U.S. patent application number 16/579799 was filed with the patent office on 2021-03-18 for improved peptide pharmaceuticals.
This patent application is currently assigned to Mederis Diabetes LLC. The applicant listed for this patent is Mederis Diabetes LLC. Invention is credited to John J. NESTOR.
Application Number | 20210077629 16/579799 |
Document ID | / |
Family ID | 1000005248082 |
Filed Date | 2021-03-18 |
![](/patent/app/20210077629/US20210077629A1-20210318-C00001.png)
![](/patent/app/20210077629/US20210077629A1-20210318-C00002.png)
![](/patent/app/20210077629/US20210077629A1-20210318-C00003.png)
![](/patent/app/20210077629/US20210077629A1-20210318-C00004.png)
![](/patent/app/20210077629/US20210077629A1-20210318-C00005.png)
![](/patent/app/20210077629/US20210077629A1-20210318-C00006.png)
![](/patent/app/20210077629/US20210077629A1-20210318-C00007.png)
![](/patent/app/20210077629/US20210077629A1-20210318-C00008.png)
![](/patent/app/20210077629/US20210077629A1-20210318-C00009.png)
![](/patent/app/20210077629/US20210077629A1-20210318-C00010.png)
![](/patent/app/20210077629/US20210077629A1-20210318-C00011.png)
View All Diagrams
United States Patent
Application |
20210077629 |
Kind Code |
A1 |
NESTOR; John J. |
March 18, 2021 |
IMPROVED PEPTIDE PHARMACEUTICALS
Abstract
Described herein are methods of syntheses and therapeutic uses
of covalently modified peptides and/or proteins. The covalently
modified peptides and/or proteins allow for improved pharmaceutical
properties of peptide and protein-based therapeutics.
Inventors: |
NESTOR; John J.; (Sugar
Land, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mederis Diabetes LLC |
Sugar Land |
TX |
US |
|
|
Assignee: |
Mederis Diabetes LLC
Sugar Land
TX
|
Family ID: |
1000005248082 |
Appl. No.: |
16/579799 |
Filed: |
September 23, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15913845 |
Mar 6, 2018 |
10420844 |
|
|
16579799 |
|
|
|
|
14118546 |
Nov 18, 2013 |
10010617 |
|
|
PCT/US12/38429 |
May 17, 2012 |
|
|
|
15913845 |
|
|
|
|
61487638 |
May 18, 2011 |
|
|
|
61487636 |
May 18, 2011 |
|
|
|
61543725 |
Oct 5, 2011 |
|
|
|
61543721 |
Oct 5, 2011 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C09D 101/28 20130101;
A61K 38/08 20130101; C07K 7/06 20130101; A61K 38/095 20190101; C07K
1/1077 20130101; A61K 38/29 20130101; A61K 38/26 20130101; A61K
47/64 20170801; A61K 38/07 20130101; C07K 5/10 20130101; A61K
47/549 20170801 |
International
Class: |
A61K 47/54 20060101
A61K047/54; C07K 1/107 20060101 C07K001/107; C07K 7/06 20060101
C07K007/06; A61K 38/08 20060101 A61K038/08; A61K 38/26 20060101
A61K038/26; A61K 38/29 20060101 A61K038/29; A61K 38/095 20060101
A61K038/095; A61K 38/07 20060101 A61K038/07; C07K 5/10 20060101
C07K005/10; A61K 47/64 20060101 A61K047/64 |
Claims
1. A peptide product comprising a surfactant X, covalently attached
to a GLP-1 receptor (GLP1R) and/or glucagon receptor (GCGR) binding
peptide, the peptide product comprising: ##STR00060## wherein X is
##STR00061## wherein A is a hydrophobic group comprising a
C.sub.1-C.sub.30 alkyl chain or an aralkyl chain; and B is a
hydrophilic saccharide group covalently attached to the peptide via
a linker amino acid U, wherein U is a natural or unnatural amino
acid.
2. The peptide product of claim 1, wherein the peptide binds GLP1R
and GCGR.
3. The peptide product of claim 1, wherein A is a substituted or
unsubstituted C.sub.3-C.sub.20 alkyl chain.
4. The peptide product of claim 1, wherein the surfactant X is an
N-linked alkyl glycoside comprising one or more hydroxyl functions
modified to be a carboxylic acid.
5. The peptide product of claim 1, wherein the the surfactant X
comprises a carboxylic acid group.
6. The peptide product of claim 1, wherein the saccharide is
selected from glucose, mannose, maltose, a glucuronic acid,
galacturonic acid, diglucuronic acid and maltouronic acid.
7. The peptide product of claim 1, wherein the surfactant is a
1-alkyl glycoside class surfactant.
8. The peptide product of claim 1, wherein the saccharide group is
attached to the peptide via an amide bond.
9. The peptide product of claim 1, wherein the surfactant X is
comprised of 1-eicosyl beta-D-glucuronic acid, 1-octadecyl
beta-D-glucuronic acid, 1-hexadecyl beta-D-glucuronic acid,
1-tetradecylbeta D-glucuronic acid, 1-dodecyl beta D-glucuronic
acid, 1-decyl beta-D-glucuronic acid, 1-octyl beta-D-glucuronic
acid, 1-eicosyl beta-D-diglucuronic acid, 1-octadecyl
beta-D-diglucuronic acid, 1-hexadecyl beta-D-diglucuronic acid,
1-tetradecyl beta-D-diglucuronic acid, 1-dodecyl
beta-D-diglucuronic acid, 1-decyl beta-D-diglucuronic acid, 1-octyl
beta-D-diglucuronic acid, or functionalized 1-ecosyl
beta-D-glucose, 1-octadecyl beta-D-glucose, 1-hexadecyl
beta-D-glucose, 1-tetradecyl beta-D-glucose, 1-dodecyl
beta-D-glucose, 1-decyl beta-D-glucose, 1-octyl beta-D-glucose,
1-eicosyl beta-D-maltoside, 1-octadecyl beta-D-maltoside,
1-hexadecyl beta-D-maltoside, 1-dodecyl beta-D-maltoside, 1-decyl
beta-D-maltoside, or 1-octyl beta-D-maltoside.
10. The peptide product of claim 1, wherein the peptide product
comprises a covalently linked N-omega-1-alkyl
.beta.-D-glucuronyl.
11. The peptide product of claim 1, wherein the peptide comprises
aa.sub.1 to aa.sub.26 of SEQ ID NO: 304;
aa.sub.1-aa.sub.2-aa.sub.3-aa.sub.4-aa.sub.5-aa.sub.6-aa.sub.7-aa.sub.8-a-
a.sub.9-aa.sub.10-aa.sub.11-aa.sub.12-aa.sub.13-aa.sub.14-aa.sub.15-aa.sub-
.16-aa.sub.17-aa.sub.18-aa.sub.19-aa.sub.20-aa.sub.21-aa.sub.22-aa.sub.23--
aa.sub.24-aa.sub.25-aa.sub.26-aa.sub.27-aa.sub.28-aa.sub.29-Z (SEQ.
ID. NO. 304) wherein: Z is OH, or --NH--R.sup.3, wherein R.sup.3 is
H, or C.sub.1-C.sub.12 substituted or unsubstituted alkyl, or a PEG
chain of less than 10 Da; aa.sub.1 is His, N--Ac-His, pGlu-His, or
N--R.sup.3-His; aa.sub.2 is Ser, Ala, Gly, Aib, Ac4c, or Ac5c;
aa.sub.3 is Gln, or Cit; aa.sub.4 is Gly, or D-Ala; aa.sub.5 is
Thr, or Ser; aa.sub.6 is Phe, Trp, F2Phe, Me2Phe, or Nal2; aa.sub.7
is Thr, or Ser; aa.sub.8 is Ser, or Asp; aa.sub.9 is Asp, or Glu;
aa.sub.10 is Tyr, Leu, Met, Nal2, Bip, or Bip2EtMeO; aa.sub.11 is
Ser, Asn, or U; aa.sub.12 is Lys, Glu, Ser, Arg, or U(X); aa.sub.13
is absent or Tyr, Gln, Cit, or U(X); aa.sub.14 is absent or Leu,
Met, Nle, or U(X); aa.sub.15 is absent or Asp, Glu, or U(X);
aa.sub.16 is absent or Ser, Gly, Glu, Aib, Ac5c, Lys, Arg, or U(X);
aa.sub.17 is absent or Arg, hArg, Gln, Glu, Cit, Aib, Ac4c, Ac5c,
or U(X); aa.sub.18 is absent or Arg, hArg, Ala, Aib, Ac4c, Ac5c, or
U(X); aa.sub.19 is absent or Ala, Val, Aib, Ac4c, Ac5c, or U(X);
aa.sub.20 is absent or Gln, Lys, Arg, Cit, Glu, Aib, Ac4c, Ac5c, or
U(X); aa.sub.21 is absent or Asp, Glu, Leu, Aib, Ac4c, Ac5c, or
U(X); aa.sub.22 is absent or Phe, Trp, Nal2, Aib, Ac4c, Ac5c, or
U(X); aa.sub.23 is absent or Val, Ile, Aib, Ac4c, Ac5c, or U(X);
aa.sub.24 is absent or Gln, Ala, Glu, Cit, or U(X); aa.sub.25 is
absent or Trp, Nal2, or U(X); aa.sub.26 is absent or Leu, or U(X);
aa.sub.27 is absent; aa.sub.28 is absent; aa.sub.29 is absent;
wherein any two of aa.sub.1-aa.sub.26 are optionally cyclized
through their side chains to form a lactam linkage; and provided
that one, or at least one of aa.sub.16, aa.sub.17, aa.sub.18,
aa.sub.19, aa.sub.20, aa.sub.21, aa.sub.22, aa.sub.23, aa.sub.24,
or aa.sub.25 is the linker amino acid U covalently attached to the
surfactant X.
12. The peptide product of claim 1, wherein the peptide comprises
SEQ ID NO: 305 TABLE-US-00047 (SEQ ID. NO. 305)
His.sub.1-aa.sub.2-aa.sub.3-Gly.sub.4-Thr.sub.5-aa.sub.6-Thr.sub.7-Ser.sub-
.8-Asp.sub.9-aa.sub.10-aa.sub.11-
aa.sub.12-aa.sub.13-aa.sub.14-aa.sub.15-aa.sub.16-aa.sub.17-aa.sub.18-aa.s-
ub.19-aa.sub.20-aa.sub.21-aa.sub.22- aa.sub.23-Z
wherein: Z is OH, or --NH--R.sup.3, wherein R.sup.3 is H or
substituted or unsubstituted C.sub.1-C.sub.12 alkyl; or a PEG chain
of less than 10 Da; aa.sub.2 is Ser, Ala, Gly, Aib, Ac4c, or Ac5c;
aa.sub.3 is Gln, or Cit; aa.sub.6 is Phe, Trp, F2Phe, Me2Phe,
MePhe, or Nal2; aa.sub.10 is Tyr, Leu, Met, Nal2, Bip, or
Bip2EtMeO; aa.sub.11 is Ser, Asn, or U(X); aa.sub.12 is Lys, Glu,
Ser, or U(X); aa.sub.13 is absent or Tyr, Gln, Cit, or U(X);
aa.sub.14 is absent or Leu, Met, Nle, or U(X); aa.sub.15 is absent
or Asp, Glu, or U(X); aa.sub.16 is absent or Ser, Gly, Glu, Aib,
Ac4c, Ac5c, Lys, R, or U(X); aa.sub.17 is absent or Arg, hArg, Gln,
Glu, Cit, Aib, Ac4c, Ac5c, or U(X); aa.sub.18 is absent or Arg,
hArg, Ala, Aib, Ac4c, Ac5c, or U(X); aa.sub.19 is absent or Ala,
Val, Aib, Ac4c, Ac5c, or U(X); aa.sub.20 is absent or Gln, Lys,
Arg, Cit, Glu, Aib, Ac4c, Ac5c, or U(X); aa.sub.21 is absent or
Asp, Glu, Leu, Aib, Ac4c, Ac5c, or U(X); aa.sub.22 is absent or
Phe, Aib, Ac4c, Ac5c, or U(X) aa.sub.23 is absent or Val, Ile, Aib,
Ac4c, Ac5c, or U(X); wherein any two of aa.sub.1-aa.sub.23 are
optionally cyclized through their side chains to form a lactam
linkage; and provided that one, or at least one of aa.sub.16,
aa.sub.17, aa.sub.18, aa.sub.19, aa.sub.20, aa.sub.21, aa.sub.22 is
the linker amino acid U covalently attached to the surfactant
X.
13. (canceled)
14. The peptide product of claim 1, wherein the surfactant X has
the structure: ##STR00062## wherein: A is a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl chain, a substituted or
unsubstituted alkoxyaryl group, a substituted or unsubstituted
aralkyl group; R.sup.1b, R.sup.1c, and R.sup.1d are each,
independently at each occurrence, H, a protecting group, or a
substituted or unsubstituted C.sub.1-C.sub.30 alkyl group; W.sup.1
is independently, at each occurrence, --CH.sub.2--,
--CH.sub.2--O--, --(C.dbd.O), --(C.dbd.O)--O--, --(C.dbd.O)--NH--,
--(C.dbd.S)--, --(C.dbd.S)--NH--, or --CH.sub.2--S--; W.sup.2 is
--O-- or --S--; R.sup.2 is a bond to U.
15. The peptide product of claim 14, wherein the surfactant X has
the structure: ##STR00063##
16. The peptide product of claim 14, wherein the surfactant X has
the structure: ##STR00064##
17. The peptide product of claim 14, wherein the surfactant X has
the structure: ##STR00065## wherein: A is a substituted
C.sub.3-C.sub.20 alkyl group; R.sup.1b, R.sup.1c, and R.sup.1d are
each, independently at each occurrence, H, a protecting group, or a
substituted or unsubstituted C.sub.1-C.sub.30 alkyl group; W.sup.1
is --(C.dbd.O)--NH--; W.sup.2 is --O--; R.sup.2 is a bond to U.
18. The peptide product of claim 14, wherein the surfactant X has
the structure: ##STR00066## wherein: A is a substituted
C.sub.3-C.sub.20 alkyl group; R.sup.1b, R.sup.1c, and R.sup.1d are
H; W.sup.1 is --(C.dbd.O)--NH--; W.sup.2 is --O--; and R.sup.2 is a
bond to U.
19. The peptide product of claim 1, wherein the hydrophobic group A
is a substituted or unsubstituted C.sub.6-C.sub.18 alkyl group and
the hydrophilic group is selected from glucose, maltose, glucuronic
acid, diglucuronic acid or maltouronic acid.
20-37. (canceled)
38. A pharmaceutical composition comprising a therapeutically
effective amount of the peptide product of claim 1, or
pharmaceutically acceptable salt thereof, and at least one
pharmaceutically acceptable carrier or excipient.
39. A method for improving the pharmaceutical and medicinal
behavior of a peptide, thereby improving its duration of action,
bioavailability or its stability in formulation, comprising
covalent attachment of a surfactant X to the peptide, wherein X is
as defined in claim 1.
40. (canceled)
Description
CROSS REFERENCE
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/118,546, which was filed Nov. 18, 2013 pursuant to 35
U.S.C. .sctn. 371 as a United States National Phase Application of
International Application Serial No. PCT/US2012/038429, filed May
17, 2012, which claims the benefit of U.S. Provisional Patent
Application Ser. No. 61/487,636, filed May 18, 2011, U.S.
Provisional Patent Application Ser. No. 61/487,638, filed May 18,
2011, U.S. Provisional Patent Application Ser. No. 61/543,725,
filed Oct. 5, 2011, and U.S. Provisional Patent Application Ser.
No. 61/543,721, filed Oct. 5, 2011, each of which are incorporated
herein by reference in their entireties.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 16, 2014, is named 38617-701-401-SeqList.txt and is 429,919
bytes in size.
FIELD OF THE INVENTION
[0003] Covalently modified peptide and protein analogs allow for
improved pharmaceutical properties of peptide and/or protein-based
therapeutics.
SUMMARY OF THE INVENTION
[0004] Peptide and/or protein pharmaceuticals suffer from several
limitations in their use in medicine (Nestor, J. J., Jr. (2007)
Comprehensive Medicinal Chemistry II 2: 573-601)--short duration of
action, poor bioavailability, and lack of receptor subtype
selectivity. In addition, peptides and/or proteins are unstable in
formulations, being subject to aggregation. In some instances,
aggregation of peptides and/or proteins leads to the development of
an immunological response to both native and foreign peptides or
proteins.
[0005] Described herein is a method and reagents for covalently
modifying peptides and/or proteins to generate products with
improved pharmaceutical properties. In some instances, covalently
modified peptides and/or proteins allow for improved stability,
bioavailability, selectivity, and duration of effect in peptide
and/or protein-based therapeutics.
[0006] Described herein are certain covalently modified peptides
and/or proteins with improved pharmaceutical properties. In some
instances, these covalently modified peptides and/or proteins allow
for improved stability, bioavailability, selectivity, and duration
of effect in peptide and/or protein-based therapeutics.
[0007] In some embodiments, the covalently modified peptides and/or
proteins described herein are attached to glycoside surfactants. In
one aspect, the covalently modified peptides and/or proteins are
attached to alkyl glycosides. In one aspect, the covalently
modified peptides and/or proteins are attached to an alkyl
glycoside surfactant wherein the peptide and/or protein is attached
to the glycoside in the surfactant and the glycoside is then
attached to a hydrophobic and/or alkyl group. Provided herein, in
some embodiments, are reagents and intermediates for the covalent
modification of peptides and/or proteins through the incorporation
of surfactants such as alkyl glycosides.
[0008] Provided herein are peptide products comprising a surfactant
X, covalently attached to a peptide, the peptide comprising a
linker amino acid U and at least one other amino acid:
##STR00001## [0009] wherein X is
[0009] ##STR00002## [0010] wherein [0011] A is a hydrophobic group;
and [0012] B is a hydrophilic group covalently attached to the
peptide via a linker amino acid U.
[0013] In some embodiments, the peptide product is synthesized by
reaction of a functionalized surfactant with the peptide as
described herein. In some embodiments, the peptide product is
synthesized by reaction of a functionalized surfactant with a
reversibly-protected linker amino acid as described herein followed
by reaction with one or more amino acids to form a
surfactant-modified peptide product. In some embodiments, U is a
terminal amino acid of the peptide. In some embodiments. U is a
non-terminal amino acid of the peptide.
[0014] In some embodiments, A is a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl chain, a substituted or unsubstituted
alkoxyaryl group, a substituted or unsubstituted aralkyl group or a
steroid nucleus containing moiety. In some embodiments, A is a
substituted or unsubstituted C.sub.8-C.sub.20 alkyl chain, a
substituted or unsubstituted 1-alkoxyaryl group, a substituted or
unsubstituted 1-aralkyl group, or a steroid nucleus containing
moiety. In some embodiments, A is a substituted or unsubstituted
C.sub.10-C.sub.20 alkyl chain, a substituted or unsubstituted
1-alkoxyaryl group, a substituted or unsubstituted 1-aralkyl group,
or a steroid nucleus containing moiety.
[0015] In some embodiments, B is a polyol. In some embodiments, the
polyol is a saccharide. In some embodiments, the saccharide is a
monosaccharide, a disaccharide, or a polysaccharide. In some
embodiments, the saccharide is selected from glucose, mannose,
maltose, a glucuronic acid, galacturonic acid, diglucuronic acid
and maltouronic acid.
[0016] In some embodiments, the surfactant is a 1-alkyl glycoside
class surfactant.
[0017] In some embodiments, the hydrophilic group in the surfactant
is attached to the peptide via an amide bond.
[0018] In some embodiments of the peptide product, the surfactant
is comprised of 1-eicosyl beta-D-glucuronic acid, 1-octadecyl
beta-D-glucuronic acid, 1-hexadecyl beta-D-glucuronic acid,
1-tetradecylbeta D-glucuronic acid, 1-dodecyl beta D-glucuronic
acid, 1-decyl beta-D-glucuronic acid, 1-octyl beta-D-glucuronic
acid, 1-eicosyl beta-D-diglucuronic acid, 1-octadecyl
beta-D-diglucuronic acid, 1-hexadecyl beta-D-diglucuronic acid,
1-tetradecyl beta-D-diglucuronic acid, 1-dodecyl
beta-D-diglucuronic acid, 1-decyl beta-D-diglucuronic acid, 1-octyl
beta-D-diglucuronic acid, or functionalized 1-ecosyl
beta-D-glucose, 1-octadecyl beta-D-glucose, 1-hexadecyl
beta-D-glucose, 1-tetradecyl beta-D-glucose, 1-dodecyl
beta-D-glucose, 1-decyl beta-D-glucose, 1-octyl beta-D-glucose,
1-eicosyl beta-D-maltoside, 1-octadecyl beta-D-maltoside,
1-hexadecyl beta-D-maltoside, 1-dodecyl beta-D-maltoside, 1-decyl
beta-D-maltoside, 1-octyl beta-D-maltoside, and the like, and the
peptide product is prepared by formation of a linkage between the
aforementioned groups and a group on the peptide (e.g., a --COOH
group in the aforementioned groups and an amino group of the
peptide).
[0019] In some embodiments, a combination of a hydrophilic with a
hydrophobic group generates a surfactant. In some embodiments, the
surfactant is an ionic surfactant. In some embodiments, the
surfactant is a non-ionic surfactant. In some embodiments, the
hydrophobic group is a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl chain or an aralkyl chain. In some
embodiments the hydrophobic group is a chain having mixed
hydrophobic and hydrophilic properties, for example a polyethylene
glycol (PEG) group.
[0020] In some embodiments, the linker amino acid is a natural D-
or L-amino acid. In some embodiments, the linker amino acid is an
unnatural amino acid. In some embodiments, the linker amino acid is
selected from Lys, Cys, Orn, Asp, Glu or an unnatural amino acid,
comprising a functional group used for covalent attachment to the
surfactant X. In some embodiments, the linker amino acid is
selected from Lys, Cys, Orn, or an unnatural amino acid, comprising
a functional group used for covalent attachment to the surfactant
X. In some embodiments, the functional group used for covalent
attachment to a surfactant is --NH.sub.2, --SH, --OH, --N.sub.3,
haloacetyl, a --(CH2).sub.m-maleimide or an acetylenic group,
wherein m is 1-10.
[0021] In some embodiments, the peptide is an opioid peptide. In
some embodiments, the peptide and/or protein product contains a
covalently linked alkyl glycoside. In some of such embodiments, the
peptide and/or protein product contains a covalently linked alkyl
glycoside that is a 1-O-alkyl glucuronic acid of alpha or beta
configuration. In some of such embodiments, the peptide and/or
protein product comprises a covalently linked alkyl glycoside that
is a 1-O-alkyl glucuronic acid and the alkyl chain is a C.sub.1 to
C.sub.20 alkyl chain.
[0022] In some embodiments, the peptide product has a structure of
Formula IA:
aa.sub.1-aa.sub.2-aa.sub.3-aa.sub.4-aa.sub.5-Z Formula IA [0023]
wherein: [0024] each of aa.sub.1, aa.sub.2, aa.sub.3, aa.sub.4, and
aa.sub.5 is independently absent, a D- or L-natural or unnatural
amino acid, an N-alkylated amino acid, an N-acetylated amino acid,
a C.alpha.R.sup.3 amino acid, a .PSI.-amino acid, or a linker amino
acid U covalently attached to the surfactant X; [0025] Z is --OH,
--NH.sub.2 or --NHR.sup.3; [0026] each R.sup.3 is independently
substituted or unsubstituted C.sub.1-C.sub.12 branched or straight
chain alkyl, a PEG chain of less than 10 Da, or substituted or
unsubstituted aralkyl chain; [0027] provided that one, or at least
one of aa.sub.1, aa.sub.2, aa.sub.3, aa.sub.4, and aa.sub.5 is the
linker amino acid U covalently attached to the surfactant X; [0028]
and further provided that not all of aa.sub.1, aa.sub.2, aa.sub.3,
aa.sub.4, and aa.sub.5 are absent.
[0029] In one aspect, provided herein is a peptide product having a
structure of Formula II:
aa.sub.1-aa.sub.2-aa.sub.3-aa.sub.4-aa.sub.5-Z Formula II [0030]
wherein: [0031] each of aa.sub.1, aa.sub.2, aa.sub.3, aa.sub.4, and
aa.sub.5 is independently absent, a D- or L-natural or unnatural
amino acid, an N-alkylated amino acid, an N-acetylated amino acid,
a C.alpha.R.sup.3 amino acid, a .PSI.-amino acid, or a linker amino
acid U covalently attached to a surfactant X; [0032] Z is --OH,
--NH.sub.2 or --NHR.sup.3; [0033] each R.sup.3 is independently
substituted or unsubstituted C.sub.1-C.sub.12 branched or straight
chain alkyl, a PEG chain of less than 10 Da, or substituted or
unsubstituted aralkyl chain; and [0034] X is
[0034] ##STR00003## [0035] wherein [0036] A is a hydrophobic group;
and [0037] B is a hydrophilic group covalently attached to the
peptide via a linker amino acid U; [0038] provided that one, or at
least one of aa.sub.1, aa.sub.2, aa.sub.3, aa.sub.4, and aa.sub.5
is the linker amino acid U covalently attached to the surfactant X;
[0039] and further provided that not all of aa.sub.1, aa.sub.2,
aa.sub.3, aa.sub.4, and aa.sub.5 are absent.
[0040] In one aspect, provided herein is a peptide product that has
a structure of Formula III:
aa.sub.1-aa.sub.2-aa.sub.3-aa.sub.4-aa.sub.5-Z Formula III (SEQ.
ID. NO. 1) [0041] wherein: [0042] aa.sub.1 is Tyr, Dmt,
N--R.sup.3-Tyr, N--R.sup.3-Dmt, N--(R.sup.3).sub.2-Tyr, or
N--(R.sup.3).sub.2-Dmt; [0043] aa.sub.2 is Pro, D-Arg, D-U(X),
D-Ala, Tic, or Tic(.PSI.[CH2-NH]); [0044] aa.sub.3 is Phe, Trp,
Tmp, D- or L-Nal(1), D- or L-Nal(2), C.alpha.MePhe, or .PSI.-Phe;
[0045] aa.sub.4 is Phe, Tmp, D- or L-Nal(1), D- or L-Nal(2), U(X),
D- or L-C.alpha.MeU(X); [0046] aa.sub.5 is absent or Pro, Aib,
U(X), D- or L-C.alpha.MeU(X); and [0047] U is a dibasic natural or
unnatural amino acid, a natural or unnatural amino acid comprising
a thiol, an unnatural amino acid comprising a --N.sub.3 group, an
unnatural amino acid comprising an acetylenic group, or an
unnatural amino acid comprising a --NH--C(.dbd.O)--CH.sub.2--Br or
a --(CH.sub.2).sub.m-maleimide, wherein m is 1-10, used for
covalent attachment to the surfactant X.
[0048] In some embodiments of peptide products of Formula I,
Formula II or Formula III described above and herein, X has the
structure:
##STR00004## [0049] wherein: [0050] A is a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl chain, a substituted or
unsubstituted alkoxyaryl group, a substituted or unsubstituted
aralkyl group, or a steroid nucleus containing moiety; [0051]
R.sup.1b, R.sup.1c, and R.sup.1d are each, independently at each
occurrence, H, a protecting group, or a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl group; [0052] W.sup.1 is
independently, at each occurrence. --CH.sub.2--, --CH.sub.2--O--,
--(C.dbd.O), --(C.dbd.O)--O--, --(C.dbd.O)--NH--, --(C.dbd.S)--,
--(C.dbd.S)--NH--, or --CH.sub.2--S--; [0053] W.sup.2 is --O--, or
--S--; [0054] R.sup.2 is a bond, C.sub.2-C.sub.4-alkene,
C.sub.2-C.sub.4-alkyne, or --(CH.sub.2).sub.m-maleimide, and [0055]
m is 1-10.
[0056] In some embodiments of peptide products of Formula I,
Formula II or Formula III described above and herein, X has the
structure:
##STR00005##
[0057] Accordingly, in the embodiment described above, R.sup.2 is a
bond.
[0058] In some embodiments of peptide products of Formula I,
Formula II or Formula III described above and herein, X has the
structure:
##STR00006##
[0059] In some embodiments of peptide products of Formula I,
Formula II or Formula III described above and herein, X has the
structure:
##STR00007## [0060] wherein: [0061] A is a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl group, or a steroid nucleus
containing moiety; [0062] R.sup.1b, R.sup.1c, and R.sup.1d are
each, independently at each occurrence, H, a protecting group, or a
substituted or unsubstituted C.sub.1-C.sub.30 alkyl group; [0063]
W.sup.1 is --(C.dbd.O)--NH--; [0064] W.sup.2 is --O--; [0065]
R.sup.2 is a bond.
[0066] In some embodiments of peptide products of Formula I,
Formula II or Formula III described above and herein, X has the
structure:
##STR00008## [0067] wherein: [0068] A is a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl group; [0069] R.sup.1b,
R.sup.1c, and R.sup.1d are H; [0070] W.sup.1 is --(C.dbd.O)--NH--;
[0071] W.sup.2 is --O--; and [0072] R.sup.2 is a bond.
[0073] In some embodiments of peptide products of Formula I,
Formula II or Formula III described above and herein, X is as
described above and A is a substituted or unsubstituted
C.sub.1-C.sub.20 alkyl group.
[0074] Also contemplated herein are alternate embodiments wherein X
in Formula I has the structure:
##STR00009##
[0075] For instance, in an exemplary embodiment of the structure of
X described above, W.sup.1 is --S--, R.sup.2 is a C.sub.1-C.sub.30
alkyl group, W.sup.2 is S, R.sup.1a is a bond between W.sup.2 and a
suitable moiety of an amino acid residue U within the peptide
(e.g., a thiol group in a cysteine residue of the peptide forms a
thioether with X).
[0076] In another exemplary alternate embodiment of the structure
of X described above, W.sup.1 is --O--, R.sup.2 is a
C.sub.1-C.sub.30 alkyl group, W.sup.2 is O, R.sup.1a is a bond
between W.sup.2 and a suitable moiety of an amino acid residue U
within the peptide (e.g., a hydroxyl group in a serine or threonine
residue of the peptide forms an ether with X).
[0077] In some embodiments, a peptide product of Formula III is a
product (SEQ. ID. NO. 149) [0078] wherein: [0079] U is a dibasic
natural or unnatural amino acid; [0080] X is a surfactant of the
1-alkyl glycoside class wherein 1-alkyl is substituted or
unsubstituted C.sub.1-C.sub.20 alkyl or a substituted or
unsubstituted alkoxyaryl substituent; [0081] Z is NH.sub.2; [0082]
aa.sub.1 is Tyr, Dmt, N.alpha.-Me-Tyr, N.alpha.-Me-Tyr,
N,N.alpha.-diMe-Tyr, or N,N.alpha.-diMe-Dmt; [0083] aa.sub.2 is
Pro, D-Arg, D-U(X), D-Ala, Tic, or Tic(.PSI.[CH2-NH]); [0084]
aa.sub.3 is Phe, Trp, Tmp, D- or L-Nal(1), D- or L-Nal(2),
C.alpha.MePhe, or .PSI.-Phe; [0085] aa.sub.4 is Phe, Tmp. D- or
L-Nal(l), D- or L-Nal(2), U(X), D- or L-C.alpha.MeU(X); [0086]
aa.sub.5 is absent or Pro, Aib, U(X), D- or L-C.alpha.MeU(X).
[0087] In some embodiments, a peptide product of Formula III is a
product (SEQ. ID. NO. 150) [0088] wherein: [0089] X is comprised of
1-alkyl glucuronic acid or 1-alkyl diglucuronic acid; [0090] Z is
NH.sub.2; [0091] aa.sub.1 is Dmt; [0092] aa.sub.2 is Pro, D-Lys(X),
Tic, or Tic(.PSI.[CH2-NH]); [0093] aa.sub.3 is Phe, Tmp, D- or
L-Nal(l), D- or L-Nal(2), or F-Phe; [0094] aa.sub.4 is Phe, D- or
L-Nal(1), D- or L-Nal(2), or Lys(X); [0095] aa.sub.5 is absent or
Pro, Aib, Lys(X), D- or L-C.alpha.MeLys(X).
[0096] In some embodiments, a peptide product of Formula III is a
product (SEQ. ID. NO. 151) [0097] wherein: [0098] aa.sub.1 is Dmt;
[0099] aa.sub.2 is Pro; [0100] aa.sub.3 is Phe, or Tmp; [0101]
aa.sub.4 is Phe, or Lys(X); [0102] aa.sub.5 is absent or Aib,
Lys(X), D- or L-C.alpha.MeLys(X).
[0103] In some embodiments, a peptide product of Formula III is a
product (SEQ. ID. NO. 152) [0104] wherein: [0105] aa.sub.1 is Dmt;
[0106] aa.sub.2 is Pro; [0107] aa.sub.3 is Phe, or Tmp; [0108]
aa.sub.4 is Phe, or Lys(X); [0109] aa.sub.5 is absent or
Lys(X).
[0110] In some embodiments, a peptide product of Formula III is a
product (SEQ. ID. NO. 153) [0111] wherein: [0112] U is a dibasic
natural or unnatural amino acid; [0113] X is a surfactant of the
1-alkyl glycoside class wherein the 1-alkyl group of the 1-alkyl
glycoside is substituted or unsubstituted C.sub.1-20 alkyl or a
substituted or unsubstituted alkoxyaryl substituent; [0114]
aa.sub.1 is Tyr, Dint, N--R.sup.3-Tyr, N--R.sup.3-Tyr,
N--(R.sup.3).sub.2-Tyr, or N--(R.sup.3).sub.2-Dmt; [0115] aa.sub.2
is D-Arg, or D-U(X); [0116] aa.sub.3 is Phe, Trp, D- or L-Nal(1),
D- or L-Nal(2), or Tmp; [0117] aa.sub.4 is Phe, Tmp, D- or
L-Nal(1), D- or L-Nal(2), U(X), D- or L-C.alpha.MeU(X); [0118]
aa.sub.5 is absent, Pro, or U(X).
[0119] In some embodiments, a peptide product of Formula II is a
product (SEQ. ID. NO. 154) [0120] wherein: [0121] X is a surfactant
of the 1-alkyl glucuronic acid or 1-alkyl diglucuronic acid class;
[0122] aa.sub.1 is Dint, N.alpha.-Me-Dint, or N,N.alpha.-Me-Dmt;
[0123] aa.sub.2 is D-Arg, D-Lys(X), or D-Orn(X); [0124] aa.sub.3 is
Phe, or Tmp; [0125] aa.sub.4 is Phe, Tmp, Lys(X), or Orn(X); [0126]
aa.sub.5 is absent or Pro, Lys(X), or Orn(X).
[0127] In some embodiments, a peptide product of Formula III is a
product (SEQ. ID. NO. 155) [0128] wherein: [0129] U is a dibasic
natural or unnatural amino acid; [0130] X is a surfactant of the
1-alkyl glycoside class wherein 1-alkyl is substituted or
unsubstituted C.sub.1-C.sub.20 alkyl or a substituted or
unsubstituted alkoxyaryloxy substituent; [0131] Z is NH.sub.2;
[0132] aa.sub.1 is Tyr, Dint, N--R.sup.3-Tyr, N--R.sup.3-Dmt,
N--(R.sup.3).sub.2-Tyr, or N--(R.sup.3).sub.2-Dmt; [0133] aa.sub.2
is Tic, or Tic(.PSI.[CH2-NH]); [0134] aa.sub.3 is .PSI.-Phe when
aa.sub.2 is Tic(.PSI.[CH2-NH]); [0135] aa.sub.4 is Phe, Tmp, D- or
L-Nal(1), D- or L-Nal(2), or U(X); [0136] aa.sub.5 is absent, Pro,
Aib, or U(X).
[0137] In some embodiments, a peptide product of Formula III is a
product (SEQ. ID. NO. 156) [0138] wherein: [0139] X is a surfactant
of the 1-alkyl glucuronic acid or 1-alkyl diglucuronic acid class;
[0140] aa.sub.1 is Tyr, Dmt, N.alpha.-Me-Tyr, N.alpha.-alkyl-Dmt,
N,N.alpha.-diMe-Tyr, or N,N.alpha.-Me-Dmt; [0141] aa.sub.2 is Tic,
or Tic(.PSI.[CH2-NH]); [0142] aa.sub.3 is .PSI.-Phe when aa.sub.2
is Tic(.PSI.[CH2-NH]), Phe, or TMP; [0143] aa.sub.4 is Phe, Tmp, D-
or L-Nal(1), D- or L-Nal(2), Lys(X), or Orn(X); [0144] aa.sub.5 is
absent or Aib, Lys(X), or Orn(X).
[0145] In some embodiments, a peptide product of Formula III is a
product (SEQ. ID. NO. 157) [0146] wherein: [0147] X is comprised of
1-alkyl glucuronic acid or 1-alkyl diglucuronic acid; [0148]
aa.sub.2 is Tic, or Tic(.PSI.[CH2-NH]); [0149] aa.sub.3 is Phe or
.PSI.-Phe; [0150] aa.sub.4 is Lys(X); [0151] aa.sub.5 is
absent.
[0152] In some embodiments, a peptide product of Formula III is a
product (SEQ. ID. NO. 158) [0153] wherein: [0154] X is comprised of
1-alkyl glucuronic acid; [0155] aa.sub.2 is Tic; [0156] aa.sub.3 is
Phe; [0157] aa.sub.4 is Lys(X); [0158] aa.sub.5 is absent.
[0159] In some embodiments, a peptide product of Formula III is a
product (SEQ. ID. NO. 159) [0160] wherein: [0161] X is comprised of
a 1-alkyl glucuronic acid selected from 1-methyl beta-D-glucuronic
acid, 1-octyl beta-D-glucuronic acid, 1-dodecyl beta-D-glucuronic
acid, 1-tetradecyl beta-D-glucuronic acid, 1-hexadecyl
beta-D-glucuronic acid, and 1-octadecyl beta-D-glucuronic acid;
[0162] aa.sub.2 is Tic; [0163] aa.sub.3 is Phe; [0164] aa.sub.4 is
Lys(X); [0165] aa.sub.5 is absent.
[0166] In some embodiments, a peptide product of Formula III is
H-Dmt-Tic-Phe-Lys(N-epsilon-1-methyl beta-D-glucuronyl)-NH.sub.2.
(SEQ. ID. NO. 78)
[0167] In some embodiments, a peptide product of Formula III is
H-Dmt-Tic-Phe-Lys(Nepsilon-1-octyl beta-D-glucuronyl)-NH.sub.2.
(SEQ. ID. NO. 80)
[0168] In some embodiments, a peptide product of Formula III is
[0169] H-Dmt-Tic-Phe-Lys(Nepsilon-1-dodecyl
beta-D-glucuronyl)-NH.sub.2. (SEQ. ID. NO. 79)
[0170] In some embodiments, a peptide product of Formula III is
H-Dmt-Tic-Phe-Lys(Nepsilon-1-tetradecyl
beta-D-glucuronyl)-NH.sub.2. (SEQ. ID. NO. 160)
[0171] In some embodiments, a peptide product of Formula III is
H-Dmt-Tic-Phe-Lys(Nepsilon-1-hexadecyl beta-D-glucuronyl)-NH.sub.2.
(SEQ. ID. NO. 82)
[0172] In some embodiments, a peptide product of Formula III is
H-Dmt-Tic-Phe-Lys(N-epsilon-1-octadecyl
beta-D-glucuronyl)-NH.sub.2. (SEQ. ID. NO. 83)
[0173] In some embodiments, the peptide product is biologically
active.
[0174] In a specific embodiment, provided herein is a compound
selected from compounds of Table 1 in FIG. 1.
[0175] Also provided herein is a pharmaceutical composition
comprising a therapeutically effective amount of a peptide product
of Formula I, II or III, or pharmaceutically acceptable salt
thereof, and at least one pharmaceutically acceptable carrier or
excipient.
[0176] Provided herein is a method of treating pain comprising
administration of a therapeutically effective amount of a peptide
product of Formula I, II or III or compounds of Table 1 in FIG.
1.
[0177] A method for improving the pharmaceutical and medicinal
behavior of a peptide, thereby improving its duration of action,
bioavailability or its stability in formulation, comprising
covalent attachment of a surfactant X to the peptide, wherein X is
as described herein.
[0178] In some embodiments, a peptide product comprising a
covalently linked alkyl glycoside described herein is an analog of
Leu-enkephalin. In some of such embodiments, the peptide product
contains a covalently linked 1-O-alkyl .beta.-D-glucuronic acid and
the peptide is an analog of Leu-enkephalin.
[0179] In some embodiments, a peptide product comprising a
covalently linked alkyl glycoside described herein is an analog of
opioid peptide DPDPE (Akiyama K, et al. (1985) Proc Natl Acad Sci
USA 82:2543-7). In some of such embodiments, the peptide product
contains a covalently linked 1-O-alkyl glucuronic acid and the
peptide is an analog of opioid peptide DPDPE.
[0180] In some embodiments, a peptide product comprising a
covalently linked alkyl glycoside described herein is an analog of
the D-amino acid containing natural product peptides dermorphin
(Melchiorri, P. and Negri, L. (1996) Gen Pharmacol 27: 1099-1107)
or deltorphin (Erspamer, V., et al. (1989) Proc Natl Acad Sci USA
86:5188-92). In some of such embodiments, the peptide product
contains a covalently linked 1-O-alkyl .beta.-D-glucumnic acid and
the peptide is an analog of dermorphin or deltorphin.
[0181] In some embodiments, a peptide product comprising a
covalently linked alkyl glycoside is an analog of endomorphin-1 or
-2. In some of such embodiments, the peptide product comprises a
covalently linked 1-O-alkyl .beta.-D-glucuronic acid and the
peptide is an analog of endomorphin (Lazarus, L. H. and Okada, Y.
(2012) Expert Opin Ther Patents 22: 1-14).
[0182] In some embodiments, a peptide product comprising a
covalently linked alkyl glycoside described herein is an analog of
opioid peptide dynorphin (James, I. F., et al. (1982) Life Sci
31:1331-4). In some of such embodiments, the peptide product
contains a covalently linked 1-O-alkyl .beta.-D-glucuronic acid and
the peptide is an analog of dynorphin.
[0183] In some embodiments side chain functional groups of two
different amino acid residues are linked to form a cyclic lactam.
For example, in some embodiments, a Lys side chain forms a cyclic
lactam with the side chain of Glu. In some embodiments such lactam
structures are reversed and are formed from a Glu and a Lys. Such
lactam linkages in some instances are known to stabilize alpha
helical structures in peptides (Condon, S. M., et al. (2002) Bioorg
Med Chem 10: 731-736).
[0184] In some embodiments side chain functional groups of two
different amino acid residues with --SH containing side chains are
linked to form a cyclic disulfide. For example, in some
embodiments, two penicillamine or two Cys side chains may be linked
to constrain the conformation of a peptide (Akiyama K. et al.
(1985) Proc Natl Acad Sci USA 82:2543-7) in order to yield greater
duration of action or greater receptor selectivity.
[0185] In a specific embodiment, the peptide products of Formula I,
Formula II or Formula III, described above and herein have the
following structure:
##STR00010##
wherein A is a C.sub.1-C.sub.20 alkyl chain as described in Table 1
of FIG. 1, R' is a peptide as described in Table 1 of FIG. 1,
W.sup.2 of Formula V is --O--, and W of Formula V is
--(C.dbd.O)NH-- and is part of an amide linkage to the peptide R'.
In some of such embodiments, A is a C.sub.6-C.sub.20 alkyl chain.
In some of such embodiments, A is a C.sub.1-C.sub.10 alkyl chain.
In some of such embodiments, A is a C.sub.12-C.sub.20 alkyl chain.
In some of such embodiments, A is a C.sub.12-C.sub.15 alkyl
chain.
[0186] In embodiments described above, an amino moiety of an amino
acid and/or a peptide R' (e.g., an amino group of an amino acid
residue such as a lysine, or a lysine within the peptide R') is
used to form a covalent linkage with a compound of structure:
##STR00011##
wherein A is a C.sub.1-C.sub.20 alkyl chain as described above and
in Table 1 of FIG. 1.
[0187] In such cases, the amino acid residue having an amino moiety
(e.g., a Lysine within the peptide R') which is used to form a
covalent linkage to the compound of Formula A described above, is a
linker amino acid U which is attached to a surfactant X.
Accordingly, as one example, Lys(C12) of Table 1 of FIG. 1 has the
following structure:
##STR00012##
[0188] Also contemplated within the scope of the embodiments
presented herein are peptide products of Formula I derived from
maltouronic acid-based surfactants prepared by covalent linkage of
a peptide to either or both carboxylic acid groups. Thus, as one
example, peptides in Table 1 of FIG. 1 comprise a lysine linker
amino acid bonded to a maltouronic acid based surfactant X and
having a structure:
##STR00013##
[0189] It will be understood that in one embodiment, compounds of
Formula I are prepared by attaching a lysine to a group X, followed
by attachment of additional amino acid residues and/or peptides are
attached to the lysine-X compound to obtain compounds of Formula I.
It will be understood that other natural or non-natural amino acids
described herein are also suitable for attachment to the surfactant
X and are suitable for attaching additional amino acid/peptides to
obtain compounds of Formula I. It will be understood that in
another embodiment, compounds of Formula I are prepared by
attaching a full length or partial length peptide to a group X,
followed by optional attachment of additional amino acid residues
and/or peptides are attached to obtain compounds of Formula I.
[0190] Also provided herein is a pharmaceutical composition
comprising a therapeutically effective amount of a peptide product
described above, or acceptable salt thereof, and at least one
pharmaceutically acceptable carrier or excipient. In some
embodiments, the carrier is an aqueous-based carrier. In some
embodiments, the carrier is a nonaqueous-based carrier. In some
embodiments, the nonaqueous-based carrier is a
hydrofluoroalkane-like solvent comprising sub-micron anhydrous
.alpha.-lactose, or other excipients.
[0191] Contemplated within the scope of embodiments presented
herein is the reaction of an amino acid and/or a peptide comprising
a linker amino acid U bearing a nucleophile, and a group X
comprising a leaving group or a functional group that can be
activated to contain a leaving group, for example a carboxylic
acid, or any other reacting group, thereby allowing for covalent
linkage of the amino acid and/or peptide to a surfactant X via the
linker amino acid U to provide a peptide product of Formula I.
[0192] Also contemplated within the scope of embodiments presented
herein is the reaction of an amino acid and/or a peptide comprising
a linker amino acid U bearing a leaving group or a functional group
that can be activated to contain a leaving group, for example a
carboxylic acid, or any other reacting group, and a group X
comprising a nucleophilic group, thereby allowing for covalent
linkage of the amino acid and/or peptide to a surfactant X via the
linker amino acid U to provide a peptide product of Formula I.
[0193] It will be understood that, in one embodiment, Compounds of
Formula I are prepared by reaction of a linker amino acid U with X,
followed by addition of further residues to U to obtain the peptide
product of Formula I. It will be understood that in an alternative
embodiment, Compounds of Formula I are prepared by reaction of a
suitable peptide comprising a linker amino acid U with X, followed
by optional addition of further residues to U, to obtain the
peptide product of Formula I.
[0194] Further provided herein are certain intermediates and/or
reagents that are suitable for synthesis of peptide products
described herein. In certain embodiments, such intermediates and/or
reagents are functionalized surfactants that allow for covalent
linkage with a peptide. In certain embodiments, such intermediates
are functionalized 1-alkyl glycoside surfactants that allow for
covalent linkage with a peptide. In certain embodiments, such
intermediates are functionalized 1-alkyl glycoside surfactants
linked to reversibly-protected linker amino acids that allow for
covalent linkage with other amino acids to form a peptide. It will
be understood that a suitably functionalized surfactant is
covalently coupled to a peptide via reaction with an appropriately
matched functional group that is on a linker amino acid.
[0195] In some embodiments, intermediates suitable for synthesis of
peptide products described herein are compounds of Formula IV.
Accordingly, provided herein are intermediates and/or compounds of
Formula IV:
##STR00014## [0196] wherein: [0197] R.sup.1a is independently at
each occurrence a bond, H, a protecting group, a natural or
unnatural amino acid, a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl hydrophobic group, a substituted or
unsubstituted alkoxyaryl group, a substituted or unsubstituted
aralkyl group, or a steroid nucleus containing moiety; [0198]
R.sup.1b, R.sup.1c, and R.sup.1d are each independently at each
occurrence a bond, H, a protecting group, a natural or unnatural
amino acid, a substituted or unsubstituted C.sub.1-C.sub.30 alkyl
hydrophobic group, a substituted or unsubstituted alkoxyaryl group,
or a substituted or unsubstituted aralkyl group; [0199] W.sup.1 is
--CH.sub.2--, --CH.sub.2--O--, --(C.dbd.O), --(C.dbd.O)--O--,
--(C.dbd.O)--NH--, --(C.dbd.S)--, --(C.dbd.S)--NH--, or
--CH.sub.2--S--; [0200] W.sup.2 is --O--, --CH.sub.2-- or --S--;
[0201] R.sup.2 is H, a protecting group, a natural or unnatural
amino acid, a substituted or unsubstituted C.sub.1-C.sub.30 alkyl
hydrophobic group, a substituted or unsubstituted alkoxyaryl group,
a substituted or unsubstituted aralkyl group, --NH.sub.2, --SH,
C.sub.2-C.sub.4-alkene, C.sub.2-C.sub.4-alkyne,
--NH(C.dbd.O)--CH.sub.2--Br, --(CH.sub.2).sub.m-maleimide, or
--N.sub.3; [0202] n is 1, 2 or 3; and [0203] m is 1-10.
[0204] In some embodiments, each natural or unnatural amino acid is
independently a reversibly protected or free linker amino acid. In
some of such embodiments, the linker amino acid is a reversibly
protected or free lysine.
[0205] In some embodiments of Formula IV, [0206] n is 1; [0207]
W.sup.1 is --(C.dbd.O)--; [0208] R.sup.1a is a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl hydrophobic group, a
substituted or unsubstituted 1-alkoxyaryl group, or a substituted
or unsubstituted 1-aralkyl group, [0209] R.sup.2 is a
reversibly-protected lysine of D- or L-configuration.
[0210] In some embodiments of Formula IV, [0211] n is 1; [0212]
W.sup.1 is --(C.dbd.O)--; [0213] R.sup.1a is a substituted or
unsubstituted C.sub.8-C.sub.30 alkyl hydrophobic group, a
substituted or unsubstituted 1-alkoxyaryl group, or a substituted
or unsubstituted 1-aralkyl group. [0214] R.sup.2 is a reversibly
protected lysine of D- or L-configuration.
[0215] In some of such embodiments, R.sup.1a is an octyl, decyl,
dodecyl, tetradecyl, or hexadecyl group.
[0216] In some embodiments of Formula IV, [0217] n is 1; [0218]
W.sup.1 is --(C.dbd.O)--NH-- or --(C.dbd.O)--O--; [0219] R.sup.2 is
a substituted or unsubstituted C.sub.1-C.sub.30 alkyl hydrophobic
group, a substituted or unsubstituted 1-alkoxyaryl group, or a
substituted or unsubstituted 1-aralkyl group, [0220] R.sup.1a is a
reversibly protected serine or threonine of D- or
L-configuration.
[0221] In some of such embodiments, R.sup.2 is an octyl, decyl,
dodecyl, tetradecyl or hexadecyl group.
[0222] In some embodiments of Formula IV, [0223] n is 1; [0224] m
is 1-6; [0225] W.sup.1 is --CH.sub.2--; [0226] R.sup.1a is a
C.sub.1-C.sub.30 alkyl hydrophobic group, a 1-alkoxyaryl group, or
a 1-aralkyl group, [0227] R.sup.2 is --N.sub.3, NH.sub.2,
--C.sub.2-alkyne, --(CH.sub.2).sub.m-maleimide,
NH--(C.dbd.O)--CH.sub.2--Br, or NH--(C.dbd.O)--CH.sub.2--I.
[0228] In some embodiments of Formula IV, [0229] n is 1; [0230]
W.sup.1 is --(C.dbd.O)--O--; [0231] R.sup.2 is H, [0232] R.sup.1a
is a substituted or unsubstituted C.sub.1-C.sub.30 alkyl
hydrophobic group.
[0233] In some embodiments of Formula IV, R.sup.2 is attached to
the peptide or is an amino acid residue in the peptide. In some of
such embodiments, R.sup.2 is a reversibly protected or free
lysine.
[0234] In some embodiments of Formula IV, n is 1. In some
embodiments of Formula IV, n is 2, and a first glycoside is
attached to a second glycoside via a bond between W.sup.2 of the
first glycoside and any one of OR.sup.1b, OR.sup.1c or OR.sup.1d of
the second glycoside.
[0235] In some embodiments of Formula IV, n is 3, and a first
glycoside is attached to a second glycoside via a bond between
W.sup.2 of the first glycoside and any one of OR.sup.1b, OR.sup.1c
or OR.sup.1d of the second glycoside, and the second glycoside is
attached to a third glycoside via a bond between W.sup.2 of the
second glycoside and any one of OR.sup.1b, OR.sup.1c or OR.sup.1d
of the third glycoside.
[0236] Provided herein is a method for synthesizing a peptide
product described above, comprising sequential steps of [0237] (a)
coupling a compound of Formula IV to the peptide; and [0238] (b)
optionally deprotecting the coupled peptide of step (a).
[0239] In some embodiments, the deprotecting comprises the use of
mild acid and or mild base treatments. In some embodiments of the
methods, the deprotecting comprises the use of strong acids.
[0240] In some embodiments, the method further comprises the steps
of chromatography, desalting of intermediates by reversed-phase,
high-performance liquid chromatography or ion exchange
chromatography of intermediates.
[0241] Provided herein is a method for improving the pharmaceutical
and medicinal behavior of a peptide, thereby improving its duration
of action, bioavailability or its stability in formulation,
comprising covalent attachment of a surfactant X to the peptide,
wherein: [0242] X is
[0242] ##STR00015## [0243] wherein [0244] A is a hydrophobic group;
and [0245] B is a hydrophilic group covalently attached to the
peptide via a linker amino acid.
[0246] In some embodiments, A is a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl chain, a substituted or unsubstituted
alkoxyaryl group or a substituted or unsubstituted aralkyl
group.
[0247] In some embodiments, A is a substituted or unsubstituted
C.sub.8-C.sub.20 alkyl chain, a substituted or unsubstituted
1-alkoxyaryl group or a substituted or unsubstituted 1-aralkyl
group.
[0248] In some embodiments, A is a substituted or unsubstituted
C.sub.10-C.sub.20 alkyl chain, a substituted or unsubstituted
1-alkoxyaryl group or a substituted or unsubstituted 1-aralkyl
group.
[0249] In some embodiments, B is a functionalized polyol.
[0250] In some embodiments, the polyol is a saccharide,
[0251] In some embodiments, the saccharide is a monosaccharide, a
disaccharide, or a polysaccharide.
[0252] In some embodiments, the saccharide is selected from
glucuronic acid, galacturonic acid, diglucuronic acid and
maltouronic acid.
[0253] The methods of synthesis described above are suitable for
synthesis of all compounds described herein, including compounds of
Formula I, II, III, 2-I-1, 2-III, 2-V, 2-VI, 2-VII, 3-I-A, 3-III-A,
3-III-B, or 3-V, and compounds in Table 1, Table 2, Table 3 and
Table 4 provided in FIG. 1, FIG. 2, FIG. 8 and FIG. 9
respectively.
[0254] Provided herein is a method for improving the pharmaceutical
and medicinal behavior of a peptide, thereby improving its duration
of action, bioavailability or its stability in formulation,
comprising covalent attachment of a surfactant X to the peptide,
wherein: [0255] X is
[0255] ##STR00016## [0256] wherein [0257] A is a substituted or
unsubstituted C.sub.1-C.sub.20 alkyl chain, a substituted or
unsubstituted alkoxyaryl group or a substituted or unsubstituted
aralkyl group; and [0258] B is a saccharide covalently attached to
the peptide via a linker amino acid.
[0259] In some embodiments, the surfactant is a 1-alkyl glycoside
class surfactant.
[0260] In some embodiments, the hydrophilic group in the surfactant
is attached to the peptide via an amide bond.
[0261] In some embodiments, the surfactant is composed of
1-hexadecyl-beta-D-glucuronic acid, 1-tetradecyl-beta-D-glucuronic
acid, 1-dodecyl-beta-D-glucuronic acid, 1-decyl-beta-D-glucuronic
acid, 1-octyl-beta-D-glucuronic acid,
1-hexadecyl-beta-D-diglucuronic acid,
1-tetradecyl-beta-D-diglucuronic acid,
1-dodecyl-beta-D-diglucuronic acid, 1-decyl-beta-D-diglucuronic
acid, 1-octyl-beta-D-diglucuronic acid.
[0262] Provided herein is a method of treating pain in an
individual in need thereof comprising administration of a
therapeutically effective amount of a peptide product described
herein, or a compound of Formula IV to an individual in need
thereof.
[0263] Also provided herein is a covalently modified peptide and/or
protein product comprising a hydrophilic group as described herein;
and a hydrophobic group covalently attached to the hydrophilic
group. In specific embodiments, the covalently modified peptide
and/or protein product comprises a hydrophilic group that is a
saccharide and a hydrophobic group that is a C.sub.1-C.sub.20 alkyl
chain or an aralkyl chain.
BRIEF DESCRIPTION OF THE FIGURES
[0264] FIG. 1 Table 1 in FIG. 1 depicts compounds that were
prepared by methods described herein. The specification provides
sequences for SEQ. ID. Nos. I and 149-169. Additionally, Table 1 of
FIG. 1 provides SEQ. ID Numbers for compounds EU-A101 to EU-A199
and EU-A600 to EU-A649 having SEQ. ID. NOs. 2-148, and SEQ. ID. NO.
645 respectively, as shown in Table 1 of FIG. 1. Compounds in Table
1 of FIG. 1, and their respective SEQ. ID. NOs. shown in Table 1 of
FIG. 1 are hereby incorporated into the specification as filed.
[0265] FIG. 2. Table 2 in FIG. 2 depicts compounds that were
prepared by methods described herein. The specification provides
sequences for SEQ. ID. Nos. 170-174 and SEQ. ID. NOs. 283-302.
Additionally. Table 2 of FIG. 2 provides SEQ. ID. Numbers for
compounds EU-201 to EU-299 and EU-900-EU-908 having SEQ. ID. NOs.
175-282 respectively, as shown in Table 2 of FIG. 2. Compounds in
Table 2 of FIG. 2, and their respective SEQ. ID. NOs. shown in
Table 2 of FIG. 2 are hereby incorporated into the specification as
filed.
[0266] FIG. 3. FIG. 3 has two panels. The top panel in FIG. 3
illustrates the interaction between PTH 1-34 or PTHrP 1-34 and the
PTH R1 receptor based on the x-ray crystal structure (Piozak. A.
A., et al. (2009) J Biol Chem 284:28382-391) of the extracellular
domain of the receptor. The critical hydrophobic interactions of
residues 23' (W or F), 24' (L) and 28' (L or I) are illustrated.
The surfactant modification on the modified peptides described
herein, in some instances, replace these critical hydrophobic
interactions. The bottom panel in FIG. 3 shows the structural
comparison between the sequence of PTH 1-34 and PTHrP 1-34 and
illustrates the strong regions of identity and homology in the
sequences and peptide helical conformation.
[0267] FIG. 4. FIG. 4 shows the cAMP responses of human cells in
culture (SaOS2) to stimulation by a representative peptide product
described herein, EU-232. The ordinate (vertical axis) shows the
response as a percentage of the maximal response shown to the
internal assay standard, i.e., PTHrP. The data illustrates a
super-agonistic response to EU-232.
[0268] FIG. 5. FIG. 5 shows the response of human cells in culture
(SaOS2) to treatment with various doses of EU-285 (a coded sample
of human PTHrP). The ordinate (vertical axis) shows the cAMP
response as a percentage of the maximal response of the internal
assay standard, i.e., PTHrP.
[0269] FIG. 6. Blood phosphate levels in rat serum were tested at
various time points after subcutaneous dosing rats with saline (G
1), 80 micrograms per kg of PTH (G2), 80 micrograms per kg of
EU-232 (G3) or 320 micrograms per kg of EU-232 (G4). This
surfactant modified analog, EU-232, demonstrates prolonged duration
of action, as evidenced by the maximal statistically significant
effect, which is seen at the last time point in the assay (i.e., 5
hrs post dosing). See Example 6.
[0270] FIG. 7. Blood calcium levels in rat serum were tested at
various time points after subcutaneous dosing in rats with saline
(G1), 80 micrograms per kg of PTH (G2), 80 micrograms per kg of
EU-232 (G3) or 320 micrograms per kg of EU-232 (G4). No groups were
statistically significantly different from control (G1). Further,
the maximally effective dose and time point for EU-232 (G4; at 5
hrs) shows no elevation and thus no indications of a propensity for
hypercalcemia at a maximally effective dose. See Example 6.
[0271] FIG. 8 Table 3 in FIG. 8 depicts compounds that were
prepared by methods described herein. The specification provides
sequences for SEQ. ID. Nos. 303-305 and SEQ. ID. Nos. 619-644.
Additionally, Table 3 of FIG. 8 provides SEQ. ID Numbers for
compounds EU-A300 to EU-A425 having SEQ. ID. NOs. 306-431
respectively, as shown in Table 3 of FIG. 8. Compounds in Table 3
of FIG. 8, and their respective SEQ. ID. NOs. shown in Table 3 of
FIG. 8 are hereby incorporated into the specification as filed.
[0272] FIG. 9 Table 4 in FIG. 9 depicts compounds that were
prepared by methods described herein. The specification provides
SEQ. ID. Nos. 303-305 and SEQ. ID. Nos. 619-644. Additionally,
Table 4 of FIG. 9 provides SEQ. ID Numbers for compounds EU-A426 to
EU-599 having SEQ. ID. NOs. 432-520 respectively, as shown in Table
4 of FIG. 9. Compounds in Table 2 of FIG. 2, and their respective
SEQ. ID. NOs. shown in Table 4 of FIG. 9 are hereby incorporated
into the specification as filed.
[0273] FIG. 10 FIG. 10 illustrates the x-ray crystal structure
(Runge, S., et al. (2008) J Biol Chem 283: 11340-7) of the binding
site of the extracellular domain of the GLP-1 receptor and
illustrates critical hydrophobic binding elements of the receptor
and the ligand exendin-4 (Val.sup.19*, Phe.sup.22*, Trp.sup.25*,
Leu.sup.26*) which are mimicked and replaced by the hydrophobic
1'-alkyl portion of the surfactant on the peptides of the
invention.
[0274] FIG. 11 FIG. 11 shows Cellular assay data of a
representative compound, EU-A178, acting on cells containing the mu
opioid receptor (MOP) and acting as an antagonist on the delta
opioid receptor (DOP) in competition with 30 nM DPDPE. The
representative compound shows potent, full agonistic behavior in
the MOP agonist assay and highly potent action as a pure antagonist
on the DOP receptor.
DETAILED DESCRIPTION OF THE INVENTION
[0275] Provided herein are modified peptides and/or proteins that
comprise a peptide and/or protein covalently attached to a
hydrophilic group, a "head" (e.g., a polyol, (e.g., a saccharide));
the hydrophilic group is covalently attached to a hydrophobic
group, a "tail", thereby generating a surfactant. In some
embodiments, use of hydrophobic-linked glycoside surfactant (e.g.,
alkyl glycoside) moieties for covalent modification of the peptides
or proteins, prolongs the duration of action of the peptides or
proteins by multiple mechanisms, including formation of depots of
the drug at the site of administration in the body and binding to
hydrophobic carrier proteins. In some embodiments, incorporation of
steric hindrance into peptide and/or protein structure can prevent
approach of proteases to the peptide and/or protein product and
thereby prevent proteolysis. In some embodiments, surfactant
modification (e.g., covalent attachment of alkyl glycoside class of
surfactants) of peptides and/or proteins as described herein,
increases the transport across mucosal barriers. Accordingly, the
modifications of the peptides and/or proteins described herein
provide desirable benefits including and not limited to, protection
from proteolysis, and slowed movement from the site of
administration, thereby leading to prolonged pharmacokinetic
behavior (e.g., prolongation of circulating t.sub.1/2) and improved
transmucosal bioavailability.
[0276] In some embodiments, interaction of the improved peptides
and/or proteins with their receptors is modified in beneficial ways
by the truncation of the sequence, introduction of constraint,
and/or the incorporation of steric hindrance. Described herein are
novel alkyl glycoside reagents that allow for incorporation of both
rigidity and steric hindrance in the modified peptides and/or
proteins. In some embodiments, steric hindrance confers receptor
selectivity to the modified peptides and/or proteins described
herein. In some embodiments, steric hindrance provides protection
from proteolysis.
[0277] Proteins and peptides undergo numerous physical and chemical
changes that may affect potency and safety. Among these are
aggregation, which includes dimerization, trimerization, and the
formation of higher-order aggregates such as amyloids. Aggregation
is a key issue underlying multiple potentially deleterious effects
for peptide and/or protein-based therapeutics, including loss of
efficacy, altered pharmacokinetics, reduced stability or product
shelf life, and induction of undesirable immunogenicity.
Bioavailability and pharmacokinetics of a self-associating peptide
can be influenced by aggregate size and the ease of disruption of
the non-covalent intermolecular interactions at the subcutaneous
site (Maji, S. K., et al. (2008) PLoS Biol 6: e17). In some
instances peptides can aggregate into subcutaneous depots that
disassociate with t.sub.1/2 of 30 or more days. Such slow
dissolution can lead to favorable effects such as delivery for one
month from a single sc injection, causes such a low blood
concentration that the peptide appears inactive in vivo. Thus
hydrophobic aggregation can appear to totally preclude a peptide's
bioavailability and effectiveness (Clodfelter, D. K., et al. (1998)
Pharm Res 15: 254-262).
[0278] Aggregation has been associated with increased
immunogenicity of the administered peptide and/or protein
therapeutic. One means to avoid this problem is to work with
solutions of lower concentration, however concentrated peptide and
protein solutions are desirable in some instances for ease of
administration. In some instances, adding polyols (e.g., mono- or
oligo-saccharides) or alkyl glycosides to the peptide and/or
protein solutions during the course of purification and
concentration, reduces or eliminates aggregation, providing greater
efficiency in the manufacturing process, and providing a final
product which has less immunogenic potential.
[0279] The FDA and other regulatory agencies have increased their
scrutiny of aggregation, especially because of this potential
linkage to undesirable immunogenicity. The immunogenicity of a
self-associating peptide can be influenced by the formation of
aggregates as a result of non-covalent intermolecular interactions.
For example, interferon has been shown to aggregate resulting in an
antibody response (Hermeling, S., et al. (2006) J Pharm Sci 95:
1084-1096). An antibody response to erythropoietin produced "pure
red cell aplasia", a potentially life threatening side effect, in a
number of patients receiving recombinant EPO (Casadevall, N., et
al. (2002) N Engl J Med 346: 469-475) following a change in
formulation that altered the serum albumin source and
concentration. Insulin loses activity due to protein aggregation
upon agitation at temperatures above those found in refrigerated
storage (Pezron, I., et al. (2002) J Pharm Sci 91: 1135-1146,
Sluzky, V., et al. (1991) Proc Natl Acad Sci USA 88: 9377-9381).
Monoclonal antibody based therapeutics are subject to inactivation
as a result of protein aggregation (King, H. D., et al. (2002) J
Med Chem 45: 4336-4343). Highly concentrated monoclonal antibody
formulations pose stability, manufacturing, and delivery challenges
related to the potential of those antibodies to aggregate. Enzymes
may also lose activity as a result of aggregation. For example
thermal inactivation of urokinase is reported to occur via
aggregation (Porter, W. R., et al. (1993) Thromb Res 71:
265-279).
[0280] Protein stabilization during lyophilization has also posed
problems. Protein therapeutics frequently lose biological activity
after lyophilization and reconstitution as a result of aggregate
formation and precipitation. In some instances, addition of
reconstitution additives (including, for example, sulfated
polysaccharides, polyphosphates, amino acids, polyethylene glycol
(PEG) and various surfactants (Zhang, M. Z., et al. (1995) Pharm
Res 12: 1447-1452, Vrkljan, M., et al. (1994) Pharm Res 11:
1004-1008) reduces aggregation. In some cases, a combination of
alcohols, or other organic solvents, is used for solubilization.
Trifluoroethanol has an effect on maintaining peptide structure and
it has been used in mixtures to stabilize various peptides
(Roccatano, D., et al. (2002) Proc Natl Acad Sci USA 99:
12179-12184). There is a danger that such agents may have a harsh
effect on mucosal tissue, causing patient discomfort or local toxic
effects. U.S. Pat. Nos. 7,390,788 and 7,425,542 describe the use of
alkyl glycosides as stabilizers because of their gentle, non-ionic
detergent-like effect. However covalent incorporation of alkyl
glycosides into a peptide and/or protein structure itself has not
been described heretofore.
[0281] Often naturally occurring oligosaccharides that are
covalently attached to proteins do not have surfactant character.
In some embodiments, peptide and/or protein products described
herein have a covalently attached saccharide and an additional
hydrophobic group that confers surfactant character to the modified
peptides, thereby allowing for tunability of bioavailability,
immunogenicity, and/or pharmacokinetic behavior of the
surfactant-modified peptides.
[0282] Proteins and peptides modified with oligosaccharides are
described in, for example, Jensen, K. J. and Brask, J. (2005)
Biopolymers 80: 747-761, through incorporation of saccharide or
oligosaccharide structures using enzymatic (Gijsen, H. J., et al.
(1996) Chem Rev 96: 443-474; Sears, P. and Wong, C. H. (1998) Cell
Mol Life Sci 54: 223-252; Guo, Z. and Shao, N. (2005) Med Res Rev
25: 655-678) or chemical approaches (Urge, L., et al. (1992)
Biochem Biophys Res Commun 184: 1125-1132; Salvador, L. A., et al.
(1995) Tetrahedron 51: 5643-5656; Kihlberg, J., et al. (1997)
Methods Enzymol 289: 221-245; Gregoriadis, G., et al. (2000) Cell
Mol Life Sci 57: 1964-1969; Chakraborty, T. K., et al. (2005)
Glycoconj J 22: 83-93; Liu, M., et al. (2005) Carbohydr Res 340:
2111-2122; Payne, R. J., et al. (2007) J Am Chem Soc 129:
13527-13536; Pedersen, S. L., et al. (2010) Chembiochem 11:
366-374). Peptides as well as proteins have been modified by
glycosylation (Filira, F., et al. (2003) Org Biomol Chem 1:
3059-3063); (Negri, L., et al. (1999) J Med Chem 42: 400-404);
(Negri, L., et al. (1998) Br J Pharmacol 124: 1516-1522); Rocchi,
R., et al. (1987) Int J Pept Protein Res 29: 250-261; Filira, F. et
al. (1990) Int J Biol Macromol 12: 41-49 Gobbo, M., et al. (1992)
Int J Pept Protein Res 40: 54-61; Urge, L., et al. (1992) Biochem
Biophys Res Commun 184: 1125-1132; Djedaini-Pilard, F., et al.
(1993) Tetrahedron Lett 34: 2457-2460; Drouillat, B., et al. (1997)
Bioorg Med Chem Lett 7: 2247-2250; Lohof. E., et al. (2000) Angew
Chem Int Ed Engl 39: 2761-2764; Gruner, S. A., et al. (2001) Org
Lett 3: 3723-3725; Pean. C., et al. (2001) Biochim Biophys Acta
1541: 150-160; Filira, F., et al. (2003) Org Biomol Chem 1:
3059-3063; Grotenbreg, G. M., et al. (2004) J Org Chem 69:
7851-7859; Biondi, L., et al. (2007) J Pept Sci 13: 179-189; Koda,
Y., et al. (2008) Bioorg Med Chem 16: 6286-6296; Lowery J. J., et
al. (2011) J Pharmacol Exptl Therap 336: 767-78; Yamamoto. T., et
al. (2009) J Med Chem 52: 5164-5175).
[0283] However, the aforementioned attempts do not describe an
additional hydrophobic group attached to the peptide-linked
oligosaccharide. Accordingly, provided herein are modified peptides
and/or proteins that incorporate a hydrophobic group attached to a
saccharide and/or oligosaccharide that is covalently attached to
the peptide and/or protein and that allow for tunability of
bioavailability, immunogenicity and pharmacokinetic behaviour.
Accordingly, also provided herein are surfactant reagents
comprising an oligosaccharide and a hydrophobic group, that allow
for modification of peptide and/or proteins.
[0284] Provided herein is the use of saccharide-based surfactants
in covalent linkage to a peptide for improvement of peptide and/or
protein properties. In some embodiments, surfactant modification
(e.g., covalent attachment of alkyl glycoside class of surfactants)
of peptides and/or proteins as described herein, increases the
transport across mucosal barriers. In some embodiments, covalent
attachment of a surfactant to a peptide and/or protein product
prevents aggregation of the peptide and/or protein.
[0285] The surfactant-modified peptides and/or proteins described
herein overcome limitations of peptide pharmaceuticals including
and not limited to short duration of action, poor bioavailability,
aggregation, immunogenicity and lack of receptor subtype
specificity through the covalent incorporation of surfactants such
as alkyl glycosides as novel peptide and protein modifiers.
[0286] In certain instances, the effects of surfactants are
beneficial with respect to the physical properties or performance
of pharmaceutical formulations, but are irritating to the skin
and/or other tissues and in particular are irritating to mucosal
membranes such as those found in the nose, mouth, eye, vagina,
rectum, buccal or sublingual areas. Additionally, in some
instances, surfactants denature proteins thus destroying their
biological function. Since surfactants exert their effects above
the critical micelle concentration (CMC), surfactants with low
CMC's are desirable so that they may be utilized with effectiveness
at low concentrations or in small amounts in pharmaceutical
formulations. Accordingly, in some embodiments, surfactants (e.g.,
alkyl glycosides) suitable for peptide modifications described
herein have the CMC's less than about 1 mM in pure water or in
aqueous solutions. By way of example only, certain CMC values for
alkyl glycosides in water are: Octyl maltoside 19.5 mM; Decyl
maltoside 1.8 mM; Dodecyl-.beta.-D-maltoside 0.17 mM; Tridecyl
maltoside 0.03 mM; Tetradecyl maltoside 0.01 mM; Sucrose
dodecanoate 0.3 mM. It will be appreciated that a suitable
surfactant could have a higher or lower CMC depending on the
peptide and/or protein that is modified. As used herein, "Critical
Micelle Concentration" or "CMC" is the concentration of an
amphiphilic component (alkyl glycoside) in solution at which the
formation of micelles (spherical micelles, round rods, lamellar
structures etc.) in the solution is initiated. In certain
embodiments, the alkyl glycosides dodecyl, tridecyl and tetradecyl
maltoside or glucoside as well as sucrose dodecanoate,
tridecanoate, and tetradecanoate are possess lower CMC's and are
suitable for peptide and/or protein modifications described
herein.
Opioid Peptides and Analogs
[0287] In some embodiments, a peptide therapeutic class amenable to
the methods of peptide modifications described herein is that of
the peptide opioids. This class derives from the endogenous peptide
opioids which have a very broad range of functions in the body,
carried out through binding to the mu (MOR), delta (DOR), and kappa
(KOR) opioid receptors (Schiller, P. W. (2005) AAPS J 7: E560-565).
Of most interest is their role in modulating and, in particular,
suppressing of transmission and perception of pain signals. In the
development of such agents the central side effects (respiratory
suppression, place preference indicating reward from self
administration) are a major concern, so peripherally acting agents
would be attractive (Stein, C., et al. (2009) Brain Res Rev 60:
90-113). Studies from a number of labs have suggested that the
optimal class of agents will have mu opioid receptor agonism with
the possibility of delta receptor antagonism (Schiller, P. W.
(2010) Life Sci 86: 598-603).
[0288] Although the endomorphins (Janecka, A., et al. (2007) Curr
Med Chem 14: 3201-3208) are primarily mu receptor specific,
judicious modification of the framework can result in molecules
with both mu and delta selectivity (Lazarus, L. H. and Okada, Y.
(2012) Expert Opin Ther Patents 22: 1-14; Keresztes, A., et al.
(2010) ChemMedChem 5: 1176-96). Derived from the dermorphin family
are the DALDA class of analogs (Schiller, P. W. (2010) Life Sci 86:
598-603). Also derived from the dermorphin family are the TIPP
family of peptides (Schiller, P. W., et al., (1999) Biopolymers
51:411-25). Described herein are certain opioid peptides that are
covalently attached to a saccharide group of an alkyl-glycoside
surfactant and have improved pharmaceutical properties.
[0289] Some of the exemplary synthetic peptide analogs described
herein are derived from endomorphins and some are derived from
dermorphin, two classes of native peptide opioid sequences. In one
aspect, the present peptide analogs of the native sequences are
endorphin related sequences such as illustrated by EU-A101 to
EU-A115. In another aspect, the peptide analogs are
dermorphin-related sequences such as EU-A107, EU-A108 and EU-A 120
to EU-A133. A related class of opioid peptide analogs is
illustrated by sequences EU-A134 to EU-A142 wherein a Tic residue
replaces the D-Ala residue seen in the dermorphin structure (TIPP
family). An additional class of specialized linkage is shown when
Tic is replaced by Tic(.PSI.[CH2-NH]) as this requires a
complementary residue .PSI.-Phe to complete the linkage.
[0290] In some embodiments, a surfactant-modified peptide product
has amino acid sequences corresponding to the general Formula
III:
aa.sub.1-aa.sub.2-aa.sub.3-aa.sub.4-aa.sub.5-Z FORMULA III (SEQ.
ID. NO. 1) [0291] wherein: [0292] aa.sub.1 is Tyr, Dint,
N-alkyl-Tyr, N-alkyl-Dmt, N-dialkyl-Tyr, N-dialkyl-Dmt and the
like; [0293] aa.sub.2 is Pro, D-Arg, D-U(X), D-Ala, Tic,
Tic(.PSI.[CH2-NH]); [0294] aa.sub.3 is Phe, Trp, Tmp, D- or
L-Nal(1), D- or L-Nal(2), C.alpha.MePhe, .PSI.-Phe; [0295] aa.sub.4
is Phe, Tmp, D- or L-Nal(1), D- or L-Nal(2), U(X), D- or
L-C.alpha.MeU(X); [0296] aa.sub.5 is absent or Pro, Aib, U(X), D-
or L-C.alpha.MeU(X) [0297] alkyl or dialkyl is independently a
substituted or unsubstituted C.sub.1-C.sub.10 branched or straight
chain, or substituted or unsubstituted aralkyl chain; [0298] U is a
linking amino acid; [0299] X is a functionalized surfactant linked
to the side chain of U; [0300] Z is --OH or NH.sub.2.
[0301] In some specific embodiments of Formula III, X has the
structure:
##STR00017## [0302] wherein: [0303] A is a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl group; [0304] R.sup.1b,
R.sup.1c, and R.sup.1d are H; [0305] W.sup.1 is --(C.dbd.O)--NH--;
[0306] W.sup.2 is --O--; and [0307] R.sup.2 is a bond.
[0308] In one embodiment, N-alkyl is N-methyl; U is a dibasic amino
acid, such as Lys or Orn; X is a modified nonionic detergent of the
1-alkyl glycoside class wherein alkyl is C.sub.1-C.sub.20 alkyl or
an alkoxyaryl substituent wherein the glycosidic linkage to the
saccharide ring is through --O-- or other heteroatom (e.g., S or
N); Z is NH.sub.2.
[0309] In another embodiment, the 1-alkyl group in the
1-alkyl-glycoside is substituted or unsubstituted
C.sub.1-C.sub.16alkyl; U is Lys, Z is NH.sub.2; [0310] aa.sub.1 is
Tyr, Dint, N.alpha.-Me-Tyr, N.alpha.-Me-Dmt; [0311] aa.sub.2 is
Pro; [0312] aa.sub.3 is Phe, Trp, Tmp; [0313] aa.sub.4 is Phe,
Lys(X); [0314] aa.sub.5 is absent or Lys(X).
[0315] In a further embodiment, 1-alkyl group in the
1-alkylglycoside is substituted or unsubstituted
C.sub.1-C.sub.20alkyl; Z is NH.sub.2; [0316] aa.sub.1 is Tyr, Dint;
[0317] aa.sub.2 is D-Arg, D-Lys(X), Tic, Tic(.PSI.[CH2-NH]); [0318]
aa.sub.3 is Phe, Trp, Tmp, C.alpha.MePhe, .PSI.-Phe; [0319]
aa.sub.4 is Phe, Tmp, Lys(X); [0320] aa.sub.5 is absent or Pro,
Aib, Lys(X), D- or L-C.alpha.MeLys(X).
[0321] The endomorphin class parent structures are:
[0322] Endomorphin 1--Tyr-Pro-Trp-Phe-NH2
[0323] Endomorphin 2--TyT-Pro-Phe-Phe-NH2
[0324] Certain analog family substitutions are as shown below:
TABLE-US-00001 Position 1 Position 2 Position 3 Position 4 Position
5 Tyr Pro Phe Phe -NH2 Dmt Trp Tmp Lys(X)-NH2 N-Alkyl-Tyr Tmp
Lys(X) Aib-NH2 N-Alkyl-Dmt C.alpha.MePhe C.alpha.MeLys(X) Pro-NH2
N-dialkyl-Tyr D- or L-Nal(1) Pro N-dialkyl-Dmt D- or L-Nal(2) X =
1-alkyl glucuronyl or 1-alkyl mannouronyl substituted with
1-(C.sub.1-C.sub.20 alkyl, aryloxyalkyl, and the like)
[0325] Certain sequences contain the following amino acids:
TABLE-US-00002 Position 1 Position 2 Position 3 Position 4 Position
5 Tyr Pro Phe Phe -NH2 Dmt Trp Tmp Lys(X)-NH2 Tmp Lys(X) X =
1-alkyl glucuronyl or 1-alkyl mannouronyl substituted with
1-(C.sub.1-C.sub.20 alkyl, aryloxyalkyl, and the like)
[0326] In specific embodiments analogs with attached surfactants
include and are not limited to:
TABLE-US-00003 Position 1 Position 2 Position 3 Position 4 EU-A101
Dmt Pro Tmp Lys(C1-glucuronyl)-NH2 EU-A102 Dmt Pro Tmp
Lys(C8-glucuronyl)-NH2 EU-A103 Dmt Pro Tmp Lys(C12-glucuronyl)-NH2
EU-A105 Dmt Pro Tmp Phe-Lys(C1-glucuronyl)-NH2 EU-A106 Dmt Pro Tmp
Phe-Lys(C12-glucuronyl)-NH2 EU-A162 Dmt Pro Phe
Lys(C1-glucuronyl)-NH2 EU-A163 Dmt Pro Phe Lys(C8-glucuronyl)-NH2
EU-A164 Dmt Pro Phe Lys(C12-glucuronyl)-NH2 EU-A189 Dmt Pro Phe
Phe-Lys(C1-glucuronyl)-NH2 EU-A190 Dmt Pro Phe
Phe-Lys(C12-glucuronyl)-NH2 X = 1-alkyl glucuronyl or 1-alkyl
mannouronyl substituted with 1-(C.sub.1-C.sub.20 alkyl,
aryloxyalkyl, and the like)
[0327] The Dermorphin class parent structure is:
[0328] Parent dermorphin--Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2
[0329] Certain analog family structures are as shown below:
TABLE-US-00004 Position 1 Position 2 Position 3 Position 4 Tyr
D-Arg Phe Phe NH2 Dmt D-Lys(X) Trp Tmp Lys(X)-NH2 N-Alkyl-Tyr D-Ala
Tmp Lys(X) Pro-NH2 N-Alkyl-Dmt Tic C.alpha.MePhe C.alpha.MeLys(X)
Aib-NH2 N-dialkyl-Tyr Tic-(.PSI.[CH.sub.2--NH]) .PSI.-Phe
N-dialkyl-Dmt D-or L-Nal(1) D- or L-Nal(1) D- or L-Nal(1) D-or
L-Nal(2) D- or L-Nal(2) D- or L-Nal(2) X = 1-alkyl glucuronyl or
1-alkyl mannouronyl substituted with 1-(C.sub.1-C.sub.20 alkyl,
aryloxyalkyl, and the like)
[0330] In some embodiments, certain analogs suitable for attachment
of surfactants include and are not limited to:
TABLE-US-00005 Position 1 Position 2 Position 3 Position 4 Tyr
D-Arg Phe Phe NH2 Dmt D-Lys(X) Trp Tmp Lys(X)-NH2 Tic Tmp Lys(X)
Pro-NH2 T10-(.PSI.[CH.sub.2--NH]) .PSI.-Phe Aib-NH2 C.alpha.MePhe
D- or L-Nal(1) D- or L-Nal(1) D- or L-Nal(2) D- or L-Nal(2) X =
1-alkyl glucuronyl or 1-alkyl mannouronyl substituted with
1-(C.sub.1-C.sub.20 alkyl, aryloxyalkyl, and the like)
[0331] In specific embodiments, analogs suitable for attachment of
surfactants include and are not limited to:
TABLE-US-00006 Position 1 Position 2 Position 3 Position 4 EU-A107
Dmt D- Lys(C1-glucuronyl) Tmp Phe-NH2 EU-A108 Dmt D-
Lys(C12-glucuronyl) Tmp Phe-NH2 EU-A120 Dmt D- Lys(C8-glucuronyl)
Nal(1) Phe-NH2 EU-A121 Dmt D- Lys(C12-glucuronyl) Nal(1) Phe-NH2
EU-A122 Dmt D- Lys(C16-glucuronyl) Nal(1) Phe-NH2 EU-A123 Dmt D-
Lys(C18-glucuronyl) Nal(1) Phe-NH2 EU-A124 Dmt D-
Lys(C20-glucuronyl) Nal(1) Phe-NH2 EU-A125 Dmt D-
Lys(C22-glucuronyl) Nal(1) Phe-NH2 EU-A126 Dmt D-
Lys(C24-glucuronyl) Nal(1) Phe-NH2 EU-A178 Dmt Tic Phe
Lys(C1-glucuronyl)-NH2 EU-A179 Dmt Tic Phe Lys(C12-glucuronyl)-NH2
EU-A180 Dmt Tic Phe Lys(C8-glucuronyl)-NH2 EU-A181 Dmt Tic Phe
Lys(C10-glucuronyl)-NH2 EU-A182 Dmt Tic Phe Lys(C16-glucuronyl)-NH2
EU-A183 Dmt Tic Phe Lys(C18-glucuronyl)-NH2 EU-A184 Dmt Tic Phe
Lys(C20-glucuronyl)-NH2 EU-A189 Dmt Tic Phe Phe-Lys(C1-glucuronyl)-
NH2 EU-A190 Dmt Tic Phe Phe-Lys(C12-glucuronyl)- NH2 EU-A191 Dmt
Tic Phe Phe-Lys(C8-glucuronyl)- NH2 EU-A600 Dmt Tic Phe
Lys(C1-glucuronyl)-Aib- NH2 EU-A601 Dmt Tic Phe
Lys(C8-glucuronyl)-Aib- NH2 EU-A603 Dmt Tic Phe
Lys(C12-glucuronyl)-Aib- NH2 EU-A615 Dmt Tic Phe
D-Lys(C1-glucuronyl)-Aib- NH2 EU-A616 Dmt Tic Phe
D-Lys(C8-glucuronyl)-Aib- NH2 EU-A618 Dmt Tic Phe
D-Lys(C12-glucuronyl)-NH2 EU-A619 Dmt Tic Phe
D-Lys(C16-glucuronyl)-NH2 EU-A620 Dmt Tic Phe Lys(C1-glucuronyl)-
NHCH.sub.2Ph EU-A621 Dmt Tic Phe Lys(C8-glucuronyl)- NHCH.sub.2Ph
EU-A623 Dmt Tic Phe Lys(C12-glucuronyl)- NHCH.sub.2Ph EU-A624 Dmt
Tic Phe Lys(C16-glucuronyl)- NHCH.sub.2Ph EU-A639 Dmt Tic Phe
D-Lys(C1-glucuronyl)- NHCH.sub.2Ph EU-A642 Dmt Tic Phe
D-Lys(C12-glucuronyl)- NHCH.sub.2Ph EU-A648 Dmt Tic Phe
Lys(C16-glucuronyl)-NH.sub.2 EU-649 Dmt Tic Phe
Lys(C14-glucuronyl)-NH.sub.2 X = 1-alkyl glucuronyl or 1-alkyl
mannouronyl substituted with 1-(C.sub.1-C.sub.20 alkyl,
aryloxyalkyl, and the like)
[0332] Contemplated within the scope of embodiments presented
herein are peptide chains substituted in a suitable position by the
substitution of the analogs claimed herein by acylation on a linker
amino acid, at for example, the .epsilon.-position of Lys, with
fatty acids such as octanoic, decanoic, dodecanoic, tetradecanoic,
hexadecanoic, octadecanoic, 3-phenylpropanoic acids and the like,
with saturated or unsaturated alkyl chains (Nestor, J. J., Jr.
(2009) Current Medicinal Chemistry 16:4399-4418; Zhang, L and
Bulaj, G. (2012) Curr Med Chem 19: 1602-18). Non-limiting,
illustrative examples of such analogs are:
TABLE-US-00007 (SEQ. ID. NO. 161)
H-Dmt-Tic-Phe-Lys(N-epsilon-acetyl)-NH.sub.2, (SEQ. ID. NO. 162)
H-Dmt-Tic-Phe-Lys(N-epsilon-dodecanoyl)-NH.sub.2, (SEQ. ID. NO.
163) H-Dmt-Tic-Phe-Lys(N-epsilon-tetradecanoyl)-NH.sub.2, (SEQ. ID.
NO. 164) H-Dmt-Tic-Phe-Lys(N-epsilon-(gamma-glutatnyl)-N-
alpha-dodecanoyl))-NH.sub.2, (SEQ. ID. NO. 165)
H-Dmt-Tic-Phe-Lys(N-epsilon-(gamma-glutamyl)-N-
alpha-tetradecanoyl))-NH.sub.2, (SEQ. ID. NO. 166)
H-Dmt-Tic-Phe-Lys(N-epsilon-acetyl)-NH-benzyl, (SEQ. ID. NO. 167)
H-Dmt-Tic-Phe-Lys(N-epsilon-dodecanoyl)-NH-benzyl, and the
like.
[0333] In other embodiments of the invention the peptide chain may
be substituted in a suitable position by reaction on a linker amino
acid, for example the sulfhydryl of Cys, with a spacer and a
hydrophobic moiety such as a steroid nucleus, for example a
cholesterol moiety. In some of such embodiments, the modified
peptide further comprises one or more PEG chains. Non-limiting
examples of such molecules are:
TABLE-US-00008 (SEQ. ID. NO. 168)
H-Dmt-Tic-Phe-Cys(S-(3-(PEG4-aminoethylacetamide-
cholesterol)))-NH.sub.2, (SEQ. ID. NO. 169)
H-Dmt-Tic-Phe-Cys(S-(3-(PEG4-aminoethylacetamide-
cholesterol)))-NH-benzyl, and the like.
[0334] The compounds of Formula I, Formula II, or Formula III are
assayed for mu opioid receptor activity in a cellular assay (MOP in
agonist and antagonist mode), and for delta2 opioid receptor
activity in cellular assay (DOP in agonist and antagonist mode) as
described in Example 12.
[0335] In certain embodiments, as shown in Example 12, a compound
having a pure MOP agonist activity along with a pure DOP
antagonistic activity, is a suitable profile for clinical
applications. Also contemplated within the scope of the disclosure
herein are compounds that have low solubility and low apparent in
vitro potency but exhibit prolonged duration of action
(pharmacodynamic action) in vivo.
[0336] Contemplated within the scope of embodiments presented
herein are peptide products of Formula I, Formula II or Formula
III, wherein the peptide product comprises one, or, more than one
surfactant groups (e.g., group X having the structure of Formula
I). In one embodiment, a peptide product of Formula I, Formula II
or Formula III, comprises one surfactant group. In another
embodiment, a peptide product of Formula I, Formula II or Formula
III, comprises two surfactant groups. In yet another embodiment, a
peptide product of Formula I, Formula II or Formula III, comprises
three surfactant groups.
PTH Peptides and Analogs
[0337] Also provided herein, in some embodiments, are reagents and
intermediates for synthesis of modified peptides and/or proteins
(e.g., modified PTH, PTHrP, or the like) through the incorporation
of surfactants.
[0338] Provided herein, in some embodiments, are peptide products
comprising a surfactant X, covalently attached to a peptide, the
peptide comprising a linker amino acid U and at least one other
amino acid:
##STR00018## [0339] wherein the surfactant X is a group of Formula
2-I:
[0339] ##STR00019## [0340] wherein: [0341] R.sup.1a is
independently, at each occurrence, a bond, H, a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl group, a substituted or
unsubstituted alkoxyaryl group, a substituted or unsubstituted
aralkyl group, or a steroid nucleus containing moiety; [0342]
R.sup.1a, R.sup.1c, and R.sup.1d are each, independently at each
occurrence, a bond, H, a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl group, a substituted or unsubstituted
alkoxyaryl group, or a substituted or unsubstituted aralkyl group;
[0343] W.sup.1 is independently, at each occurrence, --CH.sub.2--,
--CH.sub.2--O--, --(C.dbd.O), --(C.dbd.O)--O--, --(C.dbd.O)--NH--,
--(C.dbd.S)--, --(C.dbd.S)--NH--, or --CH--S--; [0344] W.sup.2 is
--O--, --CH.sub.2--, or --S--; [0345] R.sup.2 is independently, at
each occurrence, a bond, H, a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl group, a substituted or unsubstituted
alkoxyaryl group, or a substituted or unsubstituted aralkyl group,
--NH.sub.2, --SH, C.sub.2-C.sub.4-alkene, C.sub.2-C.sub.4-alkyne,
--NH(C.dbd.O)--CH.sub.2--Br, --(CH.sub.2).sub.m-maleimide, or
--N.sub.3; [0346] n is 1, 2 or 3; and [0347] m is 1-10; [0348] the
peptide is selected from Formula 2-II:
TABLE-US-00009 [0348] (SEQ. ID. NO. 170) aa.sub.1- Val.sub.2-
aa.sub.3- Glu.sub.4- aa5 aa.sub.6- aa.sub.7- aa.sub.8- His.sub.9-
aa.sub.10- aa.sub.11- aa.sub.12- aa.sub.13- aa.sub.14- aa.sub.15-
aa.sub.16- aa.sub.17- aa.sub.18- aa.sub.19- aa.sub.2- aa.sub.21-
aa.sub.22- aa.sub.23- aa.sub.24- aa.sub.25- aa.sub.26-Z Formula
2-II
[0349] wherein: [0350] Z is OH, or --NH--R.sup.3, [0351] R.sup.3 is
H, a substituted or unsubstituted C.sub.1-C.sub.12 alkyl, or a PEG
chain of less than 10 Da; [0352] aa.sub.1 is Aib, Ac5c, or Deg;
[0353] aa.sub.3 is Aib, Ac4c, or Deg; [0354] aa.sub.5 is His, or
Ile; [0355] aa.sub.6 is Gln, or Cit; [0356] aa.sub.7 is Leu, or
Phe; [0357] aa.sub.8 is Leu, or Nle; [0358] aa.sub.10 is Asp, Asn,
Gln, Glu, Cit, Ala, or Aib; [0359] aa.sub.11 is Arg, or hArg;
[0360] aa.sub.12 is Gly, Glu, Lys, Ala, Aib, or Ac5c; [0361]
aa.sub.13 is Lys, or Arg; [0362] aa.sub.14 is Ser, His, Trp, Phe,
Leu, Arg, Lys, Glu, or Nal(2); [0363] aa.sub.15 is Ile, Leu, or
Aib; [0364] aa.sub.16 is Gln, Asn, Glu, Lys, Ser, Cit, Aib, or U;
[0365] aa.sub.17 is Asp, Ser, Aib, Ac4c, Ac5c, or U; [0366]
aa.sub.18 is absent or Leu, Gln, Cit, Aib, Ac5c, Lys, Glu or U;
[0367] aa.sub.19 is absent or Arg, Glu, Aib, Ac4c, Ac5c, or U;
[0368] aa.sub.20 is absent or Arg, Glu, Lys, Aib, Ac4c, Ac5c, or U;
[0369] aa.sub.21 is absent or Arg, Val, Aib, Ac5C, Deg, or U;
[0370] aa.sub.22 is absent or Phe, Glu, Aib, Ac5C, Lys, or U;
[0371] aa.sub.23 is absent or Leu, Phe, Trp, or U; [0372] aa.sub.24
is absent or His, Arg, or U; [0373] aa.sub.25 is absent or His,
Lys, or U and [0374] aa.sub.26 is absent or Aib, Ac5c, Lys; [0375]
U is a natural or unnatural amino acid comprising a functional
group used for covalent attachment to the surfactant X; [0376]
wherein any two of aa.sub.1-aa.sub.26 are optionally cyclized
through their side chains to form a lactam linkage; and [0377]
provided that one, or at least one of aa.sub.16-aa.sub.26 is the
linker amino acid U covalently attached to X.
[0378] In some embodiments, n is 1. In some embodiments, n is 2,
and a first glycoside is attached to a second glycoside via a bond
between W.sup.2 of the first glycoside and any one of OR.sup.1b,
OR.sup.1c or OR.sup.1d of the second glycoside. In some
embodiments, n is 3, and a first glycoside is attached to a second
glycoside via a bond between W.sup.2 of the first glycoside and any
one of OR.sup.1b, OR.sup.1C or OR.sup.1d of the second glycoside,
and the second glycoside is attached to a third glycoside via a
bond between W.sup.2 of the second glycoside and any one of
OR.sup.1b, OR.sup.1c or OR.sup.1d of the third glycoside.
[0379] In one embodiment, compounds of Formula I-A are compounds
wherein X has the structure:
##STR00020## [0380] wherein: [0381] R.sup.1a is H, a protecting
group, a substituted or unsubstituted C.sub.1-C.sub.30 alkyl group,
or a steroid nucleus containing moiety; [0382] R.sup.1b, R.sup.1c,
and R.sup.1d are each, independently at each occurrence, H, a
protecting group, or a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl group; [0383] W.sup.1 is independently, at
each occurrence, --CH.sub.2--, --CH.sub.2--O--, --(C.dbd.O),
--(C.dbd.O)--O--, --(C.dbd.O)--NH--, --(C.dbd.S)--,
--(C.dbd.S)--NH--, or --CH.sub.2--S--; [0384] W.sup.2 is --O-- or
--S--; [0385] R.sup.2 is a bond, C.sub.2-C.sub.4-alkene,
C.sub.2-C.sub.4-alkyne, or --(CH.sub.2).sub.m-maleimide; and [0386]
m is 1-10.
[0387] In another embodiment, compounds of Formula I-A are
compounds wherein X has the structure:
##STR00021##
[0388] Accordingly, in the embodiment described above, R.sup.2 is a
bond.
[0389] For instance, in an exemplary embodiment of the structure of
X described above, W.sup.1 is --C(.dbd.O)NH--, R.sup.2 is a bond
between W.sup.1 and an amino acid residue U within the peptide
(e.g., an amino group in the sidechain of a lysine residue present
in the peptide).
[0390] In a further embodiment, compounds of Formula I-A are
compounds wherein X has the structure:
##STR00022##
[0391] For instance, in an exemplary embodiment of the structure of
X described above, W.sup.1 is --CH.sub.2-- and R.sup.2 is an
alkyl-linked maleimide functional group on X and R.sup.2 is
attached to a suitable moiety of an amino acid residue U within the
peptide (e.g., a thiol group in a cysteine residue of the peptide
forms a thioether with the maleimide on X).
[0392] In yet another embodiment, compounds of Formula I-A are
compounds wherein X has the structure:
##STR00023## [0393] wherein: [0394] R.sup.1a is H, a protecting
group, a substituted or unsubstituted C.sub.1-C.sub.30 alkyl group,
or a steroid nucleus containing moiety; [0395] R.sup.1b, R.sup.1c,
and R.sup.1d are each, independently at each occurrence, H, a
protecting group, or a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl group; [0396] W.sup.1 is --(C.dbd.O)--NH--;
[0397] W.sup.2 is --O--; [0398] R.sup.2 is a bond.
[0399] In an additional embodiment, compounds of Formula I-A are
compounds wherein X has the structure:
##STR00024## [0400] wherein: [0401] R.sup.1a is a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl group; [0402] R.sup.1b,
R.sup.1c, and R.sup.1d are H; [0403] W.sup.1 is --(C.dbd.O)--NH--;
[0404] W.sup.2 is --O--; and [0405] R.sup.2 is a bond.
[0406] In some embodiments described above and herein, R.sup.1a is
a substituted or unsubstituted C.sub.1-C.sub.30 alkyl group.
[0407] In some embodiments described above and herein, R.sup.1a is
a substituted or unsubstituted C.sub.6-C.sub.20 alkyl group.
[0408] Also contemplated herein are alternate embodiments wherein X
in Formula 2-I-A has the structure:
##STR00025##
[0409] For instance, in an exemplary embodiment of the structure of
X described above, W.sup.1 is --S--, R.sup.2 is a C.sub.1-C.sub.3
alkyl group, W.sup.2 is S, R.sup.1a is a bond between W.sup.2 and a
suitable moiety of an amino acid residue U within the peptide
(e.g., a thiol group in a cysteine residue of the peptide forms a
thioether with X).
[0410] In another exemplary alternate embodiment of the structure
of X described above, W.sup.1 is --O--, R.sup.2 is a
C.sub.1-C.sub.30 alkyl group, W.sup.2 is O. R.sup.1a is a bond
between W.sup.2 and a suitable moiety of an amino acid residue U
within the peptide (e.g., a hydroxyl group in a serine or threonine
residue of the peptide forms an ether with X).
[0411] In some embodiments, U is used for covalent attachment to X
and is a dibasic natural or unnatural amino acid, a natural or
unnatural amino acid comprising a thiol, an unnatural amino acid
comprising a --N.sub.3 group, an unnatural amino acid comprising an
acetylenic group, or an unnatural amino acid comprising a
--NH--C(.dbd.O)--CH.sub.2--Br or a --(CH.sub.2).sub.m-maleimide,
wherein m is 1-10.
[0412] In some embodiments of the peptide product, the surfactant
is a 1-alkyl glycoside class surfactant. In some embodiments of the
peptide product, the surfactant is attached to the peptide via an
amide bond.
[0413] In some embodiments of the peptide product, the surfactant X
is comprised of 1-eicosyl beta-D-glucuronic acid, 1-octadecyl
beta-D-glucuronic acid, 1-hexadecyl beta-D-glucuronic acid,
1-tetradecylbeta D-glucuronic acid, 1-dodecyl beta D-glucuronic
acid, 1-decyl beta-D-glucuronic acid, 1-octyl beta-D-glucuronic
acid, 1-eicosyl beta-D-diglucuronic acid, 1-octadecyl
beta-D-diglucuronic acid, 1-hexadecyl beta-D-diglucuronic acid,
1-tetradecyl beta-D-diglucuronic acid, 1-dodecyl
beta-D-diglucuronic acid, 1-decyl beta-D-diglucuronic acid, 1-octyl
beta-D-diglucuronic acid, or functionalized 1-ecosyl
beta-D-glucose, 1-octadecyl beta-D-glucose, 1-hexadecyl
beta-D-glucose, 1-tetradecyl beta-D-glucose, 1-dodecyl
beta-D-glucose, 1-decyl beta-D-glucose, 1-octyl beta-D-glucose,
1-eicosyl beta-D-maltoside, 1-octadecyl beta-D-maltoside,
1-hexadecyl beta-D-maltoside, 1-dodecyl beta-D-maltoside, 1-decyl
beta-D-maltoside, 1-octyl beta-D-maltoside, and the like, and the
peptide product is prepared by formation of a linkage between the
aforementioned groups and a group on the peptide (e.g., a --COOH
group in the aforementioned groups and an amino group of the
peptide).
[0414] In some embodiments of the peptide product, U is a terminal
amino acid of the peptide. In some embodiments of the peptide
product, U is a non-terminal amino acid of the peptide. In some
embodiments of the peptide product, U is a natural D- or L-amino
acid. In some embodiments of the peptide product, U is an unnatural
amino acid. In some embodiments of the peptide product, U is
selected from Lys, Cys, Orn, or an unnatural amino acid comprising
a functional group used for covalent attachment to the surfactant
X.
[0415] In some embodiments of the peptide product, the functional
group used for covalent attachment of the peptide to the surfactant
X is --NH.sub.2, --SH, --OH, --N.sub.3, haloacetyl, a
--(CH.sub.2).sub.m-maleimide (wherein m is 1-10), or an acetylenic
group.
[0416] In some embodiments side chain functional groups of two
different amino acid residues are linked to form a cyclic lactam.
For example a Lys.sub.14 side chain may form a cyclic lactam with
the side chain of Glu.sub.18 or a Lys.sub.18 may form a lactam with
the side chain of a Glu.sub.22. In some embodiments such lactam
structures are reversed and are formed from a Glu.sub.14 and a
Lys.sub.18, for example. Such lactam linkages, in some instances,
stabilize alpha helical structures in peptides (Condon, S. M., et
al. (2002) Bioorg Med Chem 10: 731-736).
[0417] In some embodiments, the peptide product comprising a
covalently linked alkyl glycoside is a covalently modified PTH or
analog thereof. In some of such embodiments, the peptide product
contains a covalently linked 1-O-alkyl .beta.-D-glucuronic acid and
the peptide is an analog of PTH.
[0418] In some embodiments, a peptide product comprising a
covalently linked alkyl glycoside is a covalently modified PTHrP,
or analog thereof. In some of such embodiments, the peptide product
comprises a covalently linked 1-O-alkyl .beta.-D-glucuronic acid
and the peptide is an analog of PTHrP.
[0419] In some embodiments, the peptide product has the structure
of Formula 2-III:
TABLE-US-00010 (SEQ. ID. NO. 171) aa.sub.1- Val.sub.2- Aib.sub.3-
Glu.sub.4- Ile.sub.5-Gln.sub.6- Leu.sub.7- Nle.sub.8- His.sub.9-
Gln.sub.10- hArg.sub.11- aa.sub.12- Arg.sub.13- aa.sub.14-
Ile.sub.15- aa.sub.16- aa.sub.17- aa.sub.18- aa.sub.19- aa.sub.20-
aa.sub.21- aa.sub.22- aa.sub.23- aa.sub.24- aa.sub.25- aa.sub.26-Z
Formula 2-III
[0420] wherein: [0421] Z is OH or --NH.sub.2; [0422] aa.sub.1 is
Aib, or Ac5c; [0423] aa.sub.12 is Ala, Glu, Lys, Aib, or Ac5c;
[0424] aa.sub.14 is Trp, Phe, Lys, Glu or Nal(2); [0425] aa.sub.16
is Gln, Asn, Glu, Lys, Cit. or U(X); [0426] aa.sub.17 is Asp, Ser,
Aib, Ac4c, Ac5C or U(X); [0427] aa.sub.18 is absent or Leu, Gln,
Aib, Lys, Glu or U(X); [0428] aa.sub.19 is absent or Arg, Glu, Aib,
Ac4c or Ac5c; [0429] aa.sub.20 is absent or Arg, Glu. Lys, Aib,
Ac4c, Ac5c; [0430] aa.sub.21 is absent or Arg, Val, Aib, Ac5C, or
Deg; [0431] aa.sub.22 is absent or Phe, Glu, Lys or U(X); [0432]
aa.sub.23 is absent or Leu, Phe, Trp or U(X); [0433] aa.sub.24 is
absent or His, Arg, or U(X); [0434] aa.sub.25 is absent or His,
Lys, or U(X); and [0435] aa.sub.26 is absent or Aib, Ac5c; [0436]
wherein any two of aa.sub.1-aa.sub.26 are optionally cyclized
through their side chains to form a lactam linkage; and [0437]
provided that one, or at least one of aa.sub.16, aa.sub.17,
aa.sub.18, aa.sub.22, aa.sub.23, aa.sub.24 or aa.sub.25 is the
linker amino acid U covalently attached to X.
[0438] In some embodiments of Formula 2-III. U is any linker amino
acid described herein. In some embodiments, the compound of Formula
2-III is a compound wherein aa.sub.12 and aa.sub.16 are cyclized
through their side chains to form a lactam linkage. In some
embodiments, the compound of Formula 2-III is a compound wherein
aa.sub.16 and aa.sub.20 are cyclized through their side chains to
form a lactam linkage.
[0439] In some embodiments, the peptide product has the
structure:
TABLE-US-00011 (SEQ. ID. NIO. 180) Ac5c.sub.1- Val.sub.2-
Aib.sub.3- Glu.sub.4- Ile.sub.5-Gln.sub.6- Leu.sub.7- Nle.sub.8-
His.sub.9- Gln.sub.10- hArg.sub.11- Ala.sub.12- Arg.sub.13-
TrP.sub.14- Ile.sub.15- Gln.sub.16-
Aib.sub.17-Lys(N-epsilon-1'-dodecyl beta-D-
glucuronyl).sub.18-NH.sub.2.
[0440] In some embodiments, the peptide product has the
structure:
TABLE-US-00012 (SEQ. ID. NO. 283) Ac5c.sub.1- Val.sub.2- Aib.sub.3-
Glu.sub.4- Ile.sub.5-Gln.sub.6- Leu.sub.7- Nle.sub.8- His.sub.9-
Gln.sub.10- hArg.sub.11- Ala.sub.12- Arg.sub.13- Trp.sub.14-
Ile.sub.15- Gln.sub.16- Aib.sub.17-Lys(N-epsilon-1'-alkyl beta-D-
glucuronyl).sub.18-Aib.sub.19-NH.sub.2, wherein alkyl is dodecyl,
tetradecyl, hexadecyl, or octadecyl.
[0441] In some embodiments, the peptide product has the
structure:
TABLE-US-00013 (SEQ. ID. NO. 284) Ac5c.sub.1- Val.sub.2- Aib.sub.3-
Glu.sub.4- Ile.sub.5-Gln.sub.6- Leu.sub.7- N1e.sub.8- His.sub.9-
Gln.sub.10- hArg.sub.11- Ala.sub.12- Arg.sub.13- Trp.sub.14-
Ile.sub.15- Gln.sub.16- Aib.sub.17-Lys(N-epsilon-1'-alkyl beta-D-
glucuronyl).sub.18-Ac4c.sub.19-NH.sub.2, wherein alkyl is dodecyl,
tetradecyl, hexadecyl, or octadecyl.
[0442] In some embodiments, the peptide product has the structure:
Ac5c.sub.1-Val.sub.2-Aib.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nl-
e.sub.8-His.sub.9-Gln.sub.10-hArg.sub.11-Ala.sub.12-Arg.sub.13-Trp.sub.14--
Ile.sub.15-Glu*.sub.16-Aib.sub.17-Lys(N-epsilon-1'-alkyl
beta-D-glucuronyl).sub.18-Aib.sub.19-Lys*.sub.20--NH.sub.2, wherein
Glu*.sub.16 and Lys*.sub.20 are linked through their sidechains by
a lactam and alkyl is dodecyl, tetradecyl, hexadecyl, or octadecyl.
(SEQ. ID. NO. 285)
[0443] In some embodiments, the peptide product has the structure:
Ac5c.sub.1-Val.sub.2-Aib.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nl-
e.sub.8-His.sub.9-Gln.sub.10-hArg.sub.11-Glu*.sub.12-Arg.sub.13-Trp.sub.14-
-Ile.sub.15-Lys*.sub.16-Aib.sub.17-Lys(N-epsilon-1'-alkyl
beta-D-glucuronyl).sub.18-Aib.sub.19-NH.sub.2, wherein Glu*.sub.12
and Lys*.sub.16 are linked through their sidechains by a lactam and
alkyl is dodecyl, tetradecyl, hexadecyl, or octadecyl. (SEQ. ID.
NO. 286)
[0444] In some embodiments, the peptide product has the structure:
Ac5c.sub.1-Val.sub.2-Aib.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nl-
e.sub.8-His.sub.9-Gln.sub.10-hArg.sub.11-Ala.sub.12-Arg.sub.13-Phe.sub.14--
Ile.sub.15-Gln.sub.16-Aib.sub.17-Lys(N-epsilon-1'-alkyl
beta-D-glucuronyl).sub.18-Aib.sub.19-NH.sub.2, wherein alkyl is
dodecyl, tetradecyl, hexadecyl, or octadecyl. (SEQ. ID. NO.
287)
[0445] In some embodiments, the peptide product has the structure:
Ac5c.sub.1-Val.sub.2-Aib.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nl-
e.sub.8-His.sub.9-Gln.sub.10-hArg.sub.11-Ala.sub.12-Arg.sub.13-Trp.sub.14--
Ile.sub.15-Gln.sub.16-Aib.sub.17-Lys(N-epsilon-1'-alkyl
beta-D-glucuronyl)s-Aib.sub.19-Aib.sub.20-NH.sub.2, wherein alkyl
is dodecyl, tetradecyl, hexadecyl, or octadecyl. (SEQ. ID. NO.
288)
[0446] In some embodiments, the peptide product has the structure:
Ac5c.sub.1-Val.sub.2-Aib.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nl-
e.sub.8-His.sub.9-Gln.sub.10-hArg.sub.11-Glu*.sub.12-Arg.sub.13-Trp.sub.14-
-Ile.sub.15-Lys*.sub.16-Aib.sub.7-Lys(N-epsilon-1'-alkyl
beta-D-glucuronyl)s-Aib.sub.19-Aib.sub.20-NH.sub.2, wherein
Glu*.sub.12 and Lys*.sub.16 are linked through their sidechains by
a lactam and alkyl is dodecyl, tetradecyl, hexadecyl, or octadecyl.
(SEQ. ID. NO. 289)
[0447] In some embodiments, the peptide product has the
structure:
TABLE-US-00014 (SEQ. ID. NO. 207) Ac5c.sub.1- Val.sub.2- Aib.sub.3-
Glu.sub.4- Ile.sub.5-Gln.sub.6- Leu.sub.7- Nle.sub.8- His.sub.9-
Gln.sub.10- hArg.sub.11- Ala.sub.12- Arg.sub.13- Trp.sub.14-
Ile.sub.15- Gln.sub.16- Aib.sub.17-Lys(N-epsiion-1'-dodecyl beta-D-
glucuronyl).sub.18-Aib.sub.19-NH.sub.2.
[0448] In some embodiments, the peptide product has the
structure:
TABLE-US-00015 (SEQ. ID. NO. 260) Ac5c.sub.1- Val.sub.2- Aib.sub.3-
Glu.sub.4- Ile.sub.5-G1n.sub.6- Leu.sub.7- Nle.sub.8- His.sub.9-
Gln.sub.10- hArg.sub.11- Ala.sub.12- Arg.sub.13- Trp.sub.14-
Ile.sub.15- Gln.sub.16- Aib.sub.17-Lys(N-epsilon-1'-tetradecyl
beta-D- glucuronyl).sub.18-Aib.sub.19-NH.sub.2.
[0449] In some embodiments, the peptide product has the
structure:
TABLE-US-00016 (SEQ. ID. NO. 261) Ac5c.sub.1- Val.sub.2- Aib.sub.3-
Glu.sub.4- Ile.sub.5-Gln.sub.6- Leu.sub.7- Nle.sub.8- His.sub.9-
Gln.sub.10- hArg.sub.11- Ala.sub.12- Arg.sub.13- Trp.sub.14-
Ile.sub.15- Gln.sub.16- Aib.sub.17-Lys(N-epsilon-1'-hexadecyl
beta-D- glucuronyl).sub.18-Aib.sub.19-HN2.
[0450] In some embodiments, the peptide product has the
structure:
TABLE-US-00017 (SEQ. ID. NO. 262) Ac5c.sub.1- Val.sub.2- Aib.sub.3-
Glu.sub.4- Ile.sub.5-Gln.sub.6- Leu.sub.7- Nle.sub.8- His.sub.9-
Gln.sub.10- hArg.sub.11- Ala.sub.12- Arg.sub.13- Trp.sub.14-
Ile.sub.15- Gln.sub.16- Aib.sub.17-Lys(N-epsilon-1'-octadecyl
beta-D- glucuronyl).sub.18-Aib.sub.19-NH.sub.2.
[0451] In some embodiments, the peptide product has the
structure:
TABLE-US-00018 (SEQ. ID. NO. 275) Ac5c.sub.1- Val.sub.2- Aib.sub.3-
G1u.sub.4- Ile.sub.5-Gln.sub.6- Leu.sub.7- Nle.sub.8- His.sub.9-
Gln.sub.10- hArg.sub.11- Ala.sub.12- Arg.sub.13- Trp.sub.14-
Ile.sub.15- Gln.sub.16- Aib.sub.17-Lys(N-epsilon-1'-dodecyl beta-D-
glucuronyl).sub.18-Aib.sub.19-Aib.sub.20-NH.sub.2.
[0452] In some embodiments, the peptide product has the
structure:
TABLE-US-00019 (SEQ. ID. NO. 277) Ac5c.sub.1- Val.sub.2- Aib.sub.3-
Glu.sub.4- Ile.sub.5-Gln.sub.6- Leu.sub.7- Nle.sub.8- His.sub.9-
Gln.sub.10- hArg.sub.11- Ala.sub.12- Arg.sub.13- Trp.sub.14-
Ile.sub.15- Gln.sub.16- Aib.sub.17-Lys(N-epsilon-1'-hexadecyl
beta-D- glucuronyl).sub.18-Aib.sub.19-Aib.sub.20-NH.sub.2.
[0453] In some embodiments, the peptide product is a biologically
active peptide product that binds to the PTH receptor (PTHR1).
[0454] In a specific embodiment, the peptide products of Formula
2-I-A described above and herein have the following structure:
##STR00026## [0455] wherein R.sup.1a is a C.sub.1-C.sub.2 alkyl
chain as described in Table 2 of FIG. 2, R' is a peptide as
described in Table 2 of FIG. 2, W.sup.2 of Formula I-A is --O--,
and W.sup.1 of Formula 2-I-A is --(C.dbd.O)NH-- and is part of an
amide linkage to the peptide R'. In some of such embodiments,
R.sup.1a is a C.sub.6-C.sub.20 alkyl chain. In some of such
embodiments, R.sup.1a is a C.sub.5-C.sub.20 alkyl chain. In some of
such embodiments, R.sup.1a is a C.sub.8-C.sub.20 alkyl chain. In
some of such embodiments, R.sup.1a is a C.sub.8-C.sub.18 alkyl
chain. In some of such embodiments, R.sup.1a is a C.sub.8-C.sub.16
alkyl chain.
[0456] In embodiments described above, an amino moiety of an amino
acid and/or a peptide R' (e.g., an amino group of an amino acid
residue such as a Lysine, or a lysine within the peptide R') is
used to form a covalent linkage with a compound of structure:
##STR00027##
wherein R.sup.1a is a C.sub.1-C.sub.20 alkyl chain as described in
Table 2 of FIG. 2.
[0457] In such cases, the amino acid having an amino moiety (e.g.,
a Lysine residue within the peptide R') which is used to form a
covalent linkage to the compound A described above, is a linker
amino acid U which is attached to a surfactant X having the
structure of Formula 2-A. Accordingly, as one example, Lys(C12) of
Table 2 of FIG. 2 has the following structure:
##STR00028##
[0458] Also contemplated within the scope of the embodiments
presented herein are peptide products of Formula 2-I-A derived from
maltouronic acid-based surfactants through binding at either or
both carboxylic acid functions. Thus, as one example, peptides in
Table 2 of FIG. 2 comprise a lysine linker amino acid bonded to a
maltouronic acid based surfactant X and having a structure:
##STR00029##
[0459] It will be understood that in one embodiment, compounds of
Formula 2-I-A are prepared by attaching a lysine to a group X,
followed by attachment of additional amino acid residues and/or
peptides are attached to the lysine-X compound to obtain compounds
of Formula 2-I-A. It will be understood that other natural or
non-natural amino acids described herein are also suitable for
attachment to the surfactant X and are suitable for attaching
additional amino acid/peptides to obtain compounds of Formula
2-I-A. It will be understood that in another embodiment, compounds
of Formula 2-I-A are prepared by attaching a full length or partial
length peptide to a group X, followed by optional attachment of
additional amino acid residues and/or peptides are attached to
obtain compounds of Formula 2-I-A.
[0460] In a specific embodiment, provided herein are compounds
selected from compounds of Table 2 in FIG. 2.
[0461] Also provided herein are pharmaceutical compositions
comprising a therapeutically effective amount of a peptide product
described above, or acceptable salt thereof, and at least one
pharmaceutically acceptable carrier or excipient.
[0462] In some embodiments of the pharmaceutical compositions, the
carrier is an aqueous-based carrier. In some embodiments of the
pharmaceutical compositions, the carrier is a nonaqueous-based
carrier. In some embodiments of the pharmaceutical compositions,
the nonaqueous-based carrier is a hydrofluoroalkane-like solvent
comprising sub-micron anhydrous .alpha.-lactose or other
excipients.
[0463] Contemplated within the scope of embodiments presented
herein is the reaction of an amino acid and/or a peptide comprising
a linker amino acid U bearing a nucleophile, and a group X
comprising a leaving group or a functional group that can be
activated to contain a leaving group, for example a carboxylic
acid, or any other reacting group, thereby allowing for covalent
linkage of the amino acid and/or peptide to a surfactant X via the
linker amino acid U to provide a peptide product of Formula
2-I-A.
[0464] Also contemplated within the scope of embodiments presented
herein is the reaction of an amino acid and/or a peptide comprising
a linker amino acid U bearing a leaving group or a functional group
that can be activated to contain a leaving group, for example a
carboxylic acid, or any other reacting group, and a group X
comprising a nucleophilic group, thereby allowing for covalent
linkage of the amino acid and/or peptide to a surfactant X via the
linker amino acid U to provide a peptide product of Formula
2-I-A.
[0465] It will be understood that, in one embodiment. Compounds of
Formula 2-I-A are prepared by reaction of a linker amino acid U
with X, followed by addition of further residues to U to obtain the
peptide product of Formula 2-I-A. It will be understood that in an
alternative embodiment, Compounds of Formula 2-I-A are prepared by
reaction of a suitable peptide comprising a linker amino acid U
with X, followed by optional addition of further residues to U, to
obtain the peptide product of Formula 2-I-A.
[0466] Provided herein are methods of treating hypoparathyroidism
comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide product described
above. In some embodiments, the hypoparathyroidism is associated
with bone mass reduction.
[0467] Also provided herein are methods of stimulating bone repair
or favoring the engraftment of a bone implant comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide product described above.
[0468] Also provided herein is a covalently modified PTH or PTHrP
peptide or analog thereof, comprising a hydrophilic group as
described herein; and a hydrophobic group covalently attached to
the hydrophilic group. In specific embodiments, the covalently
modified peptide and/or protein product comprises a hydrophilic
group that is a saccharide and a hydrophobic group that is a
C.sub.1-C.sub.20 alkyl chain or an aralkyl chain.
[0469] In one embodiment, provided is a method for chemically
modifying a molecule by covalent linkage to a surfactant to
increase or sustain the biological action of the composition or
molecule, for example, receptor binding or enzymatic activity. In
some embodiments, the molecule is a peptide. The method
additionally can include further modification comprising covalent
attachment of the molecule in the composition to a polymer such as
polyethylene glycol.
[0470] In another embodiment, provided is a method of reducing or
eliminating immunogenicity of a peptide and/or protein drug by
covalently linking the peptide chain to at least one alkyl
glycoside wherein the alkyl has from 1 to 30 carbon atoms.
[0471] Also provided is a method of treating hypoparathyroidism,
osteoporosis, osteopenia, post-menopausal osteoporosis, Paget's
disease, glucocorticoid induced osteoporosis, old age osteoporosis,
humoral hypercalcemia, or the like comprising administering a drug
composition comprising a peptide covalently linked to at least one
alkyl glycoside and delivered to a vertebrate, wherein the alkyl
has from 1 to 30 carbon atoms, or further in the range of 6 to 16
carbon atoms, and wherein covalent linkage of the alkyl glycoside
to the peptide increases the stability, bioavailability and/or
duration of action of the drug.
[0472] In some embodiments, the covalently modified peptides and/or
proteins are covalently modified PTH or PTHrP, or analogs thereof,
which are modified to improve their pharmaceutical and medical
properties by covalent modification with alkyl glycoside surfactant
moieties. These surfactant-modified analogs have increased steric
hindrance that hinder proteolysis, slows uptake and slows clearance
from the body.
[0473] Some studies show that approximately 50 percent of patients
suffering from osteoporosis discontinue oral bisphosphonate therapy
within the first year. Among patients who discontinue these
treatments, many do so because of side effects including
intolerance. Although truncated recombinant parathyroid hormone
1-34 (rhPTH1-34) is available commercially as a bone anabolic agent
(Brixen, K. T., et al. (2004) Basic Clin Pharmacol Toxicol 94:
260-270; Dobnig, H. (2004) Expert Opin Pharmacother 5: 1153-1162)
as teriparatide (Lilly), poor compliance is a major problem. More
recently an antibody (denosumab, Amgen) against the ligand
controlling osteoclast function has been approved, but it has
significant known side effects (serious skin infections, observed
cases of osteonecrosis of the jaw and significant suppression of
bone remodeling), some of which may have as yet unclear long-term
effects.
Bone Structure
[0474] The architecture of bone in man is maintained and elaborated
by the coordinated function of osteoclasts, which cause bone
resorption, and osteoblasts, which lay down new bone matrix. Bone
is an important depot for storage of calcium (a critical signaling
ion) in the body and a decrease in ambient extracellular Ca level
causes an increase in PTH secretion in via the Ca-sensing receptors
on the parathyroid cellular membrane. PTH binds to its receptor
(PTHR1), present on osteoblasts cell membranes, leading to
expression of the "ligand of receptor activator of nuclear
factor-KB" (RANKL). RANKL binds to its receptor, RANK, on
osteoclasts precursors, stimulating their differentiation and
proliferation (Boyce, B. F. and Xing, L. (2007) Arthritis Res Ther
9 Suppl 1: S1). This leads to bone resorption, mobilization of
calcium from the bone. PTH also acts to increase renal tubular Ca
reabsorption and indirectly to enhance intestinal Ca absorption via
its stimulatory action on renal 1-.alpha. cholecalciferol
hydroxylase (increasing circulating calcitriol). Both actions serve
to provide a longer term increase in circulating calcium ion.
[0475] The native form of human parathyroid hormone (hPTH) is an
84-amino acid peptide that plays an important role in the
maintenance of Calcium homeostasis in mammals (Rosen, C. J. and
Bilezikian, J. P. (2001) J Clin Endocrinol Metab 86: 957-964). A
structurally-related but independent hormone, parathyroid
hormone-related protein (PTHrP), plays a paracrine role, focused on
bone growth in local tissue. Both hormones bind to the same
receptor on osteoblasts, PTHR1, and cause activation of multiple
signaling pathways, including that regulated by increased cAMP
levels.
[0476] However intermittent presence of PTH(1-34) leads only to
stimulation of osteoblasts, lack of RANKL expression, and increased
bone density. PTH(1-34) exhibits potent anabolic effects on the
skeleton when given exogenously by intermittent injection. A small
group of patients received teriparatide by daily sc injections for
6-24 months (Reeve, J., et al. (1980) Br Med J 280: 1340-1344) and
paired bone biopsies revealed substantial increases in iliac
trabecular bone volume, with evidence of new bone formation and a
suggestion that there was a dissociation between bone formation and
resorption rates. Numerous studies have confirmed improvements in
bone tissue after daily injections of PTH analogs (Hodsman, A. B.,
et al. (2005) Endocr Rev 26: 688-703; Cheng, Z., et al. (2009) J
Bone Miner Res 24: 209-220). A review of the literature supports
the observation that architectural improvements do occur within the
skeleton after daily teriparatide injections, in contrast to the
skeletal architecture observed after therapy with antiresorptive
agents, which act mainly by inhibition of osteoblastic activity to
reduce bone turnover, thus preserving rather than building new
bone.
[0477] Continuous rather than intermittent administration of
exogenous PTH(1-34) results in bone absorption. Thus treatment with
infusions of PTH(1-34) for less than 6 hrs result in bone density
increases but infusion for over 8 hr or longer results in bone
resorption (Frolik, C. A., et al. (2003) Bone 33: 372-379). The
prolonged administration of PTH(1-34) causes the expression of
RANKL, activating RANK on osteoblast precursors, thus stimulating
their differentiation and proliferation (Boyce, B. F. and Xing, L.
(2007) Arthritis Res Ther 9 Suppl 1: S1). This observation has led
to the current treatment paradigm, once daily administration of
PTH(1-34) by subcutaneous injection. More recently, studies with
infusion of PTH(1-34) for one day and withdrawal for one week
showed important gains in bone density (Etoh, M. and Yamaguchi, A.
(2010) J Bone Miner Metab, 28: 641-9)). Teriparatide has a Cmax of
10 minutes and a half life of 19 minutes (Frolik, C. A., et al.
(2003) Bone 33: 372-379). A more efficacious PTH analog might be
expected to give more substantive bone density increase.
[0478] During toxicology studies with PTH(1-34), and with
PTH(1-84), it was observed that a substantial percentage of the
rats developed osteosarcomas, beginning at around 20 months
(Tashjian, A. H., Jr. and Goltzman, D. (2008) J Bone Miner Res 23:
803-811). No treatment-related sarcomas are reported in human
trials with recombinant PTH(1-34), teriparatide (Forteo.RTM.).
However, treatment with current therapy is limited to <2 yrs of
continuous daily subcutaneous injection.
PTH and PTHrP
[0479] In some embodiments, the methods and compositions described
herein comprise the use of PTH and/or PTHrP peptides and/or
proteins and/or analogs thereof. All of the biological activity of
intact human PTH (hPTH1-84) resides in the N-terminal sequence;
most clinical studies have used the 34-amino acid peptide
hPTH(1-34), known as teriparatide. The first two amino acids are
obligatory for biological activity, and it appears that the bone
anabolic properties are fully maintained by the foreshortened
fragment hPTH(1-31) or its cyclized lactam (Whitfield, J. F. and
Morley, P. (1995) Trends Pharmacol Sci 16: 382-386). More recently
studies of sequences as short as hPTH(1-11) have shown activity and
can be further modified to decrease their EC.sub.50 to the low nM
range (Shimizu, M., et al. (2000) J Biol Chem 275: 21836-21843;
Shimizu, N., et al. (2004) J Bone Miner Res 19: 2078-2086).
[0480] Studies of the interaction of the PTH and PTHrP with the
PTH1R have indicated that the ligands each have two binding
regions, one in the N-terminal 1-14 region and a second in the
C-terminal 15-34 region. The 1-14 portion has a more locally
ordered structure and interacts with the 7-transmembrane region of
the receptor while the 15-34 region is alpha helical and interacts
with the extracellular, N-terminal extension of the receptor. While
the N-terminal region of these peptides appears to have the primary
role of receptor activation through this juxtamembrane region
interaction, the C-terminal helical region has important binding
interactions (FIG. 2.) that give rise to higher potency of the
ligand (Gardella, T. J., et al. (1994) Endocrinology 135:
1186-1194; Luck, M. D., et al. (1999) Mol Endocrinol 13: 670-680)
through interaction with the extracellular region of the receptor
(Dean, T., et al. (2006) J Biol Chem 281: 32485-32495; Potetinova,
Z., et al. (2006) Biochemistry 45: 11113-11121). This leads to a
two domain model of binding that also has been extended to other
members of the family of class B GPCRs (Holtmann, M. H., et al.
(1995) J Biol Chem 270: 14394-14398; Bergwitz, C., et al. (1996) J
Biol Chem 271: 26469-26472; Runge, S., et al. (2003) J Biol Chem
278:128005-28010).
[0481] Accordingly, provided herein are covalently modified
peptides or peptide analogs that have a sequence that allows for
binding to PTH1R. Covalent modification of ligands in PTH/PTHrP
class relies on the development of N-terminal ligand sequences that
interact with the juxtamembrane portion of the PTHR1 coupled to a
C-terminal region that has been truncated and simplified through
the use of a surfactant moiety (e.g., a 1-alkyl-glucuronic acid
moiety such as the glucose-derived 1-alkylglucuronic acid). Since
much of the C-terminal region interaction involves general
hydrophobic interaction with the hydrophobic channel (Pioszak, A.
A. and Xu, H. E. (2008) Proc Natl Acad Sci USA 105: 5034-5039;
Pioszak, A. A., et al. (2009) J Biol Chem 284: 28382-28391) in the
extracellular region of the receptor, the covalent modifications
described herein allow for increased binding interaction through
low specificity interaction.
[0482] An example of the improved cellular stimulation by a
surfactant modified peptide described herein is illustrated in FIG.
4. Thus substantially greater cAMP output (125%; super-agonistic
stimulation) is shown by cells stimulated with doses of EU-232
(FIG. 5.) than by the internal standard, human PTHrP. Similarly, a
coded sample of human PTHrP (FIG. 6.; EU-285) achieves only 100% of
the maximal stimulation of the internal assay standard, human
PTHrP. EU-232 is modified with a 1-dodecyl .beta.-D-glucouronic
acid moiety in the C-terminal region. Importantly, shorter peptide
chains or peptide chains of this size unmodified with a 1-dodecyl
.beta.-D-glucouronic acid moiety can be expected to show decreased
efficacy.
[0483] Similarly, the in vivo response to this surfactant modified
analog EU-232 shows high potency and prolonged duration of action
(FIG. 7.). Blood phosphate levels were tested at various time
points after dosing rats with saline (G1), 80 micrograms per kg of
PTH (G2), 80 micrograms per kg of EU-232 (G3) or 320 micrograms per
kg of EU-232 (G4). EU-232 demonstrates prolonged duration of action
in that the maximal statistically significant effect is seen at the
last time point in the assay (5 hrs post dosing).
[0484] As shown in FIG. 7, Blood calcium levels were tested at
various time points after dosing rats with saline (G1), 80
micrograms per kg of PTH (G2), 80 micrograms per kg of EU-232 (G3)
or 320 micrograms per kg of EU-232 (G4). No groups were
statistically significantly different from control (G1).
Importantly, the maximally effective dose and time point for EU-232
(G4; at 5 hrs) shows no elevation and thus no indications of a
propensity for hypercalcemia at a maximally effective dose.
Hypercalcemia is an important side effect see following
administration of PTH 1-34 and of potent analogs of PTH.
[0485] The improvements, described above and in the figures,
associated with surfactant modification to yield the peptides
described herein have significant implications for their use in
medicine. Such molecules are suitable for use by once daily, or
less frequent, administration to give enhanced biological results
compared to treatment with short-acting native hormones such as PTH
(T& 30 min by s.c. injection) or PTHrP. Surfactant-modified
peptides such as EU-232 may be expected to show greater biological
effect when administered by intranasal insufflation due to the
well-known effects of surfactants on nasal bioavailability.
[0486] Accordingly, in some embodiments, surfactant-modified
peptide products described herein reduce the occurrence of
proteolytic degradation. In some embodiments, covalent modification
of PTH and/or PTHrP and/or analogs thereof, allows for decreased
cost of production of therapeutics, and provide favorable
pharmaceutical properties due to the presence of the covalently
attached surfactant moiety. In some embodiments, surfactant
modified PTH and/or PTHrP described herein prolong the PK and
duration of action (PD) behavior of the resulting ligands compared
to other known peptide ligands (such as those of Dean, et al.
(Dean, T., et al. (2006) J Biol Chem 281: 32485-32495)) that lack
such covalent modifications. Also contemplated within the scope of
embodiments presented herein is long term and safe administration
of surfactant modified PTH and/or PTHrP and/or analogs thereof.
[0487] In some instances, the N-terminal binding region ends at
about residue 14 and the helical region encompasses residue 16
onward. Thus a ligand optimized in the 1-14 region with a
1-alkylglucuronic acid modification in the 15 onward region will
have high specific binding (N-terminus) with high potency (1-alkyl
modification). Use of an .alpha.-helical stabilizing substitution
(Kaul, R. and Balaram, P. (1999) Bioorg Med Chem 7: 105-117) in the
C-terminal region leads to higher helical content and higher
potency. Commonly used .alpha.-helical stabilizers are Ala and the
class of 1,1-dialkyl amino acids such as Aib, Ac4c, Ac5c and the
like (see definitions below). Minimization of the PTH structure led
to shortened analogs (Shimizu, M., et al. (2000) J Biol Chem 275:
21836-21843) wherein constrained .alpha.-helical stabilizers led to
important potency increases. For example, substitution of Aib into
position 1 and 3 of PTH1-14 and PTH1-11 analogs led to increased
potency (Shimizu, N., et al. (2001) J Biol Chem 276: 49003-49012).
Incorporation of more hindered .alpha.-helical stabilizers in
position 1 lead to further potency increases (Shimizu, N., et al.
(2004) J Bone Miner Res 19: 2078-2086). Substitution of Aib into
various positions of PTH1-34 analogs also led to improvements in
potency, particularly substitutions at positions 12 and 13
(Peggion, E., et al. (2003) Biopolymers 68: 437-457). However it
was shown that Aib in positions 1 and 3 of the simple PTH1-11
sequence was not acceptable (Barazza, A., et al. (2005) J Pept Res
65: 23-35). Thus, in some embodiments, the .alpha.-helical content
of PTH and/or PTHrP is a determinant of peptide product stability
(Marx, U. C., et al. (1995) J Biol Chem 270: 15194-15202;
Schievano, E., et al. (2000) Biopolymers 54: 429-447).
[0488] In some embodiments, the use of a long side chain, such as
in the N.epsilon.-(1'-dodecyl beta-D-glucuronyl)-lysine in covalent
peptide modifications described herein, destabilizes an
.alpha.-helix. Accordingly, also contemplated within the scope of
embodiments presented herein are modifications that comprise
.alpha.-helical stabilizers. Thus in some embodiments, surfactant
modified peptide products described herein comprise a helix
stabilizer (e.g., in the position just N-Terminal and/or just
C-terminal of the surfactant substitution). In some embodiments, an
.alpha.-helix stabilizer is located at position 12 in the PTH
and/or PTHrP chain. By way of example only, the table below
describes EU-212 to EU-282 certain surfactant modified peptide
products (EU-212 to EU-282) that comprise an .alpha.-helix
stabilizer.
[0489] In one aspect, the peptides that are covalently modified and
are suitable for methods described herein are truncated analogs of
PTHrP and/or the related hormone PTH, including and not limited
to:
TABLE-US-00020 (SEQ. ID. NO. 290) hPTH(1-34):
Ser.sub.1-Val.sub.2-Ser.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-
Met.sub.8-His.sub.9-Asn.sub.10-Leu.sub.11-Gly.sub.12-Lys.sub.13-His.sub.14-
-Leu.sub.15-
Asn.sub.16-Ser.sub.17-Met.sub.18-Glu.sub.19-Arg.sub.20-Val.sub.21-Glu.sub.-
22-Trp.sub.23-
Leu.sub.24-Arg.sub.25-Lys.sub.26-Lys.sub.27-Leu.sub.28-Gln.sub.29-Asp.sub.-
30-Val.sub.31- His.sub.32-Asn.sub.33-Phe.sub.34-OH or (SEQ. ID. NO.
291) hPTHrP(1-34):
Ala.sub.1-Val.sub.2-Ser.sub.3-Glu.sub.4-His.sub.5-Gln.sub.6-Leu.sub.7-
Leu.sub.8-His.sub.9-Asp.sub.10-Lys.sub.11-Gly.sub.12-Lys.sub.13-Ser.sub.14-
-Leu.sub.15-
Gln.sub.16-Asp.sub.17-Leu.sub.18-Arg.sub.19-Arg.sub.20-Arg.sub.21-Phe.sub.-
22-Phe.sub.23-
Leu.sub.24-His.sub.25-His.sub.26-Leu.sub.27-Ile.sub.28-Ala.sub.29-Glu.sub.-
30-Ile.sub.31- His.sub.32-Thr.sub.33-Ala.sub.34-OH
[0490] In some embodiments, a peptide product described herein has
the structure of Formula 2-V:
TABLE-US-00021 FORMULA 2-V (SEQ. ID. NO. 172)
aa.sub.1-aa.sub.2-aa.sub.3-aa.sub.4-aa.sub.5
aa.sub.6-aa.sub.7-aa.sub.8-aa.sub.9-aa.sub.10-aa.sub.11-aa.sub.12-aa.sub.-
13-
aa.sub.14-aa.sub.15-aa.sub.16-aa.sub.17-aa.sub.18-aa.sub.19-aa.sub.20-aa.s-
ub.21-aa.sub.22-aa.sub.23-aa.sub.24-
aa.sub.25-aa.sub.26-aa.sub.27-aa.sub.28-aa.sub.29-aa.sub.30-aa.sub.31-aa.s-
ub.32-aa.sub.33-aa.sub.34-aa.sub.35- aa.sub.36-Z
wherein: [0491] Z is OH, or --NH--R.sup.3, wherein R.sup.3 is H or
C.sub.1-C.sub.12 alkyl; or a PEG chain of less than 10 Da. [0492]
aa.sub.1 is Ala, Ser, Val, Pro, Aib, Ac5c, or Deg; [0493] aa.sub.2
is Val; [0494] aa.sub.3 is Ser, Ala, Aib, Ac4c, or Deg; [0495]
aa.sub.4 is Glu; [0496] aa.sub.5 is His, or Ile; [0497] aa.sub.6 is
Gln, or Cit; [0498] aa.sub.7 is Leu, or Phe; [0499] aa.sub.8 is
Leu, Met, or Nle; [0500] aa.sub.9 is His; [0501] aa.sub.10 is Asp,
Asn, Gln, Glu, Cit, Ala, or Aib; [0502] aa.sub.11 is Lys, Leu, Ile,
Arg, or hArg; [0503] aa.sub.12 is Gly, Ala, Glu, Lys, Aib, or Ac5c;
[0504] aa.sub.13 is Lys, or Arg; [0505] aa.sub.14 is Ser, His, Trp,
Phe, Leu, Arg, Lys, Glu, Nal(2), or cyclized to position aa.sub.18;
[0506] aa.sub.15 is Ile, Leu, Aib; [0507] aa.sub.16 is Gln, Asn,
Glu, Lys, Ser, Cit, Aib, Ac5c, U(X); [0508] aa.sub.17 is Asp, Ser,
Aib, Ac4c, Ac5c, U(X), Z; [0509] aa.sub.18 is absent, Leu, Gln,
Cit, Aib, Ac5c, Lys, Glu, or U(X), or cyclized to position
aa.sub.14; [0510] aa.sub.19 is absent, Arg, Glu, Aib, Ac4c, Ac5c,
or U(X); [0511] aa.sub.20 is absent, Arg, Glu, Lys, Aib, Ac4c,
Ac5c, or U(X); [0512] aa.sub.21 is absent, Arg, Val, Aib, Ac5c,
Deg, or U(X); [0513] aa.sub.22 is absent, Phe, Glu, Aib, Ac5c, Lys,
U(X), or cyclized to position aa.sub.18 or aa.sub.26; [0514]
aa.sub.23 is absent, or Leu, Phe, Trp, or U(X); [0515] aa.sub.24 is
absent, His, Arg, Leu, Aib, Ac5c or U(X); [0516] aa.sub.25 is
absent, His, Lys, Arg, or U(X); [0517] aa.sub.26 is absent, His,
Lys, Arg, Aib, Ac5c or cyclized to position aa.sub.22; [0518]
aa.sub.27 is absent, Leu, or Lys; [0519] aa.sub.28 is absent, Ile,
or Leu; [0520] aa.sub.29 is absent, Ala, Gln, Cit, or Aib; [0521]
aa.sub.30 is absent, Glu, Asp, or Aib; [0522] aa.sub.31 is absent,
Ile, Val, Aib, Ac5C, or U(X); [0523] aa.sub.32 is absent, His, Aib,
Ac5C, or U(X); [0524] aa.sub.33 is absent, Thr, Asn, Aib, Ac5C, or
U(X); [0525] aa.sub.34 is absent, Ala, Phe, Aib, Ac5C, or U(X);
[0526] aa.sub.35 is absent, Aib, Ac5C, or U(X); [0527] aa.sub.36 is
absent, Aib, Ac5C, or U(X); [0528] U is a linking amino acid; and
[0529] X is a surfactant-linked to the side chain of U; [0530]
wherein any two of aa.sub.1-aa.sub.36 are optionally cyclized
through their side chains to form a lactam linkage; and provided
that one, or least one of aa.sub.1-aa.sub.36 is U.
[0531] In some embodiments, a peptide product described herein
comprises aa.sub.1-aa.sub.20 of Formula V as described above (SEQ.
ID. NO. 292). In some embodiments, a peptide product described
herein comprises aa.sub.1-aa.sub.19 of Formula V as described above
(SEQ. ID. NO. 293).
[0532] In specific embodiments, the linking amino acid U, is a
diamino acid like Lys or Orn, X is a modified surfactant from the
1-alkyl glycoside class linked to U, and Z is OH, or --NH--R.sup.3,
wherein R.sup.3 is H or C.sub.1-C.sub.12; or a PEG chain of less
than 10 Da.
[0533] In some embodiments, a peptide product described herein has
the structure of Formula 2-VI:
TABLE-US-00022 Formula 2-VI (SEQ. ID. NO. 173)
aa.sub.1-Val.sub.2-aa.sub.3-Glu.sub.4-aa.sub.5
aa.sub.6-aa.sub.7-aa.sub.8-His.sub.9-aa.sub.10-aa.sub.11-aa.sub.12-
aa.sub.13-aa.sub.14-aa.sub.15-aa.sub.16-aa.sub.17-aa.sub.18-aa.sub.19-aa.s-
ub.20-aa.sub.21-aa.sub.22-aa.sub.23-
aa.sub.24-aa.sub.25-aa.sub.26-Z
wherein: [0534] Z is OH, or --NH--R.sup.3, wherein R.sup.3 is H,
C.sub.1-C.sub.12 alkyl or a PEG chain of less than 10 Da; [0535]
aa.sub.1 is Aib, Ac5c, Deg; [0536] aa.sub.3 is Aib, Ac4c, Deg;
[0537] aa.sub.5 is His, Ile; [0538] aa.sub.6 is Gln, Cit; [0539]
aa.sub.7 is Leu, Phe; [0540] aa.sub.8 is Leu, Nle; [0541] aa.sub.10
is Asp, Asn, Gln, Glu, Cit, Ala, Aib; [0542] aa.sub.11 is Arg,
hArg; [0543] aa.sub.12 is Gly, Ala, Glu, Lys, Aib, Ac5c; [0544]
aa.sub.13 is Lys, Arg; [0545] aa.sub.14 is Ser, His, Trp, Phe, Leu,
Arg, Lys, Nal(2); [0546] aa.sub.15 is Ile, Leu, Aib; [0547]
aa.sub.16 is Gln, Asn, Glu, Lys, Ser, Cit, Aib, U(X); [0548]
aa.sub.17 is Asp, Ser, Aib, Ac4c, Ac5c, U(X); [0549] aa.sub.18 is
absent or Leu, Gln, Cit, Aib, Ac5c, Lys, Glu, U(X); [0550]
aa.sub.19 is absent or Arg, Glu, Aib, Ac4c, Ac5c, U(X); [0551]
aa.sub.20 is absent or Arg, Glu, Lys, Aib, Ac4c, Ac5c, U(X); [0552]
aa.sub.21 is absent or Arg, Val, Aib, Ac5C, Deg U(X); [0553]
aa.sub.22 is absent or Phe, Glu, Lys or U(X); [0554] aa.sub.23 is
absent or Leu, Phe, Trp or U(X); [0555] aa.sub.24 is absent or Leu,
His, Arg, or U(X); [0556] aa.sub.25 is absent or His, Lys, or U(X)
and [0557] aa.sub.26 is absent or Aib, Ac5c; [0558] U is a linking
amino acid; [0559] X is a modified surfactant from the 1-alkyl
glycoside class linked to U, wherein the 1-alkyl group is
substituted or unsubstituted C.sub.1-C.sub.20 alkyl or substituted
or unsubstituted C.sub.1-C.sub.20 aralkyl; provided that one, or
least one of aa.sub.1-aa.sub.26 is U.
[0560] In some embodiments, a peptide product described herein has
the structure of Formula 2-VII
TABLE-US-00023 Formula 2-VII (SEQ. ID. NO. 174)
aa.sub.1-Val.sub.2-Aib.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nle.s-
ub.8-His.sub.9-Gln.sub.10-
hArg.sub.11-aa.sub.12-Arg.sub.13-aa.sub.14-Ile.sub.15-aa.sub.16-aa.sub.17--
aa.sub.18-aa.sub.19-aa.sub.20-
aa.sub.21-aa.sub.22-aa.sub.23-aa.sub.24-aa.sub.25-aa.sub.26-Z
wherein: [0561] Z is OH or --NH.sub.2; [0562] aa.sub.1 is Aib,
Ac5c; [0563] aa.sub.12 is Ala, Glu, Lys, Aib, Ac5c; [0564]
aa.sub.14 is Trp, Phe, Nal(2); [0565] aa.sub.16 is Gln, Asn, Glu,
Lys, Cit, U(X); [0566] aa.sub.17 is Asp, Ser, Aib, Ac4c, Ac5c,
U(X); [0567] aa.sub.18 is absent or Leu, Gln, Aib, U(X); [0568]
aa.sub.19 is absent or Arg, Glu, Aib, Ac4c, Ac5c; [0569] aa.sub.20
is absent or Arg, Glu, Lys, Aib, Ac4c, Ac5c; [0570] aa.sub.21 is
absent or Arg, Val, Aib, Ac5C, Deg; [0571] aa.sub.22 is absent or
Phe, Glu, Lys or U(X); [0572] aa.sub.23 is absent or Leu, Phe, Trp
or U(X); [0573] aa.sub.24 is absent or His, Arg, or U; [0574]
aa.sub.25 is absent or His, Lys, or U and [0575] aa.sub.26 is
absent or Aib, Ac5c; [0576] U is a linking amino acid; and [0577] X
is a modified surfactant from the 1-alkyl glycoside class linked to
U, wherein the 1-alkyl group is substituted or unsubstituted
C.sub.1-C.sub.20 alkyl or substituted or unsubstituted
C.sub.1-C.sub.20 aralkyl; provided that one, or least one of
aa.sub.1-aa.sub.26 is U.
[0578] In some embodiments, the peptide product has the structure
of Formula 2-III:
TABLE-US-00024 Formula 2-III (SEQ. ID. NO. 171)
aa.sub.1-Val.sub.2-Aib.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nle.s-
ub.8-His.sub.9-Gln.sub.10-
hArg.sub.11-aa.sub.12-Arg.sub.13-aa.sub.14-Ile.sub.15-aa.sub.16-aa.sub.17--
aa.sub.18-aa.sub.19-aa.sub.20-
aa.sub.21-aa.sub.22-aa.sub.23-aa.sub.24-aa.sub.25-aa.sub.26-Z
wherein: [0579] Z is OH or --NH.sub.2; [0580] aa.sub.1 is Aib, or
Ac5c; [0581] aa.sub.12 is Ala, Aib, Glu, Lys, or Ac5c; [0582]
aa.sub.14 is Trp, Phe, Lys, Glu or Nal(2); [0583] aa.sub.16 is Gln,
Asn, Glu, Lys, Cit, or U(X); [0584] aa.sub.17 is Asp, Ser, Aib,
Ac4c, Ac5c, or U(X); [0585] aa.sub.18 is absent or Leu, Gln, Aib,
Lys, Glu or U(X); [0586] aa.sub.19 is absent or Arg, Glu, Aib,
Ac4c, or Ac5c; [0587] aa.sub.20 is absent or Arg, Glu, Lys, Aib,
Ac4c, Ac5c; [0588] aa.sub.21 is absent or Arg, Val, Aib, Ac5C, or
Deg; [0589] aa.sub.22 is absent or Phe, Glu, Lys or U(X); [0590]
aa.sub.23 is absent or Leu, Phe, Trp or U(X); [0591] aa.sub.24 is
absent or His, Arg, or U(X); [0592] aa.sub.25 is absent or His,
Lys, or U(X); and [0593] aa.sub.26 is absent or Aib, Ac5c; [0594]
wherein any two of aa.sub.1-aa.sub.26 are optionally cyclized
through their side chains to form a lactam linkage; and [0595]
provided that one, or at least one of aa.sub.16, aa.sub.17,
aa.sub.18, aa.sub.22, aa.sub.23, aa.sub.24 or aa.sub.25 is the
linker amino acid U covalently attached to X.
[0596] In a specific embodiment of Formula 2-III above, X has the
structure:
##STR00030## [0597] wherein: [0598] R.sup.1a is a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl group; [0599] R.sup.1b,
R.sup.1c, and R.sup.1d are H; [0600] W.sup.1 is --(C.dbd.O)--NH--;
[0601] W.sup.2 is --O--; and [0602] R.sup.2 is a bond.
[0603] In some of the embodiments described above, R.sup.1a is a
C.sub.1-C.sub.2 alkyl group, a C.sub.1-C.sub.18 alkyl group, a
C.sub.1-C.sub.16 alkyl group, or C.sub.1-C.sub.12 alkyl group. In
some embodiments of Formula 2-III, U is any linker amino acid
described herein.
[0604] Contemplated within the scope of embodiments presented
herein are peptide products of Formula 2-I-A, Formula 2-III,
Formula 2-V, Formula 2-VI or Formula 2-VI, wherein the peptide
product comprises one, or, more than one surfactant groups (e.g.,
group X having the structure of Formula I). In one embodiment, a
peptide product of Formula 2-I-A, Formula 2-III, Formula 2-V,
Formula 2-VI or Formula 2-VII comprises one surfactant group. In
another embodiment, a peptide product of Formula 2-I-A, Formula
2-III, Formula 2-V, Formula 2-VI or Formula 2-VI comprises two
surfactant groups. In yet another embodiment, a peptide product of
Formula 2-I-A, Formula 2-III, Formula 2-V, Formula 2-VI or Formula
2-VII comprises three surfactant groups.
[0605] Table 2 in FIG. 2 illustrates certain examples of peptides
that are suitable for covalent linkage with surfactants as
described herein.
[0606] Recognized herein is the importance of certain portions of
SEQ. ID. NO. 170 for the treatment of conditions associated with
bone loss and/or hyperparathyroidism including, and not limited to,
osteoporosis, osteopenia, post-menopausal osteoporosis, Paget's
disease, glucocorticoid-induced osteoporosis, inflammatory bone
loss, fixation of implants, osteonecrosis of the jaw, stem cell
proliferation, old age osteoporosis, humoral hypercalcemia, or the
like.
[0607] Accordingly, provided herein is a method of treating
conditions associated with bone loss (e.g., osteoporosis) and/or
hyperparathyroidism in an individual in need thereof comprising
administration of a therapeutically effective amount of a PTH
analog comprising amino acid residues aa.sub.1-aa.sub.17 of SEQ.
ID. NO. 170 to the individual in need thereof.
[0608] In a further embodiment, provided herein is a method of
treating conditions associated with bone loss (e.g., osteoporosis)
and/or hyperparathyroidism in an individual in need thereof
comprising administration of a therapeutically effective amount of
a PTH analog comprising amino acid residues aa.sub.1-aa.sub.18 of
SEQ. ID. NO. 170 to the individual in need thereof.
[0609] In another embodiment, provided herein is a method of
treating conditions associated with bone loss (e.g., osteoporosis)
and/or hyperparathyroidism in an individual in need thereof
comprising administration of a therapeutically effective amount of
a PTH analog comprising amino acid residues aa.sub.1-aa.sub.1 of
SEQ. ID. NO. 170 to the individual in need thereof.
[0610] In another embodiment, provided herein is a method of
treating conditions associated with bone loss (e.g., osteoporosis)
and/or hyperparathyroidism in an individual in need thereof
comprising administration of a therapeutically effective amount of
a PTH analog comprising amino acid residues aa.sub.1-aa.sub.20 of
SEQ. ID. NO. 170 to the individual in need thereof.
[0611] In an additional embodiment, the administration of the said
PTH analog described above causes increase in bone density.
[0612] Recognized herein is the importance of certain portions of
SEQ. ID. NOs. 171, 173, 174, 645 or 646 for the treatment of
conditions associated with bone loss and/or hyperparathyroidism,
including, and not limited to, osteoporosis, osteopenia,
post-menopausal osteoporosis, Paget's disease,
glucocorticoid-induced osteoporosis, inflammatory bone loss,
fixation of implants, osteonecrosis of the jaw, stem cell
proliferation, old age osteoporosis, humoral hypercalcemia, or the
like.
[0613] Accordingly, provided herein is a method of treating
conditions associated with bone loss and/or hyperparathyroidism in
an individual in need thereof comprising administration of a
therapeutically effective amount of a PTH analog comprising amino
acid residues aa.sub.1-aa.sub.17 of SEQ. ID. NOs. 171, 173, 174,
290 or 291 to the individual in need thereof.
[0614] In a further embodiment, provided herein is a method of
treating conditions associated with bone loss (e.g., osteoporosis)
and/or hyperparathyroidism in an individual in need thereof
comprising administration of a therapeutically effective amount of
a PTH analog comprising amino acid residues aa.sub.1-aa.sub.18 of
SEQ. ID. NOs. 171, 173, 174, 290 or 291 to the individual in need
thereof.
[0615] In another embodiment, provided herein is a method of
treating conditions associated with bone loss (e.g., osteoporosis)
and/or hyperparathyroidism in an individual in need thereof
comprising administration of a therapeutically effective amount of
a PTH analog comprising amino acid residues aa.sub.1-aa.sub.19 of
SEQ. ID. NOs. 171, 173, 174, 290 or 291 to the individual in need
thereof.
[0616] In another embodiment, provided herein is a method of
treating conditions associated with bone loss (e.g., osteoporosis)
and/or hyperparathyroidism in an individual in need thereof
comprising administration of a therapeutically effective amount of
a PTH analog comprising amino acid residues aa.sub.1-aa.sub.2 of
SEQ. ID. NOs. 171, 173, 174, 290 or 291 to the individual in need
thereof.
[0617] In an additional embodiment, the administration of the said
PTH analog described above causes increase in bone density.
[0618] In any of the embodiments described above, the said PTH
analog is modified with a surfactant X of Formula 2-I:
##STR00031## [0619] wherein: [0620] R.sup.1a is independently, at
each occurrence, a bond, H, a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl group, a substituted or unsubstituted
alkoxyaryl group, a substituted or unsubstituted aralkyl group, or
a steroid nucleus containing moiety; [0621] R.sup.1b, R.sup.1c, and
R.sup.1d are each, independently at each occurrence, a bond, H, a
substituted or unsubstituted C.sub.1-C.sub.30 alkyl group, a
substituted or unsubstituted alkoxyaryl group, or a substituted or
unsubstituted aralkyl group; [0622] W.sup.1 is independently, at
each occurrence, --CH.sub.2--, --CH.sub.2--O--, --(C.dbd.O),
--(C.dbd.O)--O--, --(C.dbd.O)--NH--, --(C.dbd.S)--,
--(C.dbd.S)--NH--, or --CH.sub.2--S--; [0623] W.sup.2 is --O--,
--CH.sub.2-- or --S--; [0624] R.sup.2 is independently, at each
occurrence, a bond to U, H, a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl group, a substituted or unsubstituted
alkoxyaryl group, or a substituted or unsubstituted aralkyl group,
--NH.sub.2, --SH, C.sub.2-C.sub.4-alkene, C.sub.2-C.sub.4-alkyne,
--NH(C.dbd.O)--CH.sub.2--Br, --(CH.sub.2).sub.m-maleimide, or
--N.sub.3; [0625] n is 1, 2 or 3; and [0626] m is 1-10.
[0627] In a specific embodiment, the said PTH analog is modified
with a surfactant, X having the structure:
##STR00032## [0628] wherein: [0629] R.sup.1a is a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl group; [0630] R.sup.1b,
R.sup.1c, and R.sup.1d are H; [0631] W.sup.1 is --(C.dbd.O)--NH--;
[0632] W.sup.2 is --O--; and [0633] R.sup.2 is a bond.
[0634] In some of the embodiments described above, R.sup.1a is a
C.sub.1-C.sub.20 alkyl group, a C.sub.8-C.sub.20 alkyl group,
C.sub.12-C.sub.18 alkyl group or C.sub.14-C.sub.18 alkyl group.
[0635] Modifications at the amino or carboxyl terminus may
optionally be introduced into the peptides (e.g., PTH or PTHrP)
(Nestor, J. J., Jr. (2009) Current Medicinal Chemistry 16:
4399-4418). For example, the peptides can be truncated or acylated
on the N-terminus to yield peptides analogs exhibiting low
efficacy, partial agonist and antagonist activity, as has been seen
for some peptides (Gourlet, P., et al. (1998) Eur J Pharmacol 354:
105-111, Gozes, I. and Furman, S. (2003) Curr Pharm Des 9:
483-494), the contents of which is incorporated herein by
reference). For example, deletion of the first 6 residues of bPTH
yields antagonistic analogs (Mahaffey, J. E., et al. (1979) J Biol
Chem 254: 6496-6498; Goldman, M. E., et al. (1988) Endocrinology
123: 2597-2599) and a similar operation on peptides described
herein generates potent antagonistic analogs. Other modifications
to the N-terminus of peptides, such as deletions or incorporation
of D-amino acids such as D-Phe also can give potent and long acting
agonists or antagonists when substituted with the modifications
described herein such as long chain alkyl glycosides. Such agonists
and antagonists also have commercial utility and are within the
scope of contemplated embodiments described herein.
[0636] Also contemplated within the scope of embodiments presented
herein is N-terminal truncation of PTH (e.g. 7-34 residue analogs)
or PTHrP thereby providing inverse agonists (Gardella, T. J., et
al. (1996) Endocrinology 137: 3936-3941) or antagonists. In some
embodiments, inverse agonists and/or antagonists of PTH and/or
PTHrP are useful for treatment of "humoral hypercalcemia"
associated with a wide range of tumors.
[0637] Also contemplated within the scope of embodiments described
herein are surfactants covalently attached to peptide analogs,
wherein the native peptide is modified by acetylation, acylation,
PEGylation. ADP-ribosylation, amidation, covalent attachment of a
lipid or lipid derivative, covalent attachment of
phosphotidylinositol, cross-linking, cyclization, disulfide bond
formation, demethylation, formation of covalent cross-link
formation of cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation, racemization,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins, such as arginylation, and ubiquitination. See,
for instance, (Nestor, J. J., Jr. (2007) Comprehensive Medicinal
Chemistry II 2: 573-601, Nestor, J. J., Jr. (2009) Current
Medicinal Chemistry 16: 4399-4418, Creighton, T. E. (1993, Wold, F.
(1983) Posttranslational Covalent Modification of Proteins 1-12.
Seifter, S. and Englard, S. (1990) Methods Enzymol 182: 626-646.
Rattan, S. I., et al. (1992) Ann N Y Acad Sci 663: 48-62).
[0638] Also contemplated within the scope of embodiments described
herein are peptides that are branched or cyclic, with or without
branching. Cyclic, branched and branched circular peptides result
from post-translational natural processes and are also made by
suitable synthetic methods. In some embodiments, any peptide
product described herein comprises a peptide analog described above
that is then covalently attached to an alkyl-glycoside surfactant
moiety.
[0639] Also contemplated within the scope of embodiments presented
herein are peptide chains that are substituted in a suitable
position by the modification of the analogs claimed herein, e.g.,
by acylation on a linker amino acid, at for example the
.epsilon.-position of Lys, with fatty acids such as octanoic,
decanoic, dodecanoic, tetradecanoic, hexadecanoic, octadecanoic,
3-phenylpropanoic acids and the like, or with saturated or
unsaturated alkyl chains (Zhang, L. and Bulaj, G. (2012) Curr Med
Chem 19: 1602-1618). Non-limiting, illustrative examples of such
analogs are:
TABLE-US-00025 (SEQ. ID. NO. 294)
Ac5c.sub.1-Val.sub.2-Aib.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nle-
.sub.8-His.sub.9-Gln.sub.10-
hArg.sub.11-Ala.sub.12-Arg.sub.13-Trp.sub.14-Ile.sub.15-Gln.sub.16-Aib.sub-
.17-Lys (N-epsilon-dodecanoyl).sub.18-Aib.sub.19-NH.sub.2, (SEQ.
ID. NO. 295)
Ac5c.sub.1-Val.sub.2-Aib.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nle-
.sub.8-His.sub.9-Gln.sub.10-
hArg.sub.11-Ala.sub.12-Arg.sub.13-Trp.sub.14-Ile.sub.15-Gln.sub.16-Aib.sub-
.17-Lys (N-epsilon-dodecanoyl).sub.18-NH.sub.2, (SEQ. ID. NO. 296)
Ac5c.sub.1-Val.sub.2-Aib.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nle-
.sub.8-His.sub.9-Gln.sub.10-
hArg.sub.11-Ala.sub.12-Arg.sub.13-Trp.sub.14-Ile.sub.15-Gln.sub.16-Aib.sub-
.17-Lys (N-epsilon-palmitoyl).sub.18-Aib.sub.19-NH.sub.2, (SEQ. ID.
NO. 297)
Ac5c.sub.1-Val.sub.2-Aib.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nle-
.sub.8-His.sub.9-Gln.sub.10-
hArg.sub.11-Ala.sub.12-Arg.sub.13-Trp.sub.14-Ile.sub.15-Gln.sub.16-Aib.sub-
.17-Lys (N-epsilon-dodecanoyl).sub.18-Aib.sub.19-NH.sub.2, (SEQ.
ID. NO. 298)
Ac5c.sub.1-Val.sub.2-Aib.sub.3-Gln.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nle-
.sub.8-His.sub.9-Gln.sub.10-
hArg.sub.11-Ala.sub.12-Arg.sub.13-Trp.sub.14-Ile.sub.15-Gln.sub.16-Aib.sub-
.17-Lys (N-epsilon-tetradecanoyl).sub.18-Aib.sub.19-NH.sub.2, (SEQ.
ID. NO. 299)
Ac5c.sub.1-Val.sub.2-Aib.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nle-
.sub.8-His.sub.9-Gln.sub.10-
hArg.sub.11-Ala.sub.12-Arg.sub.13-Trp.sub.14-Ile.sub.15-Gln.sub.16-Aib.sub-
.17-Lys
(N-epsilon-tetradecanoyl).sub.18-Aib.sub.19-Aib.sub.20-NH.sub.2,
(SEQ. ID. NO. 300)
Ac5c.sub.1-Val.sub.2-Aib.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nle-
.sub.8-His.sub.9-Gln.sub.10-
hArg.sub.11-Ala.sub.12-Arg.sub.13-Trp.sub.14-Ile.sub.15-Gln.sub.16-Aib.sub-
.17-Lys
(N-epsilon-(Nalpha-dodecanoyl-L-glutamyl)).sub.18-Aib.sub.19-
NH.sub.2, and the like.
[0640] In other embodiments, a peptide chain is substituted in a
suitable position by reaction on a linker amino acid, for example
the sulfhydryl of Cys, with a spacer and a hydrophobic moiety such
as a steroid nucleus, for example a cholesterol moiety. In some of
such embodiments, the modified peptide further comprises one or
more PEG chains. Non-limiting examples of such molecules are:
TABLE-US-00026 (SEQ. ID. NO. 301)
Ac5c.sub.1-Val.sub.2-Aib.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nle-
.sub.8-His.sub.9-Gln.sub.10-
hArg.sub.11-Ala.sub.12-Arg.sub.13-Trp.sub.14-Ile.sub.15-Gln.sub.16-Aib.sub-
.17-Cys(S- (3-(PEG4-aminoethylacetamide-Cholesterol))).sub.18-
Aib.sub.19-NH.sub.2, (SEQ. ID. NO. 302)
Ac5c.sub.1-Val.sub.2-Aib.sub.3-Glu.sub.4-Ile.sub.5-Gln.sub.6-Leu.sub.7-Nle-
.sub.8-His.sub.9-Gln.sub.10-
hArg.sub.11-Ala.sub.12-Arg.sub.13-Trp.sub.14-Ile.sub.15-Gln.sub.16-Aib.sub-
.17-Cys(S-
(3-(PEG4-aminoethylacetamide-Cholesterol))).sub.18-NH.sub.2, and
the like.
GLP Peptides and Analogs
[0641] Also provided herein, in some embodiments, are reagents and
intermediates for synthesis of modified peptides and/or proteins
(e.g., modified GLP-1, glucagon, analogs of glucagon or GLP-1, or
the like) through the incorporation of surfactants.
[0642] Provided herein, in some embodiments, are peptide products
comprising a surfactant X, covalently attached to a peptide, the
peptide comprising a linker amino acid U and at least one other
amino acid:
##STR00033## [0643] wherein the surfactant X is a group of Formula
I:
[0643] ##STR00034## [0644] wherein: [0645] R.sup.1a is
independently, at each occurrence, a bond, H, a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl group, a substituted or
unsubstituted alkoxyaryl group, or a substituted or unsubstituted
aralkyl group; [0646] R.sup.1b, R.sup.1c, and R.sup.1d are each,
independently at each occurrence, a bond, H, a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl group, a substituted or
unsubstituted alkoxyaryl group, or a substituted or unsubstituted
aralkyl group; [0647] W.sup.1 is independently, at each occurrence,
--CH.sub.2--, --CH.sub.2--O--, --(C.dbd.O), --(C.dbd.O)--O--,
--(C.dbd.O)--NH--, --(C.dbd.S)--, --(C.dbd.S)--NH--, or
--CH.sub.2--S--; [0648] W.sup.2 is --O--, --CH.sub.2-- or --S--;
[0649] R.sup.2 is independently, at each occurrence, a bond, H, a
substituted or unsubstituted C.sub.1-C.sub.30 alkyl group, a
substituted or unsubstituted alkoxyaryl group, or a substituted or
unsubstituted aralkyl group, --NH.sub.2, --SH,
C.sub.2-C.sub.4-alkene, C.sub.2-C.sub.4-alkyne,
--NH(C.dbd.O)--CH.sub.2--Br, --(CH.sub.2).sub.m-maleimide, or
--N.sub.3. [0650] n is 1, 2 or 3; and [0651] m is 1-10; [0652] the
peptide is selected from Formula 3-11:
TABLE-US-00027 [0652] Formula 3-II (SEQ. ID. NO. 303)
aa.sub.1-aa.sub.2-aa3-aa.sub.4-aa.sub.5-aa.sub.6-aa.sub.7-aa.sub.8-aa.sub.-
9-aa.sub.10-aa.sub.11-aa.sub.12-aa.sub.13-
aa.sub.14-aa.sub.15-aa.sub.16-aa.sub.17-aa.sub.18-aa.sub.19-aa.sub.20-aa.s-
ub.21-aa.sub.22-aa.sub.23-aa.sub.24-
aa.sub.25-aa.sub.26-aa.sub.27-aa.sub.28-aa.sub.29-aa.sub.30-aa.sub.31-aa.s-
ub.32-aa.sub.33-aa.sub.34-aa.sub.35- aa.sub.36-aa.sub.37-Z
[0653] wherein: [0654] Z is OH, or --NH--R.sup.3, wherein R.sup.3
is H or C.sub.1-C.sub.12 substituted or unsubstituted alkyl, or a
PEG chain of less than 10 Da; [0655] aa.sub.1 is His, N--Ac-His,
pGlu-His, or N--R.sup.3-His; [0656] aa.sub.2 is Ser, Ala, Gly, Aib,
Ac4c or Ac5c; [0657] aa.sub.3 is Gln, or Cit; [0658] aa.sub.4 is
Gly, or D-Ala; [0659] aa.sub.5 is Thr, or Ser; [0660] aa.sub.6 is
Phe, Trp, F2Phe, Me2Phe, or Nal2; [0661] aa.sub.7 is Thr, or Ser;
[0662] aa.sub.8 is Ser, or Asp; [0663] aa.sub.9 is Asp, or Glu;
[0664] aa.sub.10 is Tyr, Leu, Met, Nal2, Bip, or Bip2EtMeO; [0665]
aa.sub.11 is Ser, Asn, or U; [0666] aa.sub.12 is Lys, Glu, Ser,
Arg, or U; [0667] aa.sub.13 is absent or Tyr, Gln, Cit, or U;
[0668] aa.sub.14 is absent or Leu, Met, Nle, or U; [0669] aa.sub.15
is absent or Asp, Glu, or U; [0670] aa.sub.16 is absent or Ser,
Gly, Glu, Aib, Ac5c, Lys, Arg, or U; [0671] aa.sub.17 is absent or
Arg, hArg, Gln, Glu, Cit, Aib, Ac4c, Ac5c, or U; [0672] aa.sub.18
is absent or Arg, hArg, Ala, Aib, Ac4c, Ac5c, or U; [0673]
aa.sub.19 is absent or Ala, Val, Aib, Ac4c, Ac5c, or U; [0674]
aa.sub.20 is absent or Gln, Lys, Arg, Cit, Glu, Aib, Ac4c, Ac5c, or
U; [0675] aa.sub.21 is absent or Asp, Glu, Leu, Aib, Ac4c Ac5c, or
U; [0676] aa.sub.22 is absent or Phe, Trp, Nal2, Aib, Ac4c, Ac5c,
or U [0677] aa.sub.23 is absent or Val, Ile, Aib, Ac4c, Ac5c, or U;
[0678] aa.sub.24 is absent or Gln, Ala, Glu, Cit, or U; [0679]
aa.sub.25 is absent or Tip, Nal2, or U; [0680] aa.sub.26 is absent
or Leu, or U; [0681] aa.sub.27 is absent or Met, Val, Nle, Lys, or
U; [0682] aa.sub.28 is absent or Asn, Lys, or U; [0683] aa.sub.29
is absent or Thr, Gly, Aib, Ac4c, Ac5c, or U; [0684] aa.sub.30 is
absent or Lys, Aib, Ac4c, Ac5c, or U; [0685] aa.sub.31 is absent or
Arg, Aib, Ac4c, Ac5c, or U; [0686] aa.sub.32 is absent or Asn, Aib,
Ac4c, Ac5c, or U; [0687] aa.sub.33 is absent or Arg, Aib, Ac4c,
Ac5c, or U; [0688] aa.sub.34 is absent or Asn, Aib, Ac4c, Ac5c, or
U; [0689] aa.sub.35 is absent or Asn, Aib, Ac4c, Ac5c, or U; [0690]
aa.sub.36 is absent or Ile, Aib, Ac4c, Ac5C, or U; [0691] aa.sub.36
is absent or Ala, Aib, Ac4c, Ac5C, or U; [0692] aa.sub.37 absent or
U; [0693] U is a natural or unnatural amino acid comprising a
functional group used for covalent attachment to the surfactant X;
[0694] wherein any two of aa.sub.1-aa.sub.37 are optionally
cyclized through their side chains to form a lactam linkage; and
[0695] provided that one, or at least one of aa.sub.11-aa.sub.37 is
the linker amino acid U covalently attached to X.
[0696] In some embodiments, n is 1. In some embodiments, n is 2,
and a first glycoside is attached to a second glycoside via bond
between W.sup.2 of the first glycoside and any one of OR.sup.1b,
OR.sup.1c or OR.sup.1d of the second glycoside. In some
embodiments, n is 3, and a first glycoside is attached to a second
glycoside via bond between W.sup.2 of the first glycoside and any
one of OR.sup.1b, OR.sup.1c or OR.sup.1d of the second glycoside,
and the second glycoside is attached to a third glycoside via bond
between W.sup.2 of the second glycoside and any one of OR.sup.1b,
OR.sup.1c or OR.sup.1d of the third glycoside.
[0697] In one embodiment, compounds of Formula I-A are compounds
wherein X has the structure:
##STR00035## [0698] wherein: [0699] R.sup.1a is H, a protecting
group, a substituted or unsubstituted C.sub.1-C.sub.30 alkyl group,
or a steroid nucleus containing moiety; [0700] R.sup.1b, R.sup.1c,
and R.sup.1d are each, independently at each occurrence, H, a
protecting group, or a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl group; [0701] W.sup.1 is independently, at
each occurrence, --CH.sub.2--, --CH.sub.2--O--, --(C.dbd.O),
--(C.dbd.O)--O--, --(C.dbd.O)--NH--, --(C.dbd.S)--,
--(C.dbd.S)--NH--, or --CH.sub.2--S--; [0702] W.sup.2 is --O--,
--S--; [0703] R.sup.2 is a bond. C.sub.2-C.sub.4-alkene,
C.sub.2-C.sub.4-alkyne, or --(CH.sub.2).sub.m-maleimide; and [0704]
m is 1-10.
[0705] In another embodiment, compounds of Formula I-A are
compounds wherein X has the structure:
##STR00036##
[0706] Accordingly, in the embodiment described above. R.sup.2 is a
bond.
[0707] For instance, in an exemplary embodiment of the structure of
X described above, W.sup.1 is --C(.dbd.O)NH--, R.sup.2 is a bond
between W.sup.1 and an amino acid residue U within the peptide
(e.g., an amino group in the sidechain of a lysine residue present
in the peptide).
[0708] In a further embodiment, compounds of Formula I-A are
compounds wherein X has the structure:
##STR00037##
[0709] For instance, in an exemplary embodiment of the structure of
X described above, W.sup.1 is --CH.sub.2-- and R.sup.2 is an
alkyl-linked maleimide functional group on X and R.sup.2 is
attached to a suitable moiety of an amino acid residue U within the
peptide (e.g., a thiol group in a cysteine residue of the peptide
forms a thioether with the maleimide on X).
[0710] In yet another embodiment, compounds of Formula I-A are
compounds wherein X has the structure:
##STR00038## [0711] wherein: [0712] R.sup.1a is H, a protecting
group, a substituted or unsubstituted C.sub.1-C.sub.30 alkyl group,
or a steroid nucleus containing moiety; [0713] R.sup.1b, R.sup.1c,
and R.sup.1d are each, independently at each occurrence, H, a
protecting group, or a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl group; [0714] W.sup.1 is --(C.dbd.O)--NH--;
[0715] W.sup.2 is --O--; [0716] R.sup.2 is a bond.
[0717] In an additional embodiment, compounds of Formula I-A are
compounds wherein X has the structure:
##STR00039## [0718] wherein: [0719] R.sup.1a is a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl group; [0720] R.sup.1b,
R.sup.1c, and R.sup.1d are H; [0721] W.sup.1 is --(C.dbd.O)--NH--;
[0722] W.sup.2 is --O--; and [0723] R.sup.2 is a bond.
[0724] In some embodiments described above and herein, R.sup.1a is
a substituted or unsubstituted C.sub.1-C.sub.30 alkyl group.
[0725] In some embodiments described above and herein, R.sup.1a is
a substituted or unsubstituted C.sub.6-C.sub.20 alkyl group.
[0726] Also contemplated herein are alternate embodiments wherein X
in Formula 3-I-A has the structure:
##STR00040##
[0727] For instance, in an exemplary embodiment of the structure of
X described above, W.sup.1 is --S--, R.sup.2 is a C.sub.1-C.sub.30
alkyl group, W.sup.2 is S, R.sup.1a is a bond between W.sup.2 and a
suitable moiety of an amino acid residue U within the peptide
(e.g., a thiol group in a cysteine residue of the peptide forms a
thioether with X).
[0728] In another exemplary embodiment of the structure of X
described above, W.sup.1 is --O--, R.sup.2 is a C.sub.1-C.sub.3
alkyl group, W.sup.2 is O, R.sup.1a is a bond between W.sup.2 and a
suitable moiety of an amino acid residue U within the peptide
(e.g., a hydroxyl group in a serine or threonine residue of the
peptide forms an ether with X).
[0729] In some embodiments, U is used for covalent attachment to X
and is a dibasic natural or unnatural amino acid, a natural or
unnatural amino acid comprising a thiol, an unnatural amino acid
comprising a --N.sub.3 group, an unnatural amino acid comprising an
acetylenic group, or an unnatural amino acid comprising a
--NH--C(.dbd.O)--CH.sub.2--Br or a --(CH.sub.2)m-maleimide, wherein
m is 1-10.
[0730] In some embodiments of the peptide product, the surfactant
is a 1-alkyl glycoside class surfactant. In some embodiments of the
peptide product, the surfactant is attached to the peptide via an
amide bond.
[0731] In some embodiments of the peptide product, the surfactant X
is comprised of 1-eicosyl beta-D-glucuronic acid, 1-octadecyl
beta-D-glucuronic acid, 1-hexadecyl beta-D-glucuronic acid,
1-tetradecylbeta D-glucuronic acid, 1-dodecyl beta D-glucuronic
acid, 1-decyl beta-D-glucuronic acid, 1-octyl beta-D-glucuronic
acid, 1-eicosyl beta-D-diglucuronic acid, 1-octadecyl
beta-D-diglucuronic acid, 1-hexadecyl beta-D-diglucuronic acid,
1-tetradecyl beta-D-diglucuronic acid, 1-dodecyl
beta-D-diglucuronic acid, 1-decyl beta-D-diglucuronic acid, 1-octyl
beta-D-diglucuronic acid, or functionalized 1-ecosyl
beta-D-glucose, 1-octadecyl beta-D-glucose, 1-hexadecyl
beta-D-glucose, 1-tetradecyl beta-D-glucose, 1-dodecyl
beta-D-glucose, 1-decyl beta-D-glucose, 1-octyl beta-D-glucose,
1-eicosyl beta-D-maltoside, 1-octadecyl beta-D-maltoside,
1-hexadecyl beta-D-maltoside, 1-dodecyl beta-D-maltoside, 1-decyl
beta-D-maltoside, 1-octyl beta-D-maltoside, and the like, and the
peptide product is prepared by formation of a linkage between the
aforementioned groups and a group on the peptide (e.g., a --COOH
group in the aforementioned groups and an amino group of the
peptide).
[0732] In some embodiments of the peptide product, U is a terminal
amino acid of the peptide. In some embodiments of the peptide
product, U is a non-terminal amino acid of the peptide. In some
embodiments of the peptide product, U is a natural D- or L-amino
acid. In some embodiments of the peptide product, U is an unnatural
amino acid. In some embodiments of the peptide product, U is
selected from Lys, Cys, Orn, or an unnatural amino acid comprising
a functional group used for covalent attachment to the surfactant
X.
[0733] In some embodiments of the peptide product, the functional
group used for covalent attachment of the peptide to the surfactant
X is --NH.sub.2, --SH, --OH, --N.sub.3, haloacetyl, a
--(CH).sub.m-maleimide (wherein m is 1-10), or an acetylenic
group.
[0734] In some embodiments side chain functional groups of two
different amino acid residues are linked to form a cyclic lactam.
For example, in some embodiments, a Lys side chain forms a cyclic
lactam with the side chain of Glu. In some embodiments such lactam
structures are reversed and are formed from a Glu and a Lys. Such
lactam linkages in some instances are known to stabilize alpha
helical structures in peptides (Condon, S. M., et al. (2002) Bioorg
Med Chem 10: 731-736; Murage, E. N., et al (2008) Bioorg Med Chem
16: 10106-12); Murage, E. N., et al. (2010) J Med Chem 53:
6412-20). In some embodiments cysteine residues may be linked
through disulfide formation in order to accomplish a similar form
of conformational restriction and assist in the formation of
helical structures (Li, Y., et al. (2011) Peptides 32: 1400-1407.
In some embodiments side chain functional groups of two different
amino acid residues are linked to form a heterocycle generated
through a "click reaction" between side chain azide and alkyne
functional groups in order to achieve a similar form of
conformational restriction and stabilized helical conformations (Le
Chevalier Isaad A., et al. (2009) J Peptide Sci 15: 451-4).
[0735] In some embodiments, the peptide product comprising a
covalently linked alkyl glycoside is a covalently modified glucagon
or analog thereof. In some of such embodiments, the peptide product
contains a covalently linked 1-O-alkyl .beta.-D-glucuronic acid and
the peptide is an analog of glucagon.
[0736] In some embodiments, a peptide product comprising a
covalently linked alkyl glycoside is a covalently modified GLP-1,
or analog thereof. In some of such embodiments, the peptide product
comprises a covalently linked 1-O-alkyl .beta.-D-glucuronic acid
and the peptide is an analog of GLP-1.
[0737] In some embodiments, the peptide product of Formula I-A has
the structure of Formula
TABLE-US-00028 Formula 3-III-A (SEQ. ID. NO. 304)
aa.sub.1-aa.sub.2-aa.sub.3-aa.sub.4-aa.sub.5-aa.sub.6-aa7-aa.sub.8-aa.sub.-
9-aa.sub.10-aa.sub.11-aa.sub.12-aa.sub.13-
aa.sub.14-aa.sub.15-aa.sub.16-aa.sub.17-aa.sub.18-aa.sub.19-aa.sub.20-aa.s-
ub.21-aa.sub.22-aa.sub.23-aa.sub.24-
aa.sub.25-aa.sub.26-aa.sub.27-aa.sub.28-aa.sub.29-Z
[0738] wherein: [0739] Z is OH, or --NH--R.sup.3, wherein R.sup.3
is H, or C.sub.1-C.sub.12 substituted or unsubstituted alkyl, or a
PEG chain of less than 10 Da; [0740] aa.sub.1 is His, N--Ac-His,
pGlu-His, or N--R.sup.3-His; [0741] aa.sub.2 is Ser, Ala, Gly, Aib,
Ac4c, or Ac5c; [0742] aa.sub.3 is Gln, or Cit; [0743] aa.sub.4 is
Gly, or D-Ala; [0744] aa.sub.5 is Thr, or Ser; [0745] aa.sub.6 is
Phe, Trp, F2Phe, Me2Phe, or Nal2; [0746] aa.sub.7 is Thr, or Ser;
[0747] aa.sub.8 is Ser, or Asp; [0748] aa.sub.9 is Asp, or Glu;
[0749] aa.sub.10 is Tyr, Leu, Met, Nal2, Bip, or Bip2EtMeO; [0750]
aa.sub.11 is Ser, Asn, or U; [0751] aa.sub.12 is Lys, Glu, Ser,
Arg, or U(X); [0752] aa.sub.13 is absent or Tyr, Gln, Cit, or U(X);
[0753] aa.sub.14 is absent or Leu, Met, Nle, or U(X); [0754]
aa.sub.15 is absent or Asp, Glu, or U(X); [0755] aa.sub.16 is
absent or Ser, Gly, Glu, Aib, Ac5c, Lys, Arg, or U(X); [0756]
aa.sub.17 is absent or Arg, hArg, Gln, Glu, Cit, Aib, Ac4c, Ac5c,
or U(X); [0757] aa.sub.18 is absent or Arg, hArg, Ala, Aib, Ac4c,
Ac5c, or U(X); [0758] aa.sub.19 is absent or Ala, Val, Aib, Ac4c,
Ac5c, or U(X); [0759] aa.sub.20 is absent or Gln, Lys, Arg, Cit,
Glu, Aib, Ac4c, Ac5c, or U(X); [0760] aa.sub.21 is absent or Asp,
Glu, Leu, Aib, Ac4c, Ac5c, or U(X); [0761] aa.sub.22 is absent or
Phe, Trp, Nal2, Aib, Ac4c, Ac5c, or U(X); [0762] aa.sub.23 is
absent or Val, Ile, Aib, Ac4c, Ac5c, or U(X); [0763] aa.sub.24 is
absent or Gln, Ala, Glu, Cit, or U(X); [0764] aa.sub.25 is absent
or Trp, Nal2, or U(X); [0765] aa.sub.26 is absent or Leu, or U(X);
[0766] aa.sub.27 is absent or Met, Val, Nle, Lys, or U(X); [0767]
aa.sub.28 is absent or Asn, Lys, or U(X); [0768] aa.sub.29 is
absent or Thr, Gly, Aib, Ac4c, Ac5c, or U(X); [0769] wherein any
two of aa.sub.1-aa.sub.29 are optionally cyclized through their
side chains to form a lactam linkage; and [0770] provided that one,
or at least one of aa.sub.16, aa.sub.17, aa.sub.18, aa.sub.19,
aa.sub.20, aa.sub.21, aa.sub.22, aa.sub.23, aa.sub.24, aa.sub.25,
aa.sub.26, aa.sub.28, aa.sub.28 or aa.sub.29 is the natural or
unnatural amino acid U covalently attached to X.
[0771] In some embodiments, the peptide product of Formula I-A has
the structure of Formula 3-III-B:
TABLE-US-00029 Formula 3-III-B (SEQ. ID. NO. 305)
His.sub.1-aa.sub.2-aa.sub.3-Gly.sub.4-Thr.sub.5-aa.sub.6-Thr.sub.7-Ser.sub-
.8-Asp.sub.9-aa.sub.10-aa.sub.11-
aa.sub.12-aa.sub.13-aa.sub.14-aa.sub.15-aa.sub.16-aa.sub.17-aa.sub.18-aa.s-
ub.19-aa.sub.20-aa.sub.21-aa.sub.22- aa.sub.23-Z
[0772] wherein: [0773] Z is OH or --NH--R.sup.3, wherein R.sup.3 is
H or substituted or unsubstituted C.sub.1-C.sub.12 alkyl; or a PEG
chain of less than 10 Da; [0774] aa.sub.2 is Ser, Ala, Gly, Aib,
Ac4c, or Ac5c; [0775] aa.sub.3 is Gln, or Cit; [0776] aa.sub.6 is
Phe, Trp, F2Phe, Me2Phe, MePhe, or Nal2; [0777] aa.sub.10 is Tyr,
Leu, Met, Nal2, Bip, or Bip2EtMeO; [0778] aa.sub.11 is Ser, Asn, or
U(X); [0779] aa.sub.12 is Lys, Glu, Ser, or U(X); [0780] aa.sub.13
is absent or Tyr, Gln, Cit, or U(X); [0781] aa.sub.14 is absent or
Leu, Met, Nle, or U(X); [0782] aa.sub.15 is absent or Asp, Glu, or
U(X); [0783] aa.sub.16 is absent or Ser, Gly, Glu, Aib, Ac4c, Ac5c,
Lys, R, or U(X); [0784] aa.sub.17 is absent or Arg, hArg, Gln, Glu,
Cit, Aib, Ac4c, Ac5c, or U(X); [0785] aa.sub.18 is absent or Arg,
hArg, Ala, Aib, Ac4c, Ac5c, or U(X); [0786] aa.sub.19 is absent or
Ala, Val, Aib, Ac4c, Ac5c, or U(X); [0787] aa.sub.20 is absent or
Gln, Lys, Arg, Cit, Glu, Aib, Ac4c, Ac5c, or U(X); [0788] aa.sub.21
is absent or Asp, Glu, Leu, Aib, Ac4c, Ac5c, or U(X); [0789]
aa.sub.22 is absent or Phe, Aib, Ac4c, Ac5c, or U(X) [0790]
aa.sub.23 is absent or Val, Ile, Aib, Ac4c, Ac5c, or U(X); [0791]
wherein any two of aa.sub.1-aa.sub.23 are optionally cyclized
through their side chains to form a lactam linkage; and [0792]
provided that one, or at least one of aa.sub.16, aa.sub.17,
aa.sub.18, aa.sub.19, aa.sub.20, aa.sub.21, aa.sub.22, aa.sub.23 or
aa.sub.24 is the natural or unnatural amino acid U covalently
attached to X.
[0793] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, U is any linker amino acid described herein.
[0794] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, U is any linker amino acid described herein. In some
embodiments of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V,
aa.sub.12 is lysine. In some embodiments of Formula 3-I-A, 3-III-A,
3-III-B or Formula 3-V, aa.sub.14 is leucine.
[0795] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, aa.sub.12 is lysine. In some embodiments of Formula
I-A, III-A, III-B or Formula V, aa.sub.14 is leucine.
[0796] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, aa.sub.14 is a lysine residue attached to X.
[0797] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, aa.sub.17 is a homo Arginine (hArg) residue.
[0798] In some embodiments of Formula I-A, III-A, III-B or Formula
V, aa.sub.17 is a glycine residue.
[0799] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, aa.sub.2 is an Aib or Ac4c residue.
[0800] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, the peptide comprises one or more Aib residues.
[0801] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, peptide comprises one or more Aib residues at the
C-terminus.
[0802] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, the peptide product has the structure (SEQ. ID. NO.
619): [0803]
His.sub.1-aa.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.-
sub.8-Asp.sub.9-Tyr.sub.10-Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp-
.sub.15-Aib.sub.16-aa.sub.17-Lys(N-omega-1'-alkyl
beta-D-glucuronyl).sub.18-aa.sub.19-NH.sub.2; wherein [0804]
aa.sub.2 is Aib or Ac4c; [0805] aa.sub.17 is Arg, hArg or Gln;
[0806] aa.sub.19 is Aib, Ac4c or Ac5c; and [0807] alkyl is a
C.sub.8 to C.sub.20 linear alkyl chain.
[0808] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, the peptide product ha the structure (SEQ. ID. NO.
620): [0809]
His.sub.1-aa.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.-
sub.8-Asp.sub.9-Tyr.sub.10-Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp-
.sub.15-Aib.sub.16-aa.sub.17-Lys(N-omega-1'-alkyl
beta-D-glucuronyl).sub.18-aa.sub.19-aa.sub.20-NH.sub.2; wherein
[0810] aa.sub.2 is Aib or Ac4c, [0811] aa.sub.17 is Arg, hArg or
Gln, [0812] aa.sub.19 and aa.sub.20 are individually Aib, Ac4c or
Ac5c; and [0813] alkyl is a C.sub.8 to C.sub.20 linear alkyl
chain.
[0814] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, the peptide product has the structure (SEQ. ID. NO.
621): [0815]
His.sub.1-aa.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.-
sub.8-Asp.sub.9-Tyr.sub.10-Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp-
.sub.15-aa.sub.16-aa.sub.17-Lys(N-omega-1'-alkyl
beta-D-glucuronyl).sub.18-aa.sub.19-NH.sub.2; wherein [0816]
aa.sub.2 is Aib or Ac4c; [0817] aa.sub.16 is Aib or Ac4c; [0818]
aa.sub.17 is Arg, hArg or Gln; [0819] aa.sub.19 is Aib, Ac4c or
Ac5c; and [0820] alkyl is a C.sub.8 to C.sub.20 linear alkyl
chain.
[0821] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, aa.sub.16 and aa.sub.20 are cyclized to form a lactam
linkage.
[0822] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, the peptide product has the structure: (SEQ. ID. NO.
622) [0823]
His.sub.1-aa.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.-
sub.8-Asp.sub.9-Tyr.sub.10-Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp-
.sub.15-aa.sub.16-aa.sub.17-Ala.sub.18-Ala.sub.19-aa.sub.20-Glu.sub.21-Phe-
.sub.22-Ile.sub.23-Lys(N-omega-1'-alkyl
beta-D-glucuronyl).sub.24-Trp.sub.25-Leu.sub.26-aa.sub.27-Asn.sub.28-Thr.-
sub.29-NH.sub.2; wherein [0824] aa.sub.2 is Aib or Ac4c; [0825]
aa.sub.16 and aa.sub.20 are each individually either Lys or Glu and
are cyclized through their side chains to form a lactam linkage;
[0826] aa.sub.17 is Arg, hArg or Gln; [0827] aa.sub.27 is Met or
Nle; and [0828] alkyl is a C.sub.1-C.sub.20 linear alkyl chain.
[0829] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, the peptide product has the structure (SEQ. ID. NO.
623); [0830]
His.sub.1-aa.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.-
sub.8-Asp.sub.9-Tyr.sub.10-Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp-
.sub.15-cyclic(Glu.sub.16-Gln.sub.17-Ala.sub.18-Ala.sub.19-Lys.sub.20)-Glu-
.sub.21-Phe.sub.22-Ile.sub.23-Lys(N-omega-1'-alkyl
beta-D-glucuronyl).sub.24-Trp.sub.25-Leu.sub.26-Met.sub.27-Asn.sub.28-aa.-
sub.29-NH.sub.2; wherein aa.sub.2 is Aib or Ac4c, aa29 is Thr, Aib,
Ac4c, or Ac5c, and the 1'-alkyl group is selected from dodecyl,
tetradecyl, hexadecyl, or octadecyl; and the side chains on the
amino acids in position 16 and 20 are cyclized to form a side chain
lactam.
[0831] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, aa.sub.12 and aa.sub.16 are cyclized to form a lactam
linkage.
[0832] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, the peptide product has the structure (SEQ. ID. NO.
624): [0833]
His.sub.1-aa.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.-
sub.8-Asp.sub.9-Tyr.sub.10-Ser.sub.11-aa.sub.12-Tyr.sub.13-Leu.sub.14-Asp.-
sub.15-aa.sub.16-aa.sub.17-Lys(N-omega-1'-alky
beta-D-glucuronyl).sub.18-aa.sub.19-aa.sub.20-NH.sub.2; wherein
[0834] aa2 is Aib or Ac4c; [0835] aa.sub.12 and aa.sub.16 are each
individually either Lys or Glu and are cyclized through their side
chains to form a lactam linkage; [0836] aa.sub.17 is Arg, or hArg;
[0837] aa.sub.19 and aa.sub.20 are individually either Aib, Ac4c or
Ac5c; and alkyl is a C.sub.1-C.sub.20 linear alkyl chain.
[0838] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, the peptide product has the structure (SEQ. ID. NO.
625): [0839]
His.sub.1-Ac4c.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Se-
r.sub.8-Asp.sub.9-Tyr.sub.10-Ser.sub.11-cyclo(Glu.sub.12-Tyr.sub.13-Leu.su-
b.14-Asp.sub.15-Lys.sub.16)-aa.sub.17-Lys(N-omega-1'-alkyl
beta-D-glucuronyl).sub.18-Aib.sub.19-Aib.sub.20-NH.sub.2; [0840]
wherein [0841] aa.sub.12 and aa.sub.16 are cyclized through their
side chains to form a lactam linkage; [0842] aa.sub.17 is Arg or
hArg; and [0843] alkyl is a C.sub.12, C.sub.14, C.sub.16, or
C.sub.18 linear alkyl chain.
[0844] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, the peptide product has the structure (SEQ. ID. NO.
626): [0845]
His.sub.1-aa.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.-
sub.8-Asp.sub.9-Tyr.sub.10-Ser.sub.11-aa.sub.12-Tyr.sub.13-Leu.sub.14-Asp.-
sub.15-aa.sub.16-aa.sub.17-Lys(N-omega-1'-alkyl
beta-D-glucuronyl).sub.18-aa.sub.19-aa.sub.20-NH.sub.2; [0846]
wherein [0847] aa.sub.12 and aa.sub.16 are each individually either
Lys or Glu [0848] and aa.sub.12 and aa.sub.16 are cyclized through
their side chains to form a lactam linkage; [0849] aa.sub.17 is Arg
or hArg; aa.sub.19 and aa.sub.20 are individually either Aib, Ac4c
or Ac5c; and the 1'-alkyl group is selected from dodecyl,
tetradecyl, hexadecyl, or octadecyl.
[0850] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, the peptide product has the structure (SEQ. ID. NO.
627): [0851]
His.sub.1-aa.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-aa.sub.6-Thr.sub.7-Ser.s-
ub.8-Asp.sub.9-Tyr.sub.10-Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp.-
sub.15-Ser.sub.16-Aib.sub.17-Lys(N-omega-1'-dodecyl
beta-D-glucuronyl).sub.18-aa.sub.19-NH.sub.2; wherein aa.sub.2 is
Aib or Ac4c, aa.sub.6 is Me2Phe, MePhe, or Phe; and aa.sub.19 is
Aib, Ac4c, or Ac5c.
[0852] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, the peptide product has the structure (SEQ. ID. NO.
628): [0853]
His.sub.1-aa.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-aa.sub.6-Thr.sub.7-Ser.s-
ub.8-Asp.sub.9-Tyr.sub.10-Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp.-
sub.15-Ser.sub.16-aa.sub.17-Lys(N-omega-1'-dodecyl
beta-D-glucuronyl).sub.18-aa.sub.19.aa.sub.20-NH.sub.2; wherein
aa.sub.2 is Aib or Ac4c, aa.sub.6 is Me2Phe, MePhe, or Phe;
aa.sub.17 is Arg or hArg, and aa.sub.19 or aa.sub.20 is Aib, Ac4c,
or Ac5c.
[0854] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, the peptide product has the structure (SEQ. ID. NO.
629): [0855]
His.sub.1-Aib.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser-
.sub.8-Asp.sub.9-Tyr.sub.10-Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-As-
p.sub.15-cyclo(Glu.sub.16-Arg.sub.17-Ala.sub.18-Ala.sub.19-Lys.sub.20)-Lys-
(N-omega-1'-alkyl
beta-D-glucuronyl).sub.21-Phe.sub.22-aa.sub.23-NH.sub.2; wherein
aa.sub.23 is Aib, Ac4c, or Ac5c and the 1'-alkyl group is selected
from dodecyl, tetradecyl, hexadecyl, or octadecyl.
[0856] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, the peptide product has the structure (SEQ. ID. NO.
630): [0857]
His.sub.1-aa.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-aa.sub.6-Thr.sub.7-Ser.s-
ub.8-Asp.sub.9-Tyr.sub.10-Ser.sub.11-aa.sub.12-Tyr.sub.13-Leu.sub.14-Asp.s-
ub.15-aa.sub.16-aa.sub.17-aa.sub.18-Ala.sub.19-aa.sub.20-Lys(N-omega-1'-al-
kyl beta-D-glucuronyl).sub.21-Phe.sub.22-aa.sub.23-NH.sub.2; [0858]
wherein [0859] aa.sub.2 is Aib or Ac4c; [0860] aa.sub.6 is Me2Phe,
MePhe, or Phe; [0861] aa.sub.12 and aa.sub.16 are each individually
either Lys or Glu; [0862] and aa.sub.16 and aa.sub.20 are cyclized
through their side chains to form a lactam linkage; [0863]
aa.sub.17 is Arg, hArg or Gln; [0864] aa.sub.18 is Aib or Ala;
[0865] aa.sub.23 is Aib, Ac4c, or Ac5c and the 1'-alkyl group is
selected from dodecyl, tetradecyl, hexadecyl, or octadecyl.
[0866] In some embodiments of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V, the peptide product has the structure (SEQ. ID. NO.
631): [0867]
His.sub.1-aa.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-aa.sub.6-Thr.sub.7-Ser.s-
ub.8-Asp.sub.9-Tyr.sub.10-Ser.sub.11-aa.sub.12-Tyr.sub.13-Leu.sub.14-Asp.s-
ub.15-aa.sub.16-aa.sub.17-Lys(N-omega-1'-alkyl
beta-D-glucuronyl).sub.18-aa.sub.19-aa.sub.20-NH.sub.2; [0868]
wherein [0869] aa.sub.2 is Aib or Ac4c; [0870] aa.sub.6 is Phe;
[0871] aa.sub.12 and aa.sub.16 are each individually either Lys or
Glu; and aa.sub.12 and aa.sub.16 are cyclized through their side
chains to form a lactam linkage; [0872] aa.sub.17 is Arg or hArg;
[0873] aa.sub.19 is Aib, Ac4c, or Ac5c; [0874] aa.sub.20 is Aib,
Ac4c, or Ac5c and the and the 1'-alkyl group is selected from
dodecyl, tetradecyl, hexadecyl, or octadecyl.
[0875] In some embodiments, for any compound of Formula 3-I-A,
3-III-A, 3-III-B or Formula 3-V, X is comprised of a dodecyl alkyl
chain.
[0876] In some embodiments, the peptide product is a biologically
active peptide product that binds to the GLP1R and/or to the
GLCR.
[0877] In a specific embodiment, the peptide products of Formula
3-I-A, 3-III-A, 3-III-B or Formula 3-V, described above and herein
have the following structure:
##STR00041##
wherein R.sup.1a is a C.sub.1-C.sub.20 alkyl chain as described in
Table 1 of FIG. 1, R' is a peptide as described in Table 3 of FIG.
8 and Table 4 of FIG. 9, W.sup.2 of Formula I-A is --O--, and
W.sup.1 of Formula I-A is --(C.dbd.O)NH-- and is part of an amide
linkage to the peptide R'. In some of such embodiments, R.sup.1a is
a C.sub.6-C.sub.20 alkyl chain. In some of such embodiments,
R.sup.1a is a C.sub.8-C.sub.2 alkyl chain. In some of such
embodiments, R.sup.1a is a C.sub.12-C.sub.20 alkyl chain. In some
of such embodiments, R.sup.1a is a C.sub.12-C.sub.16 alkyl
chain.
[0878] In embodiments described above, an amino moiety of an amino
acid and/or a peptide R' (e.g., an amino group of an amino acid
residue such as a Lysine, or a lysine residue within the peptide
R') is used to form a covalent linkage with a compound of
structure:
##STR00042##
wherein R.sup.1a is a C.sub.1-C.sub.20 alkyl chain as described
above and in Table 3 of FIG. 8 and Table 4 of FIG. 9.
[0879] In such cases, the amino acid residue having an amino moiety
(e.g., a Lysine within the peptide R') which is used to form a
covalent linkage to the compound A described above, is a linker
amino acid U which is attached to a surfactant X having the
structure of Formula A. Accordingly, as one example, Lys(C12) of
Table 3 of FIG. 8 and Table 4 of FIG. 9 has the following
structure:
##STR00043##
[0880] Also contemplated within the scope of the embodiments
presented herein are peptide products of Formula 3-I-A derived from
maltouronic acid-based surfactants through binding at either or
both carboxylic acid functions. Thus, as one example, peptides in
Table 3 of FIG. 8 and Table 4 of FIG. 9 comprise a lysine linker
amino acid bonded to a maltouronic acid based surfactant X and
having a structure:
##STR00044##
[0881] It will be understood that in one embodiment, compounds of
Formula 3-I-A are prepared by attaching a lysine to a group X,
followed by attachment of additional amino acid residues and/or
peptides are attached to the lysine-X compound to obtain compounds
of Formula 3-I-A. It will be understood that other natural or
non-natural amino acids described herein are also suitable for
attachment to the surfactant X and are suitable for attaching
additional amino acid/peptides to obtain compounds of Formula
3-I-A. It will be understood that in another embodiment, compounds
of Formula 3-I-A are prepared by attaching a full length or partial
length peptide to a group X, followed by optional attachment of
additional amino acid residues and/or peptides are attached to
obtain compounds of Formula 3-I-A.
[0882] In a specific embodiment, provided herein are compounds
selected from compounds of Table 3 in FIG. 8 and Table 4 in FIG.
9.
[0883] Also provided herein are pharmaceutical compositions
comprising a therapeutically effective amount of a peptide product
described above, or acceptable salt thereof, and at least one
pharmaceutically acceptable carrier or excipient.
[0884] In some embodiments of the pharmaceutical compositions, the
carrier is an aqueous-based carrier. In some embodiments of the
pharmaceutical compositions, the carrier is a nonaqueous-based
carrier. In some embodiments of the pharmaceutical compositions,
the nonaqueous-based carrier is a hydrofluoroalkane-like solvent
that may comprise sub-micron anhydrous .alpha.-lactose or other
excipients.
[0885] Contemplated within the scope of embodiments presented
herein is the reaction of an amino acid and/or a peptide comprising
a linker amino acid U bearing a nucleophile, and a group X
comprising a bearing a leaving group or a functional group that can
be activated to contain a leaving group, for example a carboxylic
acid, or any other reacting group, thereby allowing for covalent
linkage of the amino acid and/or peptide to a surfactant X via the
linker amino acid U to provide a peptide product of Formula
3-I-A.
[0886] Also contemplated within the scope of embodiments presented
herein is the reaction of an amino acid and/or a peptide comprising
a linker amino acid U bearing a bearing a leaving group or a
functional group that can be activated to contain a leaving group,
for example a carboxylic acid, or any other reacting group, and a
group X comprising a nucleophilic group, thereby allowing for
covalent linkage of the amino acid and/or peptide to a surfactant X
via the linker amino acid U to provide a peptide product of Formula
I-A.
[0887] It will be understood that, in one embodiment, Compounds of
Formula 3-I-A are prepared by reaction of a linker amino acid U
with X, followed by addition of further residues to U to obtain the
peptide product of Formula 3-I-A. It will be understood that in an
alternative embodiment, Compounds of Formula 3-I-A are prepared by
reaction of a suitable peptide comprising a linker amino acid U
with X, followed by optional addition of further residues to U, to
obtain the peptide product of Formula 3-I-A.
[0888] Provided herein are methods for treating conditions
associated with insulin resistance comprising administration of a
compound of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V.
[0889] Provided herein are methods for treating diabetes, diabetic
retinopathy, diabetic neuropathy, diabetic nephropathy, wound
healing, insulin resistance, hyperglycemia, hyperinsulinemia,
metabolic syndrome, diabetic complications, elevated blood levels
of free fatty acids or glycerol, hyperlipidemia, obesity,
hypertriglyceridemia, atherosclerosis, acute cardiovascular
syndrome, infarction, ischemic reperfusion or hypertension,
comprising administering a therapeutically effective amount of a
peptide product described above and herein to an individual in need
thereof.
[0890] Provided herein are methods of reducing weight gain or
inducing weight loss comprising administering to a subject in need
thereof a therapeutically effective amount of a peptide product
described above and herein to an individual in need thereof.
[0891] Provided herein are methods for treating mammalian
conditions characterized by obesity-linked insulin resistance or
the metabolic syndrome comprising administering to a subject in
need thereof a weight loss-inducing or insulin-sensitizing amount
of a peptide product described above and herein to an individual in
need thereof.
[0892] In some embodiments, the condition to be treated is the
metabolic syndrome (Syndrome X). In some embodiments, the condition
to be treated is diabetes. In some embodiments, the condition to be
treated is hyperlipidemia. In some embodiments, the condition to be
treated is hypertension. In some embodiments, the condition to be
treated is vascular disease including atherosclerosis, or the
systemic inflammation characterized by elevated C reactive
protein.
[0893] In some embodiments of the methods, the effective amount of
the peptide product for administration is from about 0.1
.mu.g/kg/day to about 100.0 .mu.g/kg/day, or from 0.01 .mu.g/kg/day
to about 1 mg/kg/day or from 0.1 .mu.g/kg/day to about 50
mg/kg/day.
[0894] Provided herein are methods of treating the metabolic
syndrome, or its component diseases, comprising administering to a
subject in need thereof a therapeutically effective amount of a
peptide product described above. In some embodiments, the metabolic
syndrome condition has progressed to diabetes.
[0895] Also provided herein is a covalently modified GLCR and/or
GLP1R binding peptide or analog thereof, comprising a hydrophilic
group as described herein; and a hydrophobic group covalently
attached to the hydrophilic group. In specific embodiments, the
covalently modified peptide and/or protein product comprises a
hydrophilic group that is a saccharide and a hydrophobic group that
is a C.sub.1-C.sub.20 alkyl chain or an aralkyl chain.
Insulin Resistance
[0896] The risks associated with prolonged hyperglycemia include an
increased risk of microvascular complications, sensory neuropathy,
myocardial infarction, stroke, macrovascular mortality, and
all-cause mortality. Type 2 diabetes is also linked causally with
obesity, an additional global epidemic. At least $232 billion were
spent on treatment and prevention of diabetes worldwide in 2007,
with three quarters of that amount spent in industrialized
countries on the treatment of long-term complications and on
general care, such as efforts to prevent micro and macrovascular
complications. In 2007, estimated indirect costs of diabetes
(disability, lost productivity, and premature death due to
diabetes) to the United States economy were $58 billion.
[0897] Obesity leads to insulin resistance, a decreased ability of
the cells in the body to react to insulin stimulation through
decreased numbers of insulin receptors and a decreased coupling of
those receptors to critical intracellular signaling systems. The
obese state further leads to the "metabolic syndrome", a
constellation of diseases (insulin resistance, hypertension,
atherosclerosis, et al.) with very large healthcare consequences.
If insulin resistance is diagnosed early enough, overt type 2
diabetes can be prevented or delayed, with lifestyle interventions
aimed at reducing calorie intake and body fat and through drug
treatment to normalize glycemic control. Despite treatment
guidelines recommending early, aggressive intervention, many
patients fail to reach targets for glycemic control. Many factors
contribute to the failure to manage type 2 diabetes successfully
including psychosocial and economic influences and shortcomings in
the efficacy, convenience and tolerability profiles of available
antidiabetic drugs. The peptide and/or protein products described
herein are designed to overcome these shortcomings.
Incretin Effect
[0898] The "incretin effect" is used to describe the phenomenon
whereby a glucose load delivered orally produces a much greater
insulin secretion than the same glucose load administered
intravenously. This effect is mediated by at least two incretin
hormones secreted by intestinal L-cells. Glucose-dependent
insulinotropic polypeptide (GIP) and glucagon-like peptide 1
(GLP-1) were identified as incretins and it is thought that healthy
individuals may derive up to 70% of their prandial insulin
secretory response from the incretin effect.
[0899] Normally the incretin peptides are secreted as needed, in
response to ingested nutrients, and have a short plasma half-life
due to degradation by dipeptidyl peptidase IV (DPP-4) enzyme. In
people with type 2 diabetes, pancreatic responsiveness to GLP-1 is
impaired, but the insulin-secretory response can be restored with
pharmacologic doses of human GLP-1 (Kieffer, T. J., et al. (1995)
Endocrinology 136: 3585-3596). In addition, GLP-1 promotes
beta-cell neogenesis and preservation (Aaboe, K., et al. (2008)
Diabetes Obes Metab 10: 994-1003). GLP-1 has additional beneficial
effects such as on cardiac function: for example it improves left
ventricular function (Sokos. G. G., et al. (2006) J Card Fail 12:
694-699) in human subjects. GLP-1 also slows gastric emptying in
humans and reduces appetite (Toft-Nielsen, M. B., et al. (1999)
Diabetes Care 22: 1137-1143).
[0900] Treatment of diabetes patients with metabolically stable and
long-acting analogs of GLP-1 is described in, for example, Drab, S.
R. (2010) Pharmacotherapy 30: 609-624, suffers from issues related
to convenience of use and side effects such as nausea, risk of
pancreatitis and thyroid carcinoma. GLP-1 analogs provide
glucose-dependent stimulation of insulin secretion and lead to a
reduced risk of hypoglycemia. In addition, while a number of the
current treatments for diabetes cause weight gain, as described
below, GLP-1 analogs induce satiety and a mild weight loss.
Accordingly, in some embodiments, provided herein are GLP-1 analogs
that are long acting and are administered at low doses thereby
reducing side-effects associated with current treatments.
[0901] A number of peptide gut hormones are known to modulate
appetite (Sanger, G. J. and Lee, K. (2008) Nat Rev Drug Discov 7:
241-254). Several peptides are derived from tissue-specific,
enzymatic processing (prohormone convertases; PCs) of the
preproglucagon gene product: e.g. glucagon, GLP-1, glucagon-like
peptide-2 (GLP-2), glicentin and oxyntomodulin (OXM) (Drucker, D.
J. (2005) Nat Clin Pract Endocrinol Metab 1: 22-31; Sinclair, E. M.
and Drucker, D. J. (2005) Physiology (Bethesda) 20: 357-365).
GLP-1, GLP-2, glicentin and OXM are co-secreted from L-cells in the
gut in response to feeding. Preproglucagon is alternatively
processed (PC2) to produce glucagon in the alpha cells in the
pancreatic islets. The structure of OXM is essentially glucagon
with a C-terminal extension of 8 residues.
[0902] In addition to the stimulation of insulin biosynthesis and
of glucose-dependent insulin secretion, GLP-1 and its stable
mimetics (e.g. Byetta) also cause modest weight loss in animal
models (Mack, C. M., et al. (2006) Int J Obes (Lond) 30: 1332-1340)
and in Type 2 diabetic patients (DeFronzo, R. A., et al. (2005)
Diabetes Care 28: 1092-1100; Buse, J. B., et al. (2010) Diabetes
Care 33: 1255-1261). Glucagon infusion reduces food intake in man
(Geary, N., et al. (1992) Am J Physiol 262: R975-980), while
continuous glucagon treatment of adipose tissue also promotes
lipolysis (Heckemeyer, C. M., et al. (1983) Endocrinology 113:
270-276) and weight loss (Salter, J. M., et al. (1960) Metabolism
9: 753-768; Chan, E. K., et al. (1984) Exp Mol Pathol 40: 320-327).
Glucagon has wide-ranging effects on energy metabolism (Heppner, K.
M., et al. (2010) Physiol Behav)). Glucagon, or analogs, can be
used in a diagnostic mode for temporary paralysis of the intestinal
tract. Thus at least two of the products from PC processing of the
preproglucagon protein are linked to satiety and metabolic
effects.
[0903] In rodents, repeated intraperitoneal administration of OXM,
a third product of preproglucagon, has been associated with reduced
white adipose tissue and a reduction in weight compared with
controls (Dakin, C. L., et al. (2004) Endocrinology 145:
2687-2695). Oxm reduced food intake by 19.3% during an intravenous
infusion administration to normal-weight humans and this effect
continues for more than 12 hr. after infusion (Cohen, M. A., et al.
(2003) J Clin Endocrinol Metab 88: 4696-4701). Treatment of
volunteers over a 4 week period resulted in a sustained satiety
effect and weight loss reflecting a decrease in body fat (Wynne,
K., et al. (2005) Diabetes 54: 2390-2395).
[0904] OXM is structurally homologous with GLP-1 and glucagon, and
activates both the glucagon receptor (GCGR) and the GLP-1 receptor
(GLP1R), but with 10 to 100 fold less potency than the eponymous
ligands. In addition, study of OXM interactions with GLP1R suggest
it might have different effects on beta-arrestin recruitment
compared to GLP-1 (Jorgensen, R., et al. (2007) J Pharmacol Exp
Ther 322: 148-154), thus acting as a "biased" ligand. A unique
receptor for OXM was sought for a number of years, but has not yet
been elucidated and it is assumed to act through the GLP1R and GCGR
pathways. Accordingly, provided herein are methods for surfactant
modification of gut peptides that allow for induction of satiety,
weight loss, alleviation of insulin resistance and/or delay in
progression of pre-diabetes to diabetes.
GLP-1
[0905] In view of the complex and interacting behavior of the
products of the preproglucagon protein on satiety and metabolism
described above, workers from multiple groups have studied the
structure activity relationships on GLP-1 and glucagon structure.
Residues throughout the sequences were shown to accept replacement.
For example, replacement by Ala is well accepted in the N-terminal
region of GLP-1, especially at 2, 3, 5, 8, 11, and 12 (Adelhorst,
K., et al. (1994) J Biol Chem 269: 6275-6278).
[0906] It was shown that chimeric analogs with the ability to bind
to GLP1R and GLCR could be achieved by grafting C-terminal residues
from GLP-1 onto the N-terminus of glucagon (Hjorth, S. A., et al.
(1994) J Biol Chem 269: 30121-30124). The residue at position 3
(acidic Glu in GLP1 or neutral Gln in Glucagon or OXM) reduces the
affinity of glucagon (Runge. S., et al. (2003) J Biol Chem 278:
28005-28010) or OXM (Pocai, A., et al. (2009) Diabetes 58:
2258-2266) for the GlP1R. The effect on metabolic profile of
animals treated with stabilized analogs of GLP-1 or glucagon or OXM
with Gln in position 3 was studied (Day, J. W., et al. (2009) Nat
Chem Biol 5: 749-757; Druce, M. R., et al. (2009) Endocrinology
150: 1712-1722; Pocai, A., et al. (2009) Diabetes 58: 2258-2266).
These analogs were designed to have agonistic action on both GLP1R
and on GCGR (Day, J. W., et al. US 2010/0190701 A1).
[0907] Chimeric analogs should have the desirable effects of the
parent hormones acting on their receptors, and therefore similar to
the effects of OXM, which apparently acts on both GLP-1R and GLCR:
glucose-dependent insulin secretion and satiety, coupled with
lipolysis and increased burning of fat due to glucagon. The analogs
were shown to cause the desired effects of decreased weight and
increased burning of fat. Such a profile would be attractive in the
treatment of obesity, but a major challenge in obesity treatment is
compliance. Although currently known full length analogs of
glucagon and OXM, respectively, with affinity for both GLP-1R and
GLCR can result in weight loss, these analogs are not optimized for
the high bioavailability, pharmaceutical properties, and convenient
delivery to patients that are necessary for optimal drug treatment
regimens. Accordingly, provided herein are analogs of gut peptides
(e.g., GLP, OXM, glucagon or the like) that allow for high
bioavailability and/or long lasting effects for improved
therapeutic outcome in treatment of conditions such as obesity
and/or diabetes and/or the metabolic syndrome.
[0908] Additional factors for optimized treatment of the metabolic
syndrome and diabetes with OXM-like molecules relate to the
duration of treatment and the amount of glucagon action. For
example, continuous treatment with analogs that activate GLP-1 and
glucagon receptors (the OXM pharmacological profile) can result in
very large and rapid loss of fat mass (Day, J. W., et al. (2009)
Nat Chem Biol 5: 749-757), but it can also cause the loss of lean
muscle mass (Kosinski, J. R., et al. (2012) Obesity (Silver
Spring): doi: 10.1038/oby.2012.67), which is unfavorable for a
pharmaceutical in this class. For example, in the research article
by Kosinski, J. R., et al., the natural hormone Oxm is administered
continuously for 14 days from an Alzet minipump and results in a
decrease of 30% in fat mass, but also caused a 7% decrease in lean
mass (muscle).
[0909] Glucagon action is known to stimulate glycogenolysis,
lipolysis and the increased burning of fat, but can also have
catabolic effects on muscle. A successful treatment using an agent
that combines GLP-1 and glucagon action (the OXM profile) will need
to optimally cause the satiety and potentiated glucose-dependent
insulin secretion of a GLP-1 analog with a judicious amount of
glucagon action (fat burning). In addition, intermittent use of
such an agent will provide the desired clinical profile of
moderate, continuous weight loss, through loss of fat mass, with
minimized loss of lean mass. Provided herein are molecules with a
desirable combination of GLP-1 and OXM action as well as a tunable
pharmacokinetic/pharmacodynamic profile to allow optimum use in
therapy (for example in the metabolic syndrome, diabetes, obesity,
and the like).
[0910] In one embodiment, the compounds of Formula 3-I-A, 3-III-A,
3-III-B and 3-V are designed to provide either glucagon-like
activity or GLP-1 like activity. In a further embodiment, the
compounds of Formula 3-I-A, 3-III-A, 3-III-B and 3-V provide
tunable activity. For example, in one instance, the peptide
products described herein (e.g., compounds in Table 3 of FIG. 8 and
Table 4 of FIG. 9) have an EC50 of less than about 500 nM,
preferably less than about 50 nM, more preferably less than about
20 nM at receptors for both glucagon, and GLP-1. In another
instance, the peptide products described herein (e.g., compounds in
Table 3 of FIG. 8 and Table 4 of FIG. 9) are more potent (e.g.,
EC50 of less than 10 nM, preferably less than 5 nM, more preferably
about 1 nM) for the GLP-1 receptor and less potent for the glucagon
receptor (e.g., EC50 of less than 50 nM, preferably less than about
20 nM, more preferably about 5 nM) for the glucagon receptor. This
tunability of biological activity allows for some retention of a
judicious amount of glucagon action, thereby allowing for fat
burning to occur, while also retaining the beneficial effects of
potentiated glucose-dependent insulin secretion. OXM is
structurally homologous with GLP-1 and glucagon, and activates both
the glucagon receptor (GCGR) and the GLP-1 receptor (GLP1R).
Accordingly, in some embodiments, the compounds of Formula 3-I-A,
3-III-A, 3-III-B and 3-V provide a tunable OXM-like biological
activity. In some specific embodiments, the peptide products
described herein comprise a peptide having amino acid residues 1-17
of GLP-1 and/or analogs thereof (e.g., analogs comprising modified
non-natural amino acid replacements as described herein, cyclized
lactam linkages as described herein, surfactant modifications as
described herein, or a combination thereof). In some other
embodiments, the peptide products described herein comprise a
peptide having amino acid residues 1-16 of GLP-1 and/or analogs
thereof (e.g., analogs comprising modified non-natural amino acid
replacements as described herein, cyclized lactam linkages as
described herein, surfactant modifications as described herein, or
a combination thereof). In additional embodiments, the peptide
products described herein comprise a peptide having amino acid
residues 1-18 of GLP-1 and/or analogs thereof (e.g., analogs
comprising modified non-natural amino acid replacements as
described herein, cyclized lactam linkages as described herein,
surfactant modifications as described herein, or a combination
thereof). Additionally the peptide products described herein
comprise one or more residues (e.g., Aib, Ac4C) which provide helix
stabilization of the designed compounds of Formula 3-I-A, 3-III-A,
3-III-B and 3-V, and compounds in Table 3 of FIG. 8 and Table 4 of
FIG. 9.
[0911] It is believed that the glucagon subfamily of ligands bind
to their receptors in a two domain mode common to a number of the
class B of receptors (secretin class, G Protein-coupled Receptors
(GPCR)). For GLP-1 it is felt that there is a N-terminal region of
from residue 1 to about residue 16 which binds to the tops of the
transmembrane helicies (juxtomembrane region) and a helical
C-terminal region from 17 to 31 which binds to the large,
extracellular, N-terminal extension (ECD) of the receptor. The
binding of these ligands focuses on the fact that N-terminally
truncated analogs of these peptide ligands can still retain
substantial binding affinity and selectivity for just the isolated
ECD region of the receptor. Therefore it has been suggested that
the N-terminal region is responsible for receptor activation while
the C-terminal region is responsible for binding. It recently has
been shown that short, N-terminal analogs of GLP-1 can be both
potent binders as well as receptor activators (Mapelli, C., et al.
(2009) J Med Chem 52: 7788-7799; Haque, T. S., et al. (2010)
Peptides 31: 950-955; Haque, T. S., et al. (2010) Peptides 31:
1353-1360).
[0912] In addition, study of an x-ray crystal structure (Runge, S.,
et al. (2008) J Biol Chem 283: 11340-7) of the N-terminal region of
the GLP1R with a truncated antagonist analogs of the GLP-1 mimic,
exendin-4 (Byetta), bound in this region show that a critical
ligand-binding region in the ECD is of high hydrophobicity (FIG.
10). The sequence of exendin-4 beyond Glu15 interacts as an
amphiphilic helix with this very hydrophobic region (Val.sup.19*,
Phe.sup.22*, Trp.sup.25*, Leu.sup.26*). In one embodiment,
truncated N-terminal fragments of GLP-1 or glucagon are modified to
bind to GLCR and are covalently linked to a surfactant. The
hydrophobic 1'-alkyl portion of the surfactant mimics and replaces
the C-terminal region of the native hormone ligand and increases
the peptides potency, efficacy, and duration of action. In
addition, such analogs have major advantages due to their smaller
size, which reduces their complexity, synthesis costs, and
susceptibility to proteolysis. In addition smaller peptides are
more readily absorbed through the nasal mucosa or gut enterocyte
barrier.
[0913] Hypoglycemia is a condition of low blood sugar that can be
life-threatening and is increasingly seen as more aggressive
treatment of elevated blood sugar by intensive insulin treatment is
being used in more patients. Hypoglycemia is seen when blood
glucose levels drop too low to provide enough energy to the brain
and muscles for the body's activities. Glucagon can be used to
treat this condition and does so by stimulating the liver to break
down glycogen to generate glucose and cause the blood glucose
levels to rise toward the normal value. Analogs of glucagon that
retain the ability to activate the GLCR may be used to achieve this
desirable effect on blood glucose levels.
[0914] Analogs of GLP-1 that activate the GLP1R stimulate the
production and, in the presence of elevated blood glucose levels,
release of insulin from the pancreas. This action results in
efficient control and normalization of blood glucose levels, as
seen with current products such as exenatide (Byetta.RTM.). In
addition, such products appear to produce a decreased appetite and
slow the movement of food from the stomach. Thus they are effective
in treatment of diabetes through multiple mechanisms. Analogs that
combine the effects of glucagon and GLP-1 that activate both the
GLCR and the GLP1R may offer a benefit in the treatment of diabetes
through a concerted action to suppress appetite, release insulin in
a glucose-dependent fashion, assist in the protection from
hypoglycemia and accelerate the burning of fat.
[0915] Such methods for treating hyperglycemia, including diabetes,
diabetes mellitus type I, diabetes mellitus type II, or gestational
diabetes, either insulin-dependent or non-insulin dependent, are
expected to be useful in reducing complications of diabetes
including nephropathy, retinopathy and vascular disease.
Applications in cardiovascular disease encompass microvascular as
well as macrovascular disease (Davidson, M. H., (2011) Am J Cardiol
108[suppl]:33.alpha.-41B; Gejl, M., et al. (2012) J Clin Endocrinol
Metab 97:doi:10.1210/jc.2011-3456), and include treatment for
myocardial infarction. Such methods for reducing appetite or
promoting loss of body weight are expected to be useful in reducing
body weight, preventing weight gain, or treating obesity of various
causes, including drug-induced obesity, and reducing complications
associated with obesity including vascular disease (coronary artery
disease, stroke, peripheral vascular disease, ischemia reperfusion,
etc.), hypertension, onset of diabetes type II, hyperlipidemia and
musculoskeletal diseases.
[0916] As used herein, the term glucagon or GLP-1 analogs includes
all pharmaceutically acceptable salts or esters thereof.
[0917] In one aspect, the peptides that are covalently modified and
are suitable for methods described herein are truncated analogs of
glucagon and/or the related hormone GLP-1, including and not
limited to:
TABLE-US-00030 Glucagon: (SEQ. ID. NO. 632)
His.sub.1-Ser.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5
Phe.sub.6-Thr.sub.7-Ser.sub.8-Asp.sub.9-Tyr.sub.10-
Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp.sub.15-Ser.sub.16-Arg.sub.-
17-Arg.sub.18-Ala.sub.19-
Gln.sub.20-Asp.sub.21-Phe.sub.22-Val.sub.23-Gln.sub.24-Trp.sub.25-Leu.sub.-
26-Met.sub.27-Asn.sub.28- Thr.sub.29 Oxyntomodulin: (SEQ. ID. NO.
633) His.sub.1-Ser.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5
Phe.sub.6-Thr.sub.7-Ser.sub.8-Asp.sub.9-Tyr.sub.10-
Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp.sub.15-Ser.sub.16-Arg.sub.-
17-Arg.sub.18-Ala.sub.19-
Gln.sub.20-Asp.sub.21-Phe.sub.22-Val.sub.23-Gln.sub.24-Trp.sub.25-Leu.sub.-
26-Met.sub.27-Asn.sub.28-
Thr.sub.29-Lys.sub.30-Arg.sub.31-Asn.sub.32-Arg.sub.33-Asn.sub.34-Asn.sub.-
35-Ile.sub.36-Ala.sub.37 GLP-1 (using glucagon numbering): (SEQ.
ID. NO. 634) His.sub.1-Ala.sub.2-Glu.sub.3-Gly.sub.4-Thr.sub.5
Phe.sub.6-Thr.sub.7-Ser.sub.8-Asp.sub.9-Val.sub.10-
Ser.sub.11-Ser.sub.12-Tyr.sub.13-Leu.sub.14-Glu.sub.15-Gly.sub.16-Gln.sub.-
17-Ala.sub.18-Ala.sub.19-
Lys.sub.20-Glu.sub.21-Phe.sub.22-Ile.sub.23-Al.sub.a24-Trp.sub.25-Leu.sub.-
26-Val.sub.27-Lys.sub.28- Gly.sub.29-Arg.sub.30
[0918] In some embodiments, a peptide product described herein has
the structure of Formula 3-V:
TABLE-US-00031 FORMULA 3-V (SEQ. ID. NO. 635)
aa.sub.1-aa.sub.2-aa.sub.3-aa.sub.4-aa.sub.5-aa.sub.6-aa.sub.7-aa.sub.8-aa-
.sub.9-aa.sub.10-aa.sub.11-aa.sub.12-aa.sub.13-
aa.sub.14-aa.sub.15-aa.sub.16-aa.sub.17-aa.sub.18-aa.sub.19-aa.sub.20-a.su-
b.a21-aa.sub.22-aa.sub.23-aa.sub.24-
aa.sub.25-aa.sub.26-aa.sub.27-aa.sub.28-aa.sub.29-aa.sub.30-aa.sub.31-aa.s-
ub.32-aa.sub.33-aa.sub.34-aa.sub.35- aa.sub.36-aa.sub.37-Z
wherein: [0919] U is a linking amino acid; [0920] X is a
surfactant-linked to the side chain of U; [0921] Z is OH, or
--NH--R.sup.3, wherein R.sup.3 is H or C.sub.1-C.sub.12 substituted
or unsubstituted alklyl; [0922] aa.sub.1 is His, N--Ac-His,
pGlu-His or N--R.sup.3-His; [0923] aa.sub.2 is Ser, Ala, Gly, Aib,
Ac4c or Ac5c; [0924] aa.sub.3 is Gln, or Cit; [0925] aa.sub.4 is
Gly, or D-Ala; [0926] aa.sub.5 is Thr, or Ser; [0927] aa.sub.6 is
Phe, Trp, F2Phe, Me2Phe, or Nal(2); [0928] aa.sub.7 is Thr, or Ser;
[0929] aa.sub.8 is Ser, or Asp; [0930] aa.sub.9 is Asp, or Glu;
[0931] aa.sub.10 is Tyr, Leu, Met, Nal(2), Bip, or Bip2EtMeO;
[0932] aa.sub.11 is Ser, Asn, or U(X); [0933] aa.sub.12 is Lys,
Glu, Ser, Arg, or U(X); [0934] aa.sub.13 is absent, Tyr, Gln, Cit,
or U(X); [0935] aa.sub.14 is absent, Leu, Met, Nle, or U(X); [0936]
aa.sub.15 is absent, Asp, Glu, or U(X); [0937] aa.sub.16 is absent,
Ser, Gly, Glu, Aib, Ac5c, Lys, Arg, or U(X); [0938] aa.sub.17 is
absent, Arg, hArg, Gln, Glu, Cit, Aib, Ac4c, Ac5c, or U(X); [0939]
aa.sub.18 is absent, Arg, hArg, Ala, Aib, Ac4c, Ac5c, or U(X);
[0940] aa.sub.19 is absent, Ala, Val, Aib, Ac4c, Ac5c, or U(X);
[0941] aa.sub.20 is absent, Gln, Lys, Arg, Cit, Glu, Aib, Ac4c,
Ac5c, or U(X); [0942] aa.sub.21 is absent, Asp, Glu, Leu, Aib,
Ac4c, Ac5c, or U(X); [0943] aa.sub.22 is absent, Phe, Trp, Nal(2),
Aib, Ac4c, Ac5c, or U(X); [0944] aa.sub.23 is absent, Val, Ile,
Aib, Ac4c, Ac5c, or U(X); [0945] aa.sub.24 is absent, Gln, Ala,
Glu, Cit, or U(X); [0946] aa.sub.25 is absent, Trp, Nal(2), or
U(X); [0947] aa.sub.26 is absent, Leu, U(X); [0948] aa.sub.27 is
absent, Met, Val, Nle, Lys, or U(X); [0949] aa.sub.28 is absent,
Asn, Lys, or U(X); [0950] aa.sub.29 is absent, Thr, Gly, Aib, Ac4c,
Ac5c, or U(X); [0951] aa.sub.30 is absent, Lys, Aib, Ac4c, Ac5c, or
U(X); [0952] aa.sub.31 is absent, Arg, Aib, Ac4c, Ac5c, or U(X);
[0953] aa.sub.32 is absent, Asn, Aib, Ac4c, Ac5c, or U(X); [0954]
aa.sub.33 is absent, Arg, Aib, Ac5c, or U(X); [0955] aa.sub.34 is
absent, Asn, Aib, Ac4c, Ac5c, or U(X); [0956] aa.sub.35 is absent,
Asn, Aib, Ac4c, Ac5c, or U(X); [0957] aa.sub.36 is absent, Ile,
Aib, Ac4c, Ac5C, or U(X); [0958] aa.sub.36 is absent, Ala, Aib,
Ac4c, Ac5C, or U(X); [0959] aa.sub.37 absent or U(X); [0960]
provided that one, or at least one of aa.sub.11-aa.sub.37 is
U(X).
[0961] In specific embodiments, the linking amino acid U, is a
diamino acid like Lys or Orn, X is a modified surfactant from the
1-alkyl glycoside class linked to U, and Z is OH, or --NH--R.sub.2,
wherein R.sup.3 is H or C.sub.1-C.sub.12; or a PEG chain of less
than 10 Da.
[0962] In some embodiments, a peptide product described herein has
the structure of Formula III-B:
TABLE-US-00032 FORMULA 3-III-B (SEQ. ID. NO. 305)
His.sub.1-aa.sub.2-aa.sub.3-Gly.sub.4-Thr.sub.5-aa.sub.6-Thr.sub.7-Ser.sub-
.8-Asp.sub.9-aa.sub.10-aa.sub.11-
aa.sub.12-aa.sub.13-aa.sub.14-aa.sub.15-aa.sub.16-aa.sub.17-aa.sub.18-aa.s-
ub.19-aa.sub.20-aa.sub.21-aa.sub.22- aa.sub.23-Z
[0963] wherein: [0964] Z is OH, or --NH--R.sup.3, wherein R.sup.3
is H or substituted or unsubstituted C.sub.1-C.sub.12 alkyl; or a
PEG chain of less than 10 Da; [0965] aa.sub.2 is Ser, Ala, Gly,
Aib, Ac4c, or Ac5c; [0966] aa.sub.3 is Gln, or Cit; [0967] aa.sub.6
is Phe, Trp, F2Phe, Me2Phe, MePhe, or Nal2; [0968] aa.sub.10 is
Tyr, Leu, Met, Nal2, Bip, or Bip2EtMeO; [0969] aa.sub.11 is Ser,
Asn, or U; [0970] aa.sub.12 is Lys, Glu, Ser or U(X); [0971]
aa.sub.13 is absent or Tyr, Gln, Cit, or U(X); [0972] aa.sub.14 is
absent or Leu, Met, Nle, or U(X); [0973] aa.sub.15 is absent or
Asp, Glu, or U(X); [0974] aa.sub.16 is absent or Ser, Gly, Glu,
Aib, Ac4c, Ac5c, Lys, R, or U(X); [0975] aa.sub.17 is absent or
Arg, hArg, Gln, Glu, Cit, Aib, Ac4c, Ac5c, or U(X); [0976]
aa.sub.18 is absent or Arg, hArg, Ala, Aib, Ac4c, Ac5c, or U(X);
[0977] aa.sub.20 is absent or Ala, Val, Aib, Ac4c, Ac5c, or U(X);
[0978] aa.sub.21 is absent or Gln, Lys, Arg, Cit, Glu, Aib, Ac4c,
Ac5c, or U(X); [0979] aa.sub.21 is absent or Asp, Glu, Leu, Aib,
Ac4c, Ac5c, or U(X); [0980] aa.sub.22 is absent or Phe, Aib, Ac4c,
Ac5c, or U(X) [0981] aa.sub.23 is absent or Val, Ile, Aib, Ac4c,
Ac5c, or U(X); [0982] wherein any two of aa.sub.1-aa.sub.23 are
optionally cyclized through their side chains to form a lactam
linkage; and [0983] provided that one, or at least one of
aa.sub.14, aa.sub.17, aa.sub.18, aa.sub.19, aa.sub.20, aa.sub.21,
aa.sub.22, aa.sub.23 or aa.sub.24 is the natural or unnatural amino
acid U covalently attached to X.
[0984] In some specific embodiments of Formula 3-III-A, Formula
3-III-B and Formula 3-V, X has the structure:
##STR00045## [0985] wherein: [0986] R.sup.1a is a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl group; [0987] R.sup.1b,
R.sup.1c, and R.sup.1d are H; [0988] W.sup.1 is --(C.dbd.O)--NH--;
[0989] W.sup.2 is --O--; and [0990] R.sup.2 is a bond.
[0991] In some of the embodiments described above, R.sup.1a is a
C.sub.1-C.sub.2 alkyl group, a C.sub.1-C.sub.20 alkyl group,
C.sub.12-18 alkyl group or C.sub.14-C.sub.18 alkyl group.
[0992] In some embodiments of Formula 3-III-B, U is any linker
amino acid described herein. Table 3 of FIG. 8 and Table 4 of FIG.
9 illustrate certain examples of peptides that covalently linked
with surfactants as described herein.
[0993] Contemplated within the scope of embodiments presented
herein are peptide products of Formula 3-I-A, Formula 3-III-A,
Formula 3-III-B or Formula 3-V, wherein the peptide product
comprises one, or, more than one surfactant groups (e.g., group X
having the structure of Formula 3-I). In one embodiment, a peptide
product of Formula 3-I-A, Formula 3-III-A, Formula 3-III-B or
Formula 3-V, comprises one surfactant group. In another embodiment,
a peptide product of Formula 3-I-A, Formula 3-III-A, Formula
3-III-B or Formula 3-V, comprises two surfactant groups. In yet
another embodiment, a peptide product of Formula 3-I-A, Formula
3-III-A, Formula 3-III-B or Formula 3-V, comprises three surfactant
groups.
[0994] Recognized herein is the importance of certain portions of
SEQ. ID. NO. 632 for the treatment of conditions associated with
insulin resistance and/or cardiovascular conditions. Accordingly,
provided herein is a method of treating diabetes in an individual
in need thereof comprising administration of a therapeutically
effective amount of a glucagon analog comprising amino acid
residues aa.sub.1-aa.sub.17 of SEQ. ID. NO. 632 to the individual
in need thereof.
[0995] In a further embodiment, provided herein is a method of
treating diabetes in an individual in need thereof comprising
administration of a therapeutically effective amount of a glucagon
analog comprising amino acid residues aa.sub.1-aa.sub.18 of SEQ.
ID. NO. 632 to the individual in need thereof.
[0996] In another embodiment, provided herein is a method of
treating diabetes in an individual in need thereof comprising
administration of a therapeutically effective amount of a glucagon
analog comprising amino acid residues aa.sub.1-aa.sub.19 of SEQ.
ID. NO. 632 to the individual in need thereof.
[0997] In another embodiment, provided herein is a method of
treating diabetes in an individual in need thereof comprising
administration of a therapeutically effective amount of a glucagon
analog comprising amino acid residues aa.sub.1-aa.sub.20 of SEQ.
ID. NO. 632 to the individual in need thereof.
[0998] In an additional embodiment, the administration of the said
glucagon analog described above causes weight loss.
[0999] Recognized herein is the importance of certain portions of
SEQ. ID. NO. 303 for the treatment of conditions associated with
insulin resistance and/or cardiovascular conditions. Accordingly,
provided herein is a method of treating diabetes in an individual
in need thereof comprising administration of a therapeutically
effective amount of a glucagon analog comprising amino acid
residues aa.sub.1-aa.sub.17 of SEQ. ID. NO. 303 to the individual
in need thereof.
[1000] In a further embodiment, provided herein is a method of
treating diabetes in an individual in need thereof comprising
administration of a therapeutically effective amount of a glucagon
analog comprising amino acid residues aa.sub.1-aa.sub.18 of SEQ.
ID. NO. 303 to the individual in need thereof.
[1001] In another embodiment, provided herein is a method of
treating diabetes in an individual in need thereof comprising
administration of a therapeutically effective amount of a glucagon
analog comprising amino acid residues aa.sub.1-aa.sub.19 of SEQ.
ID. NO. 303 to the individual in need thereof.
[1002] In another embodiment, provided herein is a method of
treating diabetes in an individual in need thereof comprising
administration of a therapeutically effective amount of a glucagon
analog comprising amino acid residues aa.sub.1-aa.sub.20 of SEQ.
ID. NO. 303 to the individual in need thereof.
[1003] In an additional embodiment, the administration of the said
glucagon analog described above causes weight loss.
[1004] In any of the embodiments described above, the said glucagon
analog is modified with a surfactant X of Formula 3-I:
##STR00046## [1005] wherein: [1006] R.sup.1a is independently, at
each occurrence, a bond, H, a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl group, a substituted or unsubstituted
alkoxyaryl group, a substituted or unsubstituted aralkyl group, or
a steroid nucleus containing moiety; [1007] R.sup.1b, R.sup.1c, and
R.sup.1d are each, independently at each occurrence, a bond, H, a
substituted or unsubstituted C.sub.1-C.sub.30 alkyl group, a
substituted or unsubstituted alkoxyaryl group, or a substituted or
unsubstituted aralkyl group; [1008] W.sup.1 is independently, at
each occurrence. --CH.sub.2--, --CH.sub.2--O--, --(C.dbd.O),
--(C.dbd.O)--O--, --(C.dbd.O)--NH--, --(C.dbd.S)--,
--(C.dbd.S)--NH--, or --CH.sub.2--S--; [1009] W.sup.2 is --O--,
--CH.sub.2-- or --S--; [1010] R.sup.2 is independently, at each
occurrence, a bond to U, H, a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl group, a substituted or unsubstituted
alkoxyaryl group, or a substituted or unsubstituted aralkyl group,
--NH.sub.2, --SH, C.sub.2-C.sub.4-alkene, C.sub.2-C.sub.4-alkyne,
--NH(C.dbd.O)--CH.sub.2--Br, --(CH.sub.2).sub.m-maleimide, or
--N.sub.3; [1011] n is 1, 2 or 3; and [1012] m is 1-10.
[1013] In a specific embodiment, the said glucagon analog is
modified with a surfactant, X having the structure:
##STR00047## [1014] wherein: [1015] R.sup.1a is a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl group; [1016] R.sup.1b,
R.sup.1c, and R.sup.1d are H; [1017] W.sup.1 is --(C.dbd.O)--NH--;
[1018] W.sup.2 is --O--; and [1019] R.sup.2 is a bond.
[1020] In some of the embodiments described above, R.sup.1a is a
C.sub.1-C.sub.20 alkyl group, a C.sub.8-C.sub.20 alkyl group,
C.sub.12-C.sub.18 alkyl group or C.sub.14-C.sub.18 alkyl group.
[1021] Also provided herein is a method of treating a
cardiovascular disease in an individual in need thereof comprising
administration of a therapeutically effective amount of a glucagon
analog comprising amino acid residues aa.sub.1-aa.sub.17 of SEQ.
ID. NO. 632 to the individual in need thereof.
[1022] Also provided herein is a method of treating a
cardiovascular disease in an individual in need thereof comprising
administration of a therapeutically effective amount of a glucagon
analog comprising amino acid residues aa.sub.1-aa.sub.18 of SEQ.
ID. NO. 632 to the individual in need thereof.
[1023] Also provided herein is a method of treating a
cardiovascular disease in an individual in need thereof comprising
administration of a therapeutically effective amount of a glucagon
analog comprising amino acid residues aa.sub.1-aa.sub.19 of SEQ.
ID. NO. 632 to the individual in need thereof.
[1024] Also provided herein is a method of treating a
cardiovascular disease in an individual in need thereof comprising
administration of a therapeutically effective amount of a glucagon
analog comprising amino acid residues aa.sub.1-aa.sub.20 of SEQ.
ID. NO. 632 to the individual in need thereof.
[1025] In some cases for the embodiments described above, the said
glucagon analog is administered when the cardiovascular disease is
associated with an ischemic event.
[1026] Also provided herein is a method of treating a
cardiovascular disease in an individual in need thereof comprising
administration of a therapeutically effective amount of a glucagon
analog comprising amino acid residues aa.sub.1-aa.sub.17 of SEQ.
ID. NO. 303 to the individual in need thereof.
[1027] Also provided herein is a method of treating a
cardiovascular disease in an individual in need thereof comprising
administration of a therapeutically effective amount of a glucagon
analog comprising amino acid residues aa.sub.1-aa.sub.18 of SEQ.
ID. NO. 303 to the individual in need thereof.
[1028] Also provided herein is a method of treating a
cardiovascular disease in an individual in need thereof comprising
administration of a therapeutically effective amount of a glucagon
analog comprising amino acid residues aa.sub.1-aa.sub.19 of SEQ.
ID. NO. 303 to the individual in need thereof.
[1029] Also provided herein is a method of treating a
cardiovascular disease in an individual in need thereof comprising
administration of a therapeutically effective amount of a glucagon
analog comprising amino acid residues aa.sub.1-aa.sub.20 of SEQ.
ID. NO. 303 to the individual in need thereof.
[1030] In some cases for the embodiments described above, the said
glucagon analog is administered when the cardiovascular disease is
associated with an ischemic event.
[1031] In any of the embodiments described above, the said glucagon
analog is modified with a surfactant X of Formula 3-I:
##STR00048## [1032] wherein: [1033] R.sup.1a is independently, at
each occurrence, a bond, H, a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl group, a substituted or unsubstituted
alkoxyaryl group, a substituted or unsubstituted aralkyl group, or
a steroid nucleus containing moiety; [1034] R.sup.1b, R.sup.1c, and
R.sup.1d are each, independently at each occurrence, a bond, H, a
substituted or unsubstituted C.sub.1-C.sub.30 alkyl group, a
substituted or unsubstituted alkoxyaryl group, or a substituted or
unsubstituted aralkyl group; [1035] W.sup.1 is independently, at
each occurrence, --CH.sub.2--, --CH.sub.2--O--, --(C.dbd.O),
--(C.dbd.O)--O--, --(C.dbd.O)--NH--, --(C.dbd.S)--,
--(C.dbd.S)--NH--, or --CH.sub.2--S--; [1036] W.sup.2 is --O--,
--CH.sub.2-- or --S--; [1037] R.sup.2 is independently, at each
occurrence, a bond to U, H, a substituted or unsubstituted
C.sub.1-C.sub.30 alkyl group, a substituted or unsubstituted
alkoxyaryl group, or a substituted or unsubstituted aralkyl group,
--NH.sub.2, --SH, C.sub.2-C.sub.4-alkene, C.sub.2-C.sub.4-alkyne,
--NH(C.dbd.O)--CH.sub.2--Br, --(CH.sub.2).sub.m-maleimide, or
--N.sub.3; [1038] n is 1, 2 or 3; and [1039] m is 1-10.
[1040] In a specific embodiment, the said glucagon analog is
modified with a surfactant, X having the structure:
##STR00049## [1041] wherein: [1042] R.sup.1a is a substituted or
unsubstituted C.sub.1-C.sub.30 alkyl group; [1043] R.sup.1b,
R.sup.1c, and R.sup.1d are H; [1044] W.sup.1 is --(C.dbd.O)--NH--;
[1045] W.sup.2 is --O--; and [1046] R.sup.2 is a bond.
[1047] In some of the embodiments described above, R.sup.1a is a
C.sub.1-C.sub.20 alkyl group, a C.sub.1-C.sub.2 alkyl group,
C.sub.12-C.sub.18 alkyl group or C.sub.14-C.sub.18 alkyl group.
[1048] Modifications at the amino or carboxyl terminus may
optionally be introduced into the peptides (e.g., glucagon or
GLP-1) (Nestor, J. J., Jr. (2009) Current Medicinal Chemistry 16:
4399-4418). For example, the peptides can be truncated or acylated
on the N-terminus to yield peptides analogs exhibiting low
efficacy, partial agonist and antagonist activity, as has been seen
for some peptides (Gourlet, P., et al. (1998) Eur J Pharmacol 354:
105-111, Gozes, I. and Furman, S. (2003) Curr Pharm Des 9:
483-494), the contents of which is incorporated herein by
reference). For example, deletion of the first 6 residues of bPTH
yields antagonistic analogs (Mahaffey, J. E., et al. (1979) J Biol
Chem 254: 6496-6498; Goldman, M. E., et al. (1988) Endocrinology
123: 2597-2599) and a similar operation on peptides described
herein generates potent antagonistic analogs. Other modifications
to the N-terminus of peptides, such as deletions or incorporation
of D-amino acids such as D-Phe also can give potent and long acting
agonists or antagonists when substituted with the modifications
described herein such as long chain alkyl glycosides. Such agonists
and antagonists also have commercial utility and are within the
scope of contemplated embodiments described herein.
[1049] Also contemplated within the scope of embodiments described
herein are surfactants covalently attached to peptide analogs,
wherein the native peptide is modified by acetylation, acylation,
PEGylation, ADP-ribosylation, amidation, covalent attachment of a
lipid or lipid derivative, covalent attachment of
phosphotidylinositol, cross-linking, cyclization, disulfide bond
formation, demethylation, formation of covalent cross-link
formation of cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation, racemization,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins, such as arginylation, and ubiquitination. See,
for instance, (Nestor, J. J., Jr. (2007) Comprehensive Medicinal
Chemistry II 2: 573-601, Nestor, J. J., Jr. (2009) Current
Medicinal Chemistry 16: 4399-4418, Creighton, T. E. (1993, Wold, F.
(1983) Posttranslational Covalent Modification of Proteins 1-12,
Seifter, S. and Englard, S. (1990) Methods Enzymol 182: 626-646,
Rattan, S. I., et al. (1992) Ann N Y Acad Sci 663: 48-62). Also
contemplated within the scope of embodiments described herein are
peptides that are branched or cyclic, with or without branching.
Cyclic, branched and branched circular peptides result from
post-translational natural processes and are also made by suitable
synthetic methods. In some embodiments, any peptide product
described herein comprises a peptide analog described above that is
then covalently attached to an alkyl-glycoside surfactant
moiety.
[1050] Also contemplated within the scope of embodiments presented
herein are peptide chains substituted in a suitable position by the
substitution of the analogs claimed herein by acylation on a linker
amino acid, at for example the .epsilon.-position of Lys, with
fatty acids such as octanoic, decanoic, dodecanoic, tetradecanoic,
hexadecanoic, octadecanoic, 3-phenylpropanoic acids and the like,
with saturated or unsaturated alkyl chains (Zhang. L. and Bulaj, G.
(2012) Curr Med Chem 19: 1602-1618). Non-limiting, illustrative
examples of such analogs are:
TABLE-US-00033 (SEQ. ID. NO. 636)
His.sub.1-Aib.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.-
sub.8-Asp.sub.9-Tyr.sub.10-
Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp.sub.15-Ser.sub.16-Arg.sub.-
17-Lys(N- epsilon-dodecanoyl).sub.18-Aib.sub.19-NH.sub.2, (SEQ. ID.
NO. 637)
His.sub.1-Aib.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.-
sub.8-Asp.sub.9-Tyr.sub.10-
Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp.sub.15-Ser.sub.16-Arg.sub.-
17-Lys(N- epsilon-tetradecanoyl).sub.18-Ac4c.sub.19-NH.sub.2 (SEQ.
ID. NO. 638)
His.sub.1-Aib.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.-
sub.8-Asp.sub.9-Tyr.sub.10-
Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp.sub.15-Ser.sub.16-Arg.sub.-
17-Lys(N- epsilon-hexadecanoyl).sub.18-Aib.sub.19-NH.sub.2, (SEQ.
ID. NO. 639)
His.sub.1-Aib.sub.2-Gln3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.sub.8-
-Asp.sub.9-Tyr.sub.10-
Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp.sub.15-Aib.sub.16-Arg.sub.-
17-Lys(N- epsilon-dodecanoyl).sub.18-NH.sub.2, (SEQ. ID. NO. 640)
His.sub.1-Aib.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.-
sub.8-Asp.sub.9-Tyr.sub.10-
Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp.sub.15-Aib.sub.16-Arg.sub.-
17-Lys(N- epsilon-tetradecanoyl).sub.18-NH.sub.2, (SEQ. ID. NO.
641)
His.sub.1-Aib2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.sub.8-
-Asp.sub.9-Tyr.sub.10-
Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp.sub.15-Aib.sub.16-Arg.sub.-
17-Lys(N- epsilon-hexadecanoyl).sub.18-NH.sub.2, (SEQ. ID. NO. 642)
His.sub.1-Aib.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.-
sub.8-Asp.sub.9-Tyr.sub.10-
Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp.sub.15-Ser.sub.16-Arg.sub.-
17-Lys(N- epsilon-(gamma-glutamyl)-N-alpha-tetradecanoyl)).sub.18-
Aib.sub.19-NH.sub.2, and the like.
[1051] In further embodiments, a peptide chain is optionally
substituted in a suitable position by reaction on a linker amino
acid, for example the sulfhydryl of Cys, with a spacer and a
hydrophobic moiety such as a steroid nucleus, for example a
cholesterol moiety. In some of such embodiments, the modified
peptide further comprises one or more PEG chains. Non-limiting
examples of such molecules are:
TABLE-US-00034 (SEQ. ID. NO. 643)
His.sub.1-Aib.sub.2-Gln.sub.3-Gly4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.sub.8-
-Asp.sub.9-Tyr.sub.10-
Ser.sub.11-Lys.sub.12-Tyr.sub.13-Leu.sub.14-Asp.sub.15-Aib.sub.16-Arg.sub.-
17-Cys(S-(3-
(PEG4-aminoethylacetamide-Cholesteroll))).sub.18-Aib.sub.19-
NH.sub.2, (SEQ. ID. NO. 644)
His.sub.1-Aib.sub.2-Gln.sub.3-Gly.sub.4-Thr.sub.5-Phe.sub.6-Thr.sub.7-Ser.-
sub.8-Asp.sub.9-Tyr.sub.10-
Ser.sub.11-cyclo(Glu.sub.12-Tyr.sub.13-Leu.sub.14-Asp.sub.15-Lys.sub.16)-A-
rg.sub.17-Cys
(S-(3-(PEG4-aminoethylacetamide-Cholesterol))).sub.18-
NH.sub.2.
[1052] Aside from the twenty standard amino acids, there are a vast
number of "nonstandard amino acids" or unnatural amino acids that
are known to the art and that may be incorporated in the compounds
described herein, as described above. Other nonstandard amino acids
are modified with reactive side chains for conjugation (Gauthier,
M. A. and Klok, H. A. (2008) Chem Commun (Camb) 2591-2611; de
Graaf, A. J., et al. (2009) Bioconjug Chem 20: 1281-1295). In one
approach, an evolved tRNA/tRNA synthetase pair and is coded in the
expression plasmid by the amber suppressor codon (Deiters, A, et
al. (2004). Bio-org. Med. Chem. Lett. 14, 5743-5). For example,
p-azidophenylalanine was incorporated into peptides and then
reacted with a functionalized surfactant, or a PEG polymer having
an acetylene moiety in the presence of a reducing agent and copper
ions to facilitate an organic reaction known as "Huisgen [3+2]
cycloaddition." A similar reaction sequence using the reagents
described herein containing an acetylene modified alkyl glycoside
or PEG modified glycoside will result in PEGylated or alkyl
glycoside modified peptides. For peptides of less than about 50
residues, standard solid phase synthesis is used for incorporation
of said reactive amino acid residues at the desired position in the
chain. Such surfactant-modified peptides and/or proteins offer a
different spectrum of pharmacological and medicinal properties than
peptides modified by PEG incorporation alone.
[1053] The skilled artisan will appreciate that numerous
permutations of the peptide analogs are possible and, provided that
an amino acid sequence has an incorporated surfactant moiety, will
possess the desirable attributes of surfactant modified peptide
products described herein.
Certain Definitions
[1054] As used in the specification, "a" or "an" means one or more.
As used in the claim(s), when used in conjunction with the word
"comprising," the words "a" or "an" mean one or more. As used
herein, "another" means at least a second or more.
[1055] As used herein, the one- and three-letter abbreviations for
the various common amino acids are as recommended in Pure Appl.
Chem. 31, 639-645 (1972) and 40, 277-290 (1974) and comply with 37
CFR .sctn. 1.822 (55 FR 18245, May 1, 1990). The abbreviations
represent L-amino acids unless otherwise designated as D- or DL.
Certain amino acids, both natural and non-natural, are achiral,
e.g., glycine, .alpha.-amino-isobutyric acid (Aib). All peptide
sequences are presented with the N-terminal amino acid on the left
and the C-terminal amino acid on the right.
[1056] An "alkyl" group refers to an aliphatic hydrocarbon group.
Reference to an alkyl group includes "saturated alkyl" and/or
"unsaturated alkyl", i.e., an alkene or an alkyne. The alkyl group,
whether saturated or unsaturated, includes branched, straight
chain, or cyclic groups. An alkyl group is optionally substituted
with substituents including and not limited to oxo, halogen, aryl,
cycloalkyl, hydrophobic natural product such as a steroid, an
aralkyl chain (including alkoxyaryl), alkyl chain containing an
acyl moiety, or the like. In some embodiments, an alkyl group is
linked to the N.alpha.-position of a residue (e.g., Tyr or Dmt) in
a peptide. This class is referred to as N-alkyl and comprises
straight or branched alkyl groups from C.sub.1-C.sub.10, or an aryl
substituted alkyl group such as benzyl, phenylethyl and the like.
In some embodiments, an alkyl moiety is a 1-alkyl group that is in
glycosidic linkage (typically to the 1-position of, for example,
glucose) to the saccharide moiety. Such a 1-alkyl group is a
C.sub.1-C.sub.30 alkyl group.
[1057] An "aryl" group refers to an aromatic ring wherein each of
the atoms forming the ring is a carbon atom. Aryl rings described
herein include rings having five, six, seven, eight, nine, or more
than nine carbon atoms. Aryl groups are optionally substituted with
substituents selected from halogen, alkyl, acyl, alkoxy, alkylthio,
sulfonyl, dialkyl-amino, carboxyl esters, cyano or the like.
Examples of aryl groups include, but are not limited to phenyl, and
naphthalenyl.
[1058] The term "acyl" refers to a C.sub.1-C.sub.20 acyl chain.
This chain may comprise a linear aliphatic chain, a branched
aliphatic chain, a chain containing a cyclic alkyl moiety, a
hydrophobic natural product such as a steroid, an aralkyl chain, or
an alkyl chain containing an acyl moiety.
[1059] The term "steroid nucleus" refers to the core of steroids
comprising an arrangement of four fused rings designated A, B, C
and D as shown below:
##STR00050##
Examples of steroid nucleus containing moieties include, and are
not limited to, cholesterol and the like.
[1060] As used herein, a "therapeutic composition" can comprise an
admixture with an aqueous or organic carrier or excipient, and can
be compounded, for example, with the usual nontoxic,
pharmaceutically acceptable carriers for tablets, pellets,
capsules, lyophilizates, suppositories, solutions, emulsions,
suspensions, or other form suitable for use. The carriers, in
addition to those disclosed above, can include alginate, collagen,
glucose, lactose, mannose, gum acacia, gelatin, mannitol, starch
paste, magnesium trisilicate, talc, corn starch, keratin, colloidal
silica, potato starch, urea, medium chain length triglycerides,
dextrans, and other carriers suitable for use in manufacturing
preparations, in solid, semisolid, or liquid form. In addition,
auxiliary stabilizing, thickening or coloring agents can be used,
for example a stabilizing dry agent such as triulose.
[1061] As used herein, a "pharmaceutically acceptable carrier" or
"therapeutic effective carrier" is aqueous or nonaqueous (solid),
for example alcoholic or oleaginous, or a mixture thereof, and can
contain a surfactant, emollient, lubricant, stabilizer, dye,
perfume, preservative, acid or base for adjustment of pH, a
solvent, emulsifier, gelling agent, moisturizer, stabilizer,
wetting agent, time release agent, humectant, or other component
commonly included in a particular form of pharmaceutical
composition. Pharmaceutically acceptable carriers are well known in
the art and include, for example, aqueous solutions such as water
or physiologically buffered saline or other solvents or vehicles
such as glycols, glycerol, and oils such as olive oil or injectable
organic esters. A pharmaceutically acceptable carrier can contain
physiologically acceptable compounds that act, for example, to
stabilize or to increase the absorption of specific inhibitor, for
example, carbohydrates, such as glucose, sucrose or dextrans,
antioxidants, such as ascorbic acid or glutathione, chelating
agents, low molecular weight proteins or other stabilizers or
excipients.
[1062] The pharmaceutical compositions can also contain other
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions, such "substances" include,
but are not limited to, pH adjusting and buffering agents, tonicity
adjusting agents and the like, for example, sodium acetate, sodium
lactate, sodium chloride, potassium chloride, calcium chloride,
etc. Additionally, the peptide, or variant thereof, suspension may
include lipid-protective agents which protect lipids against
free-radical and lipid-peroxidative damages on storage. Lipophilic
free-radical quenchers, such as alpha-tocopherol and water-soluble
iron-specific chelators, such as ferrioxamine, are suitable.
[1063] As used herein, a "surfactant" is a surface active agent
that modifies interfacial tension of water. Typically, surfactants
have one lipophilic and one hydrophilic group or region in the
molecule. Broadly, the group includes soaps, detergents,
emulsifiers, dispersing and wetting agents, and several groups of
antiseptics. More specifically, surfactants include
stearyltriethanolamine, sodium lauryl sulfate, sodium taurocholate,
laurylaminopropionic acid, lecithin, benzalkonium chloride,
benzethonium chloride and glycerin monostearate; and hydrophilic
polymers such as polyvinyl alcohol, polyvinylpyrrolidone,
polyethyleneglycol (PEG), carboxymethylcellulose sodium,
methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose and
hydroxypropylcellulose or alkyl glycosides. In some embodiments, a
surfactant is a non-ionic surfactant (e.g., an alkyl glycoside
surfactant). In some embodiments, a surfactant is an ionic
surfactant.
[1064] As used herein, "alkyl glycoside" refers to any sugar joined
by a linkage to any hydrophobic alkyl, as is known in the art. The
hydrophobic alkyl can be chosen of any desired size, depending on
the hydrophobicity desired and the hydrophilicity of the saccharide
moiety. In one aspect, the range of alkyl chains is from 1 to 30
carbon atoms; or from 6 to 16 carbon atoms.
[1065] As used herein, "saccharide" is inclusive of
monosaccharides, oligosaccharides or polysaccharides in straight
chain or ring forms. Oligosaccharides are saccharides having two or
more monosaccharide residues. Some examples of the many possible
saccharides suitable for use in functionalized form include
glucose, galactose, maltose, maltotriose, maltotetraose, sucrose,
trehalose or the like.
[1066] As used herein. "sucrose esters" are sucrose esters of fatty
acids. Sucrose esters can take many forms because of the eight
hydroxyl groups in sucrose available for reaction and the many
fatty acid groups, from acetate on up to larger, more bulky fats
that can be reacted with sucrose. This flexibility means that many
products and functionalities can be tailored, based on the fatty
acid moiety used. Sucrose esters have food and non-food uses,
especially as surfactants and emulsifiers, with growing
applications in pharmaceuticals, cosmetics, detergents and food
additives. They are biodegradable, non-toxic and mild to the
skin.
[1067] As used herein, a "suitable" alkyl glycoside means one that
is nontoxic and nonionic. In some instances, a suitable alkyl
glycoside reduces the immunogenicity or aggregation and increases
the bioavailability of a compound when it is administered with the
compound via the ocular, nasal, nasolacrimal, sublingual, buccal,
inhalation routes or by injection routes such as the subcutaneous,
intramuscular, or intravenous routes. Suitable compounds can be
determined using the methods known to the art and those set forth
in the examples.
[1068] A "linker amino acid" is any natural or unnatural amino acid
that comprises a reactive functional group (de Graaf, A. J., et al.
(2009) Bioconjug Chem 20: 1281-1295) that is used for covalent
linkage with the functionalized surfactant. By way of example, in
some embodiments, a linker amino acid is Lys, or Orn having a
reactive functional group --NH.sub.2; or Cys, having a reactive
functional group --SH; or Asp or Glu, having a reactive functional
group --C(.dbd.O)--OH. By way of example, in some other
embodiments, a linker amino acid is any amino acid having a
reactive functional group such as --OH, --N.sub.3, haloacetyl or an
acetylenic group that is used for formation of a covalent linkage
with a suitably functionalized surfactant.
[1069] As used herein, a "functionalized surfactant" is a
surfactant comprising a reactive group suitable for covalent
linkage with a linker amino acid. By way of example, in some
embodiments, a functionalized surfactant comprises a carboxylic
acid group (e.g., at the 6-position of a monosaccharide) as the
reactive group suitable for covalent linkage with a linker amino
acid. By way of example, in some embodiments, a functionalized
surfactant comprises a --NH.sub.2 group, a --N.sub.3 group, an
acetylenic group, a haloacetyl group, a --O--NH.sub.2 group, or a
--(CH.sub.2-).sub.m-maleimide group, e.g., at the 6-position of a
monosaccharide (as shown in Scheme 6), that allows for covalent
linkage with a suitable linker amino acid. In some embodiments, a
functionalized surfactant is a compound of Formula IV as described
herein.
[1070] As used herein, the term "peptide" is any peptide comprising
two or more amino acids. The term peptide includes short peptides
(e.g., peptides comprising between 2-14 amino acids), medium length
peptides (15-50) or long chain peptides (e.g., proteins). The terms
peptide, medium length peptide and protein may be used
interchangeably herein. As used herein, the term "peptide" is
interpreted to mean a polymer composed of amino acid residues,
related naturally occurring structural variants, and synthetic
non-naturally occurring analogs thereof linked via peptide bonds,
related naturally occurring structural variants, and synthetic
non-naturally occurring analogs thereof. Synthetic peptides can be
synthesized, for example, using an automated peptide
synthesizer.
[1071] Peptides may contain amino acids other than the 20 gene
encoded amino acids. "Peptide(s)" include those modified either by
natural processes, such as processing and other post-translational
modifications, but also by chemical modification techniques. Such
modifications are well described in basic texts and in more
detailed monographs, and are well-known to those of skill in the
art. It will be appreciated that in some embodiments, the same type
of modification is present in the same or varying degree at several
sites in a given peptide. Also, a given peptide, in some
embodiments, contains more than one type of modifications.
Modifications occur anywhere in a peptide, including the peptide
backbone, the amino acid side-chains, and the amino or carboxyl
termini.
[1072] Accordingly, also contemplated within the scope of
embodiments described herein are surfactants covalently attached to
peptides that are modified, including, for example, modification
by, acetylation, acylation, PEGylation, ADP-ribosylation,
amidation, covalent attachment of a lipid or lipid derivative,
covalent attachment of phosphotidylinositol, cross-linking,
cyclization, disulfide bond formation, demethylation, formation of
covalent cross-link formation of cysteine, formation of
pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI
anchor formation, hydroxylation, iodination, methylation,
myristoylation, oxidation, proteolytic processing, phosphorylation,
prenylation, racemization, glycosylation, lipid attachment,
sulfation, gamma-carboxylation of glutamic acid residues,
hydroxylation and ADP-ribosylation, selenoylation, sulfation,
transfer-RNA mediated addition of amino acids to proteins, such as
arginylation, and ubiquitination. See, for instance, (Nestor, J.
J., Jr. (2007) Comprehensive Medicinal Chemistry II 2: 573-601,
Nestor, J. J., Jr. (2009) Current Medicinal Chemistry 16:
4399-4418, Creighton, T. E. (1993, Wold, F. (1983)
Posttranslational Covalent Modification of Proteins 1-12, Seifter,
S. and Englard, S. (1990) Methods Enzymol 182: 626-646, Rattan. S.
I., et al. (1992) Ann N Y Acad Sci 663: 48-62). Also contemplated
within the scope of embodiments described herein are peptides that
are branched or cyclic, with or without branching. Cyclic, branched
and branched circular peptides result from post-translational
natural processes and are also made by suitable synthetic
methods.
[1073] The term peptide includes peptides or proteins that comprise
natural and unnatural amino acids or analogs of natural amino
acids. As used herein, peptide and/or protein "analogs" comprise
non-natural amino acids based on natural amino acids, such as
tyrosine analogs, which includes para-substituted tyrosines,
ortho-substituted tyrosines, and meta substituted tyrosines,
wherein the substituent on the tyrosine comprises an acetyl group,
a benzoyl group, an amino group, a hydrazine, an hydroxyamine, a
thiol group, a carboxy group, a methyl group, an isopropyl group, a
C.sub.2-C.sub.20 straight chain or branched hydrocarbon, a
saturated or unsaturated hydrocarbon, an O-methyl group, a
polyether group, a halogen, a nitro group, or the like. Examples of
Tyr analogs include 2,4-dimethyl-tyrosine (Dmt),
2,4-diethyl-tyrosine, 0-4-allyl-tyrosine, 4-propyl-tyrosine,
C.alpha.-methyl-tyrosine and the like. Examples of lysine analogs
include ornithine (Orn), homo-lysine, C.alpha.-methyl-lysine
(CMeLys), and the like. Examples of phenylalanine analogs include,
but are not limited to, meta-substituted phenylalanines, wherein
the substituent comprises a methoxy group, a C.sub.1-C.sub.20 alkyl
group, for example a methyl group, an allyl group, an acetyl group,
or the like. Specific examples include, but are not limited to,
2,4,6-trimethyl-L-phenylalanine (Tmp), O-methyl-tyrosine,
3-(2-naphthyl)alanine (Nal(2)), 3-(1-naphthyl)alanine (Nal(1)),
3-methyl-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid (Tic), fluorinated phenylalanines, isopropyl-phenylalanine,
p-azido-phenylalanine, p-acyl-phenylalanine,
p-benzoyl-phenylalanine, p-iodo-phenylalanine,
p-bromophenylalanine, p-amino-phenylalanine, and
isopropyl-phenylalanine, and the like. Among the vast array of
nonstandard or unnatural amino acids known to the art and used in
peptide analog design are C-alpha-disubstituted amino acids such as
Aib, C.alpha.-diethylglycine (Deg), aminocyclopentane-1-carboxylic
acid (Ac4c), aminocyclopentane-1-carboxylic acid (Ac5c), and the
like. Such amino acids frequently lead to a restrained structure,
often biased toward an alpha helical structure (Kaul, R. and
Balaram, P. (1999) Bioorg Med Chem 7: 105-117). Additional examples
of such unnatural amino acids useful in analog design are
homo-arginine (Har), and the like. Substitution of reduced amide
bonds in certain instances leads to improved protection from
enzymatic destruction or alters receptor binding. By way of
example, incorporation of a Tic-Phe dipeptide unit with a reduced
amide bond between the residues (designated as
Tic-.PSI.[CH2-NH]-.PSI.-Phe) reduces enzymatic degradation.
Accordingly, also contemplated within the scope of embodiments
described herein are surfactants covalently attached to peptides
that comprise modified amino acids and/or peptide analogs described
above. Certain non-natural amino acids are shown below.
##STR00051## ##STR00052##
[1074] As used herein, "opioid peptides" are short sequences of
amino acids that bind to opioid receptors in the body. In some
embodiments, opioid peptides are endogenous peptides, such as, for
example, endorphins, enkephalins, endomorphins, dermorphins or the
like. In some embodiments, opioid peptides are derived from
endogenous opioid peptides (e.g., pseudo-peptides, constrained
peptides, alpha-methyl analogs, or the like). In some embodiments,
opioid peptides are exogenous and/or synthetic and comprise
modified amino acids and/or unnatural amino acids that mimic the
effects of opioid peptides.
[1075] As used herein, the term "variant" is interpreted to mean a
peptide that differs from a reference peptide, but retains
essential properties. A typical variant of a peptide differs in
amino acid sequence from another, reference peptide. Generally,
differences are limited so that the sequences of the reference
peptide and the variant are closely similar overall and, in many
regions, identical. A variant and reference peptide may differ in
amino acid sequence by one or more substitutions, additions,
deletions in any combination. A substituted or inserted amino acid
residue may or may not be one encoded by the genetic code.
Non-naturally occurring variants of peptides may be made by
mutagenesis techniques, by direct synthesis, and by other suitable
recombinant methods.
[1076] Reference now will be made in detail to various embodiments
and particular applications of the covalently modified peptides
and/or proteins described herein. While the covalently modified
peptides and/or proteins will be described in conjunction with the
various embodiments and applications, it will be understood that
such embodiments and applications are exemplary and are not
intended to limit the scope of the embodiments described herein. In
addition, throughout this disclosure various patents, patent
applications, websites and publications are referenced, and unless
otherwise indicated, each is incorporated by reference in for
relevant disclosure referenced herein.
Peptides
[1077] There are many important roles played by peptides in the
body and some commercial opportunities have been exploited (Nestor,
J. J., Jr. (2009) Current Medicinal Chemistry 16: 4399-4418;
Stevenson, C. L. (2009) Curr Pharm Biotechnol 10: 122-137). However
even these recognized targets (Tyndall, J. D., et al. (2005) Chem
Rev 105: 793-826) and products continue to suffer from deficiencies
in duration of action and bioavailability. In some embodiments, the
improved peptides described herein provide longer duration of
action and/or bioavailability and/or therapeutic efficacy compared
to currently available commercial products. Some illustrative
examples of peptides that represent attractive commercial targets
for analog design (agonists and antagonists) for clinical
development include, for example, members of the Class B, G
Protein-Coupled Receptor (GPCR) ligands and related peptides
("Secretin family"): Secretin, Parathyroid Hormone (PTH),
Parathyroid Hormone-related Protein (PTHrP), Glucagon, Glucagon
Like Protein-1 and -2 (GLP-1, GLP-2), Glucose-dependent
Insulinotropic Peptide (GIP), Oxyntomodulin, Pituitary Adenylate
Cyclase-Activating Peptide (PACAP), Vasoactive Intestinal Peptide
(VIP), Amylin (and analogs such as pramlintide, devalintide, et
al.), Calcitonin (and analogs such as salmon calcitonin, elcatonin,
et al.), calcitonin gene-related peptide (CGRP), Adrenomedulin,
Corticotrophin-Releasing Factor family (CRF, Xerecept; Urocortin),
and the like, including synthetic analogs thereof which would be
improved as clinical products through further modification by the
methods described herein. Also contemplated within the scope of
embodiments presented herein are Opioid peptide families; such
peptides would benefit from the methods of peptide modification
described herein to give increased duration of action and increased
specificity. By way of example analogs of the endomorphin,
dynorphin, enkephalin, dermorphin, casomorphin, et al. peptide
families offer attractive therapeutic approaches to the treatment
of pain, addiction, et al. Additional attractive peptide targets
for modification to yield peptide products described herein are the
hypothalamic hormones, for example gonadotrophin hormone-releasing
hormone and its analogs (for example nafarelin, goserelin,
triptorelin, leuprorelin, fertirelin, histrelin, buserelin,
ganirelix, cetrorelix, degarelix, deslorelin and the like),
adrenocorticotrophin, somatostatin (for example octreotide,
lanreotide, valpreotide et al.), thyrotropin-releasing hormone,
growth hormone-releasing hormone, and neurotensin. A further
example of attractive commercial targets are the pituitary hormones
and analogs such as vasopressin (desmopressin, and the like),
oxytocin and analogs, thyroid hormone stimulating hormone,
prolactin, growth hormone, luteinizing hormone,
follicular-stimulating hormone, alpha melanocyte-stimulating
hormone analogs (melanotan analogs), and the like, as well as their
analogs. Growth factors are an important class of molecules that
may be advantageously modified using the methods described herein
to yield improved pharmaceutical candidates, for example insulin
(and analogs such as Lispro, Levemir, glargin, et al.),
insulin-like growth factor-I (IGF-I or Somatomedin-C), Nerve Growth
Factor (NGF), Fibroblast Growth Factor (FGF; FGF-18, FGF-20,
FGF-21, and the like), Keratinocyte Growth Factor (KGF) and
Vascular Endothelial Growth Factor (VEGF), and the like. Especially
attractive targets are those peptides which control gut function
and appetite, but which have short duration of action (some of
which are mentioned above), including but not limited to, ghrelin,
Pancreatic Peptide, Peptide YY, Neuropeptide Y, Cholecystokinin
(Sincalide, et al.), Melanocortin, and the like. Additional targets
benefiting from the methods described herein are the
proinflammatory adipose tissue products relating to obesity,
including Leptin (and related analogs such as OB-3 peptide),
adipokines, adiponectin. Chemerin, visfatin, nesfatin, resistin,
tumor necrosis factor alpha, chemokines, monocyte chemotactic
protein-I (MCP-I), omentin, interleukins, and the like. Important
targets that would benefit from improved pharmaceutical and
pharmacodynamic behavior are proteins that control immune function,
among which are given the following examples, which are not meant
to be limiting, merely illustratory: members of the interferon
family (interferons-alpha, -beta, -gamma, -kappa, -omega, the IL-10
cytokine family, including IL-10, IL-19, IL-20, IL-22, IL-24,
IL-26, and the like), Thymopentin, Thymosin alpha1, and the like.
Important peptide products which control the circulatory, or blood
clotting would be improved by the methods described herein whereby
increased duration of action would be achieved, for example,
bivalirudin (Angiomax), eptifibatide (Integrelin), atrial
natriuretic peptides (ANP, Ularitide), brain natriuretic peptide, c
type natriuretic peptide, b-type natriuretic peptide (nesiritide),
angiotensin, Angiostatin Rotigaptide, thrombospondins and the like.
Peptides that stimulate stem cell proliferation and differentiation
are important agents that could be improved by the modifications
described herein. For example, erythropoietin, hematide,
thrombopoietin, macrophage colony-stimulating factor (M-CSF),
leukemia inhibitory factor (LIF), interleukin-6 (IL-6)),
granulocyte colony-stimulating factor (G-CSF),
granulocyte-macrophage-colony-stimulating factor (GM-CSF), and the
like are contemplated as peptides that are suitable for covalent
attachment to a surfactant (e.g., an alkyl glycoside surfactant).
Neurotrophic factors are another class of small proteins that would
greatly benefit from modifications described herein to increase
duration of action and efficacy. For example, Glial cell-derived
neurotrophic factor (GDNF) and family (neurturin, artemin,
persephin), neurotrophins such as nerve growth factor (NGF), BDNF
and the like. Proinflammatory and pain-causing peptides are
important peptide targets that would be improved by modifications
described herein. For example, inhibitors of bradykinin (or its
release, e.g. ecallantide) and substance P especially antagonists,
offer important therapeutic targets (icatibant, et al.) are
suitable for covalent attachment of surfactants (e.g.
alkyl-glycoside surfactants). Inhibitors of viral fusion (Fuzeon),
protein maturation (protease inhibitors) or integration suffer from
short duration of action. Modification of such inhibitors via
covalent attachment of a surfactant (e.g., an alkyl-glycoside
surfactant) will allow for longer duration of action. Many
proteases have been found to be involved in disease and inhibitors
of their action have reached the clinic (Abbenante, G. and Fairlie,
D. P. (2005) Med Chem 1: 71-104), but would be further improved by
the modifications described herein and such targets are also
contemplated within the scope of the embodiments described herein.
Additional natural peptide products and analogs thereof such as
conotoxin peptides for pain, antimicrobial defensins and the like
also suffer from lack of bioavailability and short duration of
action in physiological fluid and therefore would benefit from the
peptide modifications described herein. Those skilled in the art
will recognize many additional commercially important peptides that
are amenable to modifications described herein and provide
increased duration of action and bioavailability, and such peptides
are also contemplated within the scope of the present
disclosure.
[1078] Modifications at the amino or carboxyl terminus may
optionally be introduced into the present peptides (Nestor, J. J.,
Jr. (2009) Current Medicinal Chemistry 16: 4399-4418). For example,
the present peptides can be truncated or acylated on the N-terminus
to yield peptides exhibiting low efficacy, partial agonist and
antagonist activity, as has been seen for some peptides (Gourlet,
P., et al. (1998) Eur J Pharmacol 354: 105-111, Gozes, I. and
Furman, S. (2003) Curr Pharm Des 9: 483-494), the contents of which
is incorporated herein by reference). Other modifications to the
N-terminus of peptides, such as deletions or incorporation of
D-amino acids such as D-Phe also can give potent and long acting
agonists or antagonists when substituted with the modifications
described herein such as long chain alkyl glycosides. Such agonists
and antagonists also have commercial utility and are within the
scope of contemplated embodiments described herein.
[1079] Aside from the twenty standard amino acids, there am a vast
number of "nonstandard amino acids" or unnatural amino acids that
are known to the art and that may be incorporated in the compounds
described herein, as described above. Other nonstandard amino acids
are modified with reactive side chains for conjugation (Gauthier,
M. A. and Klok, H. A. (2008) Chem Commun (Camb) 2591-2611; de
Graaf, A. J., et al. (2009) Bioconjug Chem 20: 1281-1295). In one
approach, an evolved tRNA/tRNA synthetase pair and is coded in the
expression plasmid by the amber suppressor codon (Deiters, A, et
al. (2004). Bio-org. Med. Chem. Lett. 14, 5743-5). For example,
p-azidophenylalanine was incorporated into peptides and then
reacted with a functionalized surfactant, or a PEG polymer having
an acetylene moiety in the presence of a reducing agent and copper
ions to facilitate an organic reaction known as "Huisgen [3+2]
cycloaddition." A similar reaction sequence using the reagents
described herein containing an acetylene modified alkyl glycoside
or PEG modified glycoside will result in PEGylated or alkyl
glycoside modified peptides. For peptides of less than about 50
residues, standard solid phase synthesis is used for incorporation
of said reactive amino acid residues at the desired position in the
chain. Such surfactant-modified peptides and/or proteins offer a
different spectrum of pharmacological and medicinal properties than
peptides modified by PEG incorporation alone.
Intermediates
[1080] In one embodiment provided herein are intermediates and/or
reagents comprising a surfactant moiety and a reactive functional
group capable of forming a bond with a reactive functional group on
a natural or unnatural amino acid. These intermediates and/or
reagents allow for improvement in the bioavailability and
pharmaceutical, pharmacokinetic and/or pharmacodynamic behavior of
peptides and/or proteins of use in human and animal disease.
Covalent attachment of such intermediates and/or reagents via a
functional group on a side chain of an amino acid, for example on
an epsilon-amino function of Lys, the sulfhydryl of Cys, or at the
amino or carboxy terminus of the peptide and/or protein target
allows for synthesis of the peptide products described herein. In
specific embodiments, non-ionic surfactant moieties are mono or
disaccharides with an O-alkyl glycosidic substitution, said
glycosidic linkage being of the alpha or beta configuration. In
specific embodiments, O-alkyl chains are from C.sub.1-C.sub.20 or
from C.sub.6-C.sub.16 alkyl chains.
[1081] In another embodiment provided herein are intermediates
and/or reagents comprising a non-ionic surfactant moiety with
certain alkyl glycosidic linkage that mimic O-alkyl glycosidic
linkages and a reactive functional group capable of forming a bond
with a reactive functional group on a natural or unnatural amino
acid. Such intermediates and/or reagents contain S-linked alkyl
chains or N-linked alkyl chains and have altered chemical and/or
enzymatic stability compared to the O-linked alkyl glycoside-linked
products.
[1082] In some embodiments, an intermediate and/or reagent provided
herein is a compound wherein the hydrophilic group is a modified
glucose, galactose, maltose, glucuronic acid, diglucuronic acid,
maltouronic acid or the like. In some embodiments, the hydrophilic
group is glucose, maltose, glucuronic acid, diglucuronic acid or
maltouronic acid and the hydrophobic group is a C.sub.1-C.sub.20
alkyl chain or an aralkyl chain. In some embodiments the glycosidic
linkage to the hydrophobic group is of an alpha configuration and
in some the linkage is beta at the anomeric center on the
saccharide.
[1083] In some embodiments, the hydrophilic group is glucose,
maltose, glucuronic acid, diglucuronic acid or maltouronic acid and
the hydrophobic group is a C.sub.1-C.sub.20 alkyl or aralkyl
chain.
[1084] In some embodiments, an intermediate and/or reagent provided
herein comprises a surfactant containing a reactive functional
group that is a carboxylic acid group, an amino group, an azide, an
aldehyde, a maleimide, a sulfhydryl, a hydroxylamino group, an
alkyne or the like.
[1085] In some embodiments, the intermediate and/or reagent is an
O-linked alkyl glycoside with one of the hydroxyl functions
modified to be a carboxylic acid or amino functional group. In some
embodiments, the reagent is a 1-O-alkyl glucuronic acid of alpha or
beta configuration and the alkyl chain is from C.sub.1 to C.sub.20
in length. In some of such embodiments, the alkyl group is from
C.sub.6 to C.sub.18 in length. In some of such embodiments, the
alkyl group is from C.sub.6 to C.sub.16 in length.
[1086] In some embodiments, the reagent comprises a 1-O-alkyl
diglucuronic acid of alpha or beta configuration and the alkyl
chain is from C.sub.1 to C.sub.20 in length. In some of such
embodiments, the alkyl group is from C.sub.6 to C.sub.16 in
length.
[1087] In some embodiments, the reagent is an S-linked alkyl
glycoside of alpha or beta configuration with one of the hydroxyl
functions modified to be a carboxylic acid or amino functional
group.
[1088] In some embodiments, the reagent is an N-linked alkyl
glycoside of alpha or beta configuration with one of the hydroxyl
functions modified to be a carboxylic acid or amino functional
group.
[1089] In yet another embodiment provided herein are peptide and/or
protein products containing a covalently linked alkyl glycoside
with properties acceptable for use in human and animal disease.
Scheme 1 lists exemplary non-ionic surfactants that can be modified
to yield the reagents and/or intermediates that are useful for
synthesis of surfactant-modified peptide products described
herein.
##STR00053##
[1090] In some embodiments, the covalently modified peptides and/or
proteins described herein incorporate a surfactant moiety into the
peptide structure. In specific embodiments, the covalently modified
peptides and/or proteins described herein incorporate a non-ionic
surfactant of the alkyl, alkoxyaryl, or aralkyl glycoside class.
Alkyl glycosides are important commodities and are widely used in
the food, service and cleaning industries. Thus their production on
commercially significant scale has been the subject of extensive
study. Both enzymatic and chemical processes are available for
their production at very low cost (Park, D. W., et al. (2000)
Biotechnology Letters 22: 951-956). These alkyl glycosides can be
modified further to generate the intermediates for the synthesis of
the covalently modified peptides and/or proteins described herein.
Thus it is known that 1-dodecyl beta-D-glucoside is preferentially
oxidized on the 6-position to yield the corresponding glucuronic
acid analog in high yield when using the unprotected material and
platinum black catalyst in the presence of oxygen (van Bekkum, H.
(1990) Carbohydrates as Organic Raw Materials 289-310). Additional
chemoselective methods for oxidation of the primary alcohol at the
6 position of alkyl glucosides are available. For example, use of
catalytic amounts of 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO)
with stoichiometric amounts of the organic oxidant
[bis(acetoxy)iodo]benzene (BAIB) (De Mico, A., et al. (1997) J Org
Chem 1997: 6974-6977) gave outstanding yields of
nucleoside-5'-carboxylic acids (Epp, J. B. and Widlanski, T. S.
(1999) J Org Chem 64: 293-295) by oxidation of the primary
hydroxyl. This oxidation is chemoselective for the primary hydroxyl
even when the other, secondary hydroxyls are unprotected (Codee, J.
D., et al. (2005) J Am Chem Soc 127: 3767-3773). In a similar
manner, 1-dodecyl .beta.-D-glucopyranoside and 1-tetradecyl
.beta.-D-glucopyranoside were oxidized to the corresponding uronic
acids (1-dodecyl .beta.-D-glucuronic acid, 1-tetradecyl
.beta.-D-glucuronic acid) by oxidation with TEMPO using KBr and
sodium hypochlorite as stoichiometric oxidant (Milkereit, G., et
al. (2004) Chem Phys Lipids 127: 47-63) in water. A mild oxidation
procedure using (diacetoxyiodo)benzene (DAIB aka BAIB) is given in
the Examples. Certain of these glucuronic acid intermediates are
commercially available (for example octyl b-D-glucuronic acid;
Carbosynth, Mo. 07928) and, as indicated, a broad range are subject
to preparation by routine methods (Schamann, M. and Schafer, H. J.
(2003) Eur J Org Chem 351-358; Van den Bos, L. J., et al. (2007)
Eur J Org Chem 3963-3976) or upon request. Scheme 2 illustrates, as
examples, certain functionalized surfactant intermediates
comprising a --COOH group as a reactive functional group that are
used to prepare the intermediates and/or reagents described
herein.
##STR00054##
[1091] Similarly, aralkyl glycosides (including alkoxyaryl) can
form the basis for closely related nonionic surfactant reagents.
For example, 4-alkoxyphenyl .beta.-D-glucopyranosides are readily
synthesized by the reaction of 4-alkyloxyphenols with
penta-O-acetyl .beta.-D-glucose in the presence of boron
trifluoride etherate. Subsequent deacetylation using trimethylamine
in methanol/water and selective oxidation as described above and in
the examples, yields the alkoxyaryl glucuronic acid reagents
suitable for forming the reagents and peptides described herein
((Smits, E., et al. (1996) J Chem Soc, Perkin Trans I 2873-2877;
Smits, E., et al. (1997) Liquid Crystals 23: 481-488).
##STR00055##
[1092] The glucuronic acid class of intermediate is readily
activated by standard coupling agents for linkage to an amino acid
side chain, e.g. that of Lys. Thus Fmoc-Lys-O-Tms
(trimethylsilyl=TMS) can be reacted with octyl beta-D-glucuronic
acid in the presence of a coupling agent and the O-Tms protecting
group can then be hydrolyzed on aqueous workup to yield
Fmoc-Lys(1-octyl beta-D-glucuronamide) as shown in Scheme 4. This
reagent can be used for incorporation into the solid phase
synthesis of peptides, using standard coupling protocols, when it
is desired to incorporate the surfactant moiety near the N-terminal
region of the molecule. The secondary hydroxyl groups can be left
unprotected, due to the very much higher reactivity of the Lys
amino functional group or they can be protected by peracetylation.
If an acetyl protected form is used, the acetyl protecting groups
can be removed in high yield by treatment with either MeOH/NaOMe or
by MeOH/Et.sub.3N. Scheme 4 illustrates preparation of the reagents
described herein.
##STR00056##
[1093] In some embodiments, reagents and/or intermediates for the
preparation of the biologically active peptide products described
herein comprise a family of surfactant-modified linker amino acids
for incorporation into synthetic peptide products. Thus in one
embodiment, peptide products described herein are synthesized in a
linear fashion wherein a functionalized surfactant is attached to a
reversibly-protected linker amino acid via a functional group on a
side chain of a linker amino acid (e.g., an amino group of a lysine
residue) to yield a proprietary reagent (as shown in Scheme 4.)
which can be incorporated into the growing peptide chain and then
the remaining peptide is synthesized by attachment of further amino
acids to the cysteine residue. Protecting group suitable for
synthesis of modified peptides and/or protein described herein are
described in, for example, T. W. Green, P. G. M. Wuts, Protective
Groups in Organic Synthesis, Wiley-Interscience, New York, 1999,
503-507, 736-739, which disclosure is incorporated herein by
reference.
[1094] In another embodiment, peptide products described herein are
synthesized by covalent attachment of a functionalized surfactant
to a full-length peptide via a suitable functional group on a
linker amino acid that is in the peptide chain.
[1095] Alternatively a functionalized surfactant can be added to a
linker amino acid side chain which has been deprotected during the
course of the solid phase synthesis of the peptide. As an example,
an alkyl glucuronyl group can be added directly to a linker amino
acid side chain (e.g., a deprotected Lys side chain) during the
solid phase synthesis of the peptide. For example, use of
Fmoc-Lys(Alloc)-OH as a subunit provides orthogonal protection that
can be removed while the peptide is still on the resin. Thus
deprotection of the Lys side chain using Pd/thiobarbital or other
alloc deprotection recipe allows exposure of the amino group for
coupling with the acyl protected or unprotected 1-octyl
beta-D-glucuronic acid unit. Final deprotection with a low %
CF.sub.3CO.sub.2H (TFA) cleavage cocktail will then deliver the
desired product. Although the glycosidic linkage is labile to
strong acid, the experience here and by others is that it is
relatively stable to low % TFA cleavage conditions. Alternatively,
acyl protection (e.g. acetyl, Ac; benzoyl, Bz) or trialkylsilyl
protection on the saccharide OH functional groups may be used to
provide increased protection to the glycosidic linkage. Subsequent
deprotection by base (NH.sub.2NH.sub.2/MeOH; NH.sub.3/MeOH,
NaOMe/MeOH) yields the desired deprotected product. Scheme 4
illustrates reagents described herein. Scheme 5 illustrates peptide
intermediates described herein.
##STR00057##
[1096] Additional reagents are generated by modification of the
6-position functional group to give varied means of linkage to
amino acid side chain functional groups, as shown below in Scheme
6. Thus amino substitution can be used for linkage to Asp or Glu
side chains. Azido or alkyne substitution can be used for linkage
to unnatural amino acids containing the complementary acceptor for
Huisgen 3+2 cycloaddition (Gauthier, M. A. and Klok, H. A. (2008)
Chem Commun (Camb) 2591-2611). Aminoxy or aldehyde functional
groups can be used to link to aldehyde (i.e. oxime linkage) or to
amino functions (i.e. reductive alkylation), respectively. The
maleimide or --NH--(C.dbd.O)--CH.sub.2--Br functional group can
bind chemoselectively with a Cys or other SH functional group.
These types of linkage strategies are advantageous when used in
conjunction with the reagents described herein. Interconversion of
functional groups is widely practiced in organic synthesis and
comprehensive lists of multiple routes to each of the functional
group modifications listed herein are available (Larock, R. C.
(1999)) "Comprehensive Organic Transformations", VCH Publishers,
New York.
[1097] Thus, for example, the primary hydroxyl on position 6 of
octyl 1-.beta.-D-glucoside is converted to the azide by activation
and displacement with an azide anion, reactions such as reactions
used in carbohydrate chemistry (e.g. by tosylation followed by
NaN.sub.3). The corresponding azide is reduced to the amino
function by reduction with thiolacetic acid in pyridine (Elofsson,
M., et al. (1997) Tetrahedron 53: 369-390) or by similar methods of
amino group generation (Stangier, P., et al. (1994) Liquid Crystals
17: 589-595). Approaches to the acetylene, aminoxy, and aldehyde
moieties are best carried out on the triacetoxy form, available
from the commercially available glucoside by treatment with
Ac.sub.2O, followed by mild hydrolysis of the primary amine. This
6-hydroxy form can be selectively oxidized to the aldehyde, or
activated as a tosylate or triflate and displaced by NH.sub.2OH or
by sodium acetylide. The maleimide linkage can be through a carbon
linkage as shown or, preferably though an 0 or amide linkage, again
by displacement of the activated hydroxyl or coupling of the
glucuronic acid derivative to an amino linked maleimide reagent,
well known in the art. Additional functional group interconversions
are well known to those of average skill in the art of medicinal
chemistry and are within the scope of the embodiments described
herein.
[1098] Also contemplated within the scope of synthetic methods
described herein are surfactants wherein the saccharide and
hydrophobic chain are covalently attached via an alpha glycosidic
linkage. Synthetic routes to predominantly .alpha.-linked
glycosides are well known in the art and typically originate with
the peracetyl sugar and use acidic catalysis (e.g. SnCl.sub.4,
BF.sub.3 or HCl) to effect the .alpha.-glycosylation (Cudic, M. and
Burstein, G. D. (2008) Methods Mol Biol 494: 187-208, Vill, V., et
al. (2000) Chem Phys Lipids 104: 75-91, incorporated herein by
reference for such disclosure). Similar synthetic routes exist for
disaccharide glycosides (von Minden, H. M., et al. (2000) Chem Phys
Lipids 106: 157-179, incorporated herein by reference for such
disclosure). Functional group interconversions then proceed as
above to lead to the 6-carboxylic acid, et al. for generation of
the corresponding .alpha.-linked reagents.
[1099] Scheme 6 lists certain compounds and reagents useful in the
synthesis of the covalently modified peptides and/or proteins
described herein. Standard nomenclature using single letter
abbreviations for amino acids are used.
##STR00058##
[1100] Many alkyl glycosides can be synthesized by known
procedures, as described, e.g., in (Rosevear, P., et al. (1980)
Biochemistry 19: 4108-4115, Li, Y. T., et al. (1991) J Biol Chem
266: 10723-10726) or Koeltzow and Urfer, J. Am. Oil Chem. Soc.,
61:1651-1655 (1984), U.S. Pat. Nos. 3,219,656 and 3,839,318 or
enzymatically, as described, e.g., in (Li, Y. T., et al. (1991) J
Biol Chem 266: 10723-10726, Gopalan, V., et al. (1992) J Biol Chem
267: 9629-9638). O-alkyl linkages to natural amino acids such as
Ser can be carried out on the Fmoc-Ser-OH using peracetylglucose to
yield
N.alpha.-Fmoc-4-O-(2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyranosyl)-L-seri-
ne. This material is selectively deprotected at the primary carbon
atom (position 6) and selectively oxidized using TEMPO/BAIB as
described above to yield the corresponding 6-carboxyl function
which may be coupled to lipophilic amines to generate a new class
of nonionic surfactant and reagents (Scheme 7).
##STR00059##
[1101] The linkage between the hydrophobic alkyl and the
hydrophilic saccharide can include, among other possibilities, a
glycosidic, thioglycosidic, amide (Carbohydrates as Organic Raw
Materials, F. W. Lichtenthaler ed., VCH Publishers, New York,
1991), ureide (Austrian Pat. 386,414 (1988); Chem. Abstr.
110:137536p (1989); see Gruber, H. and Greber, G., "Reactive
Sucrose Derivatives" in Carbohydrates as Organic Raw Materials, pp.
95-116) or ester linkage (Sugar Esters: Preparation and
Application, J. C. Colbert ed., (Noyes Data Corp., New Jersey),
(1974)).
[1102] Examples from which useful alkyl glycosides can be chosen
for modification to the reagents or for the formulation of the
products described herein, include: alkyl glycosides, such as
octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl,
pentadecyl-, hexadecyl-, heptadecyl-, and octadecyl-D-maltoside,
-glucoside or -sucroside (i.e., sucrose ester) (synthesized
according to Koeltzow and Urfer; Anatrace Inc., Maumee, Ohio;
Calbiochem, San Diego, Calif.; Fluka Chemie, Switzerland); alkyl
thiomaltosides, such as heptyl, octyl, dodecyl-, tridecyl-, and
tetradecyl-.beta.-D-thiomaltoside (synthesized according to Defaye,
J. and Pederson, C., "Hydrogen Fluoride. Solvent and Reagent for
Carbohydrate Conversion Technology" in Carbohydrates as Organic Raw
Materials, 247-265 (F. W. Lichtenthaler, ed.) VCH Publishers, New
York (1991); Ferenci, T., J. Bacteriol, 144:7-11 (1980)); alkyl
thioglucosides, such as 1-dodecyl- or 1-octyl-thio .alpha.- or
.beta.-D-glucopyranoside (Anatrace, Inc., Maumee, Ohio; see Saito,
S. and Tsuchiya, T. Chem. Pharm. Bull. 33:503-508 (1985)); alkyl
thiosucroses (synthesized according to, for example, Binder, T. P.
and Robyt, J. F., Carbohydr. Res. 140-9-20 (1985)); alkyl
maltotriosides (synthesized according to Koeltzow and Urfer); long
chain aliphatic carbonic acid amides of sucrose amino-alkyl ethers;
(synthesized according to Austrian Patent 382,381 (1987); Chem.
Abstr., 108:114719 (1988) and Gruber and Greber pp. 95-116);
derivatives of palatinose and isomaltamine linked by amide linkage
to an alkyl chain (synthesized according to Kunz, M.,
"Sucrose-based Hydrophilic Building Blocks as Intermediates for the
Synthesis of Surfactants and Polymers" in Carbohydrates as Organic
Raw Materials, 127-153); derivatives of isomaltamine linked by urea
to an alkyl chain (synthesized according to Kunz); long chain
aliphatic carbonic acid ureides of sucrose amino-alkyl ethers
(synthesized according to Gruber and Greber, pp. 95-116); and long
chain aliphatic carbonic acid amides of sucrose amino-alkyl ethers
(synthesized according to Austrian Patent 382,381 (1987), Chem.
Abstr., 108:114719 (1988) and Gruber and Greber, pp. 95-116).
[1103] Some preferred glycosides include the saccharides maltose,
sucrose, glucose and galactose linked by glycosidic or ester
linkage to an alkyl chain of 6, 8, 10, 12, or 14 carbon atoms,
e.g., hexyl-, octyl-, decyl-, dodecyl- and tetradecyl-maltoside,
sucroside, glucoside and galactoside. In the body these glycosides
are degraded to non-toxic alcohol or fatty acid and an
oligosaccharide or saccharide. The above examples are illustrative
of the types of alkyl glycosides to be used in the methods claimed
herein, however the list is not intended to be exhaustive.
[1104] Generally, these surfactants (e.g., alkyl glycosides) are
optionally designed or selected to modify the biological properties
of the peptide, such as to modulate bioavailability, half-life,
receptor selectivity, toxicity, biodistribution, solubility,
stability, e.g. thermal, hydrolytic, oxidative, resistance to
enzymatic degradation, and the like, facility for purification and
processing, structural properties, spectroscopic properties,
chemical and/or photochemical properties, catalytic activity, redox
potential, ability to react with other molecules, e.g., covalently
or noncovalently, and the like.
Surfactants
[1105] The term "surfactant" comes from shortening the phrase
"surface active agent". In pharmaceutical applications, surfactants
are useful in liquid pharmaceutical formulations in which they
serve a number of purposes, acting as emulsifiers, solubilizers,
and wetting agents. Emulsifiers stabilize the aqueous solutions of
lipophilic or partially lipophilic substances. Solubilizers
increase the solubility of components of pharmaceutical
compositions increasing the concentration which can be achieved. A
wetting agent is a chemical additive which reduces the surface
tension of a fluid, inducing it to spread readily on a surface to
which it is applied, thus causing even "wetting" of the surface
with the fluids. Wetting agents provide a means for the liquid
formulation to achieve intimate contact with the mucous membrane or
other surface areas with which the pharmaceutical formulation comes
in contact. Thus surfactants may be useful additives for
stabilization of the formulation of the peptide products described
herein as well as for the modification of the properties of the
peptide itself.
[1106] In specific embodiments, alkyl glycosides which are
synthetically accessible, e.g., the alkyl glycosides dodecyl,
tridecyl and tetradecyl maltoside or glucoside as well as sucrose
dodecanoate, tridecanoate, and tetradecanoate are suitable for
covalent attachment to peptides as described herein. Similarly, the
corresponding alkylthioglycosides are stable, synthetically
accessible surfactants which are acceptable for formulation
development.
[1107] A wide range of physical and surfactant properties can be
achieved by appropriate modification of the hydrophobic or
hydrophilic regions of the surfactant (e.g., the alkyl glycoside).
For example, a study comparing the bilayer activity of dodecyl
maltoside (DM) with that of dodecyl glucoside (DG) found that of DM
to be more than three times higher than that of DG, despite having
the same length of hydrophobic tail (Lopez, O., et al. (2002)
Colloid Polym Sci 280: 352-357). In this particular instance the
identity of the polar region (disaccharide vs monosaccharide)
influences surfactant behavior. In the case of a surfactant linked
to a peptide, e.g. the peptide products described herein, the
peptide region also may contribute hydrophobic or hydrophilic
character to the overall molecule. Thus tuning of the physical and
surfactant properties may be used to achieve the particular
physical and pharmaceutical properties suitable for the individual
peptide targets.
PEG Modification
[1108] In some embodiments, surfactant-modified peptide products
described herein are further modified to incorporate one or more
PEG moieties (Veronese, F. M. and Mero, A. (2008) BioDrugs 22:
315-329). In some instances, incorporation of large PEG chains
prevents filtration of the peptide in the glomeruli in the kidney
into the dilute urine forming there (Nestor, J. J., Jr. (2009)
Current Medicinal Chemistry 16: 4399-4418, Caliceti, P. and
Veronese, F. M. (2003) Adv Drug Deliv Rev 55: 1261-1277). In some
embodiments, an optional PEG hydrophilic chain allows for balancing
the solubility and physical properties of the peptides or proteins
that have been rendered hydrophobic by the incorporation of the
longer chain alkyl glycoside moiety.
[1109] PEGylation of a protein can have potentially negative
effects as well. Thus PEGylation can cause a substantial loss of
biological activity for some proteins and this may relate to
ligands for specific classes of receptors. In such instances there
may be a benefit to reversible PEGylation (Peleg-Shulman, T., et
al. (2004) J Med Chem 47: 4897-4904, Greenwald, R. B., et al.
(2003) Adv Drug Deliv Rev 55: 217-250, Roberts, M. J. and Harris,
J. M. (1998) J Pharm Sci 87: 1440-1445).
[1110] In addition, the increased molecular mass may prevent
penetration of physiological barriers other than the glomerular
membrane barrier. For example, it has been suggested that high
molecular weight forms of PEGylation may prevent penetration to
some tissues and thereby reduce therapeutic efficacy. In addition,
high molecular weight may prevent uptake across mucosal membrane
barriers (nasal, buccal, vaginal, oral, rectal, lung delivery).
However delayed uptake may be highly advantageous for
administration of stable molecules to the lung, substantially
prolonging the duration of action. The peptide and/or protein
products described herein have increased transmucosal
bioavailability and this will allow longer chain PEG modifications
to be used in conjunction with the surfactant modification with the
achievement of commercially significant bioavailability following
intranasal or other transmucosal route.
[1111] In some embodiments, long chain PEG polymers, and short
chain PEG polymers are suitable for modification of the proteins
and peptides described herein. Administration of treatments for
diabetes by inhalation is a new approach for drug delivery and the
lung has a highly permeable barrier (e.g. Exubera). For this
application, delayed penetration of the lung barrier, preferred
forms of PEGylation are in the lower molecular weight range of
C.sub.10 to C.sub.400 (roughly 250 to 10,000 Da). Thus while a
primary route to prolongation by PEG is the achievement of an
"effective molecular weight" above the glomerular filtration
cut-off (greater than 68 kDa), use of shorter chains may be a route
for prolongation of residence in the lung for treatment of lung
diseases and other respiratory conditions. Thus PEG chains of about
500 to 3000 Da are of sufficient size to slow the entry into the
peripheral circulation, but insufficient to cause them to have a
very prolonged circulation time. In some embodiments, PEGylation is
applied to give increased local efficacy to the lung tissue with
reduced potential for systemic side effects for the the covalently
modified peptides and/or proteins described herein. In some of such
embodiments, PEG chains in the range from about 750 to about 1500
Da are referred collectively as "PEG1K."
[1112] In addition, other polymers may be used in conjunction with
the compounds of described herein in order to optimize their
physical properties. For example poly(2-ethyl 2-oxazoline)
conjugates have variable hydrophobicity and sufficient size to
enhance duration of action (Mero, A., et al. (2008) J Control
Release 125: 87-95). Linkage of such a polymer to a saccharide
yields a class of surfactant suitable for use in modification of
peptides and/or proteins described herein.
[1113] Polyethylene glycol chains are functionalized to allow their
conjugation to reactive groups on the peptide and/or protein chain.
Typical functional groups allow reaction with amino, carboxyl or
sulfhydryl groups on the peptide through the corresponding
carboxyl, amino or maleimido groups (and the like) on the
polyethylene glycol chain. In an embodiment, PEG comprises a
C.sub.10-C.sub.3000 chain. In another embodiment, PEG has a
molecular weight above 40,000 Daltons. In yet another embodiment,
PEG has a molecular weight below 10,000 Daltons. PEG as a protein
modification is well known in the art and its use is described, for
example, in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192; and 4,179,337.
[1114] A non-traditional type of PEG chain is modified to be
amphiphilic in nature. That is it has both the hydrophilic PEG
structure but is modified to contain hydrophobic regions such as
fatty acid esters and other hydrophobic components. See for example
(Miller, M. A., et al. (2006) Bioconjug Chem 17: 267-274);
Ekwuribe, et al. U.S. Pat. No. 6,309,633; Ekwuribe, et al. U.S.
Pat. No. 6,815,530; Ekwuribe, et al. U.S. Pat. No. 6,835,802).
Although these amphiphilic PEG conjugates to proteins were
originally developed to increase oral bioavailability they were
relatively ineffective in this role. However the use of such
amphiphilic PEG conjugates with amphipathic peptides will give
significantly prolonged residence in the lung to extend the useful
biological activity of these pharmaceuticals. The preferred PEG
chains are in the molecular weight range of 500 to 3000 Da.
Detailed descriptions of the methods of synthesis of these
conjugates is given in the references above, the full content of
which is incorporated herein.
[1115] A PEG entity itself does not have a functional group to be
attached to a target molecular, such as a peptide. Therefore, to
create PEG attachment, a PEG entity must be functionalized first,
then a functionalized attachment is used to attach the PEG entity
to a target molecule, such as a peptide (Greenwald, R. B., et al.
(2003) Adv Drug Deliv Rev 55: 217-250, Veronese, F. M. and Pasut,
G. (2005) Drug Discov Today 10: 1451-1458, Roberts, M. J., et al.
(2002) Adv Drug Deliv Rev 54: 459-476). In one embodiment,
site-specific PEGylation can be achieved through Cys substitution
on a peptide molecule. The target peptide can be synthesized by
solid phase synthesis, recombinant means, or other means, as
described herein.
[1116] Thus in some embodiments, a peptide product described herein
comprises a Lys or other reactive residue modified with an alkyl
glycoside and specific PEGylation on at least one Cys residue, a
Lys residue or other reactive amino acid residue elsewhere in the
molecule.
[1117] In another embodiment, a Lys or other residue residue with a
nucleophilic side chain may be used for incorporation of the PEG
residue. This may be accomplished through the use of an amide or
carbamate linkage to a PEG-carboxyl or PEG-carbonate chain. See for
example as described (Veronese, F. M. and Pasut, G. (2005) Drug
Discov Today 10: 1451-1458). An alternative approach is to modify
the Lys side chain amino function through attachment of an SH
containing residue, such as mercaptoacetyl, mercaptopropionyl
(CO--CH.sub.2--CH.sub.2--CH.sub.2--SH), and the like. Alternatively
the PEG chain may be incorporated at the C-Terminus as an amide
during the course of the synthesis. Additional methods for
attaching PEG chains utilize reaction with the side chains of His
and Trp. Other similar methods of modifying the peptide chain to
allow attachment of a PEG chain are known in the art and are
incorporated herein by reference (Roberts. M. J., et al. (2002) Adv
Drug Deliv Rev 54: 459-476).
Formulations
[1118] In one embodiment, the covalently modified peptides or
proteins as disclosed herein are provided in a formulation that
further reduces, prevents, or lessens peptide and/or protein
association or aggregation in the composition, for example, reduces
peptide and/or protein self-association or self-aggregation, or
reduces association or aggregation with other peptides or proteins
when administered to the subject.
[1119] Self-Association at high protein concentration is
problematic in therapeutic formulations. For example,
self-association increases the viscosity of a concentrated
monoclonal antibody in aqueous solution. Concentrated insulin
preparations are inactivated by self aggregation. These self
associating protein interactions, particularly at high protein
concentration, reduce, modulate or obliterate biological activity
of many therapeutics (Clodfelter, D. K., et al. (1998) Pharm Res
15: 254-262). Therapeutic proteins formulated at high
concentrations for delivery by injection or other means can be
physically unstable or become insoluble as a result of these
protein interactions.
[1120] A significant challenge in the preparation of peptide and
protein formulations is to develop manufacturable and stable dosage
forms. Physical stability properties, critical for processing and
handling, are often poorly characterized and difficult to predict.
A variety of physical instability phenomena are encountered such as
association, aggregation, crystallization and precipitation, as
determined by protein interaction and solubility properties. This
results in significant manufacturing, stability, analytical, and
delivery challenges. Development of formulations for peptide and
protein drugs requiring high dosing (on the order of mg/kg) are
required in many clinical situations. For example, using the SC
route, approximately <1.5 mL is the allowable administration
volume. This may require >100 mg/mL protein concentrations to
achieve adequate dosing. Similar considerations exist in developing
a high-concentration lyophilized formulation for monoclonal
antibodies. In general, higher protein concentrations permit
smaller injection volume to be used which is very important for
patient comfort, convenience, and compliance. The
surfactant-modified compounds described herein are designed to
minimize such aggregation events and may be further facilitated
through the use of small amounts of surfactants as herein
described.
[1121] Because injection is an uncomfortable mode of administration
for many people, other means of administering peptide therapeutics
have been sought. Certain peptide and protein therapeutics may be
administered, for example, by intranasal, buccal, oral, vaginal,
inhalation, or other transmucosal administration. Examples are
nafarelin (Synarel.RTM.) and calcitonin which are administered as
commercial nasal spray formulations. The covalently modified
peptides and/or proteins described herein are designed to
facilitate such transmucosal administration and such formulations
may be further facilitated through the use of small amounts of
surfactants as described herein.
[1122] Typical formulation parameters include selection of optimum
solution pH, buffer, and stabilizing excipients. Additionally,
lyophilized cake reconstitution is important for lyophilized or
powdered formulations. A further and significant problem comprises
changes in viscosity of the protein formulation upon
self-association. Changes in viscosity can significantly alter
delivery properties e.g., in spray (aerosol) delivery for
intranasal, pulmonary, or oral cavity sprays. Furthermore,
increased viscosity can make injection delivery by syringe or iv
line more difficult or impossible.
[1123] Many attempts to stabilize and maintain the integrity and
physiological activity of peptides have been reported. Some
attempts have produced stabilization against thermal denaturation
and aggregation, particularly for insulin pump systems. Polymeric
surfactants are described (Thurow, H. and Geisen, K. (1984)
Diabetologia 27: 212-218; Chawla, A. S., et al. (1985) Diabetes 34:
420-424). The stabilization of insulin by these compounds was
believed to be of a steric nature. Among other systems used are
saccharides (Arakawa, T. and Timasheff, S. N. (1982) Biochemistry
21: 6536-6544), osmolytes, such as amino acids (Arakawa, T. and
Timasheff, S. N. (1985) Biophys J 47: 411-414), and water structure
breakers, such as urea (Sato. S., et al. (1983) J Pharm Sci 72:
228-232). These compounds exert their action by modulating the
intramolecular hydrophobic interaction of the protein or
peptide.
[1124] Various peptides, peptides, or proteins are described herein
and may be modified with any of the covalently bound surfactant
reagents described herein. Advantageously, the peptide
modifications described herein comprise covalent attachment of a
surfactant that comprises both hydrophilic (e.g. saccharide) and
hydrophobic (e.g. alkyl chain) groups, thereby allowing for
stabilization of the peptide in physiological conditions. In some
embodiments, covalent linkage of a moiety comprising a hydrophilic
group and hydrophobic group (e.g. a glycoside surfactant) to a
peptide, and/or protein described herein eliminates the need for
modifying the amino acid sequence of the peptide, and/or protein to
enhance stability (e.g., reduce aggregation).
[1125] In some embodiments, the formulations comprise at least one
drug comprising a peptide modified with a surfactant derived
reagent described herein and in formulation additionally may be
associated with a surfactant, wherein the surfactant is further
comprised of, for example, a saccharide, an alkyl glycoside, or
other excipient and can be administered in a format selected from
the group consisting of a drop, a spray, an aerosol, a
lyophilizate, a spray dried product, an injectable, and a sustained
release format. The spray and the aerosol can be achieved through
use of the appropriate dispenser and may be administered by
intranasal, transbuccal, inhalation or other transmucosal route.
The lyophilizate may contain other compounds such as mannitol,
saccharides, submicron anhydrous .alpha.-lactose, gelatin,
biocompatible gels or polymers. The sustained release format can be
an ocular insert, erodible microparticulates, hydrolysable
polymers, swelling mucoadhesive particulates, pH sensitive
microparticulates, nanoparticles/latex systems, ion-exchange resins
and other polymeric gels and implants (Ocusert, Alza Corp.,
California; Joshi, A., S. Ping and K. J. Himmelstein, Patent
Application WO 91/19481). Significant oral bioavailability is also
achievable.
[1126] The peptide and protein modifications described herein
mitigate and, in some cases, may eliminate the need for organic
solvents. Trehalose, lactose, and mannitol and other saccharides
have been used to prevent aggregation. Aggregation of an anti-IgE
humanized monoclonal antibody was minimized by formulation with
trehalose at or above a molar ratio in the range of 300:1 to 500:1
(excipient:protein). However, the powders were excessively cohesive
and unsuitable for aerosol administration or exhibited unwanted
protein glycation during storage (Andya, J. D., et al. (1999) Pharm
Res 16: 350-358). Each of the additives discovered have limitations
as additives to therapeutics including xenobiotic metabolism,
irritation or toxicity, or high cost. Contemplated for use with the
covalently modified peptides and/or proteins described herein are
excipients that are effective, non-irritating and non-toxic, do not
require xenobiotic metabolism since they are comprised of the
natural sugars, fatty acids, or long chain alcohols, and which may
also be used to minimize aggregation in aqueous solutions or upon
aqueous reconstitution of dried peptide and/or protein formulations
in situ by physiologic aqueous reconstitution by aqueous body
fluids such as plasma or saliva.
[1127] Other formulation components could include buffers and
physiological salts, non-toxic protease inhibitors such as
aprotinin and soybean trypsin inhibitor, alpha-1-antitrypsin, and
protease-inactivating monoclonal antibodies, among others. Buffers
could include organics such as acetate, citrate, gluconate,
fumarate, malate, polylysine, polyglutamate, chitosan, dextran
sulfate, etc. or inorganics such as phosphate, and sulfate. Such
formulations may additionally contain small concentrations of
bacteriostatic agents like benzyl alcohol, and the like.
[1128] Formulations suitable for intranasal administration also
comprise solutions or suspensions of the modified peptides and/or
protein products described herein in an acceptable evaporating
solvents such as hydrofluoroalkanes. Such formulations are suitable
for administration from metered dose inhalers (MDI) and have
advantages of lack of movement from site of administration, low
irritation and absence of need for sterilization. Such formulations
may also contain acceptable excipients or bulking agents such as
submicron anhydrous .alpha.-lactose.
[1129] In yet another aspect, the covalently modified peptides
and/or proteins described herein exhibit increased shelf-life. As
used herein, the phrase "shelf life" is broadly described as the
length of time a product may be stored without becoming unsuitable
for use or consumption. The "shelf life" of the composition
described herein, can also indicate the length of time that
corresponds to a tolerable loss in quality of the composition. The
compositional shelf life as used herein is distinguished from an
expiration date; "shelf life" relates to the quality of the
composition described herein, whereas "expiration date" relates
more to manufacturing and testing requirements of the composition.
For example, a composition that has passed its "expiration date"
may still be safe and effective, but optimal quality is no longer
guaranteed by the manufacturer.
Methods
[1130] In one aspect, provided herein are methods of administering
to a subject in need thereof an effective amount of the therapeutic
compositions described herein. As used herein. "therapeutically
effective amount" is interchangeable with "effective amount" for
purposes herein, and is determined by such considerations as are
known in the art. The amount must be effective to achieve a desired
drug-mediated effect in the treated subjects suffering from the
disease thereof. A therapeutically effective amount also includes,
but is not limited to, appropriate measures selected by those
skilled in the art, for example, improved survival rate, more rapid
recovery, or amelioration, improvement or elimination of symptoms,
or other acceptable biomarkers or surrogate markers.
[1131] The compositions described herein are delivered to a
vertebrate subject in need of treatment including but not limited
to, for example, a human. Moreover, depending on the condition
being treated, these therapeutic compositions may be formulated and
administered systemically or locally. Techniques for formulation
and administration may be found in the latest edition of
"Remington's Pharmaceutical Sciences" (Mack Publishing Co, Easton
Pa.). Suitable routes may, for example, include oral or
transmucosal administration; such as intranasal; buccal; ocular,
vaginal; rectal; as well as parenteral delivery, including
intramuscular, subcutaneous, intravenous, or intraperitoneal
administration.
[1132] In some embodiments of the methods, the effective amount of
the peptide product for administration is from about 0.1
.mu.g/kg/day to about 100.0 .mu.g/kg/day, or from 0.01 .mu.g/kg/day
to about 1 mg/kg/day or from 0.1 .mu.g/kg/day to about 50
mg/kg/day. In some embodiments, the peptide product is administered
parenterally. In some embodiments, the peptide product is
administered subcutaneously. In some embodiments, the method of
administration of the peptide product is nasal insufflation.
[1133] It will be understood, however, that the specific dose level
and frequency of dosage for any particular subject in need of
treatment may be varied and will depend upon a variety of factors
including the activity of the specific compound employed, the
metabolic stability and duration of action of that compound, the
age, body weight, general health, sex, diet, mode and time of
administration, rate of excretion, drug combination, the severity
of the particular condition, and the host undergoing therapy.
[1134] In one embodiment, provided is a method for chemically
modifying a molecule by covalent linkage to a surfactant to
increase or sustain the biological action of the composition or
molecule, for example, receptor binding or enzymatic activity. In
some embodiments, the molecule is a peptide. The method
additionally can include further modification comprising covalent
attachment of the molecule in the composition to a polymer such as
polyethylene glycol.
[1135] The method(s) includes all aspects of the compositions
described herein including but not limited to compositions which
reduce or eliminate immunogenicity of peptide and/or protein drugs,
are non-irritating, have anti-bacterial or anti-fungal activity,
have increased stability or bioavailability of a drug, decrease the
bioavailability variance of that drug, avoid first pass liver
clearance and reduce or eliminate any adverse effects. As used
herein, the term "immunogenicity" is the ability of a particular
substance or composition or agent to provoke an immunological
response. The immunogenicity of the covalently modified peptides
and/or proteins described herein is confirmed by methods known in
the art.
[1136] Also provided is a method of administering a drug
composition comprising a peptide covalently linked to at least one
alkyl glycoside and delivered to a vertebrate, wherein the alkyl
has from 1 to 30 carbon atoms, or further in the range of 6 to 16
carbon atoms, and the alkyl glycoside increases the stability,
bioavailability and/or duration of action of the drug.
[1137] In another embodiment, provided is a method of reducing or
eliminating immunogenicity of a peptide and/or protein drug by
covalently linking the peptide chain to at least one alkyl
glycoside wherein the alkyl has from 1 to 30 carbon atoms.
[1138] Throughout this application, various publications are
referenced. One skilled in the art will understand that the
referenced disclosures of these publications are hereby
incorporated by reference into this application.
Methods of Treatment
[1139] Provided herein, in some embodiments are methods for
treatment of pain, including post-operative or chronic pain,
comprising administration of of a surfactant-modified peptide
and/or protein product described herein (e.g., a peptide product of
Formula I, II or III) to individuals in need thereof. In some of
such embodiments, the opioid analogs described herein are not
addictive or habit forming, and/or are administered in lower
dosages compared to current medications (e.g., codeine) and are
longer lasting compared to current medications.
[1140] Provided herein, in some embodiments are methods for
prevention and/or treatment of conditions associated with
hypoparathyroidism and/or decreases in bone mass density comprising
administration of a therapeutically effective amount of a
surfactant-modified peptide and/or protein product described herein
(e.g., a peptide product of Formula 2-I-A, 2-III, 2-V, 2-VI or
2-VII) to individuals in need thereof. In some embodiments, the
conditions characterized by decreases in bone mass density include,
and are not limited to, osteoporosis, osteopenia, post-menopausal
osteoporosis, Paget's disease, glucocorticoid induced osteoporosis,
old age osteoporosis, humoral hypercalcemia, or the like.
[1141] In some embodiments, provided herein are methods for
treatment of hypoparathyroidism comprising administration of a
therapeutically effective amount of a surfactant-modified peptide
and/or protein product described herein (e.g., a peptide product of
Formula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) to individuals in need
thereof. In some embodiments, the hypoparathyroidism is associated
with decrease in bone mass density.
[1142] Further provided herein are methods for stimulating bone
repair and/or favoring engraftment of a bone implant comprising
administration of a therapeutically effective amount of a
surfactant-modified peptide and/or protein product described herein
(e.g., a peptide product of Formula 2-I-A, 2-III, 2-V, 2-VI or
2-VII) to individuals in need thereof.
[1143] In yet further embodiments, provided herein are methods for
increasing bone density and/or reducing incidence of fractures
(e.g., vertebrae fractures, hip fractures, or the like) comprising
administration of a therapeutically effective amount of a
surfactant-modified peptide and/or protein product described herein
(e.g., a peptide product of Formula 2-I-A, 2-III, 2-V, 2-VI or
2-VII) to individuals in need thereof.
[1144] In some embodiments, provided herein are methods for
treatment of humoral hypercalcemia comprising administration of a
therapeutically effective amount of a surfactant-modified peptide
and/or protein product described herein (e.g., a peptide product of
Formula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) to individuals in need
thereof. In some embodiments, humoral hypercalcemia is associated
with tumors. In some of such embodiments, the peptide product
(e.g., a peptide product of Formula 2-I-A, 2-III, 2-V, 2-VI or
2-VII) is an inverse agonist or an antagonist of PTH or PTHrP.
[1145] In some embodiments of the methods described above, the
peptide and/or protein that is covalently attached to a surfactant
is PTH, PTHrP, or an analog thereof. In some embodiments, the
surfactant-modified peptide and/or protein (e.g., a peptide product
of Formula 2-I-A, 2-, 2-V, 2-VI or 2-VII) is administered
prophylactically and delays occurrence of any condition associated
with loss of bone density, including and not limited to
osteoporosis, osteopenia, post-menopausal osteoporosis, Paget's
disease, glucocorticoid induced osteoporosis, old age osteoporosis,
or the like. In some embodiments, the surfactant-modified peptide
and/or protein (e.g., a peptide product of Formula 2-I-A, 2-III,
2-V, 2-VI or 2-VII) is administered therapeutically and delays
progression of any condition associated with loss of bone density,
including and not limited to osteoporosis, osteopenia,
post-menopausal osteoporosis, Paget's disease, glucocorticoid
induced osteoporosis, humoral hypercalcemia, or the like. In some
embodiments, the surfactant-modified peptide and/or protein (e.g.,
a peptide product of Formula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) is
administered prophylactically and/or therapeutically and delays
progression of osteopenia to osteoporosis. In some embodiments, the
surfactant-modified peptide and/or protein (e.g., a peptide product
of Formula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) is administered
prophylactically and/or therapeutically and reduces or halts
further loss of bone density, thereby stabilizing disease.
[1146] In some embodiments, the surfactant-modified peptide and/or
protein (e.g., a peptide product of Formula 2-I-A, 2-III, 2-V, 2-VI
or 2-VII) is administered parenterally. In some embodiments, the
surfactant-modified peptide and/or protein (e.g., a peptide product
of Formula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) is administered
subcutaneously. In some embodiments, the surfactant-modified
peptide and/or protein (e.g., a peptide product of Formula 2-I-A,
2-III, 2-V, 2-VI or 2-VII) is administered by nasal
insufflation.
[1147] In some embodiments of the methods described above, the
surfactant-modified peptide and/or protein (e.g., a peptide product
of Formula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) has a longer duration
of action compared to a pharmaceutical comprising currently known
therapeutics (e.g., recombinant PTH, bisphosphonates, antibody
Denosumab, or the like). In some embodiments, the
surfactant-modified peptide and/or protein (e.g., a peptide product
of Formula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) is administered for
longer period of time (e.g., >two years) compared to a
pharmaceutical comprising currently known therapeutics (e.g.,
recombinant PTH, bisphosphonates, antibody Denosumab, or the like)
while reducing or ameliorating side-effects (e.g., osteonecrosis in
jaw, skin infections or the like) associated with currently known
therapeutics (e.g., recombinant PTH, bisphosphonates, antibody
Denosumab, or the like). In some embodiments of the methods
described above, the surfactant-modified peptide and/or protein
(e.g., a peptide product of Formula 2-I-A, 2-III, 2-V, 2-VI or
2-VII) is an agonist of PTH or PTHrP. In some embodiments of the
methods described above, the surfactant-modified peptide and/or
protein (e.g., a peptide product of Formula 2-I-A, 2-III, 2-V, 2-VI
or 2-VII) is an antagonist of PTH or PTHrP. In some embodiments of
the methods described above, the surfactant-modified peptide and/or
protein (e.g., a peptide product of Formula 2-I-A, 2-III, 2-V, 2-VT
or 2-VII) is an inverse agonist of PTH or PTHrP.
[1148] Provided herein, in some embodiments are methods for
prevention and/or treatment of conditions associated with decreases
in insulin sensitivity comprising administration of a
therapeutically effective amount of a surfactant-modified peptide
and/or protein product described herein (e.g., a peptide product of
Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) to individuals in
need thereof. In some embodiments, the conditions characterized by
decreases in insulin sensitivity include, and are not limited to,
the metabolic syndrome, obesity-related insulin resistance,
hypertension, systemic inflammation associated with high C reactive
protein, diabetes, or the like.
[1149] Also provided is a method of treating conditions associated
with insulin resistance including and not limited to obesity, the
metabolic syndrome, type 2 diabetes, hypertension, atherosclerosis
or the like, comprising administering a drug composition comprising
a peptide covalently linked to at least one alkyl glycoside and
delivered to a vertebrate, wherein the alkyl has from 1 to 30
carbon atoms, or further in the range of 6 to 18 carbon atoms
(e.g., a peptide product of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V), and wherein covalent linkage of the alkyl glycoside
to the peptide increases the stability, bioavailability and/or
duration of action of the drug.
[1150] Also provided herein are methods for treatment of insulin
resistance comprising administration of a therapeutically effective
amount of a surfactant-modified peptide and/or protein product
described herein (e.g., a peptide product of Formula 3-I-A,
3-III-A, 3-III-B or Formula 3-V) to individuals in need thereof. In
some embodiments, the insulin resistance is associated with the
metabolic syndrome (Syndrome X) and/or diabetes.
[1151] Further provided herein are methods for stimulating
resensitization of the body to insulin comprising administration of
a therapeutically effective amount of a surfactant-modified peptide
and/or protein product described herein (e.g. a peptide product of
Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) to individuals in
need thereof.
[1152] In yet further embodiments, provided herein are methods for
increasing insulin sensitivity through weight loss, comprising
administration of a therapeutically effective amount of a
surfactant-modified peptide and/or protein product described herein
(e.g. a peptide product of Formula 3-I-A, 3-III-A, 3-III-B or
Formula 3-V and in Table 1 of FIG. 1 and Table 2 of FIG. 2) to
individuals in need thereof.
[1153] Also provided herein are methods of treating diabetes or
prediabetes comprising administering to a subject in need thereof a
therapeutically effective amount of a peptide product described
above and herein and in Table 1 of FIG. 1 and Table 2 of FIG. 2 to
an individual in need thereof.
[1154] Provided herein are methods for treating or delaying the
progression or onset of conditions selected from diabetes, diabetic
retinopathy, diabetic neuropathy, diabetic nephropathy, insulin
resistance, hyperglycemia, hyperinsulinemia, metabolic syndrome,
diabetic complications, elevated blood levels of free fatty acids
or glycerol, hyperlipidemia, obesity, hypertriglyceridemia,
atherosclerosis, acute cardiovascular syndrome, infarction,
ischemic reperfusion a hypertension, comprising administering a
therapeutically effective amount of a peptide product described
herein and in Table 1 of FIG. 1 and Table 2 of FIG. 2 to an
individual in need thereof. In an additional embodiment, provided
herein are methods for treating delays in wound healing comprising
administering a therapeutically effective amount of a peptide
product described herein and in Table 1 of FIG. 1 and Table 2 of
FIG. 2 to an individual in need thereof.
[1155] In one embodiment said condition to be treated is diabetes.
In one embodiment said condition to be treated is insulin
resistance. In one embodiment said condition to be treated is the
metabolic syndrome. In one embodiment said effective amount of said
peptide is from about 0.1 .mu.g/kg/day to about 100.0
.mu.g/kg/day.
[1156] In one embodiment the method of administration is
parenteral. In one embodiment the method of administration is per
oral. In one embodiment the method of administration is
subcutaneous. In one embodiment the method of administration is
nasal insufflation.
[1157] Further provided herein is a method of reducing weight gain
or inducing weight loss comprising administering a therapeutically
effective amount of a peptide product described herein and in Table
1 of FIG. 1 and Table 2 of FIG. 2 to an individual in need thereof.
In some embodiments, the weight gain is associated with metabolic
syndrome.
[1158] Provided herein is a method of treating hypoglycemia
comprising administering a therapeutically effective amount of a
peptide product described herein and in Table 1 of FIG. 1 and Table
2 of FIG. 2 to an individual in need thereof.
[1159] Also provided herein are methods for treatment of diabetes
comprising administering a therapeutically effective amount of a
peptide product described herein and in Table 1 of FIG. 1 and Table
2 of FIG. 2 to an individual in need thereof and at least one
additional therapeutic agent; wherein said therapeutic agent is
selected from an antidiabetic agent, an anti-obesity agent, a
satiety agent, an anti-inflammatory agent, an anti-hypertensive
agent, an anti-atherosclerotic agent and a lipid-lowering
agent.
[1160] In some embodiments of the methods described above, the
peptide and/or protein that is covalently attached to a surfactant
is a glucagon or GLP-1 peptide, or an analog thereof. In some
embodiments, the surfactant-modified peptide and/or protein (e.g.,
a peptide product of Formula 3-I-A, 3-III-A, 3-III-B or Formula
3-V) is administered prophylactically and delays occurrence of any
condition associated with insulin resistance, including and not
limited to the metabolic syndrome, hypertension, diabetes, type 2
diabetes, gestational diabetes, hyperlipidemia, atherosclerosis,
systemic inflammation or the like. In some embodiments, the
surfactant-modified peptide and/or protein (e.g., a peptide product
of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administered
therapeutically and delays progression of any condition associated
with the metabolic syndrome, hypertension, diabetes, type 2
diabetes, gestational diabetes, hyperlipidemia, atherosclerosis,
systemic inflammation or the like. In some embodiments, the
surfactant-modified peptide and/or protein (e.g., a peptide product
of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administered
prophylactically and/or therapeutically and delays progression of
insulin resistance to diabetes. In some embodiments, the
surfactant-modified peptide and/or protein (e.g., a peptide product
of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administered
prophylactically and/or therapeutically and reduces or halts
further loss of insulin resistance, thereby stabilizing
disease.
[1161] In some embodiments, the surfactant-modified peptide and/or
protein (e.g., a peptide product of Formula 3-I-A, 3-III-A, 3-III-B
or Formula 3-V) is administered parenterally. In some embodiments,
the surfactant-modified peptide and/or protein (e.g., a peptide
product of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is
administered subcutaneously. In some embodiments, the
surfactant-modified peptide and/or protein (e.g., a peptide product
of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administered
by nasal insufflation.
[1162] In some embodiments of the methods described above, the
surfactant-modified peptide and/or protein (e.g., a peptide product
of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) has a longer
duration of action compared to a pharmaceutical comprising
currently known therapeutics (e.g., exenatide, metformin or the
like).
Combination Therapy with PTH Analogs
[1163] Is some embodiments of the methods described above, the
surfactant-modified peptide and/or protein (e.g., a peptide product
of Formula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) is administered in
combination with a bone resorption inhibitor including, a
bisphosphonate, (e.g. alendronate) or strontium salt; or a
substance with estrogen-like effect, e.g. estrogen; or a selective
estrogen receptor modulator, e.g. raloxifene, tamoxifene,
droloxifene, toremifene, idoxifene, or levormeloxifene; or a
calcitonin-like substance, e.g. calcitonin; or a vitamin D analog;
or a calcium salt. The therapeutic agents are optionally
administered simultaneously, or sequentially in any order. By way
of example, in some embodiments of the methods described above, a
first regimen of the surfactant-modified peptide and/or protein
(e.g., a peptide product of Formula 2-I-A, 2-III, 2-V, 2-VI or
2-VII) is administered to an individual in need thereof, and the
regimen is followed by a second regimen of bisphosphonate therapy.
By way of example, in some other embodiments, a first regimen of
the surfactant-modified peptide and/or protein (e.g., a peptide
product of Formula 2-I-A, 2-III, 2-V, 2-VI or 2-VII) is
administered to an individual in need thereof, followed by a drug
holiday, followed by a second regimen of estrogen receptor
modulators.
Combination Therapy with GLP Analogs
[1164] In some embodiments of the methods described above, the
surfactant-modified peptide and/or protein (e.g., a peptide product
of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administered
in combination with other methods of treatment of the metabolic
syndrome selected from the group comprising an antidiabetic agent,
an anti-obesity agent, an anti-hypertensive agent, an
anti-atherosclerotic agent and a lipid-lowering agent. By way of
example, efficacious antidiabetic agents suitable for
administration in combination with a surfactant-modified peptide
and/or protein product described herein include a biguanide, a
sulfonylurea, a glucosidase inhibitor a PPAR .gamma. agonist, a
PPAR .alpha./.gamma. dual agonist, an aP2 inhibitor, a DPP4
inhibitor, an insulin sensitizer, a GLP-1 analog, insulin and a
meglitinide. Additional examples include metformin, glyburide,
glimepiride, glipyride, glipizide, chlorpropamide, gliclazide,
acarbose, miglitol, pioglitazone, troglitazone, rosiglitazone,
muraglitazar, insulin, G1-262570, isaglitazone, JTT-501, NN-2344,
L895 645, YM-440, R-119702, A19677, repaglinide, nateglinide, KAD
1129, AR-HO 39242, GW-4015 44, KRP2 I 7, AC2993. LY3 I 5902,
NVP-DPP-728A and saxagliptin.
[1165] In some embodiments of the methods described above, the
surfactant-modified peptide and/or protein (e.g., a peptide product
of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administered
in combination with other methods of treatment of the metabolic
syndrome selected from the group of efficacious anti-obesity
agents. By way of example, efficacious anti-obesity agents suitable
for administration with the peptide products described herein
include beta 3 adrenergic agonist, a lipase inhibitor, a serotonin
(and dopamine) reuptake inhibitor, a thyroid receptor beta
compound, a CB-1 antagonist, a NPY-Y2 and a NPY-Y4 receptor agonist
and an anorectic agent. Specific members of these classes comprise
orlistat, AfL-962. A19671, L750355, CP331648, sibutramine,
topiramate, axokine, dexamphetamine, phentermine,
phenylpropanolamine, rimonabant (SR1 4I7164), and mazindol.
[1166] In some embodiments of the methods described above, the
surfactant-modified peptide and/or protein (e.g., a peptide product
of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administered
in combination with other methods of treatment of the metabolic
syndrome selected from the group of efficacious lipid-lowering
agents. By way of example, efficacious lipid-lowering agents
suitable for administration with the peptide products described
herein include agents selected from the group consisting of an MTP
inhibitor, cholesterol ester transfer protein, an HMG CoA reductase
inhibitor, a squalene synthetase inhibitor, a fibric acid
derivative, an upregulator of LDL receptor activity, a lipoxygenase
inhibitor, and an ACAT inhibitor. Specific examples from these
classes comprise pravastatin, lovastatin, simvastatin,
atorvastatin, cerivastatin, fluvastatin, nisvastatin, visastatin,
fenofibrate, gemfibrozil, clofibrate, avasimibe, TS-962, MD-700,
CP-529414, and LY295 427.
[1167] In some embodiments of the methods described above, the
surfactant-modified peptide and/or protein (e.g., a peptide product
of Formula 3-I-A, 3-III-A, 3-III-B or Formula 3-V) is administered
in combination with peptide hormones, and analogs thereof, that are
known to exhibit pro-satiety effects in animal models and in man.
Contemplated within the scope of embodiments presented herein is a
combination of the peptide products described herein and
long-acting satiety agents for treatment of obesity. Examples of
such peptide satiety agents include GLP-1, pancreatic polypeptide
(PP), cholecystokinin (CCK), peptide YY (PYY), amylin, calcitonin,
OXM, neuropeptide Y (NPY), and analogs thereof (Bloom, S. R., et
al. (2008) Mol Interv 8: 82-98; Field, B. C., et al. (2009) Br J
Clin Pharmacol 68: 830-843).
[1168] Also contemplated within the scope of embodiments presented
herein are methods for treatment of obesity comprising
administration of peptide products described herein in combination
with peptide hormones including and not limited to leptin, ghrelin
and CART (cocaine- and amphetamine-regulated transcript) analogs
and antagonists.
[1169] Additional peptide products in the body are associated with
fat cells or the obese state (adipokines) and are known to have
proinflammatory effects (Gonzalez-Periz, A. and Claria, J. (2010)
ScientificWorldJournal 10: 832-856). Such agents will have
additional favorable actions when used in combination with the
peptide products described herein. Examples of agents that offer a
beneficial effect when used in combination with the peptide
products described herein include analogs and antagonists of
adiponectin, chemerin, visfatin, nesfatin, omentin, resistin,
TNFalpha, IL-6 and obestatin.
Dosing
[1170] The covalently modified peptides and/or proteins described
herein may be administered in any amount to impart beneficial
therapeutic effect in a number of disease states. In some
embodiments, covalently modified peptides and/or proteins described
herein are useful in the treatment of inflammation. In an
embodiment, compounds presented herein impart beneficial activity
in the modulation of post-operative or chronic pain. In an
embodiment, the present peptides are administered to a patient at
concentrations higher or lower than that of other forms of
treatment which modulate pain. In yet another embodiment, the
present peptides are administered with other compounds to produce
synergistic therapeutic effects.
[1171] Representative delivery regimens include oral, parenteral
(including subcutaneous, intramuscular and intravenous injection),
rectal, buccal (including sublingual), transdermal, inhalation
ocular and intranasal. An attractive and widely used method for
delivery of peptides entails subcutaneous injection of a
controlled-release injectable formulation. In some embodiments,
covalently modified peptides and/or proteins described herein are
useful for subcutaneous, intranasal and inhalation
administration.
[1172] The selection of the exact dose and composition and the most
appropriate delivery regimen will be influenced by, inter alia, the
pharmacological properties of the selected peptide, the nature and
severity of the condition being treated, and the physical condition
and mental acuity of the recipient. Additionally, the route of
administration will result in differential amounts of absorbed
material. Bioavailabilities for administration of peptides through
different routes are particularly variable, with amounts from less
than 1% to near 100% being seen. Typically, bioavailability from
routes other than intravenous, intraperitoneal or subcutaneous
injection are 50% or less.
[1173] In general, covalently modified peptides and/or proteins
described herein, or salts thereof, are administered in amounts
between about 0.001 and 20 mg/kg body weight per day, between about
0.01 and 10 mg/kg body weight per day, between about 0.1 and 1000
.mu.g/kg body weight per day, or between about 0.1 to about 100
.mu.g/kg body weight per day. Routes of administration vary. For
example, covalently modified opioid peptides and/or proteins
described herein, or salts thereof, are administered in amounts
between about 0.1 and 1000 .mu.g/kg body weight per day, or between
about 0.1 to about 100 .mu.g/kg body weight per day, by
subcutaneous injection. By way of example, for a 50 kg human female
subject, the daily dose of active ingredient is from about 5 to
about 5000 .mu.g, or from about 5 to about 5000 .mu.g by
subcutaneous injection. Different doses will be needed, depending
on the route of administration, the compound potency, the
pharmacokinetic profile and the applicable bioavailability
observed, and the active agent and the disease being treated. In an
alternate embodiment where the administration is by inhalation, the
daily dose is from 1000 to about 20,000 .mu.g, twice daily. In
other mammals, such as horses, dogs, and cattle, higher doses may
be required. This dosage may be delivered in a conventional
pharmaceutical composition by a single administration, by multiple
applications, or via controlled release, as needed to achieve the
most effective results.
[1174] Pharmaceutically acceptable salts retain the desired
biological activity of the parent peptide without toxic side
effects. Examples of such salts are (a) acid addition salts formed
with inorganic acids, for example hydrochloric acid, hydrobromic
acid, sulfuric acid, phosphoric acid, nitric acid and the like; and
salts formed with organic acids such as, for example, acetic acid,
trifluoroacetic acid, tartaric acid, succinic acid, maleic acid,
fumaric acid, gluconic acid, citric acid, malic acid, ascorbic
acid, benzoic acid, tannic acid, pamoic acid, alginic acid,
polyglutamic acid, naphthalenesulfonic acids, naphthalene
disulfonic acids, polygalacturonic acid and the like; (b) base
addition salts or complexes formed with polyvalent metal cations
such as zinc, calcium, bismuth, barium, magnesium, aluminum,
copper, cobalt, nickel, cadmium, and the like; or with an organic
cation formed from N,N'-dibenzylethylenediamine or ethylenediamine;
or (c) combinations of (a) and (b), e.g., a zinc tannate salt and
the like.
[1175] Also contemplated, in some embodiments, are pharmaceutical
compositions comprising as an active ingredient covalently modified
peptides and/or proteins described herein, or pharmaceutically
acceptable salt thereof, in admixture with a pharmaceutically
acceptable, non-toxic carrier. As mentioned above, such
compositions may be prepared for parenteral (subcutaneous,
intramuscular or intravenous) administration, particularly in the
form of liquid solutions or suspensions; for oral or buccal
administration, particularly in the form of tablets or capsules;
for intranasal administration, particularly in the form of powders,
nasal drops, evaporating solutions or aerosols; for inhalation,
particularly in the form of liquid solutions or dry powders with
excipients, defined broadly; and for rectal or transdermal
administration.
[1176] The compositions may conveniently be administered in unit
dosage form and may be prepared by any of the methods well-known in
the pharmaceutical art, for example as described in Remington's
Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton,
Pa., (1985), incorporated herein by reference. Formulations for
parenteral administration may contain as excipients sterile water
or saline, alkylene glycols such as propylene glycol, polyalkylene
glycols such as polyethylene glycol, saccharides, oils of vegetable
origin, hydrogenated naphthalenes, serum albumin nanoparticles (as
used in Abraxane.TM., American Pharmaceutical Partners, Inc.
Schaumburg Ill.), and the like. For oral administration, the
formulation can be enhanced by the addition of bile salts or
acylcarnitines. Formulations for nasal administration may be solid
or solutions in evaporating solvents such as hydrofluorocarbons,
and may contain excipients for stabilization, for example,
saccharides, surfactants, submicron anhydrous .alpha.-lactose or
dextran, or may be aqueous or oily solutions for use in the form of
nasal drops or metered spray. For buccal administration typical
excipients include sugars, calcium stearate, magnesium stearate,
pregelatinated starch, and the like.
[1177] When formulated for nasal administration, the absorption
across the nasal mucous membrane may be further enhanced by
surfactants, such as for example, glycocholic acid, cholic acid,
taurocholic acid, ethocholic acid, deoxycholic acid,
chenodeoxycholic acid, dehydrocholic acid, glycodeoxycholic acid,
cyclodextrins and the like in an amount in the range between about
0.1 and 15 weight percent, between about 0.5 and 4 weight percent,
or about 2 weight percent. An additional class of absorption
enhancers reported to exhibit greater efficacy with decreased
irritation is the class of alkyl maltosides, such as
tetradecylmaltoside (Arnold, J. J., et al. (2004) J Pharm Sci 93:
2205-2213, Ahsan, F., et al. (2001) Pharm Res 18: 1742-1746) and
references therein, all of which are hereby incorporated by
reference.
[1178] When formulated for delivery by inhalation, a number of
formulations offer advantages. Adsorption of the active peptide to
readily dispersed solids such as diketopiperazines (for example
Technosphere particles; (Pfutzner, A. and Forst, T. (2005) Expert
Opin Drug Deliv 2: 1097-1106) or similar structures gives a
formulation which results in a rapid initial uptake of the
therapeutic agent. Lyophylized powders, especially glassy
particles, containing the active peptide and an excipient are
useful for delivery to the lung with good bioavailability, for
example, see Exubera.RTM. (inhaled insulin by Pfizer and Aventis
Pharmaceuticals Inc.). Additional systems for delivery of peptides
by inhalation are described (Mandal, T. K., Am. J. Health Syst.
Pharm. 62:1359-64 (2005)).
[1179] Delivery of covalently modified peptides and/or proteins
described herein to a subject over prolonged periods of time, for
example, for periods of one week to one year, may be accomplished
by a single administration of a controlled release system
containing sufficient active ingredient for the desired release
period. Various controlled release systems, such as monolithic or
reservoir-type microcapsules, depot implants, polymeric hydrogels,
osmotic pumps, vesicles, micelles, liposomes, transdermal patches,
iontophoretic devices and alternative injectable dosage forms may
be utilized for this purpose. Localization at the site to which
delivery of the active ingredient is desired is an additional
feature of some controlled release devices, which may prove
beneficial in the treatment of certain disorders.
[1180] One form of controlled release formulation contains the
peptide or its salt dispersed or encapsulated in a slowly
degrading, non-toxic, non-antigenic polymer such as
copoly(lactic/glycolic) acid, as described in the pioneering work
of Kent, Lewis, Sanders, and Tice, U.S. Pat. No. 4,675,189,
incorporated by reference herein. The compounds, or their salts,
may also be formulated in cholesterol or other lipid matrix
pellets, or silastomer matrix implants. Additional slow release,
depot implant or injectable formulations will be apparent to the
skilled artisan. See, for example, Sustained and Controlled Release
Drug Delivery Systems, J. R. Robinson ed., Marcel Dekker, Inc., New
York, 1978, and R. W. Baker, Controlled Release of Biologically
Active Agents, John Wiley & Sons, New York, 1987.
[1181] An additional form of controlled-release formulation
comprises a solution of a biodegradable polymer, such as
copoly(lactic/glycolic acid) or block copolymers of lactic acid and
PEG, is bioacceptable solvent, which is injected subcutaneously or
intramuscularly to achieve a depot formulation. Mixing of the
peptides described herein with such a polymeric formulation is
suitable to achieve very long duration of action formulations.
[1182] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each independent publication or patent application is
specifically and individually indicated to be incorporated by
reference.
[1183] The covalently modified peptides and/or proteins described
herein and the reagents for the synthesis thereof are more
particularly described in the following examples which are intended
as illustrative only since numerous modifications and variations
therein will be apparent to those of ordinary skill in the art.
EXAMPLES
Example 1: Reagents--N-.alpha.-Fmoc, N-.epsilon.-(1-octyl
.beta.-D-glucuronide-6-yl)-L-lysine
[1184] In an oven-dried 250 mL Erlenmeyer flask is placed 1-octyl
.beta.-D-glucuronic acid (Carbosynth Ltd., 3.06 g, 10 mmol), 50 mL
anhydrous DMF, and anhydrous 1-hydroxybenzotriazole (1.62 g, 12
mmol). A chilled (4.degree. C.) solution of N,
N'-dicyclohexylcarbodiimide (2.48 g, 12 mmol) in 50 mL of DMF is
added, with stirring, and the reaction is allowed to proceed for 5
min. The copious white precipitate of N,N'-dicyclohexylurea is
filtered on a fritted glass funnel and the filtrate is added to a
solution of N-.alpha.-Fmoc-L-lysine (3.68 g, 10 mmol) in 25 ml
anhydrous DMF. The reaction is allowed to proceed for 25 min with
warming to room temp or until the ninhydrin color is very faint.
The reaction mixture is filtered, stripped to dryness and
crystallized from MeOH/Et.sub.2O by dissolution in MeOH and slow
dilution to the cloud point with Et.sub.2O, followed by
refrigeration. Further purification can be achieved by silica gel
chromatography using a solvent gradient from EtOAc to
EtOAc/EtOH/AcOH.
[1185] In a similar manner, but substituting N-.alpha.-Boc-L-lysine
is obtained N-.alpha.-Boc,N-.epsilon.-(1-octyl
.beta.-D-glucuronide-6-yl)-L-lysine, suitable for N-terminal
incorporation and cleavage to a free N-Terminus. In a similar
manner, but substituting N-.alpha.-Ac-L-lysine is obtained
N-.alpha.-Ac,N-.epsilon.-(1-octyl
.beta.-D-glucuronide-6-yl)-L-lysine, suitable for incorporation at
the N-terminus of a peptide with a blocked N-terminus. In a similar
manner, but substituting the appropriate amount of
N-.alpha.-Fmoc-L-ornithine is obtained
N-.alpha.-Fmoc,N-.delta.-(1-octyl
.beta.-D-glucuronide-6-yl)-L-ornithine. In a similar manner but
substituting other N-mono-protected diamino acids one obtains the
corresponding reagents. Alternatively, use of a transient
Me.sub.3Si ester protecting group during the coupling and without
preactivation of the 1-octyl .beta.-D-glucuronic acid provides a
facile route to the formation of the reagents. The transient
Me.sub.3Si ester is produced by reaction of the Fmoc-Lys-OH with an
equimolar amount of N,O-bis(trimethylsilyl)acetamide in
dichloromethane (CH.sub.2Cl.sub.2). The organic layer contains the
desired reagent as a solution in CH.sub.2Cl.sub.2 ready for
coupling with the 1-alkyl glucoronide as above. The filtered
reaction mixture is washed with aqueous NaHSO.sub.4 to hydrolyze
the Me.sub.3Si ester, dried over MgSO.sub.4 and solvent is
removed.
[1186] Similarly, but using peracetyl or perbenzoyl 1-octyl
.beta.-D-glucuronic acid one obtains the Ac, or Bz protected form
of the reagents (e.g. 2,3,4-trisacetyl 1-octyl .beta.-D-glucuronic
acid, and the like, formed by treatment with Ac.sub.2O). Such
reagents have increased stability during acid cleavage from the
resin and are used when instability during deprotection is
detected, see (Kihlberg, J., et al. (1997) Methods Enzymol 289:
221-245) and references therein. Final deprotection of such
products is carried out by base-catalyzed transesterification after
cleavage, by use of MeOH/NH.sub.3, MeOH/NaOMe,
MeOH/NH.sub.2NH.sub.2, as described above.
Example 2: Synthetic Peptide Analogs
[1187] In general, peptide synthesis methods involve the sequential
addition of protected amino acids to a growing peptide chain.
Normally, either the amino or carboxyl group of the first amino
acid and any reactive side chain group are protected. This
protected amino acid is then either attached to an inert solid
support, or utilized in solution, and the next amino acid in the
sequence, also suitably protected, is added under conditions
amenable to formation of the amide linkage. After all the desired
amino acids have been linked in the proper sequence, protecting
groups and any solid support are removed to afford the crude
peptide. The peptide is desalted and purified
chromatographically.
[1188] A preferred method of preparing the analogs of the
physiologically active truncated peptides, having fewer than about
fifty amino acids, involves solid phase peptide synthesis. In this
method the .alpha.-amino (N.alpha.) functions and any reactive side
chains are protected by acid- or base-sensitive groups. The
protecting group should be stable to the conditions of peptide
linkage formation, while being readily removable without affecting
the extant peptide chain. Suitable .alpha.-amino protecting groups
include, but are not limited to t-butoxycarbonyl (Boc),
benzyloxycarbonyl (Cbz), o-chlorobenzyloxycarbonyl,
biphenylisopropyloxycarbonyl, t-amyloxycarbonyl (Amoc),
isobornyloxycarbonyl, .alpha.,
.alpha.-dimethyl-3,5-dimethoxybenzyloxy-carbonyl,
o-nitrophenylsulfenyl, 2-cyano-t-butoxycarbonyl,
9-fluorenyl-methoxycarbonyl (Fmoc) and the like, preferably Boc or
more preferably, Fmoc. Suitable side chain protecting groups
include, but are not limited to: acetyl, benzyl (Bzl),
benzyloxymethyl (Bom), Boc, t-butyl, o-bromobenzyloxycarbonyl,
t-butyl, t-butyldimethylsilyl, 2-chlorobenzyl (Cl-z),
2,6-dichlorobenzyl, cyclohexyl, cyclopentyl, isopropyl, pivalyl,
tetrahydropyran-2-yl, tosyl (Tos),
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf),
trimethylsilyl and trityl. A preferred N.alpha.-protecting group
for synthesis of the compounds is the Fmoc group. Preferred side
chain protecting groups are O-t-Butyl group for Glu, Tyr, Thr, Asp
and Ser; Boc group for Lys and Trp side chains; Pbf group for Arg;
Trt group for Asn, Gln, and His. For selective modification of a
Lys residue, orthogonal protection with a protecting group not
removed by reagents that cleave the Fmoc or t-butyl based
protecting groups is preferred. Preferred examples for modification
of the Lys side chain include, but are not limited to, those
removed by hydrazine but not piperidine; for example
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde)
or 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) and
allyloxycarbonyl (Alloc).
[1189] The Fmoc-Lys(ivDde) or Fmoc-Lys(Dde) protecting group scheme
is preferred in cases where side chain lactam formation is desired
(Houston, M. E., Jr., et al. (1995) J Pept Sci 1: 274-282; Murage,
E. N., et al. (2010) J Med Chem), since in this case
Fmoc-Glu(O-Allyl) and Fmoc-Lys(Alloc) can be incorporated and used
to provide transient protection, then deprotected for lactam
formation while the Lys(Dde) protecting group remains for later
removal and reaction with the functionalized surfactant.
[1190] In solid phase synthesis, the C-terminal amino acid is first
attached to a suitable resin support. Suitable resin supports are
those materials which are inert to the reagents and reaction
conditions of the stepwise condensation and deprotection reactions,
as well as being insoluble in the media used. Examples of
commercially available resins include styrene/divinylbenzene resins
modified with a reactive group, e.g., chloromethylated
co-poly-(styrene-divinylbenzene), hydroxymethylated
co-poly-(styrene-divinylbenzene), and the like. Benzylated,
hydroxymethylated phenylacetamidomethyl (PAM) resin is preferred
for the preparation of peptide acids. When the C-terminus of the
compound is an amide, a preferred resin is
p-methylbenzhydrylamino-co-poly(styrene-divinyl-benzene) resin, a
2,4 dimethoxybenzhydrylamino-based resin ("Rink amide"), and the
like. An especially preferred support for the synthesis of larger
peptides are commercially available resins containing PEG sequences
grafted onto other polymeric matricies, such as the Rink Amide-PEG
and PAL-PEG-PS resins (Applied Biosystems) or similar resins
designed for peptide amide synthesis using the Fmoc protocol. Thus
in certain cases it is desirable to have an amide linkage to a PEG
chain. It those cases it is convenient to link an
N-Fmoc-amino-PEG-carboxylic acid to the amide forming resin above
(e.g. Rink amide resin and the like). The first amino acid of the
chain can be coupled as an N-Fmoc-amino acid to the amino function
of the PEG chain. Final deprotection will yield the desired
Peptide-NH-PEG-CO--NH.sub.2 product.
[1191] Attachment to the PAM resin may be accomplished by reaction
of the N.alpha. protected amino acid, for example the Boc-amino
acid, as its ammonium, cesium, triethylammonium,
1,5-diazabicyclo-[5.4.0]undec-5-ene, tetramethylammonium, or
similar salt in ethanol, acetonitrile, N,N-dimethylformamide (DMF),
and the like, preferably the cesium salt in DMF, with the resin at
an elevated temperature, for example between about 40.degree. and
60.degree. C., preferably about 50.degree. C., for from about 12 to
72 hours, preferably about 48 hours. This will eventually yield the
peptide acid product following acid cleavage or an amide following
aminolysis.
[1192] The N.alpha.-Boc-amino acid may be attached to the
benzhydrylamine resin by means of, for example, an
N,N'-diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt)
mediated coupling for from about 2 to about 24 hours, preferably
about 2 hours at a temperature of between about 10.degree. and
50.degree. C., preferably 25.degree. C. in a solvent such as
CH.sub.2Cl.sub.2 or DMF, preferably CH.sub.2Cl.sub.2.
[1193] For Boc-based protocols, the successive coupling of
protected amino acids may be carried out by methods well known in
the art, typically in an automated peptide synthesizer. Following
neutralization with triethylamine, N,N-di-isopropylethylamine
(DIEA), N-methylmorpholine (NMM), collidine, or similar base, each
protected amino acid is introduced in approximately about 1.5 to
2.5 fold molar excess and the coupling carried out in an inert,
nonaqueous, polar solvent such as CH.sub.2Cl.sub.2, DMF,
N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), or mixtures
thereof, preferably in dichloromethane at ambient temperature. For
Fmoc-based protocols no acid is used for deprotection but a base,
preferably DIEA or NMM, is usually incorporated into the coupling
mixture. Couplings are typically done in DMF, NMP, DMA or mixed
solvents, preferably DMF. Representative coupling agents are
N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropyl-carbodiimide
(DIC) or other carbodiimide, either alone or in the presence of
HOBt, O-acyl ureas,
benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium
hexafluorophosphate (PyBop). N-hydroxysuccinimide, other
N-hydroxyimides, or oximes. Alternatively, protected amino acid
active esters (e.g. p-nitrophenyl, pentafluorophenyl and the like)
or symmetrical anhydrides may be used. Preferred coupling agents
are of the aminium/uronium (alternative nomenclatures used by
suppliers) class such as
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HBTU),
O-(7-azabenzotraiazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU),
2-(6-Chloro-1H-benzotraiazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HCTU), and the like.
[1194] A preferred method of attachment to the Fmoc-PAL-PEG-PS
resin may be accomplished by deprotection of the resin linker with
20% piperidine in DMF, followed by reaction of the N-.alpha.-Fmoc
protected amino acid, about a 5 fold molar excess of the
N-.alpha.-Fmoc-amino acid, using HBTU: di-isopropylethylamine
(DIEA) (1:2) in DMF in a microwave-assisted peptide synthesizer
with a 5 min. 75.degree. max coupling cycle.
[1195] For this Fmoc-based protocol in the microwave-assisted
peptide synthesizer, the N-.alpha.-Fmoc amino acid protecting
groups are removed with 20% piperadine in DMF containing 0.1M
1-hydroxybenzotriazole (HOBt), in a double deprotection protocol
for 30 sec and then for 3 min with a temperature maximum set at
75.degree. C. HOBt is added to the deprotection solution to reduce
aspartimide formation. Coupling of the next amino acid then employs
a five-fold molar excess using HBTU:DIEA (1:2) with a 5 min, 750
max. double-coupling cycle.
[1196] At the end of the solid phase synthesis the fully protected
peptide is removed from the resin. When the linkage to the resin
support is of the benzyl ester type, cleavage may be effected by
means of aminolysis with an alkylamine or fluoroalkylamine for
peptides with an alkylamide C-terminus, or by ammonolysis with, for
example, ammonia/methanol or ammonia/ethanol for peptides with an
unsubstituted amide C-terminus, at a temperature between about
-10.degree. and 50.degree. C., preferably about 25.degree. C., for
between about 12 and 24 hours, preferably about 18 hours. Peptides
with a hydroxy C-terminus may be cleaved by HF or other strongly
acidic deprotection regimen or by saponification. Alternatively,
the peptide may be removed from the resin by transesterification,
e.g., with methanol, followed by aminolysis or saponification. The
protected peptide may be purified by silica gel or reverse-phase
HPLC.
[1197] The side chain protecting groups may be removed from the
peptide by treating the aminolysis product with, for example,
anhydrous liquid hydrogen fluoride in the presence of anisole or
other carbonium ion scavenger, treatment with hydrogen
fluoride/pyridine complex, treatment with
tris(trifluoroacetyl)boron and trifluoroacetic acid, by reduction
with hydrogen and palladium on carbon or polyvinylpyrrolidone, or
by reduction with sodium in liquid ammonia, preferably with liquid
hydrogen fluoride and anisole at a temperature between about
-10.degree. and +10.degree. C., preferably at about 0.degree. C.,
for between about 15 minutes and 2 hours, preferably about 1.5
hours.
[1198] For peptides on the benzhydrylamine type resins, the resin
cleavage and deprotection steps may be combined in a single step
utilizing liquid hydrogen fluoride and anisole as described above
or preferably through the use of milder cleavage cocktails. For
example, for the PAL-PEG-PS resin, a preferred method is through
the use of a double deprotection protocol in the microwave-assisted
peptide synthesizer using one of the mild cleavage cocktails known
in the art, such as
TFA/water/tri-iso-propylsilane/3,6-dioxa-1,8-octanedithiol (DODT)
(92.5/2.5/2.5/2.5) for 18 min at 38.degree. C. each time. Cleavage
of alkyl glycoside containing materials have shown survival of the
alkyl glycoside linkage using protocols with TFA/water ratios in
the 9/1 to 19/1 range. A typical cocktail is 94% TFA: 2% EDT; 2%
H.sub.2O; 2% TIS. Typically the fully deprotected product is
precipitated and washed with cold (-70.degree. to 4.degree. C.)
diethylether, dissolved in deionized water and lyophilized.
[1199] The peptide solution may be desalted (e.g. with BioRad
AG-3.RTM. anion exchange resin) and the peptide purified by a
sequence of chromatographic steps employing any or all of the
following types: ion exchange on a weakly basic resin in the
acetate form; hydrophobic adsorption chromatography on
underivatized co-poly(styrene-divinylbenzene), e.g. Amberlite.RTM.
XAD; silica gel adsorption chromatography; ion exchange
chromatography on carboxymethylcellulose; partition chromatography,
e.g. on Sephadex.RTM. G-25; counter-current distribution;
supercritical fluid chromatography; or HPLC, especially
reversed-phase HPLC on octyl- or octadecylsilylsilica (ODS) bonded
phase column packing.
[1200] Also provided herein are processes for preparing covalently
modified peptides and/or proteins described herein and
pharmaceutically acceptable salts thereof, which processes comprise
sequentially condensing protected amino acids on a suitable resin
support, removing the protecting groups and resin support, and
purifying the product, to afford analogs of the physiologically
active truncated homologs and analogs of the covalently modified
peptides and/or proteins described herein. In some embodiments,
covalently modified peptides and/or proteins described herein
incorporate alkyl glycoside modifications as defined above.
[1201] Another aspect relates to processes for preparing covalently
modified peptides and/or proteins described herein and
pharmaceutically acceptable salts thereof, which processes comprise
the use of microwave-assisted solid phase synthesis-based processes
or standard peptide synthesis protocols to sequentially condense
protected amino acids on a suitable resin support, removing the
protecting groups and resin support, and purifying the product, to
afford analogs of the physiologically active peptides, as defined
above.
Example 3: N-Terminal Endomorphin-1 Analog--AcLys(1-octyl
.beta.-D-glucuronide-6-yl)endomorphin 1 (Ac-Lys(1-octyl
.beta.-D-glucuronide-6-yl)-Tyr-Pro-Trp-Phe-NH2 (AcLys(1-octyl
.beta.-D-glucuronide-6-yl)endomorphin 1)
[1202] As described above, Fmoc-Tyr(t-Bu)-Pro-Trp(Boc)-Phe-NH-Rink
amide MBHA resin is prepared as described in (Koda, Y., et al.
(2008) Bioorg Med Chem 16: 6286-6296). The resin is
N.alpha.-deprotected with piperadine/DMF solution, washed with
solvent and coupled with 2 equivalents of
N-.alpha.-Ac,N-.epsilon.-(1-octyl
.beta.-D-glucuronide-6-yl)-L-lysine using a standard coupling
mixture (e.g. HBTU/DIPEA). Following completion of the coupling,
the peptide is deprotected and cleaved from the resin using a
deprotection mixture (95% TFA/5% H.sub.2O or DODT). The solvent is
removed by a stream of nitrogen and the crude peptide is
precipitated with cold Et.sub.2O, collected, dissolved in 20%
acetonitrile and lyophilized. Purification is by reversed phase
hplc in a mobile phase containing a gradient from H.sub.2O to
H.sub.2O/acetonitrile using a 0.1% TFA or NH.sub.4OAc buffer
system. The mixture is subjected to multiple lyophilizations from
H.sub.2O.
Example 4: N-Terminal Endomorphin-1 Analog--AcLys(1-octyl
.beta.-D-glucuronide-6-yl)endomorphin 1 (Ac-Lys(1-octyl
.beta.-D-glucuronide-6-yl)-Tyr-Pro-Trp-Phe-NH2 (AcLys(1-octyl
.beta.-D-glucuronide-6-yl)endomorphin 1)
[1203] As described above,
Ac-Lys(Boc)-Tyr(t-Bu)-Pro-Trp(Boc)-Phe-NH-Rink amide MBHA resin is
prepared as described in. Following completion of the synthesis,
the peptide is deprotected and cleaved from the resin using a
deprotection mixture (95% TFA/5% H.sub.2O or DODT). The solvent is
removed by a stream of nitrogen and the crude peptide is
precipitated with cold Et.sub.2O, collected, dissolved in 20%
acetonitrile and lyophilized. The peptide, containing a deprotected
Lys .epsilon.-amino function is coupled with 2 equivalents of
1-octyl .beta.-D-glucuronic acid using a standard coupling mixture
(e.g. HBTU/DIPEA) in DMF or similar anhydrous aprotic solvent. The
solvent is removed in vacuo and the product is lyophilized from 20%
acetonitrile or H.sub.2O. Purification is by reversed phase hplc in
a mobile phase containing a gradient from H.sub.2O to
H.sub.2O/acetonitrile using a 0.1% TFA or NH.sub.4OAc buffer
system. The mixture is subjected to multiple lyophilizations from
H.sub.2O.
Example 5:
2',6'-dimethyl-L-tyrosyl-propyl-2',4',6'-trimethyl-L-phenylalan-
yl-N.epsilon.-(1'-octyl .beta.-D-glucuronyl)-L-lysine amide
(EU-A102)
[1204] A 0.3 mmol sample of Fmoc-Rink-Amide resin (0.5 mmol/g) was
coupled with the following sequence of amino acids using a standard
DIC/HOBt solid phase coupling protocol (3 equivalent of DIC/HOBt
and amino acid): Fmoc-L-lysine(N.epsilon.-Alloc);
Fmoc-2',4',6'-trimethyl-L-phenylalanine; Fmoc-L-proline;
Fmoc-2',4'-dimethyl-L-tyrosine.
[1205] The sample of
2',6'-dimethyl-L-tyrosyl-prolyl-2',4',6'-trimethyl-L-phenylalanyl-N.epsil-
on.-(Alloc)-L-lysine amide resin was deprotected on the Lys-Ne
position by incubation with Pd(PPh.sub.3).sub.4 (0.5 eq) and DMBA
(20 eq) in DMF/CH.sub.2Cl.sub.2 (1:1) overnight in the dark at room
temperature. Following washing by DMF/CH.sub.2Cl.sub.2, the Lys
side chain was acylated with 1'-octyl .beta.-D-glucuronic acid
(Carbosynth) in DMF/CH.sub.2Cl.sub.2 through the use of DIC/HOBt.
Completion of the coupling was checked by ninhydrin and the product
was washed extensively with CH.sub.2Cl.sub.2.
[1206] The product resin (0.77 g) was submitted to final
deprotection and cleavage from the resin by treatment with the
cleavage cocktail (94% TFA: 2% EDT; 2% H.sub.2O; 2% TIS) for a
period of 240 min at room temperature. The mixture was treated with
Et.sub.2O, to precipitate the product and washed extensively with
Et.sub.2O to yield 290 mg of crude title peptide product after
drying in vacuo.
[1207] Purification was carried out in two batches by
reversed-phase (C18) hplc. The crude peptide was loaded on a
4.1.times.25 cm hplc column at a flow rate of 15 mL/min (15%
organic modifier; acetic acid buffer) and eluted with a gradient
from 15-45% buffer B in 60 min at 50.degree. C. The product
fraction was lyophilized to yield 100 mg of the title product
peptide with a purity >96% by analytical hplc/mass spectrometry
(M+1 peak=911.87). The overall synthesis yield was calculated at
18%.
[1208] In a similar manner, but using the reagents 1'-methyl
.beta.-D-glucuronic acid and 1'-dodecyl .beta.-D-glucuronic acid,
were prepared the corresponding N-(1'-methyl
.beta.-D-glucuronyl)-L-lysine.sup.4 (EU-A101) and
N.epsilon.-(1'-dodecyl .beta.-D-glucuronyl)-L-lysine.sup.4
(EUA-103) analogs of the title compound.
[1209] Analysis was done by HPLC/mass spectrometery in positive ion
mode using the eluent gradients given in the table below.
TABLE-US-00035 Molecular HPLC Compound Position Wt Molecular (min;
Name 4 N.epsilon. expected Wt found elution) EU-A101 Me 812.95
812.80 13.6 [a] EU-A102 n-octyl 911.16 910.87 12.9 [b] EU-A103
n-dodecyl 967.27 966.53 12.5 [c] HPLC gradients in 0.1% TEA [a] 20
to 50% CH.sub.3CN over 30 min. [b] 35 to 65% CH.sub.3CN over 30
min. [c] 40 to 75% CH.sub.3CN over 20 min. HPLC on Phenomenex Luna
C18 5 micron 250 .times. 4.6 mm
Example 6:
2',6'-dimethyl-L-tyrosyl-propyl-2',4',6'-trimethyl-L-phenylalan-
yl-L-phenylalanyl-N.epsilon.-(1'-dodecyl
.beta.-D-glucuronyl)-L-lysine amide (EU-A106)
[1210] In a similar manner to that given for the solid phase
synthesis and lysine side chain modification in example 5, given
above, but using 1-dodecyl .beta.-D-glucuronic acid (Milkereit, G.,
et al. (2004) Chem Phys Lipids 127: 47-63) for lysine
N.epsilon.-acylation, was prepared the title peptide as a crude
product. Following reversed phase hplc purification as above one
obtains the title product as a white powder, of 96.1% purity by
hplc/mass spectrometry (M+1=1114.8).
[1211] In a similar manner, but using the reagent 1'-methyl
.beta.-D-glucuronic acid, was prepared the corresponding
N.epsilon.-(1'-methyl .beta.-D-glucuronyl)-L-lysine (EUA-105)
analog of the title compound.
[1212] Analysis was done by HPLC/mass spectrometery in positive ion
mode using the gradient eluents given in the table below.
TABLE-US-00036 Molecular HPLC Compound Position Wt Molecular (min;
Name 5 N expected Wt found elution) EU-A105 Me 960.12 959.60 8.8
[d] EU-A106 n-dodecyl 1114.44 1113.80 11.6 [e] HPLC gradients in
0.1% TFA [d] 30 to 60% CH.sub.3CN over 20 min. [e] 45 to 75%
CH.sub.3CN over 20 min. HPLC on Phenomenex Luna C18 5 micron 250
.times. 4.6 mm
Example 7: 2',6'-dimethyl-L-tyrosyl-N.epsilon.-(1'-dodecyl
.beta.-D-glucuronyl)-D-lysyl-2',4',6'-trimethyl-L-phenylalanyl-L-phenylal-
anine-amide (EU-A108)
[1213] in a similar manner as that given in Example 6 was prepared
the title peptide as a white powder of 95.8% purity by hplc/mass
spectrometry (M=1017.07).
[1214] In a similar manner, but using the reagent 1'-methyl
.beta.-D-glucuronic acid, was prepared the corresponding
N.epsilon.-(1'-methyl .beta.-D-glucuronyl)-L-lysine (EU-A107)
analog of the title compound.
[1215] Analysis was done by HPLC/mass spectrometry in positive ion
mode using the eluent gradients given in the table below.
TABLE-US-00037 Molecular HPLC Compound Position Wt Molecular (min;
Name 2 N expected Wt found elution) EU-A107 Me 862.01 862.73 11.6
[f] EU-A108 n-dodecyl 1017.32 1017.07 11.9 [g] HPLC gradients in
0.1% TFA [f] 25 to 55% CH.sub.3CN over 20 min. [g] 50 to 80%
CH.sub.3CN over 20 min. HPLC on Phenomenex Luna C18 5 micron 250
.times. 4.6 mm
Example 8:
2',6'-dimethyl-L-tyrosyl-L-1,2,3,4-tetrahydroisoquinoline-3-car-
bonyl-L-phenylalanyl-N.epsilon.-(1'-methyl
.beta.-D-glucuronyl)-L-lysyl-amide (EU-A178)
[1216] In a similar manner as that given in Example 6 was prepared
the title peptide as a white powder of 95.5% purity by hplc/mass
spectrometry (M=832.33).
[1217] In a similar manner, but using the reagent 1'-dodecyl
.beta.-D-glucuronic acid, was prepared the corresponding
N.epsilon.-(1'-dodecyl .beta.-D-glucuronyl)-L-lysine analog
(EU-A179) of the title compound. In a similar manner, but using the
corresponding 1-alkyl glucuronic acid reagents are made the
corresponding peptides of the invention, EU-A180, EU-A 181,
EU-A182, EU-A183, EU-A184, EU-A 185, and the like.
[1218] Analysis was done by HPLC/mass spectrometry in positive ion
mode using the eluent gradients given in the table below.
TABLE-US-00038 Molecular HPLC Compound Position Wt Molecular (min;
Name 4 N expected Wt found elution) EU-A178 Me 832.91 832.33 11.2
[h] EU-A179 n-dodecyl 987.23 986.47 10.7 [f] EU-A180 n-octyl 931.12
930.67 10.1 [i] HPLC gradients in 0.1% TFA [f] 25 to 55% CH.sub.3CN
over 20 min. [h] 20 to 50% CH.sub.3CN over 20 min. [i] 35 to 65%
CH.sub.3CN over 20 min HPLC on Phenomenex Luna C18 5 micron 250
.times. 4.6 mm
Example 9:
2'6'-dimethyl-L-tyrosyl-L-1,2,3,4-tetrahydroisoquinoline-3-carb-
onyl-L-phenylalanyl-L-phenylalanys-N.epsilon.-(1'-methyl
.beta.-D-glucuronyl)-L-lysyl-amide (EU-A189)
[1219] In a similar manner as that given in Example 6 was prepared
the title peptide as a white powder of 98.99% purity by hplc/mass
spectrometry (M=979.53).
[1220] In a similar manner, but using the reagent 1'-dodecyl
.beta.-D-glucuronic acid, was prepared the corresponding
N.epsilon.-(1'-dodecyl .beta.-D-glucuronyl)-L-lysine (EU-A190)
analog of the title compound.
[1221] Analysis was done by HPLC/mass spectrometry in positive ion
mode using the eluent gradients given in the table below.
TABLE-US-00039 Molecular HPLC Compound Position Wt Molecular (min;
Name 5 N expected Wt found elution) EU-A189 Me 980.09 979.53 10.9
[e] EU-A190 n-dodecyl 1134.41 1134.53 12.5 [e] HPLC gradients in
0.1% TFA [e] 45 to 75% CH.sub.3CN over 20 min. HPLC on Phenomenex
Luna C18 5 micron 250 .times. 4.6 mm
Example 10: Additional Analogs of the Invention
[1222] In a similar manner as that given in Example 6, but using
the corresponding 1-alkyl .beta.-D-glucuronic acid reagent are
prepared the additional peptides of the invention as white powders
of greater than 95% purity by hplc/mass spectrometry.
[1223] Analysis is done by HPLC/mass spectrometry in positive ion
mode using the appropriate eluent gradients such as those given in
the table below.
TABLE-US-00040 Molecular HPLC Compound Position Wt Molecular (min;
Name 3 or 4 N expected Wt found elution) EU-A600 Me 918.02 918.00
10.7 [h] EU-A601 n-octyl 1016.23 1015.87 10.1 [i] EU-A615 Me 832.91
832.67 11.0 [h] EU-A620 Me 923.06 922.73 9.5 [d] EU-A639 Me 923.06
922.80 11.5 [f] HPLC gradients in 0.1% TEA [d] 30 to 60% CH.sub.3CN
over 20 min. [e] 45 to 75% CH.sub.3CN over 20 min. [h] 20 to 50%
CH.sub.3CN over 20 min. [f] 25 to 55% CH.sub.3CN over 20 min. [i]
35 to 65% CH.sub.3CN over 20 min HPLC on Phenomenex Luna C18 5
micron 250 .times. 4.6 mm
Example 11. General Oxidation Method for Uronic Acids
[1224] To a solution of 1-dodecyl .beta.-D-glucopyranoside
(Carbosynth) [2.0 g, 5.74 mmol] in 20 mL of acetonitrile and 20 mL
of DI water was added (diacetoxyiodo)benzene (Fluka) [4.4 g, 13.7
mmol] and TEMPO (SigmaAldrich) [0.180 g, 1.15 mmol]. The resulting
mixture was stirred at room temperature for 20 h. The reaction
mixture was diluted with water and lyophilized to dryness to give
1.52 g (crude yield 73.1%) of the crude product, 1-dodecyl
.beta.-D-glucuronic acid, as a white powder, which was used
directly for the solid phase synthesis without further
purification. In a like manner, but using the corresponding
1-tetradecyl, 1-hexadecyl, and 1-octadecyl
.beta.-D-glucopyranosides (purchased from Anatrace, Maumee, Ohio)
were prepared the desired alkyl saccharide uronic acids used to
make the products and reagents described herein. This product was
previously prepared by an alternative process using NaOCl as
oxidant and also has been used for longer alkyl groups.
Example 12: Cellular Assay of the Compounds
[1225] Compounds were weighed precisely in an amount of
approximately 2 mg and assayed in standard cellular assays by the
contract research organization Cerep, Inc. (Pullman, Wash.) as
executed at their subsidiary, Cerep SA (Le Bois l'Eveque, France).
The readout is the amount of cAMP generated in the cells treated
with the test compounds, in either agonist or antagonist mode. The
assays used were the mu opioid receptor cellular assay (MOP in
agonist and antagonist mode), the delta2 opioid receptor cellular
assay (DOP in agonist and antagonist mode) and the kappa opioid
receptor cellular assay (KOP in agonist and antagonist mode). The
assays used are described in Wang, J. B., et al. (1994) FEBS Lett
338: 217-222, Law, P. Y. and Loh, H. H. (1993) Mol Pharmacol 43:
684-693, and Avidor-Reiss, T., et al. (1995) FEBS Lett 361: 70-74.
The stimulant for the cells in the DOR antagonist assay was
3.times.10E-8M DPDPE, a well-accepted DOR literature standard.
[1226] For the series of compounds EU-A101 to EU-A103, where the
hydrophobic portion of the surfactant (1-alkyl glucuronic acid)
varies in length from C1 to C12, the character of the receptor
selectivity and activation (see below) varies from full agonist
(C1) to pure antagonist (C12). No activity was seen in cells used
for DOP or KOP assays, thus showing full selectivity for the mu
opioid receptor as agonists. This behavior demonstrates the ability
of the modifications described herein to vary the fundamental
properties of the receptor interactions. Modifications elsewhere in
the molecule (e.g., using amino acid analogs) are used to further
modify the potency and character of the interaction of the drug
candidates.
TABLE-US-00041 MOP MOP agonist antagonist Compound EC.sub.50
IC.sub.50 Name Structure (nM) (nM) Characterization EU-A101 Me 13
nc pure agonist EU-A102 n-octyl 100 100 partial agonist EU-A103
n-dodecyl nc 86 pure antagonist EU-A107 Me 60 nc Agonist
[1227] DOP antagonistic activity was assessed by inhibition of the
stimulatory cAMP response of 3.times.10E-8M DPDPE.
TABLE-US-00042 MOP DOP agonist antagonist Compound EC.sub.50
IC.sub.50 Name Structure (nM) (M) Characterization EU-A178 Me 15
<10E-10 Pure MOP Ag; pure DOP antag EU-A179 n-dodecyl .sctn. NT
Pure MOP Ag; pure DOP antag EU-A180 n-octyl 36 <10E-10 Pure MOP
Ag; pure DOP antag EU-A189 Me 41 NT Pure MOP Ag; pure DOP antag
EU-A190 n-dodecyl .sctn. NT Pure MOP Ag; pure DOP antag EU-A600 Me
110 <10E-10 Pure MOP Ag; pure DOP antag EU-A601 n-octyl .sctn.
<10E-10 Pure MOP Ag; pure DOP antag EU-A615 Me 410 3.1E-10 Pure
MOP Ag; pure DOP antag EU-A620 Me 480 <10E-10 Pure MOP Ag; pure
DOP antag EU-A639 Me 280 <10E-10 Pure MOP Ag; pure DOP antag
.sctn. indicates solubility issues during dissolution, NT means not
tested, nc means not calculable
Example 13: Uses of the Compounds
[1228] The covalently modified peptides and/or proteins described
herein are useful for the prevention and treatment of a variety of
diseases depending on which class is being considered. For example,
covalently modified peptides and/or proteins described herein are
indicated for the prophylaxis and therapeutic treatment of chronic
and acute pain and other MOR- or DOR-related disease states.
Applications for sunburn, pruritus, cancer, immune function,
inflammation, cardiovascular disease also have been documented
(Lazarus, L. H., et al. (2012) Expert Opin Ther Patents 22:
1-14).
[1229] Representative delivery regimens include oral, parenteral
(including subcutaneous, intramuscular and intravenous injection),
rectal, buccal (including sublingual), transdermal, inhalation
ocular and intranasal. An attractive and widely used method for
delivery of peptides entails subcutaneous injection of a controlled
release injectable formulation. Other administration routes for the
application of the covalently modified peptides and/or proteins
described herein are subcutaneous, intranasal and inhalation
administration.
Example 14. Pharmaceutical Usage for Treatment of Pain
[1230] A human patient, with acute or chronic pain is treated with
EU-A178 by intranasal administration (200 .mu.L) from a standard
atomizer used in the art of a solution of the pharmaceutical agent
in physiological saline containing from 0.5 to 10 mg/mL of the
pharmaceutical agent and containing standard excipients such as
benzyl alcohol. The treatment is repeated as necessary for the
alleviation of pain. Alternatively a solution of EU-A178, and
selected excipients, in an evaporating solvent containing such as a
hydrofluoroalkane is administered intranasally by MDI as needed to
alleviate pain. Alternatively an aqueous solution of EU-A178, with
selected excipients, is administered by subcutaneous injection as
needed to alleviate pain.
[1231] The effect of treatment is determined from evaluation of
patients, including quality of life questionaires. Pain scales are
based on self-report, observational (behavioral), or physiological
data. Some pain scales suitable for use in clinical setting include
Alder Hey Triage Pain Score, Brief Pain Inventory (BPI), Dallas
Pain Questionnaire, Dolorimeter Pain Index (DPI), McGill Pain
Questionnaire (MPQ), Numerical 11 point box (BS-11), Numeric Rating
Scale (NRS-11), Roland-Morris Back Pain Questionnaire, Visual
analog scale (VAS) or the like.
[1232] In a similar manner, administration of an adjusted amount by
transbuccal, intravaginal, inhalation, subcutaneous, intravenous,
intraocular, or oral routes is tested to determine relief from
pain.
Example 2-1: Reagents--N-.alpha.-Fmoc, N-.epsilon.-(1-octyl
.beta.-D-glucuronide-6-yl)-L-lysine
[1233] In an oven-dried 250 mL Erlenmeyer flask is placed 1-octyl
.beta.-D-glucuronic acid (Carbosynth Ltd., 3.06 g, 10 mmol), 50 mL
anhydrous DMF, and anhydrous 1-hydroxybenzotriazole (1.62 g, 12
mmol). A chilled (4.degree. C.) solution of N,
N'-dicyclohexylcarbodiimide (2.48 g, 12 mmol) in 50 mL of DMF is
added, with stirring, and the reaction is allowed to proceed for 5
min. The copious white precipitate of N, N'-dicyclohexylurea is
filtered on a fritted glass funnel and the filtrate is added to a
solution of N-.alpha.-Fmoc-L-lysine (3.68 g, 10 mmol) in 25 ml
anhydrous DMF. The reaction is allowed to proceed for 25 min with
warming to room temp or until the ninhydrin color is very faint.
The reaction mixture is filtered, stripped to dryness and
crystallized from MeOH/Et.sub.2O by dissolution in MeOH and slow
dilution to the cloud point with Et.sub.2O, followed by
refrigeration. Further purification can be achieved by silica gel
chromatography using a solvent gradient from EtOAc to
EtOAc/EtOH/AcOH.
[1234] In a similar manner, but substituting N-.alpha.-Boc-L-lysine
is obtained N-.alpha.-Boc,N-.epsilon.-(1-octyl
.beta.-D-glucuronide-6-yl)-L-lysine, suitable for N-terminal
incorporation and cleavage to a free N-Terminus. In a similar
manner, but substituting N-.alpha.-Ac-L-lysine is obtained
N-.alpha.-Ac, N-.epsilon.-(1-octyl
.beta.-D-glucuronide-6-yl)-L-lysine, suitable for incorporation at
the N-terminus of a peptide with a blocked N-terminus. In a similar
manner, but substituting the appropriate amount of
N-.alpha.-Fmoc-L-ornithine is obtained
N-.alpha.-Fmoc,N-.delta.-(1-octyl
.beta.-D-glucuronide-6-yl)-L-ornithine. In a similar manner but
substituting other N-mono-protected diamino acids one obtains the
corresponding reagents. Alternatively, use of a transient
Me.sub.3Si ester protecting group during the coupling and without
preactivation of the 1-octyl .beta.-D-glucuronic acid provides a
facile route to the formation of the reagents. The transient
Me.sub.3Si ester is produced by reaction of the Fmoc-Lys-OH with an
equimolar amount of N,O-bis(trimethylsilyl)acetamide in
dichloromethane (CH.sub.2Cl.sub.2). The organic layer contains the
desired reagent as a solution in CH.sub.2Cl.sub.2 ready for
coupling with the 1-alkyl glucoronide as above. The filtered
reaction mixture is washed with aqueous NaHSO.sub.4 to hydrolyze
the Me.sub.3Si ester, dried over MgSO.sub.4 and solvent is
removed.
[1235] Similarly, but using peracetyl or perbenzoyl 1-octyl
.beta.-D-glucuronic acid one obtains the Ac, or Bz protected form
of the reagents (e.g. 2,3,4-trisacetyl 1-octyl .beta.-D-glucuronic
acid, and the like, formed by treatment with Ac.sub.2O). Such
reagents have increased stability during acid cleavage from the
resin and are used when instability during deprotection is
detected, see (Kihlberg, J., et al. (1997) Methods Enzymol 289:
221-245) and references therein. Final deprotection of such
products is carried out by base-catalyzed transesterification after
cleavage, by use of MeOH/NH.sub.3, MeOH/NaOMe,
MeOH/NH.sub.2NH.sub.2, as described above.
Example 2-2: Synthetic Peptide Analogs
[1236] In general, peptide synthesis methods involve the sequential
addition of protected amino acids to a growing peptide chain.
Normally, either the amino or carboxyl group of the first amino
acid and any reactive side chain group are protected. This
protected amino acid is then either attached to an inert solid
support, or utilized in solution, and the next amino acid in the
sequence, also suitably protected, is added under conditions
amenable to formation of the amide linkage. After all the desired
amino acids have been linked in the proper sequence, protecting
groups and any solid support are removed to afford the crude
peptide. The peptide is desalted and purified
chromatographically.
[1237] A preferred method of preparing the analogs of the
physiologically active truncated peptides, having fewer than about
fifty amino acids, involves solid phase peptide synthesis. In this
method the .alpha.-amino (N.alpha.) functions and any reactive side
chains are protected by acid- or base-sensitive groups. The
protecting group should be stable to the conditions of peptide
linkage formation, while being readily removable without affecting
the extant peptide chain. Suitable .alpha.-amino protecting groups
include, but are not limited to t-butyloxycarbonyl (Boc),
benzyloxycarbonyl (Cbz), o-chlorobenzyloxycarbonyl,
biphenylisopropyloxycarbonyl, t-amyloxycarbonyl (Amoc),
isobornyloxycarbonyl, .alpha.,
.alpha.-dimethyl-3,5-dimethoxybenzyloxy-carbonyl,
o-nitrophenylsulfenyl, 2-cyano-t-butoxycarbonyl,
9-fluorenyl-methoxycarbonyl (Fmoc) and the like, preferably Boc or
more preferably, Fmoc. Suitable side chain protecting groups
include, but are not limited to: acetyl, benzyl (Bzl),
benzyloxymethyl (Bom), Boc, t-butyl, o-bromobenzyloxycarbonyl,
t-butyl, t-butyldimethylsilyl, 2-chlorobenzyl (Cl-z),
2,6-dichlorobenzyl, cyclohexyl, cyclopentyl, isopropyl, pivalyl,
tetrahydropyran-2-yl, tosyl (Tos),
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf),
trimethylsilyl and trityl. A preferred N.alpha.-protecting group
for synthesis of the compounds is the Fmoc group. Preferred side
chain protecting groups are O-t-Butyl group for Glu, Tyr, Thr, Asp
and Ser, Boc group for Lys and Trp side chains; Pbf group for Arg;
Trt group for Asn Gln, and His. For selective modification of a Lys
residue, orthogonal protection with a protecting group not removed
by reagents that cleave the Fmoc or t-butyl based protecting groups
is preferred. Preferred examples for modification of the Lys side
chain include, but are not limited to, those removed by hydrazine
but not piperidine; for example
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde)
or 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) and
allyloxycarbonyl (Alloc).
[1238] The Fmoc-Lys(ivDde) or Fmoc-Lys(Dde) protecting group scheme
is preferred in cases where side chain lactam formation is desired
(Houston, M. E., Jr., et al. (1995) J Pept Sci 1: 274-282; Murage,
E. N., et al. (2010) J Med Chem), since in this case
Fmoc-Glu(O-Allyl) and Fmoc-Lys(Alloc) can be incorporated and used
to provide transient protection, then deprotected for lactam
formation while the Lys(Dde) protecting group remains for later
removal and reaction with the functionalized surfactant.
[1239] In solid phase synthesis, the C-terminal amino acid is first
attached to a suitable resin support. Suitable resin supports are
those materials which are inert to the reagents and reaction
conditions of the stepwise condensation and deprotection reactions,
as well as being insoluble in the media used. Examples of
commercially available resins include styrene/divinylbenzene resins
modified with a reactive group, e.g., chloromethylated
co-poly-(styrene-divinylbenzene), hydroxymethylated
co-poly-(styrene-divinylbenzene), and the like. Benzylated,
hydroxymethylated phenylacetamidomethyl (PAM) resin is preferred
for the preparation of peptide acids. When the C-terminus of the
compound is an amide, a preferred resin is
p-methylbenzhydrylamino-co-poly(styrene-divinyl-benzene) resin, a
2.4 dimethoxybenzhydrylamino-based resin ("Rink amide"), and the
like. An especially preferred support for the synthesis of larger
peptides are commercially available resins containing PEG sequences
grafted onto other polymeric matricies, such as the Rink Amide-PEG
and PAL-PEG-PS resins (Applied Biosystems) or similar resins
designed for peptide amide synthesis using the Fmoc protocol. Thus
in certain cases it is desirable to have an amide linkage to a PEG
chain. It those cases it is convenient to link an
N-Fmoc-amino-PEG-carboxylic acid to the amide forming resin above
(e.g. Rink amide resin and the like). The first amino acid of the
chain can be coupled as an N-Fmoc-amino acid to the amino function
of the PEG chain. Final deprotection will yield the desired
Peptide-NH-PEG-CO--NH.sub.2 product.
[1240] Attachment to the PAM resin may be accomplished by reaction
of the N.alpha. protected amino acid, for example the Boc-amino
acid, as its ammonium, cesium, triethylammonium,
1,5-diazabicyclo-[5.4.0]undec-5-ene, tetramethylammonium, or
similar salt in ethanol, acetonitrile, N,N-dimethylformamide (DMF),
and the like, preferably the cesium salt in DMF, with the resin at
an elevated temperature, for example between about 40.degree. and
60.degree. C., preferably about 50.degree. C., for from about 12 to
72 hours, preferably about 48 hours. This will eventually yield the
peptide acid product following acid cleavage or an amide following
aminolysis.
[1241] The N.alpha.-Boc-amino acid may be attached to the
benzhydrylamine resin by means of, for example, an
N,N'-diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt)
mediated coupling for from about 2 to about 24 hours, preferably
about 2 hours at a temperature of between about 10.degree. and
50.degree. C., preferably 25.degree. C. in a solvent such as
CH.sub.2Cl.sub.2 or DMF, preferably CH.sub.2Cl.sub.2.
[1242] For Boc-based protocols, the successive coupling of
protected amino acids may be carried out by methods well known in
the art, typically in an automated peptide synthesizer. Following
neutralization with triethylamine, N,N-di-isopropylethylamine
(DIEA), N-methylmorpholine (NMM), collidine, or similar base, each
protected amino acid is introduced in approximately about 1.5 to
2.5 fold molar excess and the coupling carried out in an inert,
nonaqueous, polar solvent such as CH.sub.2Cl.sub.2, DMF,
N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), or mixtures
thereof, preferably in dichloromethane at ambient temperature. For
Fmoc-based protocols no acid is used for deprotection but a base,
preferably DIEA or NMM, is usually incorporated into the coupling
mixture. Couplings are typically done in DMF, NMP, DMA or mixed
solvents, preferably DMF. Representative coupling agents are
N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropyl-carbodiimide
(DIC) or other carbodiimide, either alone or in the presence of
HOBt, O-acyl ureas,
benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium
hexafluorophosphate (PyBop), N-hydroxysuccinimide, other
N-hydroxyimides, or oximes. Alternatively, protected amino acid
active esters (e.g. p-nitrophenyl, pentafluorophenyl and the like)
or symmetrical anhydrides may be used. Preferred coupling agents
are of the aminium/uronium (alternative nomenclatures used by
suppliers) class such as
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HBTU),
O-(7-azabenzotraiazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU),
2-(6-Chloro-1H-benzotraiazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HCTU), and the like.
[1243] A preferred method of attachment to the Fmoc-PAL-PEG-PS
resin may be accomplished by deprotection of the resin linker with
20% piperidine in DMF, followed by reaction of the N-.alpha.-Fmoc
protected amino acid, about a 5 fold molar excess of the
N-.alpha.-Fmoc-amino acid, using HBTU:di-isopropylethylamine (DIEA)
(1:2) in DMF in a microwave-assisted peptide synthesizer with a 5
min. 75.degree. max coupling cycle.
[1244] For this Fmoc-based protocol in the microwave-assisted
peptide synthesizer, the N-.alpha.-Fmoc amino acid protecting
groups are removed with 20% piperidine in DMF containing 0.1M
1-hydroxybenzotriazole (HOBt), in a double deprotection protocol
for 30 sec and then for 3 min with a temperature maximum set at
75.degree. C. HOBt is added to the deprotection solution to reduce
aspartimide formation. Coupling of the next amino acid then employs
a five-fold molar excess using HBTU:DIEA (1:2) with a 5 min,
75.degree. max. double-coupling cycle.
[1245] At the end of the solid phase synthesis the fully protected
peptide is removed from the resin. When the linkage to the resin
support is of the benzyl ester type, cleavage may be effected by
means of aminolysis with an alkylamine or fluoroalkylamine for
peptides with an alkylamide C-terminus, or by ammonolysis with, for
example, ammonia/methanol or ammonia/ethanol for peptides with an
unsubstituted amide C-terminus, at a temperature between about
-10.degree. and 50.degree. C., preferably about 25.degree. C., for
between about 12 and 24 hours, preferably about 18 hours. Peptides
with a hydroxy C-terminus may be cleaved by HF or other strongly
acidic deprotection regimen or by saponification. Alternatively,
the peptide may be removed from the resin by transesterification,
e.g., with methanol, followed by aminolysis or saponification. The
protected peptide may be purified by silica gel or reverse-phase
HPLC.
[1246] The side chain protecting groups may be removed from the
peptide by treating the aminolysis product with, for example,
anhydrous liquid hydrogen fluoride in the presence of anisole or
other carbonium ion scavenger, treatment with hydrogen
fluoride/pyridine complex, treatment with
tris(trifluoroacetyl)boron and trifluoroacetic acid, by reduction
with hydrogen and palladium on carbon or polyvinylpyrrolidone, or
by reduction with sodium in liquid ammonia, preferably with liquid
hydrogen fluoride and anisole at a temperature between about
-10.degree. and +10.degree. C., preferably at about 0.degree. C.,
for between about 15 minutes and 2 hours, preferably about 1.5
hours.
[1247] For peptides on the benzhydrylamine type resins, the resin
cleavage and deprotection steps may be combined in a single step
utilizing liquid hydrogen fluoride and anisole as described above
or preferably through the use of milder cleavage cocktails. For
example, for the PAL-PEG-PS resin, a preferred method is through
the use of a double deprotection protocol in the microwave-assisted
peptide synthesizer using one of the mild cleavage cocktails known
in the art, such as
TFA/water/tri-iso-propylsilane/3,6-dioxa-1,8-octanedithiol (DODT)
(92.5/2.5/2.5/2.5) for 18 min at 38.degree. C. each time. Cleavage
of alkyl glycoside containing materials have shown survival of the
alkyl glycoside linkage using protocols with TFA/water ratios in
the 9/1 to 19/1 range. A typical cocktail is 94% TFA: 2% EDT; 2%
H.sub.2O; 2% TIS. Typically the fully deprotected product is
precipitated and washed with cold (-70.degree. to 4.degree. C.)
Et.sub.2O, dissolved in deionized water and lyophilized.
[1248] The peptide solution may be desalted (e.g. with BioRad
AG-3.RTM. anion exchange resin) and the peptide purified by a
sequence of chromatographic steps employing any or all of the
following types: ion exchange on a weakly basic resin in the
acetate form; hydrophobic adsorption chromatography on
underivatized co-poly(styrene-divinylbenzene), e.g. Amberlite.RTM.
XAD; silica gel adsorption chromatography; ion exchange
chromatography on carboxymethylcellulose; partition chromatography,
e.g. on Sephadex.RTM. G-25; counter-current distribution;
supercritical fluid chromatography; or HPLC, especially
reversed-phase HPLC on octyl- or octadecylsilylsilica (ODS) bonded
phase column packing.
[1249] Also provided herein are processes for preparing covalently
modified peptides and/or proteins described herein and
pharmaceutically acceptable salts thereof, which processes comprise
sequentially condensing protected amino acids on a suitable resin
support, removing the protecting groups and resin support, and
purifying the product, to afford analogs of the physiologically
active truncated homologs and analogs of the covalently modified
peptides and/or proteins described herein. In some embodiments,
covalently modified peptides and/or proteins described herein
incorporate alkyl glycoside modifications as defined above.
[1250] Another aspect relates to processes for preparing covalently
modified peptides and/or proteins described herein and
pharmaceutically acceptable salts thereof, which processes comprise
the use of microwave-assisted solid phase synthesis-based processes
or standard peptide synthesis protocols to sequentially condense
protected amino acids on a suitable resin support, removing the
protecting groups and resin support, and purifying the product, to
afford analogs of the physiologically active peptides, as defined
above.
Example 2-3. General Oxidation Method for Uronic Acids
[1251] To a solution of 1-dodecyl .beta.-D-glucopyranoside
(Carbosynth) [2.0 g, 5.74 mmol] in 20 mL of acetonitrile and 20 mL
of DI water was added (diacetoxyiodo)benzene (Fluka) [4.4 g, 13.7
mmol] and TEMPO (SigmaAldrich) [0.180 g, 1.15 mmol]. The resulting
mixture was stirred at room temperature for 20 h. The reaction
mixture was diluted with water and lyophilized to dryness to give
1.52 g (crude yield 73.1%) of the crude product, 1-dodecyl
.beta.-D-glucuronic acid, as a white powder, which was used
directly for the solid phase synthesis without further
purification. This product was previously prepared by an
alternative process using NaOCl as oxidant, as described in the
specification, and also has been used for longer alkyl groups. In a
like manner, but using the corresponding 1-tetradecyl, 1-hexadecyl,
and 1-octadecyl .beta.-D-glucopyranosides (purchased from Anatrace,
Maumee, Ohio) were prepared the desired alkyl saccharide uronic
acids used to make the products and reagents described herein.
Example 2-4: Preparation of PTHrP Analog EU-204
[1252] A sample of
Fmoc-Ac5c-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Arg-Ala-Arg-Trp-le-Gln-Lys(-
Alloc)-Rink amide resin was deprotected on the Lys-N-epsilon
position by incubation with Pd(PPh.sub.3).sub.4 (0.5 eq) and DMBA
(20 eq) in DMF/CH.sub.2Cl.sub.2 (1:1) overnight in the dark at room
temperature. Following washing by DMF/CH.sub.2Cl.sub.2, the Lys
side chain was acylated with 1'-octyl dodecyl .beta.-D-glucuronic
acid (Carbosynth) in DMF/CH.sub.2Cl.sub.2 through the use of
DIC/HOBt. Completion of the coupling was checked by ninhydrin and
the product was washed extensively with CH.sub.2Cl.sub.2.
[1253] The product resin was submitted to final deprotection and
cleavage from the resin by treatment with the cleavage cocktail
(94% TFA: 2% EDT; 2% H.sub.2O; 2% TIS) for a period of 240 min at
room temperature. The mixture was treated with Et.sub.2O, to
precipitate the product and washed extensively with Et.sub.2O to
yield the crude title peptide product after drying in vacuo.
[1254] Purification was carried out in two batches by reversed
phase (C18) hplc. The crude peptide was loaded on a 4.1.times.25 cm
hplc column at a flow rate of 15 mL/min (15% organic modifier;
acetic acid buffer) and eluted with a gradient from 15-45% buffer B
in 60 min at 50.degree. C. The product fraction was lyophilized to
yield the title product peptide with a purity >97% by analytical
hplc(12.0 min; 35-65% CH.sub.3CN in 0.1% TFA)/mass spectrometry
(M+1 peak=2473.9).
[1255] The corresponding 1-methyl, 1-octyl, 1-decyl, 1-dodecyl,
1-tetradecyl, 1-hexadecyl, 1-octadecyl and 1-eicosyl analogs are
prepared using the corresponding glucouronic acids, prepared as
described above. Alternatively, the 1-alkyl glucuronyl, or other
uronic acylated analogs, may be prepared by initial purification of
the deprotected or partially deprotected peptide followed by
acylation by the desired uronic acid reagent.
[1256] Analysis was done by HPLC/mass spectrometry in positive ion
mode using the eluent gradients given in the table below.
TABLE-US-00043 Molecular HPLC Compound Wt Molecular (min; Name
Expected Wt Found elution) EU-201 2278.63 2278.14 14.1 [a] EU-202
2376.86 2376.80 11.2 [bl EU-203 2432.97 2432.40 14.1 [c] EU-204
2472.00 2471.86 12.0 [d] EU-205 2343.87 2344.26 8.0 [e] EU-207
2557.12 2557.06 13.2 [c] EU-232 2642.23 2642.14 12.6 [c] EU-251
2941.56 2942.26 13.1 [cj EU-260 2967.61 2966.66 13.9 [c] EU-283
2881.47 2882.26 11.0 [c] EU-284 3640.39 3640.00 11.9 [c] EU-286
2670.47 2669.86 6.3 [e] EU-287 2698.47 2697.74 8.4 [e] EU-288
2726.47 2726.26 10.9 [f] HPLC gradients in 0.1% TFA [a] 20 to 50%
CH.sub.3CN over 30 min. [b] 25 to 55% CH.sub.3CN over 20 min. [c]
30 to 60% CH.sub.3CN over 20 min. [d] 35 to 65% CH.sub.3CN over 20
min. [e] 40 to 70% CH.sub.3CN over 20 min. [f] 45 to 75% CH.sub.3CN
over 20 min. HPLC on Phenomenex Luna C18 5 micron 250 .times. 4.6
mm.
Example 2-5: Cellular Assay of the Compounds
[1257] Compounds were weighed precisely in an amount of
approximately 1 mg and assayed in standard cellular assays (cerep
SA). The readout is the amount of cAMP generated in the cells
treated with the test compounds, in either agonist or antagonist
mode. The PTH1 cellular assay used is described in Orloff, J. J.,
et al. (1992) Endocrinol 131: 1603-1611.
[1258] For the series of compounds EU-201 to EU-203, where the
hydrophobic portion of the surfactant (1-alkyl glucuronic acid)
varies in length from C1 to C12, the cellular response increases in
potency and efficacy with the increased chain length. All of the
analogs were agonists. Further substitutions led to molecules with
an EC.sub.50 similar to PTH1-34, but with super-agonistic activity
(e.g. EU-232) and such molecules have important applications in
medicine. Additional analogs are designed to have very prolonged
duration of action in vivo (that is EU-286, EU-287, and EU-288). In
this assay, PTHrP (coded sample) had an EC50 of 2.9 nM and a
maximal response of 100% while the internal standard, PTH had EC50
of 1.4 nM and maximal response of 105%. Compounds were dissolved in
water and diluted in assay buffer containing 1% bovine serum
albumin. The following table shows potency and efficacy of certain
peptide products described herein.
TABLE-US-00044 Maximal Compound EC50 response Name Structure (nM)
(% PTHrP) Characterization EU-201 1-Me 40 115 agonist EU-202
1-octyl 25 118 agonist EU-203 1-dodecyl 25 129 agonist EU-204
1-dodecyl 20 135 agonist EU-205 1-dodecyl 40 115 agonist EU-207
1-dodecyl 40 110 agonist EU-232 1-dodecyl 2.4 145 agonist EU-251
1-dodecyl 4.3 135 agonist EU-260 1-dodecyl 2.2 120 agonist EU-283
1-dodecyl 5.2 100 agonist EU-284 1-dodecyl 62 110 agonist
[1259] When tested in antagonist mode, with added PTHrP, the
maximal effects were even greater (to 146% of PTHrP maximal for the
C-12 compound, EU-203). This behavior demonstrates the ability of
the modification described herein to vary the fundamental
properties of the receptor interactions. Modifications elsewhere in
the molecule can be used to further modify the potency and
character of the interaction of the drug candidates.
Example 2-6: In Vivo Assay of the Compounds
[1260] Following the method of Frolik, C. A., et al. (2003) Bone
33: 372-379, 20 male rats from Sino-British SIPPR/BK Lab Animal Ltd
were acclimated to standard laboratory conditions for a period of 7
days. After acclimation, the animals were sorted by age into groups
of 5. Each animal in a group was treated with a single sc injection
of either vehicle or test agent.
[1261] Animals in two test groups were treated with 80 mcg/animal
of huPTH1-34 (Bachem) or 80 mcg/animal of EU-232. A fourth group
was treated with EU-232 at 320 mcg/animal. Blood samples were
collected via retro-orbital vein at 0.5, 1, 2, 4, and 5 hrs.
post-injection and blood samples were stored on ice prior to
centrifugation and testing for blood PO.sub.4 and Ca levels.
[1262] In the 80 mcg groups (PTH and EU-232) there was a transient
but not statistically significant decrease in blood PO.sub.4 levels
in response to PTH or EU-232 and PO.sub.4 levels did not further
diminish after 1 hour. In response to treatment with EU-232, the
blood PO.sub.4 levels decreased to statistically significantly
lower levels with time and the maximal decrease (25-35% decrease
from time 0 hr. level) was seen at the 5 hr. time point indicating
a potent and prolonged duration of action for EU-232. No groups
showed a statistically different blood Ca level compared to vehicle
at any time point, thus there was no indication of a propensity for
hypercalcemia following dosing.
[1263] In a similar manner, the analogs described herein (including
compounds of Table 1 in FIG. 1) are tested to evaluate their
potency and duration of action in vivo.
[1264] The covalently modified peptides and/or proteins described
herein are useful for the prevention and treatment of a variety of
diseases. PTHR1 agonists are effective in the treatment of bone
density diseases such as postmenopausal or senile osteoporosis,
hypoparathyroidism, osteopenia, implant fixation, and certain
metastatic tumors. Antagonistic analogs are suitable for treatment
of hypercalcemia, especially as related to hyperparathyroidism or
hypercalcemia of malignancy. PTH and PTHrP agonists can be used to
mobilize proliferation of haematopoietic stem cells (HSC) in bone
marrow in vivo or in vitro for use in bone marrow transplant and in
disease syndromes related to low blood cell concentrations.
Expansion post-transplant is an attractive application as well.
Since many cells in the blood originate from HSCs, a wide range of
applications is possible. Suitably labeled surfactant modified
peptides can be used as diagnostic probes.
[1265] Representative delivery regimens include oral, parenteral
(including subcutaneous, intramuscular and intravenous injection),
rectal, buccal (including sublingual), transdermal, inhalation
ocular and intranasal. An attractive and widely used method for
delivery of peptides entails subcutaneous injection of a controlled
release injectable formulation. Other administration routes for the
application of the covalently modified peptides and/or proteins
described herein are subcutaneous, intranasal and inhalation
administration.
Example 2-7. Pharmaceutical Usage for Treatment of Osteoporosis
[1266] A human patient, with evidence of osteoporosis or osteopenia
is treated with EU-204 by intranasal administration (200 .mu.L)
from a standard atomizer used in the art of a solution of the
pharmaceutical agent in physiological saline containing from 0.5 to
10 mg/mL of the pharmaceutical agent and containing standard
excipients such as benzyl alcohol. The treatment is repeated as
necessary for the alleviation of symptoms such as bone pain,
osteopenia, low bone density, or fractures. In a similar manner, a
solution of EU-204, and selected excipients, in an evaporating
solvent containing such as a hydrofluoroalkane is administered
intranasally by metered dose inhaler (MDI) as needed to stimulate
bone accretion. The effect of treatment is measured by use of
standard tests, including the Bone Mineral Density test (BMD
test).
[1267] All of the compounds described in Table 1 of FIG. 1 are
tested using a similar protocol.
[1268] In a similar manner, administration of an adjusted amount by
transbuccal, intravaginal, inhalation, subcutaneous, intravenous,
intraocular, or oral routes is tested to determine the level of
stimulation of PTHR1 on cells in the body, and to determine
therapeutic effects.
Example 3-1: Reagents--N-.alpha.-Fmoc, N-.epsilon.-(1-octyl
.beta.-D-glucuronide-6-yl)-L-lysine
[1269] In an oven-dried 250 mL Erlenmeyer flask is placed 1-octyl
.beta.-D-glucuronic acid (Carbosynth Ltd., 3.06 g, 10 mmol), 50 mL
anhydrous DMF, and anhydrous 1-hydroxybenzotriazole (1.62 g, 12
mmol). A chilled (4.degree. C.) solution of N,
N'-dicyclohexylcarbodiimide (2.48 g, 12 mmol) in 50 mL of DMF is
added, with stirring, and the reaction is allowed to proceed for 5
min. The copious white precipitate of N, N'-dicyclohexylurea is
filtered on a fritted glass funnel and the filtrate is added to a
solution of N-.alpha.-Fmoc-L-lysine (3.68 g, 10 mmol) in 25 ml
anhydrous DMF. The reaction is allowed to proceed for 25 min with
warming to room temp or until the ninhydrin color is very faint.
The reaction mixture is filtered, stripped to dryness and
crystallized from MeOH/Et.sub.2O by dissolution in MeOH and slow
dilution to the cloud point with Et.sub.2O, followed by
refrigeration. Further purification can be achieved by silica gel
chromatography using a solvent gradient from EtOAc to
EtOAc/EtOH/AcOH.
[1270] In a similar manner, but substituting N-.alpha.-Boc-L-lysine
is obtained N-.alpha.-Boc,N-.epsilon.-(1-octyl
.beta.-D-glucuronide-6-yl)-L-lysine, suitable for N-terminal
incorporation and cleavage to a free N-Terminus. In a similar
manner, but substituting N-.alpha.-Ac-L-lysine is obtained
N-.alpha.-Ac,N-.epsilon.-(1-octyl
.beta.-D-glucuronide-6-yl)-L-lysine, suitable for incorporation at
the N-terminus of a peptide with a blocked N-terminus. In a similar
manner, but substituting the appropriate amount of
N-.alpha.-Fmoc-L-ornithine is obtained
N-.alpha.-Fmoc,N-.delta.-(1-octyl
.beta.-D-glucuronide-6-yl)-L-ornithine. In a similar manner but
substituting other N-mono-protected diamino acids one obtains the
corresponding reagents. Alternatively, use of a transient
Me.sub.3Si ester protecting group during the coupling and without
preactivation of the 1-octyl .beta.-D-glucuronic acid provides a
facile route to the formation of the reagents. The transient
Me.sub.3Si ester is produced by reaction of the Fmoc-Lys-OH with an
equimolar amount of N,O-bis(trimethylsilyl)acetamide in
dichloromethane (CH.sub.2Cl.sub.2). The organic layer contains the
desired reagent as a solution in CH.sub.2Cl.sub.2 ready for
coupling with the 1-alkyl glucoronide as above. The filtered
reaction mixture is washed with aqueous NaHSO.sub.4 to hydrolyze
the Me.sub.3Si ester, dried over MgSO.sub.4 and solvent is
removed.
[1271] Similarly, but using peracetyl or perbenzoyl 1-octyl
.beta.-D-glucuronic acid one obtains the Ac, or Bz protected form
of the reagents (e.g. 2,3,4-trisacetyl 1-octyl O-D-glucuronic acid,
and the like, formed by treatment with Ac.sub.2O). Such reagents
have increased stability during acid cleavage from the resin and
are used when instability during deprotection is detected, see
(Kihlberg, J., et al. (1997) Methods Enzymol 289: 221-245) and
references therein. Final deprotection of such products is carried
out by base-catalyzed transesterification after cleavage, by use of
MeOH/NH.sub.3, MeOH/NaOMe, MeOH/NH.sub.2NH.sub.2, as described
above.
Example 3-2: Synthetic Peptide Analogs
[1272] In general, peptide synthesis methods involve the sequential
addition of protected amino acids to a growing peptide chain.
Normally, either the amino or carboxyl group of the first amino
acid and any reactive side chain group are protected. This
protected amino acid is then either attached to an inert solid
support, or utilized in solution, and the next amino acid in the
sequence, also suitably protected, is added under conditions
amenable to formation of the amide linkage. After all the desired
amino acids have been linked in the proper sequence, protecting
groups and any solid support are removed to afford the crude
peptide. The peptide is desalted and purified
chromatographically.
[1273] A preferred method of preparing the analogs of the
physiologically active truncated peptides, having fewer than about
fifty amino acids, involves solid phase peptide synthesis. In this
method the .alpha.-amino (N.alpha.) functions and any reactive side
chains are protected by acid- or base-sensitive groups. The
protecting group should be stable to the conditions of peptide
linkage formation, while being readily removable without affecting
the extant peptide chain. Suitable .alpha.-amino protecting groups
include, but are not limited to t-butyloxycarbonyl (Boc),
benzyloxycarbonyl (Cbz), o-chlorobenzyloxycarbonyl,
biphenylisopropyloxycarbonyl, t-amyloxycarbonyl (Amoc),
isobornyloxycarbonyl, .alpha.,
.alpha.-dimethyl-3,5-dimethoxybenzyloxy-carbonyl,
o-nitrophenylsulfenyl, 2-cyano-t-butoxycarbonyl,
9-fluorenyl-methoxycarbonyl (Fmoc) and the like, preferably Boc or
more preferably, Fmoc. Suitable side chain protecting groups
include, but are not limited to: acetyl, benzyl (Bzl),
benzyloxymethyl (Bom), Boc, t-butyl, o-bromobenzyloxycarbonyl,
t-butyl, t-butyldimethylsilyl, 2-chlorobenzyl (Cl-z),
2,6-dichlorobenzyl, cyclohexyl, cyclopentyl, isopropyl, pivalyl,
tetrahydropyran-2-yl, tosyl (Tos),
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf),
trimethylsilyl and trityl. A preferred N.alpha.-protecting group
for synthesis of the compounds is the Fmoc group. Preferred side
chain protecting groups are O-t-Butyl group for Glu, Tyr, Thr, Asp
and Ser; Boc group for Lys and Trp side chains; Pbf group for Arg;
Trt group for Asn, Gln, and His. For selective modification of a
Lys residue, orthogonal protection with a protecting group not
removed by reagents that cleave the Fmoc or t-butyl based
protecting groups is preferred. Preferred examples for modification
of the Lys side chain include, but are not limited to, those
removed by hydrazine but not piperidine; for example
1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde)
or 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) and
allyloxycarbonyl (Alloc). The Fmoc-Lys(ivDde) or Fmoc-Lys(Dde)
protecting group scheme is preferred in cases where side chain
lactam formation is desired (Houston, M. E., Jr., et al. (1995) J
Pept Sci 1: 274-282; Murage, E. N., et al. (2010) J Med Chem),
since in this case Fmoc-Glu(O-Allyl) and Fmoc-Lys(Alloc) can be
incorporated and used to provide transient protection, then
deprotected for lactam formation while the Lys(Dde) protecting
group remains for later removal and reaction with the
functionalized surfactant.
[1274] The Fmoc-Lys(ivDde) or Fmoc-Lys(Dde) protecting group scheme
is preferred in cases where side chain lactam formation is desired
(Houston, M. E., Jr., et al. (1995) J Pept Sci 1: 274-282; Murage,
E. N., et al. (2010) J Med Chem), since in this case
Fmoc-Glu(O-Allyl) and Fmoc-Lys(Alloc) can be incorporated and used
to provide transient protection, then deprotected for lactam
formation while the Lys(Dde) protecting group remains for later
removal and reaction with the functionalized surfactant.
[1275] In solid phase synthesis, the C-terminal amino acid is first
attached to a suitable resin support. Suitable resin supports are
those materials which are inert to the reagents and reaction
conditions of the stepwise condensation and deprotection reactions,
as well as being insoluble in the media used. Examples of
commercially available resins include styrene/divinylbenzene resins
modified with a reactive group, e.g., chloromethylated
co-poly-(styrene-divinylbenzene), hydroxymethylated
co-poly-(styrene-divinylbenzene), and the like. Benzylated,
hydroxymethylated phenylacetamidomethyl (PAM) resin is preferred
for the preparation of peptide acids. When the C-terminus of the
compound is an amide, a preferred resin is
p-methylbenzhydrylamino-co-poly(styrene-divinyl-benzene) resin, a
2.4 dimethoxybenzhydrylamino-based resin ("Rink amide"), and the
like. An especially preferred support for the synthesis of larger
peptides are commercially available resins containing PEG sequences
grafted onto other polymeric matricies, such as the Rink Amide-PEG
and PAL-PEG-PS resins (Applied Biosystems) or similar resins
designed for peptide amide synthesis using the Fmoc protocol. Thus
in certain cases it is desirable to have an amide linkage to a PEG
chain. It those cases it is convenient to link an
N-Fmoc-amino-PEG-carboxylic acid to the amide forming resin above
(e.g. Rink amide resin and the like). The first amino acid of the
chain can be coupled as an N-Fmoc-amino acid to the amino function
of the PEG chain. Final deprotection will yield the desired
Peptide-NH-PEG-CO--NH.sub.2 product.
[1276] Attachment to the PAM resin may be accomplished by reaction
of the N.alpha. protected amino acid, for example the Boc-amino
acid, as its ammonium, cesium, triethylammonium,
1,5-diazabicyclo-[5.4.0]undec-5-ene, tetramethylammonium, or
similar salt in ethanol, acetonitrile, N,N-dimethylformamide (DMF),
and the like, preferably the cesium salt in DMF, with the resin at
an elevated temperature, for example between about 40.degree. and
60.degree. C., preferably about 50.degree. C., for from about 12 to
72 hours, preferably about 48 hours. This will eventually yield the
peptide acid product following acid cleavage or an amide following
aminolysis.
[1277] The N.alpha.-Boc-amino acid may be attached to the
benzhydrylamine resin by means of, for example, an
N,N'-diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt)
mediated coupling for from about 2 to about 24 hours, preferably
about 2 hours at a temperature of between about 10.degree. and
50.degree. C., preferably 25.degree. C. in a solvent such as
CH.sub.2Cl.sub.2 or DMF, preferably CH.sub.2Cl.sub.2.
[1278] For Boc-based protocols, the successive coupling of
protected amino acids may be carried out by methods well known in
the art, typically in an automated peptide synthesizer. Following
neutralization with triethylamine, N,N-di-isopropylethylamine
(DIEA), N-methylmorpholine (NMM), collidine, or similar base, each
protected amino acid is introduced in approximately about 1.5 to
2.5 fold molar excess and the coupling carried out in an inert,
nonaqueous, polar solvent such as CH.sub.2Cl.sub.2, DMF,
N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), or mixtures
thereof, preferably in dichloromethane at ambient temperature. For
Fmoc-based protocols no acid is used for deprotection but a base,
preferably DIEA or NMM, is usually incorporated into the coupling
mixture. Couplings are typically done in DMF, NMP, DMA or mixed
solvents, preferably DMF. Representative coupling agents are
N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropyl-carbodiimide
(DIC) or other carbodiimide, either alone or in the presence of
HOBt, O-acyl ureas,
benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium
hexafluorophosphate (PyBop). N-hydroxysuccinimide, other
N-hydroxyimides, or oximes. Alternatively, protected amino acid
active esters (e.g. p-nitrophenyl, pentafluorophenyl and the like)
or symmetrical anhydrides may be used. Preferred coupling agents
are of the aminium/uronium (alternative nomenclatures used by
suppliers) class such as
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HBTU),
O-(7-azabenzotraiazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU),
2-(6-Chloro-1H-benzotraiazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HCTU), and the like.
[1279] A preferred method of attachment to the Fmoc-PAL-PEG-PS
resin may be accomplished by deprotection of the resin linker with
20% piperidine in DMF, followed by reaction of the N-.alpha.-Fmoc
protected amino acid, about a 5 fold molar excess of the
N-.alpha.-Fmoc-amino acid, using HBTU:di-isopropylethylamine (DIEA)
(1:2) in DMF in a microwave-assisted peptide synthesizer with a 5
min. 75.degree. max coupling cycle.
[1280] For this Fmoc-based protocol in the microwave-assisted
peptide synthesizer, the N-.alpha.-Fmoc amino acid protecting
groups are removed with 20% piperidine in DMF containing 0.1M
1-hydroxybenzotriazole (HOBt), in a double deprotection protocol
for 30 sec and then for 3 min with a temperature maximum set at
75.degree. C. HOBt is added to the deprotection solution to reduce
aspartimide formation. Coupling of the next amino acid then employs
a five-fold molar excess using HBTU:DIEA (1:2) with a 5 min, 75
max. double-coupling cycle.
[1281] At the end of the solid phase synthesis the fully protected
peptide is removed from the resin. When the linkage to the resin
support is of the benzyl ester type, cleavage may be effected by
means of aminolysis with an alkylamine or fluoroalkylamine for
peptides with an alkylamide C-terminus, or by ammonolysis with, for
example, ammonia/methanol or ammonia/ethanol for peptides with an
unsubstituted amide C-terminus, at a temperature between about
-10.degree. and 50.degree. C., preferably about 25.degree. C., for
between about 12 and 24 hours, preferably about 18 hours. Peptides
with a hydroxy C-terminus may be cleaved by HF or other strongly
acidic deprotection regimen or by saponification. Alternatively,
the peptide may be removed from the resin by transesterification,
e.g., with methanol, followed by aminolysis or saponification. The
protected peptide may be purified by silica gel or reverse-phase
HPLC.
[1282] The side chain protecting groups may be removed from the
peptide by treating the aminolysis product with, for example,
anhydrous liquid hydrogen fluoride in the presence of anisole or
other carbonium ion scavenger, treatment with hydrogen
fluoride/pyridine complex, treatment with
tris(trifluoroacetyl)boron and trifluoroacetic acid, by reduction
with hydrogen and palladium on carbon or polyvinylpyrrolidone, or
by reduction with sodium in liquid ammonia, preferably with liquid
hydrogen fluoride and anisole at a temperature between about
-10.degree. and +10.degree. C., preferably at about 0.degree. C.,
for between about 15 minutes and 2 hours, preferably about 1.5
hours.
[1283] For peptides on the benzhydrylamine type resins, the resin
cleavage and deprotection steps may be combined in a single step
utilizing liquid hydrogen fluoride and anisole as described above
or preferably through the use of milder cleavage cocktails. For
example, for the PAL-PEG-PS resin, a preferred method is through
the use of a double deprotection protocol in the microwave-assisted
peptide synthesizer using one of the mild cleavage cocktails known
in the art, such as
TFA/water/tri-iso-propylsilane/3,6-dioxa-1,8-octanedithiol (DODT)
(92.5/2.5/2.5/2.5) for 18 min at 38.degree. C. each time. Cleavage
of alkyl glycoside containing materials have shown survival of the
alkyl glycoside linkage using protocols with TFA/water ratios in
the 9/1 to 19/1 range. A typical cocktail is 94% TFA: 2% EDT; 2%
H.sub.2O; 2% TIS. Typically the fully deprotected product is
precipitated and washed with cold (-70.degree. to 4.degree. C.)
Et.sub.2O, dissolved in deionized water and lyophilized.
[1284] The peptide solution may be desalted (e.g. with BioRad
AG-3.RTM. anion exchange resin) and the peptide purified by a
sequence of chromatographic steps employing any or all of the
following types: ion exchange on a weakly basic resin in the
acetate form; hydrophobic adsorption chromatography on
underivatized co-poly(styrene-divinylbenzene), e.g. Amberlite.RTM.
XAD; silica gel adsorption chromatography; ion exchange
chromatography on carboxymethylcellulose; partition chromatography,
e.g. on Sephadex.RTM. G-25; counter-current distribution;
supercritical fluid chromatography; or HPLC, especially
reversed-phase HPLC on octyl- or octadecylsilylsilica (ODS) bonded
phase column packing.
[1285] Also provided herein are processes for preparing covalently
modified peptides and/or proteins described herein and
pharmaceutically acceptable salts thereof, which processes comprise
sequentially condensing protected amino acids on a suitable resin
support, removing the protecting groups and resin support, and
purifying the product, to afford analogs of the physiologically
active truncated homologs and analogs of the covalently modified
peptides and/or proteins described herein. In some embodiments,
covalently modified peptides and/or proteins described herein
incorporate alkyl glycoside modifications as defined above. Another
aspect relates to processes for preparing covalently modified
peptides and/or proteins described herein and pharmaceutically
acceptable salts thereof, which processes comprise the use of
microwave-assisted solid phase synthesis-based processes or
standard peptide synthesis protocols to sequentially condense
protected amino acids on a suitable resin support, removing the
protecting groups and resin support, and purifying the product, to
afford analogs of the physiologically active peptides, as defined
above.
Example 3-3. General Oxidation Method for Uronic Acids
[1286] To a solution of 1-dodecyl .beta.-D-glucopyranoside
(Carbosynth) [2.0 g, 5.74 mmol] in 20 mL of acetonitrile and 20 mL
of DI water was added (diacetoxyiodo)benzene (Fluka) [4.4 g, 13.7
mmol] and TEMPO (SigmaAldrich) [0.180 g, 1.15 mmol]. The resulting
mixture was stirred at room temperature for 20 h. The reaction
mixture was diluted with water and lyophilized to dryness to give
1.52 g (crude yield 73.1%) of the crude product, 1-dodecyl
.beta.-D-glucuronic acid, as a white powder, which was used
directly for the solid phase synthesis without further
purification. This product was previously prepared by an
alternative process using NaOCl as oxidant, as described in the
specification, and also has been used for longer alkyl groups. In a
similar manner are prepared the desired alkyl saccharide uronic
acids used to make the products and reagents described herein.
[1287] In a like manner, but using the corresponding 1-tetradecyl,
1-hexadecyl, and 1-octadecyl .beta.-D-glucopyranosides (purchased
from Anatrace, Maumee, Ohio) were prepared the desired 1-alkyl
saccharide uronic acids which were used to make the products and
reagents described herein.
Example 3-4: Preparation of Analog EU-A387
[1288] A sample of
Fmoc-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Bip-Ser-Lys-Tyr-Leu-Glu-Ser-Lys(-
Alloc)-Rink amide resin was prepared by sequential addition of
N-alpha-Fmoc protected amino acids as described in Example 1 and
deprotected on the Lys-N-epsilon position by incubation with
Pd(PPh.sub.3).sub.4 (0.5 eq) and DMBA (20 eq) in
DMF/CH.sub.2Cl.sub.2 (1:1) overnight in the dark at room
temperature. Following washing by DMF/CH.sub.2Cl.sub.2, the Lys
side chain was acylated with 1'-dodecyl .beta.-D-glucuronic acid in
DMF/CH.sub.2Cl.sub.2 through the use of DIC/HOBt. Completion of the
coupling was checked by ninhydrin and the product was washed
extensively with CH.sub.2Cl.sub.2.
[1289] The product resin is submitted to final deprotection and
cleavage from the resin by treatment with the cleavage cocktail
(94% TFA: 2% EDT; 2% H.sub.2O; 2% TIS) for a period of 240 min at
room temperature. The mixture was treated with Et.sub.2O, to
precipitate the product and washed extensively with Et.sub.2O to
yield the crude title peptide product after drying in vacuo.
[1290] Purification is carried out in two batches by reversed phase
(C.sub.18) hplc. The crude peptide was loaded on a 4.1.times.25 cm
hplc column at a flow rate of 15 mL/min (15% organic modifier;
acetic acid buffer) and eluted with a gradient from 15-45% buffer B
in 60 min at 50.degree. C. The product fraction is lyophilized to
yield the title product peptide with a purity 98.03% by analytical
hplc (18.6 min; 30-60% CH.sub.3CN in 0.1% TFA)/mass spectrometry
(M+1 peak=2382.14).
[1291] The corresponding 1-methyl and 1-octyl analogs of the title
compound are prepared in a similar manner, but using the reagents
1'-methyl .beta.-D-glucuronic acid and 1'-octyl .beta.-D-glucuronic
acid (Carbosynth). The corresponding 1-decyl, 1-dodecyl,
1-tetradecyl, 1-hexadecyl, 1-octadecyl and 1-eicosyl analogs are
prepared using the corresponding glucouronic acids, prepared as
described above. Alternatively, the 1-alkyl glucuronyl, or other
uronic acylated analogs, may be prepared by initial purification of
the deprotected or partially deprotected peptide followed by
acylation by the desired uronic acid reagent.
[1292] Analysis was done by HPLC/mass spectrometry in positive ion
mode using the eluent gradients given in the table below.
TABLE-US-00045 Molecular HPLC Compound Wt Molecular (min; Name
Expected Wt found elution) EU-A387 2379.66 2380.14 18.6 [b] EU-A388
2393.69 2393.74 16.0 [a] EU-A391 2317.62 2318.26 11.2 [b] EU-A455
2988.36 2988.00 11.5 [b] EU-A474 2570.86 2570.54 11.3 [b] EU-A478
2459.75 2459.74 11.1 [b] EU-A484 2544.86 2545.06 9.6 [b] EU-A501
2904.2 2903.34 7.9 [b] EU-A502 2776.07 2776.14 8.0 [b] EU-A503
2704.98 2704.40 8.0 [b] EU-A504 2548.80 2548.00 9.1 [b] EU-A505
2392.61 2392.40 10.5 [b] EU-A506 2305.53 2305.06 10.7 [b] EU-A507
3763.23 3762.66 9.0 [b] EU-A521 2303.56 2303.60 8.2 [c] EU-A522
2315.60 2315.60 14.2 [d] EU-A523 2615.94 2616.00 8.1 [b] EU-A524
2459.75 2459.74 12.7 [d] EU-A525 2459.75 2459.06 6.0 [c] EU-A526
2473.75 2473.60 12.7 [d] EU-A527 2390.64 2390.40 14.6 [d] EU-A529
2546.83 2546.80 9.5 [b] EU-A531 2546.83 2546.80 9.5 [b] EU-A532
2559.00 2558.66 9.6 [b] EU-A533 2560.96 2560.66 9.5 [b] EU-A534
2544.99 2544.94 9.7 [b] EU-A535 2573.05 2574.00 12.0 [b] EU-A536
2602.96 2603.46 14.3 [b] EU-A538 2516.99 2516.40 10.3 [b] EU-A539
2657.20 2656.80 10.8 [b] EU-A540 2685.20 2684.94 9.8 [c] EU-A541
2713.20 2712.80 13.0 [c] EU-A544 2631.94 2632.26 10.8 [b] EU-A546
2687.94 2688.8 9.1 [c] EU-A549 2388.67 2388.66 6.3 [e] EU-A551
2444.67 2445.20 11.4 [e] EU-A552 EU-A554 2560.86 2560.40 10.3 [c]
EU-A556 2616.86 2616.40 11.7 [e] EU-A560 2570.86 2571.06 8.3 [c]
EU-A562 2626.86 2626.66 9.9 [e] EU-A563 EU-A565 2542.80 2542.54 9.5
[c] EU-A567 2598.80 2599.06 12.0 [e] HPLC gradients in 0.1% TFA [a]
35 to 65% CH.sub.3CN over 30 min. [b] 30 to 60% CH.sub.3CN over 20
min. [c] 35 to 65% CH.sub.3CN over 20 min. [d] 25 to 55% CH.sub.3CN
over 20 min. [e] 40 to 70% CH.sub.3CN over 20 min. HPLC on
Phenomenex Luna C18 5 micron 250 .times. 4.6 mm.
Example 3-5: Cellular Assay of the Compounds
[1293] Compounds were weighed precisely in an amount of
approximately 1 mg and assayed in standard cellular assays (Cerep
SA). The readout is the amount of cAMP generated in the cells
treated with the test compounds, in either agonist or antagonist
mode. The assay used was the stimulation of cAMP levels in the
glucagon and GLP-1 cellular assays. The assays are described in
Chicchi, G. G., et al. (1997) J Biol Chem 272: 7765-7769 and Runge,
S., et al. (2003) Br J Pharmacol 138: 787-794.
[1294] For compound EU-A391 the GLCR cellular response does not
change and the GLP1R cellular response rises steeply with and EC50
of 420 nM.
TABLE-US-00046 EC.sub.50 EC.sub.50 GLP-1 R glucagon R Compound
Structure (nM) (nM) EU-A391 1-dodecyl 420 n.c. EU-A455 1-dodecyl 59
770 EU-A474 1-dodecyl 3000 n.c. EU-A478 1-dodecyl n.c. n.c. EU-A484
1-dodecyl n.c. n.c. EU-A501 1-dodecyl 20000 12000 EU-A502 1-dodecyl
9400 n.c. EU-A503 1-dodecyl n.c. n.c. EU-A504 1-dodecyl 3100 1100
EU-A505 1-dodecyl 8500 6100 EU-A506 1-dodecyl 4600 1300 EU-A507
1-dodecyl 18 1 EU-A521 1-dodecyl n.c. n.c. EU-A522 1-dodecyl n.c.
9000 EU-A523 1-dodecyl n.c. n.c. EU-A524 1-dodecyl n.c. n.c.
EU-A525 1-dodecyl n.c. n.c. EU-A526 1-dodecyl n.c. n.c. EU-A527
1-dodecyl n.c. 5000 EU-A529 1-dodecyl n.c. 7000 EU-A531 1-dodecyl
2100 1100 EU-A532 1-dodecyl 5000 2600 EU-A533 1-dodecyl 770 780
EU-A534 1-dodecyl 290 1900 EU-A535 1-tetradecyl .sctn.4800 2100
EU-A536 1-hexadecyl >10000 4400 EU-A538 1-dodecyl 270 n.c.
EU-A539 1-dodecyl 860 2300 EU-A540 1-tetradecyl n.c. 8800 EU-A541
1-hexadecyl 800 5000 n.c. means EC.sub.50 not calculable
.sctn.means superagonist
Example 3-6: In Vivo Assay of Compounds
[1295] Sixty (60) diet induced obese C57BL/6J male mice are
received from JAX labs at 14 wks of age. The mice are ear notched
for identification and housed in individually and positively
ventilated polycarbonate cages with HEPA filtered air at density of
one mouse per cage. The animal room is lighted entirely with
artificial fluorescent lighting, with a controlled 12 h light/dark
cycle. The normal temperature and relative humidity ranges in the
animal rooms are 22.+-.4.degree. C. and 50.+-.15%, respectively.
Filtered tap water, acidified to a pH of 2.8 to 3.1, and high fat
diet (60 kcal %) are provided ad libitum.
[1296] Following a 2 week acclimation, 40 mice are chosen based on
desired body weight range and mice are randomized into groups
(n=10) as below. Group 1. Vehicle treated; Group 2. Low dose test
cmpd; Group 3. Mid dose test cmpd; Group 4. High dose test cmpd.
Mice are dosed via SC daily for 28 days. Body weights and cage side
observations are recorded daily. Food and water intake will be
recorded weekly. Mice undergo NMR measurements for determining
whole body fat and lean composition on days 1 (pre dose) and 26. On
days 0, 14 and 27, mice are fasted overnight for an oral glucose
tolerance test. Next day, the first blood sample is collected via
tail nick (t=0). Mice are then administered a bolus of 1.0 g/kg
glucose. Blood samples are obtained via tail nick at 5, 30, 60 and
120 min after glucose and plasma glucose will be immediately
determined using a glucometer.
[1297] Sacrifice and tissue collection: Mice are sacrificed on day
29. Terminal blood is processed to serum/plasma and aliquots are
sent for analysis of glucose, insulin and lipid profile. Fat
tissues are collected, weighed and frozen for analysis. The optimal
compound profile shows decreased glucose excursion in the OGTT,
decreased basal insulin secretion, with potentiated
glucose-dependent insulin secretion, decreased weight gain,
decreased fat mass but minimal effects on lean mass.
Example 3-7: Uses of the Compounds
[1298] The covalently modified peptides and/or proteins described
herein are useful for the prevention and treatment of a variety of
diseases related to obesity, the metabolic syndrome, cardiovascular
disease and diabetes. Suitably labeled surfactant modified peptides
can be used as diagnostic probes.
[1299] Representative delivery regimens include oral, parenteral
(including subcutaneous, intramuscular and intravenous injection),
rectal, buccal (including sublingual), transdermal, inhalation
ocular and intranasal. An attractive and widely used method for
delivery of peptides entails subcutaneous injection of a controlled
release injectable formulation. Other administration routes for the
application of the covalently modified peptides and/or proteins
described herein are subcutaneous, intranasal and inhalation
administration.
Example 3-8. Pharmaceutical Usage for Treatment of Insulin
Resistance
[1300] A human patient, with evidence of insulin or metabolic
syndrome is treated with EU-A596 by intranasal administration (200
.mu.L) from a standard atomizer used in the art of a solution of
the pharmaceutical agent in physiological saline containing from
0.5 to 10 mg/mL of the pharmaceutical agent and containing standard
excipients such as benzyl alcohol. The treatment is repeated as
necessary for the alleviation of symptoms such as obesity, elevated
blood glucose and the like. In a similar manner, a solution of
EU-A596, and selected excipients, in an evaporating solvent
containing such as a hydrofluoroalkane is administered intranasally
by metered dose inhaler (MDI) as needed to reduce insulin
resistance. The effect of treatment is determined using standard
tests including measurement of blood glucose levels, Body Mass
Index, and/or body weight and/or measurement of waist to hip
ratios.
[1301] In a similar manner, administration of an adjusted amount by
transbuccal, intravaginal, inhalation, subcutaneous, intravenous,
intraocular, or oral routes is tested to determine level of
stimulation of GLP1R and/or GLCR on cells in the body and to
determine therapeutic effects.
Sequences
[1302] Table 1 in FIG. 1 depicts compounds that were prepared by
methods described herein. The specification provides sequences for
SEQ. ID. Nos. 1 and 149-169. Additionally. Table 1 of FIG. 1
provides SEQ. ID Numbers for compounds EU-A101 to EU-A199 and
EU-A600 to EU-A649 having SEQ. ID. NOs. 2-148, and SEQ. ID. NO. 645
respectively, as shown in Table 1 of FIG. 1. Compounds in Table 1
of FIG. 1, and their respective SEQ. ID. NOs. shown in Table 1 of
FIG. 1 are hereby incorporated into the specification as filed.
[1303] Table 2 in FIG. 2 depicts compounds that were prepared by
methods described herein. The specification provides sequences for
SEQ. ID. Nos. 170-174 and SEQ. ID. NOs. 283-302. Additionally.
Table 2 of FIG. 2 provides SEQ. ID. Numbers for compounds EU-201 to
EU-299 and EU-900-EU-908 having SEQ. ID. NOs. 175-282 respectively,
as shown in Table 2 of FIG. 2. Compounds in Table 2 of FIG. 2, and
their respective SEQ. ID. NOs. shown in Table 2 of FIG. 2 are
hereby incorporated into the specification as filed.
[1304] Table 3 in FIG. 8 depicts compounds that were prepared by
methods described herein. The specification provides sequences for
SEQ. ID. Nos. 303-305 and SEQ. ID. Nos. 619-644. Additionally,
Table 3 of FIG. 8 provides SEQ. ID Numbers for compounds EU-A300 to
EU-A425 having SEQ. ID. NOs. 306-431 respectively, as shown in
Table 3 of FIG. 8. Compounds in Table 3 of FIG. 8, and their
respective SEQ. ID. NOs. shown in Table 3 of FIG. 8 are hereby
incorporated into the specification as filed.
[1305] Table 4 in FIG. 9 depicts compounds that were prepared by
methods described herein. The specification provides SEQ. ID. Nos.
303-305 and SEQ. ID. Nos. 619-644. Additionally, Table 4 of FIG. 9
provides SEQ. ID Numbers for compounds EU-A426 to EU-599 having
SEQ. ID. NOs. 432-520 respectively, as shown in Table 4 of FIG. 9.
Compounds in Table 2 of FIG. 2, and their respective SEQ. ID. NOs.
shown in Table 4 of FIG. 9 are hereby incorporated into the
specification as filed.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210077629A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210077629A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References