xaa.sub.12 is Phe or Leu; Xaa.sub.16
is Val; Xaa.sub.19 is Tyr; Xaa.sub.20 is Leu; Xaa.sub.22 is Gly,
Glu, or Lys; Xaa.sub.23 is Gln, Glu, or Arg; Xaa.sub.25 is Ala or
Val; Xaa.sub.26 is Val, His, Lys, or Arg; Xaa.sub.27 is Glu, Leu,
or Lys; Xaa.sub.30 is Ala, Glu, or Lys; Xaa.sub.31 is Trp, Lys, or
His; Xaa.sub.33 is Val; Xaa.sub.34 is Lys, Gly, Gln, Arg, His, or
absent; Xaa.sub.35 is Gly or absent; Xaa.sub.36 is Arg or absent;
Xaa.sub.37 is Gly, Lys, or absent; and Xaa.sub.38 is absent. 222.
The derivative of any of embodiments 215-221, wherein Xaa.sub.7 is
His or desamino-histidine; Xaa.sub.8 is Ala or Aib; Xaa.sub.12 is
Phe; Xaa.sub.16 is Val; Xaa.sub.19 is Tyr; Xaa.sub.20 is Leu;
Xaa.sub.22 is Gly, Glu, or Lys; Xaa.sub.23 is Gln, Glu, or Arg;
Xaa.sub.25 is Ala or Val; Xaa.sub.26 is Val, His, Lys, or Arg;
Xaa.sub.27 is Glu, Leu, or Lys; Xaa.sub.30 is Ala, Glu, or Lys;
Xaa.sub.31 is Trp, Lys, or His; Xaa.sub.33 is Val; Xaa.sub.34 is
Lys, Gly, Gln, Arg, His, or absent; Xaa.sub.35 is Gly or absent;
Xaa.sub.36 is Arg or absent; Xaa.sub.37 is Gly, Lys, or absent; and
Xaa.sub.38 is absent. 223. The derivative of any of embodiments
215-222, wherein Xaa.sub.7 is His or desamino-histidine; Xaa.sub.8
is Ala or Aib; Xaa.sub.12 is Phe; Xaa.sub.16 is Val; Xaa.sub.19 is
Tyr or Gln; Xaa.sub.20 is Leu; Xaa.sub.22 is Gly or Glu; Xaa.sub.23
is Gln; Xaa.sub.25 is Ala or Val; Xaa.sub.26 is Lys or Arg;
Xaa.sub.27 is Glu; Xaa.sub.30 is Ala; Xaa.sub.31 is Trp or Lys;
Xaa.sub.33 is Val; Xaa.sub.34 is Lys, Gln, or Arg; Xaa.sub.35 is
Gly; Xaa.sub.36 is Arg; Xaa.sub.37 is Gly; and Xaa.sub.38 is
absent. 224. The derivative of any of embodiments 215-223, wherein
Xaa.sub.7 is His. 225. The derivative of any of embodiments
215-223, wherein Xaa.sub.7 is desamino-histidine
(imidazopropionyl). 226. The derivative of any of embodiments
215-225, wherein Xaa.sub.8 is Ala. 227. The derivative of any of
embodiments 215-225, wherein Xaa.sub.8 is Ser. 228. The derivative
of any of embodiments 215-225, wherein Xaa.sub.8 is Aib. 229. The
derivative of any of embodiments 215-228, wherein Xaa.sub.12 is
Phe. 230. The derivative of any of embodiments 215-228, wherein
Xaa.sub.12 is Leu. 231. The derivative of any of embodiments
215-230, wherein Xaa.sub.16 is Val. 232. The derivative of any of
embodiments 215-231, wherein Xaa.sub.19 is Tyr. 233. The derivative
of any of embodiments 215-232, wherein Xaa.sub.20 is Leu. 234. The
derivative of any of embodiments 215-233, wherein Xaa.sub.22 is
Gly. 235. The derivative of any of embodiments 215-233, wherein
Xaa.sub.22 is Glu. 236. The derivative of any of embodiments
215-233, wherein Xaa.sub.22 is Lys. 237. The derivative of any of
embodiments 215-236, wherein Xaa.sub.23 is Gln. 238. The derivative
of any of embodiments 215-236, wherein Xaa.sub.23 is Glu. 239. The
derivative of any of embodiments 215-236, wherein Xaa.sub.23 is
Arg. 240. The derivative of any of embodiments 215-239, wherein
Xaa.sub.25 is Ala. 241. The derivative of any of embodiments
215-239, wherein Xaa.sub.25 is Val. 242. The derivative of any of
embodiments 215-241, wherein Xaa.sub.26 is His. 243. The derivative
of any of embodiments 215-241, wherein Xaa.sub.26 is Lys. 244. The
derivative of any of embodiments 215-241, wherein Xaa.sub.26 is
Arg. 245. The derivative of any of embodiments 215-244, wherein
Xaa.sub.27 is Glu. 246. The derivative of any of embodiments
215-244, wherein Xaa.sub.27 is Leu. 247. The derivative of any of
embodiments 215-244, wherein Xaa.sub.27 is Lys. 248. The derivative
of any of embodiments 215-247, wherein Xaa.sub.30 is Ala. 249. The
derivative of any of embodiments 215-247, wherein Xaa.sub.30 is
Glu. 250. The derivative of any of embodiments 215-247, wherein
Xaa.sub.30 is Lys. 251. The derivative of any of embodiments
215-250, wherein Xaa.sub.31 is Trp. 252. The derivative of any of
embodiments 215-250, wherein Xaa.sub.31 is Lys. 253. The derivative
of any of embodiments 215-250, wherein Xaa.sub.31 is His. 254. The
derivative of any of embodiments 215-253, wherein Xaa.sub.33 is
Val. 255. The derivative of any of embodiments 215-254, wherein
Xaa.sub.34 is Lys. 256. The derivative of any of embodiments
215-254, wherein Xaa.sub.34 is Gly. 257. The derivative of any of
embodiments 215-254, wherein Xaa.sub.34 is Gln. 258. The derivative
of any of embodiments 215-254, wherein Xaa.sub.34 is Arg. 259. The
derivative of any of embodiments 215-254, wherein Xaa.sub.34 is
His. 260. The derivative of any of embodiments 215-254, wherein
Xaa.sub.34 absent. 261. The derivative of any of embodiments
215-260, wherein Xaa.sub.35 is Gly. 262. The derivative of any of
embodiments 215-260, wherein Xaa.sub.35 is absent. 263. The
derivative of any of embodiments 215-262, wherein Xaa.sub.36 is
Arg. 264. The derivative of any of embodiments 215-262, wherein
Xaa.sub.36 is absent. 265. The derivative of any of embodiments
215-264, wherein Xaa.sub.37 is Gly. 266. The derivative of any of
embodiments 215-265, wherein Xaa.sub.37 is Lys. 267. The derivative
of any of embodiments 215-266, wherein Xaa.sub.37 is absent. 268.
The derivative of any of embodiments 215-267, wherein Xaa.sub.38 is
absent. 269. The derivative of any of embodiments 1-268, wherein
the analogue comprises the following amino acid changes, as
compared to GLP-1(7-37) (SEQ ID NO: 1): (i) 8Aib, 18K, 34R; (ii)
8Aib, 18K, 34Q; (iii) 8Aib, 18K, 22E, 34R; (iv) 8Aib, 18K, 22E,
34Q; (v) 8Aib, 12L, 18K, 34Q; (vi) 7Imp, 18K, 22E, 34Q; (vii) 18K,
34R; (iix) 18K, 34Q; (ix) 18K, 22E, 34R; (x) 18K, 22E, 34Q; (xi)
18K, 26R, 31K, 34R; (xii) 18K, 26H, 31K, 34R; (xiii) 18K, 26H, 27K,
34Q; (xiv) 18K, 22K, 26R, 34Q; (xv) 18K, 25V, 26R, 31K, 34R; (xvi)
18K, 22E, 26R, 31K, 34R; (xvii) 18K, 22E, 26H, 27K, 34R; (iixx)
18K, 22E, 26H, 27K, 34Q; (ixx) 18K, 22E, 26H, 27K, 31H, 34R; (xx)
18K, 22E, 26H, 27K, 31H, 34Q; (xxi) 18K, 22E, 25V, 26R, 31K, 34R;
(xxii) 18K, 22E, 25V, 26R, 31K, 34Q; (xxiii) 18K, 22E, 25V, 26R,
31K, 34G; (xxiv) 18K, 22E, 25V, 26R, 27K, 34R; (xxv) 18K, 22E, 25V,
26R, 27K, 34Q; (xxvi) 18K, 22E, 25V, 26R, 27K, 31H, 34R; (xxvii)
18K, 22E, 25V, 26R, 27K, 31H, 34Q; (iixxx) 18K, 22E, 23E, 25V, 26R,
27K, 34R; (ixxx) 18K, 22E, 23E, 25V, 26R, 27K, 34Q; (xxx) 18K, 22E,
25V, 26R, 31K, 34G, des35-37; (xxxi) 18K, 22E, 25V, 26R, 31H,
des35-37; (xxxii) 18K, 22E, 25V, 26R, 30K, 34G, des35-37; (xxxiii)
18K, 22E, 25V, 26R, 30K, 31H, 34G, des35-37; (xxxiv) 18K, 22E, 25V,
26R, 27L, 30K, 34G, des35-37); (xxxv) 18K, 22E, 26R, 31K, 34G,
des35-37; (xxxvi) 18K, 22E, 26R, 27K, 31H, 34G, des35-37; (xxxvii)
7Imp, 18K, 22E, 26R, 34R, 37K; (iixxxx) 7Imp, 18K, 22E, 26R, 27K,
31H, 34G, des35-37; (ixxxx) 7Imp, 18K, 22E, 25V, 26R, 31K, 34G,
des35-37; (xxxx) 7Imp, 8Aib, 18K, 22E, 25V, 26R, 31K, 34G,
des35-37; (xxxxi) 8S, 18K, 22E, 25V, 26R, 31K, 34G, des35-37;
(xxxxii) 8Aib, 18K, 26V, 27K, 34R; (xxxxiii) 8Aib, 18K, 26H, 30K,
34R, des36-37; (xxxxiv) 8Aib, 18K, 25V, 26R, 31K, 34R; (xxxxv)
8Aib, 18K, 22E, 34R, des36-37; (xxxxvi) 8Aib, 18K, 22E, 26R, 34R,
37K; (xxxxvii) 8Aib, 18K, 22E, 26R, 31K, 34R; (iixxxxx) 8Aib, 18K,
22E, 26R, 31K, 34G, des35-37; (ixxxxx) 8Aib, 18K, 22E, 26R, 30K,
34R, des36-37; (xxxxx) 8Aib, 18K, 22E, 26R, 30K, 34R; (xxxxxi)
8Aib, 18K, 22E, 26R, 27K, 31H, 34R, des36-37; (xxxxxii) 8Aib, 18K,
22E, 25V, 26R, 31K, des34-37; (xxxxxiii) 8Aib, 18K, 22E, 25V, 26R,
31K, 34R; (xxxxxiv) 8Aib, 18K, 22E, 25V, 26R, 31K, 34G, des35-37;
(xxxxxv) 8Aib, 18K, 22E, 25V, 26R, 30E, 31K, 34G, des35-37;
(xxxxxvi) 8Aib, 18K, 22E, 25V, 26R, 27L, des35-37; (xxxxxvii) 8Aib,
18K, 22E, 25V, 26R, 27K, 34Q; (iixxxxxx) 8Aib, 18K, 22E, 25V, 26R,
27K, 31H, 34G, des35-37; (ixxxxxx) 8Aib, 18K, 22E, 25V, 26H, 31K,
34G, des35-37; (xxxxxx) 8Aib, 18K, 22E, 23R, 25V, 26R, 31K, 34G,
des35-37; (xxxxxxi) 18K, 22E, 25V, 26R, 27L, 30K, 34G, des35-37;
(xxxxxxii) 7Imp, 18K, 22E, 26R, 27K, 34Q; (xxxxxxiii) 8Aib, 18K,
34H; (xxxxxxiv) 7Imp, 8Aib, 18K, 22E, 34Q; (xxxxxxv) 7Imp, 18K, 22E
25V, 26R, 31K, 34R; or (xxxxxxvi) 8Aib, 18K, 19Q, 22E, 34Q. 270.
The derivative of any of embodiments 1-269, wherein the analogue
has the following amino acid changes, as compared to GLP-1(7-37)
(SEQ ID NO: 1): (i) 8Aib, 18K, 34R; (ii) 8Aib, 18K, 34Q; (iii)
8Aib, 18K, 22E, 34R; (iv) 8Aib, 18K, 22E, 34Q; (v) 8Aib, 12L, 18K,
34Q; (vi) 7Imp, 18K, 22E, 34Q; (vii) 18K, 34R; (iix) 18K, 34Q; (ix)
18K, 22E, 34R; (x) 18K, 22E, 34Q; (xi) 18K, 26R, 31K, 34R; (xii)
18K, 26H, 31K, 34R; (xiii) 18K, 26H, 27K, 34Q; (xiv) 18K, 22K, 26R,
34Q; (xv) 18K, 25V, 26R, 31K, 34R; (xvi) 18K, 22E, 26R, 31K, 34R;
(xvii) 18K, 22E, 26H, 27K, 34R; (iixx) 18K, 22E, 26H, 27K, 34Q;
(ixx) 18K, 22E, 26H, 27K, 31H, 34R; (xx) 18K, 22E, 26H, 27K, 31H,
34Q; (xxi) 18K, 22E, 25V, 26R, 31K, 34R; (xxii) 18K, 22E, 25V, 26R,
31K, 34Q; (xxiii) 18K, 22E, 25V, 26R, 31K, 34G; (xxiv) 18K, 22E,
25V, 26R, 27K, 34R; (xxv) 18K, 22E, 25V, 26R, 27K, 34Q; (xxvi) 18K,
22E, 25V, 26R, 27K, 31H, 34R; (xxvii) 18K, 22E, 25V, 26R, 27K, 31H,
34Q; (iixxx) 18K, 22E, 23E, 25V, 26R, 27K, 34R; (ixxx) 18K, 22E,
23E, 25V, 26R, 27K, 34Q; (xxx) 18K, 22E, 25V, 26R, 31K, 34G,
des35-37; (xxxi) 18K, 22E, 25V, 26R, 31H, des35-37; (xxxii) 18K,
22E, 25V, 26R, 30K, 34G, des35-37; (xxxiii) 18K, 22E, 25V, 26R,
30K, 31H, 34G, des35-37; (xxxiv) 18K, 22E, 25V, 26R, 27L, 30K, 34G,
des35-37); (xxxv) 18K, 22E, 26R, 31K, 34G, des35-37; (xxxvi) 18K,
22E, 26R, 27K, 31H, 34G, des35-37; (xxxvii) 7Imp, 18K, 22E, 26R,
34R, 37K; (iixxxx) 7Imp, 18K, 22E, 26R, 27K, 31H, 34G, des35-37;
(ixxxx) 7Imp, 18K, 22E, 25V, 26R, 31K, 34G, des35-37; (xxxx) 7Imp,
8Aib, 18K, 22E, 25V, 26R, 31K, 34G, des35-37; (xxxxi) 8S, 18K, 22E,
25V, 26R, 31K, 34G, des35-37; (xxxxii) 8Aib, 18K, 26V, 27K, 34R;
(xxxxiii) 8Aib, 18K, 26H, 30K, 34R, des36-37; (xxxxiv) 8Aib, 18K,
25V, 26R, 31K, 34R; (xxxxv) 8Aib, 18K, 22E, 34R, des36-37; (xxxxvi)
8Aib, 18K, 22E, 26R, 34R, 37K; (xxxxvii) 8Aib, 18K, 22E, 26R, 31K,
34R; (iixxxxx) 8Aib, 18K, 22E, 26R, 31K, 34G, des35-37; (ixxxxx)
8Aib, 18K, 22E, 26R, 30K, 34R, des36-37; (xxxxx) 8Aib, 18K, 22E,
26R, 30K, 34R; (xxxxxi) 8Aib, 18K, 22E, 26R, 27K, 31H, 34R,
des36-37; (xxxxxii) 8Aib, 18K, 22E, 25V, 26R, 31K, des34-37;
(xxxxxiii) 8Aib, 18K, 22E, 25V, 26R, 31K, 34R; (xxxxxiv) 8Aib, 18K,
22E, 25V, 26R, 31K, 34G, des35-37; (xxxxxv) 8Aib, 18K, 22E, 25V,
26R, 30E, 31K, 34G, des35-37; (xxxxxvi) 8Aib, 18K, 22E, 25V, 26R,
27L, des35-37; (xxxxxvii) 8Aib, 18K, 22E, 25V, 26R, 27K, 34Q;
(iixxxxxx) 8Aib, 18K, 22E, 25V, 26R, 27K, 31H, 34G, des35-37;
(ixxxxxx) 8Aib, 18K, 22E, 25V, 26H, 31K, 34G, des35-37; (xxxxxx)
8Aib, 18K, 22E, 23R, 25V, 26R, 31K, 34G, des35-37; (xxxxxxi) 18K,
22E, 25V, 26R, 27L, 30K, 34G, des35-37; (xxxxxxii) 7Imp, 18K, 22E,
26R, 27K, 34Q; (xxxxxxiii) 8Aib, 18K, 34H; (xxxxxxiv) 7Imp, 8Aib,
18K, 22E, 34Q; (xxxxxxv) 7Imp, 18K, 22E 25V, 26R, 31K, 34R; or
(xxxxxxvi) 8Aib, 18K, 19Q, 22E, 34Q. 271. The derivative of any of
embodiments 1-270, wherein the analogue is modified so as to
comprise a C-terminal amide. 272. The derivative of any of
embodiments 1-271, wherein a carboxylic acid group of the
C-terminal amino acid of the analogue is converted into carboxylic
acid amide. 273. The derivative of embodiment 272, wherein the
carboxylic acid group which is converted into carboxylic acid amide
is not in the side chain of the C-terminal amino acid. 274. The
derivative of any of embodiments 1-270, wherein the analogue has a
C-terminal carboxylic acid. 275. A compound, preferably according
to any of embodiments 1-275, selected from the following: Chem. 20,
Chem. 21, Chem. 22, Chem. 23, Chem. 24, Chem. 25, Chem. 26, Chem.
27, Chem. 28, Chem. 29, Chem. 30, Chem. 31, Chem. 32, Chem. 33,
Chem. 34, Chem. 35, Chem. 36, Chem. 37, Chem. 38, Chem. 39, Chem.
40, Chem. 41, Chem. 42, Chem. 43, Chem. 44, Chem. 45, Chem. 46,
Chem. 47, Chem. 48, Chem. 49, Chem. 50, Chem. 51, Chem. 52, Chem.
53, Chem. 54, Chem. 55, Chem. 56, Chem. 57, Chem. 58, Chem. 59,
Chem. 60, Chem. 61, Chem. 62, Chem. 63, Chem. 64, Chem. 65, Chem.
66, Chem. 67, Chem. 68, Chem. 69, Chem. 70, Chem. 71, Chem. 72,
Chem. 73, Chem. 74, Chem. 75, Chem. 76, or Chem. 77; or a
pharmaceutically acceptable salt, amide, or ester thereof. 276. A
compound, preferably according to any of embodiments 1-275,
selected from the following: Chem. 20, Chem. 21, Chem. 22, Chem.
23, Chem. 24, Chem. 25, Chem. 26, Chem. 27, Chem. 29, Chem. 30,
Chem. 31, Chem. 32, Chem. 33, Chem. 34, Chem. 38, or Chem. 39; or a
pharmaceutically acceptable salt, amide, or ester thereof. 277. A
compound characterised by its name, and selected from a listing of
each of the names of the compounds of Examples 1-58 herein; or a
pharmaceutically acceptable salt, amide, or ester thereof. 278. A
compound characterised by its name, and selected from a listing of
each of the names of the compounds of Examples 1-8, 10-15, and
19-20 herein; or a pharmaceutically acceptable salt, amide, or
ester thereof. 279. The compound of embodiment 277, which is a
compound of embodiment 275. 280. The compound of embodiment 278,
which is a compound of embodiment 276. 281. The derivative of any
of embodiments 1-280, which has GLP-1 activity. 282. The derivative
of embodiment 281, wherein GLP-1 activity refers to the capability
of activating the human GLP-1 receptor. 283. The derivative of
embodiment 282, wherein activation of the human GLP-1 receptor is
measured in an in vitro assay, as the potency of cAMP production.
284. The derivative of any of embodiments 1-283, which has a
potency corresponding to an EC.sub.50 a) below 18000 pM, preferably
below 10000 pM, more preferably below 5000 pM, even more preferably
below 4000 pM, or most preferably below 3000 pM; b) below 2000 pM,
preferably below 1200 pM, more preferably below 1000 pM, even more
preferably below 800 pM, or most preferably below 600 pM; c) below
400 pM, preferably below 300 pM, more preferably below 200 pM, even
more preferably below 150 pM, or most preferably below 100 pM; or
d) below 80 pM, preferably below 60 pM, more preferably below 50
pM, even more preferably below 40 pM, or most preferably below 30
pM. 285. The derivative of embodiment 284, wherein the potency is
determined as EC.sub.50 for stimulation of the formation of cAMP in
a medium containing the human GLP-1 receptor, preferably using a
stable transfected cell-line such as BHK467-12A (tk-ts13), and/or
using for the determination of cAMP a functional receptor assay,
e.g. based on competition between endogenously formed cAMP and
exogenously added biotin-labelled cAMP, in which assay cAMP is more
preferably captured using a specific antibody, and/or wherein an
even more preferred assay is the AlphaScreen cAMP Assay, most
preferably the one described in Example 59.
286. The derivative of any of embodiments 1-285, for which the
ratio [GLP-1 receptor binding affinity (IC.sub.50 in nM) in the
presence of 2.0% HSA (high albumin), divided by GLP-1 receptor
binding affinity (IC.sub.50 in nM) in the presence of 0.005% HSA
(low albumin)] is: a) at least 1, preferably at least 10, more
preferably at least 20, even more preferably at least 30, or most
preferably at least 40; b) at least 50, preferably at least 60,
more preferably at least 70, even more preferably at least 80, or
most preferably at least 90; c) at least 100, preferably at least
200, more preferably at least 300, still more preferably at least
400, even more preferably at least 500, or most preferably at least
600; d) at least 700, preferably at least 800, more preferably at
least 900, still more preferably at least 1000, even more
preferably at least 1200, or most preferably at least 1400; or e)
at least 1500, preferably at least 1800, more preferably at least
2000, still more preferably at least 2300, even more preferably at
least 2500, or most preferably at least 2800. 287. The derivative
of any of embodiments 1-286, for which the GLP-1 receptor binding
affinity (IC.sub.50) in the presence of 0.005% HSA (low albumin) is
a) below 1000 nM, preferably below 500 nM, more preferably below
100 nM, or most preferably below 50 nM; b) below 10 nM, preferably
below 8.0 nM, still more preferably below 6.0 nM, even more
preferably below 5.0 nM, or most preferably below 2.00 nM; or c)
below 1.00 nM, preferably below 0.50 nM, even more preferably below
0.25 nM, or most preferably below 0.15 nM. 288. The derivative of
any of embodiments 1-287, for which the GLP-1 receptor binding
affinity (IC.sub.50) in the presence of 2.0% HSA (high albumin) is
a) below 1000 nM, preferably below 800 nM; b) below 700 nM,
preferably below 500 nM, more preferably below 300 nM; or c) below
200 nM, preferably below 100 nM, or more preferably below 50 nM.
289. The derivative of any of embodiments 286-288, wherein the
binding affinity to the GLP-1 receptor is measured by way of
displacement of .sup.125I-GLP-1 from the receptor, preferably using
a SPA binding assay. 290. The derivative of embodiment 289, wherein
the GLP-1 receptor is prepared using a stable, transfected cell
line, preferably a hamster cell line, more preferably a baby
hamster kidney cell line, such as BHK tk-ts13. 291. The derivative
of any of embodiments 286-290, wherein the IC.sub.50 value is
determined as the concentration which displaces 50% of
.sup.125I-GLP-1 from the receptor. 292. The derivative of any of
embodiments 1-291, which has an oral bioavailability, preferably an
absolute oral bioavailability, which is higher than that of
semaglutide. 293. The derivative of any of embodiments 1-292, which
has an oral bioavailability, preferably an absolute oral
bioavailability, which is higher than that of liraglutide. 294. The
derivative of any of embodiments 292-293, wherein oral
bioavailability is measured in vivo in rats, as exposure in plasma
after direct injection into the intestinal lumen. 295. The
derivative of any of embodiments 1-294, for which the plasma
concentration (pM) of the derivative, determined 30 minutes after
injection of a solution of the derivative in the jejunum of rat,
divided by the concentration (.mu.M) of the injected solution
(dose-corrected exposure at 30 min) is a) at least 20, preferably
at least 40, more preferably at least 45, even more preferably at
least 50, or most preferably at least 60; or b) at least 70,
preferably at least 80, or most preferably at least 100. 296. The
derivative of any of embodiments 1-295, for which the plasma
concentration (pM) of the derivative, determined 30 minutes after
injection of a solution of the derivative in the jejunum of rat,
divided by the concentration (.mu.M) of the injected solution
(dose-corrected exposure at 30 min) is at least 110, preferably at
least 120, more preferably at least 130, still more preferably at
least 140, even more preferably at least 150, or most preferably at
least 160. 297. The derivative of any of embodiments 1-296, for
which the plasma concentration (pM) of the derivative, determined
30 minutes after injection of a solution of the derivative in the
jejunum of rat, divided by the concentration (.mu.M) of the
injected solution (dose-corrected exposure at 30 min) is at least
180, preferably at least 190, more preferably at least 200, still
more preferably at least 210, even more preferably at least 220, or
most preferably at least 230. 298. The derivative of any of
embodiments 1-297, for which the plasma concentration (pM) of the
derivative, determined 30 minutes after injection of a solution of
the derivative in the jejunum of rat, divided by the concentration
(.mu.M) of the injected solution (dose-corrected exposure at 30
min) is at least 240, preferably at least 250, more preferably at
least 260, or most preferably at least 270. 299. The derivative of
any of embodiments 292-298, wherein the GLP-1 derivative is tested
in a concentration of 1000 uM in a solution of 55 mg/ml sodium
caprate. 300. The derivative of any of embodiments 1-294, for which
the AUC of the dose-corrected (i.e., divided by the dose in pmol of
injected derivative) plasma exposure curve (i.e., concentration in
plasma in pM vs time) from time 30 to 180 min is determined (i.e.,
the result is indicated in (min.times.pM/pmol) or simply in min/L).
301. The derivative of embodiment 300, wherein the AUC of the
dose-corrected plasma exposure curve is a) at least 50, preferably
at least 100, or more preferably at least 150 min/L; b) at least
200, preferably at least 250, more preferably at least 300, or most
preferably at least 320 min/L; or c) at least 1.5 times, preferably
at least 2 times, more preferably at least 3 times, or most
preferably at least 4 times the corresponding AUC value for
semaglutide. 302. The derivative of any of embodiments 1-293,
wherein oral bioavailability is measured in vivo in rats, as
exposure in plasma after oral gavage. 303. The derivative of
embodiment 302, for which the AUC of the dose-corrected (i.e.,
divided by the dose in pmol of administered derivative) plasma
exposure curve (i.e., concentration in plasma in pM vs time) from
time 30 to 180 min is determined (i.e., the result may be indicated
in (min.times.pM/pmol) or simply in min/L). 304. The derivative of
embodiment 303, wherein the AUC of the dose-corrected plasma
exposure curve is a) at least 10, preferably at least 20, or more
preferably at least 30 min/L; b) at least 40, preferably at least
50, more preferably at least 60, or most preferably at least 70
min/L; or c) at least 1.5 times, preferably at least 2 times, more
preferably at least 3 times, or most preferably at least 4 times
the corresponding AUC value for semaglutide. 305. The derivative of
any of embodiments 300-304, wherein the GLP-1 derivative is tested
in a concentration of about 1000 uM in a solution of 250 mg/ml of
sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC). 306. The
derivative of any of embodiments 292-305, wherein male Sprague
Dawley rats are used, preferably with a body weight upon arrival of
approximately 240 g. 307. The derivative of any of embodiments
292-306, wherein the rats are fasted for approximately 18 hours
before the experiment. 308. The derivative of any of embodiments
292-307, wherein the rats are and taken into general anaesthesia
after having fasted and before the injection of the derivative in
the jejunum, or the oral gavage, respectively. 309. The derivative
of any of embodiments 292-308, wherein for injection in the
intestinal lumen the derivative is administered in the proximal
part of the jejunum (10 cm distal for the duodenum) or in the
mid-intestine (50 cm proximal for the cecum), preferably in the
proximal part of the jejunum. 310. The derivative of any of
embodiments 292-309, wherein 100 .mu.l of the derivative is
injected into the jejunal lumen through a catheter with a 1 ml
syringe, and subsequently 200 .mu.l of air is pushed into the
jejunal lumen with another syringe, which is then left connected to
the catheter to prevent flow back into the catheter. 311. The
derivative of any of embodiments 292-310, wherein blood samples
(200 ul) are collected into EDTA tubes from the tail vein at
desired intervals, such as at times 0, 10, 30, 60, 120 and 240 min,
and centrifuged 5 minutes, 10000G, at 4.degree. C. within 20
minutes. 312. The derivative of any of embodiments 292-311, wherein
plasma (e.g. 75 ul) is separated, immediately frozen, and kept at
-20.degree. C. until analyzed for plasma concentration of the
derivative. 313. The derivative of any of embodiments 292-312,
wherein LOCI (Luminescent Oxygen Channeling Immunoassay) is used
for analyzing the plasma concentration of the derivative. 314. The
derivative of any of embodiments 1-313, wherein the derivative is
effective at lowering blood glucose in vivo in db/db mice. 315. The
derivative of any of embodiments 1-314, wherein the derivative is
effective at lowering body weight in vivo in db/db mice. 316. The
derivative of any of embodiments 314-316, wherein db/db mice are
treated, s.c., with a suitable range of doses of the GLP-1
derivative, and blood glucose and/or bodyweight is/are determined
at appropriate intervals. 317. The derivative of any of embodiments
313-316, wherein the dose of the GLP-1 derivative is 0.3 nmol/kg,
1.0 nmol/kg, 3.0 nmol/kg, 10 nmol/kg, 30 nmol/kg, and 100 nmol/kg,
wherein kg refers to the body weight of the mouse. 318. The
derivative of any of embodiments 313-317, wherein a control group
is treated with vehicle, s.c., preferably the medium in which the
GLP-1 derivative is dissolved, e.g. with the following composition:
50 mM sodium phosphate, 145 mM sodium chloride, 0.05% tween 80, pH
7.4. 319. The derivative of any of embodiments 313-318, wherein
blood glucose is determined, and/or the mice are weighed, at time
-1/2h (half an hour prior to dosing (t=0)), and at times 1, 2, 4,
and 8 h. 320. The derivative of any of embodiments 313-319, wherein
the glucose concentration is measured using the glucose oxidase
method. 321. The derivative of any of embodiments 313-320, wherein
[0211] (i) ED.sub.50 (body weight (BW)) is calculated as the dose
giving rise to half-maximum effect on delta (e.g., decrease) BW 8
hours following the subcutaneous administration of the derivative;
and/or [0212] (ii) ED.sub.50 (blood glucose (BG)) is calculated as
the dose giving rise to half-maximum effect on AUC (Area Under the
Curve) delta (e.g., decrease) BG 8 hours and/or 24 hours following
the subcutaneous administration of the analogue. 322. The
derivative of any of embodiments 313-321, wherein a sigmoidal
dose-response relationship exists, preferably with a clear
definition of the maximum response. 323. The derivative of any of
embodiments 313-322, wherein ED.sub.50 (BG) 8 hours is below 5.0
nmol/kg, preferably below 4.0 nmol/kg, more preferably below 3.0
nmol/kg, even more preferably below 2.0 nmol/kg, or most preferably
below 1.0 nmol/kg. 324. The derivative of any of embodiments
313-323, wherein ED.sub.50 (BW) 8 hours is a) below 10, nmol/kg,
preferably below 8 nmol/kg, even more preferably below 6.0 nmol/kg,
or most preferably below 5.0 nmol/kg; or b) below 4.0 nmol/kg,
preferably below 3.0 nmol/kg, even more preferably below 2.0
nmol/kg, or most preferably below 1.0 nmol/kg. 325. The derivative
of any of embodiments 1-324 which, in a PD study in pigs, reduces
food intake on day 1, 2, 3, and/or 4 after s.c. administration of a
single dose of the derivative, as compared to a vehicle-treated
control group. 326. The derivative of embodiment 325, wherein the
study is conducted and the data compiled and analysed as described
in Example 64. 327. The derivative of any of embodiments 1-326,
which has a more protracted profile of action than liraglutide.
328. The derivative of embodiment 327, wherein protraction means
half-life in vivo in a relevant animal species, such as db/db mice,
rat, pig, and/or, preferably, minipig; wherein the derivative is
administered i) s.c., and/or, ii) i.v.; preferably ii) i.v. 329.
The derivative of any of embodiments 1-328, wherein the terminal
half-life (T.sub.1/2) after i.v. administration in rat is higher
than that of semaglutide. 330. The derivative of any of embodiments
1-329, wherein the terminal half-life (T.sub.1/2) after i.v.
administration in rat is at least twice the terminal half-life of
semaglutide. 331. The derivative of any of embodiments 1-330,
wherein the terminal half-life (T.sub.1/2) after i.v.
administration in rat is at least three times the terminal
half-life of semaglutide. 332. The derivative of any of embodiments
1-331, wherein the terminal half-life (T.sub.1/2) after i.v.
administration in rat is at least four times the terminal half-life
of semaglutide. 333. The derivative of any of embodiments 1-332,
wherein the terminal half-life (T.sub.1/2) after i.v.
administration in rat is at least five times the terminal half-life
of semaglutide. 334. The derivative of any of embodiments 1-333,
wherein the half-life is determined in in vivo pharmacokinetic
studies in rat, for example as described in Example 65. 335. The
derivative of any of embodiments 1-334, wherein the terminal
half-life (T.sub.1/2) after i.v. administration in minipigs is a)
at least 8 hours, preferably at least 16 hours, more preferably at
least 24 hours, even more preferably at least 32 hours, or most
preferably at least 40 hours; or b) at least 50 hours, preferably
at least 58 hours, more preferably at least 70 hours, even more
preferably at least 80 hours, or most preferably at least 84 hours.
336. The derivative of embodiment 335, wherein the minipigs are
male Gottingen minipigs. 337. The derivative of any of embodiments
335-336, wherein the minipigs are 7-14 months of age, and
preferably weighing from 16-35 kg. 338. The derivative of any of
embodiments 335-337, wherein the minipigs are housed individually,
and fed once or twice daily, preferably with SDS minipig diet. 339.
The derivative of any of embodiments 335-338, wherein the
derivative is dosed, i.v., after at least 2 weeks of
acclimatisation. 340. The derivative of any of embodiments 335-339,
wherein the animals are fasted for approximately 18 h before dosing
and for at least 4 h after dosing, and have ad libitum access to
water during the whole period. 341. The derivative of any of
embodiments 335-340, wherein the GLP-1 derivative is dissolved in
50 mM sodium phosphate, 145 mM sodium chloride, 0.05% tween 80, pH
7.4 to a suitable concentration, preferably from 20-60 nmol/ml.
342. The derivative of any of embodiments 335-341, wherein
intravenous injections of the derivative are given in a volume
corresponding to 1-2 nmol/kg. 343. An intermediate product in the
form of a GLP-1 analogue which comprises the following changes as
compared to GLP-1(7-37) (SEQ ID NO: 1): (a) 7Imp, 8Aib, 18K, 22E,
34Q (SEQ ID NO: 5); (b) 7Imp, 18K, 22E 25V, 26R, 31K, 34R(SEQ ID
NO: 6); or (c) 8Aib, 18K, 19Q, 22E, 34Q (SEQ ID NO: 7); or a
pharmaceutically acceptable salt, amide, or ester of any of the
analogues of (a)-(c). 344. An intermediate product in the form of a
GLP-1 analogue selected from the following analogues of GLP-1
(7-37) (SEQ ID NO: 1): (a) 7Imp, 8Aib, 18K, 22E, 34Q (SEQ ID NO:
5); (b) 7Imp, 18K, 22E 25V, 26R, 31K, 34R(SEQ ID NO: 6); or (c)
8Aib, 18K, 19Q, 22E, 34Q (SEQ ID NO: 7); or a pharmaceutically
acceptable salt, amide, or ester of any of the analogues of
(a)-(c). 345. The analogue of any of embodiments 343-344, which
comprises a C-terminal amide. 346. The analogue of any of
embodiments 343-345, wherein a carboxylic acid group of the
C-terminal amino acid of the analogue is converted into carboxylic
acid amide. 347. The analogue of embodiment 346, wherein the
carboxylic acid group which is converted into carboxylic acid amide
is not in the side chain of the C-terminal amino acid. 348. The
analogue of any of embodiments 343-344, which comprises a
C-terminal carboxylic acid. 349. The analogue of any of embodiments
343-348, wherein the comparison with GLP-1(7-37) (SEQ ID NO: 1) is
made by handwriting and eyeballing. 350. The analogue of any of
embodiments 343-349, wherein the comparison with GLP-1(7-37) (SEQ
ID NO: 1) is made by use of a standard protein or peptide alignment
program. 351. The analogue of embodiment 350, wherein the alignment
program is a Needleman-Wunsch alignment. 352. The analogue of any
of embodiments 350-351, wherein the default scoring matrix and the
default identity matrix is used. 353. The analogue of any of
embodiments 350-352, wherein the scoring matrix is BLOSUM62. 354.
The analogue of any of embodiments 350-353, wherein the penalty for
the first residue in a gap is -10 (minus ten). 355. The analogue of
any of embodiments 350-354, wherein the penalties for additional
residues in a gap is -0.5 (minus point five). 356. A derivative
according to any of embodiments 1-342, for use as a medicament.
357. A derivative according to any of embodiments 1-342, for use in
the treatment and/or prevention of all forms of diabetes and
related diseases, such as eating disorders, cardiovascular
diseases, gastrointestinal diseases, diabetic complications,
critical illness, and/or polycystic ovary syndrome; and/or for
improving lipid parameters, improving .beta.-cell function, and/or
for delaying or preventing diabetic disease progression. 358. Use
of a derivative according to any of embodiments 1-342 in the
manufacture of a medicament for the treatment and/or prevention of
all forms of diabetes and related diseases, such as eating
disorders, cardiovascular diseases,
gastrointestinal diseases, diabetic complications, critical
illness, and/or polycystic ovary syndrome; and/or for improving
lipid parameters, improving .beta.-cell function, and/or for
delaying or preventing diabetic disease progression. 359. A method
for treating or preventing all forms of diabetes and related
diseases, such as eating disorders, cardiovascular diseases,
gastrointestinal diseases, diabetic complications, critical
illness, and/or polycystic ovary syndrome; and/or for improving
lipid parameters, improving .beta.-cell function, and/or for
delaying or preventing diabetic disease progression--by
administering a pharmaceutically active amount of a derivative
according to any of embodiments 1-342.
Additional Particular Embodiments
[0213] The following (A) are additional particular embodiments of
the invention:
A
[0214] 1. A derivative of a GLP-1 analogue,
[0215] which analogue comprises a first K residue at a position
corresponding to position 18 of GLP-1(7-37) (SEQ ID NO: 1), a
second K residue at another position, and a maximum of twelve amino
acid modifications as compared to GLP-1(7-37),
[0216] which derivative comprises two protracting moieties attached
to said first and second K residue, respectively, via a linker,
wherein
[0217] the protracting moiety is selected from Chem. A, Chem. B,
and Chem. C:
HOOC--(CH.sub.2).sub.x--CO- Chem. A:
R.sup.1--CO--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO- Chem. B:
R.sup.2--C.sub.6H.sub.4--(CH.sub.2).sub.z--CO--, Chem. C:
[0218] in which x is an integer in the range of 6-18, y is an
integer in the range of 3-11, z is an integer in the range of 1-5,
R.sup.1-- is --OH, and R.sup.2 is a group having a molar mass not
higher than 150 Da; and
[0219] the linker comprises
##STR00010##
wherein k is an integer in the range of 1-5, and n is an integer in
the range of 1-5; or a pharmaceutically acceptable salt, amide, or
ester thereof. 2. The derivative of embodiment 1, wherein Chem. D
is a first linker element. 3. The derivative of any one of
embodiments 1-2, wherein k is 1. 4. The derivative of any one of
embodiments 1-3, wherein n is 1. 5. The derivative of any one of
embodiments 1-4, wherein Chem. D is included m times, wherein m is
an integer in the range of 1-10. 6. The derivative of embodiment 5,
wherein m is an integer in the range of 1-6; preferably m is 1, 2,
4, or 6; more preferably m is 1, 2, or 4; even more preferably m is
1 or 4; or most preferably m is 2. 7. The derivative of any one of
embodiments 5-6, wherein, when m is different from 1, the Chem. 4
elements are interconnected via amide bond(s). 8. The derivative of
any one of embodiments 1-7, wherein the linker further comprises a
second linker element. 9. The derivative of embodiment 8, wherein
the second linker element is
##STR00011##
10. The derivative of embodiment 9, wherein Chem. E is included p
times, wherein p is an integer in the range of 1-3. 11. The
derivative of embodiment 10, wherein p is 1, 2, or 3; preferably 2
or 3, or most preferably 1. 12. The derivative of any one of
embodiments 9-11, wherein Chem. E is a radical of L-Glu or D-Glu,
preferably of L-Glu. 13. The derivative of any one of embodiments
10-12, wherein, when p is different from 1, the Chem. E elements
are interconnected via amide bond(s). 14. The derivative of any one
of embodiments 1-13, wherein the linker further comprises a third
linker element. 15. The derivative of embodiment 14, wherein the
third linker element is
--NH--(CH.sub.2).sub.q--CHR.sup.3--CO--, Chem. F:
in which q is an integer in the range of 2-12, and R.sup.3 is amino
(NH.sub.2). 16. The derivative of embodiment 15, wherein q is 4, 6,
or 10. 17. The derivative of any one of embodiments 15-16, wherein
Chem. F is a radical of lysine. 18. The derivative of embodiment
17, wherein the radicalised amino group is at the epsilon
position.
Still Further Additional Particular Embodiments
[0220] The following (B) are still further additional particular
embodiments of the invention:
B
[0221] 8. A derivative of a GLP-1 analogue,
[0222] which analogue comprises a first K residue at a position
corresponding to position 18 of GLP-1(7-37) (SEQ ID NO: 1), a
second K residue at another position, and a maximum of twelve amino
acid changes as compared to GLP-1 (7-37),
[0223] which derivative comprises two protracting moieties attached
to said first and second K residue, respectively, via a linker,
wherein
[0224] the protracting moiety is selected from Chem. A, Chem. B,
and Chem. C:
HOOC--(CH.sub.2).sub.x--CO--* Chem. A:
HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem. B:
R.sup.2--C.sub.6H.sub.4--(CH.sub.2).sub.z--CO--*, Chem. C:
[0225] in which x is an integer in the range of 6-18, y is an
integer in the range of 3-17, z is an integer in the range of 1-5,
and R.sup.2 is a group having a molar mass not higher than 150 Da;
and
[0226] the linker comprises
##STR00012##
wherein k is an integer in the range of 1-5, and n is an integer in
the range of 1-5; or a pharmaceutically acceptable salt, amide, or
ester thereof. 9. The derivative of any of embodiments 1, and 4-8,
wherein Chem. D is a first linker element. 10. The derivative of
embodiment 9, wherein k is 1. 11. The derivative of any of
embodiments 9-10, wherein n is 1. 12. The derivative of any of
embodiments 9-11, wherein Chem. D is included m times, wherein m is
an integer in the range of 1-10. 13. The derivative of embodiment
12, wherein m is an integer in the range of 1-6. 14. The derivative
of any of embodiments 12-13, wherein m is 1, 2, 4, or 6. 15. The
derivative of any of embodiments 12-14, wherein m is 1. 16. The
derivative of any of embodiments 12-14, wherein m is 2. 17. The
derivative of any of embodiments 12-14, wherein m is 4. 18. The
derivative of any of embodiments 12-14, wherein m is 6. 19. The
derivative of any of embodiments 12-14, and 16-18, wherein, when m
is different from 1, the Chem. D elements are interconnected via
amide bond(s). 20. The derivative of any of embodiments 8-19,
wherein the linker comprises a second, optional, linker element.
21. The derivative of embodiment 20, wherein the second linker
element is selected from Chem. E1 and Chem. E2:
##STR00013##
22. The derivative of any of embodiments 20-21, wherein the second
linker element is Chem. E1. 23. The derivative of embodiment 22,
wherein Chem. E1 is included p times, wherein p is 0, or an integer
in the range of 1-3. 24. The derivative of embodiment 23, wherein p
is 0. 25. The derivative of embodiment 23, wherein p is 1. 26. The
derivative of embodiment 23, wherein p is 2. 27. The derivative of
embodiment 23, wherein p is 3. 28. The derivative any of
embodiments 21-27, wherein Chem. E1 is a di-radical of L-Glu or
D-Glu. 29. The derivative of embodiment 28, wherein Chem. E1 is a
di-radical of L-Glu. 30. The derivative of any of embodiments
23-29, wherein, when p is different from 0 and different from 1,
the Chem. E1 elements are interconnected via amide bond(s). 31. The
derivative of any of embodiments 23-30, wherein the linker
comprises a third, optional, linker element. 32. The derivative of
embodiment 31, wherein the third linker element is
*--NH--(CH.sub.2).sub.q--CHR.sup.3--CO--*, Chem. F:
in which q is an integer in the range of 2-12, and R.sup.3 is amino
(NH.sub.2). 33. The derivative of embodiment 32, wherein q is 4.
34. The derivative of embodiment 32, wherein q is 6. 35. The
derivative of embodiment 32, wherein q is 10. 37. The derivative of
any of embodiments 32-35, wherein R.sup.3 is amino (NH.sub.2). 38.
The derivative of any of embodiments 32-37, wherein Chem. F is a
di-radical of lysine.
[0227] The combination of any of the above A or B embodiments with
any of the PARTICULAR EMBODIMENTS 1-359 listed hereinabove is
hereby specifically incorporated by reference.
EXAMPLES
[0228] This experimental part starts with a list of abbreviations,
and is followed by a section including general methods for
synthesising and characterising analogues and derivatives of the
invention. Then follows a number of examples which relate to the
preparation of specific GLP-1 derivatives, and at the end a number
of examples have been included relating to the activity and
properties of these analogues and derivatives (section headed
pharmacological methods).
[0229] The examples serve to illustrate the invention.
LIST OF ABBREVIATIONS
[0230] Aib: .alpha.-aminoisobutyric acid [0231] API: Active
Pharmaceutical Ingredient [0232] AUC: Area Under the Curve [0233]
BG: Blood Glucose [0234] BHK Baby Hamster Kidney [0235] BW: Body
Weight [0236] Boc: t-butyloxycarbonyl [0237] Bom: benzyloxymethyl
[0238] BSA: Bovine serum albumin [0239] Bzl: benzyl [0240] CAS:
Chemical Abstracts Service [0241] Clt: 2-chlorotrityl [0242]
collidine: 2,4,6-trimethylpyridine [0243] DCM: dichloromethane
[0244] Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl [0245]
DesH: des-amino histidine (may also be referred to as
imidazopropionic acid, Imp) [0246] DIC: diisopropylcarbodiimide
[0247] DIPEA: diisopropylethylamine [0248] DMEM: Dulbecco's
Modified Eagle's Medium (DMEM) [0249] EDTA:
ethylenediaminetetraacetic acid [0250] EGTA: ethylene glycol
tetraacetic acid [0251] FCS: Fetal Calf Serum [0252] Fmoc:
9-fluorenylmethyloxycarbonyl [0253] HATU:
(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluoro-phosphate) [0254] HBTU:
(2-(1H-benzotriazol-1-yl-)-1,1,3,3 tetramethyluronium
hexafluorophosphate) [0255] HEPES:
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [0256] HFIP
1,1,1,3,3,3-hexafluoro-2-propanol or hexafluoroisopropanol [0257]
HOAt: 1-hydroxy-7-azabenzotriazole [0258] HOBt:
1-hydroxybenzotriazole [0259] HPLC: High Performance Liquid
Chromatography [0260] HSA: Human Serum Albumin [0261] IBMX:
3-isobutyl-1-methylxanthine [0262] Imp: Imidazopropionic acid (also
referred to as des-amino histidine, DesH) [0263] i.v. intravenously
[0264] ivDde:
1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl [0265]
IVGTT: Intravenous Glucose Tolerance Test [0266] LCMS: Liquid
Chromatography Mass Spectroscopy [0267] LYD: Landrace Yorkshire
Duroc [0268] MALDI-MS: See MALDI-TOF MS [0269] MALDI-TOF MS:
Matrix-Assisted Laser Desorption/Ionisation Time of Flight Mass
Spectro-scopy [0270] MeOH: methanol [0271] Mmt: 4-methoxytrityl
[0272] Mtt: 4-methyltrityl [0273] NMP: N-methyl pyrrolidone [0274]
OBz: benzoyl ester [0275] OEG: 8-amino-3,6-dioxaoctanic acid [0276]
OPfp: pentafluorophenoxy [0277] OPnp: para-nitrophenoxy [0278] OSu:
O-succinimidyl esters (hydroxysuccinimide esters) [0279] OtBu: tert
butyl ester [0280] Pbf:
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl [0281] PBS:
Phosphate Buffered Saline [0282] PD: Pharmacodynamic [0283]
Pen/Strep: Pencillin/Streptomycin [0284] PK: Pharmacokinetic [0285]
RP: Reverse Phase [0286] RP-HPLC: Reverse Phase High Performance
Liquid Chromatography [0287] RT: Room Temperature [0288] Rt:
Retention time [0289] s.c.: Subcutaneously [0290] SD: Standard
Deviation [0291] SEC-HPLC: Size Exclusion High Performance Liquic
Chromatography [0292] SEM: Standard Error of Mean [0293] SPA:
Scintillation Proximity Assay [0294] SPPS: Solid Phase Peptide
Synthesis [0295] tBu: tert. butyl [0296] TFA: trifluoroacetic acid
[0297] TIS: triisopropylsilane [0298] TLC: Thin Layer
Chromatography [0299] Tos: tosylate (or pare-toluenesulfonyl)
[0300] Tris: tris(hydroxymethyl)aminomethane or
2-amino-2-hydroxymethyl-propane-1,3-diol [0301] Trt:
triphenylmethyl (trityl) [0302] Trx: tranexamic acid [0303] UPLC:
Ultra Performance Liquid Chromatography
Materials and Methods
Materials
[0303] [0304] N-.alpha.,N-.beta.-Di-Fmoc-L-2,3-Diaminopropionic
Acid (CAS 201473-90-7) [0305] 3,5-Di-tert-butyl-4-hydroxybenzoic
acid (CAS 1421-49-4) [0306] 3,5-Di-tert-butylbenzoic Acid (CAS
16225-26-6) [0307] Fmoc-8-amino-3,6-dioxaoctanoic acid (CAS
166108-71-0) [0308]
17-(9-Fluorenylmethyloxycarbonyl-amino)-9-aza-3,6,12,15-tetraoxa-10-on-he-
ptadecanoic acid (IRIS Biotech GmbH) [0309] Fmoc-L-Glutamic acid
1-tert-butyl ester (CAS 84793-07-7) [0310]
2-(2-Methoxyethoxyl)acetic acid (CAS 16024-56-9) [0311]
N-.alpha.,N-.epsilon.-Bis(9-fluorenylmethyloxycarbonyl)-L-lysine
(CAS 78081-87-5) [0312]
1-[(9H-fluoren-9-ylmethoxy)carbonyl]piperidine-4-carboxylic acid
(CAS 148928-15-8) [0313] FMOC-8-Aminocapryl acid (CAS 126631-93-4)
[0314] 4-Phenylbutyric acid (CAS 1716-12-7) [0315]
4-(4-Nitrophenyl)butyric acid (CAS 5600-62-4) [0316]
4-(4-Chlorophenyl)butyric acid (CAS 4619-18-5) [0317]
FMOC-6-Aminohexanoic acid (CAS 88574-06-5) [0318]
FMOC-12-Aminododecanoic acid (CAS 128917-74-8) [0319]
4-(9-carboxy-nonyloxy)-benzoic acid tert-butyl ester (prepared as
described in Example 25, step 1 and 2 of WO 2006/082204) [0320]
4-(8-Carboxy-octyloxy)-benzoic acid tert-butyl ester (M.p.:
71-72.degree. C.
[0321] .sup.1H NMR (300 MHz, CDCl.sub.3, .delta.H): 7.93 (d, J=8.9
Hz, 2H); 6.88 (d, J=8.9 Hz, 2H); 4.00 (t, J=6.4 Hz, 2H); 2.36 (t,
J=7.4 Hz, 2H); 1.80 (m, 2H); 1.65 (m, 2H); 1.59 (s, 9H); 1.53-1.30
(m, 8H) (prepared as described in Example 25, step 1 and 2 of WO
2006/082204, replacing methyl 10-bromodecanoate with ethyl
9-Bromononanoate (CAS 28598-81-4)) 4-(7-Carboxy-heptyloxy)-benzoic
acid tert-butyl ester (.sup.1H NMR spectrum (300 MHz, CDCl.sub.3,
.delta..sub.H): 7.93 (d, J=9.0 Hz, 2H); 6.88 (d, J=9.0 Hz, 2H);
4.00 (t, J=6.5 Hz, 2H); 2.37 (t, J=7.4 Hz, 2H); 1.80 (m, 2H); 1.64
(m, 2H); 1.59 (s, 9H); 1.53-1.33 (m, 6H)) (prepared as described in
Example 25, step 1 and 2 of WO 2006/082204, replacing methyl
10-bromodecanoate with ethyl 7-bromoheptanoate (CAS
29823-18-5))
Chemical Methods
[0322] This section is divided in two: Section A relating to
general methods (of preparation (A1); and of detection and
characterisation (A2)), and section B, in which the preparation and
characterisation of a number of specific example compounds is
described.
A. General Methods
A1. Methods of Preparation
[0323] This section relates to methods for solid phase peptide
synthesis (SPPS methods, including methods for de-protection of
amino acids, methods for cleaving the peptide from the resin, and
for its purification), as well as methods for detecting and
characterising the resulting peptide (LCMS, MALDI, and UPLC
methods). The solid phase synthesis of peptides may in some cases
be improved by the use of di-peptides protected on the di-peptide
amide bond with a group that can be cleaved under acidic conditions
such as, but not limited to, 2-Fmoc-oxy-4-methoxybenzyl, or
2,4,6-trimethoxybenzyl. In cases where a serine or a threonine is
present in the peptide, pseudoproline di-peptides may be used
(available from, e.g., Novabiochem, see also W. R. Sampson (1999),
J. Pep. Sci. 5, 403). The Fmoc-protected amino acid derivatives
used were the standard recommended: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH,
Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH,
Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH,
Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, or, Fmoc-Val-OH etc. supplied
from e.g. Anaspec, Bachem, Iris Biotech, or Novabiochem. Were
nothing else is specified the natural L-form of the amino acids are
used. The N-terminal amino acid was Boc protected at the alpha
amino group (e.g. Boc-His(Boc)-OH, or Boc-His(Trt)-OH for peptides
with His at the N-terminus). In case of modular albumin binding
moiety attachment using SPPS the following suitably protected
building blocks such as but not limited to
Fmoc-8-amino-3,6-dioxaoctanoic acid, Fmoc-tranexamic acid,
Fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester,
nonadecanedioic acid mono-tert-butyl ester, tetradecanedioic acid
mono-tert-butyl ester, or 4-(9-carboxynonyloxy) benzoic acid
tert-butyl ester were used. All operations stated below were
performed at 250-pmol synthesis scale.
1. Synthesis of Resin Bound Protected Peptide Backbone
Method: SPPS_P
[0324] SPPS_P was performed on a Prelude Solid Phase Peptide
Synthesizer from Protein Technologies (Tucson, Ariz. 85714 U.S.A.)
at 250-.mu.mol scale using six fold excess of Fmoc-amino acids (300
mM in NMP with 300 mM HOAt) relative to resin loading, e.g. low
load Fmoc-Gly-Wang (0.35 mmol/g). Fmoc-deprotection was performed
using 20% piperidine in NMP. Coupling was performed using 3:3:3:4
amino acid/HOAt/DIC/collidine in NMP. NMP and DCM top washes (7 ml,
0.5 min, 2.times.2 each) were performed between deprotection and
coupling steps. Coupling times were generally 60 minutes. Some
amino acids including, but not limited to Fmoc-Arg(Pbf)-OH,
Fmoc-Aib-OH or Boc-His(Trt)-OH were "double coupled", meaning that
after the first coupling (e.g. 60 min), the resin is drained and
more reagents are added (amino acid, HOAt, DIC, and collidine), and
the mixture allowed to react again (e.g. 60 min).
Method: SPPS_L
[0325] SPPS_L was performed on a microwave-based Liberty peptide
synthesiser from CEM Corp. (Matthews, N.C. 28106, U.S.A.) at
250-.mu.mol or 100-.mu.mol scale using six fold excess of
Fmoc-amino acids (300 mM in NMP with 300 mM HOAt) relative to resin
loading, e.g. low load Fmoc-Gly-Wang (0.35 mmol/g).
Fmoc-deprotection was performed using 5% piperidine in NMP at up to
75.degree. C. for 30 seconds where after the resin was drained and
washed with NMP and the Fmoc-deprotection was repeated this time
for 2 minutes at 75.degree. C. Coupling was performed using 1:1:1
amino acid/HOAt/DIC in NMP. Coupling times and temperatures were
generally 5 minutes at up to 75.degree. C. Longer coupling times
were used for larger scale reactions, for example 10 min. Histidine
amino acids were double coupled at 50.degree. C., or quadruple
coupled if the previous amino acid was sterically hindered (e.g.
Aib). Arginine amino acids were coupled at RT for 25 minutes and
then heated to 75.degree. C. for 5 min. Some amino acids such as
but not limited to Aib, were "double coupled", meaning that after
the first coupling (e.g. 5 min at 75.degree. C.), the resin is
drained and more reagents are added (amino acid, HOAt and DIC), and
the mixture is heated again (e.g. 5 min at 75.degree. C.). NMP
washes (5.times.10 ml) were performed between deprotection and
coupling steps.
Method: SPPS_A
[0326] The protected peptidyl resin was synthesised according to
the Fmoc strategy on an Applied Biosystems 433 peptide synthesiser
in a 250-pmol or 1000 .mu.mol scale with three or four fold excess
of Fmoc-amino acids, using the manufacturer supplied FastMoc UV
protocols which employ HBTU (2-(1H-Benzotriazol-1-yl-)-1,1,3,3
tetramethyluronium hexafluorophosphate) or HATU
(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate) mediated couplings in NMP and UV monitoring of
the deprotection of the Fmoc protection group, in some cases double
couplings were used, meaning that after the first coupling, the
resin is drained and more Fmoc-amino acids and reagents are added.
The starting resin used for the synthesis of the peptide amides was
Rink-Amide resin and either preloaded Wang (e.g. low load
Fmoc-Gly-Wang or Fmoc-Lys(Mtt)-wang) or chlorotrityl resin for
peptides with a carboxy C-terminal. The protected amino acid
derivatives used were standard Fmoc-amino acids (supplied from e.g.
Anaspec, or Novabiochem) supplied in preweighed cartridges suitable
for the AB1433A synthesiser with the exception of unnatural
aminoacids such as Fmoc-Aib-OH (Fmoc-aminoisobutyric acid). The N
terminal amino acid was Boc protected at the alpha amino group
(e.g. Boc-His(Boc)-OH or Boc-His(Trt)-OH was used for peptides with
His at the N-terminal). The epsilon amino group of lysines in the
sequence were either protected with Mtt, Mmt, Dde, ivDde, or Boc,
depending on the route for attachment of the albumin binding moiety
and spacer. The synthesis of the peptides may in some cases be
improved by the use of dipeptides protected on the dipeptide amide
bond with a group that can be cleaved under acidic conditions such
but not limited to 2-Fmoc-oxy-4-methoxybenzyl or
2,4,6-trimethoxybenzyl. In cases where a serine or a threonine is
present in the peptide, the use of pseudoproline dipeptides may be
used (see e.g. catalogue from Novobiochem 2009/2010 or newer
version, or W. R. Sampson (1999), J. Pep. Sci. 5, 403).
Method: SPPS_M
[0327] SPPS_M refers to synthesis of the protected peptidyl resin
using manual Fmoc chemistry. The coupling chemistry was
DIC/HOAt/collidine in NMP at a 4-10 fold molar excess. Coupling
conditions were 1-6 h at room temperature. Fmoc-deprotection was
performed with 20-25% piperidine in NMP (3.times.20 ml, each 10
min) followed by NMP washings (4.times.20 mL).
2. Synthesis of Side Chains
Mono Esters of Fatty Diacids
[0328] Overnight reflux of the C8, C10, C12, C14, C16 and C18
diacids with Boc-anhydride DMAP t-butanol in toluene gives
predominately the t-butyl mono ester. Obtained is after work-up a
mixture of mono acid, diacid and diester. Purification is carried
out by washing, short plug silica filtration and
crystallisation.
3. Attachment of Side Chains to Resin Bound Protected Peptide
Backbone
[0329] When an acylation is present on a lysine side chain, the
epsilon amino group of lysine to be acylated was protected with
either Mtt, Mmt, Dde, ivDde, or Boc, depending on the route for
attachment of the protracting moiety and linker. Dde- or
ivDde-deprotection was performed with 2% hydrazine in NMP
(2.times.20 ml, each 10 min) followed by NMP washings (4.times.20
ml). Mtt- or Mmt-deprotection was performed with 2% TFA and 2-3%
TIS in DCM (5.times.20 ml, each 10 min) followed by DCM (2.times.20
ml), 10% MeOH and 5% DIPEA in DCM (2.times.20 ml) and NMP
(4.times.20 ml) washings, or by treatment with
hexafluoroisopropanol/DCM (75:25, 5.times.20 ml, each 10 min)
followed by washings as above. In some cases the Mtt group was
removed by automated steps on the Liberty peptide synthesiser. Mtt
deprotection was performed with hexafluoroisopropanol or
hexafluoroisopropanol/DCM (75:25) at room temperature for 30 min
followed by washing with DCM (7 ml.times.5), followed by NMP
washings (7 ml.times.5). The protracting moiety and/or linker can
be attached to the peptide either by acylation of the resin bound
peptide or by acylation in solution of the unprotected peptide. In
case of attachment of the protracting moiety and/or linker to the
protected peptidyl resin the attachment can be modular using SPPS
and suitably protected building blocks.
Method: SC_P
[0330] The N-.epsilon.-lysine protection group was removed as
described above and the chemical modification of the lysine was
performed by one or more automated steps on the Prelude peptide
synthesiser using suitably protected building blocks as described
above. Double couplings were performed as described in SPPS_P with
3 hours per coupling.
Method: SC_L
[0331] The N-.epsilon.-lysine protection group was removed as
described above and the chemical modification of the lysine was
performed by one or more automated steps on the Liberty peptide
synthesiser using suitably protected building blocks as described
above. Double couplings were performed as described in SPPS_L.
Method: SC_A
[0332] The N-.epsilon.-lysine protection group was removed as
described above and the chemical modification of the lysine was
performed by one or more automated steps on the ABI peptide
synthesiser using suitably protected building blocks as described
in SPPS_A.
Method: SC_M1
[0333] The N-.epsilon.-lysine protection group was removed as
described above. Activated (active ester or symmetric anhydride)
protracting moiety or linker such as octadecanedioic acid
mono-(2,5-dioxo-pyrrolidin-1-yl) ester (Ebashi et al. EP511600, 4
molar equivalents relative to resin bound peptide) was dissolved in
NMP (25 mL), added to the resin and shaken overnight at room
temperature. The reaction mixture was filtered and the resin was
washed extensively with NMP, DCM, 2-propanol, methanol and diethyl
ether.
Method: SC_M2
[0334] The N-.epsilon.-lysine protection group was removed as
described above. The protracting moiety was dissolved in NMP/DCM
(1:1, 10 ml). The activating reagent such as HOBt (4 molar
equivalents relative to resin) and DIC (4 molar equivalents
relative to resin) was added and the solution was stirred for 15
min. The solution was added to the resin and DIPEA (4 molar
equivalents relative to resin) was added. The resin was shaken 2 to
24 hours at room temperature. The resin was washed with NMP
(2.times.20 ml), NMP/DCM (1:1, 2.times.20 ml) and DCM (2.times.20
ml).
Method: SC_M3
[0335] Activated (active ester or symmetric anhydride) protracting
moiety or linker such as octadecanedioic acid
mono-(2,5-dioxo-pyrrolidin-1-yl) ester (Ebashi et al. EP511600)
1-1.5 molar equivalents relative to the peptide was dissolved in an
organic solvent such as acetonitrile, THF, DMF, DMSO or in a
mixture of water/organic solvent (1-2 ml) and added to a solution
of the peptide in water (10-20 ml) together with 10 molar
equivalents of DIPEA. In case of protecting groups on the
protracting moiety such as tert-butyl, the reaction mixture was
lyophilised overnight and the isolated crude peptide deprotected
afterwards. In case of tert-butyl protection groups the
deprotection was performed by dissolving the peptide in a mixture
of trifluoroacetic acid, water and triisopropylsilane (90:5:5).
After 30 min the mixture was evaporated in vacuo and the crude
peptide purified by preparative HPLC as described later.
4. Clevage of Resin Bound Peptide with or without Attached Side
Chains and Purification
Method: CP_M1
[0336] After synthesis the resin was washed with DCM, and the
peptide was cleaved from the resin by a 2-3 hour treatment with
TFA/TIS/water (95/2.5/2.5 or 92.5/5/2.5) followed by precipitation
with diethylether. The peptide was dissolved in a suitable solvent
(such as, e.g., 30% acetic acid) and purified by standard RP-HPLC
on a C18, 5 .mu.M column, using acetonitrile/water/TFA. The
fractions were analysed by a combination of UPLC, MALDI and LCMS
methods, and the appropriate fractions were pooled and
lyophilised.
Method: CP_L1
[0337] After synthesis the resin was washed with DCM, and the
peptide was cleaved from the resin by the use of a CEM Accent
Microwave Cleavage System (CEM Corp., North Carolina). Cleavage
from the resin was performed at 38.degree. C. for 30 minutes by the
treatment with TFA/TIS/water (95/2.5/2.5) followed by precipitation
with diethylether. The peptide was dissolved in a suitable solvent
(such as, e.g., 30% acetic acid) and purified by standard RP-HPLC
on a C18, 5 .mu.M column, using acetonitrile/water/TFA. The
fractions were analysed by a combination of UPLC, MALDI and LCMS
methods, and the appropriate fractions were pooled and
lyophilized.
A2. General Methods for Detection and Characterisation
1. LC-MS Methods
Method: LCMS.sub.--1
[0338] An Agilent Technologies LC/MSD TOF (G1969A) mass
spectrometer was used to identify the mass of the sample after
elution from an Agilent 1200 series HPLC system. The de-convolution
of the protein spectra was calculated with Agilent's protein
confirmation software. Eluents: A: 0.1% Trifluoro acetic acid in
water; B: 0.1% Trifluoro acetic acid in acetonitrile. Column:
Zorbax 5 u, 300SB-C3, 4.8.times.50 mm. Gradient: 25%-95% B over 15
min.
Method: LCMS.sub.--2
[0339] A Perkin Elmer Sciex API 3000 mass spectrometer was used to
identify the mass of the sample after elution from a Perkin Elmer
Series 200 HPLC system. Eluents: A: 0.05% Trifluoro acetic acid in
water; B: 0.05% Trifluoro acetic acid in acetonitrile. Column:
Waters Xterra MS C-18.times.3 mm id 5 .mu.m. Gradient: 5%-90% B
over 7.5 min at 1.5 ml/min.
Method: LCMS.sub.--3
[0340] A Waters Micromass ZQ mass spectrometer was used to identify
the mass of the sample after elution from a Waters Alliance HT HPLC
system. Eluents: A: 0.1% Trifluoro acetic acid in water; B: 0.1%
Trifluoro acetic acid in acetonitrile. Column: Phenomenex, Jupiter
C4 50.times.4.60 mm id 5 .mu.m. Gradient: 10%-90% B over 7.5 min at
1.0 ml/min.
Method: LCMS.sub.--4
[0341] LCMS.sub.--4 was performed on a setup consisting of Waters
Acquity UPLC system and LCT Premier XE mass spectrometer from
Micromass. Eluents: A: 0.1% Formic acid in water B: 0.1% Formic
acid in acetonitrile The analysis was performed at RT by injecting
an appropriate volume of the sample (preferably 2-10 .mu.l) onto
the column which was eluted with a gradient of A and B. The UPLC
conditions, detector settings and mass spectrometer settings were:
Column: Waters Acquity UPLC BEH, C-18, 1.7 .mu.m, 2.1 mm.times.50
mm. Gradient: Linear 5%-95% acetonitrile during 4.0 min
(alternatively 8.0 min) at 0.4 ml/min. Detection: 214 nm (analogue
output from TUV (Tunable UV detector)) MS ionisation mode: API-ES
Scan: 100-2000 amu (alternatively 500-2000 amu), step 0.1 amu.
Method: LCMS_AP
[0342] A Micromass Quatro micro API mass spectrometer was used to
identify the mass of the sample after elution from a HPLC system
composed of Waters2525 binary gradient modul, Waters2767 sample
manager, Waters 2996 Photodiode Array Detector and Waters 2420 ELS
Detector. Eluents: A: 0.1% Trifluoro acetic acid in water; B: 0.1%
Trifluoro acetic acid in acetonitrile. Column: Phenomenex Synergi
MAXRP, 4 um, 75.times.4.6 mm. Gradient: 5%-95% B over 7 min at 1.0
ml/min.
2. UPLC Methods
Method: B5.sub.--1
[0343] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH130, C18, 130 .ANG., 1.7
um, 2.1 mm.times.150 mm column, 40.degree. C. The UPLC system was
connected to two eluent reservoirs containing: A: 0.2 M
Na.sub.2SO.sub.4, 0.04 M H.sub.3PO.sub.4, 10% CH.sub.3CN (pH 3.5);
B: 70% CH.sub.3CN, 30% H.sub.2O. The following linear gradient was
used: 60% A, 40% B to 30% A, 70% B over 8 minutes at a flow-rate of
0.40 ml/min.
Method: B7.sub.--1
[0344] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH130, C18, 130 .ANG., 1.7
um, 2.1 mm.times.150 mm column, 40.degree. C. The UPLC system was
connected to two eluent reservoirs containing: A: 0.2 M
Na.sub.2SO.sub.4, 0.04 M H.sub.3PO.sub.4, 10% CH.sub.3CN (pH 3.5);
B: 70% CH.sub.3CN, 30% H.sub.2O. The following linear gradient was
used: 80% A, 20% B 15 to 40% A, 60% B over 8 minutes at a flow-rate
of 0.40 ml/min.
Method: B9.sub.--1
[0345] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH130, C18, 130 .ANG., 1.7
um, 2.1 mm.times.150 mm column, 40.degree. C. The UPLC system was
connected to two eluent reservoirs containing: A: 0.2 M
Na.sub.2SO.sub.4, 0.04 M H.sub.3PO.sub.4, 10% CH.sub.3CN (pH 3.5);
B: 70% CH.sub.3CN, 30% H.sub.2O. The following linear gradient was
used: 70% A, 30% B to 20% A, 80% B over 8 minutes at a flow-rate of
0.40 ml/min.
Method: A2.sub.--1
[0346] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH130, C18, 130 .ANG., 1.7
um, 2.1 mm.times.150 mm column, 40.degree. C. The UPLC system was
connected to two eluent reservoirs containing: A: 90% H.sub.2O, 10%
CH.sub.3CN, 0.25 M ammonium bicarbonate; B: 70% CH.sub.3CN, 30%
H.sub.2O. The following linear gradient was used: 90% A, 10% B to
60% A, 40% B over 16 minutes at a flow-rate of 0.40 ml/min.
Method: A3.sub.--1
[0347] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH130, C18, 130 .ANG., 1.7
um, 2.1 mm.times.150 mm column, 40.degree. C. The UPLC system was
connected to two eluent reservoirs containing: A: 90% H.sub.2O, 10%
CH.sub.3CN, 0.25 M ammonium bicarbonate; B: 70% CH.sub.3CN, 30%
H.sub.2O. The following linear gradient was used: 75% A, 25% B to
45% A, 55% B over 16 minutes at a flow-rate of 0.40 ml/min.
Method: A4.sub.--1
[0348] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH130, C18, 130 .ANG., 1.7
um, 2.1 mm.times.150 mm column, 40.degree. C. The UPLC system was
connected to two eluent reservoirs containing: A: 90% H.sub.2O, 10%
CH.sub.3CN, 0.25 M ammonium bicarbonate; B: 70% CH.sub.3CN, 30%
H.sub.2O. The following linear gradient was used: 65% A, 35% B to
25% A, 65% B over 16 minutes at a flow-rate of 0.40 ml/min.
Method: A6.sub.--1
[0349] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH130, C18, 130 .ANG., 1.7
um, 2.1 mm.times.150 mm column, 40.degree. C. The UPLC system was
connected to two eluent reservoirs containing: A: 10 mM TRIS, 15 mM
ammonium sulphate, 80% H.sub.2O, 20% CH.sub.3CN, pH 7.3; B: 80%
CH.sub.3CN, 20% H.sub.2O. The following linear gradient was used:
95% A, 5% B to 10% A, 90% B over 16 minutes at a flow-rate of 0.35
ml/min.
Method: A7.sub.--1
[0350] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH130, C18, 130 .ANG., 1.7
um, 2.1 mm.times.150 mm column, 40.degree. C. The UPLC system was
connected to two eluent reservoirs containing: A: 10 mM TRIS, 15 mM
ammonium sulphate, 80% H.sub.2O, 20% CH.sub.3CN, pH 7.3; B: 80%
CH.sub.3CN, 20% H.sub.2O. The following linear gradient was used:
95% A, 5% B to 40% A, 60% B over 16 minutes at a flow-rate of 0.40
ml/min.
Method: B2.sub.--1
[0351] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH130, C18, 130 .ANG., 1.7
um, 2.1 mm.times.150 mm column, 40.degree. C. The UPLC system was
connected to two eluent reservoirs containing: A: 99.95% H.sub.2,
0.05% TFA; B: 99.95% CH.sub.3CN, 0.05% TFA. The following linear
gradient was used: 95% A, 5% B to 40% A, 60% B over 16 minutes at a
flow-rate of 0.40 ml/min.
Method: B4.sub.--1
[0352] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH130, C18, 130 .ANG., 1.7
um, 2.1 mm.times.150 mm column, 40.degree. C. The UPLC system was
connected to two eluent reservoirs containing: A: 99.95% H.sub.2O,
0.05% TFA; B: 99.95% CH.sub.3CN, 0.05% TFA. The following linear
gradient was used: 95% A, 5% B to 95% A, 5% B over 16 minutes at a
flow-rate of 0.40 ml/min.
Method: B10.sub.--1
[0353] The RP-analyses was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH130, C18, 130 .ANG., 1.7
um, 2.1 mm.times.150 mm column, 40.degree. C. The UPLC system was
connected to two eluent reservoirs containing: A: 0.2 M
Na.sub.2SO.sub.4, 0.04 M H.sub.3PO.sub.4, 10% CH.sub.3CN (pH 3.5);
B: 70% CH.sub.3CN, 30% H.sub.2O. The following linear gradient was
used: 40% A, 60% B to 20% A, 80% B over 8 minutes at a flow-rate of
0.40 ml/min.
Method: B14.sub.--1
[0354] The RP-analyses was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH Shield RP18, 1.7 um,
2.1 mm.times.150 mm column, 50.degree. C. The UPLC system was
connected to two eluent reservoirs containing: A: 99.95% H.sub.2,
0.05% TFA; B: 99.95% CH.sub.3CN, 0.05% TFA. The following linear
gradient was used: 70% A, 30% B to 40% A, 60% B over 12 minutes at
a flow-rate of 0.40 ml/min.
Method: B8.sub.--1
[0355] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH130, C18, 130 .ANG., 1.7
um, 2.1 mm.times.150 mm column, 40.degree. C. The UPLC system was
connected to two eluent reservoirs containing: A: 0.2 M
Na.sub.2SO.sub.4, 0.04 M H.sub.3PO.sub.4, 10% CH.sub.3CN (pH 3.5);
B: 70% CH.sub.3CN, 30% H.sub.2O. The following linear gradient was
used: 50% A, 50% B to 20% A, 80% B over 8 minutes at a flow-rate of
0.40 ml/min.
Method: B29.sub.--1
[0356] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 215 nm and 254
nm were collected using an kinetex 1.7 u C18, 100A 2.1.times.150 mm
column, 60.degree. C. The UPLC system was connected to two eluent
reservoirs containing: A: 90% water and 10% CH.sub.3CN with 0.045M
(NH.sub.4).sub.2HPO.sub.4, pH 3.6, B: 20% isopropanole, 20% water
and 60% CH.sub.3CN. The following step gradient was used: 35% B and
65% A over 2 minutes, then 35% B, 65% A to 65% B, 35% A over 15
minutes, then 65% B, 35% A to 80% B, 20% A over 3 minutes at a
flowrate of 0.5 ml/min.
Method: B31.sub.--1
[0357] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 215 nm and 254
nm were collected using an kinetex 1.7 u C18, 100A 2.1.times.150 mm
column, 60.degree. C. The UPLC system was connected to two eluent
reservoirs containing: A: 90% water and 10% MeCN with 0.045M
(NH.sub.4).sub.2HPO.sub.4, pH 3.6, B: 20% isopropanole, 20% water
and 60% CH.sub.3CN. The following step gradient was used: 25% B and
75% A over 2 minutes, then 25% B, 75% A to 55% B, 45% A over 15
minutes, then 55% B, 45% A to 80% B, 20% A over 3 minutes at a
flowrate of 0.5 ml/min.
Method: AP_B4.sub.--1
[0358] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH130, C18, 130 .ANG., 1.7
um, 2.1 mm.times.150 mm column, 30.degree. C. The UPLC system was
connected to two eluent reservoirs containing: A: 99.95% H.sub.2,
0.05% TFA; B: 99.95% CH.sub.3CN, 0.05% TFA. The following linear
gradient was used: 95% A, 5% B to 5% A, 95% B over 16 minutes at a
flow-rate of 0.30 ml/min.
Method: A9.sub.--1
[0359] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH Shield RP18, C18, 1.7
um, 2.1 mm.times.150 mm column, 60.degree. C. The UPLC system was
connected to two eluent reservoirs containing: A: 200 mM
Na.sub.2SO.sub.4+20 mM Na.sub.2HPO.sub.4+20 mM NaH.sub.2PO.sub.4 in
90% H.sub.2O/10% CH.sub.3CN, pH 7.2; B: 70% CH.sub.3CN, 30%
H.sub.2O. The following step gradient was used: 90% A, 10% B to 80%
A, 20% B over 3 minutes, 80% A, 20% B to 50% A, 50% B over 17
minutes at a flow-rate of 0.40 ml/min.
Method: B30.sub.--1
[0360] The RP-analysis was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 215 nm and 254
nm were collected using a kinetex 1.7 u C18, 100A 2.1.times.150 mm
column, 60.degree. C. The UPLC system was connected to two eluent
reservoirs containing: A: 0.09M (NH.sub.4).sub.2HPO.sub.4 and 10%
MeCN, pH 3.6, B: 20% isopropanole, 20% water and 60% CH.sub.3CN.
The following step gradient was used: 45% B and 55% A over 2
minutes, then 45% B, 55% A to 75% B, 25% A over 15 minutes, then
75% B, 25% A to 90% B, 10% A over 3 minutes at a flowrate of 0.5
ml/min.
Method: B39.sub.--2
[0361] The RP-analyses was performed using a Waters UPLC system
fitted with a dual band detector. UV detections at 214 nm and 254
nm were collected using an ACQUITY UPLC BEH C18, 1.7 um, 2.1
mm.times.50 mm column, 40.degree. C. The UPLC system was connected
to two eluent reservoirs containing: A: 99.95% H.sub.2O, 0.05% TFA;
B: 99.95% CH.sub.3CN, 0.05% TFA. The following linear gradient was
used: 70% A, 30% B to 50% A, 50% B over 3.5 minutes at a flow-rate
of 0.450 ml/min.
3. MALDI-MS Method
Method: MALDI_MS
[0362] Molecular weights were determined using matrix-assisted
laser desorption and ionisation time-of-flight mass spectroscopy,
recorded on a Microflex or Autoflex (Bruker). A matrix of
alpha-cyano-4-hydroxy cinnamic acid was used.
B. Specific Example compounds
Example 1
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxy-
tridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carbox-
ytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Lys-
.sup.18,Glu.sup.22,Gln.sup.34](SEQ ID NO:
11)-GLP-1-(7-37)-peptide
##STR00014##
[0364] Preparation Method: SPPS_L; SC_L; CP_M1
[0365] LCMS: Method: LCMS.sub.--4: Rt=2.11 min m/z: 4754.6; M/3:
1585; M/4: 1189; M/5: 951
[0366] UPLC Method: B4.sub.--1: Rt=8.25
[0367] UPLC Method: A9.sub.--1: Rt=9.95
Example 2
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(13-carboxytridecanoy-
lamino)butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(13-carboxytridecano-
ylamino)butanoyl]amino]hexanoyl]-[Lys.sup.8,Glu.sup.22,Gln.sup.34](SEQ
ID NO: 11)-GLP-1-(7-37)-peptide
##STR00015##
[0369] Preparation Method: SPPS_L; SC_L; CP_M1
[0370] UPLC method: B4.sub.--1: Rt=8.49 min
[0371] UPLC method: B9.sub.--1: Rt=4.99 min
Example 3
N.sup..epsilon.18-[2-[2-[2-[[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-[-
10-(4-carboxyphenoxyl)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]am-
ino]hexanoyl]amino]ethoxy]ethoxy]acetyl],
N.sup..epsilon.26-[2-[2-[2-[[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4--
[10-(4-carboxyphenoxyl)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]a-
mino]hexanoyl]amino]ethoxy]ethoxy]acetyl]-[Imp.sup.7,Aib.sup.8,Lys.sup.18,-
Glu.sup.22,Gln.sup.34](SEQ ID NO: 5)-GLP-1-(7-37)-peptide
##STR00016##
[0373] Preparation Method: SPPS_L; SC_L; CP_M1
[0374] UPLC Method: B4.sub.--1: Rt=8.53 min
[0375] UPLC Method: A6.sub.--1: Rt=5.02 min
[0376] LCMS method: LCMS.sub.--4: Rt=2.22 min; m/3=1715; m/4=1286;
m/5=1030
Example 4
N.sup..epsilon.18-[2-[2-[2-[[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-[-
10-(4-carboxyphenoxyl)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]am-
ino]hexanoyl]amino]ethoxy]ethoxy]acetyl],
N.sup.e31-[2-[2-[2-[[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-c-
arboxyphenoxyl)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hex-
anoyl]amino]ethoxy]ethoxy]acetyl]-[Imp.sup.7,Lys18,Glu.sup.22,Val.sup.25,A-
rg.sup.26,Lys.sup.31,Arg.sup.34](SEQ ID NO:
6)-GLP-1-(7-37)-peptide
##STR00017##
[0378] Preparation Method: SPPS_L; SC_L; CP_M1
[0379] UPLC Method: B4.sub.--1: Rt=7.72 min
[0380] UPLC Method: A9.sub.--1: Rt=11.71 min
[0381] LCMS method; LCMS.sub.--4: Rt=2.22 min; m/4=1289; m/5=1031;
m/6=860
Example 5
N.sup..epsilon.18-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[10--
(4-carboxyphenoxyl)decanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[10-
-(4-carboxyphenoxyl)decanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl]--
[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SEQ ID NO:
9)-GLP-1-(7-37)-peptide
##STR00018##
[0383] Preparation Method: SPPS_P; SC_P; CP_M1
[0384] UPLC Method: A9.sub.--1: Rt=10.7 min
[0385] UPLC Method: A6.sub.--1: Rt=5.6 min
[0386] The theoretical molecular mass of 4834.5 Da was confirmed by
Method: Maldi_MS: m/z 4833.5
Example 6
N.sup..epsilon.18-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[10--
(4-carboxyphenoxyl)decanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[10-
-(4-carboxyphenoxyl)decanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl]--
[Aib.sup.8,Lys.sup.18,Gln.sup.34](SEQ ID NO:
12)-GLP-1-(7-37)-peptide
##STR00019##
[0388] Preparation Method: SPPS_P; SC_P; CP_M1
[0389] UPLC Method: B31.sub.--1: Rt=14.0 min
[0390] UPLC Method: A6.sub.--1: Rt=6.2 min
[0391] The theoretical molecular mass of 4762 Da was confirmed by
Method: Maldi_MS: m/z 4759
Example 7
N.sup..epsilon..about.-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-
-[8-[10-(4-carboxyphenoxyl)decanoylamino]octanoylamino]butanoyl]amino]hexa-
noyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[8--
[10-(4-carboxyphenoxyl)decanoylamino]octanoylamino]butanoyl]amino]hexanoyl-
]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SEQ
ID NO: 9)-GLP-1-(7-37)-peptide
##STR00020##
[0393] Preparation Method: SPPS_P; SC_P; CP_M1
[0394] UPLC Method: B31.sub.--1: Rt=17.1 min
[0395] UPLC Method: A6.sub.--1: Rt=5.9 min
[0396] The theoretical molecular mass of 5116.9 Da was confirmed by
Method: Maldi_MS: m/z 5114
Example 8
N.sup..epsilon.18-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[12--
(4-carboxyphenoxyl)dodecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl]-
,
N.sup..epsilon.26-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[1-
2-(4-carboxyphenoxyl)dodecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoy-
l]-[Aib.sup.8,Lys.sup.18,Gln.sup.34](SEQ ID NO:
12)-GLP-1-(7-37)-peptide
##STR00021##
[0398] Preparation Method: SPPS_P; SC_P; CP_M1
[0399] UPLC Method: B31.sub.--1: Rt=17.0 min
[0400] UPLC Method: A6.sub.--1: Rt=6.9 min
[0401] The theoretical molecular mass of 4818.6 Da was confirmed by
Method: Maldi_MS: m/z 4817
Example 9
[0402] The following compound is prepared and characterised using
the above-mentioned general methods, and in analogy with the
compounds of the worked examples.
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-[[2-[2-[2-(-
15-carboxypentadecanoylamino)ethoxy]ethoxy]acetyl]amino]butanoyl]amino]eth-
oxy]ethoxy]acet yl]amino]hexanoyl],
N.sup..epsilon.31-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-[[2-[2-[2--
(15-carboxypentadecanoylamino)ethoxy]ethoxy]acetyl]amino]butanoyl]amino]et-
hoxy]ethoxy]acet
yl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.8,Glu.sup.22,Val.sup.25,Arg.sup.26,-
Lys.sup.31,Arg.sup.34](SEQ ID NO: 6)-GLP-1-(7-37)-peptide
##STR00022##
[0403] Example 10
N.sup..epsilon.18-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[10--
(4-carboxyphenoxyl)decanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[10-
-(4-carboxyphenoxyl)decanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl]--
[Aib.sup.8,Lys.sup.18,Gln.sup.19,Glu22,Gln.sup.34](SEQ ID NO:
10)-GLP-1-(7-37)-peptide
##STR00023##
[0405] Preparation Method: SPPS_P; SC_P; CP_M1
[0406] UPLC Method: B31.sub.--1: Rt=14.5 min
[0407] UPLC Method: A6.sub.--1: Rt=5.2 min
[0408] The theoretical molecular mass of 4799.4 Da was confirmed by
Method: Maldi_MS: m/z 4799.6
Example 11
N.sup..epsilon.18-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15--
carboxypentadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15-
-carboxypentadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib.su-
p.8,Lys.sup.18,Gln.sup.34](SEQ ID NO: 12)-GLP-1-(7-37)-peptide
##STR00024##
[0410] Preparation Method: SPPS_P; SC_P; CP_M1
[0411] UPLC Method: B31.sub.--1: Rt=17.9 min
[0412] UPLC Method: A6.sub.--1: Rt=7.6 min
[0413] The theoretical molecular mass of 4718.5 Da was confirmed by
Method: Maldi_MS: m/z 4716.98
Example 12
N.sup..epsilon.18-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17--
carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17-
-carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib.su-
p.8,Lys.sup.18,Gln.sup.34](SEQ ID NO: 12)-GLP-1-(7-37)-peptide
##STR00025##
[0415] Preparation Method: SPPS_P; SC_P; CP_M1
[0416] UPLC Method: B31.sub.--1: Rt=19.4 min
[0417] UPLC Method: A6.sub.--1: Rt=8.5 min
[0418] The theoretical molecular mass of 4774.6 Da was confirmed by
Method: Maldi_MS: m/z 4773.8
Example 13
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15-carboxypentadecan-
oylamino)butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15-carboxypentadeca-
noylamino)butanoyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Gln.sup.34](SEQ
ID NO: 12)-GLP-1-(7-37)-peptide
##STR00026##
[0420] Preparation Method: SPPS_P; SC_P; CP_M1
[0421] UPLC method: AP_B4.sub.--1: Rt=9.04 min
[0422] LCMS method: LCMS_AP: Rt=5.61 min; m/3=1488; m/4=1116
Example 14
N.sup..epsilon.8-[(2S)-2-amino-6-[[2-[2-[2-(15-carboxypentadecanoylamino)e-
thoxy]ethoxy]acetyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-(15-carboxypentadecanoylamino-
)ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.8,Gln.sup.34](SEQ
ID NO: 12)-GLP-1-(7-37)-peptide
##STR00027##
[0424] Preparation Method: SPPS_P; SC_P; CP_M1
[0425] UPLC method: AP_B4.sub.--1: Rt=9.35 min
[0426] LCMS method: LCMS_AP: Rt=5.71 min; m/3=1499; m/4=1124
Example 15
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxy-
pentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carbox-
ypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[A-
ib.sup.8,Lys.sup.18,Gln.sup.34](SEQ ID NO:
12)-GLP-1-(7-37)-peptide
##STR00028##
[0428] Preparation Method: SPPS_P; SC_P; CP_M1
[0429] UPLC method: AP_B4.sub.--1: Rt=9.00 min
[0430] LCMS method: LCMS_AP: Rt=5.57 min; m/3=1585; m/4=1189
Example 16
[0431] The following compound is prepared and characterised using
the above-mentioned general methods, and in analogy with the
compounds of the worked examples.
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carb-
oxyphenoxyl)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexano-
yl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-
-carboxyphenoxyl)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]h-
exanoyl]-[Aib.sup.8,Lys.sup.18,Gln.sup.34](SEQ ID NO:
12)-GLP-1-(7-37)-peptide
##STR00029##
[0432] Example 17
[0433] The following compound is prepared and characterised using
the above-mentioned general methods, and in analogy with the
compounds of the worked examples.
N.sup..epsilon.18-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[12--
(4-carboxyphenoxyl)dodecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoyl]-
,
N.sup..epsilon.26-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[1-
2-(4-carboxyphenoxyl)dodecanoylamino]butanoyl]amino]hexanoyl]amino]hexanoy-
l]-[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SEQ ID NO:
9)-GLP-1-(7-37)-peptide
##STR00030##
[0434] Example 18
[0435] The following compound is prepared and characterised using
the above-mentioned general methods, and in analogy with the
compounds of the worked examples.
N.sup..epsilon.18-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15--
carboxypentadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15-
-carboxypentadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib.su-
p.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SEQ ID NO:
9)-GLP-1-(7-37)-peptide
##STR00031##
[0436] Example 19
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[-
12-(4-carboxyphenoxyl)dodecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]-
amino]ethoxy]ethox y]acetyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4--
[12-(4-carboxyphenoxyl)dodecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl-
]amino]ethoxy]ethox
y]acetyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Gln.sup.34](SEQ ID
NO: 12)-GLP-1-(7-37)-peptide
##STR00032##
[0438] Preparation Method: SPPS_P; SC_P; CP_M1
[0439] UPLC Method: A6.sub.--1: Rt=8.8 min
[0440] The theoretical molecular mass of 5142.8 Da was confirmed by
Method: Maldi_MS: m/z 5140.6
Example 20
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(-
13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethox-
y]ethoxy]acetyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4--
(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]etho-
xy]ethoxy]acetyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Gln.sup.34](SEQ
ID NO: 12)-GLP-1-(7-37)-peptide
##STR00033##
[0442] Preparation Method SPPS_P; SC_P; CP_M1
[0443] LCMS method: LCMS_AP: Rt=6.44 m; m3=1662; m4=1247
[0444] The following compounds are prepared and characterised using
the above-mentioned general methods, and in analogy with the
compounds of the above examples.
Example 21
N.sup..epsilon.18}-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[4-carboxy-4-(13-ca-
rboxytridecanoylamino)butanoyl]amino]butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(-
13-carboxytridecanoylamino)butanoyl]amino]butanoyl]amino]hexanoyl]-[Aib.su-
p.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SEQ ID NO:
9)-GLP-1-(7-37)-peptide
##STR00034##
[0445] Example 22
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4[[(-
4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]butanoyl]amino]-
butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[-
[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]butanoyl]amin-
o]butanoyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SE-
Q ID NO: 9)-GLP-1-(7-37)-peptide
##STR00035##
[0446] Example 23
N.sup..epsilon.18-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(13--
carboxytridecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(13-
-carboxytridecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib.sup.-
8,Lys.sup.18,Glu.sup.22,Gln.sup.4](SEQ ID NO:
9)-GLP-1-(7-37)-peptide
##STR00036##
[0447] Example 24
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(13-carboxytridecanoy-
lamino)butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(13-carboxytridecano-
ylamino)butanoyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.-
34](SEQ ID NO: 9)-GLP-1-(7-37)-peptide
##STR00037##
[0448] Example 25
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4(13-carboxyt-
ridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl];
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carbox-
ytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib-
.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SEQ ID NO:
9)-GLP-1-(7-37)-peptide
##STR00038##
[0449] Example 26
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(1-
5-carboxypentadecanoylamino)butanoyl]amino]butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(-
15-carboxypentadecanoylamino)butanoyl]amino]butanoyl]amino]hexanoyl]-[Aib.-
sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SEQ ID NO:
9)-GLP-1-(7-37)-peptide
##STR00039##
[0450] Example 27
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[-
(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]butanoyl]ami-
no]butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[-
[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]butanoyl]am-
ino]butanoyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](-
SEQ ID NO: 9)-GLP-1-(7-37)-peptide
##STR00040##
[0451] Example 28
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15-carboxypentadecan-
oylamino)butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15-carboxypentadeca-
noylamino)butanoyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.su-
p.34](SEQ ID NO: 9)-GLP-1-(7-37)-peptide
##STR00041##
[0452] Example 29
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxy-
pentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carbox-
ypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[A-
ib.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SEQ ID NO:
9)-GLP-1-(7-37)-peptide
##STR00042##
[0453] Example 30
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(1-
7-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(-
17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]hexanoyl]-[Aib.-
sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SEQ ID NO:
9)-GLP-1-(7-37)-peptide
##STR00043##
[0454] Example 31
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[4-carboxy-4-[[(4S)--
4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]bu-
tanoyl]amino]hexanoyl],
N.sup..epsilon.26}-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[4-carboxy-4-[[(4S-
)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-
butanoyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SEQ
ID NO: 9)-GLP-1-(7-37)-peptide
##STR00044##
[0455] Example 32
N.sup..epsilon.18-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17--
carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17-
-carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib.su-
p.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SEQ ID NO:
9)-GLP-1-(7-37)-peptide
##STR00045##
[0456] Example 33
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17-carboxyheptadeca-
noylamino)butanoyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.su-
p.34](SEQ ID NO: 9)-GLP-1-(7-37)-peptide
##STR00046##
[0457] Example 34
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxy-
heptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carbox-
yheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[A-
ib.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SEQ ID NO:
9)-GLP-1-(7-37)-peptide
##STR00047##
[0458] Example 35
N.sup..epsilon.18-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(19--
carboxynonadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(19-
-carboxynonadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib.sup-
.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SEQ ID NO:
9)-GLP-1-(7-37)-peptide
##STR00048##
[0459] Example 36
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(19-carboxynonadecano-
ylamino)butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(19-carboxynonadecan-
oylamino)butanoyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup-
.34](SEQ ID NO: 9)-GLP-1-(7-37)-peptide
##STR00049##
[0460] Example 37
N.sup..epsilon.18-[(2S)-2-amino-6-[[(2S)-[2-[2-[[(4S)-4-carboxy-4-(19-carb-
oxynonadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],N-
.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyh-
eptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib-
.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SEQ ID NO:
9)-GLP-1-(7-37)-peptide
##STR00050##
[0461] Example 38
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[4-carboxy-4-[10-(4--
carboxyphenoxyl)decanoylamino]butanoyl]amino]butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[4-carboxy-4-[10-(4-
-carboxyphenoxyl)decanoylamino]butanoyl]amino]butanoyl]amino]hexanoyl]-[Ai-
b.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.34](SEQ ID NO:
9)-GLP-1-(7-37)-peptide
##STR00051##
[0462] Example 39
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[4-carboxy-4-[[(4S)--
4-carboxy-4-[10-(4-carboxyphenoxyl)decanoylamino]butanoyl]amino]butanoyl]a-
mino]butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[4-carboxy-4-[[(4S)-
-4-carboxy-4-[10-(4-carboxyphenoxyl)decanoylamino]butanoyl]amino]butanoyl]-
amino]butanoyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.34-
](SEQ ID NO: 9)-GLP-1-(7-37)-peptide
##STR00052##
[0463] Example 40
N.sup..epsilon.18-[(2S)-2-amino-6-[[4-carboxy-4-[[(4S)-4-carboxy-4-[12-(4--
carboxyphenoxyl)dodecanoylamino]butanoyl]amino]butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[4-carboxy-4-[[(4S)-4-carboxy-4-[12-(4-
-carboxyphenoxyl)dodecanoylamino]butanoyl]amino]butanoyl]amino]hexanoyl]-[-
Aib8,Lys18,Glu22,Gln34](SEQ ID NO: 9)-GLP-1-(7-37)-peptide
##STR00053##
[0464] Example 41
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[4-carboxy-4-[[(4S)--
4-carboxy-4-[12-(4-carboxyphenoxyl)dodecanoylamino]butanoyl]amino]butanoyl-
]amino]butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-[[4-carboxy-4-[[(4S)-
-4-carboxy-4-[12-(4-carboxyphenoxyl)dodecanoylamino]butanoyl]amino]butanoy-
l]amino]butanoyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.-
34](SEQ ID NO: 9)-GLP-1-(7-37)-peptide
##STR00054##
[0465] Example 42
N.sup..epsilon.18-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(13--
carboxytridecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(13-
-carboxytridecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib.sup.-
8,Lys.sup.18,Gln.sup.34](SEQ ID NO: 12)-GLP-1-(7-37)-peptide
##STR00055##
[0466] Example 43
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(13-carboxytridecanoy-
lamino)butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(13-carboxytridecano-
ylamino)butanoyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Gln.sup.34](SEQ
ID NO: 12)-GLP-1-(7-37)-peptide
##STR00056##
[0467] Example 44
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[4-carboxy-4-(13-carboxytride-
canoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[4-carboxy-4-(13-carboxytrid-
ecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib.sup.-
8,Lys.sup.18,Gln.sup.34](SEQ ID NO: 12)-GLP-1-(7-37)-peptide
##STR00057##
[0468] Example 45
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17-carboxyheptadeca-
noylamino)butanoyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Gln.sup.34](SEQ
ID NO: 12)-GLP-1-(7-37)-peptide
##STR00058##
[0469] Example 46
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[4-carboxy-4-(17-carboxyhepta-
decanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[4-carboxy-4-(17-carboxyhept-
adecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib.su-
p.8,Lys.sup.18,Gln.sup.34](SEQ ID NO: 12)-GLP-1-(7-37)-peptide
##STR00059##
[0470] Example 47
N.sup..epsilon.18-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(19--
carboxynonadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(19-
-carboxynonadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-[Aib.sup-
.8,Lys.sup.18,Gln.sup.34](SEQ ID NO: 12)-GLP-1-(7-37)-peptide
##STR00060##
[0471] Example 48
N.sup..epsilon.18-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(19-carboxynonadecano-
ylamino)butanoyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(19-carboxynonadecan-
oylamino)butanoyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Gln.sup.34](SEQ
ID NO: 12)-GLP-1-(7-37)-peptide
##STR00061##
[0472] Example 49
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[4-carboxy-4-(19-carboxynonad-
ecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],
N.sup..epsilon.26
[(2S)-2-amino-6-[[2-[2-[2-[[4-carboxy-4-(17-carboxyheptadecanoylamino)but-
anoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Gln-
.sup.34](SEQ ID NO: 12)-GLP-1-(7-37)-peptide
##STR00062##
[0473] Example 50
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[4-carboxy-4-[12-(4-carboxyph-
enoxyl)dodecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-
,
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[4-carboxy-4-[12-(4-carboxy-
phenoxyl)dodecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]hexanoy-
l]-[Aib.sup.8,Lys.sup.18,Gln.sup.34](SEQ ID NO:
12)-GLP-1-(7-37)-peptide
##STR00063##
[0474] Example 51
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(-
13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethox-
y]ethoxy]acetyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4--
(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]etho-
xy]ethoxy]acetyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.sup.-
34](SEQ ID NO: 9)-GLP-1-(7-37)-peptide
##STR00064##
[0475] Example 52
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(-
15)-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]et-
hoxy]ethoxy]acetyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4--
(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]et-
hoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.su-
p.34](SEQ ID NO: 9)-GLP-1-(7-37)-peptide
##STR00065##
[0476] Example 53
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(-
17)-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]et-
hoxy]ethoxy]acetyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4--
(17-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]et-
hoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.su-
p.34](SEQ ID NO: 9)-GLP-1-(7-37)-peptide
##STR00066##
[0477] Example 54
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[-
10-(4-carboxyphenoxyl)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]am-
ino]ethoxy]ethoxy]acetyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4--
[10-(4-carboxyphenoxyl)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]a-
mino]ethoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Glu.sup.22-
,Gln.sup.34](SEQ ID NO: 9)-GLP-1-(7-37)-peptide
##STR00067##
[0478] Example 55
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[-
12-(4-carboxyphenoxyl)dodecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]-
amino]ethoxy]ethoxy]acetyl]amino]hexanoyl],
N.sup.26-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[12-(4-ca-
rboxyphenoxyl)dodecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]et-
hoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Glu.sup.22,Gln.su-
p.34](SEQ ID NO: 9)-GLP-1-(7-37)-peptide
##STR00068##
[0479] Example 56
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(-
15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]eth-
oxy]ethoxy]acetyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4--
(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]et-
hoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Gln.sup.34](SEQ
ID NO: 12)-GLP-1-(7-37)-peptide
##STR00069##
[0480] Example 57
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(-
17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]eth-
oxy]ethoxy]acetyl]amino]hexanoyl],
N.sup..epsilon.26-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4--
(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]et-
hoxy]ethoxy]acetyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Gln.sup.34](SEQ
ID NO: 12)-GLP-1-(7-37)-peptide
##STR00070##
[0481] Example 58
N.sup..epsilon.18-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[-
10-(4-carboxyphenoxyl)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]am-
ino]ethoxy]ethoxy]acetyl]amino]hexanoyl],
N.sup.26-[(2S)-2-amino-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-ca-
rboxyphenoxyl)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]etho-
xy]ethoxy]acetyl]amino]hexanoyl]-[Aib.sup.8,Lys.sup.18,Gln.sup.34](SEQ
ID NO: 12)-GLP-1-(7-37)-peptide
##STR00071##
[0482] Biological Methods
Example 59
In Vitro Potency
[0483] The purpose of this example is to test the activity, or
potency, of the GLP-1 derivatives in vitro.
[0484] The potencies of the GLP-1 derivatives of Examples 1-8,
10-15, and 19 were determined as described below, i.e. as the
stimulation of the formation of cyclic AMP (cAMP) in a medium
containing membranes expressing the human GLP-1 receptor.
Principle
[0485] Purified plasma membranes from a stable transfected cell
line, BHK467-12A (tk-ts13), expressing the human GLP-1 receptor
were stimulated with the GLP-1 derivative in question, and the
potency of cAMP production was measured using the AlphaScreen.TM.
cAMP Assay Kit from Perkin Elmer Life Sciences. The basic principle
of The AlphaScreen Assay is a competition between endogenous cAMP
and exogenously added biotin-cAMP. The capture of cAMP is achieved
by using a specific antibody conjugated to acceptor beads.
Cell Culture and Preparation of Membranes
[0486] A stable transfected cell line and a high expressing clone
were selected for screening. The cells were grown at 5% CO.sub.2 in
DMEM, 10% FCS, 1% Pen/Strep (Penicillin/Streptomycin) and 1.0 mg/ml
of the selection marker G418.
[0487] Cells at approximate 80% confluence were washed 2.times.
with PBS and harvested with Versene (aqueous solution of the
tetrasodium salt of ethylenediaminetetraacetic acid), centrifuged 5
min at 1000 rpm and the supernatant removed. The additional steps
were all made on ice. The cell pellet was homogenised by the
Ultrathurax for 20-30 sec. in 10 ml of Buffer 1 (20 mM Na-HEPES, 10
mM EDTA, pH=7.4), centrifuged 15 min at 20,000 rpm and the pellet
resuspended in 10 ml of Buffer 2 (20 mM Na-HEPES, 0.1 mM EDTA,
pH=7.4). The suspension was homogenised for 20-30 sec and
centrifuged 15 min at 20,000 rpm. Suspension in Buffer 2,
homogenisation and centrifugation was repeated once and the
membranes were resuspended in Buffer 2. The protein concentration
was determined and the membranes stored at -80.degree. C. until
use.
[0488] The assay was performed in 1/2-area 96-well plates, flat
bottom (Costar cat. no:3693). The final volume per well was 50
.mu.l.
Solutions and Reagents
[0489] AlphaScreen cAMP Assay Kit from Perkin Elmer Life Sciences
(cat. No: 6760625M); containing Anti-cAMP Acceptor beads (10
U/.mu.l), Streptavidin Donor beads (10 U/.mu.l) and
Biotinylated-cAMP (133 U/.mu.l).
[0490] AlphaScreen Buffer, pH=7.4: 50 mM TRIS-HCl (Sigma, cat. no:
T3253); 5 mM HEPES (Sigma, cat. no: H3375); 10 mM MgCl.sub.2,
6H.sub.2O (Merck, cat. no: 5833); 150 mM NaCl (Sigma, cat. no:
S9625); 0.01% Tween (Merck, cat. no: 822184). The following was
added to the AlphaScreen Buffer prior to use (final concentrations
indicated): BSA (Sigma, cat. no. A7906): 0.1%; IBMX (Sigma, cat.
no. I5879): 0.5 mM; ATP (Sigma, cat. no. A7699): 1 mM; GTP (Sigma,
cat. no. G8877): 1 uM.
[0491] cAMP standard (dilution factor in assay=5): cAMP Solution: 5
.mu.L of a 5 mM cAMP-stock+495 .mu.L AlphaScreen Buffer.
[0492] Suitable dilution series in AlphaScreen Buffer were prepared
of the cAMP standard as well as the GLP-1 derivative to be tested,
e.g. the following eight concentrations of the GLP-1 compound:
10.sup.-7, 10.sup.-8, 10.sup.-9, 10.sup.-10, 10.sup.-11,
10.sup.-12, 10.sup.-13 and 10.sup.-14M, and a series from, e.g.,
10.sup.-6 to 3.times.10.sup.-11 of cAMP.
Membrane/Acceptor Beads
[0493] Use hGLP-1/BHK 467-12A membranes; 3 .mu.g/well corresponding
to 0.6 mg/ml (the amount of membranes used pr. well may vary)
[0494] "No membranes": Acceptor Beads (2 units/well final) in
AlphaScreen buffer "3 .mu.g/well membranes": membranes+Acceptor
Beads (2 units/well final) in AlphaScreen buffer
[0495] Add 10 .mu.l "No membranes" to the cAMP standard (per well
in duplicates) and the positive and negative controls
[0496] Add 10 .mu.l "3 .mu.g/well membranes" to the GLP-1
derivatives (per well in duplicates/triplicates)
[0497] Pos. Control: 10 .mu.l "no membranes"+10 .mu.l AlphaScreen
Buffer
[0498] Neg. Control: 10 .mu.l "no membranes"+10 .mu.l cAMP Stock
Solution (50.rho.M)
[0499] As the beads are sensitive to direct light, any handling was
in the dark (as dark as possible), or in green light. All dilutions
were made on ice.
Procedure
1. Make the AlphaScreen Buffer.
[0500] 2. Dissolve and dilute the GLP-1 derivatives/cAMP standard
in AlphaScreen Buffer. 3. Make the Donor Beads solution (by mixing
streptavidin donor beads (2 units/well) and biotynylated cAMP (1.2
units/well) and incubate 20-30 min. in the dark at RT. 4. Add the
cAMP/GLP-1 derivatives to the plate: 10 .mu.l per well. 5. Prepare
membrane/Acceptor Beads solution and add this to the plates: 10
.mu.l per well. 6. Add the Donor Beads: 30 .mu.l per well. 7. Wrap
the plate in aluminium foil and incubate on the shaker for 3 hours
(very slowly) at RT. 8. Count on AlphaScreen--each plate pre
incubates in the AlphaScreen for 3 minutes before counting.
Results
[0501] The EC.sub.50 [nM] values were calculated using the
Graph-Pad Prism software (version 5).
[0502] The potency of all derivatives in vitro was confirmed. 13
derivatives had an in vitro potency corresponding to an EC.sub.50
of 1200 pM or below; 11 derivatives had a still further improved
potency corresponding to an EC.sub.50 at 500 pM or below; 7
derivatives were very potent corresponding to an EC.sub.50 at 200
pM or below; and 2 derivatives had a very good potency
corresponding to an EC.sub.50 of 100 pM or below.
[0503] For comparison, compound no. 13 in Table 1 of Journal of
Medicinal Chemistry (2000), vol. 43, no. 9, p. 1664-669
(GLP-1(7-37) acylated at K.sup.26,34 with bis-C12-diacid) had an in
vitro potency corresponding to an EC.sub.50 of 1200 pM.
[0504] If desired, the fold variation in relation to GLP-1 may be
calculated as EC.sub.50 (GLP-1)/EC.sub.50 (analogue)--3693.2.
Example 60
GLP-1 Receptor Binding
[0505] The purpose of this experiment is to investigate the binding
to the GLP-1 receptor of the GLP-1 derivatives, and how the binding
is potentially influenced by the presence of albumin. This is done
in an in vitro experiment as described below.
[0506] The binding affinity of the GLP-1 derivatives of Examples
1-8, 10-15, and 19 to the human GLP-1 receptor was measured by way
of their ability to displace of .sup.125I-GLP-1 from the receptor.
In order to test the binding of the derivatives to the receptor in
the presence of albumin, the assay was performed with a low
concentration of albumin (0.005%--corresponding to the residual
amount thereof in the tracer), as well as with a high concentration
of albumin (2.0% added). A shift in the binding affinity,
IC.sub.50, is an indication that the derivative in question binds
to albumin, and thereby a prediction of a potential protracted
pharmacokinetic profile of the derivative in question in animal
models.
Conditions
[0507] Species (in vitro): Hamster
Biological End Point: Receptor Binding
Assay Method: SPA
[0508] Receptor: GLP-1 receptor Cell Line: BHK tk-ts13
Cell Culture and Preparation of Membranes
[0509] A stable transfected cell line and a high expressing clone
were selected for screening. The cells were grown at 5% CO.sub.2 in
DMEM, 10% FCS, 1% Pen/Strep (Penicillin/Streptomycin) and 1.0 mg/ml
of the selection marker G418.
[0510] Cells at approximate 80% confluence were washed 2.times.
with PBS and harvested with Versene (aqueous solution of the
tetrasodium salt of ethylenediaminetetraacetic acid), centrifuged 5
min at 1000 rpm and the supernatant removed. The additional steps
were all made on ice. The cell pellet was homogenised by the
Ultrathurax for 20-30 sec. in a suitable amount of Buffer 1 (20 mM
Na-HEPES, 10 mM EDTA, pH=7.4) but e.g. 10-20 ml, centrifuged 15 min
at 20,000 rpm and the pellet resuspended in a suitable amount of
Buffer 2 (20 mM Na-HEPES, 0.1 mM EDTA, pH=7.4) but e.g. 10-20 ml.
The suspension was homogenised for 20-30 sec and centrifuged 15 min
at 20,000 rpm. Suspension in Buffer 2, homogenisation and
centrifugation was repeated once more and the membranes were
resuspended in Buffer 2. The protein concentration was determined
and the membranes stored at -80.degree. C. until use.
SPA Binding Assay:
[0511] Test compounds, membranes, SPA-particles and
[.sup.125I]-GLP-1(7-36)NH.sub.2 were diluted in assay buffer. 25 ul
(microliter) of test compounds are added to Optiplate. HSA ("high
albumin" experiment containing 2% HSA), or buffer ("low albumin"
experiment containing 0.005% HSA), was added (50 ul). 5-10 ug
protein/sample was added (50 ul) corresponding to 0.1-0.2 mg
protein/ml (to be preferably optimised for each membrane
preparation). SPA-particles (Wheatgerm agglutinin SPA beads, Perkin
Elmer, #RPNQ0001) were added in an amount of 0.5 mg/well (50 ul).
The incubation was started with [.sup.125I]-GLP-1(7-36)NH.sub.2
(final concentration 0.06 nM corresponding to 49.880 DPM, 25 ul).
The plates were sealed with PlateSealer and incubated for 120
minutes at 30.degree. C. while shaking. The plates were centrifuged
(1500 rpm, 10 min) and counted in Topcounter.
Assay Buffer:
50 mM HEPES
5 mM EGTA
5 mM MgCl2
0.005% Tween 20
[0512] pH 7.4
HSA was SIGMA A1653
Calculations
[0513] The IC.sub.50 value was read from the curve as the
concentration which displaces 50% of .sup.125I-GLP-1 from the
receptor, and the ratio of [(IC.sub.50/nM) high
HSA]/[(IC.sub.50/nM) low HSA] was determined.
[0514] Generally, the binding to the GLP-1 receptor at low albumin
concentration should be as good as possible, corresponding to a low
IC.sub.50 value.
[0515] The IC.sub.50 value at high albumin concentration is a
measure of the influence of albumin on the binding of the
derivative to the GLP-1 receptor. As is known, the GLP-1
derivatives also bind to albumin. This is a generally desirable
effect, which extends their lifetime in plasma. Therefore, the
IC.sub.50 value at high albumin will generally be higher than the
IC.sub.50 value at low albumin, corresponding to a reduced binding
to the GLP-1 receptor, caused by albumin binding competing with the
binding to the GLP-1 receptor.
[0516] A high ratio (IC.sub.50 value (high albumin)/IC.sub.50 value
(low albumin)) may therefore be taken as an indication that the
derivative in question binds well to albumin (may have a long
half-life), and also per se binds well to the GLP-1 receptor (the
IC.sub.50 value (high albumin) is high, and the IC.sub.50 value
(low albumin) is low).
Results
[0517] The following results were obtained, where "ratio" refers to
[(IC.sub.50/nM) high HSA]/[(IC.sub.50/nM) low HSA]):
[0518] All derivatives had a ratio above 10; 13 derivatives were
above 50; 11 derivatives were above 100; 8 derivatives above 500;
and 4 derivatives had a ratio above 1000.
[0519] Furthermore as regards IC.sub.50 (low albumin), all
derivatives had an IC.sub.50 (low albumin) below 35 nM; 14
derivatives were below 15 nM; 13 derivatives were below 10 nM; 12
derivatives were below 5.0 nM; 10 derivatives were below 1.0 nM;
and 6 derivatives were below 0.50 nM.
[0520] Finally as regards IC.sub.50 (high albumin), 10 derivatives
were below 1000 nM; 7 derivatives were below 500 nM; and 3
derivatives were below 250 nM.
Example 61
Estimate of Oral Bioavailability--Gut Injection in Rat (Sodium
Caprate)
[0521] The purpose of this experiment is to estimate the oral
bioavailability of the GLP-1 derivatives. To this end, the exposure
in plasma after direct injection into the intestinal lumen of the
GLP-1 derivatives is studied in vivo in rats, as described in the
following. The GLP-1 derivatives are tested in a concentration of
1000 uM in a solution of 55 mg/ml sodium caprate.
[0522] 32 male Sprague Dawley rats with a body weight upon arrival
of approximately 240 g are obtained from Taconic (Denmark) and
assigned to the different treatments by simple randomisation, 4
rats per group. The rats are fasted for approximately 18 hours
before the experiment and taken into general anaesthesia
(Hypnorm/Dormicum).
[0523] The GLP-1 derivatives are administered in the jejunum either
in the proximal part (10 cm distal for the duodenum) or in the
mid-intestine (50 cm proximal for the cecum). A PE50-catheter, 10
cm long was inserted into the jejunum, forwarded at least 1.5 cm
into the jejunum, and secured before dosing by ligature around the
gut and the catheter with 3/0 suture distal to tip to prevent leak
or catheter displacement. Catheter is placed without syringe and
needle and 2 ml saline is administered into abdomen before closing
the incision with wound clips.
[0524] 100 .mu.l of the respective GLP-1 derivative is injected
into the jejunal lumen through the catheter with a 1 ml syringe.
Subsequently, 200 .mu.l of air is pushed into the jejunal lumen
with another syringe to "flush" the catheter. This syringe is
leaved connected to the catheter to prevent flow back into the
catheter.
[0525] Blood samples (200 ul) are collected at desired intervals
(usually at times 0, 10, 30, 60, 120 and 240 min) into EDTA tubes
from the tail vein and centrifuged 5 minutes, 10000G, at 4.degree.
C. within 20 minutes. Plasma (75 ul) is separated to Micronic
tubes, immediately frozen, and kept at -20.degree. C. until
analyzed for plasma concentration of the respective GLP-1
derivative with LOCI (Luminescent Oxygen Channeling Immunoassay),
generally as described for the determination of insulin by Poulsen
and Jensen in Journal of Biomolecular Screening 2007, vol. 12, p.
240-247. The donor beads are coated with streptavidin, while
acceptor beads are conjugated with a monoclonal antibody
recognising a mid-/C-terminal epitope of the peptide. Another
monoclonal antibody, specific for the N-terminus, is biotinylated.
The three reactants are combined with the analyte and form a
two-sited immuno-complex. Illumination of the complex releases
singlet oxygen atoms from the donor beads, which are channeled into
the acceptor beads and trigger chemiluminescence which is measured
in an Envision plate reader. The amount of light is proportional to
the concentration of the compound.
[0526] After the blood sampling the rats are sacrificed under
anaesthesia and the abdomen is opened to verify correct catheter
placement.
[0527] The mean (n=4) plasma concentrations (pmol/1) are determined
as a function of time. The ratio of plasma concentration (pmol/1)
divided by the concentration of the dosing solution (pmol/1) is
calculated for each treatment, and the results for t=30 min (30
minutes after the injection of the compound in the jejunum) are
assessed (dose-corrected exposure at 30 min) as a surrogate measure
of intestinal bioavailability. The dose-corrected exposure is
expected to correlate with the actual bioavailability.
[0528] Dose-corrected exposure at 30 min refers to (the plasma
concentration 30 minutes after injection of the compound in the
jejunum (pM)), divided by (the concentration of the compound in the
dosing solution (.mu.M)).
Example 62
Estimate of Oral Bioavailability--Gut Injection and Oral Gavage in
Rat (SNAC)
[0529] The purpose of this experiment is to estimate the oral
bioavailability of the GLP-1 derivatives in a rat model. In brief,
a liquid solution of the GLP-1 derivative in sodium
N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC)_is administered by
gut injection (to the intestines), or by oral gavage (to the
stomach), and the subsequent exposure in plasma of the GLP-1
derivative is measured.
[0530] A 250 mg/ml stock solution of SNAC was prepared by
dissolving SNAC (12.5 g) in highly pure laboratory water (MilliQ)
(50.0 ml). The pH was adjusted to about 8.5 with 1 N NaOH (aq).
[0531] Solutions with about 1000 uM (800-1200 uM) of the GLP-1
derivatives of Examples 5-8, 11-13, and 15, respectively, in 250
mg/ml SNAC were prepared by dissolving the desired amount of the
respective GLP-1 derivative in the SNAC stock solution. The
concentration of the GLP-1 derivative was determined prior to
administration by a state-of-the-art method, such as CLND-HPLC
(chemiluminescent nitrogen detection for HPLC).
[0532] 32 male Sprague Dawley rats with a body weight upon arrival
of approximately 240 g were obtained from Taconic (Denmark) and
assigned to the different treatments by simple randomisation, 8
rats per group. All rats were fasted on grids for approximately 18
hours before the experiment.
[0533] For gut injection, on the day of experiment, rats were taken
into general anaesthesia (Hypnorm/Dormicum) and remained
anaesthetized during the entire experiment. The GLP-1 derivatives
of Examples 5-7 were administered in the proximal part of the
jejunum (10 cm distal for the duodenum). A PE50-catheter, 10 cm
long, was inserted into the jejunum, forwarded at least 1.5 cm into
the jejunum, and secured before dosing by ligature around the gut.
Furthermore, the catheter was provided with a 3/0 suture distal to
tip to prevent leak or catheter displacement. The catheter was
placed without syringe and needle and 2 ml saline was administered
into abdomen before closing the incision with wound clips.
[0534] 100 .mu.l SNAC solution of the respective GLP-1 derivative
was injected into the jejunal lumen through the catheter with a 1
ml syringe. Subsequently, 200 .mu.l of air was pushed into the
jejunal lumen with another syringe to "flush" the catheter. This
syringe was leaved connected to the catheter to prevent flow back
into the catheter.
[0535] Blood samples (200 ul) were collected at desired intervals
(usually at times 0, 30, 60, 120 and 180 min) into EDTA tubes from
the tail vein.
[0536] For oral gavage, the animals were conscious during the
entire experiment.
[0537] 100 .mu.l SNAC solution of the GLP-1 derivatives of Examples
5-8, 11-13, and 15, respectively, was administered by oral gavage
directly to the stomach.
[0538] Blood samples (200 ul) were collected at desired intervals
(usually at times 0, 30, 60, 120 and 180 min) into EDTA tubes from
the sublingual plexus.
[0539] All obtained blood samples were kept on ice and centrifuged
for 5 minutes, 10000G, at 4.degree. C. within 20 minutes. Plasma
(75 ul) was separated to Micronic tubes, immediately frozen, and
kept at -20.degree. C. until analyzed for plasma concentration of
the respective GLP-1 derivative with LOCI (Luminescent Oxygen
Channeling Immunoassay), generally as described for the
determination of insulin by Poulsen and Jensen in Journal of
Biomolecular Screening 2007, vol. 12, p. 240-247. The donor beads
were coated with streptavidin, while acceptor beads were conjugated
with a monoclonal antibody recognising a mid-/C-terminal epitope of
the peptide. Another monoclonal antibody, specific for the
N-terminus, was biotinylated. The three reactants were combined
with the analyte and formed a two-sited immuno-complex.
Illumination of the complex released singlet oxygen atoms from the
donor beads, which were channeled into the acceptor beads and
triggered chemiluminescence which was measured in an Envision plate
reader. The amount of light was proportional to the concentration
of the compound.
[0540] After the blood sampling all rats were sacrificed under
anaesthesia and the abdomen of the gut injection rats was opened to
verify correct catheter placement.
[0541] The mean (n=8) plasma concentrations (pmol/1) were
determined as a function of time. The AUC of the plasma exposure
(pmol/1) vs time curve, from time 30 to 180 (min), was
dose-corrected, i.e., divided by the amount (dose) of the
derivative in the dosed solution (pmol). The thus dose-corrected
AUC of plasma exposure from time 30-180 min (having the unit of
min.times.pM/pmol=min/L) was used as a surrogate measure of
bioavailability--a measure to rank the derivatives with regards to
their absorption in the rat model.
[0542] The following results were obtained:
[0543] For the gut injection experiment, the AUC of the
dose-corrected plasma exposure from time 30 to 180 min for the
tested GLP-1 derivatives was in the range of 170 to 322
min.times.pM/pmol. Two of the three tested compounds were above 250
min.times.pM/pmol.
[0544] For the oral gavage experiment, the AUC of the
dose-corrected plasma exposure from time 30 to 180 min for the
tested GLP-1 derivatives was in the range of 18 to 75
min.times.pM/pmol. Of the eight tested compounds six were above 20,
four were above 40, and two were above 60 min.times.pM/pmol.
Example 63
Effect on Blood Glucose and Body Weight--PD Db/Db Mice
[0545] The purpose of the study is to verify the effect of the
GLP-1 derivatives on blood glucose (BG) and body weight (BW) in a
diabetic setting.
[0546] The GLP-1 derivatives are tested in a dose-response study in
an obese, diabetic mouse model (db/db mice) as described in the
following.
[0547] Fifty db/db mice (Taconic, Denmark), fed from birth with the
diet NIH31 (NIH 31M Rodent Diet, commercially available from
Taconic Farms, Inc., US, see www.taconic.com), are enrolled for the
study at the age of 7-9 weeks The mice are given free access to
standard chow (e.g. Altromin 1324, Brogaarden, Gentofte, Denmark)
and tap water and kept at 24.degree. C.
[0548] After 1-2 weeks of acclimatisation, the basal blood glucose
is assessed twice on two consecutive days (i.e. at 9 am). The 8
mice with the lowest blood glucose values are excluded from the
experiments. Based on the mean blood glucose values, the remaining
42 mice are selected for further experimentation and allocated to 7
groups (n=6) with matching blood glucose levels. The mice are used
in experiments with duration of 5 days for up to 4 times. After the
last experiment the mice are euthanised.
[0549] The seven groups receive treatment as follows:
1: Vehicle, s.c.
[0550] 2: GLP-1 derivative, 0.3 nmol/kg, s.c. 3: GLP-1 derivative,
1.0 nmol/kg, s.c. 4: GLP-1 derivative, 3.0 nmol/kg, s.c. 5: GLP-1
derivative, 10 nmol/kg, s.c. 6: GLP-1 derivative, 30 nmol/kg, s.c.
7: GLP-1 derivative, 100 nmol/kg, s.c. Vehicle: 50 mM sodium
phosphate, 145 mM sodium chloride, 0.05% tween 80, pH 7.4.
[0551] The GLP-1 derivative is dissolved in the vehicle, to
concentrations of 0.05, 0.17, 0.5, 1.7, 5.0 and 17.0 nmol/ml.
Animals are dosed s.c. with a dose-volume of 6 ml/kg (i.e. 300
.mu.l per 50 g mouse).
[0552] On the day of dosing, blood glucose is assessed at time -1/2
h (8.30 am), where after the mice are weighed. The GLP-1 derivative
is dosed at approximately 9 am (time 0). On the day of dosing,
blood glucose is assessed at times 1, 2, 4 and 8 h (10 am, 11 am, 1
pm and 5 pm).
[0553] On the following days, the blood glucose is assessed at time
24 and 48 h after dosing (and if desired also at time 72, and 96 h
after dosing), i.e. at 9 am on day 2 and 3, and if desired at 9 am
on day 4 and 5). On each day, the mice are weighed following blood
glucose sampling.
[0554] The mice are weighed individually on a digital weight.
[0555] Samples for the measurement of blood glucose are obtained
from the tail tip capillary of conscious mice. Blood, 10 .mu.l, is
collected into heparinised capillaries and transferred to 500 .mu.l
glucose buffer (EKF system solution, Eppendorf, Germany). The
glucose concentration is measured using the glucose oxidase method
(glucose analyser Biosen 5040, EKF Diagnostic, GmbH, Barleben,
Germany). The samples are kept at room temperature for up to 1 h
until analysis. If analysis had to be postponed, samples are kept
at 4.degree. C. for a maximum of 24 h.
[0556] ED.sub.50 is the dose giving rise to half-maximal effect in
nmol/kg. This value is calculated on the basis of the ability of
the derivatives to lower body weight as well as the ability to
lower blood glucose, as explained below.
[0557] ED.sub.50 for body weight is calculated as the dose giving
rise to half-maximum effect on delta BW 24 hours following the
subcutaneous administration of the derivative. For example, if the
maximum decrease in body weight after 24 hours is 4.0 g, then
ED.sub.50 bodyweight would be that dose in nmol/kg which gives rise
to a decrease in body weight after 24 hours of 2.0 g. This dose
(ED.sub.50 body weight) may be read from the dose-response
curve.
[0558] ED.sub.50 for blood glucose is calculated as the dose giving
rise to half-maximum effect on AUC delta BG 8 hours following the
subcutaneous administration of the analogue.
[0559] The ED.sub.50 value may only be calculated if a proper
sigmoidal dose-response relationship exists with a clear definition
of the maximum response. Thus, if this would not be the case the
derivative in question is re-tested in a different range of doses
until the sigmoidal dose-response relationship is obtained.
Example 64
Effect on Food Intake--PD LYD Pigs
[0560] The purpose of this experiment is to investigate the effect
of GLP-1 derivatives on food intake in pigs. This is done in a
pharmacodynamic (PD) study as described below, in which food intake
is measured 1, 2, 3, and 4 days after administration of a single
dose of the GLP-1 derivative, as compared to a vehicle-treated
control group.
[0561] Female Landrace Yorkshire Duroc (LYD) pigs, approximately 3
months of age, weighing approximately 30-35 kg were used (n=3-4 per
group). The animals were housed in a group for 1-2 weeks during
acclimatisation to the animal facilities. During the experimental
period the animals were placed in individual pens from Monday
morning to Friday afternoon for measurement of individual food
intake. The animals were fed ad libitum with pig fodder
(Svinefoder, Antonio) at all times both during the acclimatisation
and the experimental period. Food intake was monitored on line by
logging the weight of fodder every 15 minutes. The system used was
Mpigwin (Ellegaard Systems, Faaborg, Denmark).
[0562] The GLP-1 derivatives were dissolved in a phosphate buffer
(50 mM phosphate, 0.05% tween 80, pH 8) at concentrations of 12,
40, 120, 400 or 1200 nmol/ml corresponding to doses of 0.3, 1, 3,
10 or 30 nmol/kg. The phosphate buffer served as vehicle. Animals
were dosed with a single subcutaneous dose of the GLP-1 derivative
or vehicle (dose volume 0.025 ml/kg) on the morning of day 1, and
food intake was measured for 3-4 days after dosing. On the last day
of each study, 3-4 days after dosing, a blood sample for
measurement of plasma exposure of the GLP-1 derivative was taken
from the heart in anaesthetized animals. The animals were
thereafter euthanized with an intra-cardial overdose of
pentobarbitone. Plasma content of the GLP-1 derivatives was
analysed using ELISA or a similar antibody based assay.
[0563] Food intake was calculated as mean.+-.SEM 24 h food intake
on each of the 3-4 study days. Statistical comparisons of the 24
hour food intake in the vehicle vs. GLP-1 derivative group on the 4
days were done using one-way or two-way-ANOVA repeated measures,
followed by Bonferroni post-test.
[0564] The derivatives of Examples 1, 5, and 6 were tested as
described above in a dosage of 3 nM/kg. Two of the derivatives
significantly reduced the food intake as compared to the
vehicle-treated group on day 1 (0-24 h) and day 2 (24-48 h). One of
these also significantly reduced the food intake on day 3 (48-72 h)
and day 4 (72-96 h). As expected, the food intake reducing effect
of the derivatives diminished from day to day in the study period,
depending on the terminal half-life of the derivative.
Example 65
Half-Life in Rat--PK Rat
[0565] The purpose of this Example is to investigate half-life in
vivo in rat.
[0566] In vivo pharmacokinetic studies in rats were performed with
three GLP-1 derivatives (compounds of the Examples 5, 7, and 11),
as described in the following.
[0567] Male Sprague Dawley rats of same age with a body weight from
400 to 600 g were obtained from Taconic (Denmark) and assigned to
the treatments by simple randomisation on body weight,
approximately 3-6 rats per group, so that all animals in each group
were of similar body weight.
[0568] The GLP-1 derivatives (approximately 6 nmole/ml) were
dissolved in 50 mM sodium phosphate, 145 mM sodium chloride, 0.05%
tween 80, pH 7.4. Intravenous injections (1.0 ml/kg) of the
compounds were given through a catheter implanted in the right
jugular vein. Blood was sampled from vena sublingualis for 5 days
post dosing. Blood samples (200 .mu.l) were collected in EDTA
buffer (8 mM) and then centrifuged at 4.degree. C. and 10000G for 5
minutes. Plasma samples were kept at -20.degree. C. until analyzed
for plasma concentration of the respective GLP-1 compound.
[0569] The plasma concentrations of the GLP-1 compounds were
determined using a Luminescence Oxygen Channeling Immunoasssay
(LOCI), generally as described for the determination of insulin by
Poulsen and Jensen in Journal of Biomolecular Screening 2007, vol.
12, p. 240-247. The donor beads were coated with streptavidin,
while acceptor beads were conjugated with a monoclonal antibody
recognising a mid-/C-terminal epitope of the peptide. Another
monoclonal antibody, specific for the N-terminus, was biotinylated.
The three reactants were combined with the analyte and formed a
two-sited immuno-complex. Illumination of the complex released
singlet oxygen atoms from the donor beads, which were channeled
into the acceptor beads and triggered chemiluminescence which was
measured in an Envision plate reader. The amount of light was
proportional to the concentration of the compound.
[0570] Plasma concentration-time profiles were analyzed using
WinNonlin (ver. 5.0, Pharsight Inc., Mountain View, Calif., USA),
and the half-life (T.sub.1/2) calculated using individual plasma
concentration-time profiles from each animal.
[0571] All tested derivatives had a half-life above 10 hours, two
were above 15, and one was above 20 hours.
Example 66
Half-Life in Minipigs--PK Minipig
[0572] The purpose of this study was to determine the protraction
in vivo of the GLP-1 derivatives after i.v. administration to
minipigs, i.e. the prolongation of their time of action. This was
done in a pharmacokinetic (PK) study, where the terminal half-life
of the derivative in question was determined. By terminal half-life
was generally meant the period of time it takes to halve a certain
plasma concentration, measured after the initial distribution
phase.
[0573] Male Gottingen minipigs (Ellegaard Gottingen Minipigs A/S,
Dalmose, Denmark), approximately 7-14 months of age and weighing
from approximately 16-35 kg, were used in the studies. The minipigs
were housed individually and fed restrictedly once or twice daily
with SDS minipig diet (Special Diets Services, Essex, UK). After at
least 2 weeks of acclimatisation two permanent central venous
catheters were implanted in vena cava caudalis or cranialis in each
animal. The animals were allowed 1 week recovery after the surgery,
and were then used for repeated pharmacokinetic studies with a
suitable wash-out period between dosings.
[0574] The animals were fasted for approximately 18 h before dosing
and for at least 4 h after dosing, but has ad libitum access to
water during the whole period.
[0575] The GLP-1 derivatives of Examples 5, 6, 8, and 11 were
dissolved in 50 mM sodium phosphate, 145 mM sodium chloride, 0.05%
tween 80, pH 7.4 to a concentration of usually from 20-60
nmol/ml.
[0576] Intravenous injections (the volume corresponding to usually
1-2 nmol/kg) of the compounds were given through one catheter, and
blood was sampled at predefined time points for up till 13 days
post dosing (preferably through the other catheter). Blood samples
were collected in EDTA buffer (8 mM) and then centrifuged at
4.degree. C. and 1942G for 10 minutes.
[0577] Plasma was pippetted into Micronic tubes on dry ice, and
kept at -20.degree. C. until analyzed for plasma concentration of
the respective GLP-1 compound using ELISA or a similar antibody
based assay or LC-MS. Individual plasma concentration-time profiles
were analyzed by a non-compartmental model in WinNonlin v. 5.0
(Pharsight Inc., Mountain View, Calif., USA), and the resulting
terminal half-lives (harmonic mean) determined.
[0578] The following results were obtained: All derivatives had a
terminal half-life of above 5 hours; 3 derivatives above 25 hours;
and 1 derivative above 50 hours.
[0579] While certain features of the invention have been
illustrated and described herein, many modifications,
substitutions, changes, and equivalents will now occur to those of
ordinary skill in the art. It is, therefore, to be understood that
the appended claims were intended to cover all such modifications
and changes as fall within the true spirit of the invention.
Sequence CWU 1
1
12131PRTHomo sapiens 1His 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 228PRTArtificial SequenceSynthetic
2His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Glu Glu 1
5 10 15 Gln Ala Ala Arg Lys Phe Ile Ala His Leu Val Gly 20 25
331PRTArtificial SequenceSynthetic 3His Xaa Glu Gly Thr Phe Thr Ser
Asp Val Ser Xaa Tyr Leu Glu Xaa 1 5 10 15 Gln Ala Xaa Xaa Glu Phe
Ile Ala Xaa Leu Val Xaa Gly Arg Gly 20 25 30 431PRTArtificial
SequenceSynthetic 4Xaa Xaa Glu Gly Thr Xaa Thr Ser Asp Xaa Ser Lys
Xaa Xaa Glu Xaa 1 5 10 15 Xaa Ala Xaa Xaa Xaa Phe Ile Xaa Xaa Leu
Xaa Xaa Xaa Xaa Xaa 20 25 30 531PRTArtificial SequenceSynthetic
5Xaa Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Xaa Tyr Leu Glu Xaa 1
5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg Gly
20 25 30 631PRTArtificial SequenceSynthetic 6Xaa Ala Glu Gly Thr
Phe Thr Ser Asp Val Ser Xaa Tyr Leu Glu Xaa 1 5 10 15 Gln Ala Xaa
Xaa Glu Phe Ile Ala Xaa Leu Val Xaa Gly Arg Gly 20 25 30
731PRTArtificial SequenceSynthetic 7His Xaa Glu Gly Thr Phe Thr Ser
Asp Val Ser Xaa Xaa Leu Glu Xaa 1 5 10 15 Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu Val Xaa Gly Arg Gly 20 25 30 832PRTArtificial
SequenceSynthetic 8Xaa Xaa Glu Gly Thr Xaa Thr Ser Asp Xaa Ser Lys
Xaa Xaa Glu Xaa 1 5 10 15 Xaa Ala Xaa Xaa Xaa Phe Ile Xaa Xaa Leu
Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 931PRTArtificial SequenceSynthetic
9His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Xaa Tyr Leu Glu Gly 1
5 10 15 Gln Ala Ala Lys Glu Xaa Ile Ala Trp Leu Val Xaa Gly Arg Gly
20 25 30 1031PRTArtificial SequenceSynthetic 10His Xaa Glu Gly Thr
Phe Thr Ser Asp Val Ser Xaa Xaa Leu Glu Xaa 1 5 10 15 Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg Gly 20 25 30
1131PRTArtificial SequenceSynthetic 11His Ala Glu Gly Thr Phe Thr
Ser Asp Val Ser Xaa Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu
Xaa Ile Ala Trp Leu Val Xaa Gly Arg Gly 20 25 30 1231PRTArtificial
SequenceSynthetic 12His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Xaa
Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
Val Xaa Gly Arg Gly 20 25 30
* * * * *
References