U.S. patent application number 17/024264 was filed with the patent office on 2020-12-31 for egf(a) analogues with fatty acid substituents.
The applicant listed for this patent is Jianhe Chen, Patrick William Garibay, Janos Tibor Kodra, Jesper F. Lau, Lars Linderoth, Salka Elboel Rasmussen, Henning Thoegersen, Birgit Wieczorek. Invention is credited to Jianhe Chen, Patrick William Garibay, Janos Tibor Kodra, Jesper F. Lau, Lars Linderoth, Salka Elboel Rasmussen, Henning Thoegersen, Birgit Wieczorek.
Application Number | 20200407409 17/024264 |
Document ID | / |
Family ID | 1000005090780 |
Filed Date | 2020-12-31 |
United States Patent
Application |
20200407409 |
Kind Code |
A1 |
Chen; Jianhe ; et
al. |
December 31, 2020 |
EGF(A) ANALOGUES WITH FATTY ACID SUBSTITUENTS
Abstract
The invention relates to compounds derived from the EGF(A)
domain of LDL-R, in particular compounds comprising a peptide
analogue of the wild-type EGF(A) (LDL-R(293-332)) sequence and at
least one substituent comprising at least one fatty acid group. The
invention also relates to a pharmaceutical composition thereof and
use a medicament. The novel EGF(A) compounds of the invention are
useful as treatment e.g. in the field of cholesterol lowering,
dyslipidaemia and cardiovascular disease.
Inventors: |
Chen; Jianhe; (Beijing,
CN) ; Lau; Jesper F.; (Farum, DK) ; Kodra;
Janos Tibor; (Koebenhavn OE, DK) ; Wieczorek;
Birgit; (Koebenhavn N, DK) ; Linderoth; Lars;
(Hilleroed, DK) ; Thoegersen; Henning; (Farum,
DK) ; Rasmussen; Salka Elboel; (Vaerloese, DK)
; Garibay; Patrick William; (Holte, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chen; Jianhe
Lau; Jesper F.
Kodra; Janos Tibor
Wieczorek; Birgit
Linderoth; Lars
Thoegersen; Henning
Rasmussen; Salka Elboel
Garibay; Patrick William |
Beijing
Farum
Koebenhavn OE
Koebenhavn N
Hilleroed
Farum
Vaerloese
Holte |
|
CN
DK
DK
DK
DK
DK
DK
DK |
|
|
Family ID: |
1000005090780 |
Appl. No.: |
17/024264 |
Filed: |
September 17, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16069932 |
Jul 13, 2018 |
10822385 |
|
|
PCT/EP2017/050668 |
Jan 13, 2017 |
|
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17024264 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/485 20130101; C07K 14/705 20130101; A61K 38/177
20130101 |
International
Class: |
C07K 14/485 20060101
C07K014/485; A61K 38/17 20060101 A61K038/17; C07K 14/705 20060101
C07K014/705 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 13, 2016 |
CN |
PCT/CN2016/070791 |
Mar 17, 2016 |
CN |
PCT/CN2016/076580 |
Oct 27, 2016 |
EP |
16195965.5 |
Claims
1. An EGF(A) derivative comprising an EGF(A) peptide analogue of
the EGF(A) domain of LDL-R defined by SEQ ID NO: 1, comprising
301Leu and a substituent comprising at least one fatty acid
group.
2. The EGF(A) derivative according to claim 1, wherein said
substituent comprises a carboxylic acid, a sulphonic acid, a
tetrazole moiety, a methylsulfonylcarbamoylamino moiety or a
3-hydroxy-isoxazole moiety and 8-20 consecutive --CH.sub.2--
groups.
3. The EGF(A) derivative according to claim 1, wherein said
substituent has Formula I:
Z.sub.1--Z.sub.2--Z.sub.3--Z.sub.4--Z.sub.5--Z.sub.6--Z.sub.7--Z.sub.8--Z-
.sub.9--Z.sub.10-- [I] wherein Z.sub.1 is selected from:
HOOC--(CH.sub.2).sub.n--CO--*, Chem. 1:
tetrazolyl-(CH.sub.2).sub.n--CO--*, Chem. 2:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, Chem. 3:
HOS(O).sub.2--(CH.sub.2).sub.n--CO--*, Chem. 4:
MeS(O).sub.2NH(CO)N--(CH.sub.2).sub.n--CO--* and Chem. 5:
3-HO-Isoxazole-(CH.sub.2).sub.n--CO--* Chem. 6: wherein n is an
integer in the range of 8-20, m is an integer in the range of 8-11,
the --COOH group in Chem. 3 can be attached to position 2, 3 or 4
on the phenyl ring, the symbol * indicates the attachment point to
the nitrogen in Z.sub.2 or, if Z.sub.2 is a bond, to the nitrogen
on the neighbouring Z element; Z.sub.2 is selected from
*--NH--SO.sub.2--(CH.sub.2).sub.3--CO--*, Chem. 7:
*--NH--CH.sub.2--(C.sub.6H.sub.10)--CO--* and Chem. 8: a bond;
Z.sub.3 is selected from: .gamma.Glu, Glu and a bond; Z.sub.4,
Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8, Z.sub.9 are selected,
independently of each other, from: Glu, .gamma.Glu, Gly, Ser, Ala,
Thr, Ado, Aeep, Aeeep, TtdSuc and a bond; and Z.sub.10 is selected
from: *--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2--* and a bond.
Chem. 14:
4. The EGF(A) derivative according to claim 1, wherein the EGF(A)
derivative comprises one or two substituent(s) selected from the
group consisting of: HOOC--(CH.sub.2).sub.18--CO-gGlu-2xADO-
HOOC--(CH.sub.2).sub.18--CO--NH--CH.sub.2--(C.sub.6H.sub.10)--CO-gGlu-2xA-
DO- HOOC--(CH.sub.2).sub.16--CO-gGlu-2xADO-
HOOC--(CH.sub.2).sub.16--CO-gGlu-2xADO--NH--CH.sub.2--(C.sub.6H.sub.4)--C-
H.sub.2-- HOOC--(CH.sub.2).sub.16--CO-gGlu-
HOOC--(CH.sub.2).sub.16--CO--NH--CH.sub.2--(C.sub.6H.sub.10)--CO-gGlu-2xA-
DO- HOOC--(CH.sub.2).sub.14--CO-gGlu-2xADO-
HOOC--(CH.sub.2).sub.14--CO-gGlu-
HOOC--(CH.sub.2).sub.14--CO-gGlu-2xADO-
HOOC--(CH.sub.2).sub.12--CO-gGlu-2xADO-
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-2xADO-
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-3xADO-
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO--2xgGlu-
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-3xGly-
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO--2xgGlu-2xADO-
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-TtdSuc-
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9--CO--
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-4xADO-
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO--NH--CH.sub.2--(C.sub.-
6H.sub.10)--CO-gGlu-2xADO-
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9--CO-gGlu-2xADO-
3-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9--CO-gGlu-2xADO-
3-HO-Isoxazole-(CH.sub.2).sub.12--CO-gGlu-2xADO-
HOS(O).sub.2--(CH.sub.2).sub.15--CO-gGlu-2xADO--NH--CH.sub.2--(C.sub.6H.s-
ub.4)--CH.sub.2-- HOS(O).sub.2--(CH.sub.2).sub.13--CO-gGlu-2xADO-
Tetrazolyl-(CH.sub.2).sub.15--CO--NH--SO.sub.2--(CH.sub.2).sub.3--CO-ADO--
ADO-NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2--
Tetrazolyl-(CH.sub.2).sub.12--CO-gGlu-2xADO-
Tetrazolyl-(CH.sub.2).sub.15--CO-gGlu-2xADO- and
MeS(O).sub.2NH(CO)NH--(CH.sub.2).sub.12--CO-gGlu-2xADO--.
5. The EGF(A) derivative according to claim 1, wherein the EGF(A)
peptide analogue comprises one, two, three, four or all five of the
following (wild type) amino acid residue(s): 295Asn, 296Glu,
298Leu, 302Gly and 310Asp.
6. The EGF(A) derivative according to claim 1, wherein the EGF(A)
peptide analogue comprises the wild-type residue 310Asp.
7. The EGF(A) derivative according to claim 1, wherein the EGF(A)
peptide analogue comprises the wild-type residue 295Asn.
8. The EGF(A) derivative according to claim 1, wherein at least one
substituent is attached to a Lys residue in the EGF(A) peptide
analogue selected from the group consisting of: 292Lys, 293Lys,
294Lys, 296Lys, 299Lys, 300Lys, 303Lys, 305Lys, 306Lys, 309Lys,
311Lys, 312Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys,
321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys,
329Lys, 330Lys, 332Lys and 333Lys.
9. The EGF(A) derivative according to claim 1, wherein the EGF(A)
derivative is selected from the group of EGF(A) derivatives
consisting of: Example compounds 1-44, 46-47, 51-55, 57, 60-64,
66-69, 71-102 and 106-159.
10. An EGF(A) peptide analogue of the EGF(A) domain of LDL-R
defined by SEQ ID NO: 1, wherein the EGF(A) peptide analogue
comprises 301Leu, 310Asp and an amino acid substitution of
312Lys.
11. An EGF(A) peptide analogue of the EGF(A) domain defined by SEQ
ID NO: 1, wherein the EGF(A) peptide analogue comprises 301Leu and
310Asp and wherein the peptide does not have a substitution of
299Asp to Glu, Val or His.
12. The EGF(A) peptide analogue according to claim 10, wherein the
EGF(A) peptide analogue comprises Cys residues 297Cys, 304Cys,
308Cys, 317Cys, 319Cys and 331Cys.
13. The EGF(A) peptide analogue according to claim 10, further
comprising one, two, three, four or all five of the following (wild
type) amino acid residue(s): 295Asn, 296Glu, 298Leu, 302Gly and
310Asp.
14. The EGF(A) peptide analogue according to claim 10, EGF(A)
further comprising at least one Lys substitution selected from the
group consisting of: 292Lys, 293Lys, 294Lys, 296Lys, 299Lys,
300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 313Lys, 314Lys,
315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys,
325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and
333Lys.
15. The EGF(A) peptide analogue according to claim 10, wherein said
peptide sequence is selected from the group consisting of: SEQ ID
NOs: 2-47 and 49-106.
16. The EGF(A) peptide analogue according to claim 11, wherein the
EGF(A) peptide analogue comprises Cys residues 297Cys, 304Cys,
308Cys, 317Cys, 319Cys and 331Cys.
17. The EGF(A) peptide analogue according to claim 11, further
comprising one, two, three, four or all five of the following (wild
type) amino acid residue(s): 295Asn, 296Glu, 298Leu, 302Gly and
310Asp.
18. The EGF(A) peptide analogue according to claim 11, EGF(A)
further comprising at least one Lys substitution selected from the
group consisting of: 292Lys, 293Lys, 294Lys, 296Lys, 299Lys,
300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 313Lys, 314Lys,
315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys,
325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and
333Lys.
19. The EGF(A) peptide analogue according to claim 12, further
comprising one, two, three, four or all five of the following (wild
type) amino acid residue(s): 295Asn, 296Glu, 298Leu, 302Gly and
310Asp.
20. The EGF(A) peptide analogue according to claim 12, EGF(A)
further comprising at least one Lys substitution selected from the
group consisting of: 292Lys, 293Lys, 294Lys, 296Lys, 299Lys,
300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 313Lys, 314Lys,
315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys,
325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation application of U.S.
patent application Ser. No. 16/069,932, filed Jul. 13, 2018, which
is a 35 U.S.C. .sctn. 371 National Stage application of
International Application PCT/EP2017/050668 (WO 2017/121850), filed
Jan. 13, 2017, which claims priority to Chinese Patent Applications
PCT/CN2016/070791, filed Jan. 13, 2016 and PCT/CN2016/076580, filed
Mar. 17, 2016, and European Patent Application 16195965.5, filed
Oct. 27, 2016; the contents of which are incorporated herein by
reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Sep. 11, 2020, is named 150089US02_SeqList.txt and is 48
kilobytes in size.
TECHNICAL FIELD OF THE INVENTION
[0003] The present invention relates to EGF(A) analogues and
derivatives thereof, more particularly to EGF(A) peptide analogues
with a fatty acid substituent, and their pharmaceutical use.
BACKGROUND
[0004] High LDL-C (Low Density Lipoprotein cholesterol) levels and
dyslipidaemia are well-recognised drivers of cardiovascular
disease.
[0005] Statins have been approved for the treatment of dyslipidemia
for 25 years. This class has demonstrated substantial and
consistent reduction of cardiovascular events with an acceptable
safety profile. The best-selling statin, atorvastatin (Lipitor.TM.)
was the world's best-selling drug of all time, with more than $125
billion in sales from 1996 to 2012.
[0006] Despite the availability and widespread use of statins and
other lipid lowering agents, many patients do not reach their
target LDL-C levels and remain at high risk for developing
cardiovascular disease. PCSK9 (Proprotein Convertase
Subtilisin/Kexin type 9) promotes hepatic LDL-R (LDL receptor)
degradation, thereby reducing hepatic LDL-R surface expression and
consequently clearance of LDL particles. Conversely, blocking PCSK9
increase the clearance of LDL-C as well as other atherogenic
lipoproteins. Indeed, LDL receptors contribute to the clearance of
atherogenic lipoproteins other than LDL, such as
intermediate-density lipoproteins and remnant particles. Increased
intermediate-density lipoproteins and remnant particle clearance
may have therapeutic benefits beyond that provided by LDL
reduction.
[0007] Statins increase the expression of both LDL-R and PCSK9 via
the SREBP2 transcription factor. The increased expression of PCSK9
may diminish the effect of statins on LDL-C clearance from the
circulation. By inhibiting the binding of PCSK9 to the LDL-R and
thereby preventing LDL-R degradation the efficacy of statins is
enhanced. Taken together, PCSK9 inhibition offers a novel approach
to lipid management.
[0008] Two anti-PCSK9 antibodies, alirocumab/Praluent.RTM. and
evolocumab/Repatha.RTM., have recently been approved for the
treatment of high LDL-C levels. These are administered by 1 ml
subcutaneous injections every two weeks. However, compliance with
this dose regimen of a subcutaneously administered drug, especially
for an asymptomatic condition could be questioned.
[0009] The EGF(A) (Epidermal Growth Factor-like domain A) sequence
(40 amino acids) of the LDL-R (LDL-R-(293-332)) is well recognized
as the site for PCSK9 binding. The isolated wild-type EGF(A)
peptide has been shown to inhibit the binding of PCSK9 to the LDL-R
with an IC.sub.50 in the low .mu.M range (Biochemical and
Biophysical Research Communications 375 (2008) 69-73). This poor
potency will prevent a practical pharmaceutical use of the EGF(A)
peptide. Furthermore, the half-life of such peptides would be
expected to be too short to be of therapeutic use.
[0010] WO2012177741 and J. Mol. Biol. (2012) 422, 685-696 disclose
analogues of the EGF(A) and Fc-Fusion thereof.
[0011] There is still a need to improve patients treatment, for
example in terms of efficacy, also or alternatively in terms of
convenience, comfort for the patients, such as comfort and
convenience of the administration mode, and thereby compliance.
SUMMARY
[0012] The present invention relates to novel EGF(A) compounds
which have potential for improved patient treatments, in particular
in the field of cholesterol lowering, dyslipidaemia and
cardiovascular diseases.
[0013] In one aspect, the invention provides compounds with
improved pharmacokinetic (PK) properties. In particular, the
compounds of the invention have long half-lives and still show good
ability to inhibit PCSK9 in binding to the LDL-R.
[0014] Also or alternatively, in another aspect, the invention
provides EGF(A) compounds with improved ability to inhibit PCSK9
binding to the LDL-R or alternatively, in another aspect, the
invention provides compounds with improved binding capacity to
PCSK9. Also or alternatively, in another aspect, the invention
provides EGF(A) compounds with prolonged half-life. Also or
alternatively, in another aspect, the invention provides EGF(A)
compounds with prolonged half-life and no loss or no substantial
loss of ability to inhibit PCSK9 binding to the LDL-R. Also or
alternatively, in another aspect, the invention provides EGF(A)
compounds with prolonged half-life and preserved binding capacity.
In an aspect the invention provides EGF(A) compounds with a high
liquid stability suitable for liquid formulations. In an aspect the
invention provides EGF(A) compounds with a high in vivo stability.
Also or alternatively, in another aspect, the invention provides
compounds with potential for oral administration. Also or
alternatively, in another aspect, the invention provides EGF(A)
compounds with potential for a more convenient treatment for the
patient. Also or alternatively, in another aspect, the invention
provides compounds with potential for improved patient compliance.
The invention may also solve further problems that will be apparent
from the disclosure of the exemplary embodiments.
[0015] In one aspect, the invention relates to a compound
comprising an EGF(A) peptide analogue of the EGF(A) peptide defined
by sequence SEQ ID NO: 1:
Gly-Thr-Asn-Glu-Cys-Leu-Asp-Asn-Asn-Gly-Gly-Cys-Ser-His-Val-Cys-As-
n-Asp-Leu-Lys-Ile-Gly-Tyr-Glu-Cys-Leu-Cys-Pro-Asp-Gly-Phe-Gln-Leu-Val-Ala--
Gln-Arg-Arg-Cys- Glu, wherein the peptide analogue comprises
301Leu.
[0016] In one aspect, the invention relates to an EGF(A) derivative
comprising an EGF(A) peptide analogue comprising 301Leu and at
least one substituent comprising at least one fatty acid group.
[0017] In one embodiment the EGF(A) derivative, comprises an EGF(A)
peptide analogue wherein, as describe above amino acid 301 is Leu
(L), while the peptide further comprises the wild type residue(s)
in one or more of positions 295 (Asn/N), 296 (Glu/E), 298 (Leu/L),
302 (Gly/G) and 310 (Asp/D).
[0018] In further embodiments the EGF(A) peptide analogue of the
EGF(A) derivative has 1-15 amino acid substitutions compared to SEQ
ID NO.: 1.
[0019] In a further embodiment the substituent of the EGF(A)
derivative is not attached to the EGF(A) peptide analogue via an
amino acid residue in any the positions 295, 298, 301, 302, 307 and
310.
[0020] In a further embodiment the substituent is attached to the
EGF(A) peptide analogue via an amino acid residue other than the
positions 295, 298, 301, 302, 307 and 310.
[0021] In an aspect the invention relates to an EGF(A) peptide
analogue of the EGF(A) domain of LDL-R defined by SEQ ID NO.: 1,
wherein the peptide analogue comprises 301Leu and 310Asp and
wherein the peptide analogue has an amino acid substitution of
312Lys or where in the peptide analogue does not have a
substitution of 299Asp to Glu, Val or His.
[0022] In further embodiments the EGF(A) peptide analogues have
one, two, three, four or all five of the following (wild type)
amino acid residue(s) 295Asn, 296Glu, 298Leu, 302Gly and
310Asp/D).
[0023] In a further embodiment said peptide analogue comprises
three disulphide bridges in positions 297Cys-308Cys, 304Cys-317Cys
and 319Cys-331Cys.
[0024] In another aspect, the invention relates to a pharmaceutical
composition comprising a compound according to the invention.
[0025] In another aspect, the invention relates to a compound
according to the invention for use as a medicament.
[0026] In another aspect, the invention relates to medical use of
the compounds according to the invention.
BRIEF DESCRIPTION OF DRAWINGS
[0027] FIG. 1 shows hepatic LDL-R expression levels in mice
measured by Western Blot, presented as scatter plot for the
individual animals.
[0028] FIG. 2 shows plasma LDL cholesterol in hamsters treated with
vehicle or with protracted EGF(A) compounds of example 2.
[0029] FIG. 3 shows hepatic LDL-R expression in livers of hamsters
treated with vehicle or with protracted EGF(A) compounds of example
2 measured by Western Blot.
BRIEF DESCRIPTION OF SEQUENCE LISTING
[0030] The amino acid sequence of wild-type EGF(A) (LDL-R(293-332))
is included in the sequence listing as SEQ ID NO: 1. SEQ ID NO's
2-78 are the amino acid sequences of the EGF(A) peptides of
specific EGF(A) compounds of the invention.
Description
[0031] In what follows, Greek letters may be represented by their
symbol or the corresponding written name, for example:
.alpha.=alpha; .beta.=beta; .epsilon.=epsilon; .gamma.=gamma;
.delta.=delta; .omega.=omega; etc. Also, the Greek letter of .mu.
may be represented by "u", e.g. in .mu.l=ul, or in .mu.M=uM.
[0032] In what follows, "a" means "one or more". Unless otherwise
indicated in the specification, terms presented in singular form
also include the plural situation.
[0033] An asterisk (*) in a chemical formula designates i) a point
of attachment, ii) a radical, and/or iii) an unshared electron.
[0034] In its first aspect the invention relates to a compound
comprising a peptide analogue of SEQ ID NO.: 1, and at least one
substituent comprising at least one fatty acid group, or a
pharmaceutically acceptable salt, amide, or ester thereof.
[0035] In a second aspect, the invention relates to a peptide
analogue of SEQ ID NO.: 1, which may be considered an intermediate
product for the preparation of the derivatives of the
invention.
[0036] In its third aspect, the invention relates to a
pharmaceutical composition comprising a compound of the invention
and a pharmaceutically acceptable excipient, in particular suitable
for oral administration; and the use of the compound of the
invention as a medicament. Further aspects of the invention are
described below.
Structural Features
EGF(A) Compound
[0037] The term "EGF(A) compound" is used herein to generally refer
to a compound comprising an EGF(A) peptide, encompassing
wt-LDL-R(293-332) as defined by SEQ ID NO: 1 and analogues hereof.
The term EGF(A) compound encompasses derivatives of EGF-(A) peptide
and analogue thereof i.e. EGF(A) peptide analogues with a
substituent as described herein is a typical example of an EGF(A)
compound.
EGF(A) Peptides
[0038] The term "peptide", as e.g. used in the context of the
invention, refers to a compound which comprises a series of amino
acids interconnected by amide (or peptide) bonds. In a particular
embodiment the peptide consists of amino acids interconnected by
peptide bonds.
[0039] The peptide of the invention comprises at least 35, such as
36, 37, 38, 39 or at least 40 amino acids. In a particular
embodiment the peptide is composed of 36, such as 38 or 40 amino
acids. In an additional particular embodiment the peptide consists
of 35, 36, 37, 38, 39 or 40 amino acids.
[0040] In the presence of amino acid additions, referred to herein
as N-terminal and C-terminal elongations, the peptide of the
invention may comprise up to 140 amino acids. In an embodiment, the
peptide of the invention may comprise or consist of 41 amino acid
residues. In a particular embodiment, it comprises 40-140, 40-120,
40-100, 40-80, 40-60 or 40-50 amino acids.
[0041] The terms "EGF(A) domain of the LDL-R", "LDL-R (293-332)",
"native LDL-R (293-332), "EGF(A) (293-332)", "wild-type EGF(A)",
"wt-EGF(A)" or "native EGF(A)" as used herein refer to a peptide
consisting of the sequence SEQ ID NO: 1.
TABLE-US-00001 SEQ ID NO: 1 is:
Gly-Thr-Asn-Glu-Cys-Leu-Asp-Asn-Asn-Gly-Gly-Cys-
Ser-His-Val-Cys-Asn-Asp-Leu-Lys-Ile-Gly-Tyr-Glu-
Cys-Leu-Cys-Pro-Asp-Gly-Phe-Gln-Leu-Val-Ala-Gln-
Arg-Arg-Cys-Glu.
[0042] In this formula the numbering of the amino acid residues
follows the numbering for the EGF(A) domain of the LDL-R
(LDL-R-(293-332)), wherein the first (N-terminal) amino acid
residue is numbered or accorded position no. 293, and the
subsequent amino acid residues towards the C-terminus are numbered
294, 295, 296 and so on, until the last (C-terminal) amino acid
residue, which in the EGF(A) domain of the LDL-R is Glu with number
332.
[0043] The numbering is done differently in the sequence listing,
where the first amino acid residue of SEQ ID NO: 1 (Gly) is
assigned no. 1, and the last (Glu) no. 40. The same applies for the
other sequences of the sequence listing, i.e. the N-terminal amino
acid assigned is no. 1 irrespective of its positioning relative to
293Gly or 293 substituting amino acid residue by reference to
LDL-R(293-332). However, herein the numbering of amino acid
positions is with reference to LDL-R(293-332), as explained
above.
[0044] The present invention relates to analogues of the EGF(A)
peptide identified by SEQ ID NO:1 and derivatives of such EGF(A)
peptide analogues of the wild-type EGF(A) domain of LDLR defined by
SEQ ID NO: 1.
[0045] The term "analogue" generally refers to a peptide, the
sequence of which has one or more amino acid changes when compared
to a reference amino acid sequence.
[0046] The terms "analogue of the invention", "peptide analogue of
the invention", "LDL-R(293-332) analogue", "EGF(A) analogue" or
"analogue of SEQ ID NO: 1" as used herein may be referred to as a
peptide, the sequence of which comprises amino acid substitutions,
i.e. amino acid replacement, relative to sequence SEQ ID NO: 1. An
"analogue" may also include amino acid elongations in the
N-terminal and/or C-terminal positions and/or truncations in the
N-terminal and/or C-terminal positions.
[0047] The level of identity to SEQ ID NO.:1 can be calculated by
determining the number of amino acids that are not changed relative
to SEQ ID NO 1. SEQ ID NO: 1 consists of 40 amino acid residues and
if three amino acid substitutions are introduced the level of
identity is 37/40%=92.5%. If 5 amino acid residues are changed the
level of identity is 87, 5%. If the peptide is N-terminal or
C-terminal elongated that part is usually not included in the
comparison, whereas a deletion of one or more amino acids shortens
the comparator. For instance, in the examples above, if the
N-terminal amino acid is deleted the level of identity is slightly
reduced to 36/39.times.100% and 34/39.times.100%, respectively.
When discussing identity of the back-bone sequence of a derivative
the amino acid residue of the substituent e.g. the residue to which
the substituent is attached, also termed the amino acid residue of
the substituent, may be either a wild type (wt) or a substituted
amino acid. If the amino acid residue of the substituent is a wild
type residue, such as the N-term Gly or 312K this residue is
included in the calculation of identity level, whereas a Lys in any
other position from 293 to 332 would be an amino acid substitution
and not included when calculated amino acid identity to SEQ ID
NO.:1.
[0048] In one embodiment the EGF(A) peptide analogue has 1-15 amino
acid substitutions compared to SEQ ID NO.: 1. In one embodiments
the EGF(A) peptide analogue has 1-10 amino acid substitutions
compared to SEQ ID NO.: 1. In one embodiments the EGF(A) peptide
analogue has 1-8 amino acid substitutions compared to SEQ ID NO.:
1, such as 1-7, 1-6, 1-5 amino acid substitutions compared to SEQ
ID NO.: 1. In a particular embodiment, up to 7 amino acid
substitutions may be present, for example up to 6, 5, 4, 3, 2 or 1
amino acid substitutions may be present in the EGF-1 peptide
analogue.
[0049] In one embodiment the analogue of the invention has at least
75% identity, such as 80%, such as 85, such as 90 or even 95%
identity to SEQ ID NO.:1 corresponding to up to 10, 8, 6, 4 and 2
amino acid substitutions relative to SEQ ID NO 1, respectively in
case of no truncation.
[0050] Each of the peptide analogues of the invention may be
described by reference to i) the number of the amino acid residue
in the native EGF(A) (LDL-R(293-332)) which corresponds to the
amino acid residue which is changed (i.e., the corresponding
position in native LDL-R(293-332) EGF(A)), and to ii) the actual
change.
[0051] In other words, the peptide analogues of the invention may
be described by reference to the native LDL-R(293-332) EGF(A)
peptide, namely as a variant thereof in which a number of amino
acid residues have been changed when compared to native
LDL-R(293-332) EGF(A) (SEQ ID NO: 1). These changes may represent,
independently, one or more amino acid substitutions.
[0052] The followings are non-limiting examples of suitable
analogue nomenclature: The EGF(A) peptide incorporated in the
derivative of Example 2 herein may be referred to as the following
LDL-R(293-332) EGF(A) analogue: (301Leu, 309Arg) LDL-R(293-332)
EGF(A), or (Leu301, Arg309)-LDL-R(293-332) EGF(A) or (301L,309R)
LDL-R(293-332) or (L301,R309) LDL-R(293-332). This means that when
this analogue is aligned with native LDL-R(293-332), it has i) a
Leu at the position in the analogue which corresponds, according to
the alignment, to position 301 in native LDL-R(293-332) EGF(A), ii)
an Arg at the position in the analogue which corresponds to
position 309 in native LDL-R(293-332) EGF(A).
[0053] Analogues "comprising" certain specified changes may
comprise further changes, when compared to SEQ ID NO: 1.
[0054] In a particular embodiment, the analogue "has" or
"comprises" the specified changes. In a particular embodiment, the
analogue "consists of" the changes. When the term "consists" or
"consisting" is used in relation to an analogue e.g. an analogue
consists or consisting of a group of specified amino acid
substitutions, it should be understood that the specified amino
acid substitutions are the only amino acid substitutions in the
peptide analogue. In contrast an analogue "comprising" a group of
specified amino acid substitutions may have additional
substitutions.
[0055] As is apparent from the above examples, amino acid residues
may be identified by their full name, their one-letter code, and/or
their three-letter code. These three ways are fully equivalent.
[0056] The expressions "a position equivalent to" or "corresponding
position" may be used to characterise the site of change in a
variant LDL-R(293-332) EGF(A) sequence by reference to the
reference sequence native LDL-R(293-332) EGF(A) (SEQ ID NO: 1).
Equivalent or corresponding positions, as well as the number of
changes, are easily deduced, e.g. by simple handwriting and
eyeballing; and/or a standard protein or peptide alignment program
may be used, such as "align" which is based on a Needleman-Wunsch
algorithm.
[0057] In what follows, it may occur that a chemical formula is
defined such that two subsequent chemical groups may both be
selected to be "a bond". In such instances, the two subsequent
chemical groups would actually be absent, and just one bond would
connect the surrounding chemical groups.
[0058] Amino acids are molecules containing an amino group and a
carboxylic acid group, and, optionally, one or more additional
groups, often referred to as a side chain.
[0059] The term "amino acid" includes proteinogenic (or natural)
amino acids (amongst those the 20 standard amino acids), as well as
non-proteinogenic (or non-natural) amino acids. Proteinogenic amino
acids are those which are naturally incorporated into proteins. The
standard amino acids are those encoded by the genetic code.
Non-proteinogenic amino acids are either not found in proteins, or
not produced by standard cellular machinery (e.g., they may have
been subject to post-translational modification). Non-limiting
examples of non-proteinogenic amino acids are Aib
(.alpha.-aminoisobutyric acid, or 2-aminoisobutyric acid),
norleucine, norvaline as well as the D-isomers of the proteinogenic
amino acids.
[0060] In what follows, each amino acid of the peptides of the
invention for which the optical isomer is not stated is to be
understood to mean the L-isomer (unless otherwise specified).
Peptide Analogues of the Invention
[0061] An aspect of the invention relates to an analogue of a
peptide of SEQ ID NO: 1.
[0062] The peptide analogues of the invention may be defined as
peptides comprising an amino acid sequence which is an analogue of
SEQ ID NO: 1. The peptide analogues of the invention have the
ability to bind to PCSK9. In a specific embodiment, the analogues
of the invention have an improved ability to bind to PCSK9, for
example compared to native LDL-R(293-332) (native EGF-(A)) or to
other PCSK9-binding compounds.
[0063] The peptide analogues of the invention have the ability to
inhibit PCSK9 binding to the LDL-R. In one embodiment the peptide
is a PCSK9 inhibitor. In one embodiment the peptide inhibits PCSK9
binding to human Low Density Lipoprotein Receptor (LDL-R). Such
binding may be assessed using the assay described in Example D.1.1
herein. In one embodiment the peptide analogues and peptide
derivatives of the invention are PCSK9 inhibitor peptides or simply
PCSK9 inhibitors. In one embodiment the invention relates to a
peptide analogue of SEQ ID NO.:1, wherein peptide analogue is a
capable of inhibiting PCSK9 binding to human Low Density
Lipoprotein Receptor (LDL-R).
[0064] In one embodiment the peptide analogues, compounds or PCSK9
inhibitors of the invention have an improved ability to bind PCSK9
compared to EGF(A), LDL-R(293-332) (SEQ ID 1).
[0065] In one embodiment the peptide analogues, compounds or PCSK9
inhibitors of the invention have an improved ability to bind PCSK9
compared to Ex. 48 (SEQ ID 2).
[0066] In one embodiment the K.sub.i of the peptide analogues,
compounds or PCSK9 inhibitors as described herein as measured in
the PCSK9-LDL-R binding competitive ELISA assay is below 10 nM,
such as below 8 nM or such as below 5 nM.
[0067] Functionality of EGF-(A) analogues and derivatives hereof
may be further characterized by their ability to improve LDL
uptake, such as described in Example D1.2 herein. In one embodiment
the peptide analogues, compounds or PCSK9 inhibitors of the
invention increases LDL uptake in the presence of PCSK9. In one
embodiment the peptide analogues, compounds or PCSK9 inhibitors of
the invention are capable of reversing or reducing PCSK9 mediated
reduction of LDL uptake.
[0068] In one embodiment the peptide analogues, compounds or PCSK9
inhibitors of the invention have a EC50 as measured in the LDL
uptake assay of below 1500 nM, such as below 1000 nM or such as
below 500 nM.
[0069] In an embodiment, a peptide analogue of the invention may be
defined as comprising at least 1 amino acid substitution compared
to SEQ ID NO: 1, and optionally an elongation. In an embodiment, a
peptide analogue of the invention may be defined as comprising up
to 15, up to 14, up to 13, up to 12, up to 11, up to 10, up to 9,
up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or 1
amino acid(s) substitution(s) compared to SEQ ID NO: 1, and
optionally an elongation. This means that a peptide comprising an
elongation in the N-terminal and/or in the C-terminal may comprise
up to 15 amino acids substitutions in positions from 293 to 332 in
addition to said elongation.
[0070] An amino acid "elongation" may also be referred to as
"extension". In an embodiment, peptide analogues of the invention
comprise an elongation. Said elongation may be an addition of up to
50 amino acid residues in position N-terminal of SEQ ID NO: 1 or an
analogue thereof, also referred to as an N-terminal elongation,
meaning that a peptide of the invention may comprise up to 50 amino
acids from position 292 down to, for example position 242.
Additionally or alternatively, said elongation may be an addition
of up to 50 amino acid residues in position C-terminal of SEQ ID
NO: 1 or analogue thereof, also referred to as a C-terminal
elongation, meaning that a peptide of the invention may comprise up
to 50 amino acids from position 333 up to, for example position
383.
[0071] Said elongation may be present either in N-terminal, in
C-terminal or both. Said elongation may also be of any length
between 0 and 50 amino acids on each side, independently of each
other. In one embodiment, the peptide analogues of the invention
comprise a N-terminal elongation of 1-50, 1-40, 10-40, 1-30, 10-30,
20-30, 20-40, 20-50, 30-50, 1-10, 11-20, 21-30, 31-40 or 41-50
amino acid residues or of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49 or 50 amino acid residues. In addition or alternatively,
the peptide analogues of the invention may comprise a C-terminal
elongation of 1-50, 1-40, 10-40, 1-30, 10-30, 20-30, 20-40, 20-50,
30-50, 1-10, 11-20, 21-30, 31-40 or 41-50 amino acid residues or of
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid
residues.
[0072] An elongation may in some situation be referred to a
substitution as a new amino acid residue is introduced, such as the
292A, 292Lys or 333Lys exemplified herein.
[0073] Minor truncations at the N-terminal and/or C-terminal of the
EGF(A) peptide may be present in the EGF(A) peptide analogue.
[0074] In one embodiment the EGF(A) peptide comprise at least 35
amino acid residues, such as 36 amino acid residues, such as 37
amino acid residues, such as 38 amino acid residues or such as such
as 39 amino acid residues. In one embodiment the EGF(A) peptide
analogue according comprises an N-terminal truncation of 1-2amino
acid residues. In one embodiment one or two N-terminal amino acid
residues are deleted. In further embodiments the EGF(A) peptide
analogue accordingly comprises an N-terminal truncation deleting at
least or specifically amino acid 293Gly.
[0075] In further embodiments the EGF(A) peptide analogue comprises
an N-terminal truncation deleting at least or specifically
293Gly-294Thr.
[0076] In one embodiment the EGF(A) peptide analogue comprises a
C-terminal truncation of 1 amino acid residue. In one embodiment a
single C-terminal amino acid residue is deleted. In on embodiment
the peptide analogue comprises a C-terminal truncation deleting
specifically amino acid 332Glu.
[0077] In addition or alternatively, a peptide analogue of the
invention may comprise at least one amino acid elongation in the
N-terminal or the C-terminal for example in position 292 and/or
333.
[0078] The EGF(A) peptide analogue of the invention comprises the
amino acid substitution of amino acid residue 301 from Asn to Leu,
also described by Asn301Leu or simply 301Leu. In a specific
embodiment, the EGF(A) peptide analogue comprises the substitution
301Leu.
[0079] In addition or alternatively the EGF(A) peptide analogue
comprises the amino acid residues 297Cys, 304Cys, 308Cys, 317Cys,
319Cys and 331Cys. Those Cys residues are wild type residues which
may be engaged in disulphide bridges, such as the disulphide
bridges between 297Cys and 308Cys, between 304Cys and 317Cys and
between 319Cys and 331Cys.
[0080] In one embodiment, the EGF(A) peptide analogue comprises
301Leu and a number of further amino acid substitutions, as
described above.
[0081] In one embodiment the EGF(A) peptide analogue comprises
301Leu, 310Asp and an amino acid substitution of 312Lys.
[0082] In one embodiment, the EGF(A) peptide analogue comprises
301Leu and 310Asp and wherein the peptide analogue does not have a
substitution of 299Asp to Glu, Val or His. In one embodiment the
EGF(A) peptide analogue comprises 301Leu, 309Arg and 312Glu.
[0083] In one embodiment the EGF(A) peptide analogue comprises
301Leu and 309Arg with a proviso that the peptide analogue does not
have a substitution of 310Asp to 310Lys or
[0084] In one embodiment the EGF(A) peptide analogue comprises
301Leu and 309Arg with a proviso that the peptide analogue does not
have a substitution of 299Asp to Glu, Val or His.
[0085] In a further embodiment the peptide analogue does not have
any of the substitutions D310K, D310N, D310Q, D310Q, D310R and
D310A or even any substitution of 310Asp.
[0086] In one embodiment the EGF(A) peptide analogue comprises one,
two, three or all four wild type residues: 295Asn, 296Glu, 298Leu
and 302Gly.
[0087] In one embodiment the EGF(A) peptide analogue comprises one,
two, three, four or all five wild type residues: 295Asn, 296Glu,
298Leu, 302Gly and 310Asp.
[0088] In one embodiment the peptide has 295Asn.
[0089] In one embodiment the peptide analogue has 296Glu. In one
embodiment the peptide analogue has 298Leu. In one embodiment the
peptide analogue has 302Gly. In one embodiment the peptide analogue
has 310Asp.
[0090] In one embodiment the peptide analogue has two or more of
310Asp, 295Asn and 296Glu. In one embodiment the peptide analogue
has all three of 310Asp, 295Asn and 296Glu.
[0091] The EGF(A) peptide analogue may comprise further amino acid
substitutions as described herein. In one embodiment the analogue
of the invention may further comprise one or more amino acid
substitution in a position(s) selected from the group of positions:
293, 294, 296, 299, 300, 303, 305, 306, 309, 311, 312, 313, 314,
315, 316, 318, 320, 321, 322, 323, 324, 325, 326, 328, 329, 330 and
332.
[0092] In one embodiment the analogue of the invention may further
comprise one or more amino acid substitution(s) in a position(s)
selected from the group of positions: 293, 294, 299, 300, 303, 305,
306, 309, 311, 312, 313, 314, 316, 318, 321, 322, 323, 324, 325,
326, 328, 329, 330, 331 and 332.
[0093] In one embodiment the analogue of the invention may further
comprise one or more amino acid substitution(s) in a position(s)
selected from the 294, 299, 300, 303, 309, 312, 313, 314, 316, 318,
321, 322, 323, 324, 325, 326, 328, 329, 330 and 332.
[0094] In one embodiment the analogue of the invention may further
comprise one or more amino acid substitution(s) in a position(s)
selected from the 299, 300, 309, 313, 316, 318, 321, 322, 323, 324,
326, 328, 329, 330 and 332.
[0095] In one embodiment the analogue of the invention may further
comprise one or further amino acid substitution(s) in a position(s)
selected from the group of positions: 309, 312, 313, 321, 324, 328
and 332.
[0096] In a further embodiment the peptide analogue comprise either
the wt amino acid residue or a different residue i.e. an amino acid
substitution, in certain specific positions in addition to the
amino acid residues specified herein above.
[0097] In one such embodiment the analogue of the invention
comprises the amino acid residue Gly(G) or Asn(N) in position
293.
[0098] In one such embodiment the analogue of the invention
comprises the amino acid residue Trp (W), Thr(T) or Gly(G) in
position 294.
[0099] In one such embodiment the analogue of the invention
comprises the amino acid residue Asp(D), Gly(G), Pro(P), Arg(R),
Lys(K), Ser(S), Thr(T), Asn(N), Gln(Q), Ala(A), Ile(I), Leu(L),
Met(M), Phe(F), Tyr(Y) or Trp(W) in position 299.
[0100] In one such embodiment the analogue of the invention
comprises the amino acid residue Asp(D), Gly(G), Pro (P), Arg(R),
Lys(K), Ser(S), Thr(T), Asn(N), Gln(Q), Ala(A), Met(M), Phe(F),
Tyr(Y) or Trp(W) in position 299.
[0101] In one such embodiment the analogue of the invention
comprises the amino acid residue Asp(D), Ser (S), Arg(R), Leu (L),
Ala (A), Lys(K) or Tyr(Y) in position 299.
[0102] In one such embodiment the analogue of the invention
comprises the amino acid residue Asp(D) or Ala(A) in position
299.
[0103] In one such embodiment the analogue of the invention
comprises the amino acid residue His(H) or Asn(N) in position
300.
[0104] In one such embodiment the analogue of the invention
comprises the amino acid residue Val(V), Ser(S), Thr (T) or Ile (I)
in position 307.
[0105] In one such embodiment the analogue of the invention
comprises the amino acid residue Val(V) or Ile (I) in position
307.
[0106] In one such embodiment the analogue of the invention
comprises Ser (S), Thr (T) or Ile (I) in position 307.
[0107] In one such embodiment the analogue of the invention
comprises Ile (I) in position 307.
[0108] In one such embodiment the analogue of the invention
comprises the amino acid residue Asn(N), Glu (E), His (H,) Arg (R),
Ser (S) or Lys (K) in position 309.
[0109] In one such embodiment the analogue of the invention
comprises the amino acid residue Asn(N), Arg (R), Ser (S) or Lys
(K) in position 309.
[0110] In one such embodiment the analogue of the invention
comprises the amino acid residue Asn(N), Arg (R) or Ser (S) in
position 309.
[0111] In one such embodiment the analogue of the invention
comprises the amino acid residue Asn(N) or Arg (R) in position
309.
[0112] In one such embodiment the analogue of the invention
comprises the amino acid residue Lys(K) or Arg (R) in position
309.
[0113] The EGF(A) peptide analogue may comprise several amino acid
substitutions as described herein, such as one or more amino acid
substitutions selected from the group of: 299Ala, 307Ile and
321Glu.
[0114] In further embodiments, the EGF(A) peptide analogue
comprises the amino acid residue Asp(D), Lys (K) or Glu(E) in
position 321.
[0115] In further embodiments, the EGF(A) peptide analogue
comprises the amino acid residue Asp(D) or Glu(E) in position
321.
[0116] In further embodiments, the EGF(A) peptide analogue
comprises the amino acid residue Glu(E) in position 321.
[0117] In further embodiments, the EGF(A) peptide analogue
comprises the amino acid residue Gln (Q) or Gly (G) in position
324.
[0118] In further embodiments, the EGF(A) peptide analogue
comprises the amino acid residue Arg (R) or His (H) in position
329.
[0119] In further embodiments, the EGF(A) peptide analogue does not
have a substitution of 300Asn(N) to Pro(P).
[0120] The EGF(A) domain of LDL-R includes a Lysine in position 312
which may be useful for substitution as described herein. In
embodiments where attachment of the substituent to 312 is not
wanted 312Lys may be substituted by another amino acid as described
herein.
[0121] In one embodiment, Lys in position 312 is substituted by an
amino acid residue selected from: Gly, Pro, Asp, Glu, Arg, His,
Ser, Thr, Asn, Gln, Ala, Val, Ile, Leu, Met, Phe and Tyr. In one
embodiment, Lys in position 312 is substituted by an amino acid
residue selected from: Gly, Asp, Glu, Ser, Thr, Asn, Ala, Val, Ile,
Leu, Phe and Tyr. In one embodiment, Lys in position 312 is
substituted by an amino acid residue selected from: Asp, Glu, Thr,
Asn, Ile, Leu, Phe and Tyr. In one embodiment, 312Lys is
substituted by 312Asp, 312Glu, 312Thr, 312Asn, 312Ile or 312Phe. In
one embodiment, 312Lys is substituted by 312Glu, 312Asp, 312Gln or
312Arg.
[0122] In one embodiment, 312Lys is substituted by 312Glu, 312Thr,
312Asn, 312Ile, 312Phe or 312Tyr. In one embodiment, 312Lys is
substituted by 312Glu, 312Asn or 312Ile,
[0123] In one embodiment, 312Lys is substituted by 312Glu or
312Arg. In one embodiment 312Lys is substituted by 312Arg. In one
embodiment, 312Lys is substituted by 312Glu.
[0124] To include an option for attaching the substituent in
various positions (see further below), a Lys may be introduced by
amino acid substitution of a wild type residue of SEQ ID NO.: 1 or
by a peptide elongation of SEQ ID NO.: 1, such as a 292Lys or a
333Lys.
[0125] In cases where more than one substituent is desired one may
be via 312Lys while the second is via a Lys introduced by peptide
elongation or substitution in SEQ ID NO.: 1.
[0126] In one embodiment the peptide analogue of SEQ ID NO: 1
comprises at least one Lys residue in a position selected from the
group of: 292Lys, 293Lys, 294Lys, 296Lys, 299Lys, 300Lys, 303Lys,
305Lys, 306Lys, 309Lys, 311Lys, 312Lys, 313Lys, 314Lys, 315Lys,
316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys,
326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys.
[0127] In one embodiment the peptide analogue of SEQ ID NO: 1
comprises at least one Lys residue in a position selected from the
group of: 292Lys, 293Lys, 294Lys, 299Lys, 300Lys, 303Lys, 305Lys,
306Lys, 309Lys, 311Lys, 312Lys, 313Lys, 314Lys, 315Lys, 316Lys,
318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys,
327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys.
[0128] In one embodiment the peptide analogue of SEQ ID NO: 1
comprises at least one Lys residue in a position selected from the
group of: 292Lys, 293Lys, 294Lys, 300Lys, 303Lys, 305Lys, 306Lys,
309Lys, 311Lys, 312Lys, 313Lys, 314Lys, 316Lys, 318Lys, 321Lys,
322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys,
330Lys, 332Lys and 333Lys.
[0129] In one embodiment the peptide analogue of SEQ ID NO: 1
comprises at least one Lys residue in a position selected from the
group of: 292Lys, 293Lys, 294Lys, 300Lys, 303Lys, 305Lys, 306Lys,
311Lys, 312Lys, 313Lys, 314Lys, 316Lys, 318Lys, 322Lys, 323Lys,
324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and
333Lys.
[0130] In one embodiment the peptide analogue of SEQ ID NO: 1
comprises at least one Lys residue in a position selected from the
group of: 292Lys, 293Lys, 294Lys, 300Lys, 303Lys, 305Lys, 306Lys,
311Lys, 313Lys, 314Lys, 316Lys, 318Lys, 322Lys, 323Lys, 324Lys,
325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and
333Lys.
[0131] In addition or alternatively, the peptide analogue of the
invention comprises at least one amino acid substitution selected
from 292Lys, 293Lys, 294Lys, 295Lys, 296Lys, 298Lys, 299Lys,
301Lys, 302Lys, 303Lys, 305Lys, 306Lys, 307Lys, 309Lys, 310Lys,
311Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys, 321Lys,
322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys,
330Lys, 332Lys and 333Lys.
[0132] In a further embodiment, the EGF(A) peptide analogue of the
invention comprises at least one amino acid substitution selected
from: 292Lys, 293Lys, 294Lys, 295Lys, 296Lys, 298Lys, 299Lys,
302Lys, 303Lys, 305Lys, 306Lys, 307Lys, 309Lys, 311Lys, 313Lys,
314Lys, 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys,
324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and
333Lys.
[0133] In a further embodiment, the EGF(A) peptide analogue of the
invention comprises at least one amino acid substitution selected
from 292Lys, 293Lys, 294Lys, 295Lys, 296Lys, 298Lys, 299Lys,
303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 313Lys, 314Lys, 315Lys,
316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys,
326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys.
[0134] In a further embodiment, the EGF(A) peptide analogue of the
invention comprises at least one amino acid substitution selected
from 292Lys, 293Lys, 294Lys, 295Lys, 296Lys, 299Lys, 303Lys,
305Lys, 306Lys, 309Lys, 311Lys, 313Lys, 314Lys, 315Lys, 316Lys,
318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys,
327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys.
[0135] In a further embodiment, the EGF(A) analogue peptide of the
invention comprises at least one amino acid substitution selected
from 292Lys, 293Lys, 294Lys, 296Lys, 299Lys, 303Lys, 305Lys,
306Lys, 309Lys, 311Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys,
320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys,
328Lys, 329Lys, 330Lys, 332Lys and 333Lys.
[0136] In a further embodiment, the EGF(A) peptide analogue of the
invention comprises at least one amino acid substitution selected
from 292Lys, 293Lys, 294Lys, 299Lys, 303Lys, 305Lys, 306Lys,
309Lys, 311Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys,
321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys,
329Lys, 330Lys, 332Lys and 333Lys.
[0137] In a further embodiment, the EGF(A) peptide analogue of the
invention comprises at least one amino acid substitution selected
from 292Lys, 293Lys, 294Lys, 299Lys, 303Lys, 305Lys, 306Lys,
309Lys, 311Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys,
321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys,
329Lys, 330Lys, 332Lys and 333Lys.
[0138] In a further embodiment, the EGF(A) peptide analogue of the
invention comprises at least one amino acid substitution selected
from 292Lys, 293Lys, 294Lys, 299Lys, 303Lys, 305Lys, 306Lys,
310Lys, 311Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys,
321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys,
329Lys, 330Lys, 332Lys and 333Lys.
[0139] In a further embodiment, the EGF(A) peptide analogue of the
invention comprises at least one amino acid substitution selected
from 292Lys, 293Lys, 294Lys, 299Lys, 303Lys, 305Lys, 306Lys,
309Lys, 310Lys, 311Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys,
321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys,
329Lys, 330Lys, 332Lys and 333Lys.
[0140] In a further embodiment, the EGF(A) peptide analogue of the
invention comprises at least one amino acid substitution selected
from 292Lys, 293Lys, 294Lys, 303Lys, 305Lys, 306Lys, 310Lys,
311Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 321Lys, 322Lys,
323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys,
332Lys and 333Lys. In one embodiment, the peptide analogues of the
invention do not comprise any of the following substitutions: 296K,
298K, 301K, 302K and 307K.
[0141] In one embodiment, the peptide analogues of the invention do
not comprise any of the following substitution: 296K, 298K, 301K,
302K, 307K and 310K.
[0142] In one embodiment, the peptide analogues of the invention do
not comprise any of the following substitution: 296K, 298K, 301K,
302K, 307, and 295K.
[0143] In one embodiment, the peptide analogues of the invention do
not comprise any of the following substitution: 296K, 298K, 301K,
302K, 307K and 295D.
[0144] In a particular embodiment, the peptide analogue of the
invention comprises 1 or 2, of such Lys substitutions.
[0145] In addition or alternatively, the peptide of the invention
may comprise 312Lys.
[0146] In one embodiment the peptide analogue of the invention
comprises two Lys residues. In one embodiment the peptide analogue
of the invention comprises two Lys residues selected from the pairs
consisting of:
TABLE-US-00002 i. 293K and 294K xiv. 313K and 321K ii. 293K and
312K xv. 313K and 324K iii. 293K and 333K xvi. 313K and 328K iv.
309K and 313K xvii. 313K and 332K v. 309K and 324K xviii. 313K and
333K vi. 309K and 328K xix. 314K and 333K vii. 309K and 332K xx.
321K and 332K viii. 309K and 333K xxi. 321K and 333K ix. 311K and
313K xxii. 324K and 333K x. 312K and 333K xxiii. 324K and 328K xi.
312K and 313K xxiv. 328K and 333K xii. 312K and 314K xxv. 330K and
333K and xiii. 313K and 314K xxvi. 332K and 333K.
[0147] As seen herein above various peptide analogues are provided
by the present invention. In a further embodiment the EGF(A)
peptide analogue according to the invention comprises at least two
amino acid substitutions identified by any of the groups i-xxiv
shown below compared to SEQ ID NO.:1.
[0148] In a still further embodiment, the EGF(A) peptide analogue
of the invention consists of the amino acid substitutions
identified by any of the groups i-xxiv as shown below.
[0149] In a further embodiment the EGF(A) peptide analogue
according to the invention comprises at least two amino acid
substitutions identified by any of the groups i-xvi shown below
compared to SEQ ID NO.:1.
[0150] In a still further embodiment, the EGF(A) peptide analogue
of the invention consists of the amino acid substitutions
identified by any of the groups i-xvi as shown below. [0151] i.
301Leu and 309Arg [0152] ii. 301Leu, 309Arg, 312Glu [0153] iii.
301Leu, 307Ile and 309Arg [0154] iv. 301Leu, 307Ile, 309Arg and
312Glu [0155] v. 301Leu, 309Arg and 321Glu [0156] vi. 301Leu,
309Arg, 321Glu and 312Glu [0157] vii. 301Leu, 307Ile, 309Arg and
299Ala [0158] viii. 301Leu, 307Ile, 309Arg, 299Ala and 312Glu
[0159] ix. 301Leu and 309Arg and at least one Lys substitution
[0160] x. 301Leu, 309Arg, 312Glu and at least one Lys substitution
[0161] xi. 301Leu, 307Ile and 309Arg and at least one Lys
substitution [0162] xii. 301Leu, 307Ile, 309Arg and 312Glu and at
least one Lys substitution [0163] xiii. 301Leu, 309Arg and 321Glu
and at least one Lys substitution [0164] xiv. 301Leu, 309Arg,
321Glu and 312Glu and at least one Lys substitution [0165] xv.
301Leu, 307Ile, 309Arg and 299Ala and at least one Lys substitution
or [0166] xvi. 301Leu, 307Ile, 309Arg, 299Ala and 312Glu and at
least one Lys substitution.
[0167] In a further embodiment the EGF(A) peptide analogue
according to the invention comprises at least two amino acid
substitutions identified by any of the groups xvii-xx shown below
compared to SEQ ID NO.: 1.
[0168] In a still further embodiment, the EGF(A) peptide analogue
of the invention consists of at the amino acid substitutions
identified by any of the groups xvii-xx as shown below. [0169]
xvii. 301Leu and 309Lys [0170] xviii. 301Leu, 309Lys and 312Glu
[0171] xix. 301Leu and 309Lys and at least one further Lys
substitution [0172] xx. 301Leu, 309Lys and 312Glu and at least one
further Lys substitution.
[0173] In a further embodiment the EGF(A) peptide analogue
according to the invention comprises at least two amino acid
substitutions identified by any of the groups xxi-xxiv shown below
compared to SEQ ID NO.: 1.
[0174] In a still further embodiment, the EGF(A) peptide analogue
of the invention consists of the amino acid substitution identified
by any of the groups xxi-xxiv as shown below [0175] xxi. 301Leu and
307Ile, [0176] xxii. 301Leu, 307Ile and 312Glu [0177] xxiii. 301Leu
and 307Ile and at least one further Lys substitution and [0178]
xxiv. 301Leu, 3307Ile and 312Glu and at least one further Lys
substitution.
[0179] In further specific embodiments the peptide analogue or the
peptide analogue of the compounds according to the invention
comprises or consists of anyone of the amino acid sequences
identified by SEQ ID 1 to 106.
[0180] In one embodiment the peptide analogue comprises or consists
of anyone of the amino acid sequences identified by SEQ ID NO.:
2-106.
[0181] In one embodiment the peptide analogue comprises or consists
of anyone of the amino acid sequences identified by SEQ ID NO.:
2-47 and 49-106.
[0182] In one embodiment the peptide analogue comprises or consists
of anyone of the amino acid sequences identified by anyone of the
amino acid sequences SEQ ID NO.: 2-44, 46, 47 and 49-106.
[0183] In one embodiment the peptide analogue comprises or consists
of anyone of the amino acid sequences identified by of SEQ ID NO.:
2-44, 46, 47, 49-53, 55, 58-106.
[0184] In one embodiment the peptide analogue comprises or consists
of anyone of the amino acid sequences identified by SEQ ID NO.:
2-4, 6-44, 46, 47, 49-53, 55, 58-106.
[0185] In one embodiment the peptide analogue comprises or consists
of anyone of the amino acid sequences identified by SEQ ID NO.:
2-4, 6-19, 21-44, 46, 47, 49-53, 55, 58-106.
Intermediate Compounds
[0186] The present invention also relates to peptide analogues
which may be incorporated in the derivatives of the invention. Such
peptide analogues may be referred to as "intermediate product" or
"intermediate compound". They are in the form of novel
LDL-R(293-332) analogues, which as described above can be
incorporated in EGF(A) derivatives of the invention as further
describe below. Such peptide analogues are as defined in the above
section.
[0187] In particular, a peptide analogue, or intermediate peptide,
according to the present invention may be referred to as a peptide
analogue of sequence SEQ ID NO: 1.
[0188] In one aspect the invention relates to a EGF(A) peptide
analogue as described herein for use in the manufacture of a EGF(A)
compound, such as a EGF(A) derivative.
[0189] Other features, definitions, aspects and embodiments
disclosed herein in connection with peptide analogues of the
invention may also be applicable to the intermediates products of
the invention.
EGF(A) Derivatives
[0190] The peptides analogues of the invention may further comprise
a substituent and thereby become derivative compounds.
[0191] The term "derivative" generally refers to a compound which
may be prepared from a native peptide or an analogue thereof by
chemical modification, in particular by covalent attachment of one
or two substituents.
[0192] The terms "derivative of the invention", "EGF(A)
derivative", "EGF(A) derivative or "LDL-R(293-332) derivative" or
"derivative of a LDL-R(293-332) analogue" as used herein refers to
as a peptide to which one or two substituents are attached. Each of
these may, also or alternatively, be referred to as a side chain.
In other words, a "derivative of the invention" comprises a peptide
i.e. a peptide sequence, which herein is an EGF(A) peptide
analogue, and at least one, including such as one or two,
substituent(s).
[0193] The terms "substituent" is used to describe a moiety
covalently bond to the EGF(A) peptide e.g. the substituent is a
moiety not part of the EGF(A) peptide itself.
[0194] In one embodiment the one or more substituent(s) is/are
attached to a nitrogen atom of the EGF(A) peptide analogue. In one
embodiment the one or more substituent(s) is/are attached to an
amino group of the EGF(A) peptide analogue. In one embodiment the
one or more substituent(s) is/are attached to the N-terminal amino
acid of the EGF(A) peptide analogue or to a Lys residue of the
EGF(A) peptide analogue. In one embodiment the one or more
substituent(s) is/are attached to the N-terminal amino acid of the
EGF(A) peptide analogue. In one embodiment the one or more
substituent(s) is/are attached to the alpha-nitrogen of the
N-terminal amino acid residue of the EGF(A) peptide analogue In one
embodiment the one or more substituent(s) is/are attached to a Lys
residue in the EGF(A) peptide analogue. In one embodiment the one
or more substituent(s) is/are attached to the epsilon-nitrogen of a
Lys residue in the EGF(A) peptide analogue.
[0195] Examples of substituents are various and further described
below.
[0196] In one aspect, the invention relates to an EGF(A) derivative
comprising an EGF(A) peptide analogue and at least one substituent.
In one embodiment the substituent of the derivative comprises at
least one fatty acid group. For all embodiments the term EGF(A)
derivative also encompasses any pharmaceutically acceptable salt,
amide, or ester thereof.
Substituents
[0197] A substituent is a moiety attached to an EGF(A) peptide
analogue. According to the invention it is preferred that the
moiety e.g. the substituent has no or minimal effect on the
functionality of the EGF(A) peptide while adding other beneficial
properties, such as longer half-life and/or improved exposure after
oral dosing.
[0198] It follows that the derivatives, as well as the analogues of
the invention described above, have the ability to bind to PCSK9.
Such binding to PCSK9 inhibits PCSK9 binding to the LDL-R, thereby
preventing LDL-R degradation hence increasing the clearance of
LDL-C and atherogenic lipoproteins.
[0199] In a specific embodiment, the derivatives and analogues of
the invention have an improved ability to bind to PCSK9, for
example compared to native LDL-R(293-332) or to other PCSK9-binding
compounds. The analogues and derivatives of the invention can for
example be tested for their ability to inhibit PCSK9 binding to
LDL-R using the assay described in Example D.1.1 herein.
[0200] In an embodiment the substituent is aimed at improving the
functionality of the peptides.
[0201] In one embodiment the substituent increase half-life of the
peptide analogue in a way that the plasma half-live of a derivative
comprising a backbone peptide and a substituent have an increase
half-life compared to the half-life of the backbone peptide as
illustrated by Example 1 and 48 (Section D2, table 7). Methods for
determining half-life in different species are well known in the
art and exemplified herein for mice and dogs (Section D2 and
D5).
[0202] In one embodiment the EGF(A) derivative according to the
invention has a half-life above 4 hours.
[0203] In one embodiment the EGF(A) derivative according to the
invention has a half-life above 6 hours, such as above 8 hours or
such as above 10 hours in mice measured after either subcutaneously
or intravenously dosing.
[0204] In one embodiment the EGF(A) derivative according to the
invention has a half-life above 25 hours in dogs.
[0205] In one embodiment the EGF(A) derivative according to the
invention has a half-life above 50 hours, such as above 100 hours
or such as above 150 hours in dogs.
[0206] In one embodiment, a half-life extending substituent is a
protein moiety. In a further such embodiment the protein moiety may
include human albumin, an Fc-domain or an unstructured protein
extension. In a further embodiment the protein moiety may by fused
to the peptide analogue. In a further embodiment, the protein
moiety is Fc domain and the Fc domain is fused to the peptide
analogue. When an Fc fusion is prepared the resulting compound will
usually be divalent as two Fc-polypeptides will form one
Fc-domain.
[0207] In one embodiment the substituent is not a protein moiety.
In one embodiment the substituent is not a protein moiety fused to
the EGF(A) peptide analogue. In one embodiment the protein moiety
is not an Fc domain.
[0208] In another embodiment the substituent is a non-protein
moiety.
[0209] In a particular embodiment, the substituent is capable of
forming non-covalent complexes with albumin, thereby promoting the
circulation of the derivative within the blood stream, and also
having the effect of protracting the time of action of the
derivative. In a particular embodiment, the substituent is capable
of protracting the time of action of the EGF(A) compound without
substantially decreasing its binding capacity to PCSK9.
[0210] In one embodiment the EGF(A) derivative comprises a
half-life extending substituent. Various half-life extending
substituents are well-known in the art and include in particular
albumin binders comprising a fatty acid group as described further
below, and such albumin binders are non-protein substituents.
[0211] The substituent comprises at least one fatty acid group.
[0212] In a particular embodiment, the fatty acid group comprises a
carbon chain which contains at least 8 consecutive --CH.sub.2--
groups. In one embodiment the fatty acid group comprise at least 10
consecutive --CH.sub.2-- groups, such as least 12 consecutive
--CH.sub.2-- groups, at least 14 consecutive --CH.sub.2-- groups,
at least 16 consecutive --CH.sub.2-- groups, at least 18
consecutive --CH.sub.2-- groups.
[0213] In one embodiment the fatty acid group comprises 8-20
consecutive --CH.sub.2-- groups. In one embodiment the fatty acid
group comprises 10-18 consecutive --CH.sub.2-- groups. In one
embodiment the fatty acid group comprises 12-18 consecutive
--CH.sub.2-- groups. In one embodiment the fatty acid group
comprises 14-18 consecutive --CH.sub.2-- groups.
[0214] In situations where the derivative comprise two
substituents, an increased half-life may be obtained with shorter
fatty acid groups, thus in an embodiment where the derivate
comprise two substituents the fatty acid groups may comprise at
least 8 consecutive --CH.sub.2-- groups, such as least 10
consecutive --CH.sub.2-- groups, such as least 12 consecutive
--CH.sub.2-- groups, at least 14 consecutive --CH.sub.2-- groups,
at least 16 consecutive --CH.sub.2-- groups.
[0215] In a further embodiment where the derivative comprises two
substituents, the substituents each comprise a fatty acid group
comprising 8-18 consecutive --CH.sub.2-- groups. In further such
embodiments the fatty acid groups comprise 10-18 consecutive
--CH.sub.2-- groups, such as 12-18 consecutive --CH.sub.2-- groups,
such as 14-18 consecutive --CH.sub.2-- groups. The term "fatty acid
group" as used herein may be referred to as chemical group
comprising at least one functional group being a Bronsted-Lowry
acid with a pKa<7. Non-limiting examples of such functional
groups that are Bronsted-Lowry acids include a carboxylic acid
(including also carboxyphenoxy), a sulphonic acid, a tetrazole
moiety.
[0216] In one embodiment said fatty acid group comprises a
functional group selected from a carboxylic acid, a sulphonic acid,
a tetrazole moiety, a methylsulfonylcarbamoylamino (MSU) moiety and
a 3-Hydroxy-isoxazolelsoxazole moiety. Accordingly the half-life
extending substituent of the invention in an embodiment comprises a
carboxylic acid, a sulphonic acid, a tetrazole moiety, a
methylsulfonylcarbamoylamino moiety or a hydroxy-isoxazolelsoxazole
moiety further including 8-20 consecutive --CH.sub.2-- groups as
defined by:
[0217] Chem. 1: HOOC--(CH.sub.2).sub.n--CO--* wherein n is an
integer in the range of 8-20, which may also be referred to as a
C(n+2) diacid or as
##STR00001##
wherein n is an integer in the range of 8-20,
[0218] Chem. 2: 5-tetrazolyl-(CH.sub.2).sub.n--CO--* wherein n is
an integer in the range of 8-20, which may also be referred to
as
##STR00002##
wherein n is an integer in the range of 8-20.
[0219] Chem. 3: HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*
wherein n is an integer in the range of 8-20, which may also be
referred to as
##STR00003##
wherein the carboxy group is in position 2, 3 or 4 of the
(C.sub.6H.sub.4) group of Chem. 3 and wherein m is an integer in
the range of 8-11
[0220] Chem. 4: HO--S(O).sub.2--(CH.sub.2).sub.n--CO--* wherein n
is an integer in the range of 8-20, which may also be referred to
as
##STR00004##
wherein n is an integer in the range of 8-20,
[0221] Chem. 5: MeS(O).sub.2NH(CO)NH--(CH.sub.2).sub.n--CO--*
wherein n is an integer in the range of 8-20, which may also be
referred to as.
##STR00005##
wherein n is an integer in the range of 8-20,
[0222] Chem. 6: 3-HO-Isoxazole-(CH.sub.2).sub.n--CO--* wherein n is
an integer in the range of 8-20, which may also be referred to
as
##STR00006##
wherein n is an integer in the range of 8-20.
[0223] The term functional group in its acidic form is referred to
as FG-H and its form as conjugated base referred to as FG.sup.-.
The term "functional group with a pKa<7" as used herein may be
referred to as a Bronsted-Lowry acid which in the form of its
methyl derivative (CH.sub.3--FG-H) in aqueous solution has a
equilibrium pKa of below 7, wherein the pKa is the -log to the
equilibrium constant (Ka) of the equilibrium shown below:
CH.sub.3--FG-H+H.sub.2O.revreaction.CH.sub.3--FG.sup.-+H.sub.3O.sup.+.
[0224] Methods for the determination of pKa are well known in the
art. Such a method has for example been described by Reijenga et
al. in Anal Chem Insights 2013 (2013; 8: 53-71).
[0225] Substituents according to the invention in an embodiment
comprise one or more linker elements. The linker elements may be
linked to the fatty acid group by amide bonds and referred to as
Z.sub.2-Z.sub.10. As further defined herein below the number of
linker elements may be at most 10.
[0226] In a specific embodiment, the substituent is of Formula
I:
Z.sub.1--Z.sub.2--Z.sub.3--Z.sub.4--Z.sub.5--Z.sub.6--Z.sub.7--Z.sub.8---
Z.sub.9-Z.sub.10-- [I] wherein
[0227] Z.sub.1 is selected from:
HOOC--(CH.sub.2).sub.n--CO--* or Chem. 1:
##STR00007## 5-tetrazolyl-(CH.sub.2).sub.n--CO--* or Chem. 2:
##STR00008## HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--* or
Chem. 3:
##STR00009##
wherein the carboxy group is in position 2, 3 or 4 of
--(C.sub.6H.sub.4)--,
HOS(O).sub.2--(CH.sub.2).sub.n--CO--* or Chem. 4:
##STR00010## MeS(O).sub.2NH.sub.2N(CO)NHN--(CH.sub.2).sub.n--CO--*
or Chem. 5:
##STR00011##
and
3-HO-Isoxazole-(CH.sub.2).sub.n--CO--* or Chem. 6:
##STR00012##
[0228] wherein n is an integer in the range of 8-20 and m is an
integer in the range of 8-11.
[0229] In a particular embodiment, n is 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20 in Chem. 1 or 1b. In a particular
embodiment, n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
in Chem. 2 or 2b. In a particular embodiment, n is 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 in Chem. 4 or 4b. In a
particular embodiment, m is 8, 9, 10 or 11 in Chem. 3 or 3b.
[0230] In a particular embodiment, n is 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20 in Chem. 5 or 5b.
[0231] In a particular embodiment, n is 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20 in Chem. 6 or 6b.
[0232] In a particular embodiment, the symbol * indicates the
attachment point to the nitrogen in Z.sub.2. In another embodiment,
where Z.sub.2 is a bond, the symbol * indicates the attachment
point to the nitrogen of the neighbouring Z element.
[0233] The term "bond" as used in the context of Formula I means a
covalent bond. When a component of Formula I (Z.sub.1--Z.sub.10) is
defined as a bond, it is equivalent to a formula I wherein said
component is absent.
[0234] The indication herein below that any of Z.sub.2--Z.sub.10 is
a bond may also be read as any of Z.sub.2-Z.sub.10 being absent.
Logically "a bond" cannot follow "a bond". The indication "a bond"
here thus means that the previous Z element is covalently linked to
the next Z element that is not "a bond" (or absent).
[0235] The linker elements Z.sub.2--Z.sub.10 are selected from
chemical moieties that are capable of forming amide bounds,
including amino acid like moieties, such as Glu, .gamma.Glu (also
termed gamma) Glu or gGlu and defined by
*--NH--CH--(COOH)--CH.sub.2--CH.sub.2--CO--*), Gly, Ser, Ala, Thr,
Ado, Aeep, Aeeep and TtdSuc and further moieties defined below.
[0236] Z.sub.2 is selected from
*--NH--SO.sub.2--(CH.sub.2).sub.3--CO--* or Chem. 7:
##STR00013## *--NH--CH.sub.2--(C.sub.6H.sub.10)--CO--* or Chem.
8:
##STR00014##
and
[0237] a bond.
[0238] Z.sub.3 is selected from .gamma.Glu, Glu, or a bond.
[0239] Z.sub.3 is selected from .gamma.Glu, Glu, or a bond when
Z.sub.2 is Chem. 7 or Chem. 7b.
[0240] Z.sub.3 is selected from .gamma.Glu, Glu, or a bond,
provided that Z.sub.3 is selected from .gamma.Glu, Glu when Z.sub.2
is Chem. 8.
[0241] Z.sub.3 is selected from .gamma.Glu and Glu when Z.sub.2 is
Chem. 8.
[0242] Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8, Z.sub.9 are
selected, independently of each other, from Glu, .gamma.Glu, Gly,
Ser, Ala, Thr, Ado, Aeep, Aeeep, TtdSuc and a bond.
[0243] Glu, Gly, Ser, Ala, Thr are amino acid residues as well
known in the art.
[0244] .gamma.Glu is of formula Chem. 9:
*--NH--CH(COOH)--(CH.sub.2).sub.2--CO--* which is the same as Chem.
9b:
##STR00015##
and may also be referred to as gGlu.
[0245] TtdSuc is of formula Chem. 10:
[0246]
*--NH--(CH.sub.2).sub.3--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.20--
-(CH.sub.2).sub.3--NHCO* or
[0247]
*--NH--CH.sub.2CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OC-
H.sub.2CH.sub.2CH.sub.2NHCO* which is the same as
##STR00016##
[0248] Ado is of formula Chem. 11:
*--NH--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--CH.sub.2--CO--*
may also be referred to as 8-amino-3,6-dioxaoctanoic acid and which
is the same as
##STR00017##
[0249] Aeep is of formula Chem. 12:
*NH--CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2CO*, which
may also be referred to as
##STR00018##
[0250] Aeeep is of formula Chem. 13:
*NH--CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2C-
O*, which may also be referred to as
##STR00019##
[0251] Z.sub.10 is selected from a bond, and Chem. 14:
*--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2--*, which may also be
referred to as
##STR00020##
[0252] In a particular embodiment, when Z.sub.10 is Chem. 14, the
substituent is attached to the N-terminal amino group of said
peptide.
[0253] In another embodiment, when Z.sub.10 is a bond, said
substituent is attached to the epsilon position of a Lys residue
present in said peptide or to the N-terminal amino acid residue of
said peptide.
[0254] In one embodiment the derivative comprises two substituents.
In one such embodiment the two substituents are identical. In one
such embodiment the two substituents are different. In one
embodiment the two substituents are attached to nitrogen atoms of
the EGF(A) peptide analogue. In one embodiment the two substituents
are attached to amino groups of the EGF(A) peptide analogue. In one
embodiment the two substituents are attached to the N-terminal
amino acid EGF(A) and to a Lys residue of the EGF(A) peptide
analogue. In one embodiment one substituent is attached the
alpha-nitrogen of the N-terminal amino acid residue of the EGF(A)
peptide analogue and one substituent is attach to a Lys residue of
the EGF(A) peptide analogue. In one embodiment two substituents are
attached to the N-terminal amino acid of the EGF(A) peptide
analogue. In one embodiment the two substituents are attached to
different Lys residues of the EGF(A) peptide analogue. In one
embodiment the two substituents are attached to the
epsilon-nitrogen's of different Lys residues in the EGF(A) peptide
analogue.
[0255] In one embodiment where two substituents are present,
Z.sub.10 is Chem. 14 in one substituent which is attached to the
N-terminal amino group of a peptide analogue and Z.sub.10 is a bond
in the other substituent which is attached to the epsilon position
of a Lys residue present in said peptide analogue.
[0256] In another embodiment where two substituents are present,
Z.sub.10 is a bond in one substituent which is attached to the
N-terminal amino group of a peptide analogue and Z.sub.10 is a bond
in the other substituent which is attached to the epsilon position
of a Lys residue present in said peptide analogue.
[0257] In another embodiment where two substituents are present,
Z.sub.10 is a bond in both substituents and each of the two
substituents is attached to the epsilon position of different Lys
residues present in a peptide analogue.
[0258] In a particular embodiment, the derivatives of the invention
may be prepared from a EGF(A) peptide analogue by covalent
attachment of one or two substituent(s).
[0259] In a particular embodiment, the two substituents are of
Formula I:
Z.sub.1--Z.sub.2--Z.sub.3--Z.sub.4--Z.sub.5--Z.sub.6-Z.sub.7--Z.sub.8--Z.-
sub.9-Z.sub.10-- [I]. Z.sub.1 to Z.sub.10 are as defined above. In
a particular embodiment, the two substituents are of formula I and
are identical, meaning that selected Z.sub.1 to Z.sub.10 are the
same in both substituents. In another embodiment, the two
substituents are of formula I and are different, meaning that one
or more of selected Z.sub.1 to Z.sub.10 are different between one
substituent and the other.
Specific Substituents
[0260] As seen above various substituents can be prepared by the
persons skilled in the art. The substituents include in the present
application are thus not to be considered limiting to the
invention.
[0261] In one embodiment the one or two substituent(s) is/are
selected from the group of substituents consisting of: [0262]
HOOC--(CH.sub.2).sub.18--CO-gGlu-2xADO [0263]
HOOC--(CH.sub.2).sub.18--CO--NH--CH.sub.2--(C.sub.6H.sub.10)--CO-gGlu-2xA-
DO [0264] HOOC--(CH.sub.2).sub.16--CO-gGlu-2xADO [0265]
HOOC--(CH.sub.2).sub.16--CO-gGlu-2xADO--NH--CH.sub.2--(C.sub.6H.sub.4)--C-
H.sub.2 [0266] HOOC--(CH.sub.2).sub.16--CO-gGlu [0267]
HOOC--(CH.sub.2).sub.16--CO--NH--CH.sub.2--(C.sub.6H.sub.10)--CO-gGlu-2xA-
DO [0268] HOOC--(CH.sub.2).sub.14--CO-gGlu-2xADO [0269]
HOOC--(CH.sub.2).sub.14--CO-gGlu- [0270]
HOOC--(CH.sub.2).sub.14--CO-gGlu-2xADO- [0271]
HOOC--(CH.sub.2).sub.12--CO-gGlu-2xADO [0272]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-2xADO
[0273]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-3xADO
[0274] 4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu
[0275] 4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO--2xGlu
[0276]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-3xGly
[0277]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO--2xgGlu-2xADO
[0278]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-TtdSuc
[0279] 4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9-.phi. [0280]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-4xADO
[0281]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO--NH--CH.sub.2--(C.sub.-
6H.sub.10)--CO-gGlu-2xADO [0282]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9--CO-gGlu-2xADO [0283]
3-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9--CO-gGlu-2xADO [0284]
3-HO-Isoxazole-(CH.sub.2).sub.12--CO-gGlu-2xADO [0285]
HOS(O).sub.2--(CH.sub.2).sub.15--CO-gGlu-2xADO--NH--CH.sub.2--(C.sub.6H.s-
ub.4)--CH.sub.2 [0286]
HOS(O).sub.2--(CH.sub.2).sub.13--CO-gGlu-2xADO [0287]
Tetrazolyl-(CH.sub.2).sub.15--CO--NH--SO.sub.2--(CH.sub.2).sub.3---
CO-ADO-ADO-NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2 [0288]
Tetrazolyl-(CH.sub.2).sub.12--CO-gGlu-2xADO [0289]
Tetrazolyl-(CH.sub.2).sub.15--CO-gGlu-2xADO and [0290]
MeS(O).sub.2NH(CO)NH--(CH.sub.2).sub.12--CO-gGlu-2xADO.
[0291] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 1: HOOC--(CH.sub.2).sub.n--CO--*, wherein n is 16;
Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; two of Z.sub.4, Z.sub.5,
Z.sub.6, Z.sub.7, Z.sub.8, Z.sub.9 are Ado and the remaining four
are bonds; Z.sub.10 is Chem. 14:
*--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2--*.
[0292] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 1: HOOC--(CH.sub.2).sub.n--CO--*, wherein n is 16;
Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; two of Z.sub.4, Z.sub.5,
Z.sub.6, Z.sub.7, Z.sub.8, and Z.sub.9 are Ado and the remaining
four are bonds; Z.sub.10 is a bond.
[0293] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 1: HOOC--(CH.sub.2).sub.n--CO--*, wherein n is 14
or 16; Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; and all of
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are bonds;
Z.sub.10 is a bond.
[0294] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 1: HOOC--(CH.sub.2).sub.n--CO--*, wherein n is 16
or 18; Z.sub.2 is Chem 8 (Trx); Z.sub.3 is .gamma.Glu; two of
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado and
the remaining four are bonds; Z.sub.10 is a bond.
[0295] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem 2: Tetrazolyl-(CH.sub.2).sub.n--CO--*, wherein n is
15; Z.sub.2 is Chem 7 (sulfonimide); Z.sub.3 is a bond; two of
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado and
the remaining four are bonds; Z.sub.10 is Chem. 14:
*--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2--*.
[0296] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem 2: Tetrazolyl-(CH.sub.2).sub.n--CO--*, wherein n is
15; Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; two of Z.sub.4,
Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado and the
remaining four are bonds; Z.sub.10 is a bond.
[0297] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem 2: Tetrazolyl-(CH.sub.2).sub.n--CO--*, wherein n is
12; Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; two of Z.sub.4,
Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado and the
remaining four are bonds; Z.sub.10 is a bond.
[0298] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, wherein m is
10; Z.sub.2 is a bond; Z.sub.3 is a bond; and all off Z.sub.4,
Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are bonds; Z.sub.10
is a bond.
[0299] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, wherein m is
10; Z.sub.2 is a bond; Z.sub.3 is a .gamma.Glu; and all off
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are bonds;
Z.sub.10 is a bond.
[0300] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, wherein m is
10; Z.sub.2 is a bond; Z.sub.3 is a .gamma.Glu; and one off
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 is a
.gamma.Glu and the remaining five are bonds; Z.sub.10 is a
bond.
[0301] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, wherein m is
10; Z.sub.2 is a bond; Z.sub.3 is a .gamma.Glu; and one off
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 is a
.gamma.Glu and two are Ado and the remaining three are bonds;
Z.sub.10 is a bond.
[0302] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, wherein m is
10; Z.sub.2 is a bond; Z.sub.3 is a .gamma.Glu; and three off
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Gly and
the remaining three are bonds; Z.sub.10 is a bond.
[0303] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, wherein m is
10; Z.sub.2 is a bond; Z.sub.3 is a .gamma.Glu; and two off
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado and
the remaining four are bonds; Z.sub.10 is a bond.
[0304] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, wherein m is
10; Z.sub.2 is a bond; Z.sub.3 is a .gamma.Glu; and three off
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado and
the remaining three are bonds; Z.sub.10 is a bond.
[0305] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, wherein m is
10; Z.sub.2 is a bond; Z.sub.3 is a .gamma.Glu; and four off
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado and
the remaining two are bonds; Z.sub.10 is a bond.
[0306] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, wherein m is
10; Z.sub.2 is a bond; Z.sub.3 is a .gamma.Glu; and one off
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 is a TtdSuc
and the remaining five are bonds; Z.sub.10 is a bond.
[0307] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, wherein m is
10; Z.sub.2 is Chem 8 (Trx); Z.sub.3 is a .gamma.Glu; and two off
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado and
the remaining four are bonds; Z.sub.10 is a bond.
[0308] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, wherein m is 9;
Z.sub.2 is a bond; Z.sub.3 is a .gamma.Glu; and one off Z.sub.4,
Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 is a TtdSuc and the
remaining five are bonds; Z.sub.10 is a bond.
[0309] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, wherein m is
10; Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; two of Z.sub.4,
Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and
[0310] Z.sub.9 are Ado, the remaining four are bonds; Z.sub.10 is a
bond.
[0311] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, wherein m is
10; Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; two of Z.sub.4,
Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado, the
remaining four are bonds; Z.sub.10 is a bond.
[0312] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 4: HO--S(O).sub.2--(CH.sub.2).sub.n--CO--*,
wherein n is 15; Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; two of
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado,
the remaining four are bonds; Z.sub.10 is a bond.
[0313] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 4: HO--S(O).sub.2--(CH.sub.2).sub.n--CO--*,
wherein n is 15; Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; two of
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado,
the remaining four are bonds; Z.sub.10 is Chem. 14:
*--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2--*.
[0314] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 5: MeS(O).sub.2NH(CO)NH--(CH.sub.2).sub.n--CO--*,
wherein n is 12; Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; two of
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado,
the remaining four are bonds; Z.sub.10 is a bond.
[0315] In one embodiment, the substituent is of Formula I wherein
Z.sub.1 is Chem. 6: 3-0H-Isoxazole-(CH.sub.2).sub.12--CO--*,
wherein n is 12; Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; two of
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado,
the remaining four are bonds; Z.sub.10 is a bond.
Specific Substituent Combinations:
[0316] In one embodiment, the compound of the invention comprises
or has two substituents of Formula I wherein Z.sub.1 is Chem. 1:
HOOC--(CH.sub.2).sub.n--CO--*, wherein n is 16; Z.sub.2 is a bond;
Z.sub.3 is .gamma.Glu; two of Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7,
Z.sub.8, Z.sub.9 are Ado and the remaining four are bonds; Z.sub.10
is a bond.
[0317] In one embodiment, the compound of the invention comprises
or has two substituents of Formula I wherein Z.sub.1 is Chem. 1:
HOOC--(CH.sub.2).sub.n--CO--*, wherein n is 14; Z.sub.2 is a bond;
Z.sub.3 is .gamma.Glu; two of Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7,
Z.sub.8, Z.sub.9 are Ado and the remaining four are bonds; Z.sub.10
is a bond.
[0318] In one embodiment, the compound of the invention comprises
or has two substituents of Formula I wherein Z.sub.1 is Chem. 1:
HOOC--(CH.sub.2).sub.n--CO--*, wherein n is 14; Z.sub.2 is a bond;
Z.sub.3 is .gamma.Glu; all four of Z.sub.4, Z.sub.5, Z.sub.6,
Z.sub.7, Z.sub.8, Z.sub.9 are bonds; Z.sub.10 is a bond.
[0319] In one embodiment, the compound of the invention comprises
or has two substituents of Formula I wherein Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, wherein m is
10; Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; two of Z.sub.4,
Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado, the
remaining four are bonds; Z.sub.10 is a bond.
[0320] In one embodiment, the compound of the invention comprises
or has two substituents, one being of Formula I wherein Z.sub.1 is
Chem. 1: HOOC--(CH.sub.2).sub.n--CO--*, wherein n is 16; Z.sub.2 is
a bond; Z.sub.3 is .gamma.Glu; two of Z.sub.4, Z.sub.5, Z.sub.6,
Z.sub.7, Z.sub.8, Z.sub.9 are Ado and the remaining four are bonds;
Z.sub.10 is Chem. 14:
*--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2--*; the other
substituent being of Formula I wherein Z.sub.1 is Chem. 1:
HOOC--(CH.sub.2).sub.n--CO--*, wherein n is 16; Z.sub.2 is a bond;
Z.sub.3 is .gamma.Glu; two of Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7,
Z.sub.8, Z.sub.9 are Ado and the remaining four are bonds; Z.sub.10
is a bond.
[0321] In one embodiment, the compound of the invention comprises
or has two substituents, one being of Formula I wherein Z.sub.1 is
Chem. 1: HOOC--(CH.sub.2).sub.n--CO--*, wherein n is 16; Z.sub.2 is
a bond; Z.sub.3 is .gamma.Glu; two of Z.sub.4, Z.sub.5, Z.sub.6,
Z.sub.7, Z.sub.8, Z.sub.9 are Ado and the remaining four are bonds;
Z.sub.10 is Chem. 14:
*--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2--*; the other
substituent being of Formula I wherein Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, wherein m is
10; Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; two of Z.sub.4,
Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado, the
remaining four are bonds; Z.sub.10 is a bond.
[0322] In one embodiment, the compound of the invention comprises
or has two substituents, one being of Formula I wherein Z.sub.1 is
Chem. 1: HOOC--(CH.sub.2).sub.n--CO--*, wherein n is 16; Z.sub.2 is
a bond; Z.sub.3 is .gamma.Glu; two of Z.sub.4, Z.sub.5, Z.sub.6,
Z.sub.7, Z.sub.8, Z.sub.9 are Ado and the remaining four are bonds;
Z.sub.10 is a bond; the other substituent being of Formula I
wherein Z.sub.1 is Chem. 3:
HOOC--(C.sub.6H.sub.4.)--O--(CH.sub.2).sub.m--CO--*, wherein m is
10; Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; two of Z.sub.4,
Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado, the
remaining four are bonds; Z.sub.10 is a bond.
[0323] In one embodiment, the compound of the invention comprises
or has two substituents, one being of Formula I wherein Z.sub.1 is
Chem. 1: HOOC--(CH.sub.2).sub.n--CO--*, wherein n is 16; Z.sub.2 is
a bond; Z.sub.3 is .gamma.Glu; two of Z.sub.4, Z.sub.5, Z.sub.6,
Z.sub.7, Z.sub.8, Z.sub.9 are Ado and the remaining four are bonds;
Z.sub.10 is a bond; and the other substituent is of formula I
wherein Z.sub.1 is Chem. 4: HOS(O).sub.2--(CH.sub.2).sub.n--CO--*,
wherein m is 15; Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; two of
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado,
the remaining four are bonds; Z.sub.10 is Chem. 14:
*--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2--*.
[0324] In one embodiment, the compound of the invention comprises
or has two substituents, one being of Formula I wherein Z.sub.1 is
Chem. 3: HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*,
wherein m is 10; Z.sub.2 is a bond; Z.sub.3 is .gamma.Glu; two of
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8 and Z.sub.9 are Ado,
the remaining four are bonds; Z.sub.10 is a bond; the other
substituent being of Formula I wherein Z.sub.1 is Chem. 4:
HOS(O).sub.2--(CH.sub.2).sub.n--CO--*, wherein m is 15; Z.sub.2 is
a bond; Z.sub.3 is .gamma.Glu; two of Z.sub.4, Z.sub.5, Z.sub.6,
Z.sub.7, Z.sub.8 and Z.sub.9 are Ado, the remaining four are bonds;
Z.sub.10 is Chem. 14:
*--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2--*.
Peptide and Attachment Site
[0325] An EGF(A) derivative or compound according to the invention
comprises a EGF(A) peptide analogue of the EGF(A) domain of LDL-R
as defined by SEQ ID NO.: 1. Such peptide sequence have been
described in details herein above and the peptide of the derivative
or compound of the invention may be described and defined by
identical terms. The EGF(A) derivative or compound further has at
least one substituent as described herein above which is linked to
the peptide sequence.
[0326] In the compounds of the invention, the substituent is
covalently attached to the peptide, meaning to one amino acid
residue of the peptide sequence.
[0327] In one embodiment the EGF(A) derivative of the invention,
comprise a substituent which is not attached to any one of the
following positions: 295, 296, 298, 301, 302 and 307.
[0328] In a further embodiment the substituent is not attached to
any one of the following positions: 295, 296, 298, 301, 302, 307
and 310. In further such embodiments, it is also not attached to
any one of the following positions: 299 and 320.
[0329] In a particular embodiment a substituent is attached via any
position from 292 to 333 except in any or the positions 297, 304,
308, 317, 319 and 331.
[0330] In a particular embodiment a substituent attached via any
position from 292 to 333 except in any of the positions 297, 298,
301, 302, 304, 307, 308, 317, 319 and 331.
[0331] In a particular embodiment a substituent attached via any
position from 292 to 333 except in any of the positions 295, 296,
297, 298, 301, 302, 304, 307, 308, 317, 319 and 331. In a
particular embodiment a substituent attached via in any position
from 292 to 333 except in any of the positions 295, 296, 297, 298,
301, 302, 304, 307, 308, 310, 317, 319, 320 and 331. In a
particular embodiment a substituent attached via any position from
292 to 333 except in any of the positions 295, 296, 297, 298, 301,
302, 304, 307, 308, 309, 310, 317, 319, 320 and 331.
[0332] In one embodiment, the substituent(s) is/are attached to any
one or two of the positions 292, 293, 294, 299, 300, 303, 305, 306,
309, 311, 312, 313, 314, 315, 316, 318, 320, 321, 322, 323, 324,
325, 326, 327, 328, 329, 330, 332 and 333 of the EGF(A) peptide
analogue.
[0333] In one embodiment, the substitution(s) is/are attached to
any one or two of the positions 292, 293, 294, 300, 303, 305, 306,
309, 311, 312, 313, 314, 315, 316, 318, 321, 322, 323, 324, 325,
326, 327, 328, 329, 330, 332 and 333 of the EGF(A) peptide
analogue.
[0334] In one embodiment, the substitution(s) is/are attached to
any one or two of the positions 292, 293, 294, 300, 303, 305, 306,
311, 312, 313, 314, 315, 316, 318, 321, 322, 323, 324, 325, 326,
327, 328, 329, 330, 332 and 333 of the EGF(A) peptide analogue.
[0335] In one embodiment, the substituent is attached to the
N-terminal amino acid of the peptide sequence. In a particular
embodiment, the N-terminal amino acid is Gly. In a particular
embodiment, the N-terminal amino acid is 293Gly. In a particular
embodiment, the N-terminal amino acid is 293Lys. In a particular
embodiment, the N-terminal amino acid is 292Lys. It may also be a
Lys or a Gly or another amino acid residue in the N-terminal
position which may be 293 or any position further down from the
N-terminus, such as 294Thr, 294Gly or 294Lys or 295Asn. In a
particular embodiment, the substituent is attached to the
alpha-nitrogen of the N-terminal amino acid residue of the peptide
analogue. In another embodiment, if the N-terminal amino acid
residue is Lys, the substituent may be covalently linked to the
alpha-nitrogen or to the epsilon amino group of the lysine
residue.
[0336] In a particular embodiment, a substituent is attached to the
.epsilon.-amino group of a Lys residue present in the peptide.
[0337] In another embodiment, a substituent is attached to a Lys in
C-terminal position which may be position 332, 333 or any position
further towards the C-terminus.
[0338] In embodiments wherein the peptides of the invention
comprise an elongation, either in N-terminal or C-terminal, the
substituent(s) may be attached to an amino acid residue of said
elongation(s). In the presence of a N-terminal elongation, a
substituent may be attached to the N-terminal amino acid of said
elongation or to a Lys present within the elongation sequence. In
the presence of a C-terminal elongation, a substituent may be
attached to a Lys residue in C-terminal position or to a Lys
present within the elongation sequence.
[0339] In yet another embodiment, the substituent is attached to an
amino acid present in the peptide sequence. In a particular
embodiment, the substituent is linked to a lysine residue present
in the peptide. In a particular embodiment, the substituent is
linked to the epsilon amino group of a lysine residue present in
the peptide. The lysine residue to which the substituent is linked
may be located in any position of the LDL-R(293-332) EGF(A)
analogue including the N-terminal position or C-terminal position
of the peptide, any position within or at the N-terminal end
residue of a N-terminal elongation if present, any position within
or at the C-terminal end residue of a C-terminal elongation if
present.
[0340] As described herein above the EGF(A) peptide analogue may
have one or more Lys residues; and those residues are useful for
attachment of substituents.
[0341] In a particular embodiment, the lysine(s) to which the
substituent(s) is/are linked is selected from the group of: 292Lys,
293Lys, 294Lys, 299Lys, 300Lys, 303Lys, 305Lys, 306Lys, 309Lys,
311Lys, 312Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys,
321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys,
329Lys, 330Lys, 332Lys and 333Lys.
[0342] In a particular embodiment, the lysine(s) to which the
substituent(s) is/are linked is selected from 293Lys, 294Lys,
295Lys, 296Lys, 298Lys, 299Lys, 301Lys, 302Lys, 303Lys, 305Lys,
306Lys, 307Lys, 309Lys, 310Lys, 311Lys, 312Lys, 313Lys, 314Lys,
315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys,
325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and
333Lys.
[0343] In a particular embodiment, the lysine(s) to which the
substituent(s) is/are linked is selected from 293Lys, 294Lys,
300Lys, 303Lys, 306Lys, 309Lys, 311Lys, 312Lys, 313Lys, 314Lys,
315Lys, 316Lys, 318Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys,
326Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys.
[0344] In another embodiment, the lysine(s) to which the
substituent(s) is/are linked is selected from 293Lys, 294Lys,
298Lys, 299Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 312Lys,
313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys,
323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys,
332Lys and 333Lys.
[0345] In another embodiment, the lysine(s) to which the
substituent(s) is/are linked is selected from: 292Lys, 293Lys,
294Lys, 299Lys, 300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys,
313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys,
323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys,
332Lys and 333Lys.
[0346] In another embodiment, the lysine(s) to which the
substituent(s) is/are linked is selected from: 292Lys, 293Lys,
294Lys, 300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 313Lys,
314Lys, 316Lys, 318Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys,
326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys.
[0347] In another embodiment, the lysine(s) to which the
substituent(s) is/are linked is selected from: 293Lys, 294Lys,
300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 313Lys, 314Lys,
316Lys, 318Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys,
327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys.
[0348] In another embodiment, the lysine(s) to which the
substituent(s) is/are linked is selected from: 293Lys, 294Lys,
300Lys, 303Lys, 305Lys, 306Lys, 311Lys, 313Lys, 314Lys, 316Lys,
318Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys,
328Lys, 329Lys, 330Lys, 332Lys and 333Lys.
[0349] In embodiments where the substituent is attached to a
C-terminal elongation, the lysine to which the substituent is
linked may be selected from anyone of 333Lys to 242Lys position
and/or to anyone of 333Lys to 383Lys position.
[0350] In embodiments where compounds of the invention have two
substituents, the substituents may be linked independently of each
other as defined above, meaning that either one may be attached to
the N-terminal amino acid of the peptide, to the C-terminal amino
acid of the peptide, or to an amino acid within the amino acid
sequence of the peptide.
[0351] In embodiments where a Lys is present in N-terminal
position, two substituents may be both linked to the N-terminal Lys
of the peptide. One may be linked to the N-terminal alpha-amine of
said Lys while the other may be linked to the epsilon nitrogen of
said Lys. When two substituents are present, one may be linked to
the N-terminal amino acid of the peptide while the other
substituent is linked to an amino acid, such as a Lys, within the
peptide. Alternatively, one substituent may be linked to a Lys in
position C-terminal of the peptide while the other substituent is
linked to an amino acid, such as a Lys, in the peptide.
Alternatively, one substituent may be linked to an amino acid
residue, such as a Lys, within the peptide, including elongations,
the other substituent being linked to another amino acid residue,
such as a Lys, within the peptide, including elongations.
[0352] In an embodiment, the compounds of the invention have one
substituent, said substituent is linked to the peptide at the
N-terminal; or said substituent is linked to the peptide in
position 292Lys; or said substituent is linked to the peptide in
position 293Lys, or said substituent is linked to the peptide in
position 299Lys; or said substituent is linked to the peptide in
position 300Lys; or said substituent is linked to the peptide in
position 309Lys; or said substituent is linked to the peptide in
position 311Lys; or said substituent is linked to the peptide in
position 312Lys; or said substituent is linked to the peptide in
position 313Lys; or said substituent is linked to the peptide in
position 314Lys; or said substituent is linked to the peptide in
position 315Lys; or said substituent is linked to the peptide in
position 316Lys; or said substituent is linked to the peptide in
position 318Lys; or said substituent is linked to the peptide in
position 320Lys; or said substituent is linked to the peptide in
position 321Lys; or said substituent is linked to the peptide in
position 322Lys; or said substituent is linked to the peptide in
position 323Lys; or said substituent is linked to the peptide in
position 324Lys; or said substituent is linked to the peptide in
position 325Lys; or said substituent is linked to the peptide in
position 326Lys; or said substituent is linked to the peptide in
position 328Lys; or said substituent is linked to the peptide in
position 329Lys; or said substituent is linked to the peptide in
position 330Lys; or said substituent is linked to the peptide in
position 332Lys; or said substituent is linked to the peptide in
position 333Lys.
[0353] In an embodiment where the derivative of the invention have
two substituents, said substituents may be linked to the peptide
via the N-terminal and any of the above mention Lys positions, such
as 293Lys, 309Lys, 313Lys, 324Lys, 328Lys, 330Lys, 332Lys and
333Lys.
[0354] In further embodiments where the derivative comprises two
substituents, they may be linked to two different Lys residues,
such as any of the following pairs of Lys residues
TABLE-US-00003 i. 293K and 294K xiv. 313K and 321K ii. 293K and
312K xv. 313K and 324K iii. 293K and 333K xvi. 313K and 328K iv.
309K and 313K xvii. 313K and 332K v. 309K and 324K xviii. 313K and
333K vi. 309K and 328K xix. 314K and 333K vii. 309K and 332K xx.
321K and 332K viii. 309K and 333K xxi. 321K and 333K ix. 311K and
313K xxii. 324K and 333K x. 312K and 333K xxiii. 324K and 328K xi.
312K and 313K xxiv. 328K and 333K xii. 312K and 314K xxv. 330K and
333K and xiii. 313K and 314K xxvi. 332K and 333K.
[0355] In one embodiment the two substituents are attached via
333Lys and a Lys selected from 293Lys, 309Lys, 312Lys, 313Lys,
314Lys, 321Lys, 324Lys, 328Lys, 330Lys and 332Lys.
[0356] In one embodiment the two substituents are attached via
333Lys and a Lys selected from 312Lys, 313Lys, 314Lys, 321Lys,
324Lys, 328Lys and 330Lys.
[0357] In one embodiment the two substituents are attached via
333Lys and a Lys selected from 313Lys, 324Lys and 328Lys.
[0358] As described above the peptide may have one or more amino
acid substitutions which may be combined with specific amino acid
residues in specific positions as described herein. Such specific
amino acid residues may be wt amino acid residues that should be
maintained, such as the cysteines which may in a series of
preferred embodiments e.g. in combination with other features
described herein, be present in the peptide analogue. In such
embodiments the peptide analogue comprises three disulphide bridges
in positions 297Cys-308Cys, 304Cys-317Cys and 319Cys-331Cys. In a
further example of such embodiments the peptide analogue of a
peptide derivative comprises three disulphide bridges in positions
297Cys-308Cys, 304Cys-317Cys and 319Cys-331Cys and at least one
substituent, wherein the substituent(s) is not attached to a
positions selected from 295, 296, 298, 301, 302 and 307 of said
peptide analogue, The skilled person will understand that
combinations of peptide sequence information may be combined with
information on position and identity of the substituent to define
various specific embodiments of the present invention.
[0359] In an embodiment, the peptide analogue comprises no Lys in
other positions than the positions to which a substituent is
linked.
[0360] In an embodiment, the compounds of the invention have one
substituent, said substituent is linked either in position
N-terminal or to a Lys in any position, and the peptide analogue
comprises no Lys in all other positions. In an embodiment, the
compounds of the invention have one substituent, said substituent
is linked to a Lys in any position other than position 312, and the
peptide analogue comprises an Arg in position 312Arg.
[0361] In an embodiment, the compounds of the invention have two
substituents, and the peptide analogue comprises no Lys in
positions other than positions to which the substituents are
linked.
[0362] In one embodiment the EGF(A) derivative according to the
invention is selected from the group of EGF(A) derivative
consisting of: Examples 1-47, 51-102 and 106-159.
[0363] In further embodiments the EGF(A) derivative according to
the invention is individually selected from the group of EGF(A)
derivative consisting of: Examples 1-47, 51-102 and 106-159.
[0364] In one embodiment the EGF(A) derivative according to the
invention is selected from the group of EGF(A) derivative
consisting of: Examples 1-44, 46-47, 51-55, 57, 60-64, 66-69,
71-102 and 106-159.
[0365] In one embodiment the EGF(A) derivative according to the
invention is selected from the group of EGF(A) derivative
consisting of: Examples 31, 95, 128, 133, 143, 144, 150, 151, 152
and 153.
Pharmaceutical Composition
[0366] The invention also relates to pharmaceutical compositions
comprising a compound of the invention, including e.g. a peptide
analogue of the invention, or a pharmaceutically acceptable salt,
amide, or ester thereof, and a pharmaceutically acceptable
excipient. Such compositions may be prepared as is known in the
art.
[0367] The term "excipient" broadly refers to any component other
than the active therapeutic ingredient(s). The excipient may be an
inert substance, an inactive substance, and/or a not medicinally
active substance. The excipient may serve various purposes, e.g. as
a carrier, vehicle, diluent, tablet aid, and/or to improve
administration, and/or absorption of the active substance.
Non-limiting examples of excipients are: solvents, diluents,
buffers, preservatives, tonicity regulating agents, chelating
agents, and stabilisers. The formulation of pharmaceutically active
ingredients with various excipients is known in the art, see e.g.
Remington: The Science and Practice of Pharmacy (e.g. 19th edition
(1995), and any later editions).
[0368] A composition of the invention may be in the form of a
liquid formulation, i.e. aqueous formulation comprising water. A
liquid formulation may be a solution, or a suspension.
Alternatively, it may be a solid formulation, e.g. a freeze-dried
or spray-dried composition.
[0369] A pharmaceutical composition of the invention may further
comprise a second active ingredient, such as a therapeutic agent,
which may simplify administration in case of combination
treatments.
[0370] A composition of the invention may be an oral composition,
and the route of administration is per oral. The compounds of the
invention and in particular the protracted compounds, i.e. the
derivative compounds, are suitable for oral administration. The
peptides and compounds of the invention may according to the
invention be comprised by an oral formulation i.e. a composition
suited for oral administration and capable of providing a suitable
level of bioavailability. Oral formulations technologies know in
the art may be used. This includes use of salts of
N-(8-(2-hydroxybenzoyl)amino)caprylic acid, in particular sodium
N-(8-(2-hydroxybenzoyl)amino)caprylate (SNAC) as described in
WO96/30036 and WO2008/028859 and GIPET formulations including
sodium caprate such as described in EP1154761 and U.S. Pat. No.
8,053,429.
[0371] In order to provide compounds for oral compositions the
inventors confirmed that a EGF(A) peptide derivatives according to
the invention display gastrointestinal absorption in rats (Table
10).
[0372] Alternatively, a composition of the invention may be for
parenteral administration, e.g. performed by subcutaneous,
intramuscular, intraperitoneal, or intravenous injection.
Naturally, compounds aimed for subcutaneous administration may not
need to display gastrointestinal absorption while other features
such as high stability in liquid formulation may be desired.
Combination Treatment
[0373] Treatment with a EGF(A) peptide analogue or derivative
thereof according to the present invention may also be combined
with one or more additional pharmacologically active substances,
e.g. selected from anti-diabetic agents, anti-obesity agents,
appetite regulating agents, antihypertensive agents, agents for the
treatment and/or prevention of complications resulting from or
associated with diabetes and agents for the treatment and/or
prevention of complications and disorders resulting from or
associated with obesity.
[0374] Examples of these pharmacologically active substances are:
GLP-1 receptor agonists, insulin, DPP-IV (dipeptidyl peptidase-IV)
inhibitors, amylin agonists and leptin receptor agonists.
Particular examples of such active substances are the GLP-1
receptor agonists liraglutide and semaglutide and insuling
degludec.
Pharmaceutical Indications
[0375] In one aspect the invention relates to the use of an EGF(A)
peptide analogue or an EGF(A) derivative as described herein for
use in the manufacture of a medicament.
[0376] The invention also relates to a compound of the invention,
e.g. a peptide analogue or a derivative according to the invention,
or a pharmaceutical composition thereof for use as a medicament or
in the manufacture of a medicament.
[0377] In an embodiment, a compound of the invention or a
composition thereof may be used for (i) improving lipid parameters,
such as prevention and/or treatment of dyslipidemia, lowering total
serum lipids; lowering LDL-C, increasing HDL; lowering small, dense
LDL; lowering VLDL; lowering triglycerides; lowering cholesterol;
lowering plasma levels of lipoprotein a (Lp(a)); inhibiting
generation of apolipoprotein A (apo(A)); (ii) the prevention and/or
the treatment of cardiovascular diseases, such as cardiac syndrome
X, atherosclerosis, myocardial infarction, coronary heart disease,
reperfusion injury, stroke, cerebral ischemia, an early cardiac or
early cardiovascular disease, left ventricular hypertrophy,
coronary artery disease, hypertension, essential hypertension,
acute hypertensive emergency, cardiomyopathy, heart insufficiency,
exercise intolerance, acute and/or chronic heart failure,
arrhythmia, cardiac dysrhythmia, syncopy, angina pectoris, cardiac
bypass and/or stent reocclusion, intermittent claudication
(atheroschlerosis oblitterens), diastolic dysfunction, and/or
systolic dysfunction; and/or the reduction of blood pressure, such
as reduction of systolic blood pressure; the treatment of
cardiovascular disease.
[0378] The invention also relates to a method for (i) improving
lipid parameters, such as prevention and/or treatment of
dyslipidemia, lowering total serum lipids; increasing HDL-C;
lowering LDL-C, lowering small, dense LDL-C; lowering VLDL-C;
lowering triglycerides; lowering cholesterol; lowering plasma
levels of lipoprotein a (Lp(a)); inhibiting generation of
apolipoprotein A (apo(A)); (ii) prevention and/or treatment of
cardiovascular diseases, such as cardiac syndrome X,
atherosclerosis, myocardial infarction, coronary heart disease,
reperfusion injury, stroke, cerebral ischemia, an early cardiac or
early cardiovascular disease, left ventricular hypertrophy,
coronary artery disease, hypertension, essential hypertension,
acute hypertensive emergency, cardiomyopathy, heart insufficiency,
exercise intolerance, acute and/or chronic heart failure,
arrhythmia, cardiac dysrhythmia, syncopy, angina pectoris, cardiac
bypass and/or stent reocclusion, intermittent claudication
(atheroschlerosis oblitterens), diastolic dysfunction, and/or
systolic dysfunction; and/or reduction of blood pressure, such as
reduction of systolic blood pressure; the treatment of
cardiovascular disease; wherein a pharmaceutically active amount of
a compound according to the invention, e.g. a peptide analogue or a
derivative according to the invention, is administered.
[0379] While certain features of the invention have been
illustrated and described herein, many modifications,
substitutions, changes, and equivalents will now occur to those of
ordinary skill in the art. It is, therefore, to be understood that
the appended embodiments are intended to cover all such
modifications and changes as fall within the true spirit of the
invention.
EMBODIMENTS
[0380] 1. A EGF(A) peptide analogue of the EGF(A) domain of the
LDL-R defined by SEQ ID NO 1, wherein the peptide analogue
comprises 301Leu. [0381] 2. The EGF(A) peptide analogue according
to embodiment 1, wherein the peptide analogue comprises the
wild-type cys residues 297Cys, 304Cys, 308Cys, 317Cys, 319Cys and
331Cys. [0382] 3. The EGF(A) peptide analogue according any of the
previous embodiments, wherein the peptide analogue comprises one or
more of the (wild-type) amino acid residues 295Asn, 296Glu, 298Leu,
302Gly and 310Asp. [0383] 4. The EGF(A) peptide analogue according
any of the previous embodiments, wherein the peptide analogue
comprises the residue Asn(N) in position 295. [0384] 5. The EGF(A)
peptide analogue according any of the previous embodiments, wherein
the peptide analogue comprises the residue Glu(E) in position 296.
[0385] 6. The EGF(A) peptide analogue according any of the previous
embodiments, wherein the peptide analogue comprises the residue
Leu(L) in position 298. [0386] 7. The EGF(A) peptide analogue
according any of the previous embodiments, wherein the peptide
analogue comprises the residue Gly(G) in position 302. [0387] 8.
The EGF(A) peptide analogue according any of the previous
embodiments, wherein the peptide analogue comprises the residue
Asp(D) in position 310. [0388] 9. The EGF(A) peptide analogue
according any of the previous embodiments, wherein the peptide
analogue comprises the wild-type residues in positions 295 (Asn/N)
and 310 (Asp/D). [0389] 10. The EGF(A) peptide analogue according
any of the previous embodiments, wherein the peptide has 1-15 amino
acid substitution(s) compared to SEQ ID NO.: 1. [0390] 11. The
EGF(A) peptide analogue according any of the previous embodiments,
wherein the peptide analogue comprises one or more amino acid
substitution(s) in a position(s) selected from the group of
positions: 293, 294, 296, 299, 300, 303, 305, 306, 309, 311, 312,
313, 314, 315, 316, 318, 320, 321, 322, 323, 324, 325, 326, 328,
329, 330, 332. [0391] 12. The EGF(A) peptide analogue according any
of the previous embodiments, wherein the peptide analogue comprises
one or more amino acid substitution(s) in a position(s) selected
from the group of positions: 294, 299, 300, 303, 309, 312, 313,
314, 316, 318, 321, 322, 323, 324, 325, 326, 328, 329, 330, 332.
[0392] 13. The EGF(A) peptide analogue according any of the
previous embodiments, wherein the peptide analogue comprises one or
more further amino acid substitution(s) in a position(s) selected
from the group of positions: 309, 312, 313, 321, 324, 328, 332.
[0393] 14. The EGF(A) peptide analogue according any of the
previous embodiments, wherein the peptide analogue comprises the
amino acid residue Gly(G) or Asn(N) in position 293. [0394] 15. The
EGF(A) peptide analogue according any of the previous embodiments,
wherein the peptide analogue comprises the amino acid residue
Thr(T) or Gly(G) in position 294. [0395] 16. The EGF(A) peptide
analogue according any of the previous embodiments, wherein the
peptide analogue comprises the amino acid residue Asp(D), Gly(G),
Pro(P), Arg(R), Lys(K), Ser(S), Thr(T), Asn(N), Gln(Q), Ala(A),
Ile(I), Leu(L), Met(M), Phe(F), Tyr(Y) or Trp(W) in position 299.
[0396] 17. The EGF(A) peptide analogue according any of the
previous embodiments, wherein the peptide analogue comprises the
amino acid residue Asp(D), Gly(G), Pro (P), Arg(R), Lys(K), Ser(S),
Thr(T), Asn(N), Gln(Q), Ala(A), Ile(I), Leu(L), Met(M), Phe(F),
Tyr(Y) or Trp(W) in position 299. [0397] 18. The EGF(A) peptide
analogue according any of the previous embodiments, wherein the
peptide analogue comprises the amino acid residue Asp(D), Ser (S),
Arg(R), Leu (L), Ala (A), Lys(K) or Tyr(Y) in position 299. [0398]
19. The EGF(A) peptide analogue according any of the previous
embodiments, wherein the peptide analogue comprises the amino acid
residue Asp(D) or Ala(A) in position 299. [0399] 20. The EGF(A)
peptide analogue according any of the previous embodiments, wherein
the peptide analogue comprises the amino acid residue His(H) or
Asn(N) in position 300. [0400] 21. The EGF(A) peptide analogue
according any of the previous embodiments, wherein the peptide
analogue comprises the amino acid residue Val(V), Ser(S), Thr (T)
or Ile (I) in position 307. [0401] 22. The EGF(A) peptide analogue
according any of the previous embodiments, wherein the peptide
analogue comprises the amino acid residue Val(V) or Ile (I) in
position 307. [0402] 23. The EGF(A) peptide analogue according any
of the previous embodiments, wherein the peptide analogue comprises
Ser(S), Thr (T) or Ile (I) in position 307. [0403] 24. The EGF(A)
peptide analogue according any of the previous embodiments, wherein
the peptide analogue comprises Ile (I) in position 307. [0404] 25.
The EGF(A) peptide analogue according any of the previous
embodiments, wherein the peptide analogue comprises the amino acid
residue Asn(N), Glu (E), His (H,) Arg (R), Ser (S) or Lys (K) in
position 309. [0405] 26. The EGF(A) peptide analogue according any
of the previous embodiments, wherein the peptide analogue comprises
the amino acid residue Asn(N), Arg (R), Ser (S) or Lys (K) in
position 309. [0406] 27. The EGF(A) peptide analogue according any
of the previous embodiments, wherein the peptide analogue comprises
the amino acid residue Asn(N), Arg (R) or Ser (S) in position 309.
[0407] 28. The EGF(A) peptide analogue according any of the
previous embodiments, wherein the peptide analogue comprises the
amino acid residue Asn(N) or Arg (R) in position 309. [0408] 29.
The EGF(A) peptide analogue according any of the previous
embodiments, wherein the peptide analogue comprises the amino acid
residue Lys(K) or Arg (R) in position 309. [0409] 30. The EGF(A)
peptide analogue according any of the previous embodiments, wherein
the peptide analogue comprises the amino acid residue Arg (R) in
position 309. [0410] 31. The EGF(A) peptide analogue according any
of the previous embodiments, wherein the peptide analogue comprises
the amino acid residue Lys(K), Glu(E), Asp(D), Gln(Q) or Arg (R) in
position 312. [0411] 32. The EGF(A) peptide analogue according any
of the previous embodiments, wherein the peptide analogue comprises
an amino acid substitution of Lys(K) in position 312. [0412] 33.
The EGF(A) peptide analogue according embodiment 32, wherein 312Lys
is substituted by an amino acid selected from the group consisting
of: 312Gly, 312Pro, 312Asp, 312Glu, 312Arg, 312His, 312Ser, 312Thr,
312Asn, 312Gln, 312Ala, 312Val, 312Ile, 312Leu, 312Met, 312Phe and
312Tyr. [0413] 34. The EGF(A) peptide analogue according embodiment
32, wherein 312Lys is substituted by an amino acid selected from
the group consisting of: 312Asp, 312Glu, 312Thr, 312Asn, 312Ile,
312Phe and 312Tyr. [0414] 35. The EGF(A) peptide analogue according
embodiment 32, wherein 312Lys is substituted by an amino acid
selected from the group consisting of: 312Asp, 312Glu, 312Thr,
312Asn, 312Ile and 312Phe. [0415] 36. The EGF(A) peptide analogue
according embodiment 32, wherein 312Lys is substituted by an amino
acid selected from the group consisting of: 312Glu, 312Asp, 312Gln
and 312Arg. [0416] 37. The EGF(A) peptide analogue according any of
the previous embodiments, wherein the peptide analogue comprises
the amino acid residue Asp(D), Lys (K) or Glu(E) in position 321.
[0417] 38. The EGF(A) peptide analogue according any of the
previous embodiments, wherein the peptide analogue comprises the
amino acid residue Asp(D) or Glu(E) in position 321. [0418] 39. The
EGF(A) peptide analogue according any of the previous embodiments,
wherein the peptide analogue comprises the amino acid residue
Glu(E) in position 321. [0419] 40. The EGF(A) peptide analogue
according any of the previous embodiments, wherein the peptide
analogue comprises the amino acid residue Gln (Q) or Gly (G) in
position 324. [0420] 41. The EGF(A) peptide analogue according any
of the previous embodiments, wherein the peptide analogue comprises
the amino acid residue Arg (R) or His (H) in position 329. [0421]
42. The EGF(A) peptide analogue according any of the previous
embodiments, wherein the peptide does not have a substitution of
299Asp(D) to Glu(E), Val(V) or His (H). [0422] 43. The EGF(A)
peptide analogue according any of the previous embodiments, wherein
the peptide does not have a substitution of 300Asn(N) to Pro(P).
[0423] 44. The EGF(A) peptide analogue according to any of the
previous embodiments, wherein the peptide analogue comprises at
least one Lys amino acid residue. [0424] 45. The EGF(A) peptide
analogue according to any of the previous embodiments, wherein the
peptide analogue comprises a Lys substitution. [0425] 46. The
EGF(A) peptide analogue according to any of the previous
embodiments, wherein the peptide analogue comprises a Lys
substitution and wt Lys in position 312. [0426] 47. The EGF(A)
peptide analogue according to any of the previous embodiments,
wherein the peptide analogue comprises a Lys substitution and a non
Lys amino acid residue in position 312. [0427] 48. The EGF(A)
peptide analogue according to any of the previous embodiments,
wherein the peptide analogue comprises a Lys substitution and a Glu
(E), Asp (D), Gln (Q) or Arg (R) in position 312. [0428] 49. The
EGF(A) peptide analogue according to any of the previous
embodiments, wherein the peptide analogue comprises a Lys
substitution and a Glu(E) in position 312. [0429] 50. The EGF(A)
peptide analogue according to any of the previous embodiments,
wherein the peptide analogue comprises one or more Lys
substitution(s). [0430] 51. The EGF(A) peptide analogue according
to any of the previous embodiments, wherein said peptide has at
least two amino acid substitutions comprising and/or consisting of:
[0431] i. 301Leu and 309Arg [0432] ii. 301Leu, 309Arg, 312Glu
[0433] iii. 301Leu, 307Ile and 309Arg [0434] iv. 301Leu, 307Ile,
309Arg and 312Glu [0435] v. 301Leu, 309Arg and 321Glu [0436] vi.
301Leu, 309Arg, 321Glu and 312Glu [0437] vii. 301Leu, 307Ile,
309Arg and 299Ala [0438] viii. 301Leu, 307Ile, 309Arg, 299Ala and
312Glu [0439] ix. 301Leu and 309Arg and at least one Lys
substitution [0440] x. 301Leu, 309Arg, 312Glu and at least one Lys
substitution [0441] xi. 301Leu, 307Ile and 309Arg and at least one
Lys substitution [0442] xii. 301Leu, 307Ile, 309Arg and 312Glu and
at least one Lys substitution [0443] xiii. 301Leu, 309Arg and
321Glu and at least one Lys substitution [0444] xiv. 301Leu,
309Arg, 321Glu and 312Glu and at least one Lys substitution [0445]
xv. 301Leu, 307Ile, 309Arg and 299Ala and at least one Lys
substitution or [0446] xvi. 301Leu, 307Ile, 309Arg, 299Ala and
312Glu and at least one Lys substitution. [0447] 52. The EGF(A)
peptide analogue according to any of the previous embodiments 1-50,
wherein said peptide has at least two amino acid substitutions
comprising and/or consisting of: [0448] xvii. 301Leu and 309Lys
[0449] xviii. 301Leu, 309Lys and 312Glu [0450] xix. 301Leu and
309Lys and at least one further Lys substitution or [0451] xx.
301Leu, 309Lys and 312Glu and at least one further Lys
substitution. [0452] 53. The EGF(A) peptide analogue according to
any of the previous embodiments 1-50, wherein said peptide has at
least two amino acid substitutions comprising and/or consisting of:
[0453] xxi. 301Leu and 307Ile, [0454] xxii. 301Leu, 307Ile and
312Glu [0455] xxiii. 301Leu and 307Ile and at least one further Lys
substitution or [0456] xxiv. 301Leu, 3307Ile and 312Glu and at
least one further Lys substitution. [0457] 54. The EGF(A) peptide
analogue according to any of the previous embodiments, wherein the
peptide analogue comprises an N-terminal and/or C-term elongation.
[0458] 55. The EGF(A) peptide analogue according to any of the
previous embodiments, wherein the peptide analogue comprises an
N-terminal elongation of 1-10 amino acid residues. [0459] 56. The
EGF(A) peptide analogue according to any of the previous
embodiments, wherein the peptide analogue comprises an N-terminal
elongation comprising an amino acid residue in position 292, such
as 292 Ala (A) or 292 (K). [0460] 57. The EGF(A) peptide analogue
according to any of the previous embodiments, wherein the peptide
analogue comprises a C-terminal elongation of 1-10 amino acid
residues. [0461] 58. The EGF(A) peptide analogue according to any
of the previous embodiments, wherein the peptide analogue comprises
an C-terminal elongation comprising an amino acid residue in
position 333, such as 333 Ala (A) or 333 (K). [0462] 59. The EGF(A)
peptide analogue according to any of the previous embodiments,
wherein the peptide analogue comprises at least one Lys residue
selected from the group consisting of: 292Lys, 293Lys, 294Lys,
296Lys, 299Lys, 300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys,
312Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys, 321Lys,
322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys,
330Lys, 332Lys and 333Lys. [0463] 60. The EGF(A) peptide analogue
according to any of the previous embodiments, wherein the peptide
analogue comprises at least one Lys residue selected from the group
consisting of: 292Lys, 293Lys, 294Lys, 299Lys, 300Lys, 303Lys,
305Lys, 306Lys, 309Lys, 311Lys, 312Lys, 313Lys, 314Lys, 315Lys,
316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys,
326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys. [0464]
61. The EGF(A) peptide analogue according to any of the previous
embodiments, wherein the peptide analogue comprises at least one
Lys residue selected from the group consisting of: 292Lys, 293Lys,
294Lys, 300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 312Lys,
313Lys, 314Lys, 316Lys, 318Lys, 321Lys, 322Lys, 323Lys, 324Lys,
325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys.
[0465] 62. The EGF(A) peptide analogue according to any of the
previous embodiments, wherein the peptide analogue comprises at
least one Lys residue selected from the group consisting of:
292Lys, 293Lys, 294Lys, 300Lys, 303Lys, 305Lys, 306Lys, 311Lys,
312Lys, 313Lys, 314Lys, 316Lys, 318Lys, 322Lys, 323Lys, 324Lys,
325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys.
[0466] 63. The EGF(A) peptide analogue according to any of the
previous embodiments, wherein the peptide analogue comprises at
least one Lys residue selected from the group consisting of:
292Lys, 293Lys, 294Lys, 300Lys, 303Lys, 305Lys, 306Lys, 311Lys,
313Lys, 314Lys, 316Lys, 318Lys, 322Lys, 323Lys, 324Lys, 325Lys,
326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys. [0467]
64. The EGF(A) peptide analogue according to any of the previous
embodiments, wherein the peptide analogue comprises at least one
Lys residue selected from the group consisting of: 313Lys, 324Lys,
328Lys and 333Lys. [0468] 65. The EGF(A) peptide analogue according
to any of the previous embodiments, wherein the peptide analogue
comprises two Lys residues selected from any of the groups defined
in embodiments 59-65. [0469] 66. The EGF(A) peptide analogue
according to any of the previous embodiments, wherein the peptide
analogue comprises two Lys residues selected from the pairs
consisting of:
TABLE-US-00004 [0469] i. 293K and 294K xiv. 313K and 321K ii. 293K
and 312K xv. 313K and 324K iii. 293K and 333K xvi. 313K and 328K
iv. 309K and 313K xvii. 313K and 332K v. 309K and 324K xviii. 313K
and 333K vi. 309K and 328K xix. 314K and 333K vii. 309K and 332K
xx. 321K and 332K viii. 309K and 333K xxi. 321K and 333K ix. 311K
and 313K xxii. 324K and 333K x. 312K and 333K xxiii. 324K and 328K
xi. 312K and 313K xxiv. 328K and 333K xii. 312K and 314K xxv. 330K
and 333K and xiii. 313K and 314K xxvi. 332K and 333K.
[0470] 67. The EGF(A) peptide analogue according to any of the
previous embodiments, wherein the peptide analogue comprises an
N-terminal or C-term truncation. [0471] 68. The EGF(A) peptide
analogue according to any of the previous embodiments, wherein the
peptide analogue comprises an N-terminal truncation of 1-10 amino
acid residues. [0472] 69. The EGF(A) peptide analogue according to
any of the previous embodiments, wherein the peptide analogue
comprises an N-terminal truncation deleting at least or
specifically amino acid 293Gly. [0473] 70. The EGF(A) peptide
analogue according to any of the previous embodiments, wherein the
peptide analogue comprises a C-terminal truncation of 1-2 amino
acid residues. [0474] 71. The EGF(A) peptide analogue according to
any of the previous embodiments, wherein the peptide analogue
comprises a C-terminal truncation deleting at least or specifically
amino acid 332Glu. 72. The EGF(A) peptide analogue according to any
of the previous embodiments, wherein said peptide sequence is
identified by any one of SEQ ID 2 to 106. [0475] 73. The EGF(A)
peptide analogue according to any of the previous embodiments,
wherein said peptide sequence is identified by any one of SEQ ID
NO.: 2-47 and 49-106. [0476] 74. The EGF(A) peptide analogue
according to any of the previous embodiments, wherein said peptide
sequence is identified by any one of SEQ ID NO.: 2-44, 46, 47 and
49-106. [0477] 75. The EGF(A) peptide analogue according to any of
the previous embodiments, wherein said peptide sequence is
identified by any one of SEQ ID NO.: 2-44, 46, 47, 49-53, 55,
58-106. [0478] 76. The EGF(A) peptide analogue according to any of
the previous embodiments, wherein said peptide sequence is
identified by any one of SEQ ID NO.: 2-4, 6-44, 46, 47, 49-53, 55,
58-106. [0479] 77. The EGF(A) peptide analogue according to any of
the previous embodiments, wherein said peptide sequence is
identified by any one of SEQ ID NO.: 2-4, 6-19, 21-44, 46, 47,
49-53, 55, 58-106. [0480] 78. An EGF(A) derivative comprising an
EGF(A) peptide analogue and a substituent. [0481] 79. The EGF(A)
derivative according to embodiment 78, wherein the EGF(A)
derivative comprise at least one substituent. [0482] 80. The EGF(A)
derivative according to embodiment 78 or 79, wherein the
substituent is a half-life extending substituent. [0483] 81. The
EGF(A) derivative according to embodiment 78 or 80, wherein the
EGF(A) peptide analogue is defined as in any of the above
embodiments 1-77. [0484] 82. The EGF(A) derivative according to any
of the embodiments 78-81, wherein one or two substituent(s) is/are
attached to a nitrogen atom of the EGF(A) peptide analogue. [0485]
83. The EGF(A) derivative according to any of the embodiments
78-81, wherein one or two substituent(s) is/are attached to an
amino group of the EGF(A) peptide. [0486] 84. The EGF(A) derivative
according to any of the embodiments 78-81, wherein one or more
substituent(s) is/are attached to the N-terminal amino acid of the
EGF(A) peptide or to a Lys residue of the EGF(A) peptide [0487] 85.
The EGF(A) derivative according to any of the embodiments 78-81,
wherein one or two substituent(s) is/are attached to the N-terminal
amino acid of the EGF(A) peptide. [0488] 86. The EGF(A) derivative
according to any of the embodiments 78-81, wherein one or two
substituent(s) is/are attached to the alpha-nitrogen of the
N-terminal amino acid residue of the EGF(A) peptide. [0489] 87. The
EGF(A) derivative according to any of the embodiments 78-81,
wherein one or two substituent(s) is/are attached to a Lys residue
in the EGF(A) peptide. [0490] 88. The EGF(A) derivative according
to any of the embodiments 78-81, wherein one or two substituent(s)
is/are attached to the epsilon-nitrogen of a Lys residue in the
EGF(A) peptide. [0491] 89. The EGF(A) derivative according to any
of the embodiments 78-81, wherein the EGF(A) derivative comprises
two substituents. [0492] 90. The EGF(A) derivative according to
embodiment 89, wherein the two substituents are identical. [0493]
91. The EGF(A) derivative according to embodiment 89, wherein the
two substituents are attached to nitrogen atoms of the EGF(A)
peptide analogue. [0494] 92. The EGF(A) derivative according to
embodiment 89, wherein the two substituents are attached to amino
groups of the EGF(A) peptide analogue. [0495] 93. The EGF(A)
derivative according to embodiment 89, wherein the two substituents
are attached to the N-terminal amino acid of the EGF(A) peptide and
to a Lys residue of the EGF(A) peptide analogue. [0496] 94. The
EGF(A) derivative according to embodiment 89, wherein one
substituent is attached to the alpha-nitrogen of the N-terminal
amino acid residue of the EGF(A) peptide analogue and one
substituent is attach to a Lys residue of the EGF(A) peptide
analogue. [0497] 95. The EGF(A) derivative according to embodiment
89, wherein the two substituents are attached to the N-terminal
amino acid of the EGF(A) peptide analogue. [0498] 96. The EGF(A)
derivative according to embodiment 89, wherein the two substituents
are attached to Lys residues of the EGF(A) peptide analogue. [0499]
97. The EGF(A) derivative according to embodiment 89, wherein the
two substituents are attached to the epsilon-nitrogen's of Lys
residues in the EGF(A) peptide analogue. [0500] 98. The EGF(A)
derivative according to any of the embodiment 78-97, wherein one or
more substituent(s) is/are attached to a Lys residue in the EGF(A)
peptide analogue selected from the group consisting of: 292Lys,
293Lys, 294Lys, 296Lys, 299Lys, 300Lys, 303Lys, 305Lys, 306Lys,
309Lys, 311Lys, 312Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys,
320Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys,
328Lys, 329Lys, 330Lys, 332Lys and 333Lys. [0501] 99. The EGF(A)
derivative according to any of the embodiment 78-97, wherein one or
more substituent(s) is/are attached to a Lys residue in the EGF(A)
peptide analogue selected from the group consisting of: 292Lys,
293Lys, 294Lys, 299Lys, 300Lys, 303Lys, 305Lys, 306Lys, 309Lys,
311Lys, 312Lys, 313Lys, 314Lys, 315Lys, 316Lys, 318Lys, 320Lys,
321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys, 327Lys, 328Lys,
329Lys, 330Lys, 332Lys and 333Lys. [0502] 100. The EGF(A)
derivative according to any of the embodiment 78-97, wherein one or
more substituent(s) is/are attached to a Lys residue in the EGF(A)
peptide analogue selected from the group consisting of: 292Lys,
293Lys, 294Lys, 300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys,
312Lys, 313Lys, 314Lys, 316Lys, 318Lys, 321Lys, 322Lys, 323Lys,
324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and
333Lys. [0503] 101. The EGF(A) derivative according to any of the
embodiment 78-100, wherein a substituent is attached to 312K in the
EGF(A) peptide analogue. [0504] 102. The EGF(A) derivative
according to any of the embodiment 78-100, wherein a substituent is
attached to a substituted Lys residue in the EGF(A) peptide
analogue. [0505] 103. The EGF(A) derivative according to embodiment
102, wherein the derivative comprises two substituents and one is
attached to a substituted Lys residue and one is attached to 312K
in the EGF(A) peptide analogue. [0506] 104. The EGF(A) derivative
according to any of the embodiment 102 and 103, wherein the
derivative comprises two substituents and both are attached to
substituted Lys residues in the EGF(A) peptide analogue. [0507]
105. The EGF(A) derivative according to any of the embodiments
102-104, wherein one or two substituents is/are attached to a
substituted Lys residue in the EGF(A) peptide analogue selected
from the group consisting of: 292Lys, 293Lys, 294Lys, 296Lys,
299Lys, 300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 313Lys,
314Lys, 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys,
324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and
333Lys. [0508] 106. The EGF(A) derivative according to any of the
embodiment 102-104, wherein one or two substituents is/are attached
to a substituted Lys residue in the EGF(A) peptide analogue
selected from the group consisting of: 292Lys, 293Lys, 294Lys,
299Lys, 300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 313Lys,
314Lys, 315Lys, 316Lys, 318Lys, 320Lys, 321Lys, 322Lys, 323Lys,
324Lys, 325Lys, 326Lys, 327Lys, 328Lys, 329Lys, 330Lys, 332Lys and
333Lys. [0509] 107. The EGF(A) derivative according to any of the
embodiment 102-104, wherein one or two substituents is/are attached
to a substituted Lys residue in the EGF(A) peptide analogue
selected from the group consisting of: 292Lys, 293Lys, 294Lys,
300Lys, 303Lys, 305Lys, 306Lys, 309Lys, 311Lys, 313Lys, 314Lys,
316Lys, 318Lys, 321Lys, 322Lys, 323Lys, 324Lys, 325Lys, 326Lys,
327Lys, 328Lys, 329Lys, 330Lys, 332Lys and 333Lys. [0510] 108. The
EGF(A) derivative according to any of the embodiment 102-104,
wherein one or two substituents is/are attached to a substituted
Lys residue in the EGF(A) peptide analogue selected from the group
consisting of: 313Lys, 324Lys, 328Lys and 333Lys. [0511] 109. The
EGF(A) derivative according to any of the embodiment 78-107,
wherein a substituent is not attached to the EGF(A) peptide
analogue via an amino acid residue in any of the positions 295,
298, 301, 302, 307 and 310. [0512] 110. The EGF(A) derivative
according to any of the embodiment 78-107, wherein a substituent is
not attached to the EGF(A) peptide analogue via an amino acid
residue in any the positions 295, 296, 298, 301, 302, 307, 310.
[0513] 111. The EGF(A) derivative according to any of the
embodiments 78-110, wherein the substituent is not an Fc domain.
[0514] 112. The EGF(A) derivative according to any of the
embodiments 78-110, wherein the substituent is not fused with the
EGF (A) peptide. [0515] 113. The EGF(A) derivative according to any
of the embodiment 78-112, wherein the substituent comprises at
least one fatty acid group. [0516] 114. The EGF(A) derivative
according to embodiment 113, wherein said fatty acid group is a
chemical group comprising at least one functional group (FG) with a
pKa<7 and a carbon chain which contains at least 8 consecutive
--CH.sub.2-- groups. [0517] 115. The EGF(A) derivative according to
embodiment 113, wherein said fatty acid group comprise a functional
group selected from a carboxylic acid, a sulphonic acid, a
tetrazole moiety, a methylsulfonylcarbamoylamino moiety or a
3-hydroxy-isoxazole moiety. [0518] 116. The EGF(A) derivative
according to embodiment 113, wherein said substituent comprises a
carboxylic acid, a sulphonic acid, a tetrazole moiety, a
methylsulfonylcarbamoylamino moiety or a
hydroxyisoxazole3-hydroxyisoxazole moiety including 8-20
consecutive --CH.sub.2-- groups. [0519] 117. The EGF(A) derivative
according to embodiment 113, wherein said substituent has Formula
I:
[0519]
Z.sub.1--Z.sub.2--Z.sub.3--Z.sub.4--Z.sub.5--Z.sub.6--Z.sub.7--Z.-
sub.8--Z.sub.9--Z.sub.10-- [I] [0520] wherein [0521] Z.sub.1 is
selected from:
[0521] HOOC--(CH.sub.2).sub.n--CO--*, Chem. 1:
tetrazolyl-(CH.sub.2).sub.n--CO--*, Chem. 2:
HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.m--CO--*, Chem. 3:
HOS(O).sub.2--(CH.sub.2).sub.n--CO--*, Chem. 4:
MeS(O).sub.2NH(CO)NH--(CH.sub.2).sub.n--CO--* and Chem. 5:
3-HO-Isoxazole-(CH.sub.2).sub.n--CO--* Chem. 6: wherein n is an
integer in the range of 8-20, m is an integer in the range of 8-11,
the --COOH group in Chem. 3 can be attached to position 2, 3 or 4
on the phenyl ring, the symbol * indicates the attachment point to
the nitrogen in Z.sub.2 or, if Z.sub.2 is a bond, to the nitrogen
on the neighbouring Z element; [0522] Z.sub.2 is selected from
[0522] *--NH--SO.sub.2--(CH.sub.2).sub.3--CO--*, Chem. 7:
*--NH--CH.sub.2--(C.sub.6H.sub.10)--CO--* and Chem. 8: a bond;
[0523] Z.sub.3 is selected from: .gamma.Glu, Glu and a bond; [0524]
Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8, Z.sub.9 are selected,
independently of each other, from: Glu, .gamma.Glu, Gly, Ser, Ala,
Thr, Ado, Aeep, Aeeep, TtdSuc and a bond; [0525] Z.sub.10 is
selected from:
[0525] *--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2--* and a bond.
Chem. 7: [0526] 118. The EGF(A) derivative according to embodiment
117, wherein [0527] .gamma.Glu is of formula Chem. 9:
*NH--CH(COOH)--(CH.sub.2).sub.2--CO--*, [0528] TtdSuc is of formula
Chem. 10:
*NH--CH.sub.2CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.su-
b.2CH.sub.2CH.sub.2NHCO*, [0529] Ado is of formula Chem. 11:
*NH--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--CH.sub.2--CO--*,
[0530] Aeep is of formula Chem. 12
*NH--CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2CO*, and
[0531] Aeeep is of formula Chem. 13
*NH--CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2C-
O*. [0532] 119. The EGF(A) derivative according to embodiment 117,
wherein said substituent has Formula I:
[0532]
Z.sub.1--Z.sub.2--Z.sub.3--Z.sub.4--Z.sub.5--Z.sub.6--Z.sub.7--Z.-
sub.8--Z.sub.9--Z.sub.10-- [I] [0533] wherein [0534] Z.sub.1 is
selected from
[0534] ##STR00021## [0535] wherein [0536] n in Chem. 1b, 2b, 4b, 5b
or 6b is an integer in the range of 8-20, [0537] m in Chem. 3b is
an integer in the range of 8-11, the --COOH group in Chem. 3b can
be attached to position 2, 3 or 4 on the phenyl ring, [0538] the
symbol * indicates the attachment point to the nitrogen in Z.sub.2
or, if Z.sub.2 is a bond, to the nitrogen on the neighbouring Z
element; [0539] Z.sub.2 is selected from
##STR00022##
[0539] and [0540] a bond; [0541] Z.sub.3 is selected from [0542]
.gamma.Glu, Glu, and a bond; [0543] Z.sub.4, Z.sub.5, Z.sub.6,
Z.sub.7, Z.sub.8, Z.sub.9 are selected, independently of each
other, from: [0544] Glu, .gamma.Glu, Gly, Ser, Ala, Thr, Ado,
TtdSuc and a bond; [0545] Z.sub.10 is selected from
##STR00023##
[0545] and a bond; [0546] provided that [0547] when Z.sub.10 is
Chem. 14b, said substituent is attached to the alpha-nitrogen of
the N-terminal amino group of said peptide; and [0548] when
Z.sub.10 is a bond, said substituent is attached to the epsilon
position of a Lys residue present in said peptide or to the
alpha-nitrogen of the N-terminal amino acid residue of said
peptide. [0549] 120. The EGF(A) derivative according to embodiment
117, wherein Z.sub.1 is formula Chem. 1b:
[0549] ##STR00024## [0550] and wherein n is an integer in the range
of 8-20. [0551] 121. A compound according to any of embodiments
117, wherein Z.sub.1 is formula Chem. 2b:
[0551] ##STR00025## [0552] and wherein n is an integer in the range
of 8-20. [0553] 122. The EGF(A) derivative according to embodiment
117, wherein Z.sub.1 is formula Chem. 4b:
[0553] ##STR00026## [0554] and wherein n is an integer in the range
of 8-20. [0555] 123. The EGF(A) derivative according to embodiment
117, wherein Z.sub.1 is formula Chem. 5b:
[0555] ##STR00027## [0556] and wherein n is an integer in the range
of 8-20. [0557] 124. The EGF(A) derivative according to embodiment
117, wherein Z.sub.1 is formula Chem. 6b:
[0557] ##STR00028## [0558] and wherein n is an integer in the range
of 8-20. [0559] 125. The EGF(A) derivative according to embodiment
117, wherein Z.sub.1 is formula Chem. 3b:
[0559] ##STR00029## [0560] and wherein m is an integer in the range
of 8-11. [0561] 126. The EGF(A) derivative according to embodiment
125, wherein m is 8, 9, 10 or 11. [0562] 127. The EGF(A) derivative
according to embodiment 125, wherein m is 10 or 11. [0563] 128. The
EGF(A) derivative according to any of the embodiments 120-124,
wherein n is in the range of 10-18, 10-14, 15-18, 8-15 or 16-20.
[0564] 129. The EGF(A) derivative according to any of the
embodiments 120-124, wherein n is 8, 9, 10, 11 or 12. [0565] 130.
The EGF(A) derivative according to any of the embodiments 120-124,
wherein n is 13, 14, 15 or 16. [0566] 131. The EGF(A) derivative
according to any of the embodiments 120-124, wherein n is 14, 15,
16, 17 or 18. [0567] 132. The EGF(A) derivative according to any of
the embodiments 120-124, wherein n is 17, 18, 19 or 20. [0568] 133.
The EGF(A) derivative according to any of the embodiments 117 and
132, wherein Z.sub.2 is Chem. 7 or Chem. 7b and Z.sub.3 is selected
from .gamma.Glu, Glu and a bond. [0569] 134. The EGF(A) derivative
according to any of the embodiments 117 and 132, wherein Z.sub.2 is
Chem. 8 or Chem. 8b and Z.sub.3 is selected from .gamma.Glu and
Glu. [0570] 135. The EGF(A) derivative according to any of the
embodiments 117 and 134, wherein the derivative has one or two
substituents selected from the group consisting of: [0571]
HOOC--(CH.sub.2).sub.18--CO-gGlu-2xADO [0572]
HOOC--(CH.sub.2).sub.18--CO--NH--CH.sub.2--(C.sub.6H.sub.10)--CO-gGlu-2xA-
DO [0573] HOOC--(CH.sub.2).sub.16--CO-gGlu-2xADO [0574]
HOOC--(CH.sub.2).sub.16--CO-gGlu-2xADO--NH--CH.sub.2--(C.sub.6H.sub.4)--C-
H.sub.2 [0575] HOOC--(CH.sub.2).sub.16--CO-gGlu [0576]
HOOC--(CH.sub.2).sub.16--CO--NH--CH.sub.2--(C.sub.6H.sub.10)--CO-gGlu-2xA-
DO [0577] HOOC--(CH.sub.2).sub.14--CO-gGlu-2xADO [0578]
HOOC--(CH.sub.2).sub.14--CO-gGlu- [0579]
HOOC--(CH.sub.2).sub.14--CO-gGlu-2xADO- [0580]
HOOC--(CH.sub.2).sub.12--CO-gGlu-2xADO [0581]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-2xADO
[0582]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-3xADO
[0583] 4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu
[0584] 4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO--2xgGlu
[0585]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-3xGly
[0586]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO--2xgGlu-2xADO
[0587]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-TtdSuc
[0588] 4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9-.phi. [0589]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-gGlu-4xADO
[0590]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO--NH--CH.sub.2--(C.sub.-
6H.sub.10)--CO-gGlu-2xADO [0591]
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9--CO-gGlu-2xADO [0592]
3-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9--CO-gGlu-2xADO [0593]
3-HO-Isoxazole-(CH.sub.2).sub.12--CO-gGlu-2xADO [0594]
HOS(O).sub.2--(CH.sub.2).sub.15--CO-gGlu-2xADO--NH--CH.sub.2--(C.sub.6H.s-
ub.4)--CH.sub.2 [0595]
HOS(O).sub.2--(CH.sub.2).sub.13--CO-gGlu-2xADO [0596]
Tetrazolyl-(CH.sub.2).sub.15--CO--NH--SO.sub.2--(CH.sub.2).sub.3---
CO-ADO-ADO-NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2 [0597]
Tetrazolyl-(CH.sub.2).sub.12--CO-gGlu-2xADO [0598]
Tetrazolyl-(CH.sub.2).sub.15--CO-gGlu-2xADO and [0599]
MeS(O).sub.2NH(CO)NH--(CH.sub.2).sub.12--CO-gGlu-2xADO. [0600] 136.
The EGF(A) derivative according to embodiment 78, wherein the
EGF(A) derivative is selected from the group of EGF(A) derivatives
consisting of: Example compounds 1-47, 51-102 and 106-159. [0601]
137. The EGF(A) derivative according to embodiment 78 wherein the
EGF(A) derivative is selected from the group of EGF(A) derivatives
consisting of: Example compounds 1-44, 46-47, 51-55, 57, 60-64,
66-69, 71-102 and 106-159. [0602] 138. The EGF(A) derivative
according to embodiment 78, wherein the EGF(A) derivative is
selected from the group of EGF(A) derivatives consisting of: the
Example compounds 31, 95, 128, 133, 143, 144, 150, 151, 152 and
153. [0603] 139. The EGF(A) derivative according to embodiment 78,
wherein the EGF(A) derivative is individually selected from the
group of EGF(A) derivatives consisting of: the Examples 1-47,
51-102 and 106-159. [0604] 140. The EGF(A) peptide analogue or
EGF(A) derivative according to any of the previous embodiments
wherein the peptide or derivative is a PCSK9 inhibitor. [0605] 141.
The EGF(A) peptide analogue or EGF(A) derivative according to
Embodiment 135, wherein the PCSK9 inhibitor is a capable of
inhibiting PCSK9 binding to human Low Density Lipoprotein Receptor
(LDL-R). [0606] 142. The EGF(A) peptide analogue or EGF(A)
derivative according to Embodiment 135, wherein the PCSK9 inhibitor
decreases PCSK9 binding to human Low Density Lipoprotein Receptor
(LDL-R). [0607] 143. The EGF(A) peptide analogue or EGF(A)
derivative according to Embodiment 135, wherein the PCSK9 inhibitor
has an apparent binding affinity (K) below 10 nM, such as below 8
nM, 6 nM, 5 nM, 4 nM, 3 nM or such as below 2 nM as measured in a
competitive ELISA. [0608] 144. The EGF(A) peptide analogue or
EGF(A) derivative according to Embodiment 135, wherein the PCSK9
inhibitor has an apparent binding affinity (K) below 10 nM, such as
below 8 nM, 6 nM, 5 .mu.M, 4 nM, 3 nM or such as below 2 nM as
measured in the competitive ELISA described in D1.1. [0609] 145.
The EGF(A) derivative according to any of the above embodiments,
wherein the derivative has a half-life above 6 hours, such as 8
hours or such as 10 hours in mice. The EGF(A) derivative according
to any of the above embodiments, wherein the derivative has a
half-life above 50 hours, such as 100 hours or such as 150 hours in
dogs. [0610] 146. A EGF(A) peptide analogue or EGF(A) derivative
according to any of the previous embodiments 1-145 for use as a
medicament. [0611] 147. A EGF(A) peptide analogue or EGF(A)
derivative according to any of the previous embodiments 1-145 for
use in a method of treatment. [0612] 148. A EGF(A) peptide analogue
or EGF(A) derivative according to any of the previous embodiments
1-145 for use in a method of prevention or treatment of a
cardiovascular disease. [0613] 149. A EGF(A) peptide analogue or
EGF(A) derivative according to any of the previous embodiments
1-145 for use in a method for improving lipid parameters. [0614]
150. A EGF(A) peptide analogue or EGF(A) derivative according to
any of the previous embodiments 1-145 for use in a method of
treatment for [0615] i. improving lipid parameters, such as
prevention and/or treatment of dyslipidemia, lowering total serum
lipids, increasing HDL-C, lowering LDL-C, lowering small, dense
LDL-C, lowering VLDL-C, lowering triglycerides, lowering
cholesterol, lowering plasma levels of lipoprotein a (Lp(a)) or
inhibiting generation of apolipoprotein A (apo(A)); [0616] ii.
prevention and/or treatment of cardiovascular diseases, such as
cardiac syndrome X, atherosclerosis, myocardial infarction,
coronary heart disease, reperfusion injury, stroke, cerebral
ischemia, an early cardiac or early cardiovascular disease, left
ventricular hypertrophy, coronary artery disease, hypertension,
essential hypertension, acute hypertensive emergency,
cardiomyopathy, heart insufficiency, exercise intolerance, acute
and/or chronic heart failure, arrhythmia, cardiac dysrhythmia,
syncopy, angina pectoris, cardiac bypass and/or stent reocclusion,
intermittent claudication (atheroschlerosis oblitterens), diastolic
dysfunction, and/or systolic dysfunction; and/or reduction of blood
pressure, such as reduction of systolic blood pressure; the
treatment of cardiovascular disease. [0617] 151. Use of EGF(A)
peptide analogue or EGF(A) derivative according to any of the
previous embodiments 1-145 for [0618] i. improving lipid
parameters, such as prevention and/or treatment of dyslipidemia,
lowering total serum lipids, increasing HDL, lowering LDL-C,
lowering small dense LDL-C, lowering VLDL-C, non-HDL-C, lowering
triglycerides, lowering cholesterol, lowering plasma levels of
lipoprotein a (Lp(a)), inhibiting generation of apolipoprotein A
(apo(A)); [0619] ii. prevention and/or treatment of cardiovascular
diseases, such as cardiac syndrome X, atherosclerosis, myocardial
infarction, coronary heart disease, reperfusion injury, stroke,
cerebral ischemia, an early cardiac or early cardiovascular
disease, left ventricular hypertrophy, coronary artery disease,
hypertension, essential hypertension, acute hypertensive emergency,
cardiomyopathy, heart insufficiency, exercise intolerance, acute
and/or chronic heart failure, arrhythmia, cardiac dysrhythmia,
syncopy, angina pectoris, cardiac bypass and/or stent reocclusion,
intermittent claudication (atheroschlerosis oblitterens), diastolic
dysfunction, and/or systolic dysfunction; and/or reduction of blood
pressure, such as reduction of systolic blood pressure; the
treatment of cardiovascular disease. [0620] 152. A pharmaceutical
composition comprising a EGF(A) peptide analogue or EGF(A)
derivative according to any of the previous embodiments, and a
pharmaceutically acceptable excipient. [0621] 153. A pharmaceutical
composition according to embodiment 152 for subcutaneous
administration. [0622] 154. A pharmaceutical composition according
to embodiment 152 for oral administration. [0623] 155. A method for
improving lipid parameters comprising a step of administering a
pharmaceutically active amount of a EGF(A) peptide analogue or
EGF(A) derivative according to any of the previous embodiments
1-145. [0624] 156. A method for improving lipid parameters
comprising a step of administering a pharmaceutically active amount
of a EGF(A) peptide analogue or EGF(A) derivative according to any
of the previous embodiments 1-145 wherein improving lipid
parameters, is such as prevention and/or treatment of dyslipidemia,
lowering total serum lipids; increasing HDL; lowering LDL-C;
lowering small, dense LDL-C; lowering VLDL-C; non_HDL-C; lowering
triglycerides; lowering cholesterol; lowering plasma levels of
lipoprotein a (Lp(a)); inhibiting generation of apolipoprotein A
(apo(A)). [0625] 157. A method for prevention and/or treatment of a
cardiovascular disease comprising a step of administering a
pharmaceutically active amount of a EGF(A) peptide analogue or
EGF(A) derivative according to any of the previous embodiments
1-145. [0626] 158. A method for prevention and/or treatment of a
cardiovascular disease comprising a step of administering a
pharmaceutically active amount of a EGF(A) peptide analogue or
EGF(A) derivative according to any of the previous embodiments
1-145 wherein a cardiovascular disease is such as cardiac syndrome
X, atherosclerosis, myocardial infarction, coronary heart disease,
reperfusion injury, stroke, cerebral ischemia, an early cardiac or
early cardiovascular disease, left ventricular hypertrophy,
coronary artery disease, hypertension, essential hypertension,
acute hypertensive emergency, cardiomyopathy, heart insufficiency,
exercise intolerance, acute and/or chronic heart failure,
arrhythmia, cardiac dysrhythmia, syncopy, angina pectoris, cardiac
bypass and/or stent reocclusion, intermittent claudication
(atheroschlerosis oblitterens), diastolic dysfunction, and/or
systolic dysfunction; and/or reduction of blood pressure, such as
reduction of systolic blood pressure. [0627] 159. A methods for
producing a EGF(A) peptide analogue or EGF(A) derivative according
to any of the previous embodiments 1-145, wherein the EGF(A)
peptide is produce recombinantly and optionally linked with a
substituent.
EXAMPLES
[0628] This experimental part starts with a list of abbreviations,
and is followed by a section including general methods for
synthesising and characterising analogues and derivatives of the
invention. Then follows a number of examples which relate to the
preparation of specific EGF(A) compounds of the invention,
including analogues and derivatives, and at the end a number of
examples have been included relating to the activity and properties
of these compounds (section headed pharmacological methods).
[0629] The examples serve to illustrate the invention.
List of Abbreviations
[0630] AcOH: acetic acid [0631] Ado: 8-amino-3,6-dioxaoctanoic acid
[0632] Aeep: 9-Amino-4,7-Dioxanonanoic acid [0633] Aeeep:
12-Amino-4,7,10-trioxa-dodecanoic acid [0634] Alloc:
Allyloxycarbonyl [0635] API: Active Pharmaceutical Ingredient
[0636] AUC: Area Under the Curve [0637] BG: Blood Glucose [0638]
Boc: t-butyloxycarbonyl [0639] BSA: Bovine serum albumin [0640]
Clt: 2-chlorotrityl [0641] collidine: 2,4,6-trimethylpyridine
[0642] DCM: dichloromethane [0643] Dde:
1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl [0644] DIC:
diisopropylcarbodiimide [0645] DIPEA: diisopropylethylamine [0646]
DMAP: 4-dimethylaminopyridine [0647] EGF: Epidermal growth
factor-like [0648] EGF(A): Epidermal growth factor-like domain A
[0649] F (table 5): Bio-availability [0650] Fmoc:
9-fluorenylmethyloxycarbonyl [0651] HDL: High density lipoprotein
[0652] HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
[0653] HFIP 1,1,1,3,3,3-hexafluoro-2-propanol or
hexafluoroisopropanol [0654] HOAt: 1-hydroxy-7-azabenzotriazole
[0655] HOBt: 1-hydroxybenzotriazole [0656] hPCKS9: human PCSK9
[0657] HPLC: High Performance Liquid Chromatography [0658] HSA:
Human Serum Albumin [0659] IC.sub.50: half maximum inhibitory
concentration [0660] Inp: isonipecotic acid [0661] i.v.
intravenously [0662] ivDde:
1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl [0663]
LCMS: Liquid Chromatography Mass Spectroscopy [0664] LDL-R or LDLr:
LDL receptor [0665] LDL: low density lipoprotein [0666] LDL-C: LDL
cholesterol [0667] MeOH: methanol [0668] Mmt: 4-methoxytrityl
[0669] Mtt: 4-methyltrityl [0670] MRT: Mean residence time [0671]
MSU: Methylsulfonylcarbamoylamino [0672] NMP: N-methyl pyrrolidone
[0673] OBz: benzoyl ester [0674] Ado: 8-amino-3,6-dioxaoctanoic
acid [0675] OSu: 0-succinimidyl esters (hydroxysuccinimide esters)
[0676] OtBu: tert butyl ester [0677] Oxyma Pure.RTM.:
Cyano-hydroxyimino-acetic acid ethyl ester [0678] Pbf:
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl [0679] PBS:
Phosphate Buffered Saline [0680] PD: Pharmacodynamic [0681] PK:
Pharmacokinetic [0682] QC: Quality control [0683] RP: Reverse Phase
[0684] RP-HPLC: Reverse Phase High Performance Liquid
Chromatography [0685] RT: Room Temperature [0686] Rt: Retention
time [0687] s.c.: Subcutaneously [0688] SD: Standard Deviation
[0689] SEM: Standard Error of Mean [0690] SPPS: Solid Phase Peptide
Synthesis [0691] tBu: tert. butyl [0692] TFA: trifluoroacetic acid
[0693] TIS or TIPS: triisopropylsilane [0694] Tmax: time to reach
Cmax [0695] Tris: tris(hydroxymethyl)aminomethane or
2-amino-2-hydroxymethyl-propane-1,3-diol [0696] Trt:
triphenylmethyl (trityl) [0697] Trx: tranexamic acid [0698] UPLC:
Ultra Performance Liquid Chromatography [0699] TBS-T: Tris buffered
saline
Chemical Methods
[0700] This section is divided in three: Section A relating to
general methods of preparation of compounds of the invention,
section B relating to the preparation of a number of specific
compounds of the invention, and section C relating to methods of
detection and characterisation of compounds of the invention and
the results for a number of specific example compounds.
A. Methods of Preparation
[0701] The compounds of the invention may be prepared by the method
described below.
Preparation of the Peptide, i.e. the EGF(A) Peptide of SEQ ID NO: 1
or Analogues Thereof:
SPPS General Methods:
[0702] The Fmoc-protected amino acids to be used may be the
standard recommended: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH,
Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(BOC)-OH, Fmoc-Met-OH,
Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH,
Fmoc-Trp(BOC)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH and
Fmoc-Lys(Mtt)-OH or Fmoc-Lys(Alloc)-OH supplied from e.g. Anaspec,
Bachem, Iris Biotech or NovabioChem. SPPS may be performed using
Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer
from Protein Technologies (Tucson, Ariz. 85714 U.S.A.). A suitable
resin for the preparation of C-terminal carboxylic acids is a Wang
resin preloaded with an amino acid such as Fmoc-Glu(tBu)-Wang resin
(Low Load, 0.35 mmol/g). In cases where the substituent is attached
to a C-terminal lysine, a suitable resin is a preloaded
Fmoc-Lys(Mtt)-Wang. A suitable resin for the preparation of
C-terminal peptide amides is H-Rink Amide-ChemMatrix resin (loading
e.g. 0.52 nmol/g) or Rink Amide AM polystyrene resin (Novabiochem,
loading e.g. 0.62 mmol/g) or the like. Fmoc-deprotection is
achieved with 20% piperidine in NMP. Peptide couplings are
performed by using either DIC/HOAt/collidine or DIC/Oxyma Pure with
or without collidine with or without preactivation or using DEPBt
(3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one)/DIPEA for
suppression of epimization of eg. His during coupling. Amino
acid/HOAt or amino acid/Oxyma Pure solutions (0.3 M/0.3 M in NMP at
a molar excess of 3-10 fold) are added to the resin followed by the
same molar equivalent of DIC (3 M in NMP) followed by collidine (3
M in NMP). For example, the following amounts of 0.3 M amino
acid/HOAt solution can be used per coupling for the following scale
reactions: Scale/mL, 0.05 mmol/1.5 mL, 0.10 mmol/3.0 mL, 0.25
mmol/7.5 mL.
[0703] If Fmoc-Lys(Mtt)-OH is used, the Mtt group may be removed by
washing the resin with HFIP/DCM (75:25) (2.times.2 min), washing
with DCM and suspending the resin in HFIP/DCM (75:25)(2.times.20
min) and subsequent washing before the substituent can be
introduced at the epsilon-position of the lysine moiety.
[0704] If Fmoc-Lys(Alloc)-OH is used, the Alloc group may be
removed by treating the resin with Pd(PPh.sub.3).sub.4 (0.02 equiv)
in the presence of one or more scavengers in combination, e.g.
morpholine (6.0 equiv) and/or dimethyl borane complex (18.0 equiv)
(30 min). The resin is then washed with MeOH, NMP or DMF and IPA
(isopropyl alcohol), respectively, before the substituent can be
introduced at the epsilon-position of the lysine moiety.
Attachment of the Substituent (Acylation)
[0705] The substituent can be introduced in a stepwise procedure by
the Prelude peptide synthesizer as described above using suitably
protected building blocks, such as the standard amino acids
described above, Fmoc-8-amino-3,6-dioxaoctanoic acid or
Fmoc-Glu-OtBu. Introduction of the substituent can be achieved
using a building block, such as, but not limited to,
octadecanedioic acid mono-tert-butyl-ester. After each coupling
step, unreacted peptide intermediate can be capped using acetic
acid anhydride and collidine in excess (>10 eq.).
[0706] The introduction of a substituent on the epsilon-nitrogen of
a lysine is achieved using a lysine protected with Mtt
(Fmoc-Lys(Mtt)-OH), Alloc (Fmoc-Lys(Alloc)-OH) or an ivDde group
(Fmoc-Lys(ivDde)-OH). The incorporation of .gamma.Glu moieties in
the substituent may be achieved by coupling with the amino acid
Fmoc-Glu-OtBu.
[0707] Introduction of each moiety in the substituent can be
achieved using prolonged coupling time (1.times.6 hours) followed
by capping with acetic anhydride or alternatively acetic
acid/DIC/HOAt/collidine.
Cleavage from the Resin
[0708] After synthesis the resin is washed with DCM, and the
peptide is cleaved from the resin by a 2-3 hour treatment with
TFA/TIPS/water (95/2.5/2.5) or TFA/EDT (1,2-ethanedithiol)/water
(90/5/5) followed by precipitation with Et.sub.2O (diethyl ether).
The precipitate is washed with Et.sub.2O.
Oxidative Folding
[0709] The precipitate from the step above is dissolved in DMSO and
added to a solution consisting of: [0710] 50 mM TRIS [0711] 5 mM
CaCl.sub.2) [0712] 3 mM Cysteine [0713] 0.3 mM Cystine [0714] in MQ
water, pH 8 to 8.8
[0715] The reaction mixture is kept overnight at room temperature
or until LCMS shows complete reaction.
Purification and Quantification
[0716] The crude peptide (derivative) is acidified with TFA to pH
2-3 and purified by reversed-phase preparative HPLC (Waters
Deltaprep 4000 or Gilson) on a column comprising C8- or C18-silica
gel. Elution is performed with an increasing gradient of MeCN in
water comprising 0.1% TFA. Relevant fractions are checked by
analytical HPLC or UPLC. Fractions comprising the pure target
peptide derivative are mixed. An additional purification step may
be introduced using another gradient, e.g. containing 0.05M
NH.sub.4HCO.sub.3. The resulting solution is analyzed (HPLC, LCMS)
and the product (i.e. the derivative) is quantified using a
chemiluminescent nitrogen specific HPLC detector (Antek 8060
HPLC-CLND) or by measuring UV-absorption at 280 nm. The product is
dispensed into glass vials. The vials are capped with Millipore
glassfibre prefilters. Freeze-drying affords the peptide
trifluoroacetate as a white solid.
Attachment of the Substituent (Reductive Alkylation)
[0717] The purified peptide analogue can be subjected to reductive
alkylation using a suitable albumin binding substituent derivatized
with an aldehyde functionality.
[0718] The peptide analogue is dissolved in citric acid pH=5.5 and
a suitable aldehyde is dissolved in water that may contain
cyclodextrin to increase the solubility. A reducing agent such as
borane pyridine complex dissolved in MeOH is added and the mixture
is gently shaken overnight. Subsequent addition of excess of the
aldehyde and reducing agent may be required for optimal yield. The
mixture is purified using the procedure described above.
B. Synthesis of Compounds of the Invention
[0719] The compounds of the invention were prepared by a method not
essentially different from the general methods described below.
Method A
Synthesis of LDL-R(293-332) Peptide Analogues (without
Substituent)
[0720] The Fmoc-protected amino acids used were the standard
recommended: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH,
Fmoc-Leu-OH, Fmoc-Lys(BOC)-OH, BOC-Lys(Fmoc)-OH Fmoc-Phe-OH,
Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(BOC)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from
e.g. Anaspec, Bachem, Iris Biotech or NovabioChem. SPPS was
performed using Fmoc based chemistry on a Prelude Solid Phase
Peptide Synthesizer from Protein Technologies (Tucson, Ariz. 85714
U.S.A.). A Wang resin preloaded with an amino acid such as
Fmoc-Glu(tBu)-Wang resin (Low Load, 0.35 mmol/g) or the like was
used. Fmoc-deprotection was achieved with 20% piperidine in NMP.
Peptide couplings were performed by using DIC/Oxyma Pure with
collidine. Amino acid/Oxyma Pure solutions (0.3 M/0.3 M in DMF at a
molar excess of 3-10 fold) was added to the resin followed by the
same molar equivalent of DIC (3 M in NMP) followed by collidine (3
M in NMP).
Cleavage from the Resin
[0721] After synthesis the resin was washed with DCM, and the
peptide was cleaved from the resin by a 2-3 hour treatment with
TFA/TIPS/DTT/water (92.5/2.5/2.5/2.5) followed by precipitation
with diethyl ether. The precipitate was subsequently washed with
diethyl ether.
Oxidative Folding
[0722] The precipitate from the step above was dissolved in DMSO
and added to a solution consisting of: [0723] 50 mM TRIS [0724] 5
mM CaCl.sub.2) [0725] 3 mM Cysteine [0726] 0.3 mM Cystine [0727] in
MQ water, pH 8.0 to 8.8
[0728] The reaction mixture was kept overnight at room temperature
or until LCMS showed complete reaction.
Purification and Quantification
[0729] The crude peptide was acidified with TFA to pH 2-3 and
purified by reversed-phase preparative HPLC (Waters Deltaprep 4000
or Gilson) on a column comprising C8- or C18-silica gel. Elution
was performed with an increasing gradient of MeCN in water
comprising 0.1% TFA. Relevant fractions were analyzed using UPLC.
Fractions comprising the pure target peptide were pooled. The
resulting solution was analyzed (UPLC, LCMS) and the peptide
derivative was quantified using a chemiluminescent nitrogen
specific HPLC detector (Antek 8060 HPLC-CLND) or by measuring
UV-absorption at 280 nm. The product was dispensed into glass
vials. The vials were capped with Millipore glassfibre prefilters.
Freeze-drying afforded the trifluoroacetate salt of the peptide as
a white solid.
Method B
Synthesis of Derivatives of LDL-R(293-332) EGF(A) Analogues (with
Substituent) (on Resin)
[0730] Synthesis of the resin bound EGF(A) peptide proceded as
described above.
[0731] The introduction of a substituent on the epsilon-nitrogen of
a lysine in the N-terminus of the sequence was achieved using
Boc-Lys(Fmoc)-OH. Introduction of the substituent at the
alpha-position of the N-terminal amino acid was accomplished using
a standard Fmoc-protected amino acid i.e. Fmoc-Gly-OH.
[0732] For the introduction of a substituent on the
epsilon-nitrogen of a lysine in other positions, Fmoc-Lys(Mtt)-OH
were used. The Mtt group was removed by treatment with HFIP/DCM
(75:25) (2.times.2 min), followed by a wash with DCM. The resin was
then resuspended in HFIP/DCM (75:25)(2.times.20 min or 2.times.30
min) and subsequently washed with DCM before the substituent was
introduced at the epsilon-position of the lysine moiety.
[0733] The moieties of the substituent were introduced in a
stepwise procedure by a Prelude peptide synthesizer as described
under method A, using suitably protected building blocks, such as
the standard Fmoc-protected amino acids described under method A,
Fmoc-8-amino-3,6-dioxaoctanoic acid or Fmoc-Glu-OtBu. Introduction
of the fatty acid group was achieved using the suitable building
block, such as but not limited to, octadecanedioic acid
mono-tert-butyl-ester. In some cases the coupling time was
increased or the coupling step for each building block was
repeated.
[0734] Cleavage, oxidative folding, purification and quantification
were performed as described under method A.
Method C
Attachment of the Substituent in Solution (Via Reductive
Alkylation)
[0735] The purified peptide obtained from method A was subjected to
reductive alkylation using a suitable substituent derivatized with
an aldehyde functionality.
[0736] The freeze-dried peptide powder was dissolved in a citric
acid buffer (40 mM, pH 5.55; peptide concentration: 4 mg/mL). A
solution comprising, the selected substituent (10 eq., 10 mg/mL) in
40% (w/v) aqueous cyclodextrin was added to the peptide solution
and gently mixed by inversion of the reaction vial. To this
solution was added borane pyridine complex (100 eq., 80 mg/mL
solution in MeOH) in small aliquots, followed by gentle inversion
of the reaction vial. The reaction solution was gently shaken at
room temperature overnight. The progress of the reaction was
monitored by LC-MS. The next morning, the reaction solution was
acidified to pH 2-3 using TFA and purified using the procedure
described above under method A.
Method D
Attachment of the Substituent (N-Terminal Acylation of the Folded
Peptide in Solution)
[0737] The freeze-dried peptide powder was dissolved in
K.sub.2HPO.sub.4 buffer (20 mM, pH 8.15) to a target concentration
of 5 mg/mL. A solution of
18-[[(1S)-1-carboxy-4-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxy--
2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-4-oxo-buty-
l]amino]-18-oxo-octadecanoic acid in DMSO (4 eq.; 4 mg/mL) was
added in four aliquots. After addition of each aliquot the peptide
solution was gently mixed by inversion of the reaction vial.
Subsequently, the pH value of the reaction solution was measured
and adjusted to pH 8.0-8.3 by adding small portions of
N,N-diisopropylethylamine, after which the solution was left
standing at room temperature. The progression of the reaction was
followed by LC-MS. After three hours the solution was acidified to
pH 5.9 with TFA and purified using the procedure described
above.
B.1. Synthesis of Protractors and Linker Elements
[0738] For synthesis of octadecanedioic acid mono-tert-butyl ester:
see patent application WO 2010102886. The corresponding
mono-tert-bytyl esters of C14-, C16- and C20 diacid can be prepared
accordingly. For synthesis of 14-sulfo-hexadecanoic acid og
16-sulfo-hexadecanoic acid sulfonsyre see WO2015071355. For
synthesis of 16-(1H-tetrazol-5-yl)hexadecanoic acid and
13-(1H-tetrazol-5-yl)hexadecanoic acid see WO2006005667.
13-(methylsulfonylcarbamoylamino)tridecanoic Acid
[0739] Chem. 6-OH, wherein n=12:
##STR00030##
[0740] This molecule was made using a modified procedure from
Luckhurst et al. Tetrahedron Letters Volume 48, Issue 50, 2007,
Pages 8878-8882 http://dx.doi.org/10.1016/j.tetlet.2007.10.046
[0741] Triethylamine (4.46 mL, 32.0 mmol) and ethyl chloroformate
(3.05 mL, 32.0 mmol) were subsequently added to a solution of the
14-(tert-butoxy)-14-oxotetradecanoic acid (1, C14(OtBu)-OH, 6.29 g,
20.0 mmol) in acetone (176 mL) at 0.degree. C. After 30 minutes at
0.degree. C., a solution of sodium azide (2.60 g, 40.0 mmol) in
water (12 mL) was added and the mixture was stirred for 2 hours at
0.degree. C. The mixture was concentrated in vacuo (at 30.degree.
C.) and poured into water with ice (300 mL). The resulting mixture
was extracted with ethyl acetate (3.times.250 mL); the organic
extracts were combined and washed with water (200 mL), 10% aqueous
solution of sodium hydrogencarbonate (200 mL) and water (200 mL);
dried over anhydrous magnesium sulfate and evaporated to dryness to
give mixture of tert-butyl 14-azido-14-oxotetradecanoate and
tert-butyl 14-isocyanato-14-oxotetradecanoate as pale yellow
oil.
[0742] Methanesulfonamide (1.52 g, 16.0 mmol), potassium carbonate
(6.63 g, 48.0 mmol) were added to a solution of mixture tert-butyl
14-azido-14-oxotetradecanoate and tert-butyl
14-isocyanato-14-oxotetradecanoate (5.43 g, 16.0 mmol) in dry
toluene (50 mL). The reaction mixture was heated at 85.degree. C.
overnight. Water (100 mL) was added followed by 1 M aqueous
hydrochloric acid (pH was adjusted to pH=4). The mixture was
extracted with diethyl ether (4.times.150 mL), dried over anhydrous
magnesium sulfate and evaporated in vacuo to give tert-butyl
13-(3-(methylsulfonyl)ureido)tridecanoate.
[0743] 1H NMR spectrum (300 MHz, DMSO, dH): 10.01 (s, 1H); 6.42 (t,
J=4.7 Hz, 1H); 3.20 (s, 3H); 3.02 (q, J=6.7 Hz, 2H); 2.16 (t, J=7.3
Hz, 2H); 1.52-1.33 (m, 13H); 1.30-1.11 (m, 16H).
[0744] Trifluoroacetic acid (21.0 mL) and water (2.50 mL) were
added dropwise to a solution of tert-butyl
13-(3-(methylsulfonyl)ureido)tridecanoate (3, 6.30 g, 15.5 mmol) in
dichloromethane (30 mL). Reaction mixture was stirred for 3 hours
at room temperature. The solvent was evaporated under reduced
pressure, affording 13-(3-(methylsulfonyl)ureido)tridecanoic
acid.
[0745] 1H NMR spectrum (300 MHz, DMSO, dH): 10.02 (s, 1H); 6.43 (t,
J=4.5 Hz, 1H); 3.20 (s, 3H); 3.02 (q, J=6.6 Hz, 2H); 2.18 (t, J=7.3
Hz, 2H); 1.56-1.33 (m, 4H); 1.24 (s, 16H).
13-(3-Hydroxyisoxazol-5-yl)tridecanoic Acid
[0746] Chem. 5-OH, wherein n=12:
##STR00031##
[0747] This molecule was made using a modified procedure from
Sorensen et al. J. Org. Chem., 2000, 65 (4), pp 1003-1007. DOI:
10.1021/jo991409d
[0748] 14-(tert-Butoxy)-14-oxotetradecanoic acid (1, 30.0 g, 95.4
mmol), N,N'-dicyclohexylcarbodiimide (43.3 g, 209 mmol) and
4-dimethylaminopyridine (25.6 g, 20.9 mmol) were dissolved in
anhydrous dichloromethane (700 mL) and
2,2-dimethyl-1,3-dioxane-4,6-dione (2, 20.6 g, 143 mmol) was added
to the solution. The reaction mixture was stirred at room
temperature for 16 hours. Volatiles were then evaporated and the
mixture was diluted with diethyl ether (500 mL) and white
precipitate was filtered off. Filtrate was concentrated under
reduced pressure, diluted with diethyl ether (300 mL) and extracted
with 1 M aqueous hydrochloric acid (3.times.100 mL) and brine
(1.times.100 mL). Organic portion was dried with sodium sulfate and
evaporated under reduced pressure to give tert-butyl
14-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)-14-oxotetradecanoate as
yellow oil. The crude product was used for the next step without
further purification.
[0749] 1H NMR spectrum (300 MHz, CDCl3, dH): 3.07 (t, J=7.5 Hz,
2H); 2.20 (t, J=7.5 Hz, 2H); 1.74 (s, 6H); 1.71-1.51 (m, 4H); 1.45
(s, 9H); 1.36-1.23 (m, 16H).
[0750] The crude product from above was dissolved in ethanol (300
mL) and the resulting solution was stirred at 80 C for 3 hours and
then overnight at room temperature. Solvent was removed under
reduced pressure and the residue was purified by flash column
chromatography (Silicagel 60, 0.040-0.063 mm; eluent:
cyclohexane/ethyl acetate 9:1) to give 1-ethyl 16-methyl
3-oxohexadecanedioate as colorless oil
[0751] 1H NMR spectrum (300 MHz, CDCl3, dH): 4.21 (q, J=7.2 Hz,
2H); 3.44 (s, 2H); 2.54 (t, J=7.5 Hz, 2H); 2.21 (t, J=7.5 Hz, 2H);
1.67-1.51 (m, 4H); 1.45 (s, 9H); 1.38-1.21 (m, 19H).
[0752] Sodium hydroxide (1.09 g, 27.3 mmol) was dissolved in
methanol (40.0 mL) and water (10.0 mL) at -30 C under argon
atmosphere. The above ester (4, 10.0 g, 26.0 mmol) was dissolved in
methanol (40 mL) and dimethoxyethane (50 mL) and added dropwise to
the reaction mixture at -30 C. After 20 minutes, solution of
hydroxylamine hydrochloride (3.61 g, 52.0 mmol) and sodium
hydroxide (2.18 g, 54.6 mmol) in dimethoxyethane (10 mL) and water
(10.0 mL) was added dropwise and the reaction mixture was stirred
for 3 hours at -30 C. The mixture was then quenched with acetone (5
mL) and after 5 minutes poured at once into concentrated
hydrochloric acid (70 mL) and heated to 80 C for 70 minutes. All
volatiles were then removed under reduced pressure, solids were
dissolved with dichloromethane (400 mL) and extracted with
distilled water (100 mL) and brine (70 mL). Organic portion was
dried with sodium sulfate. The crude product was purified by flash
column chromatography (Silicagel 60, 0.040-0.063 mm; eluent:
cyclohexane/ethyl acetate 3:1) to give methyl
13-(3-hydroxyisoxazol-5-yl)tridecanoate as white solid.
[0753] 1H NMR spectrum (300 MHz, CDCl3, dH): 5.66 (s, 1H); 3.67 (s,
3H); 2.63 (t, J=7.6 Hz, 2H); 2.31 (t, J=7.6 Hz, 1H); 1.72-1.55 (m,
4H); 1.40-1.19 (m, 16H).
[0754] Methyl 13-(3-hydroxyisoxazol-5-yl)tridecanoate (5, 6.20 g,
19.9 mmol) was dissolved in methanol (60.0 mL) and water (20.0 mL),
lithium hydroxide monohydrate (4.04 g, 96.3 mmol) was added and
reaction mixture was stirred for 16 hours at room temperature.
Volatiles were then removed under reduced pressure and water (50.0
mL) was added followed by 1 M aqueous hydrochloric acid (50.0 mL).
Precipitate was filtered off and washed with water (2.times.100 mL)
and then dried under reduced pressure to give
13-(3-hydroxyisoxazol-5-yl)tridecanoic acid as a beige solid.
[0755] 1H NMR spectrum (300 MHz, DMSO-d6, dH): 5.74 (s, 1H); 2.57
(t, J=7.5 Hz, 2H); 2.18 (t, J=7.5 Hz, 2H); 1.63-1.41 (m, 4H);
1.34-1.14 (m, 16H).
B.2 Synthesis of Intermediate Substituent Precursors for Reductive
Alkylation
18-[[(1 S)-1-carboxy-4-[2-[2-[2-[2-[2-[2-[(4-formyl
phenyl)methylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]e-
thylamino]-4-oxo-butyl]amino]-18-oxo-octadecanoic acid
##STR00032##
[0757] 2-Chlorotrityl resin 100-200 mesh (42.6 g, 42.6 mmol) was
left to swell in dry dichloromethane (205 mL) for 20 min. A
solution of
{2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic
acid (13.7 g, 35.5 mmol) and N,N-diisopropylethylamine (23.5 mL,
135 mmol) in dry dichloromethane (30 mL) was added to resin and the
mixture was shaken for 3 hrs. Resin was filtered and treated with a
solution of N,N-diisopropylethylamine (12.4 mL, 70.9 mmol) in
methanol/dichloromethane mixture (4:1, 250 mL, 2.times.5 min). Then
resin was washed with N,N-dimethylformamide (2.times.150 mL),
dichloromethane (3.times.150 mL) and N,N-dimethylformamide
(3.times.150 mL). Fmoc group was removed by treatment with 20%
piperidine in dimethylformamide (1.times.5 min, 1.times.30 min,
2.times.150 mL). Resin was washed with N,N-dimethylformamide
(3.times.150 mL), 2-propanol (2.times.150 mL) and dichloromethane
(200 mL, 2.times.150 mL). Solution of
{2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic
acid (20.5 g, 53.2 mmol),
O-(6-chloro-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TCTU, 18.9 g, 53.2 mmol) and
N,N-diisopropylethylamine (16.7 mL, 95.7 mmol) in
N,N-dimethylformamide (100 mL) and dichloromethane (50 mL) was
added to resin and mixture was shaken for 1 hr. Resin was filtered
and washed with N,N-dimethylformamide (2.times.150 mL),
dichloromethane (3.times.150 mL) and N,N-dimethylformamide (155
mL). Fmoc group was removed by treatment with 20% piperidine in
dimethylformamide (1.times.5 min, 1.times.30 min, 2.times.150 mL).
Resin was washed with N,N-dimethylformamide (3.times.150 mL),
2-propanol (2.times.150 mL) and dichloromethane (200 mL,
2.times.150 mL). Solution of Fmoc-Glu-OtBu (22.6 g, 53.2 mmol),
O-(6-chloro-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TCTU, 18.9 g, 53.2 mmol) and
N,N-diisopropylethylamine (16.7 mL, 95.7 mmol) in
N,N-dimethylformamide (155 mL) was added to resin and mixture was
shaken for 1 hr. Resin was filtered and washed with
N,N-dimethylformamide (2.times.150 mL), dichloromethane
(2.times.150 mL) and N,N-dimethylformamide (150 mL). Fmoc group was
removed by treatment with 20% piperidine in dimethylformamide
(1.times.5 min, 1.times.30 min, 2.times.150 mL). Resin was washed
with N,N-dimethylformamide (3.times.150 mL), 2-propanol
(2.times.150 mL) and dichloromethane (200 mL, 2.times.150 mL).
Solution of octadecanedioic acid mono-tert-butyl ester (19.7 g,
53.2 mmol),
O-(6-chloro-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TCTU, 18.9 g, 53.2 mmol) and
N,N-diisopropylethylamine (16.7 mL, 95.7 mmol) in
N,N-dimethylformamide/dichloromethane mixture (1:4, 200 mL) was
added to resin. Resin was shaken for 2 hrs, filtered and washed
with N,N-dimethylformamide (3.times.150 mL), dichloromethane
(2.times.150 mL), methanol (2.times.150 mL) and dichloromethane
(300 mL, 6.times.150 mL). The product was cleaved from resin by
treatment with 2,2,2-trifluoroethanol (200 mL) for 19 hrs. Resin
was filtered off and washed with dichloromethane (2.times.150 mL),
2-propanol/dichloromethane mixture (1:1, 2.times.150 mL),
2-propanol (150 mL) and dichloromethane (2.times.150 mL). Solutions
were combined; solvent evaporated and crude product was purified by
flash column chromatography (Silicagel 60, 0.040-0.060 mm; eluent:
dichloromethane/methanol 1:0-9:1). Pure product was dried in vacuo
and obtained as yellow oil.
[0758] Yield of
17-{(S)-1-tert-Butoxycarbonyl-3-[2-(2-{[2-(2-carboxymethoxy-ethoxy)-ethyl-
carbamoyl]-methoxy}-ethoxy)-ethylcarbamoyl]-propylcarbamoyl}-heptadecanoic
acid tert-butyl ester: 25.85 g (86%).
[0759] RF (SiO2, chloroform/methanol 85:15): 0.25.
[0760] 1H NMR spectrum (300 MHz, CDCl3, dH): 7.38 (bs, 1H); 7.08
(bs, 1H); 6.61 (d, J=7.5 Hz, 1H); 4.43 (m, 1H); 4.15 (s, 2H); 4.01
(s, 2H); 3.78-3.39 (m, 16H); 2.31 (t, J=6.9 Hz, 2H); 2.27-2.09 (m,
5H); 2.01-1.84 (m, 1H); 1.69-1.50 (m, 4H); 1.46 (s, 9H); 1.43 (s,
9H); 1.24 (bs, 24H).
[0761] LC-MS m/z: 846.6 (M+H)+.
[0762] (4-Formyl-benzyl)-carbamic acid tert-butyl ester
(Boc-aminomethylbenzaldehyde, 1.54 g, 6.60 mmol) was dissolved in
dichloromethane (50 mL) and solution of hydrochloric acid in
dioxane (3.8 M, 20 mL, 76 mmol) was added. The mixture was stirred
for 16 hrs and solid material precipitated from the solution. All
solvents were removed by evaporation.
17-{(S)-1-tert-Butoxycarbonyl-3-[2-(2-{[2-(2-carboxymethoxy-ethoxy)-ethyl-
carbamoyl]-methoxy}-ethoxy)-ethylcarbamoyl]-propylcarbamoyl}-heptadecanoic
acid tert-butyl ester (5.08 g, 6.00 mmol),
ethyl-(N',N'-dimethylamino)propylcarbodiimide hydrochloride (EDC,
1.73 g, 9.00 mmol), N,N-dimethylaminopyridine (DMAP, 0.037 g, 0.30
mmol) and dichloromethane (50 mL) were added. The mixture was
stirred and diisopropylethylamine (2 mL, 11.6 mmol) was added in 3
portions. The reaction mixture was stirred for 2 hrs and the
solvents were evaporated. The residue was dissolved in
dichloromethane (10 mL) and a solution of hydrochloric acid was
added dropwise until pH was lower than 5. The solution was
submitted to column chromatography (Silicagel 60, 0.040-0.060 mm;
eluent: dichloromethane/methanol 95:5) to provide the substituent
as a yellow oil.
[0763] Yield: 3.15 g (54%).
[0764] 1H NMR spectrum (300 MHz, CDCl3, dH): 9.99 (s, 1H); 7.85 (d,
J=7.9 Hz, 2H); 7.54-7.43 (m, 3H); 7.06 (t, J=5.5 Hz, 1H); 6.86 (t,
J=5.6 Hz, 1H); 6.48 (d, J=7.7 Hz, 1H); 4.58 (d, J=6.2 Hz, 2H);
4.45-4.36 (m, 1H); 4.09 (s, 2H); 3.94 (s, 2H); 3.73-3.37 (m, 16H);
2.32-2.05 (m, 7H); 1.99-1.80 (m, 1H); 1.69-1.51 (m, 4H); 1.45 (s,
9H); 1.44 (s, 9H); 1.33-1.20 (m, 24H).
[0765] LC-MS m/z: 963.5 (M+H)+.
(2S)-5-[2-[2-[2-[2-[2-[2-[(4-formylphenyl)methylamino]-2-oxo-ethoxy]ethoxy-
]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-5-oxo-2-(16-sulfohexadecanoyl-
amino)pentanoic acid
##STR00033##
[0767] 2-Chlorotrityl resin 100-200 mesh 1.8 mmol/g (1, 8.40 g,
14.3 mmol) was left to swell in dry dichloromethane (150 mL) for 30
minutes. A solution of Fmoc-Ado-OH (2.82 g, 9.50 mmol) and
N,N-diisopropylethylamine (6.30 mL, 36.1 mmol) in dry
dichloromethane (.about.150 mL) was added to resin and the mixture
was shaken for 24 hours. Resin was filtered and treated with a
solution of N,N-diisopropylethylamine (3.30 mL, 19.0 mmol) in
methanol/dichloromethane mixture (4:1, 2.times.150 mL, 2.times.5
min). Then resin was washed with N,N-dimethylformamide (3.times.150
mL), dichloromethane (3.times.150 mL) and N,N-dimethylformamide
(3.times.150 mL). Fmoc group was removed by treatment with 20%
piperidine in N,N-dimethylformamide (1.times.5 min, 1.times.30 min,
2.times.150 mL). Resin was washed with N,N-dimethylformamide
(3.times.150 mL), 2-propanol (3.times.150 mL) and dichloromethane
(3.times.150 mL). Solution of
{2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic
acid (Fmoc-Ado-OH, 4.80 g, 16.2 mmol),
O-(6-chloro-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TCTU, 5.74 g, 16.2 mmol) and
N,N-diisopropylethylamine (4.47 mL, 25.7 mmol) in
N,N-dimethylformamide (150 mL) was added to resin and mixture was
shaken for 2 hours. Resin was filtered and washed with
N,N-dimethylformamide (3.times.150 mL), dichloromethane
(3.times.150 mL) and N,N-dimethylformamide (3.times.150 mL). Fmoc
group was removed by treatment with 20% piperidine in
N,N-dimethylformamide (1.times.5 min, 1.times.30 min, 2.times.150
mL). Resin was washed with N,N-dimethylformamide (3.times.150 mL),
2-propanol (3.times.150 mL) and dichloromethane (3.times.150 mL).
Solution of
(S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-pentanedioic acid
1-tert-butyl ester (Fmoc-Glu-OtBu, 6.87 g, 16.2 mmol),
O-(6-chloro-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TCTU, 5.74 g, 16.2 mmol) and
N,N-diisopropylethylamine (4.47 mL, 25.7 mmol) in
N,N-dimethylformamide (150 mL) was added to resin and mixture was
shaken for 2 hours. Resin was filtered and washed with
N,N-dimethylformamide (3.times.150 mL), dichloromethane
(3.times.150 mL) and N,N-dimethylformamide (3.times.150 mL). Fmoc
group was removed by treatment with 20% piperidine in
N,N-dimethylformamide (1.times.5 min, 1.times.30 min, 2.times.150
mL). Resin was washed with N,N-dimethylformamide (3.times.150 mL),
2-propanol (3.times.150 mL) and dichloromethane (3.times.150 mL). A
solution of
16-((4-((tert-butoxycarbonyl)amino)-2,2-dimethylbutoxy)sulfonyl)hexadecan-
oic acid (6.62 g, 12.4 mmol),
O-(6-chloro-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TCTU, 4.39 g, 12.4 mmol) and
N,N-diisopropylethylamine (4.47 mL, 25.7 mmol) in
N,N-dimethylformamide/dichloromethane mixture (1/1, 150 mL) was
added to resin and mixture was shaken for 2 hours. Resin was
filtered and washed with N,N-dimethylformamide (3.times.150 mL),
methanol (5.times.150 mL) and dichloromethane (10.times.150 mL).
The product was cleaved from resin by treatment with
2,2,2-trifluoroethanol (150 mL) for 24 hours. Resin was filtered
off and washed with dichloromethane (3.times.150 mL). Solutions
were combined, solvents were evaporated and crude product (7.80 g)
was purified by flash column chromatography (Silicagel 60,
0.040-0.060 mm; eluent: dichloromethane/methanol 100:2 to
dichloromethane/methanol 100:10) to give the intermediate compounds
as a white solid.
[0768] Yield: 4.00 g (42%).
[0769] RF (SiO2, dichloromethane/methanol 8:1): 0.50.
[0770] 1H NMR spectrum (300 MHz, CDCl3, dH): 7.79-7.65 (m, 1H);
7.36-7.20 (m, 1H); 6.86 (d, J=7.5 Hz, 1H); 4.49-4.63 (m, 1H);
4.44-4.29 (m, 1H); 4.07-3.93 (m, 4H); 3.90 (s, 2H); 3.77-3.35 (m,
16H); 3.25-3.02 (m, 4H); 2.44-1.75 (m, 8H); 1.72-1.38 (m, 22H);
1.38-1.18 (m, 22H); 1.00 (m, 6H).
[0771] LC-MS m/z: 1012.3 (M+H)+.
[0772] The intermediate compound from above (3.77 g, 3.73 mmol),
N,N-diisopropylethylamine (1.75 mL, 10.1 mmol),
[1,2,3]triazolo[4,5-b]pyridin-1-ol (HOAt, 0.51 g, 3.73 mmol),
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC
HCl, 1.43 g, 7.46 mmol) were dissolved in dichloromethane (120 mL).
4-Formyl-benzyl-ammonium chloride (5, 0.77 g, 4.48 mmol) was added.
The mixture was stirred at room temperature for 24 hours. After
this time reaction mixture was evaporated, dissolved in ethyl
acetate (300 mL) and washed with 0.5 M aqueous solution of
hydrochloric acid (200 mL). Organic phase was separated, washed
with water (200 mL) and dried over magnesium sulfate. Ethyl acetate
was evaporated and the crude mixture was purified by flash column
chromatography (Silicagel 60, 0.040-0.060 mm; eluent:
dichloromethane to dichloromethane/methanol 100:5) to give the
protected aldehyde as a white solid.
[0773] Yield: 3.00 g (71%).
[0774] RF (SiO2, dichloromethane/methanol 10:1): 0.70.
[0775] 1H NMR spectrum (300 MHz, CDCl3, dH): 9.99 (s, 1H); 7.85 (d,
J=8.1 Hz, 2H); 7.64-7.38 (m, 3H); 7.14-6.97 (m, 1H); 6.91-6.76 (m,
1H); 6.54-6.43 (m, 1H); 4.58 (d, J=6.2 Hz, 2H); 4.47-4.32 (m, 1H);
4.09 (s, 2H); 3.88 (s, 2H); 3.81-3.30 (m, 16H); 3.22-3.02 (m, 4H);
2.38-2.08 (m, 4H); 1.96-1.71 (m, 5H); 1.71-1.16 (m, 41H); 0.99 (m,
6H).
[0776] LC-MS m/z: 1129.5 (M+H)+.
[0777] The protected aldehyde from above (3.00 g, 2.66 mmol) was
stirred with trifluoroacetic acid (15 mL) and water (1 mL) mixture
for 3 hours. After this time the mixture was evaporated several
times with dichloromethane and toluene under reduced pressure. The
residue was poured into water/acetonitrile mixture (1/1, 15 mL). pH
was adjusted to 8.0 with saturated aqueous solution of trisodium
phosphate and the resulting solution was stirred for 20 minutes at
50.degree. C. pH was adjusted to 6.0 with saturated aqueous
solution of potassium hydrogen sulfate. The residue was desalinated
by reverse-phase chromatography (DeltaPak, C18, 15 mm 50
mm.times.500 mm, acetonitrile/water 5-15%/15 min., 5-55%/180
min.+0.05% TFA). Solvents were removed by freeze-drying to give the
substituent as a white powder.
[0778] Yield: 0.66 g (28%).
[0779] H NMR spectrum (300 MHz, D2O, dH): 9.81 (s, 1H); 7.79 (d,
J=7.7 Hz, 2H); 7.41 (d, J=7.9 Hz, 2H); 4.44 (s, 2H); 4.32-4.19 (m,
1H); 4.05 (s, 2H); 3.90 (s, 2H); 3.74-3.43 (m, 12H); 3.38-3.18 (m,
4H); 2.83-2.60 (m, 2H); 2.37-1.76 (m, 6H); 1.76-1.37 (m, 4H);
1.34-0.91 (m, 22H).
[0780] LC-MS m/z: 873.8 (M+H)+.
N-((1-(4-Formylphenyl)-3,12,21-trioxo-5,8,14,17-tetraoxa-2,11,20-triazatet-
racosan-24-yl)sulfonyl)-16-(1H-tetrazol-5-yl)hexadecanamide
##STR00034##
[0782] Reaction Scheme:
##STR00035##
[0783] Wang resin 0.68 mmol/g (20.5 g, 13.9 mmol) was left to swell
in tetrahydrofuran (200 mL) for 20 minutes. A solution of of
{2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic
acid (Fmoc-Ado-OH, 16.1 g, 41.8 mmol) and 4-dimethylaminopyridine
(DMAP, 0.17 g, 1.39 mmol) and N,N'-diisopropylcarbodiimide (DIC,
6.47 mL, 41.8 mmol) in tetrahydrofuran (200 mL) was added to resin
and the mixture was shaken for 18 hours. Then resin was filtered
and washed with N,N-dimethylformamide (2.times.180 mL),
dichloromethane (2.times.180 mL) and N,N-dimethylformamide
(2.times.180 mL). Resin was treated with a solution of acetic
anhydride (13.2 mL, 139 mmol) and pyridine (11.3 mL, 139 mmol) in
N,N-dimethylformamide (180 mL). Then resin was filtered and washed
with N,N-dimethylformamide (2.times.180 mL), dichloromethane
(2.times.180 mL) and N,N-dimethylformamide (2.times.180 mL). Fmoc
group was removed by treatment with 20% piperidine in
N,N-dimethylformamide (1.times.5 min, 1.times.30 min, 2.times.180
mL). Resin was filtered and washed with N,N-dimethylformamide
(2.times.180 mL), dichloromethane (2.times.180 mL) and
N,N-dimethylformamide (2.times.180 mL). A solution of of
{2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic
acid (Fmoc-Ado-OH, 10.8 g, 27.9 mmol),
5-chloro-1-((dimethylamino)(dimethyliminio)methyl)-1H-benzo[d][1,2,3]tria-
zole 3-oxide tetrafluoroborate (TCTU, 9.91 g, 27.9 mmol) and
N,N-diisopropylethylamine (7.28 mL, 41.8 mmol) in
N,N-dimethylformamide (180 mL) was added to resin and mixture was
shaken for 2 hours. Then resin was filtered and washed with
N,N-dimethylformamide (2.times.180 mL), dichloromethane
(2.times.180 mL) and N,N-dimethylformamide (2.times.180 mL). Fmoc
group was removed by treatment with 20% piperidine in
N,N-dimethylformamide (1.times.5 min, 1.times.30 min, 2.times.180
mL). Resin was filtered and washed with N,N-dimethylformamide
(2.times.180 mL), dichloromethane (2.times.180 mL) and
N,N-dimethylformamide (2.times.180 mL). Solution of of
4-(N-(16-(1H-tetrazol-5-yl)hexadecanoyl)sulfamoyl)butanoic acid
(THA-SBA-OH, 8.91 g, 18.8 mmol),
1-((dimethylamino)(dimethyliminio)methyl)-1H-[1,2,3]triazolo[4,5-b]pyridi-
ne 3-oxide hexafluorophosphate (HATU, 7.16 g, 18.8 mmol) and
N,N-diisopropylethylamine (5.71 mL, 32.8 mmol) in mixture of
N,N-dimethylformamide (90 mL) and dichloromethane (90 mL) was added
to resin and mixture was shaken for 18 hours.
[0784] Resin was filtered and washed with N,N-dimethylformamide
(2.times.180 mL), dichloromethane (2.times.180 mL), 2-propanol
(2.times.180 mL) and dichloromethane (10.times.180 mL). The product
was cleaved from the resin by the treatment with mixture of
trifluoracetic acid (150 mL) and water (7.5 mL) for 1 hour. Resin
was filtered and washed with dichloromethane (2.times.150 mL). The
solvent was removed under reduced pressure and the residue was
treated with diethyl ether (100 mL). To a solution of the
intermediate (7.90 g, 10.3 mmol) in tetrahydrofuran (100 mL) was
added lithium hydroxide monohydrate (1.74 g, 41.4 mmol) in water
(100 mL). The solution was stirred for 18 hours. The solution was
acidified by 10% aqueous solution potassium hydrogen sulfate until
pH=3 was achieved, followed by saturation with sodium chloride.
Organic phase was removed, aqueous phase was extracted by ethyl
acetate (1.times.300 mL). Combined organic phases were dried over
anhydrous sodium sulfate. The solvent was removed under reduced
pressure to give the intermediate as white powder.
[0785] Yield: 5.50 g (52%).
[0786] 1H NMR spectrum (300 MHz, AcOD-d4, dH): 4.22 (s, 2H); 4.12
(s, 2H); 3.83-3.61 (m, 12H); 3.59-3.43 (m, 6H); 3.02 (t, J=7.4 Hz,
2H); 2.49 (t, J=7.3 Hz, 2H); 2.41 (t, J=7.5 Hz, 2H); 2.22-2.09 (m,
2H); 1.89-1.75 (m, 2H); 1.74-1.60 (m, 2H); 1.47-1.26 (m, 22H).
[0787] A solution of above compound (2.90 g, 3.80 mmol),
4-aminomethylbenzaldehyde hydrochloride (0.78 g, 4.56 mmol),
N,N'-dicyclohexylcarbodiimide (DCC, 0.78 g, 3.80 mmol) and
4-dimethylaminopyridine (DMAP, 1.02 g, 8.35 mmol) in dry
dichloromethane (100 mL) was stirred for 18 hours. The precipitate
was filtered-off and the solution was washed with 10% aqueous
solution of potassium hydrogen sulfate (2.times.100 mL). The
solvent was removed under reduced pressure and the residue was
crystallized from tetrahydrofuran (30 mL). Purification by flash
column chromatography (Silicagel 60, 0.040-0.063 mm; eluent:
dichloromethane/methanol, 20:1-5:1) gave final product as pale
yellow powder.
[0788] Yield: 1.51 g (45%).
[0789] 1H NMR spectrum (300 MHz, AcOD-d4, dH): 9.96 (s, 1H); 7.91
(d, J=7.9 Hz, 2H); 7.92 (d, J=7.9 Hz, 2H); 4.63 (s, 2H); 4.21 (s,
2H); 4.08 (s, 2H); 3.81-3.57 (m, 12H); 3.54-3.41 (m, 6H); 3.02 (t,
J=7.4 Hz, 2H); 2.47 (t, J=7.3 Hz, 2H); 2.38 (t, J=7.4 Hz, 2H);
2.19-2.07 (m, 2H); 1.86-1.72 (m, 2H); 1.70-1.57 (m, 2H); 1.43-1.23
(m, 22H).
[0790] LC-MS purity: 100%.
[0791] LC-MS Rt (Kinetex 4.6 mm.times.50 mm, acetonitrile/water
20:50 to 100:0+0.1% FA): 3.58 min.
[0792] LC-MS m/z: 882.0 (M+H)+.
[0793] A total of 29 substituents were prepared and are listed
herein below specifying the Z1-Z10 elements of the individual
substituents.
TABLE-US-00005 Z1(protractor) Z2 Z3 Z3-Z9 Z10 1.
HOOC--(CH.sub.2).sub.18--CO-- -gGlu- -ADO--ADO-- 2.
HOOC--(CH.sub.2).sub.18--CO--
--NH--CH.sub.2--(C.sub.6H.sub.10)--CO-- -gGlu- -ADO--ADO-- 3.
HOOC--(CH.sub.2).sub.16--CO-- -gGlu- -ADO--ADO-- 4.
HOOC--(CH.sub.2).sub.16--CO-- -gGlu- -ADO--ADO--
--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2-- 5.
HOOC--(CH.sub.2).sub.16--CO-- -gGlu- 6.
HOOC--(CH.sub.2).sub.16--CO--
--NH--CH.sub.2--(C.sub.6H.sub.10)--CO-- -gGlu- -ADO--ADO-- 7.
HOOC--(CH.sub.2).sub.14--CO-- -gGlu- -ADO--ADO-- 8.
HOOC--(CH.sub.2).sub.14--CO-- -gGlu- 9.
HOOC--(CH.sub.2).sub.14--CO-- -gGlu- -ADO--ADO-- 10.
HOOC--(CH.sub.2).sub.12--CO-- -gGlu- -ADO--ADO-- 11.
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-- -gGlu-
-ADO--ADO-- 12.
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-- -gGlu-
-ADO--ADO--ADO-- 13.
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-- -gGlu- 14.
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-- 2xgGlu- 15.
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-- -gGlu-
-Gly-Gly-Gly- 16.
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-- 2xgGlu-
-ADO--ADO-- 17.
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-- -gGlu-
-TtdSuc- 18. 4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO--
19. 4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO-- -gGlu-
-ADO--ADO--ADO--ADO-- 20.
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--CO--
--NH--CH.sub.2--(C.sub.6H.sub.10)--CO-- -gGlu- -ADO--ADO-- 21.
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9--CO-- -gGlu-
-ADO--ADO-- 22. 4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9--CO--
-gGlu- -ADO--ADO-- 23. 3-HO-Isoxazole-(CH.sub.2).sub.12--CO--
-gGlu- -ADO--ADO-- 24. HOS(O).sub.2--(CH.sub.2).sub.15--CO-- -gGlu-
-ADO--ADO-- --NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2-- 25.
HOS(O).sub.2--(CH.sub.2).sub.13--CO-- -gGlu- -ADO--ADO-- 26.
Tetrazolyl-(CH.sub.2).sub.15--CO--
--NH--SO.sub.2--(CH.sub.2).sub.3--CO-- -ADO--ADO--
--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2-- 27.
Tetrazolyl-(CH.sub.2).sub.12--CO-- -gGlu- -ADO--ADO-- 28.
Tetrazolyl-(CH.sub.2).sub.15--CO-- -gGlu- -ADO--ADO-- 29.
MeS(O).sub.2NH(CO)NH--(CH.sub.2).sub.12--CO -gGlu- -ADO--ADO--
Methods for Detection and Characterization
LCMS Methods
LCMS01 (See Table 1)
TABLE-US-00006 [0794] TABLE 1 LC-system: Waters Acquity UPLC.
Linear gradient: 5% to 95% B. System LC-system: Waters Acquity UPLC
Column: : Waters Acquity UPLC BEH, C-18, 1.7 .mu.m, 2.1 mm .times.
50 mm Detector: : Waters (Micromass) LCT Premier XE Detector setup
Ionisation method: ES Scanning range: 500-2000 amu Operating mode:
W mode positive/negative: positive mode Cone Voltage: 50 V Scantime
1 Interscandelay: 0.0 Conditions Linear gradient: 5% to 95% B
Gradient run-time: 4.0 minutes Total run-time: 7.0 minutes Flow
rate: 0.4 ml/min Column temperature: 40.degree. C. Eluents Solvent
A: 99.90% MQ-water, 0.1% formic acid Solvent B: 99.90%
acetonitrile, 0.1% formic acid Solvent C: NA Results Mass found is
the mass found of the compound specification M/z found is the
molecular ion found ((M + z)/z) of the and validation compound
Calculated Mass is the molecular weight of the desired compound
Calculated M/z is the molecular weight (M + z)/z of the desired
compound Purity: Total ion current (TIC) AUC of analyte peak, in
percent of total AUC excl solvent peak, as reported by system
software. Identity: Mass of each analyte mass peak expressed as m/z
from highest to lowest. Scanning range is the range scanned in the
method used. Detection method is e.g linear reflector
LCMS027 (See Table 2)
TABLE-US-00007 [0795] TABLE 2 Agilent 1290 infinity series UPLC,
LC/MSD TOF, 6 min, 5% to 95% B, 100-3200 amu, C18 System System:
Agilent 1290 infinity series UPLC Column: Eclipse C18+ 2.1 .times.
50 mm 1.8 u Detector: Agilent Technologies LC/MSD TOF 6230 (G6230A)
Detector setup Ionisation method: Agilent Jet Stream source
Scanning range: m/z min. 100, m/z max. 3200 linear reflector mode
positive mode Conditions Linear gradient: 5% to 95% B Gradient
run-time: 6 minutes 0-4.5 min 5-95% B, 4.5-5 95% B, 5-5.5 95-5% B
5.5-6 5% B Flow rate: 0.40 ml/min fixed Column temperature:
40.degree. C. Eluents Solvent A: 99.90% H.sub.2O, 0.02% TFA Solvent
B: 99.90% CH.sub.3CN, 0.02% TFA Solvent C: NA Results Mass found is
either m/z ((m + z)/z) of the compound for specification compounds
with m < 4000 or mass (average) as the result and validation of
a deconvolution using Masshunter Workstation Software Version
B.05.00 Build 5.0.519.13 SP1 (Agilent). Calculated Mass is the
average molecular weight of the desired compound Calculated m/z is
the molecular weight (m + z)/z of the desired compound
LCMS029 (See Table 3):
TABLE-US-00008 [0796] TABLE 3 Waters Acquity UPLC system, 6 min
(3.5 min), 5-(15-35)-100-100-5% B System System: Waters Acquity
UPLC system Column: ACQUITY UPLC BEH C18, 1.7 .mu.m, 2.1 mm .times.
50 mm column Detectors: Waters Acquity TUV Detector Detector setup
214 nm or 254 nm Conditions Step gradient: Gradient run 0.0-0.5 min
5-15% B 0.5-4.0 min 15-35% B 4.0-4.5 min 35-100% B 4.5-5.0 min
100-100% B 5.0-5.5 min 100-5% B 5.5-6.0 min 5-5%B Total run-time:
6.0 minutes Flow rate: 0.45 ml/min fixed Column temperature:
40.degree. C. Eluents Solvent A: 99.95% Water, 0.05%
Trifluoroacetic acid Solvent B: 99.95% Acetonitrile, 0.05%
Trifluoroacetic acid Results Purity defined as peak AUC in relation
to total AUC excl. solvent specification peak (in percent) as
reported by system software for each UV and validation wavelenght.
Retention time between 2.8 and 4.2 min, baseline separation of
analyte peak required. Peak AU value between 0.5 and 1.5. Results
uploaded are based on 214 nm
C. Example Compounds
C.1. Preparation of Example Compounds
Example 1
[0797]
N{293}-[4-[[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptad-
ecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acety-
l]amino]methyl]phenyl]methyl-[Ala299,
Leu301,Ile307,Arg309,Lys310]-LDL-R-(293-332)-peptide
##STR00036##
The peptide is SEQ ID NO: 2.
Compound Prepared by General Method a and C
[0798] LCMS029: Found m/3=1743.9; Found m/4=1308.1; Found
m/5=1046.7; Calc. mass=5229.1; Found mass=5229.6
Example 2
N{293}-[4-[[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-
amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino-
]methyl]phenyl]methyl-[Leu301,Arg309]-LDL-R-(293-332)-peptide
##STR00037##
[0799] The peptide is SEQ ID NO: 3.
Compound Prepared by General Method a and C
[0800] LCMS029: Found m/3=1749.5; Found m/4=1312.4; Found
m/5=1050.1; Calc. mass=5246.0; Found mass=5246.4
Example 3
N{Alpha}([Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-[2--
[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]ami-
no]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00038##
[0802] The peptide is SEQ ID NO: 4.
Compound Prepared by General Method B
[0803] LCMS01: Found m/4=1314.6; Found m/5=1052.1; Calc
mass=5255.9
Example 4
N{Epsilon-312}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309]-LDL-R-(293-332)-peptide
##STR00039##
[0804] The peptide is SEQ ID NO: 3.
Compound Prepared by General Method B
[0805] LCMS01: Found m/4=1282.3; Found m/5=1026.3; Calc
mass=5126.8
Example 5
N{293}-[4-[[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-
amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino-
]methyl]phenyl]methyl-[Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00040##
[0806] The peptide is SEQ ID NO: 6.
Compound Prepared by General Method a and C
[0807] LCMS029: Calc. mass=5246.92; Found mass=5247.37
Example 6
N{Epsilon-299}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Lys299,Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00041##
[0808] The peptide is SEQ ID NO: 7.
Compound Prepared by General Method B
[0809] LCMS01: Found m/3=1714.2; Found m/4=1286.1; Calc
mass=5140.85
Example 7
N{Epsilon-330}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys330]-LDL-R-(293-332)-peptide
##STR00042##
[0810] The peptide is SEQ ID NO: 8.
Compound Prepared by General Method B
[0811] LCMS01: Found m/3=1700.8; Found m/4=1275.8; Found
m/5=1020.9, Calc. mass 5099.7; Found mass=5099.75
Example 8
N{293}-[4-[[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(16-sulfohexadecanoylami-
no)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]me-
thyl]phenyl]methyl-[Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00043##
[0812] 23: The peptide is SEQ ID NO: 6.
Compound Prepared by General Method a and C
[0813] LCMS029: Found m/3=1757.1; Found m/4=1318.04; Calc.
mass=5268.95; Found mass=5269.39
Example 9
N{293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino-
)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N{Epsilon-
-330}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-b
carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethox-
y]ethoxy]acetyl]-[Leu301,Arg309,Glu312,Lys330]-LDL-R-(293-332)-peptide
##STR00044##
[0814] 24: The peptide is SEQ ID NO: 8.
Compound Prepared by General Method B
[0815] LCMS029: Found m/3=1939.2; Found m/4=1454.2; Calc.
mass=5815.6; Found mass=5816.1
Example 10
N{Epsilon-332}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys332]-LDL-R-(293-332)-peptide
##STR00045##
[0816] The peptide is SEQ ID NO: 11.
Compound Prepared by General Method B
[0817] LCMS01: Found m/4=1282.6; Found m/5=1026.3; Calc
mass=5126.8
Example 11
N{Epsilon-293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Lys293,Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00046##
[0818] The peptide is SEQ ID NO: 12.
Compound Prepared by General Method B
[0819] LCMS01: Found m/4=1300.6; Found m/5=1040.5; Calc
mass=5198.9
Example 12
N{Alpha}(N{Epsilon-293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyh-
eptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]-
acetyl]-[Lys293,Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptidyl)-N{Epsilon}-
[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butano-
yl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00047##
[0820] The peptide is SEQ ID NO: 13.
Compound Prepared by General Method B
[0821] LCMS029: Found m/2=3022.4; Found m/3=2015.3; Found
m/4=1511.8; Found m/5=1209.6; Found mass=6043.6; Calc.
mass=6042.9
Example 13
N{Alpha}(N{Epsilon-293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[Lys293,Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptidyl)-N{-
Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undeca-
noylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]L-
ys
##STR00048##
[0822] The peptide is SEQ ID NO: 13.
Compound Prepared by General Method B
[0823] LCMS029: Found m/2=3030.4; Found m/3=2020.7; Found
m/4=1515.7; Found m/5=1212.8; Found mass=6059.7; Calc.
mass=6058.8
Example 14
N{Alpha}(N{Epsilon-332}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[Leu301,Arg309,Glu312,Lys332]-LDL-R-(293-332)-peptidyl)-N{-
Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undeca-
noylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]L-
ys
##STR00049##
[0824] The peptide is SEQ ID NO: 15.
Compound Prepared by General Method B
[0825] LCMS01: Found m/4=1497.4; Found m/5=1198.3; Calc
mass=5986.7
Example 15
N{Alpha}(N{Epsilon-330}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[Leu301,Arg309,Glu312,Lys330]-LDL-R-(293-332)-peptidyl)-N{-
Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undeca-
noylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]L-
ys
##STR00050##
[0826] The peptide is SEQ ID NO: 16.
Compound Prepared by General Method B
[0827] LCMS01: Found m/3=1987.7; Found m/4=1491.0; Found
m/5=1193.0; Calc mass=5959.7
Example 16
N{Alpha}(N{Epsilon-321}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[Leu301,Arg309,Glu312,Lys321]-LDL-R-(293-332)-peptidyl)-N{-
Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undeca-
noylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]L-
ys
##STR00051##
[0828] The peptide is SEQ ID NO: 17.
Compound Prepared by General Method B
[0829] LCMS01: Found m/4=1500.9; Found m/5=1201.2; Calc
mass=6000.8
Example 17
N{Alpha}(N{Epsilon-312}-[2-[2-[[2-[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[Leu301,Arg309]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-[2--
[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]buta-
noyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00052##
[0830] The peptide is SEQ ID NO: 18.
Compound Prepared by General Method B
[0831] LCMS01: Found m/4=1498.2; Found m/5=1198.7; Calc
mass=5986.7
Example 18
N{Alpha}([Leu301,Arg309,Glu312,Glu321]-LDL-R-(293-332)-peptidyl)-N{Epsilon-
}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butan-
oyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00053##
[0832] The peptide is SEQ ID NO: 19.
Compound Prepared by General Method B
[0833] LCMS027: Found m/2=2635.7; Found m/3=1757.5; Found
m/4=1318.4; Found m/5=1054.9; Calc. mass=5270.0; Found
mass=5270.5
Example 19
N{293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino-
)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Leu301,A-
rg309,Glu312]-LDL-R-(293-332)-peptide
##STR00054##
[0834] The peptide is SEQ ID NO: 6.
Compound Prepared by General Method A and D
[0835] LCMS01: Found m/1=5127.8; Found m/3=1710.0; Found
m/4=1282.3; Found m/5=1026.5; Calc. mass=5127.8
Example 20
[0836]
N{Epsilon-321}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyhep-
tadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ac-
etyl]-[Leu301,Arg309,Glu312,Lys321]-LDL-R-(293-332)-peptide
##STR00055##
The peptide is SEQ ID NO: 21.
Compound Prepared by General Method B
[0837] LCMS01: Found m/4=1286.1; Found m/5=1029.1; Calc
mass=5140.9
Example 21
[0838]
N{Epsilon-324}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyhep-
tadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ac-
etyl]-[Leu301,Arg309,Glu312,Lys324]-LDL-R-(293-332)-peptide
##STR00056##
The peptide is SEQ ID NO: 22.
Compound Prepared by General Method B
[0839] LCMS01: Found m/4=1282.9; Found m/5=1026.5; Calc
mass=5127.8
Example 22
N{293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino-
)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Leu301,A-
rg309,Gln312]-LDL-R-(293-332)-peptide
##STR00057##
[0840] The peptide is SEQ ID NO: 23.
Compound Prepared by General Method B
[0841] LCMS029: Found m/3=1709.8; Found m/4=1282.6; Calc.
mass=5126.8; Found mass=5127.3
Example 23
N{Epsilon-332}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Glu321,Lys332]-LDL-R-(293-332)-peptide
##STR00058##
[0842] The peptide is SEQ ID NO: 24.
Compound Prepared by General Method B
[0843] LCMS029: Found m/2=2571.2; Found m/3=1714.5; Found
m/4=1286.1; Found m/z=5141.4; Calc. mass=5140.9
Example 24
N{Epsilon-293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Lys293,Leu301,Arg309,Glu312,Glu321]-LDL-R-(293-332)-peptide
##STR00059##
[0844] The peptide is SEQ ID NO: 25.
Compound Prepared by General Method B
[0845] LCMS029: Found m/2=2607.2; Found m/3=1738.5; Found
m/4=1304.1; Found mass=5213.5; Calc. mass=5212.9
Example 25
N{Alpha-293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoy-
lamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N{E-
psilon-293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-
amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Lys-
293,Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00060##
[0846] The peptide is SEQ ID NO: 12.
Compound Prepared by General Method B
[0847] LCMS029: Found m/4=1479.7; Calc. mass=5914.8 Da; Found
mass=5914.3
Example 26
N{Epsilon-300}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Lys300,Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00061##
[0848] The peptide is SEQ ID NO: 27.
Compound Prepared by General Method B
[0849] LCMS01: Found m/4=1286.9; Found m/5=1029.7; Calc
mass=5141.8
Example 27
N{Epsilon-293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl],N{Epsilon-294}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Lys293,Lys294,Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00062##
[0850] The peptide is SEQ ID NO: 28.
Compound Prepared by General Method B
[0851] LCMS01: Found m/1=5957.6; Found m/4=1490.4; Found
m/5=1192.3; Calc m/1=5957.7
Example 28
N{Epsilon-293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl],N{Epsilon-312}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Lys293,Leu301,Arg309]-LDL-R-(293-332)-peptide
##STR00063##
[0852] The peptide is SEQ ID NO: 29.
Compound Prepared by General Method B
[0853] LCMS01: Found m/1=5929.4; Found m/4=1483.3; Found
m/5=1186.8; Calc m/1=5929.7
Example 29
N{Epsilon-309}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Lys309,Glu312]-LDL-R-(293-332)-peptide
##STR00064##
[0854] The peptide is SEQ ID NO: 30.
Compound Prepared by General Method B
[0855] LCMS01: Found m/3=1700.8; Found m/4=1275.8; Found
m/5=1020.9; Calc mass=5099.8 (1A)
Example 30
N{Epsilon-318}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys318]-LDL-R-(293-332)-peptide
##STR00065##
[0856] The peptide is SEQ ID NO: 31. LCMS01: Found m/4=1286.5;
Found m/5=1029.5; Calc mass=5142.8
Example 31
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-peptidyl)-N{-
Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undeca-
noylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]L-
ys
##STR00066##
[0857] The peptide is SEQ ID NO: 32.
Compound Prepared by General Method B
[0858] LCMS01: Found m/1=6002.8; Found m/4=1501.6; Found
m/5=1201.5; Calc m/1=6002.7
Example 32
N{Epsilon-326}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys326]-LDL-R-(293-332)-peptide
##STR00067##
[0859] The peptide is SEQ ID NO: 33.
Compound Prepared by General Method B
[0860] LCMS01: Found m/3=1719.8; Found m/4=1290.1; Found
m/5=1032.3; Calc mass=5156.8
Example 33
N{Epsilon-325}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys325]-LDL-R-(293-332)-peptide
##STR00068##
[0861] The peptide is SEQ ID NO: 34.
Compound Prepared by General Method B
[0862] LCMS01: Found m/3=1715.1; Found m/4=1286.6; Found
m/5=1029.5; Calc mass=5142.8
Example 34
N{Epsilon-323}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys323]-LDL-R-(293-332)-peptide
##STR00069##
[0863] The peptide is SEQ ID NO: 35.
Compound Prepared by General Method B
[0864] LCMS01: Found m/1=5108.8; Found m/3=1703.8; Found
m/4=1278.1; Found m/5=1022.5
Example 35
N{Epsilon-322}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys322]-LDL-R-(293-332)-peptide
##STR00070##
[0865] The peptide is SEQ ID NO: 36.
Compound Prepared by General Method B
[0866] LCMS01: Found m/1=5198.9; Found m/3=1733.8; Found
m/4=1300.6; Found m/5=1040.7
Example 36
N{Epsilon-320}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys320]-LDL-R-(293-332)-peptide
##STR00071##
[0867] The peptide is SEQ ID NO: 37.
Compound Prepared by General Method B
[0868] LCMS01: Found m/3=1720.4; Found m/4=1290.3; Found
m/5=1032.5; Calc mass=5158.8
Example 37
N{Epsilon-329}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys329]-LDL-R-(293-332)-peptide
##STR00072##
[0869] The peptide is SEQ ID NO: 38.
Compound Prepared by General Method B
[0870] LCMS029: Found m/2=2550.7; Found m/3=1700.8; Found
m/4=1275.9; Calc. mass=5099.8; Found mass=5100.5
Example 38
N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-peptide
##STR00073##
[0871] The peptide is SEQ ID NO: 39.
Compound Prepared by General Method B
[0872] LCMS01: Found m/1=5143.0; Found m/4=1286.0; Found
m/5=1029.0; Calc m/1=5142.8
Example 39
N{Epsilon-328}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys328]-LDL-R-(293-332)-peptide
##STR00074##
[0873] The peptide is SEQ ID NO: 40.
Compound Prepared by General Method B
[0874] LCMS029: Found m/2=2564.7; Found m/3=1710.2; Found
m/4=1282.9; Found 5127.8; Calc. mass=5128.5
Example 40
N{Epsilon-316}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys316]-LDL-R-(293-332)-peptide
##STR00075##
[0875] The peptide is SEQ ID NO: 41.
Compound Prepared by General Method B
[0876] LCMS01: Found m/3=1709.7; Found m/4=1282.3; Found
m/5=1026.1; Calc mass=5126.8
Example 41
N{Epsilon-315}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys315]-LDL-R-(293-332)-peptide
##STR00076##
[0877] The peptide is SEQ ID NO: 42.
Compound Prepared by General Method B
[0878] LCMS01: Found m/3=1698.3; Found m/4=1273.8; Found
m/5=1019.3; Calc m/1=5092.8
Example 42
N{Alpha}([His300,Leu301,Arg309,Arg312]-LDL-R-(293-332)-peptidyl)-N{Epsilon-
}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butan-
oyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00077##
[0879] The peptide is SEQ ID NO: 43.
Compound Prepared by General Method B
[0880] LCMS01: Found m/1=5306.3; Found m/3=1768.7; Found
m/4=1327.1; Found m/5=1061.7
Example 43
N{Epsilon-314}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys314]-LDL-R-(293-332)-peptide
##STR00078##
[0881] The peptide is SEQ ID NO: 44.
Compound Prepared by General Method B
[0882] LCMS01: Found m/4=1300.2; Found m/5=1040.2; Calc
mass=5198.9
Example 44
N{Epsilon-311}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Lys311,Glu312]-LDL-R-(293-332)-peptide
##STR00079##
[0883] The peptide is SEQ ID NO: 45.
Compound Prepared by General Method B
[0884] LCMS01: Found m/3=1714.9; Found m/4=1286.2; Found
m/5=1029.2; Calc mass=5142.8
Example 45
N{Epsilon-307}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Lys307,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00080##
[0885] The peptide is SEQ ID NO: 46.
Compound Prepared by General Method B
[0886] LCMS01: Found m/3=1919.8; Found m/4=1290.1; Found
m/5=1032.3; Calc mass=5156.8
Example 46
N{Alpha}([Leu301,Ser309,Arg312]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-[2--
[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]ami-
no]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00081##
[0887] The peptide is SEQ ID NO: 47.
Compound Prepared by General Method B
[0888] LCMS01: Found m/3=1738.8; Found m/4=1304.1; Found
m/5=1043.5; Found mass=5214.3
Example 47
N{Alpha}([Leu301,Ser309,Glu312]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-[2--
[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]ami-
no]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00082##
[0889] The peptide is SEQ ID NO: 48.
Compound Prepared by General Method B
[0890] LCMS01: Found m/1=5187.2; Found m/3=1729.7; Found
m/4=1297.2; Found m/5=1038.4; Calc m/1=5186.8
Example 48
Ala299,Leu301,Ile307,Arg309,Lys310]-LDL-R-(293-332)-peptide
##STR00083##
[0891] The peptide is SEQ ID NO: 2.
Compound Prepared by General Method A
[0892] LCMS01: Found m/3=1465.3; Found m/4=1099.3; Found m/5=879.6;
Calc=4391.0
Example 49
[Leu301,Arg309]-LDL-R-(293-332)-peptide
##STR00084##
[0893] The peptide is SEQ ID NO: 3.
Compound Prepared by General Method A.
[0894] LCMS01: Found m/3=1470.3; Found m/4=1103.0; Found m/5=882.6;
Calc=4407.9
Example 50
[Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00085##
[0895] The peptide is SEQ ID NO: 6.
Compound Prepared by General Method A.
[0896] LCMS01: Found m/3=1471.3; Found m/4=1103.7; Found m/5=883.2;
Calc=4411.9
Example 51
N{293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino-
)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Leu301,T-
yr306,Ser309,Glu312]-LDL-R-(293-332)-peptide
##STR00086##
[0897] The peptide is SEQ ID NO: 49.
Compound Prepared by General Method B
[0898] LCMS029: Found m/3=1695.8; Calc mass=5085.1
Example 52
N{Alpha-293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoy-
lamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[As-
n293,Leu301,Ser309,Glu312]-LDL-R-(293-332)-peptide
##STR00087##
[0899] The peptide is SEQ ID NO: 50. LCMS29: Found m/3=1706.1, Calc
mass=5115.7
Compound Prepared by General Method B
Example 53
N{Epsilon-306}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Lys306,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00088##
[0900] The peptide is SEQ ID NO: 51.
Compound Prepared by General Method B
[0901] LCMS01: Found m/3=1707.0; Found m/4=1280.3; Found
m/5=1024.4; Calc mass=5118.8
Example 54
N{Epsilon-305}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Lys305,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00089##
[0902] The peptide is SEQ ID NO: 52.
Compound Prepared by General Method B
[0903] LCMS01: Found m/3=1723.8; Found m/4=1292.8; Found
m/5=1034.4; Calc mass=5168.8
Example 55
N{Epsilon-303}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Lys303,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00090##
[0904] The peptide is SEQ ID NO: 53.
Compound Prepared by General Method B
[0905] LCMS01: Found m/3=1733.7; Found m/4=1300.3; Found
m/5=1040.5; Calc mass=5198.9
Example 56
N{Epsilon-302}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Lys302,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00091##
[0906] The peptide is SEQ ID NO: 54.
Compound Prepared by General Method B
[0907] LCMS01: Found m/3=1733.7; Found m/4=1300.3; Found
m/5=1040.5; Calc mass=5198.9
Example 57
N{Alpha}([Asn293,His300,Leu301,Arg309,Arg312]-LDL-R-(293-332)-peptidyl)-N{-
Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamin-
o)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00092##
[0908] The peptide is SEQ ID NO: 55.
Compound Prepared by General Method B
[0909] LCMS01: Found m/4=1341.5; Found m/5=1073.3; Calc
mass=5363
Example 58
N{Epsilon-301}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Lys301,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00093##
[0910] The peptide is SEQ ID NO: 56.
Compound Prepared by General Method B
[0911] LCMS01: Found m/3=1715.2; Found m/4=1286.6; Found
m/5=1029.5; Calc mass=5142.8
Example 59
N{Epsilon-298}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Lys298,Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00094##
[0912] The peptide is SEQ ID NO: 57.
Compound Prepared by General Method B
[0913] LCMS01: Found m/3=1715.1; Found m/4=1286.3; Found
m/5=1029.3; Calc m/z=5142.8
Example 60
N{Alpha}([Asn293,
Leu301,Arg309,Arg312]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-[2-[2-[[2-[2-
-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy-
]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00095##
[0914] The peptide is SEQ ID NO: 58.
Compound Prepared by General Method B
[0915] LCMS01: Found m/3=1780.7; Found m/4=1335.5; Found
m/5=1068.4; Calc mass=5340.1
Example 61
N{Epsilon-332}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Ile307,Lys332]-LDL-R-(293-332)-peptide
##STR00096##
[0916] The peptide is SEQ ID NO: 59.
Compound Prepared by General Method B
[0917] LCMS01: Found m/3=1700.0; Found m/4=1275.1; Found
m/5=1020.3; Calc mass=5097.8
Example 62
N{Epsilon-332}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Tyr306,Glu312,Lys332]-LDL-R-(293-332)-peptide
##STR00097##
[0918] The peptide is SEQ ID NO: 60.
Compound Prepared by General Method B
[0919] LCMS01: Found m/3=1704.3; Found m/4=1278.5; Found
m/5=1030.4; Calc m/z=5110.8
Example 63
N{Epsilon-332}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Ile307,Glu312,Lys332]-LDL-R-(293-332)-peptide
##STR00098##
[0920] The peptide is SEQ ID NO: 61.
Compound Prepared by General Method B
[0921] LCMS01: Found m/3=1700.3; Found m/4=1275.2; Found m/5=Calc
mass=5098.8
Example 64
N{293}-[4-[[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl-
amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino-
]methyl]phenyl]methyl-[His300,Leu301,Arg309]-LDL-R-(293-332)-peptide
##STR00099##
[0922] The peptide is SEQ ID NO: 62.
Compound Prepared by General Method A+C
[0923] LCMS01: Found m/3=1757.1; Found m/4=1318.1; Found
m/5=1054.2; Calc mass=5269.0
Example 65
N{293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino-
)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Pro300,L-
eu301,Ile307,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00100##
[0924] The peptide is SEQ ID NO: 5.
Compound Prepared by General Method B
[0925] LCMS029: Found m/3=1709.1; Calc mass=5124.8
Example 66
N{Alpha}([Asn293,Leu301,Ile307,Arg309,Asp312]-LDL-R-(293-332)-peptidyl)-N{-
Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamin-
o)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00101##
[0926] The peptide is SEQ ID NO: 9.
Compound Prepared by General Method B
[0927] LCMS029: Found m/4=1329.1; Calc mass=5313.0
Example 67
N{Alpha}([Asn293,Leu301,Arg309,Asp312]-LDL-R-(293-332)-peptidyl)-N{Epsilon-
}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butan-
oyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00102##
[0928] The peptide is SEQ ID NO: 10.
Compound Prepared by General Method B
[0929] LCMS029: Found m/4=1325.6; Calc mass=5299.0
Example 68
N{293}-[4-[[[2-[2-[2-[[2-[2-[2-[4-[16-(1H-tetrazol-5-yl)hexadecanoylsulfam-
oyl]butanoylamino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]me-
thyl]phenyl]methyl-[Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00103##
[0930] The peptide is SEQ ID NO: 6.
Compound Prepared by General Method C
[0931] LCMS029: Found m/4=1320.1; Calc mass=5276.98
Example 69
N{Epsilon-328}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys328,His329]-LDL-R-(293-332)-peptide
##STR00104##
[0932] The peptide is SEQ ID NO: 14.
Compound Prepared by General Method B
[0933] LCMS029: Found m/4=1278.09; Calc mass=5108.8 Da
Example 70
N{Epsilon-332}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Asp295,Leu301,Arg309,Glu312,Lys332]-LDL-R-(293-332)-peptide
##STR00105##
[0934] The peptide is SEQ ID NO: 20.
Compound Prepared by General Method B
[0935] LCMS029: Found m/4=1282.84; Calc mass=5127.8 Da
Example 71
N{Epsilon-312}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
His300,Leu301,Arg309]-LDL-R-(293-332)-peptide
##STR00106##
[0936] The peptide is SEQ ID NO: 62.
Compound Prepared by General Method B
[0937] LCMS01: Found m/3=1717.5 Found m/4=1288.2 Found
m/5=1030.4-Calc mass=5149.9-
Example 72
N{293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino-
)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[His300,L-
eu301,Ile307,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00107##
[0938] The peptide is SEQ ID NO: 26.
Compound Prepared by General Method B
[0939] LCMS029: Found m/4=1292.1; Calc mass=5164.8 Da
Example 73
N{Epsilon-296}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Lys296,Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00108##
[0940] The peptide is SEQ ID NO: 63.
Compound Prepared by General Method B
[0941] LCMS01: Found m/3=1709.9; Found m/4=1282.6; Calc mass
5126.8
Example 74
N{Epsilon-294}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Lys294,Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00109##
[0942] The peptide is SEQ ID NO: 64.
Compound Prepared by General Method B
[0943] LCMS01: Found m/4=1289.7; Found m/5=1031.7; Calc
mass=5154.8
Example 75
N{Epsilon}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyla-
mino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-Lys[L-
eu301,Arg309,Glu312]-LDL-R-(293-332)-peptide
##STR00110##
[0944] The peptide is SEQ ID NO: 65.
Compound Prepared by General Method B
[0945] LCMS01: Found m/3=1752.9; Found m/4=1315.0; Calc
mass=5255.9
Example 76
N{Epsilon-328}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Gly294,Leu301,Arg309,Glu312,Lys328],des-Gly293-LDL-R-(294-332)-peptide
##STR00111##
[0946] The peptide is SEQ ID NO: 66.
Compound Prepared by General Method B
[0947] LCMS01: Found m/3=1676.6 Found m/4=1257.7 Found
m/5=1006)-Calc. mass=5026.7
Example 77
N{Alpha}([Leu301,Asp306,Arg309,Glu312,Gly324]-LDL-R-(293-332)-peptidyl)-N{-
Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamin-
o)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00112##
[0948] The peptide is SEQ ID NO: 67.
Compound Prepared by General Method B
[0949] LCMS029: Found m/3=1721.8; Calc mass=5162.8 Da
Example 78
N{Alpha}(N{293}-[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-car-
boxyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]etho-
xy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Leu301,Asp306,Arg309,Glu312]-
-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-
-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl-
]amino]ethoxy]ethoxy]acetyl]Lys
##STR00113##
[0950] The peptide is SEQ ID NO: 68.
Compound Prepared by General Method B
[0951] LCMS029: Found m/3=1528.7; Calc mass=6110.8 Da
Example 79
N{Alpha}(N{Epsilon-321}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carbo-
xyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Arg309,Glu312,Lys321]-LDL-R-(293-332)-peptidyl)-N{Ep-
silon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoyl-
amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00114##
[0952] The peptide is SEQ ID NO: 17.
Compound Prepared by General Method B
[0953] LCMS01: Found m/4=1493.9; Found m/5=1195.5; Calc
mass=5972.7
Example 80
N{Alpha}([Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-[2--
[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]ami-
no]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00115##
[0954] The peptide is SEQ ID NO: 4.
Compound Prepared by General Method B
[0955] LCMS01: Found m/3=1743.6; Found m/4=1307.9; Found m/5=1046.4
Calc mass=5227.9
Example 81
N{Alpha}([Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-[2--
[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecanoylamino)butanoyl]amin-
o]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00116##
[0956] The peptide is SEQ ID NO: 4.
Compound Prepared by General Method B
[0957] LCMS01: Found m/3=1762.3; Found m/4=1321.7; Found
m/5=1057.8; Calc mass=5284.0
Example 82
N{Alpha}([Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[(4S)--
4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]Lys
##STR00117##
[0958] The peptide is SEQ ID NO: 4.
Compound Prepared by General Method B
[0959] LCMS01: Found m/3=1656.2; Found m/4=1242.4; Found m/5=994.0;
Calc mass=4965.6
Example 83
N{Alpha}(N{Epsilon-321}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxyt-
ridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ac-
etyl]-[Leu301,Arg309,Glu312,Lys321]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-
-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]a-
mino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00118##
[0960] The peptide is SEQ ID NO: 17.
Compound Prepared by General Method B
[0961] LCMS01: Found m/3=1958.5; Found m/4=1468.9; Found
m/5=1175.3; Calc mass=5872.7
Example 84
N{Alpha}(N{Epsilon-321}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxyp-
entadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]-
acetyl]-[Leu301,Arg309,Glu312,Lys321]-LDL-R-(293-332)-peptidyl)-N{Epsilon}-
[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butano-
yl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00119##
[0962] The peptide is SEQ ID NO: 17
Compound Prepared by General Method B
[0963] LCMS01: Found m/4=1483.1; Found m/5=1186.6; Calc
mass=5928.8
Example 85
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carbo-
xyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[His300,Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-peptidy-
l)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)d-
ecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acety-
l]Lys
##STR00120##
[0964] The peptide is SEQ ID NO: 69.
Compound Prepared by General Method B
[0965] LCMS01: Found m/4=1500.1 Found m/5=1200.3 Found m/z=1000.
Calc mass=5997.7
Example 86
N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl],N{Epsilon-328}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Arg309,Glu312,Lys313,Lys328]-LDL-R-(293-332)-peptide
##STR00121##
[0966] The peptide is SEQ ID NO: 70.
Compound Prepared by General Method B
[0967] LCMS01: Found m/4=1469.3; Found m/5=1175.8; Calc
mass=5874.6
Example 87
N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl],N{Epsilon-324}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Arg309,Glu312,Lys313,
Lys324]-LDL-R-(293-332)-peptide
##STR00122##
[0968] The peptide is SEQ ID NO:71.
Compound Prepared by General Method B
[0969] LCMS01: Found m/4=1469.1; Found m/5=1175.5; Calc
mass=5874.6
Example 88
N{293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecan-
oylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N-
{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)-
undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ac-
etyl]-[Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-peptide
##STR00123##
[0970] The peptide is SEQ ID NO: 39.
Compound Prepared by General Method B
[0971] LCMS01: Found m/4=1469.3; Found m/5=1175.7; Calc
mass=5874.5
Example 89
N{Alpha}(N{Epsilon-324}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[Leu301,Arg309,Glu312,Lys324]-LDL-R-(293-332)-peptidyl)-N{-
Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undeca-
noylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]L-
ys
##STR00124##
[0972] The peptide is SEQ ID NO:72.
Compound Prepared by General Method B
[0973] LCMS01: Found m/4=1497.6; Found m/5=1198.3; Calc
mass=5987.7
Example 90
N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl],N{Epsilon-321}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Arg309,Glu312,Lys313,Lys321]-LDL-R-(293-332)-peptide
##STR00125##
[0974] The peptide is SEQ ID NO: 73.
Compound Prepared by General Method B
[0975] LCMS01: Found m/4=1472.6; Found m/5=1178.3; Calc
mass=5887.6
Example 91
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carbo-
xyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[His300,Leu301,Arg309,Glu312,Lys313],des-Gly293-LDL-R-(294-3-
32)-peptidyl)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carbo-
xyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]Lys
##STR00126##
[0976] The peptide is SEQ ID NO: 74.
Compound Prepared by General Method B
[0977] LC-MS: Found m/3=1981, m/4=1486: Calculated mass=5940.6
Example 92
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[His300,Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-pepti-
dyl)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl]Lys
##STR00127##
[0978] The peptide is SEQ ID NO: 69.
Compound Prepared by General Method B
[0979] LCMS01: Found m/4=1507.3 Found m/5=1205.9 Calc
mass=6025.7
Example 93
N{292}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecan-
oylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N-
{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)-
undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ac-
etyl]-Ala[Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-peptide
##STR00128##
[0980] The peptide is SEQ ID NO: 75.
Compound Prepared by General Method B
[0981] LCMS01: Found m/4=1487.1; Found m/5=1190.0; Calc
mass=5945.6
Example 94
N{294}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecan-
oylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N-
{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)-
undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ac-
etyl]-[Leu301,Arg309,Glu312,Lys313],
des-Gly293-LDL-R-(294-332)-peptide
##STR00129##
[0982] The peptide is SEQ ID NO: 76.
Compound Prepared by General Method B
[0983] LCMS01: Found m/4=1455.1; Calc mass=5817.5
Example 95
N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[-
Leu301,Arg309,Glu312,Lys313],des-Gly293-LDL-R-(294-332)-peptide
##STR00130##
[0984] The peptide is SEQ ID NO: 76.
Compound Prepared by General Method B
[0985] LCMS01: Found m/4=1272.4; Found m/5=1017.9; Calc
mass=5085.7
Example 96
N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl],N{Epsilon-332}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Arg309,Glu312,Lys313,
Lys332]-LDL-R-(293-332)-peptide
##STR00131##
[0986] The peptide is SEQ ID NO: 77.
Compound Prepared by General Method B
[0987] LCMS01: Found m/4=1469.1; Found m/5=1175.5; Calc
mass=5873.6
Example 97
N{Alpha}(N{Epsilon-328}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[Leu301,Arg309,Glu312,Lys328]-LDL-R-(293-332)-peptidyl)-N{-
Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undeca-
noylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]L-
ys
##STR00132##
[0988] The peptide is SEQ ID NO: 78.
Compound Prepared by General Method B
[0989] LCMS28: Found m/3=1996.9; Found m/4=1497.9; Calc
mass=5987.7
Example 98
N{Alpha}(N{Epsilon-313}-[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoyl-
amino]butanoyl]-[Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-peptidyl)-N{-
Epsilon}[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]L-
ys
##STR00133##
[0990] The peptide is SEQ ID NO:32.
Compound Prepared by General Method B
[0991] LCMS01: Found m/3=1808.1; Found m/4=1356.4; Found
m/5=1085.3; Calc mass=5422.1
Example 99
N{Alpha}(N{Epsilon-313}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[11-(4-carbox-
yphenoxy)undecanoylamino]butanoyl]amino]butanoyl]-[Leu301,Arg309,Glu312,Ly-
s313]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[(4S)-4-carboxy-4-[[(4S)-4-carbo-
xy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]butanoyl]Lys
##STR00134##
[0992] The peptide is SEQ ID NO: 32.
Compound Prepared by General Method B
[0993] LCMS01: Found m/3=1894.2; Found m/4=1420.9; Found
m/5=1136.9; Calc mass=5680.3
Example 100
N{Alpha}(N{Epsilon-313}-[2-[[2-[[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenox-
y)undecanoylamino]butanoyl]amino]acetyl]amino]acetyl]amino]acetyl]-[Leu301-
,Arg309,Glu312,Lys313]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-[[2-[[2-[[(4-
S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]acetyl-
]amino]acetyl]amino]acetyl]Lys
##STR00135##
[0994] The peptide is SEQ ID NO: 32.
Compound Prepared by General Method B
[0995] LCMS01: Found m/3=1922.2; Found m/4=1441.9; Found
m/5=1153.7; Calc mass=5764.4
Example 101
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-car-
boxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]butanoyl]amino-
]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Leu301,Arg309,Glu312,Ly-
s313]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carb-
oxy-4-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]am-
ino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00136##
[0996] The peptide is SEQ ID NO: 32.
Compound Prepared by General Method B
[0997] LCMS01: Found m/4=1566.2; Found m/5=1252.9; Calc
mass=6260.9
Example 102
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(3-carbo-
xyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-peptidyl)-N{Ep-
silon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(3-carboxyphenoxy)decanoyl-
amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00137##
[0998] The peptide is SEQ ID NO: 32.
Compound Prepared by General Method B
[0999] LCMS01: Found m/4=1494.6; m/5=1195.9 Calc mass=5974.6
Example 103
Ala299,Leu301,Ile307,Arg309-LDL-R-(293-332)-peptide
##STR00138##
[1000] The peptide is SEQ ID NO: 79.
Compound Prepared by General Method A.
[1001] LCMS01: Found m/2=2191.4; Found m/3=1461.0; Calc=4381.0
Example 104
Leu301,Arg309,Lys310-LDL-R-(293-332)-peptide
##STR00139##
[1002] The peptide is SEQ ID NO: 80.
Compound Prepared by General Method A
[1003] LCMS01: Found m/3=1475.3; Found m/4=1107.0; Calc=4424.0
Example 105
Leu301-LDL-R-(293-332)-peptide
##STR00140##
[1004] The peptide is SEQ ID NO: 81.
Compound Prepared by General Method A
[1005] LCMS01: Found m/3=1456.3; Found m/4=1217.0; Calc=4368.9
Example 106
N{Alpha}([His300,Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptidyl)-N{Epsilon-
}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butan-
oyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00141##
[1006] The peptide back-bone is SEQ ID NO:82.
Compound Prepared by General Method B
[1007] LCMS027: Found m/3=1760.5; Found m/4=1320.6; Found
m/5=1056.7; Calc mass=5279.0
Example 107
N{Alpha}(N{293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenox-
y)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]-
acetyl]-[Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-[2-[-
2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)
undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl]Lys
##STR00142##
[1008] The peptide back-bone is SEQ ID NO: 4
Compound Prepared by General Method B
[1009] LCMS29: Found m/3=1996.9; Found m/4=1497.9; Found
m/5=1198.6; Calc mass=5987.7
Example 108
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carbo-
xyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[His300,Leu301,Arg309,Glu312,Lys313]-LDL-R-(295-332)-peptidy-
l)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)d-
ecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acety-
l]Lys
##STR00143##
[1010] The peptide back-bone is SEQ ID NO: 83
Compound Prepared by General Method B
[1011] LCMS01: Found m/4=1460.8; Found m/5=1168.7; Calc
mass=5839.5
Example 109
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[13-(3-hydro-
xy-1,2-oxazol-5-yl)tridecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]am-
ino]ethoxy]ethoxy]acetyl]-[His300,Leu301,Arg309,Glu312,Lys313]-LDL-R-(293--
332)-peptidyl)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[13-(3-hydr-
oxy-1,2-oxazol-5-yl)tridecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]a-
mino]ethoxy]ethoxy]acetyl]Lys
##STR00144##
[1012] The peptide back-bone is SEQ ID NO: 69
Compound Prepared by General Method B
[1013] LCMS01: Found m/4=1495.0; Found m/5=1196.0; Calc
mass=5975.7
Example 110
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[13-(3-hydro-
xy-1,2-oxazol-5-yl)tridecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]am-
ino]ethoxy]ethoxy]acetyl]-[Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-pe-
ptidyl)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[13-(3-hydroxy-1,2-
-oxazol-5-yl)tridecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]et-
hoxy]ethoxy]acetyl]Lys
##STR00145##
[1014] The peptide back-bone is SEQ ID NO: 32
Compound Prepared by General Method B
[1015] LCMS01: Found m/4=1489.0; Found m/5=1191.0; Calc
mass=5952.7
Example 111
N{Alpha}(N{Epsilon-309}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[Leu301,Lys309,Glu312]-LDL-R-(293-332)-peptidyl)-N{Epsilon-
}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylami-
no]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00146##
[1016] The peptide back-bone is SEQ ID NO:84
Compound Prepared by General Method B
[1017] LCMS29: Found m/3=1987.6; Found m/4=1490.9; Found
m/5=1193.0; Calc mass=5959.7
Example 112
N{Alpha}(N{Epsilon-324}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[Leu301,Tyr306,Glu312,Lys324]-LDL-R-(293-332)-peptidyl)-N{-
Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undeca-
noylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]L-
ys
##STR00147##
[1018] The peptide back-bone is SEQ ID NO: 85
Compound Prepared by General Method B
[1019] LCMS29: Found m/3=1991.6; Found m/4=1493.9; Found
m/5=1195.1; Calc mass=5971.7
Example 113
N{Alpha}(N{Epsilon-314}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[His300,Leu301,Arg309,Glu312,Lys314]-LDL-R-(293-332)-pepti-
dyl)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl]Lys
##STR00148##
[1020] The peptide back-bone is SEQ ID NO: 86
Compound Prepared by General Method B
[1021] LCMS29: Found m/3=2028.3; Found m/4=1521.5; Calc
mass=6081.8
Example 114
N{Alpha}(N{293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenox-
y)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]-
acetyl]-[Trp294,Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptidyl)-N{Epsilon}-
[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamin-
o]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00149##
[1022] The peptide back-bone is SEQ ID NO: 87
Compound Prepared by General Method B
[1023] LCMS29: Found m/3=2025.3; Found m/4=1519.2; Found
m/5=1215.6; Calc mass=6072.8
Example 115
N{Epsilon-309}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl],N{Epsilon-328}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Lys309,Glu312,Lys328]-LDL-R-(293-332)-peptide
##STR00150##
[1024] The peptide back-bone is SEQ ID NO: 88
Compound Prepared by General Method B
[1025] LCMS27: Found m/2=2916.7; Found m/3=1944.9; Found
m/4=1458.9; Calc mass=5831.5
Example 116
N{Epsilon-309}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl]-N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Lys309,Glu312,Lys313]-LDL-R-(293-332)-peptide
##STR00151##
[1026] The peptide back-bone is SEQ ID NO: 89
Compound Prepared by General Method B
[1027] LCMS29: Found m/2=2924.1; Found m/3=1949.6 Found m/4=1462.4;
Calc mass=5846.5
Example 117
N{Alpha}(N{294}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenox-
y)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]-
acetyl]-[Leu301,Arg309,Glu312],des-Gly293-LDL-R-(294-332)-peptidyl)-N{Epsi-
lon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoyl-
amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00152##
[1028] The peptide back-bone is SEQ ID NO: 90
Compound Prepared by General Method B
[1029] LCMS29: Found m/3=1977.6; Found m/4=1483.5; Found
m/5=1187.2; Calc mass=5930.6
Example 118
N{Epsilon-324}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl],N{Epsilon-328}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Arg309,Glu312,Lys324,Lys328]-LDL-R-(293-332)-peptide
##STR00153##
[1030] The peptide back-bone is SEQ ID NO: 91
Compound Prepared by General Method B
[1031] LCMS27: Found m/2=2930.4; Found m/3=1953.9; Found
m/4=1465.7; Calc mass=5859.6
Example 119
N{Alpha}(N{292}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenox-
y)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]-
acetyl]-Ala[Leu301,Arg309,Glu312]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-[-
2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]bu-
tanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00154##
[1032] The peptide back-bone is SEQ ID NO: 92
Compound Prepared by General Method B
[1033] LCMS29: Found m/3=2020.6; Found m/4=1515.7; Found
m/5=1212.8; Calc mass=6058.8
Example 120
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[Leu301,Tyr306,Arg309,Glu312,Lys313]-LDL-R-(293-332)-pepti-
dyl)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl]Lys
##STR00155##
[1034] The peptide back-bone is SEQ ID NO: 93
Compound Prepared by General Method B
[1035] LCMS29: Found m/3=2010.2; Found m/4=1508.2; Found
m/5=1206.8; Calc mass=6028.7
Example 121
N{293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecan-
oylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N-
{Epsilon-332}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)-
undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ac-
etyl]-[Leu301,Arg309,Glu312,Lys332]-LDL-R-(293-332)-peptide
##STR00156##
[1036] The peptide back-bone is SEQ ID NO: 11
Compound Prepared by General Method B
[1037] LCMS27: Found m/2=2930.3; Found m/3=1953.7; Found
m/4=1465.8; Calc mass=5858.6
Example 122
N{293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecan-
oylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N-
{Epsilon-328}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)-
undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ac-
etyl]-[Leu301,Arg309,Glu312,Lys328]-LDL-R-(293-332)-peptide
##STR00157##
[1038] The peptide back-bone is SEQ ID NO: 40
Compound Prepared by General Method B
[1039] LCMS27: Found m/2=2930.1; Found m/3=1953.9; Found
m/4=1465.7; Calc mass=5859.6
Example 123
N{293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecan-
oylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N-
{Epsilon-324}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)-
undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ac-
etyl]-[Leu301,Arg309,Glu312,Lys324]-LDL-R-(293-332)-peptide
##STR00158##
[1040] The peptide back-bone is SEQ ID NO: 22
Compound Prepared by General Method B
[1041] LCMS29: Found m/2=2930.9; Found m/3=1954.3; Found
m/4=1465.9; Calc mass=5859.6
Example 124
N{Epsilon-309}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl],N{Epsilon-332}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Lys309,Glu312,Lys332]-LDL-R-(293-332)-peptide
##STR00159##
[1042] The peptide back-bone is SEQ ID NO: 94
Compound Prepared by General Method B
[1043] LCMS29: Found m/2=2916.1; Found m/3=1944.2; Found
m/4=1458.4; Calc mass=5830.6
Example 125
N{Epsilon-309}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl],N{Epsilon-324}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Lys309,Glu312,Lys324]-LDL-R-(293-332)-peptide
##STR00160##
[1044] The peptide back-bone is SEQ ID NO: 106
Compound Prepared by General Method B
[1045] LCMS29: Found m/2=2916.6; Found m/3=1944.5; Found
m/4=1458.9; Calc mass=5831.5
Example 126
N{293}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecan-
oylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N-
{Epsilon-309}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)-
undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ac-
etyl]-[Leu301,Lys309,Glu312]-LDL-R-(293-332)-peptide
##STR00161##
[1046] The peptide back-bone is SEQ ID NO: 30
Compound Prepared by General Method B
[1047] LCMS29: Found m/2=2916.7; Found m/3=1944.6; Found
m/4=1458.7; Calc mass=5831.5
Example 127
N{Epsilon-321}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl],N{Epsilon-332}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Arg309,Glu312,Lys321,
Lys332]-LDL-R-(293-332)-peptide
##STR00162##
[1048] The peptide back-bone is SEQ ID NO: 95
Compound Prepared by General Method B
[1049] LCMS29: Found m/3=1958.3; Found m/4=1469.0; Calc
mass=5871.6
Example 128
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxyp-
entadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]-
acetyl]-[Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-peptidyl)-N{Epsilon}-
[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butano-
yl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00163##
[1050] The peptide back-bone is SEQ ID NO: 32
Compound Prepared by General Method B
[1051] LCMS29: Found m/2=2966.28; Found m/3=1978.0; Found
m/4=1483.5; Calc mass=5930.7
Example 129
N{Alpha}(N{Epsilon-313}-[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)bu-
tanoyl]-[Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-peptidyl)-N{Epsilon}-
[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]Lys
##STR00164##
[1052] The peptide back-bone is SEQ ID NO: 32
Compound Prepared by General Method B
[1053] LCMS29: Found m/2=2676.0; Found m/3=1784.2; Found
m/4=1338.4; Calc mass=5330.1
Example 130
N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl],N{Epsilon-332}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[His300,Leu301,Arg309,Glu312,Lys313,
Lys332]-LDL-R-(293-332)-peptide
##STR00165##
[1054] The peptide back-bone is SEQ ID NO: 96
Compound Prepared by General Method B
[1055] LCMS29: Found m/3=1966.7; Found m/4=1475.0; Calc
mass=5896.6
Example 131
N{Alpha}(N{Epsilon-313}-[4-[3-[2-[2-[3-[[(4S)-4-carboxy-4-[11-(4-carboxyph-
enoxy)undecanoylamino]butanoyl]amino]propoxy]ethoxy]ethoxy]propylamino]-4--
oxobutanoyl]-[Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-peptidyl)-N{Eps-
ilon}[4-[3-[2-[2-[3-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylami-
no]butanoyl]amino]propoxy]ethoxy]ethoxy]propylamino]-4-oxobutanoyl]Lys
##STR00166##
[1056] The peptide back-bone is SEQ ID NO: 32
Compound Prepared by General Method B
[1057] LCMS29: Found m/3=2009.9; Found m/4=1507.7; Calc
mass=6026.8
Example 132
N{Epsilon-313}-[4-[3-[2-[2-[3-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)und-
ecanoylamino]butanoyl]amino]propoxy]ethoxy]ethoxy]propylamino]-4-oxobutano-
yl],N{Epsilon-332}-[4-[3-[2-[2-[3-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]propoxy]ethoxy]ethoxy]propylamino]-4-oxobu-
tanoyl]-[Leu301,Arg309,Glu312,Lys313,Glu321,
Lys332]-LDL-R-(293-332)-peptide
##STR00167##
[1058] The peptide back-bone is SEQ ID NO: 97
Compound Prepared by General Method B
[1059] LCMS29: Found m/3=1971.3; Found m/4=1478.9; Calc
mass=5911.7
Example 133
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[Leu301,Arg309,Glu312,Lys313,Glu321]-LDL-R-(293-332)-pepti-
dyl)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl]Lys
##STR00168##
[1060] The peptide back-bone is SEQ ID NO: 98
Compound Prepared by General Method B
[1061] LCMS01: Found m/4=1505.0; Found m/5=1204.3; Calc
mass=6016.7
Example 134
N{Alpha}([Leu301,Arg309,Glu312,Glu321]-LDL-R-(293-332)-peptidyl)-N{Epsilon-
}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecanoylamino)butano-
yl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00169##
[1062] The peptide back-bone is SEQ ID NO: 19
Compound Prepared by General Method B
[1063] LCMS01: Found m/4=1766.7; Found m/5=1325.3; Calc
mass=5258.0
Example 135
N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl],N{Epsilon-314}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Arg309,Glu312,Lys313,
Lys314]-LDL-R-(293-332)-peptide
##STR00170##
[1064] The peptide back-bone is SEQ ID NO: 99
Compound Prepared by General Method B
[1065] LCMS01: Found m/4=1487.3; Found m/5=1190.0; Calc
mass=5945.6
Example 136
N{Epsilon-312}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl],N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Arg309, Lys313]-LDL-R-(293-332)-peptide
##STR00171##
[1066] The peptide back-bone is SEQ ID NO: 100
Compound Prepared by General Method B
[1067] LCMS01: Found m/4=1469.3; Found m/5=1175.5; Calc
mass=5873.6
Example 137
N{Epsilon-312}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl]-N{Epsilon-314}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Arg309,Lys314]-LDL-R-(293-332)-peptide
##STR00172##
[1068] The peptide back-bone is SEQ ID NO: 101
Compound Prepared by General Method B
[1069] LCMS01: Found m/4=1483.3; Found m/5=1186.8; Calc
mass=5929.7
Example 138
N{Epsilon-311}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy-
)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]a-
cetyl]-N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]-[Leu301,Arg309,Lys311,Glu312,Lys313]-LDL-R-(293-332)-peptide
##STR00173##
[1070] The peptide back-bone is SEQ ID NO: 102
Compound Prepared by General Method B
[1071] LCMS01: Found m/4=1473.0; Found m/5=1178.6; Calc
mass=5889.5
Example 139
N{Alpha}(N{Epsilon-313}-11-(4-carboxyphenoxy)undecanoyl-[His300,Leu301,Arg-
309,Glu312,Lys313]-LDL-R-(293-332)-peptidyl)-N{Epsilon}11-(4-carboxyphenox-
y)undecanoylLys
##STR00174##
[1072] The peptide back-bone is SEQ ID NO: 69
Compound Prepared by General Method B
[1073] LCMS01: Found m/4=1297.4; Found m/5=1038.2; Calc
mass=5186.9
Example 140
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[13-(1H-tetr-
azol-5-yl)tridecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethox-
y]ethoxy]acetyl]-[Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-peptidyl)-N-
{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[13-(1H-tetrazol-5-yl)tride-
canoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl-
]Lys
##STR00175##
[1074] The peptide back-bone is SEQ ID NO: 32
Compound Prepared by General Method B
[1075] LCMS01: Found m/4=1481.6; Found m/5=1185.3; Calc
mass=5922.7
Example 141
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(14-sulfotet-
radecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ac-
etyl]-[Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-
-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(14-sulfotetradecanoylamino)butanoyl]a-
mino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00176##
[1076] The peptide back-bone is SEQ ID NO: 32
Compound Prepared by General Method B
[1077] LCMS027: Found m/3=1992.6; Found m/4=1494.7; Found
m/5=1196.0; Calc mass=5974.8
Example 142
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[13-(methyls-
ulfonylcarbamoylamino)tridecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl-
]amino]ethoxy]ethoxy]acetyl]-[Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-
-peptidyl)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[13-(methylsulf-
onylcarbamoylamino)tridecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]am-
ino]ethoxy]ethoxy]acetyl]Lys
##STR00177##
[1078] The peptide back-bone is SEQ ID NO: 32
Compound Prepared by General Method B
[1079] LCMS027: Found m/3=2020.7; Found m/4=1515.8; Found
m/5=1212.8; Calc mass=6058.8
Example 143
N{Alpha}(N{Epsilon-313}-[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoyl-
amino]butanoyl]-[Leu301,Arg309,Glu312,Lys313,Glu321]-LDL-R-(293-332)-pepti-
dyl)-N{Epsilon}[(4S)-4-carboxy-4-[11-(4-carboxyphenoxy)undecanoylamino]but-
anoyl]Lys
##STR00178##
[1080] The peptide back-bone is SEQ ID NO: 98
Compound Prepared by General Method B
[1081] LCMS29: Found m/2=2719.0; Found m/3=1812.8; Found
m/4=1359.8; Calc mass=5436.1
Example 144
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxyp-
entadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]-
acetyl]-[Leu301,Arg309,Glu312,Lys313,Glu321]-LDL-R-(293-332)-peptidyl)-N{E-
psilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino-
)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00179##
[1082] The peptide back-bone is SEQ ID NO: 98
Compound Prepared by General Method B
[1083] LCMS29: Found m/3=1982.2; Found m/4=1486.9; Found
m/5=1189.7; Calc mass=5944.7
Example 145
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[16-(1H-tetr-
azol-5-yl)hexadecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]etho-
xy]ethoxy]acetyl]-[Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-peptidyl)--
N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[16-(1H-tetrazol-5-yl)hexa-
decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acet-
yl]Lys
##STR00180##
[1084] The peptide back-bone is SEQ ID NO: 32
Compound Prepared by General Method B
[1085] LCMS29: Found m/3=2003.3; Found m/4=1502.7; Found
m/5=1202.2; Calc mass=6006.8
Example 146
N{Alpha}(N{Epsilon-313}-[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)bu-
tanoyl]-[Leu301,Arg309,Glu312,Lys313,Glu321]-LDL-R-(293-332)-peptidyl)-N{E-
psilon}[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]Lys
##STR00181##
[1086] The peptide back-bone is SEQ ID NO: 98
Compound Prepared by General Method B
[1087] LCMS29: Found m/3=1788.8; Found m/4=1341.9; Found
m/5=1073.7; Calc mass=5364.1
Example 147
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[His300,Leu301,Arg309,Glu312,Lys313,Glu321]-LDL-R-(293-332-
)-peptidyl)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carboxy-
phenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]e-
thoxy]acetyl]Lys
##STR00182##
[1088] The peptide back-bone is SEQ ID NO: 103
Compound Prepared by General Method B
[1089] LCMS29: Found m/2=3020.8; Found m/3=2014.3; Found
m/4=1510.9; Calc mass=6039.8
Example 148
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-ca-
rboxy-4-
[11-(4-carboxyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethox-
y]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethox-
y]ethoxy]acetyl]-[Leu301,Arg309,Glu312,Lys313]-LDL-R-(293-332)-peptidyl)-N-
{Epsilon}[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4--
carboxyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]e-
thoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-
Lys
##STR00183##
[1090] The peptide back-bone is SEQ ID NO: 32
Compound Prepared by General Method B
[1091] LCMS027: Found m/3=2195.5; Found m/4=1646.9; Found
m/5=1317.7; Calc mass=6583.3
Example 149
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-carbo-
xyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy-
]ethoxy]acetyl]-[His300,Leu301,Arg309,Glu312,Lys313],des-Gly293-LDL-R-(294-
-332)-peptidyl)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[11-(4-car-
boxyphenoxy)undecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]etho-
xy]ethoxy]acetyl]Lys
##STR00184##
[1092] The peptide back-bone is SEQ ID NO: 74
Compound Prepared by General Method B
[1093] LCMS027: Found m/3=1990.6; Found m/4=1493.2; Found
m/5=1191.1; Calc mass=5968.7
Example 150
N{Alpha}(N{Epsilon-328}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxyp-
entadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]-
acetyl]-[Leu301,Arg309,Glu312,Lys328]-LDL-R-(293-332)-peptidyl)-N{Epsilon}-
[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butano-
yl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00185##
[1094] The peptide back-bone is SEQ ID NO: 78
Compound Prepared by General Method B
[1095] LCMS27: Found m/2=2958.7; Found m/3=1973.0; Found
m/4=1480.0; Calc mass=5915.7
Example 151
N{Alpha}(N{Epsilon-328}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxyp-
entadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]-
acetyl]-[Leu301,Arg309,Glu312,Glu321,Lys328]-LDL-R-(293-332)-peptidyl)-N{E-
psilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino-
)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00186##
[1096] The peptide back-bone is SEQ ID NO: 104
Compound Prepared by General Method B
[1097] LCMS01: Found m/4=1483.2; Found m/5=1186.8; Calc
mass=5930.0
Example 152
N{Alpha}(N{Epsilon-324}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxyp-
entadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]-
acetyl]-[Leu301,Arg309,Glu312,Lys324]-LDL-R-(293-332)-peptidyl)-N{Epsilon}-
[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butano-
yl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00187##
[1098] The peptide back-bone is SEQ ID NO: 72
Compound Prepared by General Method B
[1099] LCMS34: Found m/4=1974.6; Found m/5=1183.9; Calc
mass=5915.7
Example 153
N{Alpha}(N{Epsilon-324}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxyp-
entadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]-
acetyl]-[Leu301,Arg309,Glu312,Glu321,Lys324]-LDL-R-(293-332)-peptidyl)-N{E-
psilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino-
)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00188##
[1100] The peptide back-bone is SEQ ID NO: 105
Compound Prepared by General Method B
[1101] LCMS01: Found m/4=1483.3 Found m/5=1186.8 Calc
mass=5929.8
Example 154
N{Alpha}(N{Epsilon-328}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyh-
eptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]-
acetyl]-[Leu301,Arg309,Glu312,Glu321,Lys328]-LDL-R-(293-332)-peptidyl)-N{E-
psilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino-
)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys
##STR00189##
[1102] The peptide back-bone is SEQ ID NO: 104
Compound Prepared by General Method B
[1103] LCMS01: Found m/4=1497.3; Found m/5=1198.2; Calc
mass=5985.9
Example 155
N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecan-
oylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N-
{Epsilon-321}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecano-
ylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[L-
eu301,Arg309,Glu312,Lys313,Lys321]-LDL-R-(293-332)-peptide
##STR00190##
[1104] The peptide back-bone is SEQ ID NO:73
Compound Prepared by General Method B
[1105] LCMS01: Found m/4=1454.7; Found m/5=1164.0 Calc
mass=5815.6
Example 156
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[4-[[11-(4--
carboxyphenoxy)undecanoylamino]methyl]cyclohexanecarbonyl]amino]butanoyl]a-
mino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Leu301,Arg309,Glu31-
2,Lys313]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[(4S)-4--
carboxy-4-[[4-[[1-(4-carboxyphenoxy)undecanoylamino]methyl]cyclohexanecarb-
onyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-
Lys
##STR00191##
[1106] The peptide back-bone is SEQ ID NO: 32
Compound Prepared by General Method B
[1107] LCMS27: Found m/3=2094.6; Found m/4=1571.2; Calc
mass=6281.1
Example 157
N{Alpha}(N{Epsilon-313}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[4-[[11-(4--
carboxyphenoxy)undecanoylamino]methyl]cyclohexanecarbonyl]amino]butanoyl]a-
mino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Leu301,Arg309,Glu31-
2,Lys313,Glu321]-LDL-R-(293-332)-peptidyl)-N{Epsilon}[2-[2-[2-[[2-[2-[2-[[-
(4S)-4-carboxy-4-[[4-[[1-(4-carboxyphenoxy)undecanoylamino]methyl]cyclohex-
anecarbonyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]-
acetyl]Lys
##STR00192##
[1108] The peptide back-bone is SEQ ID NO: 98
Compound Prepared by General Method B
[1109] LCMS27: Found m/3=2099.3; Found m/4=1574.7; Calc
mass=6295.1
Example 158
N{Alpha}([Leu301,Arg309,Glu312,Glu321]-LDL-R-(293-332)-peptidyl)-N{Epsilon-
}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)m-
ethyl]cyclohexanecarbonyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]-
ethoxy]ethoxy]acetyl]Lys
##STR00193##
[1110] The peptide back-bone is SEQ ID NO: 19
Compound Prepared by General Method B
[1111] LCMS27: Found m/3=1813.2; Found m/4=1360.2; Found
m/5=1088.3; Calc mass=5437.2
Example 159
N{Alpha}([Leu301,Arg309,Glu312,Glu321]-LDL-R-(293-332)-peptidyl)-N{Epsilon-
}[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[4-[(17-carboxyheptadecanoylamino)-
methyl]cyclohexanecarbonyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino-
]ethoxy]ethoxy]acetyl]Lys
##STR00194##
[1112] The peptide back-bone is SEQ ID NO: 19
Compound Prepared by General Method B
[1113] LCMS27: Found m/3=1803.9; Found m/4=1353.1; Found
m/5=1082.7; Calc mass=5409.2
TABLE-US-00009 TABLE 4 Summary table of Example compounds 1-159 Ex-
am- ple Sequence Attachment no. modifications Substituent sites 1
299A, 301L, 307I, HOOC--(CH.sub.2).sub.16--O--gGlu- N- 309R, 310K
2xADO--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2-- terminal 2 301L,
309R HOOC--(CH.sub.2).sub.16--O--gGlu- N-
2xADO--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2-- terminal 3 301L,
309R, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 333K 333K 4
301L, 309R HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 312K 5 301L,
309R, 312E HOOC--(CH.sub.2).sub.16--O--gGlu- N-
2xADO--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2-- terminal 6 299K,
301L, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 299K 312E 7
301L, 309R, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 330K 330K
8 301L, 309R, 312E HOS(C).sub.2--(CH.sub.2).sub.15--O--gGlu- N-
2xADO--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2-- terminal 9 301L,
309R, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO N- 330K
terminal, 330K 10 301L, 309R, 312E,
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 332K 332K 11 293K, 301L,
309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 293K 312E 12 293K,
301L, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 293K, 312E, 333K
333K 13 293K, 301L, 309R,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 293K,
312E, 333K 333K 14 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 332K,
332K, 333K 333K 15 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 330K,
330K, 333K 333K 16 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 321K,
321K, 333K 333K 17 301L, 309R, 333K
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 312K,
333K 18 301L, 309R, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO
333K 321E, 333K 19 301L, 309R, 312E
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO N- terminal 20 301L, 309R,
312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 321K 321K 21 301L,
309R, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 324K 324K 22
301L, 309R, 312Q HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO N- terminal
23 301L, 309R, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 332K
321E, 332K 24 293K, 301L, 309R,
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 293K 312E, 321E 25 293K,
301L, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO N- 312E
terminal, 293K 26 300K, 301L, 309R,
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 300K 312E 27 293K, 294K,
301L, 4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO
293K, 309R, 312E 294K 28 293K, 301L, 309R
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 293K,
312K 29 301L, 309K, 312E HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO
309K 30 301L, 309R, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO
318K 318K 31 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 313K,
313K, 333K 333K 32 301L, 309R, 312E,
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 326K 326K 33 301L, 309R,
312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 325K 325K 34 301L,
309R, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 323K 323K 35
301L, 309R, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 322K 322K
36 301L, 309R, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 320K
320K 37 301L, 309R, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO
329K 329K 38 301L, 309R, 312E,
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 313K 313K 39 301L, 309R,
312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 328K 328K 40 301L,
309R, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 316K 316K 41
301L, 309R, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 315K 315K
42 300H, 301L, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 333K
312R, 333K 43 301L, 309R, 312E,
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 314K 314K 44 301L, 309R,
311K, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 311K 312E 45 301L,
307K, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 307K 312E 46
301L, 309S, 312R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 333K 333K
47 301L, 309S, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 333K
333K 48 299A, 301L, 307I, 309R, 310K 49 301L, 309R 50 301L, 309R,
312E 51 301L, 306Y, 309S, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO N-
312E terminal 52 293N, 301L, 309S,
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO N- 312E terminal 53 301L,
306K, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 306K 312E 54
301L, 305K, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 305K 312E
55 301L, 303K, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 303K
312E 56 301L, 302K, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO
302K 312E 57 293N, 300H, 301L,
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 333K 309R, 312R, 333K 58
301K, 309R, 312E HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 301K 59
298K, 301L, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 298K 312E
60 293N, 301L, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 333K
312R, 333K 61 301L, 307I, 332K
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 332K 62 301L, 306Y, 312E,
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 332K 332K 63 301L, 307I,
312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 332K 332K 64 300H,
301L, 309R HOOC--(CH.sub.2).sub.16--O--gGlu- N-
2xADO--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2-- terminal 65 300P,
301L, 307I, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO N- 309R, 312E
terminal 66 293N, 301L, 307I,
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 333K 309R, 312D, 333K 67
293N, 301L, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 333K 312D,
333K 68 301L, 309R, 312E
Tetrazolyl--(CH.sub.2).sub.15--O--NH-SO.sub.2--(CH.sub.2).sub.3--O--ADO-
N- ADO--NH--CH.sub.2--(C.sub.6H.sub.4)--CH.sub.2-- terminal 69
301L, 309R, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 328K 328K,
329H 70 295D, 301L, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO
332K 312E, 332K 71 300H, 301L, 309R
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 312K 72 300H, 301L, 307I,
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO N- 309R, 312E terminal 73
296K, 301L, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 296K 312E
74 294K, 301L, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 294K
312E 75 292K, 301L, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO
292K 312E 76 des293, 294G, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO
328K 301L, 309R, 312E, 328K 77 301L, 306D, 309R,
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 333K 312E, 324G, 333K 78
301L, 306D, 309R,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-3xADO and
N- 312E, 333K
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO
terminal, 333K 79 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9--O--gGlu-2xADO 321K,
321K, 333K 333K 80 301L, 309R, 312E,
HOOC--(CH.sub.2).sub.14-O--gGlu-2xADO 333K 333K 81 301L, 309R,
312E, HOOC--(CH.sub.2).sub.18--O--gGlu-2xADO 333K 333K 82 301L,
309R, 312E, HOOC--(CH.sub.2).sub.16--O--gGlu 333K 333K 83 301L,
309R, 312E, HOOC--(CH.sub.2).sub.12--O--gGlu-2xADO 321K, 321K, 333K
333K 84 301L, 309R, 312E, HOOC--(CH.sub.2).sub.14-O--gGlu-2xADO
321K, 321K, 333K 333K 85 300H, 301L, 309R,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9--O--gGlu-2xADO 313K,
312E, 313K, 333K 333K 86 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 313K,
313K, 328K 328K 87 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 313K,
313K, 324K 324K 88 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO N-
313K terminal, 313K 89 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 324K,
324K, 333K 333K 90 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 313K,
313K, 321K 321K 91 des293, 300H,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9--O--gGlu-2xADO 313K,
301L, 309R, 312E, 333K 313K, 333K 92 300H, 301L, 309R,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 313K,
312E, 313K, 333K 333K 93 292A, 301L, 309R,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO N-
312E, 313K terminal, 313K 94 des293, 301L,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO N-
309R, 312E, 313K terminal, 313K 95 des293, 301L,
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 313K 309R, 312E, 313K 96
301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 313K,
313K, 332K 332K 97 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 328K,
328K, 333K 333K 98 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu 313K, 313K,
333K 333K 99 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--2xgGlu 313K,
313K, 333K 333K 100 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-3xGly 313K,
313K, 333K 333K 101 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--2xgGlu-2xADO
313K, 313K, 333K 333K 102 301L, 309R, 312E,
3-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9--O--gGlu-2xADO 313K,
313K, 333K 333K 103 299A, 301L, 307I, 309R 104 301L, 309R, 310K 105
301L 106 300H, 301L, 309R, HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO
333K 312E, 333K 107 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO N-
333K terminal, 333K 108 des293-294, 300H,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9--O--gGlu-2xADO 313K,
301L, 309R, 312E, 333K 313K, 333K 109 300H, 301L, 309R,
3-HO-Isoxazole--(CH.sub.2).sub.12--O--gGlu-2xADO 313K, 312E, 313K,
333K 333K 110 301L, 309R, 312E,
3-HO-Isoxazole--(CH.sub.2).sub.12--O--gGlu-2xADO 313K, 313K, 333K
333K 111 301L, 309K, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 309K,
333K 333K 112 301L, 306Y, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 324K,
324K, 333K 333K 113 300H, 301L, 309R,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 314K,
312E, 314K, 333K 333K 114 294W, 301L, 309R,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO N-
312E, 333K terminal, 333K 115 301L, 309K, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 309K,
328K 328K 116 301L, 309K, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 309K,
313K 313K 117 des293, 301L,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO N-
309R, 312E, 333K terminal, 333K 118 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 324K,
324K, 328K 328K 119 292A, 301L, 309R,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO N-
312E, 333K terminal, 333K 120 301L, 306Y, 309R,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 313K,
312E, 313K, 333K 333K 121 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO N-
332K terminal, 332K 122 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO N-
328K terminal, 328K 123 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O-- N- 324K
gGlu-2xADO terminal, 324K 124 301L, 309K, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 309K,
332K 332K 125 301L, 309K, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 309K,
324K 324K 126 301L, 309K, 312E
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO N-
terminal, 309K 127 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 321K,
321K, 332K 332K 128 301L, 309R, 312E,
HOOC--(CH.sub.2).sub.14-O--gGlu-2xADO 313K, 313K, 333K 333K 129
301L, 309R, 312E, HOOC--(CH.sub.2)14-O--gGlu 313K, 313K, 333K 333K
130 300H, 301L, 309R,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 313K,
312E, 313K, 332K 332K 131 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-TtdSuc
313K, 313K, 333K 333K 132 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-TtdSuc
313K, 313K, 321E, 332K 332K 133 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 313K,
313K, 321E, 333K 333K 134 301L, 309R, 312E,
HOOC--(CH.sub.2).sub.18--O--gGlu-2xADO 333K 321E, 333K 135 301L,
309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 313K,
313K, 314K 314K 136 301L, 309R, 313K
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 312K,
313K 137 301L, 309R, 314K
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 312K,
314K 138 301L, 309R, 311K,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 311K,
312E, 313K 313K 139 300H, 301L, 309R,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.9--CO 313K, 312E, 313K,
333K 333K 140 301L, 309R, 312E,
Tetrazolyl--(CH.sub.2).sub.12--O--gGlu-2xADO 313K, 313K, 333K 333K
141 301L, 309R, 312E,
HOS(C).sub.2--(CH.sub.2).sub.13--O--gGlu-2xADO 313K, 313K, 333K
333K 142 301L, 309R, 312E,
MeS(C).sub.2NH(CO)NH--(CH.sub.2).sub.12--O--gGlu-2xADO 313K, 313K,
333K 333K 143 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu 313K, 313K,
321E, 333K 333K 144 301L, 309R, 312E,
HOOC--(CH.sub.2).sub.14-O--gGlu-2xADO 313K, 313K, 321E, 333K 333K
145 301L, 309R, 312E, Tetrazolyl--(CH.sub.2).sub.15--O--gGlu-2xADO
313K, 313K, 333K 333K 146 301L, 309R, 312E,
HOOC--(CH.sub.2).sub.14-O--gGlu 313K, 313K, 321E, 333K 333K 147
300H, 301L, 309R,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 313K,
312E, 313K, 321E, 333K 333K 148 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-4xADO 313K,
313K, 333K 333K 149 des293, 300H,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--gGlu-2xADO 313K,
301L, 309R, 312E, 333K 313K, 333K 150 301L, 309R, 312E,
HOOC--(CH.sub.2).sub.14-O--gGlu-2xADO 328K, 328K, 333K 333K 151
301L, 309R, 312E, HOOC--(CH.sub.2).sub.14-O--gGlu-2xADO 328K, 321E,
328K, 333K 333K 152 301L, 309R, 312E,
HOOC--(CH.sub.2).sub.14-O--gGlu-2xADO 324K, 324K, 333K 333K 153
301L, 309R, 312E, HOOC--(CH.sub.2).sub.14-O--gGlu-2xADO 324K, 321E,
324K, 333K 333K 154 301L, 309R, 312E,
HOOC--(CH.sub.2).sub.16--O--gGlu-2xADO 328K, 321E, 328K, 333K 333K
155 301L, 309R, 312E, HOOC--(CH.sub.2).sub.14-O--gGlu-2xADO 313K,
313K, 321K 321K 156 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--Trx-gGlu-2xADO
313K, 313K, 333K 333K 157 301L, 309R, 312E,
4-HOOC--(C.sub.6H.sub.4)--O--(CH.sub.2).sub.10--O--Trx-gGlu-2xADO
313K, 313K, 321E, 333K 333K 158 301L, 309R, 312E,
HOOC--(CH.sub.2).sub.18--O--Trx-gGlu-2xADO 333K 321E, 333K 159
301L, 309R, 312E, HOOC--(CH.sub.2).sub.16--O--Trx-gGlu-2xADO 333K
321E, 333K
D. General Methods for Characterisation
D.1.1 PCSK9-LDL-R Binding Competitive (ELISA)
[1114] The aim of this assay is to measure the apparent binding
affinity of EGF(A) compounds to PCSK9.
[1115] Due to their ability to inhibit the interaction of PCSK9
with LDL-R, compounds of the invention may also be referred to as
PCSK9 inhibitors.
[1116] The day before the experiment, recombinant human Low Density
Lipoprotein Receptor (rhLDL-R; NSO-derived; R & D systems
#2148-LD) was dissolved at 1 .mu.g/ml in 50 mM sodium carbonate, pH
9.6, and then 100 .mu.l of the solution was added to each well of
the assay plates (Maxisorp 96, NUNC #439454) and coated overnight
at 4.degree. C. On the day of the experiments, 8 point
concentration curves of the EGF(A) compounds containing
Biotinylated PCSK9 (0.5 ug/ml, BioSite/BPSBioscience cat #71304)
were made in duplicate. EGF(A) compound and biotinylated PCSK9
mixtures were prepared an incubated for 1 hour at room temperature
in assay buffer containing 25 mM Hepes, pH 7.2 (15630-056, 100 ml,
1M), 150 mM NaCl (Emsure 1.06404.1000) 1% HSA (Sigma A1887-25G)
0.05% Tween 20 (Calbiochem 655205) 2 mM CaCl.sub.2 (Sigma
223506-500G). The coated assay plates were then washed 4.times. in
200 .mu.l assay buffer, and then 100 .mu.l of the mixture of EGF(A)
compounds and biotinylated PCSK9 was added to the plates and
incubated 2 h at room temperature. The plates were washed 4.times.
in 200 .mu.l assay buffer and then incubated with Streptevadin-HRP
(25 ng/ml; VWR #14-30-00) for 1 h at room temperature. The reaction
is detected by adding 50 .mu.l TMB-on (KEM-EN-TEC) and incubated 10
min in the dark. Then the reaction was stopped by adding 50 .mu.l 4
M H.sub.3PO.sub.4 to the mixture, added by electronic multi
pipetting. The plates were then read in a Spectramax at 450 and 620
nm within 1 h. The 620 nm read was used for background subtraction.
IC50 values were calculated using Graphpad Prism, by nonlinear
regression log(inhibitor) vs. response-variable slope (four
parameters), and converted into Ki values using the following
formula: Ki=IC50/(1+(Biotin-PCSK9)/(kd(Biotin-PCSK9))), where Kd of
the biotin-PCSK9 is 1.096727714 .mu.g/ml and [Biotin-PCSK9]=0.5
(.mu.g/ml).
[1117] The results are shown in Table 5.1-5.5 below. Higher Ki
values reflects lower apparent binding affinities to PCSK9 and vice
versa. It is noticed that few of the compounds display a Ki which
is substantially higher than the value measured for EGF66, such as
a value above 500 nM, which indicate that the observed binding is
not specific. Both the amino acid substitutions of the peptide
and/or the one or more side-chain derivation may contribute to the
loss of binding to LDL-R. In general a large number of the tested
EGF(A) compounds displayed the ability to inhibit PCSK9 in binding
to the hLDL-R.
PCSK9 Inhibitor Peptides
[1118] Initially a group of peptides include various amino acids
substitutions were tested as described in section D1.1 and the
results are shown in table 5.1.
TABLE-US-00010 TABLE 5.1 Apparent binding affinity (Ki) for PCSK9
peptides Ki Ex. No. Peptide variant (nM) WT -- 48 299A, 301L, 307I,
309R, 310K 9.4 103 299A, 301L, 307I, 309R 0.9 104 301L, 309R, 310K
7.3 49 301L, 309R 1.2 105 301L 2.8 50 301L, 309R, 312E 1.1
[1119] EGF66, identified as the most potent peptide variant in WO
2012177741, has 5 mutations. As seen above the inventors of the
present case found that several of these mutations were not of
great importance for the EC50 value determined in the assay
described in D1.1. In particular the inventors found that compounds
including the wild type residue Asp (D) in position 310 had higher
potencies than compounds with 310K. It also appeared that the key
amino substitution is 301L preferably in combination with 309R.
Finally 307I and 299A contributed only modestly to the affinity of
the peptides.
N-Terminal Attachment of Substituent
[1120] In a subsequent experiment it was tested if attachment of a
half-life protractor e.g. a substituent to the peptides influences
the EC50 as determined by the assay described in D.1.1. As
described herein a substituent may be attached by different
technologies and the inventors initially decide to apply attachment
via a nitrogen atom using the N-terminal amino acid of the
peptides. This was as described in section B accomplished by
acylation (in solution or on resin) and alkylation.
[1121] As seen in Table 5.2 all the tested compounds have an Ki
value below 3.0 suggesting that the various protractor and linker
elements are well tolerated. This was unusual as potency is usually
negatively influence by attachment of a side chain as previously
observer for peptides like GLP-1.
TABLE-US-00011 TABLE 5.2 Apparent Ki for N-terminal substituted
PCSK9 peptides Ki Ex. No. Peptide variant Attachment (nM) 1. 299A,
301L, 307I, 309R, 310K Alkylation nd 2 301L, 309R Alkylation 1.7 64
300H, 301L, 309R Alkylation 0.7 5 301L, 309R, 312E Alkylation 1.3 8
301L, 309R, 312E Alkylation 1.2 19 301L, 309R, 312E Acylation 1.7
68 301L, 309R, 312E Alkylation 0.8 22 301L, 309R, 312Q Acylation
2.6 51 301L, 306Y, 309S, 312E Acylation 1.6 52 293N, 301L, 309S,
312E Acylation 2.1 65 300P, 301L, 307I, 309R, 312E Acylation
>1000 72 300H, 301L, 307I, 309R, 312E Acylation 2.8
Lys Attachment of Substituent
[1122] In order to evaluate alternative positions for linkage of a
substituent to a PCSK9 inhibitor peptide a series of compounds were
prepared. A back-bone peptide including three amino acid
substitutions; N301L, N309R and K312E were used except in Ex. 58,
29 and 4 in combination with a Lys substitution at various
positions. All compounds tested included the 6 cysteine amino acids
in positions 297, 304, 308, 317, 319, 331 which are usually engaged
in cysteine disulfide bridges. The 312E was included to ensure site
specific substitution except in example 4 where attachment to wt
312K is obtained. Extension of the peptide with one Lys is also
tested (Ex. 75 and 3). The same substituent as described above
including a C18 diacid protractor and a gGlu-2xAdo linker was used
in all compounds and attached via acylation. The results are
included in Table 5.3.
TABLE-US-00012 TABLE 5.3 Apparent Ki for PCSK derivatives with a
substituent attached via a Lys residue Attachment Ki Ex. No.
Peptide variant site (nM) 75 292K, 301L, 309R, 312E 292K 1.5 11
293K, 301L, 309R, 312E 293K 2.4 74 294K, 301L, 309R, 312E 294K 1.4
73 296K, 301L, 309R, 312E 296K 8.9 59 298K, 301L, 309R, 312E 298K
610.7 6 299K, 301L, 309R, 312E 299K 3.3 26 300K, 301L, 309R, 312E
300K 1.3 58 301K, 309R, 312E 301K 1000.0 56 301L, 302K, 309R, 312E
302K 1032.0 55 301L, 303K, 309R, 312E 303K 1.7 54 301L, 305K, 309R,
312E 305K 2.1 53 301L, 306K, 309R, 312E 306K 1.7 45 301L, 307K,
309R, 312E 307K 1000.0 29 301L, 309K, 312E 309K 0.8 44 301L, 309R,
311K, 312E 311K 1.0 4 301L, 309R 312K 1.2 38 301L, 309R, 312E, 313K
313K 0.8 43 301L, 309R, 312E, 314K 314K 0.9 41 301L, 309R, 312E,
315K 315K 3.0 40 301L, 309R, 312E, 316K 316K 1.6 30 301L, 309R,
312E, 318K 318K 2.0 36 301L, 309R, 312E, 320K 320K 5.5 20 301L,
309R, 312E, 321K 321K 2.0 35 301L, 309R, 312E, 322K 322K 1.5 34
301L, 309R, 312E, 323K 323K 1.7 21 301L, 309R, 312E, 324K 324K 0.9
33 301L, 309R, 312E, 325K 325K 1.4 32 301L, 309R, 312E, 326K 326K
1.4 39 301L, 309R, 312E, 328K 328K 0.9 37 301L, 309R, 312E, 329K
329K 1.0 7 301L, 309R, 312E, 330K 330K 1.4 10 301L, 309R, 312E,
332K 332K 1.1 3 301L, 309R, 312E, 333K 333K 0.8
[1123] The analysis showed that the majority of the PCSK9 inhibitor
peptide maintain functionality. The exceptions were Lys
substitution and derivation in either of position 298, 301, 302 and
307 which gave rise to non-functional peptides. It was also
observed that Lys introduction and substitution in position 296,
299,315 and 320K reduced the apparent affinity.
[1124] The data thus also confirm the result from table 5.1
indicating that the amino acid substitution of Asn(N) 301 to Leu
(L) is essential for the binding.
[1125] No data was observed for Lys introduction and substitution
in position 295 and 310. As described above it was previously found
that maintenance of Asp in 310 was preferred above the 310K
substitution. As seen below it was also found that binding is
abolished by introduction of Asp (D) in position 295 (Ex. 70).
[1126] In summary it was concluded that compounds which do not
comprise a substituent attached in any of the positions 295, 298,
302, 307 and 310 or in any of the positions 295, 296, 298, 299,
302, 307, 310, 315 and 320 of the PCSK9 peptide are generally
functional. It was further concluded that an amino acid
substitution in any of the positions 295, 298, 302, and 310 is
generally not attractive. As seen from table 5.1 and 5.2 the V307I
mutation none the less seem to be acceptable or even attractive in
combination with 301Leu.
[1127] It is further considered that peptides with amino acid
substitution in one of the positions 295, 296, 298, 302, 310 are
likely to have a lower functionality, while substitutions in 299,
315 and 320 only seems to lower functionality slightly. This on the
other hand also suggests that a high degree of flexibility may
exist for the remaining amino acid residues as Lys substitution and
attachment of a sidechain will influence the peptides as much as
most other amino acid substitutions.
PCSK9 Inhibitors with Two Substituents
[1128] A series of compound with two substituents were prepared.
Double substitution may be obtained by acylation, alkylation or a
combination at the N-terminal or at Lys (K) residues. Again the
N-terminal may be amino acid 293G or a variant amino acid residue
such as 292A, 293G, 293K and 294T (in cases where 293G is deleted).
The compounds were prepared with different substituents, although
the two substituents on the individual compounds are identical. The
back-bone used in this study again included the N301L amino acid
substitution in combination with N309R and various N-terminal
and/or Lys substitutions as required to obtain the specific
acylation/alkylation.
TABLE-US-00013 TABLE 5.4 Apparent Ki for double substituted PCSK9
inhibitors Ex- am- ple Variant Attachment Ki No. 301L, 309R, +
sites (nM) 9 312E, 330K N-terminal, 330K 2.7 12 293K, 312E, 333K
293K, 333K 2.7 13 293K, 312E, 333K 293K, 333K 2.1 14 312E, 332K,
333K 332K, 333K 1.2 15 312E, 330K, 333K 330K, 333K 1.5 16 312E,
321K, 333K 321K, 333K 1.1 17 333K 312K, 333K 1.8 25 293K, 312E
N-terminal, 293K 2.0 27 293K, 294K, 312E 293K, 294K 0.9 28 293K
293K, 312K 0.8 31 312E, 313K, 333K 313K, 333K 0.5 78 306D, 312E,
333K N-terminal, 333K 2.3 79 312E, 321K, 333K 321K, 333K 1.5 83
312E, 321K, 333K 321K, 333K 1.5 84 312E, 321K, 333K 321K, 333K 1.8
85 300H, 312E, 313K, 333K 313K, 333K 0.9 86 312E, 313K, 328K 313K,
328K 1.1 87 312E, 313K, 324K 313K, 324K 1.0 88 312E, 313K
N-terminal, 313K 1.2 89 312E, 324K, 333K 324K, 333K 1.0 90 312E,
313K, 321K 313K, 321K 1.6 91 des293, 300H, 312E, 313K, 333K 313K,
333K 0.9 92 300H, 312E, 313K, 333K 313K, 333K 1.0 93 292A, 312E,
313K N-terminal (292A), 1.2 313K 94 des293, 312E, 313K N-terminal
(294T), 0.9 313K 96 312E, 313K, 332K 313K, 332K 1.2 97 312E, 328K,
333K 328K, 333K 1.2 98 312E, 313K, 333K 313K, 333K 0.9 99 312E,
313K, 333K 313K, 333K 1.3 100 312E, 313K, 333K 313K, 333K 1.4 101
312E, 313K, 333K 313K, 333K 0.6 102 312E, 313K, 333K 313K, 333K 0.8
107 312E, 333K N-terminal, 333K 2.6 108 des293-294, 300H, 312E,
313K 313K, 333K 3.8 333K 109 300H, 312E, 313K, 333K 313K, 333K 1.0
110 312E, 313K, 333K 313K, 333K 1.7 113 300H, 312E, 314K, 333K
314K, 333K 1.6 114 294W, 312E, 333K N-terminal, 333K 3.1 117
des293, 312E, 333K N-terminal, 333K 2.5 118 312E, 324K, 328K 324K,
328K 1.2 119 292A, 312E, 333K N-terminal, 333K 2.1 120 306Y, 312E,
313K, 333K 313K, 333K 1.6 121 312E, 332K N-terminal, 332K 2.1 122
312E, 328K N-terminal, 328K 2.2 123 312E, 324K N-terminal, 324K 2.0
127 312E, 321K, 332K 321K, 332K 2.4 128 312E, 313K, 333K 313K, 333K
1.0 129 312E, 313K, 333K 313K, 333K 2.6 130. 300H, 312E, 313K, 332K
313K, 332K 1.8 131. 312E, 313K, 333K 313K, 333K 2.6 132. 312E,
313K, 321E, 332K 313K, 332K 1.9 133. 301L, 309R, 312E, 313K, 321E
313K, 333K 1.6 333K 134. 312E, 321E, 333K 333K 1.9 135. 312E, 313K,
314K 313K, 314K 3.6 136. 313K 312K, 313K 2.8 137. 314K 312K, 314K
4.7 138. 311K, 312E, 313K 311K, 313K 2.5 139. 300H, 312E, 313K,
333K 313K, 333K 3.3 140. 312E, 313K, 333K 313K, 333K 1.7 141. 312E,
313K, 333K 313K, 333K 2.2 142. 312E, 313K, 333K 313K, 333K 1.7 143.
312E, 313K, 321E, 333K 313K, 333K 1.9 144. 312E, 313K, 321E, 333K
313K, 333K 2.09 145. 312E, 313K, 333K 313K, 333K 2.6 146. 312E,
313K, 321E, 333K 313K, 333K 3.0 147. 300H, 312E, 313K, 321E, 333K
313K, 333K 1.5 148. 312E, 313K, 333K 313K, 333K 2.5 149. des293,
300H, 312E, 313K, 333K 313K, 333K 1.9 150. 312E, 328K, 333K 328K,
333K 2.3 151. 312E, 321E, 328K, 333K 328K, 333K 1.8 152. 312E,
324K, 333K 324K, 333K 1.9 153. 312E, 321E, 324K, 333K 324K, 333K
2.0 154. 312E, 321E, 328K, 333K 328K, 333K 1.8 155. 312E, 313K,
321K 313K, 321K 1.4 156. 312E, 313K, 333K 313K, 333K 1.2 157. 312E,
313K, 321E, 333K 313K, 333K 1.3 Ex- am- ple Variant Attachment Ki
No. 301L + sites (nM) 111 309K, 312E, 333K 309K, 333K 1.6 112 306Y,
312E, 324K, 333K 324K, 333K 1.5 115 309K, 312E, 328K 309K, 328K 1.0
116 309K, 312E, 313K 309K, 313K 1.1 124 309K, 312E, 332K 309K, 332K
1.2 125 309K, 312E, 324K 309K, 324K 1.4 126 309K, 312E N-terminal,
309K 2.8
[1129] Again the inventors concluded that the substituents are very
well tolerated in a variety of positions and combinations.
Further PCSK9 Inhibitor Derivatives
[1130] To explore further the role of various amino acid
substitutions in the PCSK9 peptides further compounds were prepared
and tested as shown in table 5.5. All compounds include one
substituent which is attached via a Lys residue introduced by amino
acid substitution or extension with 333K. The back-bone peptides
all include the N301L amino acid substitution and optionally one or
more of N309R and I312E. The substituents all includes a fatty
diacid comprising 16-20 carbon atoms and a linker which is either
gGlu alone or extended with Ado-Ado and/or a tranexamic acid (Trx)
moiety.
TABLE-US-00014 TABLE 5.5 Apparent Ki for further PCSK9 derivatives.
Example Variant Attachment Ki No. 301L 309R 312E + sites (nM) 18
321E, 333K 333K 1.5 23 321E, 332K 332K 0.9 24 293K, 321E 293K 1.8
69 328K, 329H 328K 1.3 70 295D, 332K 332K 1325 76 des293, 294G,
328K 328K 1.3 77 306D, 324G, 333K 333K 2.2 80 333K 333K 1.9 81 333K
333K 1.4 82 333K 333K 1.9 106 300H, 333K 333K 1.0 134 321E, 333K
333K 1.9 158 321E, 333K 333K 2.3 159 321E, 333K 333K 1.9 Example
Variant Attachment Ki No. 301L 309R + sites (nM) 22 312Q N-term 2.6
42 300H, 312R, 333K 333K 0.7 57 293N, 300H, 312R, 333K 333K 0.5 60
293N, 312R, 333K 333K 1.0 66 293N, 307I, 312D, 333K 333K 2.1 67
293N, 312D, 333K 333K 2.0 71 300H 312K 0.9 Example Variant
Attachment No. 301L, 312E, + site Ki 47 309S, 333K 333K 2.7 62
306Y, 332K 332K 0.6 63 307I, 332K 332K 1.4 Example Variant
Attachment No. 301L, + site Ki 46 309S, 312R, 333K 333K 1.3 61
307I, 332K 332K 0.7
[1131] The results in table 5.5 above shows that the internal wt
lysine in position 312 can be substituted with Glu (E) as well as
Gln (Q), Arg (R) or Asp (D).Based on this variation it is
contemplated that a broad range of amino acid residues will be
tolerated in position 312 without interfering with the inhibitory
function of the peptide.
[1132] Several other amino acid substitutions were also proven to
be well tolerated including G293N, T294G, D299A, N300H, H306Y,
H306D, N3095, Q324G and R329H, while as mentioned above N295D and
N300P are none attractive amino acid substitutions.
D.1.2 LDL Uptake Assay in HepG2 Cells
[1133] An alternative assay to determine the inhibitory potency of
the PCSK9 peptides and derivatives thereof measuring uptake of LDL
in HepG2 cells is described here below.
[1134] Assay Principle:
[1135] LDL uptake is primarily mediated by the endogenously
expressed hLDLRs, and thus LDL uptake capacity is an indirect
measure of LDLR expression. The hLDLRs can be down-regulated by
incubation with exogenous PCSK9 in a dose dependent fashion. Thus
PCSK9 incubation will decrease the ability of cells to take up LDL
molecules. This down-regulation of LDL uptake can then be
antagonized by the addition of compounds neutralizing or inhibiting
the PCSK9/LDLR binding. Consequently PCSK9 inhibitors can be
characterized based on their capacity to increase LDL uptake in the
presence of PCSK9 and e.g. counter act the PCSK9 mediated hLDLR
down-regulation.
[1136] The assay is performed using HepG2 cells (Sigma Aldrich
ECACC: Acc no. 85011430) grown in 10% Lipoprotein deficient Foetal
Calf Serum (Sigma Aldrich #S5394) and the capacity of the cells to
take up BODIPY fluorescently labelled LDL particles (Life
technologies Europe BV #L3483) is measured.
[1137] Assay protocol: The 96 well plates (Perkin Elmer,
ViewPlate-96 Black #60005182) were coated with Poly-D-Lysin (10
mg/L, Sigma Aldrich #P6407 dissolved in PBS Gibco #14190-094) for 1
hour at 37.degree. C. in incubator. Then the plates were washed
2.times. in 100 .mu.l PBS (Gibco #14190-094). Test compositions for
8 point concentration curves of the EGF(A) compounds were prepared
all containing PCSK9 (10 ug/ml) diluted in Assay medium (DMEM
(Gibco #31966-021), 10% Lipoprotein deficient Foetal Calf Serum
(Sigma Aldrich #S5394) and 1% Pen Strep (Cambrex #DE17-602E)), and
added on to the plates in a volume of 50 ul/well.
[1138] After 30-60 minutes 50.000 HepG2 cells (Sigma-Aldrich:
ECACC: Atcc no. 85011430 lot: 13B023), diluted in Assay medium were
added in a volume of 50 .mu.l/well, and the plates were incubated
20 hours (at 37.degree. C., 5% CO2) in CO2 permeable plastic bags
(Antalis Team, LDPE bag 120/35.times.300.times.0,025 mm #281604).
Hereafter, the plates were emptied and immediately hereafter 50
.mu.l FL-LDL (Life technologies Europe BV #L3483) in a
concentration of 10 .mu.g/ml in Assay Medium was added to each
well, and the plates were incubated for 2 hours (at 37.degree. C.,
5% CO2) in CO2 permeable plastic bag using the black cover on the
lid to protect from light. The plates were emptied and washed 2
times with 100 .mu.l of PBS (Gibco #14190-094). Then 100 .mu.l of
PBS (Gibco #14190-094) was added and within 15 min hereafter, the
plates were read (bottom read) using the following filters Ex (515
nm)/Em (520 nm) on a SpecktraMax M4 (Molecular Probes, Invitrogen
Detection Technologies).
[1139] Finally, EC50 values were calculated using GraphPad Prism,
nonlinear regression curve fit, sigmoidal dose-response (variable
slope).
[1140] The results are shown in Table 6 below. Lower EC50 values
reflects higher capacity to reverse the PCSK9 mediated
down-regulation of LDL uptake, and inversely a high EC50 value is
indicative for a compound with low capacity to inhibit the PCSK9
mediated down-regulation of LDL uptake.
[1141] As can be seen most compounds display an EC50 in the LDL
uptake assay of 100-500 nM which is indicative of compounds with a
high capacity to reverse the PCSK9 mediated down-regulation of LDL
uptake.
TABLE-US-00015 TABLE 6 LDL uptake data in HepG2 cells (EC.sub.50)
Exam- LDL ple uptake No. EC.sub.50 (nM) 1. ND 2. 255 3. 168 4. 302
5. 220 6. 413 7. 304 8. 130 9. ND 10. 199 11. 401 12. ND 13. 280
14. 161 15. 211 16. 144 17. 199 18. 172 19. 206 20. 198 21. 174 22.
357 23. 143 24. 160 25. ND 26. 358 27. ND 28. ND 29. 163 30. 182
31. 170 32. 224 33. 245 34. 232 35. 252 36. ND 37. 188 38. 149 39.
156 40. 231 41. ND 42. 324 43. 499 44. 237 45. ND 46. ND 47. 1102
48. 1278 49. 398 50. 164 51. ND 52. ND 53. ND 54. 526 55. ND 56. ND
57. 438 58. ND 59. ND 60. 261 61. 347 62. 411 63. 197 64. 590 65.
10000 66. 248 67. 384 68. 124 69. 311 70. ND 71. 217 72. 222 73. ND
74. 123 75. 239 76. 272 77. 2044 78. 546 79. ND 80. 248 81. 617 82.
203 83. 165 84. 337 85. 157 86. 248 87. 185 88. 298 89. 139 90. 380
91. 114 92. 147 93. 267 94. 375 95. 257 96. 261 97. 138 98. 203 99.
167 100. 174 101. 129 102. 112 103. ND 104. ND 105. ND 106. 195
107. 486 108. 2555 109. 572 110. 465 111. 316 112. 539 113. 1383
114. 739 115. 247 116. 330 117. 316 118. 191 119. 327 120. 300 121.
201 122. 241 123. 351 124. 264 125. 334 126. 489 127. 245 128. 351
129. 892 130. 259 131. 218 132. 195 133. 220 134. 180 135. 1505
136. 455 137. 2070 138. 480 139. 546 140. 226 141. 210 142. 126
143. 299 144. 484 145. 329 146. 718 147. 246 148. 204 149. 233 150.
ND 151. ND 152. ND 153. ND 154. 148 155. 391 156. 167 157. ND 158.
303 159. 178
D.2. PK in Mice
[1142] The aim of this study was to measure the PK profile of PCSK9
inhibitors as identified above.
Method:
[1143] Female C57bl/J mice from Taconic (Ry, Denmark) were
used.
[1144] Dosing of Compound: Compounds were dosed either
subcutaneously (s.c., 500 nmol/kg) or intravenously (i.v., 250
nmol/kg) in a volume of 5 .mu.L per gram body weight.
[1145] Blood Sampling: Blood was sparse sampled at 2 min, 15 min,
30 min, 60 min, 2 hours, 4 hours, 6 hours, 8 hours, 18 hours, 24
hours, 30 hours and 48 hours. Blood (200 .mu.L) was taken from the
sublingual vein and transferred to EDTA-coated tubes
(Microvette.RTM. VetMed 200 K3E, Sarstedt nr 09.1293.100). Plasma
was isolated and used for quantification of anti-PCSK9
peptides.
[1146] Quantification: Plasma samples were used for quantification
of PCSK9 inhibitors using LC-MS.
Sampling and Analysis:
[1147] Plasma was pipetted into Micronic tubes on dry ice, and kept
at -20.degree. C. until analysed for plasma concentration of the
respective PCSK9 inhibitors using LC-MS. The plasma samples
(including standard curve and QC samples used for quantitation of
unknowns and prepared from blank plasma spiked with PCSK9
inhibitors at a concentration range of 0.5-1000 nM) were protein
precipitated using three volumes of 100% methanol or acetonitrile
with 1% formic acid (depending on anti-PCSK9 peptide) and
centrifuged (16000.times.g, 4.degree. C., 20 min). The supernatants
were injected into the chromatographic system (TurboFlow Transcend
1250 & 10 valve VIM, Thermo Fisher Scientific) which consisted
of an initial Turboflow Cyclone purification column 0.5.times.50 mm
(Thermo Fischer Scientific) and an eluting Aeris peptide 3.6 .mu.m
XB-C18 column 2.1.times.50 mm (Phenomenex) kept at 60.degree. C.
The anti-PCSK9 peptide was eluted using a chromatographic gradient
with mobile phases consisting of mixtures of water and acetonitrile
with 0.1% or 1% formic acid (depending on EGF(A) analogue or
derivative). The anti-PCSK9 peptide was detected and quantified
after on-line infusion of the LC flow to the LTQ OrbiTrap or the Q
Exactive mass spectrometer (Thermo Fischer Scientific) equipped
with an electrospray interface operated in positive mode, ESI+.
Calculation of PK Properties:
[1148] Plasma concentration-time profiles were analysed by a
non-compartmental pharmacokinetics analysis using the software
Phoenix WinNonlin 6.4. Calculations for both the I.V. and S.C. data
were performed using Linear Trapezoidal Linear Interpolation, with
the weighting 1/Y{circumflex over ( )}Y. The bioavailability was
calculated dividing AUC/Dose for the S.C. profile with the AUC/Dose
for the I.V. profile.
Results:
[1149] The results are shown in Table 7. In Table 7, Tmax indicates
the time to reach the maximum plasma concentration of the tested
EGF(A) analogue or derivative. T1/2 is the half-life of the EGF(A)
analogue or derivative. MRT is mean residence time. F (s.c.) is the
bio-availability of the EGF(A) analogue or derivative after
subcutaneous injection. Higher T1/2 values reflect longer half-life
of the tested compound.
[1150] The results show that PCSK9 inhibitors of the invention, in
particular LDL-R(293-332) analogues substituted with a fatty acid
substituent show prolonged half-lifes.
TABLE-US-00016 TABLE 7 Pharmacokinetic properties of LDL-R
(293-332) analogues and derivatives in mice T.sub.max i.v.
T.sub.1/2 s.c. T1/2 MRT F (s.c.) Substituent (hrs) (hrs) (hrs)
(hrs) (%) Example 1 Yes (N-term) 2 16 12 15 99 Example 48 No 0.3
0.2 0.4 0.2 76 Example 2 Yes (N-term) 2 14 14 19 100 Example 3 Yes
4 14 14 19 87 (via 333K) Example 5 Yes (N-term) 4 13 17 17 94
Example 6 Yes (via 6 11 11 16 100 299K) Example 13 Yes 2 6 7.5 11
96 (via 293K and 333K) Example 19 Yes (N-term) 2 13 14 18 100
Example 4 Yes (via 8 14.3 12.8 20.3 54 312K)
D.3. hPCSK9 Challenge Model
[1151] The aim of this study was to show the change in the LDL
receptor expression level in mouse liver in response to inhibiting
the action of intravenously injected hPCSK9 with an anti-PCSK9
peptide.
Method
[1152] Healthy male BalBC or NMRI mice (Charles River, Germany) are
injected with an anti-PCSK9 peptide, either s.c. or i.v. 15-120
minutes before injecting hPCSK9 (Sino Biologicals, China)
intravenously in the tail vein at a dose of 0.4 mg/kg. Sixty
minutes after the injection of hPCSK9, the animals are
anaesthetised in isoflurane and euthanised by cervical dislocation.
The liver is then quickly excised and snapfrozen in liquid
nitrogen. The livers are kept at -80 degrees celsius until
analysis.
LDL-R Western Blotting:
[1153] Liver tissue samples (100 mg) were homogenized in 500 .mu.l
lysis buffer (Life Technology, FNN0011) containing phosphatase
inhibitor cocktail; PhosStop (Roche, 04 906 837 001) and protease
inhibitor cocktail; complete (Roche, 04 693 159 001). After adding
1 steel bead tissues were homogenized for 2.5 min at 30 Hz. After
centrifugation at 5000.times.g for 5 min, total protein content was
determined using BCA Protein Assay Kit (Pierce, 23225). Equal
amounts of proteins (60 .mu.g) in sample buffer (Life Technology,
NP0007) were boiled for 10 min and spun for 2 min at 14000 rpm
before loaded onto Criterion XT 3-8% Tris-Acetate gels (BioRad
#345-0131) and subjected to SDS-PAGE. The proteins were transferred
to nitrocellulose membranes (iBlot 2 NC Regular stacks, novex
#IB23001) according to manufacturer's instructions (Life
Technology). Equal protein transfer was confirmed by Ponceau S
(Sigma, P7170) staining of the membranes and the membranes were
further blocked in blocking buffer (TBS-T, 2% Tween). LDL-r
proteins were detected with Primary rabbit anti LDLr antibody
(Cayman Chemical Company #10012422), whereas beta-actin proteins
were detected using Primary rabbit anti beta-actin antibody (abcam
#ab6276). Both proteins were further visualized with
peroxidase-conjugated goat anti-rabbit secondary antibodies (Biorad
#170-6516) using the WesternBright Quantum Chemiluminscent
(Advansta #K-12042-D10) and imaged using a CCD camera (LAS3000,
FujiFilm). Quantitative analysis of chemiluminescent signals from
Western blots was done with MultiGauge software (Fujifilm).
Results
[1154] FIG. 1 shows hepatic LDL-R expression levels measured by
Western Blot, presented as scatter plot for the individual animals,
n=3-6."Vehicle-vehicle" is the group of healthy controls (baseline
level), "vehicle-hPCSK9" is the group injected with hPCSK9 alone.
The results show that hPCSK9 decreases the expression level of
LDL-R and this effect is inhibited by the PCSK9 inhibitors
tested.
[1155] In Table 8, data are presented as percentage change in
relation to the window between baseline level in healthy control
animals (set to 100%) and the level after down regulation by hPCSK9
alone (set to 0%).
[1156] All 6 tested examples are able to inhibit the action of
hPCSK9 on the LDL-R expression level and the level of inhibition
observed is similar to the level of inhibition observed using the
control molecule Alirocumab.
TABLE-US-00017 TABLE 8 Percentage of Dose of inhibitor
Group/Example baseline (%) (nmol/kg) Vehicle-Vehicle 100 0
Vehicle-hPCSK9 0 0 Example 2-hPCSK9 110 300 Example 3-hPCSK9 113
300 Example 5-hPCSK9 123 300 Example 6-hPCSK9 96 300 Example
13-hPCSK9 175 300 Example 19-hPCSK9 190 300 Alirocumab-hPCSK9 157
22
Conclusion
[1157] Several compound examples have shown efficacy in inhibiting
the down-regulation of the LDL-R expression levels by hPCSK9.
D.4. LDL-Cholesterol Reduction in Hamsters
[1158] The aim of the study was to evaluate the effects of PCSK9
inhibitors on LDL-C in Golden Syrian hamsters fed a standard chow
diet.
Method
[1159] Male Golden Syrian Hamsters (Janvier Elevage, Saint Isle,
France), 6 weeks of age (91-100 g) were used in the study. After 1
week of acclimatisation, 4-hour fasted hamsters (fasting starts at
.about.08:00 am) were weighed and bled (100 .mu.L/EDTA) by
retro-orbital bleeding under isoflurane anesthesia at .about.noon
to measure total cholesterol, LDL-cholesterol and HDL-cholesterol.
Hamsters were randomized into 5 homogenous groups (n=10/group)
according to their 1) LDL-cholesterol, 2) HDL-cholesterol and 3)
total cholesterol. After randomization, hamsters were treated by
subcutaneous injection once daily for 5 days. Body weight was
measured daily during the treatment period.
[1160] At 3 days of treatment, 4-hour fasted hamsters were weighed
and bled (100 .mu.L/EDTA) by retro-orbital bleeding under
isoflurane anesthesia at .about.1 hour after the morning doses (at
.about.noon) to measure total cholesterol, LDL-cholesterol and
HDL-cholesterol.
[1161] At 5 days of treatment, 4-hour fasted hamsters were weighed
and bled (maximal blood volume/EDTA) by retro-orbital bleeding
under isoflurane anesthesia at .about.1 hour after the morning
doses (at .about.noon).
[1162] Plasma was immediately isolated. For each individual, a
.about.15 .mu.L plasma volume was kept to measure total
cholesterol, LDL-cholesterol and HDL-cholesterol. Another plasma
volume (.about.50 .mu.L) of each individual was then used to make a
plasma pool for each treatment group (i.e. 1 pool of .about.500
.mu.L per group, 5 pools) for FPLC total cholesterol profile.
Hamsters were then sacrificed under isoflurane anesthesia by
cervical dislocation and exsanguinated. Liver was harvested,
weighed and 2 liver samples (.about.50 mg and .about.100 mg, weight
not recorded) were flash frozen in liquid nitrogen and then stored
at -80.degree. C.
[1163] The .about.50 mg samples were used to evaluate hepatic
LDL-receptor and pan-cadherin (loading control) protein expression
by Western Blot and densitometry analysis (Image J software). Data
are presented as mean+/-SEM. A 1-way or 2-way ANOVA w/Dunnett or
Bonferroni post-test, respectively, were used for statistical
analysis. A p<0.05 was considered significant.
Results
[1164] FIG. 2 shows plasma LDL-cholesterol during the treatment
period in hamsters treated by subcutaneous injection once daily for
5 days with vehicle or 10 nmol/kg, 30 nmol/kg, 100 nmol/kg or 300
nmol/kg of Example 2. (**p<0.01 and ***p<0.001 vs. test
vehicle, two way ANOVA, Dunnetts post hoc analysis).
[1165] FIG. 3 shows hepatic LDL-R expression to loading control
pan-cadherin from liver samples of hamsters treated by subcutaneous
injection once daily for 5 days with vehicle or with Example 2 10
nmol/kg, Example 2 30 nmol/kg, Example 2 100 nmol/kg or Example 2
300 nmol/kg) (*p<0.05, **p<0.01 and ***p<0.001 vs.
vehicle, One way ANOVA, Dunnetts post hoc analysis).
[1166] Compared with vehicle body weight and body weight gain were
not affected in any treatment (data not shown). All doses reduced
LDL-cholesterol (see FIG. 2). This effect was not significant for
the lowest dose of Example 2, but the higher doses 100 and 300
nmol/kg reduced LDL-cholesterol levels by up to 35% at day 5. These
trends were further confirmed by FPLC analysis, which showed
substantial reductions in total cholesterol levels in fractions
corresponding to LDL and HDL when hamsters were treated with test
items Example 2 (data not shown). A concomitant dose-dependent
increase in the LDL-R expression levels in livers was also
demonstrated (see FIG. 2 and FIG. 3).
Conclusion
[1167] The dose response study demonstrates that it is possible to
obtain significant effect on LDL cholesterol at least with a dose
of 30 nmol/kg after 3 and 5 days of dosing in Golden Syrian
Hamsters on normal chow. The effect on LDL cholesterol is
concomitant with significantly higher hepatic LDL-receptor
expression levels.
D.5 Dog i.v. PK Study
[1168] For dog i.v. PK profile determination, 3-4 beagle dogs
(male, 10-16 kg) was dosed i.v. (2 nmol/kg, 0.1 ml/kg) with single
or multiple PCSK9 analogues in 70 mM sodium chloride; 50 mM
phosphate, 70 ppm polysorbate 20; pH=7.4. Before dosing, dogs were
fasted overnight with free access to tap water. Analogues were
dosed through saphenous or cephalic vein by single injection
through a needle (20 G) or sequential dosing through an inserted
venflon. A 0.8 ml of blood sample will be collected into each
EDTA-coated tube at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 24,
48, 72, 120, 144, 168, 192, 216, 240, 288 hours after dosing. For
the first 4 hour sampling, blood was collected through an inserted
venflon when the dogs were restrained on a platform. The rest of
sampling points after 4 hours were collected through the jugular
vein by single needle punch (20 G). Immediately after blood
collection, each sample was gently inversed for 3-4 times and
quickly transferred on an ice box before plasma preparation (10
min, 4.degree. C., 4000 rpm). Plasma samples were kept at
-20.degree. C. before bioanalysis. The maximal deviation for blood
sampling is 1 min on the day of dosing until 120-min post-dosing, 5
min for 4- to 10-hr time points, and within 1 hour for the rest of
the days.
Plasma Analysis:
[1169] Plasma from the co-dosing study was pipetted into Micronic
tubes on dry ice, and kept at -20.degree. C. until analysed for
plasma concentration of the respective PCSK9 derivatives using
liquid chromatography mass spectrometry (LC-MS). The plasma samples
(including standard curve and QC samples used for quantitation of
unknowns and prepared from blank plasma spiked with PCSK9
derivatives at a nominal concentration range of 0.5-500 nM) were
protein precipitated using three volumes of methanol (including
Example 4 as internal standard) and centrifuged (16000.times.g,
4.degree. C., 30 min). The supernatants were injected into the
chromatographic system (TurboFlow Transcend 1250 & 10 valve
VIM, Thermo Fisher Scientific) which consisted of an initial
Turboflow Cyclone purification column 0.5.times.50 mm (Thermo
Fischer Scientific) and an eluting Aeris peptide 3.6 .mu.m XB-C18
column 2.1.times.50 mm (Phenomenex) kept at 60.degree. C. The PCSK9
derivatives were eluted using a chromatographic gradient with
mobile phases consisting of mixtures of water and
acetonitrile/methanol 50/50 v/v % with 1 v/v % formic acid. The
PCSK9 derivatives were detected and quantified after on-line
infusion of the LC flow to the Q Exactive mass spectrometer (Thermo
Fischer Scientific) equipped with an electrospray interface
operated in positive mode, ESI+. During bioanalysis of plasma
samples, a varying degree of isomerization was observed for
different PCSK9 derivatives. The isomers all have identical
monoisotopic masses and are quantitated together.
[1170] PK parameters of each tested analogue (eg. T.sub.1/2) were
analyzed by non-compartmental analysis (NCA) using Phoenix
WinNonlin software, and half-lifes calculations are based on
exposure levels of a total of all isomers with the same molecular
mass.
TABLE-US-00018 TABLE 9 Half-lives of EGF(A) derivatives in dogs
after i.v. dosing Example Dog iv PK No. Peptide variant co-dosing
T.sub.1/2 (h) Example 3 301L, 309R, 312E, 333K 122 Example 31 301L,
309R, 312E, 313K, 333K 117 Example 81 301L, 309R, 312E, 333K 209
Example 91 des293, 300H, 301L, 309R, 312E, 313K, 333K 34 Example 95
des293, 301L, 309R, 312E, 313K 116 Example 301L, 309R, 312E, 313K,
333K 128 190 Example 301L, 309R, 312E, 313K, 321E, 333K 133 115
Example 301L, 309R, 312E, 313K, 321E, 333K 143 89 Example 301L,
309R, 312E, 313K, 321E, 333K 193 144
D.6 Oral Uptake Study in Rats
[1171] The current studies investigated gastrointestinal absorption
of co-formulated peptides dosed perorally to healthy rats.
Animals:
[1172] Male Sprague Dawley rats from Taconic, Denmark, 250 g at
arrival. Rats were acclimatised at least one week at Animal Unit,
Novo Nordisk A/S, prior to study. Bodyweight at study start was
approximately 280-300 g. The rats were fasted for 18 h on grid
prior to dosing.
Co-Formulation of Peptides
[1173] Preparation of liquid formulations for oral co-dosing of
PCSK9i analogues in vivo (rats) was carried out as described
below.
[1174] Target EGF(A) peptide concentration was 200 .mu.M of each
analogue, formulated in a target concentration of 55 mg/ml sodium
decanoate and water. Five to six different peptide analogues were
formulated together in the same formulation.
[1175] In short, a stock solution (110 mg/mL) of sodium decanoate
was prepared using ultrapure water and pH of the solution was
adjusted to 8.0using HCl.
[1176] The APIs were transferred into a 20 mL glass vial and 5 g of
ultrapure water was added (assuming 1 mg/mL density) and the APIs
were left to dissolve at room temperature on a roller mixer. The pH
of the solution was subsequently adjusted to 8.0 with NaOH until
the pH stabilized, after which 6.5 g of the sodium decanoate stock
(final concentration 55 mg/mL) was added followed by pH adjustment
to pH 8.0. The solution was then kept at room temperature on a
roller mixer overnight (protected from light). The next day a final
pH adjustment to pH 8.0 was performed if required using NaOH.
[1177] The final formulation weight was set to 13 g using ultrapure
water and subsequently filtered through a 0.22 .mu.m filter. API
and sodium decanoate content was determined on the final
formulation to ensure an accurate dosing. Formulations were stored
at 4.degree. C. until further use.
[1178] The concentration of each API in the liquid formulation was
determined by UV absorbance at 215 nm. LC methods were developed to
ensure that each co-dosing API eluted separately from each other.
Standards of known concentrations (determined by CLND) for each API
were mixed together, and in total five concentrations of standards
were used to generate calibration curves. The final determined
concentration was an average taken from three samples, each with
two experimental repeats. Caprate concentration in the liquid
formulation was determined in a similar fashion, using a
calibration curve consisting of three concentrations of
standards.
Dosing:
[1179] The animals were dosed perorally by gavage with a target
dose of 1000 nmol/kg of each peptide and a volume of 5 ml/kg at
time=0
Blood Sampling and Plasma Separation
[1180] Blood samples were taken at times: 15, 30, 60 and 120 min
after dosing. Blood samples (200 .mu.l) were collected into
EDTA-coated tubes by puncturing the tongue vein in conscious rats.
Samples were centrifuged for 5 minutes at 8000G by 4.degree. C.
Plasma (60-75 .mu.l) was separated and pipetted into micronic tubes
(75 .mu.l) and immediately frozen at -20.degree. C.
Plasma Analysis:
[1181] Plasma from the co-dosing study was pipetted into Micronic
tubes on dry ice, and kept at -20.degree. C. until analysed for
plasma concentration of the respective PCSK9 derivatives using
liquid chromatography mass spectrometry (LC-MS). The plasma samples
(including standard curve and QC samples used for quantitation of
unknowns and prepared from blank plasma spiked with PCSK9
derivatives at a nominal concentration range of 0.5-500 nM) were
protein precipitated using three volumes of methanol or
acetonitrile with 1 v/v % formic acid (including Example 4 as
internal standard) and centrifuged (16000.times.g, 4.degree. C., 30
min). The supernatants were injected into the chromatographic
system (TurboFlow Transcend 1250 & 10 valve VIM, Thermo Fisher
Scientific) which consisted of an initial Turboflow Cyclone
purification column 0.5.times.50 mm (Thermo Fischer Scientific) and
an eluting Aeris peptide 3.6 .mu.m XB-C18 column 2.1.times.50 mm
(Phenomenex) kept at 60.degree. C. The PCSK9 derivatives were
eluted using a chromatographic gradient with mobile phases
consisting of mixtures of water and acetonitrile/methanol 50/50 v/v
% with 1 v/v % formic acid. The PCSK9 derivatives were detected and
quantified after on-line infusion of the LC flow to the Q Exactive
or LTQ OrbiTrap Discovery mass spectrometer (Thermo Fischer
Scientific) equipped with an electrospray interface operated in
positive mode, ESI+. During bioanalysis of plasma samples, a
varying degree of isomerization was observed for different PCSK9
derivatives. The isomers all have identical monoisotopic masses and
are quantitated together.
Data Calculations:
[1182] From the plasma concentrations determined by LC-MS, maximal
plasma concentrations (Cmax) were extracted for each peptide in
each rat and Cmax/dose was calculated as mean values.+-.SD for
n=6-8 rats. The dose was calculated as the injection volume,
adjusted for body weight, multiplied with the actual concentration
of the peptide, the unit being pmol/kg.
[1183] In each co-formulation group a reference peptide was
included (example 3). In below table, Cmax/dose (kg/I) is listed
for 8 different peptides together with the Cmax/dose (kg/l) for the
reference peptide (Example 3). Cmax calculations are based on
exposure levels of a total of all isomers with the same molecular
mass. The results show that the EGF(A) derivatives are generally
well absorbed.
TABLE-US-00019 TABLE 10 Plasma concentrations divided by dose in
rats after oral co-dosing of EGF(A) derivatives Example no
Cmax/dose (kg/l) Cmax/dose for ref. Example 3 (kg/l) Example 31
0.108 .+-. 0.086 0.053 .+-. 0.050 Example 81 0.024 .+-. 0.005 0.079
.+-. 0.013 Example 91 0.116 .+-. 0.032 0.083 .+-. 0.023 Example 95
0.106 .+-. 0.029 0.087 .+-. 0.023 Example 128 0.130 .+-. 0.013
0.087 .+-. 0.019 Example 133 0.071 .+-. 0.017 0.057 .+-. 0.015
Example 143 0.151 .+-. 0.038 0.096 .+-. 0.029 Example 144 0.100
.+-. 0.025 0.057 .+-. 0.015
[1184] While certain features of the invention have been
illustrated and described herein, many modifications,
substitutions, changes, and equivalents will now occur to those of
ordinary skill in the art. It is, therefore, to be understood that
the appended claims are intended to cover all such modifications
and changes as fall within the true spirit of the invention.
Sequence CWU 1
1
106140PRTArtificial SequenceSynthetic sequence 1Gly Thr Asn Glu Cys
Leu Asp Asn Asn Gly Gly Cys Ser His Val Cys1 5 10 15Asn Asp Leu Lys
Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala
Gln Arg Arg Cys Glu 35 40240PRTArtificial SequenceSynthetic
sequence 2Gly Thr Asn Glu Cys Leu Ala Asn Leu Gly Gly Cys Ser His
Ile Cys1 5 10 15Arg Lys Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp
Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu 35
40340PRTArtificial SequenceSynthetic sequence 3Gly Thr Asn Glu Cys
Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Lys
Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala
Gln Arg Arg Cys Glu 35 40441PRTArtificial SequenceSynthetic
sequence 4Gly Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His
Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp
Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu Lys 35
40540PRTArtificial SequenceSynthetic 5Gly Thr Asn Glu Cys Leu Asp
Pro Leu Gly Gly Cys Ser His Ile Cys1 5 10 15Arg Asp Leu Glu Ile Gly
Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg
Arg Cys Glu 35 40640PRTArtificial SequenceSynthetic sequence 6Gly
Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10
15Arg Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30Leu Val Ala Gln Arg Arg Cys Glu 35 40740PRTArtificial
SequenceSynthetic sequence 7Gly Thr Asn Glu Cys Leu Lys Asn Leu Gly
Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu Cys
Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu
35 40840PRTArtificial SequenceSynthetic sequence 8Gly Thr Asn Glu
Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu
Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val
Ala Gln Arg Lys Cys Glu 35 40941PRTArtificial SequenceSynthetic
9Asn Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Ile Cys1 5
10 15Arg Asp Leu Asp Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe
Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu Lys 35
401041PRTArtificial SequenceSynthetic sequence 10Asn Thr Asn Glu
Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu
Asp Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val
Ala Gln Arg Arg Cys Glu Lys 35 401140PRTArtificial
SequenceSynthetic sequence 11Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Lys 35 401240PRTArtificial SequenceSynthetic sequence 12Lys Thr Asn
Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp
Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu
Val Ala Gln Arg Arg Cys Glu 35 401341PRTArtificial
SequenceSynthetic sequence 13Lys Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu Lys 35 401440PRTArtificial SequenceSynthetic sequence 14Gly Thr
Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg
Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25
30Leu Val Ala Lys His Arg Cys Glu 35 401541PRTArtificial
SequenceSynthetic sequence 15Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Lys Lys 35 401641PRTArtificial SequenceSynthetic sequence 16Gly Thr
Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg
Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25
30Leu Val Ala Gln Arg Lys Cys Glu Lys 35 401741PRTArtificial
SequenceSynthetic sequence 17Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu
Cys Leu Cys Pro Lys Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu Lys 35 401841PRTArtificial SequenceSynthetic sequence 18Gly Thr
Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg
Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25
30Leu Val Ala Gln Arg Arg Cys Glu Lys 35 401941PRTArtificial
SequenceSynthetic sequence 19Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu
Cys Leu Cys Pro Glu Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu Lys 35 402040PRTArtificial SequenceSynthetic 20Gly Thr Asp Glu
Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu
Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val
Ala Gln Arg Arg Cys Lys 35 402140PRTArtificial SequenceSynthetic
sequence 21Gly Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His
Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Lys
Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu 35
402240PRTArtificial SequenceSynthetic sequence 22Gly Thr Asn Glu
Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu
Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Lys 20 25 30Leu Val
Ala Gln Arg Arg Cys Glu 35 402340PRTArtificial SequenceSynthetic
sequence 23Gly Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His
Val Cys1 5 10 15Arg Asp Leu Gln Ile Gly Tyr Glu Cys Leu Cys Pro Asp
Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu 35
402440PRTArtificial SequenceSynthetic sequence 24Gly Thr Asn Glu
Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu
Glu Ile Gly Tyr Glu Cys Leu Cys Pro Glu Gly Phe Gln 20 25 30Leu Val
Ala Gln Arg Arg Cys Lys 35 402540PRTArtificial SequenceSynthetic
sequence 25Lys Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His
Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Glu
Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu 35
402640PRTArtificial SequenceSynthetic sequence 26Gly Thr Asn Glu
Cys Leu Asp His Leu Gly Gly Cys Ser His Ile Cys1 5 10 15Arg Asp Leu
Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val
Ala Gln Arg Arg Cys Glu 35 402740PRTArtificial SequenceSynthetic
sequence 27Gly Thr Asn Glu Cys Leu Asp Lys Leu Gly Gly Cys Ser His
Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp
Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu 35
402840PRTArtificial SequenceSynthetic sequence 28Lys Lys Asn Glu
Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu
Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val
Ala Gln Arg Arg Cys Glu 35 402940PRTArtificial SequenceSynthetic
sequence 29Lys Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His
Val Cys1 5 10 15Arg Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp
Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu 35
403040PRTArtificial SequenceSynthetic sequence 30Gly Thr Asn Glu
Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Lys Asp Leu
Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val
Ala Gln Arg Arg Cys Glu 35 403140PRTArtificial SequenceSynthetic
sequence 31Gly Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His
Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu Cys Lys Cys Pro Asp
Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu 35
403241PRTArtificial SequenceSynthetic sequence 32Gly Thr Asn Glu
Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu
Glu Lys Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val
Ala Gln Arg Arg Cys Glu Lys 35 403340PRTArtificial
SequenceSynthetic sequence 33Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Lys Ala Gln Arg Arg Cys
Glu 35 403440PRTArtificial SequenceSynthetic sequence 34Gly Thr Asn
Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp
Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Lys
Val Ala Gln Arg Arg Cys Glu 35 403540PRTArtificial
SequenceSynthetic sequence 35Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Lys Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu 35 403640PRTArtificial SequenceSynthetic sequence 36Gly Thr Asn
Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp
Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Lys Phe Gln 20 25 30Leu
Val Ala Gln Arg Arg Cys Glu 35 403740PRTArtificial
SequenceSynthetic sequence 37Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu
Cys Leu Cys Lys Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu 35 403840PRTArtificial SequenceSynthetic sequence 38Gly Thr Asn
Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp
Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu
Val Ala Gln Lys Arg Cys Glu 35 403940PRTArtificial
SequenceSynthetic sequence 39Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Lys Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu 35 404040PRTArtificial SequenceSynthetic sequence 40Gly Thr Asn
Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp
Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu
Val Ala Lys Arg Arg Cys Glu 35 404140PRTArtificial
SequenceSynthetic sequence 41Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Lys
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu 35 404240PRTArtificial SequenceSynthetic sequence 42Gly Thr Asn
Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp
Leu Glu Ile Gly Lys Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu
Val Ala Gln Arg Arg Cys Glu 35 404341PRTArtificial
SequenceSynthetic sequence 43Gly Thr Asn Glu Cys Leu Asp His Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Arg Ile Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu Lys 35 404440PRTArtificial SequenceSynthetic sequence 44Gly Thr
Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg
Asp Leu Glu Ile Lys Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25
30Leu Val Ala Gln Arg Arg Cys Glu 35 404540PRTArtificial
SequenceSynthetic sequence 45Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Lys Glu Ile Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu 35 404640PRTArtificial SequenceSynthetic sequence 46Gly Thr Asn
Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Lys Cys1 5 10 15Arg Asp
Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu
Val Ala Gln Arg Arg Cys Glu 35 404741PRTArtificial
SequenceSynthetic sequence 47Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Ser Asp Leu Arg Ile Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu Lys 35 404841PRTArtificial SequenceSynthetic sequence 48Gly Thr
Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Ser
Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25
30Leu Val Ala Gln Arg Arg Cys Glu Lys 35 404940PRTArtificial
SequenceSynthetic sequence 49Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser Tyr Val Cys1 5 10 15Ser Asp Leu Glu Ile Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu 35 405040PRTArtificial SequenceSynthetic sequence 50Asn Thr Asn
Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Ser Asp
Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu
Val Ala Gln Arg Arg Cys Glu 35 405140PRTArtificial
SequenceSynthetic sequence 51Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser Lys Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu 35
405240PRTArtificial SequenceSynthetic sequence 52Gly Thr Asn Glu
Cys Leu Asp Asn Leu Gly Gly Cys Lys His Val Cys1 5 10 15Arg Asp Leu
Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val
Ala Gln Arg Arg Cys Glu 35 405340PRTArtificial SequenceSynthetic
sequence 53Gly Thr Asn Glu Cys Leu Asp Asn Leu Gly Lys Cys Ser His
Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp
Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu 35
405440PRTArtificial SequenceSynthetic sequence 54Gly Thr Asn Glu
Cys Leu Asp Asn Leu Lys Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu
Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val
Ala Gln Arg Arg Cys Glu 35 405541PRTArtificial SequenceSynthetic
sequence 55Asn Thr Asn Glu Cys Leu Asp His Leu Gly Gly Cys Ser His
Val Cys1 5 10 15Arg Asp Leu Arg Ile Gly Tyr Glu Cys Leu Cys Pro Asp
Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu Lys 35
405640PRTArtificial SequenceSynthetic sequence 56Gly Thr Asn Glu
Cys Leu Asp Asn Lys Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu
Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val
Ala Gln Arg Arg Cys Glu 35 405740PRTArtificial SequenceSynthetic
sequence 57Gly Thr Asn Glu Cys Lys Asp Asn Leu Gly Gly Cys Ser His
Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp
Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu 35
405841PRTArtificial SequenceSynthetic sequence 58Asn Thr Asn Glu
Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu
Arg Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val
Ala Gln Arg Arg Cys Glu Lys 35 405940PRTArtificial
SequenceSynthetic sequence 59Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Ile Cys1 5 10 15Asn Asp Leu Lys Ile Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Lys 35 406040PRTArtificial SequenceSynthetic sequence 60Gly Thr Asn
Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser Tyr Val Cys1 5 10 15Asn Asp
Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu
Val Ala Gln Arg Arg Cys Lys 35 406140PRTArtificial
SequenceSynthetic sequence 61Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Ile Cys1 5 10 15Asn Asp Leu Glu Ile Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Lys 35 406240PRTArtificial SequenceSynthetic sequence 62Gly Thr Asn
Glu Cys Leu Asp His Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp
Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu
Val Ala Gln Arg Arg Cys Glu 35 406340PRTArtificial
SequenceSynthetic sequence 63Gly Thr Asn Lys Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu 35 406440PRTArtificial SequenceSynthetic sequence 64Gly Lys Asn
Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp
Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu
Val Ala Gln Arg Arg Cys Glu 35 406541PRTArtificial
SequenceSynthetic sequence 65Lys Gly Thr Asn Glu Cys Leu Asp Asn
Leu Gly Gly Cys Ser His Val1 5 10 15Cys Arg Asp Leu Glu Ile Gly Tyr
Glu Cys Leu Cys Pro Asp Gly Phe 20 25 30Gln Leu Val Ala Gln Arg Arg
Cys Glu 35 406639PRTArtificial SequenceSynthetic sequence 66Gly Asn
Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys Arg1 5 10 15Asp
Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu 20 25
30Val Ala Lys Arg Arg Cys Glu 356741PRTArtificial SequenceSynthetic
sequence 67Gly Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser Asp
Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp
Gly Phe Gly 20 25 30Leu Val Ala Gln Arg Arg Cys Glu Lys 35
406841PRTArtificial SequenceSynthetic sequence 68Gly Thr Asn Glu
Cys Leu Asp Asn Leu Gly Gly Cys Ser Asp Val Cys1 5 10 15Arg Asp Leu
Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val
Ala Gln Arg Arg Cys Glu Lys 35 406941PRTArtificial
SequenceSynthetic sequence 69Gly Thr Asn Glu Cys Leu Asp His Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Lys Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu Lys 35 407040PRTArtificial SequenceSynthetic sequence 70Gly Thr
Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg
Asp Leu Glu Lys Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25
30Leu Val Ala Lys Arg Arg Cys Glu 35 407140PRTArtificial
SequenceSynthetic sequence 71Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Lys Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Lys 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu 35 407241PRTArtificial SequenceSynthetic sequence 72Gly Thr Asn
Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp
Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Lys 20 25 30Leu
Val Ala Gln Arg Arg Cys Glu Lys 35 407340PRTArtificial
SequenceSynthetic sequence 73Gly Thr Asn Glu Cys Leu Asp Asn Leu
Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Lys Gly Tyr Glu
Cys Leu Cys Pro Lys Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu 35 407440PRTArtificial SequenceSynthetic sequence 74Thr Asn Glu
Cys Leu Asp His Leu Gly Gly Cys Ser His Val Cys Arg1 5 10 15Asp Leu
Glu Lys Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu 20 25 30Val
Ala Gln Arg Arg Cys Glu Lys 35 407541PRTArtificial
SequenceSynthetic sequence 75Ala Gly Thr Asn Glu Cys Leu Asp Asn
Leu Gly Gly Cys Ser His Val1 5 10 15Cys Arg Asp Leu Glu Lys Gly Tyr
Glu Cys Leu Cys Pro Asp Gly Phe 20 25 30Gln Leu Val Ala Gln Arg Arg
Cys Glu 35 407639PRTArtificial SequenceSynthetic sequence 76Thr Asn
Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys Arg1 5 10 15Asp
Leu Glu Lys Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu 20 25
30Val Ala Gln Arg Arg Cys Glu 357740PRTArtificial SequenceSynthetic
sequence 77Gly Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His
Val Cys1 5 10 15Arg Asp Leu Glu Lys Gly Tyr Glu Cys Leu Cys Pro Asp
Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Lys 35
407841PRTArtificial SequenceSynthetic sequence 78Gly Thr Asn Glu
Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu
Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val
Ala Lys Arg Arg Cys Glu Lys 35 407940PRTArtificial
SequenceSynthetic sequence 79Gly Thr Asn Glu Cys Leu Ala Asn Leu
Gly Gly Cys Ser His Ile Cys1 5 10 15Arg Asp Leu Lys Ile Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys
Glu 35 408040PRTArtificial SequenceGTNECLDNLG GCSHVCRKLK IGYECLCPDG
FQLVAQRRCE 80Gly Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser
His Val Cys1 5 10 15Arg Lys Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro
Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu 35
408140PRTArtificial SequenceSynthetic sequence 81Gly Thr Asn Glu
Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Asn Asp Leu
Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val
Ala Gln Arg Arg Cys Glu 35 408241PRTArtificial Sequencesynthetic
82Gly Thr Asn Glu Cys Leu Asp His Leu Gly Gly Cys Ser His Val Cys1
5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe
Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu Lys 35
408339PRTArtificial Sequencesynthetic 83Asn Glu Cys Leu Asp His Leu
Gly Gly Cys Ser His Val Cys Arg Asp1 5 10 15Leu Glu Lys Gly Tyr Glu
Cys Leu Cys Pro Asp Gly Phe Gln Leu Val 20 25 30Ala Gln Arg Arg Cys
Glu Lys 358441PRTArtificial Sequencesynthetic 84Gly Thr Asn Glu Cys
Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Lys Asp Leu Glu
Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala
Gln Arg Arg Cys Glu Lys 35 408541PRTArtificial Sequencesynthetic
85Gly Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser Tyr Val Cys1
5 10 15Asn Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe
Lys 20 25 30Leu Val Ala Gln Arg Arg Cys Glu Lys 35
408641PRTArtificial Sequencesynthetic 86Gly Thr Asn Glu Cys Leu Asp
His Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Ile Lys
Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg
Arg Cys Glu Lys 35 408741PRTArtificial Sequencesynthetic 87Gly Trp
Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg
Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25
30Leu Val Ala Gln Arg Arg Cys Glu Lys 35 408840PRTArtificial
Sequencesynthetic 88Gly Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys
Ser His Val Cys1 5 10 15Lys Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys
Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Lys Arg Arg Cys Glu 35
408940PRTArtificial Sequencesynthetic 89Gly Thr Asn Glu Cys Leu Asp
Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Lys Asp Leu Glu Lys Gly
Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg
Arg Cys Glu 35 409040PRTArtificial Sequencesynthetic 90Thr Asn Glu
Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys Arg1 5 10 15Asp Leu
Glu Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu 20 25 30Val
Ala Gln Arg Arg Cys Glu Lys 35 409140PRTArtificial
Sequencesynthetic 91Gly Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys
Ser His Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys
Pro Asp Gly Phe Lys 20 25 30Leu Val Ala Lys Arg Arg Cys Glu 35
409242PRTArtificial Sequencesynthetic 92Ala Gly Thr Asn Glu Cys Leu
Asp Asn Leu Gly Gly Cys Ser His Val1 5 10 15Cys Arg Asp Leu Glu Ile
Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe 20 25 30Gln Leu Val Ala Gln
Arg Arg Cys Glu Lys 35 409341PRTArtificial Sequencesynthetic 93Gly
Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser Tyr Val Cys1 5 10
15Arg Asp Leu Glu Lys Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30Leu Val Ala Gln Arg Arg Cys Glu Lys 35 409440PRTArtificial
Sequencesynthetic 94Gly Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys
Ser His Val Cys1 5 10 15Lys Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys
Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Lys 35
409540PRTArtificial Sequencesynthetic 95Gly Thr Asn Glu Cys Leu Asp
Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Ile Gly
Tyr Glu Cys Leu Cys Pro Lys Gly Phe Gln 20 25 30Leu Val Ala Gln Arg
Arg Cys Lys 35 409640PRTArtificial Sequencesynthetic 96Gly Thr Asn
Glu Cys Leu Asp His Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp
Leu Glu Lys Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu
Val Ala Gln Arg Arg Cys Lys 35 409740PRTArtificial
Sequencesynthetic 97Gly Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys
Ser His Val Cys1 5 10 15Arg Asp Leu Glu Lys Gly Tyr Glu Cys Leu Cys
Pro Glu Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Lys 35
409841PRTArtificial Sequencesynthetic 98Gly Thr Asn Glu Cys Leu Asp
Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Lys Gly
Tyr Glu Cys Leu Cys Pro Glu Gly Phe Gln 20 25 30Leu Val Ala Gln Arg
Arg Cys Glu Lys 35 409940PRTArtificial Sequencesynthetic 99Gly Thr
Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg
Asp Leu Glu Lys Lys Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25
30Leu Val Ala Gln Arg Arg Cys Glu 35 4010040PRTArtificial
Sequencesynthetic 100Gly Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly
Cys Ser His Val Cys1 5 10 15Arg Asp Leu Lys Lys Gly Tyr Glu Cys Leu
Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu 35
4010140PRTArtificial Sequencesynthetic 101Gly Thr Asn Glu Cys Leu
Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Lys Ile
Lys Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30Leu Val Ala Gln
Arg Arg Cys Glu 35 4010240PRTArtificial Sequencesynthetic 102Gly
Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10
15Arg Asp Lys Glu Lys Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30Leu Val Ala Gln Arg Arg Cys Glu 35 4010341PRTArtificial
Sequencesynthetic 103Gly Thr Asn Glu Cys Leu Asp His Leu Gly Gly
Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Lys Gly Tyr Glu Cys Leu
Cys Pro Glu Gly Phe Gln 20 25 30Leu Val Ala Gln Arg Arg Cys Glu Lys
35
4010441PRTArtificial Sequencesynthetic 104Gly Thr Asn Glu Cys Leu
Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Arg Asp Leu Glu Ile
Gly Tyr Glu Cys Leu Cys Pro Glu Gly Phe Gln 20 25 30Leu Val Ala Lys
Arg Arg Cys Glu Lys 35 4010541PRTArtificial Sequencesynthetic
105Gly Thr Asn Glu Cys Leu Asp Asn Leu Gly Gly Cys Ser His Val Cys1
5 10 15Arg Asp Leu Glu Ile Gly Tyr Glu Cys Leu Cys Pro Glu Gly Phe
Lys 20 25 30Leu Val Ala Gln Arg Arg Cys Glu Lys 35
4010640PRTArtificial sequenceSynthetic 106Gly Thr Asn Glu Cys Leu
Asp Asn Leu Gly Gly Cys Ser His Val Cys1 5 10 15Lys Asp Leu Glu Ile
Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Lys 20 25 30Leu Val Ala Gln
Arg Arg Cys Glu 35 40
* * * * *
References