U.S. patent application number 12/115976 was filed with the patent office on 2009-06-11 for glp-1 analogues.
This patent application is currently assigned to Biomeasure, Incorporated. Invention is credited to David H. Coy, Zheng Xin Dong.
Application Number | 20090149378 12/115976 |
Document ID | / |
Family ID | 26808707 |
Filed Date | 2009-06-11 |
United States Patent
Application |
20090149378 |
Kind Code |
A1 |
Dong; Zheng Xin ; et
al. |
June 11, 2009 |
GLP-1 ANALOGUES
Abstract
The present invention is directed to peptide analogues of
glucagon-like peptide-1, the pharmaceutically-acceptable salts
thereof, to methods of using such analogues to treat mammals and to
pharmaceutical compositions useful therefor comprising said
analogues.
Inventors: |
Dong; Zheng Xin; (Holliston,
MA) ; Coy; David H.; (New Orleans, LA) |
Correspondence
Address: |
Biomeasure, Incorporated
27 Maple Street
Milford
MA
01757-3650
US
|
Assignee: |
Biomeasure, Incorporated
Milford
MA
The Administrators of the Tulane Educational Fund
New Orleans
LA
|
Family ID: |
26808707 |
Appl. No.: |
12/115976 |
Filed: |
May 6, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09856676 |
Jul 16, 2001 |
7368427 |
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PCT/US99/28929 |
Dec 7, 1999 |
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12115976 |
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09206833 |
Dec 7, 1998 |
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09856676 |
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60111186 |
Dec 7, 1998 |
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Current U.S.
Class: |
514/7.2 ;
530/324 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 9/04 20180101; A61P 9/10 20180101; A61P 3/10 20180101; A61P
19/02 20180101; A61K 38/00 20130101; C07K 14/605 20130101; A61P
19/10 20180101; A61P 3/00 20180101; A61P 25/28 20180101; A61P 9/12
20180101; A61P 3/04 20180101; A61P 1/14 20180101; A61P 1/16
20180101; A61P 3/08 20180101; A61P 19/00 20180101; A61P 13/12
20180101; Y02P 20/55 20151101; A61P 11/00 20180101; A61P 5/48
20180101 |
Class at
Publication: |
514/12 ;
530/324 |
International
Class: |
A61K 38/26 20060101
A61K038/26; C07K 14/605 20060101 C07K014/605; A61P 3/00 20060101
A61P003/00; A61P 3/04 20060101 A61P003/04; A61P 19/00 20060101
A61P019/00; A61P 25/00 20060101 A61P025/00; A61P 3/10 20060101
A61P003/10 |
Claims
1. A compound of formula (I),
(R.sup.2R.sup.3)-A.sup.7-A.sup.8-A.sup.9-A.sup.10-A.sup.11-A.sup.12-A.sup-
.13-A.sup.14-A.sup.15-A.sup.16-A.sup.17-A.sup.18-A.sup.19-A.sup.20-A.sup.2-
1-A.sup.22-A.sup.23-A.sup.24-A.sup.25-A.sup.26-A.sup.27-A.sup.28-A.sup.29--
A.sup.30-A.sup.31-A.sup.32-A.sup.33-A.sup.34-A.sup.35-A.sup.36-A.sup.37-R.-
sup.1, (I) wherein A.sup.7 is L-His, Ura, Paa, Pta, D-His, Tyr,
3-Pal, 4-Pal, Hppa, Tma-His, Amp or deleted, provided that when
A.sup.7 is Ura, Paa, Pta or Hppa then R.sup.2 and R.sup.3 are
deleted; A.sup.8 is Ala, D-Ala, Aib, Acc, N-Me-Ala, N-Me-D-Ala, Arg
or N-Me-Gly; A.sup.9 is Glu, N-Me-Glu, N-Me-Asp or Asp; A.sup.10 is
Gly, Acc, Ala, D-Ala, Phe or Aib; A.sup.11 is Thr or Ser; A.sup.12
is Phe, Acc, Aic, Aib, 3-Pal, 4-Pal, .beta.-Nal, Cha, Trp or
X.sup.1-Phe; A.sup.13 is Thr or Ser; A.sup.14 is Ser, Thr, Ala or
Aib; A.sup.15 is Asp, Ala, D-Asp or Glu; A.sup.16 is Val, D-Val,
Acc, Aib, Leu, Ile, Tle, Nle, Abu, Ala, D-Ala, Tba or Cha; A.sup.17
is Ser, Ala, D-Ala, Aib, Acc or Thr; A.sup.13 is Ser, Ala, D-Ala,
Aib, Acc or Thr; A.sup.19 is Tyr, D-Tyr, Cha, Phe, 3-Pal, 4-Pal,
Acc, .beta.-Nal, Amp or X.sup.1-Phe; A.sup.20 is Leu, Ala, Acc,
Aib, Nle, Ile, Cha, Tle, Val, Phe or X.sup.1-Phe; A.sup.21 is Glu,
Ala or Asp; A.sup.22 is Gly, Acc, Ala, D-Ala, .beta.-Ala or Aib;
A.sup.23 is Gln, Asp, Ala, D-Ala, Aib, Acc, Asn or Glu; A.sup.24 is
Ala, Aib, Val, Abu, Tle or Acc; A.sup.25 is Ala, Aib, Val, Abu,
Tle, Acc, Lys, Arg, hArg, Orn,
HN--CH((CH.sub.2).sub.n--NR.sup.10R.sup.11)--C(O) or
HN--CH((CH.sub.2).sub.e--X.sup.3)--C(O); A.sup.26 is Lys, Ala,
3-Pal, 4-Pal, Arg, hArg, Orn, Amp,
HN--CH((CH.sub.2).sub.n--NR.sup.10R.sup.11)--C(O) or
HN--CH((CH.sub.2).sub.e--X.sup.3)--C(O); A.sup.27 is Glu, Ala,
D-Ala or Asp; A.sup.28 is Phe, Ala, Pal, .beta.-Nal, X.sup.1-Phe,
Aic, Acc, Aib, Cha or Trp; A.sup.29 is Ile, Acc, Aib, Leu, Nle,
Cha, Tle, Val, Abu, Ala, Tba or Phe; A.sup.30 is Ala, Aib, Acc or
deleted; A.sup.31 is Trp, Ala, .beta.-Nal, 3-Pal, 4-Pal, Phe, Acc,
Aib, Cha, Amp or deleted; A.sup.32 is Leu, Ala, Acc, Aib, Nle, Ile,
Cha, Tle, Phe, X.sup.1-Phe, Ala or deleted; A.sup.33 is Val, Acc,
Aib, Leu, Ile, Tle, Nle, Cha, Ala, Phe, Abu, X.sup.1-Phe, Tba, Gaba
or deleted; A.sup.34 is Lys, Arg, hArg, Orn, Amp, Gaba,
HN--CH((CH.sub.2), --NR.sup.10R.sup.11)--C(O),
HN--CH((CH.sub.2).sub.e--X.sup.3)--C(O) or deleted; A.sup.35 is Gly
or deleted; A.sup.36 is L- or D-Arg, D- or L-Lys, D- or L-hArg, D-
or L-Orn, Amp, HN--CH((CH.sub.2), --NR.sup.10R.sup.11)--C(O),
HN--CH((CH.sub.2).sub.e--X.sup.3)--C(O) or deleted; A.sup.37 is Gly
or deleted; X.sup.1 for each occurrence is independently selected
from the group consisting of (C.sub.1-C.sub.6)alkyl, OH and halo;
R.sup.1 is OH, NH.sub.2, (C.sub.1-C.sub.12)alkoxy, or
NH--X.sup.2--CH.sub.2-Z.sup.0, wherein X.sup.2 is a
(C.sub.1-C.sub.12)hydrocarbon moiety, and Z.sup.0 is H, OH,
CO.sub.2H or CONH.sub.2; X.sup.3 is ##STR00011## or
--C(O)--NHR.sup.12, wherein X.sup.4 for each occurrence is
independently --C(O)--, --NH--C(O)-- or --CH.sub.2--, and f for
each occurrence is independently an integer from 1 to 29; each of
R.sup.2 and R.sup.3 is independently selected from the group
consisting of H, (C.sub.1-C.sub.30)alkyl,
(C.sub.2-C.sub.30)alkenyl, phenyl(C.sub.1-C.sub.30)alkyl,
naphthyl(C.sub.1-C.sub.30)alkyl, hydroxy(C.sub.1-C.sub.30)alkyl,
hydroxy(C.sub.2-C.sub.30)alkenyl,
hydroxyphenyl(C.sub.1-C.sub.30)alkyl, and
hydroxynaphthyl(C.sub.1-C.sub.30)alkyl; or one of R.sup.2 and
R.sup.3 is C(O)X.sup.5 in which X.sup.5 is (C.sub.1-C.sub.30)alkyl,
(C.sub.2-C.sub.30)alkenyl, phenyl(C.sub.1-C.sub.30)alkyl,
naphthyl(C.sub.1-C.sub.30)alkyl, hydroxy(C.sub.1-C.sub.30)alkyl,
hydroxy(C.sub.2-C.sub.30)alkenyl,
hydroxyphenyl(C.sub.1-C.sub.30)alkyl,
hydroxynaphthyl(C.sub.1-C.sub.30)alkyl, ##STR00012## where Y is H
or OH, r is 0 to 4 and q is 0 to 4; e for each occurrence is
independently an integer from 1 to 4; n for each occurrence is
independently an integer from 1-5; and R.sup.10 and R.sup.11 for
each occurrence is each independently H, (C.sub.1-C.sub.30)alkyl,
(C.sub.1-C.sub.30)acyl, (C.sub.1-C.sub.30)alkylsulfonyl,
--C((NH)(NH.sub.2)) or ##STR00013## provided that when R.sup.10 is
(C.sub.1-C.sub.30)acyl, (C.sub.1-C.sub.30)alkylsulfonyl,
--C((NH)(NH.sub.2)) or ##STR00014## R.sup.11 is H or
(C.sub.1-C.sub.30)alkyl; and R.sup.12 is (C.sub.1-C.sub.30)alkyl;
with the proviso that: (i) at least one amino acid of a compound of
formula (I) is not the same as the native sequence of hGLP-1 (7-36,
or -37)NH.sub.2 (SEQ ID NOS: 1, 2) or hGLP-1 (7-36, or -37)OH (SEQ
ID NOS: 3, 4); (ii) a compound of formula (I) is not an analogue of
hGLP-1(7-36, or -37)NH.sub.2 (SEQ ID NOS: 1,2) or hGLP-1(7-36, or
-37)OH (SEQ ID NOS: 3, 4) wherein a single position has been
substituted by Ala; (iii) a compound of formula (I) is not
[Lys.sup.26(N.sup..epsilon.-alkanoyl)]hGLP-1(7-36, or -37)-E (SEQ
ID NOS: 5-8), [Lys.sup.34(N.sup..epsilon.-alkanoyl)]hGLP-1(7-36, or
-37)-E (SEQ ID NOS: 9-12),
[Lys.sup.26,34-bis(N.sup..epsilon.-alkanoyl)]hGLP-1(7-36, or -37)-E
(SEQ ID NOS: 13-16), [Arg.sup.26,
Lys.sup.34(NE-alkanoyl)]hGLP-1(8-36, or -37)-E (SEQ ID NOS: 17-20),
or [Arg.sup.26,34,
Lys.sup.36(N.sup..epsilon.-alkanoyl)]hGLP-1(7-36, or -37)-E,
wherein E is --OH or --NH.sub.2 (SEQ ID NOS: 21-24); (iv) a
compound of formula (I) is not Z.sup.1-hGLP-1(7-36, or -37)-OH,
Z.sup.1-hGLP-1(7-36, or -37)-NH.sub.2, where Z.sup.1 is selected
from the group consisting of (a) [Arg.sup.26] (SEQ ID NOS: 25-28),
[Arg.sup.34] (SEQ ID NOS: 29-32), [Arg.sup.26,34] (SEQ ID NOS:
33-36), [Lys.sup.36] (SEQ ID NOS: 37-40), [Arg.sup.26, Lys.sup.36]
(SEQ ID NOS: 41-44), [Arg.sup.34, Lys.sup.36] (SEQ ID NOS: 45-46),
[D-Lys.sup.36], [Arg.sup.36] (SEQ ID NOS: 37-40), [D-Arg.sup.36],
[Arg.sup.26,34, Lys.sup.36] (SEQ ID NOS: 49-52), or [Arg.sup.26,36,
Lys.sup.34] (SEQ ID NOS: 25-28); (b) [Asp.sup.21] (SEQ ID NOS:
53-56); (c) at least one of [Aib.sup.8] (SEQ ID NOS: 57-60),
[D-Ala.sup.8] and [Asp.sup.9] (SEQ ID NOS: 61-64); and (d)
[Tyr.sup.7] (SEQ ID NOS: 65-68), [N-acyl-His.sup.7] (SEQ ID NOS:
69-72), [N-alkyl-His.sup.7] (SEQ ID NOS: 73-76),
[N-acyl-D-His.sup.7] or [N-alkyl-D-His.sup.7]; (v) a compound of
formula (I) is not a combination of any two of the substitutions
listed in groups (a) to (d); and (vi) a compound of formula (I) is
not [N-Me-Ala.sup.8]hGLP-1(8-36 or -37) (SEQ ID NOS: 77, 78),
[Glu.sup.15]hGLP-1(7-36 or -37) (SEQ ID NOS: 79, 80),
[Asp.sup.21]hGLP-1(7-36 or -37) (SEQ ID NOS: 53, 54) or
[Phe.sup.31]hGLP-1(7-36 or -37) (SEQ ID NOS: 81, 82).
2. A compound according to claim 1 or a pharmaceutically acceptable
salt thereof wherein A.sup.11 is Thr; A.sup.13 is Thr; A.sup.14 is
Ser, Aib or Ala; A.sup.17 is Ser, Ala, Aib or D-Ala; A.sup.18 is
Ser, Ala, Aib or D-Ala; A.sup.21 is Glu or Ala; A.sup.23 is Gln,
Glu, or Ala; and A.sup.27 is Glu or Ala.
3. A compound according to claim 2 or a pharmaceutically acceptable
salt thereof wherein A.sup.9 is Glu, N-Me-Glu or N-Me-Asp; A.sup.12
is Phe, Acc or Aic; A.sup.16 is Val, D-Val, Acc, Aib, Ala, Tle or
D-Ala; A.sup.19 is Tyr, 3-Pal, 4-Pal or D-Tyr; A.sup.20 is Leu,
Acc, Cha, Ala or Tle; A.sup.24 is Ala, Aib or Acc; A.sup.25 is Ala,
Aib, Acc, Lys, Arg, hArg, Orn, HN--CH((CH.sub.2),
--NH--R.sup.10)--C(O); A.sup.28 is Phe or Ala; A.sup.29 is Ile, Acc
or Tle; A.sup.30 is Ala, Aib or deleted; A.sup.31 is Trp, Ala,
3-Pal, 4-Pal or deleted; A.sup.32 is Leu, Acc, Cha, Ala or deleted;
A.sup.33 is Val, Acc, Ala, Gaba, Tle or deleted.
4. A compound according to claim 3 or a pharmaceutically acceptable
salt thereof wherein A.sup.8 is Ala, D-Ala, Aib, A6c, A5c,
N-Me-Ala, N-Me-D-Ala or N-Me-Gly; A.sup.10 is Gly, Ala, D-Ala or
Phe; A.sup.12 is Phe, A6c or A5c; A.sup.16 is Val, Ala, Tle, A6c,
A5c or D-Val; A.sup.20 is Leu, A6c, A5c, Cha, Ala or Tle; A.sup.22
is Gly, Aib, .beta.-Ala, L-Ala or D-Ala; A.sup.24 is Ala or Aib;
A.sup.29 is Ile, A6c, A5c or Tle; A.sup.32 is Leu, A6c, A5c, Cha,
Ala or deleted; A.sup.33 is Val, A6c, A5c, Ala, Gaba, Tle or
deleted.
5. A compound according to claim 4 or a pharmaceutically acceptable
salt thereof wherein R.sup.1 is OH or NH.sub.2.
6. A compound according to claim 5 or a pharmaceutically acceptable
salt thereof wherein R.sup.2 is H and R.sup.3 is
(C.sub.1-C.sub.30)alkyl, (C.sub.2-C.sub.30)alkenyl,
(C.sub.1-C.sub.30)acyl, ##STR00015##
7. A compound according to claim 1 wherein said compound is
[D-Ala.sup.8, Ala.sup.17,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]-GLP-1(7-34)NH.sub.2; [D-Ala.sup.8,23,27,
3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2; [Ala.sup.18,23,27,
3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2 (SEQ ID NO: 83);
[Ala.sup.16,23,27, 3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2 (SEQ ID
NO: 84); [Ala.sup.14,23,27, 3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2
(SEQ ID NO: 85); [Ala.sup.22,23,27,
3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2 (SEQ ID NO: 86);
[Hppa.sup.7]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 87);
[Ala.sup.15,23,27, 3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2 (SEQ ID
NO: 88); [Ala.sup.17,23,27, 3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2
(SEQ ID NO: 89); [Ala.sup.22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 90);
[Ala.sup.15,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 91);
[Ala.sup.17,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 92);
[Ala.sup.18,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 93);
[Ala.sup.21,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 94);
[Ala.sup.22,23,26,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 95);
[Ala.sup.22,23,27,32, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 96);
[Ala.sup.22,23,26,27, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 97);
[Ala.sup.22,23,27,31, 3-Pal.sup.19,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 98);
[Ala.sup.22,23,27,28, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 99);
[Ala.sup.22,23,27,29, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 100); [Ala.sup.23,27,
3-Pal.sup.19,31, Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO:
101); [Ala.sup.20,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 102);
[Ala.sup.22,23,27, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 103);
[Ala.sup.17,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 104); [D-Ala.sup.10,
Ala.sup.22,23,27, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2; [D-Ala.sup.8, Ala.sup.17,23,27,
3-Pal.sup.19,31]hGLP-1(7-34)-NH.sub.2; [Ala.sup.17,23,27,
3-Pal.sup.19,26,31]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 105);
[D-Ala.sup.8, Ala.sup.17, 3-Pal.sup.19,31]hGLP-1(7-34)-NH.sub.2;
[Ala.sup.17,23,27, 3-Pal.sup.19,31]hGLP-1(7-34)-NH.sub.2 (SEQ ID
NO: 106); [D-Ala.sup.8, Ala.sup.17,23,27, 3-Pal.sup.19,31,
Tle.sup.29]hGLP-1(7-34)-NH.sub.2; [D-Ala.sup.8, Ala.sup.17,23,27,
3-Pal.sup.19,31, Tle.sup.16]hGLP-1(7-34)-NH.sub.2; [D Ala.sup.8,
Ala.sup.17,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; [D-Ala.sup.22, Ala.sup.17,23,27,
3-Pal.sup.19,31, Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; [Aib.sup.8,
Ala.sup.17,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 107); [D-Ala.sup.8,
Ala.sup.17,22,23,27, 3-Pal.sup.19,31]hGLP-1(7-33)-NH.sub.2;
[Aib.sup.8, Ala.sup.17,22,23,27,
3-Pal.sup.19,31]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 108);
[Ala.sup.17,18,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 109);
[Ala.sup.17,23,27, 3-Pal.sup.19,31, Tle.sup.33,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 110); [Tle.sup.16,
Ala.sup.17,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 111);
[N-Me-D-Ala.sup.8, Ala.sup.17,22,23,27,
3-Pal.sup.19,31]hGLP-1(7-33)-NH.sub.2; [Aib.sup.8,
Ala.sup.17,18,23,27, 3-Pal.sup.19,31]hGLP-1(7-33)-NH.sub.2 (SEQ ID
NO: 112); [Ala.sup.17,18,22,23,27, 3-Pal.sup.19,31, Tle.sup.16,20,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 113); [D-Ala.sup.8,
Ala.sup.17,18,22,23,27, 3-Pal.sup.19,31, Tle.sup.16,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; [D-Ala.sup.8,22,
Ala.sup.17,18,23,27,3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; [D-Ala.sup.8,18,
Ala.sup.17,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; [D-Ala.sup.8,17,
Ala.sup.18,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; or [D-Ala.sup.8,
Ala.sup.17,18,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; or a pharmaceutically acceptable
salt thereof.
8. A compound according to claim 1 wherein said compound is
[Aib.sup.8, A6c.sup.32]hGLP-1(7-36)NH.sub.2 (SEQ ID NO: 114);
[A6c.sup.20,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 115);
[Aib.sup.8]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 116);
[(Tma-His).sup.7]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 117);
[A6c.sup.8]hGLP-1(8-36)-NH.sub.2 (SEQ ID NO: 118);
[A6c.sup.8]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 119);
[A6c.sup.16,20]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 120);
[A6c.sup.29,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 121); [A6c.sup.20,
Aib.sup.24]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 122); [Aib.sup.24,
A6c.sup.29,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 123);
[A6c.sup.16,29,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 124);
[Ura.sup.7]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 125);
[Paa.sup.7]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 126);
[Pta.sup.7]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 127);
[N-Me-Ala.sup.8]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 128);
[N-Me-Ala.sup.8]hGLP-1(8-36)-NH.sub.2 (SEQ ID NO: ______);
[N-Me-D-Ala.sup.8]hGLP-1(7-36)-NH.sub.2;
[N-Me-D-Ala.sup.8]hGLP-1(8-36)-NH.sub.2;
[N-Me-Gly.sup.8]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 129);
[A5c.sup.8]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 130);
[N-Me-Glu.sup.9]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 131); [A5c.sup.8,
A6c.sup.20,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 132); [Aib.sup.8,
A6c.sup.32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 133);
[Aib.sup.8,25]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 134);
[Aib.sup.824]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 135);
[Aib.sup.8.30]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 136); [Aib.sup.8,
Cha.sup.20]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 137); [Aib.sup.8,
Cha.sup.32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 138); [Aib.sup.8,
Glu.sup.23]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 139); [Aib.sup.8,
A6c.sup.20]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 140); [Aib.sup.8,
A6c.sup.20.32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 141);
[Aib.sup.8,22]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 142); [Aib.sup.8,
.beta.-Ala.sup.22]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 143);
[Aib.sup.8, Lys.sup.25]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 144);
[Aib.sup.8, A6c.sup.12]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 145);
[Aib.sup.8, A6c.sup.29]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 146);
[Aib.sup.8, A6c.sup.33]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 147);
[Aib.sup.8,14]hGLP-1(7-36)NH.sub.2 (SEQ ID NO: 148);
[Aib.sup.8,18]hGLP-1(7-36)NH.sub.2 (SEQ ID NO: 149);
[Aib.sup.8,17]hGLP-1(7-36)NH.sub.2 (SEQ ID NO: 150); or [Aib.sup.8,
D-Arg;.sup.26]hGLP-1(7-36)NH.sub.2; or a pharmaceutically
acceptable salt thereof.
9. A pharmaceutical composition comprising an effective amount of a
compound according to claim 1 or a pharmaceutically acceptable salt
thereof and a pharmaceutically acceptable carrier or diluent.
10. A method of eliciting an agonist effect from a GLP-1 receptor
in a subject in need thereof which comprises administering to said
subject an effective amount of a compound according to claim 1 or a
pharmaceutically acceptable salt thereof.
11. A method of treating a disease selected from the group
consisting of Type I diabetes, Type II diabetes, obesity,
glucagonomas, secretory disorders of the airway, metabolic
disorder, arthritis, osteoporosis, central nervous system disease,
restenosis, neurodegenerative disease, renal failure, congestive
heart failure, nephrotic syndrome, cirrhosis, pulmonary edema, and
hypertension, in a subject in need thereof which comprises
administering to said subject an effective amount of a compound
according to claim 1 or a pharmaceutically acceptable salt
thereof.
12. A method according to claim 11 wherein said disease is Type I
diabetes or Type II diabetes.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention is directed to peptide analogues of
glucagon-like peptide-1, the pharmaceutically-acceptable salts
thereof, to methods of using such analogues to treat mammals and to
pharmaceutical compositions useful therefor comprising said
analogues.
[0002] Glucagon-like peptide-1 (7-36) amide (GLP-1) (SEQ ID NO: 1)
is synthesized in the intestinal L-cells by tissue-specific
post-translational processing of the glucagon precursor
pre-proglucagon (Varndell, J. M., et al., J. Histochem Cytochem,
1985:33:1080-6) and is released into the circulation in response to
a meal. The plasma concentration of GLP-1 rises from a fasting
level of approximately 15 pmol/L to a peak postprandial level of 40
pmol/L. It has been demonstrated that, for a given rise in plasma
glucose concentration, the increase in plasma insulin is
approximately threefold greater when glucose is administered orally
compared with intravenously (Kreymann, B., et al., Lancet 1987:2,
1300-4). This alimentary enhancement of insulin release, known as
the incretin effect, is primarily humoral and GLP-1 is now thought
to be the most potent physiological incretin in humans. In addition
to the insulinotropic effect, GLP-1 suppresses glucagon secretion,
delays gastric emptying (Wettergren A., et al., Dig Dis Sci
1993:38:665-73) and may enhance peripheral glucose disposal
(D'Alessio, D. A. et al., J. Clin Invest 1994:93:2293-6).
[0003] In 1994, the therapeutic potential of GLP-1 was suggested
following the observation that a single subcutaneous (s/c) dose of
GLP-1 could completely normalize postprandial glucose levels in
patients with non-insulin-dependent diabetes mellitus (NIDDM)
(Gutniak, M. K., et al., Diabetes Care 1994:17:1039-44). This
effect was thought to be mediated both by increased insulin release
and by a reduction in glucagon secretion. Furthermore, an
intravenous infusion of GLP-1 has been shown to delay postprandial
gastric emptying in patients with NIDDM (Williams, B., et al., J.
Clin Endo Metab 1996:81:327-32). Unlike sulfonylureas, the
insulinotropic action of GLP-1 is dependent on plasma glucose
concentration (Holz, G. G. 4.sup.th, et al., Nature
1993:361:362-5). Thus, the loss of GLP-1-mediated insulin release
at low plasma glucose concentration protects against severe
hypoglycemia. This combination of actions gives GLP-1 unique
potential therapeutic advantages over other agents currently used
to treat NIDDM.
Numerous studies have shown that when given to healthy subjects,
GLP-1 potently influences glycemic levels as well as insulin and
glucagon concentrations (Orskov, C, Diabetologia 35:701-711, 1992;
Holst, J. J., et al., Potential of GLP-1 in diabetes management in
Glucagon III, Handbook of Experimental Pharmacology, Lefevbre P J,
Ed. Berlin, Springer Verlag, 1996, p. 311-326), effects which are
glucose dependent (Kreymann, B., et al., Lancet ii: 1300-1304,
1987; Weir, G. C., et al., Diabetes 38:338-342, 1989). Moreover, it
is also effective in patients with diabetes (Gutniak, M., N. Engl J
Med 226:1316-1322, 1992; Nathan, D. M., et al., Diabetes Care
15:270-276, 1992), normalizing blood glucose levels in type 2
diabetic subjects (Nauck, M. A., et al., Diagbetologia 36:741-744,
1993), and improving glycemic control in type 1 patients
(Creutzfeldt, W. O., et al., Diabetes Care 19:580-586, 1996),
raising the possibility of its use as a therapeutic agent.
[0004] GLP-1 is, however, metabolically unstable, having a plasma
half-life (t.sub.1/2) of only 1-2 min in vivo. Exogonously
administered GLP-1 is also rapidly degraded (Deacon, C. F., et al.,
Diabetes 44:1126-1131, 1995). This metabolic instability limits the
therapeutic potential of native GLP-1. Hence, there is a need for
GLP-1 analogues that are more active or are more metabolically
stable than native GLP-1.
SUMMARY OF THE INVENTION
[0005] In one aspect, the present invention is directed to a
compound of formula (I),
(R.sup.2R.sup.3)-A.sup.7-A.sup.8-A.sup.9-A.sup.10-A.sup.11-A.sup.12-A.su-
p.13-A.sup.14-A.sup.15-A.sup.16-A.sup.17-A.sup.18-A.sup.19-A.sup.20-A.sup.-
21-A.sup.22-A.sup.23-A.sup.24-A.sup.25-A.sup.26-A.sup.27-A.sup.28-A.sup.29-
-A.sup.30-A.sup.31-A.sup.32-A.sup.33-A.sup.34-A.sup.35-A.sup.36-A.sup.37-R-
.sup.1, (I)
wherein A.sup.7 is L-His, Ura, Paa, Pta, D-His, Tyr, 3-Pal, 4-Pal,
Hppa, Tma-His, Amp or deleted, provided that when A.sup.7 is Ura,
Paa, Pta or Hppa then R.sup.2 and R.sup.3 are deleted;
A.sup.8 is Ala, D-Ala, Aib, Acc, N-Me-Ala, N-Me-D-Ala, Arg or
N-Me-Gly;
A.sup.9 is Glu, N-Me-Glu, N-Me-Asp or Asp;
A.sup.10 is Gly, Acc, Ala, D-Ala, Phe or Aib;
A.sup.11 is Thr or Ser;
A.sup.12 is Phe, Acc, Aic, Aib, 3-Pal, 4-Pal, .beta.-Nal, Cha, Trp
or X.sup.1-Phe;
A.sup.13 is Thr or Ser;
A.sup.14 is Ser, Thr, Ala or Aib;
A.sup.15 is Asp, Ala, D-Asp or Glu;
A.sup.16 is Val, D-Val, Acc, Aib, Leu, Ile, Tle, Nle, Abu, Ala,
D-Ala, Tba or Cha;
A.sup.17 is Ser, Ala, D-Ala, Aib, Acc or Thr;
A.sup.18 is Ser, Ala, D-Ala, Aib, Acc or Thr;
A.sup.19 is Tyr, D-Tyr, Cha, Phe, 3-Pal, 4-Pal, Acc, .beta.-Nal,
Amp or X.sup.1-Phe;
A.sup.20 is Leu, Ala, Acc, Aib, Nle, Ile, Cha, Tle, Val, Phe or
X.sup.1-Phe;
A.sup.21 is Glu, Ala or Asp;
A.sup.22 is Gly, Acc, Ala, D-Ala, .beta.-Ala or Aib;
A.sup.23 is Gln, Asp, Ala, D-Ala, Aib, Acc, Asn or Glu;
A.sup.24 is Ala, Aib, Val, Abu, Tle or Acc;
[0006] A.sup.25 is Ala, Aib, Val, Abu, Tle, Acc, Lys, Arg, hArg,
Orn, HN--CH((CH.sub.2), --NR.sup.10R.sup.11)--C(O) or
HN--CH((CH.sub.2).sub.e--X.sup.3)--C(O); A.sup.26 is Lys, Ala,
3-Pal, 4-Pal, Arg, hArg, Orn, Amp,
HN--CH((CH.sub.2).sub.n--NR.sup.10R.sup.11)--C(O) or
HN--CH((CH.sub.2).sub.e--X.sup.3)--C(O);
A.sup.27 is Glu, Ala, D-Ala or Asp;
A.sup.23 is Phe, Ala, Pal, .beta.-Nal, X.sup.1-Phe, Aic, Acc, Aib,
Cha or Trp;
A.sup.29 is Ile, Acc, Aib, Leu, Nle, Cha, Tle, Val, Abu, Ala, Tba
or Phe;
[0007] A.sup.30 is Ala, Aib, Acc or deleted; A.sup.31 is Trp, Ala,
.beta.-Nal, 3-Pal, 4-Pal, Phe, Acc, Aib, Cha, Amp or deleted;
A.sup.32 is Leu, Ala, Acc, Aib, Nle, Ile, Cha, Tle, Phe,
X.sup.1-Phe, Ala or deleted; A.sup.33 is Val, Acc, Aib, Leu, Ile,
Tle, Nle, Cha, Ala, Phe, Abu, X.sup.1-Phe, Tba, Gaba or deleted;
A.sup.34 is Lys, Arg, hArg, Orn, Amp, Gaba, HN--CH((CH.sub.2),
--NR.sup.10R.sup.11)--C(O), HN--CH((CH.sub.2).sub.e--X.sup.3)--C(O)
or deleted; A.sup.35 is Gly or deleted; A.sup.36 is L- or D-Arg, D-
or L-Lys, D- or L-hArg, D- or L-Orn, Amp, HN--CH((CH.sub.2),
--NR.sup.10R.sup.11)--C(O), HN--CH((CH.sub.2).sub.e--X.sup.3)--C(O)
or deleted; A.sup.37 is Gly or deleted; [0008] X.sup.1 for each
occurrence is independently selected from the group consisting of
(C.sub.1-C.sub.6)alkyl, OH and halo; [0009] R.sup.1 is OH,
NH.sub.2, (C.sub.1-C.sub.12)alkoxy, or
NH--X.sup.2--CH.sub.2-Z.sup.0, wherein X.sup.2 is a
(C.sub.1-C.sub.12)hydrocarbon moiety, and Z.sup.0 is H, OH,
CO.sub.2H or CONH.sub.2; [0010] X.sup.3 is
##STR00001##
[0010] or --C(O)--NHR.sup.12, wherein X.sup.4 for each occurrence
is independently --C(O)--, --NH--C(O)-- or --CH.sub.2--, and f for
each occurrence is independently an integer from 1 to 29; [0011]
each of R.sup.2 and R.sup.3 is independently selected from the
group consisting of H, (C.sub.1-C.sub.30)alkyl,
(C.sub.2-C.sub.30)alkenyl, phenyl(C.sub.1-C.sub.30)alkyl,
naphthyl(C.sub.1-C.sub.30)alkyl, hydroxy(C.sub.1-C.sub.30)alkyl,
hydroxy(C.sub.2-C.sub.30)alkenyl,
hydroxyphenyl(C.sub.1-C.sub.30)alkyl, and
hydroxynaphthyl(C.sub.1-C.sub.30)alkyl; or one of R.sup.2 and
R.sup.3 is C(O)X.sup.5 in which X is (C.sub.1-C.sub.30)alkyl,
(C.sub.2-C.sub.30)alkenyl, phenyl(C.sub.1-C.sub.30)alkyl,
naphthyl(C.sub.1-C.sub.30)alkyl, hydroxy(C.sub.1-C.sub.30)alkyl,
hydroxy(C.sub.2-C.sub.30)alkenyl,
hydroxyphenyl(C.sub.1-C.sub.30)alkyl,
hydroxynaphthyl(C.sub.1-C.sub.30)alkyl,
[0011] ##STR00002## [0012] where Y is H or OH, r is 0-4 and q is
0-4; [0013] n for each occurrence is independently an integer from
1-5; and [0014] R.sup.10 and R.sup.11 for each occurrence is each
independently H, (C.sub.1-C.sub.30)alkyl, (C.sub.1-C.sub.30)acyl,
(C.sub.1-C.sub.30)alkylsulfonyl, --C((NH)(NH.sub.2)) or
##STR00003##
[0014] provided that when R.sup.10 is (C.sub.1-C.sub.30)acyl,
(C.sub.1-C.sub.30)alkylsulfonyl, --C((NH)(NH.sub.2)) or
##STR00004## [0015] R.sup.11 is H or (C.sub.1-C.sub.30)alkyl; and
[0016] R.sup.12 is (C.sub.1-C.sub.30)alkyl; with the proviso that:
(i) at least one amino acid of a compound of formula (I) is not the
same as the native sequence of hGLP-1(7-36, or -37)NH.sub.2 (SEQ ID
NOS: 1, 2) or hGLP-1(7-36, or -37)OH (SEQ ID NOS: 3, 4); (ii) a
compound of formula (I) is not an analogue of hGLP-1(7-36, or
-37)NH.sub.2 (SEQ ID NOS: 1, 2) or hGLP-1(7-36, or -37)OH (SEQ ID
NOS: 3, 4) wherein a single position has been substituted by Ala;
(iii) a compound of formula (I) is not
[Lys.sup.26(N.sup..epsilon.-alkanoyl)]hGLP-1(7-36, or -37)-E (SEQ
ID NOS: 5-8), [Lys.sup.34(N.sup..epsilon.-alkanoyl)]hGLP-1(7-36, or
-37)-E (SEQ ID NOS: 9-12),
[Lys.sup.26,34-bis(N.sup..epsilon.-alkanoyl)]hGLP-1(7-36, or -37)-E
(SEQ ID NOS: 13-16), [Arg.sup.26,
Lys.sup.34(N.sup..epsilon.-alkanoyl)]hGLP-1(8-36, or -37)-E (SEQ ID
NOS: 17-20), or [Arg.sup.26,34,
Lys.sup.36(N.sup..epsilon.-alkanoyl)]hGLP-1(7-36, or -37)-E,
wherein E is --OH or --NH.sub.2 (SEQ ID NOS: 21-24); (iv) a
compound of formula (I) is not Z.sup.1-hGLP-1(7-36, or -37)-OH,
Z.sup.1-hGLP-1(7-36, or -37)-NH.sub.2, where Z.sup.1 is selected
from the group consisting of [0017] (a) [Arg.sup.26] (SEQ ID NOS:
25-28), [Arg.sup.34] (SEQ ID NOS: 29-32), [Arg.sup.23,34] (SEQ ID
NOS: 33-36), [Lys.sup.36] (SEQ ID NOS: 37-40), [Arg.sup.26,
Lys.sup.36] (SEQ ID NOS: 41-44), [Arg.sup.3, Lys.sup.36] (SEQ ID
NOS: 45-48), [D-Lys.sup.36], [Arg.sup.36] (SEQ ID NOS: 3,4,1,2),
[D-Arg.sup.36], [Arg.sup.26,34, Lys.sup.36] (SEQ ID NOS: 49-52), or
[Arg.sup.26,36, Lys.sup.34] (SEQ ID NOS: 25-28); [0018] (b)
[Asp.sup.21] (SEQ ID NOS: 53-56); [0019] (c) at least one of
[Aib.sup.3] (SEQ ID NOS: 57-60), [D-Ala.sup.8] and [Asp.sup.9] (SEQ
ID NOS: 61-64); and [0020] (d) [Tyr.sup.7] (SEQ ID NOS: 65-68),
[N-acyl-His.sup.7] (SEQ ID NOS: 69-72), [N-alkyl-His.sup.7],
[N-acyl-D-His.sup.7] (SEQ ID NOS: 73-76) or [N-alkyl-D-His.sup.7];
(v) a compound of formula (I) is not a combination of any two of
the substitutions listed in groups (a) to (d); and (vi) a compound
of formula (I) is not [N-Me-Ala.sup.8]hGLP-1(8-36 or -37) (SEQ ID
NOS: 75, 78), [Glu.sup.1]hGLP-1(7-36 or -37) (SEQ ID NOS: 79, 80),
[Asp.sup.21]hGLP-1(7-36 or -37) (SEQ ID NOS: 53, 54) or
[Phe.sup.31]hGLP-1(7-36 or -37) (SEQ ID NOS: 81, 82).
[0021] A preferred compound of the immediately foregoing compound
of formula (I) is where A.sup.11 is Thr; A.sup.13 is Thr; A.sup.14
is Ser, Aib or Ala; A.sup.17 is Ser, Ala, Aib or D-Ala; A.sup.18 is
Ser, Ala, Aib or D-Ala; A.sup.21 is Glu or Ala; A.sup.23 is Gln,
Glu, or Ala; and A.sup.27 is Glu or Ala; or a pharmaceutically
acceptable salt thereof.
[0022] A preferred compound of the immediately foregoing compound
of formula (I) is where A.sup.9 is Glu, N-Me-Glu or N-Me-Asp;
A.sup.12 is Phe, Acc or Aic; A.sup.16 is Val, D-Val, Acc, Aib, Ala,
Tle or D-Ala; A.sup.19 is Tyr, 3-Pal, 4-Pal or D-Tyr; A.sup.20 is
Leu, Acc, Cha, Ala or Tle; A.sup.24 is Ala, Aib or Acc; A.sup.25 is
Ala, Aib, Acc, Lys, Arg, hArg, Orn, HN--CH((CH.sub.2),
--NH--R.sup.10)--C(O); A.sup.28 is Phe or Ala; A.sup.29 is Ile, Acc
or Tle; A.sup.30 is Ala, Aib or deleted; A.sup.31 is Trp, Ala,
3-Pal, 4-Pal or deleted; A.sup.32 is Leu, Acc, Cha, Ala or deleted;
A.sup.33 is Val, Acc, Ala, Gaba, Tle or deleted; or a
pharmaceutically acceptable salt thereof.
[0023] A preferred compound of the immediately foregoing compound
of formula (I) is where A.sup.8 is Ala, D-Ala, Aib, A6c, A5c,
N-Me-Ala, N-Me-D-Ala or N-Me-Gly; A.sup.10 is Gly, Ala, D-Ala or
Phe; A.sup.12 is Phe, A6c or A5c; A.sup.16 is Val, Ala, Tle, A6c,
A5c or D-Val; A.sup.20 is Leu, A6c, A5c, Cha, Ala or Tle; A.sup.22
is Gly, Aib, .beta.-Ala, L-Ala or D-Ala; A.sup.24 is Ala or Aib;
A.sup.29 is Ile, A6c, A5c or Tle; A.sup.32 is Leu, A6c, A5c, Cha,
Ala or deleted; A.sup.33 is Val, A6c, A5c, Ala, Gaba, Tle or
deleted; or a pharmaceutically acceptable salt thereof.
[0024] A preferred compound of the immediately foregoing compound
of formula (I) is where R.sup.1 is OH or NH.sub.2 or a
pharmaceutically acceptable salt thereof.
[0025] A preferred compound of the immediately foregoing compound
of formula (I) or a pharmaceutically acceptable salt thereof is
where R.sup.2 is H and R.sup.3 is (C.sub.1-C.sub.30)alkyl,
(C.sub.2-C.sub.30)alkenyl, (C.sub.1-C.sub.30)acyl,
##STR00005##
[0026] A most preferred compound of formula (I) is where said
compound is [D-Ala.sup.8, Ala.sup.17,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]-GLP-1(7-34)NH.sub.2; [D-Ala.sup.8,23,27,
3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2; [Ala.sup.18,23,27,
3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2 (SEQ ID NO: 83);
[Ala.sup.16,23,27, 3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2 (SEQ ID
NO: 84); [Ala.sup.14,23,27
3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2(SEQ ID NO: 85);
[Ala.sup.22,23,27, 3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2 (SEQ ID
NO: 86); [Hppa.sup.7]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 87);
[Ala.sup.23,27, 3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2 (SEQ ID NO:
88); [Ala.sup.7,23,27, 3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2 (SEQ
ID NO: 89); [Ala.sup.22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 90);
[Ala.sup.15,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 91);
[Ala.sup.17,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 92);
[Ala.sup.8,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 93);
[Ala.sup.21,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 94);
[Ala.sup.22,23,26,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 95);
[Ala.sup.22,23,27,32, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 96);
[Ala.sup.22,23,26,27, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 97);
[Ala.sup.22,23,27,31, 3-Pal.sup.19,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 98);
[Ala.sup.22,23,27,28, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 99);
[Ala.sup.22,23,27,29, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 100); [Ala.sup.23,27,
3-Pal.sup.19,31, Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO:
101); [Ala.sup.20,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 102);
[Ala.sup.22,23,27, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 103);
[Ala.sup.17,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 104); [D-Ala.sup.10,
Ala.sup.22,23,27, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2; [D-Ala.sup.8, Ala.sup.17,23,27,
3-Pal.sup.19,31]hGLP-1(7-34)-NH.sub.2; [Ala.sup.17,23,27,
3-Pal.sup.19,26,31]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 105);
[D-Ala.sup.8, Ala.sup.17, 3-Pal.sup.19,31]hGLP-1(7-34)-NH.sub.2;
[Ala.sup.17,23,27, 3-Pal.sup.19,31]hGLP-1(7-34)-NH.sub.2 (SEQ ID
NO: 106); [D-Ala.sup.8, Ala.sup.17,23,27, 3-Pal.sup.31,
Tle.sup.29]hGLP-1(7-34)-NH.sub.2; [D-Ala.sup.8, Ala.sup.17,23,27,
3-Pal.sup.19,31, Tle.sup.16]hGLP-1(7-34)-NH.sub.2; [D-Ala.sup.8,
Ala.sup.17,23,27, 3-Pal.sup.19,31, Gaba.sup.34]hGLP
(7-34)-NH.sub.2; [D-Ala.sup.22, Ala.sup.17,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; [Aib.sup.8, Ala.sup.17,23,27,
3-Pal.sup.19,31, Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO:
107); [D-Ala.sup.8, Ala.sup.17,22,23,27,
3-Pal.sup.19,31]hGLP-1(7-33)-NH.sub.2; [Aib.sup.8,
Ala.sup.17,22,23,27, 3-Pal.sup.193]hGLP-1(7-33)-NH.sub.2 (SEQ ID
NO: 108); [Ala.sup.17,18,23,27, 3-Pal.sup.9,3,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 109);
[Ala.sup.17,23,27, 3-Pal.sup.19,31, Tle.sup.33,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 110); [Tle.sup.16,
Ala.sup.17,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 111);
[N-Me-D-Ala.sup.8, Ala.sup.17,22,23,27,
3-Pal.sup.19,31]hGLP-1(7-33)-NH.sub.2; [Aib.sup.8,
Ala.sup.17,22,23,27, 3-Pal.sup.19,31]hGLP-1(7-33)-NH.sub.2 (SEQ ID
NO: 112); [Ala.sup.17,18,22,23,27, 3-Pal.sup.19,31, Tle.sup.16,20,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 113); [D-Ala.sup.6,
Ala.sup.17,18,22,23,27, 3-Pal.sup.19,31, Tle.sup.16,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; [D-Ala.sup.8,22
Ala.sup.17,18,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; [D-Ala.sup.8,18,
Ala.sup.17,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; [D-Ala.sup.8,17,
Ala.sup.18,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(73)-NH.sub.2; [Ala.sup.17,18,22,23,27,
3-Pal.sup.19,31, Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; or a
pharmaceutically acceptable salt thereof.
[0027] Another most preferred compound of formula (I) is wherein
said compound is
[Aib.sup.8, A6c.sup.32]hGLP-1(7-36)NH.sub.2 (SEQ ID NO: 114);
[A6c.sup.20,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 115);
[Aib.sup.9]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 116);
[(Tma-His).sup.7]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 117);
[A6c.sup.8]hGLP-1(8-36)-NH.sub.2 (SEQ ID NO:118);
[A6c.sup.8]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 119);
[A6c.sup.16,20]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 120);
[A6c.sup.29,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 121); [A6c.sup.20,
Aib.sup.24]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 122); [Aib.sup.24,
A6c.sup.29,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 123);
[A6c.sup.16,29,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 124);
[Ura.sup.7]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 125);
[Paa.sup.7]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 126);
[Pta.sup.7]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 127);
[N-Me-Ala.sup.8]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 128);
[N-Me-Ala.sup.8]hGLP-1(8-36)-NH.sub.2; (SEQ ID NO.)
[N-Me-D-Ala.sup.8]hGLP-1(7-36)-NH.sub.2;
[N-Me-D-Ala.sup.8]hGLP-1(8-36)-NH.sub.2;
[N-Me-Gly.sup.8]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 129);
[A5c.sup.8]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 130);
[N-Me-Glu.sup.9]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 131); [A5c.sup.8,
A6c.sup.20,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 132); [Aib.sup.8,
A6c.sup.32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 133);
[Aib.sup.8,25]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 134);
[Aib.sup.8,24]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 135);
[Aib.sup.8,30]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 136); [Aib.sup.8,
Cha.sup.20]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 137); [Aib.sup.8,
Cha.sup.32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 138); [Aib.sup.8,
Glu.sup.23]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 139); [Aib.sup.8,
A6c.sup.20]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 140); [Aib.sup.8,
A6c.sup.20,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 141);
[Aib.sup.8,22]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 142); [Aib.sup.8,
.beta.-Ala.sup.22]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 143);
[Aib.sup.8, Lys.sup.25]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 144);
[Aib.sup.8, A6c.sup.12]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 145);
[Aib.sup.8, A6c.sup.29]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 146);
[Aib.sup.8, A6c.sup.33]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 147);
[Aib.sup.8,14]hGLP-1(7-36)NH.sub.2 (SEQ ID NO: 148);
[Aib.sup.8,18]hGLP-1(7-36)NH.sub.2 (SEQ ID NO: 149); or
[Aib.sup.8,17]hGLP-1(7-36)NH.sub.2 (SEQ ID NO: 150); or a
pharmaceutically acceptable salt thereof. In another aspect, the
present invention provides a pharmaceutical composition comprising
an effective amount of a compound of formula (I) as defined
hereinabove or a pharmaceutically acceptable salt thereof and a
pharmaceutically acceptable carrier or diluent.
[0028] In still another aspect, the present invention provides a
method of eliciting an agonist effect from a GLP-1 receptor in a
subject in need thereof which comprises administering to said
subject an effective amount of a compound of formula (I) as defined
hereinabove or a pharmaceutically acceptable salt thereof.
[0029] In yet a further aspect, this invention provides a method of
treating a disease selected from the group consisting of Type I
diabetes, Type II diabetes, obesity, glucagonomas, secretory
disorders of the airway, metabolic disorder, arthritis,
osteoporosis, central nervous system disease, restenosis and
neurodegenerative disease, renal failure, congestive heart failure,
nephrotic syndrome, cirrhosis, pulmonary edema, hypertension, and
disorders wherein the reduction of food intake is desired, in a
subject in need thereof which comprises administering to said
subject an effective amount of a compound of formula (I) as defined
hereinabove or a pharmaceutically acceptable salt thereof.
Preferred of the foregoing method is where the disease is Type I
diabetes or Type II diabetes.
[0030] With the exception of the N-terminal amino acid, all
abbreviations (e.g. Ala) of amino acids in this disclosure stand
for the structure of --NH--CH(R)--CO--, wherein R is the side chain
of an amino acid (e.g., CH.sub.3 for Ala). For the N-terminal amino
acid, the abbreviation stands for the structure of
(R.sup.2R.sup.3)--N--CH(R)--CO--, wherein R is a side chain of an
amino acid and R.sup.2 and R.sup.3 are as defined above except in
the case where A.sup.7 is Ura, Paa, Pta or Hppa in which case
R.sup.2 and R.sup.3 are not present since Ura, Paa, Pta and Hppa
are considered here as des-amino amino acids. The abbreviations:
.beta.-Nal, Nle, Cha, Amp, 3-Pal, 4-Pal and Aib stand for the
following .alpha.-amino acids: .beta.-(2-naphthyl)alanine,
norleucine, cyclohexylalanine, 4-amino-phenylalanine,
.beta.-(3-pyridinyl)alanine, .beta.-(4-pyridinyl)alanine and
.alpha.-aminoisobutyric acid, respectively. Other amino acid
definitions are: Ura is urocanic acid; Pta is (4-pyridylthio)
acetic acid; Paa is trans-3-(3-pyridyl)acrylic acid; Tma-His is
N,N-tetramethylamidino-histidine; N-Me-Ala is N-methyl-alanine;
N-Me-Gly is N-methyl-glycine; N-Me-Glu is N-methyl-glutamic acid;
Tle is tert-butylglycine; Abu is .alpha.-aminobutyric acid; Tba is
tert-butylalanine; Orn is ornithine; Aib is .alpha.-aminoisobutyric
acid; .beta.-Ala is .beta.-alanine; Gaba is .gamma.-aminobutyric
acid; Ava is 5-aminovaleric acid; and Aic is
2-aminoindane-2-carboxylic acid.
[0031] What is meant by Acc is an amino acid selected from the
group of 1-amino-1-cyclopropanecarboxylic acid (A3c);
1-amino-1-cyclobutanecarboxylic acid (A4c);
1-amino-1-cyclopentanecarboxylic acid (A5c);
1-amino-1-cyclohexanecarboxylic acid (A6c);
1-amino-1-cycloheptanecarboxylic acid (A7c);
1-amino-1-cyclooctanecarboxylic acid (A8c); and
1-amino-1-cyclononanecarboxylic acid (A9c). In the above formula,
hydroxyalkyl, hydroxyphenylalkyl, and hydroxynaphthylalkyl may
contain 1-4 hydroxy substituents. COX.sup.5 stands for
--C.dbd.O.X.sup.5. Examples of --C.dbd.O.X.sup.5 include, but are
not limited to, acetyl and phenylpropionyl.
[0032] What is meant by Lys(N.sup..epsilon.-alkanoyl) is
represented by the following structure:
##STR00006##
[0033] What is meant by Lys(N.sup..epsilon.-alkylsulfonyl) is
represented by the following structure:
##STR00007##
What is meant by
Lys(N.sup..epsilon.-(2-(4-alkyl-1-piperazine)-acetyl)) is
represented by the following structure:
##STR00008##
What is meant by Asp(1-(4-alkyl-piperazine)) is represented by the
following structure:
##STR00009##
What is meant by Asp(1-alkylamino) is represented by the following
structure:
##STR00010##
The variable n in the foregoing structures is 1 to 30.
[0034] The full names for other abbreviations used herein are as
follows: Boc for t-butyloxycarbonyl, HF for hydrogen fluoride, Fm
for formyl, Xan for xanthyl, Bzl for benzyl, Tos for tosyl, DNP for
2,4-dinitrophenyl, DMF for dimethylformamide, DCM for
dichloromethane, HBTU for
2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate, DIEA for diisopropylethylamine, HOAc for
acetic acid, TFA for trifluoroacetic acid, 2CIZ for
2-chlorobenzyloxycarbonyl and OcHex for Q-cyclohexyl.
[0035] A peptide of this invention is also denoted herein by
another format, e.g., [A5c.sup.8]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO:
130), with the substituted amino acids from the natural sequence
placed between the set of brackets (e.g., A5c.sup.8 for Ala.sup.8
in hGLP-1). The abbreviation GLP-1 means glucagon-like peptide-1,
and hGLP-1 means human glucagon-like peptide-1. The numbers between
the parentheses refer to the number of amino acids present in the
peptide (e.g., hGLP-1(7-36) (SEQ ID NO: 3) is amino acids 7 through
36 of the peptide sequence for human GLP-1). The sequence for
hGLP-1(7-37) (SEQ ID NO: 4) is listed in Mojsov, S., Int. J.
Peptide Protein Res., 40, 1992, pp. 333-342. The designation
"NH.sub.2" in hGLP-1(7-36)NH.sub.2 (SEQ ID NO: 1) indicates that
the C-terminus of the peptide is amidated. hGLP-1(7-36) (SEQ ID NO:
2) means that the C-terminus is the free acid.
DETAILED DESCRIPTION
[0036] The peptides of this invention can be prepared by standard
solid phase peptide synthesis. See, e.g., Stewart, J. M., et al.,
Solid Phase Synthesis (Pierce Chemical Co., 2d ed. 1984). The
substituents R.sup.2 and R.sup.3 of the above generic formula can
be attached to the free amine of the N-terminal amino acid by
standard methods known in the art. For example, alkyl groups, e.g.,
(C.sub.1-C.sub.30)alkyl, can be attached using reductive
alkylation. Hydroxyalkyl groups, e.g.,
(C.sub.1-C.sub.30)hydroxyalkyl, can also be attached using
reductive alkylation wherein the free hydroxy group is protected
with a t-butyl ester. Acyl groups, e.g., COE.sup.1, may be attached
by coupling the free acid, e.g., E.sup.1COOH, to the free amine of
the N-terminal amino acid by mixing the completed resin with 3
molar equivalents of both the free acid and diisopropylcarbodiimide
in methylene chloride for one hour. If the free acid contains a
free hydroxy group, e.g., p-hydroxyphenylpropionic acid, then the
coupling should be performed with an additional 3 molar equivalents
of HOBT.
[0037] When R.sup.1 is NH--X.sup.2--CH.sub.2--CONH.sub.2 (i.e.,
Z.sup.0=CONH.sub.2), the synthesis of the peptide starts with
BocHN--X.sup.2--CH.sub.2--COOH which is coupled to the MBHA resin.
If R.sup.1 is NH--X.sup.2--CH.sub.2--COOH (i.e., Z.sup.0=COOH) the
synthesis of the peptide starts with
Boc-HN--X.sup.2--CH.sub.2--COOH which is coupled to PAM resin.
[0038] The following describes a synthetic method for making a
peptide of this invention, which method is well-known to those
skilled in the art. Other methods are also known to those skilled
in the art.
[0039] Benzhydrylamine-polystyrene resin (Advanced ChemTech, Inc.,
Louisville, Ky.) (0.9 g, 0.3 mmole) in the chloride ion form is
placed in a reaction vessel of an Advanced ChemTech Peptide
Synthesizer Model 200 programmed to perform the following reaction
cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in
methylene chloride (2 times for 1 and 15 min each); (c) methylene
chloride; (d) ethanol; (e) methylene chloride; (f) 10%
diisopropylethylamine in methylene chloride.
[0040] The neutralized resin is stirred with Boc-protected amino
acid which is to be the C-terminal amino acid of the desired
peptide to be synthesized and diisopropylcarbodiimide (3 mmole
each) in methylene chloride for 1 hour and the resulting amino acid
resin is then cycled through steps (a) through (f) in the above
wash program. The other amino acids (3 mmol) of the desired peptide
are then coupled successively by the same procedure. The finished
peptide is cleaved from the resin by mixing it with anisole (5 ml),
dithiothreitol (100 mg) and anhydrous hydrogen fluoride (35 ml) at
about 0.degree. C. and stirring it for about 45 min. Excess
hydrogen fluoride is evaporated rapidly under a stream of dry
nitrogen and free peptide precipitated and washed with ether. The
crude peptide is then dissolved in a minimum volume of dilute
acetic acid and eluted on a column (2.5.times.25 cm) of VYDAC.RTM.
octadecylsilane silica (10 mM) and eluted with a linear gradient of
20-60% acetonitrile over about 1 h in 0.1% trifluoroacetic acid in
water. Fractions are examined by thin layer chromatography and
analytical high performance liquid chromatography (40-70% B at
1%/min, solution B is 80% acetonitrile/water containing 0.1% TFA)
and pooled to give maximum purity rather than yield. Repeated
lyophilization of the solution from water gives the product as a
white, fluffy powder.
[0041] The product peptide is analyzed by HPLC. Amino acid analysis
of an acid hydrolysate of the product peptide can confirm the
composition of the peptide. Laser desorption MS is used to
determine the molecular weight of the peptide.
[0042] The protected amino acid
1-[N-tert-butoxycarbonyl-amino]-1-cyclohexane-carboxylic acid
(Boc-A6c-OH) was synthesized as follows. 19.1 g (0.133 mol) of
1-amino-1-cyclohexanecarboxylic acid (Acros Organics, Fisher
Scientific, Pittsburgh, Pa.) was dissolved in 200 ml of dioxane and
100 ml of water. To it was added 67 ml of 2N NaOH. The solution was
cooled in an ice-water bath. 32.0 g (0.147 mol) of
di-tert-butyl-dicarbonate was added to this solution. The reaction
mixture was stirred overnight at room temperature. Dioxane was then
removed under reduced pressure. 200 ml of ethyl acetate was added
to the remaining aqueous solution. The mixture was cooled in an
ice-water bath. The pH of the aqueous layer was adjusted to about 3
by adding 4N HCl. The organic layer was separated. The aqueous
layer was extracted with ethyl acetate (1.times.100 ml). The two
organic layers were combined and washed with water (2.times.150
ml), dried over anhydrous MgSO.sub.4, filtered, and concentrated to
dryness under reduced pressure. The residue was recrystallized in
ethyl acetate/hexanes. 9.2 g of the pure product was obtained. 29%
yield.
[0043] Boc-A5c-OH was synthesized in an analogous manner to that of
Boc-A6c-OH. Other protected Acc amino acids can be prepared in an
analogous manner by a person of ordinary skill in the art as
enabled by the teachings herein.
[0044] In the synthesis of a peptide of this invention containing
A5c, A6c and/or Aib, the coupling time is about 2 hrs. for these
residues and the residue immediately following them. For the
synthesis of [Tma-His.sup.7]hGLP-1(7-36)NH.sub.2 (SEQ ID NO: 117),
HBTU (2 mmol) and DIEA (1.0 ml) in 4 ml DMF were used to react with
the N-terminal free amine of the peptide-resin in the last coupling
reaction; the coupling time is about 2 hours.
[0045] The full names for the abbreviations used above are as
follows: Boc for t-butyloxycarbonyl, HF for hydrogen fluoride, Fm
for formyl, Xan for xanthyl, Bzl for benzyl, Tos for tosyl, DNP for
2,4-dinitrophenyl, DMF for dimethylformamide, DCM for
dichloromethane, HBTU for
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate, DIEA for diisopropylethylamine, HOAc for
acetic acid, TFA for trifluoroacetic acid, 2CIZ for
2-chlorobenzyloxycarbonyl, 2BrZ for 2-bromobenzyloxycarbonyl and
OcHex for O-cyclohexyl.
[0046] The substituents R.sup.2 and R.sup.3 of the above generic
formula can be attached to the free amine of the N-terminal amino
acid by standard methods known in the art. For example, alkyl
groups, e.g., (C.sub.1-C.sub.30)alkyl, may be attached using
reductive alkylation. Hydroxyalkyl groups, e.g.,
(C.sub.1-C.sub.30)hydroxyalkyl, can also be attached using
reductive alkylation wherein the free hydroxy group is protected
with a t-butyl ester. Acyl groups, e.g., COX.sup.1, can be attached
by coupling the free acid, e.g., X.sup.1COOH, to the free amine of
the N-terminal amino acid by mixing the completed resin with 3
molar equivalents of both the free acid and diisopropylcarbodiimide
in methylene chloride for about one hour. If the free acid contains
a free hydroxy group, e.g., p-hydroxyphenylpropionic acid, then the
coupling should be performed with an additional 3 molar equivalents
of HOBT.
[0047] A compound of the present invention can be tested for
activity as a GLP-1 binding compound according to the following
procedure.
Cell Culture:
[0048] RIN 5F rat insulinoma cells (ATCC-# CRL-2058, American Type
Culture Collection, Manassas, Va.), expressing the GLP-1 receptor,
were cultured in Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal calf serum, and maintained at about 37.degree.
C. in a humidified atmosphere of 5% CO.sub.2/95% air.
Radioligand Binding:
[0049] Membranes were prepared for radioligand binding studies by
homogenization of the RIN cells in 20 ml of ice-cold 50 mM Tris-HCl
with a Brinkman Polytron (Westbury, N.Y.) (setting 6, 15 sec). The
homogenates were washed twice by centrifugation (39,000 g/10 min),
and the final pellets were re-suspended in 50 mM Tris-HCl,
containing 2.5 mM MgCl.sub.2, 0.1 mg/ml bacitracin (Sigma Chemical,
St. Louis, Mo.), and 0.1% BSA. For assay, aliquots (0.4 ml) were
incubated with 0.05 nM [.sup.125I]GLP-1(7-36) (SEQ ID NO: 151)
(.about.2200 Ci/mmol, New England Nuclear, Boston, Mass.), with and
without 0.05 ml of unlabeled competing test peptides. After a 100
min incubation (25.degree. C.), the bound [.sup.125I]GLP-1(7-36)
(SEQ ID NO: 151) was separated from the free by rapid filtration
through GF/C filters (Brandel, Gaithersburg, Md.), which had been
previously soaked in 0.5% polyethyleneimine. The filters were then
washed three times with 5 ml aliquots of ice-cold 50 mM Tris-HCl,
and the bound radioactivity trapped on the filters was counted by
gamma spectrometry (Wallac L K B, Gaithersburg, Md.). Specific
binding was defined as the total [.sup.125I]GLP-1(7-36) (SEQ ID NO:
151) bound minus that bound in the presence of 1000 nM GLP-1(7-36)
(SEQ ID NO: 3) (Bachem, Torrence, Calif.).
[0050] The peptides of this invention can be provided in the form
of pharmaceutically acceptable salts. Examples of such salts
include, but are not limited to, those formed with organic acids
(e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic,
benzoic, methanesulfonic, toluenesulfonic, or pamoic acid),
inorganic acids (e.g., hydrochloric acid, sulfuric acid, or
phosphoric acid), and polymeric acids (e.g., tannic acid,
carboxymethyl cellulose, polylactic, polyglycolic, or copolymers of
polylactic-glycolic acids). A typical method of making a salt of a
peptide of the present invention is well known in the art and can
be accomplished by standard methods of salt exchange. Accordingly,
the TFA salt of a peptide of the present invention (the TFA salt
results from the purification of the peptide by using preparative
HPLC, eluting with TFA containing buffer solutions) can be
converted into another salt, such as an acetate salt by dissolving
the peptide in a small amount of 0.25 N acetic acid aqueous
solution. The resulting solution is applied to a semi-prep HPLC
column (Zorbax, 300 SB, C-8). The column is eluted with (1) 0.1N
ammonium acetate aqueous solution for 0.5 hrs., (2) 0.25N acetic
acid aqueous solution 0.5 hrs. and (3) a linear gradient (20% to
100% of solution B over 30 min.) at a flow rate of 4 ml/min
(solution A is 0.25N acetic acid aqueous solution; solution B is
0.25N acetic acid in acetonitrile/water, 80:20). The fractions
containing the peptide are collected and lyophilized to
dryness.
[0051] As is well known to those skilled in the art, the known and
potential uses of GLP-1 is varied and multitudinous [See, Todd, J.
F., et al., Clinical Science, 1998, 95, pp. 325-329; and Todd, J.
F. et al., European Journal of Clinical Investigation, 1997, 27,
pp. 533-536]. Thus, the administration of the compounds of this
invention for purposes of eliciting an agonist effect can have the
same effects and uses as GLP-1 itself. These varied uses of GLP-1
may be summarized as follows, treatment of: Type I diabetes, Type
II diabetes, obesity, glucagonomas, secretory disorders of the
airway, metabolic disorder, arthritis, osteoporosis, central
nervous system diseases, restenosis and neurodegenerative diseases.
GLP-1 analogues of the present invention that elicit an antagonist
effect from a subject can be used for treating the following:
hypoglycemia and malabsorption syndrome associated with
gastroectomy or small bowel resection.
[0052] Accordingly, the present invention includes within its scope
pharmaceutical compositions comprising, as an active ingredient, at
least one of the compounds of formula (I) in association with a
pharmaceutically acceptable carrier or diluent.
[0053] The dosage of active ingredient in the compositions of this
invention may be varied; however, it is necessary that the amount
of the active ingredient be such that a suitable dosage form is
obtained. The selected dosage depends upon the desired therapeutic
effect, on the route of administration, and on the duration of the
treatment. In general, an effective dosage for the activities of
this invention is in the range of 1.times.10.sup.-7 to 200
mg/kg/day, preferably 1.times.10.sup.-4 to 100 mg/kg/day, which can
be administered as a single dose or divided into multiple
doses.
[0054] The compounds of this invention can be administered by oral,
parenteral (e.g., intramuscular, intraperitoneal, intravenous or
subcutaneous injection, or implant), nasal, vaginal, rectal,
sublingual or topical routes of administration and can be
formulated with pharmaceutically acceptable carriers to provide
dosage forms appropriate for each route of administration.
[0055] Solid dosage forms for oral administration include capsules,
tablets, pills, powders and granules. In such solid dosage forms,
the active compound is admixed with at least one inert
pharmaceutically acceptable carrier such as sucrose, lactose, or
starch. Such dosage forms can also comprise, as is normal practice,
additional substances other than such inert diluents, e.g.,
lubricating agents such as magnesium stearate. In the case of
capsules, tablets and pills, the dosage forms may also comprise
buffering agents. Tablets and pills can additionally be prepared
with enteric coatings.
[0056] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups, the elixirs containing inert diluents commonly used in the
art, such as water. Besides such inert diluents, compositions can
also include adjuvants, such as wetting agents, emulsifying and
suspending agents, and sweetening, flavoring and perfuming
agents.
[0057] Preparations according to this invention for parenteral
administration include sterile aqueous or non-aqueous solutions,
suspensions, or emulsions. Examples of non-aqueous solvents or
vehicles are propylene glycol, polyethylene glycol, vegetable oils,
such as olive oil and corn oil, gelatin, and injectable organic
esters such as ethyl oleate. Such dosage forms may also contain
adjuvants such as preserving, wetting, emulsifying, and dispersing
agents. They may be sterilized by, for example, filtration through
a bacteria-retaining filter, by incorporating sterilizing agents
into the compositions, by irradiating the compositions, or by
heating the compositions. They can also be manufactured in the form
of sterile solid compositions which can be dissolved in sterile
water, or some other sterile injectable medium immediately before
use.
[0058] Compositions for rectal or vaginal administration are
preferably suppositories which may contain, in addition to the
active substance, excipients such as coca butter or a suppository
wax.
[0059] Compositions for nasal or sublingual administration are also
prepared with standard excipients well known in the art.
[0060] Further, a compound of this invention can be administered in
a sustained release composition such as those described in the
following patents and patent applications. U.S. Pat. No. 5,672,659
teaches sustained release compositions comprising a bioactive agent
and a polyester. U.S. Pat. No. 5,595,760 teaches sustained release
compositions comprising a bioactive agent in a gelable form. U.S.
application Ser. No. 08/929,363 filed Sep. 9, 1997, teaches
polymeric sustained release compositions comprising a bioactive
agent and chitosan. U.S. application Ser. No. 08/740,778 filed Nov.
1, 1996, teaches sustained release compositions comprising a
bioactive agent and cyclodextrin. U.S. application Ser. No.
09/015,394 filed Jan. 29, 1998, teaches absorbable sustained
release compositions of a bioactive agent. U.S. application Ser.
No. 09/121,653 filed Jul. 23, 1998, teaches a process for making
microparticles comprising a therapeutic agent such as a peptide in
an oil-in-water process. U.S. application Ser. No. 09/131,472 filed
Aug. 10, 1998, teaches complexes comprising a therapeutic agent
such as a peptide and a phosphorylated polymer. U.S. application
Ser. No. 09/184,413 filed Nov. 2, 1998, teaches complexes
comprising a therapeutic agent such as a peptide and a polymer
bearing a non-polymerizable lactone. The teachings of the foregoing
patents and applications are incorporated herein by reference.
[0061] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Also,
all publications, patent applications, patents and other references
mentioned herein are incorporated by reference.
[0062] The following examples describe synthetic methods for making
a peptide of this invention, which methods are well-known to those
skilled in the art. Other methods are also known to those skilled
in the art. The examples are provided for the purpose of
illustration and is not meant to limit the scope of the present
invention in any manner.
EXAMPLE 1
[D-Ala.sup.8, Ala.sup.17,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]-GLP-1(7-34)NH.sub.2
[0063] Benzhydrylamine-polystyrene resin (Advanced ChemTech, Inc.
Louisville, Ky.) (0.9 g, 0.3 mmole) in the chloride ion form was
placed in a reaction vessel of an Advanced ChemTech peptide
synthesizer Model 200 programmed to perform the following reaction
cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in
methylene chloride (2 times for 1 and 15 min each); (c) methylene
chloride; (d) ethanol; (e) methylene chloride; (f) 10%
diisopropylethylamine in methylene chloride.
[0064] The neutralized resin was stirred with Boc-Gaba and
diisopropylcarbodiimide (3 mmole each) in methylene chloride for 1
hour and the resulting amino acid resin was then cycled through
steps (a) to (f) in the above wash program. The following amino
acids (3 mmole) were then coupled successively by the same
procedure: Boc-Val, Boc-Leu, Boc-3-Pal, Boc-Ala, Boc-Ile, Boc-Phe,
Boc-Ala, Boc-Lys(2-Cl-Z), Boc-Ala, Boc-Ala, Boc-Ala, Boc-Ala,
Boc-Glu(Bzl), Boc-Leu, Boc-3-Pal, Boc-Ser(Bzl), Boc-Ala, Boc-Val,
Boc-Asp(Bzl), Boc-Ser(Bzl), Boc-Thr(Bzl), Boc-Phe, Boc-Thr(Bzl),
Boc-Gly, Boc-Glu(Bzl), Boc-D-Ala, Boc-His(Bom).
[0065] The resin with the completed peptide sequence was mixed with
anisole (5 ml), dithiothreitol (100 mg) and anhydrous hydrogen
fluoride (35 ml) at about 0.degree. C. and stirred for about 45
min. Excess hydrogen fluoride was evaporated rapidly under a stream
of dry nitrogen and free peptide precipitated and washed with
ether. The crude peptide was then dissolved in a minimum volume of
dilute acetic acid and eluted on a column (2.5.times.25 cm) of
VYDAC.RTM. octadecylsilane silica (10 mM) and eluted with a linear
gradient of 20-60% acetonitrile over about 1 h in 0.1%
trifluoroacetic acid in water. Fractions were examined by thin
layer chromatography and analytical high performance liquid
chromatography (40-70% B at 1%/min; r.t.: 14.1 min) and pooled to
give maximum purity rather than yield. Repeated lyophilization of
the solution from water gives the product (49.9 mg) as a white,
fluffy powder.
[0066] The product was found to be homogeneous by HPLC and tlc.
Amino acid analysis of an acid hydrolysate confirms the composition
of the peptide. Laser desorption MS gave a MW of 2880 (Calc. M+H
2873).
EXAMPLE 2
Synthesis of Peptide Lower-Alkylamides
[0067] Peptides are assembled on O-benzyl-polystyrene resin (often
referred to as Merrifield resin) using the Boc amino acid protocol
described in Example 1, except that Asp and Glu amino acid carboxyl
side-chains are protected with an Fm (fluorenylmethyl ester) group.
Completed peptide-resins are suspended in dilute DMF solutions of
an appropriate lower alkylamine (such as ethylamine, propylamine,
phenethylamine, 1,2-diaminoethane, etc.) and stirred at about
60.degree. C. (for about 18 hrs) whereupon filtration, removal of
solvents under reduced pressure and trituration of cleaved peptide
oil with ether gives a solid, protected alkylamide peptide. This is
then subjected to HF cleavage to remove additional side chain
protecting groups and HPLC purification as described in Example
1.
EXAMPLES 3-5
[0068] Examples 3-5 can be synthesized substantially according to
the procedure described in Example 1 using the appropriate
protected amino acids to yield the noted peptides.
Example 3: [Aib.sup.8, D-Ala.sup.17, Ala.sup.18,22,23,27,
3-Pal.sup.19,31, Tle.sup.16, Gaba.sup.34]-GLP-1(7-34)NH.sub.2
Example 4: [Aib.sup.3, D-Ala.sup.17, Ala.sup.22,23,27,
3-Pal.sup.19,31, Tle.sup.16]-GLP-1(7-33)NH.sub.2
Example 5: [Aib.sup.8 D-Ala.sup.17 Ala.sup.22,23,27,
3-Pal.sup.19,31, Tle.sup.16,20]-GLP-1(7-33)NH.sub.2
EXAMPLES 6-51
[0069] Examples 6-51 were made substantially according to the
procedure described for Example 1 but using the appropriate
protected amino acid to yield the noted peptide. MS were obtained
by laser desorption MS (NA means not available).
Example 6: [D-Ala.sup.8,23,27,
3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2; MS=2971.0; Calc. MW=2974.4.
Example 7: [Ala.sup.18,23,27, 3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2
(SEQ ID NO: 83); MS=2954.4; Calc. MW=2958.4. Example 8:
[Ala.sup.16,23,27, 3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2 (SEQ ID
NO: 84); MS=2943.0; Calc. MW=2946.3. Example 9: [Ala.sup.14,23,27,
3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2 (SEQ ID NO: 85); MS=2956.0;
Calc. MW=2958.4. Example 10: [Ala.sup.22,23,27,
3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2 (SEQ ID NO: 86); MS=2981.0;
Calc. MW=2988.4. Example 11: [Hppa.sup.7]hGLP-1(7-36)-NH.sub.2 (SEQ
ID NO: 87); MS=NA Example 12: [Ala.sup.15,23,27,
3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2 (SEQ ID NO: 88); MS=2928.0;
Calc. MW=2930.4. Example 13: [Ala.sup.17,23,27,
3-Pal.sup.19,31]hGLP-1(7-35)-NH.sub.2 (SEQ ID NO: 89); MS=2955.0;
Calc. MW=2958.4. Example 14: [Ala.sup.22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 90); MS=2896.0; Calc.
MW=2888.3. Example 15: [Ala.sup.15,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 91); MS=2852.0; Calc.
MW=2844.3. Example 16: [Ala.sup.17,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 92); MS=2880.0; Calc.
MW=2872.3. Example 17: [Ala.sup.18,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 93); MS=2870.0; Calc.
MW=2872.3. Example 18: [Ala.sup.21,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 94); MS=NA. Example
19: [Ala.sup.22,23,26,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 95); MS=2832.0; Calc.
MW=2831.2. Example 20: [Ala.sup.22,23,27,32, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 96); MS=2855.0; Calc.
MW=2846.2. Example 21: [Ala.sup.22,23,26,27, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 97); MS=2729.0; Calc.
MW=2732.0. Example 22: [Ala.sup.22,23,27,31, 3-Pal.sup.19,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 98); MS=2711.6; Calc.
MW=2712.0. Example 23: [Ala.sup.22,23,27,28, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 99); MS=2712.0; Calc.
MW=2713.0. Example 24: [Ala.sup.22,23,27,29, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 100); MS=2746.9;
Calc. MW=2747.1. Example 25: [Ala.sup.23,27, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO: 101); MS=2777.0;
Calc. MW=2,775.1. Example 26: [Ala.sup.20,22,23,27,
3-Pal.sup.19,31, Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO:
102); MS=2742.0; Calc. MW=2747.1. Example 27: [Ala.sup.22,23,27,
3-Pal.sup.19,31, Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO:
103); MS=2786.7; Calc. MW=2789.1. Example 28: [Ala.sup.17,22,23,27,
3-Pal.sup.19,31, Gaba.sup.33]hGLP-1(7-33)-NH.sub.2 (SEQ ID NO:
104); MS=2771.0; Calc. MW=2773.1. Example 29: [D-Ala.sup.10,
Ala.sup.22,23,27, 3-Pal.sup.19,31,
Gaba.sup.33]hGLP-1(7-33)-NH.sub.2; MS=2802.0; Calc. MW=2803.2.
Example 30: [D-Ala.sup.8, Ala.sup.17,23,27,
3-Pal.sup.19,31]hGLP-1(7-34)-NH.sub.2; MS=2905.0; Calc. MW=2901.3.
Example 31: [Ala.sup.17,23,27,
3-Pal.sup.19,26,31]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 105);
MS=2920.0; Calc. MW=2921.3. Example 32: [D-Ala.sup.8, Ala.sup.17,
3-Pal.sup.19,31]hGLP-1(7-34)-NH.sub.2; MS=2908.0 (Na.sup.+ salt);
Calc. MW=2885.3. Example 33: [Ala.sup.17,23,27,
3-Pal.sup.19,31]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 106); MS=2907.0;
Calc. MW=2901.3. Example 34: [D-Ala.sup.8, Ala.sup.17,23,27,
3-Pal.sup.19,31, Tle.sup.29]hGLP-1(7-34)-NH.sub.2; MS=2906.0; Calc.
MW=2901.3. Example 35: [D-Ala.sup.8, Ala.sup.17,23,27,
3-Pal.sup.19,31, Tle.sup.16]hGLP-1(7-34)-NH.sub.2; MS=2914.0; Calc.
MW=2915.4. Example 36: [D-Ala.sup.8, Ala.sup.17,23,27,
3-Pal.sup.19,31, Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; MS=2856.8;
Calc. MW=2858.2. Example 37: [D-Ala.sup.22, Ala.sup.17,23,27,
3-Pal.sup.19,31, Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; MS=2871.0;
Calc. MW=2872.3. Example 38: [Aib.sup.8, Ala.sup.17,23,27,
3-Pal.sup.19,31, Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO:
107); MS=2875.0; Calc. MW=2872.3. Example 39: [D-Ala.sup.8,
Ala.sup.17,22,23,27, 3-Pal.sup.19,31]hGLP-1(7-33)-NH.sub.2;
MS=2786.0; Calc. MW=2787.2. Example 40: [Aib.sup.8
Ala.sup.17,22,23,27, 3-Pal.sup.19,31]hGLP-1(7-33)-NH.sub.2 (SEQ ID
NO: 108); MS=2800.0; Calc. MW=2801.2. Example 41:
[Ala.sup.17,18,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 109); MS=2842.5;
Calc. MW=2842.2. Example 42: [Ala.sup.17,23,27, 3-Pal.sup.19,31,
Tle.sup.33, Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 110);
MS=2871.0; Calc. MW=2872.3. Example 43: [Tle.sup.16,
Ala.sup.17,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 111); MS=2870.0;
Calc. MW=2872.3. Example 44: [N-Me-D-Ala.sup.8,
Ala.sup.17,22,23,27, 3-Pal.sup.19,31]hGLP-1(7-33)-NH.sub.2;
MS=2795.0; Calc. MW=2801.2. Example 45: [Aib.sup.8,
Ala.sup.17,18,22,23,27, 3-Pal.sup.19,31]hGLP-1(7-33)-NH.sub.2 (SEQ
ID NO: 112); MS=2784.2; Calc. MW=2785.2. Example 46:
[Ala.sup.17,18,22,23,27, 3-Pal.sup.19,31, Tle.sup.16,20
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2 (SEQ ID NO: 113); MS=2871.9;
Calc. MW=2870.3. Example 47: [D-Ala.sup.8, Ala.sup.17,18,22,23,27,
3-Pal.sup.19,31, Tle.sup.16, Gaba.sup.34]hGLP-1(7-34)-NH.sub.2;
MS=2870.0; Calc. MW=2870.3. Example 48: [D-Ala.sup.8,22,
Ala.sup.17,18,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; MS=2856.3; Calc. MW=2856.3.
Example 49: [D-Ala.sup.8,18, Ala.sup.17,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; MS=NA. Example 50: [D-Ala.sup.8,
Ala.sup.17,18,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; MS=NA. Example 51: [D-Ala.sup.8,
Ala.sup.17,18,22,23,27, 3-Pal.sup.19,31,
Gaba.sup.34]hGLP-1(7-34)-NH.sub.2; MS=2861.6; Calc. MW=2856.3.
EXAMPLE 52
[Aib.sup.8, A6c.sup.32]hGLP-1(7-36)NH.sub.2 (SEQ ID NO: 114)
[0070] The title peptide was synthesized on an Applied Biosystems
(Foster City, Calif.) model 430A peptide synthesizer which was
modified to do accelerated Boc-chemistry solid phase peptide
synthesis. See Schnolzer, et al., Int. J. Peptide Protein Res.,
40:180 (1992). 4-Methylbenzhydrylamine (MBHA) resin (Peninsula,
Belmont, Calif.) with the substitution of 0.91 mmol/g was used. The
Boc amino acids (Bachem, Calif., Torrance, Calif.; Nova Biochem.,
LaJolla, Calif.) were used with the following side chain
protection: Boc-Ala-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHex)-OH,
Boc-Tyr(2BrZ)-OH, Boc-His(DNP)--OH, Boc-Val-OH, Boc-Leu-OH,
Boc-Gly-OH, Boc-Gln-OH, Boc-Ile-OH, Boc-Lys(2CIZ)-OH,
Boc-Thr(Bzl)-OH, Boc-A6c-OH, Ser(Bzl)-OH, Boc-Phe-OH, Boc-Aib-OH,
Boc-Glu(OcHex)-OH and Boc-Trp(Fm)--OH. The synthesis was carried
out on a 0.20 mmol scale. The Boc groups were removed by treatment
with 100% TFA for 2.times.1 min. Boc amino acids (2.5 mmol) were
pre-activated with HBTU (2.0 mmol) and DIEA (1.0 ml) in 4 ml of DMF
and were coupled without prior neutralization of the peptide-resin
TFA salt. Coupling times were about 5 min except for the Boc-Aib-OH
and Boc-A6c-OH residues and the following residues, Boc-Trp(Fm)-OH
and Boc-His(DNP)--OH wherein the coupling times were about 2
hours.
[0071] At the end of the assembly of the peptide chain, the resin
was treated with a solution of 20% mercaptoethanol/10% DIEA in DMF
for 2.times.30 min to remove the DNP group on the His side chain.
The N-terminal Boc group was then removed by treatment with 100%
TFA for 2.times.2 min. After neutralization of the peptide-resin
with 10% DIEA in DMF (1.times.1 min), the formyl group on the side
of the chain of Trp was removed by treatment with a solution of 15%
ethanolamine/15% water/70% DMF for 2.times.30 min. The
partially-deprotected peptide-resin was washed with DMF and DCM and
dried under reduced pressure. The final cleavage was done by
stirring the peptide-resin in 10 ml of HF containing 1 ml of
anisole and dithiothreitol (24 mg) at 0.degree. C. for about 75
min. HF was removed with a flow of nitrogen. The residue was washed
with ether (6.times.10 ml) and extracted with 4N HOAc (6.times.10
ml).
[0072] The peptide mixture in the aqueous extract was purified on a
reverse-phase preparative high pressure liquid chromatography
(HPLC) using a reverse phase VYDAC.RTM. C.sub.18 column (Nest
Group, Southborough, Mass.). The column was eluted with a linear
gradient (20% to 50% of solution B over 105 min) at a flow rate of
10 ml/min (Solution A=water containing 0.1% TFA; Solution
B=acetonitrile containing 0.1% of TFA). Fractions were collected
and checked on analytical HPLC. Those containing pure product were
combined and lyophilized to dryness. 92 mg of a white solid was
obtained. Purity was >99% based on analytical HPLC analysis.
Electro-spray mass spectrometer analysis gave the molecular weight
at 3324.2 (the calculated molecular weight is 3323.7).
[0073] The synthesis of other compounds of the present invention
can be carried out in the same manner as described for the
synthesis of [Aib.sup.8, A6c.sup.32]hGLP-1(7-36)NH.sub.2 (SEQ ID
NO: 114) in Example 52 above but using the appropriate protected
amino acids depending on the desired peptide.
[0074] [(N.sup..alpha.-HEPES-His).sub.7]hGLP-1(7-36)NH.sub.2 (SEQ
ID NO: 152) {HEPES is
(4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid)} can be
synthesized as follows: After assembly of the peptide long chain on
MBHA resin (0.20 mmol), the peptide-resin is treated with 100% TFA
(2.times.2 min.) and washed with DMF and DCM. The resin is then
neutralized with 10% DIEA in DMF for about 2 min. After washing
with DMF and DCM, the resin is treated with 0.23 mmol of
2-chloro-1-ethanesulfonyl chloride and 0.7 mmol of DIEA in DMF for
about 1 hour. The resin is washed with DMF and DCM and treated with
1.2 mmol of 2-hydroxyethylpiperazine for about 2 hours. The resin
is washed with DMF and DCM and treated with different reagents ((1)
20% mercaptoethanol/10% DIEA in DMF and (2) 15% ethanolamine/15%
water/70% DMF) to remove the DNP group from the His side chain and
formyl group on the Trp side chain as described above before the
final HF cleavage of the peptide from the resin.
[0075] [(N.sup..alpha.-HEPA-His).sub.7]hGLP-1(7-36)NH.sub.2 (SEQ ID
NO: 153)
([(4-(2-hydroxyethyl)-1-piperazineacetyl)-His.sup.7]hGLP-1(7-36)NH.s-
ub.2) can be made substantially according to the procedure
described immediately above for making
[(N.sup..alpha.-HEPES-His).sup.7]hGLP-1(7-36)NH.sub.2 (SEQ ID NO:
152) except that 2-bromo-acetic anhydride is used in place of
2-chloro-1-ethanesulfonyl chloride.
EXAMPLES 53-90 and 104
[0076] Examples 53-90 and 104 were made substantially according to
Example 52 but using the appropriate protected amino acid.
Example 53: [A6c.sup.20,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO:115);
MS=3322.3; Calc. MW=3321.7. Example 54:
[Aib.sup.8]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 116); MS=3311.7; Calc.
MW=3311.7. Example 55: [(Tma-His).sup.7]hGLP-1(7-36)-NH.sub.2 (SEQ
ID NO: 117); MS=3395.9; Calc. MW=3396.9. Example 56:
[A6c.sup.8]hGLP-1(8-36)-NH.sub.2 (SEQ ID NO: 118); MS=3214.5; Calc.
MW=3214.7. Example 57: [A6c.sup.8]hGLP-1(7-36)-NH.sub.2 (SEQ ID
NO:119); MS=3351.5; Calc. MW=3351.8. Example 58:
[A6c.sup.16,20]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 120); MS=3335.9;
Calc. MW=3335.8. Example 59: [A6c.sup.29,32]hGLP-1(7-36)-NH.sub.2
(SEQ ID NO: 121); MS=3321.7; Calc. MW=3321.7. Example 60:
[A6c.sup.20, Aib.sup.24]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 122);
MS=3323.6; Calc. MW=3323.7. Example 61: [Aib.sup.24,
A6c.sup.29,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 123); MS=3335.7;
Calc. MW=3335.8. Example 62:
[A6c.sup.16,29,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 124);
MS=3347.7; Calc. MW=3347.8. Example 63:
[Ura.sup.7]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 125); MS=3279.5; Calc.
MW=3280.7. Example 64: [Paa.sup.7]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO:
126); MS=3290.9; Calc. MW=3291.8. Example 65:
[Pta.sup.7]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 127); MS=3311.2; Calc.
MW=3311.8. Example 66: [N-Me-Ala.sup.8]hGLP-1(7-36)-NH.sub.2 (SEQ
ID NO: 128); MS=3311.4; Calc. MW=3311.7. Example 67:
[N-Me-D-Ala.sup.8]hGLP-1(7-36)-NH.sub.2; MS=3311.6; Calc.
MW=3311.7. Example 68: [N-Me-D-Ala.sup.8]hGLP-1(8-36)-NH.sub.2;
MS=3174.0; Calc. MW=3174.6. Example 69:
[N-Me-Gly.sup.8]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 129); MS=3297.3;
Calc. MW=3297.7. Example 70: [A5c.sup.8]hGLP-1(7-36)-NH.sub.2 (SEQ
ID NO: 130); MS=3337.3; Calc. MW=3337.8. Example 71:
[N-Me-Glu.sup.9]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 131); MS=3311.4;
Calc. MW=3311.7. Example 72; [A5c.sup.8,
A6c.sup.20,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 132); MS=3361.4;
Calc. MW=3361.8. Example 73: [Aib.sup.8,
A6c.sup.32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 133); MS=3323.2; Calc.
MW=3323.7. Example 74: [Aib.sup.8,25]hGLP-1(7-36)-NH.sub.2 (SEQ ID
NO: 134); MS=3325.8; Calc. MW=3325.7. Example 75:
[Aib.sup.8,24]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 135); MS=3325.8;
Calc. MW=3325.7. Example 76: [Aib.sup.8,30]hGLP-1(7-36)-NH.sub.2
(SEQ ID NO: 136); MS=3326.1; Calc. MW=3325.7. Example 77:
[Aib.sup.8, Cha.sup.20]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 137);
MS=3351.8; Calc. MW=3351.8. Example 78: [Aib.sup.8,
Cha.sup.32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 138); MS=3352.0; Calc.
MW=3351.8. Example 79: [Aib.sup.8, Glu.sup.23]hGLP-1(7-36)-NH.sub.2
(SEQ ID NO: 139); MS=3311.7; Calc. MW=3312.7. Example 80:
[Aib.sup.8, A6c.sup.20]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 140);
MS=3323.6; Calc. MW=3323.7. Example 81: [Aib.sup.8,
A6c.sup.20,32]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 141); MS=3335.3;
Calc. MW=3335.7. Example 82: [Aib.sup.8,
Lys.sup.25]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 142); MS=3339.8; Calc.
MW=3339.8. Example 83: [Aib.sup.8,
.beta.-Ala.sup.22]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 143);
MS=3325.6; Calc. MW=3325.8. Example 84: [Aib.sup.8,
Lys.sup.25]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 144); MS=3369.0; Calc.
MW=3368.8. Example 85: [Aib.sup.8, A6c.sup.12]hGLP-1(7-36)-NH.sub.2
(SEQ ID NO: 145); MS=3289.8; Calc. MW=3289.7. Example 86:
[Aib.sup.8, A6c.sup.29]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO:146);
MS=3323.9; Calc. MW=3323.7. Example 87: [Aib.sup.8,
A6c.sup.3]hGLP-1(7-36)-NH.sub.2 (SEQ ID NO: 147); MS=3338.0; Calc.
MW=3337.8. Example 88: [Aib.sup.8,14]hGLP-1(7-36)NH.sub.2 (SEQ ID
NO: 148); MS=3309.8; Calc. MW=3309.7. Example 89:
[Aib.sup.8,18]hGLP-1(7-36)NH.sub.2 (SEQ ID NO: 149); MS=3309.7;
Calc. MW=3309.7. Example 90: [Aib.sup.8,7]hGLP-1(7-36)NH.sub.2 (SEQ
ID NO; 150); MS=3309.4; Calc. MW=3309.7. Example 104: [Aib.sup.8,
D-Arg;.sup.26]hGLP-1(7-36)NH.sub.2; MS=3310.7; Calc.
MW=3311.73.
EXAMPLE 91
[Aib.sup.8, A5c.sup.33]hGLP-1(7-36)NH.sub.2 (SEQ ID NO: 154)
[0077] The title compound can be made substantially according to
Example 52 using the appropriate protected amino acids.
EXAMPLE 92
[Aib.sup.8, A6c.sup.32,
Lys.sup.36(N.sup..epsilon.-tetradecanoyl)]hGLP-1(7-36)NH.sub.2 (SEQ
ID NO: 155)
[0078] The Boc amino acids to be used are the same as those in the
synthesis of [Aib.sup.8, A6c.sup.32]hGLP-1(7-36)NH.sub.2 (SEQ ID
NO: 114) (Example 52) except that Fmoc-Lys(Boc)-OH is used here for
the Lys.sup.36(N.sup..epsilon.-tetradecanoyl) residue. The first
amino acid residue is coupled to the resin manually on a shaker.
2.5 mmol of Fmoc-Lys(Boc)-OH is dissolved in 4 ml of 0.5N HBTU in
DMF. To the solution is added 1 ml of DIEA. The mixture is shaken
for about 2 min. To the solution is then added 0.2 mmol of MBHA
resin (substitution=0.91 mmol/g). The mixture is shaken for about 1
hr. The resin is washed with DMF and treated with 100% TFA for
2.times.2 min to remove the Boc protecting group. The resin is
washed with DMF. Myristic acid (2.5 mmol) is pre-activated with
HBTU (2.0 mmol) and DIEA (1.0 ml) in 4 ml of DMF for 2 min and is
coupled to the Fmoc-Lys-resin. The coupling time is about 1 hr. The
resin is washed with DMF and treated with 25% piperidine in DMF for
2.times.20 min to remove the Fmoc protecting group. The resin is
washed with DMF and transferred to the reaction vessel of the
peptide synthesizer. The remainder of the synthesis and
purification procedures of the peptide are the same as those in the
synthesis of [Aib.sup.8, A6c.sup.32]hGLP-1(7-36)NH.sub.2. (SEQ ID
NO: 114)
[0079] The syntheses of other compounds containing
Lys(N.sup..epsilon.-alkanoyl) residue are carried out in an
analogous manner as described for the synthesis of [Aib.sup.8,
A6c.sup.32,
Lys.sup.36(N.sup..epsilon.-tetradecanoyl)]hGLP-1(7-36)NH.sub.2 SEQ
ID NO: 155). Fmoc-Lys(Boc)-OH amino acid is used for the residue of
Lys(N.sup..epsilon.-alkanoyl) in the peptide, while
Boc-Lys(2CIZ)-OH amino acid is used for the residue of Lys. If the
Lys(N.sup..epsilon.-alkanoyl) residue is not at the C-terminus, the
peptide fragment immediately prior to the
Lys(N.sup..epsilon.-alkanoyl) residue is assembled on the resin on
the peptide synthesizer first.
EXAMPLES 93-98
[0080] Examples 93-98 can be made substantially according to the
procedure described for Example 92 using the appropriate amino
acids.
Example 93: [Aib.sup.8, A6c.sup.32,
Lys.sup.36(N.sup..epsilon.-tetradecanoyl)]hGLP-1(7-36)NH.sub.2 (SEQ
ID NO: 155) Example 94: [Aib.sup.8, Arg.sup.26,34, A6c.sup.32,
Lys.sup.36 (N.sup..epsilon.-tetradecanoyl)]hGLP-1(7-36)NH.sub.2
(SEQ ID NO: 156) Example 95: [Aib.sup.8, Arg.sup.28, A6c.sup.32,
Lys.sup.34(N.sup..epsilon.-tetradecanoyl)]hGLP-1(7-36)NH.sub.2 (SEQ
ID NO: 157) Example 96: [Aib.sup.8,
Lys.sup.26(N.sup..epsilon.-tetradecanoyl), A6c.sup.32,
Arg.sup.34]hGLP-1(7-36)NH.sub.2 (SEQ ID NO: 158) Example 97:
[Aib.sup.8,
Lys.sup.36(N.sup..epsilon.-octanoyl)]hGLP-1(7-36)NH.sub.2 (SEQ ID
NO: 159) Example 98: [Aib.sup.8, A6c.sup.20,32,
Lys.sup.36(N.sup..epsilon.-octanoyl)]hGLP-1(7-36)NH.sub.2 (SEQ ID
NO: 160)
EXAMPLE 99
[Aib.sup.8, Arg.sup.26,34, A6c.sup.32,
Lys.sup.36(N.sup..epsilon.-tetradecanoyl)]hGLP-1(7-36)-OH (SEQ ID
NO: 161)
[0081] The Boc amino acids to be used are the same as those used in
the synthesis of [Aib.sup.8, A6c.sup.32,
Lys.sup.36(N.sup..epsilon.-tetradecanoyl)]hGLP-1(7-36)NH.sub.2 (SEQ
ID NO: 162) (Example 92). Fmoc-Lys(Boc)-OH (2.5 mmol) is
pre-activated with HBTU (2.0 mmol), HOBt (2.0 mmol) and DIEA (2.5
ml) in DMF (4 ml) for about 2 min. This amino acid is coupled to
235 mg of PAM resin (Chem-Impex, Wood Dale, Ill.; substitution=0.85
mmol/g) manually on a shaker. The coupling time is about 8 hrs. The
remainder of the synthesis and purification procedures for making
the peptide are the same as those described in Example 52.
[0082] The syntheses of other analogs of hGLP-1(7-36)-OH (SEQ ID
NO: 3) and hGLP-1(7-37)-OH, (SEQ ID NO: 4) which contain
Lys(N.sup..epsilon.-alkanoyl) residue, are carried out in an
analogous manner as described for the synthesis of [Aib.sup.8,
Arg.sup.26,34, A6c.sup.32,
Lys.sup.36(N.sup..epsilon.-tetradecanoyl)]hGLP-1(7-36)-OH (SEQ ID
NO: 161). Fmoc-Lys(Boc)-OH amino acid is used for the residue of
Lys(N.sup..epsilon.-alkanoyl) in the peptide, while
Boc-Lys(2CIZ)-OH amino acid is used for the residue of Lys.
EXAMPLES 100-103
[0083] Examples 100-103 can be made substantially according to the
procedure described for Example 99 using the appropriate amino
acids.
Example 100: [Aib.sup.8, Arg.sup.26, A6c.sup.32,
Lys.sup.34(NF-tetradecanoyl)]hGLP-1(7-36)-OH (SEQ ID NO: 162)
Example 101: [Aib.sup.8, Lys.sup.26(N.sup..epsilon.-tetradecanoyl),
A6c.sup.32, Arg.sup.34]hGLP-1(7-36)-OH (SEQ ID NO: 163) Example
102: [Aib.sup.8, Arg.sup.26,34-A6c.sup.32,
Lys.sup.36(N.sup..epsilon.-tetradecanoyl)]hGLP-1(7-37)-OH (SEQ ID
NO: 164) Example 103: [Aib.sup.8, Arg.sup.26, A6c.sup.32,
Lys.sup.34(N.sup..epsilon.-tetradecanoyl)]hGLP-1(7-37)-OH (SEQ ID
NO: 165)
Sequence CWU 1
1
165130PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 1His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 30231PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 2His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
Val Lys Gly Arg Gly 20 25 30330PRTHomo sapiens 3His Ala Glu Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30431PRTHomo
sapiens 4His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg Gly 20 25 30530PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 5His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg 20 25 30631PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 6His Ala Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala
Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25
30730PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 7His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 30831PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 8His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu
Val Lys Gly Arg Gly 20 25 30930PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 9His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile
Ala Trp Leu Val Xaa Gly Arg 20 25 301031PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 10His Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg Gly 20 25
301130PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 11His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Xaa
Gly Arg 20 25 301231PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 12His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
Val Xaa Gly Arg Gly 20 25 301330PRTArtificial SequenceDescription
of Artificial Sequence Mutagen 13His Ala Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe
Ile Ala Trp Leu Val Xaa Gly Arg 20 25 301431PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 14His Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg Gly 20 25
301530PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 15His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Xaa
Gly Arg 20 25 301631PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 16His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu
Val Xaa Gly Arg Gly 20 25 301729PRTArtificial SequenceDescription
of Artificial Sequence Mutagen 17Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly Gln 1 5 10 15Ala Ala Arg Glu Phe Ile
Ala Trp Leu Val Xaa Gly Arg 20 251830PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 18Ala Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln 1 5 10 15Ala
Ala Arg Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg Gly 20 25
301929PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 19Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
Gly Gln 1 5 10 15Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Xaa Gly
Arg 20 252030PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 20Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
Leu Glu Gly Gln 1 5 10 15Ala Ala Arg Glu Phe Ile Ala Trp Leu Val
Xaa Gly Arg Gly 20 25 302130PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 21His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile
Ala Trp Leu Val Arg Gly Xaa 20 25 302231PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 22His Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Xaa Gly 20 25
302330PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 23His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg
Gly Xaa 20 25 302431PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 24His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu
Val Arg Gly Xaa Gly 20 25 302530PRTHomo sapiens 25His Ala Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala
Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 302631PRTHomo
sapiens 26His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg Gly 20 25 302730PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 27His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg 20 25 302831PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 28His Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25
302930PRTHomo sapiens 29His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser
Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp
Leu Val Arg Gly Arg 20 25 303031PRTHomo sapiens 30His Ala Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 20 25
303130PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 31His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
Gly Arg 20 25 303231PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 32His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
Val Arg Gly Arg Gly 20 25 303330PRTHomo sapiens 33His Ala Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala
Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg 20 25 303431PRTHomo
sapiens 34His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg
Gly Arg Gly 20 25 303530PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 35His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile
Ala Trp Leu Val Arg Gly Arg 20 25 303631PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 36His Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 20 25
303730PRTHomo sapiens 37His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser
Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp
Leu Val Lys Gly Lys 20 25 303831PRTHomo sapiens 38His Ala Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala
Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Lys Gly 20 25
303930PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 39His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Lys 20 25 304031PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 40His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
Val Lys Gly Lys Gly 20 25 304130PRTHomo sapiens 41His Ala Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala
Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Gly Lys 20 25 304231PRTHomo
sapiens 42His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys
Gly Lys Gly 20 25 304330PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 43His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile
Ala Trp Leu Val Lys Gly Lys 20 25 304431PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 44His Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Gly Lys Gly 20 25
304530PRTHomo sapiens 45His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser
Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp
Leu Val Arg Gly Lys 20 25 304631PRTHomo sapiens 46His Ala Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Lys Gly 20 25
304730PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 47His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
Gly Lys 20 25 304831PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 48His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
Val Arg Gly Lys Gly 20 25 304930PRTHomo sapiens 49His Ala Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala
Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Lys 20 25 305031PRTHomo
sapiens 50His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg
Gly Lys Gly 20 25 305130PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 51His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile
Ala Trp Leu Val Arg Gly Lys 20 25 305231PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 52His Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Lys Gly 20 25
305330PRTHomo sapiens 53His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser
Ser Tyr Leu Asp Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp
Leu Val Lys Gly Arg 20 25 305431PRTHomo sapiens 54His Ala Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly 1 5 10 15Gln Ala
Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25
305530PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 55His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Asp Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 305631PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 56His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Asp Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
Val Lys Gly Arg Gly 20 25 305730PRTArtificial SequenceDescription
of Artificial Sequence Mutagen 57His Xaa Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu Val Lys Gly Arg 20 25 305831PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 58His Xaa Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25
305930PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 59His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 306031PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 60His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
Val Lys Gly Arg Gly 20 25 306130PRTHomo sapiens 61His Ala Asp Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala
Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 306231PRTHomo
sapiens 62His Ala Asp Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg Gly 20 25 306330PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 63His Ala Asp Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg
20 25 306431PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 64His Ala Asp Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
Val Lys Gly Arg Gly 20 25 306530PRTHomo sapiens 65Tyr Ala Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala
Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 306631PRTHomo
sapiens 66Tyr Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg Gly 20 25 306730PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 67Tyr Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg 20 25 306831PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 68Tyr Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25
306930PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 69Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 307031PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 70Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
Val Lys Gly Arg Gly 20 25 307130PRTArtificial SequenceDescription
of Artificial Sequence Mutagen 71Xaa Ala Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu Val Lys Gly Arg 20 25 307231PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 72Xaa Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25
307330PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 73Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 307431PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 74Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
Val Lys Gly Arg Gly 20 25 307530PRTArtificial SequenceDescription
of Artificial Sequence Mutagen 75Xaa Ala Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu Val Lys Gly Arg 20 25 307631PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 76Xaa Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25
307729PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 77Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
Gly Gln 1 5 10 15Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly
Arg 20 257830PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 78Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
Leu Glu Gly Gln 1 5 10 15Ala Ala Lys Glu Phe Ile Ala Trp Leu Val
Lys Gly Arg Gly 20 25 307930PRTHomo sapiens 79His Ala Glu Gly Thr
Phe Thr Ser Glu Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 308031PRTHomo
sapiens 80His Ala Glu Gly Thr Phe Thr Ser Glu Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg Gly 20 25 308130PRTHomo sapiens 81His Ala Glu Gly Thr Phe
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys
Glu Phe Ile Ala Phe Leu Val Lys Gly Arg 20 25 308231PRTHomo sapiens
82His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1
5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Phe Leu Val Lys Gly Arg Gly
20 25 308329PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 83His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ala
Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu
Val Lys Gly 20 258429PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 84His Ala Glu Gly Thr Phe Thr Ser Asp
Ala Ser Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile
Ala Xaa Leu Val Lys Gly 20 258529PRTArtificial SequenceDescription
of Artificial Sequence Mutagen 85His Ala Glu Gly Thr Phe Thr Ala
Asp Val Ser Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe
Ile Ala Xaa Leu Val Lys Gly 20 258629PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 86His Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala
Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Lys Gly 20
258730PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 87Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 308829PRTArtificial SequenceDescription of Artificial
Sequence Mutagen 88His Ala Glu Gly Thr Phe Thr Ser Ala Val Ser Ser
Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu
Val Lys Gly 20 258929PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 89His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ala Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile
Ala Xaa Leu Val Lys Gly 20 259028PRTArtificial SequenceDescription
of Artificial Sequence Mutagen 90His Ala Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe
Ile Ala Xaa Leu Val Xaa 20 259128PRTArtificial SequenceDescription
of Artificial Sequence Mutagen 91His Ala Glu Gly Thr Phe Thr Ser
Ala Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe
Ile Ala Xaa Leu Val Xaa 20 259228PRTArtificial SequenceDescription
of Artificial Sequence Mutagen 92His Ala Glu Gly Thr Phe Thr Ser
Asp Val Ala Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe
Ile Ala Xaa Leu Val Xaa 20 259328PRTArtificial SequenceDescription
of Artificial Sequence Mutagen 93His Ala Glu Gly Thr Phe Thr Ser
Asp Val Ser Ala Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe
Ile Ala Xaa Leu Val Xaa 20 259428PRTArtificial SequenceDescription
of Artificial Sequence Mutagen 94His Ala Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Xaa Leu Ala Ala 1 5 10 15Ala Ala Ala Lys Ala Phe
Ile Ala Xaa Leu Val Xaa 20 259528PRTArtificial SequenceDescription
of Artificial Sequence Mutagen 95His Ala Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Ala Ala Phe
Ile Ala Xaa Leu Val Xaa 20 259628PRTArtificial SequenceDescription
of Artificial Sequence Mutagen 96His Ala Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe
Ile Ala Xaa Ala Val Xaa 20 259727PRTArtificial SequenceDescription
of Artificial Sequence Mutagen 97His Ala Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Ala Ala Phe
Ile Ala Xaa Leu Xaa 20 259827PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 98His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile
Ala Ala Leu Xaa 20 259927PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 99His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Ala Ile
Ala Xaa Leu Xaa 20 2510027PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 100His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ala
Ala Xaa Leu Xaa 20 2510127PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 101His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile
Ala Xaa Leu Xaa 20 2510227PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 102His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Xaa Ala Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile
Ala Xaa Leu Xaa 20 2510327PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 103His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile
Ala Xaa Leu Xaa 20 2510427PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 104His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ala Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile
Ala Xaa Leu Xaa 20 2510528PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 105His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ala Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Xaa Ala Phe Ile
Ala Xaa Leu Val Lys 20 2510628PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 106His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ala Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile
Ala Xaa Leu Val Lys 20 2510728PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 107His Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ala Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile
Ala Xaa Leu Val Xaa 20 2510827PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 108His Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ala Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile
Ala Xaa Leu Val 20 2510928PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 109His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ala Ala Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile
Ala Xaa Leu Val Xaa 20 2511028PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 110His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ala Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile
Ala Xaa Leu Xaa Xaa 20 2511128PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 111His Ala Glu Gly Thr Phe Thr Ser Asp
Xaa Ala Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile
Ala Xaa Leu Val Xaa 20 2511227PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 112His Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ala Ala Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile
Ala Xaa Leu Val 20 2511328PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 113His Ala Glu Gly Thr Phe Thr Ser Asp
Xaa Ala Ala Xaa Xaa Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile
Ala Xaa Leu Val Xaa 20 2511430PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 114His Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile
Ala Trp Xaa Val Lys Gly Arg 20 25 3011530PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 115His Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Xaa Val Lys Gly Arg 20 25
3011630PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 116His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 3011730PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 117Xaa Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg 20 25 3011829PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 118Xaa Glu Gly
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln 1 5 10 15Ala
Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20
2511930PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 119His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 3012030PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 120His Ala Glu Gly Thr Phe Thr Ser Asp
Xaa Ser Ser Tyr Xaa Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg 20 25 3012130PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 121His Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Xaa Ala Trp Xaa Val Lys Gly Arg 20 25
3012230PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 122His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa
Glu Gly 1 5 10 15Gln Xaa Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 3012330PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 123His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Xaa Ala Lys Glu Phe Xaa
Ala Trp Xaa Val Lys Gly Arg 20 25 3012430PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 124His Ala Glu
Gly Thr Phe Thr Ser Asp Xaa Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Xaa Ala Trp Xaa Val Lys Gly Arg 20 25
3012530PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 125Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 3012630PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 126Xaa Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25
3012730PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 127Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 3012830PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 128His Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg 20 25 3012930PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 129His Xaa Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25
3013030PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 130His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 3013130PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 131His Ala Xaa Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg 20 25 3013230PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 132His Xaa Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Xaa Val Lys Gly Arg 20 25
3013330PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 133His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Xaa Val Lys
Gly Arg 20 25 3013430PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 134His Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Xaa Lys Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg 20 25 3013530PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 135His Xaa Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Xaa Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25
3013630PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 136His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Xaa Trp Leu Val Lys
Gly Arg 20 25 3013730PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 137His Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Xaa Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg 20 25 3013830PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 138His Xaa Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Xaa Val Lys Gly Arg 20 25
3013930PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 139His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Glu Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 3014030PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 140His Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Xaa Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg 20 25 3014130PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 141His Xaa Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Xaa Val Lys Gly Arg 20 25
3014230PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 142His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Xaa 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 3014330PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 143His Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Xaa 1 5 10 15Gln Ala Ala Lys Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg 20 25 3014430PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 144His Xaa Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Lys Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25
3014530PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 145His Xaa Glu Gly Thr Xaa Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 3014630PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 146His Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Xaa
Ala Trp Leu Val Lys Gly Arg 20 25 3014730PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 147His Xaa Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Xaa Lys Gly Arg 20 25
3014830PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 148His Xaa Glu Gly Thr Phe Thr Xaa Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 3014930PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 149His Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ser Xaa Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg 20 25 3015030PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 150His Xaa Glu
Gly Thr Phe Thr Ser Asp Val Xaa Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25
3015130PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 151His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg 20 25 3015230PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 152Xaa Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile
Ala Trp Leu Val Lys Gly Arg 20 25 3015330PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 153Xaa Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25
3015430PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 154His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Xaa Lys
Gly Arg 20 25 3015530PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 155His Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile
Ala Trp Xaa Val Lys Gly Xaa 20 25 3015630PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 156His Xaa Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Arg Glu Phe Ile Ala Trp Xaa Val Arg Gly Xaa 20 25
3015730PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 157His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Xaa Val Xaa
Gly Arg 20 25 3015830PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 158His Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile
Ala Trp Xaa Val Arg Gly Arg 20 25 3015930PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 159His Xaa Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Xaa 20 25
3016030PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 160His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Xaa Val Lys
Gly Xaa 20 25 3016130PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 161His Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile
Ala Trp Xaa Val Arg Gly Xaa 20 25 3016230PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 162His Xaa Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Arg Glu Phe Ile Ala Trp Xaa Val Xaa Gly Arg 20 25
3016330PRTArtificial SequenceDescription of Artificial Sequence
Mutagen 163His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile Ala Trp Xaa Val Arg
Gly Arg 20 25 3016431PRTArtificial SequenceDescription of
Artificial Sequence Mutagen 164His Xaa Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile
Ala Trp Xaa Val Arg Gly Xaa Gly 20 25 3016531PRTArtificial
SequenceDescription of Artificial Sequence Mutagen 165His Xaa Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln
Ala Ala Arg Glu Phe Ile Ala Trp Xaa Val Xaa Gly Arg Gly 20 25
30
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