CN112074289A - Thioether cyclic peptide amylin receptor modulators - Google Patents

Thioether cyclic peptide amylin receptor modulators Download PDF

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CN112074289A
CN112074289A CN201980027903.XA CN201980027903A CN112074289A CN 112074289 A CN112074289 A CN 112074289A CN 201980027903 A CN201980027903 A CN 201980027903A CN 112074289 A CN112074289 A CN 112074289A
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S·欣克
W·简
R·帕奇
R·张
S·郑
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Janssen Pharmaceutica NV
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Abstract

The present invention relates to amylin mimetic peptide analogs, and derivatives thereof, in which the N-terminus of each peptide is covalently linked to an internal amino acid side chain thiol functional group through a nonpeptidyl cyclization bridge element. Thioether-cyclized amylin mimetic analogs and derivatives thereof may comprise one or more alterations (including substitutions, insertions, deletions, and modifications), and may optionally comprise a serum albumin binding element such as an alkyl chain of at least 14 carbon atoms, optionally with additional pendant carboxylate moieties or half-life extending biological moieties such as HSA, non-targeting mabs, or Fc. In addition, the present invention relates to compositions thereof and methods of treating conditions responsive to amylin receptor modulation.

Description

Thioether cyclic peptide amylin receptor modulators
This application claims the benefit of U.S. provisional patent application serial No. 62/662,492, filed 2018, 4, 25, which is hereby incorporated by reference in its entirety.
Sequence listing
This application contains a sequence listing that has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created in 2019 on 16.4.2019, named PRD3471WOPCT1_ sl. txt, and was 55,747 bytes in size.
Technical Field
The present invention relates to thioether-cyclized analogs of amylin, pramlintide and davalintide and derivatives thereof (referred to herein as "amylin mimetics") that act as agonists of the amylin receptor and are therefore useful in the treatment of metabolic diseases and disorders, such as obesity, type 2 diabetes, metabolic syndrome, insulin resistance and dyslipidemia.
Background
Amylin is a naturally occurring 37 amino acid peptide that is a structurally related member of the calcitonin family of peptides, including Calcitonin (CT), calcitonin gene-related peptide (CGRP), Adrenomedullin (AM) and intermediums (AM 2). It is synthesized and secreted by the pancreas in response to the inflow of nutrients into the gastrointestinal tract. After release into the circulation, amylin binds with high affinity to specific class B GPCRs located predominantly in the rearmost region of the hindbrain region of the central nervous system. Thus, it is a centrally acting neuroendocrine hormone used to regulate glucose homeostasis by inhibiting gastric emptying, inhibiting glucagon release and inducing satiety (reviewed in HayDL et al, Pharmacol Rev 2015; 67: 564-. Amylin has been found to interact in vitro with three target receptor subtypes, AMY1R, AMY2R and AMY3R, which are heterodimeric structures consisting of a calcitonin receptor (CTR) and a receptor modifying protein (RAMP 1, RAMP2 and RAMP3, respectively) (Christopoulos G et al, Mol Pharmacol 1999; 56: 235-42). The association of CTR with these RAMPs results in an increase in amylin affinity relative to CTR alone and provides the basis for selective receptor pharmacology of amylin (relative to CT) (Bower RL and Hay DL, Br J Pharmacol 2016; 173: 1883-98). While it is generally accepted that amylin is involved in high affinity/potency interactions, particularly with AMY1R and AMY3R, simultaneous agonism at all receptor subtypes may not necessarily be necessary for pharmacologically beneficial effects. (Hay DL et al, Br J Pharmacol 2018; 175: 3-17; Hay DL, Headache 2017; 57: 89-96).
Human amylin, also known as amylin islet amyloid polypeptide (IAPP), has several physicochemical properties that make it unsuitable for use as a pharmaceutical agent, including most notably its low water solubility and a tendency to self-aggregate and adhere to surfaces. Pramlintide, an equivalent amylin analog, 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 result in improved physicochemical properties (reduced tendency to aggregate) relative to amylin. Pramlintide reduces food intake (Smith SR et al, AAm J Physiol Endocrinol Metab 2007; 293: E620-7) and body weight (Aronne L et al, J Clin Endocrinol Metab 2007; 92:2977-83) in obese subjects. It is approved as an adjuvant therapy for insulin for the treatment of adult patients with type 1 diabetes, and as an adjuvant therapy for insulin alone or for concurrent treatment with metformin and/or sulfonylureas for adult patients with type 2 diabetes (Pullman J et al, Vasc Health Risk Manag 2006; 2: 203-12). The darvan peptide is related synthetic peptide with the length of 32 amino acids, and the structure of the darvan peptide is a chimera of pramlintide and salmon calcitonin primary sequence. Thus, it lacks the amyloid gene residues of human amylin (Westermark P et al, Proc Natl Acad Sci USA 1990; 87: 5036-40). It is a potent agonist of both the amylin and calcitonin receptors, which exhibits stronger pharmacological properties than native (rat) amylin in reducing food intake and body weight in rats (Mack CM et al, Int J Obes 2010; 34: 385-95). Amylin, pramlintide and davalin each have a very short in vivo half-life (<0.75h), which limits their practical therapeutic utility (Roth JD et al, Immun endo metals 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 requires a multiple daily administration regimen to achieve clinical effects. Thus, it would be desirable to obtain amylin agonist peptides or derivatives thereof having improved metabolic stability and pharmacokinetic characteristics.
One technique for extending the half-life of these peptides involves conjugation to a biological carrier, such as albumin, a suitable mAb or antigen-binding fragment thereof, or a mAb-derived crystalline fragment domain (Fc) protein. Such bioconjugation chemistry proceeds through the reaction of selectively reactive thiol functional groups on the bio-carrier molecule with complementary electrophilic sites engineered into the peptide of interest. Although the maleimide functional group incorporated onto the peptide molecule has served as a suitable electrophile for bioconjugation chemistry, the resulting bioconjugate can undergo a reverse conjugation reaction (reverse michael reaction) in vivo, resulting in the loss of the peptide from the biocontainer. Thus, the more stable thioacetamide bond formed by coupling to the reactive haloacetamide-derived peptide serves to avoid the possibility of such reverse conjugation. However, the conditions required for the bromoacetamide conjugation reaction cannot be successfully used with its selectively bioconjugated disulfide-containing amylin analogs due to the unhindered reactivity of the disulfide ring. Such attempted chemistry results in concomitant ring opening and associated complex side reactions. It has long been recognized that AN intact disulfide ring is a key molecular feature required for receptor activation and biological function (Roberts AN et al, Proc Natl Acad Sci USA 1989; 86: 9662-. Herein, an N-terminal cyclic thioether alternative for the dithiocysteine linkage of pramlintide and davalintide was identified that unexpectedly maintained amylin receptor agonist activity and was chemically stable to bioconjugation reactions.
Disclosure of Invention
In one general aspect, the present invention relates to novel amylin mimetic compounds. Also provided herein are amylin mimetic derivatives of pramlintide or davalintide and conjugates thereof comprising a monoclonal antibody or antigen-binding fragment thereof coupled to an amylin mimetic peptide.
In one aspect, the invention is represented by: a compound of formula I or a derivative thereof (SEQ ID NO:53)
Figure BDA0002739484940000031
Wherein
n is 1 or 2;
Z2is a direct bond, serine or glycine;
Z4is T or
Figure BDA0002739484940000041
Z5Is A, beta-alanine,
Figure BDA0002739484940000042
Figure BDA0002739484940000043
Z6Is T or
Figure BDA0002739484940000044
Z10Is Q or E;
Z11is R or K, wherein-amine of K is optionally substituted by-C (═ NH) NH2Substitution;
Z12is L or
Figure BDA0002739484940000045
Z16Is L or
Figure BDA0002739484940000046
Z25Is P or K, wherein-amine of said K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000047
Figure BDA0002739484940000048
Figure BDA0002739484940000051
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z26is I or K, wherein-amine of said K is optionally substituted with-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000052
Figure BDA0002739484940000053
Figure BDA0002739484940000054
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
x is ATZ10Z11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34(SEQ ID NO:54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55);
Z29is P or K, wherein-amine of said K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000055
Figure BDA0002739484940000056
Figure BDA0002739484940000061
Figure BDA0002739484940000062
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z34is S or K, wherein-amine of K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000063
Figure BDA0002739484940000064
Figure BDA0002739484940000065
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
and pharmaceutically acceptable salts thereof.
In certain embodiments, the invention is represented by: a compound of formula I or a derivative thereof, wherein:
n is 1 or 2;
Z2is a direct bond;
Z4is T or
Figure BDA0002739484940000071
Z5Is beta-alanine,
Figure BDA0002739484940000072
Z6Is T;
Z10is Q or E;
Z11is R or K, wherein-amine of K is optionally substituted by-C (═ NH) NH2Substitution;
Z12is L or
Figure BDA0002739484940000073
Z16Is L or
Figure BDA0002739484940000074
Z25Is P or K, wherein-amine of said K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000075
Figure BDA0002739484940000076
Figure BDA0002739484940000077
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z26is I or K, wherein-amine of said K is optionally substituted with-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000081
Figure BDA0002739484940000082
Figure BDA0002739484940000083
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
x is ATZ10Z11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34(SEQ ID NO:54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55);
Z29is P or K, wherein-amine of said K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000084
Figure BDA0002739484940000085
Figure BDA0002739484940000091
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z34is S or K, wherein-amine of K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000092
Figure BDA0002739484940000093
Figure BDA0002739484940000094
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
and pharmaceutically acceptable salts thereof.
In certain embodiments, the invention is represented by: a compound of formula I or a derivative thereof, wherein:
n is 1 or 2;
Z2is a direct bond or serine;
Z4is T or
Figure BDA0002739484940000095
Z5Is A, beta-alanine,
Figure BDA0002739484940000101
Figure BDA0002739484940000102
Z6Is T or
Figure BDA0002739484940000103
Z10Is Q or E;
Z11is R or K, wherein-amine of K is optionally substituted by-C (═ NH) NH2Substitution;
Z12is L or
Figure BDA0002739484940000104
Z16Is L;
Z25is P or K, wherein-amine of said K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000105
Figure BDA0002739484940000106
Figure BDA0002739484940000107
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z26is I or K, wherein-amine of said K is optionally substituted with-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000108
Figure BDA0002739484940000109
Figure BDA0002739484940000111
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
x is ATZ10Z11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34(SEQ ID NO:54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55);
Z29is P or K, wherein-amine of said K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000112
Figure BDA0002739484940000113
Figure BDA0002739484940000114
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z34is S or K, wherein-amine of K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000115
Figure BDA0002739484940000116
Figure BDA0002739484940000117
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
and pharmaceutically acceptable salts thereof.
In certain embodiments, the invention is represented by: a compound of formula I or a derivative thereof, wherein:
n is 1 or 2;
Z2is a direct bond;
Z4is T or
Figure BDA0002739484940000121
Z5Is beta-alanine,
Figure BDA0002739484940000122
Z6Is T;
Z10is Q or E;
Z11is R or K, wherein-amine of K is optionally substituted by-C (═ NH) NH2Substitution;
Z12is L or
Figure BDA0002739484940000123
Z16Is L;
Z25is P or K, wherein-amine of said K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000124
Figure BDA0002739484940000125
Figure BDA0002739484940000126
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond, such that the mAb is present thereonTwo identical compounds of formula I;
Z26is I or K, wherein-amine of said K is optionally substituted with-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000127
Figure BDA0002739484940000128
Figure BDA0002739484940000131
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
x is ATZ10Z11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34(SEQ ID NO:54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55);
Z29is P or K, wherein-amine of said K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000132
Figure BDA0002739484940000133
Figure BDA0002739484940000134
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z34is S or K, wherein-amine of K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000135
Figure BDA0002739484940000136
Figure BDA0002739484940000137
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
and pharmaceutically acceptable salts thereof.
In certain embodiments, the invention is represented by: a compound of formula I or a derivative thereof, wherein:
n is 1 or 2;
Z2is a direct bond or serine;
Z4is T or
Figure BDA0002739484940000141
Z5Is A, beta-alanine,
Figure BDA0002739484940000142
Figure BDA0002739484940000143
Z6Is T or
Figure BDA0002739484940000144
Z10Is Q or E;
Z11is R or K, wherein-amine of K is-C (═ NH) NH2Substitution;
Z12is L or
Figure BDA0002739484940000145
Z16Is L;
Z25is P or K, wherein-amine of said K is-C (═ O) CH3Or
Figure BDA0002739484940000146
SubstitutionWherein the mAb is substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z26is I or K, wherein-amine of said K is-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000147
Figure BDA0002739484940000151
(ii) substitution, wherein the mAb is substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
x is ATZ10Z11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34(SEQ ID NO:54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55);
Z29is P or K, wherein-amine of said K is-C (═ O) CH3Substitution;
Z34is S or K, wherein-amine of said K is-C (═ O) CH3Substitution;
and pharmaceutically acceptable salts thereof.
In certain embodiments, the invention is represented by: a compound of formula I or a derivative thereof, wherein:
n is 1 or 2;
Z2is a direct bond;
Z4is T or
Figure BDA0002739484940000152
Z5Is beta-alanine,
Figure BDA0002739484940000153
Z6Is T;
Z10is Q or E;
Z11is R or K, wherein K is-amines by-C (═ NH) NH2Substitution;
Z12is L or
Figure BDA0002739484940000154
Z16Is L;
Z25is P or K, wherein-amine of said K is-C (═ O) CH3Or
Figure BDA0002739484940000155
(ii) substitution, wherein the mAb is substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z26is I or K, wherein-amine of said K is-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000161
Figure BDA0002739484940000162
(ii) substitution, wherein the mAb is substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
x is ATZ10Z11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34(SEQ ID NO:54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55);
Z29is P or K, wherein-amine of said K is-C (═ O) CH3Substitution;
Z34is S or K, wherein-amine of said K is-C (═ O) CH3Substitution;
and pharmaceutically acceptable salts thereof.
In certain embodiments, the invention is represented by: a compound of formula I or a derivative thereof, wherein:
n is 1 or 2;
Z2is a direct bond or serine;
Z4is T or
Figure BDA0002739484940000163
Z5Is A, beta-alanine,
Figure BDA0002739484940000164
Figure BDA0002739484940000165
Z6Is T or
Figure BDA0002739484940000166
Z10Is Q or E;
Z11is R or K, wherein-amine of K is-C (═ NH) NH2Substitution;
Z12is composed of
Figure BDA0002739484940000171
Z16Is L;
Z25is P or K, wherein-amine of said K is-C (═ O) CH3Or
Figure BDA0002739484940000172
(ii) substitution, wherein the mAb is substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z26is I or K, wherein-amine of said K is-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure BDA0002739484940000173
Figure BDA0002739484940000174
Substitution wherein the mAb is via another thioether bond(ii) substitution to a second compound of formula I such that there are two identical compounds of formula I on the mAb;
x is ATZ10Z11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34(SEQ ID NO:54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55);
Z29is P or K, wherein-amine of said K is-C (═ O) CH3Substitution;
Z34is S or K, wherein-amine of said K is-C (═ O) CH3Substitution;
and pharmaceutically acceptable salts thereof.
In certain embodiments, the compound is selected from SEQ ID NOS 4-42, or a pharmaceutically acceptable salt thereof.
In certain embodiments, the monoclonal antibody or antigen binding fragment thereof is covalently attached to the amylin mimetic peptide at a lysine residue of the amylin mimetic peptide via a linker. Non-limiting examples of joints include: PEG chain of 2-24 PEG units, (OEG)(0-4)-γ-Glu)、(OEG(1-4)) Or an alkyl chain containing 2 to 10 carbon atoms, wherein the linker may comprise a group such as, but not limited to, acetyl.
In certain embodiments, Z in formula I25、Z26、Z29And Z34Is a lysine, and the lysine is covalently linked via a linker to an engineered cysteine residue of the monoclonal antibody or antigen binding fragment thereof.
Another embodiment of the invention is a pharmaceutical composition comprising a compound of formula I or a compound selected from SEQ ID NOS 4-42 and a pharmaceutically acceptable carrier.
In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining region 1(HCDR1), HCDR2, HCDR3, and light chain complementarity determining region 1(LCDR1), LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NOs: 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 SEQ ID NO 43 and a light chain variable domain (VL) having the polypeptide sequence SEQ ID NO 45. In certain embodiments, the isolated monoclonal antibody further comprises an 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.
The present invention also provides a conjugate comprising a monoclonal antibody or antigen-binding fragment thereof coupled to an amylin mimetic peptide, wherein the monoclonal antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining region 1(HCDR1), HCDR2, HCDR3 and light chain complementarity determining region 1(LCDR1), LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NOs 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 polypeptide sequence SEQ ID NO 43 and a light chain variable domain (VL) having polypeptide sequence SEQ ID NO 45, and more preferably the monoclonal antibody comprises a Heavy Chain (HC) having polypeptide sequence SEQ ID NO 44 and a Light Chain (LC) having polypeptide sequence SEQ ID NO 46; the amylin mimetic 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 to the amylin mimetic peptide at residue 25, 26, 29, or 34 of the amylin mimetic peptide, preferably at lysine residue 25 or 26 of the amylin mimetic peptide, either directly or via a linker.
The invention also provides methods of making the conjugates of the invention. The method comprises reacting an electrophile, preferably bromoacetamide, introduced onto a side chain of, or a linker on, the side chain, preferably a side chain of a lysine residue of the amylin mimetic peptide, with a thiol group of a cysteine residue of SEQ ID NO:49 of the monoclonal antibody or antigen-binding fragment thereof, thereby forming a covalent bond between the amylin mimetic peptide and the monoclonal antibody or antigen-binding fragment thereof.
The invention also provides a pharmaceutical composition comprising a conjugate of the invention and a pharmaceutically acceptable carrier.
The present invention also provides a method for treating or preventing a disease or disorder in a subject in need thereof, wherein the 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 Hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unmanaged cholesterol and/or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease, and eczema. The method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition of the invention.
The invention also provides methods of reducing food intake in a subject in need thereof. The method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition of the invention.
The invention also provides methods of modulating amylin receptor activity in a subject in need thereof. The method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition of the invention.
The present invention also provides methods of modulating amylin receptor activity in a subject in need thereof, wherein the amylin receptor comprises AMY1R, and/or AMY2R, and/or AMY 3R. The method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition of the invention.
The present invention also provides a method of modulating the activity of an amylin receptor in a subject in need thereof, wherein the amylin receptor is AMY 1R.
The present invention also provides a method of modulating the activity of an amylin receptor in a subject in need thereof, wherein the amylin receptor is AMY 3R.
The method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition of the invention.
In certain embodiments, the pharmaceutical composition is administered via injection. In certain embodiments, the pharmaceutical composition is administered in combination with at least one antidiabetic agent. The antidiabetic agent may be, for example, a glucagon-like peptide-1 receptor modulator. In certain embodiments, the pharmaceutical composition is administered in combination with liraglutide.
The invention also provides a conjugate comprising the invention, preferably also liraglutide and a device for injection.
The invention also provides a method for preparing the pharmaceutical composition of the invention. The invention includes combining the conjugate with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.
Other aspects, features and advantages of the present invention will become better understood from a reading of the following detailed description of the invention and the claims.
Drawings
The foregoing summary, as well as the following detailed description of preferred embodiments of the present patent 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
Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles and the like which has been included in this specification is intended to provide a 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 invention disclosed or claimed.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Otherwise, certain terms used herein have the meanings described in the specification.
It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
Unless otherwise indicated, any numerical value, such as concentrations or concentration ranges set forth herein, is to be understood as being modified in all instances by the term "about". Accordingly, numerical values typically include the stated values ± 10%. For example, a concentration of 1mg/mL includes 0.9mg/mL to 1.1 mg/mL. Also, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, unless the context clearly indicates otherwise, a numerical range used explicitly includes all possible subranges, all individual numerical values within the range, including integers within such range and fractions within the range.
The term "at least" preceding a series of elements is to be understood as referring to each element in the series, unless otherwise indicated. 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 present invention.
As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains" or any other variation thereof, are to be construed to mean that a specified integer or group of integers is included, but that no other integer or group of integers is excluded, and that it is non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Furthermore, unless explicitly stated to the contrary, "or" means an inclusive "or" rather than an exclusive "or". For example, condition a or B is satisfied by either: 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).
It should also be understood that when referring to dimensions or characteristics of components of the preferred invention, "about", "approximately", "substantially" and similar terms are used herein to indicate that the described dimensions/characteristics are not strict boundaries or parameters and do not exclude minor variations that are functionally identical or similar, as will be understood by those skilled in the art. At the very least, such reference to include numerical parameters is intended to include variations that do not alter the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).
In two or more nucleic acid or polypeptide sequences (e.g., amylin mimetics)3-36Polypeptide sequences, antibody light or heavy chain sequences), the term "identical" or percent "identity" refers 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, or that are the same, when compared and aligned for maximum correspondence, according to the present disclosure, when compared using one of the following sequence comparison algorithms or by visual inspection using methods known in the art.
For sequence alignment, one sequence is typically used as a reference sequence to which test sequences are aligned. When using a sequence alignment algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are designated (if necessary), and program parameters of the sequence algorithm are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence relative to the reference sequence based on the specified program parameters.
Optimal alignment of sequences for comparison can be carried out, for example, by the local homology algorithm of Smith & Waterman, high-end application mathematics, volume 2: page 482(1981) (adv. appl. Math.2:482(1981)) by using the homology alignment algorithm of Needleman & Wunsch, journal of molecular biology, volume 48: page 443(1970) (J mol. biol. 48: 443(1970)), by a method of searching for similarities of Pearson & Lipman, journal of the national academy of sciences of the united states, volume 85: on page 2444(1988) (Proc. Nat' l. Acad. Sci. USA 85: 2444(1988)), by these algorithms (GAP, BESTFIT, FASTA and TFASTA, in the state of Wisconsin genetics software package, genetics computing group, Wisconsin, Madison, Wis., Science No. 575 (Science Dr., Madison, Wis.), computerized implementation, or by visual inspection (see generally, molecular biology laboratory Manual, F.M. Ausubel et al, edited by laboratory Manual, Greeny publishing Association and the Joint venture of Williams (supplementary 1995) (Ausubel)).
Examples of algorithms 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) journal of molecular biology, Vol.215, p.403-: page 3389-3402 (Nucleic Acids Res.25: 3389-3402). Software for performing BLAST analysis is publicly available through the national center for biotechnology information.
Another 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, e.g., 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.
As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human. As used herein, the term "mammal" 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, and the like, and more preferably, humans.
With respect to the methods of the present invention, the term "administering" means a method of therapeutically or prophylactically preventing, treating, or ameliorating a syndrome, disorder, or disease described herein by using the conjugates of the present invention or forms, compositions, or medicaments thereof. Such methods include administering an effective amount of the conjugate, form, composition or medicament thereof at different times during the course of treatment, or simultaneously in combination. The methods of the invention are understood to encompass all known therapeutic treatment regimens.
The term "effective amount" means that amount of active conjugate 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 prevention, treatment, or amelioration of the syndrome, disorder, or disease being treated, or the symptoms of the syndrome, disorder, or disease being treated.
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 combination of the specified ingredients in the specified amounts.
The term "coupled" as used herein refers to the association or linking together of two or more objects. When referring to a chemical or biological compound, coupling may refer to covalent linkage between two or more chemical or biological compounds. By way of non-limiting example, an antibody of the invention can be coupled to a peptide of interest (e.g., an amylin mimetic peptide of the invention) to form an antibody-coupled peptide. In certain embodiments, an antibody of the invention can be covalently coupled to a peptide of the invention via a linker. The linker may, for example, be first covalently linked to the antibody or peptide and then covalently linked to the peptide or antibody. Antibody-coupled peptides can be formed by specific chemical reactions designed to conjugate antibodies to peptides. By way of example, mAb-coupled amylin mimetic peptide conjugates may be formed by a conjugation reaction. The conjugation reaction may, for example, involve reacting an electrophilic group (e.g., bromoacetamide or maleimide) with the thiol group of a cysteine residue on the mAb. The electrophilic groups may, for example, be introduced onto the side chains of the amino acid residues of the amylin mimetic peptides. The reaction of the electrophilic group with the thiol group results in the formation of covalent thioether bonds.
As used herein, the term "linker" refers to a chemical moiety comprising a covalent chain or atomic chain that covalently links an antibody to a peptide. Linkers can, for example, include, but are not limited to, peptide linkers, hydrocarbon linkers, polyethylene glycol (PEG) linkers, polypropylene glycol (PPG) linkers, polysaccharide linkers, polyester linkers, mixed linkers consisting of PEG and embedded heterocycles, and hydrocarbon chains.
The term "conjugated" as used herein refers to the covalent coupling of an antibody or fragment thereof to a pharmaceutically active moiety. The term "conjugated to" means that the antibody or fragment thereof of the invention is covalently bonded or covalently linked, either directly or indirectly through a linker, to a pharmaceutically active moiety, preferably a therapeutic peptide. By way of non-limiting example, the antibody may be a monoclonal antibody of the invention and the pharmaceutically active moiety may be a therapeutic peptide, such as an amylin mimetic peptide of interest.
Antibodies
As used herein, in the context of an antibody, the term "non-targeted" 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 a binding at 1 × 108M or less, preferably 5X 109M or less, 1X 10-9M or less, 5X 10-10M or less, or 1X 10-10An antibody with a KD of M or less that binds to a target antigen. The term "KD" refers to the dissociation constant obtained from the ratio of KD to Ka (i.e., KD/Ka) and expressed as molar concentration (M). In accordance with the present disclosure, the KD value of an antibody can be determined using methods in the art. For example, the KD of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system (e.g., using a biosensor system)
Figure BDA0002739484940000241
System) or by using a bio-layer interferometry technique, such as the OctetRED96 system. The smaller the value of antibody KD, the higher the affinity of the antibody for binding to the target antigen.
Monoclonal antibodies, either intact or fragments thereof, can be used as half-life extending moieties. Monoclonal antibodies are well studied proteins that have been exploited and characterized for in vivo use, and therefore, the mechanisms to achieve their prolonged in vivo half-life and their in vivo elimination mechanisms are well known. In addition, spatial separation and display of the two "arms" of a monoclonal antibody may be advantageous for efficient bivalent display of therapeutic moieties (i.e., therapeutic peptides). Therapeutic agents have been developed in which toxins or other small molecule drugs are chemically bonded to monoclonal antibodies, but they typically utilize monoclonal antibodies that bind to a particular antigen and target the antibody-drug conjugate to the tissue/cell of interest, which preferably expresses the antigen, and typically the drug/small molecule is linked to the antibody in a manner that does not affect the antigen binding of the antibody.
For therapeutic peptide-mAb conjugates, antigen-specific binding of half-life extending monoclonal antibodies is not desired. Thus, pairs of Heavy (HC) and Light (LC) variable (V) domains that do not specifically bind any target would be expected to be useful in the preparation of non-targeted monoclonal antibodies capable of conjugation in accordance with the present invention. To obtain a non-targeted monoclonal antibody capable of conjugation, the cysteine residue is engineered into one of the Complementarity Determining Regions (CDRs) of the selected non-targeted antibody. The pharmaceutically active moiety (e.g., therapeutic peptide/compound) may comprise a suitable chemical moiety to allow conjugation of the pharmaceutically active moiety to the engineered cysteine residue of the non-targeted monoclonal antibody.
As used herein, the term "antibody" is broad and includes non-human (e.g., murine, rat), human adapted, humanized and chimeric monoclonal antibodies; an antibody fragment; bispecific or multispecific antibodies; dimeric, tetrameric or multimeric antibodies; and single chain antibodies.
The light chain of an antibody of any vertebrate species can be assigned to one of two completely different types, κ and λ, based on the amino acid sequence of its constant domains. Thus, an antibody of the invention may contain a kappa or lambda light chain constant domain. According to a particular embodiment, the antibody of the invention comprises heavy and/or light chain constant regions from a mouse or human antibody. In addition to the heavy and light chain constant domains, antibodies comprise an antigen binding region consisting of a light chain variable region and a heavy chain variable region, each variable region comprising three domains (i.e., complementarity determining regions 1-3; (CDR1, CDR2, and CDR 3)). The light chain variable regions are alternatively referred to as LCDR1, LCDR2, and LCRD3, and the heavy chain variable regions are alternatively referred to as HCDR1, HCRD2, and HCDR 3.
Depending on the heavy chain constant domain amino acid sequence, immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM. IgG is the most stable of the five classes of immunoglobulins, with a serum half-life in humans of about 23 days. IgA and IgG are further sub-classified as isotypes IgA1、IgA2、IgG1、IgG2、IgG3And IgG4. Each of the four IgG subclasses has a different biological function, which is referred to as an effector function. These effector functions are typically mediated by interaction with Fc receptors (Fc γ R) or by binding to C1q and fixing complement. Binding to Fc γ R results in antibody-dependent cell-mediated lysis, while binding to complement factors results in complement-mediated lysis. The antibodies of the invention utilized for their ability to extend the half-life of a therapeutic peptide have no or minimal effector function, but retain its ability to bind FcRn, which can be the primary means by which an antibody has an extended half-life in vivo.
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 a fully human Ig germline V gene sequence and a heavy chain variable region having a fully human Ig germline V gene sequence, except that HCDR3 has the amino acid sequence SEQ ID NO:49 and a pharmaceutically active moiety conjugated thereto (e.g., an amylin mimetic peptide of the present invention), wherein the antibody or antigen-binding fragment thereof does not specifically bind to any human antigen in vivo. In the present disclosure, the phrase "a conjugate comprising an antibody or an antigen-binding fragment thereof and a pharmaceutically active moiety conjugated thereto" is used interchangeably with the phrase "an antibody or an antigen-binding fragment thereof conjugated to a pharmaceutically active moiety" in relation to an antibody or an antigen-binding fragment thereof according to embodiments of the present invention.
As used herein, the term "antigen-binding fragment" refers to antibody fragments, such as diabodies, Fab ', F (ab')2, Fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv)2Bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), single domain antibodies (sdab), scFv dimers (diabodies), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, camelized single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds an antigen but does not comprise a complete antibody structure. The antigen binding fragment is capable of binding to the same antigen as the parent antibody or the antigen to which the parent antibody fragment binds. According to the specific implementationIn one embodiment, the antigen binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment (i.e., the portion of the heavy chain that is comprised in the Fab fragment). According to other specific embodiments, the antigen binding fragment comprises Fab and F (ab').
As used herein, the term "single chain antibody" refers to a single chain antibody as is conventional in the art, which comprises a heavy chain variable region and a light chain variable region linked by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single domain antibody" refers to a single domain antibody as is conventional in the art, which comprises a heavy chain variable region and a heavy chain constant region or only a heavy chain variable region.
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 that specifically binds a target antigen is substantially free of antibodies that do not specifically bind the target antigen). Furthermore, an isolated antibody or antibody fragment can be substantially free of other cellular material and/or chemicals.
The antibody variable region consists of a "framework" region interrupted by three "antigen binding sites". Different terms are used to define antigen binding sites: (i) three Complementarity Determining Regions (CDRs) 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-. (ii) Three "hypervariable regions", "HVRs" or "HV" in VH (H1, H2, H3) and three in VL (L1, L2, L3) refer to regions of 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-CDR" (Lefranc et al, Dev company Immunol 27:55-77,2003) and "specificity determining residue usage" (SDRU) (Almagro, Mol Recognit 17: 132-. The International Immunogenetics (IMGT) database (http:// www-mgt _ org) provides a standardized numbering and definition of antigen binding sites. The correspondence between CDR, HV and IMGT descriptions is described in Lefranc et al, Dev company Immunol 27:55-77,2003.
"framework" or "framework sequence" is the remaining sequence of the variable region except for those sequences defined as antigen binding sites. Because the antigen binding site can be defined by various terms as described above, the exact amino acid sequence of the framework depends on how the antigen binding site is defined.
In one embodiment of the invention, the isolated antibody or antigen-binding fragment thereof comprises a light chain variable region having LCDR1, LCDR2 and LCDR3 having the amino acid sequences SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:52, respectively, and a heavy chain variable region having HCDR1, HCDR2 and HCDR3 having the amino acid sequences SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49, respectively.
In another embodiment, the isolated antibody further comprises an Fc region derived from the Fc region of human IgG 4. The human IgG4Fc region has a reduced ability to bind Fc γ R and complement factors compared to other IgG subtypes. Preferably, the Fc region comprises a substituted human IgG4Fc region with an elimination of effector function. Thus, the isolated antibody further comprises an Fc region having a modified human IgG4Fc region comprising one or more of the following substitutions: proline for glutamic acid at residue 233, alanine or valine for phenylalanine at residue 234, and alanine or glutamic acid for leucine at residue 235 (EU numbering, Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5 th edition, U.S. Dept. of Health and Human Services, Bethesda, Md., NIH publication No. 91-3242). Removal of the N-bonded glycosylation site in the IgG4Fc region by replacement of Asn with Ala at residue 297(EU numbering) is another way to ensure elimination of residual effector activity.
Preferably, the antibodies of the invention may exist as dimers held together by disulfide bonds and various non-covalent interactions. Thus, the Fc portion useful in the antibodies of the invention may be a human IgG4Fc region comprising a substitution, such as a serine to proline substitution at position 228(EU numbering), that stabilizes heavy chain dimer formation and prevents the formation of the hemi IgG4Fc chain.
In another embodiment, the C-terminal Lys residue in the heavy chain is removed, as is common in recombinantly produced monoclonal antibodies.
"human antibody" refers to an antibody having a heavy chain variable region and a light chain variable region, wherein both the framework and the antigen-binding site are derived from sequences of human origin. If the antibody comprises a constant region, the constant region is also derived from a sequence of human origin.
A human antibody comprises a heavy chain variable region or a light chain variable region "derived" from a sequence of human origin if the variable regions of the human antibody are derived from a system using human germline immunoglobulins or rearranged immunoglobulin genes. Such systems include human immunoglobulin gene libraries displayed on phage, as well as transgenic non-human animals, such as mice bearing human immunoglobulin loci as described herein. A "human antibody" may comprise amino acid differences resulting from, for example, naturally occurring somatic mutations or deliberate introduction of substitutions in the framework or antigen-binding site when compared to human germline or rearranged immunoglobulin sequences. Typically, the amino acid sequence of a "human antibody" has 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% identity to the amino acid sequence encoded by a human germline or rearranged immunoglobulin gene. In some cases, a "human antibody" can comprise a consensus framework sequence derived from human framework sequence analysis (e.g., as described in Knappik et al, J Mol Biol 296:57-86,2000); or binding to synthetic HCDR3 in a human immunoglobulin gene library displayed on phage (e.g., as described in Shi et al, J Mol Biol 397: 385-. Antibodies whose antigen-binding sites are derived from non-human species are not included in the definition of "human antibodies".
The isolated humanized antibody may be synthetic. Although human antibodies are derived from human immunoglobulin sequences, they can be generated using systems such as phage display in conjunction with 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 occur within the full complement of human antibody germline in vivo.
As used herein, the term "recombinant antibody" includes all antibodies prepared, expressed, formed or isolated by recombinant methods, such as antibodies isolated from animals (e.g., mice), i.e., transgenic or transchromosomes of human immunoglobulin genes, or antibodies isolated from hybridomas prepared therefrom; an antibody 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 method involving the splicing of human immunoglobulin gene sequences with other DNA sequences, or antibodies generated in vitro using Fab arm swapping.
As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of a single molecular composition. The monoclonal antibody of the present invention can be prepared by a hybridoma method, a phage display technique, a single lymphocyte gene cloning technique, or by a recombinant DNA method. For example, a monoclonal antibody can be produced by a hybridoma that includes a B cell obtained from a transgenic non-human animal, such as a transgenic mouse or rat, having a genome comprising a human heavy chain transgene and a light chain transgene.
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 deleted.
As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecules are derived from two or more species. The variable regions of both the light and heavy chains often correspond to those of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions correspond to sequences in an antibody derived from another mammalian species (e.g., human) in order to avoid eliciting an immune response in that species.
As used herein, the term "multispecific antibody" refers to an antibody comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality of immunoglobulin variable domain sequences has a binding specificity for a first epitope or comprises a germline sequence lacking any known binding specificity, and a second immunoglobulin variable domain sequence of the plurality of immunoglobulin variable domain sequences has a binding specificity for a second epitope or comprises a germline sequence lacking any known binding specificity, and wherein the first immunoglobulin variable domain and/or the second immunoglobulin variable domain optionally comprise a conjugated pharmaceutically active moiety (e.g., a therapeutic peptide). In one embodiment, the first epitope and the second epitope are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In one embodiment, the first epitope and the second epitope overlap or substantially overlap. In one embodiment, the first epitope and the second epitope do not overlap or do not substantially overlap. In one embodiment, the first epitope and the second epitope are on different antigens, such as different proteins (or different subunits of a multimeric protein). In one embodiment, the first immunoglobulin variable domain and the second immunoglobulin variable domain comprise the same conjugated pharmaceutically active moiety. In one embodiment, the first immunoglobulin variable domain and the second immunoglobulin variable domain comprise different pharmaceutically active portions. In one embodiment, only the first immunoglobulin variable domain comprises a conjugated pharmaceutically active moiety. In one embodiment, only the second immunoglobulin variable domain comprises a conjugated pharmaceutically active moiety. In one embodiment, the multispecific antibody comprises a third, fourth or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody or a tetraspecific antibody molecule.
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 moieties). The bispecific antibody is characterized in that the first immunoglobulin variable domain sequence has a binding specificity for the first epitope or comprises a germline sequence lacking any known binding specificity and the second immunoglobulin variable domain sequence has a binding specificity for the second epitope or comprises a germline sequence lacking any known binding specificity and wherein the first immunoglobulin variable domain and/or the second immunoglobulin variable domain optionally comprises a conjugated pharmaceutically active moiety. In one embodiment, the first epitope and the second epitope are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In one embodiment, the first epitope and the second epitope overlap or substantially overlap. In one embodiment, the first epitope and the second epitope are on different antigens, such as different proteins (or different subunits of a multimeric protein). In one embodiment, the first immunoglobulin variable domain and the second immunoglobulin variable domain comprise the same conjugated pharmaceutically active moiety. In one embodiment, the first immunoglobulin variable domain and the second immunoglobulin variable domain comprise different pharmaceutically active portions. In one embodiment, only the first immunoglobulin variable domain comprises a conjugated pharmaceutically active moiety. In one embodiment, only the second immunoglobulin variable domain comprises a conjugated pharmaceutically active moiety. In one embodiment, the bispecific antibody comprises a first heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope or comprising a germline sequence lacking any known binding specificity, and a second heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope or comprising a germline sequence lacking any known binding specificity, and wherein the first heavy chain variable domain and/or the second heavy chain variable domain optionally comprises a conjugated pharmaceutically active moiety. In one embodiment, the first heavy chain variable domain and the second heavy chain variable domain comprise the same conjugated pharmaceutically active moiety. In one embodiment, the first heavy chain variable domain and the second heavy chain variable domain comprise different conjugated pharmaceutically active moieties. In one embodiment, only the first heavy chain variable domain comprises a conjugated pharmaceutically active moiety. In one embodiment, only the second heavy chain variable domain comprises a conjugated pharmaceutically active moiety.
As used herein, "full-length antibody" refers to an antibody having two full-length antibody heavy chains and two full-length antibody light chains. The full length antibody Heavy Chain (HC) consists of the well known variable and constant regions of the heavy chain VH, CH1, CH2 and CH 3. Full-length antibody Light Chains (LCs) consist of the well-known variable and constant regions of the light chain, VL and CL. Full-length antibodies may lack a C-terminal lysine (K) in one or both heavy chains.
The term "Fab arm" or "half molecule" refers to a heavy chain-light chain pair that specifically binds to an antigen.
A full-length bispecific antibody can be generated, for example, using Fab arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies by: substitutions are introduced at the heavy chain CH3 interface in each half molecule to facilitate heterodimer formation of two antibody halves with different specificities in an in vitro cell-free environment or using co-expression. The Fab arm exchange reaction is the result of disulfide bond isomerization and dissociation-association of the CH3 domain. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. The resulting free cysteine of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with the cysteine residue of a second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and reformed by dissociation-association. The CH3 domain of the Fab arm can be engineered to favor heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half-molecules, each binding a different epitope.
As used herein, with respect to antibodies, "homodimerization" refers to the interaction of two heavy chains having the same CH3 amino acid sequence. As used herein, with respect to antibodies, "homodimers" refers to antibodies having two heavy chains with the same CH3 amino acid sequence.
As used herein, with respect to antibodies, "heterodimerization" refers to the interaction of two heavy chains having different CH3 amino acid sequences. As used herein, with respect to antibodies, "heterodimer" refers to an antibody having two heavy chains with different CH3 amino acid sequences.
The "knob-in-hole" strategy (see, e.g., PCT international publication WO 2006/028936) can be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the boundary of the CH3 domain in human IgG may be mutated at positions that affect the CH3 domain interaction, thereby promoting heterodimer formation. Amino acids with small side chains (knobs) are introduced into the heavy chain of an antibody that specifically binds a first antigen, and amino acids with large side chains (knobs) are introduced into the heavy chain of an antibody that specifically binds a second antigen. Upon co-expression of both antibodies, heterodimers are formed due to the preferential interaction of the heavy chain with the "button" with the heavy chain with the "button". An exemplary CH3 substitution pair (denoted as modification position in the first CH3 domain of the first heavy chain/modification position in the second CH3 domain of the second heavy chain) that forms a button and clasp is: T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S _ L368A _ Y407V.
Other strategies may also be used, such as promoting heavy chain heterodimerization using electrostatic interactions by replacing positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface, as described in U.S. patent publication US 2010/0015133; U.S. patent publication US 2009/0182127; U.S. patent publication US2010/028637 or U.S. patent publication US 2011/0123532. In other strategies, heterodimerization may be promoted by the 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, T366I _ K392M _ T394W/F405A _ Y407V, T366L _ K392M _ T394W/F405A _ Y407V, L351Y _ Y407A/T366A _ K409F, L351Y _ Y407A/T366V _ K409F, Y407A/T366A _ K409F, or T350V _ L351Y _ F405A _ Y407V/T350V _ T366 _ V _ K V _ T394 363672 as described in US patent publication US 2012/V or US patent publication US 2013/V.
In addition to the above methods, bispecific antibodies can be generated in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and forming bispecific heterodimeric antibodies from the two parent monospecific homodimeric antibodies under reducing conditions that allow disulfide bond isomerization as described in international patent publication WO 2011/131746. In the method, the first monospecific bivalent antibody and the second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that promote heterodimer stability; incubating the antibodies together under reducing conditions sufficient to disulfide isomerization of cysteines in the hinge region; thereby generating bispecific antibodies by Fab arm exchange. The incubation conditions are optimally restored to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), Dithiothreitol (DTT), Dithioerythritol (DTE), glutathione, tris (2-carboxyethyl) phosphine (TCEP), L-cysteine and β -mercaptoethanol, preferably a reducing agent selected from 2-mercaptoethylamine, dithiothreitol and tris (2-carboxyethyl) phosphine. For example, the following conditions may be used: incubating at a pH of 5-8, e.g., pH7.0 or pH7.4, in the presence of at least 25mM 2-MEA or in the presence of at least 0.5mM dithiothreitol at a temperature of at least 20 ℃ for at least 90 minutes.
Unless otherwise specifically indicated, the numbering of amino acid residues in the constant region of an antibody is performed according to the EU index as described in Kabat et al, Sequences of Proteins of Immunological Interest, published Health Service 5 th edition, National Institutes of Health, Bethesda, Md. (1991).
Conjugates
In another general aspect, the invention relates to conjugates comprising an antibody of the invention covalently conjugated to a pharmaceutically active moiety, such as a synthetic therapeutic peptide (e.g., an amylin mimetic 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 of 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 Hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unmanaged cholesterol and/or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease, eczema, and the like.
In certain embodiments, the antibodies of the invention are modified to include at least one cysteine residue substitution, thereby enabling conjugation to a pharmaceutically active moiety to extend/increase the half-life of the pharmaceutically active moiety. In certain embodiments, at least one cysteine residue substitution is comprised in a complementarity determining region of an antibody. In certain embodiments, at least one cysteine residue is substituted in the heavy chain complementarity determining region (HCDR). In certain embodiments, at least one cysteine residue is substituted in HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID No. 49. In certain embodiments, an antibody comprising HCDR3 having the amino acid sequence of SEQ ID No. 49 has at least one additional cysteine substitution capable of being conjugated to a pharmaceutically active moiety.
In certain embodiments, the pharmaceutically active moiety may comprise a linker. The linker may be chemically modified to allow conjugation of the antibody to a pharmaceutically active moiety. The linker may, 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 mixed linker composed of PEG and embedded heterocycles, or a hydrocarbon chain. The PEG linker may, for example, comprise 2-24 PEG units.
In certain embodiments, a monoclonal antibody of the invention is conjugated to one, two, three, four, five, or six pharmaceutically active moieties of interest (e.g., therapeutic peptides). In a preferred embodiment, the non-targeted monoclonal antibody is conjugated to two pharmaceutically active moieties of interest. In certain embodiments where a monoclonal antibody is conjugated to at least two pharmaceutically active moieties of interest, the pharmaceutically active moieties of interest may be the same pharmaceutically active moiety or may be different pharmaceutically active moieties.
Methods of conjugating an antibody of the invention to a pharmaceutically active moiety of the invention are known in the art. Briefly, an antibody 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 to a pharmaceutically active moiety (e.g., by providing the reduced antibody with a lyophilized peptide under conditions that allow conjugation). Following the conjugation reaction, the conjugate can be purified by ion exchange chromatography or Hydrophobic Interaction Chromatography (HIC), with the final purification step being protein a adsorption. In certain embodiments, the antibodies of the invention may be purified prior to reduction using the HIC method. For a more detailed description of the conjugation procedure see, e.g., example 103 and Dennler et al, Antibodies 4:197-224 (2015).
In certain embodiments, the amylin mimetic peptide is a derivative of the amylin mimetic peptide of formula I, or a pharmaceutically acceptable salt thereof, that is modified by one or more methods selected from the group consisting of: amidation, lipidation and pegylation.
In certain embodiments, the conjugates comprise a monoclonal antibody or fragment thereof conjugated to an amylin mimetic peptide, wherein the amylin mimetic peptide is selected from SEQ ID NOs 4-28.
In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof is covalently attached to the amylin mimetic peptide at a lysine residue of the amylin mimetic peptide via a linker. The linker may for example comprise a linker selected from the group consisting of: a PEG chain of 2 to 24 PEG units, an alkyl chain containing 2-10 carbon atoms, or a bond.
In certain embodiments, Z in formula I25、Z26、Z29And Z34Is a lysine, and the lysine is covalently linked via a linker to an engineered cysteine residue of the monoclonal antibody or antigen binding fragment thereof. In a preferred embodiment, a monoclonal antibody or antigen-binding fragment thereof according to an embodiment of the invention is conjugated to an amylin mimetic peptide at residue 25 or 26 of the amylin mimetic. In another preferred embodiment, an electrophile, such as bromoacetamide, is introduced into the amylin mimetic at residue 25 or 26 of the amylin mimeticAnd site-specifically reacting the electrophile with a thiol group of a Cys residue engineered into a CDR of the monoclonal antibody or fragment thereof (preferably HCDR3), thereby forming a covalent bond between the amylin mimetic peptide and the monoclonal antibody or fragment thereof. More preferably, the amylin mimetic peptide is selected from the group consisting of SEQ ID NO6, 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 directly onto the side chain of the amylin mimetic. In another embodiment, the electrophile is introduced indirectly via a linker to the side chain of the amylin mimetic.
The invention also provides pharmaceutical compositions comprising the conjugates of the invention and further comprising a pharmaceutically acceptable carrier.
Also provided herein are amylin mimetic peptides that exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to pramlintide or davalintide. As an example of a method for determining sequence identity between two analogues, two peptides (pramlintide (SEQ ID NO:2) and (SEQ ID NO:4)) were aligned.
Figure BDA0002739484940000341
The sequence identity of an analog 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 not present. N3 is now replaced, but remains in its original position. Thus, in this example, the sequence identity is (37-2)/37X 100.
In the case where the compound according to the invention is an amylin mimetic peptide coupled to a mAb, it is expected that the mAb will have two copies of the peptide coupled thereto. It will be appreciated by those skilled in the art that the reaction product may comprise a partial conjugation product, thereby producing an amylin mimetic peptide coupled to the mAb. It is to be understood that all such mono-coupling compounds, di-coupling compounds and mixtures thereof are encompassed within the scope of the present invention.
Amylin mimetic peptides
In view of their role in controlling appetite and food intake, pramlintide and/or davalintide may be effective in the treatment of obesity. However, the therapeutic utility of pramlintide and/or davalintide as a treatment agent is limited by its rapid metabolism and short circulatory half-life. Thus, the present invention relates generally to modified pramlintide and/or davalintide conjugates that extend the half-life of amylin mimetic peptides and reduce the metabolism of the peptides in vivo.
In certain embodiments of the invention, the modified pramlintide and/or davalin peptide is an amylin mimetic peptide. The terms "amylin mimetic peptide," "amylin mimetic analog," and "amylin mimetic peptide analog" are used interchangeably.
The peptide sequences described herein are written according to common practice with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although the isomeric form of an amino acid is known, it is the L form of the amino acid represented, unless specifically indicated otherwise. For convenience in describing the molecules of the invention, conventional and non-conventional abbreviations for the 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 below: a ═ Ala ═ alanine; r ═ Arg ═ arginine; n ═ Asn ═ asparagine; d ═ Asp ═ aspartic acid; β a ═ β Ala ═ β -alanine; C-Cys-cysteine; hC ═ hCys ═ homocysteine; e ═ Glu ═ glutamic acid; q ═ Gln ═ glutamic acid; g ═ Gly ═ glycine; H-His-histidine; i ═ Ile ═ isoleucine; l ═ Leu ═ leucine; k ═ Lys ═ lysine; nle ═ norleucine; phe ═ phenylalanine; p ═ Pro ═ proline; s ═ Ser ═ serine; t ═ Thr ═ threonine; w ═ Trp ═ tryptophan; y ═ Tyr ═ tyrosine and V ═ Val ═ valine.
Additional amino acid abbreviations used herein are listed below: ACPC ═ 2-aminocyclopentanecarboxylic acid; β -Aib ═ β aminoisobutyric acid ═ 3-amino-2-methylpropionic acid; β -hA ═ β -ala ═ β -homoalanine ═ 3-aminobutyric acid; α -MeL ═ α -MeLeu ═ α -methylleucine; β -hpo ═ β -homoproline; β -hT ═ β -hThr ═ β -homothreonine;
for convenience, the numbering convention used to designate amino acid residues of the amylin mimetic peptides of the present invention follows that of pramlintide and/or davalinin. Specific amino acid substitutions that have been introduced into these peptides are indicated by appropriate amino acid codes relative to the natural residue at the corresponding position in pramlintide and/or davalintide, followed by the position of the substitution. Thus, "hC 7" in the amylin mimetic peptide refers to a peptide in which the homocysteine has replaced the corresponding native Cys7 residue of pramlintide. Similarly, "K (Ac) 26" in amylin mimetics refers to those in which the-amine is CH-substituted3C (O) -substituted lysine has replaced the peptide of the corresponding native Ile26 residue of pramlintide. According to this convention, additional amino acid substitutions that occur within the amylin mimetic peptide are described and will likewise be recognized by those skilled in the art.
For convenience, the naming convention for amylin mimetic peptides used in the present invention binds the amino acid residues involved in the loop, along with the linking group between them, in a left to right direction, starting from the N-terminal residue involved in the loop. In all cases, the N-terminal amino acid residue of the loop is attached with its alpha-amino function to an acetyl linker which in turn is attached to the thiol side chain residue of the amino acid at position 7 of the amylin mimetic peptide. Thus, "Ring- (N3-COCH)2-hC7) "is used to describe the loop of amylin mimetics in which the native Lys1 and Cys2 residues have been deleted from the sequence and the a-amino function of Asn3 has been acylated with an acetyl residue whose methyl group is further linked to the side chain of the hCys7 residue by a thioether bond. Similarly, "Ring- (S2-COCH)2-hC7) "is used to describe the loop of amylin mimetics in which the native Lys1 residue has been deleted, the native Cys2 residue has been replaced by Ser2, the a-amino function of said Ser2 has been acylated with an acetyl group which is in turn linked to the hCys7 residue by a thioether bondSide chains.
Lysine residues can be incorporated at various positions in the sequence of the amylin mimetic to provide a convenient functional handle for further derivatization. Lysine residues may be modified to couple directly or indirectly to monoclonal antibodies. In indirect coupling to a monoclonal antibody, the lysine residues may be modified to include a linker that will allow the amylin mimetic peptide to be coupled to the monoclonal antibody. Those skilled in the art will recognize that related orthologs may also be used so effectively and are contemplated herein.
The term "K (γ -Glu)" appearing in the peptide sequence denotes a lysyl residue whose side chain-amino group has been acylated by the γ -carboxyl group of glutamic acid.
The term "k (ac)" denotes a lysyl residue whose side chain-amino group has been replaced by an acetyl group.
The term "k (alloc)" denotes a lysyl residue whose side chain-amino group has been replaced by an allyloxycarbonyl group.
The term "K (OEG)2-Pal) "denotes a lysyl residue whose side chain-amino group has been replaced by 17-amino-10-oxo-3, 6,12, 15-tetraoxa-9-azaheptadecanoic acid through an amide bond between the 17-amino group and palmitic acid, wherein the 17-amino group has also been replaced by palmitic acid.
Term "(OEG)2"denotes two OEG units linked together in series by an amide bond (i.e., 17-amino-10-oxo-3, 6,12, 15-tetraoxa-9-azaheptadecanoic acid).
The term "K (OEG)2"denotes a lysyl residue whose side chain-amino group has been acylated with 17-amino-10-oxo-3, 6,12, 15-tetraoxa-9-azaheptadecanoic acid.
The term "K (OEG)2- γ -Glu-Pal) "denotes a lysyl residue whose side chain-amino group has been acylated with (22S) -22-amino-10, 19-dioxo-3, 6,12, 15-tetraoxa-9, 18-diazicosyldiacid via its 1-carboxylic acid function, and wherein said 22-amino group is amidated with palmitic acid.
The term "dPEGx" refers to a discrete oligomer comprising x ethylene glycol units attached to propionic acid at one end and comprising a terminal amino functional group at the distal end, which may be further functionalized.
The term "K (dPEG 12)" denotes a lysyl residue whose side chain-amino group has been acylated with 1-amino-3, 6,9,12,15,18,21,24,27,30,33, 36-dodecaoxatriacontane-39-acid via its 39-carboxylic acid function.
The term "K (dPEG 12-AcBr)" denotes a lysyl residue whose side chain-amino group has been acylated with 1-amino-3, 6,9,12,15,18,21,24,27,30,33, 36-dodecaoxatriacontan-39-acid via its 39-carboxylic acid function, and wherein the acid is substituted on its 1-amine group with-C (O) CH2Amidating the Br group.
Half-life extending moieties
In addition to the antibodies or antigen-binding fragments thereof of the invention, the conjugates of the invention can bind one or more other moieties for the purpose of extending the half-life of a pharmaceutically active moiety (e.g., amylin mimetic peptide), e.g., via covalent interactions. Exemplary other half-life extending moieties include, but are not limited to, albumin variants, albumin-binding proteins and/or domains, transferrin, fragments thereof, and analogs 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, such as laurate, myristate, stearate, arachinate, behenate, oleate, arachidonic acid, suberic acid, tetradecanedioic acid, octadecanedioic acid, behenic acid, etc., polylysine, octane, carbohydrates (dextran, cellulose, oligosaccharides or polysaccharides) in order to obtain the desired properties. These portions can be fused directly to the protein scaffold coding sequence and can be produced by standard cloning and expression techniques. Alternatively, well-known chemical coupling methods can be used to attach these moieties to the recombinantly and chemically prepared conjugates of the invention.
The pegyl moiety may for example be added to the peptide molecule of the invention by binding a cysteine residue to the C-terminus of the molecule and linking the pegyl group to the cysteine using well known methods.
The functionality of the peptide molecules of the invention incorporating additional moieties can be compared by a variety of well-known assays. For example, the biological or pharmacokinetic activity of a therapeutic peptide of interest, alone or in the form of a conjugate according to the invention, can be determined and compared using known in vitro or in vivo assays.
Pharmaceutical composition
In another general aspect, the invention relates to pharmaceutical compositions 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 and a pharmaceutically acceptable carrier. The conjugates and compounds of the invention, and compositions comprising them, are also useful in the manufacture of medicaments for the therapeutic applications described herein.
As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid-containing vesicle, microsphere, liposome encapsulation, or other material known in the art for use in pharmaceutical formulations. It will be appreciated 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 effect of, or the biological activity of, the composition according to the present invention. According to the present disclosure, any pharmaceutically acceptable carrier suitable for use in antibody pharmaceutical compositions may be used in the present invention, according to a specific embodiment.
Pharmaceutically acceptable acid/anion salts for use in the present invention include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camphorsulfonate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, etonate, ethanesulfonate, fumarate, glucoheptonate, gluconate, glutamate, p-hydroxyacetaminophenylarsonate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, methanesulfonate, methylbromide, methylnitrate, methylsulfate, mucate, naphthalenesulfonate, nitrate, pamoate, pantothenate, phosphate/diphosphate, salts of sodium, Polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theachlorate, tosylate, and triethyliodide. Organic or inorganic acids also include, without limitation, hydroiodic acid, perchloric acid, sulfuric acid, phosphoric acid, propionic acid, glycolic acid, methanesulfonic acid, hydroxyethanesulfonic acid, oxalic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, saccharinic acid, or trifluoroacetic acid.
Pharmaceutically acceptable base/cationic salts include, without limitation, aluminum, 2-amino-2-hydroxymethyl-propane-1, 3-diol (also known as TRIS (hydroxymethyl) aminomethane, trometamol or "TRIS"), ammonia, benzathine, tert-butylamine, chloroprocaine, choline, cyclohexylamine, diethanolamine, ethylenediamine, lithium, L-lysine, magnesium, meglumine, N-methyl-D-glucamine, piperidine, potassium, procaine, quinine, sodium, triethanolamine or zinc.
In some embodiments of the invention, pharmaceutical formulations are provided comprising a conjugate of the invention in an amount from about 0.001mg/ml to about 100mg/ml, from about 0.01mg/ml to about 50mg/ml, or from about 0.1mg/ml to about 25 mg/ml. The pharmaceutical formulation has a pH of about 3.0 to about 10, for example about 3 to about 7, or about 5 to about 9. The formulation may further comprise at least one ingredient selected from the group consisting of: a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, and a surfactant.
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., 21 st edition (2005) and any subsequent editions). Non-limiting examples of additional ingredients include: buffers, diluents, solvents, tonicity adjusting agents, preservatives, stabilizers and chelating agents. One or more pharmaceutically acceptable carriers may be used to formulate the pharmaceutical compositions of the present invention.
In one embodiment of the invention, the pharmaceutical composition is a liquid formulation. One preferred example of a liquid formulation is an aqueous formulation, i.e. a formulation comprising water. Liquid formulations may comprise solutions, suspensions, emulsions, microemulsions, gels, and the like. The 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 water.
In one embodiment, the pharmaceutical composition may be formulated as an injectable, which may be injected, for example, via an injection device (e.g., a syringe or infusion pump). Injection may be delivered, for example, subcutaneously, intramuscularly, intraperitoneally, or intravenously.
In another embodiment, the pharmaceutical composition is a solid formulation, e.g., a freeze-dried or spray-dried composition, which may be used as such, or with the addition of solvents and/or diluents by a physician or patient prior to use. Solid dosage forms may include tablets, such as compressed tablets and/or coated tablets, and capsules (e.g., hard gelatin capsules or soft gelatin capsules). The pharmaceutical compositions may also be in the form of sachets, dragees, powders, granules, lozenges or powders, for example for reconstitution.
The dosage forms may be immediate release, in which case they may comprise a water-soluble or water-dispersible carrier, or they may be delayed, sustained or modified release, in which case they may comprise a water-insoluble polymer which modulates the dissolution rate of the dosage form in the gastrointestinal tract.
In other embodiments, the pharmaceutical composition may be delivered intranasally, buccally or sublingually.
The pH in the aqueous formulation may be between pH3 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 7.0.
In another embodiment of the present invention, the pharmaceutical composition comprises a buffering agent. Non-limiting examples of buffers include: arginine, aspartic acid, dihydroxyethylglycine, 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, triazine, and tris (hydroxymethyl) aminomethane, and mixtures thereof. The buffer may be present alone or in the aggregate at a concentration of about 0.01mg/ml to about 50mg/ml, for example about 0.1mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each of these specific buffers constitute an alternative embodiment of the present invention.
In another embodiment of the invention, the pharmaceutical composition comprises a preservative. Non-limiting examples of buffers include: benzethonium chloride, benzoic acid, benzyl alcohol, bromonitropropanediol, 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, thimerosal, and mixtures thereof. Preservatives may be present alone or in the aggregate at a concentration of from about 0.01mg/ml to about 50mg/ml, for example from about 0.1mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each of these particular preservatives constitute alternative embodiments of the present invention.
In another embodiment of the invention, the pharmaceutical composition comprises an isotonic agent. Non-limiting examples of this embodiment include salts (such as sodium chloride), amino acids (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), sugar alcohols (such as glycerol, 1, 2-propanediol, propylene glycol), 1, 3-propanediol, and 1, 3-butanediol), polyethylene glycols (e.g., PEG400), and mixtures thereof. Another example of an isotonic agent includes sugars. Non-limiting examples of sugars can 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 carboxymethyl cellulose. Another example of an isotonicity agent is a sugar alcohol, where 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, hexitol, xylitol, and arabitol. Pharmaceutical compositions comprising each of the isotonic agents listed in this paragraph constitute alternative embodiments of the present invention. The isotonic agent may be present alone or in the aggregate at a concentration of about 0.01mg/ml to about 50mg/ml, for example about 0.1mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each of these specific isotonic agents constitute alternative embodiments of the present invention.
In another embodiment of the present invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include salts of citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. The chelating agent may be present alone or in the aggregate at a concentration of about 0.01mg/ml to about 50mg/ml, for example about 0.1mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each of these specific chelating agents constitute an alternative embodiment of the present invention.
In another embodiment of the present 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.
In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, wherein said stabilizer is carboxy-/hydroxycellulose and derivatives thereof (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrin, 2-methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (such as sodium chloride), sulfur-containing substances such as thioglycerol or thioglycolic acid. The stabilizer may be present alone or in the aggregate at a concentration of about 0.01mg/ml to about 50mg/ml, for example about 0.1mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each of these particular stabilizers constitute an alternative embodiment of the present invention.
In another embodiment of the invention, the pharmaceutical composition comprises one or more surfactants, preferably one surfactant, at least one surfactant or two different surfactants. The term "surfactant" refers to any molecule or ion that consists of a water-soluble part (hydrophilic) and a fat-soluble and partly (lipophilic). For example, the surfactant is selected from: anionic surfactants, cationic surfactants, nonionic surfactants, and/or zwitterionic surfactants. The surfactant may be present alone or in the aggregate at a concentration of about 0.1mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each of these specific surfactants constitute an alternative embodiment of the present invention.
In another embodiment of the invention, the pharmaceutical composition comprises one or more protease inhibitors, such as, for example, EDTA and/or benzamidine hydrochloride (HCl). The protease inhibitor may be present alone or in the aggregate at a concentration of about 0.1mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each of these specific protease inhibitors constitute alternative embodiments of the present invention.
The pharmaceutical compositions of the invention may comprise an amount of amino acid bases sufficient to reduce aggregation 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 analogs thereof. Any amino acid may be present in its free base form or its salt form. Any stereoisomer of the amino acid base (i.e., L, D or mixtures thereof) may be present. The amino acid bases may be present alone or in combination with other amino acid bases, at a concentration of about 0.01mg/ml to about 50mg/ml, for example about 0.1mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each of these specific amino acid bases constitute alternative embodiments of the invention.
It will also be apparent to those skilled in the art that the therapeutically effective dose of the conjugates of the invention or pharmaceutical compositions thereof will vary depending on the desired effect. Thus, the optimal dosage to be administered can 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 formulation, 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 dosage to the appropriate therapeutic level.
For all indications, the conjugates of the invention are preferably peripherally administered in a dose of about 1 μ g to about 50mg 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 in a dose of about 0.01 μ g/kg to about 500 μ g/kg, more preferably about 0.05 μ g/kg to about 250 μ g/kg, most preferably less than about 50 μ g/kg. Dosages within these ranges will, of course, vary with the potency of each agonist and are readily determined by those skilled in the art. Thus, the above dosages are exemplary of the general case. Of course, there may be individual instances where higher or lower dosage ranges should be used, and such are within the scope of this invention.
In certain embodiments, the conjugates of the invention are administered at a dose of about 1 μ g to about 5mg, or at a dose of about 0.01 μ g/kg to about 500 μ g/kg, more preferably at a dose of about 0.05 μ g/kg to about 250 μ g/kg, most preferably at a dose of less than about 50 μ g/kg, with a second therapeutic agent (e.g., liraglutide) at a dose of about 1 μ g/kg to about 5mg, or at a dose of about 0.01 μ g/kg to about 500 μ g/kg, more preferably at a dose of about 0.05 μ g/kg to about 250 μ g/kg, most preferably at a dose of less than about 50 μ g/kg.
Pharmaceutically acceptable salts of the conjugates of the invention include conventional non-toxic salts or quaternary ammonium salts 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 calcium and magnesium salts, organic base salts such as dicyclohexylamine salts, and salts of amino acids such as arginine. In addition, the basic nitrogen-containing groups can be quaternized with, for example, alkyl halides.
The pharmaceutical compositions of the present invention may be administered by any means that achieves their intended purpose. Examples include administration by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal or ocular routes. Administration may be by the oral route. Formulations suitable for parenteral administration include aqueous solutions of the active conjugate in water-soluble form (e.g., a water-soluble salt), acidic solutions, alkaline solutions, aqueous dextrose, isotonic carbohydrate solutions, and cyclodextrin inclusion complexes.
The invention also encompasses a method of making a pharmaceutical composition comprising mixing a pharmaceutically acceptable carrier with any one of the conjugates of the invention. In addition, the invention includes pharmaceutical compositions prepared by mixing one or more pharmaceutically acceptable carriers with any of the conjugates of the invention.
Furthermore, the conjugates of the invention may have one or more polymorphs or amorphous crystalline forms and are therefore intended to be included within the scope of the present invention. Furthermore, these conjugates can form solvates with, for example, water (i.e., hydrates) or common organic solvents. The term "solvate" as used herein means a physical association of a conjugate of the invention with one or more solvent molecules. The physical association involves varying degrees of ionic bonding and covalent bonding, including hydrogen bonding. In some cases, the solvate will be able to separate out when, for example, one or more solvent molecules are incorporated into 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.
The present invention is intended to include within its scope polymorphs and solvates of the conjugates of the invention. Thus, in the methods of treatment of the present invention, the term "administering" shall encompass methods for treating, ameliorating or preventing the syndromes, conditions or diseases described herein using the conjugates of the present invention or polymorphs or solvates thereof, which although not specifically disclosed, are expressly included within the scope of the present invention.
In another embodiment, the invention relates to a conjugate according to the invention for use as a medicament.
The present invention includes within its scope prodrugs of the conjugates of the invention. Generally, such prodrugs will be functional derivatives of the conjugates that can be readily converted in vivo to the desired conjugates. Thus, in the methods of treatment of the present invention, the term "administering" shall encompass the use of specifically disclosed conjugates or conjugates not specifically disclosed to treat the various disorders described, and such non-specifically disclosed conjugates can be converted in vivo to the designated conjugates following administration to a patient. Conventional methods for selecting and preparing suitable prodrug derivatives are described, for example, in "Design of produgs" edited by h.
Furthermore, within the scope of the present invention, any element (especially when mentioned in relation to the conjugates of the invention) is intended to be intended to comprise all isotopes or isotopic mixtures (naturally occurring or synthetically prepared) of said element in its natural abundance or in its isotopically enriched form. For example, references to hydrogen include within their scope1H、2H, (D) and3h (T). Similarly, references to carbon and oxygen include within their scope 12C, respectively,13C and14c and16o and18and O. The isotope may be radioactive or non-radioactive. The radiolabeled conjugates of the invention may comprise a compound selected from3H、11C、18F、122I、123I、125I、131I、75Br、76Br、77Br and82a radioisotope of Br. Preferably, the radioisotope is selected from3H、11C and18F。
some conjugates of the invention may exist in the form of atropisomers. Atropisomers are stereoisomers obtained by hindered rotation about a single bond, wherein the steric strain barrier to rotation is sufficiently high to allow separation of conformers. It is to be understood that all such conformers and mixtures thereof are encompassed within the scope of the present invention.
When the conjugates according to the invention have at least one stereocenter, they may accordingly exist as enantiomers or diastereomers. It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention.
If the process for preparing the conjugates according to the invention yields a mixture of stereoisomers, these isomers may be separated by conventional techniques such as preparative chromatography. The conjugates may be prepared in racemic form, or the individual enantiomers may be prepared by enantiospecific synthesis or by resolution. For example, the conjugates can be resolved into their component enantiomers by standard techniques, such as by 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 conjugate can also be resolved by formation of diastereomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the conjugate can be resolved by High Performance Liquid Chromatography (HPLC) or SFC using a chiral column. In some cases, rotamers of the conjugate may be present, which can be passed through1H NMR observation, thereby resulting in1Complex multiplets and peak integration in H NMR spectra.
In any of the methods for preparing the conjugates of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups in any of the molecules concerned. This can be achieved by conventional protecting groups, such as protective groups in organic synthesis, edited by j.f.w.mcomie, plenem press, 1973; (Protective Groups in Organic Chemistry, ed.J.F.W.McOmie, Plenum Press, 1973; and T.W.Greene and P.G.M.Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons,1991 (Protective Groups in Organic Synthesis, John Wiley & Sons,1991), each of which is incorporated herein by reference in its entirety for all purposes.
Application method
The present invention relates to a method for preventing, treating or ameliorating an amylin receptor mediated syndrome, disorder or disease 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 present invention.
The invention also provides a method for preventing, treating or ameliorating the onset of a disorder, disease or condition or ameliorating any one or more symptoms of said disorder, disease or condition in a subject in need thereof, said method comprising administering to the subject in need thereof an effective amount of a conjugate, compound or pharmaceutical composition of the invention.
According to a particular embodiment, the disease, disorder or condition is selected from: 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 Hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unmanaged cholesterol and/or lipid levels, osteoporosis, inflammation, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), kidney disease, and/or eczema.
According to particular embodiments, a therapeutically effective amount refers to a therapeutic amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or ameliorating the severity of or symptoms associated with a disease, disorder, or condition to be treated; (ii) reducing the duration of the disease, disorder or condition being treated or symptoms associated therewith; (iii) preventing the development of the disease, disorder or condition being treated or symptoms associated therewith; (iv) causing regression of the disease, disorder or condition being treated or symptoms associated therewith; (v) preventing the development or onset of the disease, disorder or condition being treated or symptoms associated therewith; (vi) preventing the recurrence of the disease, disorder or condition being treated or symptoms associated therewith; (vii) reducing hospitalization of the subject with the treated disease, disorder or condition or symptoms associated therewith; (viii) reducing the length of hospitalization of a subject having the treated disease, disorder or condition or symptoms associated therewith; (ix) increasing survival of a subject having the treated disease, disorder, or condition or symptoms associated therewith; (xi) Inhibiting or reducing the disease, disorder or condition being treated or symptoms associated therewith in a subject; and/or (xii) enhances or improves the prophylactic or therapeutic effect of the other therapy.
A therapeutically effective amount or dose may vary depending on various factors, such as the disease, disorder or condition to be treated, the mode of administration, the target site, the physiological state of the subject (including, for example, age, weight, health), whether the subject is human or animal, other drugs administered, and whether prophylactic or therapeutic treatment is employed. Therapeutic doses are optimally titrated to optimize safety and efficacy.
As used herein, the terms "treatment," "treating," and "therapy" are all intended to mean an improvement or reversal of at least one measurable physical parameter associated with a disease, disorder, or condition, which is not necessarily identifiable in a subject, but which is identifiable in a subject. The terms "treat" and "treating" may also refer to causing regression, preventing progression, or at least delaying progression of a disease, disorder, or condition. In a particular embodiment, "treating," "treatment," and "therapy" refer to alleviating, preventing the development or onset of, or shortening the duration of one or more symptoms associated with a disease, disorder, or condition. In particular embodiments, "treating" and "treatment" refer to preventing the recurrence of a disease, disorder, or condition. In particular embodiments, "treating" and "treatment" refer to an increase in survival of a subject having a disease, disorder, or condition. In particular embodiments, "treating" and "treatment" refer to the elimination of a disease, disorder, or condition in a subject.
In one embodiment, the present invention provides a method for preventing, treating or delaying the onset of obesity, or ameliorating obesity, or any one or more symptoms of obesity, 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 present invention. In some embodiments, prior to administration of any of the conjugates, compounds, pharmaceutical compositions, forms or medicaments of the invention described herein, the amount of the conjugate, compound, pharmaceutical composition, form or medicament in the subject is, relative to the body weight of the subject, or a reduction in the body weight of the subject as compared to a control subject that does not receive any of the conjugates, compositions, forms, medicaments or combinations of the invention described herein, for example, between about 0.01% and about 0.1%, between about 0.1% and about 0.5%, between about 0.5% and about 1%, between about 1% and about 5%, between about 2% and about 3%, between about 5% and about 10%, between about 10% and about 15%, between about 15% and about 20%, between about 20% and about 25%, between about 25% and about 30%, between about 30% and about 35%, between about 35% and about 40%, between about 40% and about 45%, or between about 45% and about 50%.
In some embodiments, the reduction in body weight is sustained for, e.g., about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, or about 20 years.
The present invention provides methods 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 Hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unmanaged cholesterol and/or lipid levels, osteoporosis, inflammation, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), kidney disease, and eczema, comprising administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention.
As used herein, metabolic syndrome refers to a subject having any one or more of the following: hyperglycemia (e.g., high fasting blood glucose), hypertension, abnormal cholesterol levels (e.g., low HDL levels), abnormal triglyceride levels (e.g., high triglycerides), large waist circumference (i.e., waist circumference), increased abdominal fat, insulin resistance, glucose intolerance, elevated levels of C-reactive protein (i.e., proinflammatory state), and increased levels of plasminogen activator inhibitor-1 and fibrinogen (i.e., prothrombotic state).
The present invention provides a method of reducing food intake in a subject in need thereof, the method comprising administering to a subject in need thereof an effective amount of a conjugate, compound or pharmaceutical composition of the invention. In some embodiments, prior to administration of any of the conjugates, compounds, compositions, forms, medicaments, or combinations of the invention described herein, the subject's food intake is reduced, e.g., 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% relative to the subject's food intake, or compared to a control subject that does not receive any of the conjugates, compounds, compositions, forms, medicaments, or combinations of the invention described herein, between about 40% and about 45% or between about 45% and about 50%.
In some embodiments, the decrease in food intake lasts, for example, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, or about 20 years.
The present invention provides a method of reducing glycated hemoglobin (AIC) 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 present invention. In some embodiments, prior to administration of any of the conjugates, compounds, compositions, forms, medicaments, or combinations of the invention described herein, the subject's A1C is reduced relative to A1C of the subject, or compared to a control subject that does not receive any of the conjugates, compounds, compositions, forms, medicaments, or combinations of the invention described herein, e.g., about 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 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 4% and about 4% of the subject, 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%.
In other embodiments, there is provided a method for reducing fasting blood glucose levels 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. Prior to administration of any of the conjugates, compounds, compositions, forms, medicaments, or combinations of the invention described herein, the fasting blood glucose level may be lowered to less than about 140 to about 150mg/dL, less than about 140 to about 130mg/dL, less than about 130 to about 120mg/dL, less than about 120 to about 110mg/dL, less than about 110 to about 100mg/dL, less than about 100 to about 90mg/dL, or less than about 90 to about 80mg/dL relative to the fasting blood glucose level of the subject, or compared to a control subject that has not received any of the conjugates, compounds, compositions, forms, medicaments, or combinations of the invention described herein.
The present invention provides a method of modulating amylin receptor activity 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 present invention. As used herein, "modulate" refers to increasing or decreasing receptor activity.
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 a day, twice a day, three times a day, four times a day, five times a day, six times a day, seven times a day, or eight times a day. In other embodiments, an effective amount of a conjugate or compound of the invention or a form, composition, or medicament thereof is administered once every other day, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, two times a month, three times a month, or four times a month to a subject in need thereof.
Another embodiment of the invention includes a method of preventing, treating, delaying the onset of, or ameliorating a disease, disorder or syndrome, or one or more symptoms of the disease, disorder or syndrome, in a subject in need thereof, comprising administering to the subject in need thereof 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.
As used herein, the term "combination" in the context of administering two or more therapies to a subject refers to the use of more than one therapy.
As used herein, combination therapy involves administering one or more additional therapeutic agents, or one or more surgical therapies to a subject in need thereof concurrently with an effective amount of a conjugate or compound of the invention, or a form, composition, or medicament thereof. In some embodiments, one or more additional therapeutic agents or surgical therapies can be administered with an effective amount of a conjugate of the invention on the same day, and in other embodiments, one or more additional therapeutic agents or surgical therapies can be administered with an effective amount of a conjugate or compound of the invention for the same week or month.
In certain embodiments, wherein the disease or disorder is selected from: obesity, type II diabetes, metabolic syndrome, insulin resistance, and dyslipidemia, and the second therapeutic agent may be an anti-diabetic agent. In certain embodiments, the antidiabetic agent may be a glucagon-like peptide-1 (GLP-1) receptor modulator.
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 using a combination therapy comprising administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the invention in combination with any one or more of the following therapeutic agents: dipeptidyl peptidase-4 (DPP-4) inhibitors (e.g., sitagliptin, saxagliptin, linagliptin, alogliptin, etc.); GLP-1 receptor agonists (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 extended release exenatide, abiratetide, dolaglutide); sodium-glucose cotransporter-2 (SGLT-2) inhibitors (e.g., canaglifozin (canaglifozin), dapaglifozin (dapaglifozin), empaglifozin (empaglifozin)); bile acid sequestrants (e.g., colesevelam, etc.); dopamine receptor agonists (e.g., immediate release bromocriptine); biguanides (e.g., metformin, etc.); insulin; oxyntomodulin; sulfonylureas (e.g., chlorpropamide, glimepiride, glipizide, glyburide, glibornuride, glipizide, glipiride, tolazamide, tolbutamide, acetylbenzenesulfonylcyclohexamide, carbutamide, etc.); and thiazolidinediones (e.g., pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, darglitazone, englitazone, nateglinide, rosiglitazone, troglitazone, etc.). In some embodiments, the dosage of one or more additional therapeutic agents is reduced when provided in combination with a conjugate or compound of the invention. In some embodiments, the additional therapeutic agent may be used at a lower dosage when used in combination with the conjugates or compounds of the invention than when each is used alone.
In certain embodiments, wherein the disease or disorder is selected from: 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 Hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unmanaged cholesterol and/or lipid levels, osteoporosis, inflammation, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), kidney disease, and eczema, and the second therapeutic agent may be liraglutide.
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 using a combination therapy comprising administering to a subject in need thereof an effective amount of a conjugate, compound, or pharmaceutical composition of the invention in combination with a surgical therapy. In certain embodiments, the surgical therapy can be bariatric surgery (e.g., gastric bypass surgery, such as a biliary jejunal gastric bypass surgery, gastric sleeve debulking, adjustable gastric banding, biliopancreatic diversion, duodenal switch, intragastric balloon, gastric plication, and combinations thereof).
In embodiments where one or more additional therapeutic agents or surgical therapies are administered on the same day as an effective amount of a conjugate or compound of the invention, the conjugate or compound of the invention can be administered before, after, or simultaneously with the additional therapeutic agent or surgical therapy. The use of the term "in combination" does not limit the order in which the 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), concurrently with, or after (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 second therapy is administered to the subject.
Other embodiments of the invention include additional proteolytic stabilizing modifications that incorporate the amylin agonist peptide, preferably in the region of the peptide between residues 10 and 17. Such proteolytic stabilizing modifications include, but are not limited to, amino acid substitutions 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 proteolytic stabilizing modification are contemplated herein.
In a further embodiment of the invention, the thioether-cyclized amylin mimetic peptide or derivative thereof comprises at least one amino acid residue derived from a half-life extending moiety. Such half-life extending moieties are introduced at suitable (tolerant) sites via conjugation to residues appropriately mutated at these positions. Examples of half-life extending moieties include, but are not limited to, albumin binding lipids, such as palmitic acid or similar fatty acids, and protein bioconjugates, such as HSA, mAb or Fc conjugates.
Other embodiments of the invention include amino acid substitutions and/or peptide modifications introduced to improve the physicochemical properties of the thioether cyclized amylin peptide. In some cases, the native amylin amino acid residues may be mutated to residues that reduce the pI of the peptide, thereby making it easier to formulate for administration while maintaining amylin receptor potency. In other cases, derivatization with a water-soluble functional group (such as, but not limited to, polyethylene glycol) is contemplated as a means of improving the solubility of the thioether-cyclized amylin analog in a suitable formulation vehicle.
Detailed description of the preferred embodiments
The present invention also provides the following non-limiting embodiments.
Embodiment 1 is a conjugate comprising a monoclonal antibody or antigen-binding fragment thereof coupled to an amylin mimetic peptide, wherein the amylin mimetic peptide is represented by formula I or a derivative or a pharmaceutically acceptable salt thereof (SEQ ID NO: 53):
Figure BDA0002739484940000521
wherein
n is 1 or 2;
Z2is a direct bond, serine or glycine;
Z4is T or
Figure BDA0002739484940000522
Z5Is A, beta-alanine,
Figure BDA0002739484940000523
Figure BDA0002739484940000524
Z6Is T or
Figure BDA0002739484940000525
Z10Is Q or E;
Z11is R or K, wherein-amine of K is optionally substituted by-C (═ NH) NH2Substitution;
Z12is L or
Figure BDA0002739484940000526
Z16Is L or
Figure BDA0002739484940000527
Z25Is P or K;
Z26is I or K;
x is ATZ10Z11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34(SEQ ID NO:54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55);
Z29is P or K;
Z34is S or K;
wherein the derivative is a compound of formula I modified by one or more methods selected from: amidation, glycosylation, carbamylation, sulfation, phosphorylation, cyclization, lipidation, and pegylation.
Embodiment 2 is the conjugate of embodiment 1 wherein the amylin mimetic peptide is a derivative of an amylin mimetic peptide of formula I, or a pharmaceutically acceptable salt thereof, modified by one or more methods selected from the group consisting of: amidation, lipidation and pegylation.
Embodiment 3 is the conjugate of embodiment 1, wherein the amylin mimetic peptide is represented by formula I or a derivative, or a pharmaceutically acceptable salt thereof, wherein:
Z2is a direct bond;
Z5is beta-alanine,
Figure BDA0002739484940000531
Z6Is T.
Embodiment 4 is the conjugate of embodiment 1, wherein the amylin mimetic peptide is represented by formula I or a derivative, or a pharmaceutically acceptable salt thereof, wherein:
Z16is L;
Z12is composed of
Figure BDA0002739484940000532
Embodiment 5 is the conjugate of embodiment 1, wherein the amylin mimetic peptide is represented by formula I or a derivative, or a pharmaceutically acceptable salt thereof, wherein:
Z11is R;
Z12is composed of
Figure BDA0002739484940000533
Embodiment 6 is the conjugate of embodiment 1, wherein the amylin mimetic peptide is selected from SEQ ID NOs 4-28.
Embodiment 7 is the conjugate of any one of embodiments 1 to 6, wherein the monoclonal antibody or antigen binding fragment thereof is covalently attached to the amylin mimetic peptide at a lysine residue of the amylin mimetic peptide via a linker.
Embodiment 8 is the conjugate of any one of embodiments 1 to 7, wherein the monoclonal antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining region 1(HCDR1), HCDR2, HCDR3, and light chain complementarity determining region 1(LCDR1), LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NOs: 47, 48, 49, 50, 51, and 52, respectively.
Embodiment 9 is the conjugate of embodiment 8, wherein the isolated monoclonal antibody comprises a heavy chain variable domain (VH) having the polypeptide sequence SEQ ID NO 43 and a light chain variable domain (VL) having the polypeptide sequence SEQ ID NO 45.
Embodiment 10 is the conjugate of embodiment 9, further comprising an Fc moiety.
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.
Embodiment 12 is a method of making a conjugate according to any one of embodiments 1 to 11 comprising reacting an electrophile, preferably a bromoacetamide-derived linker, on a side chain of the amylin mimetic peptide, preferably on an amino side chain of a lysine residue of the amylin mimetic peptide, with a thiol group of a cysteine residue of SEQ ID NO:49 of the monoclonal antibody, or antigen-binding fragment thereof, thereby forming a covalent bond between the amylin mimetic peptide and the monoclonal antibody, or antigen-binding fragment thereof.
Embodiment 13 is a pharmaceutical composition comprising a conjugate according to any one of embodiments 1 to 11 and a pharmaceutically acceptable carrier.
Embodiment 14 is a method for treating or preventing obesity in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition according to embodiment 13.
Embodiment 15 is the method of embodiment 14, wherein administering an effective amount of the pharmaceutical composition to a 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% compared to the body weight of the subject prior to administration of the pharmaceutical composition.
Embodiment 16 is a method for treating or preventing a disease or disorder in a subject in need thereof, wherein the 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 Hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unmanaged cholesterol and/or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease, and eczema, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to embodiment 13.
Embodiment 17 is the method of embodiment 16, wherein the disease or disorder is obesity.
Embodiment 18 is the method of embodiment 16, wherein the disease or disorder is type I diabetes.
Embodiment 19 is the method of embodiment 16, wherein the disease or disorder is type II diabetes.
Embodiment 20 is the method of embodiment 16, wherein the disease or disorder is metabolic syndrome.
Embodiment 21 is the method of embodiment 16, wherein the disease or disorder is kidney disease.
Embodiment 22 is the method of embodiment 16, wherein the disease or disorder is nonalcoholic steatohepatitis (NASH).
Embodiment 23 is the method of embodiment 16, wherein the disease or disorder is non-alcoholic fatty liver disease (NAFLD).
Embodiment 24 is a method of reducing food intake in a subject in need thereof, the method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of embodiment 13.
Embodiment 25 is the method of embodiment 24, wherein administering an effective amount of the pharmaceutical composition to a subject in need thereof results in a decrease 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 50% compared to the food intake of the subject prior to administration of the pharmaceutical composition.
Embodiment 26 is a method of modulating amylin receptor activity in a subject in need thereof comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of embodiment 13.
Embodiment 27 is the method of any one of embodiments 14 to 26, wherein the pharmaceutical composition is administered via injection.
Embodiment 28 is the method of embodiment 27, wherein the injection is delivered subcutaneously, intramuscularly, intraperitoneally, or intravenously.
Embodiment 29 is the method of any one of embodiments 14 to 28, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.
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.
Embodiment 31 is the method of embodiment 30, wherein the anti-diabetic agent is a glucagon-like peptide-1 receptor modulator.
Embodiment 32 is the method of embodiment 29, wherein the second therapeutic agent is liraglutide.
Embodiment 33 is the method of any one of embodiments 14 to 32, wherein the pharmaceutical composition is administered to the subject in need thereof daily, weekly, or monthly.
Embodiment 34 is the method of embodiment 33, wherein the pharmaceutical composition is administered once, twice, three times, four times, five times, or six times daily.
Embodiment 35 is the method of embodiment 33, wherein the pharmaceutical composition is administered once, twice, three times, four times, five times, or six times per week.
Embodiment 36 is the method of embodiment 33, wherein the pharmaceutical composition is administered once, twice, three times, or four times a month.
Embodiment 37 is a kit comprising a conjugate according to any one of embodiments 1 to 14 or a pharmaceutical composition according to embodiment 13, preferably the kit further comprises an effective amount of a second therapeutic agent, more preferably the kit further comprises an effective amount of liraglutide.
Embodiment 38 is the kit of embodiment 37, wherein the kit further comprises an injection device.
Embodiment 39 is a method of making a pharmaceutical composition comprising a compound selected from the group consisting of SEQ ID NOS 4-42 and a pharmaceutically acceptable carrier.
Synthesis of
The compounds or conjugates of the invention can be synthesized according to general synthetic methods known to those skilled in the art. The following description of the synthesis is for illustrative purposes and is in no way intended to be a limitation of the present invention.
The thioether-cyclized amylin mimetic peptides or derivatives of the present invention can be synthesized by a variety of known conventional methods for forming a continuous peptide bond between amino acids, and are preferably performed by Solid Phase Peptide Synthesis (SPPS), as generally described in Merrifield (J.am.chem.1963, 85, 2149-. Conventional methods for peptide synthesis involve condensation between the free amino group of one amino acid residue whose other reactive functional groups have been suitably protected and the free carbonyl group of the other amino acid whose reactive functional group has also been suitably protected. Examples of condensation reagents commonly utilized for peptide bond formation include Diisopropylcarbodiimide (DIC) with or without 1-Hydroxybenzotriazole (HOBT), or ethyl cyano (isonitroso) acetate (Oxyma Pure), 2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethylammonium Hexafluorophosphate (HBTU), 2- (1H-7-azabenzotriazol-1-yl) -1,1,3, 3-tetramethylammonium Hexafluorophosphate (HATU), 2- (6-chloro-1H-benzotriazol-1-yl) -1,1,3, 3-tetramethylammonium Hexafluorophosphate (HCTU), 1-cyano-2-ethoxy-2-oxoethylaminooxy-trispyrrolidinylhexafluorophosphate phosphonium salt; (PyOxim), 2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyltetrafluoroborate ammonium salt (TBTU), trispyrrolidinyl phosphonium hexafluorophosphate bromide (PyBroP), and the like.
Automated peptide synthesis processes may be performed at room temperature (rt) or at elevated temperatures, preferably by applying microwave heating, as described by: yu (J.org.chem.,1992,57,4781-4784)) and as newly improved by Palasek (J.Pept.Sci.,2007, Vol.13, p.143-148) (J.Pept.Sci.,2007,13, 143-47148)).
The compounds of the invention (C-terminal amides) can be conveniently prepared using the N- α -FMOC protected amino acid procedure, whereby the carboxy terminus of a suitably protected N- α -FMOC protected amino acid is coupled to 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, etc. The resin bound FMOC-amino acid can then be deprotected by exposure to 20% piperidine in DMF or NMP, which treatment serves to selectively remove the FMOC protecting group. Additional FMOC-protected amino acids are then sequentially coupled and deprotected to yield the desired resin-bound protected peptide. In some cases, it may be necessary to use an orthogonal reactive protecting group for another amine in the peptide sequence that will tolerate FMOC deprotection conditions. Protecting groups, such 4-methyltrityl (Mtt) or 4-methoxytrityl (Mmt), both of which can be removed by treatment with 1% TFA/DCM, or preferably allyloxycarbonyl (alloc; accessible by Pd (PPh)3)4/PhSiH3Treatment removal), 1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) ethyl (Dde; removable by treatment with 2-3% hydrazine/DMF) and 1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) -3-methylbutyl (ivDde; removable by treatment with 2-3% hydrazine/DMF) can be effectively used in this case.
In conventional peptide synthesis methods, the reactive side chains of alpha amino acids are typically protected throughout the synthesis with suitable protecting groups to render them inert to coupling and deprotection protocols. Although a number of protecting groups are known in the art for amino acid side chains, the following protecting groups are most preferred herein: tert-butyl (t-Bu) for serine, threonine, glutamic acid, aspartic acid, and tyrosine; trityl (Trt) for asparagine, glutamine, cysteine, homocysteine and histidine; t-butyloxycarbonyl (Boc) for the-amino group of tryptophan and lysine; and 2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine. These protecting groups are removed upon strong acid treatment, such as high concentrations of trifluoroacetic acid (TFA).
Upon completion of the SPPS, the resin-bound, side chain-protected peptide is deprotected and concomitantly cleaved from the resin using a cleavage mixture consisting essentially of (TFA) in combination with various combinations of carbenium scavengers such as Triisopropylsilane (TIPS), water, phenol, and anisole. The crude solid peptide was then isolated by precipitation of the peptide/mixture filtrate with cold ether. The crude peptide thus obtained is then dissolved in a predominantly aqueous solvent system comprising an organic co-solvent, such as acetonitrile or ethanol, at low concentrations (about <5 mg/mL). 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 analog of the present invention. The thioether-cyclized amylin analog so formed may be purified using purification techniques generally known in the art. The preferred method of peptide purification for use herein is reverse phase High Performance Liquid Chromatography (HPLC). The purified peptide was then characterized by liquid chromatography/mass spectrometry (LC/MS).
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 suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
Abbreviations
Herein and throughout the specification, the following abbreviations may be used:
aq aqueous
alloc allyloxycarbonyl radical
Boc tert-butyloxycarbonyl group
BSA bovine serum albumin
CDI 1, 1' -carbonyldiimidazole
CT calcitonin
CTR calcitonin receptor
DCM dichloromethane
Dde 1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) ethyl
DIC diisopropylcarbodiimide
DIEA diisopropylethylamine
DMA N, N-dimethylacetamide
DMEM Darbeike modified eagle's medium
DMF N, N-dimethylformamide
EDC N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide
EDTA ethylene diamine tetraacetic acid
Et Ethyl group
EtOAc ethyl acetate
EtOH ethanol
FBS fetal bovine serum
FMOC 9-fluorenylmethyloxycarbonyl
g
h hours
HATU 2- (1H-7-azabenzotriazol-1-yl) -1,1,3, 3-tetramethylammonium hexafluorophosphate
HBSS Hank balanced salt solution
HBTU 2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate)
Ammonium HCTU 2- (6-chloro-1H-benzotriazol-1-yl) -1,1,3, 3-tetramethylhexafluorophosphate
HCl hydrochloric acid
HEPES 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid;
HIC hydrophobic interaction chromatography
HOBT 1-hydroxybenzotriazole
HPLC high performance liquid chromatography
HTRF homogeneous time-resolved fluorescence
IBMX 3-isobutyl-1-methylxanthine
ivDde 1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) -3-methylbutyl
LCMS high performance liquid chromatography-mass spectrometry
Me methyl group
MeCN acetonitrile
mg of
min for
mL of
Mmt 4-methoxytrityl
Mtt 4-Methyltriphenylmethyl
NMP 1-methyl-2-pyrrolidone
OEG 8-amino-3, 6-dioxaoctanoyl
ORF open reading frame
Oxyma cyano (Isonitroso) acetic acid ethyl ester
Pal palmitoyl radical
Pbf 2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl
PBS phosphate buffered saline
Pd(PPh3)4Tetrakis (triphenylphosphine) palladium (0)
PhSiH3Phenyl silane
PyBroP trispyrrolidinylphosphonium hexafluorophosphate Bromide
Pyroxim 1-cyano-2-ethoxy-2-oxoethylaminooxy-tripyrrolidinyl hexakis
Fluorophosphoric acid phosphonium salts
RAMP receptor activity modified protein
rt Room temperature
Retention time of RT
sat'd saturation
SPPS solid phase peptide Synthesis
t-Bu tert-butyl
TBTU 2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyltetrafluoroborate ammonium salt
TFA trifluoroacetic acid
TIPS Tri-isopropyl silyl
Tris Tris (hydroxymethyl) aminomethane
Trt Triphenylmethyl
Examples
Example 1: synthesis of thioether-cyclized amylin mimetics (scheme 1)
Step A: synthesis of resin-bound C-terminal amide peptidesBecome into
The protected peptidyl resin was synthesized on a CEM Liberty Blue Microwave peptide synthesizer using FMOC strategy as described above, using a low load Rink amide resin, preferably FMOC-PAL-PEG PS resin (approximately 0.16-0.2meq/g, supplied by Applied Biosystems), on a 0.1mmol scale, as described in scheme 1. Standard FMOC protected amino acids (supplied by Novabiochem (EMD Millipore), Bachem, Peptides International, Sigma-Aldrich or Chem-Impex) were coupled for 4 minutes at 5-fold excess over resin loading using DIC/Oxyma as coupling reagent and reaction temperature of about 90 ℃. FMOC-Arg (Pbf) -OH was double coupled at 90 ℃ for 4 min each, and FMOC-His (Trt) -OH was coupled using a two-stage protocol: at room temperature for 4 minutes, then at 50 ℃ for 8 minutes. MonoFMOC deprotection was performed in 20% piperidine in DMF (deprotection solution) at 90 ℃ for 1.5 min.
And B: protocol for Bromoacetylation of resin-bound peptides (scheme 2)
The FMOC deprotected peptide resin (0.1mmol) was treated with a solution of bromoacetic anhydride (6-20 equivalents) in DMF (5mL) in a microwave reactor at 50 ℃ for 5 minutes, at which time the reaction was generally determined to be complete according to the Kaiser ninhydrin test. In case it was determined that the coupling was incomplete, the coupling was repeated using fresh reagents.
And C: protocol for cleaving peptides from resins
Upon completion of SPPS, the resin was washed thoroughly with DMF, then DCM, and dried. The resin was then treated with a cleavage mixture (10mL/0.1mmol scale) consisting of TFA/water/TIPS (95:2.5:2.5) (cleavage mixture A), or more preferably TFA/water/phenol/TIPS (88:5:5:2) (cleavage mixture B), and heated in a microwave reactor at 38 ℃ for 40 minutes, followed by filtration. The resin was washed with TFA, and the combined filtrates were concentrated under a stream of nitrogen to a volume of about 2.5mL, and the peptide was precipitated by addition of cold diethyl ether (40 mL). The peptide/ether suspension was centrifuged and the ether layer decanted. The peptide pellet was resuspended in ether, centrifuged and decanted, and the process repeated three times. The crude peptide thus obtained was dried under a gentle stream of nitrogen.
Step D: protocol for peptide cyclization (thioether formation)
To be provided with<The crude cysteine-or homocysteine-containing peptide was dissolved in deoxygenated MeCN/water (50-60% MeCN) or EtOH/water (50-60% EtOH) at a concentration of 4 mg/mL. Then by adding solid NaHCO3、NaHCO3The pH of the peptide solution was raised to about 7-9 with saturated aqueous solution or 1M Tris aqueous buffer (pH 7.5), and the resulting solution was stirred at room temperature for 3-16 hours. Typically, cyclization is complete within 1 hour as determined by LC/MS analysis.
Step E: protocol for peptide purification
The cyclization reaction mixture was acidified to pH 1.5-3 by addition of TFA and the solution was concentrated to remove most of the organic co-solvent (MeCN or EtOH) to the point where slight turbidity appeared. A minimum amount of co-solvent was added back if necessary to homogenize the mixture, and the resulting solution was then directly purified by preparative HPLC in multiple injections. Purification was performed on an Agilent PrepStar HPLC system or a Gilson HPLC 2020 personal purification system using a reverse phase C18 or C8 column selected from: varian Pursuit XRs C18(21x250mm,
Figure BDA0002739484940000621
5μm)；Varian Pursuit XRs Diphenyl(30x100mm,
Figure BDA0002739484940000623
5μm)；Zorbax 300 SB-C8(21x250mm,
Figure BDA0002739484940000622
5μm)；Waters Atlantis T3 C18(19x250mm,
Figure BDA0002739484940000625
5μm)；Agilent Polaris 5 C18-A(30x250mm,
Figure BDA0002739484940000624
5 μm). The mobile phase consisted of buffer A (0.1% TFA in water) and buffer B (0.1 in MeCN)% TFA) ranging from an initial concentration of 10-20% B to a final concentration of 40-90% B, run time ranging between 36-80 min. UV detection was monitored at 220nm and 254 nm. The product containing fractions were purified using the fractions from above (4.6x250mm,
Figure BDA0002739484940000626
5 μm) was analyzed by analytical HPLC on an Agilent 1100 HPLC system. Pure fractions were combined, concentrated to remove most of the organic phase, and then lyophilized. TFA/HCl salt exchange was then performed by triple lyophilization from 2mM HCl, according to the procedure described by Andrushchenko et al (j.pept.sci.,2006,13, 37-43).
Figure BDA0002739484940000631
Wherein LG is a leaving group
X is ATZ10Z11Z12ANFLVHSSNNFGZ25Z26LPZ29 TNVGZ34Or
VLGRLSQELHRLQTYPRTNTGS
Protected amino acid
Scheme 1: synthesis of thioether-cyclized amylin mimetics as follows:
amylin/pramlintide/davalinin analogs (scheme 1 discloses SEQ ID NOs 56, 56-58 and 55, respectively) Listed in order of occurrence, respectively
Example 2: synthesis of Lipidated amylin mimetics (scheme 2)
Step A: for introducing derivatized lysine residues into peptide sequences stacked on standard Rink amide resins Protocol (1)
Resin-bound C-terminal amide peptide (set to before the desired point of derivatization) was added under microwave conditions (either manually or on a Liberty Blue peptide synthesizer) using the DIC/Oxyma coupling procedure as described in example 1AAnd prepared as described in example 1A) sequentially couple Dde-lys (FMOC) -OH or ivDde-lys (FMOC) -OH, followed by FMOC-OEG-OH (coupling one or two units in series), optionally followed by FMOC-Glu-OtBu. Deprotection of FMOC followed by application of lipophilic acid [ e.g., palmitic acid ] in microwave conditions at 90 deg.C](5-10 equiv.), DIC (5-10 equiv.), and HOBT or Oxyma (5-10 equiv.) the resin was treated in DMF for 10 minutes. The reaction was then drained and the resin was washed with DMF. Scheme 2 shows that at position Z25Lipidated lysine was introduced. Those skilled in the art will recognize that a similar approach will be at Z26、Z29Or Z3To produce lipidated lysine.
Figure BDA0002739484940000651
Scheme 2: protocol for the introduction of derivatized lysine residues into amylin mimetic peptides (scheme 2 discloses SEQ ID NOs 59, 60 and 61, listed in order of occurrence respectively)
And B: protocol for deprotection of Dde-or ivDde-protected lysyl peptides
The derivatized lysyl peptide resin was treated with a solution of 3% hydrazine in DMF (6mL/0.1mmol resin) under microwave conditions at 90 ℃ for 3.5 minutes. The reaction was drained and the procedure repeated two more times. The reaction was drained and the resin was washed thoroughly with DMF and then DCM.
And C: protocol for direct incorporation of FMOC-Lys (Pal-Glu-OtBu) -OH residue
In the case of incorporation of a palmitoylated-gamma Glu-lysyl residue into this sequence, FMOC-Lys (Pal-Glu-OtBu) -OH (available from Peptides International or Activepeptide) can be used directly in the protocol described in example 1A.
Example 3: synthesis of BrAc-dPEGx-derived amylin mimetics (scheme 3)
Thioether cyclized peptide obtained from example 1E (wherein Z25、Z26、Z29Or Z34One of them is K (NH)2) A solution (0.3mL) of (7. mu. mol) and BrAc-dPEGx-OTFP (3 equiv.) in DMA was treated with DIEA (7 equiv.) and the resulting solution was stirred at room temperature. After completion of the reaction (ca.1 h), the mixture was acidified with TFA, diluted with water (0.1% TFA), and purified by reverse phase chromatography as described in example 1E. Scheme 3 shows that at position Z25Lipidated lysine was introduced. Those skilled in the art will recognize that a similar approach will be at Z26、Z29Or Z34To produce a BrAc-dPEGx-derived lysine.
Figure BDA0002739484940000671
Scheme 3: protocol for the synthesis of BrAc-dPEGx-derived amylin mimetic peptides (scheme 3 discloses SEQ ID) NO62 and 62-65, listed in order of occurrence respectively)
Example 4: synthesis of thioether-cyclized amylin mimetic peptide-mAb conjugates
Expression and purification of mAbs
Fully human monoclonal antibodies (mabs) can be recombinantly expressed in mammalian expression hosts and purified from cell culture supernatants using standard methods known in the art. For example, cDNA sequences encoding the Light Chain (LC) and Heavy Chain (HC) of a mAb, each comprising an appropriate single peptide that causes secretion, can be cloned into separate mammalian expression vectors or into a single expression vector using standard molecular biology methods. The expression vector used may be a commercially available vector such as pEE12.4, pcDNATM3.1(+) or pIRESpuro3 or any custom-made expression vector with similar functions. In such vectors, transcription of the heavy and light chains of the mAb is each driven by any known effective promoter, such as the hCMV-MIE promoter. Transfection grade Plasmid DNA of separate LC and HC expression constructs or a single construct expressing both LC and HC was prepared using standard methods such as QIAGEN Plasmid Midi kit.
Following the manufacturer's instructions, a lipid-based transfection reagent such as Freestyle was usedTMMax transfection reagents purified plasmid DNA was prepared and then transfected into standard mammalian expression host cell lines, such as CHO-S or HEK 293-F. If the mAbs LC and HC are encoded by separate expression constructs, both constructs are transfected simultaneously. Mammalian cells for maintenance or for mAb expression are cultured according to standard cell culture methods before and after transfection, whereby the range of cell densities maintained, the culture medium used and other cell culture conditions followed are determined by the particular mammalian host cell line utilized. These parameters are usually documented by the supplier of the cell line or in the scientific literature. For example, CHO-S cells are cultured in CHO FreestyleTMSuspension was maintained in medium at 37 ℃ and 8% CO2Was shaken at 125RPM and the cell concentration was 1.5 and 2.0X10 per ml6The other cells divide.
Cell culture supernatants from transiently transfected mammalian cells expressing mabs were harvested several days post transfection, clarified by centrifugation and filtered. The duration of expression of CHO-S cells is typically four days, but can be adjusted and may vary for different mammalian host cell lines. Large scale transfections (>10 liters) were concentrated 10-fold using a concentrator such as Centramate. The mAb is purified from the clarified supernatant using a protein a affinity column such as HiTrap MabSelect Sure using standard methods for binding the mAb to a protein a resin, washing the resin, and eluting the protein using low pH buffer. The protein fractions were immediately neutralized by elution into tubes containing pH7 buffer, and the peak fractions were pooled, filtered and dialyzed overnight at 4 ℃ against pH7.2 Phosphate Buffered Saline (PBS). After dialysis, the mAb was filtered again (0.2 μ filter) and the protein concentration was determined by absorbance at 280 nm. The quality of the purified mAb protein was assessed by SDS-polyacrylamide gel electrophoresis (PAGE) and analytical size exclusion HPLC, and endotoxin content was measured using Limulus Amoebocyte Lysate (LAL) assay. The purified mAb was stored at 4 ℃.
Expression and purification of MSCB97 transiently transfected into CHO cells
MSCB97 was transfected transiently into cells with purified plasmid DNA of the MSCB97 expression construct according to the manufacturer' S recommendations at ExpicCHO-STMExpressed in cells (ThermoFisher Scientific, Waltham, Mass.; Cat. No. A29127). Briefly, 8% CO at 37 ℃ was set2And 125RPM in a shaking incubator ExpicHO-STMCells in ExpicHOTMSuspension was maintained in expression medium (ThermoFisher Scientific, Cat. No. A29100). Cells were passaged so that dilution to 6.0X 10 per ml could be achieved on the day of transfection6Individual cells, maintaining cell viability of 98% or better. Expifeacmine was usedTMTransient transfection was performed using the CHO transfection kit (ThermoFisher Scientific Cat. No. A29131). For each ml of diluted cells to be transfected, one mg of plasmid DNA was used and diluted to OptiPROTMSFM complex medium. Expifeacmine was used at a 1:3 ratio (v/v, DNA: reagent)TMCHO reagent, and also diluted to OptiPROTMIn (1). The diluted DNA and transfection reagent were combined for one minute, allowing DNA/lipid complexes to form, and then added to the cells. After overnight incubation, expihcho was addedTMFeed and ExpifeacamineTMThe CHO enhancer was added to the cells. The cells were cultured with shaking at 32 ℃ for five days, after which the culture supernatant was harvested.
Transient transfection ExpicHO-S was harvested by centrifugation (30min,6000rpm) for clarification followed by filtration (0.2. mu. PES membrane, Corning)TMCulture supernatant of cells. Large-scale transfections (5 to 20 liters) were first concentrated 10-fold using a Pall Centramate Tangential Flow Filtration system. 10 XDPBS (Darbeike phosphate buffered saline), pH7.2, was added to the supernatant to 1 Xfinal concentration and then loaded onto an equilibrated (DPBS, pH 7.2) HiTrap MabSelect Sure Protein A column (GE Healthcare; Little Chalfount, United Kingdom) at a relative concentration of 20mg Protein per ml resin using an AKTA FPLC chromatography system. After loading, the column was washed with 10 column volumes of DPBS pH 7.2. Washing with 10 column volumes of 0.1M sodium acetate pH3.5And (4) deproteinizing. Protein fractions were immediately neutralized by elution to tubes containing 2.0M Tris, pH7, at 20% elution fraction volume. The peak fractions were combined and the pH adjusted to about 5.5 with additional Tris if necessary. The purified protein (0.2 μ) was filtered and the concentration determined by absorbance at 280nm on a BioTek SynergyHTTM spectrophotometer. The quality of the purified proteins was assessed by SDS-PAGE and analytical size exclusion HPLC (Dionex HPLC system). LAL determination by nephelometry (
Figure BDA0002739484940000691
Associates of Cape Cod) endotoxin levels were measured.
Conjugation of MSCB97 to amylin mimetics
To mAb (1.2mL,19mg/mL) was added 4 equivalents of TCEP followed by EDTA (100 mM; 12. mu.L). After 2 hours at room temperature, LCMS analysis indicated complete reduction of the disulfide adduct at position C102. The reduced mAb was treated with a Zebra desalting spin column (7x10mL, 7K MWCO, pre-equilibrated with Tris-acetate 100mM pH 5.6) to remove the released cysteine/GSH. To this reduced mAb was added a solution of bromoacetylated amylin mimetic peptide from example 3 in Milli grade Q water (7 equivalents relative to mAb, 30-35mg/mL), followed by EDTA (100 mM; 13.5. mu.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 room temperature with gentle stirring. Then saturated with (NH)4)2SO4The reaction was diluted (10% v/v) and the crude conjugate was purified by hydrophobic interaction chromatography (TOSOH TSKgel Phenyl HIC) with a linear gradient (40-100% B/A, solvent A: 5% i-PrOH,1M (NH)4)2SO4100mM 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 (NaOAc, 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 obtain the final sample.
Example 5: peptide and peptide-bioconjugate analysis and characterization
The method A comprises the following steps: on a Hewlett Packard series 1100 MSD system configured with HP 1100 series HPLC, a Waters Atlantis T3C 18(4.6x250mm,
Figure BDA0002739484940000701
5 μm) column, purified peptide was analyzed by LC/MS. Depending on the polar/non-polar nature of the peptide, one of two linear gradients (buffer A: water + 0.1% TFA; buffer B: MeCN + 0.1% TFA) was used at a flow rate of 1mL/min and a column temperature of 35 deg.C [ method A1: 15-60% B, for 22 min; method A2: 40-90% B, for 22min]. Electrospray analysis (ES-API, positive ion scan) provides mass analysis for each peptide. In all cases, a number of charged species were observed, of which 1/3[ M +3 ]]+ and 1/4[ M +4 ]]The + ions are the characteristic, most prominently observed ions. All products produced their desired multiply charged ions within acceptable limits. The results of mass spectrometry analysis of the peptides and the Retention Time (RT) of the LC observed are shown in Table 1.
The method B comprises the following steps: on the Shimadzu 10AVP system, YMC-Pack-ODS-A (4.6x250mm,
Figure BDA0002739484940000702
5 μm) column, and the purified peptide was analyzed by HPLC. A linear solvent gradient (20-80% B over 30min) was used at a flow rate of 1mL/min (buffer A: water + 0.05% TFA; buffer B: MeCN + 0.05% TFA). Mass spectra were obtained on a Waters Xevo G2 ToF spectrometer (ToF MS ES, positive ion scan). In all cases, a number of charged species were observed, of which 1/3[ M +3 ]]+ and 1/4[ M +4 ]]The + ions are the characteristic, most prominently observed ions. All products produced their desired multiply charged ions within acceptable limits. The results of mass spectrometry analysis of the peptides and the Retention Time (RT) of the LC observed are shown in Table 1.
The method C comprises the following steps: on a Hewlett Packard series 1100 MSD system configured with HP 1100 series HPLC, MAbPac HIC-10(4.6x1000mm,
Figure BDA0002739484940000712
5 μm) column, purified amylin molds analyzed by Hydrophobic Interaction Chromatography (HIC)A peptidomimetic-mAb conjugate. A linear solvent gradient (0-100% B over 30min) was used at a flow rate of 0.5mL/min (buffer A: 5% i-PrOH,1.5M (NH)4)2SO4100mM phosphate buffer, pH 6.0; and (3) buffer solution B: 20% i-PrOH,100mM phosphate buffer, pH 6.0). Complete mass measurements were obtained on a Waters Xevo G2-XS QToF spectrometer (TOF MS ES, positive ion scan). The results of analytical characterization of amylin mimetic peptide-mAb conjugates are shown in table 1.
Table 1: analytical data for thioether-cyclized amylin mimetic Compounds。
Figure BDA0002739484940000711
Figure BDA0002739484940000721
Example 6: human calcitonin/RAMP 3 receptor cAMP assay (AMY3R assay)
The method for testing the in vitro potency of amylin mimetic analogs is a cell-based assay designed to measure cAMP produced by adenylate cyclase by modulating the human calcitonin G protein-coupled receptor through its interaction with the receptor activity modifying protein 3(CTR/RAMP 3). cAMP production in 1321N1 astrocytoma cells (discover x) transfected with human AMY3R was induced in a dose-dependent manner by amylin mimetic analogs and controls and measured in a LANCE FRET-based competitive cAMP immunoassay (PerkinElmer).
Cells were cultured in DMEM, 10% FBS, 2.5. mu.g/ml puromycin and 800. mu.g/. mu.l G418. For the assay, cells were collected by removing the medium, washing with PBS and verene to lift the cells (Life Technologies). Cells were centrifuged at 450 Xg for 5min and the supernatant aspirated. The cells were incubated at 0.5X 106Each cell/ml was resuspended in 1 XHBSS (Life Technologies), 5mM HEPES (Life Technologies), 0.1% BSA (Perkin Elmer), 1.0mM 3-isobutyl-1-methylxanthine(IBMX) (Sigma) and 10 μ Ι _ of suspended cells were added to each well of 384-well white optimal plate (PerkinElmer) to a final density of 5000 cells/well. Dilutions of amylin analogs and controls were prepared in 1x HBSS, 5mM HEPES, 0.1% BSA, and 10 μ Ι/well of each sample was added to the designated wells. The plates were incubated at room temperature for 30min with shaking. Then 20. mu.L/well of LANCE cAMP detection reagent mix (PerkinElmer) was added to each assay plate, which was incubated at room temperature for 2 to 24h with shaking. Plates were read on a Perkin Elmer Envision plate reader using a protocol based on the manufacturer's recommendations included in the LANCE Ultra cAMP kit. Four replicate measurements were made for all samples. Data were analyzed using the crubile internal 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 concentration. Use of a non-linear weighted least squares application (open source http:// cran. us. R-project. org/, by Janssen R) within an R environment&Non-clinical statistics and computational department implementation of D), data were fitted with a 4-P model.
The potency of the amylin mimetic analogs of the present invention relative to pramlintide used as a control in the same assay is presented in table 2 below:
table 2: AMY3 receptor potency of thioether-cyclized amylin mimetic Compounds and Pramlintide (seq.2)
Figure BDA0002739484940000731
Figure BDA0002739484940000741
Example 7: human calcitonin/RAMP 1 Complex cAMP assay (AMY1R assay)
In vitro potency and selectivity of amylin mimetic analogs were assessed using a cell-based assay designed to measure cAMP production following modulation of human CTR or CTR/RAMP1 complex (AMY 1R). cAMP production in human CTR or AMY1R transiently transfected COS7 cells was induced in a dose-dependent manner by amylin mimetic analogs and controls and measured using HTRF cAMP kit (CisBio cAMP Dynamic kit, catalog No. 62AM4 PEC).
A plasmid encoding the HA-labeled human calcitonin receptor was generated by subcloning the human CTR ORF (ENST00000426151.5) labeled with 3xHA immediately after the signal peptide into pcDNA3.1(+) using EcoRV and XhoI. A plasmid encoding Flag-tagged human RAMP1 was generated by subcloning the human RAMP1 ORF (ENST00000254661.4) into pcDNA3.1(+) using EcoRV and XhoI, the human RAMP1 ORF being tagged with a Flag-tag immediately after the signal peptide at the N-terminus (DYKDDDDK (SEQ ID NO: 66)).
COS-7 cells were cultured in DMEM (ThermoFisher Scientific #11965092) containing 10% FBS (Hyclone # SH30070.03) and 1% penicillin-streptomycin (ThermoFisher Scientific #15140122) and transfected in 384-well white poly-D-lysine coated plates (Corning #356663) using Fugene HD (Promega # E2312). For each condition, DNA (μ g): Fugene HD (μ L) mix ratio was 1:3, and 10,000 cells/well were added at 40 μ L on top of 10 μ L DNA: Fugene mixture. The plates were incubated at 37 ℃ in CO2Incubate in incubator for 48 hours. CTR: RAMP1 cDNA transfection ratio (2:9) was optimized to favor the formation of amylin-1 receptor (AMY1R) and the amount of CTR cDNA transfected was optimized so that the expression of calcitonin and amylin receptor on the cell surface was not significantly different as assessed by ELISA for HA labeling.
On the day of assay, the media was replaced with assay buffer containing calcium and magnesium-containing HBSS, 20mM HEPES and 0.1% fatty acid-free BSA, pH7.4, and the cells were starved for 1h at 37 ℃. The assay buffer was then replaced with fresh assay buffer containing 500 μ M IBMX and the compound was added to the assay buffer (without IBMX). The plates were incubated at room temperature for 30min with shaking. cAMP was detected according to the manufacturer's protocol (CisBio cAMP Dynamic kit, Cat # 62AM4 PEC). Fluorescence was read with a pheasar plate reader using excitation at 337nm and emission at 620nm and 665 nm. Data were normalized for the maximal response of pramlintide. Emax and EC50 determinations were performed from agonist response curves analyzed with a curve fitting program using the 4 parameter logistic dose response equation in Graphpad Prism 7.0. The data presented represent three independent experiments with four replicate measurements for each compound. Data are presented as mean values. The AMY1R potency of the compound relative to pramlintide (seq2) is expressed as fold change. The potency of the compound at AMY1R relative to the potency of the compound at CTR is also shown as a fold difference (table 3).
For cell surface receptor expression assays, cells from the same transfection as used for cAMP assays were seeded into 96-well plates, fixed with 4% paraformaldehyde and blocked with PBS + 1% FBS. Rat anti-HA-peroxidase (clone 3F10, Roche Bioscience #12013819001) was administered at 0.5mg/L for 30 min. After washing with blocking buffer and PBS, chemiluminescence was detected using SuperSignal substrate (Pierce, Rockford, IL, USA) and a PHERAstar plate reader.
Table 3: AMY1 and CT receptor potency of thioether-cyclized amylin mimetic Compounds and Pramlintide (seq.2)
Figure BDA0002739484940000751
Example 8: in vivo efficacy studies
Gastric emptying: acetaminophen (AAP) uptake in lean C57Bl/6N mice
Male lean C57BL/6 mice (6-8 weeks old) were obtained from Taconic laboratories. Mice were placed as one mouse per cage in a 12-hour light/dark-cycled temperature-controlled chamber bedding with AlphaDri. Mice were exposed to water ad libitum and maintained on a normal diet (laboratory diet catalog: 5K 75). Animals were acclimated to the facility for at least one week prior to the start of the experiment.
The day prior to dosing, mice were divided into groups of ten animals based on individual body weight. On the next day, 5:00-6:00pm, animals were taken off the food and treated with vehicle (PBS, pH7.4) or test compound at a dose of 30nmol/kg (3nmol/mL) via subcutaneous administration. After 18h, the acetaminophen (AAP) suspension mixture [ AAP (10 mg/mL); HPMC (5 mg/mL); acacia gum (50mg/mL) ] was administered to the animals by oral drench (10 mL/kg). Whole blood samples (tail incisions; -25 μ L) were collected into DMPK-C dried blood filter paper sheets at time points of 5, 10, 15, 30, and 60 min. The pieces of hemofilter paper were completely dried and placed in individual bags with desiccant to be ready for LC/MS analysis by standard techniques. Statistical analysis was performed using one-way ANOVA in Prism using Dunnett's post-hoc test. All data are shown as mean values.
Food intake of fasted lean C57BL6N mice: acute administration
Male C57BL/6 mice (6-8 weeks old) were obtained from Taconic laboratories. Mice were placed as one mouse per cage in a 12-hour light/dark-cycled temperature-controlled chamber bedding with AlphaDri. Mice were exposed to water ad libitum and maintained on a normal diet (laboratory diet catalog: 5K 75). Animals were acclimated in BioDAQ cages (Research Diets, inc., New Brunswick, NJ) not less than 72 hours prior to starting the experiment.
Once acclimated in the BioDAQ cage, mice were divided into groups of ten animals based on their individual body weights and food intake over the previous 24 hours. At 4:00-5:00pm, animals were weighed and treated with vehicle (PBS, ph7.4) or test compound at a dose of 30nmol/kg (3nmol/mL) via subcutaneous administration. After a subsequent overnight fasting period (16-18 hours), the food weight of each cage was continuously recorded by the BioDAQ automated monitoring system for the next 48 hours. Debris was removed daily from the hopper and surrounding area of the cage using vacuum. Supplementing food when necessary. The percentage of mean cumulative food intake relative to vehicle over the 12-48 hour period post-dose was calculated and reported in table 4. Statistical analysis was performed using two-way ANOVA in Prism using Dunnett's post-hoc test. All data are shown as mean values.
Table 4: in vivo efficacy studies of thioether-cyclized amylin mimetic compounds
Figure BDA0002739484940000771
Not determined ND
*p<0.05；**p<0.01；***p<0.001；§p<0.0001
Example 9: PK study of Male C57BL6N mice
In a 12-hour light/dark-cycling temperature-controlled chamber, male C57BL/6N mice (6-8 weeks old, Taconic) were individually housed with AlphaDri bedding and were allowed to freely reach labdie 5K75 rodent diet and drinking water. Mice were grouped based on body weight fed (N ═ 3); the compounds were formulated in PBS (1nmol/mL) and administered subcutaneously at a dose of 10 nmol/kg. Whole blood samples (tail-cut; -50. mu.L) were collected at 4h, 24h, 72h, 96h and 7d time points into EDTA-coated Sarstedt containing a protease inhibitor cocktail (Roche complete protease inhibitor and Millipore DPPIV inhibitor; 2.5. mu.L)
Figure BDA0002739484940000772
Tubes were placed on ice. The final blood draw (7d) is a terminal blood draw with a target volume of 500. mu.L (25. mu.L of protease inhibitor cocktail). The samples were then centrifuged (10,000rpm) for 10 minutes at 4 ℃; the plasma was then transferred to 96-well plates (. about.25. mu.L/well plasma) which were stored at-80 ℃ to be bioanalyzed.
Plasma samples were analyzed using an LC-MS/MS assay to quantify representative peptides. In this assay, analytes are extracted from plasma using immunoaffinity capture by anti-human IgGFc antibodies, followed by protease digestion (trypsin or pepsin) and reverse phase LC-MS/MS analysis. Multiple Reaction Monitoring (MRM) MS analysis was performed on an API5000 triple quadrupole mass spectrometer operating in positive electrospray mode. Peptides derived from the N-terminal region of the trypsin mimetic sequence were monitored as surrogate for the quantitative active conjugate, while peptides located on the Fc region of the mAb were monitored as surrogate for the total level of mAb. Standard curve and quality control samples were prepared by doping plasma with reference standards of amylin mimetic conjugates and treated simultaneously using the same protocol as the study samples. The data are shown in table 5.
Table 5: pharmacokinetic parameters of thioether-cyclized amylin mimetic Compounds in Male C57BL/6N mice
Figure BDA0002739484940000781
Estimation of finite time point samples taken during the cancellation phase
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 particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention, as defined by the present specification.
All documents cited herein are incorporated by reference.
Exemplary amylin mimetic sequences or conjugates thereof of the present invention include:
SEQ ID NO:1
name: amylin (1-37)
The structure is as follows:
Figure BDA0002739484940000782
SEQ ID NO:2
name: pramlintide (1-37)
The structure is as follows:
Figure BDA0002739484940000791
SEQ ID NO:3
name: davallin peptide (1-32)
The structure is as follows:
Figure BDA0002739484940000792
SEQ ID NO:4
name: [ Ring- (N3-COCH)2-C7)]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000793
SEQ ID NO:5
name: [ Ring- (N3-COCH)2-hC7),K(Ac)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000794
SEQ ID NO:6
name: [ Ring- (N3-COCH)2-hC7),β-A5]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000801
SEQ ID NO:7
name: [ Ring- (N3-COCH)2-hC7),β-A5,K(Ac)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000802
SEQ ID NO:8
name: [ Ring- (N3-COCH)2-hC7),β-A5,K(Alloc)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000803
SEQ ID NO:9
name: [ Ring- (N3-COCH)2-C7),β-A5,K(Ac)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000811
SEQ ID NO:10
name: [ Ring- (N3-COCH)2-C7),β-A5,K(Alloc)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000812
SEQ ID NO:11
name: [ Ring- (N3-COCH)2-C7),Abu5,K(Ac)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000813
SEQ ID NO:12
name: [ Ring- (N3-COCH)2-hC7),Abu5,K(Ac)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000821
SEQ ID NO:13
name: [ Ring- (N3-COCH)2-hC7),(S)-β-Aib5,K(Ac)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000822
SEQ ID NO:14
name: [ Ring- (N3-COCH)2-hC7),(R)-β-Aib5,K(Ac)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000823
SEQ ID NO:15
name: [ Ring- (N3-COCH)2-hC7),β-hA5,K(Ac)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000831
SEQ ID NO:16
name: [ Ring- (N3-COCH)2-hC7),β-hP5,K(Ac)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000832
SEQ ID NO:17
name: [ Ring- (N3-COCH)2-hC7), { pyrrolidinyl- (3S) -carboxy }5, k (ac)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000833
SEQ ID NO:18
name: [ Ring- (N3-COCH)2-hC7),{(1R,2R)-ACPC}5,K(Ac)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000841
SEQ ID NO:19
name: [ Ring- (N3-COCH)2-hC7),β-hT4,K(Ac)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000842
SEQ ID NO:20
name: [ Ring- (N3-COCH)2-C7),β-hT4,K(Ac)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000843
SEQ ID NO:21
name: [ Ring- (N3-COCH)2-hC7),β-hT6,K(Ac)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000851
SEQ ID NO:22
name: [ Ring- (N3-COCH)2-C7),β-hT6,K(Ac)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000852
SEQ ID NO:23
name: [ Ring- (S2-COCH)2-hC7),β-A5,K(Ac)26]-pramlintide 2-37
The structure is as follows:
Figure BDA0002739484940000853
SEQ ID NO:24
name: [ Ring- (S2-COCH)2-C7),β-A5,K(Ac)26]-pramlintide 2-37
The structure is as follows:
Figure BDA0002739484940000861
SEQ ID NO:25
name: [ Ring- (N3-COCH)2-hC7),β-A5,K(Ac)25]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000862
SEQ ID NO:26
name: [ Ring- (N3-COCH)2-hC7),β-A5,K(Ac)29]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000863
SEQ ID NO:27
name: [ Ring- (N3-COCH)2-hC7),β-A5,K(Ac)34]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000871
SEQ ID NO:28
name: [ Ring- (N3-COCH)2-hC7),β-A5]-Davallin peptide 3-32
The structure is as follows:
Figure BDA0002739484940000872
SEQ ID NO:29
name: [ Ring- (N3-COCH)2-hC7),β-A5,K(OEG2-Pal)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000873
SEQ ID NO:30
name: [ Ring- (N3-COCH)2-hC7),β-A5,K(OEG2-γ-Glu-Pal)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000881
SEQ ID NO:31
name: [ Ring- (N3-COCH)2-hC7),β-A5,α-MeL12,K(dPEG12-AcBr)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000882
SEQ ID NO:32
name: [ Ring- (N3-COCH)2-hC7),β-A5,hR11,K(dPEG12-AcBr)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000883
SEQ ID NO:33
name: [ Ring- (N3-COCH)2-hC7),β-A5,E10,K(dPEG12-AcBr)26]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000891
SEQ ID NO:34
name: [ Ring- (N3-COCH)2-hC7),β-A5,α-MeL12,K(dPEG12-AcBr)25]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000892
SEQ ID NO:35
name: [ Ring- (N3-COCH)2-hC7),β-A5,hR11,K(dPEG12-AcBr)25]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000893
SEQ ID NO:36
name: [ Ring- (N3-COCH)2-hC7),β-A5,E10,K(dPEG12-AcBr)25]-pramlintide 3-37
The structure is as follows:
Figure BDA0002739484940000901
SEQ ID NO:37
name: [ Ring- (N3-COCH)2-hC7),β-A5,α-MeL12,K(dPEG12)26]-pramlintide 3-37mAb homodimer conjugates
The structure is as follows:
Figure BDA0002739484940000902
SEQ ID NO:38
name: [ Ring- (N3-COCH)2-hC7),β-A5,hR11,K(dPEG12)26]-pramlintide 3-37mAb homodimer conjugates
The structure is as follows:
Figure BDA0002739484940000903
SEQ ID NO:39
name: [ Ring- (N3-COCH)2-hC7),β-A5,E10,K(dPEG12)26]-pramlintide 3-37mAb homodimer conjugates
The structure is as follows:
Figure BDA0002739484940000911
SEQ ID NO:40
name:[ Ring- (N3-COCH)2-hC7),β-A5,α-MeL12,K(dPEG12)25]-pramlintide 3-37mAb homodimer conjugates
The structure is as follows:
Figure BDA0002739484940000912
SEQ ID NO:41
name: [ Ring- (N3-COCH)2-hC7),β-A5,hR11,K(dPEG12)25]-pramlintide 3-37mAb homodimer conjugates
The structure is as follows:
Figure BDA0002739484940000921
SEQ ID NO:42
name: [ Ring- (N3-COCH)2-hC7),β-A5,E10,K(dPEG12)25]-pramlintide 3-37mAb homodimer conjugates
The structure is as follows:
Figure BDA0002739484940000922
SEQ ID NO:43
name: MSCB97 VH (variable heavy chain)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYDGCYGELDFWGQGTLVTVSS
SEQ ID NO:44
Name: MSCB97 HC (heavy chain)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYDGCYGELDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO:45
Name: MSCB97 VL (light chain variable region)
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIK
SEQ ID NO:46
Name: MSCB97 LC (light chain)
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:47
Name: MSCB97 HCDR1
SYAMS
SEQ ID NO:48
Name: MSCB97 HCDR2
AISGSGGSTYYADSVKG
SEQ ID NO:49
Name: MSCB97 HCDR3
YDGCYGELDF
SEQ ID NO:50
Name: MSCB97 LCDR1
RASQSVSSYLA
SEQ ID NO:51
Name: MSCB97 LCDR2
DASNRAT
SEQ ID NO:52
Name: MSCB97 LCDR3
QQRSNWPLT
Figure IDA0002739484970000011
Figure IDA0002739484970000021
Figure IDA0002739484970000031
Figure IDA0002739484970000041
Figure IDA0002739484970000051
Figure IDA0002739484970000061
Figure IDA0002739484970000071
Figure IDA0002739484970000081
Figure IDA0002739484970000091
Figure IDA0002739484970000101
Figure IDA0002739484970000111
Figure IDA0002739484970000121
Figure IDA0002739484970000131
Figure IDA0002739484970000141
Figure IDA0002739484970000151
Figure IDA0002739484970000161
Figure IDA0002739484970000171
Figure IDA0002739484970000181
Figure IDA0002739484970000191
Figure IDA0002739484970000201
Figure IDA0002739484970000211
Figure IDA0002739484970000221
Figure IDA0002739484970000231
Figure IDA0002739484970000241
Figure IDA0002739484970000251
Figure IDA0002739484970000261
Figure IDA0002739484970000271
Figure IDA0002739484970000281
Figure IDA0002739484970000291
Figure IDA0002739484970000301
Figure IDA0002739484970000311
Figure IDA0002739484970000321
Figure IDA0002739484970000331
Figure IDA0002739484970000341
Figure IDA0002739484970000351
Figure IDA0002739484970000361
Figure IDA0002739484970000371
Figure IDA0002739484970000381
Figure IDA0002739484970000391
Figure IDA0002739484970000401
Figure IDA0002739484970000411
Figure IDA0002739484970000421
Figure IDA0002739484970000431
Figure IDA0002739484970000441
Figure IDA0002739484970000451

Claims (22)

1. A compound of formula I (SEQ ID NO:53)
Figure FDA0002739484930000011
Wherein
n is 1 or 2;
Z2is a direct bond, serine or glycine;
Z4is T or
Figure FDA0002739484930000012
Z5Is A, beta-alanine,
Figure FDA0002739484930000013
Figure FDA0002739484930000014
Z6Is T or
Figure FDA0002739484930000015
Z10Is Q or E;
Z11is R or K, wherein-amine of K is optionally substituted by-C (═ NH) NH2Substitution;
Z12is L or
Figure FDA0002739484930000016
Z16Is L or
Figure FDA0002739484930000017
Z25Is P or K, wherein-amine of said K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure FDA0002739484930000021
Figure FDA0002739484930000022
Figure FDA0002739484930000023
Substitution, wherein the mAb is optionally substituted by another thioether bondTo a second compound of formula I such that there are two identical compounds of formula I on the mAb;
Z26is I or K, wherein-amine of said K is optionally substituted with-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure FDA0002739484930000024
Figure FDA0002739484930000025
Figure FDA0002739484930000026
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
x is ATZ10Z11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34(SEQ ID NO:54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55);
Z29is P or K, wherein-amine of said K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure FDA0002739484930000031
Figure FDA0002739484930000032
Figure FDA0002739484930000033
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z34is S or K, wherein-amine of K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure FDA0002739484930000034
Figure FDA0002739484930000035
Figure FDA0002739484930000041
Figure FDA0002739484930000042
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
and pharmaceutically acceptable salts thereof.
2. The compound of claim 1, wherein:
Z2is a direct bond;
Z5is beta-alanine,
Figure FDA0002739484930000043
Z6Is T;
and pharmaceutically acceptable salts thereof.
3. The compound of claim 1, wherein:
Z16is L;
Z25is P or K, wherein-amine of said K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure FDA0002739484930000044
Figure FDA0002739484930000045
Figure FDA0002739484930000046
Substitution, wherein the mAbOptionally substituted onto a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z26is I or K, wherein-amine of said K is optionally substituted with-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure FDA0002739484930000047
Figure FDA0002739484930000051
Figure FDA0002739484930000052
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z29is P or K, wherein-amine of said K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure FDA0002739484930000053
Figure FDA0002739484930000054
Figure FDA0002739484930000055
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z34is S or K, wherein-amine of K is optionally substituted by-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure FDA0002739484930000056
Figure FDA0002739484930000057
Figure FDA0002739484930000058
(ii) substitution, wherein the mAb is optionally substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
and pharmaceutically acceptable salts thereof.
4. A compound according to claim 3, wherein:
Z2is a direct bond;
Z5is beta-alanine,
Figure FDA0002739484930000061
Z6Is T;
and pharmaceutically acceptable salts thereof.
5. A compound according to claim 3, wherein:
Z11is R or K, wherein-amine of K is-C (═ NH) NH2Substitution;
Z25is P or K, wherein-amine of said K is-C (═ O) CH3Or
Figure FDA0002739484930000062
(ii) substitution, wherein the mAb is substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z26is I or K, wherein-amine of said K is-C (═ O) CH3、-C(O)CH2CH＝CH2、
Figure FDA0002739484930000063
Figure FDA0002739484930000064
Figure FDA0002739484930000065
(ii) substitution, wherein the mAb is substituted to a second compound of formula I by another thioether bond such that there are two identical compounds of formula I on the mAb;
Z29is P or K, wherein-amine of said K is-C (═ O) CH3Substitution;
Z34is S or K, wherein-amine of said K is-C (═ O) CH3Substitution;
and pharmaceutically acceptable salts thereof.
6. The compound of claim 5, wherein:
Z2is a direct bond;
Z5is beta-alanine,
Figure FDA0002739484930000066
Z6Is T;
and pharmaceutically acceptable salts thereof.
7. The compound of claim 5, wherein:
Z12is composed of
Figure FDA0002739484930000071
And pharmaceutically acceptable salts thereof.
8. The compound of claim 5, selected from (SEQ ID NOs 4-42, listed in order of occurrence, respectively):
Figure FDA0002739484930000072
Figure FDA0002739484930000081
Figure FDA0002739484930000091
Figure FDA0002739484930000101
Figure FDA0002739484930000111
Figure FDA0002739484930000121
Figure FDA0002739484930000131
and pharmaceutically acceptable salts thereof.
9. The compound of claim 1, wherein the compound is selected from SEQ ID NOs 4-42, or a pharmaceutically acceptable salt thereof.
10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.
11. A method for treating or preventing a disease or disorder in a subject in need thereof, wherein the 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 Hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with unmanaged cholesterol and/or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease, and eczema, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to claim 10.
12. A method of reducing food intake in a subject in need thereof, the method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 10.
13. A method of modulating amylin receptor activity in a subject in need thereof comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 10.
14. The method according to claim 13 wherein the amylin receptor comprises AMY1R, and/or AMY2R and/or AMY 3R.
15. The method according to claim 14 wherein the amylin receptor is AMY 1R.
16. The method according to claim 14 wherein the amylin receptor is AMY 3R.
17. The method of any one of claims 11 to 14, wherein the pharmaceutical composition is administered via injection.
18. The method of any one of claims 11-16, wherein the pharmaceutical composition is administered in combination with at least one antidiabetic agent.
19. The method of claim 18, wherein the anti-diabetic agent is a glucagon-like peptide-1 receptor modulator.
20. The method of claim 18, wherein the pharmaceutical composition is administered in combination with liraglutide.
21. A kit comprising the conjugate according to any one of claims 1 to 8, preferably further comprising liraglutide and a device for injection.
22. A process for preparing a pharmaceutical composition according to claim 10.
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