Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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THIOETHER CYCLIC PEPTIDE AMYLIN RECEPTOR MODULATORS
This application claims the benefit of U.S. Provisional Patent Application
Serial
No. 62/662,492, filed April 25, 2018, which is hereby incorporated by
reference in its
entirety.
SEQUENCE LISTING
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 April 16, 2019, is named PRD3471W0PCT1 SL.txt and
is
55,747 bytes in size.
FIELD OF THE INVENTION
[0001] This invention relates to thioether-cyclized analogues of amylin,
pramlintide and
davalintide and derivatives thereof (herein referred to as "amylinomimetic
peptides"),
which function as agonists of amylin receptors and as such are useful for the
treatment of
metabolic diseases and disorders, such as obesity, type 2 diabetes, metabolic
syndrome,
insulin resistance and dyslipidemia.
BACKGROUND OF THE INVENTION
[0002] Amylin is a naturally-occurring, 37-amino acid-containing peptide that
is a
structurally related member of the calcitonin family of peptides, which
includes
calcitonin (CT), calcitonin gene-related peptide (CGRP), adrenomedullin (AM)
and
intermedin (AM2). It is synthesized in and secreted by the pancreas in
response to
nutrient influx into the GI tract. Following release into circulation, amylin
binds with
high affinity to specific Class B GPCRs, located primarily in the hindbrain
area postrema
region of the central nervous system. As such, it is a centrally acting
neuroendocrine
hormone that serves to regulate glucose homeostasis by inhibiting gastric
emptying,
inhibiting the release of glucagon and inducing satiety (reviewed in Hay DL et
al.,
Pharmacol Rev 2015;67:564-600). Amylin has been found to interact in vitro
with three
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target receptor subtypes - AMY1R, AMY2R and AMY3R, which are heterodimeric
structures comprised of the calcitonin receptor (CTR) and a receptor modifying
protein
(RAMP1, RAMP2 and RAMP3, respectively) (Christopoulos G et al., Mol Pharmacol
1999;56:235-42). Association of the CTR with these RAMPs confers an increase
in
amylin affinity relative to that at CTR alone, and provides a basis for the
selective
receptor pharmacology of amylin (vs. CT) (Bower RL and Hay DL, Br J Pharmacol
2016;173:1883-98). While it has been generally accepted that amylin
participates in
high-affinity/potency interactions, particularly with AMY1R and AMY3R,
simultaneous
agonism at all receptor subtypes may not necessarily be required for
pharmacological
benefit. (Hay DL et al., Br J Pharmacol 2018;175:3-17; Hay DL, Headache
2017;57:89-
96).
[0003] Human amylin, also known as islet amyloid polypeptide (TAPP), has
several
physicochemical properties that make it unsuitable as a pharmaceutical agent,
including
most notably, its low aqueous solubility and tendency to self-aggregate and
adhere to
surfaces. Pramlintide, an equipotent amylin analogue was developed by
incorporating
three specific residue mutations (A25P, S28P and S29P) into the amylin
sequence
(Young AA et al., Drug Dev Res 1996;37:231-48). These mutations confer
improved
physicochemical properties (reduced aggregation propensity) relative to
amylin.
Pramlintide reduces food intake (Smith SR et al., Am J Physiol Endocrinol
Metab
2007;293:E620-7) and body weight (Aronne L et al., J Clin Endocrin Metab
2007;92:2977-83) in obese subjects. It is approved for the treatment of adult
patients
with type 1 diabetes as an adjunctive therapy to insulin, and for adult
patients with type 2
diabetes as an adjunctive therapy to either insulin alone, or concurrently
with metformin
and/or sulfonylureas (Pullman J et al., Vasc Health Risk Manag 2006;2:203-12).
Davalintide is a related synthetic peptide, 32 amino acids in length, whose
structure is a
chimera of the primary sequences of pramlintide and salmon calcitonin. As
such, it is
devoid of the amyloidogenic residues of human amylin (Westermark P et al.,
Proc Natl
Acad Sci USA 1990;87:5036-40). It is a highly potent agonist at both amylin
and
calcitonin receptors, demonstrating enhanced pharmacological properties over
native (rat)
amylin at reducing food intake and body weight in rats (Mack CM et al., Int J
Obes 2010;
34:385-95). Amylin, pramlintide and davalintide each have extremely short half-
lives in
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vivo (< 0.75 h), which limit their practical therapeutic utilities (Roth JD et
al., Immun
Endoc Metab Agents in Med Chem 2008;8:317-24; Mack CM et al., Diabetes Obes
Metab 2011;13:1105-13). In the case of pramlintide, this short half-life
necessitates a
regimen of multiple daily administration for achieving clinical effectiveness.
Thus, it is
desirable to obtain amylin agonist peptides or derivatives thereof with
improved
metabolic stabilities and pharmacokinetic profiles.
[0004] One technique used for extending the half-lives of peptides involves
conjugation
to a biological carrier, such as albumin, a suitable mAb or antigen-binding
fragment
thereof, or a mAb-derived crystallization fragment domain (Fc) protein. Such
bioconjugation chemistry is performed by the reaction of a selectively
reactive sulfhydryl
functionality on the biologic carrier molecule and a complementary
electrophilic site
engineered into the peptide of interest. While maleimide functionalities
incorporated
onto peptide molecules have served as suitable electrophiles for
bioconjugation
chemistry, the resultant bioconjugates may undergo a reverse-conjugation
reaction (retro-
Michael reaction) in vivo, leading to a loss of the peptide from the biologic
carrier. More
stable thioacetamide linkages, formed by way of coupling to a reactive
haloacetamido-
derivatized peptide are therefore used to avoid this reverse conjugation
potential.
However, the conditions required for bromoacetamide conjugation reaction
cannot be
used successfully with disulfide containing amylin analogues for their
selective
bioconjugation, due to the reactivity of the unhindered disulfide ring. Such
attempted
chemistry leads to concomitant ring opening and associated complicating side
reactions.
It has long been recognized that an intact disulfide loop is a critical
molecular feature that
is required for receptor activation and biological function (Roberts AN et
al., Proc Natl
Acad Sci USA 1989;86:9662-9666; Cornish J et al., Am. J. Physiol.
1998;274:E827-
E833; Bower RL and Hay DL., Br. J. Pharmacol. 2016;173:1883-1898). Herein, an
N-
terminal cyclic thioether replacement for the cysteine disulfide connectivity
of
pramlintide and davalintide is identified which unexpectedly maintains amylin
receptor
agonist activity and is stable towards bioconjugation reaction chemistry.
SUMMARY OF THE INVENTION
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[0005] In one general aspect, the invention relates to novel amylinomimetic
compounds. Also provided herein are amylinomimetic derivatives of pramlintide
or
davalintide and their conjugates comprising a monoclonal antibody or an
antigen binding
fragment thereof coupled to an amylinomimetic peptide.
[0006] In one aspect, the invention is represented by a compound of Formula I
or a
derivative thereof (SEQ ID NO: 53):
H
0,, N,
Z2N Z4Z5Z6¨ N
X-NTY-CONH2
)n
Formula I
wherein
n is 1, or 2;
Z2 is a direct bond, serine, or glycine;
k N
.'1 =
Z4 iS T, or 01-1
H H
N N ,35,N
Z5 is A, f3-alanine, 0 , 0 , 0 , = 0
H
N
/N
0 0 , or
' =
Z6 iS T, or ''01-1
Zio is Q, or E;
Zii is R, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=NH)NH2;
SN
Z12 1S L, or 0 =
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SN
Z16 1S L, or 0 =
Z25 is P, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=0)CH3,
0
(CH2)(12-20)CH3
C(0)CH2CH=CH2,HO 0
0 0
\,,"2/(12-20)C1-13
- (0-4) o H
0 -
kjON (CH2)(12_20)CH3
_
0
HO 00
0
N (CH2)(12_20)CH3
- (3-24) a
0 - 0 -
Br mAb
- (3-24) 0 - (3-24) 8
, or ,
wherein said
mAb is optionally substituted through another thioether bond to a second
compound of
Formula I, so that there are two identical compounds of Formula I on the mAb;
Z26 is I, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=0)CH3, -
0
(CH2)(12-20)CH3
C(0)CH2CH=CH2,HO 0
0 0
)=0c)N
N k,--2/(l2_20)0H3
- (0-4) o
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0
µjON (CH2)(12-2o)CH3
- (3-2;)Thr
0
HO 0
0 0
N)L(CH2)(12-20)CH3
- (3-24) 0
O - 0 -
N Brs/mAb
- (3-24) 0 - (3-24) 8
, or , wherein said
mAb is optionally substituted through another thioether bond to a second
compound of
Formula I, so that there are two identical compounds of Formula I on the mAb;
X is ATZ1oZ11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34 (SEQ ID NO: 54), or
VLGRLSQELEIRLQTYPRTNTGS (SEQ ID NO: 55);
Z29 is P, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=0)CH3, -
0
(CH2)(12-20)CH3
C(0)CH2CH=CH2, 0
0 0
IN kµa..2i(12_20)CH3
(0-4) H
0
O -
- (3-24) II
0
HO 0
0 0
N)L(CH2)(12-20)CH3
- (3-24) 0
O 0 -
s/mAb
- (3-24) 0 (3_24) 8
, or , wherein said
mAb is optionally substituted through another thioether bond to a second
compound of
Formula I, so that there are two identical compounds of Formula I on the mAb;
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Z34 is S, or K, wherein the 6-amine of said K is optionally substituted
with¨C(=0)CH3, -
0
(CH2)(12-20)CH3
C(0)CH2CH=CH2, 0
0 0
)=0c)N
k,-,..2,(12_20)CH3
0 -
H
ON(CH)CH3
- (3-24-)
0
HO 0 n
0
õ, \
IN krs,-,112/(12-20)CH3
0
,mAb
, or ,
wherein said
mAb is optionally substituted through another thioether bond to a second
compound of
Formula I, so that there are two identical compounds of Formula I on the mAb;
and pharmaceutically acceptable salts thereof.
[0007] In certain embodiments, the invention is represented by a compound of
Formula
I or a derivative thereof, wherein:
n is 1, or 2;
Z2 is a direct bond;
kN.,r`v
'''OH =
Z4 iS T, or
7
H
)C.N ,kNA)c
Z5 is f3-alanine, 0 , 0 , or 0 ;
Z6 is T;
Zio is Q, or E;
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Zii is R, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=NH)NH2;
SN
Z12 is L, or 0 =
$N
Z16 is L, or 0 =
Z25 is P, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=0)CH3, -
0
(CH2)(12-20)CH3
C(0)CH2CH=CH2, 0
0 HO 0
N k.2/(l2_20)CH3
0
0 -
(CH2)(12-20)CH3
0
0
HO 00
N-k(CF12)(12-20)CH3
0 0
Br oN mAb
, or ,
wherein said
mAb is optionally substituted through another thioether bond to a second
compound of
Formula I, so that there are two identical compounds of Formula I on the mAb;
Z26 is I, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=0)CH3, -
0
(CH2)(12-20)CH3
C(0)CH2CH=CH2, 0
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0 0
0
0
kjON (CH2)(12-20)CH3
-
0
HO 0
0 0
N)1.--(CF12)(12-20)CH3
Br s/mAb
, or , wherein said
mAb is optionally substituted through another thioether bond to a second
compound of
Formula I, so that there are two identical compounds of Formula I on the mAb;
X is ATZ1oZ11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34 (SEQ ID NO: 54), or
VLGRLSQELEIRLQTYPRTNTGS (SEQ ID NO: 55);
Z29 is P, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=0)CH3, -
0
)0
(CH2)(12-20)CH3
C(0)CH2CH=CH2, 0
0 HO 0
)=0c)N
N (=--2)(12_20)0H3
0
0 -
0--"\---N \Tr (cH2)(12_20,cH3
- (3-24)
0
HO 0
0 0
NA"(CH2)(12-20)CH3
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--.01\11rBr oN mAb
, or , wherein said
mAb is optionally substituted through another thioether bond to a second
compound of
Formula I, so that there are two identical compounds of Formula I on the mAb;
Z34 is S, or K, wherein the 6-amine of said K is optionally substituted
with¨C(=0)CH3, -
0
(CH)( 2-20)C H3
C(0)CH2CH=CH2, 0 2i
0 0
).=0c)N
N k,-..2)(12-20)CH3
0
(CH2)(12-2o)CH3
- (3-24-)Thr
0
0
HO 0
H Nit(CH
2)(12-20)CH3
0 - 0
Br oN mAb
, or , wherein said
mAb is optionally substituted through another thioether bond to a second
compound of
Formula I, so that there are two identical compounds of Formula I on the mAb;
and pharmaceutically acceptable salts thereof.
[0008] In certain embodiments, the invention is represented by a compound of
Formula
I or a derivative thereof, wherein:
n is 1, or 2;
Z2 is a direct bond, or serine;
' =
Z4 iS T, or ''OHC)
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H H H
N kN µ35..N µkN
Z5 is A, f3-alanine, 0 , 0 , 0 , z 0
H
cinrµ /rµc
0 0 , or ;
kN(
'''01-1 =
Z6 is T, or
Zio is Q, or E;
Zii is R, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=NH)NH2;
$
N
Z12 is L, or 0 =
Z16 is L;
Z25 is P, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=0)CH3,
0
21I
C(0)CH2CH=CH2, 0
HO 0
H
N)L(CH2)14CH3 Br,
0 mAb , or
0 -
- 12 0 , wherein said mAb is
optionally substituted through
another thioether bond to a second compound of Formula I, so that there are
two identical
compounds of Formula I on the mAb;
Z26 is I, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=0)CH3, -
0
21I
C(0)CH2CH=CH2, 0
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HO 0
N 2
N)L(CH2)14CH3 1r Br,
0
0 -
)0N1r
- 12 or 0 , wherein said mAb is optionally substituted
through
another thioether bond to a second compound of Formula I, so that there are
two identical
compounds of Formula I on the mAb;
X is ATZ10Z11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34 (SEQ ID NO: 54), or
VLGRLSQELEIRLQTYPRTNTGS (SEQ ID NO: 55);
Z29 is P, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=0)CH3, -
0
(CF12)14CH3
C(0)CH2CH=CH2, 0
H
N 2
N)L"(CF12)14CH3 Br,
12 0
0
0 -
)0N1r mAb
- 12 10 or 0 , wherein said mAb is optionally substituted
through
another thioether bond to a second compound of Formula I, so that there are
two identical
compounds of Formula I on the mAb;
Z34 is S, or K, wherein the 6-amine of said K is optionally substituted
with¨C(=0)CH3, -
0
N =-=;ir (CF12)14CH3
C(0)CH2CH=CH2, 0
0 0 0
N
N )L(C 15 F12)14CH3 1r Br,
20
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0 -
II H mAb
- or 120 , wherein said mAb is optionally substituted
through
another thioether bond to a second compound of Formula I, so that there are
two identical
compounds of Formula I on the mAb;
and pharmaceutically acceptable salts thereof.
[0009] In certain embodiments, the invention is represented by a compound of
Formula
I or a derivative thereof, wherein:
n is 1, or 2;
Z2 is a direct bond;
Z4 is T, or '''OH =
H H
1/2.N.rµ= µ30Ar41-
Z5 is f3-alanine, 0 , 0 , or 0 ;
Z6 is T;
Zio is Q, or E;
Zii is R, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=NH)NH2;
N
Z12 iS L, or 0 =
Z16 is L;
Z25 is P, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=0)CH3, -
0
- 2 I
C(0)CH2CH=CH2, 0
:_::x0
0 0 0
N )L(C H2)14C H3 k).L'O ).r Br
, or
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0 -
mAb
k)ONIrS
- 12 0 , wherein said mAb is optionally substituted
through
another thioether bond to a second compound of Formula I, so that there are
two identical
compounds of Formula I on the mAb;
Z26 is I, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=0)CH3,
0
21I
C(0)CH2CH=CH2, 0
H
N
(CH)iCH 0NBr,
- 12
20 0
0
mAb
µ')01\11-rs
- 12
or 0 , wherein said mAb is optionally substituted
through
another thioether bond to a second compound of Formula I, so that there are
two identical
compounds of Formula I on the mAb;
X is ATZ1oZ11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34 (SEQ ID NO: 54), or
VLGRLSQELEIRLQTYPRTNTGS (SEQ ID NO: 55);
Z29 is P, or K, wherein the 6-amine of said K is optionally substituted with
¨C(=0)CH3, -
0
(CE12)14CH3
21I
C(0)CH2CH=CH2, 0
HO 0
N 2
N)L(CF12)14CH3 0N1.r Br,
0
0
mAb
N 1rS
- 12
or 0 , wherein said mAb is optionally substituted
through
another thioether bond to a second compound of Formula I, so that there are
two identical
compounds of Formula I on the mAb;
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Z34 is S, or K, wherein the 6-amine of said K is optionally substituted
with¨C(=0)CH3, -
0
- 2 I
C(0)CH2CH=CH2, 0
HO 0
0 0 0
)0c)N
2 N (CH2)14CH3 lr Br,
0
0
mAb
- or 120 , wherein said mAb is optionally substituted
through
another thioether bond to a second compound of Formula I, so that there are
two identical
compounds of Formula I on the mAb;
and pharmaceutically acceptable salts thereof.
[0010] In certain embodiments, the invention is represented by a compound of
Formula
I or a derivative thereof, wherein:
n is 1, or 2;
Z2 is a direct bond, or serine;
r`z2z-.
'''OH =
Z4 iS T, or
H H
N kN ,IceN Ar'N. µkN
Z5 is A, f3-alanine, 0 , 0 , 0 , z 0
H
õ -
0 0 , or NO
kN
Z6 is T, or ."OHC) =
Zio is Q, or E;
Zii is R, or K, wherein the 6-amine of said K is substituted with ¨C(=NH)NH2;
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SN
Z12 1S L, or 0 =
Z16 is L;
Z25 is P, or K, wherein the 6-amine of said K is substituted with ¨C(=0)CH3,
or
0
II H mAb
)0N1r
- 12 0
, wherein said mAb is substituted through another
thioether bond to a second compound of Formula I, so that there are two
identical
compounds of Formula I on the mAb;
Z26 is I, or K, wherein the 6-amine of said K is substituted with ¨C(=0)CH3, -
-0
21I
C(0)CH2CH=CH2, 0
HO 0
H
2 N kL'H2)14CI-13
)r Br,
- 12
0 0
0 -
mAb
k)ONIr
- 10 or 120 , wherein said mAb is substituted
through another
thioether bond to a second compound of Formula I, so that there are two
identical
compounds of Formula I on the mAb;
X is ATZ1oZ11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34 (SEQ ID NO: 54), or
VLGRLSQELEIRLQTYPRTNTGS (SEQ ID NO: 55);
Z29 is P, or K, wherein the 6-amine of said K is substituted with ¨C(=0)CH3;
Z34 is S, or K, wherein the 6-amine of said K is substituted with ¨C(=0)CH3;
and pharmaceutically acceptable salts thereof.
[0011] In certain embodiments, the invention is represented by a compound of
Formula
I or a derivative thereof, wherein:
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n is 1, or 2;
Z2 is a direct bond;
'''01-1 =
Z4 is T, or
H H
SN=r\-- 1/2.N.rµ= µ30Ar41-
Z5 is f3-alanine, 0 , 0 ,or 0 ;
Z6 is T;
Zio is Q, or E;
Zii is R, or K, wherein the 6-amine of said K is substituted with ¨C(=NH)NH2;
Z12 iS L, or 0 =
Z16 is L;
Z25 is P, or K, wherein the 6-amine of said K is substituted with ¨C(=0)CH3,
or
0 -
mAb
k)01\11rs
- 12 0
, wherein said mAb is substituted through another
thioether bond to a second compound of Formula I, so that there are two
identical
compounds of Formula I on the mAb;
Z26 is I, or K, wherein the 6-amine of said K is substituted with ¨C(=0)CH3, -
-0
N--..,õ(CH2)14CH3
- 2 I
C(0)CH2CH=CH2, 0
HO 0
)c(30N 2
N)L(CH2)14CH3 k)'0 N )r Br,
0
0 -
mAb
k)C01\')rs
- or 120 , wherein said mAb is substituted through
another
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thioether bond to a second compound of Formula I, so that there are two
identical
compounds of Formula I on the mAb;
X is ATZ1oZ11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34 (SEQ ID NO: 54), or
VLGRLSQELEIRLQTYPRTNTGS (SEQ ID NO: 55);
Z29 is P, or K, wherein the 6-amine of said K is substituted with ¨C(=0)CH3;
Z34 is S, or K, wherein the 6-amine of said K is substituted with ¨C(=0)CH3;
and pharmaceutically acceptable salts thereof.
[0012] In certain embodiments, the invention is represented by a compound of
Formula
I or a derivative thereof, wherein:
n is 1, or 2;
Z2 is a direct bond, or serine;
'''OHC) =
Z4 iS T, or
H H
N k N .iµ2za! ,35,N
Z5 is A, f3-alanine, 0 , 0 , 0 , z 0
H
N
,
0 0 , or
N
' =
Z6 is T, or ''01-1
Zio is Q, or E;
Zii is R, or K, wherein the 6-amine of said K is substituted with ¨C(=NH)NH2;
. H
Z12 1S 0 =
Z16 is L;
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Z25 is P, or K, wherein the 6-amine of said K is substituted with ¨C(=0)CH3,
or
0 -
mAb
k)01\11-r`s
- 12 0 , wherein said mAb is substituted through
another
thioether bond to a second compound of Formula I, so that there are two
identical
compounds of Formula I on the mAb;
Z26 is I, or K, wherein the 6-amine of said K is substituted with ¨C(=0)CH3, -
-0
21I
C(0)CH2CH=CH2, 0
HO 0
)0c)N 2
N)L(CH2)14CH3 0N
Br,
0
0 -
mAb
)0N)r
- 12
or 0 , wherein said mAb is substituted through
another
thioether bond to a second compound of Formula I, so that there are two
identical
compounds of Formula I on the mAb;
X is ATZ10Z11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34 (SEQ ID NO: 54), or
VLGRLSQELEIRLQTYPRTNTGS (SEQ ID NO: 55);
Z29 is P, or K, wherein the 6-amine of said K is substituted with ¨C(=0)CH3;
Z34 is S, or K, wherein the 6-amine of said K is substituted with ¨C(=0)CH3;
and pharmaceutically acceptable salts thereof.
[0013] In certain embodiments, the compound is selected from the group
consisting of
SEQ ID NOs: 4-42, or a pharmaceutically acceptable salt thereof.
[0014] In certain embodiments, the monoclonal antibody or the antigen binding
fragment thereof is covalently linked to the amylinomimetic peptide at a
lysine residue of
the amylinomimetic peptide via a linker. Non-limiting examples of the linker
comprise
aa PEG chain of 2-24 PEG units, (0EG(o-4)-y-Glu), (0EG(1-4)), or an alkyl
chain
containing 2-10 carbon atoms, wherein said linker may include a group, such as
but not
limited to, acetyl.
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[0015] In certain embodiments, only one of Z25, Z26, Z29 and Z34 in Formula I
is lysine,
and the lysine is covalently linked to an engineered cysteine residue of the
monoclonal
antibody or the antigen binding fragment thereof via the linker.
[0016] Another embodiment of the invention is a pharmaceutical composition
comprising the compound of Formula I, or a compound selected from the group
consisting of SEQ ID NOs:4-42, and a pharmaceutically acceptable carrier.
[0017] In certain embodiments, the monoclonal antibody or the antigen binding
fragment thereof comprises a heavy chain complementarity determining region 1
(HCDR1), HCDR2, HCDR3, and a light chain complementarity determining region 1
(LCDR1), LCDR2, and LCDR3, having the polypeptide sequences of SEQ ID NO: 47,
48, 49, 50, 51, and 52, respectively. In certain embodiments, the isolated
monoclonal
antibody comprises a heavy chain variable domain (VH) having the polypeptide
sequence
of SEQ ID NO:43, and a light chain variable domain (VL) having the polypeptide
sequence of SEQ ID NO:45. In certain embodiments, the isolated monoclonal
antibody
further comprises a Fc portion. In certain embodiments, the isolated
monoclonal
antibody comprises a heavy chain (HC) having the polypeptide sequence of SEQ
ID
NO:44 and a light chain (LC) having the polypeptide sequence of SEQ ID NO:46.
[0018] Also provided are conjugates comprising a monoclonal antibody or an
antigen
binding fragment thereof coupled to a amylinomimetic peptide, wherein the
monoclonal
antibody or the antigen binding fragment thereof comprises a heavy chain
complementarity determining region 1 (HCDR1), HCDR2, HCDR3, and a light chain
complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, having the
polypeptide sequences of SEQ ID NO: 47, 48, 49, 50, 51, and 52, respectively,
preferably
the monoclonal antibody or antigen binding fragment thereof comprises a heavy
chain
variable domain (VH) having the polypeptide sequence of SEQ ID NO:43, and a
light
chain variable domain (VL) having the polypeptide sequence of SEQ ID NO:45,
and
more preferably, the monoclonal antibody a heavy chain (HC) having the
polypeptide
sequence of SEQ ID NO:44 and a light chain (LC) having the polypeptide
sequence of
SEQ ID NO:46; the amylinomimetic peptide comprises a polypeptide sequence
selected
from the group consisting of SEQ ID NOs: 4-28, or a pharmaceutically
acceptable salt
thereof; and the monoclonal antibody or antigen binding fragment thereof is
conjugated
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to the amylinomimetic peptide at residue 25, 26, 29, or 34 of the
amylinomimetic peptide,
preferably at lysine residue 25 or 26 of the amylinomimetic peptide, directly
or via a
linker.
[0019] Also provided are methods of producing the conjugates of the invention.
The
methods comprise reacting an electrophile, preferably bromoacetamide
introduced onto a
sidechain of the amylinomimetic peptide, or a linker on said sidechain,
preferably the
sidechain of a lysine residue of the amylinomimetic peptide, with the
sulfhydryl group of
the cysteine residue of SEQ ID NO:49 of the monoclonal antibody or antigen-
binding
fragment thereof, thereby creating a covalent linkage between the
amylinomimetic
peptide and the monoclonal antibody or antigen-binding fragment thereof.
[0020] Also provided are pharmaceutical compositions comprising the conjugates
of
the invention and a pharmaceutically acceptable carrier.
[0021] Also provided are methods for treating or preventing a disease or
disorder in a
subject in need thereof, wherein said disease or disorder is selected from the
group
consisting of obesity, type I or type II diabetes, metabolic syndrome, insulin
resistance,
impaired glucose tolerance, hyperglycemia, hyperinsulinemia,
hypertriglyceridemia,
hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia,
atherosclerosis,
diabetic nephropathy, and other cardiovascular risk factors such as
hypertension and
cardiovascular risk factors related to unmanaged cholesterol and/or lipid
levels,
osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-
alcoholic
steatohepatitis (NASH), renal disease, and eczema. The methods comprise
administering
to the subject in need thereof an effective amount of the pharmaceutical
compositions of
the invention.
[0022] Also provided are methods of reducing food intake in a subject in need
thereof.
The methods comprise administering to the subject in need thereof an effective
amount of
the pharmaceutical composition of the invention.
[0023] Also provided are methods of modulating amylin receptor activity in a
subject
in need thereof. The methods comprise administering to the subject in need
thereof an
effective amount of the pharmaceutical composition of the invention.
[0024] Also provided are methods of modulating amylin receptor activity in a
subject
in need thereof, wherein said amylin receptor comprises AMY1R, and/or AMY2R
and/or
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AMY3R. The methods comprise administering to the subject in need thereof an
effective
amount of the pharmaceutical composition of the invention.
[0025] Also provided are methods of modulating amylin receptor activity in a
subject
in need thereof, wherein said amylin receptor is AMY1R.
[0026] Also provided are methods of modulating amylin receptor activity in a
subject
in need thereof, wherein said amylin receptor is AMY3R.
[0027] The methods comprise administering to the subject in need thereof an
effective
amount of the pharmaceutical composition of the invention.
[0028] In certain embodiments, the pharmaceutical composition is administered
via an
injection. In certain embodiments, the pharmaceutical composition is
administered in a
combination with at least one antidiabetic agent. The antidiabetic agent can,
for example,
be a glucagon-like-peptide-1 receptor modulator. In certain embodiments, the
pharmaceutical composition is administered in combination with liraglutide.
[0029] Also provided are kits comprising the conjugates of the invention,
preferably
further comprising a liraglutide and a device for injection.
[0030] Also provided are methods of producing the pharmaceutical compositions
of the
invention. The methods comprise combining the conjugate with a
pharmaceutically
acceptable carrier to obtain the pharmaceutical composition.
[0031] Further aspects, features and advantages of the present invention will
be better
appreciated upon a reading of the following detailed description of the
invention and
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] The foregoing summary, as well as the following detailed description of
preferred embodiments of the present application, will be better understood
when read in
conjunction with the appended drawings. It should be understood, however, that
the
application is not limited to the precise embodiments shown in the drawings.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Various publications, articles and patents are cited or described in
the
background and throughout the specification; each of these references is
herein
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incorporated by reference in its entirety. Discussion of documents, acts,
materials,
devices, articles or the like which has been included in the present
specification is for the
purpose of providing context for the invention. Such discussion is not an
admission that
any or all of these matters form part of the prior art with respect to any
inventions
disclosed or claimed.
[0034] Unless defined otherwise, all technical and scientific terms used
herein have the
same meaning as commonly understood to one of ordinary skill in the art to
which this
invention pertains. Otherwise, certain terms used herein have the meanings as
set forth
in the specification.
.. [0035] It must be noted that as used herein and in the appended claims, the
singular
forms "a," "an," and "the" include plural reference unless the context clearly
dictates
otherwise.
[0036] Unless otherwise stated, any numerical values, such as a concentration
or a
concentration range described herein, are to be understood as being modified
in all
instances by the term "about." Thus, a numerical value typically includes
10% of the
recited value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to
1.1
mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v)
to
11% (w/v). As used herein, the use of a numerical range expressly includes all
possible
subranges, all individual numerical values within that range, including
integers within
such ranges and fractions of the values unless the context clearly indicates
otherwise.
[0037] Unless otherwise indicated, the term "at least" preceding a series of
elements is
to be understood to refer to every element in the series. Those skilled in the
art will
recognize, or be able to ascertain using no more than routine experimentation,
many
equivalents to the specific embodiments of the invention described herein.
Such
equivalents are intended to be encompassed by the invention.
[0038] As used herein, the terms "comprises," "comprising," "includes,"
"including,"
"has," "having," "contains" or "containing," or any other variation thereof,
will be
understood to imply the inclusion of a stated integer or group of integers but
not the
exclusion of any other integer or group of integers and are intended to be non-
exclusive
.. or open-ended. For example, a composition, a mixture, a process, a method,
an article, or
an apparatus that comprises a list of elements is not necessarily limited to
only those
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elements but can include other elements not expressly listed or inherent to
such
composition, mixture, process, method, article, or apparatus. Further, unless
expressly
stated to the contrary, "or" refers to an inclusive or and not to an exclusive
or. For
example, a condition A or B is satisfied by any one of the following: A is
true (or
present) and B is false (or not present), A is false (or not present) and B is
true (or
present), and both A and B are true (or present).
[0039] It should also be understood that the terms "about," "approximately,"
"generally," "substantially" and like terms, used herein when referring to a
dimension or
characteristic of a component of the preferred invention, indicate that the
described
dimension/ characteristic is not a strict boundary or parameter and does not
exclude
minor variations therefrom that are functionally the same or similar, as would
be
understood by one having ordinary skill in the art. At a minimum, such
references that
include a numerical parameter would include variations that, using
mathematical and
industrial principles accepted in the art (e.g., rounding, measurement or
other systematic
errors, manufacturing tolerances, etc.), would not vary the least significant
digit.
[0040] The terms "identical" or percent "identity," in the context of two or
more nucleic
acids or polypeptide sequences (e.g., amylinomimetic3-36 polypeptide
sequences, antibody
light chain or heavy chain sequences), refer to two or more sequences or
subsequences
that are the same or have a specified percentage of amino acid residues or
nucleotides
that are the same, when compared and aligned for maximum correspondence, as
measured using one of the following sequence comparison algorithms or by
visual
inspection using methods known in the art in view of the present disclosure.
[0041] For sequence comparison, typically one sequence acts as a reference
sequence,
to which test sequences are compared. When using a sequence comparison
algorithm,
test and reference sequences are input into a computer, subsequence
coordinates are
designated, if necessary, and sequence algorithm program parameters are
designated.
The sequence comparison algorithm then calculates the percent sequence
identity for the
test sequence(s) relative to the reference sequence, based on the designated
program
parameters.
[0042] Optimal alignment of sequences for comparison can be conducted, e.g.,
by the
local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981),
by the
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homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443
(1970), by
the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci.
USA
85:2444 (1988), by computerized implementations of these algorithms (GAP,
BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer
Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally,
Current
Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols,
a joint
venture between Greene Publishing Associates, Inc. and John Wiley & Sons,
Inc., (1995
Supplement) (Ausubel)).
[0043] Examples of algorithms that are suitable for determining percent
sequence
identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which
are
described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et
al. (1997)
Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST
analyses is publicly available through the National Center for Biotechnology
Information.
[0044] A further indication that two nucleic acid sequences or polypeptides
are
substantially identical is that the polypeptide encoded by the first nucleic
acid is
immunologically cross reactive with the polypeptide encoded by the second
nucleic acid,
as described below. Thus, a polypeptide is typically substantially identical
to a second
polypeptide, for example, where the two peptides differ only by conservative
substitutions. Another indication that two nucleic acid sequences are
substantially
identical is that the two molecules hybridize to each other under stringent
conditions.
[0045] As used herein, "subject" means any animal, preferably a mammal, most
preferably a human. The term "mammal" as used herein, encompasses any mammal.
Examples of mammals include, but are not limited to, cows, horses, sheep,
pigs, cats,
dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably
a human.
[0046] The term "administering" with respect to the methods of the invention,
means a
method for therapeutically or prophylactically preventing, treating or
ameliorating a
syndrome, disorder or disease as described herein by using a conjugate of the
invention
or a form, composition or medicament thereof. Such methods include
administering an
effective amount of said conjugate, a form, composition or medicament thereof
at
different times during the course of a therapy or concurrently in a
combination form. The
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methods of the invention are to be understood as embracing all known
therapeutic
treatment regimens.
[0047] The term "effective amount" means that amount of active conjugate or
pharmaceutical agent that elicits the biological or medicinal response in a
tissue system,
animal or human, that is being sought by a researcher, veterinarian, medical
doctor, or
other clinician, which includes preventing, treating or ameliorating a
syndrome, disorder,
or disease being treated, or the symptoms of a syndrome, disorder or disease
being
treated.
[0048] As used herein, the term "composition" is intended to encompass a
product
comprising the specified ingredients in the specified amounts, as well as any
product
which results, directly or indirectly, from combinations of the specified
ingredients in the
specified amounts.
[0049] As used herein the term "coupled" refers to the joining or connection
of two or
more objects together. When referring to chemical or biological compounds,
coupled can
refer to a covalent connection between the two or more chemical or biological
compounds. By way of a non-limiting example, an antibody of the invention can
be
coupled with a peptide of interest (e.g., amylinomimetic peptides of the
invention) to
form an antibody coupled peptide. In certain embodiments, an antibody of the
invention
can be covalently coupled with a peptide of the invention through a linker.
The linker
can, for example, be first covalently connected to the antibody or the
peptide, then
covalently connected to the peptide or the antibody. An antibody coupled
peptide can be
formed through specific chemical reactions designed to conjugate the antibody
to the
peptide. By way of an example, a mAb coupled amylinomimetic peptide conjugate
can
be formed through a conjugation reaction. The conjugation reaction can, for
example,
comprise reacting an electrophilic group (e.g., a bromoacetamide or a
maleimide) with
the sulfhydryl group of a cysteine residue on the mAb. The electrophilic group
can, for
example, be introduced onto a sidechain of an amino acid residue of an
amylinomimetic
peptide. The reaction of the electrophilic group with the sulfhydryl group
results in the
formation of a covalent thioether bond.
[0050] As used herein, the term "linker" refers to a chemical module
comprising a
covalent or atomic chain that covalently attaches an antibody to the peptide.
The linker
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can, for example, include, but is not limited to, a peptide linker, a
hydrocarbon linker, a
polyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker, a
polysaccharide
linker, a polyester linker, a hybrid linker consisting of PEG and an embedded
heterocycle, and a hydrocarbon chain.
[0051] As used herein, the term "conjugate" refers to an antibody or a
fragment thereof
covalently coupled to a pharmaceutically active moiety. The term "conjugated
to" refers
to an antibody or a fragment thereof of invention covalently linked to or
covalently
connected to a pharmaceutically active moiety, preferably a therapeutic
peptide, directly
or indirectly via a linker. By way of a non-limiting example, the antibody can
be a
monoclonal antibody of the invention and the pharmaceutically active moiety
can be a
therapeutic peptide, such as a amylinomimetic peptide of interest.
[0052] Antibodies
[0053] As used herein, the term "non-targeting" in the context of an antibody
refers to
an antibody that does not specifically bind to any target in vivo. As used
herein, an
antibody that "specifically binds to a target" refers to an antibody that
binds to a target
antigen, with a KD of 1 x10-8 M or less, preferably 5x10-9 M or less, 1x10-9 M
or less,
Sx 10-1 M or less, or 1x10-10 M or less. The term "KD" refers to the
dissociation
constant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is
expressed as a
molar concentration (M). KD values for antibodies can be determined using
methods in
the art in view of the present disclosure. For example, the KD of an antibody
can be
determined by using surface plasmon resonance, such as by using a biosensor
system,
e.g., a Biacore system, or by using bio-layer interferometry technology, such
as a Octet
RED96 system. The smaller the value of the KD of an antibody, the higher
affinity that
the antibody binds to a target antigen.
[0054] Monoclonal antibodies, complete or a fragment thereof, can be used as a
half-
life extending moiety. Monoclonal antibodies are well-studied proteins that
have been
utilized and characterized for uses in vivo, and as such, the mechanisms that
enable their
protracted half-life in vivo and the mechanisms for their elimination in vivo
are well
understood. Additionally, the spatial separation and presentation of the two
"arms" of the
monoclonal antibody can be advantageous for effective bivalent presentation of
a
therapeutic moiety (i.e., a therapeutic peptide). Therapeutics in which toxins
or other
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small molecule drugs are chemically linked to a monoclonal antibody have been
developed but typically utilize a monoclonal antibody that binds to a specific
antigen and
targets the antibody-drug conjugate to a tissue/cell of interest, which
preferentially
expressed the antigen, and typically the drug/small molecule is attached to
the antibody in
a manner that does not impact antigen binding of the antibody.
[0055] For therapeutic peptide-mAb conjugates, antigen specific binding by the
half-
life extending monoclonal antibody is not desired. Because of this, a heavy
chain (HC)
and light chain (LC) variable (V) domain pair not expected to specifically
bind any target
are used for preparing the coupling-enabled, non-targeting monoclonal antibody
of the
invention. To obtain a coupling-enabled, non-targeting monoclonal antibody, a
cysteine
residue is engineered into one of the complementarity determining regions
(CDRs) of a
selected non-targeting antibody. The pharmaceutically active moiety (e.g.,
therapeutic
peptide/compound) can contain the appropriate chemical moiety to allow for the
conjugation of the pharmaceutically active moiety to the engineered cysteine
residue of
the non-targeting monoclonal antibody.
[0056] The term "antibodies" as used herein is meant in a broad sense and
includes
non-human (e.g., murine, rat), human, human-adapted, humanized and chimeric
monoclonal antibodies, antibody fragments, bispecific or multispecific
antibodies,
dimeric, tetrameric or multimeric antibodies, and single chain antibodies.
[0057] Antibody light chains of any vertebrate species can be assigned to one
of two
clearly distinct types, namely kappa (K) and lambda (X), based on the amino
acid
sequences of their constant domains. Accordingly, the antibodies of the
invention can
contain a kappa or lambda light chain constant domain. According to particular
embodiments, the antibodies of the invention include heavy and/or light chain
constant
regions from mouse or human antibodies. In addition to the heavy and light
constant
domains, antibodies contain an antigen-binding region that is made up of a
light chain
variable region and a heavy chain variable region, each of which contains
three domains
(i.e., complementarity determining regions 1-3; (CDR1, CDR2, and CDR3)). The
light
chain variable region domains are alternatively referred to as LCDR1, LCDR2,
and
LCRD3, and the heavy chain variable region domains are alternatively referred
to as
HCDR1, HCRD2, and HCDR3.
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[0058] Immunoglobulins can be assigned to five major classes, namely IgA, IgD,
IgE,
IgG and IgM, depending on the heavy chain constant domain amino acid sequence.
IgG
is the most stable of the five types of immunoglobulins, having a serum half-
life in
humans of about 23 days. IgA and IgG are further sub-classified as the
isotypes IgAi,
IgA2, IgGi, IgG2, IgG3 and IgG4. Each of the four IgG subclasses has different
biological
functions known as effector functions. These effector functions are generally
mediated
through interaction with the Fc receptor (FcyR) or by binding Clq and fixing
complement. Binding to FcyR can lead to antibody dependent cell mediated
cytolysis,
whereas binding to complement factors can lead to complement mediated cell
lysis. An
antibody of the invention utilized for its ability to extend half-life of a
therapeutic peptide
has no or minimal effector function, but retains its ability to bind FcRn, the
binding of
which can be a primary means by which antibodies have an extended in vivo half-
life.
[0059] In certain embodiments, the invention relates to a conjugate comprising
an
isolated antibody or antigen binding fragment thereof comprising a light chain
variable
region having completely human Ig germline V gene sequences, and a heavy chain
variable region having completely human Ig germline V gene sequences except
HCDR3
having the amino acid sequence of SEQ ID NO:49 and a pharmaceutically active
moiety
(e.g., a amylinomimetic peptide of the invention) conjugated thereto, wherein
the
antibody or antigen binding fragment thereof does not specifically bind to any
human
antigen in vivo. In the present disclosure, with respect to an antibody or
antigen binding
fragment thereof according to an embodiment of the invention, the phrase "a
conjugate
comprising an antibody or antigen binding fragment thereof and a
pharmaceutically
active moiety conjugated thereto" is used interchangeably with the phrase "an
antibody or
antigen binding fragment thereof conjugated to a pharmaceutically active
moiety."
[0060] As used herein, the term "antigen-binding fragment" refers to an
antibody
fragment such as, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fy
fragment, a
disulfide stabilized Fy fragment (dsFv), a (dsFv)2, a bispecific dsFy (dsFy-
dsFy'), a
disulfide stabilized diabody (ds diabody), a single-chain antibody molecule
(scFv), a
single domain antibody (sdab) an scFy dimer (bivalent diabody), a
multispecific antibody
formed from a portion of an antibody comprising one or more CDRs, a camelized
single
domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or
any
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other antibody fragment that binds to an antigen but does not comprise a
complete
antibody structure. An antigen-binding fragment is capable of binding to the
same
antigen to which the parent antibody or a parent antibody fragment binds.
According to
particular embodiments, the antigen-binding fragment comprises a light chain
variable
region, a light chain constant region, and an Fd segment (i.e., portion of the
heavy chain
which is included in the Fab fragment). According to other particular
embodiments, the
antigen-binding fragment comprises Fab and F(ab').
[0061] As used herein, the term "single-chain antibody" refers to a
conventional single-
chain antibody in the field, which comprises a heavy chain variable region and
a light
chain variable region connected by a short peptide of about 15 to about 20
amino acids.
As used herein, the term "single domain antibody" refers to a conventional
single domain
antibody in the field, which comprises a heavy chain variable region and a
heavy chain
constant region or which comprises only a heavy chain variable region.
[0062] The phrase "isolated antibody or antibody fragment" refers to an
antibody or
antibody fragment that is substantially free of other antibodies having
different antigenic
specificities (e.g., an isolated antibody specifically binding a target
antigen is
substantially free of antibodies that specifically do not bind the target
antigen). Moreover,
an isolated antibody or antibody fragment can be substantially free of other
cellular
material and/or chemicals.
[0063] An antibody variable region consists of a "framework" region
interrupted by
three "antigen binding sites". The antigen binding sites are defined using
various terms:
(i) Complementarity Determining Regions (CDRs), three in the VH (HCDR1, HCDR2,
HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3), are based on sequence
variability (Wu and Kabat J Exp Med 132:211-50, 1970; Kabat et al Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service, National
Institutes of
Health, Bethesda, Md., 1991). (ii) "Hypervariable regions," "HVR," or "HV,"
three in the
VH (H1, H2, H3) and three in the VL (L1, L2, L3), refer to the regions of an
antibody
variable domains which are hypervariable in structure as defined by Chothia
and Lesk
(Chothia and Lesk Mol Biol 196:901-17, 1987). Other terms include "IMGT-CDRs"
(Lefranc et al., Dev Comparat Immunol 27:55-77, 2003) and "Specificity
Determining
Residue Usage" (SDRU) (Almagro Mol Recognit 17:132-43, 2004). The
International
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ImMunoGeneTics (IMGT) database (http://www mgt org) provides a standardized
numbering and definition of antigen-binding sites. The correspondence between
CDRs,
HVs and IMGT delineations is described in Lefranc et al., Dev Comparat Immunol
27:55-77, 2003.
[0064] "Framework" or "framework sequences" are the remaining sequences of a
variable region other than those defined to be antigen binding sites. Because
the antigen
binding sites can be defined by various terms as described above, the exact
amino acid
sequence of a framework depends on how the antigen-binding site was defined.
[0065] In one embodiment of the invention, an isolated antibody or antigen
binding
fragment thereof comprises a light chain variable region having the LCDR1,
LCDR2 and
LCDR3 of the amino acid sequence of SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID
NO: 52, respectively, and a heavy chain variable region having the HCDR1,
HCDR2 and
HCDR3 of the amino acid sequences of SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID
NO: 49, respectively.
[0066] In another embodiment, the isolated antibody further comprises a Fc
region
derived from human IgG4 Fc region. Human IgG4 Fc region has reduced ability to
bind
FcyR and complement factors compared to other IgG sub-types. Preferably, the
Fc region
contains human IgG4 Fc region having substitutions that eliminate effector
function.
Thus, an isolated antibody further comprises a Fc region having a modified
human IgG4
Fc region containing one or more of the following substitutions: substitution
of proline
for glutamate at residue 233, alanine or valine for phenylalanine at residue
234 and
alanine or glutamate for leucine at residue 235 (EU numbering, Kabat, E. A. et
al. (1991)
Sequences of Proteins of Immunological Interest, 5th Ed. U.S. Dept. of Health
and Human
Services, Bethesda, Md., NTH Publication no. 91-3242). Removing the N-linked
glycosylation site in the IgG4 Fc region by substituting Ala for Asn at
residue 297 (EU
numbering) is another way to ensure that residual effector activity is
eliminated.
[0067] Preferably, an antibody of the invention can exist as a dimer joined
together by
disulfide bonds and various non-covalent interactions. Thus, the Fc portion
useful for the
antibody of the invention can be human IgG4 Fc region containing a
substitution, such as
serine to proline at position at 228 (EU numbering), that stabilizes heavy
chain dimer
formation and prevents the formation of half-IgG4 Fc chains.
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[0068] In another embodiment, the C-terminal Lys residue in the heavy chain is
removed, as commonly seen in recombinantly produced monoclonal antibodies.
[0069] "Human antibody" refers to an antibody having heavy and light chain
variable
regions in which both the framework and the antigen binding sites are derived
from
sequences of human origin. If the antibody contains a constant region, the
constant region
also is derived from sequences of human origin.
[0070] Human antibody comprises heavy or light chain variable regions that are
"derived from" sequences of human origin if the variable regions of the
antibody are
obtained from a system that uses human germline immunoglobulin or rearranged
immunoglobulin genes. Such systems include human immunoglobulin gene libraries
displayed on phage, and transgenic non-human animals such as mice carrying
human
immunoglobulin loci as described herein. "Human antibody" may contain amino
acid
differences when compared to the human germline or rearranged immunoglobulin
sequences due to for example naturally occurring somatic mutations or
intentional
introduction of substitutions in the framework or antigen binding sites.
Typically,
"human antibody" is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in
amino acid sequence to an amino acid sequence encoded by a human germline or
rearranged immunoglobulin gene. In some cases, "human antibody" may contain
consensus framework sequences derived from human framework sequence analyses,
for
example as described in Knappik et al., J Mol Biol 296:57-86, 2000), or
synthetic
HCDR3 incorporated into human immunoglobulin gene libraries displayed on
phage, for
example as described in Shi et al., J Mol Biol 397:385-96, 2010 and Intl. Pat.
Publ. No.
W02009/085462). Antibodies in which antigen binding sites are derived from a
non-
human species are not included in the definition of "human antibody".
[0071] Isolated humanized antibodies may be synthetic. Human antibodies, while
derived from human immunoglobulin sequences, may be generated using systems
such as
phage display incorporating synthetic CDRs and/or synthetic frameworks, or can
be
subjected to in vitro mutagenesis to improve antibody properties, resulting in
antibodies
that do not naturally exist within the human antibody germline repertoire in
vivo.
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[0072] The term "recombinant antibody" as used herein, includes all antibodies
that are
prepared, expressed, created or isolated by recombinant means, such as
antibodies
isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal
for human
immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated
from a
.. host cell transformed to express the antibody, antibodies isolated from a
recombinant,
combinatorial antibody library, and antibodies prepared, expressed, created or
isolated by
any other means that involve splicing of human immunoglobulin gene sequences
to other
DNA sequences, or antibodies that are generated in vitro using Fab arm
exchange.
[0073] The term "monoclonal antibody" as used herein refers to a preparation
of
antibody molecules of a single molecular composition. The monoclonal
antibodies of the
invention can be made by the hybridoma method, phage display technology,
single
lymphocyte gene cloning technology, or by recombinant DNA methods. For
example,
the monoclonal antibodies can be produced by a hybridoma which includes a B
cell
obtained from a transgenic nonhuman animal, such as a transgenic mouse or rat,
having a
genome comprising a human heavy chain transgene and a light chain transgene.
[0074] In certain embodiments, the term "mAb" refers to a monoclonal antibody
having a variable heavy chain (VH) sequence comprising SEQ ID NO:43 and a
variable
light chain (VL) sequence comprising SEQ ID NO:45. In certain embodiments the
mAb
is a fully human monoclonal antibody having a heavy chain (HC) sequence
comprising
SEQ ID NO:44 and a light chain (LC) sequence comprising SEQ ID NO:46. In
certain
embodiments, the lysine residue at position 446 of SEQ ID NO:44 is optionally
missing.
[0075] As used herein, the term "chimeric antibody" refers to an antibody
wherein the
amino acid sequence of the immunoglobulin molecule is derived from two or more
species. The variable region of both the light and heavy chains often
corresponds to the
variable region of an antibody derived from one species of mammal (e.g.,
mouse, rat,
rabbit, etc.) having the desired specificity, affinity, and capability, while
the constant
regions correspond to the sequences of an antibody derived from another
species of
mammal (e.g., human) to avoid eliciting an immune response in that species.
[0076] As used herein, the term "multispecific antibody" refers to an antibody
that
comprises a plurality of immunoglobulin variable domain sequences, wherein a
first
immunoglobulin variable domain sequence of the plurality has binding
specificity for a
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first epitope or comprises germline sequences lacking any known binding
specificity and
a second immunoglobulin variable domain sequence of the plurality has binding
specificity for a second epitope or comprises germline sequences lacking any
known
binding specificity, and wherein the first and/or second immunoglobulin
variable domain
optionally include a conjugated pharmaceutically active moiety (e.g., a
therapeutic
peptide). In an embodiment, the first and second epitopes are on the same
antigen, e.g.,
the same protein (or subunit of a multimeric protein). In an embodiment, the
first and
second epitopes overlap or substantially overlap. In an embodiment, the first
and second
epitopes do not overlap or do not substantially overlap. In an embodiment, the
first and
second epitopes are on different antigens, e.g., the different proteins (or
different subunits
of a multimeric protein). In an embodiment, the first and second
immunoglobulin
variable domains include the same conjugated pharmaceutically active moiety.
In an
embodiment, the first and second immunoglobulin variable domains include
different
pharmaceutically active moieties. In an embodiment, only the first
immunoglobulin
variable domain includes a conjugated pharmaceutically active moiety. In an
embodiment, only the second immunoglobulin variable domain includes a
conjugated
pharmaceutically active moiety. In an embodiment, a multispecific antibody
comprises a
third, fourth, or fifth immunoglobulin variable domain. In an embodiment, a
multispecific antibody is a bispecific antibody molecule, a trispecific
antibody, or a
.. tetraspecific antibody molecule.
[0077] As used herein, the term "bispecific antibody" refers to a
multispecific antibody
that binds no more than two epitopes or two antigens and/or comprises two
conjugated
pharmaceutically active moieties (e.g., the same or different pharmaceutically
active
moiety). A bispecific antibody is characterized by a first immunoglobulin
variable
domain sequence which has binding specificity for a first epitope or comprises
germline
sequences lacking any known binding specificity and a second immunoglobulin
variable
domain sequence that has binding specificity for a second epitope or comprises
germline
sequences lacking any known binding specificity, and wherein the first and/or
second
immunoglobulin variable domain optionally include a conjugated
pharmaceutically active
moiety. In an embodiment, the first and second epitopes are on the same
antigen, e.g.,
the same protein (or subunit of a multimeric protein). In an embodiment, the
first and
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second epitopes overlap or substantially overlap. In an embodiment the first
and second
epitopes are on different antigens, e.g., the different proteins (or different
subunits of a
multimeric protein). In an embodiment, the first and second immunoglobulin
variable
domains include the same conjugated pharmaceutically active moiety. In an
embodiment, the first and second immunoglobulin variable domains include
different
pharmaceutically active moieties. In an embodiment, only the first
immunoglobulin
variable domain includes a conjugated pharmaceutically active moiety. In an
embodiment, only the second immunoglobulin variable domain includes a
conjugated
pharmaceutically active moiety. In an embodiment a bispecific antibody
comprises a
first heavy chain variable domain sequence and light chain variable domain
sequence
which have binding specificity for a first epitope or comprise germline
sequences lacking
any known binding specificity and a second heavy chain variable domain
sequence and
light chain variable domain sequence which have binding specificity for a
second epitope
or comprise germline sequences lacking any known binding specificity, and
wherein the
first and/or second heavy chain variable domains optionally include a
conjugated
pharmaceutically active moiety. In an embodiment, the first and second heavy
chain
variable domains include the same conjugated pharmaceutically active moiety.
In an
embodiment, the first and second heavy chain variable domains include
different
conjugated pharmaceutically active moieties. In an embodiment, only the first
heavy
chain variable domain includes a conjugated pharmaceutically active moiety. In
an
embodiment, only the second heavy chain variable domain includes a conjugated
pharmaceutically active moiety.
[0078] "Full length antibody" as used herein refers to an antibody having two
full
length antibody heavy chains and two full length antibody light chains. A full-
length
.. antibody heavy chain (HC) consists of well-known heavy chain variable and
constant
domains VH, CHL CH2, and CH3. A full-length antibody light chain (LC) consists
of
well-known light chain variable and constant domains VL and CL. The full-
length
antibody may be lacking the C-terminal lysine (K) in either one or both heavy
chains.
[0079] The term "Fab-arm" or "half molecule" refers to one heavy chain-light
chain
pair that specifically binds an antigen.
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[0080] Full length bispecific antibodies can be generated for example using
Fab arm
exchange (or half molecule exchange) between two monospecific bivalent
antibodies by
introducing substitutions at the heavy chain CH3 interface in each half
molecule to favor
heterodimer formation of two antibody half molecules having distinct
specificity either in
vitro in cell-free environment or using co-expression. The Fab arm exchange
reaction is
the result of a disulfide-bond isomerization reaction and dissociation-
association of CH3
domains. The heavy-chain disulfide bonds in the hinge regions of the parent
monospecific antibodies are reduced. The resulting free cysteines of one of
the parent
monospecific antibodies form an inter heavy-chain disulfide bond with cysteine
residues
of a second parent monospecific antibody molecule and simultaneously CH3
domains of
the parent antibodies release and reform by dissociation-association. The CH3
domains of
the Fab arms may be engineered to favor heterodimerization over
homodimerization. The
resulting product is a bispecific antibody having two Fab arms or half
molecules which
each can bind a distinct epitope.
[0081] "Homodimerization" as used herein, with respect to the antibodies,
refers to an
interaction of two heavy chains having identical CH3 amino acid sequences.
"Homodimer" as used herein, with respect to the antibodies, refers to an
antibody having
two heavy chains with identical CH3 amino acid sequences.
[0082] "Heterodimerization" as used herein, with respect to the antibodies,
refers to an
interaction of two heavy chains having non-identical CH3 amino acid sequences.
"Heterodimer" as used herein, with respect to the antibodies, refers to an
antibody having
two heavy chains with non-identical CH3 amino acid sequences.
[0083] The "knob-in-hole" strategy (see, e.g., PCT Intl. Publ. No. WO
2006/028936)
can be used to generate full length bispecific antibodies. Briefly, selected
amino acids
forming the interface of the CH3 domains in human IgG can be mutated at
positions
affecting CH3 domain interactions to promote heterodimer formation. An amino
acid
with a small side chain (hole) is introduced into a heavy chain of an antibody
specifically
binding a first antigen and an amino acid with a large side chain (knob) is
introduced into
a heavy chain of an antibody specifically binding a second antigen. After co-
expression
of the two antibodies, a heterodimer is formed as a result of the preferential
interaction of
the heavy chain with a "hole" with the heavy chain with a "knob". Exemplary
CH3
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substitution pairs forming a knob and a hole are (expressed as modified
position in the
first CH3 domain of the first heavy chain/modified position in the second CH3
domain of
the second heavy chain): T366Y/F405A, T366W/F405W, F405W/Y407A,
T394W/Y407T, T394S/Y407A, T366W/T394S, F405W/T394S and
T366W/T366S L368A Y407V.
[0084] Other strategies such as promoting heavy chain heterodimerization using
electrostatic interactions by substituting positively charged residues at one
CH3 surface
and negatively charged residues at a second CH3 surface may be used, as
described in US
Pat. Publ. No. US2010/0015133; US Pat. Publ. No. US2009/0182127; US Pat. Publ.
No.
US2010/028637 or US Pat. Publ. No. US2011/0123532. In other strategies,
heterodimerization may be promoted by following substitutions (expressed as
modified
position in the first CH3 domain of the first heavy chain/modified position in
the second
CH3 domain of the second heavy chain): L351Y F405A Y407V/T394W,
T3 661 K392M T394W/F405A Y407V, T366L K392M T394W/F405A Y407V,
L3 51Y Y407A/T366A K409F, L3 51Y Y407A/T366V K409F, Y407A/T366A K409F,
or T350V L351Y F405A Y407V/T350V T366L K392L T394W as described in U.S.
Pat. Publ. No. U52012/0149876 or U.S. Pat. Publ. No. U52013/0195849.
[0085] In addition to methods described above, bispecific antibodies can be
generated
in vitro in a cell-free environment by introducing asymmetrical mutations in
the CH3
regions of two monospecific homodimeric antibodies and forming the bispecific
heterodimeric antibody from two parent monospecific homodimeric antibodies in
reducing conditions to allow disulfide bond isomerization according to methods
described in Intl. Pat. Publ. No. W02011/131746. In the methods, the first
monospecific
bivalent antibody and the second monospecific bivalent antibody are engineered
to have
certain substitutions at the CH3 domain that promoter heterodimer stability;
the
antibodies are incubated together under reducing conditions sufficient to
allow the
cysteines in the hinge region to undergo disulfide bond isomerization; thereby
generating
the bispecific antibody by Fab arm exchange. The incubation conditions may
optimally
be restored to non-reducing. Exemplary reducing agents that may be used are 2-
mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE),
glutathione,
tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol,
preferably
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a reducing agent selected from the group consisting of: 2-mercaptoethylamine,
dithiothreitol and tris(2-carboxyethyl)phosphine. For example, incubation for
at least 90
min at a temperature of at least 20 C. in the presence of at least 25 mM 2-
MEA or in the
presence of at least 0.5 mM dithiothreitol at a pH of from 5-8, for example at
pH of 7.0 or
at pH of 7.4 may be used.
[0086] The numbering of amino acid residues in the antibody constant region
throughout the specification is performed according to the EU index as
described in
Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health
Service, National Institutes of Health, Bethesda, Md. (1991), unless otherwise
explicitly
stated.
[0087] Conjugates
[0088] In another general aspect, the invention relates to a conjugate
comprising an
antibody of the invention covalently conjugated to a pharmaceutically active
moiety, such
as a synthetic therapeutic peptide (e.g., an amylinomimetic peptide), in a
site-specific
manner, such that the antibody coupled peptide has an extended/increased half-
life
compared to the peptide alone. The invention also relates to pharmaceutical
compositions and methods for use thereof. The conjugates are useful for
preventing,
treating, or ameliorating diseases or disorders, such as obesity, type 2
diabetes, metabolic
syndrome (i.e., Syndrome X), insulin resistance, impaired glucose tolerance
(e.g., glucose
intolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia,
hypoglycemia due
to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic
nephropathy,
and other cardiovascular risk factors such as hypertension and cardiovascular
risk factors
related to unmanaged cholesterol and/or lipid levels, osteoporosis,
inflammation, non-
alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH),
renal
disease, and eczema, among others.
[0089] In certain embodiments, the antibody of the invention is modified to
comprise at
least one cysteine residue substitution that is capable of being conjugated to
the
pharmaceutically active moiety to extend/increase the half-life of the
pharmaceutically
active moiety. In certain embodiments, the at least one cysteine residue
substitution is
comprised in a complementarity determining region of the antibody. In certain
embodiments, the at least one cysteine residue substitution is in a heavy
chain
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complementarity determining region (HCDR). In certain embodiments, the at
least one
cysteine residue substitution is in an HCDR3, wherein the HCDR3 comprises an
amino
acid sequence of SEQ ID NO:49. In certain embodiments, the antibody comprising
an
HCDR3 comprising an amino acid sequence of SEQ ID NO:49 has at least one
additional
cysteine substitution that is capable of being conjugated to a
pharmaceutically active
moiety.
[0090] In certain embodiments the pharmaceutically active moiety can comprise
a
linker. The linker can be modified chemically to allow for the conjugation of
the
antibody to the pharmaceutically active moiety. The linker can, for example,
include, but
is not limited to, a peptide linker, a hydrocarbon linker, a polyethylene
glycol (PEG)
linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, a
polyester linker, a
hybrid linker consisting of PEG and an embedded heterocycle, or a hydrocarbon
chain.
The PEG linkers can, for example, comprise 2-24 PEG units.
[0091] In certain embodiments, a monoclonal antibody of the invention is
conjugated to
one, two, three, four, five, or six pharmaceutically active moieties (e.g.,
therapeutic
peptide(s)) of interest. In preferred embodiments, the non-targeting
monoclonal antibody
is conjugated to two pharmaceutically active moieties of interest. In certain
embodiments
where the monoclonal antibody is conjugated to at least two pharmaceutically
active
moieties of interest, the pharmaceutically active moieties of interest can be
the same
pharmaceutically active moiety or can be different pharmaceutically active
moieties.
[0092] Methods of conjugating antibodies of the invention with the
pharmaceutically
active moieties of the invention are known in the art. Briefly, the antibodies
of the
invention can be reduced with a reducing agent (e.g., TCEP (tris(2-
carboxyethyl)
phosphine), purified (e.g., by protein A adsorption or gel filtration), and
conjugated with
the pharmaceutically active moiety (e.g., by providing a lyophilized peptide
to the
reduced antibody under conditions that allow for conjugation). After the
conjugation
reaction, the conjugate can be purified by ion exchange chromatography or
hydrophobic
interaction chromatography (RIC) with a final purification step of protein A
adsorption.
In certain embodiments, the antibodies of the invention can be purified prior
to being
reduced utilizing RIC methods. For more detailed description of the
conjugation
methods, see, e.g., Example 103 and Dennler et al., Antibodies 4:197-224
(2015).
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[0093] In certain embodiments, the amylinomimetic peptide is a derivative of
the
amylinomimetic peptide of Formula I that is modified by one or more processes
selected
from the group consisting amidation, lipidation, and pegylation, or a
pharmaceutically
acceptable salt thereof.
[0094] In certain embodiments, a conjugate comprises a monoclonal antibody or
a
fragment thereof conjugated to an amylinomimetic peptide, wherein the
amylinomimetic
peptide is selected from the group consisting of SEQ ID NOs: 4-28.
[0095] In certain embodiments, a monoclonal antibody or the antigen binding
fragment
thereof is covalently linked to the amylinomimetic peptide at a lysine residue
of the
amylinomimetic peptide via a linker. The linker can, for example, comprise a
linker
selected from the group consisting of a PEG chain of 2-24 PEG units, an alkyl
chain
containing 2-10 carbon atoms, or a bond.
[0096] In certain embodiments, only one of Z25, Z26, Z29 and Z34 in Formula I
is lysine,
and the lysine is covalently linked to an engineered cysteine residue of the
monoclonal
antibody or the antigen binding fragment thereof via the linker. In a
preferred
embodiment, a monoclonal antibody or the antigen binding fragment thereof
according to
an embodiment of the invention is conjugated to an amylinomimetic peptide at
residue 25
or 26 of the amylinomimetic. In another preferred embodiment, an electrophile,
such as
bromoacetamide is introduced onto a sidechain of a amylinomimetic, such as the
amino
side chain of a lysine at residue 25 or 26 of the amylinomimetic, and the
electrophile
reacts site specifically with the sulfhydryl group of the Cys residue
engineered into a
CDR, preferably HCDR3, of the monoclonal antibody or fragment thereof, thereby
creating a covalent linkage between the amylinomimetic peptide and the
monoclonal
antibody or fragment thereof. More preferably, the amylinomimetic peptide is
selected
from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID
NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID
NO: 27, and SEQ ID NO: 28. In one embodiment, the electrophile is introduced
onto the
sidechain of an amylinomimetic directly. In another embodiment, the
electrophile is
introduced onto the sidechain of an amylinomimetic indirectly via a linker.
[0097] Also provided are pharmaceutical compositions comprising the conjugates
of
the invention and further comprising a pharmaceutically acceptable carrier.
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[0098] Also provided herein are amylinomimetic peptides exhibiting at least
70%, 75%
80%, 85%, 90%, 95%, or 99% sequence identity to pramlintide or davalintide. As
an
example of a method for determination of the sequence identity between two
analogues
the two peptides, pramlintide (SEQ ID NO: 2) and (SEQ ID NO: 4) are
aligned.
H-K-NcNTAT-171-ATQRLANF LVHSSNNFGPI LPPTNVGSNTY-NH,
Pramlintide (SEQ ID NO: 2)
s
NTAT-N-ATQR LANF LVHSSNNFGP1 LPPTNVGSNTY
-NH2
0
(SEQ ID NO: 4)
[0099] The sequence identity of the analogue relative to pramlintide is given
by the
total number of aligned residues minus the number of different residues (i.e.
the number
of aligned identical residues) divided by the total number of residues in
pramlintide. In
this example the different residues are K1 and C2 which are absent. N3 is now
substituted, but is retained in its original position. Accordingly, in said
example the
sequence identity is (37-2)/37 X 100.
[00100] Where the compounds according to this invention are amylinomimetic
peptides
coupled to a mAb, it is expected that said mAb will have two copies of said
peptide
coupled to it. It is understood by those skilled in the art that reaction
products may
include partial conjugation products, resulting in one amylinomimetic peptide
coupled to
the mAb. It is to be understood that all such mono-coupled compounds, di-
coupled
compounds and mixtures thereof are encompassed within the scope of the present
invention.
Amylinomimetic peptides
[00101] Given its role in controlling appetite and food intake, pramlintide
and/or
davalintide may be effective in treating obesity. However, the therapeutic
utility of
pramlintide and/or davalintide as a treatment agent is limited by its rapid
metabolism and
short circulating half-life. Thus, the present invention is generally directed
to modified
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pramlintide and/or davalintide conjugates, which extend the half-life of the
amylinomimetic peptide and reduce the metabolism of the peptide in vivo.
[00102] In certain embodiments of the invention, the modified pramlintide
and/or
davalintide peptides are amylinomimetic peptides. The terms "amylinomimetic
peptide,"
"amylinomimetic analog," and "amylinomimetic peptide analog" can be used
interchangeably.
[00103] The peptide sequences described herein are written according to the
usual
convention whereby the N-terminal region of the peptide is on the left and the
C-terminal
region is on the right. Although isomeric forms of the amino acids are known,
it is the L-
.. form of the amino acid that is represented unless otherwise expressly
indicated. For
convenience in describing the molecules of this invention, conventional and
non-
conventional abbreviations for various amino acids (both single and three-
letter codes)
and functional moieties are used. These abbreviations are familiar to those
skilled in the
art, but for clarity are listed as follows: A = Ala = alanine; R = Arg =
arginine; N = Asn
= asparagine; D = Asp = aspartic acid; f3A = f3Ala = beta-alanine; C = Cys =
cysteine; hC
= hCys = homocysteine; E = Glu = glutamic acid; Q = Gln = glutamine; G = Gly =
glycine; H = His = histidine; I = Ile = isoleucine; L = Leu = leucine; K = Lys
= lysine;
Nle = norleucine; F = Phe = phenylalanine; P = Pro = proline; S = Ser =
serine; T = Thr =
threonine; W = Trp = tryptophan; Y = Tyr = tyrosine and V = Val = valine.
[00104] Additional amino acid abbreviations used herein are listed as follows:
ACPC
= 2-aminocyclopentanecarboxylic acid; f3-Aib = f3-aminoisobutyric acid = 3-
amino-2-
methylpropionic acid; f3-hA = f3-hAla = f3-homoalanine = 3-aminobutyric acid;
a-MeL =
a-MeLeu = a-methylleucine; f33¨hP = f33¨hPro = f33¨homoproline; f3-hT =
f3¨hThr = f3-
homothreonine;
[00105] For convenience, the amino acid residue numbering convention used in
naming
the amylinomimetic peptides of the present invention follows that of
pramlintide and/or
davalintide. Specific amino acid replacements that have been introduced into
the
peptides, relative to the native residues at the corresponding positions in
pramlintide
and/or davalintide, are indicated by the appropriate amino acid code, followed
by the
position of the substitution. Thus, "hC7" in the amylinomimetic peptide refers
to a
peptide in which homocysteine has replaced the corresponding native Cys7
residue of
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pramlintide. Similarly, "K(Ac)26" in the amylinomimetic peptide refers to a
peptide in
which lysine substituted on the 6¨amine with CH3C(0)- has replaced the
corresponding
native 11e26 residue of pramlintide. Additional amino acid replacements
occurring within
amylinomimetic peptides are described according to this convention and will be
recognized as such by one skilled in the art.
[00106] Also for convenience, the naming convention used for the
amylinomimetic
peptides of the present invention incorporates the amino residues involved in
the cycle
along with the linking group(s) between them in a left-to-right direction,
starting from the
N-terminal residue involved in the cycle. In all cases, the N-terminal amino
acid residue
of the cycle links by way of its a¨amino functionality to an acetyl linking
group, which
in turn connects to the thiol side chain residue of the amino acid at position
7 of the
amylinomimetic peptide. Thus, "cyclo-(N3-COCH2- hC7)" is used to describe the
cycle
of an amylinomimetic peptide in which native Lysl and Cys2 residues have been
deleted
from the sequence, and the a¨amino functionality of Asn3 is acylated with an
acetyl
residue, whose methyl group is further linked by way of a thioether bond to
the side chain
of a hCys7 residue. Similarly, "cyclo-(52- COCH2- hC7)" is used to describe
the cycle
of an amylinomimetic peptide, in which the native Lysl residue has been
deleted, the
native Cys2 residue has been replaced by 5er2 whose a-amino functionality is
acylated
by an acetyl group, which in turn, is linked by way of a thioether bond to the
side chain
of a hCys7 residue.
[00107] Lysine residues can be incorporated at various positions of the
amylinomimetic
sequence to provide a convenient functional handle for further derivatization.
The lysine
residues can be modified to be coupled to the monoclonal antibody either
directly or
indirectly. In an indirect coupling to the monoclonal antibody, the lysine
residue can be
modified to comprise a linker which will allow for the amylinomimetic peptide
to be
coupled to the monoclonal antibody. One skilled in the art will recognize that
related
orthologues could also be effectively employed as such and are contemplated
herein.
[00108] The term, "K(y-Glu)", appearing in the peptide sequence, represents a
lysinyl
residue whose side chain 6¨amino group has been acylated by the y-carboxyl
group of
glutamic acid.
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[00109] The term, "K(Ac)" represents a lysinyl residue whose side chain 6-
amino
group has been substituted with an acetyl group.
[00110] The term, "K(Alloc)" represents a lysinyl residue whose side chain 6-
amino
group has been substituted with an allyloxycarbonyl group.
[00111] The term, "K(OEG2-Pal)" represents a lysinyl residue whose side chain
6¨amino group has been substituted with 17-amino-10-oxo-3,6,12,15-tetraoxa-9-
azaheptadecanoic acid, wherein the 17-amino group is further substituted with
palmitic
acid, by means of an amide bond between the 17-amino group and the palmitic
acid.
[00112] The term, "(0EG)2" represents two OEG units linked together in
succession
.. via an amide linkage (i.e., 17-amino-10-oxo-3,6,12,15-tetraoxa-9-
azaheptadecanoic acid).
[00113] The term, "K(OEG)2" represents a lysinyl residue whose side chain 6-
amino
group has been acylated by 17-amino-10-oxo-3,6,12,15-tetraoxa-9-
azaheptadecanoic
acid.
[00114] The term, "K(OEG2-y-Glu-Pal)" represents a lysinyl residue whose side
chain
6-amino group has been acylated by (22S)-22-amino-10,19-dioxo-3,6,12,15-
tetraoxa-
9,18-diazatricosanedioic acid via its 1-carboxylic acid functionality, and
wherein said 22-
amino group is further amidated with palmitic acid.
[00115] The term, "dPEGx" refers to a discrete oligomer containing x
ethyleneglycol
units linked to propanoic acid at one end and containing a terminal amino
functionality
on the distal end, which may be further functionalized.
[00116] The term, "K(dPEG12)" represents a lysinyl residue whose side chain 6-
amino
group has been acylated by 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-
dodecaoxanonatriacontan-39-oic acid via its 39-carboxylic acid functionality.
[00117] The term, "K(dPEG12-AcBr)" represents a lysinyl residue whose side
chain E-
.. amino group has been acylated by 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-
dodecaoxanonatriacontan-39-oic acid via its 39-carboxylic acid functionality,
and
wherein said acid is amidated on its 1-amine group with a -C(0)CH2Br group.
[00118] Half-life extending moieties
[00119] In addition to the antibody of the present invention or an antigen
binding
fragment thereof, the conjugates of the invention can incorporate one or more
other
moieties for extending the half-life of the pharmaceutical active moiety
(e.g., the
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amylinomimetic peptide), for example via covalent interaction. Exemplary other
half-life
extending moieties include, but not limited to, albumin, albumin variants,
albumin-
binding proteins and/or domains, transferrin and fragments and analogues
thereof.
Additional half-life extending moieties that can be incorporated into the
conjugates of the
invention include, for example, polyethylene glycol (PEG) molecules, such as
PEG5000
or PEG20,000, fatty acids and fatty acid esters of different chain lengths,
for example
laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate,
octanedioic acid,
tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like,
polylysine,
octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides) for
desired
properties. These moieties can be direct fusions with the protein scaffold
coding
sequences and can be generated by standard cloning and expression techniques.
Alternatively, well known chemical coupling methods can be used to attach the
moieties
to recombinantly and chemically produced conjugates of the invention.
[00120] A pegyl moiety can, for example, be added to the peptide molecules of
the
invention by incorporating a cysteine residue to the C-terminus of the
molecule and
attaching a pegyl group to the cysteine using well known methods.
[00121] Peptide molecules of the invention incorporating additional moieties
can be
compared for functionality by several well-known assays. For example, the
biological or
pharmacokinetic activities of a therapeutic peptide of interest, alone or in a
conjugate
according to the invention, can be assayed using known in vitro or in vivo
assays and
compared.
[00122] Pharmaceutical Compositions
[00123] In another general aspect, the invention relates to a pharmaceutical
composition, comprising the conjugates and compounds of the invention and a
pharmaceutically acceptable carrier. The term "pharmaceutical composition" as
used
herein means a product comprising a conjugate of the invention together with a
pharmaceutically acceptable carrier. Conjugates and compounds of the invention
and
compositions comprising them are also useful in the manufacture of a
medicament for
therapeutic applications mentioned herein.
[00124] As used herein, the term "carrier" refers to any excipient, diluent,
filler, salt,
buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle,
microsphere, liposomal
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encapsulation, or other material well known in the art for use in
pharmaceutical
formulations. It will be understood that the characteristics of the carrier,
excipient or
diluent will depend on the route of administration for a particular
application. As used
herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic
material that
.. does not interfere with the effectiveness of a composition according to the
invention or
the biological activity of a composition according to the invention. According
to
particular embodiments, in view of the present disclosure, any
pharmaceutically
acceptable carrier suitable for use in an antibody pharmaceutical composition
can be used
in the invention.
[00125] Pharmaceutically acceptable acidic/anionic salts for use in the
invention
include, and are not limited to acetate, benzenesulfonate, benzoate,
bicarbonate,
bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate,
dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate,
gluconate,
glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,
hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate,
malate,
maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate,
mucate,
napsylate, nitrate, pamoate, pantothenate, phosphate/diphosphate,
polygalacturonate,
salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate,
teoclate, tosylate and
triethiodide. Organic or inorganic acids also include, and are not limited to,
hydriodic,
perchloric, sulfuric, phosphoric, propionic, glycolic, methanesulfonic,
hydroxyethanesulfonic, oxalic, 2-naphthalenesulfonic, p-toluenesulfonic,
cyclohexanesulfamic, saccharinic or trifluoroacetic acid.
[00126] Pharmaceutically acceptable basic/cationic salts include, and are not
limited to
aluminum, 2-amino-2-hydroxymethyl-propane-1,3-diol (also known as
tris(hydroxymethyl)aminomethane, tromethane or "TRIS"), ammonia, benzathine,
t-butylamine, calcium, chloroprocaine, choline, cyclohexylamine,
diethanolamine,
ethylenediamine, lithium, L-lysine, magnesium, meglumine, N-methyl-D-
glucamine,
piperidine, potassium, procaine, quinine, sodium, triethanolamine, or zinc.
[00127] In some embodiments of the invention, pharmaceutical formulations are
provided comprising the conjugates of the invention in an amount from about
0.001
mg/ml to about 100 mg/ml, from about 0.01 mg/ml to about 50 mg/ml, or from
about 0.1
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mg/ml to about 25 mg/ml. The pharmaceutical formulation may have a pH from
about
3.0 to about 10, for example from about 3 to about 7, or from about 5 to about
9. The
formulation may further comprise at least one ingredient selected from the
group
consisting of a buffer system, preservative(s), tonicity agent(s), chelating
agent(s),
stabilizer(s) and surfactant(s).
[00128] The formulation of pharmaceutically active ingredients with
pharmaceutically
acceptable carriers is known in the art, e.g., Remington: The Science and
Practice of
Pharmacy (e.g. 21st edition (2005), and any later editions). Non-limiting
examples of
additional ingredients include: buffers, diluents, solvents, tonicity
regulating agents,
preservatives, stabilizers, and chelating agents. One or more pharmaceutically
acceptable
carrier may be used in formulating the pharmaceutical compositions of the
invention.
[00129] In one embodiment of the invention, the pharmaceutical composition is
a
liquid formulation. A preferred example of a liquid formulation is an aqueous
formulation, i.e., a formulation comprising water. The liquid formulation may
comprise a
solution, a suspension, an emulsion, a microemulsion, a gel, and the like. An
aqueous
formulation typically comprises at least 50% w/w water, or at least 60%, 70%,
75%,
80%, 85%, 90%, or at least 95% w/w of water.
[00130] In one embodiment, the pharmaceutical composition may be formulated as
an
injectable which can be injected, for example, via an injection device (e.g.,
a syringe or
an infusion pump). The injection may be delivered subcutaneously,
intramuscularly,
intraperitoneally, or intravenously, for example.
[00131] In another embodiment, the pharmaceutical composition is a solid
formulation,
e.g., a freeze-dried or spray-dried composition, which may be used as is, or
whereto the
physician or the patient adds solvents, and/or diluents prior to use. Solid
dosage forms
may include tablets, such as compressed tablets, and/or coated tablets, and
capsules (e.g.,
hard or soft gelatin capsules). The pharmaceutical composition may also be in
the form
of sachets, dragees, powders, granules, lozenges, or powders for
reconstitution, for
example.
[00132] The dosage forms may be immediate release, in which case they may
comprise
a water-soluble or dispersible carrier, or they may be delayed release,
sustained release,
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or modified release, in which case they may comprise water-insoluble polymers
that
regulate the rate of dissolution of the dosage form in the gastrointestinal
tract.
[00133] In other embodiments, the pharmaceutical composition may be delivered
intranasally, intrabuccally, or sublingually.
[00134] The pH in an aqueous formulation can be between pH 3 and pH 10. In one
embodiment of the invention, the pH of the formulation is from about 7.0 to
about 9.5. In
another embodiment of the invention, the pH of the formulation is from about
3.0 to
about 7Ø
[00135] In another embodiment of the invention, the pharmaceutical composition
comprises a buffer. Non-limiting examples of buffers include: arginine,
aspartic acid,
bicine, citrate, disodium hydrogen phosphate, fumaric acid, glycine,
glycylglycine,
histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate,
sodium
dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tricine, and
tris(hydroxymethyl)-aminomethane, and mixtures thereof. The buffer may be
present
individually or in the aggregate, in a concentration from about 0.01 mg/ml to
about 50
mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical
compositions comprising each one of these specific buffers constitute
alternative
embodiments of the invention.
[00136] In another embodiment of the invention, the pharmaceutical composition
comprises a preservative. Non-limiting examples of buffers include:
benzethonium
chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate,
chlorobutanol, chlorocresol, chlorohexidine, chlorphenesin, o-cresol, m-
cresol, p-cresol,
ethyl 4-hydroxybenzoate, imidurea, methyl 4-hydroxybenzoate, phenol, 2-
phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium
dehydroacetate,
thiomerosal, and mixtures thereof. The preservative may be present
individually or in the
aggregate, in a concentration from about 0.01 mg/ml to about 50 mg/ml, for
example
from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising
each
one of these specific preservatives constitute alternative embodiments of the
invention.
[00137] In another embodiment of the invention, the pharmaceutical composition
comprises an isotonic agent. Non-limiting examples of the embodiment include a
salt
(such as sodium chloride), an amino acid (such as glycine, histidine,
arginine, lysine,
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isoleucine, aspartic acid, tryptophan, and threonine), an alditol (such as
glycerol, 1,2-
propanediol propyleneglycol), 1,3-propanediol, and 1,3-butanediol),
polyethyleneglycol
(e.g. PEG400), and mixtures thereof. Another example of an isotonic agent
includes a
sugar. Non-limiting examples of sugars may be mono-, di-, or polysaccharides,
or
water-soluble glucans, including for example fructose, glucose, mannose,
sorbose,
xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin,
cyclodextrin, alpha
and beta- HPCD, soluble starch, hydroxyethyl starch, and sodium
carboxymethylcellulose. Another example of an isotonic agent is a sugar
alcohol,
wherein the term "sugar alcohol" is defined as a C(4-8) hydrocarbon having at
least one
¨OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol,
inositol, galactitol, dulcitol, xylitol, and arabitol. Pharmaceutical
compositions
comprising each isotonic agent listed in this paragraph constitute alternative
embodiments of the invention. The isotonic agent may be present individually
or in the
aggregate, in a concentration from about 0.01 mg/ml to about 50 mg/ml, for
example
from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising
each
one of these specific isotonic agents constitute alternative embodiments of
the invention.
[00138] In another embodiment of the invention, the pharmaceutical composition
comprises a chelating agent. Non-limiting examples of chelating agents include
citric
acid, aspartic acid, salts of ethylenediaminetetraacetic acid (EDTA), and
mixtures
thereof. The chelating agent may be present individually or in the aggregate,
in a
concentration from about 0.01 mg/ml to about 50 mg/ml, for example from about
0.1
mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each one of
these
specific chelating agents constitute alternative embodiments of the invention.
[00139] In another embodiment of the invention, the pharmaceutical composition
comprises a stabilizer. Non-limiting examples of stabilizers include one or
more
aggregation inhibitors, one or more oxidation inhibitors, one or more
surfactants, and/or
one or more protease inhibitors.
[00140] In another embodiment of the invention, the pharmaceutical composition
comprises a stabilizer, wherein said stabilizer is carboxy-/hydroxycellulose
and derivates
thereof (such as HPC, HPC-SL, EIPC-L and EIPMC), cyclodextrins, 2-
methylthioethanol,
polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinyl
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pyrrolidone, salts (such as sodium chloride), sulphur-containing substances
such as
monothioglycerol), or thioglycolic acid. The stabilizer may be present
individually or in
the aggregate, in a concentration from about 0.01 mg/ml to about 50 mg/ml, for
example
from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising
each
one of these specific stabilizers constitute alternative embodiments of the
invention.
[00141] In further embodiments of the invention, the pharmaceutical
composition
comprises one or more surfactants, preferably a surfactant, at least one
surfactant, or two
different surfactants. The term "surfactant" refers to any molecules or ions
that are
comprised of a water-soluble (hydrophilic) part, and a fat-soluble
(lipophilic) part. The
surfactant may, for example, be selected from the group consisting of anionic
surfactants,
cationic surfactants, nonionic surfactants, and/or zwitterionic surfactants.
The surfactant
may be present individually or in the aggregate, in a concentration from about
0.1 mg/ml
to about 20 mg/ml. Pharmaceutical compositions comprising each one of these
specific
surfactants constitute alternative embodiments of the invention.
[00142] In a further embodiment of the invention, the pharmaceutical
composition
comprises one or more protease inhibitors, such as, e.g., EDTA, and/or
benzamidine
hydrochloric acid (HC1). The protease inhibitor may be present individually or
in the
aggregate, in a concentration from about 0.1 mg/ml to about 20 mg/ml.
Pharmaceutical
compositions comprising each one of these specific protease inhibitors
constitute
alternative embodiments of the invention.
[00143] The pharmaceutical composition of the invention may comprise an amount
of
an amino acid base sufficient to decrease aggregate formation of the
polypeptide during
storage of the composition. The term "amino acid base" refers to one or more
amino
acids (such as methionine, histidine, imidazole, arginine, lysine, isoleucine,
aspartic acid,
tryptophan, threonine), or analogues thereof. Any amino acid may be present
either in its
free base form or in its salt form. Any stereoisomer (i.e., L, D, or a mixture
thereof) of the
amino acid base may be present. The amino acid base may be present
individually or in
the combination with other amino acid bases, in a concentration from about
0.01 mg/ml
to about 50 mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml.
Pharmaceutical compositions comprising each one of these specific amino acid
bases
constitute alternative embodiments of the invention.
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[00144] It is also apparent to one skilled in the art that the therapeutically
effective dose
for conjugates of the present invention or a pharmaceutical composition
thereof will vary
according to the desired effect. Therefore, optimal dosages to be administered
may be
readily determined by one skilled in the art and will vary with the particular
conjugate
used, the mode of administration, the strength of the preparation, and the
advancement of
the disease condition. In addition, factors associated with the particular
subject being
treated, including subject age, weight, diet and time of administration, will
result in the
need to adjust the dose to an appropriate therapeutic level.
[00145] For all indications, the conjugates of the invention are preferably
administered
peripherally at a dose of about 1 pg to about 50 mg per day in single or
divided doses
(e.g., a single dose can be divided into 2, 3, 4, 5, 6, 7, 8, 9, or 10 sub-
doses), or at about
0.01 p.g/kg to about 500 p.g/kg per dose, more preferably about 0.05 p.g/kg to
about 250
p.g/kg, most preferably below about 50 p.g/kg. Dosages in these ranges will
vary with the
potency of each agonist, of course, and are readily determined by one of skill
in the art.
The above dosages are thus exemplary of the average case. There can, of
course, be
individual instances where higher or lower dosage ranges are merited, and such
are
within the scope of this invention.
[00146] In certain embodiments, the conjugates of the invention are
administered at a
dose of about 1 pg to about 5 mg, or at a dose of about 0.01 p.g/kg to about
500 p.g/kg,
more preferably at a dose of about 0.05 p.g/kg to about 250 p.g/kg, most
preferably at a
dose below about 50 p.g/kg with a dose of a second therapeutic agent (e.g.,
liraglutide) at
a dose of about 1 pg to about 5 mg, or at a dose of about 0.01 p.g/kg to about
500 p.g/kg,
more preferably at a dose of about 0.05 p.g/kg to about 250 p.g/kg, most
preferably at a
dose below about 50 p.g/kg.
[00147] The pharmaceutically-acceptable salts of the conjugates of the
invention
include the conventional non-toxic salts or the quaternary ammonium salts
which are
formed from inorganic or organic acids or bases. Examples of such acid
addition salts
include acetate, adipate, benzoate, benzenesulfonate, citrate, camphorate,
dodecylsulfate,
hydrochloride, hydrobromide, lactate, maleate, methanesulfonate, nitrate,
oxalate,
pivalate, propionate, succinate, sulfate and tartrate. Base salts include
ammonium salts,
alkali metal salts such as sodium and potassium salts, alkaline earth metal
salts such as
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calcium and magnesium salts, salts with organic bases such as
dicyclohexylamino salts
and salts with amino acids such as arginine. Also, the basic nitrogen-
containing groups
may be quaternized with, for example, alkyl halides.
[00148] The pharmaceutical compositions of the invention may be administered
by any
means that accomplish their intended purpose. Examples include administration
by
parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal,
transdermal, buccal
or ocular routes. Administration may be by the oral route. Suitable
formulations for
parenteral administration include aqueous solutions of the active conjugates
in water-
soluble form, for example, water-soluble salts, acidic solutions, alkaline
solutions,
dextrose-water solutions, isotonic carbohydrate solutions and cyclodextrin
inclusion
complexes.
[00149] The present invention also encompasses a method of making a
pharmaceutical
composition comprising mixing a pharmaceutically acceptable carrier with any
of the
conjugates of the present invention. Additionally, the present invention
includes
pharmaceutical compositions made by mixing one or more pharmaceutically
acceptable
carriers with any of the conjugates of the present invention.
[00150] Furthermore, the conjugates of the present invention may have one or
more
polymorph or amorphous crystalline forms and as such are intended to be
included in the
scope of the invention. In addition, the conjugates may form solvates, for
example with
water (i.e., hydrates) or common organic solvents. As used herein, the term
"solvate"
means a physical association of the conjugates of the present invention with
one or more
solvent molecules. This physical association involves varying degrees of ionic
and
covalent bonding, including hydrogen bonding. In certain instances, the
solvate will be
capable of isolation, for example when one or more solvent molecules are
incorporated in
the crystal lattice of the crystalline solid. The term "solvate" is intended
to encompass
both solution-phase and isolatable solvates. Non-limiting examples of suitable
solvates
include ethanolates, methanolates, and the like.
[00151] It is intended that the present invention include within its scope
polymorphs
and solvates of the conjugates of the present invention. Thus, in the methods
of treatment
of the present invention, the term "administering" shall encompass the means
for treating,
ameliorating or preventing a syndrome, disorder or disease described herein
with the
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conjugates of the present invention or a polymorph or solvate thereof, which
would
obviously be included within the scope of the invention albeit not
specifically disclosed.
[00152] In another embodiment, the invention relates to the conjugates of the
invention
for use as a medicament.
[00153] The present invention includes within its scope prodrugs of the
conjugates of
this invention. In general, such prodrugs will be functional derivatives of
the conjugates
which are readily convertible in vivo into the required conjugate. Thus, in
the methods of
treatment of the present invention, the term "administering" shall encompass
the
treatment of the various disorders described with the conjugate specifically
disclosed or
with a conjugate which may not be specifically disclosed, but which converts
to the
specified conjugate in vivo after administration to the patient. Conventional
procedures
for the selection and preparation of suitable prodrug derivatives are
described, for
example, in "Design of Prodrugs", Ed. H. Bundgaard, Elsevier, 1985.
[00154] Furthermore, it is intended that within the scope of the present
invention, any
element, in particular when mentioned in relation to the conjugates of the
invention, shall
comprise all isotopes and isotopic mixtures of said element, either naturally
occurring or
synthetically produced, either with natural abundance or in an isotopically
enriched form.
For example, a reference to hydrogen includes within its scope 11-1, 2H (D),
and 3H (T).
Similarly, references to carbon and oxygen include within their scope
respectively 12C,
13C and "C and 160 and 180. The isotopes may be radioactive or non-
radioactive.
Radiolabeled conjugates of the invention may comprise a radioactive isotope
selected
, ,
nc 18F 1221, 1231, 1251, 131-,
from the group of 3H, 75Br, 76Br, 77Br and 'Br.
Preferably,
the radioactive isotope is selected from the group of 3H, 11C and 18F.
[00155] Some conjugates of the present invention may exist as atropisomers.
Atropisomers are stereoisomers resulting from hindered rotation about single
bonds
where the steric strain barrier to rotation is high enough to allow for the
isolation of the
conformers. It is to be understood that all such conformers and mixtures
thereof are
encompassed within the scope of the present invention.
[00156] Where the conjugates according to this invention have at least one
stereo
center, they may accordingly exist as enantiomers or diastereomers. It is to
be
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understood that all such isomers and mixtures thereof are encompassed within
the scope
of the present invention.
[00157] Where the processes for the preparation of the conjugates according to
the
invention give rise to mixture of stereoisomers, these isomers may be
separated by
conventional techniques such as preparative chromatography. The conjugates may
be
prepared in racemic form, or individual enantiomers may be prepared either by
enantiospecific synthesis or by resolution. The conjugates may, for example,
be resolved
into their component enantiomers by standard techniques, such as the formation
of
diastereomeric pairs by salt formation with an optically active acid, such as
(-)-di-p-
.. toluoyl-D-tartaric acid and/or (+)-di-p-toluoyl-L-tartaric acid followed by
fractional
crystallization and regeneration of the free base. The conjugates may also be
resolved by
formation of diastereomeric esters or amides, followed by chromatographic
separation
and removal of the chiral auxiliary. Alternatively, the conjugates may be
resolved using
a chiral column via high performance liquid chromatography (HPLC) or SFC. In
some
instances, rotamers of conjugates may exist which are observable by 11-I NMR
leading to
complex multiplets and peak integration in the 11-I NMR spectrum.
[00158] During any of the processes for preparation of the conjugates of the
present
invention, it may be necessary and/or desirable to protect sensitive or
reactive groups on
any of the molecules concerned. This may be achieved by means of conventional
protecting groups, such as those described in Protective Groups in Organic
Chemistry,
ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P.G.M. Wuts,
Protective
Groups in Organic Synthesis, John Wiley & Sons, 1991, each of which is herein
incorporated by reference in its entirety for all purposes. The protecting
groups may be
removed at a convenient subsequent stage using methods known from the art.
[00159] Methods of Use
[00160] The present invention is directed to a method for preventing, treating
or
ameliorating an amylin receptor mediated syndrome, disorder or disease in a
subject in
need thereof comprising administering to the subject in need thereof an
effective amount
of a conjugate, compound, or pharmaceutical composition of the invention.
[00161] The present invention also provides a method for preventing, treating,
delaying
the onset of, or ameliorating a disorder, disease, or condition or any one or
more
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symptoms of said disorder, disease, or condition in a subject in need thereof,
comprising
administering to the subject in need thereof an effective amount of a
conjugate,
compound, or pharmaceutical composition of the invention.
[00162] According to particular embodiments, the disease disorder, or
condition is
selected from the group consisting of obesity, type I or II diabetes,
metabolic syndrome
(i.e., Syndrome X), insulin resistance, impaired glucose tolerance (e.g.,
glucose
intolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia,
hypoglycemia due
to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic
nephropathy,
and other cardiovascular risk factors such as hypertension and cardiovascular
risk factors
related to unmanaged cholesterol and/or lipid levels, osteoporosis,
inflammation, non-
alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH),
renal
disease, and/or eczema.
[00163] According to particular embodiments, a therapeutically effective
amount refers
to the amount of therapy which is sufficient to achieve one, two, three, four,
or more of
the following effects: (i) reduce or ameliorate the severity of the disease,
disorder, or
condition to be treated or a symptom associated therewith; (ii) reduce the
duration of the
disease, disorder or condition to be treated, or a symptom associated
therewith; (iii)
prevent the progression of the disease, disorder or condition to be treated,
or a symptom
associated therewith; (iv) cause regression of the disease, disorder or
condition to be
treated, or a symptom associated therewith; (v) prevent the development or
onset of the
disease, disorder or condition to be treated, or a symptom associated
therewith; (vi)
prevent the recurrence of the disease, disorder or condition to be treated, or
a symptom
associated therewith; (vii) reduce hospitalization of a subject having the
disease, disorder
or condition to be treated, or a symptom associated therewith; (viii) reduce
hospitalization length of a subject having the disease, disorder or condition
to be treated,
or a symptom associated therewith; (ix) increase the survival of a subject
with the
disease, disorder or condition to be treated, or a symptom associated
therewith; (xi)
inhibit or reduce the disease, disorder or condition to be treated, or a
symptom associated
therewith in a subject; and/or (xii) enhance or improve the prophylactic or
therapeutic
effect(s) of another therapy.
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[00164] The therapeutically effective amount or dosage can vary according to
various
factors, such as the disease, disorder or condition to be treated, the means
of
administration, the target site, the physiological state of the subject
(including, e.g., age,
body weight, health), whether the subject is a human or an animal, other
medications
administered, and whether the treatment is prophylactic or therapeutic.
Treatment
dosages are optimally titrated to optimize safety and efficacy.
[00165] As used herein, the terms "treat," "treating," and "treatment" are all
intended to
refer to an amelioration or reversal of at least one measurable physical
parameter related
the disease, disorder, or condition, which is not necessarily discernible in
the subject, but
can be discernible in the subject. The terms "treat," "treating," and
"treatment," can also
refer to causing regression, preventing the progression, or at least slowing
down the
progression of the disease, disorder, or condition. In a particular
embodiment, "treat,"
"treating," and "treatment" refer to an alleviation, prevention of the
development or
onset, or reduction in the duration of one or more symptoms associated with
the disease,
disorder, or condition. In a particular embodiment, "treat," "treating," and
"treatment"
refer to prevention of the recurrence of the disease, disorder, or condition.
In a particular
embodiment, "treat," "treating," and "treatment" refer to an increase in the
survival of a
subject having the disease, disorder, or condition. In a particular
embodiment, "treat,"
"treating," and "treatment" refer to elimination of the disease, disorder, or
condition in
the subject.
[00166] In one embodiment, the invention provides a method for preventing,
treating,
delaying the onset of, or ameliorating obesity, or any one or more symptoms of
obesity in
a subject in need thereof, the method comprising administering to the subject
in need
thereof an effective amount of a conjugate, compound, or pharmaceutical
composition of
the invention. In some embodiments, the body weight of a subject is reduced,
for
example, by between about 0.01% to about 0.1%, between about 0.1% to about
0.5%,
between about 0.5% to about 1%, between about 1% to about 5%, between about 2%
to
about 3%, between about 5% to about 10%, between about 10% to about 15%,
between
about 15% to about 20%, between about 20% to about 25%, between about 25% to
about
30%, between about 30% to about 35%, between about 35% to about 40%, between
about 40% to about 45%, or between about 45% to about 50%, relative to the
body
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weight of a subject prior to administration of any of the conjugates,
compounds,
pharmaceutical compositions, forms, or medicaments of the invention described
herein,
or compared to control subjects not receiving any of the conjugates,
compositions, forms,
medicaments, or combinations of the invention described herein.
.. [00167] In some embodiments, the reduction in body weight is maintained for
about 1
week, for about 2 weeks, for about 3 weeks, for about 1 month, for about 2
months, for
about 3 months, for about 4 months, for about 5 months, for about 6 months,
for about 7
months, for about 8 months, for about 9 months, for about 10 months, for about
11
months, for about 1 year, for about 1.5 years, for about 2 years, for about
2.5 years, for
about 3 years, for about 3.5 years, for about 4 years, for about 4.5 years,
for about 5
years, for about 6 years, for about 7 years, for about 8 years, for about 9
years, for about
10 years, for about 15 years, or for about 20 years, for example.
[00168] The present invention provides a method of preventing, treating,
delaying the
onset of, or ameliorating a syndrome, disorder or disease, or any one or more
symptoms
of said syndrome, disorder, or disease in a subject in need thereof, wherein
said
syndrome, disorder or disease is selected from the group consisting of
obesity, type I or
type II diabetes, metabolic syndrome (i.e., Syndrome X), insulin resistance,
impaired
glucose tolerance (e.g., glucose intolerance), hyperglycemia,
hyperinsulinemia,
hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI),
dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular
risk factors
such as hypertension and cardiovascular risk factors related to unmanaged
cholesterol
and/or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver
disease
(NAFLD), non-alcoholic steatohepatitis (NASH), renal disease, and eczema,
comprising
administering to the subject in need thereof an effective amount of a
conjugate,
compound, or pharmaceutical composition of the invention.
[00169] As used herein, metabolic syndrome refers to a subject having any one
or more
of the following: high blood sugar (e.g., high fasting blood sugar), high
blood pressure,
abnormal cholesterol levels (e.g., low EIDL levels), abnormal triglyceride
levels (e.g.,
high triglycerides), a large waistline (i.e., waist circumference), increased
fat in the
abdominal area, insulin resistance, glucose intolerance, elevated C-reactive
protein levels
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(i.e., a proinflammatory state), and increased plasma plasminogen activator
inhibitor-1
and fibrinogen levels (i.e., a prothrombotic state).
[00170] The present invention provides a method of reducing food intake in a
subject in
need thereof, the method comprising administering to the subject in need
thereof an
effective amount of a conjugate, compound, or pharmaceutical composition of
the
invention. In some embodiments, food intake of a subject is reduced, for
example, by
between about 0.01% to about 0.1%, between about 0.1% to about 0.5%, between
about
0.5% to about 1%, between about 1% to about 5%, between about 2% to about 3%,
between about 5% to about 10%, between about 10% to about 15%, between about
15%
to about 20%, between about 20% to about 25%, between about 25% to about 30%,
between about 30% to about 35%, between about 35% to about 40%, between about
40%
to about 45%, or between about 45% to about 50%, relative to food intake of a
subject
prior to administration of any of the conjugates, compounds, compositions,
forms,
medicaments, or combinations of the invention described herein, or compared to
control
subjects not receiving any of the conjugates, compounds, compositions, forms,
medicaments, or combinations of the invention described herein.
[00171] In some embodiments, the reduction in food intake is maintained for
about 1
week, for about 2 weeks, for about 3 weeks, for about 1 month, for about 2
months, for
about 3 months, for about 4 months, for about 5 months, for about 6 months,
for about 7
months, for about 8 months, for about 9 months, for about 10 months, for about
11
months, for about 1 year, for about 1.5 years, for about 2 years, for about
2.5 years, for
about 3 years, for about 3.5 years, for about 4 years, for about 4.5 years,
for about 5
years, for about 6 years, for about 7 years, for about 8 years, for about 9
years, for about
10 years, for about 15 years, or for about 20 years, for example.
[00172] The present invention provides a method of reducing glycated
hemoglobin
(Al C) in a subject in need thereof, the method comprising administering to
the subject in
need thereof an effective amount of a conjugate, compound, or pharmaceutical
composition of the invention. In some embodiments, Al C of a subject is
reduced, for
example, by between about 0.001% and about 0.01%, between about 0.01% and
about
0.1%, between about 0.1% and about 0.2%, between about 0.2% and about 0.3%,
between about 0.3% and about 0.4%, between about 0.4% and about 0.5%, between
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about 0.5% and about 1%, between about 1% and about 1.5%, between about 1.5%
and
about 2%, between about 2% and about 2.5%, between about 2.5% and about 3%,
between about 3% and about 4%, between about 4% and about 5%, between about 5%
and about 6%, between about 6% and about 7%, between about 7% and about 8%,
between about 8% and about 9%, or between about 9% and about 10% relative to
the
Al C of a subject prior to administration of any of the conjugates, compounds,
compositions, forms, medicaments, or combinations of the invention described
herein, or
compared to control subjects not receiving any of the conjugates, compounds,
compositions, forms, medicaments, or combinations of the invention described
herein.
[00173] In other embodiments, methods are provided for reducing fasting blood
glucose levels in a subject in need thereof, the methods comprising
administering to the
subject in need thereof an effective amount of a conjugate, compound, or
pharmaceutical
composition of the invention. Fasting blood glucose levels may be reduced to
less than
about 140 to about 150 mg/dL, less than about 140 to about 130 mg/dL, less
than about
130 to about 120 mg/dL, less than about 120 to about 110 mg/dL, less than
about 110 to
about 100 mg/dL, less than about 100 to about 90 mg/dL, or less than about 90
to about
80 mg/dL, relative to the fasting blood glucose levels of a subject prior to
administration
of any of the conjugates, compounds, compositions, forms, medicaments, or
combinations of the invention described herein, or compared to control
subjects not
receiving any of the conjugates, compounds, compositions, forms, medicaments,
or
combinations of the invention described herein.
[00174] The present invention provides a method of modulating amylin receptor
activity in a subject in need thereof, the method comprising administering to
the subject
in need thereof an effective amount of a conjugate, compound, or
pharmaceutical
composition of the invention. As used herein, "modulating" refers to
increasing or
decreasing receptor activity.
[00175] In some embodiments, an effective amount of a conjugate or compound of
the
invention or a form, composition or medicament thereof is administered to a
subject in
need thereof once daily, twice daily, three times daily, four times daily,
five times daily,
six times daily, seven times daily, or eight times daily. In other
embodiments, an
effective amount of a conjugate or compound of the invention or a form,
composition or
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medicament thereof is administered to a subject in need thereof once every
other day,
once per week, twice per week, three times per week, four times per week, five
times per
week, six times per week, two times per month, three times per month, or four
times per
month.
.. [00176] Another embodiment of the invention comprises a method of
preventing,
treating, delaying the onset of, or ameliorating a disease, disorder or
syndrome, or one or
more symptoms of any of said diseases, disorders, or syndromes in a subject in
need
thereof, the method comprising administering to the subject in need thereof an
effective
amount of a conjugate, compound, or pharmaceutical composition of the
invention in a
combination therapy. In certain embodiments, the combination therapy is a
second
therapeutic agent. In certain embodiments, the combination therapy is a
surgical therapy.
[00177] As used herein, the term "in combination," in the context of the
administration
of two or more therapies to a subject, refers to the use of more than one
therapy.
[00178] As used herein, combination therapy refers to administering to a
subject in
.. need thereof one or more additional therapeutic agents, or one or more
surgical therapies,
concurrently with an effective amount of a conjugate or compound of the
invention or a
form, composition or medicament thereof. In some embodiments, the one or more
additional therapeutic agents or surgical therapies can be administered on the
same day as
an effective amount of a conjugate of the invention, and in other embodiments,
the one or
more additional therapeutic agents or surgical therapies may be administered
in the same
week or the same month as an effective amount of a conjugate or compound of
the
invention.
[00179] In certain embodiments, wherein the disease or disorder is selected
from the
group consisting of obesity, type II diabetes, metabolic syndrome, insulin
resistance and
dyslipidemia, the second therapeutic agent can be an antidiabetic agent. In
certain
embodiments, the antidiabetic agent can be a glucagon-like peptide-1 (GLP-1)
receptor
modulator.
[00180] The present invention also contemplates preventing, treating, delaying
the
onset of, or ameliorating any of the diseases, disorders, syndromes, or
symptoms
described herein in a subject in need thereof with a combination therapy that
comprises
administering to the subject in need thereof an effective amount of a
conjugate,
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compound, or pharmaceutical composition of the invention, in combination with
any one
or more of the following therapeutic agents: a dipeptidyl peptidase-4 (DPP-4)
inhibitor
(e.g., sitagliptin, saxagliptin, linagliptin, alogliptin, etc.); a GLP-1
receptor agonist (e.g.,
short-acting GLP-1 receptor agonists such as exenatide and lixisenatide;
intermediate-
acting GLP-1 receptor agonists such as liraglutide; long-acting GLP-1 receptor
agonists
such as exenatide extended-release, albiglutide, dulaglutide); a sodium-
glucose co-
transporter-2 (SGLT-2) inhibitors (e.g., canaglifozin, dapaglifozin,
empaglifozin, etc.);
bile acid sequestrants (e.g., colesevelam, etc.); dopamine receptor agonists
(e.g.,
bromocriptine quick-release); biguanides (e.g., metformin, etc.); insulin;
oxyntomodulin;
sulfonylureas (e.g., chlorpropamide, glimepiride, glipizide, glyburide,
glibenclamide,
glibornuride, glisoxepide, glyclopyramide, tolazamide, tolbutamide,
acetohexamide,
carbutamide, etc.); and thiazolidinediones (e.g; pioglitazone, rosiglitazone,
lobeglitazone,
ciglitazone, darglitazone, englitazone, netoglitazone, rivoglitazone,
troglitazone, etc.). In
some embodiments, the dose of the additional therapeutic agent(s) is reduced
when given
.. in combination with a conjugate or compound of the invention. In some
embodiments,
when used in combination with a conjugate or compound of the invention, the
additional
therapeutic agent(s) may be used in lower doses than when each is used singly.
[00181] In certain embodiments, wherein the disease or disorder is selected
from the
group consisting of obesity, type I or type II diabetes, metabolic syndrome
(i.e.,
Syndrome X), insulin resistance, impaired glucose tolerance (e.g., glucose
intolerance),
hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to
congenital
hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy,
and other
cardiovascular risk factors such as hypertension and cardiovascular risk
factors related to
unmanaged cholesterol and/or lipid levels, osteoporosis, inflammation, non-
alcoholic
fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), renal
disease, and
eczema, the second therapeutic agent can be liraglutide.
[00182] The present invention contemplates preventing, treating, delaying the
onset of,
or ameliorating any of the diseases, disorders, syndromes, or symptoms
described herein
in a subject in need thereof, with a combination therapy that comprises
administering to
the subject in need thereof an effective amount of a conjugate, compound, or
pharmaceutical composition of the invention in combination with a surgical
therapy. In
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certain embodiments, the surgical therapy can be bariatric surgery (e.g.,
gastric bypass
surgery, such as Roux-en-Y gastric bypass surgery; sleeve gastrectomy;
adjustable gastric
band surgery; biliopancreatic diversion with duodenal switch; intragastric
balloon; gastric
plication; and combinations thereof).
[00183] In embodiments in which the one or more additional therapeutic agents
or
surgical therapies is administered on the same day as an effective amount of a
conjugate
or compound of the invention, the conjugate or compound of the invention may
be
administered prior to, after, or simultaneously with the additional
therapeutic agent or
surgical therapy. The use of the term "in combination" does not restrict the
order in
which therapies are administered to a subject. For example, a first therapy
(e.g., a
composition described herein) can be administered prior to (e.g., 5 minutes,
15 minutes,
30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours,
24 hours,
48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6
weeks, 8
weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5
minutes, 15
minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours,
16 hours, 24
hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks, 6
weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a
subject.
[00184] Further embodiments of the present invention include the incorporation
of
additional proteolysis-stabilizing modifications of amylin agonist peptides,
preferably in
the peptide region between residues 10 and 17. Such proteolysis¨stabilizing
modifications include, but are not limited to amino acid substitution by one
or more non-
proteinogenic amino acids, C-alpha-alkylated amino acids, homologous amino
acids or
synthetic amino acids, and the like. Combinations of more than one such
proteolysis-
stabilizing modification are contemplated herein.
[00185] In further embodiments of the present invention, the thioether-
cyclized
amylinomimetic peptides or derivatives thereof contain at least one amino acid
residue
which is derivatized with a half-life extension moiety. Such half-life
extension moieties
are introduced at suitable (tolerant) sites via conjugation onto appropriately
mutated
residues at these positions. Examples of half-life extension moieties include,
but are not
limited to albumin-binding lipids, such as palmitate or similar fatty acids,
and protein
bioconjugates, such as HSA, mAb, or Fc conjugates.
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[00186] Further embodiments of the present invention include amino acid
substitutions
and/or peptide modifications that are introduced to improve upon the
physicochemical
properties of the thioether-cyclized amylin peptides. In certain instances,
native amylin
amino acid residues may be mutated to residues that reduce the pI of the
peptide, thereby
making it more readily formulatable for administration, while at the same time
maintaining amylin receptor potency. In other instances, derivatization with
water-
soluble functional groups, such as, but not limited to polyethylene glycols is
contemplated as a means of improving the solubility of the thioether-cyclized
amylin
analogues in suitable formulation vehicles.
EMBODIMENTS
[00187] The invention provides also the following non-limiting embodiments.
[00188] Embodiment 1 is a conjugate comprising a monoclonal antibody or an
antigen
binding fragment thereof coupled to an amylinomimetic peptide, wherein the
amylinomimetic peptide is represented by Formula I or a derivative or
pharmaceutically
acceptable salt thereof (SEQ ID NO: 53):
N, H
Z2NZ4Z5Z6¨NX¨NTY¨CONH2
)n
Formula I
wherein
n is 1, or 2;
Z2 is a direct bond, serine, or glycine;
N
'''01-1 =
Z4 iS T, or
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H
kN µ35..N µkN
Z5 is A, f3-alanine, 0 , 0 , 0 , z 0
H
N
,
0 0 , or
'''01-1 =
Z6isT,or
Zio is Q, or E;
.. Zii is R, or K, wherein the 6-amine of said K is optionally substituted
with ¨C(=NH)NH2;
$N
Z12 iS L, or 0 =
SN
Z16 iS L, or 0 =
Z25 is P, or K;
Z26 is I, or K;
X is ATZ10Z11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34 (SEQ ID NO: 54), or
VLGRLSQELEIRLQTYPRTNTGS (SEQ ID NO: 55);
Z29 is P, or K;
Z34 is S, or K;
wherein the derivative is the compound of Formula I that is modified by one or
more
processes selected from the group consisting of amidation, glycosylation,
carbamylation,
sulfation, phosphorylation, cyclization, lipidation, and pegylation.
[00189] Embodiment 2 is the conjugate of embodiment 1, wherein the
amylinomimetic
peptide is a derivative of the amylinomimetic peptide of Formula I that is
modified by
one or more processes selected from the group consisting amidation,
lipidation, and
pegylation, or a pharmaceutically acceptable salt thereof.
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[00190] Embodiment 3 is the conjugate of embodiment 1, wherein the
amylinomimetic
peptide is represented by Formula I or the derivative or pharmaceutically
acceptable salt
thereof, wherein:
Z2 is a direct bond;
7
H
SN )C.N '3CNAr.-Lq.
Z5 is f3-alanine, 0 ; 0 , or 0 ;
Z6 is T.
[00191] Embodiment 4 is the conjugate of embodiment 1, wherein the
amylinomimetic
peptide is represented by Formula I or the derivative or pharmaceutically
acceptable salt
thereof, wherein:
Z16 is L;
SN
. H
Z12 1S 0
[00192] Embodiment 5 is the conjugate of embodiment 1, wherein the
amylinomimetic
peptide is represented by Formula I or the derivative or pharmaceutically
acceptable salt
thereof, wherein:
Zii is R;
SNc. H
Z12 1S 0
[00193] Embodiment 6 is the conjugate of embodiment 1, wherein the
amylinomimetic
peptide is selected from the group consisting of SEQ ID NOs:4-28.
[00194] Embodiment 7 is the conjugate of any one of embodiments 1-6, wherein
the
monoclonal antibody or the antigen binding fragment thereof is covalently
linked to the
amylinomimetic peptide at a lysine residue of the amylinomimetic peptide via a
linker.
[00195] Embodiment 8 is the conjugate of any one of embodiments 1-7, wherein
the
monoclonal antibody or the antigen binding fragment thereof comprises a heavy
chain
complementarity determining region 1 (HCDR1), HCDR2, HCDR3, and a light chain
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complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, having the
polypeptide sequences of SEQ ID NO: 47, 48, 49, 50, 51, and 52, respectively
[00196] Embodiment 9 is the conjugate of embodiment 8, wherein the isolated
monoclonal antibody comprises a heavy chain variable domain (VH) having the
polypeptide sequence of SEQ ID NO:43, and a light chain variable domain (VL)
having
the polypeptide sequence of SEQ ID NO:45.
[00197] Embodiment 10 is the conjugate of embodiment 9, further comprising a
Fc
portion.
[00198] Embodiment 11 is the conjugate of embodiment 10, comprising a heavy
chain
(HC) having the polypeptide sequence of SEQ ID NO:44 and a light chain (LC)
having
the polypeptide sequence of SEQ ID NO:46.
[00199] Embodiment 12 is a method of producing the conjugate of any one of
embodiments 1-11, comprising reacting an electrophile, preferably a
bromoacetamide
derivatized linker on a sidechain of the amylinomimetic peptide, preferably
the amino
sidechain of a lysine residue of the amylinomimetic peptide, with the
sulfhydryl group of
the cysteine residue of SEQ ID NO:49 of the monoclonal antibody or antigen-
binding
fragment thereof, thereby creating a covalent linkage between the
amylinomimetic
peptide and the monoclonal antibody or antigen-binding fragment thereof.
[00200] Embodiment 13 is a pharmaceutical composition comprising the conjugate
of
any one of embodiments 1-11 and a pharmaceutically acceptable carrier.
[00201] Embodiment 14 is a method for treating or preventing obesity in a
subject in
need thereof, comprising administering to the subject in need thereof an
effective amount
of the pharmaceutical composition of embodiment 13.
[00202] Embodiment 15 is the method of embodiment 14, wherein administration
of
the effective amount of the pharmaceutical composition to the subject in need
thereof
results in a reduction in body weight of about 5% to about 10%, about 10% to
about 15%,
about 15% to about 20%, or about 20% to about 25% as compared to the body
weight of
the subject prior to administration of the pharmaceutical composition.
[00203] Embodiment 16 is a method for treating or preventing a disease or
disorder in a
subject in need thereof, wherein said disease or disorder is selected from the
group
consisting of obesity, type I or type II diabetes, metabolic syndrome, insulin
resistance,
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impaired glucose tolerance, hyperglycemia, hyperinsulinemia,
hypertriglyceridemia,
hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia,
atherosclerosis,
diabetic nephropathy, and other cardiovascular risk factors such as
hypertension and
cardiovascular risk factors related to unmanaged cholesterol and/or lipid
levels,
osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-
alcoholic
steatohepatitis (NASH), renal disease, and eczema, the method comprising
administering
to the subject in need thereof an effective amount of the pharmaceutical
composition of
embodiment 13.
[00204] Embodiment 17 is the method of embodiment 16, wherein said disease or
disorder is obesity.
[00205] Embodiment 18 is the method of embodiment 16, wherein said disease or
disorder is type I diabetes.
[00206] Embodiment 19 is the method of embodiment 16, wherein said disease or
disorder is type II diabetes
[00207] Embodiment 20 is the method of embodiment 16, wherein said disease or
disorder is metabolic syndrome.
[00208] Embodiment 21 is the method of embodiment 16, wherein said disease or
disorder is a renal disease.
[00209] Embodiment 22 is the method of embodiment 16, wherein said disease or
disorder is non-alcoholic steatohepatitis (NASH).
[00210] Embodiment 23 is the method of embodiment 16, wherein said disease or
disorder is non-alcoholic fatty liver disease (NAFLD).
[00211] Embodiment 24 is a method of reducing food intake in a subject in need
thereof, the method comprising administering to the subject in need thereof an
effective
amount of the pharmaceutical composition of embodiment 13.
[00212] Embodiment 25 is the method of embodiment 24, wherein administration
of
the effective amount of the pharmaceutical composition to the subject in need
thereof
results in a reduction in food intake of about 5% to about 10%, about 10% to
about 15%,
about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about
30%
to about 35%, about 35% to about 40%, about 40% to about 45%, or about 45% to
about
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50% as compared to the food intake of the subject prior to administration of
the
pharmaceutical composition.
[00213] Embodiment 26 is a method of modulating amylin receptor activity in a
subject
in need thereof, the method comprising administering to the subject in need
thereof an
effective amount of the pharmaceutical composition of embodiment 13.
[00214] Embodiment 27 is the method of any one of embodiments 14-26, wherein
the
pharmaceutical composition is administered via an injection.
[00215] Embodiment 28 is the method of embodiment 27, wherein the injection is
delivered subcutaneously, intramuscularly, intraperitoneally, or
intravenously.
[00216] Embodiment 29 is the method of any one of embodiments 14-28, wherein
the
pharmaceutical composition is administered in a combination with a second
therapeutic
agent.
[00217] Embodiment 30 is the method of embodiment 29, wherein the disease or
disorder is selected from the group consisting of obesity, type 2 diabetes,
metabolic
syndrome, insulin resistance and dyslipidemia and the second therapeutic agent
is at least
one antidiabetic agent.
[00218] Embodiment 31 is the method of embodiment 30, wherein said
antidiabetic
agent is a glucagon-like-peptide-1 receptor modulator.
[00219] Embodiment 32 is the method of embodiment 29, wherein the second
therapeutic agent is liraglutide.
[00220] Embodiment 33 is the method of any one of embodiments 14-32, wherein
the
pharmaceutical composition is administered daily, weekly, or monthly to the
subject in
need thereof.
[00221] Embodiment 34 is the method of embodiment 33, wherein the
pharmaceutical
composition is administered once, twice, three, four, five, or six times per
day.
[00222] Embodiment 35 is the method of embodiment 33, wherein the
pharmaceutical
composition is administered once, twice, three, four, five, or six times per
week.
[00223] Embodiment 36 is the method of embodiment 33, wherein the
pharmaceutical
composition is administered once, twice, three, or four times per month.
[00224] Embodiment 37 is a kit comprising the conjugate of any one of
embodiments
1-14 or a pharmaceutical composition of embodiment 13, preferably the kit
further
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comprising an effective amount of a second therapeutic agent, more preferably,
the kit
further comprising an effective amount of liraglutide.
[00225] Embodiment 38 is the kit of embodiment 37, wherein the kit further
comprises
an injection device.
[00226] Embodiment 39 is a method of producing a pharmaceutical composition
comprising a compound selected from the group consisting of SEQ ID NOs:4-42,
and a
pharmaceutically acceptable carrier.
SYNTHESIS
[00227] Compounds or conjugates of the present invention can be synthesized
in
accordance with the general synthetic methods known to those who are skilled
in the art.
The following description of the synthesis is for exemplary purposes and is in
no way
meant to be a limit of the invention.
[00228] The thioether-cyclized amylinomimetic peptides or derivatives of this
invention may be synthesized by a variety of known, conventional procedures
for the
formation of successive peptide linkages between amino acids, and are
preferentially
carried out by solid phase peptide synthesis (SPPS), as generally described by
Merrifield
(J. Am. Chem. Soc., 1963, 85, 2149-2154), using an automated peptide
synthesizer,
traditional bench synthesis, or a combination of both approaches. Conventional
.. procedures for peptide synthesis involve the condensation between the free
amino group
of one amino acid residue, whose other reactive functionalities have been
suitably
protected, and the free carboxyl group of another amino acid, whose reactive
functionalities have also been suitably protected. Examples of condensation
agents
typically utilized for peptide bond formation include diisopropylcarbodiimide
(DIC) with
or without 1-hydroxybenztriazole (HOBT) or ethyl cyano(hydroxyimino)acetate
(Oxyma
Pure), 2-(1H-benzotriazol-1-y1)-1,1,3,3-tetramethylaminium hexafluorophosphate
(EIBTU), 2-(1H-7-azabenztriazol-1-y1)-1,1,3,3-tetramethylaminium
hexafluorophosphate
(HATU), 2-(6-chloro-1H-benztriazol-1-y1)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HCTU), 1-Cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-
pyrrolidino-phosphonium hexafluorophosphate (PyOxim), 2-(1H-benzotriazole-1-
y1)-
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1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) bromo-tris-pyrrolidino-
phosphonium hexafluorophosphate (PyBroP), and the like.
[00229] The automated peptide synthetic methodology may be carried out at room
temperature (rt), or at elevated temperatures, preferably through the
application of
microwave heating, as described by Yu (J. Org. Chem., 1992, 57, 4781-4784) and
as
more recently refined by Palasek (J. Pept. Sci., 2007, 13, 143-148).
[00230] Compounds of the present invention (C-terminal amides) can be
conveniently
prepared using N-a-FMOC protected amino acid methodology, whereby the carboxy
terminus of a suitably protected N-a-FMOC protected amino acid is coupled onto
a
conventional solid phase resin using a suitable coupling agent. Suitable
conventional,
commercially-available solid phase resins include Rink amide MBHA resin, Rink
amide
AM resin, Tentagel S RAM Resin, FMOC-PAL-PEG PS resin, SpheriTide Rink amide
resin, ChemMatrix Rink resin, Sieber amide resin, TG Sieber resin and the
like. The
resin-bound FMOC-amino acid may then be deprotected by exposure to 20%
piperidine
in either DMF or NMP, treatment of which serves to selectively remove the FMOC
protecting group. Additional FMOC-protected amino acids are then subsequently
coupled and deprotected sequentially, thereby generating the desired resin-
bound
protected peptide. In certain instances, it may be necessary to utilize an
orthogonally
reactive protecting group for another amine in the peptide sequence that would
withstand
the FMOC deprotection conditions. Protecting groups such 4-methyltrityl (Mtt)
or 4-
methoxytrityl (Mmt), both removable by 1% TFA/DCM treatments, or preferably
allyloxycarbonyl (alloc; removable by Pd(PPh3)4/PhSiH3 treatment), 1-(4,4-
dimethy1-2,6-
dioxocyclohex-1-yliden)ethyl (Dde; removable by treatment with 2-3 %
hydrazine/DMF)
and 1-(4,4-dimethy1-2,6-dioxocyclohex-1-yliden)-3-methylbutyl (ivDde;
removable by
treatment with 2-3 % hydrazine/DMF) can be used effectively in such instances.
[00231] In conventional peptide synthetic methodologies, reactive side chains
of alpha
amino acids are generally protected throughout the synthesis with suitable
protecting
groups to render them inert to the coupling and deprotection protocols. While
multiple
protecting groups for amino acid side chains are known in the art, herein the
following
protecting groups are most preferred: tert-butyl (t-Bu) for serine, threonine,
glutamic
acid, aspartic acid and tyrosine; trityl (Trt) for asparagine, glutamine,
cysteine,
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homocysteine and histidine; tert-butyloxycarbonyl (Boc) for tryptophan and the
6-amino
group of lysine; and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf)
for
arginine. These protecting groups are removed upon strong acid treatment, such
as with
high concentrations of trifluoroacetic acid (TFA).
[00232] Upon completion of the SPPS, the resin-bound, side chain-protected
peptide is
deprotected and concomitantly cleaved from the resin using a cleavage cocktail
that
consists predominantly of (TFA) along with various combinations of carbocation
scavengers, such as triisopropylsilane (TIPS), water, phenol and anisole. The
crude solid
peptide is then isolated by precipitation of the peptide/cocktail filtrate
with cold ether.
The crude peptide thus obtained is then dissolved at low concentration (ca., <
5 mg/mL)
in a largely aqueous solvent system containing an organic co-solvent such as
acetonitrile
or ethanol. Upon raising the pH of the solution to > 7, the peptide then
undergoes an
intramolecular cyclization reaction to form the corresponding crude thioether-
cyclized
amylin analogue of the present invention. Thioether-cyclized amylin analogues
thus
formed may be purified using purification techniques generally known in the
art. A
preferable method of peptide purification used herein is reverse phase high
performance
liquid chromatography (HPLC). Purified peptides are then characterized by
liquid
chromatography/mass spectrometry (LC/MS).
[00233] It is understood that the following examples and embodiments described
herein
are for illustrative purposes only and that various modifications or changes
in light
thereof will be suggestive to persons skilled in the art and are to be
included within the
spirit and purview of this application and the scope of the appended claims.
All
publications, patents, and patent applications cited herein are hereby
incorporate by
reference in their entirety for all purposes.
ABBREVIATIONS
Herein and throughout the specification, the following abbreviations may be
used:
aq aqueous
alloc allyloxycarbonyl
Boc tert-butoxycarbonyl
BSA bovine serum albumin
CDI 1,1'-carbonyldiimidazole
CT calcitonin
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CTR calcitonin receptor
DCM dichloromethane
Dde 1-(4,4-dimethy1-2,6-dioxocyclohex-1-yliden)ethyl
DIC diisopropylcarbodiimide
DIEA diisopropylethylamine
DMA N, N-dimethylacetamide
DMEM Dulbecco's modified eagle's medium
DMF N,N-dimethylformamide
EDC N-(3-dimethylaminopropy1)-N'-ethylcarbodiimide
EDTA ethylenediaminetetraacetic acid
Et ethyl
Et0Ac ethyl acetate
Et0H ethyl alcohol
FBS fetal bovine serum
FMOC 9-fluorenylmethyloxycarbonyl
g gram(s)
h hour(s)
HATU 2-(1H-7-azabenztriazol-1-y1)-1,1,3,3-tetramethylaminium-
hexafluorophosphate
MSS Hank's balanced salt solution
liBTU 2-(1H-Benzotriazole-1-y1)-1,1,3,3-tetramethyluronium-
hexafluorophosphate)
HCTU 2-(6-chloro-1H-benztriazol-1-y1)-1,1,3,3-tetramethylaminium-
hexafluorophosphate
HC1 hydrochloric acid
HEPE S 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIC hydrophobic interaction chromatography
HOBT 1-hydroxybenztriazole
HPLC high performance liquid chromatography
HTRF homogeneous time resolved fluorescence
IBMX 3-isobuty1-1-methylxanthine
ivDde 1-(4,4-dimethy1-2,6-dioxocyclohex-1-yliden)-3-methylbutyl
LCMS high performance liquid chromatography with mass spectrometer
Me methyl
MeCN acetonitrile
mg milligram
min minute(s)
mL milliliter
Mmt 4-methoxytrityl
MU 4-methyltrityl
NMP 1-methy1-2-pyrrolidone
OEG 8-amino-3,6-dioxaoctanoyl
ORF open reading frame
Oxyma ethyl cyano(hydroxyimino)acetate
Pal palmitoyl
Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
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PBS phosphate buffered saline
Pd(PPh3)4 tetrakis(triphenylphosphine)palladium(0)
PhSiH3 phenylsilane
PyBroP bromo-tris-pyrrolidino-phosphonium hexafluorophosphate
Pyoxim 1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidino-
phosphonium hexafluorophosphate
RAMP receptor activity-modifying protein
rt room temperature
RT retention time
sat'd saturated
SPPS solid phase peptide synthesis
t-Bu tert-butyl
TBTU 2-(1H-benzotriazole-1-y1)-1,1,3,3-tetramethylaminium
tetrafluoroborate
TFA trifluoroacetic acid
TIPS triisopropylsilane
Tris tris(hydroxymethyl)aminomethane
Trt triphenylmethyl
EXAMPLES
Example 1: Synthesis of Thioether-cyclized Amylinomimetic Peptides (Scheme 1)
[00234] Step A: Synthesis of Resin-bound C-terminal Amide Peptide
[00235] The protected peptidyl resin was synthesized using FMOC strategy as
described above on a CEM Liberty Blue Microwave peptide synthesizer using low
loading Rink amide resins, preferably, FMOC-PAL-PEG PS resin (ca., 0.16 ¨ 0.2
meq/g,
supplied by Applied Biosystems) on a scale of 0.1 mmol, as depicted in Scheme
1.
Standard FMOC-protected amino acids (supplied by Novabiochem (EMD Millipore),
Bachem, Peptides International, Sigma-Aldrich or Chem-Impex) were coupled in 5-
fold
excess relative to resin loading using DIC/Oxyma as the coupling agents and a
reaction
temperature of ca., 90 C for 4 min. FMOC-Arg(Pbf)-OH was double coupled at 90
C
for 4 min each and FMOC-His(TrO-OH was coupled using a two-stage protocol: 4
min
at rt followed by 8 min at 50 C. Single FMOC deprotections were carried out
using
20% piperidine in DMF (deprotection solution) at 90 C for 1.5 min.
[00236] Step B: Procedure for Bromoacetylation of Resin-bound Peptide (Scheme
2)
[00237] The FMOC-deprotected peptide-resin (0.1 mmol) was treated with a
solution
of bromoacetic anhydride (6-20 eq.) in DMF (5 mL) in a microwave reactor at 50
C for
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min, by which time the reaction was generally determined to be complete as per
a
Kaiser ninhydrin test. In cases where the coupling was determined to be
incomplete, the
coupling was repeated with fresh reagents.
[00238] Step C: Procedure for Peptide Cleavage from Resin
5 [00239] Upon completion of the SPPS, the resin was washed extensively
with DMF
and then with DCM and dried. The resin was then treated with a cleavage
cocktail (10
mL / 0.1 mmol scale) consisting of either TFA/water/TIPS (95:2.5:2.5)
(Cleavage
Cocktail A) or more preferably with TFA/water/phenol/TIPS (88:5:5:2) (Cleavage
Cocktail B) and heated in a microwave reactor at 38 C for 40 min, then
filtered. The
resin was washed with TFA and the combined filtrates were concentrated under a
stream
of nitrogen to a volume of ca. 2.5 mL and the peptide was precipitated by the
addition of
cold diethyl ether (40 mL). The peptide/ether suspension was centrifuged and
the ether
layer was decanted. The peptide pellet was re-suspended in ether, centrifuged
and
decanted, and this process was repeated a third time. The crude peptide thus
obtained
was dried under a mild nitrogen stream.
[00240] Step D: Procedure for Peptide Cyclization (Thioether Formation)
[00241] The crude cysteine- or homocysteine-containing peptide was dissolved
in
deoxygenated MeCN/water (50-60% MeCN) or Et0H/water (50-60% Et0H) at a
concentration of < 4 mg/mL. The pH of the peptide solution was then raised to
ca. 7 ¨ 9
through the addition of either solid NaHCO3, sat'd aq. NaHCO3 or 1M aq. Tris
buffer
(pH 7.5) and the resulting solution was stirred at rt for 3-16 h. Typically,
the cyclizations
were complete within 1 h, as determined by analytical LC/MS.
[00242] Step E: Procedure for Peptide Purification
[00243] The cyclization reaction mixture was acidified to pH 1.5 ¨ 3 by the
addition of
TFA, and the solution was concentrated to remove most of the organic co-
solvent (MeCN
or Et0H) to a point where slight clouding occurred. A minimal amount of the co-
solvent
was added back as necessary to render the mixture homogeneous and the
resultant
solution was then purified directly by preparative EIPLC in multiple
injections.
Purifications were performed on either an Agilent PrepStar EIPLC system or a
Gilson
EIPLC 2020 Personal Purification System using a reverse phase C18 or C8 column
selected from the following: Varian Pursuit XRs C18 (21x250 mm, 100 A, 5 lam);
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Varian Pursuit XRs Diphenyl (30x100 mm, 100 A, 5 nm); Zorbax 300 SB-C8 (21x250
mm, 300 A, 5 nm); Waters Atlantis T3 C18 (19x250 mm, 100 A, 5 nm); Agilent
Polaris
C18-A (30x250 mm, 180 A, 5 nm). The mobile phase consisted of gradient
elutions of
buffer A (0.1% TFA in water) and Buffer B (0.1% TFA in MeCN) ranging in
initial
5 concentration of 10 - 20 % B to final concentrations of 40 - 90 % B with
run times
ranging between 36 ¨ 80 min. UV detection was monitored at 220 and 254 nm.
Product-
containing fractions were analyzed by analytical HPLC on an Agilent 1100 HPLC
system
using an appropriate column type from above (4.6x250 mm, 300 A, 5 nm). Pure
fractions were combined, concentrated to remove most of the organic phase, and
then
lyophilized. TFA/HC1 salt exchange was subsequently carried out by triple
lyophilization
from 2 mM HC1, according to the procedure described by Andrushchenko, et al.,
(J. Pept.
Sci., 2006, 13, 37-43).
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Fmoc-Rink Resin (PAL-PEG PS resin; ¨0.16-0.2 mmoi/g)
IAutomated SPPS
eq. Fmoc-AA(Protected)-0H. DIC/Oxyma or H13TU/DIEA
H2N-Z2NZ4Z5Z6Z7-X-NTY-Resin*
1 15eq. (LG-CH2C0)20, it-wave, 50 C, 10 min
LG-CH2COHN-Z2NZ4Z5Z6Z7-X-NTY-Resin*
11, TFA/H20/Phenol/TIS (88:5:5:2)
COHN-Z2NZ4Z5Z6Z7-X-NTY-CONH2
LLG HS "- )n
II, MeCN/H20 or Et0H/H20, pH ¨ 7 - 9
[<4 mg/mL]
COHN-Z2NZ4Z5Z6Z7-X-NTY-CONH2
)
n
wherein LG is a leaving group
X is ATZi oZi 1 Zi2ANFLVHSSNNFGZ25Z26LPZ29TNVGZ34, or
VLGRLSQELHRLQTYPRTNTGS
*Amino acids are protected
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Scheme 1: Synthesis of Thioether-cyclized Amylinomimetic Peptides:
Amylin/Pramlintide/Davalintide Analogues (Scheme 1 discloses SEQ ID NOS 56,
56,
56-58, and 55, respectively, in order of appearance)
Example 2: Synthesis of Lipidated Amylinomimetic Peptides (Scheme 2)
[00244] Step A: Procedure for Introducing Derivatized Lysine Residues into
Peptide
Sequences Built on Standard Rink Amide Resin
[00245] To a resin-bound C-terminal amide peptide, elaborated to the point
preceding
the desired point of derivatization and prepared as described in Example 1A,
were
sequentially coupled either Dde-Lys(FMOC)-OH or ivDde-Lys(FMOC)-OH and then
FMOC-OEG-OH (one or two units coupled in tandem), optionally followed by FM0C-
Glu-OtBu under microwave conditions (either manually or on the Liberty Blue
Peptide
Synthesizer) using DIC/Oxyma coupling methods as described in Example 1A.
Following FMOC deprotection, the resin was treated with a solution of the
lipophilic acid
[ex., palmitic acid] (5-10 eq.), DIC (5-10 eq.) and either HOBT or Oxyma (5-10
eq.) in
DMF under microwave conditions at 90 C for 10 min. The reaction was then
drained
and the resin was washed with DMF. Scheme 2 shows the introduction of
lipidated
lysine at position Z25. Those skilled in the art will recognize that a similar
approach will
yield lipidated lysine at Z26. Z29, or Z34.
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Fmoc-Rink Resin (PAL-PEG PS resin; ¨0.16-0.2 mmol/g)
I Automated SPPS
eq. Fmoc-AA(Protected)-0H, DIC/Oxyma,
90 C, 4 min
ivDde-K(FMOC)-Z26LPZ29TNVGZ34NTY-Resin*
1) 5 eq. FM0C-OEG-0O2H, DIC/Oxyma,
ti-wave, 90 C, 4 min
1 or 2 units
[5 eq. FM0C-Glu-0O2tBu, DIC/Oxyma,
-wave, 90 C, 4 min] optional
2) 5 eq. Lipophilic acid, DIC/Oxyma or HBTU/DIEA
pt-wave, 75 C, 15 min
ivDde-Z25Z26LPZ29TNVGZ34NTY-Resin*
I(manual)
[2-3% H2NNH2 in DIVfF; 90 C, 3.5min] x 3
H2N-Z25Z26LPZ29TNVGZ34NTY-Resin*
I (automated)
5 eq. Fmoc-AA, DIC/Oxyma, ti-wave,
90 C, 4 min
H2N-Z2NZ4Z5Z6Z7ATZ1 oZliZi2ANFLVHSSNNFGZ25Z26LPZ29TNVGZ34NTY-Resin*
* Amino acids are protected
Scheme 2: Procedure for Introducing Derivatized Lysine Residues into
Amylinomimetic
Peptides (Scheme 2 discloses SEQ ID NOS 59, 60, 60, and 61, respectively, in
order of
appearance)
5 [00246]
[00247] Step B: Procedure for Deprotecting Dde- or ivDde-protected Lysinyl
Peptide
[00248] The derivatized lysinyl peptide resin was treated with a solution of 3
%
hydrazine in DMF (6 mL / 0.1 mmol resin) under microwave conditions at 90 C
for 3.5
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min. The reaction was drained and this procedure was repeated two additional
times.
The reaction was drained and the resin was washed extensively with DMF and
then with
DCM.
[00249] Step C: Procedure for Direct Incorporation of FM0C-Lys(Pal-Glu-
OtBu)-OH Residue
[00250] In cases where the palmitoylated-y-Glu-Lysinyl residue is to be
incorporated
into the sequence, FM0C-Lys(Pal-Glu-OtBu)-OH (available from Peptides
International
or ActivePeptide) may be used directly in the procedure described in Example
1A.
Example 3: Synthesis of BrAc-dPEGx-Derivatized Amylinomimetic Peptides
(Scheme 3)
[00251] A solution of the thioether-cyclized peptide obtained from Example 1E,
in
which one of Z25, Z26, Z29 or Z34 was K(NH2) (7 [tmol), and BrAc-dPEGx-OTFP (3
eq.)
in DMA (0.3 mL) was treated with DIEA (7 eq) and the resultant solution was
stirred at
rt. Upon completion of the reaction (ca., 1h), the mixture was acidified with
TFA, diluted
with water (0.1% TFA) and purified by reversed phase chromatography, as
described in
Example 1E. Scheme 3 shows the introduction of lipidated lysine at position
Z25. Those
skilled in the art will recognize that a similar approach will yield BrAc-
dPEGx-
derivatized lysine at Z26. Z29, or Z34.
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Fmoc-Rink Resin (PAL-PEG PS resin; -0.16-0.2 mmol/g)
IAutomated SPPS
eq. Fmoc-AA(Protected)-0H. DIC/Oxyma or HBTU/DIEA
H2N-Z2NZ4Z5Z6Z7ATZ1 0Z11Z12ANFLVHSSNNFGK(BOC)Z26LPZ29TNVGZ34NTY-Resin*
1 15eq. (LG-CH2C0)20, -wave. 50 C, 10 min
LG-CH2COHN-Z2NZ4Z5Z6Z7ATZ1 oZiiZi2ANFLVHSSNNFGK(BOC)Z26LPZ29TNVGZ34NTY-Resin*
1 TFA/H20/Phenol/TIS (88:5:5:2)
COHN-Z2NZ4Z5Z6Z7ATZ1 oZiiZi2ANFLVHSSNNFGK(NH2)Z26LPZ29TNVGZ34NTY-coNn2
[----- LG HS---+ )n MeCN/H20 or Et0H/H20,
pH - 7 - 9
[< 4 mg/mL]
COHN-Z2NZ4Z5Z6Z7ATZ10Z1 iZi2ANFLVHSSNNFGK(NH2)Z261-F3Z29TNVGZ34NTY-coNH2
1-------s---(- ) BrAc-dPEGx-OT, DIEA,
DMA
n
COHN-Z2NZ4Z5Z6Z7ATZ1 oZi iZi2ANFLVHSSNNFGK(NH-dPEGx-AcBr)Z26LPZ29TNVGZ34NTY-
coNn2
1---------s---+ )n
*Amino acids are protected
LG is a leaving group
Scheme 3: Procedure for Synthesizing BrAc-dPEGx-Derivatized Amylinomimetic
Peptides (Scheme 3 discloses SEQ ID NOS 62 and 62-65, respectively, in order
of
appearance)
5
Example 4: Synthesis of Thioether-Cyclized Amylinomimetic Peptide-mAb
Conjugate
[00252] Expression and Purification of the mAb
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[00253] The fully human monoclonal antibody (mAb) can be recombinantly
expressed
in a mammalian expression host and purified from the cell culture supernatant
using
standard methods that are known in the field. For example, a cDNA sequence
encoding
the light (LC) and heavy chains (HC) of the mAb, each including an appropriate
signal
peptide to enable secretion, can be cloned into separate mammalian expression
vectors or
into a single expression vector using standard molecular biology methods.
Expression
vectors used can be any of those commercially available such as pEE12.4,
pcDNATm3.1(+) or pIRESpuro3 or any custom expression vector with similar
functional ities. In such vectors transcription of the heavy and light chains
of the mAb are
each driven by any of the known effective promoters such as the hCMV-MIE
promoter.
Transfection grade plasmid DNA is prepared for separate LC and HC expression
constructs or a single construct expressing both LC and HC using standard
methods such
as a QIAGEN Plasmid Midi Kit.
[00254] Purified plasmid DNA is prepared for transfection with a lipid-based
transfection reagent such as FreestyleTM Max transfection reagent, following
manufacturer's instructions, and is then transfected into a standard mammalian
expression host cell line, such as CHO-S or FIEK 293-F. If the mAb LC and HC
are
encoded by separate expression constructs, the two constructs are
simultaneously
transfected. Prior to and after transfection, mammalian cells are cultured for
maintenance
or for mAb expression following standard cell culture methods whereby the cell
density
ranges to maintain, the culture media to use, and the other cell culture
conditions
followed are determined by the specific mammalian host cell line utilized.
These
parameters are typically documented by the vendor from which the cell line was
obtained
or in the scientific literature. For example, CHO-S cells are maintained in
CHO
FreestyleTM media in suspension, shaking at 125 RPM in a humidified incubator
set at 37
C and 8% CO2, and split when the cell concentration is between 1.5 and 2.0 x
106 cells
per ml.
[00255] Cell culture supernatants from the transiently transfected mammalian
cells
expressing the mAb are harvested several days after transfection, clarified by
centrifugation and filtered. Duration of expression for CHO-S cells is
typically four days
but can be adjusted and can differ for different mammalian host cell lines.
Large scale
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transfections (>10 liters) are concentrated 10-fold using a concentrator such
as a
Centramate. The mAb is purified from the clarified supernatant using a Protein
A
affinity column such as the HiTrap MabSelect Sure utilizing standard methods
for
binding mAb to Protein A resin, washing the resin and eluting the protein
using low pH
buffer. The protein fractions are neutralized immediately by elution into
tubes containing
pH 7 buffer and peak fractions are pooled, filtered and dialyzed against
phosphate
buffered saline (PBS), pH 7.2 overnight at 4 C. After dialysis the mAb is
filtered again
(0.41 filter) and the protein concentration is determined by absorbance at
280nm.
Quality of the purified mAb protein is assessed by SDS-polyacrylamide gel
electrophoresis (PAGE) and analytical size exclusion 1-1PLC and endotoxin
levels are
measured using a limulus amebocyte lysate (LAL) assay. Purified mAb is stored
at 4 C.
[00256] Expression and Purification of MSCB97 from transiently transfected
CHO cells
[00257] MSCB97 was expressed in ExpiCHO-STM cells (ThermoFisher Scientific,
Waltham, MA; Cat # A29127) by transient transfection of the cells with
purified plasmid
DNA of a MSCB97 expression construct following manufacturer's recommendations.
Briefly, ExpiCHO-STM cells were maintained in suspension in ExpiCHOTM
expression
medium (ThermoFisher Scientific, Cat # A29100) in a shaking incubator set at
37 C, 8%
CO2 and 125 RPM. The cells were passaged so that on the day of transfection,
dilution
down to 6.0 x 106 cells per ml could be achieved, maintaining cell viability
at 98% or
better. Transient transfections were done using the ExpiFectamineTM CHO
transfection
kit (ThermoFisher Scientific Cat # A29131). For each ml of diluted cells to be
transfected, one microgram of plasmid DNA is used and diluted into OptiPROTM
SFM
complexation medium. ExpiFectamineTM CHO reagent is used at a 1:3 ratio (v/v,
DNA:reagent) and also diluted into OptiPROTM. The diluted DNA and transfection
reagent were combined for one minute, allowing DNA/lipid complex formation,
and then
added to the cells. After overnight incubation, ExpiCHOTM feed and
ExpiFectamineTM
CHO enhancer were added to the cells. Cells were cultured with shaking at 32
C for five
days prior to harvesting the culture supernatants.
[00258] Culture supernatants from the transiently transfected ExpiCHO-STM
cells were
harvested by clarifying through centrifugation (30 min, 6000 rpm) followed by
filtration
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(0.2 PES membrane, Corning). Large scale transfections (5 to 20 liters) were
first
concentrated 10-fold using a Pall Centramate Tangential Flow Filtration
system. 10x
DPBS (Dulbecco's phosphate buffered saline), pH7.2 was added to the
supernatant to lx
final concentration prior to loading onto an equilibrated (DPBS, pH 7.2)
HiTrap
MabSelect Sure Protein A column (GE Healthcare; Little Chalfont, United
Kingdom) at a
relative concentration of ¨20 mg protein per ml of resin, using an AKTA FPLC
chromatography system. After loading, the column was washed with 10 column
volumes
of DPBS, pH7.2. The protein was eluted with 10 column volumes of 0.1 M Na-
Acetate,
pH 3.5. Protein fractions were neutralized immediately by elution into tubes
containing
2.0 M Tris, pH 7 at 20% the elution fraction volume. Peak fractions were
pooled and the
pH adjusted to ¨5.5 with additional Tris, if necessary. The purified protein
was filtered
(0.2p) and the concentration was determined by absorbance at 280nm on a BioTek
SynergyHTTM spectrophotometer. The quality of the purified protein was
assessed by
SDS-PAGE and analytical size exclusion I-IPLC (Dionex I-IPLC system). The
endotoxin
level was measured using a turbidometric LAL assay (Pyrotell -T, Associates of
Cape
Cod).
[00259] Conjugation of MSCB97 to amylinomimetic peptides
[00260] To the mAb (1.2 mL, 19 mg/mL) was added 4 eq. TCEP followed by EDTA
(100mM; 12 L). After 2 h at rt, LCMS analysis indicated that the disulfide
adducts at
position C102 had been completely reduced. The reduced mAb was treated with
Zebra
desalting spin column (7x10mL, 7K MVVCO, pre-equilibrated with Tris-acetate
100mM
pH 5.6) to remove the liberated cysteines/GSH. To this reduced mAb was added a
solution of the bromoacetylated amylinomimetic peptide from Example 3 in Milli
Q
grade water (7 eq vs mAb, 30-35 mg/mL) followed by EDTA (100mM; 13.5 L). The
pH of the reaction was adjusted to 7.9 by dropwise addition of 1M Tris-acetate
buffer
(pH ¨ 9). The reaction was allowed to proceed overnight at rt with gentle
agitation. The
reaction was then diluted with sat'd (NH4)2504 (10% v/v) and the crude
conjugate was
purified by hydrophobic interaction chromatography (TOSOH TSKgel Phenyl BIC),
eluting with a linear gradient (40-100% B/A, solvent A: 5% i-PrOH, 1M
(NH4)2504,
100mM phosphate buffer, pH 6.0; solvent B: 20% i-PrOH, 100mM phosphate buffer,
pH
6.0). Final purification was achieved by protein A adsorption (PBS buffer) and
elution
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(Na0Ac, pH 3.5). The pH of the product was adjusted to 6 with 2.5M Tris (pH
7.7; 10
v%) and dialyzed against PBS to give the final sample.
Example 5: Peptide and Peptide-Bioconjugate Analysis and Characterization
[00261] Method A: Purified peptides were analyzed by LC/MS on an Hewlett
Packard
Series 1100 MSD system configured with an HP 1100 series HPLC using a Waters
Atlantis T3 C18 (4.6x250 mm, 300 A, 5 lam) column. Depending on the polar/non-
polar
nature of the peptide, one of two linear gradients was used (buffer A: Water +
0.1% TFA;
buffer B: MeCN + 0.1% TFA) at a flow rate of 1 mL/min and a column temperature
of
35 C [Method Al: 15 ¨60 %B over 22 min; Method A2: 40 ¨ 90 %B over 22 min].
Electrospray analysis (ES-API, positive ion scan) provided mass analysis for
each
peptide. In all cases, multiple charged species were observed with 1/3[M+3]+
and
1/4[M+4]+ ions being the characteristic, most prominently observed ions. All
products
yielded their expected multi-charged ions within acceptable limits. Results of
the mass
spectral analyses of the peptides and observed LC retention times (RT) are
shown in
Table 1.
[00262] Method B: Purified peptides were analyzed by HPLC on a Shimadzu 10AVP
system using a YMC-Pack-ODS-A (4.6x250 mm, 200A, 5 lam) column. A linear
solvent
gradient (20 ¨ 80 %B over 30 min) was used (buffer A: Water + 0.05% TFA;
buffer B:
MeCN + 0.05% TFA) at a flow rate of 1 mL/min. Mass spectra were obtained on a
Waters Xevo G2 ToF spectrometer (TOF MS ES, positive ion scan). In all cases,
multiple charged species were observed with 1/3[M+3]+ and 1/4[M+4]+ ions being
the
characteristic, most prominently observed ions. All products yielded their
expected
multi-charged ions within acceptable limits. Results of the mass spectral
analyses of the
peptides and observed LC retention times (RT) are shown in Table 1.
[00263] Method C: Purified amylinomimetic peptide-mAb conjugates were analyzed
by hydrophobic interaction chromatography (MC) on an Hewlett Packard Series
1100
MSD system configured with an HP 1100 series HPLC using a MAbPac MC-10
(4.6x1000 mm, 1000 A, 5 lam) column. A linear solvent gradient (0 ¨ 100 %B
over 30
min) was used (buffer A: 5% i-PrOH, 1.5M (NH4)2504, 100mM phosphate buffer, pH
.. 6.0; buffer B20% i-PrOH, 100mM phosphate buffer, pH 6.0) at a flow rate of
0.5
mL/min. Intact mass measurements were obtained on a Waters Xevo G2-XS QToF
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spectrometer (TOF MS ES, positive ion scan). Results of the analytical
characterization
of the amylinomimetic peptide-mAb conjugates are shown in Table 1.
Table 1: Analytical data for Thioether-cyclized Amylinomimetic Compounds.
Seq.
MW MVV 1/3[M+3]+ 1/3[M+3] + 1/4[M+4] + 1/4[M+4] + 1-1113LC RT
I.D.
Found (Calc' d) (Found) (Calc' d) (Found)
Method (min)
No.
4 3760.2 1254.4 1254.3 941.1 941.0 B
15.3
3831.2 1278.1 1277.8 958.8 958.7 Al 15.5
6 3774.2 1259.1 1258.9 944.6 944.4 B
14.2
7 3831.3 1278.1 1278.0 958.8 958.7 B
11.4
8 3873.3 1292.1 1291.7 969.3 969.2 Al
16.0
9 3817.2 1273.4 1273.2 955.3 955.2 B
13.7
3859.3 1287.4 1287.0 965.8 965.7
Al 16.3
11 3831.3 1278.1 1278.0 958.8 958.8 B
11.4
12 3845.3 1282.8 1282.7 962.3 962.3 B
13.0
13 3845.3 1282.8 1282.7 962.3
962.2 B 12.3
14 3845.3 1282.8 1282.7 962.3 962.3 B
11.8
3845.3 1282.8 1282.5 962.3 962.2 B
14.4
16 3871.3 1291.4 1291.3 968.8 968.8 B
14.9
17 3857.3 1286.8 1286.7 965.3 965.3 B
12.7
18 3871.3 1291.4 1291.3 968.8 968.7 B
13.8
19 3845.3 1282.8 1282.5 962.3 962.1 B
13.2
3831.3 1278.1 1278.0 958.8 958.7 B
13.2
21 3845.3 1282.8 1282.7 962.3
962.2 B 13.1
22 3831.3 1278.1 1278.0 958.8 958.7 B
14.6
23 3918.4 1307.1 1306.9 980.6
980.4 B 12.7
24 3904.3 1302.4 1302.3 977.1 977.0 B
15.3
3847.3 1283.4 1283.4 962.8 962.8 B
12.4
26 3847.3 1283.4 1283.4 962.8 962.8 B
12.2
27 3857.4 1286.8 1286.7 965.4 965.3 B
12.4
28 3448.9 1150.6 1150.2 863.2 862.9 B
12.9
29 4317.9 1440.3 1440.1
1080.5 1080.5 A2 11.1
4447.1 1483.4 1483.2
1112.8 1112.6 A2 10.3
31 4523.9 1509.0 1508.8 1132.0 1131.8 Al
16.4
32 4523.9 1509.0 1508.8
1132.0 1131.9 Al 15.9
33 4510.8
1504.6 1504.4 1128.7 1128.6 Al 16.2
34 4539.9 1514.3 1514.1
1136.0 1135.8 Al 17.1
4539.9
1514.3 1514.2 1136.0 1135.9 Al 16.6
36 4526.9 1510.0 1509.9
1132.7 1132.7 Al 16.9
37 155396 155394 C
25.1
38 155396 155393 C
23.4
39 155369 155363 C
22.4
155428 155418 C
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41 155428 155418 C
27.2
42 155402 155388 C
26.2
Example 6: Human Calcitonin/RAMP3 Receptor cAMP Assay (AMY3R Assay)
[00264] The method used to test the potency of amylinomimetic analogs in vitro
was a
cell based assay designed to measure cAMP produced by adenylate cyclase
through
modulation of the human calcitonin G-protein coupled receptor through its
interaction
with receptor activity-modifying protein 3 (CTR/RAMP3). Production of cAMP in
human AMY3R-transfected 1321N1 astrocytoma cells (DiscoverX) was induced by
amylinomimetic analogs and controls in a dose-dependent manner, and measured
in the
LANCE FRET-based competitive cAMP immunoassay (PerkinElmer).
[00265] Cells were cultured in DMEM, 10% FBS, 2.5 Kg/m1 puromycin and 800
[Ig/[11
of G418. For assay, the cells were collected by removing the media, washing
with PBS
and versene to lift the cells (Life Technologies). Cells were centrifuged at
450 x g for 5
min, and supernatants were aspirated. Cells were resuspended in lx HMS (Life
Technologies), 5 mM HEPES (Life Technologies), 0.1% BSA (Perkin Elmer), 1.0 mM
3-
Isobuty1-1-methylxanthine (IBMX) (Sigma) at 0.5 x 106 cells/ml, and 10 [IL of
suspended cells was added to each well of a 384-well white opti-plate
(PerkinElmer) to a
final density of 5000 cells/well. Dilutions of amylin analogs and controls
were prepared
in lx HMS, 5 mM HEPES, 0.1% BSA, and 10 [IL/well of each sample were added to
designated wells. Plates were incubated with shaking at rt for 30 min. Then 20
[IL/well
LANCE cAMP detection reagent mix (PerkinElmer) was added to each assay plate,
which was incubated with shaking at rt for 2 h to 24 h. Plates were read on a
Perkin
Elmer Envision plate reader using a protocol based on the manufacturer
recommendations included in the LANCE Ultra cAMP Kit. All samples were
measured
in quadruplicate. Data were analyzed using the Crucible in-house data analysis
software,
designed by Eudean Shaw, to derive parameters such as EC50, LogEC50, HillSlope
(nH),
top, and bottom, by plotting raw LANCE cAMP values versus log compound
concentrations. Data were fitted with 4-P model using a non-linear weighted
least
squares application within R environment (Open Source http://cran.us.r-
project.org/)
implemented by the NonClinical Statistics & Computing department at Janssen
R&D.
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[00266] The potencies of the amylinomimetic analogues of the present invention
relative to that of pramlintide, which was used as a control in the same
assay, are
presented in Table 2 below:
Table 2: AMY3 Receptor Potencies of Thioether-cyclized Amylinomimetic
Compounds
and Pramlintide (Seq. 2)
AMY3R AMY3R Relative
Seq. I.D. EC50 (pM) EC50 (pM) potency (fold
No. change rel. to
Seq 4-42 Seq 2 Seq 2)
4 2.9 0.49 -6.0
5 33 2.4 -13.8
6 0.28 0.36 1.3
7 0.43 0.36 -1.2
8 1.12 0.38 -2.9
9 447 0.56 -794
10 198 0.38 -521
11 78 0.56 -139
12 11 0.49 -22
13 0.16 0.40 2.4
14 0.42 0.43 1.0
15 39 0.49 -79
16 22 0.49 -45
17 1800 0.45 -4027
18 33 0.49 -68
19 0.23 0.41 1.8
20 4.9 0.45 -11
21 18 0.45 -39
22 471 0.56 -845
23 14 0.45 -32
24 183 0.56 -326
25 0.37 0.41 1.1
26 2.2 0.40 -5.5
27 0.32 0.36 1.1
28 0.059 0.36 6.1
29 0.064 0.41 6.4
30 0.028 0.41 14.6
31 ND
32 ND
33 ND
34 ND
35 ND
36 ND
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37 8.6 0.19 -45
38 313 0.19 -1647
39 1485 0.19 -7816
40 11.4 0.19 -60
41 430 0.19 -2263
42 1840 0.19 -9684
Example 7: Human Calcitonin/RAMP1 Complex cAMP Assay (AMY1R Assay)
[00267] The potency and selectivity of amylinomimetic analogs in vitro was
assessed
using a cell based assay designed to measure cAMP production following
modulation of
the human CTR or CTR/RAMP1 complex (AMY1R). Production of cAMP in human
CTR or A1V1Y1R transiently transfected COS7 cells was induced by
amylinomimetic
analogs and controls in a dose-dependent manner, and measured using a HTRF
cAMP kit
(CisBio cAMP Dynamic kit kit, Cat # 62AM4PEC).
[00268] The plasmid encoding the HA-tagged human calcitonin receptor was
generated
by sub-cloning human CTR ORF (ENST00000426151.5), tagged at its N-terminus
immediately after the signal peptide with 3xHA, into pcDNA3.1(+) using EcoRV
and
XhoI. The plasmid encoding Flag-tagged human RAMP1 was generated by sub-
cloning
human RAMP1 ORF (ENST00000254661.4), tagged at the N-terminus immediately
after
the signal peptide with a Flag-tag (DYKDDDDK (SEQ ID NO: 66)), into
pcDNA3.1(+)
using EcoRV and XhoI.
[00269] COS-7 cells were cultured in DMEM (ThermoFisher Scientific # 11965092)
containing 10% FBS (Hyclone # 5H3 0070.03) and 1% Penicillin-Streptomycin
(ThermoFisher Scientific # 15140122) and transfected using Fugene HD (Promega
#
E2312) in 384-well white poly-D-lysine coated plates (Corning # 356663). For
each
condition, the DNA (p,g):Fugene HD (pL) mix ratio was 1:3 and 10,000
cells/well were
added in 40 [IL on top of 10 [IL DNA:Fugene mix. Plates were incubated at 37
C in a
CO2 incubator for 48 h. The CTR:RAMP1 cDNA transfection ratio (2:9) was
optimized
to favor the formation of the amylin-1 receptor (AMY1R) and the amount of CTR
cDNA
transfected was optimized such that the expression of the calcitonin and
amylin receptors
at the cell surface were not significantly different, as assessed by ELISA
against the HA
tag.
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[00270] The day of the assay, the culture media was replaced with assay buffer
containing HMS with calcium and magnesium, 20 mM HEPES and 0.1% Fatty acid
free
BSA, pH 7.4, and cells were starved for 1 h at 37 C. The assay buffer was
then replaced
with fresh assay buffer containing 500 [IM IBMX, and compounds were added in
assay
.. buffer (no IBMX). Plates were incubated with shaking at rt for 30 min. cAMP
was
detected according to the manufacturer's protocol (CisBio cAMP Dynamic kit,
Cat #
62AM4PEC). Fluorescence was read with a PHERAstar plate reader using an
excitation
of 337 nm and emissions of 620 and 665 nm. Data were normalized on the maximal
response of pramlintide. Emax and EC50 determinations were made from an
agonist-
response curve analyzed with a curve fitting program using a 4-parameter
logistic dose
response equation in Graphpad Prism 7Ø Data presented are representative of
three
independent experiments performed in quadruplicate for each compound. Data are
represented as averages. AMY1R potencies of compounds relative to that of
pramlintide
(seq 2) are represented as the fold change. Potencies of compounds at the
AMY1R
relative to their potencies at the CTR are also presented as fold differences
(Table 3).
[00271] For cell surface receptor expression determinations, cells from the
same
transfection as that used for the cAMP assay were plated into 96-well plates,
fixed with
4% paraformaldehyde and blocked with PBS + 1% FBS. Rat anti-HA-Peroxidase
(clone
3F10, Roche Bioscience # 12013819001) was applied for 30 min at 0.5 mg/L.
After
washes with blocking buffer and PBS, chemoluminescence was detected using
SuperSignal substrate (Pierce, Rockford, IL, USA) and a PHERAstar plate
reader.
Table 3: AMY1 and CT Receptor Potencies of Thioether-cyclized Amylinomimetic
Compounds and Pramlintide (Seq. 2)
AMY1R
AMY1R CTR Relative
Relative potency
Seq. I.D. ECso (PM) potency (fold EC50(nM) AMY1R vs
No. change rel. to CTR (fold
Seq 37-42 Seq 37-42
Seq 2) difference)
2 2.3 1 0.061 27
37 64 -27 3.5 55
38 82 -35 36.8 448
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39 197 -85 >500 >1000
40 55 -24 1.6 29
41 85 -37 31.2 367
42 287 -123 >500 >1000
Example 8: Efficacy Studies In Vivo
[00272] Gastric Emptying: Acetaminophen (AAP) Absorption in Lean C57B1/6N Mice
[00273] Male lean C57BL/6 mice (6-8 weeks of age) were obtained from Taconic
Laboratory. Mice were housed one mouse per cage with AlphaDri bedding in a
temperature-controlled room with 12-h light/dark cycle. Mice were allowed ad
libitum
access to water and maintained on a regular diet (Lab Diet Cat: 5K75). Animals
were
acclimated to the facility for at least one week prior to the start of the
experiment.
[00274] The day prior to dosing, mice were grouped into cohorts of ten animals
based
on individual body weights. At 5:00 - 6:00 pm the following day, animals were
deprived
of food and treated with either vehicle (PBS, pH 7.4) or test compound at a
dose of 30
nmol/kg (3 nmol/mL) via subcutaneous administration. After 18 h, an
acetaminophen
(AAP) suspension mixture [AAP (10 mg/mL); EIPMC (5 mg/mL); acacia gum (50
mg/mL)] was administered to the animals (10 mL/kg) by oral gavage. Whole blood
.. samples (tail snip; ¨ 25 L) were collected at 5, 10, 15, 30 and 60 min
time points into
DMPK-C Dry Blood Spot Cards. Blood dot cards were dried completely and placed
into
individual bags with desiccant pending LC/MS analysis by standard techniques.
Statistical analyses were performed using one-way ANOVA with Dunnett's post-
test in
Prism. All data are presented as the mean.
.. [00275] Food Intake in Fasted Lean C57BL6N Mice: Acute Dosing
[00276] Male C57BL/6 mice (6-8 weeks of age) were obtained from Taconic
Laboratory. Mice were housed one mouse per cage with AlphaDri bedding in a
temperature-controlled room with 12-h light/dark cycle. Mice were allowed ad
libitum
access to water and maintained on a regular diet (Lab Diet Cat: 5K75). Animals
were
acclimated in BioDAQ cages (Research Diets, Inc., New Brunswick, NJ) no less
than 72
h prior to the start of the experiment.
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[00277] Once acclimated in the BioDAQ cages, mice were grouped into cohorts of
ten
animals based on their individual body weights and food intake over the
previous 24 h.
At 4:00-5:00 pm, animals were weighed and treated with either vehicle (PBS, pH
7.4) or
test compound at a dose of 30 nmol/kg (3 nmol/mL) via subcutaneous
administration.
Following a subsequent overnight fasting period (16-18 h), changes in food
weight for
each cage were recorded continuously by the BioDAQ automated monitoring system
for
the next 48 h. Crumbs were removed daily from hoppers and the areas around the
cages
with a vacuum. Food was replenished as necessary. The percentage of mean
cumulative
food intake relative to vehicle over the 12-48 h period following dosing was
calculated
and is reported in Table 4. Statistical analyses were performed using two-way
ANOVA
with Dunnett's post-test in Prism. All data are presented as the mean.
Table 4: In Vivo Efficacy Studies of Thioether-cyclized Amylinomimetic
Compounds
AAP Absorption AAP Absorption
Seq. I.D. C57BL6 Mice C57BL6 Mice Food Intake in Food Intake in
Fasted Lean Mice Fasted Lean Mice
No. [AAP] @15 min AUCo-90
% Change (12-36h) % Change (12-48h)
(}1M) (uM=min)
Vehicle 489 20835
40 320 13292 -20.2***
41 437* 18904 -2.7 -8.8
42 444* 18325 ND ND
ND = not determined
*p< 0.05; **p< 0.01; ***p< 0.001; p< 0.0001
Example 9: PK Studies in Male C57BL6N Mice
[00278] Male C57BL/6N mice (6-8 weeks of age, Taconic) were single-housed with
AlphaDri bedding in a temperature-controlled room with 12-hour light/dark
cycle and
given free access to LabDiet5K75 rodent diet and drinking water. Mice were
grouped (N
= 3) based upon fed body weight; compounds were formulated in PBS (1 nmol/mL)
and
administered subcutaneously at a dose of 10 nmol/kg. Whole blood samples (tail
snip; ¨
50 [IL) were collected at 4h, 24h, 72h, 96h and 7d time points into EDTA-
coated Sarstedt
Microvette tubes containing a protease inhibitor cocktail (Roche complete
protease
inhibitors and Millipore DPPIV inhibitors; 2.5 [IL) and placed on ice. The
final bleed (7
d) was a terminal bleed with a target volume of 500 [IL (25 [IL of protease
inhibitor
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cocktail). Samples were then centrifuged (10,000 rpm) at 4 C for 10 minutes;
plasma
was then transferred to a 96-well plate (-25 L/well of plasma) which was
stored at -80
C pending bioanalysis.
[00279] Plasma samples were analyzed using an LC-MS/MS assay of surrogate
peptides for quantitation. In this assay, the analytes were extracted from
plasma using
immuno-affinity capture by an anti-human IgG Fc antibody, followed by protease
digestion (trypsin or pepsin) and reversed phase LC-MS/MS analysis. The
multiple
reaction monitoring (MRM) MS analysis was conducted on an API5000 triple
quadruple
mass spectrometer operated in positive electrospray mode. The peptide derived
from N-
terminal region of the amylinomimetic sequence was monitored as a surrogate
for
quantitation of active conjugate, while a peptide located on Fc region of the
mAb was
monitored as a surrogate for the total level of mAb. Standard curve and
quality control
samples were prepared by spiking the reference standards of the amylinomimetic
conjugates in plasma and were processed simultaneously using the same
procedure as the
study samples. Data are shown in Table 5.
Table 5: Pharmacokinetic Parameters of Thioether-cyclized Amylinomimetic
Compounds in Male C57BL/6N mice
Seq. I.D. t112 Cmax AUC0-168
No. (days) (nmol/L) (h=nmol/L)
37 4.5 86.5 10033
38 2.7 73.5 5743
39 2.4 64.8 4701
40 1.9* 137.3 15765
41 2.4 81.3 6656
42 2.7 83.2 6544
*estimated from limited time-point sampling taken during elimination phase
[00280] It will be appreciated by those skilled in the art that
changes could be
made to the embodiments described above without departing from the broad
inventive
concept thereof. It is understood, therefore, that this invention is not
limited to the
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particular embodiments disclosed, but it is intended to cover modifications
within the
spirit and scope of the present invention as defined by the present
description.
[00281] .. All documents cited herein are incorporated by reference.
[00282] Exemplary amylinomimetic sequences or conjugates thereof of the
invention include:
SEQ ID NO: 1
Name: Amy lin(1-37)
.. Structure:
H¨K¨N/--- sNIA sT¨N ATQRLANFLVHSSNNFGAI LSSINVGSNTY¨NH2
H I H
0 0
SEQ ID NO: 2
Name: Pramlintide(1-37)
Structure:
s __________________ S
H¨K¨N NTAT¨N ATQRLANF LVHSSNNFGPI LPPTNVGSNTY¨NH2
H H
o o
SEQ ID NO: 3
Name: Davalintide(1-32)
Structure:
s ________________ s
H¨K¨N NTAT¨N O VLGRLSQELHRLQTYPRTNIGSNTY¨NH2
H H
o
SEQ ID NO: 4
Name: [cyclo-(N3- COCH2- C7)]-Pramlintide3-37
Structure:
ol/ s
NTAT:))-FATQR LANF LVHSSNNFGP1 LPPTNVGSNT y¨NFI,
H
o
SEQ ID NO: 5
Name: [cyclo-(N3- COCH2- hC7), K(Ac)26]-Pramlintide3-37
Structure:
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0
HN).L
/
{¨S
o
NTAT¨N
ATQRLANFLVHSSNNFGP-N LPPTNVGSNT y¨NH,
H H
0 o
SEQ ID NO: 6
Name: [cyclo-(N3- COCH2- hC7), f3-A5]-Pramlintide3-37
Structure:
___________ s ___
o...-_-,/
N
T¨it....."--1¨T-11 ATQRLANFLVHSSNNFGPI LPPTNVGSNT Y¨NH2
II
o o
SEQ ID NO: 7
Name: [cyclo-(N3- COCH2- hC7), f3-A5, K(Ac)26]-Pramlintide3-37
Structure:
0
HN).L
___________ S __
NT¨N.....7"-I¨Til ATQRLANFLVHSSNNFGP1 LPPTNVGSNTY¨NH2
H
0 0 o
SEQ ID NO: 8
Name: [cyclo-(N3- COCH2- hC7), f3-A5, K(Alloc)26]-Pramlintide3-37
Structure:
o
HNAO'''''':-.7-
___________ S __
0.._-õ/
NT-11-...../'IrTil ATQRLANFLVHSSNNFGP1 LPPTNVGSNTY¨NH2
o o o
SEQ ID NO: 9
Name: [cyclo-(N3- COCH2- C7), f3-A5, K(Ac)26]-Pramlintide3-37
Structure:
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0
HN)L
NT¨ENT¨ENI
ATQR LANFLVHSSNNFGP1 LPPTNVGSNT y¨NH2
o o o
SEQ ID NO: 10
Name: [cyclo-(N3- COCH2- C7), f3-A5, K(Alloc)26]-Pramlintide3-37
Structure:
0
HNAo""
0L-...,7 ______ s
NT¨NT¨hl ATQR LANF
LVHSSNNFGPifil LPPTNVGSNTY¨NH2
H
o o o
SEQ ID NO: 11
Name: [cyclo-(N3- COCH2- C7), Abu5, K(Ac)26]-Pramlintide3-37
Structure:
0
HN)..
______________ S
NT'NT¨N ATQR
LANFLVHSSNNFGP¨N LPPTNVGSNTY
, -NH,
H H H
0 0 0
SEQ ID NO: 12
Name: [cyclo-(N3- COCH2- hC7), Abu5, K(Ac)26]-Pramlintide3-37
Structure:
0
HN).
____________ s ___
NTT-1\11 ATQR LANF
LVHSSNNFGP¨il LPPINVGSNTY¨NH2
H 0 0 0
SEQ ID NO: 13
Name: [cyclo-(N3- COCH2- hC7), (5)-(3-Aib5, K(Ac)26]-Pramlintide3-37
Structure:
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0
HN).
os _______________
NT-ii LPPTNVGSNTY¨NH,
SEQ ID NO: 14
Name: [cyclo-(N3- COCH2- hC7), (R)- f3-Aib5, K(Ac)26]-Pramlintide3-37
Structure:
HN
os _______________
N
ATQRLANFLVHSSNNFGP-N LPPTNVGSNTY¨NH2
H H
SEQ ID NO: 15
Name: [cyclo-(N3- COCH2- hC7), f3-hA5, K(Ac)26]-Pramlintide3-37
Structure:
0
HN)L
NT-1 LPPTNVGSNTY
. ¨NH2
o 0
SEQ ID NO: 16
Name: [cyclo-(N3- COCH2- hC7), f3-hP5, K(Ac)26]-Pramlintide3-37
Structure:
0
HN)L
N T¨NT¨NA TO R
LANF LVHSSNNFGPil LPPTNVGSNT y¨NH2
(,) 0 0 0
SEQ ID NO: 17
Name: [cyclo-(N3- COCH2- hC7), {pyrrolidinyl-(3S)-carboxyl} 5, K(Ac)26]-
Pramlintide3-37
Structure:
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FIN).L
os _______________
1-Th
ATQR LANF LVHSSNNFGPifil
LPPTNVGSNTY¨NH2
0 0 0
SEQ ID NO: 18
Name: [cyclo-(N3- COCH2- hC7), {(1R,2R)-ACPC} 5, K(Ac)26]-Pramlintide3-37
Structure:
0
HN)
0
N T¨N ATQR LANF LVHSSNNFGP¨N
LPPTNVGSNTY¨NH,
0 0
SEQ ID NO: 19
Name: [cyclo-(N3- COCH2- hC7), f3-hT4, K(Ac)26]-Pramlintide3-37
Structure:
0
HN)L
os
A T¨N ATORLANF LVHSSNNFGP¨N
LPPTNVGSNTY¨NH2
8 H a H 0
SEQ ID NO: 20
Name: [cyclo-(N3- COCH2- C7), f3-hT4, K(Ac)26]-Pramlintide3-37
Structure:
(:)
HN)L
AT0RLANELVHSSNNFGP¨N
LPPTNVGSNTY¨NE12
H 0 H 0
SEQ ID NO: 21
Name: [cyclo-(N3- COCH2- hC7), f3-hT6, K(Ac)26]-Pramlintide3-37
Structure:
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0
HN)L
___________ S __
01/
H
NTA-N,..-õ.,-ri
-- N ATQRLANF LVHSSNNFGP-
N LPPTNVGSNTY-NH2
H
0 H
0
0
OH
SEQ ID NO: 22
Name: [cyclo-(N3- COCH2- C7), f3-hT6, K(Ac)26]-Pramlintide3-37
Structure:
0
H N--11",
_____________ s
01/
H
NTA-N,,. N ATQRLANF
LVHSSNNFGP-N LPPTNVGSNTY-NH2
H H
0 0
0
SEQ ID NO: 23
Name: [cyclo-(52- COCH2- hC7), f3-A5, K(Ac)26]-Pramlintide2-37
Structure:
0
)NH
S __________________
0..--,-..õ..----
H
SNT- _
N-----7)7T¨N ATQRLANF LVHSSNNFGP-N LPPTNVGSNTY-NH2
H H
0 0 0
SEQ ID NO: 24
Name: [cyclo-(52- COCH2- C7), f3-A5, K(Ac)26]-Pramlintide2-37
Structure:
0
NH
S
0...--,....õ/ __
H
SNT-N
----.Z)i-T-N ATQRLANF LVHSSNNFGP-N LPPTNVGSNTY-NH2
H H
0 0 0
SEQ ID NO: 25
Name: [cyclo-(N3- COCH2- hC7), f3-A5, K(Ac)25]-Pramlintide3-37
Structure:
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0
H N)L
____________ S __
0_,...._,../
NT¨EN1i-fl¨T¨E1
ATQR LANFLVHSSNNFG1 I LPPTNVGSNTY¨NH,
0 0 0
SEQ ID NO: 26
Name: [cyclo-(N3- COCH2- hC7), f3-A5, K(Ac)29]-Pramlintide3-37
Structure:
0
H N)L
____________ S __
NT¨ft-fir-T-11
ATQR LANFLVHSSNNFGPI LP1 TNVGSNT y¨NH2
o o o
SEQ ID NO: 27
Name: [cyclo-(N3- COCH2- hC7), f3-A5, K(Ac)34]-Pramlintide3-37
Structure:
0
H N)L
____________ S __
N T¨[1.-_,-----r-T¨INI
ATQRLANFLVHSSNNFGPI LPPTNVG1 NTY¨NH,
0 0 0
SEQ ID NO: 28
Name: [cyclo-(N3- COCH2- hC7), f3-A5]-Dayalintide3-32
Structure:
s ___________________
o__-_ ________
H
N T¨N
T¨N VLGRLSQELHRLQTYPRTNTGSNT Y¨N H2
H I
0 0
SEQ ID NO: 29
Name: [cyclo-(N3- COCH2- hC7), f3-A5, K(OEG2-Pa1)26]-Pramlintide3-37
Structure:
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0
NH
L.
0
N ATQRLANF LVHSSNNFGP-N LPPTNVGSNTY-NH2
0 0 0
SEQ ID NO: 30
Name: [cyclo-(N3- COCH2- hC7), f3-A5, K(OEG2-y-G1u-Pa1)26]-Pramlintide3-37
Structure:
0HO.O
0
2 NH
0
0
N N
T A TQRL ANF LVHSSNNFGP- LPP TNVGSNT Y-
8H2
0 0 0
SEQ ID NO: 31
Name: [cyclo-(N3- COCH2- hC7), f3-A5, a-MeL12, K(dPEG12-AcBr)26]-Pramlintide3-
37
Structure:
BrO
0NH
0
H
NT-N N ATQR-N * ANF LVHSSNNFGR-FLPPTNVGSNTY-NH2
0 0 0 0
SEQ ID NO: 32
Name: [cyclo-(N3- COCH2- hC7), f3-A5, hR11, K(dPEG12-AcBr)26]-Pramlintide3-37
Structure:
NH2
Br( NO 12",
H NN H 0
0/
A TQ-N _________________________________________________ L ANF LVHSSNNFGP-N
LRRTNVGSNTY-NH2
H I
0 0 0 0
SEQ ID NO: 33
Name: [cyclo-(N3- COCH2- hC7), f3-A5, El 0, K(dPEG12-AcBr)26]-Pramlintide3-37
Structure:
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BryN
0
0NH
O
NT¨N N
ATERLANFLVHSSNNFGP¨N LPPTNVGSNTY¨NH2
0 0 0
SEQ ID NO: 34
Name: [cyclo-(N3- COCH2- hC7), f3-A5, a-MeL12, K(dPEG12-AcBr)25]-Pramlintide3-
37
Structure:
BrO
os
NT ATQR-VNIFANF LVHSSNNFG-NILPPTNVGSNTY-NH2
0 0 0 0
SEQ ID NO: 35
Name: [cyclo-(N3- COCH2- hC7), f3-A5, hR11, K(dPEG12-AcBr)25]-Pramlintide3-37
Structure:
NH2
HANH 0
ONH
0
H
N T-N N
ATQ-N LANF LVHSSNNFG-N _______ I LPPTNVGSNTY-NH2
H
0 0 0
SEQ ID NO: 36
Name: [cyclo-(N3- COCH2- hC7), f3-A5, El 0, K(dPEG12-AcBr)25]-Pramlintide3-37
Structure:
12
0
NH
01/
N T-N N
ATE NFL LVHSSNNFG-N ______ I LPPTNVGSNTY-NH2
H
0 0 0
SEQ ID NO: 37
Name: [cyclo-(N3- COCH2- hC7), f3-A5, a-MeL12, K(dPEG12)26]-Pramlintide3-37
mAb homodimer conjugate
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Structure:
H
N A T Q R-N T ANF LVHSSNNFGP2O-LPPTNVGSNTY-NH2
HN,e0
____________________________________________________________________________
mAb
12 H 2
SEQ ID NO: 38
Name: [cyclo-(N3- COCH2- hC7), f3-A5, hR11, K(dPEG12)26]-Pramlintide3-37 mAb
homodimer conjugate
Structure:
HI"NH
I H
N
AT _____________________________ LANF LVHSSNNFGP-LPPTNVGSNTY-NH2
0 0
H Ny0
_____________________________________________________________________________
mAb
____________________________________________________________________________ 2
SEQ ID NO: 39
Name: [cyclo-(N3- COCH2- hC7), f3-A5, E10, K(dPEG12)26]-Pramlintide3-37 mAb
homodimer conjugate
Structure:
0
N ATERLANF LVHSSNNFGP¨Qi¨LPP TNVGSNTY¨NH,
0 0
HN,,e0 0
S __ mAb
12 H
2
SEQ ID NO: 40
Name: [cyclo-(N3- COCH2- hC7), f3-A5, a-MeL12, K(dPEG12)25]-Pramlintide3-37
mAb homodimer conjugate
Structure:
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01 H
N
ATQR¨INI
ANFLVHSSNNFG¨EN1J¨ILPPTNVGSNTY¨NH2
0 0 0
HN 0
______________________________________________________________________________
mAb
12 H 2
SEQ ID NO: 41
Name: [cyclo-(N3- COCH2- hC7), f3-A5, hR11, K(dPEG12)25]-Pramlintide3-37 mAb
homodimer conjugate
Structure:
NH2
HNANH
01
N T¨N N
ATQ¨N LANF LVHSSNNFG-0 ____________________________
I LPPTNVGSNTY-NH2
0 0 0
0
______________________________________________________________________________
mAb
12 H ________________________________________________________________________
2
SEQ ID NO: 42
Name: [cyclo-(N3- COCH2- hC7), f3-A5, E10, K(dPEG12)25]-Pramlintide3-37 mAb
homodimer conjugate
Structure:
o
N T¨ T¨H N N
----7.)F ATE RLANF LVHSSNNFG-0 ________________________________
I LPPTNVGSNTY¨NH2
0 0
0
S ____________________________________________________________________________
mAb
12 H ________________________________________________________________________
2
SEQ ID NO: 43
Name: MSCB97 VEI (heavy chain variable region)
EVQL LES GGGLVQP GGS LRL S CAAS GET FS SYAMSWVRQAPGKGLEWVSAI S GS GGS
TYYADSVKGRFT I S
RDNS KNT LYLQMNS LRAEDTAVYYCAKYDGCYGELDFWGQ GT LVTVS S
SEQ ID NO: 44
103
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Name: MSCB97 HC (heavy chain)
EVQLLES GGGLVQP GGS LRL S CAAS GET FS SYAMSWVRQAPGKGLEWVSAI S GS GGS
TYYADSVKGRFT I S
RDNSKNTLYLQMNSLRAEDTAVYYCAKYDGCYGELDFWGQGTLVTVS SAS TKGP SVFPLAPCS RST S ESTA
ALGCLVKDYFPEPVTVSWNS GALT S GVHT FPAVLQS S GLYSLS SVVTVPSSSLGTKTYTCNVDHKPSNTKV
DKRVESKYGP PCP P CPAPEAAGGP SVFLFP PKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH
NAKT KPREEQ FNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGL PS SI EKT I S KAKGQ PRE PQVYTLP
PSQE
EMTKNQVS LT CLVKGFYP S D IAVEWESNGQ PENNYKT T P PVLD S DGS FFLYS RLTVDKS
RWQEGNVFS CSV
MHEALHNHYTQKS L S LS LGK
SEQ ID NO: 45
Name: MSCB97 VL (light chain variable region)
EIVLTQS PAT L S L S PGERATLSCRASQSVS SYLAWYQQKP GQAPRLL I YDASNRATGI PARES GS
GS GTDF
TLT I S S LEPEDFAVYYCQQRSNWPLT FGQGTKVEI K
SEQ ID NO: 46
Name: MSCB97 LC (light chain)
EIVLTQS PAT L S L S PGERATLSCRASQSVS SYLAWYQQKP GQAPRLL I YDASNRATGI PARES GS
GS GTDF
TLT I SSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIKRTVAAPSVFI FP P S DEQLKS GTASVVCLLNNFYP
R
EAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGE
c
SEQ ID NO: 47
Name: MSCB97 HCDR1
S YAMS
SEQ ID NO: 48
Name: MSCB97 HCDR2
AI S GS GGSTYYADSVKG
SEQ ID NO: 49
Name: MSCB97 HCDR3
YDGCYGELDF
SEQ ID NO: 50
Name: MSCB97 LCDR1
RASQSVS SYLA
SEQ ID NO: 51
Name: MSCB97 LCDR2
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DASN RAT
SEQ ID NO: 52
Name: MSCB97 LCDR3
QQRSNWPLT
105
