U.S. patent application number 12/406267 was filed with the patent office on 2009-10-08 for conjugation of peptides.
This patent application is currently assigned to Novo Nordisk A/S. Invention is credited to Florencio Zaragoza Dorwald, Niels Peter Fiil, Nils Langeland Johansen, Bernd Peschke, Henning Ralf Stennicke, Magali A. Zundel.
Application Number | 20090253166 12/406267 |
Document ID | / |
Family ID | 46045529 |
Filed Date | 2009-10-08 |
United States Patent
Application |
20090253166 |
Kind Code |
A1 |
Zundel; Magali A. ; et
al. |
October 8, 2009 |
Conjugation of Peptides
Abstract
Methods for the selective conjugation of peptides which
comprises an enzymatic incorporation of a functional group at the
C-terminal end of a peptide followed by reaction with a second
compound comprising the moiety to be conjugated to the peptide,
wherein said second compound comprises a functional group which
selectively reacts with the incorporated functional group.
Inventors: |
Zundel; Magali A.; (Soborg,
DK) ; Peschke; Bernd; (Malov, DK) ; Dorwald;
Florencio Zaragoza; (Visp, CH) ; Fiil; Niels
Peter; (Frederiksberg, DK) ; Johansen; Nils
Langeland; (Copenhagen, DK) ; Stennicke; Henning
Ralf; (Kokkedal, DK) |
Correspondence
Address: |
NOVO NORDISK, INC.;INTELLECTUAL PROPERTY DEPARTMENT
100 COLLEGE ROAD WEST
PRINCETON
NJ
08540
US
|
Assignee: |
Novo Nordisk A/S
Bagsvaerd
DK
|
Family ID: |
46045529 |
Appl. No.: |
12/406267 |
Filed: |
March 18, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11395784 |
Mar 31, 2006 |
7524813 |
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12406267 |
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PCT/DK2004/000685 |
Oct 8, 2004 |
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11395784 |
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60510892 |
Oct 14, 2003 |
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Current U.S.
Class: |
435/68.1 ;
530/324; 530/409; 564/164; 564/165 |
Current CPC
Class: |
C12P 21/00 20130101;
A61K 38/00 20130101; C07K 1/1077 20130101; C07K 14/61 20130101;
C07C 323/60 20130101; C07K 1/1075 20130101; C12P 21/02 20130101;
C07C 237/20 20130101; C07C 237/22 20130101 |
Class at
Publication: |
435/68.1 ;
530/409; 564/164; 530/324; 564/165 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C07K 14/00 20060101 C07K014/00; C07C 233/51 20060101
C07C233/51; C07K 14/47 20060101 C07K014/47; C07C 235/34 20060101
C07C235/34 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 10, 2003 |
DK |
PA 2003 01496 |
Claims
1. A method for preparing a conjugated peptide, said method
comprising the steps of i) reacting in one or more steps a peptide
with a first compound bearing one or more functional groups, which
are not accessible in any of the amino acids residues constituting
said peptide, in the presence of an enzyme capable of catalysing
the incorporation of said first compound into the C-terminal of
said peptide to form a transacylated peptide, and ii) reacting in
one or more steps said transacylated peptide with a second compound
comprising one or more functional groups, wherein said functional
group(s) do not react with functional groups accessible in the
amino acid residues constituting said peptide, and wherein said
functional group(s) in said second compound is capable of reacting
with said functional group(s) in said first compound so that one or
more covalent bond between said transacylated peptide and said
second compound is formed.
2. A method according to claim 1, wherein a peptide P is reacted in
one or more steps with a first compound, which is an .alpha.-amino
acid amide represented by the formula ##STR00201## in the presence
of carboxypeptidase to form a transacylated peptide of the formula
##STR00202## said transacylated peptide being further reacted in
one or more steps with a second compound of the formula Y-E-Z to
form a conjugated peptide of the formula ##STR00203## wherein R
represents a linker or a bond; P' represents a peptide formed by
removing the C-terminal amino acid from the peptide P; X represents
a radical comprising one or more functional group not accessible in
the amino acid residues constituting the peptide P'; Y represents a
radical comprising one or more functional group which functional
group(s) do not react with functional groups accessible in the
amino acid residues constituting the peptide P', and which
functional group(s) react with said functional group(s) present in
X; E represents a linker or a bond; wherein A represents the moiety
formed by the reaction between the functional groups comprised in X
and Y; and Z comprises the moiety to be conjugated to the peptide,
wherein said moiety decrease the clearance of compounds of formula
[a] in comparison with the clearance of P.
3. The method according to claim 2, wherein A represents oxime,
hydrazone, phenylhydrazone, semicarbazone or triazole moieties.
4. The method according to claim 2, wherein the functional group
present in X is selected from the group consisting of: keto-,
aldehyde-, --NH--NH.sub.2, --O--C(O)--NH--NH.sub.2,
--NH--C(O)--NH--NH.sub.2, --NH--C(S)--NH--NH.sub.2,
--NHC(O)--NH--NH--C(O)--NH--NH.sub.2, --NH--NH--C(O)--NH--NH.sub.2,
--NH--NH--C(S)--NH--NH.sub.2,
--NH--C(O)--C.sub.6H.sub.4--NH--NH.sub.2, --C(O)--NH--NH.sub.2,
--O--NH.sub.2, --C(O)--O--NH.sub.2, --NH--C(O)--O--NH.sub.2,
--NH--C(S)--O--NH.sub.2, alkyne, nitril-oxide and azide.
5. The method according to claim 2, wherein the functional group
present in Y is selected from the group consisting of: keto-,
aldehyde-, --NH--NH.sub.2, --O--C(O)--NH--NH.sub.2,
--NH--C(O)--NH--NH.sub.2, --NH--C(S)--NH--NH.sub.2,
--NHC(O)--NH--NH--C(O)--NH--NH.sub.2, --NH--NH--C(O)--NH--NH.sub.2,
--NH--NH--C(S)--NH--NH.sub.2,
--NH--C(O)--C.sub.6H.sub.4--NH--NH.sub.2, --C(O)--NH--NH.sub.2,
--O--NH.sub.2, --C(O)--O--NH.sub.2, --NH--C(O)--O--NH.sub.2,
--NH--C(S)--O--NH.sub.2, alkyne, nitril-oxide and azide.
6. The method according to claim 2, wherein X is selected from the
group consisting of keto- and aldehyde-derivatives, and Y is
selected from the group consisting of --NH--NH.sub.2,
--O--C(O)--NH--NH.sub.2, --NH--C(O)--NH--NH.sub.2,
--NH--C(S)--NH--NH.sub.2, --NHC(O)--NH--NH--C(O)--NH--NH.sub.2,
--NH--NH--C(O)--NH--NH.sub.2, --NH--NH--C(S)--NH--NH.sub.2,
--NH--C(O)--C.sub.6H.sub.4--NH--NH.sub.2, --C(O)--NH--NH.sub.2,
--O--NH.sub.2, --C(O)--O--NH.sub.2, --NH--C(O)--O--NH.sub.2, and
--NH--C(S)--O--NH.sub.2.
7. The method according to claim 2, wherein X represents alkyne,
and Y represents azide or nitril-oxide.
8. The method according to claim 2, wherein X represents azide or
nitril-oxide, and Y represents azide.
9. The method according to claim 2, wherein R and E each
independently represents a bi-radical of straight, branched and/or
cyclic C.sub.1-10alkane, C.sub.2-10alkene, C.sub.2-10alkyne,
C.sub.1-10heteroalkane, C.sub.2-10heteroalkene, or
C.sub.2-10heteroalkyne, wherein one or more homocyclic aromatic
compound bi-radical or heterocyclic compound biradical may be
inserted.
10. The method according to claim 2, wherein said .alpha.-amino
acid amide represents a compound selected from the group consisting
of: 2-amino-3-oxo-butyramide,
2-amino-6-(4-oxo-pentanoylamino)-hexanoic acid amide,
2-amino-3-(2-oxo-2-phenyl-ethylsulfanyl)-propionamide,
2-amino-5-oxo-hexanoic acid amide, 2-amino-3-oxo-propionamide,
2-amino-6-(4-acetylbenzoylamino)hexanoic acid amide,
2-amino-3-oxopropionic acid amide,
(2S)-Amino-3-[4-(2-oxopropoxy)phenyl]propionamide,
(2S)-Amino-3-[4-(2-oxobutoxy)phenyl]propionamide,
(2S)-Amino-3-[4-(2-oxopentoxy)phenyl]propionamide,
(2S)-Amino-3-[4-(4-oxopentoxy)phenyl]propionamide,
(2S)-2-Amino-6-(4-oxo-4-phenylbutyrylamino)hexanoic acid amide,
4-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide,
(2S)-2-Amino-6-(4-oxo-4-(4-chlorophenylbutyrylamino)hexanoic acid
amide, 3-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide,
2-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide,
(2S)-2-amino-3-(4-(prop-2-ynyloxy)phenyl)propionamide, and
(S)-2-aminopent-4-ynoicacid amide and S-phenylacylcysteine
amide.
11. A method according to claim 2, wherein Z comprises one or more
polyethylene glycol or methoxy polyethylene glycol radicals and
amino derivatives thereof; straight, branched and/or cyclic
C.sub.1-22alkyl, C.sub.2-22alkenyl, C.sub.2-22alkynyl,
C.sub.1-22heteroalkyl, C.sub.2-22heteroalkenyl,
C.sub.2-22heteroalkynyl, wherein one or more homocyclic aromatic
compound biradical or heterocyclic compound biradical may be
inserted, and wherein said C.sub.1-C.sub.22 or C.sub.2-C.sub.22
radicals may optionally be substituted with one or more
substituents selected from hydroxyl, halogen, carboxyl and aryl,
wherein said aryl may optionally be further substituted by one or
more substituents selected from hydroxyl, halogen, and carboxyl;
steroid radicals; lipid radicals; polysaccharide radicals; dextran
radicals; polyamide radicals; polyamino acid radicals; PVP
radicals; PVA radicals; poly(1-3-dioxalane) radicals;
poly(1,3,6-trioxane) radicals; ethylene/maleic anhydride polymer
radicals; Cibacron dye stuff radicals; and Cibacron Blue 3GA
radical.
12. A method according to claim 11, wherein Z comprises one or more
polyethylene glycol or methoxy polyethylene glycol radical with a
molecular weight of about 10, 20, 30 or 40 kDa.
13. A method according to claim 11, wherein Z comprises one or more
C.sub.10-20alkyl, optionally substituted with a carboxy group.
14. A method according to claim 11, wherein Z represents a
undecanoic acid radical.
15. The method according to claim 11, wherein Z comprises one or
more of C.sub.15alkyl, C.sub.17alkyl, Cibacron Blue 3GA or radical
of the formula ##STR00204##
16. The method according to claim 11, wherein Z comprises one or
more moieties that bind to albumin.
17. The method according to claim 1, wherein the enzyme is
Carboxypeptidase Y.
18. The method according to claim 2, wherein P represents a peptide
selected from the group consisting of: insulin, GLP-1, GLP-2,
growth hormone, cytokines, TFF, melanocortin receptor modifiers and
Factor VII.
19. The method according to claim 2, which further comprises the
step of formulating said conjugated peptide in a pharmaceutical
composition.
20. A conjugated peptide according to the formula ##STR00205##
wherein P', R, A, E and Z are as defined in claim 2, and wherein
##STR00206## is attached to P' at the C-terminal end of P' via a
peptide bond.
21. A peptide according to claim 20 selected from the group
consisting of:
Lys.sup..epsilon.(4-((2-(1-(mPEGcarbonyl)piperidin-4yl)ethoxy)imino)p-
entanoyl) 192)hGH(1-192) amide, in which mPEG has a molecular
weight of 20 kDa;
(Lys.sup..epsilon.(4-((3-(palmitoylamino)propoxy)imino)pentanoyl)192-
)hGH(1-192) amide;
(Lys.sup..epsilon.(4-((3-((2S)-2,6-mPEGcarbonylamino)hexanoylamino)propox-
y)imino)pentanoyl)34)GLP-2(1-34) amide, in which mPEG has a
molecular weight of 20 kDa;
(Lys.sup..epsilon.(4-(1-(2-(3-(mPEG)propanoylamino)hydrazino)ethyl)benzoy-
l)192)hGH(1-92) amide, in which mPEG has a molecular weight of 10
kDa;
(S)-3-(4-((3-(3-Chlorophenyl)isoxazol-5-yl)methoxy)phenyl)-2-([Glu.sup.3,-
Leu.sup.10]GLP-2ylleucinylamino)propionic amide;
(S)-3-(4-((3-(3-Chlorophenyl)isoxazol-5-yl)methoxy)phenyl)-2-([Glu.sup.3]-
GLP-2ylleucinylamino)propionic amide;
3-(3-(3-((4-((S)-2-Carbamoyl-3-([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamin-
o)ethyl)phenoxyl)methyl)isoxazol-3-yl)benzylcarbamoyl)propionic
acid;
11-(4-(4-((2S)-2-Carbamoyl-2-(([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamino-
)ethyl)pheoxymethyl)-1,2,3-triazolyl)undecanoic acid;
11-(5-(4-((2S)-2-Carbamoyl-2-(([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamino-
)ethyl)pheoxymethyl)-1,2,3-triazolyl)undecanoic acid
1-(4-(4-((S)-2-carbamoyl-2-([Glu.sup.3]GLP-2ylleucinylamino))phenoxymethy-
l)-1H-1,2,3-triazol-1-yl)undecanoic acid;
11-(5-(4-((S)-2-carbamoyl-2-([Glu.sup.3]GLP-2ylleucinylamino))phenoxymeth-
yl)-1H-1,2,3-triazol-1-yl)undecanoic acid;
2-([Glu.sup.3]GLP-2ylleucinyl)-3-(4-((1-((N-(mPeg20kDayl)carbamoyl)decany-
l)-1H-1,2,3-tetrazol-4-yl)methoxy)phenyl)propionamide; and
2-([Glu.sup.3]GLP-2ylleucinyl)-3-(4-((1-((N-(mPeg20kDayl)carbamoyl)decany-
l)-1H-1,2,3-tetrazol-5-yl)methoxy)phenyl)propionamide.
22. A pharmaceutical composition comprising one or more peptides
according to claim 21.
23. A compound according for formula I ##STR00207## wherein A and E
independently represent C.sub.1-6alkylene, C.sub.2-6alkenylene,
C.sub.2-6alkynylene or arylene, all of which may optionally be
substituted with one or more substituents selected from halogen,
amino, cyano and nitro; B and D represents --C(O)-- or --NH-- with
the proviso that when B represents --C(O)-- then D must represent
--NH--, and when B represents --NH-- then D must represent
--C(O)--; and F represents hydrogen or C.sub.1-6alkylene,
C.sub.2-6alkenylene, C.sub.2-6alkynylene or arylene, all of which
may optionally be substituted with one or more substituents
selected from halogen, amino, cyano and nitro.
24. A compound according to claim 23 selected from the group
consisting of: (2S)-2-Amino-6-(4-oxo-4-phenylbutyrylamino)hexanoic
acid amide, 4-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide,
(2S)-2-Amino-6-(4-oxo-4-(4-chlorophenylbutyrylamino)hexanoic acid
amide, 3-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide, and
2-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide.
25. A compound according to formula II ##STR00208## wherein J and L
independently represent C.sub.1-6alkylene, C.sub.2-6alkenylene,
C.sub.2-6alkynylene or arylene, all of which may optionally be
substituted with one or more substituents selected from halogen,
amino, cyano and nitro; and M represents hydrogen or
C.sub.1-6alkyl.
26. A compound according to formula 25 selected from the group
consisting of: (2S)-Amino-3-[4-(2-oxopropoxy)phenyl]propionamide,
(2S)-Amino-3-[4-(2-oxobutoxy)phenyl]propionamide,
(2S)-Amino-3-[4-(2-oxopentoxy)phenyl]propionamide, and
(2S)-Amino-3-[4-(4-oxopentoxy)phenyl]propionamide.
27. A compound according to formula III ##STR00209## wherein Q
represents represent C.sub.1-6alkylene, C.sub.2-6alkenylene,
C.sub.2-6alkynylene or arylene, all of which may optionally be
substituted with one or more substituents selected from halogen,
amino, cyano and nitro; and T represents hydrogen or
C.sub.1-6alkyl.
28. A compound according to formula IV ##STR00210## wherein J'' and
L'' independently represent C.sub.1-6alkylene or arylene, all of
which may optionally be substituted with one or more substituents
selected from halogen amino, cyano and nitro.
29. The compound according to claim 28, which is
(S)-2-amino-3-(4-(propargyloxy)phenyl)propionyl amide.
30. A conjugated peptide obtainable by a method according to claim
2.
31. A method for conjugating a first peptide, wherein said peptide
comprises an alkyne group, the method comprising reacting said
first peptide with a second compound, wherein said second compound
comprises a nitri-oxide to form a conjugated peptide, wherein said
first peptide is bonded to said second compound via a isoxazoline
moiety.
32. A method for conjugating a first peptide, wherein said peptide
comprises an nitril-oxide group, the method comprising reacting
said first peptide with a second compound, wherein said second
compound comprises an alkyne to form a conjugated peptide, wherein
said first peptide is bonded to said second compound via a
isoxazoline moiety.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/395,784, filed Mar. 31, 2006, which is a
continuation of International Patent Application No.
PCT/DK2004/000685, filed Oct. 8, 2004, which claims priority from
Danish Patent Application No. PA 2003 01496, filed Oct. 10, 2003;
and to U.S. Patent Application No. 60/510,892, filed Oct. 14,
2003.
FIELD OF THE INVENTION
[0002] The present invention relates to a novel method for
post-translational conjugation of peptides. Said conjugated
peptides have altered characteristics and may thus be of use in
therapeutic applications or they may ease the analysis or isolation
and purification of said peptides.
BACKGROUND OF THE INVENTION
[0003] It is well-known to modify the properties and
characteristics of peptides by conjugating groups to the peptide
which duly changes the properties of the peptide. Such conjugation
generally requires some functional group in the peptide to react
with another functional group in a conjugating group. Typically,
amino groups, such as the N-terminal amino group or the
.epsilon.-amino group in lysines, have been used in combination
with a suitable acylating reagent. It is often desired or even
required to be able to control the conjugation reaction, i.e. to
control where the conjugating compounds are attached and to control
how many conjugating groups are attached. This is often referred to
as specificity.
[0004] It is an object of the present invention to provide a method
by which peptides may be conjugated with a high degree of
specificity. In general terms, the method exploits an enzyme
capable of incorporating a compound comprising a suitable
functional group into the C-terminal end of a peptide, where said
functional group is subsequently used as a point where to
conjugate.
[0005] The use of carboxypeptidases to modify the C-terminal of
peptides has been described earlier. WO 92/05271 discloses the use
of carboxypeptidases and nucleophilic compounds to amidate the
C-terminal carboxy group, and WO 98/38285 discloses variants of
carboxypeptidase Y particular apt for this purpose.
[0006] The grafting of PEG or PEG based chains have been amply
describes in the literature. By way of example, U.S. Pat. No.
5,739,208 discloses the use of a PEG with a sulfone group which
reacts with thioles present in the peptide.
[0007] EP 605 963 discloses the grafting of aqueous polymers which
form an oxime linkage with an aldehyde group on a protein. None of
the natural amino acid comprises an aldehyde, so a hydroxyl group
thus has to be oxidized as a first step in the conjugating
process.
[0008] EP 243 929 discloses the use of carboxypeptidase to
incorporate polypeptides, reporter groups or cytotoxic agents into
the C-terminal of proteins or polypeptides.
SUMMARY OF THE INVENTION
[0009] The present inventors have surprisingly found that enzymes,
e.g. carboxypeptidases may be used to incorporate into the
C-terminal of peptides a first compound comprising one or more
functional groups, which are not accessible in the peptide, to form
a transacylated compound, and that this transacylated compound may
subsequently be reacted with another compound comprising one or
more functional groups which react with the functional group of the
first compound but not with other functional groups accessible in
the peptide. Such method provides a high degree of specificity in
that the enzyme is chosen so that it only catalyses the
incorporation at the C-terminal, and the two functional groups are
selected so that they only react with each other, not with other
functional groups accesible in the peptide. In this way, the
conjugating group is only attached at one locus, and by selecting
the functional groups, the number of conjugated groups can be
controlled.
[0010] Accordingly, in one embodiment, the present invention
provides a method for conjugating peptides, said method comprising
the steps of [0011] i) reacting in one or more steps a peptide with
a first compound bearing one or more functional groups, which are
not accessible in any of the amino acids constituting said peptide,
in the presence of an enzyme capable of catalysing the
incorporation of said first compound into the C-terminal of said
peptide to form a transacylated peptide, and [0012] ii) reacting in
one or more steps said transacylated peptide with a second compound
comprising one or more functional groups, wherein said functional
group(s) do not react with functional groups accessible in the
amino acid residues constituting said peptide, and wherein said
functional group(s) in said second compound is capable of reacting
with said functional group(s) in said first compound so that a
covalent bond between said transacylated peptide and said second
compound is formed.
[0013] It is also an objective of the present invention to provide
peptides conjugated by the method of the present invention.
[0014] It is a further objective of the present invention to
provide peptides which are modified in a way to make them better
suited for the method of the present invention.
[0015] It is a still further objective of the present invention to
provide reagents and enzymes suitable for use in the methods of the
present invention.
[0016] In a still further embodiment, the present invention
provides the use of peptides conjugated by methods of the present
invention in therapy.
[0017] It is a still further objective of the present invention to
provide compositions, e.g. pharmaceutical compositions comprising
peptides conjugated by methods of the present invention.
[0018] It is a still further objective of the present invention to
provide therapeutic methods for the treatment of diseases
comprising the administration of conjugated peptides prepared
according to the methods of the present invention.
[0019] It is a still further objective of the present invention to
provide a use of conjugated peptides prepared according to the
methods of the present invention in the manufacture of
medicaments.
[0020] It is a still further objective of the present invention to
provide a method for improving the properties of a peptide by
conjugation said peptide according to the methods of the present
invention.
DEFINITIONS
[0021] In the present context, the term "transacylation" is
intended to indicate a reaction in which a leaving group is
exchanged for a nucleophile, wherein a nucleophile is understood to
be an electron-rich reagent that tends to attack the nucleus of
carbons. Transpeptidation is one example of a transacylation.
[0022] In the present context, the term "not accessible" is
intended to indicate that something is absent or de facto absent in
the sense that it cannot be reached. When it is stated that
functional groups are not accessible in a peptide to be conjugated
it is intended to indicate that said functional group is absent
from the peptide or, if present, in some way prevented from taking
part in reactions. By way of example, said functional group could
be buried deep in the structure of the peptide so that it is
shielded from participating in the reaction. It is recognised that
whether or not a functional group is accessible depends on the
reaction conditions. It may be envisaged that in the presence of
denaturing agents or at elevated temperatures the peptide may
unfold to expose otherwise not accessible functional groups. It is
to be understood that "not accessible" means "not accessible at the
reaction condition chosen for the particular reaction of
interest".
[0023] In the present context, the term "oxime bond" is intended to
indicate a moiety of the formula --C.dbd.N--O--.
[0024] In the present context, the term "hydrazone bond" is
intended to indicate a moiety of the formula --C.dbd.N--N--.
[0025] In the present context, the term "phenylhydrazone bond" is
intended to indicate a moiety of the formula
##STR00001##
[0026] In the present context, the term "semicarbazone bond" is
intended to indicate a moiety of the formula
--C.dbd.N--N--C(O)--N--.
[0027] The term "alkane" is intended to indicate a saturated,
linear, branched and/or cyclic hydrocarbon. Unless specified with
another number of carbon atoms, the term is intended to indicate
hydrocarbons with from 1 to 30 (both included) carbon atoms, such
as 1 to 20 (both included), such as from 1 to 10 (both included),
e.g. from 1 to 5 (both included). The terms alkyl and alkylene
refer to the corresponding radical and bi-radical,
respectively.
[0028] The term "alkene" is intended to indicate linear, branched
and/or cyclic hydrocarbons comprising at least one carbon-carbon
double bond. Unless specified with another number of carbon atoms,
the term is intended to indicate hydrocarbons with from 2 to 30
(both included) carbon atoms, such as 2 to 20 (both included), such
as from 2 to 10 (both included), e.g. from 2 to 5 (both included).
The terms alkenyl and alkenylene refer to the corresponding radical
and biradical, respectively.
[0029] The term "alkyne" is intended to indicate linear, branched
and/or cyclic hydrocarbons comprising at least one carbon-carbon
triple bond, and it may optionally comprise one or more
carbon-carbon double bonds. Unless specified with another number of
carbon atoms, the term is intended to indicate hydrocarbons with
from 2 to 30 (both included) carbon atoms, such as from 2 to 20
(both included), such as from 2 to 10 (both included), e.g. from 2
to 5 (both included). The terms alkynyl and alkynylene refer to the
corresponding radical and bi-radical, respectively.
[0030] The term "homocyclic aromatic compound" is intended to
indicate aromatic hydrocarbons, such as benzene and
naphthalene.
[0031] The term "heterocyclic compound" is intended to indicate a
cyclic compound comprising 5, 6 or 7 ring atoms from which 1, 2, 3
or 4 are hetero atoms selected from N, O and/or S. Examples include
heterocyclic aromatic compounds, such as thiophene, furan, pyran,
pyrrole, imidazole, pyrazole, isothiazole, isooxazole, pyridine,
pyrazine, pyrimidine, pyridazine, as well as their partly or fully
hydrogenated equivalents, such as piperidine, pirazolidine,
pyrrolidine, pyroline, imidazolidine, imidazoline, piperazine and
morpholine.
[0032] The terms "hetero alkane", "hetero alkene" and "hetero
alkyne" is intended to indicate alkanes, alkenes and alkynes as
defined above, in which one or more hetero atom or group have been
inserted into the structure of said moieties. Examples of hetero
groups and atoms include --O--, --S--, --S(O)--, --S(O).sub.2--,
--C(O)-- --C(S)-- and --N(R*)--, wherein R* represents hydrogen or
C.sub.1-C.sub.6-alkyl. Examples of heteroalkanes include.
##STR00002##
[0033] The term "radical" or "biradical" is intended to indicate a
compound from which one or two, respectively, hydrogen atoms have
been removed. When specifically stated, a radical may also indicate
the moiety formed by the formal removal of a larger group of atoms,
e.g. hydroxyl, from a compound.
[0034] The term "halogen" is intended to indicate members of the
seventh main group of the periodic table, i.e. F, Cl, Br and I.
[0035] The term "PEG" is intended to indicate polyethylene glycol
of a molecular weight between 500 and 150,000 Da, including
analogues thereof, wherein for instance the terminal OH-group has
been replaced by a methoxy group (referred to as mPEG).
[0036] In the present context, the words "peptide" and "protein"
are used interchangeably and are intended to indicate the same. The
term "peptide" is intended to indicate a compound with two or more
amino acid residues linked by a peptide bond. The amino acids may
be natural or unnatural. The term is also intended to include said
compounds substituted with other peptides, saccharides, lipids, or
other organic compound, as well as compounds wherein one or more
amino acid residue have been chemically modified and peptides
comprising a prosthetic group.
[0037] In the present context, the term "aryl" is intended to
indicate a carbocyclic aromatic ring radical or a fused aromatic
ring system radical wherein at least one of the rings are aromatic.
Typical aryl groups include phenyl, biphenylyl, naphthyl, and the
like.
[0038] The term "heteroaryl", as used herein, alone or in
combination, refers to an aromatic ring radical with for instance 5
to 7 member atoms, or to a fused aromatic ring system radical with
for instance from 7 to 18 member atoms, wherein at least one ring
is aromatic, containing one or more heteroatoms as ring atoms
selected from nitrogen, oxygen, or sulfur heteroatoms, wherein
N-oxides and sulfur monoxides and sulfur dioxides are permissible
heteroaromatic substitutions. Examples include furanyl, thienyl,
thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,
thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl,
isothiazolyl, pyridinyl, pyridazinyl, pyrazinyl, pyrimidinyl,
quinolinyl, isoquinolinyl, benzofuranyl, benzothiophenyl, indolyl,
and indazolyl, and the like.
[0039] The term "conjugate" as a noun is intended to indicate a
modified peptide, i.e. a peptide with a moiety bonded to it to
modify the properties of said peptide. As a verb, the term is
intended to indicate the process of bonding a moiety to a peptide
to modify the properties of said peptide.
[0040] As used herein, the term "prodrug" indicates biohydrolyzable
amides and biohydrolyzable esters and also encompasses a) compounds
in which the biohydrolyzable functionality in such a prodrug is
encompassed in the compound according to the present invention, and
b) compounds which may be oxidized or reduced biologically at a
given functional group to yield drug substances according to the
present invention. Examples of these functional groups include
1,4-dihydropyridine, N-alkylcarbonyl-1,4-dihydropyridine,
1,4-cyclohexadiene, tert-butyl, and the like.
[0041] As used herein, the term "biohydrolyzable ester" is an ester
of a drug substance (in casu, a compound according to the
invention) which either a) does not interfere with the biological
activity of the parent substance but confers on that substance
advantageous properties in vivo such as duration of action, onset
of action, and the like, or b) is biologically inactive but is
readily converted in vivo by the subject to the biologically active
principle. The advantage is, for example increased solubility or
that the biohydrolyzable ester is orally absorbed from the gut and
is transformed to a compound according to the present invention in
plasma. Many examples of such are known in the art and include by
way of example lower alkyl esters (e.g., C.sub.1-C.sub.4), lower
acyloxyalkyl esters, lower alkoxyacyloxyalkyl esters, alkoxyacyloxy
esters, alkyl acylamino alkyl esters, and choline esters.
[0042] As used herein, the term "biohydrolyzable amide" is an amide
of a drug substance (in casu, a compound according to the present
invention) which either a) does not interfere with the biological
activity of the parent substance but confers on that substance
advantageous properties in vivo such as duration of action, onset
of action, and the like, or b) is biologically inactive but is
readily converted in vivo by the subject to the biologically active
principle. The advantage is, for example increased solubility or
that the biohydrolyzable amide is orally absorbed from the gut and
is transformed to a compound according to the present invention in
plasma. Many examples of such are known in the art and include by
way of example lower alkyl amides, .alpha.-amino acid amides,
alkoxyacyl amides, and alkylaminoalkylcarbonyl amides.
[0043] In the present context, the term "pharmaceutically
acceptable salt" is intended to indicate salts which are not
harmful to the patient. Such salts include pharmaceutically
acceptable acid addition salts, pharmaceutically acceptable metal
salts, ammonium and alkylated ammonium salts. Acid addition salts
include salts of inorganic acids as well as organic acids.
Representative examples of suitable inorganic acids include
hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric
acids and the like. Representative examples of suitable organic
acids include formic, acetic, trichloroacetic, trifluoroacetic,
propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic,
maleic, malic, malonic, mandelic, oxalic, picric, pyruvic,
salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric,
ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic,
gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic,
p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids
and the like. Further examples of pharmaceutically acceptable
inorganic or organic acid addition salts include the
pharmaceutically acceptable salts listed in J. Pharm. Sci. 1977,
66, 2, which is incorporated herein by reference. Examples of metal
salts include lithium, sodium, potassium, magnesium salts and the
like. Examples of ammonium and alkylated ammonium salts include
ammonium, methylammonium, dimethylammonium, trimethylammonium,
ethylammonium, hydroxyethylammonium, diethylammonium,
butylammonium, tetramethylammonium salts and the like.
[0044] A "therapeutically effective amount" of a compound as used
herein means an amount sufficient to cure, alleviate or partially
arrest the clinical manifestations of a given disease and its
complications. An amount adequate to accomplish this is defined as
"therapeutically effective amount". Effective amounts for each
purpose will depend on the severity of the disease or injury as
well as the weight and general state of the subject. It will be
understood that determining an appropriate dosage may be achieved
using routine experimentation, by constructing a matrix of values
and testing different points in the matrix, which is all within the
ordinary skills of a trained physician or veterinary.
[0045] The term "treatment" and "treating" as used herein means the
management and care of a patient for the purpose of combating a
condition, such as a disease or a disorder. The term is intended to
include the full spectrum of treatments for a given condition from
which the patient is suffering, such as administration of the
active compound to alleviate the symptoms or complications, to
delay the progression of the disease, disorder or condition, to
alleviate or relief the symptoms and complications, and/or to cure
or eliminate the disease, disorder or condition as well as to
prevent the condition, wherein prevention is to be understood as
the management and care of a patient for the purpose of combating
the disease, condition, or disorder and includes the administration
of the active compounds to prevent the onset of the symptoms or
complications. The patient to be treated is preferably a mammal, in
particular a human being, but it may also include animals, such as
dogs, cats, cows, sheep and pigs.
DESCRIPTION OF THE INVENTION
[0046] In principle, any enzyme capable of catalysing the
incorporation of a compound into a peptide is useful in the methods
of the present invention. By way of example, useful enzymes include
carboxypeptidases, which constitute a group of peptide hydrolases
belonging to the classification groups E.C. 3.4.16, 3.4.17 and
3.4.18. The in vivo reaction catalysed by said enzymes is the
hydrolysis of the C-terminal amino acid residue. Various
carboxypeptidases are known and they differ in what terminal amino
acid residue they are capable of cleaving off. During the catalytic
cycle an enzyme-substrate complex is formed which under normal in
vivo conditions is subjected to a nucleophilic attack by a water
molecule, which eventually leads to the hydrolysis of the peptide
bond. In the methods of the present invention, however, a
nucleophilic reagent is added, which can out compete water as a
nucleophile. Moreover, the water activity may be reduced by running
the reaction in solvents or in aqueous solvents. In the methods of
the present invention, said nucleophile attacks the
enzyme-substrate complex eventually forming a transacylated
compound. On top of being a nuclophile, said reagent also has to
comprise one or more functional groups, which are not accessible in
the peptide to be conjugated.
[0047] Other enzymes which could be applied in the methods of the
present invention include trypsin.
[0048] The reaction of the peptide and the nucleophile affords a
transacylated peptide wherein the C-terminal amino acid residue has
been exchanged with the nucleophilic compound, which comprises one
or more functional groups which are not accessible in the peptide
to be conjugated. The overall result of this reaction (or this
series of reactions) is an incorporation of one or more functional
groups into the peptide which are present at only one locus in the
peptide. A subsequent reaction (or series of reactions) of this
transacylated peptide with a compound comprising the moiety to be
conjugated to the peptide and one or more functional groups, which
only react with the functional groups added to the peptide in the
transacylation reaction, effects a selective conjugation of the
peptide to be conjugated.
[0049] Compared to other conjugation methods which take advantage
of functional groups already present in the peptide, e.g.
N-terminal amino groups or .epsilon.-amino groups of lysines, the
method of the present invention offers the advantage of improved
selectivity. The incorporation of one or more functional groups not
accessible in the peptide ensures that the conjugation takes place
at only specified loci.
[0050] As mentioned earlier, any enzyme capable of catalysing the
incorporation of a compound into a peptide may be used in the
methods of the present invention, and in particular
carboxypeptidases are useful. Examples of particular useful
carboxypeptidease are serine-type carboxypeptidases, such as
lysosomalPro-X carboxypeptidase (also known as proline
carboxypeptidase, angiotensinase C, lysosomal carboxypeptidase C
and prolylcarboxypeptidase), serine-type D-Ala-D-Ala
carboxypeptidase (also known as D-alanyl-D-alanine
carboxypeptidase, DD-peptidase and DD-transpeptidase),
carboxypeptidase C (also known as Serine-type carboxypeptidase I,
cathepsin A, carboxypeptidase Y and lysosomal protective protein)
and carboxypeptidase D (also known as carboxypeptidase KEX1 and
carboxypeptidase S1); metallocarboxypeptidases, such as
carboxypeptidase A, carboxypeptidase B (also known as protaminase),
lysine(arginine) carboxypeptidase (also known as carboxypeptidase
N), and Gly-X carboxypeptidase (also known as carboxypeptidase S);
and cysteine-type carboxypeptidase (also known as lysosomal
carboxypeptidase B, cathepsin B2, Cathepsin Iv and acid
carboxypeptidase). It is also well-known that amino acid residues
may be changed, added or deleted in the sequence of
carboxypeptidases to modify the catalytic properties of the enzyme.
Such modified carboxypeptidases are disclosed in, e.g. WO 98/38285,
which is incorporated herein by reference. Particular mentioning is
made of carboxypeptidase Y as a useful enzyme.
[0051] Many nucleophilic compounds are known which could be
incorporated into peptides according to the methods of the present
invention, and .alpha.-amino acids is one such type of nucleophilic
compounds. For the purpose of the present invention, it is,
however, preferred to select the nucleophilic compound so that the
transacylated compound formed is not itself a substrate for the
enzyme applied. Stated differently, it is preferred to apply a
nucleophilic compound which effectively blocks any further reaction
of the enzyme. One example of such compounds is amides of
.alpha.-amino acids as carboxy amidated peptides are not substrates
for carboxypeptidases.
[0052] It is recognised that whether or not a compound is a
substrate for a given enzyme in principle depends on the
conditions, e.g. the time frame, under which the reaction takes
place. Given sufficient time, many compounds are, in fact,
substrates for an enzyme although they are not under normal
conditions regarded as such. When it is stated above that the
transacylated compound itself should not be a substrate of the
enzyme it is intended to indicate that the tranacylated compound
itself is not a substrate for the enzyme to an extent where the
following reactions in the method of the present invention are
disturbed. If the transacylated compound is, in fact, a substrate
for the enzyme, the enzyme may be removed or inactivated, e.g. by
enzyme inhibitors, following the transacylation reaction.
[0053] In one embodiment, the invention relates to a method of
conjugating peptides, wherein a peptide P is reacted in one or more
steps with a first compound, which is an .alpha.-amino acid amide
represented by the formula
##STR00003##
in the presence of carboxypeptidase to form a transacylated peptide
of the formula
##STR00004##
said transacylated peptide being further reacted in one or more
steps with a second compound of the formula
Y-E-Z
to form a conjugated peptide of the formula
##STR00005##
wherein R represents a linker or a bond; wherein P' represents the
peptide obtained when the C-terminal amino acid is removed from the
peptide P; X represents a radical comprising a functional group not
accessible in the amino acid residues constituting the peptide P';
Y represents a radical comprising one or more functional groups
which groups react with functional groups present in X, and which
functional groups do not react with functional groups accessible in
the peptide P'; E represents a linker or a bond; A represents the
moiety formed by the reaction between the functional groups
comprised in X and Y; and Z is the moiety to be conjugated to the
peptide, wherein said moiety decreases the clearance of the
compound of formula [a] in comparison with the clearance of P.
[0054] In a further embodiment, the invention relates to methods of
conjugating peptides as disclosed above, which further comprises
the step of formulating the resulting conjugated peptide in a
pharmaceutical composition.
[0055] Following the conjugation, the conjugated peptide may be
isolated and purified by techniques well-known in the art. The
conjugated peptide may also be converted into a pharmaceutically
acceptable salt or prodrug, if relevant.
[0056] The moiety, A, formed in the reaction between the functional
groups of X and Y may in principle be of any kind depending on what
properties of the final conjugated peptide is desired. In some
situation it may be desirable to have a labile bond which can be
cleaved at some later stage, e.g. by some enzymatic action or by
photolysis. In other situations, it may be desirable to have a
stable bond, so that a stable conjugated peptide is obtained.
Particular mentioning is made of the type of moieties formed by
reactions between amine derivatives and carbonyl groups, such as
oxime, hydrazone, phenylhydrazone and semicarbazone moieties.
[0057] In one embodiment the functional groups of X and Y are
selected from amongst carbonyl groups, such as keto and aldehyde
groups, and amino derivatives, such as
hydrazine derivatives --NH--NH.sub.2, hydrazine carboxylate
derivatives --O--C(O)--NH--NH.sub.2, semicarbazide derivatives
--NH--C(O)--NH--NH.sub.2, thiosemicarbazide derivatives
--NH--C(S)--NH--NH.sub.2, carbonic acid dihydrazide derivatives
--NHC(O)--NH--NH--C(O)--NH--NH.sub.2, carbazide derivatives
--NH--NH--C(O)--NH--NH.sub.2, thiocarbazide derivatives
--NH--NH--C(S)--NH--NH.sub.2, aryl hydrazine derivatives
--NH--C(O)--C.sub.6H.sub.4--NH--NH.sub.2, and hydrazide derivatives
--C(O)--NH--NH.sub.2; oxylamine derivatives, such as --O--NH.sub.2,
--C(O)--O--NH.sub.2, --NH--C(O)--O--NH.sub.2 and
--NH--C(S)--O--NH.sub.2.
[0058] It is to be understood, that if the functional group
comprised in X is a carbonyl group, then the functional group
comprised in Y is an amine derivative, and vice versa. Due to the
presence of --NH.sub.2 groups in most peptides, a better
selectivity is believed to be obtained if X comprises a keto- or an
aldehyde-functionality.
[0059] Another example of a suitable pair of X and Y is azide
derivatives (--N.sub.3) and alkynes which react to form a triazole
moiety.
[0060] Another example of a suitable pair of X and Y is alkyne and
nitril-oxide, which reacts to form a isooxazolidine moiety.
[0061] In particular, the group to be transacylated,
##STR00006##
may be selected from amongst 2-amino-3-oxo-butyramide,
2-amino-6-(4-oxo-pentanoylamino)-hexanoic acid amide,
2-amino-3-(2-oxo-2-phenyl-ethylsulfanyl)-propionamide,
2-amino-5-oxo-hexanoic acid amide, 2-amino-3-oxo-propionamide,
2-amino-6-(4-acetylbenzoylamino)hexanoic acid amide,
2-amino-3-oxopropionic acid amide,
(2S)-Amino-3-[4-(2-oxopropoxy)phenyl]propionamide,
(2S)-Amino-3-[4-(2-oxobutoxy)phenyl]propionamide,
(2S)-Amino-3-[4-(2-oxopentoxy)phenyl]propionamide,
(2S)-Amino-3-[4-(4-oxopentoxy)phenyl]propionamide,
(2S)-2-Amino-6-(4-oxo-4-phenylbutyrylamino)hexanoic acid amide,
4-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide,
(2S)-2-Amino-6-(4-oxo-4-(4-chlorophenylbutyrylamino)hexanoic acid
amide, 3-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide,
2-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide,
(2S)-2-amino-3-(4-(prop-2-ynyloxy)phenyl)propionamide,
(S)-2-aminopent-4-ynoicacid amide and S-phenylacylcysteine
amide.
[0062] Both the compound to be transacylated and the compound to be
reacted with the transacylated peptide comprises a linker, R and E,
respectively. These linkers, which are independent of each other,
may be absent or selected from amongst alkane, alkene or alkyne
diradicals and hetero alkane, hetero alkene and hetero alkyne
diradicals, wherein one or more optionally substituted aromatic
homocyclic biradical or biradical of a heterocyclic compound, e.g.
phenylene or piperidine biradical may be inserted into the
aforementioned biradicals. It is to be understood that said linkers
may also comprise substitutions by groups selected from amongst
hydroxyl, halogen, nitro, cyano, carboxyl, aryl, alkyl and
heteroaryl.
[0063] Both E and R represent bonds or linkers, and in the present
context the term "linker" is intended to indicate a moiety
functioning as a means to separate Y from Z and X from
NH.sub.2--C(O)--C(NH.sub.2)--, respectively. One function of the
linkers E and R may be to provide adequate flexibility in the
linkage between the peptide and the conjugated moiety Z. Typical
examples of E and R include bi-radicals of straight, branched
and/or cyclic C.sub.1-10alkane, C.sub.2-10alkene, C.sub.2-10alkyne,
C.sub.1-10heteroalkane, C.sub.2-10heteroalkene,
C.sub.2-10heteroalkyne, wherein one or more homocyclic aromatic
compound biradical or heterocyclic compound biradical may be
inserted. Particular examples of E and R include
##STR00007##
[0064] A need for modifying peptides may arise for any number of
reasons, and this is also reflected in the kind of compounds that
may be conjugated to peptides according to the methods of the
present invention. It may be desirable to conjugate peptides to
alter the physicochemical properties of the peptide, such as e.g.
to increase (or to decrease) solubility to modify the
bioavailability of therapeutic peptides. In another embodiment, it
may be desirable to modify the clearance rate in the body, e.g. by
conjugating compounds to the peptide which binds to plasma
proteins, such as e.g. albumin, or which increase the size of the
peptide to prevent or delay discharge through the kidneys. In
another embodiment, it may be desirable to conjugate a label to
facilitate analysis of the peptide. Examples of such label include
radioactive isotopes, fluorescent markers and enzyme substrates. In
still another embodiment, a compound is conjugated to a peptide to
facilitate isolation of the peptide. For example, a compound with a
specific affinity to a particular column material may be conjugated
to the peptide. It may also be desirable to modify the
immunogenecity of a peptide, e.g. by conjugating a peptide so as to
hide, mask or eclipse one or more immunogenic epitopes at the
peptide.
[0065] In particular, the methods of the present invention may be
used to decrease the clearance in order to increase the plasma
half-life of the modified peptide compared to the corresponding
un-modified peptide. The term "plasma half-life" is used in its
ordinary meaning, i.e. the time at which 50% of the biological
activity of the peptide is present in the plasma prior to being
cleared. Alternative terms include serum half-life, circulating
half-life, circulatory half-life, serum clearance, plasma
clearance, and clearance half-life.
[0066] The term "increased" used in connection with plasma
half-life is used to indicate that the half-life of the conjugated
peptide is significantly increased relative to the half-life of the
corresponding un-modified peptide. For instance, the half-life may
be increased by at least 25%, at least 50%, at least 100%, at least
150%, at least 200% or even at least 500%.
[0067] In one embodiment, the present invention relates to methods
of conjugating peptides as disclosed above, which further comprises
the step of measuring whether an increase in the plasma half-life
has been effected.
[0068] Particular examples of Z which gives rise to a decrease in
clearance of compounds of formula [a] in comparison with the
clearance of P include organic moieties, such as PEG or mPEG
radicals and amino derivatives thereof; straight, branched and/or
cyclic C.sub.1-22alkyl, C.sub.2-22alkenyl, C.sub.2-22alkynyl,
C.sub.1-22heteroalkyl, C.sub.2-22heteroalkenyl,
C.sub.2-22heteroalkynyl, wherein one or more homocyclic aromatic
compound biradical or heterocyclic compound biradical may be
inserted, and wherein said C.sub.1-C.sub.22 or C.sub.2-C.sub.22
radicals may optionally be substituted with one or more
substituents selected from hydroxyl, halogen, carboxyl, heteroaryl
and aryl, wherein said aryl or heteroaryl may optionally be further
substituted by one or more substituents selected from hydroxyl,
halogen, and carboxyl; steroid radicals; lipid radicals;
polysaccharide radicals, e.g. dextrans; polyamide radicals e.g.
polyamino acid radicals; PVP radicals; PVA radicals;
poly(1-3-dioxalane); poly(1,3,6-trioxane); ethylene/maleic
anhydride polymer; Cibacron dye stuffs, such as Cibacron Blue 3GA,
and polyamide chains of specified length, as disclosed in WO
00/12587, which is incorporated herein by reference.
[0069] Particular mentioning is made of C.sub.10-20alkyl, such as
C.sub.15 and C.sub.17, and benzophenone derivatives of the
formula
##STR00008##
[0070] The PEG conjugated to a peptide according to the present
invention may be of any molecular weight. In particular the
molecular weight may be between 500 and 100,000 Da, such as between
500 and 60,000 Da, such as between 1000 and 40,000 Da, such as
between 5000 and 40,000 Da. In particular, PEG with molecular
weights of 10000 Da, 20000 Da, 30000 Da or 40000 Da may be used in
the present invention.
[0071] In one embodiment, Z comprises one or more moieties that are
known to bind to plasma proteins, such as e.g. albumin. The ability
of a compound to bind to albumin may be determined as described in
J. Med. Chem., 43, 2000, 1986-1992, which is incorporated herein by
reference. In the present context, a compound is defined as binding
to albumin if Ru/Da is above 0.05, such as above 0.10, such as
above 0.12 or even above 0.15.
[0072] In another embodiment of the invention the albumin binding
moiety is a peptide, such as a peptide comprising less than 40
amino acid residues. A number of small peptides which are albumin
binding moieties are disclosed in J. Biol. Chem. 277, 38 (2002)
35035-35043, which is incorporated herein by reference.
[0073] Z may be branched so that Z comprises more than one of the
above mentioned labels or radicals.
[0074] Particular examples of compounds of the formula Y-E-Z
include
##STR00009##
[0075] wherein mPEG has a molecular weight of 20 kDa,
##STR00010##
[0076] wherein mPEG has a molecular weight of 20 kDa,
##STR00011##
[0077] wherein mPEG has a molecular weight of 20 kDa,
##STR00012##
[0078] wherein mPEG has a molecular weight of 20 kDa,
##STR00013##
[0079] wherein mPEG has a molecular weight of 20 kDa,
##STR00014##
[0080] wherein mPEG has a molecular weight of 20 kDa,
##STR00015##
[0081] wherein mPEG has a molecular weight of 20 kDa,
##STR00016##
[0082] wherein mPEG has a molecular weight of 20 kDa,
##STR00017##
[0083] wherein mPEG has a molecular weight of 20 kDa,
##STR00018##
[0084] wherein mPEG has a molecular weight of 20 kDa,
##STR00019##
[0085] wherein mPEG has a molecular weight of 20 kDa,
##STR00020##
[0086] wherein mPEG has a molecular weight of 20 kDa,
##STR00021##
[0087] wherein mPEG has a molecular weight of 20 kDa,
##STR00022##
[0088] wherein mPEG has a molecular weight of 20 kDa,
##STR00023##
[0089] wherein mPEG has a molecular weight of 20 kDa,
##STR00024##
[0090] wherein mPEG has a molecular weight of 20 kDa,
##STR00025##
[0091] wherein mPEG has a molecular weight of 20 kDa,
##STR00026##
[0092] wherein mPEG has a molecular weight of 20 kDa,
##STR00027##
[0093] wherein mPEG has a molecular weight of 20 kDa,
##STR00028##
[0094] wherein mPEG has a molecular weight of 20 kDa,
##STR00029##
[0095] wherein mPEG has a molecular weight of 20 kDa,
##STR00030##
[0096] wherein mPEG has a molecular weight of 20 kDa,
##STR00031##
[0097] wherein mPEG has a molecular weight of 20 kDa,
##STR00032##
[0098] wherein mPEG has a molecular weight of 20 kDa,
##STR00033##
[0099] wherein mPEG has a molecular weight of 20 kDa,
##STR00034##
[0100] wherein mPEG has a molecular weight of 20 kDa,
##STR00035##
[0101] wherein mPEG has a molecular weight of 10 kDa,
##STR00036##
[0102] wherein mPEG has a molecular weight of 10 kDa,
##STR00037##
[0103] wherein mPEG has a molecular weight of 10 kDa,
##STR00038##
[0104] wherein mPEG has a molecular weight of 10 kDa,
##STR00039##
[0105] wherein mPEG has a molecular weight of 10 kDa,
##STR00040##
[0106] wherein mPEG has a molecular weight of 10 kDa,
##STR00041##
[0107] wherein mPEG has a molecular weight of 10 kDa,
##STR00042##
[0108] wherein mPEG has a molecular weight of 10 kDa,
##STR00043##
[0109] wherein mPEG has a molecular weight of 10 kDa,
##STR00044##
[0110] wherein mPEG has a molecular weight of 10 kDa,
##STR00045##
[0111] wherein mPEG has a molecular weight of 10 kDa,
##STR00046##
[0112] wherein mPEG has a molecular weight of 10 kDa,
##STR00047##
[0113] wherein mPEG has a molecular weight of 10 kDa,
##STR00048##
[0114] wherein mPEG has a molecular weight of 10 kDa,
##STR00049##
[0115] wherein mPEG has a molecular weight of 10 kDa,
##STR00050##
[0116] wherein mPEG has a molecular weight of 10 kDa,
##STR00051##
[0117] wherein mPEG has a molecular weight of 10 kDa,
##STR00052##
[0118] wherein mPEG has a molecular weight of 10 kDa,
##STR00053##
[0119] wherein mPEG has a molecular weight of 10 kDa,
##STR00054##
[0120] wherein mPEG has a molecular weight of 10 kDa,
##STR00055##
[0121] wherein mPEG has a molecular weight of 10 kDa,
##STR00056##
[0122] wherein mPEG has a molecular weight of 10 kDa,
##STR00057##
[0123] wherein mPEG has a molecular weight of 10 kDa,
##STR00058##
[0124] wherein mPEG has a molecular weight of 10 kDa,
##STR00059##
[0125] wherein mPEG has a molecular weight of 10 kDa,
##STR00060##
[0126] wherein mPEG has a molecular weight of 10 kDa,
##STR00061##
[0127] wherein mPEG has a molecular weight of 10 kDa,
##STR00062## ##STR00063## ##STR00064##
[0128] wherein mPEG has a molecular weight of 20 kDa,
##STR00065##
[0129] wherein mPEG has a molecular weight of 20 kDa,
##STR00066##
[0130] wherein mPEG has a molecular weight of 20 kDa,
##STR00067##
[0131] wherein mPEG has a molecular weight of 20 kDa,
##STR00068##
[0132] wherein mPEG has a molecular weight of 20 kDa,
##STR00069##
[0133] wherein mPEG has a molecular weight of 20 kDa,
##STR00070##
[0134] wherein mPEG has a molecular weight of 20 kDa,
##STR00071##
[0135] wherein mPEG has a molecular weight of 20 kDa,
##STR00072##
[0136] wherein mPEG has a molecular weight of 20 kDa,
##STR00073##
[0137] wherein mPEG has a molecular weight of 20 kDa,
##STR00074##
[0138] wherein mPEG has a molecular weight of 30 kDa,
##STR00075##
[0139] wherein mPEG has a molecular weight of 30 kDa,
##STR00076##
[0140] wherein mPEG has a molecular weight of 30 kDa,
##STR00077##
[0141] wherein mPEG has a molecular weight of 30 kDa,
##STR00078##
[0142] wherein mPEG has a molecular weight of 30 kDa,
##STR00079##
[0143] wherein mPEG has a molecular weight of 30 kDa,
##STR00080##
[0144] wherein mPEG has a molecular weight of 30 kDa,
##STR00081##
[0145] wherein mPEG has a molecular weight of 30 kDa,
##STR00082##
[0146] wherein mPEG has a molecular weight of 30 kDa,
##STR00083##
[0147] wherein mPEG has a molecular weight of 30 kDa,
##STR00084##
[0148] wherein mPEG has a molecular weight of 20 kDa,
##STR00085##
[0149] wherein mPEG has a molecular weight of 20 kDa, and
##STR00086##
[0150] wherein mPEG has a molecular weight of 20 kDa.
[0151] As explained above, the catalytic action of
carboxypeptidases causes the C-terminal amino acid residue to be
exchanged with, e.g. the compound of the formula
##STR00087##
If it is desired to maintain the entire sequence of the peptide to
be conjugated it is thus necessary to elongate the sequence of the
peptide with one amino acid residue. Means for doing so are
well-known to persons skilled in the art, e.g. by recombinant
techniques or by protein synthetic methods. Another reason for
wanting to elongate the sequence of the peptide could be to make
the peptide a substrate for the particular carboxypeptidase at
hand. As explained earlier, the difference between
carboxypeptidases mainly resides with the kind of amino acid
residue they are able to cleave off. It may thus be necessary to
add one or more amino acid residue to make a given peptide a
substrate for a given carboxypeptidase. The added amino acid
residues may either be natural or unnatural.
[0152] It is recognised that some peptides, e.g. insulin and Factor
VII, comprise more than one chain, which in turn means that they
have more than one C-terminal. In some cases it might be possible
to distinguish between the C-terminals by a proper selection of the
carboxypeptidase used. In other cases it might be necessary to
introduce a difference between the C-terminals, e.g. by adding or
deleting one or more amino acid residues from one of the
C-terminals to effect a conjugation at only a limited number of the
C-terminals present. In still other cases it might be useful to
conjugate the peptide at all C-terminals.
[0153] Any peptide can be conjugated by the methods of the present
invention, such as e.g. enzymes, peptide hormones, growth factors,
antibodies, cytokines, receptors, lymphokines and vaccine
antigenes, and particular mentioning is made of therapeutic
peptides, such as insulin, glucagon like-peptide 1 (GLP-1),
glucagon like-peptide 2 (GLP-2), growth hormone, cytokines, trefoil
factor peptides (TFF), peptide melanocortin receptor modifiers and
factor VII compounds.
[0154] Particular applicable insulin is human insulin. In the
present context the term "human insulin" refers to naturally
produced insulin or recombinantly produced insulin. Recombinant
human insulin may be produced in any suitable host cell, for
example the host cells may be bacterial, fungal (including yeast),
insect, animal or plant cells. Many insulin compounds have been
disclosed in the literature, and they too are particular useful in
the methods of the present invention. By "insulin compound" (and
related expressions) is meant human insulin in which one or more
amino acids have been deleted and/or replaced by other amino acids,
including non-codeable amino acids, and/or human insulin comprising
additional amino acids, i.e. more than 51 amino acids, and/or human
insulin in which at least one organic substituent is bound to one
or more of the amino acids.
[0155] The following patent documents are mentioned as disclosures
of insulin compounds particular applicable in the methods provided
by the present invention.
[0156] WO 97/31022 (Novo Nordisk), which is incorporated herein by
reference, discloses insulin compounds with a protracted activity
profile wherein the amino group of the N-terminal amino acid of the
B-chain and/or the .epsilon.-amino group of Lys.sup.B29 has a
carboxylic acid containing lipophilic substituent. Particular
mentioning is made of
N.sup..epsilon.B29--(CO--(CH.sub.2).sub.14--COOH) human insulin;
N.sup..epsilon.B29--(CO--(CH.sub.2).sub.16--COOH) human insulin;
N.sup..epsilon.B29--(CO--(CH.sub.2).sub.18--COOH) human insulin;
N.sup..epsilon.B29--(CO--(CH.sub.2).sub.2O--COOH);
N.sup..epsilon.B29--(CO--(CH.sub.2).sub.22--COOH) human insulin;
N.sup..epsilon.B29--(CO--(CH.sub.2).sub.14--COOH) Asp.sup.B28-human
insulin; N.sup..epsilon.B29--(CO--(CH.sub.2).sub.16--COOH)
Asp.sup.B28-human insulin;
N.sup..epsilon.B29--(CO--(CH.sub.2).sub.18--COOH) Asp.sup.B28-human
insulin; N.sup..epsilon.B29--(CO--(CH.sub.2).sub.20--COOH)
Asp.sup.B28-human insulin;
N.sup..epsilon.B29--(CO--(CH.sub.2).sub.22--COOH) Asp.sup.B28-human
insulin; N.sup..epsilon.B30--(CO--(CH.sub.2).sub.14--COOH)
Thr.sup.B29Lys.sup.B30-human insulin;
N.sup..epsilon.B30--(CO--(CH.sub.2).sub.16--COOH)
Thr.sup.B29Lys.sup.B30-human insulin;
N.sup..epsilon.B30-(CO--(CH.sub.2).sub.18--COOH)
Thr.sup.B29Lys.sup.B30-human insulin;
N.sup.B30--(CO--(CH.sub.2).sub.20--COOH)
Thr.sup.B29Lys.sup.B30-human insulin;
N.sup..epsilon.B30--(CO--(CH.sub.2).sub.22--COOH)
Thr.sup.B29Lys.sup.B30-human insulin;
[0157] N.sup..epsilon.B28-(CO--(CH.sub.2).sub.14--COOH)
Lys.sup.B28Pro.sup.B29-human insulin; N.sup..epsilon.B28
(CO--(CH.sub.2).sub.16--COOH) Lys.sup.B28Pro.sup.B29-human insulin;
N.sup..epsilon.B28--(CO--(CH.sub.2).sub.18--COOH)
Lys.sup.B28Pro.sup.B29-human insulin;
N.sup..epsilon.B28--(CO--(CH.sub.2).sub.20--COOH)
Lys.sup.B28Pro.sup.B29-human insulin;
N.sup..epsilon.B28--(CO--(CH.sub.2).sub.22--COOH)
Lys.sup.B28Pro.sup.B29-human insulin;
N.sup..epsilon.B29--(CO--(CH.sub.2).sub.14--COOH) desB30 human
insulin; N.sup..epsilon.B29--(CO--(CH.sub.2).sub.16--COOH) desB30
human insulin; N.sup..epsilon.B29--(CO--(CH.sub.2).sub.18--COOH)
desB30 human insulin;
N.sup..epsilon.B29--(CO--(CH.sub.2).sub.20--COOH) desB30 human
insulin; and N.sup..epsilon.B29--(CO--(CH.sub.2).sub.22COOH) desB30
human insulin.
[0158] WO 96/29344 (Novo Nordisk), which is incorporated herein by
reference, discloses insulin compounds with a protracted activity
profile wherein either the amino group of the N-terminal amino acid
of the B-chain has a lipophilic substituent comprising from 12 to
40 carbon atoms attached, or wherein the carboxylic acid group of
the C-terminal amino acid of the B-chain has a lipophilic
substituent comprising from 12 to 40 carbon atoms attached.
[0159] WO 95/07931 (Novo Nordisk), which is incorporated herein by
reference, discloses insulin compounds with a protracted activity
profile, wherein the .epsilon.-amino group of Lys.sup.B29 has a
lipophilic substituent. Particular mentioning is made of
N.sup..epsilon.B29-tridecanoyl des(B30) human insulin,
N.sup..epsilon.B29-tetradecanoyl des(B30) human insulin,
N.sup..epsilon.B29-decanoyl des(B30) human insulin,
N.sup..epsilon.B29-dodecanoyl des(B30) human insulin,
N.sup..epsilon.B29-tridecanoyl Gly.sup.A21 des(B30) human insulin,
N.sup..epsilon.B29-tetradecanoyl Gly.sup.A21 des(B30) human
insulin, N.sup..epsilon.B29-decanoyl Gly.sup.A21 des(B30) human
insulin, N.sup..epsilon.B29-dodecanoyl Gly.sup.A21 des(B30) human
insulin, N.sup..epsilon.B29-tridecanoyl Gly.sup.A21 Gln.sup.B3
des(B30) human insulin, N.sup..epsilon.B29-tetradecanoyl
Gly.sup.A21 Gln.sup.B3 des(B30) human insulin,
N.sup..epsilon.B29-decanoyl Gly.sup.A21 Gln.sup.B3 des(B30) human
insulin, N.sup..epsilon.B29-dodecanoyl Gly.sup.A21 Gln.sup.B3
des(B30) human insulin, N.sup..epsilon.B29-tridecanoyl Ala.sup.A21
des(B30) human insulin, N.sup..epsilon.B29-tetradecanoyl
Ala.sup.A21 des(B30) human insulin, N.sup..epsilon.B29-decanoyl
Ala.sup.A21 des(B30) human insulin, N.sup..epsilon.B29-dodecanoyl
Ala.sup.A21 des(B30) human insulin, N.sup..epsilon.B29-tridecanoyl
Ala.sup.A21 Gln.sup.B3 des(B30) human insulin,
N.sup..epsilon.B29-tetradecanoyl Ala.sup.A21 Gln.sup.B3 des(B30)
human insulin, N.sup..epsilon.B29-decanoyl Ala.sup.A21 Gln.sup.B3
des(B30) human insulin, N.sup..epsilon.B29-dodecanoyl Ala.sup.A21
Gln.sup.B3 des(B30) human insulin, N.sup..epsilon.B29-tridecanoyl
Gln.sup.B3 des(B30) human insulin, N.sup..epsilon.B29-tetradecanoyl
Gln.sup.B3 des(B30) human insulin, N.sup..epsilon.B29-decanoyl
Gln.sup.B3 des(B30) human insulin, N.sup..epsilon.B29-dodecanoyl
Gln.sup.B3 des(B30) human insulin, N.sup..epsilon.B29-tridecanoyl
Gly.sup.A21 human insulin, N.sup..epsilon.B29-tetradecanoyl
Gly.sup.A21 human insulin, N.sup..epsilon.B29-decanoyl Gly.sup.A21
human insulin, N.sup..epsilon.B29-dodecanoyl Gly.sup.A21 human
insulin, N.sup..epsilon.B29-tridecanoyl Gly.sup.A21 Gln.sup.B3
human insulin, N.sup..epsilon.B29-tetradecanoyl Gly.sup.A21
Gln.sup.B3 human insulin, N.sup..epsilon.B29-decanoyl Gly.sup.A21
Gln.sup.B3 human insulin, N.sup..epsilon.B29-dodecanoyl Gly.sup.A21
Gln.sup.B3 human insulin, N.sup..epsilon.B29-tridecanoyl
Ala.sup.A21 human insulin, N.sup..epsilon.B29-tetradecanoyl
Ala.sup.A21 human insulin, N.sup..epsilon.B29-decanoyl Ala.sup.A21
human insulin, N.sup..epsilon.B29-dodecanoyl Ala.sup.A21 human
insulin, N.sup..epsilon.B29-tridecanoyl Ala.sup.A21 Gln.sup.B3
human insulin, N.sup..epsilon.B29-tetradecanoyl Ala.sup.A21
Gln.sup.B3 human insulin, N.sup.29-decanoyl Ala.sup.A21 Gln.sup.B3
human insulin, N.sup..epsilon.B29-dodecanoyl Ala.sup.A21 Gln.sup.B3
human insulin, N.sup..epsilon.B29-tridecanoyl Gln.sup.B3 human
insulin, N.sup..epsilon.B29-tetradecanoyl Gln.sup.B3 human insulin,
N.sup..epsilon.B29-decanoyl Gln.sup.B3 human insulin,
N.sup..epsilon.B29-dodecanoyl Gln.sup.B3 human insulin,
N.sup..epsilon.B29-tridecanoyl Glu.sup.B30 human insulin,
N.sup..epsilon.B29-tetradecanoyl Glu.sup.B30 human insulin,
N.sup..epsilon.B29-decanoyl Glu.sup.B30 human insulin,
N.sup..epsilon.B29-dodecanoyl Glu.sup.B30 human insulin,
N.sup..epsilon.B29-tridecanoyl Gly.sup.A21 Glu.sup.B30 human
insulin, N.sup..epsilon.B29-tetradecanoyl Gly.sup.A21 Glu.sup.B30
human insulin, N.sup..epsilon.B29-decanoyl Gly.sup.A21 Glu.sup.B30
human insulin, N.sup..epsilon.B29-dodecanoyl Gly.sup.A21
Glu.sup.B30 human insulin, N.sup..epsilon.B29-tridecanoyl
Gly.sup.A21 Gln.sup.B3 Glu.sup.B30 human insulin,
N.sup..epsilon.B29-tetradecanoyl Gly.sup.A21 Gln.sup.B3 Glu.sup.B30
human insulin, N.sup..epsilon.B29-decanoyl Gly.sup.A21 Gln.sup.B3
Glu.sup.B30 human insulin, N.sup..epsilon.B29-dodecanoyl
Gly.sup.A21 Gln.sup.B3 Glu.sup.B30 human insulin,
N.sup..epsilon.B29-tridecanoyl Ala.sup.A21 Glu.sup.B30 human
insulin, N.sup..epsilon.B29-tetradecanoyl Ala.sup.A21 Glu.sup.B30
human insulin, N.sup..epsilon.B29-decanoyl Ala.sup.A21 Glu.sup.B30
human insulin, N.sup..epsilon.B29-dodecanoyl Ala.sup.A21
Glu.sup.B30 human insulin, N.sup..epsilon.B29-tridecanoyl
Ala.sup.A21 Gln.sup.B3 Glu.sup.B30 human insulin,
N.sup..epsilon.B22-tetradecanoyl Ala.sup.A21 Gln.sup.B3 Glu.sup.B30
human insulin, N.sup..epsilon.B21-decanoyl Ala.sup.1 Gln.sup.B3
Glu.sup.B30 human insulin, N.sup..epsilon.B2-dodecanoyl Ala.sup.A21
Gln.sup.B3 Glu.sup.B30 human insulin,
N.sup..epsilon.B29-tridecanoyl Gln.sup.B3 Glu.sup.B30 human
insulin, N.sup..epsilon.B29-tetradecanoyl Gln.sup.B3 Glu.sup.B30
human insulin, N.sup..epsilon.B29-decanoyl Gln.sup.B3 Glu.sup.B30
human insulin and N.sup..epsilon.B29-dodecanoyl Gln.sup.B3
Glu.sup.B30 human insulin.
[0160] WO 97/02043 (Novo Nordisk), which is incorporated herein by
reference discloses hormonally inactive insulin compounds which are
useful in insulin prophylaxis, and in particular such analogues of
human insulin are selected from amongst desA1 human insulin;
des(A1-A2) human insulin; des(A1-A3) human insulin; desA21 human
insulin; des(B1-B5) human insulin; des(B1-B6) human insulin;
des(B23-B30) human insulin; des(B24-B30) human insulin;
des(B25-B30) human insulin; Gly.sup.A2 human insulin; Ala.sup.A2
human insulin; Nle.sup.A2 human insulin; Thr.sup.A2 human insulin;
Pro.sup.A2 human insulin; D-allo Ile.sup.A2 human insulin;
Nva.sup.A3 human insulin; Nle.sup.A3 human insulin; Leu.sup.A3
human insulin; Val.sup.A2,Ile.sup.A3 human insulin;
Abu.sup.A2,Abu.sup.A3 human insulin; Gly.sup.A2,Gly.sup.A3 human
insulin; D-Cys.sup.A6 human insulin; D-Cys.sup.A6,D-Cys.sup.A11
human insulin; Ser.sup.A6,Ser.sup.A11, des(A8-A10) human insulin;
D-Cys.sup.A7 human insulin; D-Cys.sup.A11 human insulin;
Leu.sup.A19 human insulin; Gly.sup.B6 human insulin; Glu.sup.B12
human insulin; Asn.sup.B12 human insulin; PheB12 human insulin;
D-Ala.sup.B12 human insulin; and Asp.sup.B25 human insulin are
applicable in the methods of the present invention.
[0161] WO 92/15611 (Novo nordisk), which is incorporated herein by
reference, discloses analogues of human insulin with a fast
association rate constants in the insulin receptor binding process
and characterised by comprising a tyrosine in position A13 and/or a
phenylalanin, tryptophane or tyrosine in position B17. In
particular, such analogues are selected from amongst Tyr.sup.A13
human insulin, Phe.sup.B17 human insulin, Trp.sup.B17 human
insulin, Tyr.sup.B17 human insulin, Tyr.sup.A13,Phe.sup.B17 human
insulin, Tyr.sup.A13, Trp.sup.B17 human insulin, Tyr.sup.A13,
Tyr.sup.B17 human insulin, Phe.sup.13,Phe.sup.B17 human insulin,
Phe.sup.A13,Trp.sup.B17 human insulin, Phe.sup.A13,Tyr.sup.B17
human insulin, Trp.sup.A13,Phe.sup.B17 human insulin,
Trp.sup.A13,Trp.sup.B17 human insulin and Trp.sup.A13,Tyr.sup.B17
human insulin.
[0162] WO 92/00322 (Novo Nordisk), which is incorporated herein by
reference, discloses analogues of human insulin which are capable
of being targeted to specific tissues, and which are characterized
by having in the A13 position and/or in the B17 position in the
insulin molecule a naturally occurring amino acid residue different
from leucine and/or by having in the B18 position in the insulin
molecule a naturally occurring amino acid residue different from
valine. In particular, such analogues are selected from amongst
Ala.sup.B17 human insulin, Ala.sup.B18 human insulin, Asn.sup.A13
human insulin, Asn.sup.A13,Ala.sup.B17human insulin,
Asn.sup.A13,Asp.sup.B17 human insulin, Asn.sup.B13,Glu.sup.B17
human insulin, Asn.sup.B18 human insulin, Asp.sup.A13 human
insulin, Asp.sup.A13,Ala.sup.B17 human insulin,
Asp.sup.A13,Asp.sup.B17 human insulin, Asp.sup.A13,Glu.sup.B17
human insulin, Asp.sup.B18 human insulin, Gln.sup.A13 human
insulin, Gln.sup.A13,Ala.sup.B17 human insulin,
Gln.sup.A13,Asp.sup.B17 human insulin, Gln.sup.B18 human insulin,
Glu.sup.A13 human insulin, Glu.sup.A13,Ala.sup.B17 human insulin,
Glu.sup.A13,Asp.sup.B17 human insulin, Glu.sup.A13, Glu.sup.B17
human insulin, Glu.sup.B18 human insulin, Gly.sup.A13 human
insulin, Gly.sup.A13,Ala.sup.B17 human insulin,
Gly.sup.A13,Asn.sup.B17 human insulin, Gly.sup.A13,Asp.sup.B17
human insulin, Gly.sup.A13,Glu.sup.B17 human insulin, Gly.sup.B18
human insulin, Ser.sup.A13 human insulin,
Ser.sup.A13,Gln.sup.A17,Glu.sup.B10,Gln.sup.B17-des(Thr.sup.B30)
human insulin, Ser.sup.A13,Ala.sup.B17 human insulin,
Ser.sup.A13,Asn.sup.B17 human insulin, Ser.sup.A13,Asp.sup.B17
human insulin, Ser.sup.A13,Gln.sup.B17 human insulin,
Ser.sup.A13,Glu.sup.B17 human insulin, Ser.sup.A13,Thr.sup.B17
human insulin, Ser.sup.B14,Asp.sup.B17 human insulin, Ser.sup.B18
human insulin, Thr.sup.A13 human insulin or Thr.sup.B18 human
insulin.
[0163] WO 90/01038 (Novo Nordisk), which is incorporated herein by
reference, discloses analogues of human insulin with high
biological activity and characterized by having Phe.sup.B25
substituted by His or Tyr, by having substitutions in one or more
of positions A4, A8, A17, A21, B9, B10, B12, B13, B21, B26, B27,
B28 and B30, and by having the amino acid residue at position B30
optionally absent. In particular, such analogues are selected from
amongst Tyr.sup.B25 human insulin, Tyr.sup.B25, Asp.sup.B28 human
insulin, His.sup.B25 human insulin, His.sup.B25, Asp.sup.B21 human
insulin, Tyr.sup.B25 human insulin-B30-amide and His.sup.B25 human
insulin-B30-amide.
[0164] WO 86/05496 (Nordisk Gentofte) discloses analogues of human
insulin with a protracted action and characterized by having a
blocked B30 carboxylic group, and by having one to four blocked
carboxylic groups in the amino acid residues at positions A4, A17,
A21, B13 and B21. In particular, such analogues are selected from
amongst insulin-B30-octyl ester, insulin-B30-dodecyl amide,
insulin-B30-hexadecyl amide, insulin-(B21,B30)-dimethyl ester,
insulin-(B17,B30)-dimethyl ester, insulin-(A4,B30) diamide,
insulin-A17amide-B30-octyl ester,
insulin-(A4,B13)-diamide-B30-hexylamide,
insulin-(A4,A17,B21,B30)-tetraamide, insulin-(A17,B30)-diamide,
A4-Ala-insulin-B30-amide and B30-Leu-insulin-(A4,B30)-diamide.
[0165] WO 86/05497 (Nordisk Gentofte), which is incorporated herein
by reference, discloses insulin compounds in which one or more of
the four amino acid residues in positions A4, A17, B13 and B21
comprises an uncharged side chain. Particular mentioning is made of
human insulin A17-Gln, human insulin A4-Gln, porcine insulin
B21-Gln, human insulin B13-Gln, human insulin (A17,B21)-Gln, human
insulin A4-Ala, human insulin B21-Thr, human insulin B13-Val, human
insulin-Thr-A17-Gln, human insulin B21-methyl ester and human
insulin A17-methyl ester.
[0166] WO 92/00321 (Novo Nordisk), which is incorporated herein by
reference, discloses insulin compounds with prolonged activity
wherein a positive charge in the N-terminal end of the B-chain has
been introduced. Particular mentioning is made of
Arg.sup.B5,Ser.sup.A21,Thr.sup.B30-NH.sub.2 human insulin,
Arg.sup.B5,Pro.sup.B6,Ser.sup.A21,Thr.sup.B30-NH.sub.2 human
insulin, Arg.sup.B5,Gly.sup.A21,Thr.sup.B30-NH.sub.2 human insulin,
Arg.sup.B5,Pro.sup.B6,Gly.sup.A21,Thr.sup.B30-NH.sub.2 human
insulin, Arg.sup.B2,Ser.sup.A21,Thr.sup.B30-NH.sub.2 human insulin,
Arg.sup.B2,Pro.sup.B3,Ser.sup.A21,Thr.sup.B30-NH.sub.2 human
insulin, Arg.sup.B2,Gly.sup.A21, Thr.sup.B30-NH.sub.2 human
insulin, Arg.sup.B2,Pro.sup.B3,Gly.sup.A21,Thr.sup.B30-NH.sub.2
human insulin,
Arg.sup.B2,Arg.sup.B3,Ser.sup.A21,Thr.sup.B30-NH.sub.2 human
insulin, Arg.sup.B2,Arg.sup.B3,Ser.sup.A21 human insulin,
Arg.sup.B4,Pro.sup.B5,Ser.sup.A21,Thr.sup.B30-NH.sub.2 human
insulin, Arg.sup.B4,Arg.sup.B5,Pro.sup.B6,Gly.sup.A2,Thr.sup.B30
human insulin, Arg.sup.B3,Gly.sup.A21,Thr.sup.B30-NH.sub.2 human
insulin, Arg.sup.B3,Ser.sup.A21,Thr.sup.B30-NH.sub.2 human insulin,
Arg.sup.B4,Gly.sup.A21,Thr.sup.B30-NH.sub.2 human insulin,
Arg.sup.B4,Ser.sup.A21,Thr.sup.B30-NH.sub.2 human insulin and
Arg.sup.B1,Pro.sup.B2,Gly.sup.A21,Thr.sup.B30-NH.sub.2 human
insulin.
[0167] WO 90/07522 (Novo Nordisk), which is incorporated herein by
reference, discloses insulin compounds exhibiting a low ability to
associate in solution wherein there is a positively charged amino
acid residue, i.e. Lys or Arg in the position B28. Particular
mentioning is made of des[Phe.sup.B25]-human insulin,
des[Tyr.sup.B26]-human insulin, des[Thr.sup.B27]-human insulin,
des[Pro.sup.B28]-human insulin, des[Phe.sup.B25]-porcine insulin,
des[Pro.sup.B28]-porcine insulin, des[Pro.sup.B28]-rabbit insulin,
des[Phe.sup.B25],des[Thr.sup.B30]-human insulin,
des[Tyr.sup.B26],des[Thr.sup.B30]-human insulin,
[Ser.sup.A21]-des[Pro.sup.B28]-human insulin,
[Gly.sup.A21]-des[Pro.sup.B28]-human insulin,
[Gly.sup.A21]-des[Phe.sup.B25]-human insulin,
[Asp.sup.A21]-des[Phe.sup.B25]-human insulin,
[His.sup.B25]-des[Tyr.sup.B26],des[Thr.sup.B30]-human insulin,
[Asn.sup.B25]-des[Tyr.sup.B26],des[Thr.sup.B30]-human insulin,
[Asp.sup.A21]-des[Phe.sup.B25],des[Thr.sup.B30]-human insulin,
[Asp.sup.B28]-des[Phe.sup.B25]-human insulin,
[Asp.sup.B30]-des[Phe.sup.B25]-human insulin, [Lys.sup.B28-human
insulin, [Lys.sup.B28,Thr.sup.B29]-human insulin and
[Arg.sup.B28]-des[Lys.sup.B29]-human insulin.
[0168] WO 90/11290 (Novo Nordisk), which is incorporated herein by
reference discloses insulin compounds with a prolonged activity.
Particular mentioning is made of [Arg.sup.A0]-human
insulin-(B30-amide), [Arg.sup.A0,Gln.sup.B13]-human
insulin-(B30-amide), [Arg.sup.A0,Gln.sup.A4,Asp.sup.A21]-human
insulin-(B30-amide), [Arg.sup.A0,Ser.sup.A21]-human
insulin-(B30-amide) and
[Arg.sup.A0,Arg.sup.B27]-des[Thr.sup.B30]-human insulin.
[0169] WO 90/10645 (Novo Nordisk), which is incorporated herein by
reference discloses glycosylated insulins. Particular mentioning is
made of Phe(B1) glucose human insulin, Phe(B1) mannose human
insulin, Gly(A1) mannose human insulin, Lys(B29) mannose human
insulin, Phe(B1) galactose human insulin, Gly(A1) galactose human
insulin, Lys(B29) galactose human insulin, Phe(B1) maltose human
insulin, Phe(B1) lactose human insulin, Gly(A1) glucose human
insulin, Gly(A1) maltose human insulin, Gly(A1) lactose human
insulin, Lys(B29) glucose human insulin, Lys(B29) maltose human
insulin, Lys(B29) lactose human insulin, Gly(A1), Phe(B1) diglucose
human insulin, Gly(A1), Lys(B29) diglucose human insulin, Phe(B1),
Lys(B29) diglucose human insulin, Phe(B1) isomaltose human insulin,
Gly(A1) isomaltose human insulin, Lys(B29) isomaltose human
insulin, Phe(B1) maltotriose human insulin, Gly(A1) maltotriose
human insulin, Lys(B29) maltotriose human insulin, Gly(A1), Phe(B1)
dimaltose human insulin, Gly(A1), Lys(B29) dimaltose human insulin,
Phe(B1), Lys(B29) dimaltose human insulin, Gly(A1), Phe(B1)
dilactose human insulin, Gly(A1), Lys(B29) dilactose human insulin,
Phe(B1), Lys(B29) dilactose human insulin, Gly(A1), Phe(B1)
dimaltotriose human insulin, Gly(A1), Lys(B29) dimaltotriose human
insulin, Phe(B1), Lys(B29) dimaltotriose human insulin, Phe(B1),
Gly(A1) dimannose human insulin, Phe(B1), Lys(B29) dimannose human
insulin, Gly(A1), Lys(B29) dimannose human insulin, Phe(B1),
Gly(A1) digalactose human insulin, Phe(B1), Lys(B29) digalactose
human insulin, Gly(A1), Lys(B29) digalactose human insulin,
Phe(B1), Gly(A1) diisomaltose human insulin, Phe(B1), Lys(B29)
diisomaltose human insulin, Gly(A1), Lys(B29) diisomaltose human
insulin, Phe(B1) glucose [Asp.sup.B10] human insulin and Gly(A1),
Phe(B1) diglucose [Asp.sup.B10] human insulin.
[0170] WO 88/065999 (Novo Nordisk), which is incorporated herein by
reference, discloses stabilized insulin compounds, wherein
Ans.sup.21A has been substituted with other amino acid residues.
Particular mentioning is made of Gly.sup.A21 human insulin,
Ala.sup.A21 human insulin, Ser.sup.A21 human insulin, Thr.sup.A21
human insulin and hSer.sup.A21 human insulin.
[0171] EP 254516 (Novo Nordisk), which is incorporated herein by
reference, discloses insulin compounds with a prolonged action,
wherein basic amino acid residues have been substituted by neutral
amino acid residues. Particular mentioning is made of
Gly.sup.A21,Lys.sup.B27,Thr.sup.B30-NH.sub.2 human insulin,
Ser.sup.A21, Lys.sup.B27,Thr.sup.B30-NH.sub.2 human insulin,
Thr.sup.A21,Lys.sup.B27,Thr.sup.B30-NH.sub.2human insulin,
Ala.sup.B21,Lys.sup.B27,Thr.sup.B30-NH.sub.2human insulin,
His.sup.A21,Lys.sup.B27,Thr.sup.B30-NH.sub.2human insulin,
Asp.sup.B21,Lys.sup.B27,Thr.sup.B30-NH.sub.2human Insulin,
Gly.sup.A21,Arg.sup.B21,Thr.sup.B30-NH.sub.2 human insulin,
Ser.sup.A21,Arg.sup.B27,Thr.sup.B30-NH.sub.2 human insulin,
Thr.sup.A21,Arg.sup.B27,Thr.sup.B30-NH.sub.2 human insulin,
Ala.sup.B21,Arg.sup.B27,Thr.sup.B30-NH.sub.2human insulin,
His.sup.A21,Arg.sup.B27,Thr.sup.B30-NH.sub.2 human insulin,
Asp.sup.B21,Arg.sup.B27,Thr.sup.B30-NH.sub.2 human insulin,
Gln.sup.B13,Gly.sup.A21,Arg.sup.B27,Thr.sup.B30-NH.sub.2 human
insulin, Gln.sup.B13,Ser.sup.A21,Thr.sup.B30-NH.sub.2 human
insulin,
Gln.sup.B13,Ser.sup.A21,Arg.sup.B27,Thr.sup.B30-NH.sub.2human
insulin,
Gln.sup.B13,Thr.sup.A21,Arg.sup.B27,Thr.sup.B30-NH.sub.2human
insulin, Gln.sup.B13,Ala.sup.A21,Arg.sup.B27,Thr.sup.B30-NH.sub.2
human insulin,
Gln.sup.B13,His.sup.A21,Arg.sup.B27,Thr.sup.B30-NH.sub.2human
insulin, Gln.sup.B13,Asp.sup.A21,Arg.sup.B27,Thr.sup.B30-NH.sub.2
human insulin, Gln.sup.B13,Gly.sup.A21,Lys.sup.B27,Thr.sup.B30-NH
human insulin,
Gln.sup.B13,Ser.sup.A21,Lys.sup.B27,Thr.sup.B30-NH.sub.2 human
insulin,
Gln.sup.B13-Thr.sup.A21,Lys.sup.B27,Thr.sup.B30-NH.sub.2human
insulin, Gln.sup.B13,Ala.sup.A21,Lys.sup.B27, Thr.sup.B30-NH.sub.2
human insulin, Gly.sup.A21,Arg.sup.B27 human insulin,
Gln.sup.B13,Asp.sup.A21,Lys.sup.B27,Thr.sup.B30-NH.sub.2 human
insulin, Asn.sup.A21,Lys.sup.B27 human insulin,
Ser.sup.A21,Lys.sup.B27 human insulin, Thr.sup.A21,Lys.sup.B27
human insulin, Ala.sup.A21,Lys.sup.B27 human insulin,
His.sup.A21,Lys.sup.B27 human insulin, Asp.sup.A21,Lys.sup.B27
human insulin, Gly.sup.A21,Lys.sup.B27 human insulin,
Asn.sup.A21,Arg.sup.B27 human insulin, Ser.sup.A21,Arg.sup.B27
human insulin, Thr.sup.A21,Arg.sup.B27 human insulin,
Ala.sup.A21,Arg.sup.B27 human insulin, His.sup.A21, Arg.sup.B27
human insulin, Asp.sup.A21,Arg.sup.B27 human insulin,
Gly.sup.A21,Arg.sup.B27 human insulin,
Gln.sup.A17,Asn.sup.A21,Arg.sup.B27 human insulin,
Gln.sup.A17,Ser.sup.A21,Arg.sup.B27 human insulin,
Gln.sup.A17,Thr.sup.A21,Arg.sup.B27human insulin,
Gln.sup.A17,Ala.sup.A21,Arg.sup.B27 human insulin,
Gln.sup.A17,His.sup.A21,Arg.sup.B27 human insulin,
Gln.sup.A17,Ala.sup.A21,Arg.sup.B27 human insulin, Gln.sup.A17,
Gly.sup.A21,Arg.sup.B27 human insulin,
Gln.sup.A17,Asn.sup.A21,Gln.sup.B13 human insulin,
Gln.sup.A17,Ser.sup.A21,Gln.sup.B13 human insulin,
Gln.sup.A17,Thr.sup.A21,Gln.sup.B13 human insulin,
Gln.sup.A17,Ala.sup.A21,Gln.sup.B13 human insulin,
Gln.sup.A17,His.sup.A21,Gln.sup.B13 human insulin,
Gln.sup.A17,Asp.sup.A21,Gln.sup.B13 human insulin,
Gln.sup.A17,Gly.sup.A21,Gln.sup.B13human insulin,
Arg.sup.A27,Asn.sup.A21,Gln.sup.B13 human insulin,
Arg.sup.A27,Ser.sup.A21,Gln.sup.B13 human insulin,
Arg.sup.A27,Thr.sup.A21,Gln.sup.B13 human insulin,
Arg.sup.A27,Ala.sup.A21,Gln.sup.B13 human insulin,
Arg.sup.A27,His.sup.A21,Gln.sup.B13 human insulin,
Arg.sup.A27,Asp.sup.A21,Gln.sup.B13 human insulin,
Arg.sup.A27,Gly.sup.A21,Gln.sup.B13 human insulin,
Gln.sup.A17,Asn.sup.A21,Lys.sup.B27 human insulin,
Gln.sup.A17,Ser.sup.A21,Lys.sup.B27 human insulin,
Gln.sup.A17,Thr.sup.A21,Lys.sup.B27 human insulin,
Gln.sup.A17,Ala.sup.A21,Lys.sup.B27 human insulin,
Gln.sup.A17,His.sup.A21,Lys.sup.B27 human insulin,
Gln.sup.A17,Asp.sup.A21,Lys.sup.B27human insulin,
Gln.sup.A17,Gly.sup.A21,Lys.sup.B27 human insulin,
Gln.sup.B13,Asn.sup.A21,Lys.sup.B27human insulin, Gln.sup.B13
Ser.sup.A21,Lys.sup.B27human insulin,
Gln.sup.B13,Thr.sup.A21,Lys.sup.B27 human insulin,
Gln.sup.B13,Ala.sup.A21,Lys.sup.B27 human insulin,
Gln.sup.B13,His.sup.A2,Lys.sup.B27 human insulin,
Gln.sup.B13,Asp.sup.A21,Lys.sup.B27human insulin, and
Gln.sup.B13,Gly.sup.A21,Lys.sup.B27human insulin.
[0172] EP 214826 (Novo Nordisk), which is incorporated herein by
reference, discloses rapid onset insulin compounds.
[0173] EP 194864 (Novo Nordisk), which is incorporated herein by
reference, discloses insulin compounds with a prolonged action,
wherein basic amino acid residues have been substituted by neutral
amino acid residues. Particular mentioning is made of
Gln.sup.A17,Arg.sup.B27,Thr.sup.B31-NH.sub.2 human insulin,
Gln.sup.A17,Gln.sup.B13,Thr.sup.B30-NH.sub.2 human insulin,
Gln.sup.A17,Lys.sup.B27,Thr.sup.B30--NH.sub.2 human insulin,
Gln.sup.A17,Lys.sup.B27-NH.sub.2 human insulin, Gln.sup.A17,
Gln.sup.A17,Thr.sup.B30-NH.sub.2 human insulin,
Gln.sup.B13,Arg.sup.B27,Thr.sup.B30-NH.sub.2 human insulin,
Gln.sup.B13,Lys.sup.B27,Thr.sup.B30-NH.sub.2 human insulin,
Gln.sup.B13,Lys.sup.B30-NH.sub.2 human insulin,
Gln.sup.B13,Thr.sup.B30-NH.sub.2 human insulin,
Arg.sup.B27,Arg.sup.B30-NH.sub.2 human insulin,
Arg.sup.B27,Lys.sup.B30-NH.sub.2 human insulin,
Arg.sup.B27,Thr.sup.B30-NH.sub.2 human insulin,
Lys.sup.B27,Arg.sup.B30-NH.sub.2 human insulin,
Lys.sup.B27,Lys.sup.B30-NH.sub.2 human insulin,
Lys.sup.B27,Thr.sup.B30-NH.sub.2 human insulin,
Lys.sup.B29-NH.sub.2,des-(B30) human insulin, Thr.sup.B30-NH.sub.2
human insulin, Lys.sup.B30-NH.sub.2 human insulin,
Lys.sup.B30(Lau)-NH.sub.2 human insulin,
Lys.sup.B30,Arg.sup.B31-NH.sub.2 human insulin,
Lys.sup.B30,Lys.sup.B31-NH.sub.2 human insulin,
Arg.sup.B30-NH.sub.2 human insulin,
Arg.sup.B30,Arg.sup.B31-NH.sub.2 human insulin, and
Arg.sup.B30,Lys.sup.B31-NH.sub.2 human insulin.
[0174] U.S. Pat. No. 3,528,960 (Eli Lilly), which is incorporated
herein by reference, discloses N-carboxyaroyl insulin compounds in
which one, two or three primary amino groups of the insulin
molecule has a carboxyaroyl group.
[0175] GB Patent No. 1.492.997 (Nat. Res. Dev. Corp.), which is
incorporated herein by reference, discloses insulin compounds with
a carbamyl substitution at N.sup..epsilon.B29 with an improved
profile of hypoglycaemic effect.
[0176] JP laid-open patent application No. 1-254699 (Kodama Co.,
Ltd.), which is incorporated herein by reference, discloses insulin
compounds, wherein an alkanoyl group is bound to the amino group of
Phe.sup.B1 or to the .epsilon.-amino group of Lys.sup.B29 or to
both of these.
[0177] JP laid-open patent application No. 57-067548 (Shionogi),
which is incorporated herein by reference discloses insulin
compounds, in which the B30 position have an amino acid having at
least five carbon atoms which cannot necessarily be coded for by a
triplet of nucleotides.
[0178] WO 03/053339 (Eli Lilly), which is incorporated herein by
reference, disclose insulin compounds, wherein the A-chain in the
N-terminal has been extended with two amino acid residues, A-1 and
A0, wherein the B-chain has been extended at the N-terminal with
two amino acid residues, B-1 and B0, wherein the amino acid
residues at positions B28, B29 and B39 may be substituted, and
wherein the .epsilon.-amino group of Lys at position B28 or B29 is
covalently bound to the .alpha.-carboxyl group of a positively
charged amino acid to form a Lys-N.epsilon.-aminoacid derivative.
Particular mentioning is made of said analogues, wherein A-1 and
B-1 are both absent, and wherein A0 represent Arg and B0 represents
Arg or is absent.
[0179] Insulin compounds selected from the group consisting of
[0180] i. An analogue wherein position B28 is Asp, Lys, Leu, Val,
or Ala and position B29 is Lys or Pro; and [0181] ii. des(B28-B30),
des(B27) or des(B30) human insulin. are also applicable for the
methods of the present invention, and in particular, the insulin
compound wherein position B28 is Asp or Lys, and position B29 is
Lys or Pro.
[0182] des(B30) human insulin is also applicable in the methods of
the present invention.
[0183] Other applicable insulin compounds are selected from the
group consisting of B29-N.sup..epsilon.-myristoyl-des(B30) human
insulin, B29-N.sup..epsilon.-palmitoyl-des(B30) human insulin,
B29-N.sup..epsilon.-myristoyl human insulin,
B29-N.sup..epsilon.-palmitoyl human insulin,
B28-N.sup..epsilon.-myristoyl Lys.sup.B28Pro.sup.B29 human insulin,
B28-N.sup..epsilon.-palmitoyl Lys.sup.B28Pro.sup.B29 human insulin,
B30-N.sup..epsilon.-myristoyl-Thr.sup.B29Lys.sup.B30 human insulin,
B30-N.sup..epsilon.-palmitoyl-Thr.sup.B29Lys.sup.B30 human insulin,
B29-N.sup..epsilon.-(N-palmitoyl-.gamma.-glutamyl)-des(B30) human
insulin,
B29-N.sup..epsilon.-(N-lithocholyl-.gamma.-glutamyl)-des(B30) human
insulin,
B29-N.sup..epsilon.-(.omega.-carboxyheptadecanoyl)-des(B30) human
insulin, B29-N.sup..epsilon.-(o-carboxyheptadecanoyl) human insulin
and B29-N.sup..epsilon.-myristoyl-des(B30) human insulin.
[0184] Examples of GLP-1 applicable in the methods of the present
invention include human GLP-1 and GLP-1 compounds. Human GLP-1 is a
37 amino acid residue peptide originating from preproglucagon which
is synthesised i.a. in the L-cells in the distal ileum, in the
pancreas and in the brain. GLP-1 is an important gut hormone with
regulatory function in glucose metabolism and gastrointestinal
secretion and metabolism. Processing of preproglucagon to give
GLP-1(7-36)-amide, GLP-1(7-37) and GLP-2 occurs mainly in the
L-cells. The fragments GLP-1(7-36)-amide and GLP-1(7-37) are both
glucose-dependent insulinotropic agents. In the past decades a
number of structural analogues of GLP-1 were isolated from the
venom of the Gila monster lizards (Heloderma suspectum and
Heloderma horridum). Exendin-4 is a 39 amino acid residue peptide
isolated from the venom of Heloderma horridum, and this peptide
shares 52% homology with GLP-1. Exendin-4 is a potent GLP-1
receptor agonist which has been shown to stimulate insulin release
and ensuring lowering of the blood glucose level when injected into
dogs. The group of GLP-1(1-37) and exendin-4(1-39) and certain
fragments, analogues and derivatives thereof (designated GLP-1
compounds herein) are potent insulinotropic agents, and they are
all applicable in the method of the present invention.
Insulinotropic fragments of GLP-1(1-37) are insulinotropic peptides
for which the entire sequence can be found in the sequence of
GLP-1(1-37) and where at least one terminal amino acid has been
deleted. Examples of insulinotropic fragments of GLP-1(1-37) are
GLP-1(7-37) wherein the amino acid residues in positions 1-6 of
GLP-1(1-37) have been deleted, and GLP-1(7-36) where the amino acid
residues in position 1-6 and 37 of GLP-1(1-37) have been deleted.
Examples of insulinotropic fragments of exendin-4(1-39) are
exendin-4(1-38) and exendin-4(1-31). The insulinotropic property of
a compound may be determined by in vivo or in vitro assays well
known in the art. For instance, the compound may be administered to
an animal and monitoring the insulin concentration over time.
Insulinotropic analogs of GLP-1(1-37) and exendin-4(1-39) refer to
the respective molecules wherein one or more of the amino acids
residues have been exchanged with other amino acid residues and/or
from which one or more amino acid residues have been deleted and/or
from which one or more amino acid residues have been added with the
proviso that said analogue either is insulinotropic or is a prodrug
of an insulinotropic compound. Examples of insulinotropic analogs
of GLP-1(1-37) is e.g. Met.sup.8-GLP-1(7-37) wherein the alanine in
position 8 has been replaced by methionine and the amino acid
residues in position 1 to 6 have been deleted, and
Arg.sup.34-GLP-1(7-37) wherein the valine in position 34 has been
replaced with arginine and the amino acid residues in position 1 to
6 have been deleted. An example of an insulinotropic analog of
exendin-4(1-39) is Ser.sup.2Asp.sup.3-exendin-4(1-39) wherein the
amino acid residues in position 2 and 3 have been replaced with
serine and aspartic acid, respectively (this particular analog also
being known in the art as exendin-3). Insulinotropic derivatives of
GLP-1(1-37), exendin-4(1-39) and analogs thereof are what the
person skilled in the art considers to be derivatives of these
peptides, i.e. having at least one substituent which is not present
in the parent peptide molecule with the proviso that said
derivative either is insulinotropic or is a prodrug of an
insulinotropic compound. Examples of substituents are amides,
carbohydrates, alkyl groups and lipophilic substituents. Examples
of insulinotropic derivatives of GLP-1(1-37), exendin-4(1-39) and
analogs thereof are GLP-1(7-36)-amide, Arg.sup.34,
Lys.sup.26(N.sup..epsilon.-(.gamma.-Glu(N-hexadecanoyl)))-GLP-1(7-37)
and Tyr.sup.31-exendin-4(1-31)-amide. Further examples of
GLP-1(1-37), exendin-4(1-39), insulinotropic fragments thereof,
insulinotropic analogs thereof and insulinotropic derivatives
thereof are described in WO 98/08871, WO 99/43706, U.S. Pat. No.
5,424,286 and WO 00/09666, which are all enclosed herein by
reference.
[0185] GLP-2 and GLP-2 compounds may also be modified by the
methods provided by the present invention. In the present context a
GLP-2 compound binds to a GLP-2 receptor, preferably with an
affinity constant (K.sub.D) or a potency (EC.sub.50) of below 1
.mu.M, e.g. below 100 nM. The term "GLP-2 compound" is intended to
indicate human GLP-2 in which one or more amino acid residue has
been deleted and/or replaced by another amino acid residue, natural
or unnatural, and/or human GLP-2 comprising additional amino acid
residues, and/or human GLP-2 in which at least one organic
substituent is bound to one or more of the amino acid residues. In
particular, those peptides are considered, which amino acid
sequence exhibit at any sequence of 33 consecutive amino acids more
than 60% of the amino acid sequence of human GLP-2. Also those
peptides are considered, which amino acid sequence exhibit at any
sequence of 37 consecutive amino acids more than 60% of the amino
acid sequence of human GLP-2 when up to four amino acids are
deleted from the amino acid sequence. Also those peptides are
considered, which amino acid sequence exhibit at any sequence of 31
consecutive amino acids more than 60% of the amino acid sequence of
GLP-2, when up to two amino acids are added to their amino acid
sequence. The term "GLP compounds" also includes natural allelic
variations that may exist and occur from one individual to another.
Also, degree and location of glycosylation or other
post-translation modifications may vary depending on the chosen
host cells and the nature of the host cellular environment.
[0186] Candidate GLP-2 compounds, which may be used according to
the present invention include the GLP-2 compounds described in WO
96/32414, WO 97/39031, WO 98/03547, WO 96/29342, WO 97/31943, WO
98/08872, which are all incorporated herein by reference.
[0187] In particular, the following GLP-2 compounds are applicable
in the methods of the present invention: A2G-GLP-2(1-33);
K30R-GLP-2(1-33); S5K-GLP-2(1-33); S7K-GLP-2(1-33);
D8K-GLP-2(1-33); E9K-GLP-2(1-33); M10K-GLP-2(1-33);
N11K-GLP-2(1-33); T12K-GLP-2(1-33); 113K-GLP-2(1-33);
L14K-GLP-2(1-33); D15K-GLP-2(1-33); N16K-GLP-2(1-33);
L17K-GLP-2(1-33); A18K-GLP-2(1-33); D21K-GLP-2(1-33);
N24K-GLP-2(1-33); Q28K-GLP-2(1-33); S5K/K30R-GLP-2(1-33);
S7K/K30R-GLP-2(1-33); D8K/K30R-GLP-2(1-33); E9K/K30R-GLP-2(1-33);
M10K/K30R-GLP-2(1-33); N11K/K30R-GLP-2(1-33);
T12K/K30R-GLP-2(1-33); 113K/K30R-GLP-2(1-33);
L14K/K30R-GLP-2(1-33); D15K/K30R-GLP-2(1-33);
N16K/K30R-GLP-2(1-33); L17K/K30R-GLP-2(1-33);
A18K/K30R-GLP-2(1-33); D21K/K30R-GLP-2(1-33);
N24K/K30R-GLP-2(1-33); Q28K/K30R-GLP-2(1-33);
K30R/D33K-GLP-2(1-33); D3E/K30R/D33E-GLP-2(1-33);
D3E/S5K/K30R/D33E-GLP-2(1-33); D3E/S7K/K30R/D33E-GLP-2(1-33);
D3E/D8K/K30R/D33E-GLP-2(1-33); D3E/E9K/K30R/D33E-GLP-2(1-33);
D3E/M10K/K30R/D33E-GLP-2(1-33); D3E/N11K/K30R/D33E-GLP-2(1-33);
D3E/T12K/K30R/D33E-GLP-2(1-33); D3E/I13K/K30R/D33E-GLP-2(1-33);
D3E/L14K/K30R/D33E-GLP-2(1-33); D3E/D15K/K30R/D33E-GLP-2(1-33);
D3E/N16K/K30R/D33E-GLP-2(1-33); D3E/L17K/K30R/D33E-GLP-2(1-33);
D3E/A18K/K30R/D33E-GLP-2(1-33); D3E/D21K/K30R/D33E-GLP-2(1-33);
D3E/N24K/K30R/D33E-GLP-2(1-33); and
D3E/Q28K/K30R/D33E-GLP-2(1-33).
[0188] In one embodiment of the invention the GLP-2 compound is
selected from GLP-2(1-33), 34R-GLP-2(1-34), A2G-GLP-2(1-33),
A2G/34R-GLP-2(1-34), K30R-GLP-2(1-33); S5K-GLP-2(1-33);
S7K-GLP-2(1-33); D8K-GLP-2(1-33); E9K-GLP-2(1-33);
M10K-GLP-2(1-33); N11K-GLP-2(1-33); T12K-GLP-2(1-33);
113K-GLP-2(1-33); L14K-GLP-2(1-33); D15K-GLP-2(1-33);
N16K-GLP-2(1-33); L17K-GLP-2(1-33); A18K-GLP-2(1-33);
D21K-GLP-2(1-33); N24K-GLP-2(1-33); Q28K-GLP-2(1-33);
S5K/K30R-GLP-2(1-33); S7K/K30R-GLP-2(1-33); D8K/K30R-GLP-2(1-33);
E9K/K30R-GLP-2(1-33); M10K/K30R-GLP-2(1-33); N11K/K30R-GLP-2(1-33);
T12K/K30R-GLP-2(1-33); 113K/K30R-GLP-2(1-33);
L14K/K30R-GLP-2(1-33); D15K/K30R-GLP-2(1-33);
N16K/K30R-GLP-2(1-33); L17K/K30R-GLP-2(1-33);
A18K/K30R-GLP-2(1-33); D21K/K30R-GLP-2(1-33);
N24K/K30R-GLP-2(1-33); Q28K/K30R-GLP-2(1-33);
K30R/D33K-GLP-2(1-33); D3E/K30R/D33E-GLP-2(1-33);
D3E/S5K/K30R/D33E-GLP-2(1-33); D3E/S7K/K30R/D33E-GLP-2(1-33);
D3E/D8K/K30R/D33E-GLP-2(1-33); D3E/E9K/K30R/D33E-GLP-2(1-33);
D3E/M10K/K30R/D33E-GLP-2(1-33); D3E/N11K/K30R/D33E-GLP-2(1-33);
D3E/T12K/K30R/D33E-GLP-2(1-33); D3E/113K/K30R/D33E-GLP-2(1-33);
D3E/L14K/K30R/D33E-GLP-2(1-33); D3E/D15K/K30R/D33E-GLP-2(1-33);
D3E/N16K/K30R/D33E-GLP-2(1-33); D3E/L17K/K30R/D33E-GLP-2(1-33);
D3E/A18K/K30R/D33E-GLP-2(1-33); D3E/D21K/K30R/D33E-GLP-2(1-33);
D3E/N24K/K30R/D33E-GLP-2(1-33); D3E/Q28K/K30R/D33E-GLP-2(1-33).
[0189] GLP-2 derivatives with only one lipophilic substituent
attached to the GLP-2 peptide are also applicable in the methods of
the present invention, such as GLP-2 derivatives wherein the
lipophilic substituent comprises from 4 to 40 carbon atoms, such as
from 8 to 25 carbon atoms, e.g. from 12 to 20 carbon atoms.
[0190] The lipophilic substituent may be attached to an amino acid
residue in such a way that a carboxyl group of the lipophilic
substituent forms an amide bond with an amino group of the amino
acid residue.
[0191] By way of example, the lipophilic substituent is attached to
a Lys residue.
[0192] The lipophilic substituent may be attached to an amino acid
residue in such a way that an amino group of the lipophilic
substituent forms an amide bond with a carboxyl group of the amino
acid residue.
[0193] The lipophilic substituent may also be attached to the GLP-2
peptide by means of a spacer, and said spacer may be selected from
amongst .beta.-alanine, gamma-aminobutyric acid (GABA),
.gamma.-glutamic acid, Lys, Asp, Glu, a dipeptide containing Asp, a
dipeptide containing Glu, or a dipeptide containing Lys. In one
embodiment of the invention the spacer is .beta.-alanine.
[0194] A carboxyl group of the parent GLP-2 peptide may also form
an amide bond with an amino group of a spacer, and the carboxyl
group of the amino acid or dipeptide spacer forms an amide bond
with an amino group of the lipophilic substituent.
[0195] An amino group of the parent GLP-2 peptide may also form an
amide bond with a carboxylic group of a spacer, and an amino group
of the spacer forms an amide bond with a carboxyl group of the
lipophilic substituent.
[0196] In one embodiment of the invention the lipophilic
substituent is a straight-chain or branched alkyl group. In one
embodiment of the invention the lipophilic substituent is the acyl
group of a straight-chain or branched fatty acid.
[0197] In one embodiment of the invention the lipophilic
substituent is an acyl group of a straight-chain or branched alkane
.alpha.,.omega.-dicarboxylic acid.
[0198] In one embodiment of the invention the GLP-2 derivative has
one lipophilic substituent. In one embodiment of the invention the
GLP-2 derivative has two lipophilic substituents. In one embodiment
of the invention the GLP-2 derivative has three lipophilic
substituents. In one embodiment of the invention the GLP-2
derivative has four lipophilic substituents.
[0199] The following list contains GLP-2 derivatives which are
particular applicable in the methods of the present invention.
S5K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
S7K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
D8K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
E9K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
M10K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
N11K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
T12K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
I13K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
L14K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
D15K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
N16K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
L17K(3-(octanoylamino)propionyl)-GLP-2(1-33);
L17K(3-(nonanoylamino)propionyl)-GLP-2(1-33);
L17K(3-(decanoylamino)propionyl)-GLP-2(1-33);
L17K(3-(undecanoylamino)propionyl)-GLP-2(1-33);
L17K(3-(dodecanoylamino)propionyl)-GLP-2(1-33);
L17K(3-(tridecanoylamino)propionyl)-GLP-2(1-33);
L17K(3-(tetradecanoylamino)propionyl)-GLP-2(1-33);
L17K(3-(pentadecanoylamino)propionyl)-GLP-2(1-33);
L17K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
L17K(3-(heptadecanoylamino)propionyl)-GLP-2(1-33);
L17K(3-(octadecanoylamino)propionyl)-GLP-2(1-33);
L17K(3-(nonadecanoylamino)propionyl)-GLP-2(1-33);
L17K(3-(eicosanoylamino)propionyl)-GLP-2(1-33);
L17K((S)-4-carboxy-4-(octanoylamino)butanoyl)-GLP-2(1-33);
L17K((S)-4-carboxy-4-(nonanoylamino)butanoyl)-GLP-2(1-33);
L17K((S)-4-carboxy-4-(decanoylamino)butanoyl)-GLP-2(1-33);
L17K((S)-4-carboxy-4-(undecanoylamino)butanoyl)-GLP-2(1-33);
L17K((S)-4-carboxy-4-(dodecanoylamino)butanoyl)-GLP-2(1-33);
L17K((S)-4-carboxy-4-(tridecanoylamino)butanoyl)-GLP-2(1-33);
L17K((S)-4-carboxy-4-(tetradecanoylamino)butanoyl)-GLP-2(1-33);
L17K((S)-4-carboxy-4-(pentadecanoylamino)butanoyl)-GLP-2(1-33);
L17K((S)-4-carboxy-4-(hexadecanoylamino)butanoyl)-GLP-2(1-33);
L17K((S)-4-carboxy-4-(heptadecanoylamino)butanoyl)-GLP-2(1-33);
L17K((S)-4-carboxy-4-(octadecanoylamino)butanoyl)-GLP-2(1-33);
L17K((S)-4-carboxy-4-(nonadecanoylamino)butanoyl)-GLP-2(1-33);
L17K((S)-4-carboxy-4-(eicosanoylamino)butanoyl)-GLP-2(1-33);
L17K(4-(octanoylamino)butanoyl)-GLP-2(1-33);
L17K(4-(nonanoylamino)butanoyl)-GLP-2(1-33);
L17K(4-(decanoylamino)butanoyl)-GLP-2(1-33);
L17K(4-(undecanoylamino)butanoyl)-GLP-2(1-33);
L17K(4-(dodecanoylamino)butanoyl)-GLP-2(1-33);
L17K(4-(tridecanoylamino)butanoyl)-GLP-2(1-33);
L17K(4-(tetradecanoylamino)butanoyl)-GLP-2(1-33);
L17K(4-(pentadecanoylamino)butanoyl)-GLP-2(1-33);
L17K(4-(hexadecanoylamino)butanoyl)-GLP-2(1-33);
L17K(4-(heptadecanoylamino)butanoyl)-GLP-2(1-33);
L17K(4-(octadecanoylamino)butanoyl)-GLP-2(1-33);
L17K(4-(nonadecanoylamino)butanoyl)-GLP-2(1-33);
L17K(4-(eicosanoylamino)butanoyl)-GLP-2(1-33);
A18K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
D21K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
N24K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
Q28K(3-(hexadecanoylamino)propionyl)-GLP-2(1-33);
S5K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33);
S7K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33);
D8K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33);
E9K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33);
M10K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33);
N11K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33);
T12K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33);
I13K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33);
L14K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33);
D15K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33); N
16K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33);
L17K(3-(octanoylamino)propionyl)/K30R-GLP-2(1-33);
L17K(3-(nonanoylamino)propionyl)/K30R-GLP-2(1-33);
L17K(3-(decanoylamino)propionyl)/K30R-GLP-2(1-33);
L17K(3-(undecanoylamino)propionyl)/K30R-GLP-2(1-33);
L17K(3-(dodecanoylamino)propionyl)/K30R-GLP-2(1-33);
L17K(3-(tridecanoylamino)propionyl)/K30R-GLP-2(1-33);
L17K(3-(tetradecanoylamino)propionyl)/K30R-GLP-2(1-33);
L17K(3-(pentadecanoylamino)propionyl)/K30R-GLP-2(1-33);
L17K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33);
L17K(3-(heptadecanoylamino)propionyl)/K30R-GLP-2(1-33);
L17K(3-(octadecanoylamino)propionyl)/K30R-GLP-2(1-33);
L17K(3-(nonadecanoylamino)propionyl)/K30R-GLP-2(1-33);
L17K(3-(eicosanoylamino)propionyl)/K30R-GLP-2(1-33);
L17K((S)-4-carboxy-4-(octanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K((S)-4-carboxy-4-(nonanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K((S)-4-carboxy-4-(decanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K((S)-4-carboxy-4-(undecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K((S)-4-carboxy-4-(dodecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K((S)-4-carboxy-4-(tridecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K((S)-4-carboxy-4-(tetradecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K((S)-4-carboxy-4-(pentadecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K((S)-4-carboxy-4-(hexadecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K((S)-4-carboxy-4-(heptadecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K((S)-4-carboxy-4-(octadecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K((S)-4-carboxy-4-(nonadecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K((S)-4-carboxy-4-(eicosanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K(4-(octanoylamino)butanoyl)/K30R-GLP-2(1-33); L
17K(4-(nonanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K(4-(decanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K(4-(undecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K(4-(dodecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K(4-(tridecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K(4-(tetradecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K(4-(pentadecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K(4-(hexadecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K(4-(heptadecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K(4-(octadecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K(4-(nonadecanoylamino)butanoyl)/K30R-GLP-2(1-33);
L17K(4-(eicosanoylamino)butanoyl)/K30R-GLP-2(1-33);
A18K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33);
D21K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33);
N24K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33);
Q28K(3-(hexadecanoylamino)propionyl)/K30R-GLP-2(1-33);
D3E/S5K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/S7K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/D8K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/E9K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/M10K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/N11K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/T12K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/I13K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/L14K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/D15K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/N16K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(3-(octanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/L117K(3-(nonanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(3-(decanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(3-(undecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(3-(dodecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(3-(tridecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(3-(tetradecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(3-(pentadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(3-(heptadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(3-(octadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(3-(nonadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(3-(eicosanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K((S)-4-carboxy-4-(octanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K((S)-4-carboxy-4-(nonanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K((S)-4-carboxy-4-(decanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K((S)-4-carboxy-4-(undecanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33)-
;
D3E/L17K((S)-4-carboxy-4-(dodecanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33-
);
D3E/L17K((S)-4-carboxy-4-(tridecanoylamino)butanoyl)/K30R/D33E-GLP-2(1--
33);
D3E/L17K((S)-4-carboxy-4-(tetradecanoylamino)butanoyl)/K30R/D33E-GLP--
2(1-33);
D3E/L17K((S)-4-carboxy-4-(pentadecanoylamino)butanoyl)/K30R/D33E--
GLP-2(1-33);
D3E/L17K((S)-4-carboxy-4-(hexadecanoylamino)butanoyl)/K30R/D33E-GLP-2(1-3-
3);
D3E/L17K((S)-4-carboxy-4-(heptadecanoylamino)butanoyl)/K30R/D33E-GLP-2-
(1-33);
D3E/L17K((S)-4-carboxy-4-(octadecanoylamino)butanoyl)/K30R/D33E-GL-
P-2(1-33);
D3E/L17K((S)-4-carboxy-4-(nonadecanoylamino)butanoyl)/K30R/D33E-
-GLP-2(1-33);
D3E/L17K((S)-4-carboxy-4-(eicosanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33)-
; D3E/L17K(4-(octanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(4-(nonanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(4-(decanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(4-(undecanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(4-(dodecanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(4-(tridecanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(4-(tetradecanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(4-(pentadecanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(4-(hexadecanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(4-(heptadecanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(4-(octadecanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(4-(nonadecanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/L17K(4-(eicosanoylamino)butanoyl)/K30R/D33E-GLP-2(1-33);
D3E/A18K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/D21K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33);
D3E/N24K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33); and
D3E/Q28K(3-(hexadecanoylamino)propionyl)/K30R/D33E-GLP-2(1-33).
[0200] Factor VII compounds applicable in the methods of the
present invention encompasses wild-type Factor VII (i.e., a
polypeptide having the amino acid sequence disclosed in U.S. Pat.
No. 4,784,950), as well as variants of Factor VII exhibiting
substantially the same or improved biological activity relative to
wild-type Factor VII, Factor VII-related polypeptides as well as
Factor VII derivatives and Factor VII conjugates. The term "Factor
VII compounds" is intended to encompass Factor VII polypeptides in
their uncleaved (zymogen) form, as well as those that have been
proteolytically processed to yield their respective bioactive
forms, which may be designated Factor VIIa. Typically, Factor VII
is cleaved between residues 152 and 153 to yield Factor VIIa. Such
variants of Factor VII may exhibit different properties relative to
human Factor VII, including stability, phospholipid binding,
altered specific activity, and the like.
[0201] As used herein, "Factor VII-related polypeptides"
encompasses polypeptides, including variants, in which the Factor
VIIa biological activity has been substantially modified or reduced
relative to the activity of wild-type Factor VIIa. These
polypeptides include, without limitation, Factor VII or Factor VIIa
into which specific amino acid sequence alterations have been
introduced that modify or disrupt the bioactivity of the
polypeptide.
[0202] The term "Factor VII derivative" as used herein, is intended
to designate wild-type Factor VII, variants of Factor VII
exhibiting substantially the same or improved biological activity
relative to wild-type Factor VII and Factor VII-related
polypeptides, in which one or more of the amino acids of the parent
peptide have been chemically modified, e.g. by alkylation,
PEGylation, acylation, ester formation or amide formation or the
like. This includes but are not limited to PEGylated human Factor
VIIa, cysteine-PEGylated human Factor VIIa and variants
thereof.
[0203] The term "PEGylated human Factor VIIa" means human Factor
VIIa, having a PEG molecule conjugated to a human Factor VIIa
polypeptide. It is to be understood, that the PEG molecule may be
attached to any part of the Factor VIIa polypeptide including any
amino acid residue or carbohydrate moiety of the Factor VIIa
polypeptide. The term "cysteine-PEGylated human Factor VIIa" means
Factor VIIa having a PEG molecule conjugated to a sulfhydryl group
of a cysteine introduced in human Factor VIIa.
[0204] The biological activity of Factor VIIa in blood clotting
derives from its ability to (i) bind to tissue factor (TF) and (ii)
catalyze the proteolytic cleavage of Factor IX or Factor X to
produce activated Factor IX or X (Factor IXa or Xa, respectively).
For purposes of the invention, Factor VIIa biological activity may
be quantified by measuring the ability of a preparation to promote
blood clotting using Factor VII-deficient plasma and
thromboplastin, as described, e.g., in U.S. Pat. No. 5,997,864. In
this assay, biological activity is expressed as the reduction in
clotting time relative to a control sample and is converted to
"Factor VII units" by comparison with a pooled human serum standard
containing 1 unit/ml Factor VII activity. Alternatively, Factor
VIIa biological activity may be quantified by (i) measuring the
ability of Factor VIIa to produce of Factor Xa in a system
comprising TF embedded in a lipid membrane and Factor X. (Persson
et al., J. Biol. Chem. 272:19919-19924, 1997); (ii) measuring
Factor X hydrolysis in an aqueous system; (iii) measuring its
physical binding to TF using an instrument based on surface plasmon
resonance (Persson, FEBS Letts. 413:359-363, 1997) and (iv)
measuring hydrolysis of a synthetic substrate.
[0205] Factor VII variants having substantially the same or
improved biological activity relative to wild-type Factor VIIa
encompass those that exhibit at least about 25%, preferably at
least about 50%, more preferably at least about 75% and most
preferably at least about 90% of the specific activity of Factor
VIIa that has been produced in the same cell type, when tested in
one or more of a clotting assay, proteolysis assay, or TF binding
assay as described above. Factor VII variants having substantially
reduced biological activity relative to wild-type Factor VIIa are
those that exhibit less than about 25%, preferably less than about
10%, more preferably less than about 5% and most preferably less
than about 1% of the specific activity of wild-type Factor VIIa
that has been produced in the same cell type when tested in one or
more of a clotting assay, proteolysis assay, or TF binding assay as
described above. Factor VII variants having a substantially
modified biological activity relative to wild-type Factor VII
include, without limitation, Factor VII variants that exhibit
TF-independent Factor X proteolytic activity and those that bind TF
but do not cleave Factor X.
[0206] Variants of Factor VII, whether exhibiting substantially the
same or better bioactivity than wild-type Factor VII, or,
alternatively, exhibiting substantially modified or reduced
bioactivity relative to wild-type Factor VII, include, without
limitation, polypeptides having an amino acid sequence that differs
from the sequence of wild-type Factor VII by insertion, deletion,
or substitution of one or more amino acids.
[0207] The terms "variant" or "variants", as used herein, is
intended to designate Factor VII having the sequence of wild-type
factor VII, wherein one or more amino acids of the parent protein
have been substituted by another amino acid and/or wherein one or
more amino acids of the parent protein have been deleted and/or
wherein one or more amino acids have been inserted in protein
and/or wherein one or more amino acids have been added to the
parent protein. Such addition can take place either at the
N-terminal end or at the C-terminal end of the parent protein or
both. The "variant" or "variants" within this definition still have
FVII activity in its activated form. In one embodiment a variant is
70% identical with the sequence of wild-type Factor VII. In one
embodiment a variant is 80% identical with the sequence of
wild-type factor VII. In another embodiment a variant is 90%
identical with the sequence of wild-type factor VII. In a further
embodiment a variant is 95% identical with the sequence of
wild-type factor VII.
[0208] Non-limiting examples of Factor VII variants having
substantially the same biological activity as wild-type Factor VII
include S52A-FVIIa, S60A-FVIIa (Lino et al., Arch. Biochem.
Biophys. 352: 182-192, 1998); FVIIa variants exhibiting increased
proteolytic stability as disclosed in U.S. Pat. No. 5,580,560;
Factor VIIa that has been proteolytically cleaved between residues
290 and 291 or between residues 315 and 316 (Mollerup et al.,
Biotechnol. Bioeng. 48:501-505, 1995); oxidized forms of Factor
VIIa (Kornfelt et al., Arch. Biochem. Biophys. 363:43-54, 1999);
FVII variants as disclosed in PCT/DK02/00189; and FVII variants
exhibiting increased proteolytic stability as disclosed in WO
02/38162 (Scripps Research Institute); FVII variants having a
modified Gla-domain and exhibiting an enhanced membrane binding as
disclosed in WO 99/20767 (University of Minnesota) and WO 00/66753
(University of Minnesota); and FVII variants as disclosed in WO
01/58935 (Maxygen ApS), WO 03/93465 (Maxygen ApS) and WO 04/029091
(Maxygen ApS) all of which are incorporated herein by
reference.
[0209] Particular mentioning is made of FVII variants having
increased biological activity compared to wild-type FVIIa include
FVII variants as disclosed in WO 01/83725, WO 02/22776, WO
02/077218, PCT/DK02/00635, WO 2004/029090, WO 2003/037932; WO
02/38162 (Scripps Research Institute); and FVIIa variants with
enhanced activity as disclosed in JP 2001061479
(Chemo-Sero-Therapeutic Res Inst.), all of which are incorporated
herein by reference, all of which are incorporated herein by
reference.
[0210] Examples of Factor VII variants having substantially reduced
or modified biological activity relative to wild-type Factor VII
include R152E-FVIIa (Wildgoose et al., Biochem 29:3413-3420, 1990),
S344A-FVIIa (Kazama et al., J. Biol. Chem. 270:66-72, 1995),
FFR-FVIIa (Holst et al., Eur. J. Vasc. Endovasc. Surg. 15:515-520,
1998), and Factor VIIa lacking the Gla domain, (Nicolaisen et al.,
FEBS Letts. 317:245-249, 1993), all of which are incorporated
herein by reference.
[0211] Examples of variants of factor VII, factor VII or factor
VII-related polypeptides include wild-type Factor VII, L305V-FVII,
L305V/M306D/D309S-FVII, L3051-FVII, L305T-FVII, F374P-FVII,
V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII,
V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII,
V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII,
K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII,
V158D/M298K-FVII, and S336G-FVII, L305V/K337A-FVII,
L305V/V158D-FVII, L305V/E296V-FVII, L305V/M298Q-FVII,
L305V/V158T-FVII, L305V/K337A/V1158T-FVII, L305V/K337A/M298Q-FVII,
L305V/K337A/E296V-FVII, L305V/K337V/V158D-FVII,
L305V/V158D/M298Q-FVII, L305V/V158D/E296V-FVII,
L305V/V158T/M298Q-FVII, L305V/V 158T/E296V-FVII,
L305V/E296V/M298Q-FVII, L305V/V158D/E296V/M298Q-FVII,
L305V/V158T/E296V/M298Q-FVII, L305V/V158T/K337A/M298Q-FVII,
L305V/V158T/E296V/K337A-FVII, L305V/V158D/K337A/M298Q-FVII,
L305V/V158D/E296V/K337A-FVII, L305V/V158D/E296V/M298Q/K337A-FVII,
L305V/V 158T/E296V/M298Q/K337A-FVII, S314E/K316H-FVII,
S314E/K316Q-FVII, S314E/L305V-FVII, S314E/K337A-FVII,
S314E/V158D-FVII, S314E/E296V-FVII, S314E/M298Q-FVII,
S314E/V158T-FVII, K316H/L305V-FVII, K316H/K337A-FVII,
K316H/V158D-FVII, K316H/E296V-FVII, K316H/M298Q-FVII,
K316H/V158T-FVII, K316Q/L305V-FVII, K316Q/K337A-FVII,
K316Q/V158D-FVII, K316Q/E296V-FVII, K316Q/M298Q-FVII,
K316Q/V158T-FVII, S314E/L305V/K337A-FVII, S314E/L305V/V158D-FVII,
S314E/L305V/E296V-FVII, S314E/L305V/M298Q-FVII,
S314E/L305V/V158T-FVII, S314E/L305V/K337A/V158T-FVII,
S314E/L305V/K337A/M298Q-FVII, S314E/L305V/K337A/E296V-FVII,
S314E/L305V/K337A/V158D-FVII, S314E/L305V/V158D/M298Q-FVII,
S314E/L305V/V158D/E296V-FVII, S314E/L305V/V158T/M298Q-FVII,
S314E/L305V/V158T/E296V-FVII, S314E/L305V/E296V/M298Q-FVII,
S314E/L305V/V158D/E296V/M298Q-FVII,
S314E/L305V/V158T/E296V/M298Q-FVII,
S314E/L305V/V158T/K337A/M298Q-FVII,
S314E/L305V/V158T/E296V/K337A-FVII,
S314E/L305V/V158D/K337A/M298Q-FVII,
S314E/L305V/V158D/E296V/K337A-FVII,
S314E/L305V/V158D/E296V/M298Q/K337A-FVII,
S314E/L305V/V158T/E296V/M298Q/K337A-FVII, K316H/L305V/K337A-FVII,
K316H/L305V/V158D-FVII, K316H/L305V/E296V-FVII,
K316H/L305V/M298Q-FVII, K316H/L305V/V158T-FVII,
K316H/L305V/K337A/V158T-FVII, K316H/L305V/K337A/M298Q-FVII,
K316H/L305V/K337A/E296V-FVII, K316H/L305V/K337A/V158D-FVII,
K316H/L305V/V158D/M298Q-FVII, K316H/L305V/V158D/E296V-FVII,
K316H/L305V/V158T/M298Q-FVII, K316H/L305V/V158T/E296V-FVII,
K316H/L305V/E296V/M298Q-FVII, K316H/L305V/V158D/E296V/M298Q-FVII,
K316H/L305V/V158T/E296V/M298Q-FVII,
K316H/L305V/V158T/K337A/M298Q-FVII,
K316H/L305V/V158T/E296V/K337A-FVII,
K316H/L305V/V158D/K337A/M298Q-FVII,
K316H/L305V/V158D/E296V/K337A-FVII,
K316H/L305V/V158D/E296V/M298Q/K337A-FVII,
K316H/L305V/V158T/E296V/M298Q/K337A-FVII, K316Q/L305V/K337A-FVII,
K316Q/L305V/V158D-FVII, K316Q/L305V/E296V-FVII,
K316Q/L305V/M298Q-FVII, K316Q/L305V/V158T-FVII,
K316Q/L305V/K337A/V158T-FVII, K316Q/L305V/K337A/M298Q-FVII,
K316Q/L305V/K337A/E296V-FVII, K316Q/L305V/K337A/V158D-FVII,
K316Q/L305V/V158D/M298Q-FVII, K316Q/L305V/V158D/E296V-FVII,
K316Q/L305V/V158T/M298Q-FVII, K316Q/L305V/V158T/E296V-FVII,
K316Q/L305V/E296V/M298Q-FVII, K316Q/L305V/V158D/E296V/M298Q-FVII,
K316Q/L305V/V158T/E296V/M298Q-FVII,
K316Q/L305V/V158T/K337A/M298Q-FVII,
K316Q/L305V/V158T/E296V/K337A-FVII,
K316Q/L305V/V158D/K337A/M298Q-FVII,
K316Q/L305V/V158D/E296V/K337A-FVII,
K316Q/L305V/V158D/E296V/M298Q/K337A-FVII,
K316Q/L305V/V158T/E296V/M298Q/K337A-FVII, F374Y/K337A-FVII,
F374Y/V158D-FVII, F374Y/E296V-FVII, F374Y/M298Q-FVII,
F374Y/V158T-FVII, F374Y/S314E-FVII, F374Y/L305V-FVII,
F374Y/L305V/K337A-FVII, F374Y/L305V/V158D-FVII,
F374Y/L305V/E296V-FVII, F374Y/L305V/M298Q-FVII,
F374Y/L305V/V158T-FVII, F374Y/L305V/S314E-FVII,
F374Y/K337A/S314E-FVII, F374Y/K337A/V158T-FVII,
F374Y/K337A/M298Q-FVII, F374Y/K337A/E296V-FVII,
F374Y/K337A/V158D-FVII, F374Y/V158D/S314E-FVII,
F374Y/V158D/M298Q-FVII, F374Y/V158D/E296V-FVII,
F374Y/V158T/S314E-FVII, F374Y/V158T/M298Q-FVII,
F374Y/V158T/E296V-FVII, F374Y/E296V/S314E-FVII,
F374Y/S314E/M298Q-FVII, F374Y/E296V/M298Q-FVII,
F374Y/L305V/K337A/V158D-FVII, F374Y/L305V/K337A/E296V-FVII,
F374Y/L305V/K337A/M298Q-FVII, F374Y/L305V/K337A/V158T-FVII,
F374Y/L305V/K337A/S314E-FVII, F374Y/L305V/V158D/E296V-FVII,
F374Y/L305V/V158D/M298Q-FVII, F374Y/L305V/V158D/S314E-FVII,
F374Y/L305V/E296V/M298Q-FVII, F374Y/L305V/E296V/V158T-FVII,
F374Y/L305V/E296V/S314E-FVII, F374Y/L305V/M298Q/V158T-FVII,
F374Y/L305V/M298Q/S314E-FVII, F374Y/L305V/V158T/S314E-FVII,
F374Y/K337A/S314E/V158T-FVII, F374Y/K337A/S314E/M298Q-FVII,
F374Y/K337A/S314E/E296V-FVII, F374Y/K337A/S314E/V158D-FVII,
F374Y/K337A/V158T/M298Q-FVII, F374Y/K337A/V158T/E296V-FVII,
F374Y/K337A/M298Q/E296V-FVII, F374Y/K337A/M298Q/V158D-FVII,
F374Y/K337A/E296V/V158D-FVII, F374Y/V158D/S314E/M298Q-FVII,
F374Y/V158D/S314E/E296V-FVII, F374Y/V158D/M298Q/E296V-FVII,
F374Y/V158T/S314E/E296V-FVII, F374Y/V158T/S314E/M298Q-FVII,
F374Y/V158T/M298Q/E296V-FVII, F374Y/E296V/S314E/M298Q-FVII,
F374Y/L305V/M298Q/K337A/S314E-FVII,
F374Y/L305V/E296V/K337A/S314E-FVII,
F374Y/E296V/M298Q/K337A/S314E-FVII,
F374Y/L305V/E296V/M298Q/K337A-FVII,
F374Y/L305V/E296V/M298Q/S314E-FVII,
F374Y/V158D/E296V/M298Q/K337A-FVII,
F374Y/V158D/E296V/M298Q/S314E-FVII,
F374Y/L305V/V158D/K337A/S314E-FVII,
F374Y/V158D/M298Q/K337A/S314E-FVII,
F374Y/V158D/E296V/K337A/S314E-FVII,
F374Y/L305V/V158D/E296V/M298Q-FVII,
F374Y/L305V/V158D/M298Q/K337A-FVII,
F374Y/L305V/V158D/E296V/K337A-FVII,
F374Y/L305V/V158D/M298Q/S314E-FVII,
F374Y/L305V/V158D/E296V/S314E-FVII,
F374Y/V158T/E296V/M298Q/K337A-FVII,
F374Y/V158T/E296V/M298Q/S314E-FVII,
F374Y/L305V/V158T/K337A/S314E-FVII,
F374Y/V158T/M298Q/K337A/S314E-FVII,
F374Y/V158T/E296V/K337A/S314E-FVII,
F374Y/L305V/V158T/E296V/M298Q-FVII,
F374Y/L305V/V158T/M298Q/K337A-FVII,
F374Y/L305V/V158T/E296V/K337A-FVII,
F374Y/L305V/V158T/M298Q/S314E-FVII,
F374Y/L305V/V158T/E296V/S314E-FVII,
F374Y/E296V/M298Q/K337A/V158T/S314E-FVII,
F374Y/V158D/E296V/M298Q/K337A/S314E-FVII,
F374Y/L305V/V158D/E296V/M298Q/S314E-FVII,
F374Y/L305V/E296V/M298Q/V158T/S314E-FVII,
F374Y/L305V/E296V/M298Q/K337A/V158T-FVII,
F374Y/L305V/E296V/K337A/V158T/S314E-FVII,
F374Y/L305V/M298Q/K337A/V158T/S314E-FVII,
F374Y/L305V/V158D/E296V/M298Q/K337A-FVII,
F374Y/L305V/V158D/E296V/K337A/S314E-FVII,
F374Y/L305V/V158D/M298Q/K337A/S314E-FVII,
F374Y/L305V/E296V/M298Q/K337A/V158T/S314E-FVII,
F374Y/L305V/V158D/E296V/M298Q/K337A/S314E-FVII, S52A-Factor VII,
S60A-Factor VII; R152E-Factor VII, S344A-Factor VII, Factor VIIa
lacking the Gla domain; and P11Q/K33E-FVII, T106N-FVII,
K143N/N145T-FVII, V253N-FVII, R290N/A292T-FVII, G291N-FVII,
R315N/V317T-FVII, K143N/N145T/R315N/V317T-FVII; and FVII having
substitutions, additions or deletions in the amino acid sequence
from 233Thr to 240Asn, FVII having substitutions, additions or
deletions in the amino acid sequence from 304Arg to 329Cys.
[0212] Growth hormone applicable in the methods of the present
invention includes human growth hormone (hGH), which sequence and
characteristics are set froth in, e.g. Hormone Drugs, Gueriguian,
U.S.P. Covention, Rockvill, 1982 and growth hormone compounds. The
term "growth hormone compound" is intended to indicate human growth
hormone (hGH) in which one or more amino acid residues have been
deleted and/or replaced by other amino acid residues, natural or
unnatural, and/or hGH comprising addition amino acid residues,
natural or unnatural, and/or hGH in which at least one organic
substituent is bound to one or more organic substituent. Particular
mentioning is made of the 191 native amino acid sequence
(somatropin) and the 192 amino acid N-terminal methionine species
(somatrem).
[0213] Other examples of growth hormone compound applicable in the
present invention include wherein amino acid No 172, 174, 176 and
178 as a group are replaced by one of the following groups of amino
acids (R, S, F, R); (R, A, Y, R), (K, T, Y, K); (R, S, Y, R); (K,
A, Y, R); (R, F, F, R); (K, Q, Y, R); (R, T, Y, H); (Q, R, Y, R);
(K, K, Y, K); (R, S, F, S) or (K, S, N, R) as disclosed in WO
92/09690 (Genentech), which is incorporated herein by
reference.
[0214] Other examples of growth hormone compound applicable in the
present invention include hGH with the following substitutions
G120R, G120K, G120Y, G120F and G120E, as disclosed in U.S. Pat. No.
6,004,931 (Genentech), which is incorporated herein by
reference.
[0215] Other examples of growth hormone compound applicable in the
present invention include hGH with the following set of
substitutions R167N, D171S, E174S, F176Y and 1179T; R176E, D171S,
E174S and F176Y; F10A, M14W, H18D and H.sub.21N; F10A, M14W, H18D,
H.sub.21N, R167N, D171S, E174S, F176Y, 1179T; F10A, M14W, H18D,
H.sub.21N, R167N, D171A, E174S, F176Y, 1179T; F10H, M14G, H.sub.18N
and H.sub.21N; F10A, M14W, H18D, H.sub.21N, R167N, D171A, T175T and
1179T; and F101, M14Q, H18E, R167N, D171S and 1179T, as disclosed
in U.S. Pat. No. 6,143,523 (Genentech), which is incorporated
herein by reference.
[0216] Other examples of growth hormone compound applicable in the
present invention include hGH with the following set of
substitutions H18A, Q22A, F25A, D26A, Q29A, E65A, K168A, E174A and
G120K as disclosed in U.S. Pat. No. 6,136,536 (Genentech), which is
incorporated herein by reference.
[0217] Other examples of growth hormone compound applicable in the
present invention include hGH with the following set of
substitutions H18D, H.sub.21N, R167N, K168A, D171S, K172R, E174S,
I179T and wherein G120 is further substituted with either R, K, W,
Y, F or E, as disclosed in U.S. Pat. No. 6,057,292 (Genentech),
which is incorporated herein by reference.
[0218] Other examples of growth hormone compound applicable in the
present invention include hGH with the following set of
substitutions H18D, H.sub.21N, R167N, K168A, D171S, K172R, E174S
and 1179T, as disclosed in U.S. Pat. No. 5,849,535 (Genentech),
which is incorporated herein by reference.
[0219] Other examples of growth hormone compound applicable in the
present invention include hGH with the following set of
substitutions H18D, H21D, R167N, K168A, D171S, K172R, E174S and
1179T; and H18A, Q22A, F25A, D26A, Q29A, E65A, K168A and E174A, as
disclosed in WO 97/11178 (Genentech), which is incorporated herein
by reference.
[0220] Other examples of growth hormone compound applicable in the
present invention include hGH with the following set of
substitutions K168A and E174A; R178N and 1179M; K172A and F176A;
and H.sub.54F, S56E, L581, E62S, D63N and Q66E as disclosed in WO
90/04788 (Genentech), which is incorporated herein by
reference.
[0221] Examples of cytokines which could be modified using the
method of the present invention include erythropoietin (EPO),
thrombopoietin, INF-.alpha., IFN-.beta., IFN-.gamma., TNF-.alpha.,
interleukin-1.beta. (IL-1-.beta.), IL-3, IL-4, IL-5, IL-10, IL-12,
IL-15, IL-18, IL-19, IL-20, IL-21 IL-24, grannolyte
colony-stimulating factor (G-CSF), GM-CSF, and chemokines such as
machrophage inflammatory protein-1 (MIP-1) gamma interferon
inducible protein and monokines induced by IFN.gamma. (MIG).
[0222] Particular examples of IL-19 applicable in the methods of
the present invention include those disclosed WO 98/08870 (Human
Genome Science), which is incorporated herein by reference.
Particular mentioning is made of the peptide disclosed as SEQ ID
NO:2 in WO 98/08870.
[0223] Particular examples of applicable IL-20 include those
disclosed in WO 99/27103 (Zymo-genetics), which is incorporated
herein by reference. In the present context, IL-20 is intended to
indicate IL-20 itself and fragments thereof as well as polypeptides
being at least 90% identical to IL-20 or fragments thereof.
Proteins particular applicable in the methods of the present
invention includes those disclosed in WO 99/27103 as SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ
ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,
SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID
NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and
SEQ ID NO:35.
[0224] Examples of IL-21 applicable in the methods of the present
invention include those disclosed in WO 00/53761 (Zymogenetics),
which is incorporated herein by reference. particular mentioning is
made of the peptide disclosed as SEQ ID NO:2 in WO 00/53761.
[0225] TTF are applicable in the methods of the present invention.
TTF peptides are a family of peptides found mainly in association
with the gastrointestinal tract. Particular mentioning is made of
breast cancer associated pS2 peptide (TFF-1), which is known from
human, mouse, and rat, spasmolytical polypeptide (TFF-2), which is
known from human, pig, rat, and mouse and intestinal trefoil factor
(TFF-3), known from human, rat and mouse.
[0226] Other peptides from the TFF family applicable in the methods
of the present invention include those disclosed in WO 02/46226
(Novo Nordisk), which is included herein by reference. Particular
mentioning is made of a TFF-2 peptide wherein a TFF2 peptide with
an amino acid as disclosed in SEQ ID NO:1 of WO 02/46226 comprising
disulphide bonds between Cys6-Cys104, Cys8-Cys35, Cys19-Cys34,
Cys29-Cys46, Cys58-Cys84, Cys68-Cys83, and Cys78-Cys95 and wherein
a moiety X independently selected from sugar residues and
oligosaccharides is covalently attached to Asn15.
[0227] Other peptides of the TFF family include TFF-1 and TFF-3
dimers as those disclosed in WO 96/06861 (Novo Nordisk), which is
incorporated herein by reference.
[0228] Several melanorcortin receptors are known, and particular
mentioning of peptides applicable for the methods of the present
invention is made of peptidic melanocortin-4 receptor agonists,
which are known to have an appetite suppressive effect. Particular
mentioning is made of peptides or proteins disclosed in the
following patent documents, which are all incorporated herein by
reference: U.S. Pat. No. 6,054,556 (Hruby), WO 00/05263 (William
Harvey Research), WO 00/35952 (Melacure), WO 00/35952 (Melacure),
WO 00/58361 (Procter & Gamble), WO 01/52880 (Merck), WO
02/26774 (Procter & Gamble), WO 03/06620 (Palatin), WO 98/27113
(Rudolf Magnus Institute) and WO 99/21571 (Trega).
[0229] Other classes of peptides or proteins which are applicable
in the methods of the present invention include enzymes. Many
enzymes are used for various industrial purposes, and particular
mentioning is made of hydrolases (proteases, lipases, cellulases,
esterases), oxidoreductases (laccases, peroxidaxes, catalases,
superoxide dismutases, lipoxygenases), transferases and
isomerases.
[0230] Other peptides or proteins applicable in the methods of the
present invention include ACTH, corticotropin-releasing factor,
angiotensin, calcitonin, insulin and fragments and analogues
thereof, glucagon, IGF-1, IGF-2, enterogastrin, gastrin,
tetragastrin, pentagastrin, urogastrin, epidermal growth factor,
secretin, nerve growth factor, thyrotropin releasing hormone,
somatostatin, growth hormone releasing hormone, somatomedin,
parathyroid hormone, thrombopoietin, erythropoietin, hypothalamic
releasing factors, prolactin, thyroid stimulating hormones,
endorphins, enkephalins, vasopressin, oxytocin, opiods and
analogues thereof, asparaginase, arginase, arginine deaminase,
adenosine deaminase and ribonuclease.
[0231] Peptides to be modified according to the methods of the
present invention may either be isolated from natural sources (e.g.
plants, animals or micro-organisms, such as yeast, bacteria, fungi
or vira) or they may be synthesised. Peptides form natural sources
also include peptides form transgenic sources, e.g. sources which
have been genetically modified to express or to increase the
expression of a peptide, wherein said peptide may be "natural" in
the sense that it exists in nature or "unnatural" in the sense that
it only exists due to human intervention. Peptides isolated form
natural sources may also be subjected to synthetic modification
prior to the conjugation of the present invention.
[0232] In one embodiment, the invention relates to conjugated
peptides obtainable according to the methods of the present
invention. If the conjugated peptide obtained by the methods of the
present invention is a therapeutic peptide, the invention also
provides the use of such compounds in therapy, and pharmaceutical
compositions comprising such compounds.
[0233] In one embodiment, the invention provides conjugated
peptides of the formula
##STR00088##
wherein P', R, A, E and Z are as defined above, and wherein the
group
##STR00089##
is bonded to the C-terminal of P' via a peptide bond.
[0234] Particular examples of such compounds include [0235]
Lys.sup..epsilon.(4-((2-(1-(mPEGcarbonyl)piperidin-4-yl)ethoxy)imino)pent-
anoyl) 192)hGH(1-192) amide, in which mPEG has a molecular weight
of 20 kDa; [0236]
(Lys.sup..epsilon.(4-((3-(palmitoylamino)propoxy)imino)pentanoyl)192)hGH(-
1-192) amide; [0237]
(Lys.sup..epsilon.(4-((3-((2S)-2,6-mPEGcarbonylamino)hexanoylamino)propox-
y)imino)pentanoyl)34)GLP-2(1-34) amide, in which mPEG has a
molecular weight of 20 kDa; [0238]
(Lys.sup..epsilon.(4-(1-(2-(3-(mPEG)propanoylamino)hydrazino)ethyl)benzoy-
l)192)hGH(1-92) amide, in which mPEG has a molecular weight of 10
kDa; [0239]
(S)-3-(4-((3-(3-Chlorophenyl)isoxazol-5-yl)methoxy)phenyl)-2-([Glu-
.sup.3, Leu.sup.10]GLP-2-ylleucinylamino)propionic amide; [0240]
(S)-3-(4-((3-(3-Chlorophenyl)isoxazol-5-yl)methoxy)phenyl)-2-([Glu.sup.3]-
GLP-2ylleucinylamino)propionic amide; [0241]
3-(3-(3-((4-((S)-2-Carbamoyl-3-([Glu.sup.3,Leu.sup.10]GLP-2-ylleucinylami-
no)ethyl)phenoxyl)methyl)isoxazol-3-yl)benzylcarbamoyl)propionic
acid; [0242]
11-(4-(4-((2S)-2-Carbamoyl-2-(([Glu.sup.3,Leu.sup.10]GLP-2ylleucin-
ylamino)ethyl)pheoxymethyl)-1,2,3-triazolyl)undecanoic acid; [0243]
1'-(5-(4-((2S)-2-Carbamoyl-2-(([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamino-
)ethyl)pheoxymethyl)-1,2,3-triazolyl)undecanoic acid
1'-(4-(4-((S)-2-carbamoyl-2-([Glu.sup.3]GLP-2ylleucinylamino))phenoxymeth-
yl)-1H-1,2,3-triazol-1-yl)undecanoic acid; [0244]
11-(5-(4-((S)-2-carbamoyl-2-([Glu.sup.3]GLP-2ylleucinylamino))phenoxymeth-
yl)-1H-1,2,3-triazol-1-yl)undecanoic acid; [0245]
2-([Glu.sup.3]GLP-2ylleucinyl)-3-(4-((1-((N-(mPeg20kDayl)carbamoyl)decany-
l)-1H-1,2,3-tetrazol-4-yl)methoxy)phenyl)propionamide; and [0246]
2-([Glu.sup.3]GLP-2ylleucinyl)-3-(4-((1-((N-(mPeg20kDayl)carbamoyl)decany-
l)-1H-1,2,3-tetrazol-5-yl)methoxy)phenyl)propionamide.
[0247] Insulin is used to treat or prevent diabetes, and in one
embodiment, the present invention thus provides a method of
treating type 1 or type 2 diabetes, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of an insulin or insulin compound conjugate
according to the present invention.
[0248] In another embodiment, the invention provides the use of an
insulin or insulin compound conjugate according to the present
invention in the manufacture of a medicament used in the treatment
of type 1 or type 2 diabetes.
[0249] GLP-1 may be used in the treatment of hyperglycemia, type 2
diabetes, impaired glucose tolerance, type 1 diabetes, obesity,
hypertension, syndrome X, dyslipidemia, .beta.-cell apoptosis,
.beta.-cell deficiency, inflammatory bowel syndrome, dyspepsia,
cognitive disorders, e.g. cognitive enhancing, neuroprotection,
atheroschlerosis, coronary heart disease and other cardiovascular
disorders. In one embodiment, the present invention thus provides a
method of treating said diseases, the method comprising
administering to a subject in need thereof a therapeutically
effective amount of a GLP-1 or GLP-1 compound conjugate according
to the present invention.
[0250] In another embodiment, the invention provides the use of a
GLP-1 or GLP-1 compound conjugate according to the present
invention in the manufacture of a medicament used in the treatment
of the above mentioned diseases.
[0251] GLP-2 may be used in the treatment of intestinal failure
leading to malabsorption of nutrients in the intestines, and in
particular GLP-2 may be used in the treatment of small bowel
syndrome, Inflammatory bowel syndrome, Crohns disease, colitis
including collagen colitis, radiation colitis, post radiation
atrophy, non-tropical (gluten intolerance) and tropical sprue,
damaged tissue after vascular obstruction or trauma, tourist
diarrhea, dehydration, bacteremia, sepsis, anorexia nervosa,
damaged tissue after chemotherapy, premature infants, schleroderma,
gastritis including atrophic gastritis, postantrectomy atrophic
gastritis and helicobacter pylori gastritis, ulcers, enteritis,
cul-de-sac, lymphatic obstruction, vascular disease and
graft-versus-host, healing after surgical procedures, post
radiation atrophy and chemotherapy, and osteoporosis. It is
therefore an intension of the present invention to provide methods
of treating the above diseases, the method comprising administering
to a subject in need thereof a therapeutically effective amount of
a GLP-2 or GLP-2 compound conjugate according to this
invention.
[0252] In another embodiment, the present invention provides the
use of a GLP-2 or GLP-2 compound conjugate according to this
invention in the manufacture of a medicament used in the treatment
of the above mentioned diseases.
[0253] Growth hormone has been implicated in the treatment of
diseases benefiting from an increase in the plasma level of growth
hormone. In one embodiment, the invention provides a method for the
treatment of growth hormone deficiency (GHD); Turner Syndrome;
Prader-Willi syndrome (PWS); Noonan syndrome; Down syndrome;
chronic renal disease, juvenile rheumatoid arthritis; cystic
fibrosis, HIV-infection in children receiving HAART treatment
(HIV/HALS children); short children born short for gestational age
(SGA); short stature in children born with very low birth weight
(VLBW) but SGA; skeletal dysplasia; hypochondroplasia;
achondroplasia; idiopathic short stature (ISS); GHD in adults;
fractures in or of long bones, such as tibia, fibula, femur,
humerus, radius, ulna, clavicula, matacarpea, matatarsea, and
digit; fractures in or of spongious bones, such as the scull, base
of hand, and base of food; patients after tendon or ligament
surgery in e.g. hand, knee, or shoulder; patients having or going
through distraction oteogenesis; patients after hip or discus
replacement, meniscus repair, spinal fusions or prosthesis
fixation, such as in the knee, hip, shoulder, elbow, wrist or jaw;
patients into which osteosynthesis material, such as nails, screws
and plates, have been fixed; patients with non-union or mal-union
of fractures; patients after osteatomia, e.g. from tibia or
1.sup.st toe; patients after graft implantation; articular
cartilage degeneration in knee caused by trauma or arthritis;
osteoporosis in patients with Turner syndrome; osteoporosis in men;
adult patients in chronic dialysis (APCD); malnutritional
associated cardiovascular disease in APCD; reversal of cachexia in
APCD; cancer in APCD; chronic abstractive pulmonal disease in APCD;
HIV in APCD; elderly with APCD; chronic liver disease in APCD,
fatigue syndrome in APCD; Crohn's disease; impaired liver function;
males with HIV infections; short bowel syndrome; central obesity;
HIV-associated lipodystrophy syndrome (HALS); male infertility;
patients after major elective surgery, alcohol/drug detoxification
or neurological trauma; aging; frail elderly; osteo-arthritis;
traumatically damaged cartilage; erectile dysfunction;
fibromyalgia; memory disorders; depression; traumatic brain injury;
subarachnoid haemorrhage; very low birth weight; metabolic
syndrome; glucocorticoid myopathy; or short stature due to
glucucorticoid treatment inchildren, the method comprising
administering to a patient in need thereof an effective amount of a
growth hormone compound conjugate according to the present
invention.
[0254] In one aspect, the invention provides a method for the
acceleration of the healing of muscle tissue, nervous tissue or
wounds; the acceleration or improvement of blood flow to damaged
tissue; or the decrease of infection rate in damaged tissue, the
method comprising administration to a patient in need thereof an
effective amount of a growth hormone compound onjugayte according
to the present invention.
[0255] In one aspect, the invention provides the use of growth
hormone compound conjugates according to the present invention in
the manufacture of medicaments for the treatment of the above
mentioned diseases.
[0256] Cytokines are implicated in the etiology of a host of
diseases involving the immune system. In particular it is mentioned
that IL-20 could be involved in psoriasis and its treatment, and
I-21 is believed to be involved in cancer and could constitute a
treatment to this disease. In one embodiment, the invention
provides a method for the treatment of psoriasis comprising the
administration of IL-20 conjugates according to the present
invention. In another embodiment, the invention relates to the use
of an IL-20 conjugate of the present invention in the manufacture
of a medicament used in the treatment of psoriasis.
[0257] In another embodiment, the present invention relates to a
method of treating cancer, the method comprising administration of
an IL-21 conjugate of the present invention to a subject in need
thereof.
[0258] In another embodiment, the invention relates to the use of
an IL-21 conjugate according to the present invention in the
manufacture of a medicament used in the treatment of cancer.
[0259] TTF peptides may be used to increase the viscosity of muscus
layers in subject, to reduce secretion of salvia, e.g. where the
increase salvia secretion is caused by irradiation therapy,
treatment with anticholinergics or Sjogren's syndrome, to treat
allergic rhinitis, stress induced gastric ulcers secondary to
trauma, shock, large operations, renal or liver diseases, treatment
with NSAID, e.g. aspirin, steroids or alcohol. TTF peptides may
also be used to treat Chrohn's disease, ulcerative colitis,
keratoconjunctivitis, chronic bladder infections, intestinal
cystitis, papillomas and bladder cancer. In one embodiment, the
invention thus relates the a method of treating the above mention
diseases or states, the method comprising administering to a
subject patient in need thereof a therapeutically effective amount
of a TTF conjugate according to the present invention.
[0260] In another embodiment, the invention relates the use of a
TTF conjugate of the present invention in the manufacture of a
medicament for the treatment of the above mentioned diseases or
states.
[0261] Melanocortin receptor modifiers, and in particular
melanorcortin 4 receptor agonists have been implicated the
treatment and prevention of obesity and related diseases. In one
embodiment, the present invention provides a method for preventing
or delaying the progression of impaired glucose tolerance (IGT) to
non-insulin requiring type 2 diabetes, for preventing or delaying
the progression of non-insulin requiring type 2 diabetes to insulin
requiring diabetes, for treating obesity and for regulating the
appetite. Melanocortin 4 receptor agonists have also been
implicated in the treatment of diseases selected from
atherosclerosis, hypertension, diabetes, type 2 diabetes, impaired
glucose tolerance (IGT), dyslipidemia, coronary heart disease,
gallbladder disease, gall stone, osteoarthritis, cancer, sexual
dysfunction and the risk of premature death. In one embodiment, the
invention thus provides a method of treating the above diseases or
states, the method comprising administering to a subject in need
thereof a therapeutically effective amount of an melanocortin 4
receptor agonist conjugate of the present invention.
[0262] In still another embodiment, the invention relates to the
use of a melanocortin 4 receptor agonist conjugate of the present
invention in the manufacture of a medicament for the treatment of
the above mentioned diseases or states.
[0263] Factor VII compounds have been implicated in the treatment
of disease related to coagulation, and biological active Factor VII
compounds in particular have been implicated in the treatment of
hemophiliacs, hemophiliacs with inhibitors to Factor VIII and IX,
patients with thrombocytopenia, patients with thrombocytopathies,
such as Glanzmann's thrombastenia platelet release defect and
strorage pool defects, patient with von Willebrand's disease,
patients with liver disease and bleeding problems associated with
traumas or surgery. Biologically inactive Factor VII compounds have
been implicated in the treatment of patients being in
hypercoagluable states, such as patients with sepsis, deep-vein
thrombosis, patients in risk of myocardial infections or thrombotic
stroke, pulmonary embolism, patients with acute coronary syndromes,
patients undergoing coronary cardiac, prevention of cardiac events
and restenosis for patient receiving angioplasty, patient with
peripheral vascular diseases, and acute respiratory distress
syndrome. In one embodiment, the invention thus provides a method
for the treatment of the above mentioned diseases or states, the
method comprising administering to a subject in need thereof a
therapeutically effective amount of a Factor VII compound conjugate
according to the present invention.
[0264] In another embodiment, the invention provides the use of a
Factor VII compound conjugate according to the present invention in
the manufacture of a medicament used in the treatment of the above
mentioned diseases or states.
[0265] Many diseases are treated using more than one medicament in
the treatment, either concomitantly administered or sequentially
administered. It is therefore within the scope of the present
invention to use the peptide conjugates of the present invention in
therapeutic methods for the treatment of one of the above mentioned
diseases in combination with one or more other therapeutically
active compound normally used to in the treatment said disease. By
analogy, it is also within the scope of the present invention to
use the peptide conjugates of the present invention in combination
with other therapeutically active compounds normally used in the
treatment of one of the above mentioned diseases in the manufacture
of a medicament for said disease.
[0266] In another embodiment, the present invention provides the
use of conjugated peptides of the present invention in
diagnostics.
[0267] .alpha.-amino acid amides are, as mentioned previously,
particular well-suited as a nucleophile in the methods of the
present invention. In one embodiment, the invention thus provides
compounds according to formula (I)
##STR00090##
wherein A and E independently represent C.sub.1-6alkylene,
C.sub.2-6alkenylene, C.sub.2-6alkynylene or arylene, all of which
may optionally be substituted with one or more substituents
selected from halogen, amino, cyano and nitro; B and D represents
--C(O)-- or --NH-- with the proviso that when B represents --C(O)--
then D must represent --NH--, and when B represents --NH-- then D
must represent --C(O)--; and G represents hydrogen or
C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynyl or aryl, all of
which may optionally be substituted with one or more substituents
selected from halogen, amino, cyano and nitro.
[0268] In one embodiment, A and E independently represent
C.sub.1-6alkylene, such as methylene, ethylene, propylene,
butylenes, pentylene or hexylene, or arylene, such as
phenylene.
[0269] In one embodiment, G represents hydrogen or methyl, ethyl,
propyl or butyl.
[0270] Particular examples of a compound of formula I include
[0271] (2S)-2-Amino-6-(4-oxo-4-phenylbutyrylamino)hexanoic acid
amide, [0272] 4-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide,
[0273] (2S)-2-Amino-6-(4-oxo-4-(4-chlorophenylbutyrylamino)hexanoic
acid amide, [0274]
3-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide, and [0275]
2-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide
[0276] In another embodiment, the invention provides compounds
according to formula II
##STR00091##
wherein J and L independently represent C.sub.1-6alkylene,
C.sub.2-6alkenylene, C.sub.2-6alkynylene or arylene, all of which
may optionally be substituted with one or more substituents
selected from halogen, amino, cyano and nitro; and M represents
hydrogen or C.sub.1-6alkyl.
[0277] In one embodiment, J and L independently represent
C.sub.1-6alkylene, such as methylene, ethylene, propylene,
butylenes, pentylene or hexylene, or arylene, such as
phenylene.
[0278] In one embodiment, M represents hydrogen or methyl, ethyl,
propyl or butyl.
[0279] In one embodiment, the compounds of formula II are selected
from amongst [0280]
(2S)-Amino-3-[4-(2-oxopropoxy)phenyl]propionamide, [0281]
(2S)-Amino-3-[4-(2-oxobutoxy)phenyl]propionamide, [0282]
(2S)-Amino-3-[4-(2-oxopentoxy)phenyl]propionamide, and [0283]
(2S)-Amino-3-[4-(4-oxopentoxy)phenyl]propionamide.
[0284] In still another embodiment, the invention provides
compounds according to formula III
##STR00092##
wherein Q represents represent C.sub.1-6alkylene,
C.sub.2-6alkenylene, C.sub.2-6alkynylene or arylene, all of which
may optionally be substituted with one or more substituents
selected from halogen, amino, cyano and nitro; and T represents
hydrogen or C.sub.1-6alkyl.
[0285] In one embodiment, Q represents C.sub.1-6alkylene, such as
methylene, ethylene, propylene, butylenes, pentylene or hexylene,
or arylene, such as phenylene.
[0286] In one embodiment, T represents hydrogen or methyl, ethyl,
propyl or butyl.
[0287] In still another embodiment, the invention provides
compounds according to formula IV
##STR00093##
wherein J'' and L'' independently represent C.sub.1-6alkylene or
arylene, all of which may optionally be substituted with one or
more substituents selected from halogen amino, cyano and nitro.
[0288] In one embodiment J and L independently represent methylene
or ethylene.
[0289] In one embodiment, the compounds of formula IV are selected
from amongst [0290] (S)-2-amino-3-(4-(propargyloxy)phenyl)propionyl
amide.
Pharmaceutical Compositions
[0291] Another object of the present invention is to provide a
pharmaceutical composition comprising a compound of formula
[a]which is present in a concentration from 10.sup.-12 mg/ml to 200
mg/ml, such as e.g. 10.sup.-10 mg/ml to 5 mg/ml and wherein said
composition has a pH from 2.0 to 10.0. The composition may further
comprise a buffer system, preservative(s), tonicity agent(s),
chelating agent(s), stabilizers and surfactants. In one embodiment
of the invention the pharmaceutical composition is an aqueous
composition, i.e. composition comprising water. Such composition is
typically a solution or a suspension. In a further embodiment of
the invention the pharmaceutical composition is an aqueous
solution. The term "aqueous composition" is defined as a
composition comprising at least 50% w/w water. Likewise, the term
"aqueous solution" is defined as a solution comprising at least 50%
w/w water, and the term "aqueous suspension" is defined as a
suspension comprising at least 50% w/w water.
[0292] In another embodiment the pharmaceutical composition is a
freeze-dried composition, whereto the physician or the patient adds
solvents and/or diluents prior to use.
[0293] In another embodiment the pharmaceutical composition is a
dried composition (e.g. freeze-dried or spray-dried) ready for use
without any prior dissolution.
[0294] In a further aspect the invention relates to a
pharmaceutical composition comprising an aqueous solution of a
compound of formula [a], and a buffer, wherein said compound of
formula [a] is present in a concentration from 0.1-100 mg/ml or
above, and wherein said composition has a pH from about 2.0 to
about 10.0.
[0295] In another embodiment of the invention the pH of the
composition is selected from the list consisting of 2.0, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,
3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,
4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,
6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,
7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and
10.0.
[0296] In a further embodiment of the invention the buffer is
selected from the group consisting of sodium acetate, sodium
carbonate, citrate, glycylglycine, histidine, glycine, lysine,
arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate,
sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine,
tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric
acid, aspartic acid or mixtures thereof. Each one of these specific
buffers constitutes an alternative embodiment of the invention.
[0297] In a further embodiment of the invention the composition
further comprises a pharmaceutically acceptable preservative. In a
further embodiment of the invention the preservative is selected
from the group consisting of phenol, o-cresol, m-cresol, p-cresol,
methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,
2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl
alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid,
imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol,
ethyl p-hydroxybenzoate, benzethonium chloride, chlorphenesine
(3p-chlorphenoxypropane-1,2-diol) or mixtures thereof. In a further
embodiment of the invention the preservative is present in a
concentration from 0.1 mg/ml to 20 mg/ml. In a further embodiment
of the invention the preservative is present in a concentration
from 0.1 mg/ml to 5 mg/ml. In a further embodiment of the invention
the preservative is present in a concentration from 5 mg/ml to 10
mg/ml. In a further embodiment of the invention the preservative is
present in a concentration from 10 mg/ml to 20 mg/ml. Each one of
these specific preservatives constitutes an alternative embodiment
of the invention. The use of a preservative in pharmaceutical
compositions is well-known to the skilled person. For convenience
reference is made to Remington: The Science and Practice of
Pharmacy, 20.sup.th edition, 2000.
[0298] In a further embodiment of the invention the composition
further comprises an isotonic agent. In a further embodiment of the
invention the isotonic agent is selected from the group consisting
of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an
amino acid (e.g. L-glycine, L-histidine, arginine, lysine,
isoleucine, aspartic acid, tryptophan, threonine),
[0299] an alditol (e.g. glycerol (glycerine), 1,2-propanediol
(propyleneglycol), 1,3-propanediol, 1,3-butanediol)
polyethyleneglycol (e.g. PEG400), or 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, soluble starch, hydroxyethyl starch and
carboxymethylcellulose-Na may be used. In one embodiment the sugar
additive is sucrose. Sugar alcohol is defined as a C.sub.4-C.sub.8
hydrocarbon having at least one --OH group and includes, for
example, mannitol, sorbitol, inositol, galactitol, dulcitol,
xylitol, and arabitol. In one embodiment the sugar alcohol additive
is mannitol. The sugars or sugar alcohols mentioned above may be
used individually or in combination. There is no fixed limit to the
amount used, as long as the sugar or sugar alcohol is soluble in
the liquid preparation and does not adversely effect the
stabilizing effects obtained using the methods of the invention. In
one embodiment, the sugar or sugar alcohol concentration is between
about 1 mg/ml and about 150 mg/ml. In a further embodiment of the
invention the isotonic agent is present in a concentration from 1
mg/ml to 50 mg/ml. In a further embodiment of the invention the
isotonic agent is present in a concentration from 1 mg/ml to 7
mg/ml. In a further embodiment of the invention the isotonic agent
is present in a concentration from 8 mg/ml to 24 mg/ml. In a
further embodiment of the invention the isotonic agent is present
in a concentration from 25 mg/ml to 50 mg/ml. Each one of these
specific isotonic agents constitutes an alternative embodiment of
the invention. The use of an isotonic agent in pharmaceutical
compositions is well-known to the skilled person. For convenience
reference is made to Remington: The Science and Practice of
Pharmacy, 20.sup.th edition, 2000.
[0300] In a further embodiment of the invention the composition
further comprises a chelating agent. In a further embodiment of the
invention the chelating agent is selected from salts of
ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic
acid, and mixtures thereof. In a further embodiment of the
invention the chelating agent is present in a concentration from
0.1 mg/ml to 5 mg/ml. In a further embodiment of the invention the
chelating agent is present in a concentration from 0.1 mg/ml to 2
mg/ml. In a further embodiment of the invention the chelating agent
is present in a concentration from 2 mg/ml to 5 mg/ml. Each one of
these specific chelating agents constitutes an alternative
embodiment of the invention. The use of a chelating agent in
pharmaceutical compositions is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 20.sup.th edition, 2000.
[0301] In a further embodiment of the invention the composition
further comprises a stabilizer. The use of a stabilizer in
pharmaceutical compositions is well-known to the skilled person.
For convenience reference is made to Remington: The Science and
Practice of Pharmacy, 20.sup.th edition, 2000.
[0302] More particularly, compositions of the invention are
stabilized liquid pharmaceutical compositions whose therapeutically
active components include a protein that possibly exhibits
aggregate formation during storage in liquid pharmaceutical
compositions. By "aggregate formation" is intended a physical
interaction between the protein molecules that results in formation
of oligomers, which may remain soluble, or large visible aggregates
that precipitate from the solution. By "during storage" is intended
a liquid pharmaceutical composition or composition once prepared,
is not immediately administered to a subject. Rather, following
preparation, it is packaged for storage, either in a liquid form,
in a frozen state, or in a dried form for later reconstitution into
a liquid form or other form suitable for administration to a
subject. By "dried form" is intended the liquid pharmaceutical
composition or composition is dried either by freeze drying (i.e.,
lyophilization; see, for example, Williams and Polli (1984) J.
Parenteral Sci. Technol. 38:48-59), spray drying (see Masters
(1991) in Spray-Drying Handbook (5th ed; Longman Scientific and
Technical, Essez, U.K.), pp. 491-676; Broadhead et al. (1992) Drug
Devel. Ind. Pharm. 18:1169-1206; and Mumenthaler et al. (1994)
Pharm. Res. 11:12-20), or air drying (Carpenter and Crowe (1988)
Cryobiology 25:459-470; and Roser (1991) Biopharm. 4:47-53).
Aggregate formation by a protein during storage of a liquid
pharmaceutical composition can adversely affect biological activity
of that protein, 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 protein-containing pharmaceutical composition is
administered using an infusion system.
[0303] The pharmaceutical compositions of the invention may further
comprise an amount of an amino acid base sufficient to decrease
aggregate formation by the protein during storage of the
composition. By "amino acid base" is intended an amino acid or a
combination of amino acids, where any given amino acid is present
either in its free base form or in its salt form. Where a
combination of amino acids is used, all of the amino acids may be
present in their free base forms, all may be present in their salt
forms, or some may be present in their free base forms while others
are present in their salt forms. In one embodiment, amino acids to
use in preparing the compositions of the invention are those
carrying a charged side chain, such as arginine, lysine, aspartic
acid, and glutamic acid. Any stereoisomer (i.e., L or D isomer, or
mixtures thereof) of a particular amino acid (methionine,
histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan,
threonine and mixtures thereof) or combinations of these
stereoisomers or glycine or an organic base such as but not limited
to imidazole, may be present in the pharmaceutical compositions of
the invention so long as the particular amino acid or organic base
is present either in its free base form or its salt form. In one
embodiment the L-stereoisomer of an amino acid is used. In one
embodiment the D-stereoisomer is used. Compositions of the
invention may also be formulated with analogues of these amino
acids. By "amino acid analogue" is intended a derivative of the
naturally occurring amino acid that brings about the desired effect
of decreasing aggregate formation by the protein during storage of
the liquid pharmaceutical compositions of the invention. Suitable
arginine analogues include, for example, aminoguanidine, ornithine
and N-monoethyl L-arginine, suitable methionine analogues include
ethionine and buthionine and suitable cysteine analogues include
S-methyl-L cysteine. As with the other amino acids, the amino acid
analogues are incorporated into the compositions in either their
free base form or their salt form. In a further embodiment of the
invention the amino acids or amino acid analogues are used in a
concentration, which is sufficient to prevent or delay aggregation
of the protein.
[0304] In a further embodiment of the invention methionine (or
other sulphuric amino acids or amino acid analogous) may be added
to inhibit oxidation of methionine residues to methionine sulfoxide
when the protein acting as the therapeutic agent is a protein
comprising at least one methionine residue susceptible to such
oxidation. By "inhibit" is intended minimal accumulation of
methionine oxidized species over time. Inhibiting methionine
oxidation results in greater retention of the protein in its proper
molecular form. Any stereoisomer of methionine (L or D isomer) or
any combinations thereof can be used. The amount to be added should
be an amount sufficient to inhibit oxidation of the methionine
residues such that the amount of methionine sulfoxide is acceptable
to regulatory agencies. Typically, this means that the composition
contains no more than about 10% to about 30% methionine sulfoxide.
Generally, this can be obtained by adding methionine such that the
ratio of methionine added to methionine residues ranges from about
1:1 to about 1000:1, such as 10:1 to about 100:1.
[0305] In a further embodiment of the invention the composition
further comprises a stabilizer selected from the group of high
molecular weight polymers or low molecular compounds. In a further
embodiment of the invention the stabilizer is selected from
polyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PVA),
polyvinylpyrrolidone, carboxy-/hydroxycellulose or derivates
thereof (e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins,
sulphur-containing substances as monothioglycerol, thioglycolic
acid and 2-methylthioethanol, and different salts (e.g. sodium
chloride). Each one of these specific stabilizers constitutes an
alternative embodiment of the invention.
[0306] The pharmaceutical compositions may also comprise additional
stabilizing agents, which further enhance stability of a
therapeutically active protein therein. Stabilizing agents of
particular interest to the present invention include, but are not
limited to, methionine and EDTA, which protect the protein against
methionine oxidation, and a nonionic surfactant, which protects the
protein against aggregation associated with freeze-thawing or
mechanical shearing.
[0307] In a further embodiment of the invention the composition
further comprises a surfactant. In a further embodiment of the
invention the surfactant is selected from a detergent, ethoxylated
castor oil, polyglycolized glycerides, acetylated monoglycerides,
sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block
polymers (e.g. poloxamers such as Pluronice F68, poloxamer 188 and
407, Triton X-100), polyoxyethylene sorbitan fatty acid esters,
polyoxyethylene and polyethylene derivatives such as alkylated and
alkoxylated derivatives (tweens, e.g. Tween-20, Tween-40, Tween-80
and Brij-35), monoglycerides or ethoxylated derivatives thereof,
diglycerides or polyoxyethylene derivatives thereof, alcohols,
glycerol, lectins and phospholipids (e.g. phosphatidyl serine,
phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl
inositol, diphosphatidyl glycerol and sphingomyelin), derivates of
phospholipids (e.g. dipalmitoyl phosphatidic acid) and
lysophospholipids (e.g. palmitoyl lysophosphatidyl-L-serine and
1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline,
serine or threonine) and alkyl, alkoxyl (alkyl ester), alkoxy
(alkyl ether)-derivatives of lysophosphatidyl and
phosphatidylcholines, e.g. lauroyl and myristoyl derivatives of
lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and
modifications of the polar head group, that is cholines,
ethanolamines, phosphatidic acid, serines, threonines, glycerol,
inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP,
lysophosphatidylserine and lysophosphatidylthreonine, and
glycerophospholipids (e.g. cephalins), glyceroglycolipids (e.g.
galactopyransoide), sphingoglycolipids (e.g. ceramides,
gangliosides), dodecylphosphocholine, hen egg lysolecithin, fusidic
acid derivatives--(e.g. sodium tauro-dihydrofusidate etc.),
long-chain fatty acids and salts thereof C.sub.6-C.sub.12 (e.g.
oleic acid and caprylic acid), acylcarnitines and derivatives,
N-acylated derivatives of lysine, arginine or histidine, or
side-chain acylated derivatives of lysine or arginine, N-acylated
derivatives of dipeptides comprising any combination of lysine,
arginine or histidine and a neutral or acidic amino acid,
N-acylated derivative of a tripeptide comprising any combination of
a neutral amino acid and two charged amino acids, DSS (docusate
sodium, CAS registry no [577-11-7]), docusate calcium, CAS registry
no [128-49-4]), docusate potassium, CAS registry no [7491-09-0]),
SDS (sodium dodecyl sulphate or sodium lauryl sulphate), sodium
caprylate, cholic acid or derivatives thereof, bile acids and salts
thereof and glycine or taurine conjugates, ursodeoxycholic acid,
sodium cholate, sodium deoxycholate, sodium taurocholate, sodium
glycocholate,
N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, anionic
(alkyl-aryl-sulphonates) monovalent surfactants, zwitterionic
surfactants (e.g. N-alkyl-N,N-dimethylammonio-1-propanesulfonates,
3-cholamido-1-propyldimethylammonio-1-propanesulfonate, cationic
surfactants (quaternary ammonium bases) (e.g.
cetyl-trimethylammonium bromide, cetylpyridinium chloride),
non-ionic surfactants (e.g. Dodecyl .beta.-D-glucopyranoside),
poloxamines (e.g. Tetronic's), which are tetrafunctional block
copolymers derived from sequential addition of propylene oxide and
ethylene oxide to ethylenediamine, or the surfactant may be
selected from the group of imidazoline derivatives, or mixtures
thereof. Each one of these specific surfactants constitutes an
alternative embodiment of the invention.
[0308] The use of a surfactant in pharmaceutical compositions is
well-known to the skilled person. For convenience reference is made
to Remington: The Science and Practice of Pharmacy, 20.sup.th
edition, 2000.
[0309] It is possible that other ingredients may be present in the
pharmaceutical composition of the present invention. Such
additional ingredients may include wetting agents, emulsifiers,
antioxidants, bulking agents, tonicity modifiers, chelating agents,
metal ions, oleaginous vehicles, proteins (e.g., human serum
albumin, gelatine or proteins) and a zwitterion (e.g., an amino
acid such as betaine, taurine, arginine, glycine, lysine and
histidine). Such additional ingredients, of course, should not
adversely affect the overall stability of the pharmaceutical
composition of the present invention.
[0310] Pharmaceutical compositions containing a compound of formula
[a]according to the present invention may be administered to a
patient in need of such treatment at several sites, for example, at
topical sites, for example, skin and mucosal sites, at sites which
bypass absorption, for example, administration in an artery, in a
vein, in the heart, and at sites which involve absorption, for
example, administration in the skin, under the skin, in a muscle or
in the abdomen.
[0311] Administration of pharmaceutical compositions according to
the invention may be through several routes of administration, for
example, lingual, sublingual, buccal, in the mouth, oral, in the
stomach and intestine, nasal, pulmonary, for example, through the
bronchioles and alveoli or a combination thereof, epidermal,
dermal, transdermal, vaginal, rectal, ocular, for examples through
the conjunctiva, uretal, and parenteral to patients in need of such
a treatment.
[0312] Compositions of the present invention may be administered in
several dosage forms, for example, as solutions, suspensions,
emulsions, microemulsions, multiple emulsion, foams, salves,
pastes, plasters, ointments, tablets, coated tablets, rinses,
capsules, for example, hard gelatine capsules and soft gelatine
capsules, suppositories, rectal capsules, drops, gels, sprays,
powder, aerosols, inhalants, eye drops, ophthalmic ointments,
ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal
ointments, injection solution, in situ transforming solutions, for
example in situ gelling, in situ setting, in situ precipitating, in
situ crystallization, infusion solution, and implants.
[0313] Compositions of the invention may further be compounded in,
or attached to, for example through covalent, hydrophobic and
electrostatic interactions, a drug carrier, drug delivery system
and advanced drug delivery system in order to further enhance
stability of the compound of formula [a], increase bioavailability,
increase solubility, decrease adverse effects, achieve
chronotherapy well known to those skilled in the art, and increase
patient compliance or any combination thereof. Examples of
carriers, drug delivery systems and advanced drug delivery systems
include, but are not limited to, polymers, for example cellulose
and derivatives, polysaccharides, for example dextran and
derivatives, starch and derivatives, poly(vinyl alcohol), acrylate
and methacrylate polymers, polylactic and polyglycolic acid and
block co-polymers thereof, polyethylene glycols, carrier proteins,
for example albumin, gels, for example, thermogelling systems, for
example block co-polymeric systems well known to those skilled in
the art, micelles, liposomes, microspheres, nanoparticulates,
liquid crystals and dispersions thereof, L2 phase and dispersions
there of, well known to those skilled in the art of phase behaviour
in lipid-water systems, polymeric micelles, multiple emulsions,
self-emulsifying, self-microemulsifying, cyclodextrins and
derivatives thereof, and dendrimers.
[0314] Compositions of the present invention are useful in the
composition of solids, semisolids, powder and solutions for
pulmonary administration of compound of formula [a], using, for
example a metered dose inhaler, dry powder inhaler and a nebulizer,
all being devices well known to those skilled in the art.
[0315] Compositions of the current invention are specifically
useful in the composition of controlled, sustained, protracting,
retarded, and slow release drug delivery systems. More
specifically, but not limited to, compositions are useful in
composition of parenteral controlled release and sustained release
systems (both systems leading to a many-fold reduction in number of
administrations), well known to those skilled in the art. Even more
preferably, are controlled release and sustained release systems
administered subcutaneous. Without limiting the scope of the
invention, examples of useful controlled release system and
compositions are hydrogels, oleaginous gels, liquid crystals,
polymeric micelles, microspheres, nanoparticles,
[0316] Methods to produce controlled release systems useful for
compositions of the current invention include, but are not limited
to, crystallization, condensation, co-crystallization,
precipitation, co-precipitation, emulsification, dispersion, high
pressure homogenisation, encapsulation, spray drying,
microencapsulating, coacervation, phase separation, solvent
evaporation to produce microspheres, extrusion and supercritical
fluid processes. General reference is made to Handbook of
Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker,
New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99:
Protein Composition and Delivery (MacNally, E. J., ed. Marcel
Dekker, New York, 2000).
[0317] Parenteral administration may be performed by subcutaneous,
intramuscular, intraperitoneal or intravenous injection by means of
a syringe, optionally a pen-like syringe. Alternatively, parenteral
administration can be performed by means of an infusion pump. A
further option is a composition which may be a solution or
suspension for the administration of the Compound of formula [a] in
the form of a nasal or pulmonal spray. As a still further option,
the pharmaceutical compositions containing the compound of formula
[a]of the invention can also be adapted to transdermal
administration, e.g. by needle-free injection or from a patch,
optionally an iontophoretic patch, or transmucosal, e.g. buccal,
administration.
[0318] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference in
their entirety and to the same extent as if each reference were
individually and specifically indicated to be incorporated by
reference and were set forth in its entirety herein (to the maximum
extent permitted by law).
[0319] All headings and sub-headings are used herein for
convenience only and should not be construed as limiting the
invention in any way.
[0320] The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
[0321] The citation and incorporation of patent documents herein is
done for convenience only and does not reflect any view of the
validity, patentability, and/or enforceability of such patent
documents.
[0322] This invention includes all modifications and equivalents of
the subject matter recited in the claims appended hereto as
permitted by applicable law.
EXAMPLES
[0323] Following abbreviations are used for chemical groups:
##STR00094##
[0324] Following other abbreviations were used:
[0325] DMSO: Dimethylsulfoxide
[0326] CHCA: 4-Hydroxy-alpha-cyanocinnamic acid
##STR00095##
CPY: Carboxypeptidase Y.
HPLC-Methods:
Method 02-B4-4:
[0327] The RP-analyses was performed using an Alliance Waters 2695
system fitted with a Waters 2487 dualband detector. UV detections
at 214 nm and 254 nm were collected using a Symmetry300 C18, 5 um,
3.9 mm.times.150 mm column, 42.degree. C. The compounds are eluted
with a linear gradient of 5-95% acetonitrile in water which is
buffered with 0.05% trifluoroacetic acid over 15 minutes at a
flow-rate of 1.0 min/min.
Method 03-B1-1:
[0328] The RP-analysis was performed using a Waters 2690 systems
fitted with a Waters 996 diode array detector. UV detections were
collected at 214, 254, 276, and 301 nm on a 218TP54 4.6
mm.times.250 mm 5.mu. C-18 silica column (The Seperations Group,
Hesperia), which was eluted at 1 ml/min at 42.degree. C. The column
was equilibrated with 5% acetonitrile, which was buffered with 0.1%
trifluoroacetic acid, in a 0.1% aqueous solution of trifluoroacetic
acid in water. After injection, the sample was eluted by a gradient
of 0% to 90% acetonitrile, which was buffered with 0.1%
trifluoroacetic acid, in a 0.1% aqueous solution of trifluoroacetic
acid in water during 50 min.
[0329] Mass spectra for peptides were obtained on an Agilent 1100
Series in the range of 500-1800 Da or on Perkin Elmer PE API 100 in
the range of 500-2000 Da. Typically the found signals for m/z
correspond to a series of any of z=1, 2, 3, 4, 5, or 6. MALDI-TOF
spectra were obtained on a Bruker Daltonix autoflex.
[0330] The transacylating compound, e.g. the compound of the
formula
##STR00096##
and the conjugating moiety, Y-E-Z, may either be acquired
commercially or synthesized according to the following guidelines
in general methods below.
General Method (A):
[0331] A compound of the general formula
##STR00097##
wherein R' and R'' independently represents C.sub.1-15alkylene,
C.sub.2-15alkenylene, C.sub.2-15alkynylene,
C.sub.11-15heteroalkylene, C.sub.2-15heteroalkenylene,
C.sub.2-15heteroalkynylene, wherein one or more homocyclic aromatic
compound biradical or heterocyclic compound biradical may be
inserted, may be prepared from a suitable amino acid methyl ester
which is protected at the alpha-amino group by a suitable
protecting group PG as described in the literature (e.g. T. W.
Greene, P. G. M. Wuts, Protective groups in organic synthesis,
2.sup.nd ed., 1991 John Wiley & Sons, Inc. New York)
##STR00098##
by an acylation method, e.g. using an suitable acid, in which X may
or may not be protected by a suitable protective group, as
described in the literature (e.g. T. W. Greene, P. G. M. Wuts,
Protective groups in organic synthesis, 2.sup.nd ed., 1991 John
Wiley & Sons, Inc. New York)
##STR00099##
and a coupling reagent such as e.g. 1-hydroxybenzotriazole,
3,4-dihydro-3-hydroxybenzotriazin-4-one or 7-azabenzotriazole in
combination with e.g. a carbodiimide such as e.g.
diisopropylcarbodiimide or
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the
presence or absence of a suitable base such as e.g. triethylamine
or ethyldiisopropylamine to form the ester of type
##STR00100##
The ester may be transformed into the corresponding amide by
reaction with e.g. ammonia in a suitable solvent or mixture of
solvents such as e.g. water or N,N-dimethylformamide.
##STR00101##
The removal of all protective groups may be performed in one or
several steps by methods as described in the literature (e.g. T. W.
Greene, P. G. M. Wuts, Protective groups in organic synthesis,
2.sup.nd ed., 1991 John Wiley & Sons, Inc. New York)
##STR00102##
As defined in General Method (A)
##STR00103##
Amino acid methyl esters are generally commercially available, or
they may be synthesized by well-known methods.
General Method (B):
[0332] A compound of the general formula
##STR00104##
wherein R' and R'' are defined as above, may be prepared from a
suitable amino acid methyl ester which is protected at the
alpha-amino group by a suitable protecting group PG, as described
in the literature (e.g. T. W. Greene, P. G. M. Wuts, Protective
groups in organic synthesis, 2.sup.nd ed., 1991 John Wiley &
Sons, Inc. New York)
##STR00105##
by an alkylation of the aromatic hydroxyl group using an suitable
alcohol, in which X may or may not be protected by a suitable
protective group, as described in the literature (e.g. T. W.
Greene, P. G. M. Wuts, Protective groups in organic synthesis,
2.sup.nd ed., 1991 John Wiley & Sons, Inc. New York)
##STR00106##
under conditions which effect alkylation, as described in the
literature, e.g. Mitsunobu conditions such as e.g.
triphenylphosphine and ethyl azodicarboxylate to form the ester of
type
##STR00107##
The ester may be transformed into the corresponding amide by
reaction with e.g. ammonia in a suitable solvent or mixture of
solvents such as e.g. water or N,N-dimethylformamide.
##STR00108##
The removal of all protective groups may be performed in one or
several steps by methods as described in the literature (e.g. T. W.
Greene, P. G. M. Wuts, Protective groups in organic synthesis,
2.sup.nd ed., 1991 John Wiley & Sons, Inc. New York)
##STR00109##
[0333] As defined in General Method (B)
##STR00110##
General Method (C):
[0334] A compound of the general formula
##STR00111##
wherein R' and R'' are defined as above, may be prepared from a
suitable amino acid methyl ester which is protected at the
alpha-amino group by a suitable protecting group PG, as and
described in the literature, e.g. in T. W. Greene, P. G. M. Wuts,
Protective groups in organic synthesis, 2.sup.nd ed., 1991 John
Wiley & Sons, Inc. New York)
##STR00112##
by an alkylation of the aromatic hydroxyl group, using an suitable
alkylation reagent
##STR00113##
in which the anion of LG' is a suitable leaving group such as
halogenide or sulfonate and X may or may not be protected by a
suitable protective group as described in the literature, e.g. in
T. W. Greene, P. G. M. Wuts, Protective groups in organic
synthesis, 2.sup.nd ed., 1991 John Wiley & Sons, Inc. New York.
The reaction may take place under basic conditions, applying bases
such as e.g. potassium carbonate, diazabicylo[5,4,0]undec-5-ene, or
tert-butyltetramethyluanidine at a suitable temperature, typically
between -78.degree. C. and 200.degree. C.
##STR00114##
The ester may be transformed into the corresponding amide by
reaction with e.g. ammonia in a suitable solvent or mixture of
solvents such as e.g. water or N,N-dimethylformamide.
##STR00115##
The removal of all protective groups may be performed in one or
several steps by methods as described in the literature, e.g. in T.
W. Greene, P. G. M. Wuts, Protective groups in organic synthesis,
2.sup.nd ed., 1991 John Wiley & Sons, Inc. New York
##STR00116##
As defined in General Method (C)
##STR00117##
General Method (D):
[0335] A compound of the general formula
##STR00118##
wherein R' and R'' are defined as above, may be prepared from a
suitable acid which is protected at the alpha-amino group by a
suitable protecting group PG, as described in the literature, e.g.
in T. W. Greene, P. G. M. Wuts, Protective groups in organic
synthesis, 2.sup.nd ed., 1991 John Wiley & Sons, Inc. New
York
##STR00119##
by reaction with a suitable primary or secondary amine, in which X
may or may not be protected by a suitable protecting group, using
acylation conditions known to a person skilled in the art e.g. a
coupling reagent such as e.g. 1-hydroxybenzotriazole,
3,4-dihydro-3-hydroxybenzotriazin-4-one or 7-azabenzotriazole in
combination with e.g. a carbodiimide such as e.g.
diisopropylcarbodiimide or
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the
presence or absence of a suitable base such as e.g. triethylamine
or ethyldiisopropylamine to form an amide
##STR00120##
The removal of all protective groups may be performed in one or
several steps as described in the literature, T. W. Greene, P. G.
M. Wuts, Protective groups in organic synthesis, 2.sup.nd ed., 1991
John Wiley & Sons, Inc. New York
##STR00121##
As defined in general Method (D)
##STR00122##
General Method (E): Synthesis of Ketogroup-Containing Amino Acid
Amides from Cysteine
[0336] A conveniently N-protected cysteine derivative (for instance
an ester, N-(2,4-dimethoxybenzyl)amide or N-bis(cyclopropyl)methyl
amide) or conveniently N-protected cysteine amide is treated with a
carbonyl-group-containing alkylating agent
(R.sup.50CO(CH.sub.2).sub.nLG'', LG''=leaving group for
nucleophilic displacement selected from halogen, sulfonate
(--O--SO.sub.2--R.sup.51), dialkylsulfonium, phenyliodonium, or
hydroxy, wherein R.sup.51 represents C.sub.1-6alkyl, partially or
completely fluorinated C.sub.1-6alkyl, or aryl, optionally
substituted with alkyl, halogen, nitro, cyano, or acetamido, and
R.sup.50 represents hydrogen, alkyl, aryl, or heteroaryl, said aryl
or heteroaryl being optionally substituted once or several times
with C.sub.1-6alkoxy, hydroxy, halogen, cyano, acyl, alkyl, or
nitro, under suitable reaction conditions to yield an S-alkylated
cysteine derivative. This derivative is converted into an amino
acid amide by conversion of the acid derivative into an amide and
deprotection of the alpha-amino group. Suitable N-protecting groups
are for instance trityl, phthaloyl, or alkoxycarbonyl groups, such
as tert-butyloxycarbonyl
##STR00123##
wherein n represents an integer from 1 to 10. General Method (F):
Synthesis of Ketogroup-Containing Amino Acid Amides from Aspartic
or Glutamic Acid
[0337] Aspartic or glutamic acids can be selectively protected by
treatment of an N-alkoxycarbonyl derivative with formaldehyde, to
yield cyclic esters as shown below:
##STR00124##
These derivatives, in which R.sup.60 represents tert-butyl, benzyl,
2-chlorobenzyl, allyl, 2-(trimethylsilyl)ethyl,
2,2,2-trichloroethyl, or benzhydryl, can be converted to protected,
ketone-containing amino acid derivatives by activation of the
carboxylic acid (LvG representing halogen, aryloxy, or
heteroaryloxy) and reaction with a carbon nucleophile
R.sup.80-M.sup.1, in which R.sup.80 represents alkyl, aryl, or
heteroaryl, said aryl or heteroaryl being optionally substituted
once or several times with C.sub.1-6alkoxy, hydroxy, halogen,
cyano, acyl, alkyl, or nitro, and in which M.sup.1 represents an
alkali metal, Mg, Zn, Ti, Zr, Mn, Cu, Ce, or Ca, optionally in the
presence of a suitable catalyst. Reaction of the product with
ammonia and deprotection will yield the desired amino acid
amide
##STR00125##
Similarly, reaction of N-alkoxycarbonyl pyroglutamic acid esters,
in which R.sup.70 represents tert-butyl, benzyl, 2-chlorobenzyl,
allyl, 2-(trimethylsilyl)ethyl, 2,2,2-trichloroethyl, or
benzhydryl, and R.sup.80 represents lower alkyl, with nucleophilic
carbon reagents can yield protected, keto-group-containing amino
acid derivatives. Reaction of the product with ammonia and
deprotection will yield the desired amino acid amide:
##STR00126##
Similarly, suitably N-protected glutamic acid diesters as those
shown below, in which R.sup.90 represents lower alkyl, can be
selectively acylated at carbon to yield, after hydrolysis and
decarboxylation, protected derivatives of keto-group-containing
amino acids, which can be converted into amino acid amides using
standard procedures
##STR00127##
General Method (G)
[0338] A compound of the general formula
##STR00128##
wherein R''' represents C.sub.1-15alkylene, C.sub.2-15alkenylene,
C.sub.2-15alkynylene, C.sub.1-15heteroalkylene,
C.sub.2-15heteroalkenylene, C.sub.2-15heteroalkynylene, wherein one
or more homocyclic aromatic compound biradical or heterocyclic
compound biradical may be inserted, may be prepared from a suitable
protected primary or secondary amine
##STR00129##
in which PG may be a suitable protection group, as described in the
literature, e.g. in T. W. Greene, P. G. M. Wuts, Protective groups
in organic synthesis, 2.sup.nd ed., 1991 John Wiley & Sons,
Inc. New York, and wherein the anion of LG''' is a leaving group,
such as e.g. halogenide or sulfonate.
[0339] This amine is reacted with a suitable protected
hydroxylamine
##STR00130##
wherein PG' is a protecting group, which is chosen in a way that PG
can be removed from an amine without removal of PG' from the
hydroxylamine. Examples for that can be found in the literature,
e.g. in T. W. Greene, P. G. M. Wuts, Protective groups in organic
synthesis, 2.sup.nd ed., 1991 John Wiley & Sons, Inc. New
York.
[0340] The two components are reacted under basic conditions such
as e.g. sodium hydride at a suitable temperature such as e.g.
-78.degree. C. to 200.degree. C.
##STR00131##
The protecting group of the amine may be removed selectively with a
method described in the literature
##STR00132##
The amine may be acylated with a suitable acid and a coupling
reagent such as e.g. 1-hydroxybenzotriazole,
3,4-dihydro-3-hydroxybenzotriazin-4-one or 7-azabenzotriazole in
combination with e.g. a carbodiimide such as e.g.
diisopropylcarbodiimide or
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the
presence or absence of a suitable base such as e.g. triethylamine
or ethyldiisopropylamine to give an amide.
##STR00133##
Finally, the protecting group of the hydroxylamine may be removed
by a method described in the literature, e.g. in T. W. Greene, P.
G. M. Wuts, Protective groups in organic synthesis, 2.sup.nd ed.,
1991 John Wiley & Sons, Inc. New York
##STR00134##
General Method (H)
[0341] A compound of the general formula
##STR00135##
may be prepared from a suitable ester, in which R.sup.IV is
C.sub.1-10alkyl in a suitable solvent such as ethanol by addition
of hydrazine hydrate.
##STR00136##
General Method (J) Transacylation Reaction
[0342] At a suitable temperature such as e.g. 5-50.degree. C. or
room temperature, a solution of the peptide in question (final
concentration 1-10 mM) and the nucleophile in question (final
concentration 10 mM-2M) is dissolved or suspended in water
containing low concentrations of EDTA.
[0343] Organic solvents may be added to improve the solubility of
the reactants. The mixture may be buffered to a suitable pH-value
such as e.g. between pH 1 and pH 14, between e.g. between pH 3.5
and pH 9, between pH 6 and pH 8.5, with a suitable buffer such as
e.g. phosphate buffer or HEPES, or the pH can be maintained by
addition of base or acid. A suitable enzyme e.g. carboxypeptidase Y
is added to the said mixture of peptide and nucleophile. The
reaction may be stopped after a suitable time e.g. between 5 min
and 10 days, by changing temperature or pH-value, by adding organic
solvents, or by dialysis or gel filtration.
##STR00137##
[0344] The pH of choice is determined e.g. by the solubility of the
peptide to be conjugated and the activity of the enzyme to be used.
Solubility of peptides is to a large extent determined by the pKa
of the peptide. Normally, the solubility of a given peptide is at
its minimum when pH equals pKa of the peptide. It lies within the
skills of a skilled person to choose a pH at which to run the
reaction taking due care to the above considerations.
General Method (K) Oxime Formation
[0345] An oxime moiety may be formed by dissolving the
transacylated peptide in question, in which R.sup.V may be a
substituted or unsubstituted aromatic ring, a substituted or an
unsubstituted heteroaromatic ring, hydrogen, or C.sub.1-10alkyl, in
water. Organic solvents may be added to increase solubility. The
solution is buffered to a suitable pH-value such as e.g. between pH
0 and pH 14, between pH 3 and pH 6, or pH 5 and kept at a suitable
temperature such as e.g. 0-60.degree. C. The hydroxylamine in
question is added, and oxime moiety is formed according to the
reaction scheme below
##STR00138##
[0346] The pH of choice is determined e.g. by the solubility of the
peptide to be. Solubility of peptides is to a large extent
determined by the pKa of the peptide. Normally, the solubility of a
given peptide is at its minimum when pH equals pKa of the peptide.
It lies within the skills of a skilled person to choose a pH at
which to run the reaction taking due care to the above
consideration.
General Method (L) Hydrazone Formation
Hydrazone Formation (I)
[0347] An hydrazone moiety is formed by dissolving the
transacylated peptide in question, in which R.sup.VI may be a
substituted or unsubstituted aromatic ring, a substituted or an
unsubstituted heteroaromatic ring, hydrogen, or C.sub.1-10alkyl, in
water. The solution is buffered to a suitable pH-value such as e.g.
between pH 2 and pH 14 or between pH 0 and pH 4 and kept at a
suitable temperature such as e.g. 0-60.degree. C. The hydrazide in
question is added, whereby the hydrazone is formed
##STR00139##
Hydrazone Formation (II)
[0348] An hydrazone is formed by dissolving the transacylated
peptide in question, in which R.sup.VII may be a substituted or
unsubstituted aromatic ring, a substituted or an unsubstituted
heteroaromatic ring, hydrogen, or C.sub.1-10alkyl, in water. The
solution is buffered to a suitable pH-value such as e.g. between pH
2 and pH 14 or between pH 0 and pH 4 and kept at a suitable
temperature such as e.g. 0-60.degree. C. The hydrazine in question
is added, whereby the hydrazone is formed
##STR00140##
General Method (M) Isoxazole Formation
[0349] An isoxazole can be formed by reaction between a
nitril-oxide and an alkyne. The nitril-oxide is formed by addition
of a suitable oxidation-reagent such as e.g. bleach to an excess of
a suitable oxime. A solution of an excess of the freshly formed
nitrile-oxide may be added to the peptide in question.
##STR00141##
General Method (N) Triazole Formation
[0350] A triazole can be formed by reaction between an azide which
is attached to the group Z and an alkyne, which is attached to the
peptide in question, in the presence of Cu(I)-ions in a suitable
solvent such as water or a mixture of water and an organic solvent
such as e.g. acetonitrile. The triazole may be formed in two
possible regioisomers.
##STR00142##
General Method (O) Triazole Formation
[0351] A triazole can be formed by reaction between an alkyne which
is attached to the group Z and an azide, which is attached to the
peptide in question, in the presence of Cu(I)-ions in a suitable
solvent such as water or a mixture of water and an organic solvent
such as e.g. acetonitrile. The triazole may be formed in two
possible regioisomers.
##STR00143##
General Method (P) Amide Formation
[0352] An amide can be regioselectively formed by reaction of an
azide, which is covalently attached to a peptide with an ester,
containing a triphenylphosphine-moiety as it is described in e.g.
Tetrahedron Lett. 2003, 44, 4515-4518.
##STR00144##
General Method (Q) Amide Formation
[0353] An amide can be regioselectively formed by reaction of an
azide, which is covalently attached to a peptide with a thioester,
containing a diphenylphosphine-moiety as it is described in e.g. J.
Org. Chem. 2002, 67, 4993-4996.
##STR00145##
General Method (R) Arylalkyne Formation
[0354] An arylalkyne can be formed by reaction between an alkyne,
which is covalently attached to a peptide and a haloaryl compound
in the presence of a palladium catalyst, which is water-soluable,
as described in e.g. Bioconjugate Chemistry, 2004, 15, 231-234. The
haloaryl compound may be exchanged with the corresponding aryl
trifluorosulfonate.
##STR00146##
General Method (S) Arylalkyne Formation
[0355] An arylalkyne might be formed by reaction between a
haloaryl-moiety, which is covalently attached to a peptide and an
alkyne in the presence of a palladium catalyst, which is
water-soluable, as described in e.g. Bioconjugate Chemistry, 2004,
15, 231-234. Instead of the haloaryl-moiety a
trifluorosulfonyloxyaryl-moiety, which is attached to a peptide can
be used as well.
##STR00147##
General Method (T)
[0356] A compound of the general formula
##STR00148##
wherein R' and R'' are as defined above may be prepared from a
suitable amino acid, which is protected at the alpha-amino group,
with an acid-labile protecting group PG.sup.1 such as e.g. BOC or
trityl, and which is protected at the omega-amino group with a
base-labile protecting group PG.sup.2 such as e.g. Fmoc. The acid
may be attached to a Rink-amide resin using standard coupling
conditions known to a person skilled in the art, such as e.g. use
of a carbodiimide e.g. diisopropylcarbodiimide in the presence or
absence of a reagent such as e.g. 1-hydroxybenzotriazole,
1-hydroxy-7-azabenzotriazole or
3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazin and in the presence
or absence of a base such as e.g. triethylamine or
ethyldiisopropylamine. The protecting group at the omega-amine
PG.sup.2, may be removed under basic conditions described for the
particular protecting group in the literature such as e.g. T. W.
Greene, P. G. M. Wuts, Protective groups in organic synthesis,
2.sup.nd ed., 1991 John Wiley & Sons, Inc. New York.
##STR00149##
An acid can be attached to the omega amino moiety using standard
coupling conditions, such as e.g. use of a carbodiimide e.g.
diisopropylcarbodiimide in the presence or absence of a reagent
such as e.g. 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole
or 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazin and in the
presence or absence of a base such as e.g. triethylamine or
ethyldiisopropylamine. The intermediate may be cleaved from the
solid support under acidic conditions such as e.g. trifluoroacetic
acid or a 20-70% solution of trifluoroacetic acid in
dichloromethane to give the desired aminamide.
##STR00150##
General Method (U)
[0357] A compound of the general formula
##STR00151##
wherein R' and R'' are defined as above, may be prepared from a
suitable amino acid, which is protected with an acid labile
protecting group PG.sup.1, such as e.g. Boc or trityl, which is
reacted with an excess of ammonia in the presence of a coupling
reagent, such as e.g. a carbodiimide e.g. diisopropylcarbodiimide
in the presence or absence of a reagent such as e.g.
1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole or
3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazin.
##STR00152##
The phenolic hydroxyl group may be alkylated with a suitable
halogenide or sulfonate, in which R.sup.a is any suitable
substituted alkyl or aryl radical, in the presence of a suitable
base such as e.g. potassium carbonate or tetramethylguanidine. The
protecting group PG.sup.1 may be removed from the alpha amino acid
under acidic conditions and described in the literature for the
particular protecting group chosen e.g. in T. W. Greene, P. G. M.
Wuts, Protective groups in organic synthesis, 2.sup.nd ed., 1991
John Wiley & Sons, Inc. New York, to give the desired amino
amide.
##STR00153##
General Method (V) PEG-Reagent
[0358] A reagent of the general formula
##STR00154##
in which
##STR00155##
is E, as defined above, may be prepared from a suitable acid, which
may be activated by reaction with a suitable reagent or a
combination of reagents, such as e.g.
2-succinimido-1,1,3,3,-tetramethyluronium tetrafluoroborate (TSTU)
in a suitable solvent such as e.g. N,N-dimethylformamide. The
activated acid e.g. the obtained 2,5-dioxopyrrodin-1yl ester of
said acid may be reacted with commercially available PEG-reagents,
which are functionalized with a primary amine, optionally in the
presence of a suitable base such as e.g. ethyldiisopropylamine or
triethylamine.
##STR00156##
Example 1
(2S)-2-Amino-6-(4-oxo-4-phenylbutyrylamino)hexanoic Acid Amide
##STR00157##
[0359] Step A:
(2S)-2-tert-(Butoxycarbonylamino)-6-(4-oxo-4-phenylbutyrylamino)hexanoic
Acid Methyl Ester
##STR00158##
[0361] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(3.23 g, 16.8 mmol) was added to a solution of benzoylpropionic
acid (3.00 g, 16.8 mmol) and
3,4-dihydro-3-hydroxybenzotriazin-4-one (2.75 g, 16.8 mmol) in a
mixture of N,N-dimethylformamide (20 ml) and dichloromethane (20
ml). The reaction mixture was stirred for 20 min at room
temperature. The hydrochloride salt of BOC-Lys-OMe (5.00 g, 16.8
mmol) and ethyldiisopropylamine (8.65 ml, 50.5 mmol) were added
successively. The reaction mixture was stirred for 16 h. It was
diluted with ethyl acetate (300 ml) and washed with a
half-concentrated solution of sodium hydrogencarbonate (2.times.300
ml). The organic layer was dried over magnesium sulphate. The
solvent was removed in vacuo. The crude product was purified by
flash chromatography on silica (90 g), using ethyl acetate/heptane
2:1 as eluent, to give 2.41 g of
(2S)-2-tert-(butoxycarbonylamino)-6-(4-oxo-4-phenylbutyrylamino)hexanoic
acid methyl ester.
[0362] .sup.1H-NMR (CDCl.sub.3): .delta. 1.30-1.90 (m, 6H); 1.44
(s, 9H); 2.61 (t, 2H); 3.20 (q, 2H); 3.37 (t, 2H); 4.25 (m, 1H);
5.20 (br, 1H); 5.90 (br, 1H); 7.46 (m, 2H); 7.50 (m, 1H); 8.00 (d,
2H).
Step B:
[(1S)-1-Carbamoyl-5-(4-oxo-4-phenylbutyrylamino)pentyl]carbamic
Acid Tert-Butyl Ester
##STR00159##
[0364] A 25% solution of ammonia in water (25 ml) was added to
(2S)-2-tert-(butoxycarbonylamino)-6-(4-oxo-4-phenylbutyrylamino)hexanoic
acid methyl ester (0.70 g, 1.67 mmol). The reaction mixture was
stirred for 2 days at room temperature. The solvent was removed in
vacuo to give 0.56 g of
[(1S)-1-carbamoyl-5-(4-oxo-4-phenylbutyrylamino)pentyl]carbamic
acid tert-butyl ester.
[0365] .sup.1H-NMR (CDCl.sub.3): .delta. 0.90 (m, 6H); 2.75 (t,
2H); 3.20-3.50 (m, 4H); 4.15 (m, 1H); 7.35-7.60 (m, 3H); 8.00 (d,
2H).
Step C:
[0366] Trifluoroacetic acid (25 ml) was added to a solution of
[(1S)-1-carbamoyl-5-(4-oxo-4-phenylbutyrylamino)pentyl]carbamic
acid tert-butyl ester (0.56 g, 1.38 mmol) in dichloromethane (25
ml). The reaction mixture was stirred for 1 h at room temperature.
The solvent was removed. The crude product was purified by HPLC on
a RP-18 column, using a gradient of 20-45% acetonitrile in water,
containing 0.1% of trifluoracetic acid as buffer to give 92 mg of
the title compound with a purity of approx. 85%, which was used for
the further experiments.
[0367] .sup.1H-NMR (CDCl.sub.3): .delta. 1.40 (m, 4H); 1.70 (m,
2H); 2.46 (t, 2H); 3.00 (q, 2H); 3.23 (t, 2H); 3.70 (m, 1H); 7.53
(m, 3H); 7.65 (t, 1H); 7.83 (br, 1H); 7.90 (t, 1H); 8.00 (d, 2H);
8.05 (br, 3H). MS: m/z=306 [M+1].sup.+
4-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide
##STR00160##
[0368] Step A:
[0369] 1.65 g of the title compound was prepared as described for
(2S)-2-amino-6-(4-oxo-4-phenylbutyrylamino)hexanoic acid amide,
using 4-acetylbenzoic acid instead of benzoylpropionic acid.
[0370] .sup.1H-NMR (CDCl.sub.3): .delta. 1.40 (m, 2H); 1.60 (m,
2H); 1.80 (m, 2H); 2.62 (s, 3H); 3.30 (q, 2H); 3.75 (q, 1H); 7.55
(br, 1H); 7.85 (br, 1H); 7.97 (d, 2H); 8.05 (d, 2H); 8.10 (br, 3H);
8.65 (t, 1H).
S-Phenacylcysteine Amide Hydrochloride
Step A: S-Phenacyl-N-Boc-Cystein Methyl Ester
##STR00161##
[0372] To a solution of the N-Boc cysteine methyl ester (2.05 ml,
9.93 mmol) in MeCN (20 ml) at 0.degree. C. were added DIPEA (3.55
ml, 20.1 mmol), NaI (0.48 g, 3.20 mmol), and then a solution of
phenacyl bromide (2.41 g, 12.1 mmol) in MeCN (4 ml). The mixture
was stirred at room temperature for 19 h. Water (100 ml) and 1N
aqueous HCl (30 ml) was added, and the product was extracted
(3.times.AcOEt). The combined extracts were washed with brine,
dried (MgSO.sub.4), and concentrated under reduced pressure to
yield 4.37 g of an oil. Crystallization from AcOEt (approx 10 ml)
and heptane (approx 40 ml) at -20.degree. C. overnight yielded 3.49
g (99%) of the title methyl ester as a brown solid.
[0373] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.37 (s, 9H), 2.74 (dd,
J=9 Hz, 13 Hz, 1H), 2.89 (dd, J=5.5 Hz, 13 Hz, 1H), 3.62 (s, 3H),
4.03 (d, J=15 Hz, 1H), 4.14 (d, J=15 Hz, 1H), 4.22 (m, 1H), 7.33
(br d, J=8 Hz, 1H), 7.52 (m, 2H), 7.64 (m, 1H), 7.99 (m, 2H).
Step B: S-Phenacyl-N-Boc Cysteine Amide
##STR00162##
[0375] To a solution of S-phenacyl-N-Boc-cystein methyl ester (1.77
g, 5.01 mmol) in MeCN (30 ml) was added aqueous ammonia (50 ml,
25%; 12.5 g NH.sub.3). After stirring at room temperature for 71 h
no more starting material could be detected by TLC. The mixture was
concentrated under reduced pressure, and the residue was
resuspended in toluene and ethanol and concentrated again.
stripping with PhMe+EtOH. Crystallization from cold methanol
yielded 0.86 g (50%) of the title amide.
[0376] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.37 (s, 9H), 2.66 (dd,
J=9 Hz, 13 Hz, 1H), 2.83 (dd, J=5.5 Hz, 13 Hz, 1H), 4.07 (d, J=15
Hz, 1H), 4.10 (m, 1H), 4.12 (d, J=15 Hz, 1H), 6.88 (br d, J=8 Hz,
7.12 (br s, 1H), 7.35 (br s, 1H), 7.52 (t, J=8 Hz, 2H), 7.64 (m,
1H), 7.96 (m, 2H).
Step C S-Phenacylcysteine Amide Hydrochloride
##STR00163##
[0378] S-Phenacyl-N-Boc cysteine amide (0.70 g, 2.07 mmol) was
mixed with DCM (10 ml) and TFA (20 ml). After 30 min the mixture
was concentrated, and the residue was mixed with toluene and MeCN
and concentrated again. The residue was mixed with 1N HCl (1.5 ml),
ethanol, MeCN, and toluene and concentrated again. The residue was
suspended in boiling EtOH (approx 5 ml). Filtration and drying
yielded 0.18 g (32%) of the title hydrochloride as a light brown
solid. LCMS: only one product (HPLC, 210 nm), MH+=221
(product--water).
[0379] .sup.1H NMR (DMSO-d.sub.6): .delta. 2.93 (dd, J=7 Hz, 13 Hz,
1H), 3.06 (dd, J=6 Hz, 13 Hz, 1H), 3.97 (m, 1H), 4.33 (br s, 2H),
7.58 (m, 2H), 7.68 (m, 1H), 8.02 (m, 2H), 8.32 (br s, 3H).
Example 4
4-Acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide
##STR00164##
[0381] Rink-amide-resin (loading: 0.43 mmol/g, 6.66 g, 2.86 mmol)
was swelled with dichloromethane (50 ml). The solvent was removed.
A 20% solution of piperidine in N-methylpyrrolidinone was added (50
ml). The reactor was shaken for 20 min. The liquid was removed. The
resin was washed with N-methylpyrrolidinone (3.times.50 ml) and
dichloromethane (5.times.50 ml). A solution of BOC-Lys(FMOC)-OH
(5.37 g, 11.5 mmol) in N-methylpyrrolidinone (50 ml) and a solution
of 1-hydroxybenzotriazole (1.75 g, 11.5 mmol) in
N-methylpyrrolidinone (20 ml) were added successively.
Diisopropylcarbodiimide (1.79 ml, 11.5 mmol) and
ethyldiisopropylamine (1.96 ml, 11.5 mmol) were added. The reactor
was shaken at room temperature for 16 h. The liquid was removed.
The resin was washed with N-methylpyrrolidinone (3.times.50 ml) and
dichloromethane (3.times.50 ml). A solution of 4-acetylbenzoic acid
(2.82 g, 11.5 mmol) in N-methylpyrrolidinone (50 ml) and a solution
of 1-hydroxybenzotriazole (1.75 g, 11.5 mmol) in
N-methylpyrrolidinone (20 ml) were added successively.
Diisopropylcarbodiimide (1.79 ml, 11.5 mmol) and
ethyldiisopropylamine (1.96 ml, 11.5 mmol) were added. The reactor
was shaken at room temperature for 16 h. The resin was washed with
N-methylpyrrolidinone (3.times.50 ml) and dichloromethane
(3.times.50 ml). A solution of 50% of trifluoroacetic acid and 10%
triisopropylsilane in dichloromethane (50 ml) was added to the
resin. The reaction vessel was shaken for 1 h at room temperature.
The liquid was collected. The solvent was removed in vacuo. The
residue was redissolved in toluene (50 ml). The solvent was removed
in vacuo.
[0382] The crude products of 6 runs of the procedure described
above were combined. They were purified by HPLC-chromatography on a
C.sub.18-reversed phase column, using a gradient of 3-23% of
acetonitrile in water in a 0.1% buffer of trifluoroacetic acid to
afford 1.07 g of the trifluoroacetic acid salt of
4-acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide.
Example 5
1-[4-(2-(Aminooxy)ethyl)piperidin-1-yl]hexadecan-1-one
##STR00165##
[0383] Step 1:
4-[2-(Toluene-4-sulfonyloxy)ethyl]piperidine-1-carboxylic Acid
Tert-Butyl Ester
##STR00166##
[0385] Tosyl chloride (4.16 g, 21.8 mmol) was added to a solution
of commercially available 4-(2-hydroxyethyl)piperidine-1-carbocylic
ester tert-butyl ester (e.g. Aldrich 54, 724-7, 5.0 g, 21.8 mmol)
and triethylamine (4.25 ml, 30.5 mmol) in dichloromethane (100 ml).
The reaction mixture was stirred at room temperature for 16 h. It
was diluted with ethyl acetate (300 ml) and washed with a 10%
aqueous solution of sodium hydrogensulphate (200 ml). The aqueous
phase was extracted with ethyl acetate (150 ml). The combined
organic layers were washed with a saturated aqueous solution of
sodium hydrogencarbonate (250 ml) and dried over magnesium
sulphate. The solvent was removed in vacuo. The crude product was
purified by flash chromatography on silica (80 g), using ethyl
acetate/heptane first: 1:2 then 1:1 as eluent, to give 6.04 g of
4-[2-(toluene-4-sulfonyloxy)ethyl]piperidine-1-carboxylic acid
tert-butyl ester.
[0386] .sup.1H-NMR (CDCl.sub.3): .delta. 1.05 (m, 2H); 1.45 (s,
9H); 1.55 (m, 5H); 2.50 (s, 3H); 2.65 (t, 2H); 4.05 (m, 4H); 7.35
(d, 2H); 7.80 (d, 2H).
Step 2:
4-[2-(1,3-Dioxo-1,3-dihydroisoindol-2-yloxy)ethyl]piperidine-1-carboxylic
Acid Tert-Butyl Ester
##STR00167##
[0388] At 0.degree. C., a 60% suspension of sodium hydride in
mineral oil (0.69 g, 17.2 mmol) was added to a solution of
N-hydroxyphthalimide (2.80 g, 17.2 mmol) in N,N-dimethylformamide
(20 ml). The reaction mixture was stirred for 45 min at 0.degree.
C. A solution of
4-[2-(toluene-4-sulfonyloxy)ethyl]piperidine-1-carboxylic acid
tert-butyl ester (5.99 g, 15.6 mmol) in N,N-dimethylformamide (15
ml) and tetrabutylammonium iodide (0.17 g, 0.47 mmol) were added
successively. The reaction mixture was heated to 60.degree. C. for
2 days and cooled to room temperature. Water (5 ml) was added
carefully. The reaction mixture was diluted with ethyl acetate (250
ml) and washed with a 10% aqueous solution of sodium
hydrogensulphate (200 ml). The aqueous phase was extracted with
ethyl acetate (200 ml). The combined organic layers were washed
with a saturated aqueous solution of sodium hydrogencarbonate (150
ml) and dried over magnesium sulphate. The solvent was removed in
vacuo. The crude product was purified by flash chromatography on
silica (80 g), using ethyl acetate/heptane 1:1 as eluent to give
4.36 g of
4-[2-(1,3-dioxo-1,3-dihydroisoindol-2-yloxy)ethyl]piperidine-1-carboxylic
acid tert-butyl ester.
[0389] .sup.1H-NMR (CDCl.sub.3): .delta. 1.15 (m, 2H); 1.50 (s,
9H); 1.75 (m, 5H); 2.75 (m, 2H); 4.10 (m, 2H); 4.30 (t, 2H); 7.80
(m, 4H).
Step 3:
2-(2-(Piperidin-4-yl)ethoxy)isoindole-1,3-dione
##STR00168##
[0391] Trifluoroacetic acid (20 ml) was added to a solution of
4-[2-(1,3-dioxo-1,3-dihydroisoindol-2-yloxy)ethyl]piperidine-1-carboxylic
acid tert-butyl ester (4.26 g, 11.4 mmol) in dichloromethane (20
ml). The reaction mixture was stirred at room temperature for 50
min. The solvent was removed in vacuo. The residue was dissolved in
dichloromethane (50 ml) and the solvent was removed in vacuo. The
latter procedure was repeated twice to give 6.46 g of the crude
trifluoroacetate salt of
2-(2-(piperidin-4-yl)ethoxy)isoindole-1,3-dione.
[0392] MS: m/z=275 [M+1.sup.+]
[0393] .sup.1H-NMR (DMSO-d.sub.6): .delta. 1.30 (m, 2H); 1.65 (m,
2H); 1.90 (m, 3H); 2.90 (q, 2H); 3.30 (d, 2H); 4.20 (t, 2H); 7.90
(s, 4H); 8.30 (br, 1H); 8.65 (br, 1H).
Step 4:
2-[2-(1-(Hexadecanoyl)piperidin-4-yl)ethoxy]isoindole-1,3-dione
##STR00169##
[0395] At 0.degree. C.,
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.04
g, 5.44 mmol) was added to a solution of palmic acid (1.40 g, 5.44
mmol) and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazole (0.89 g,
5.44 mmol) in N,N-dimethylformamide (20 ml) and dichloromethane (20
ml). The reaction mixture was stirred at 0.degree. C. for 20 min. A
solution of the trifluoroacetate salt of
2-(2-(piperidin-4-yl)ethoxy)isoindole-1,3-dione (2.11 g, 5.44 mmol)
in N,N-dimethylformamide (5 ml) and ethyldiisopropylamine (6.19 ml,
38.1 mmol) were added successively. The reaction mixture was
stirred for 16 h, while it was warming up to room temperature. It
was diluted with ethyl acetate (150 ml) and was washed with a 10%
aqueous solution of sodium hydrogensulphate (150 ml). The aqueous
phase was extracted with ethyl acetate. The combined organic layers
were washed with a mixture of water (50 ml) and a saturated aqueous
solution of sodium hydrogencarbonate (50 ml) and dried over
magnesium sulphate. The crude product was purified by flash
chromatography on silica (40 g), using ethyl acetate/heptane 1:1 as
eluent to give 1.52 g of
2-[2-(1-(hexadecanoyl)piperidin-4-yl)ethoxy]isoindole-1,3-dione.
[0396] MS: m/z=513 [M+1.sup.+]
[0397] .sup.1H-NMR (DMSO-d.sub.6): .delta. 0.90 (t, 3H); 1.10 (m,
2H); 1.25 (m, 26H); 1.45 (m, 2H); 1.65 (m, 1H); 1.80 (m, 2H); 2.30
(t, 2H); 2.95 (t, 1H); 3.85 (m, 3H); 4.20 (t, 2H); 4.40 (d, 1H);
7.90 (s, 4H).
Step 5:
[0398] Hydrazine hydrate (0.14 ml, 2.96 mmol) was added to a
solution of
2-[2-(1-(hexadecanoyl)piperidin-4-yl)ethoxy]isoindole-1,3-dione
(1.52 g, 2.96 mmol) in ethanol (30 ml). The reaction mixture was
heated to reflux for 75 min and cooled to room temperature. The
formed precipitation was removed by filtration. The solvent of the
filtrate was removed in vacuo. The crude product was purified by
flash chromatography on silica (30 g), using a mixture of
dichloromethane/methanol/25% aqueous ammonia (100:10:1) as eluent,
to give 800 mg of
1-[4-(2-(aminooxy)ethyl)piperidin-1-yl]hexadecan-1-one.
[0399] MS: m/z=383 [M+1.sup.+]
[0400] .sup.1H-NMR (CDCl.sub.3): .delta. 0.80 (t, 3H); 1.25 (m,
2H); 1.60 (m, 26H); 1.70 (m, 4H); 1.65 (m, 3H); 2.708t, 2H); 2.60
(t, 1H); 3.05 (t, 1H); 3.80 (m, 3H); 4.60 (d, 1H).
Example 6
(S)-2-Aminopent-4-ynoic Acid Amide
##STR00170##
[0401] Step 1:
((S)-1-Carbamoylbut-3-ynyl)carbamic Acid Tert-Butyl Ester
[0402] At 0.degree. C.,
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (563
mg, 2.94 mmol) was added to a solution of commercially available
(S)-2-(tert-butyoxycarbonylaminopent-4-ynoic acid (e.g. Acros, 626
mg, 2.94 mmol) and 1-hydroxybenzotriazole (397 mg, 2.94 mmol) in
N,N-dimethylformamide (20 ml). The reaction mixture was stirred at
0.degree. C. for 20 min. A 25% aqueous solution of ammonia (2.38
ml) was added. The reaction mixture was stirred for 16 h, while
warming up to room temperature. It was diluted with ethyl acetate
(150 ml) and washed with a 10% aqueous solution of sodium
hydrogensulphate (150 ml). The aqueous phase was extracted with
ethyl acetate (2.times.100 ml). The combined organic layers were
washed with brine (250 ml) and dried over magnesium sulphate. The
solvent was removed in vacuo. The residue was dissolved in ethyl
acetate (100 ml) and washed with a mixture of brine (75 ml) and
water (75 ml). The aqueous phase was extracted with ethyl acetate
(2.times.50 ml). The combined organic layers were dried over
magnesium sulphate. The solvent was removed in vacuo. The crude
product was purified by flash chromatography on silica (50 g),
using dichloromethane/methanol (10:1) as eluent, to give 138 mg of
((S)-1-carbamoylbut-3-ynyl)carbamic acid tert-butyl ester.
[0403] .sup.1H-NMR (CDCl.sub.3): .delta. 1.40 (s, 9H); 2.15 (t,
1H); 2.70 (m, 1H); 2.90 (m, 1H); 4.40 (m, 1H); 5.70 (d, 1H); 6.50
(br, 1H); 6.90 (br, 1H).
Step 2
[0404] Trifluoroacetic acid (3 ml) was added to a solution of
((S)-1-carbamoylbut-3-ynyl)carbamic acid tert-butyl ester (138 mg,
0.65 mmol) in dichloromethane (3 ml). The reaction mixture was
stirred for 1.25 h at room temperature. The solvent was removed in
vacuo. The residue was dissolved in dichloromethane (40 ml) and the
solvent was removed in vacuo. The latter procedure was repeated
twice to give crude trifluoroacetate salt of
(S)-2-aminopent-4-ynoic acid amide, which was used for the
following experiments.
[0405] MS: m/z=113 [M+1.sup.+]
[0406] .sup.1H-NMR (DMSO-d.sub.6): .delta. 2.70 (m, 2H); 3.15 (t,
1H); 3.85 (m, 1H); 7.65 (s, 1H); 7.85 (s, 1H); 8.20 (br, 3H).
Example 7
(S)-2-(([Leu.sup.37]GLP-1-(7-37)yl)amino)pent-4-ynoic Amide
##STR00171##
[0408] A solution of [Leu.sup.37]GLP-1(7-37)ylalanine (0.348 mg,
100 nmol), trifluoroacetate salt of (S)-2-aminopent-4-ynoic acid
amide (2.26 mg, 10000 nmol), and hydroxypropyl-beta-cyclodextrin (4
mg) in a buffer of 250 mM HEPES and 5 mM EDTA (0.085 ml), which had
been adjusted to pH 7.5 prior its use, a 25% aqueous solution of
ammonia and 1 N hydrochloric acid (together 0.011 ml) was prepared,
having a pH of 7.96. A solution of CPY (1.0 U) in water (0.005 ml)
was added. The reaction mixture was left at room temperature. After
40 min a mass corresponding to
(S)-2-(([Leu.sup.37]GLP-1-(7-37)yl)amino)pent-4-ynoic amide could
be found in the MALDI-TOF besides masses corresponding to
[Leu.sup.37]GLP-1-(7-37)ylalanine, to [Leu.sup.37]GLP-1-(7-37)
peptide, and to
(S)-2-{(S)-2-(([Leu.sup.37]GLP-1-(7-37)yl)amino)pent-4-ynoylamino}-
pent-4-ynoyl amide.
[0409] MALDI-TOF (CHCA): m/z=3508, 3485, 3604, 3413.
Example 8
(2S)-2-Amino-3-(4-(prop-2-ynyloxy)phenyl)propionamide
##STR00172##
[0410] Step 1:
[(S)-1-Carbamoyl-2-(4-hydroxyphenyl)ethyl]-carbamic Acid Tert-Butyl
Ester
##STR00173##
[0412] Di-tert-butyl dicarbonate (15 g, 69 mmol) was added to a
solution of the hydrochloride salt of tyrosine amide (15 g, 69
mmol) in dioxane (140 ml) and a 1 N aqueous solution of sodium
hydroxide (140 ml). The reaction mixture was stirred for 16 h at
room temperature. It was diluted with a 10% aqueous solution of
sodium hydrogensulphate (200 ml) and extracted with ethyl acetate
(3.times.200 ml). The combined organic layers were washed with a
saturated aqueous solution of sodium hydrogencarbonate (100 ml) and
dried over magnesium sulphate. The solvent was removed in vacuo.
The crude product was purified by flash chromatography on silica
(400 g), using a mixture of dichloromethane/methanol (10:1) to give
8.17 g of [(S)-1-carbamoyl-2-(4-hydroxyphenyl)ethyl]-carbamic acid
tert-butyl ester.
[0413] MS: m/z=303 (M+Na).sup.+.
[0414] .sup.1H-NMR (DMSO-d.sub.6): .delta. 1.31 (s 9H); 2.80 (dd,
1H); 2.83 (dd, 1H); 4.00 (m, 1H); 6.62 (d, 2H); 6.70 (d, 1H); 6.97
(br, 1H); 7.03 (d, 2H); 7.31 (br, 1H); 9.14 (s, 1H).
Step 2:
[(S)-1-Carbamoyl-2-(4-(prop-2-ynyloxy)phenyl)ethyl]carbamic Acid
Tert-Butyl Ester
##STR00174##
[0416] A mixture of
[(S)-1-carbamoyl-2-(4-hydroxyphenyl)ethyl]-carbamic acid tert-butyl
ester (1.0 g, 3.57 mmol), tetrabutylammonium iodide (65 mg, 0.17
mmol), potassium carbonate (3.94 g, 29 mmol), propargyl bromide
(0.38 ml, 4.28 mmol) and N,N-dimethylformamide (15 ml) was heated
to 60.degree. C. for 16 h. It was cooled to room temperature,
diluted with water (30 ml) and acidified with a 10% aqueous
solution of sodium hydrogensulphate. The mixture was extracted with
ethyl acetate (2.times.100 ml). The combined organic layers were
washed with a saturated aqueous solution of sodium
hydrogencarbonate (200 ml) and dried over magnesium sulphate. The
solvent was removed in vacuo. The crude product was purified by
flash chromatography on silica (100 g), using a mixture of
dichloromethane/methanol (10:1) as eluent, to give 998 mg of
[(S)-1-carbamoyl-2-(4-(prop-2-ynyloxy)phenyl)ethyl]carbamic acid
tert-butyl ester.
[0417] MS: m/z=341 (M+Na).sup.+.
[0418] .sup.1H-NMR (DMSO-d.sub.6) .delta. 1.31 (s, 9H); 2.50 (s,
1H); 2.67 (dd, 1H); 2.91 (dd, 1H); 4.03 (m, 1H); 4.74 (s, 2H); 6.77
(d, 1H); 6.86 (d, 2H); 6.99 (s, 1H), 7.17 (d, 2H); 7.35 (s,
1H).
[0419] Trifluoroacetic acid (10 ml) was added to a solution of
[(S)-1-carbamoyl-2-(4-(prop-2-ynyloxy)phenyl)ethyl]carbamic acid
tert-butyl ester (998 mg, 3.13 mmol) in dichloromethane (10 ml).
The reaction mixture was stirred for 1.5 h at room temperature. The
solvent was removed. The residue was dissolved in dichloromethane
(30 ml). The solvent was removed. The latter procedure was repeated
twice to give 1.53 g of the trifluoroacetate salt of
(2S)-2-amino-3-(4-(prop-2-ynyloxy)phenyl)propionamide.
[0420] HPLC (method 02-B4-4): R.sub.f=5.62 min.
[0421] MS: m/z=219(M+1).sup.+.
[0422] .sup.1H-NMR (CDCl.sub.3) .delta. 2.51 (s, 1H); 3.02 (m, 2H);
3.90 (m, 1H); 4.78 (s, 2H); 6.95 (d, 2H); 7.20 (d, 2H); 7.56 (s,
1H); 7.87 (s, 1H); 8.10 (br, 3H).
Example 9
(S)-2-([Leu37]GLP-1(7-37)ylamino)-3-(4-(prop-2-ynyl)phenyl)propionamide
##STR00175##
[0423] Step 1:
[0424] [Leu.sup.37]GLP-1(7-37)ylalanine
[0425] [Leu.sup.37]GLP-1(7-37)ylalanine was prepared on an Applied
Biosystems 433A Peptide Synthesizer by standard Fmoc-strategy
starting with a commercially available Fmoc-Ala-Wang resin.
Following amino acid derivatives were used:
TABLE-US-00001 coupling no. amino acid derivative 1 Fmoc-Leu-OH 2
Fmoc-Arg(Pmc)-OH 3 Fmoc-Gly-OH 4 Fmoc-Lys(Boc)-OH 5 Fmoc-Val-OH 6
Fmoc-Leu-OH 7 Fmoc-Trp(Boc)-OH 8 Fmoc-Ala-OH 9 Fmoc-Ile-OH 10
Fmoc-Phe-OH 11 Fmoc-Glu(OtBu)-OH 12 Fmoc-Lys(Boc)-OH 13 Fmoc-Ala-OH
14 Fmoc-Ala-OH 15 Fmoc-Gln(Trt)-OH 16 Fmoc-Gly-OH 17
Fmoc-Glu(OtBu)-OH 18 Fmoc-Leu-OH 19 Fmoc-Tyr(tBu)-OH 20
Fmoc-Ser(tBu)-OH 21 Fmoc-Ser(tBu)-OH 22 Fmoc-Val-OH 23
Fmoc-Asp(OtBu)-OH 24 Fmoc-Ser(tBu)-OH 25 Fmoc-Thr(tBu)-OH 26
Fmoc-Phe-OH 27 Fmoc-Thr(tBu)-OH 28 Fmoc-Gly-OH 29 Fmoc-Glu(OtBu)-OH
30 Fmoc-Ala-OH 31 Fmoc-His(Trt)-OH
[0426] A mixture of trifluoroacetic acid (10 ml), water (0.265 ml)
and triisopropylsilane (0.265 ml) was added to the resin. It was
shaken for 1.5 h. The liquid was collected. The resin was washed
with trifluoroacetic acid (1 ml). The liquids were combined. The
solution was concentrated under a stream of nitrogen. Ether (40 ml)
was added. The precipitation was isolated by centrifugation. The
crude product was purified on a reversed phase C.sub.1B-column on a
HPLC, using a gradient of 37-65% acetonitrile in water in a buffer
of 0.1% trifluoroacetic acid, as eluent.
Step 2:
CPY-Reaction of
(2S)-2-Amino-3-(4-(prop-2-ynyloxy)phenyl)propionamide with
[Leu.sup.37]GLP-1(7-37)alanine
[0427] A mixture (0.100 ml final volume) of
[Leu.sup.37]GLP-1(7-37)ylalanine (1 mM final concentration) and the
trifluoroacetate salt of
(2S)-2-amino-3-(4-(prop-2-ynyloxy)phenyl)propionamide (100 mM final
concentration) and hydroxypropyl-beta-cyclodextrin (4 mg) in a
buffer, consisting of 250 mM HEPES and 5 mM EDTA, was adjusted to
pH 8, using a 1 N aqueous solution of sodium hydroxide. A solution
of carboxypeptidase Y (CPY, 200 U/ml, 0.005 ml, 1 U) was added to
obtain the desired final volume and concentrations. The mixture was
left for 3 h at room temperature.
[0428] MALDI-TOF (Matrix assisted laser desorption/ionization time
of flight mass spectroscopy): m/z=3612
((S)-2-([Leu37]GLP-1(7-37)ylamino)-3-(4-(prop-2-ynyl)phenyl)propionamide)
along with 3412 ([Leu.sup.37]GLP-1 peptide).
[0429] MS (electrospray): 1205 (M).sup.3+.
Example 10
(2S)-2-([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamino)-3-(4-(prop-2-ynyloxy)p-
henyl)propionamide
##STR00176##
[0430] Step 1:
[0431] [Glu.sup.3,Leu.sup.10]GLP-2ylleuccinylalanine
[0432] [Glu.sup.3,Leu.sup.10]GLP-2ylleuccinylalanine was prepared
on an Applied Biosystems 433A Peptide Synthesizer by standard
Fmoc-strategy starting with a commercially available Fmoc-Ala-Wang
resin. Following amino acid derivatives were used:
TABLE-US-00002 coupling no. amino acid derivative 1 Fmoc-Leu-OH 2
Fmoc-Asp(OtBu)-OH 3 Fmoc-Thr(tBu)-OH 4 Fmoc-Ile-OH 5
Fmoc-Lys(Boc)-OH 6 Fmoc-Thr(tBu)-OH 7 Fmoc-Gln(Trt)-OH 8
Fmoc-Ile-OH 9 Fmoc-Leu-OH 10 Fmoc-Trp(Boc)-OH 11 Fmoc-Asn(Trt)-OH
12 Fmoc-Ile-OH 13 Fmoc-Phe-OH 14 Fmoc-Asp(OtBu)-OH 15
Fmoc-Arg(Pmc)-OH 16 Fmoc-Ala-OH 17 Fmoc-Ala-OH 18 Fmoc-Leu-OH 19
Fmoc-Asn(Trt)-OH 20 Fmoc-Asp(OtBu)-OH 21 Fmoc-Leu-OH 22 Fmoc-Ile-OH
23 Fmoc-Thr(tBu)-OH 24 Fmoc-Asn(Trt)-OH 25 Fmoc-Leu-OH 26
Fmoc-Glu(OtBu)-OH 27 Fmoc-Asp(OtBu)-OH 28 Fmoc-Ser(tBu)-OH 29
Fmoc-Phe-OH 30 Fmoc-Ser(tBu)-OH 31 Fmoc-Gly-OH 32 Fmoc-Glu(OtBu)-OH
33 Fmoc-Ala-OH 34 Fmoc-His(Trt)-OH
[0433] A mixture of trifluoroacetic acid (10 ml), water (0.265 ml)
and triisopropylsilane (0.265 ml) was added to the resin. It was
shaken for 1.5 h. The liquid was collected. The resin was washed
with trifluoroacetic acid (1 ml). The liquids were combined. The
solution was concentrated under a stream of nitrogen. Ether (40 ml)
was added. The precipitation was isolated by centrifugation. The
crude product was purified on a reversed phase C.sub.1B-column on a
HPLC, using a gradient of 37-65% acetonitrile in water in a buffer
of 0.1% trifluoroacetic acid, as eluent.
[0434] HPLC: 8.81 min (method 02-B4-4).
[0435] MALDI-TOF: m/z=3946
[0436] MS: m/z=1317. 988, 790.
Step 2:
[0437] CPY-Reaction of
(2S)-2-Amino-3-(4-(prop-2-ynyloxy)phenyl)propionamide with
(([Glu.sup.3,Leu.sup.10]GLP-2yl)leucinyl)alanine
[0438] A mixture (1.5 ml final volume) of
(([Glu.sup.3,Leu.sup.10]GLP-2yl)leucinyl)alanine (1 mM final
concentration) and the trifluoroacetate salt of
(2S)-2-amino-3-(4-(prop-2-ynyloxy)phenyl)propionamide (6 mg, 150 mM
final concentration) and hydroxypropyl-beta-cyclodextrin (61 mg) in
a buffer, consisting of 250 mM HEPES and 5 mM EDTA, was adjusted to
pH 8, using a 1 N aqueous solution of sodium hydroxide. A solution
of carboxypeptidase Y (CPY, 800 U/ml, 0.019 ml, 15 U) was added to
obtain the desired final volume and concentrations. The mixture was
left for 3.5 h at room temperature. The mixture was diluted with
water to a volume of 10 ml. The product was isolated by
HPLC-purification, using a C.sub.1B-column and a gradient of 39-67%
acetonitrile in water, which was acidified with 0.1%
trifluoroacetic acid, to give
(2S)-2-([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamino)-3-(4-(prop-2-ynyloxy)-
phenyl)propionamide. Using an absorption coefficient of 1500000 at
214 nm, a yield of 2.5 mg was determined.
[0439] MALDI-TOF: 4073.
[0440] HPLC (system 02-b4-4): 9.14 min.
[0441] MS (electrospray): m/z=815, 1120, 1359.
Example 11
(S)-3-(4-((3-(3-Chlorophenyl)isoxazol-5-yl)methoxy)phenyl)-2-([Glu.sup.3
Leu.sup.10]GLP-2ylleucinylamino)propionic Amide
##STR00177##
[0443] Step 1:
3-Chlorobenzaldehyde Oxime
##STR00178##
[0445] A solution of hydroxylamine hydrochloride (3.68 g, 53 mmol)
in water (5 ml) was added to a solution of 3-chlorobenzaldehyde
(5.00 ml, 44 mmol) in ethanol (20 ml). A solution of sodium
hydroxide (2.64 g, 66 mmol) in water (5 ml) was added. The reaction
mixture was stirred at room temperature for 48 h. The reaction
mixture was given onto water/ice (150 ml). The formed precipitation
was isolated by filtration and dissolved in dichloromethane (200
ml). This solution was dried over magnesium sulphate. The solvent
was removed to give 3.88 g of 3-chlorobenzaldehyde oxime, which was
used without further purification.
[0446] Step 2:
[0447] A 10% solution of sodium hypochlorite (0.008 ml) was added
to a suspension of 3-chlorobenzaldehyde oxime (4.2 mg, 0.027 mmol)
in water (0.5 ml). The solution was left for 10 min at room
temperature. A solution of
(2S)-2-([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamino)-3-(4-(prop-2ynyloxy)p-
henyl)propionamide (1.1 mg, 0.00027 mmol) and triethylamine (0.003
ml) in water (0.5 ml) was added. The reaction mixture was left at
room temperature for 16 h. The crude product was purified on a
reversed phase C.sub.18-HPLC, using a gradient of 43-75%
acetonitrile in water in a 0.1% buffer of TFA.
[0448] HPLC (method 02-b4-4): 9.56 min.
[0449] MS (EI): m/z=1410 (M.sup.3+), 1054 (M.sup.4+) and 844
(M.sup.5+)
Example 12
(S)-2-Amino-3-[4-(2-oxopropoxy)phenyl]propionamide
##STR00179##
[0451] Step 1:
[(S)-1-Carbamoyl-2-(4-hydroxyphenyl)ethyl]-carbamic Acid Tert-Butyl
Ester
##STR00180##
[0453] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(6.82 g, 35.5 mmol) was added to a solution of BOC-protected
tyrosine (10.0 g, 35.5 mmol) and 1-hydroxybenzotriazole (5.44 g,
35.5 mmol) in a mixture of N,N-dimethylformamide (10 ml) and
dichloromethane (10 ml). The reaction mixture was stirred for 20
min. A 25% aqueous solution of ammonia in water was added. The
reaction mixture was stirred at room temperature for 16 h. It was
diluted with ethyl acetate (100 ml) and washed with water
(3.times.100 ml) and subsequently with a saturated aqueous solution
of sodium hydrogencarbonate (100 ml). It was dried over magnesium
sulphate. The solvent was removed in vacuo to give 4.24 g of
[(S)-1-carbamoyl-2-(4-hydroxyphenyl)ethyl]-carbamic acid tert-butyl
ester.
[0454] .sup.1H-NMR (DMSO-d.sub.6): .delta. 1.31 (s 9H); 2.80 (dd,
1H); 2.83 (dd, 1H); 4.00 (m, 1H); 6.62 (d, 2H); 6.70 (d, 1H); 6.97
(br, 1H); 7.03 (d, 2H); 7.31 (br, 1H); 9.14 (s, 1H).
[0455] Step 2:
{(S)-1-Carbamoyl-2-[4-(2-oxopropoxy)phenyl]ethyl}carbamic Acid
Tert-Butyl Ester
##STR00181##
[0457] To a mixture of
[(S)-1-carbamoyl-2-(4-hydroxyphenyl)ethyl]-carbamic acid tert-butyl
ester (3.00 g, 10.7 mmol) and potassium carbonate (7.40 g, 53.5
mmol) in N,N-dimethylformamide (50 ml) were added subsequently
chloroacetone (1.02 ml, 12.8 mmol) and tetrabutylammonium iodide
(197 mg, 0.54 mmol). The reaction mixture was heated to 90.degree.
C. for 16 h and cooled to room temperature. It was diluted with
water (100 ml) and acidified with a 10% solution of sodium
hydrogensulphate to pH 2. Ethyl acetate (300 ml) was added. The
phases were separated. The organic layer was washed with water
(3.times.150 ml) and dried over magnesium sulphate. The solvent was
removed in vacuo to give 2.65 g of
{(S)-1-carbamoyl-2-[4-(2-oxopropoxy)phenyl]ethyl}carbamic acid
tert-butyl ester.
[0458] MS: m/z=359 (M+Na.sup.+)
[0459] .sup.1H-NMR (DMSO-d.sub.6) .delta. 1.30 (s, 9H); 2.10 (s,
3H); 2.70 (dd, 1H); 2.90 (dd, 1H); 3.95 (br, 1H); 4.00 (m, 1H);
4.75 (s, 2H); 6.80 (d, 2H); 7.00 (br, 1H); 7.20 (d, 2H); 7.35 (br,
1H).
[0460] Step 3:
[0461] Trifluoroacetic acid (50 ml) was added to a solution of
{(S)-1-carbamoyl-2-[4-(2-oxopropoxy)phenyl]ethyl}carbamic acid
tert-butyl ester (2.65 g, 7.88 mmol) in dichloromethane (50 ml).
The reaction mixture was stirred for 1 h at room temperature. The
solvent was removed in vacuo. The residue was dissolved in
dichloromethane (50 ml) and the solvent was removed in vacuo. The
latter procedure was repeated once. The crude product was purified
by C-18 reversed phase chromatography on a HPLC, using a gradient
of 13-33% acetonitrile in water in a buffer of trifluoroacetic acid
(0.1%) to give 460 mg of
(S)-2-amino-3-[4-(2-oxopropoxy)phenyl]propionamide.
[0462] MS: m/z=237 (M.sup.+)
[0463] .sup.1H-NMR (DMSO-d.sub.6, TFA-salt) .delta. 2.20 (s, 3H);
2.80-3.10 (m, 2H); 3.90 (m, 1H); 4.80 (s, 2H); 6.90 (d, 2H); 7.20
(d, 2H); 7.55 (br, 1H); 7.90 (br, 1H); 8.10+ (br, 3H).
Example 13
(S)-2-([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamino)-3-(4-(2-oxopropoxy)phen-
yl)propionic Amide
##STR00182##
[0465] A solution of
(([Glu.sup.3,Leu.sup.10]GLP-2yl)leucinyl)alanine (0.50 mg, 127
pmol) in water (0.040 ml) and an 1 N aqueous solution of sodium
hydroxide (0.003 ml) was added to a solution of the
trifluoroacetate salt of
(S)-2-amino-3-[4-(2-oxopropoxy)phenyl]propionamide (13.3 mg, 0.038
mmol) in an aqueous buffer containing 250 mM HEPES and 5 mM EDTA,
which had been adjusted to pH 8 with sodium hydroxide. The solution
was adjusted to pH 8 with a 1 N aqueous solution of sodium
hydroxide. The solution was diluted to a final volume of 0.127 ml
with an aqueous buffer containing 250 mM HEPES and 5 mM EDTA, which
had been adjusted to pH 8 with sodium hydroxide. A solution of CPY
in water (0.005 ml, 1 U) was added. The reaction mixture was left
at room temperature for 16 h. The MS analysis showed the formation
of the product with the desired mass.
[0466] MALDI-TOF: m/z=4090.321
[0467] MS: m/z=1365, 1024
[0468] HPLC (Method 03-b6-1): 30.69 min.
Example 14
(2S)-2-([Glu.sup.3]GLP-2ylleucinylamino)-3-(4-(prop-2-ynyloxy)phenyl)propi-
onamide
##STR00183##
[0470] Step 1:
[0471] (([Glu.sup.3]GLP-2yl)leucinyl)alanine
[0472] (([Glu.sup.3]GLP-2yl)leucinyl)alanine was prepared as
described for (([Glu.sup.3,Leu.sup.10]GLP-2yl)leucinyl)alanine from
commercially available Fmoc-Ala-Wang resin. Following amino acid
derivatives were used:
TABLE-US-00003 coupling no. amino acid derivative 1 Fmoc-Leu-OH 2
Fmoc-Asp(OtBu)-OH 3 Fmoc-Thr(tBu)-OH 4 Fmoc-Ile-OH 5
Fmoc-Lys(Boc)-OH 6 Fmoc-Thr(tBu)-OH 7 Fmoc-Gln(Trt)-OH 8
Fmoc-Ile-OH 9 Fmoc-Leu-OH 10 Fmoc-Trp(Boc)-OH 11 Fmoc-Asn(Trt)-OH
12 Fmoc-Ile-OH 13 Fmoc-Phe-OH 14 Fmoc-Asp(OtBu)-OH 15
Fmoc-Arg(Pmc)-OH 16 Fmoc-Ala-OH 17 Fmoc-Ala-OH 18 Fmoc-Leu-OH 19
Fmoc-Asn(Trt)-OH 20 Fmoc-Asp(OtBu)-OH 21 Fmoc-Leu-OH 22 Fmoc-Ile-OH
23 Fmoc-Thr(tBu)-OH 24 Fmoc-Asn(Trt)-OH 25 Fmoc-Met-OH 26
Fmoc-Glu(OtBu)-OH 27 Fmoc-Asp(OtBu)-OH 28 Fmoc-Ser(tBu)-OH 29
Fmoc-Phe-OH 30 Fmoc-Ser(tBu)-OH 31 Fmoc-Gly-OH 32 Fmoc-Glu(OtBu)-OH
33 Fmoc-Ala-OH 34 Fmoc-His(Trt)-OH
[0473] HPLC: 8.60 min (method 02-B4-4).
[0474] MALDI-TOF: m/z=3964.17.
[0475] Step 2:
[0476] CPY-Reaction of
(2S)-2-Amino-3-(4-(prop-2-ynyloxy)phenyl)propionamide with
(([Glu.sup.3]GLP-2yl)leucinyl)alanine:
[0477] (([Glu.sup.3]GLP-2yl)leucinyl)alanine was prepared by
standard solid phase peptide synthesis on an ABI-433A Peptide
Synthesizer using a FMOC-strategy, known to a person skilled in the
art. A mixture (7 ml final volume) of
(([Glu.sup.3]GLP-2yl)leucinyl)alanine (1 mM final concentration)
and the trifluoroacetate salt of
(2S)-2-amino-3-(4-(prop-2-ynyloxy)phenyl)propionamide (28 mg, 150
mM final concentration) and hydroxypropyl-beta-cyclodextrin (284
mg) in a buffer, consisting of 250 mM HEPES and 5 mM EDTA, was
adjusted to pH 8, using a 1 N aqueous solution of sodium hydroxide.
A solution of carboxypeptidase Y (CPY, 800 U/ml, 0.088 ml, 70 U)
was added to obtain the desired final volume and concentrations.
The mixture was left for 100 min at room temperature. The mixture
was diluted with water to a volume of 10 ml. The product was
isolated by HPLC-purification, using a C.sub.18-column and a
gradient of 36-75% acetonitrile in water, which was acidified with
0.1% trifluoroacetic acid, to give
(2S)-2-([Glu.sup.3]GLP-2ylleucinylamino)-3-(4-(prop-2-ynyloxy)phenyl)prop-
ionamide. Using an absorption coefficient of 1500000 at 214 nm, a
yield of 9.9 mg was determined.
[0478] MALDI-TOF: 4096 (M.sup.+)
[0479] HPLC (system 02-b4-4): 8.97 min
[0480] MS (electrospray): m/z=1366 (M.sup.3+), 1024 (M.sup.4+), and
819 (M.sup.5+).
Example 15
(S)-3-(4-((3-(3-Chlorophenyl)isoxazol-5-yl)methoxy)phenyl)-2-([Glu.sup.3]G-
LP-2ylleucinylamino)propionic Amide
##STR00184##
[0482] A 10% solution of sodium hypochlorite (0.062 ml) was added
to a suspension of 3-chlorobenzaldehyde oxime (32 mg, 0.205 mmol)
in water (4.2 ml). The mixture was left for 10 min at room
temperature and added to a solution of
(2S)-2-([Glu.sup.3]GLP-2ylleucinylamino)-3-(4-(prop-2-ynyloxy)phenyl)prop-
ionamide (8.4 mg, 0.0021 mmol) and triethylamine (0.025 ml) in
water (4.7 ml). The reaction mixture was left at room temperature
for 16 h. The crude product was purified on a reversed phase
C.sub.18-HPLC, using a gradient of 40-80% acetonitrile in water in
a 0.1% buffer of TFA. Using an absorption coefficient of 1500000 at
214 nm, a yield of 0.132 mg was determined.
[0483] MALDI-TOF: 4244 (M.sup.+)and 4228 (M-O.sup.+)
[0484] HPLC (method 02-b4-4): 9.41 min.
[0485] MS (EI): m/z=1417 (M.sup.3+) and 1062 (M.sup.4+).
Example 16
3-(3-(3-((4-((S)-2-Carbamoyl-3-([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamino-
)ethyl)phenoxyl)methyl)-isoxazol-3-yl)benzylcarbamoyl)propionic
Acid
##STR00185##
[0486] Step 1:
(3-Hydroxymethylbenzyl)carbamic Acid Tert Butylester
##STR00186##
[0488] At 0.degree. C., ethyl chloroformate (1.93 ml, 20 mmol) was
added to a solution of 3-(tert-butoxycarbonylaminomethyl)benzoic
acid (5.0 g, 20 mmol) and triethylamine (3.33 ml, 24 mmol) in
tetrahydrofuran (30 ml). The reaction mixture was stirred for 40
min at 0.degree. C., and the formed precipitation was filtered off.
The filtrate was cooled to 0.degree. C. A 2.0 M solution of lithium
borohydride in THF (25 ml, 50 mmol) was added. The reaction mixture
was stirred for 16 h, while warming up to room temperature. Water
was added carefully, until no gas was formed. A 10% solution of
sodium hydrogensulphate (10 ml) was added. A saturated solution of
sodium hydrogen carbonate (200 ml) was added. The mixture was
extracted with ethyl acetate (200 and 100 ml). The combined organic
layers were dried over magnesium sulphate. The solvent was removed
in vacuo. The crude product was purified by flash chromatography on
silica (80 g), using ethyl acetate/heptane 1:1 as eluent, to give
3.73 g of (3-hydroxymethylbenzyl)carbamic acid tert butylester.
[0489] MS: m/z=260 (M+23.sup.+)
[0490] .sup.1H-NMR (CDCl.sub.3): .delta. 1.48 (s, 9H); 4.30 (br,
2H); 4.70 (s, 2H); 4.85 (br, 1H); 7.15-7.35 (m, 5H).
Step 2:
(3-(Aminomethyl)phenyl)methanol
##STR00187##
[0492] Trifluoroacetic acid (5 ml) was added to a solution of
(3-hydroxymethylbenzyl)carbamic acid tert butylester (1.70 g, 7.17
mmol) in dichloromethane (5 ml). The reaction mixture was stirred
for 40 min. The solvent was removed in vacuo. The residue was
dissolved in dichloromethane (40 ml). The solvent was removed in
vacuo. The latter procedure was repeated twice. The residue was
dissolved in water (50 ml) and an 1 N aqueous solution of sodium
hydroxide (100 ml). It was washed with tert-butyl methyl ether
(3.times.100 ml). It was saturated with sodium chloride and
extracted with dichloromethane (3.times.75 ml). The combined
dichloromethane-phases were dried over magnesium sulphate. The
solvent was removed in vacuo to give 328 mg of crude
(3-(aminomethyl)phenyl)methanol, which was used for the further
steps without purification.
[0493] .sup.1H-NMR (DMSO-d.sub.6): .delta. 3.30 (br, 2H); 3.70 (s,
2H); 4.45 (s, 2H); 5.15 (br, 1H); 7.10-7.30 (m, 4H).
Step 3:
N-(3-(Hydroxymethyl)benzyl)succinamic Acid Tert-Butyl Ester
##STR00188##
[0495] At 0.degree. C.,
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (451
mg, 2.35 mmol) was added to a solution of mono-tert-butyl succinate
(410 mg, 2.35 mmol) and
3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazin (384 mg, 2.35 mmol)
in a mixture of N,N-dimethylformamide (5 ml) and dichloromethane (5
ml). The reaction mixture was stirred for 25 min at 0.degree. C. A
solution of crude (3-(aminomethyl)phenyl)methanol (340 mg, 2.48
mmol) in N,N-dimethylformamide (5 ml) and ethyldiisopropylamine
(0.40 ml, 2.48 mmol) were added successively. The reaction mixture
was stirred for 16 h, while it was slowly warming up to room
temperature. It was diluted with ethyl acetate (150 ml) and washed
with a 10% aqueous solution of sodium hydrogensulphate (100 ml).
The aqueous phase was extracted with ethyl acetate (50 ml). The
combined organic layers were washed with a saturated aqueous
solution of sodium hydrogencarbonate (150 ml) and dried over
magnesium sulphate. The solvent was removed in vacuo. The crude
product was purified by flash chromatography on silica (60 g),
using a mixture of ethyl acetate and heptane (2:1) as eluent to
give 372 mg of N-(3-(hydroxymethyl)benzyl)succinamic acid
tert-butyl ester.
[0496] MS: m/z=316 (M+23.sup.+)
[0497] .sup.1H-NMR (CDCl.sub.3): .delta. 1.45 (s, 9H); 2.45 (t,
2H); 2.60 (t, 2H); 4.45 (d, 2H); 4.70 (s, 2H); 6.15 (br, 1H);
7.15-7.35 (m, 5H).
Step 4:
N-(3-Formylbenzyl)succinamic Acid Tert-Butyl Ester
##STR00189##
[0499] At -78.degree. C., oxalyl chloride (0.142 ml, 1.63 mmol) was
added dropwise to a solution of dimethyl sulphoxide (0.232 ml. 3.26
mmol) in dichloromethane (5 ml). The reaction mixture was stirred
for 10 min at -78.degree. C. A solution of
N-(3-(hydroxymethyl)benzyl)succinamic acid tert-butyl ester (372
mg, 1.55 mmol) in dichloromethane (5 ml) was added. The reaction
mixture was stirred at -78.degree. C. for 10 min. Triethylamine
(1.08 ml, 7.77 mmol) was added. The reaction mixture was stirred at
-78.degree. C. for 5 min and then warmed to room temperature. It
was stirred at room temperature for 40 min and diluted with ethyl
acetate (100 ml). It was washed with a 10% aqueous solution of
sodium hydrogensulphate (100 ml). The aqueous phase was extracted
with ethyl acetate (2.times.50 ml). The combined organic layers
were washed with a saturated aqueous solution of sodium
hydrogencarbonate (150 ml) and dried over magnesium sulphate. The
solvent was removed to give 312 mg of crude
N-(3-formylbenzyl)succinamic acid tert-butyl ester, which was used
for the next step without further purification.
[0500] MS: m/z=314 (M+23.sup.+)
[0501] .sup.1H-NMR (CDCl.sub.3): .delta. 1.35 (s, 9H); 2.45 (t,
2H); 2.55 (t, 2H); 4.45 (d, 2H); 6.20 (br, 1H); 7.44 (t, 1H); 7.50
(d, 1H); 7.75 (m, 2H); 9.95 (s, 1H).
Step 5:
N-[3-((Hydroxyimino)methyl)benzyl]succinamic Acid Tert-Butyl
Ester
##STR00190##
[0503] A 3.2 M aqueous solution of sodium hydroxide (0.5 ml, 1.60
mmol) was added to a solution of N-(3-formylbenzyl)succinamic acid
tert-butyl ester (312 mg, 1.07 mmol) and hydroxylamine
hydrochloride (89 mg, 1.29 mmol) in ethanol (2.5 ml) and water (0.5
ml). The reaction mixture was stirred at room temperature for 3
days. A 10% aqueous solution of sodium hydrogensulphate (20 ml) and
water (50 ml) were added. The mixture was extracted with ethyl
acetate (3.times.50 ml). The combined organic layers were dried
over magnesium sulphate. The solvent was removed in vacuo to give
249 mg of crude N-[3-((hydroxyimino)methyl)benzyl]succinamic acid
tert-butyl ester, which was used without further purification in
the next step.
[0504] MS: m/z=329 (M+23.sup.+), 307 (M+1.sup.+)
[0505] .sup.1H-NMR (DMSO-d.sub.6): .delta. 1.35 (s, 9H); 2.40 (m,
4H); 4.30 (d, 2H); 7.25 (d, 1H); 7.35 (t, 1H); 7.50 (m, 2H); 8.10
(s, 1H); 8.40 (t, 1H); 11.20 (s, 1H).
Step 6:
N-[3-(Hydroxyiminomethyl)benzyl]succinamic Acid
##STR00191##
[0507] Trifluoroacetic acid (7 ml) was added to a solution of crude
N-[3-((hydroxyimino)methyl)benzyl]succinamic acid tert-butyl ester
(249 mg, 0.81 mmol) in dichloromethane (7 ml). The reaction mixture
was stirred for 55 min at room temperature. The solvent was removed
in vacuo. The residue was redissolved in dichloromethane (50 ml).
The solvent was removed in vacuo. The latter procedure was repeated
twice to give 294 mg of crude
N-[3-(hydroxyiminomethyl)benzyl]succinamic acid, which was used in
the next step without further purification.
[0508] MS: m/z=273 (M+23.sup.+), 251 (M+1.sup.+)
[0509] .sup.1H-NMR (DMSO-d.sub.6): .delta. 2.45 (A.sub.2B.sub.2,
4H); 4.30 (d, 2H); 7.20-7.50 (m, 4H); 8.10 (s, 1H); 8.40 (t, 1H);
11.20 (br, 1H).
Step 7:
[0510] A 10% aqueous solution of sodium hypochlorite (0.0015 ml,
2600 pmol) was added to a solution of crude
N-[3-(hydroxyiminomethyl)benzyl]succinamic acid (1.29 mg, 5150
pmol) in a mixture of water (0.11 ml) and a saturated aqueous
solution of sodium hydrogencarbonate (0.01 ml). The reaction
mixture was left for 10 min at room temperature. A solution of
(2S)-2-([Glu.sup.3,Leu.sup.10]GLP-2-ylleucinylamino)-3-(4-(prop-2ynyloxy)-
phenyl)propionamide (0.210 mg, 51 pmol) and triethylamine (0.0006
ml) in water (0.11 ml) was added. The reaction mixture was shaken
at room temperature. After 1 h, the MALDI-TOF showed small amounts
of m/z=4323, corresponding to the mass of
3-(3-(3-((4-((S)-2-carbamoyl-3-([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamin-
o)ethyl)phenoxyl)methyl)isoxazol-3-yl)benzylcarbamoyl)propionic
acid, along with majour amounts of m/z=4076, corresponding to the
mass of
(2S)-2-([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamino)-3-(4-(prop-2ynyloxy)p-
henyl)propionamide. After 2 h, the LC-MS electrospray showed masses
of m/z=1442, 1082, and 866, corresponding to (M.sup.3+), (M.sup.4+)
and (M.sup.5+) respectively of
3-(3-(3-((4-((S)-2-carbamoyl-3-([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamin-
o)ethyl)phenoxyl)methyl)isoxazol-3-yl)benzylcarbamoyl)propionic
acid along with masses of m/z=1359, 1020, and 816, corresponding to
(M.sup.3+), (M.sup.4+) and (M.sup.5+) respectively of
(2S)-2-([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamino)-3-(4-(prop-2ynyloxy)p-
henyl)propionamide. After 8 h, the LC-MS electrospray showed small
amounts of masses of m/z=1442 and 1082, corresponding to (M.sup.3+)
and (M.sup.4+) respectively of
3-(3-(3-((4-((S)-2-carbamoyl-3-([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamin-
o)ethyl)phenoxyl)methyl)isoxazol-3-yl)benzylcarbamoyl)propionic
acid along with major amounts of masses of m/z=1360 and 1020,
corresponding to (M.sup.3+) and (M.sup.4+) respectively of
(2S)-2-([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamino)-3-(4-(prop-2ynyloxy)p-
henyl)propionamide.
Example 17
11-(4-(4-((2S)-2-Carbamoyl-2-(([Glu.sup.3
Leu.sup.10]GLP-2ylleucinylamino)ethyl)pheoxymethyl)-1,2,3-triazolyl)undec-
anoic acid and
11-(5-(4-((2S)-2-Carbamoyl-2-(([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamino-
)ethyl)pheoxymethyl)-1,2,3-triazolyl)undecanoic Acid
##STR00192##
[0511] Step 1:
Methyl 11-azidoundecanoate
##STR00193##
[0513] Sodium azide (4.66 g, 72 mmol) and tetrabutylammonium iodide
(66 mg, 0.18 mmol) were successively added to a solution of methyl
11-bromoundecanoate (commercially available at Aldrich, 5.00 g,
17.9 mmol) in N,N-dimethylformamide (50 ml). The reaction mixture
was heated to 60.degree. C. for 16 h and cooled to room
temperature. It was diluted with water (200 ml) and extracted with
ethyl acetate (200 ml). The aqueous phase was washed with water
(2.times.200 ml). The organic phase was dried over sodium sulphate.
The solvent was removed in vacuo to give 4.28 g of methyl
11-azidoundecanoate.
[0514] MS: m/z=264 (M+23.sup.+), 214 (M-N.sub.2.sup.+)
Step 2:
11-Azidoundecanoic Acid
##STR00194##
[0516] Crunched sodium hydroxide (709 mg, 17.7 mmol) was added to a
solution of methyl 11-azidoundecanoate (4.03 g, 17.7 mmol) in
methanol (75 ml). The reaction mixture was stirred for 16 h at room
temperature. Water (50 ml) was added. The mixture was acidified to
pH 2 by addition of a 10% aqueous solution of sodium
hydrogensulphate and was extracted with ethyl acetate (3.times.50
ml). The combined organic layers were dried over sodium sulphate.
The solvent was removed in vacuo. The residue was dissolved in
methanol (50 ml). Crunched sodium hydroxide (1.42 g, 35.4 mmol) was
added. The reaction mixture was stirred at room temperature for 16
h. Water (50 ml) was added. The mixture was acidified to pH 2 by
addition of a 10% aqueous solution of sodium hydrogensulphate and
was extracted with ethyl acetate (3.times.50 ml). The combined
organic layers were dried over sodium sulphate. The solvent was
removed in vacuo to give 3.13 g of 11-azidoundecanoic acid.
[0517] MS: m/z=250 (M+23.sup.+), 200 (M-N.sub.2.sup.+).
[0518] .sup.1H-NMR (CDCl.sub.3): .delta. 1.30 (m, 12H); 1.65 (m,
4H); 2.40 (t, 2H); 3.20 (t, 2H); 9.00-10.80 (br, 1H).
Step 3
[0519] A solution of 11-azidoundecanoic acid (0.116 mg, 510 nmol)
in acetonitrile (0.055 ml) was added to a solution of
(2S)-2-([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamino)-3-(4-(prop-2ynyloxy)p-
henyl)propionamide (0.210 mg, 51 nmol) and 2,6-lutidine (0.0012 ml,
10200 nmol) in water (0.105 ml). A solution of copper(1) iodide
(0.001 mg, 5 nmol) in acetonitrile (0.050 ml) was added. The
reaction mixture was kept at room temperature. After 4 h, a
solution of copper(1) iodide (0.098 mg, 500 nmol) in acetonitrile
was added. The reaction mixture was kept at room temperature for 16
h. A 2.5% solution of ammonia in water (0.200 ml) was added. The
reaction mixture was kept at room temperature and room atmosphere
for 4 h. The masses found by MS and MALDI-TOF were in
correspondence with the expectations for the mass found for
11-(4-(4-((2S)-2-Carbamoyl-2-(([Glu.sup.3,Leu.sup.10]GLP-2ylleucinylamino-
)ethyl)pheoxymethyl)-1,2,3-triazolyl)undecanoic acid and
11-(5-(4-((2S)-2-Carbamoyl-2-(([Glu.sup.3,
Leu.sup.10]GLP-2ylleucinylamino)ethyl)pheoxymethyl)-1,2,3-triazolyl)undec-
anoic acid.
[0520] HPLC: 9.43 min (method 02-B4-4).
[0521] MS: m/z=1435, 1077.
[0522] MALDI-TOF: 4303.
Example 18
1'-(4-(4-((S)-2-carbamoyl-2-([Glu.sup.3]GLP-2ylleucinylamino))phenoxymethy-
l)-1H-1,2,3-triazol-1-yl)undecanoic acid and
11-(5-(4-((S)-2-carbamoyl-2-([Glu.sup.3]GLP-2ylleucinylamino))phenoxymeth-
yl)-1H-1,2,3-triazol-1-yl)undecanoic Acid
##STR00195##
[0524] 2,6-Lutidine was added to a mixture of
(2S)-2-([Glu.sup.3]GLP-2ylleucinylamino)-3-(4-(prop-2-ynyloxy)phenyl)prop-
ionamide (1.0 mg, 244 pmol) in water (0.5 ml) to give a clear
solution. A solution of 11-azidoundecanoic acid (0.554 mg, 0.0025
mmol) in acetonitrile (0.25 ml) and a solution of copper(I) iodide
(0.467 mg, 0.0025 mmol) in acetonitrile (0.25 ml) were added
successively. The reaction mixture was left for 16 h at room
temperature. It was fractionated on a reversed phase C.sub.18
column on a HPLC, using a gradient of 35-75% acetonitrile in water
in a buffer of 0.1% trifluoroacetic acid to give approx. 0.3 mg of
11-(4-(4-((S)-2-carbamoyl-2-([Glu.sup.3]GLP-2ylleucinylamino))phenoxymeth-
yl)-1,2,3-triazolyl)undecanoic acid or
11-(5-(4-((S)-2-carbamoyl-2-([Glu.sup.3]GLP-2ylleucinylamino))phenoxymeth-
yl)-1H-1,2,3-triazol-1-yl)undecanoic acid or a mixture thereof.
[0525] HPLC: 9.27 min (method 02-B4-4).
[0526] MS: m/z=1441.8, 1081.3, 865.2, 721.2, 618.9.
[0527] MALDI-TOF: m/z=4317
Example 19
2-([Glu.sup.3]GLP-2ylleucinyl)-3-(4-((1-((N-(mPeg20kDayl)carbamoyl)decanyl-
)-1H-1,2,3-tetrazol-4-yl)methoxy)phenyl)propionamide and
2-([Glu.sup.3]GLP-2ylleucinyl)-3-(4-((1-((N-(mPeg20kDayl)carbamoyl)decany-
l)-1H-1,2,3-tetrazol-5-yl)methoxy)phenyl)propionamide
##STR00196##
[0528] Step 1:
11-Azidoundecanoic Acid 2,5-dioxopyrroldin-1-yl Ester
##STR00197##
[0530] N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uranium
tetrafluoroborate (1.32 g, 4.40 mmol) was added to a solution of
11-azidoundecanoic acid (1.00 g, 4.40 mmol) and triethylamine (0.61
ml, 4.40 mmol) in N,N-dimethylformamide (10 ml). The reaction
mixture was stirred for 2 h at room temperature. It was diluted
with ethyl acetate (50 ml) and washed with water (3.times.50 ml).
The organic phase was dried over sodium sulphate. The solvent was
removed in vacuo to give 1.40 g of crude 11-azidoundecanoic acid
2,5-dioxopyrroldin-1-yl ester, which was used in the next steps
without further purification.
[0531] MS: m/z=347 [M+Na.sup.+]
[0532] .sup.1H-NMR (CDCl3): .delta. 1.35 (m, 12H); 1.60 (quintett,
2H); 1.75 (quintett, 2H); 2.60 (t, 2H); 1.85 (m, 4H); 3.25 (t,
2H).
Step 2:
[0533] 11-AzidoundecanoylaminomPEG20kDa
##STR00198##
[0534] A solution of 11-azidoundecanoic acid
2,5-dioxopyrroldin-1-yl ester (227 mg, 0.7 mmol) was added to a
solution of commercially available mPEG20000DA-amine (Nektar
2M2U0P01, 5.00 g, 0.25 mmol) and triethylamine (0.174 ml, 1.25
mmol) in dichloromethane (50 ml). The reaction mixture was stirred
at room temperature for 16 h. Ether (800 ml) was added. The formed
precipitation was isolated by filtration and washed with ether
(2.times.100 ml). It was dried in vacuo to give 4.58 g of
11-azidoundecanoylaminomPEG20kDa.
Step 3:
[0535] A solution of ascorbic acid (1.72 mg, 9766 nmol) and
2,6-lutidine (0.0024 ml) in water (0.10 ml) was added to a solution
of copper(II) sulphate pentahydrate (0.49 mg, 1954 nmol) in water
(0.1 ml). This solution was kept for 5 min at room temperature. A
part of the resulting mixture (0.025 ml) was added to a solution of
(S)-2-([Glu.sup.3]GLP-2ylleucinyl)-3-(4-propargyloxyphenyl)propionamide
(0.1 mg, 24 nmol), 2,6-lutidine (0.0012 ml) and
11-azidoundecanoylaminomPEG20kDa (0.049 mg, 240 nmol) in water
(0.075 ml). The reaction mixture was kept at room temperature.
After 24 h, a SDS-gel electrophoreses applying a 10% Bis-Tirs Gel
of NuPAGE (Invitrogen) and a SilverXpress.RTM. silver staining
method, showed the formation of high-molecular peptide, in
accordance with the expectations for
2-([Glu.sup.3]GLP-2ylleucinyl)-3-(4-((1-((N-(mPeg20kDayl)carbamoyl)de-
canyl)-1H-1,2,3-tetrazol-4-yl)methoxy)phenyl)propionamide and
2-([Glu.sup.3]GLP-2ylleucinyl)-3-(4-((1-((N-(mPeg20kDayl)carbamoyl)decany-
l)-1H-1,2,3-tetrazol-5-yl)methoxy)phenyl)propionamide.
Example 20
N--((S)-5-([Leu.sup.37]GLP-1(7-37)ylamino)-5-carbamoylpentyl)-4-acetyl
benzamide
##STR00199##
[0536] Step 1:
[0537] [Leu.sup.37]GLP-1(7-37)ylalanine was prepared as in example
9
Step 2:
[0538] CPY-catalyzed transpeptidation of
4-acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide with
[Leu.sup.37]GLP-1(7-37)ylalanine:
[0539] To a mixture of
4-acetyl-N-((5S)-5-amino-5-carbamoylpentyl)benzamide (final
concentration 100 mM) and hydroxypropyl-.beta.-cyclodextrin (4% w/v
final concentration) in solution in HEPES buffer 250 mM pH8
containing 5 mM EDTA was added [Leu.sup.37]GLP-1(7-37)ylalanine (1
mM final concentration) in solution in HEPES buffer 250 mM pH8
containing 5 mM EDTA. The pH was adjusted to 8.1 by addition of
diisopropylethylamine. The reaction was started by addition of the
enzyme in solution in water (10 U/ml final concentration). The
reaction is monitored by HPLC.
HPLC Method:
Column: Vydac C18 (218TP53) 250.times.4.6
A: (NH.sub.4).sub.2SO.sub.4 50 mM, 0.5% CH.sub.3CN, pH2.5 B:
CH.sub.3CN/TFA 0.1%
[0540] 1.5 ml/min 5 to 45% B over 20 min detection at 214 nm
40 C
[0541] After 6 h30 at 30.degree. C., the reaction mixture content
was about 22% of the remaining starting compound
[Leu.sup.37]GLP-1(7-37)ylalanine (retention time: 18.1 min), 70% of
the transpeptidation product (retention time: 18.3 min) and 8% of
the hydrolysis product [Leu.sup.37]GLP-1(7-37) (retention time:
18.4 min).
[0542] MALDI-TOF: m/z=3684
(S)-5-[Leu.sup.37]GLP-1(7-37)ylamino)-5-carbamoylpentyl) 4-acetyl
benzamide), 3482 ([Leu.sup.37]GLP-1(7-37)ylalanine), 3411
([Leu.sup.37]GLP-1(7-37)) and 1162 and 1742
([Leu.sup.37]GLP-1(7-37)ylalanine).
[0543] MS (electrospray): m/z=1844 and 1229
((S)-5-[Leu37]GLP-1(7-37)ylamino)-5-carbamoylpentyl) 4-acetyl
benzamide), 1139 ad 1702 ([Leu.sup.37]GLP-1(7-37)) and 1162 and
1742 ([Leu.sup.37]GLP-1(7-37)ylalanine).
Example 21
N--((S)-5-([Leu.sup.37]GLP-1(7-37)ylamino)-5-carbamoylpentyl)-4-[1-[2-(1-(-
hexadecanoyl)piperidin-4-yl))ethoxyimino]ethyl]benzamide
##STR00200##
[0545] To a solution of
N--((S)-5-([Leu.sup.37]GLP-1(7-37)ylamino)-5-carbamoylpentyl)-4-acetyl
benzamide in acetate buffer 50 mM pH4 (final concentration 0.3 mM)
was added 1-[4-(2-(aminooxy)ethyl)piperidin-1-yl]hexadecan-1-one
(final concentration 3 mM) in solution in acetonitrile (final
acetonitrile concentration: 18% v/v). The reaction was run at
30.degree. C. and followed by HPLC.
HPLC Method:
Column: Vydac C18 (218TP53) 250.times.4.6
A: H.sub.2O/TFA 0.1%
B: CH.sub.3CN/TFA 0.1%
[0546] 10% B for 5 min, then 10 to 91% B over 27 min 1 ml/min
40 C
Detection at 214 and 280 nm
[0547] Retention time of
N--((S)-5-([Leu.sup.37]GLP-1(7-37)ylamino)-5-carbamoylpentyl)-4-acetyl
benzamide: 18.4 min, retention times of products: 26.5 and 27.1
min.
[0548] More than 90% yield was obtained after 4 h reaction
time.
[0549] MS (electrospray): m/z=1351.4 (calc: 1350.9)
[0550] MALDI-TOF: m/z=4048 (calc: 4049.8).
[0551] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference in
their entirety and to the same extent as if each reference were
individually and specifically indicated to be incorporated by
reference and were set forth in its entirety herein (to the maximum
extent permitted by law).
[0552] All headings and sub-headings are used herein for
convenience only and should not be construed as limiting the
invention in any way.
[0553] The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
[0554] The citation and incorporation of patent documents herein is
done for convenience only and does not reflect any view of the
validity, patentability, and/or enforceability of such patent
documents.
[0555] This invention includes all modifications and equivalents of
the subject matter recited in the claims appended hereto as
permitted by applicable law.
* * * * *