JP2021522231A - Thioether Cyclic Peptide Amyrin Receptor Regulator - Google Patents

Abstract

The present invention relates to an amilinomimetic peptide analog and its derivatives in which the N-terminus of each peptide is covalently attached to the internal amino acid side chain thiol functional group by a non-peptidyl cyclization cross-linking element. The thioether cyclized amilinomimetic analog and its derivatives may contain one or more modifications, including substitutions, insertions, deletions, and modifications. Also, thioether cyclized amilinomimetic analogs and derivatives thereof are optionally serum albumin bonds, such as an alkyl chain of at least 14 carbon atoms having an optional additional pendant carboxylate moiety. It may include elements or may include extended half-life biological moieties such as HSA, non-targeted mAbs, or Fc. Furthermore, the present invention relates to compositions of the above analogs and derivatives thereof that respond to the regulation of amyrin receptors and methods of treating pathological conditions.

Description

This application claims the priority benefit to US Patent Provisional Application No. 62 / 662,492 filed on April 25, 2018, the provisional patent application of which is hereby incorporated by reference in its entirety. Be incorporated.

(Sequence list)
This application has been submitted electronically in ASCII format and includes a sequence listing, which is incorporated herein by reference in its entirety. A copy of the ASCII was made on April 16, 2019, and the file name is PRD3471WOPCT1_SL. It is txt and its size is 55.747 bytes.

(Field of invention)
The present invention relates to thioether cyclized analogs of amylin, pramlintide, and davalintide, as well as derivatives thereof (referred to herein as "amillinomic peptides"), which function as agonists of the amylin receptor. Therefore, it is useful for the treatment of metabolic diseases and disorders such as obesity, type 2 diabetes, metabolic syndrome, insulin resistance, and dyslipidemia.

Amylin is a naturally occurring member of the calcitonin family of peptides, including calcitonin (CT), calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and intermedin (AM2) 37. It is an amino acid-containing peptide. Amyrin is synthesized in the pancreas in response to the influx of nutrients into the gastrointestinal tract and is secreted by the pancreas. After being released into the circulatory system, amylin binds with high affinity to specific class B G protein-coupled receptors (GPCRs) located predominantly in the vomiting central region of the retencephalic area of the central nervous system. .. Thus, amyrin is a centrally active neuroendocrine hormone that plays a role in regulating glucose homeostasis by inhibiting gastric emptying, inhibiting glucagon release, and inducing satiety (Hay DL et al. , Pharmacol Rev 2015; 67: 564-600). Amylin is a heterodimer structure consisting of calcitonin receptor (CTR) and receptor modifying proteins (RAMP1, RAMP2, and RAMP3) with three target receptor subtypes (AMY1R, AMY2R, and AMY3R) and in vitro. It has been shown that they interact with each other (Christopolos G et al., Mol Pharmacol 1999; 56: 235-42). The association of CTRs with these RAMPs provides an increased affinity for amyrin compared to that of CTR alone and provides a pharmacological basis for selecting amyrin receptors (in preference to CT). RL and Hay DL, Br J Pharmacol 2016; 173: 1883-98). Although it is generally accepted that amyrin is involved in high-affinity / strong interactions, especially with AMY1R and AMY3R, co-agonism in all receptor subtypes is for pharmacological benefit. May not always be required. (Hay DL et al., Br J Pharmacol 2018; 175: 3-17; Hay DL, Headache 2017; 57: 89-96).

Human amyloid, also known as islet amyloid polypeptide (IAPP), has several physicochemical properties that make it unsuitable for pharmaceuticals, but most notably its low water solubility, In addition, there is a tendency for self-aggregation and a tendency for adhesion to the surface. Pramlintide, an isogenic amyrin analog, was developed by incorporating three specific residue mutations (A25P, S28P, and S29P) into the amyrin sequence (Young AA et al., Drug Dev Res 1996; 37: 231). -48). These mutations confer improved physicochemical properties (reduced agglutination tendency) for amyrin. Pramlintide is a food intake for obese subjects (Smith SR et al., Am J Physiol Endocrine Mol tab 2007; 293: E620-7) and body weight (Aronne L et al., J Clin Endocrine Metab 2007). To reduce. Plumlintide is approved as an adjunct therapy in parallel with insulin treatment for the treatment of adult patients with type 1 diabetes, and for adult patients with type 2 diabetes, insulin alone or metformin insulin. And / or approved as an adjunct therapy in parallel with treatment with sulfonylurea (Pullman Jet al., Vasc Health Risk Manag 2006; 2: 203-12). Davalintide is a related synthetic peptide, 32 amino acids in length, the structure of which is a chimera of the primary sequences of pramlintide and salmon calcitonin. Therefore, davalintide lacks amyloid-forming residues of human amyloid (Westermark Pet al., Proc Natl Acad Sci USA 1990; 87: 5036-40). Davalintide is a highly potent agonist at both the amyrin and calcitonin receptors and exhibits improved pharmacological properties over native (rat) amyrin in reducing food intake and body weight in rats. (Mac CM et al., Int J Obes 2010; 34: 385-95). Amyrin, pramlintide and davalintide each have a very short half-life (less than 0.75 hours) in vivo, which limits their therapeutic utility (Roth JD et al., Immuno Endoc Matab 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 regimen of multiple daily doses to achieve clinical efficacy. Therefore, it is desirable to obtain an amyrin agonist peptide or derivative thereof having improved metabolic stability and pharmacokinetic profile.

One technique used to extend the half-life of a peptide is a conjugated reaction with a biological carrier such as albumin, a suitable mAb or an antigen-binding fragment thereof, or a crystallized fragment domain (Fc) protein derived from a mAb. Accompanied by. Such a bioconjugated chemical reaction is carried out by the reaction of a selectively reactive sulfhydryl functional group on a biological carrier molecule with a complementary electrophilic site modified to the peptide of interest. The maleimide functional group incorporated into the peptide molecule functions as a suitable electrophile for a bioconjugated chemical reaction, but the resulting substance formed by the bioconjugated reaction undergoes an inverse conjugation reaction in vivo. The peptide is lost from the biological carrier in response to the (retro-Michael reaction). Therefore, a more stable thioacetamide bond formed by coupling to the reactive haloacetamide derivatized peptide is used to avoid this possibility of inverse conjugation. However, the conditions required for bromoacetamide conjugation cannot be used with disulfide-containing amyrin analogs to successfully obtain their selective bioconjugation because of the reactivity of the unhindered disulfide ring. .. Such attempted chemical reactions result in simultaneous ring opening reactions and related side reactions that accompany them. It has long been recognized that the perfect disulfide loop is a crucial molecular feature required for receptor activation and biological function (Roberts AN et al., Proc Natl). Acad Sci USA 1989; 86: 9662-9666; Cornish J et al., Am. J. Physiol. 1998; 274: E827-E833; Bower RL and Hay DL., Br. J. Pharmacol. 2016; 173: 1383- 1898). It is specified herein that the N-terminal cyclic thioether substitution for the cysteine disulfide bond of pramlintide and davalintide unexpectedly maintains amylin receptor agonist activity and is stable towards a bioconjugated chemical reaction.

In one general aspect, the invention relates to a novel amilinomimetic compound. Also provided herein are conjugates comprising an amilinomimetic derivative of pramlintide or davalintide, and a monoclonal antibody or antigen-binding fragment thereof bound to an amilinomimetic peptide.

In one aspect, the invention is represented by a compound of formula I or a derivative thereof (SEQ ID NO: 53):

（式中、
ｎは、１又は２であり、
Ｚ_２は、直接結合、セリン、又はグリシンであり、
Ｚ_４は、Ｔ又は

(During the ceremony,
n is 1 or 2
Z ₂ is a direct bond, serine, or glycine,
Z ₄ is T or

であり、
Ｚ_５は、Ａ、β−アラニン、

And
Z ₅ is A, β-alanine,

であり、
Ｚ_６は、Ｔ又は

And
Z ₆ is T or

であり、
Ｚ_１０は、Ｑ又はＥであり、
Ｚ_１１は、Ｒ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝ＮＨ）ＮＨ_２によって置換されたものであり、
Ｚ_１２は、Ｌ又は

And
Z ₁₀ is Q or E,
Z ₁₁ is R or K, in which the ε-amine of K is optionally replaced by _{-C (= NH) NH 2.}
Z ₁₂ is L or

であり、
Ｚ_１６は、Ｌ又は

And
Z ₁₆ is L or

であり、
Ｚ_２５は、Ｐ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

And
Z ₂₅ is P or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在し、
Ｚ_２６は、Ｉ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb. death,
Z ₂₆ is I or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在し、
Ｘは、ＡＴＺ_１０Ｚ_１１Ｚ_１２ＡＮＦＺ_１６ＶＨＳＳＮＮＦＧＺ_２５Ｚ_２６ＬＰＺ_２９ＴＮＶＧＺ_３４（配列番号５４）、又はＶＬＧＲＬＳＱＥＬＨＲＬＱＴＹＰＲＴＮＴＧＳ（配列番号５５）であり、
Ｚ_２９は、Ｐ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb. death,
X is ATZ ₁₀ Z ₁₁ Z ₁₂ ANFZ ₁₆ VHSSNNFGZ ₂₅ Z ₂₆ LPZ ₂₉ TNVGZ ₃₄ (SEQ ID NO: 54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55).
Z ₂₉ is P or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在し、
Ｚ_３４は、Ｓ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb. death,
Z ₃₄ is S or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在する）、
及びその薬学的に許容される塩である。

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb. do),
And its pharmaceutically acceptable salt.

In certain embodiments, the present invention is represented by a compound of formula I or a derivative thereof (in the formula,
n is 1 or 2
Z ₂ is a direct bond,
Z ₄ is T or

であり、
Ｚ_５は、β−アラニン、

And
Z ₅ is β-alanine,

であり、
Ｚ_６は、Ｔであり、
Ｚ_１０は、Ｑ又はＥであり、
Ｚ_１１は、Ｒ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝ＮＨ）ＮＨ_２によって置換されたものであり、
Ｚ_１２は、Ｌ又は

And
Z ₆ is T
Z ₁₀ is Q or E,
Z ₁₁ is R or K, in which the ε-amine of K is optionally replaced by _{-C (= NH) NH 2.}
Z ₁₂ is L or

であり、
Ｚ_１６は、Ｌ又は

And
Z ₁₆ is L or

であり、
Ｚ_２５は、Ｐ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

And
Z ₂₅ is P or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在し、
Ｚ_２６は、Ｉ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb. death,
Z ₂₆ is I or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在し、
Ｘは、ＡＴＺ１０Ｚ１１Ｚ１２ＡＮＦＺ１６ＶＨＳＳＮＮＦＧＺ２５Ｚ２６ＬＰＺ２９ＴＮＶＧＺ３４（配列番号５４）、又はＶＬＧＲＬＳＱＥＬＨＲＬＱＴＹＰＲＴＮＴＧＳ（配列番号５５）であり、
Ｚ_２９は、Ｐ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb. death,
X is ATZ10Z11Z12ANFZ16VHSSNNFGZ25Z26LPZ29TNVGZ34 (SEQ ID NO: 54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55).
Z ₂₉ is P or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在し、
Ｚ_３４は、Ｓ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb. death,
Z ₃₄ is S or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在する）、
及びその薬学的に許容される塩である。

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb. do),
And its pharmaceutically acceptable salt.

In certain embodiments, the present invention is represented by a compound of formula I or a derivative thereof (in the formula,
n is 1 or 2
Z ₂ is a direct bond or serine
Z ₄ is T or

であり、
Ｚ_５は、Ａ、β−アラニン、

And
Z ₅ is A, β-alanine,

であり、
Ｚ_６は、Ｔ又は

And
Z ₆ is T or

であり、
Ｚ_１０は、Ｑ又はＥであり、
Ｚ_１１は、Ｒ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝ＮＨ）ＮＨ_２によって置換されたものであり、
Ｚ_１２は、Ｌ又は

And
Z ₁₀ is Q or E,
Z ₁₁ is R or K, in which the ε-amine of K is optionally replaced by _{-C (= NH) NH 2.}
Z ₁₂ is L or

であり、
Ｚ_１６は、Ｌであり、
Ｚ_２５は、Ｐ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

And
Z ₁₆ is L,
Z ₂₅ is P or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在し、
Ｚ_２６は、Ｉ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb. death,
Z ₂₆ is I or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在し、
Ｘは、ＡＴＺ_１０Ｚ_１１Ｚ_１２ＡＮＦＺ_１６ＶＨＳＳＮＮＦＧＺ_２５Ｚ_２６ＬＰＺ_２９ＴＮＶＧＺ_３４（配列番号５４）、又はＶＬＧＲＬＳＱＥＬＨＲＬＱＴＹＰＲＴＮＴＧＳ（配列番号５５）であり、
Ｚ_２９は、Ｐ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb. death,
X is ATZ ₁₀ Z ₁₁ Z ₁₂ ANFZ ₁₆ VHSSNNFGZ ₂₅ Z ₂₆ LPZ ₂₉ TNVGZ ₃₄ (SEQ ID NO: 54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55).
Z ₂₉ is P or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式ＩＩの化合物が存在し、
Ｚ_３４は、Ｓ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula II compounds on the mAb. death,
Z ₃₄ is S or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在する）、
及びその薬学的に許容される塩である。

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb. do),
And its pharmaceutically acceptable salt.

In certain embodiments, the present invention is represented by a compound of formula I or a derivative thereof (in the formula,
n is 1 or 2
Z ₂ is a direct bond,
Z ₄ is T or

であり、
Ｚ_５は、β−アラニン、

And
Z ₅ is β-alanine,

であり、
Ｚ_６は、Ｔであり、
Ｚ_１０は、Ｑ又はＥであり、
Ｚ_１１は、Ｒ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝ＮＨ）ＮＨ_２によって置換されたものであり、
Ｚ_１２は、Ｌ又は

And
Z ₆ is T
Z ₁₀ is Q or E,
Z ₁₁ is R or K, in which the ε-amine of K is optionally replaced by _{-C (= NH) NH 2.}
Z ₁₂ is L or

であり、
Ｚ_１６は、Ｌであり、
Ｚ_２５は、Ｐ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

And
Z ₁₆ is L,
Z ₂₅ is P or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式ＩＩの化合物が存在し、
Ｚ_２６は、Ｉ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula II compounds on the mAb. death,
Z ₂₆ is I or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在し、
Ｘは、ＡＴＺ_１０Ｚ_１１Ｚ_１２ＡＮＦＺ_１６ＶＨＳＳＮＮＦＧＺ_２５Ｚ_２６ＬＰＺ_２９ＴＮＶＧＺ_３４（配列番号５４）、又はＶＬＧＲＬＳＱＥＬＨＲＬＱＴＹＰＲＴＮＴＧＳ（配列番号５５）であり、
Ｚ_２９は、Ｐ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb. death,
X is ATZ ₁₀ Z ₁₁ Z ₁₂ ANFZ ₁₆ VHSSNNFGZ ₂₅ Z ₂₆ LPZ ₂₉ TNVGZ ₃₄ (SEQ ID NO: 54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55).
Z ₂₉ is P or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在し、
Ｚ_３４は、Ｓ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb. death,
Z ₃₄ is S or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、任意選択で、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在する）、
及びその薬学的に許容される塩である。

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb. do),
And its pharmaceutically acceptable salt.

In certain embodiments, the present invention is represented by a compound of formula I or a derivative thereof (in the formula,
n is 1 or 2
Z ₂ is a direct bond or serine
Z ₄ is T or

であり、
Ｚ_５は、Ａ、β−アラニン、

And
Z ₅ is A, β-alanine,

であり、
Ｚ_６は、Ｔ又は

And
Z ₆ is T or

であり、
Ｚ_１０は、Ｑ又はＥであり、
Ｚ_１１は、Ｒ又はＫであり、Ｋのε−アミンが、−Ｃ（＝ＮＨ）ＮＨ_２によって置換されたものであり、
Ｚ_１２は、Ｌ又は

And
Z ₁₀ is Q or E,
Z ₁₁ is R or K, in which the ε-amine of K is replaced by _{-C (= NH) NH 2.}
Z ₁₂ is L or

であり、
Ｚ_１６は、Ｌであり、
Ｚ_２５は、Ｐ又はＫであり、Ｋのε−アミンが、−Ｃ（＝Ｏ）ＣＨ_３又は

And
Z ₁₆ is L,
Z ₂₅ is P or K, and the ε-amine of K is -C (= O) CH ₃ or

によって置換されたものであり、ｍＡｂが、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在し、
Ｚ_２６は、Ｉ又はＫであり、Ｋのε−アミンが、−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

The mAb was substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb.
Z ₂₆ is I or K, and the ε-amine of K is -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在し、
Ｘは、ＡＴＺ_１０Ｚ_１１Ｚ_１２ＡＮＦＺ_１６ＶＨＳＳＮＮＦＧＺ_２５Ｚ_２６ＬＰＺ_２９ＴＮＶＧＺ_３４（配列番号５４）、又はＶＬＧＲＬＳＱＥＬＨＲＬＱＴＹＰＲＴＮＴＧＳ（配列番号５５）であり、
Ｚ_２９は、Ｐ又はＫであり、Ｋのε−アミンが、−Ｃ（＝Ｏ）ＣＨ_３によって置換されたものであり、
Ｚ_３４は、Ｓ又はＫであり、Ｋのε−アミンが、−Ｃ（＝Ｏ）ＣＨ_３によって置換されたものである）、
及びその薬学的に許容される塩である。

The mAb was substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb.
X is ATZ ₁₀ Z ₁₁ Z ₁₂ ANFZ ₁₆ VHSSNNFGZ ₂₅ Z ₂₆ LPZ ₂₉ TNVGZ ₃₄ (SEQ ID NO: 54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55).
Z ₂₉ is P or K, where the ε-amine of K is replaced by _{-C (= O) CH 3.}
Z ₃₄ is S or K, where the ε-amine of K is replaced by _{-C (= O) CH 3),}
And its pharmaceutically acceptable salt.

In certain embodiments, the present invention is represented by a compound of formula I or a derivative thereof (in the formula,
n is 1 or 2
Z ₂ is a direct bond,
Z ₄ is T or

であり、
Ｚ_５は、β−アラニン、

And
Z ₅ is β-alanine,

であり、
Ｚ_６は、Ｔであり、
Ｚ_１０は、Ｑ又はＥであり、
Ｚ_１１は、Ｒ又はＫであり、Ｋのε−アミンが、−Ｃ（＝ＮＨ）ＮＨ_２によって置換されたものであり、
Ｚ_１２は、Ｌ又は

And
Z ₆ is T
Z ₁₀ is Q or E,
Z ₁₁ is R or K, in which the ε-amine of K is replaced by _{-C (= NH) NH 2.}
Z ₁₂ is L or

であり、
Ｚ_１６は、Ｌであり、
Ｚ_２５は、Ｐ又はＫであり、Ｋのε−アミンが、−Ｃ（＝Ｏ）ＣＨ_３又は

And
Z ₁₆ is L,
Z ₂₅ is P or K, and the ε-amine of K is -C (= O) CH ₃ or

によって置換されたものであり、ｍＡｂが、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在し、
Ｚ_２６は、Ｉ又はＫであり、Ｋのε−アミンが、−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

The mAb was substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb.
Z ₂₆ is I or K, and the ε-amine of K is -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在し、
Ｘは、ＡＴＺ_１０Ｚ_１１Ｚ_１２ＡＮＦＺ_１６ＶＨＳＳＮＮＦＧＺ_２５Ｚ_２６ＬＰＺ_２９ＴＮＶＧＺ_３４（配列番号５４）、又はＶＬＧＲＬＳＱＥＬＨＲＬＱＴＹＰＲＴＮＴＧＳ（配列番号５５）であり、
Ｚ_２９は、Ｐ又はＫであり、Ｋのε−アミンが、−Ｃ（＝Ｏ）ＣＨ_３によって置換されたものであり、
Ｚ_３４は、Ｓ又はＫであり、Ｋのε−アミンが、−Ｃ（＝Ｏ）ＣＨ_３によって置換されたものである）、
及びその薬学的に許容される塩である。

The mAb was substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb.
X is ATZ ₁₀ Z ₁₁ Z ₁₂ ANFZ ₁₆ VHSSNNFGZ ₂₅ Z ₂₆ LPZ ₂₉ TNVGZ ₃₄ (SEQ ID NO: 54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55).
Z ₂₉ is P or K, where the ε-amine of K is replaced by _{-C (= O) CH 3.}
Z ₃₄ is S or K, where the ε-amine of K is replaced by _{-C (= O) CH 3),}
And its pharmaceutically acceptable salt.

In certain embodiments, the present invention is represented by a compound of formula I or a derivative thereof (in the formula,
n is 1 or 2
Z ₂ is a direct bond or serine
Z ₄ is T or

であり、
Ｚ_５は、Ａ、β−アラニン、

And
Z ₅ is A, β-alanine,

であり、
Ｚ_６は、Ｔ又は

And
Z ₆ is T or

であり、
Ｚ_１０は、Ｑ又はＥであり、
Ｚ_１１は、Ｒ又はＫであり、Ｋのε−アミンが、−Ｃ（＝ＮＨ）ＮＨ_２によって置換されたものであり、
Ｚ_１２は、

And
Z ₁₀ is Q or E,
Z ₁₁ is R or K, in which the ε-amine of K is replaced by _{-C (= NH) NH 2.}
Z ₁₂ is

であり、
Ｚ_１６は、Ｌであり、
Ｚ_２５は、Ｐ又はＫであり、Ｋのε−アミンが、−Ｃ（＝Ｏ）ＣＨ_３又は

And
Z ₁₆ is L,
Z ₂₅ is P or K, and the ε-amine of K is -C (= O) CH ₃ or

によって置換されたものであり、ｍＡｂが、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式ＩＩの化合物が存在し、
Ｚ_２６は、Ｉ又はＫであり、Ｋのε−アミンが、−Ｃ（＝Ｏ）ＣＨ_３、−Ｃ（Ｏ）ＣＨ_２ＣＨ＝ＣＨ_２、

The mAb was substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula II compounds on the mAb.
Z ₂₆ is I or K, and the ε-amine of K is -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

によって置換されたものであり、ｍＡｂが、式Ｉの第２の化合物への別のチオエーテル結合を介して置換され、その結果、ｍＡｂ上に２つの同一の式Ｉの化合物が存在し、
Ｘは、ＡＴＺ_１０Ｚ_１１Ｚ_１２ＡＮＦＺ_１６ＶＨＳＳＮＮＦＧＺ_２５Ｚ_２６ＬＰＺ_２９ＴＮＶＧＺ_３４（配列番号５４）、又はＶＬＧＲＬＳＱＥＬＨＲＬＱＴＹＰＲＴＮＴＧＳ（配列番号５５）であり、
Ｚ_２９は、Ｐ又はＫであり、Ｋのε−アミンが、−Ｃ（＝Ｏ）ＣＨ_３によって置換されたものであり、
Ｚ_３４は、Ｓ又はＫであり、Ｋのε−アミンが、−Ｃ（＝Ｏ）ＣＨ_３によって置換されたものである）、
及びその薬学的に許容される塩である。

The mAb was substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb.
X is ATZ ₁₀ Z ₁₁ Z ₁₂ ANFZ ₁₆ VHSSNNFGZ ₂₅ Z ₂₆ LPZ ₂₉ TNVGZ ₃₄ (SEQ ID NO: 54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55).
Z ₂₉ is P or K, where the ε-amine of K is replaced by _{-C (= O) CH 3.}
Z ₃₄ is S or K, where the ε-amine of K is replaced by _{-C (= O) CH 3),}
And its pharmaceutically acceptable salt.

In certain embodiments, the compound is selected from the group consisting of SEQ ID NOs: 4-42, or pharmaceutically acceptable salts thereof.

In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof is covalently attached to the amilinomimetic peptide at the lysine residue of the amilinomimetic peptide via a linker. Non-limiting examples of linkers include aa PEG chains of 2 to 24 PEG units, (OEG _(0-4) -γ-Glu), (OEG _(1-4) ), or 2 to 10 PEG chains. Alkyl chains containing carbon atoms can be mentioned, and the linker may contain groups such as, but not limited to, acetyl.

In certain embodiments, _{only one of Z 25} , Z ₂₆ , Z ₂₉ , and Z ₃₄ in Formula I is lysine, which is a linker of the monoclonal antibody or antigen-binding fragment thereof. Covalently attached to the modified cysteine residue.

Another embodiment of the invention is a pharmaceutical composition comprising a compound of formula I, or a compound selected from the group consisting of SEQ ID NOs: 4-42, and a pharmaceutically acceptable carrier.

In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises the polypeptide sequences of SEQ ID NOs: 47, 48, 49, 50, 51, and 52, respectively, heavy chain complementarity determining regions 1 (HCDR1), HCDR2. , And HCDR3, and light chain complementarity determining regions 1 (LCDR1), LCDR2, and LCDR3. In certain embodiments, the isolated monoclonal antibody comprises a heavy chain variable domain (VH) having the polypeptide sequence of SEQ ID NO: 43 and a light chain variable domain (VL) having the polypeptide sequence of SEQ ID NO: 45. including. 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.

Also provided are conjugates comprising a monoclonal antibody bound to an amilinomimetic peptide or an antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof is SEQ ID NO: 47, 48, 49, 50, 51, and 52. Contains heavy chain complementarity determination regions 1 (HCDR1), HCDR2, and HCDR3, and light chain complementarity determination regions 1 (LCDR1), LCDR2, and LCDR3, respectively, each having the polypeptide sequence of, preferably this monoclonal antibody or The antigen-binding fragment comprises a heavy chain variable domain (VH) having the polypeptide sequence of SEQ ID NO: 43 and a light chain variable domain (VL) having the polypeptide sequence of SEQ ID NO: 45, and more preferably this monoclonal. The 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, and the amilinomimetic peptides are from SEQ ID NOs: 4-28. A polypeptide sequence selected from the group consisting of, or a pharmaceutically acceptable salt thereof, the monoclonal antibody or antigen-binding fragment thereof comprises residues 25, 26, 29, or 34 of the amilinomimetic peptide, preferably 34. At the lysine residue 25 or 26 of the amilinomimetic peptide, it is conjugated to the amilinomimetic peptide either directly or via a linker.

Also provided is a method for producing a conjugate of the present invention. The method is a monoclonal antibody, preferably a bromoacetamide, introduced on the side chain of an amilinomimetic peptide or a linker on that side chain, preferably on the side chain of a lysine residue of an amilinomimetic peptide. It involves reacting the sulfhydryl group of the cysteine residue of SEQ ID NO: 49 of the antibody or antigen-binding fragment thereof with a sulfhydryl group thereby creating a covalent bond between the amilinomimetic peptide and the monoclonal antibody or antigen-binding fragment thereof.

The present invention also provides a pharmaceutical composition comprising the conjugate of the present invention and a pharmaceutically acceptable carrier.

Methods of treating or preventing a disease or disorder in a subject in need thereof are also provided, the disease or disorder being obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, dyslipidemia, hyperglycemia. , Hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and uncontrolled cholesterol and / or lipid levels From the group consisting of other cardiovascular risk factors such as related hypertension and cardiovascular risk factors, osteoporosis, inflammation, non-alcoholic steatohepatitis (NAFLD), non-alcoholic steatohepatitis (NASH), renal disease, and eczema. Be selected. The method comprises administering an effective amount of the pharmaceutical composition of the invention to a subject in need thereof.

Also provided is a method of reducing food intake to those in need. The method comprises administering an effective amount of the pharmaceutical composition of the invention to a subject in need thereof.

Also provided is a method of regulating amyrin receptor activity in a subject in need thereof. The method comprises administering an effective amount of the pharmaceutical composition of the invention to a subject in need thereof.

Also provided is a method of regulating amyrin receptor activity in a subject in need thereof, the amyrin receptor comprising AMY1R, AMY2R, and / or AMY3R. The method comprises administering an effective amount of the pharmaceutical composition of the invention to a subject in need thereof.

Also provided is a method of regulating amyrin receptor activity in a subject in need thereof, the amyrin receptor being AMY1R.

Also provided is a method of regulating amyrin receptor activity in a subject in need thereof, the amyrin receptor being AMY3R.

The method comprises administering an effective amount of the pharmaceutical composition of the invention to a subject in need thereof.

In certain embodiments, the pharmaceutical composition is administered by injection. In certain embodiments, the pharmaceutical composition is administered in combination with at least one anti-diabetic agent. The anti-diabetic drug can be, for example, a glucagon-like peptide-1 receptor regulator. In certain embodiments, the pharmaceutical composition is administered in combination with liraglutide.

Kits comprising the conjugates of the invention, preferably further including liraglutide and an injectable device, are also provided.

Also provided is a method for producing the pharmaceutical composition of the present invention. The method comprises combining the conjugate with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

Further aspects, features, and advantages of the invention will be better understood by reading "Detailed Description of the Invention" and "Claims".

The above outline and the following detailed description of preferred embodiments of the present application will be better understood by reading in conjunction with the accompanying drawings. However, it should be understood that the application is not limited to the embodiment itself shown in the drawings.

In the background art, and throughout the specification, various publications, treatises and patents are cited or described. Each of these references is incorporated herein by reference in its entirety. The discussion of documents, operations, materials, devices, articles, etc. contained herein is for the purpose of providing the context of the present invention. Such considerations do not allow any or all of these things to form part of the prior art for any invention disclosed or claimed.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Otherwise, certain terms used herein have the meanings described herein.

It should be noted that the singular forms "a", "an" and "the" as used herein and in the appended claims include a plurality of referents unless the context clearly dictates otherwise. ..

Unless otherwise stated, any numerical value, such as the concentration or concentration range described herein, should be understood as being modified by the term "about" in all cases. Therefore, the numbers typically include ± 10% of the stated values. For example, a concentration of 1 mg / mL comprises 0.9-1.1 mg / mL. Similarly, the concentration range of 1-10% (w / v) includes 0.9-11% (w / v). As used herein, the use of numerical ranges is all possible subranges, all within that range, including integers and fractions of values within that range, unless explicitly stated in the context. Explicitly include the individual numbers of.

Unless otherwise stated, the term "at least" preceding a set of elements should be understood to refer to all elements in series. One of ordinary skill in the art will be able to recognize or confirm a large number of equivalents for the particular procedure described herein by using merely conventional experimental procedures. Such equivalents are intended to be included by the present invention.

As used herein, the terms "comprises," "comprising," "includes," "including," "has," and "having." , "Contains", or "containing", or any other variant thereof, includes the specified integers or groups of integers, but excludes any other integers or groups of integers. It is understood not to suggest doing so and is intended to be non-exclusive or unlimited. For example, a composition, mixture, process, method, article, or device comprising a set of elements is not necessarily limited to those elements alone and is not explicitly listed, or such a composition, mixture,. It may include other elements that are not inherent in the process, method, article, or device. Furthermore, unless explicitly stated in the opposite direction, "or" refers to inclusive or, not exclusive or. For example, in conditions A or B, if A is true (or exists) and B is false (or nonexistent), then A is false (or nonexistent) and B is true (or exists). If, and if both A and B are true (or exist), then one of them is satisfied.

Terms such as "about," "approximately," "approximately," and "substantially" used herein when referring to the dimensions or features of components of a preferred invention will be understood by those skilled in the art. As such, it is also appreciated to indicate that the dimensions / features described are not exact boundaries or parameters and do not exclude slight differences from them that are functionally the same or similar. At a minimum, such references, including numerical parameters, use mathematical and industrial principles accepted in the art (eg, rounding, measurement, or other systematic errors, manufacturing tolerances, etc.). The minimum significant figures will include variants that do not change.

The term "identical" or "identity" percent is used in _{the context of two or more nucleic acid or polypeptide sequences (eg, aminomimetic 3-36} polypeptide sequences, light or heavy chain sequences of antibodies). , When measured using one of the following sequence comparison algorithms or by visual inspection using methods known in the art in light of the present disclosure, the maximum match is compared and aligned. In the case, it refers to two or more sequences or subsequences having a specified proportion of amino acid residues or nucleotides that are identical or identical.

For sequence comparison, typically one sequence acts as a reference sequence to which the test sequences are compared. When using the sequence comparison algorithm, test and reference sequences are entered into the computer, subsequence coordinates are specified, and sequence algorithm program parameters are specified, if necessary. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence (s) relative to the reference sequence based on the specified program parameters.

Optimal alignment of sequences for comparison is described, for example, in Smith & Waterman, Adv. Apple. Math. 2: 482 (1981) Local Homology Algorithm, Needleman & Wunsch, J. Mol. Mol. Biol. 48: 443 (1970) Homology Alignment Algorithm, Pearson & Lipman, Proc. Nat'l. Acad. Sci. Similar Search Methods in USA 85: 2444 (1988), Computerized Implementations of These Algorithms (Wisconsin Computers Software Package, Genetics Computer Group, 575 Science Dr., Madison, FASTA, FASTA, WI, GAP, BEST) or visual inspection (see generally, Current Protocols in Molecular Biology, F.M.Ausubel et al., eds., Current Protocols, a joint venture between Greene Protocols, a joint venture between Greene publishing Associates, inc.and John Wiley & It can be done by Sons, Inc., (see 1995 Implementation) (Ausube).

Examples of suitable algorithms for determining percent sequence identity and sequence similarity are described in Altschul et al., Respectively. , (1990) J.M. mol. Biol. 215: 403-410 and Altschul et al. , (1997) Nucleic Acids Res. 25: 3389-3402, BLAST and BLAST 2.0 algorithms. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

A further indicator that the two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is the poly encoded by the second nucleic acid, as described below. It is immunologically cross-reactive with peptides. Thus, a polypeptide is typically substantially identical to, for example, a second polypeptide in which the two peptides differ only by conservative substitution. Another indicator that the two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

As used herein, "subject" means any animal, preferably a mammal, most preferably a human. As used herein, the term "mammal" includes 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 more preferably humans.

The term "administration" with respect to the methods of the invention therapeutically describes a syndrome, disorder, or disease described herein by using a conjugate of the invention, or a product, composition, or agent thereof. Or prophylactically, it means a method of preventing, treating, or ameliorating. Such methods include administering an effective amount of the conjugate, its formation, composition, or agent at different times during the course of treatment or simultaneously in combination. The methods of the invention are understood to include all known therapeutic treatment regimens.

The term "effective amount" refers to a biological or medical response (syndrome, disorder, or treatment to be treated) in a tissue system, animal, or human that is being sought by researchers, veterinarians, doctors, or other clinicians. It means the amount of active conjugate or medicinal agent that elicits a disease, or a syndrome, disorder, or symptom of the disease to be treated, including preventing, treating, or ameliorating.

As used herein, the term "composition" is a product that comprises a particular component in a particular amount, as well as any product that directly or indirectly results from a combination of a particular amount of a particular component. Shall be included.

As used herein, the term "combined" refers to joining or connecting two or more objects together. When referring to a chemical or biological compound, the term "bound" can refer to a covalent bond between two or more chemical or biological compounds. As a non-limiting example, the antibody of the present invention can be bound to a peptide of interest (eg, the amilinomimetic peptide of the present invention) to form an antibody-binding peptide. In certain embodiments, the antibodies of the invention can be covalently linked to the peptides of the invention by a linker. The linker can be, for example, first covalently attached to an antibody or peptide and then covalently attached to a peptide or antibody. Antibody-binding peptides can be formed by specific chemical reactions designed to conjugate the antibody to the peptide. As an example, mAb-bound amilinomimetic peptide conjugates can be formed by a conjugated reaction. Conjugated reactions can include, for example, reacting an electrophilic group (eg, bromoacetamide or maleimide) with a sulfhydryl group of a cysteine residue on mAb. The electrophilic group can be introduced, for example, on the side chain of the amino acid residue of the amilinomimetic peptide. A covalent thioether bond is formed by the reaction of the electrophilic group with the sulfhydryl group.

As used herein, the term "linker" refers to a chemical module that comprises a covalent bond or atomic chain that covalently binds an antibody to a peptide. Examples of the linker include a peptide linker, a hydrocarbon linker, a polyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, a polyester linker, a hybrid linker composed of PEG and an embedded heterocycle, and a hydrocarbon. Chains can be mentioned, but are not limited to these.

As used herein, the term "conjugate" refers to an antibody or fragment thereof that is covalently bound to a pharmaceutically active moiety. The term "conjugated" means that an antibody or fragment thereof of the invention is covalently linked to a pharmaceutically active moiety (preferably a therapeutic peptide), either directly or indirectly via a linker. Or it means that they are connected by a covalent bond. As a non-limiting example, the antibody may be the monoclonal antibody of the invention, and the pharmaceutically active moiety may be a therapeutic peptide, such as the amilinometric peptide of interest.

Antibodies When used herein in the context of antibodies, the term "non-targeting" refers to antibodies that do not specifically bind to any target in vivo. As used herein, when the term "target-specifically bound" antibody is used, it is 1 x ^10-8 M or less, preferably 5 x ^10-9 M or less, 1 x ^10-9 M or less. Refers to an antibody that binds to a target antigen with a KD of 5, ^10-10 M or less, or 1 x ^{10-10 M or less.} The term "KD" refers to the dissociation constant obtained from the Kd to Ka ratio (ie, Kd / Ka) and expressed as molarity (M). The KD value of an antibody can be determined using methods in the art in light of the present disclosure. For example, antibody KD can be obtained by using surface plasmon resonance, such as by using a biosensor system, such as the Biacore® system, or by using biolayer interferometry techniques such as the Octet RED96 system. Can be decided. The smaller the KD value of an antibody, the higher the affinity for the antibody to bind to the target antigen.

Monoclonal antibodies (whole or fragments thereof) can be used as a half-life extension portion. Monoclonal antibodies are well-studied proteins that are utilized in vivo and characterized for in vivo use, and thus the mechanisms and raw that allow them to prolong their half-life in vivo. The mechanisms for their removal in the body are well understood. In addition, the spatial separation and presentation of the two "arms" of the monoclonal antibody can be advantageous for effective divalent presentation of the therapeutic moiety (ie, the therapeutic peptide). Therapeutic agents in which toxins or other small molecule drugs are chemically linked to monoclonal antibodies have been developed, but typically antibodies-drugs that bind to a specific antigen and preferentially express the antigen. Utilizing a monoclonal antibody that targets the conjugate to the tissue / cell of interest, the drug / small molecule is typically bound to the antibody in a manner that does not affect the antigen binding of the antibody.

For therapeutic peptide-mAb conjugates, antigen-specific binding by a half-life extended monoclonal antibody is not desired. Thus, heavy chain (HC) and light chain (LC) variable (V) domain pairs that are not expected to specifically bind to any target to prepare a bindable non-targeted monoclonal antibody of the invention. Used for. To obtain a bindable non-targeted monoclonal antibody, the cysteine residue is modified to one of the complementarity determining regions (CDRs) of the selected non-target antibody. The pharmaceutically active moiety (eg, therapeutic peptide / compound) contains the appropriate chemical moiety that allows conjugation of the pharmaceutically active moiety to the modified cysteine residue of the non-targeted monoclonal antibody. be able to.

As used herein, the term "antibody" has a broad meaning and is non-human (eg, mouse, rat), human, human-adapted, humanized, and chimeric monoclonal antibody, antibody fragment, two. Includes multispecific or multispecific antibodies, dimers, tetramers, or multimeric antibodies, as well as single chain antibodies.

Antibody light chains of any vertebrate species can be assigned to one of two distinct types, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain. Therefore, the antibody of the present invention can contain a κ or λ light chain constant domain. According to certain embodiments, the antibodies of the invention comprise heavy and / or light chain constant regions from mouse or human antibodies. In addition to the heavy and light constant domains, the antibody contains antigen binding regions consisting of light chain variable regions and heavy chain variable regions, each of which has three domains (ie, complementarity determining regions 1-3 (CDR1, CDR2). , And CDR3). Light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCRD3, and heavy chain variable region domains are alternatively referred to as HCDR1, HCRD2, and HCDR3.

Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy chain constant domain. IgG is the most stable of the five immunoglobulins with a serum half-life of about 23 days in humans. IgA and IgG are further subdivided into _{isotypes IgA 1} , IgA ₂ , IgG ₁ , IgG ₂ , IgG ₃ , and IgG _4. Each of the four IgG subclasses has a different biological function known as an effector function. These effector functions are generally mediated by interaction with the Fc receptor (FcγR) or by binding of C1q and fixation of complement. Binding to FcγR can result in antibody-dependent cell-mediated cytolysis, whereas binding to complement factors can result in complement-mediated cytolysis. The antibodies of the invention utilized for their ability to prolong the half-life of therapeutic peptides have no or minimal effector function, but retain the ability to bind FcRn, the binding of which is the antibody. Can be the primary means of having an extended half-life in vivo.

In certain embodiments, the present invention has a light chain variable region having a fully human Ig germline V gene sequence and a heavy chain having a fully human Ig germline V gene sequence except HCDR3 having the amino acid sequence of SEQ ID NO: 49. For a conjugate containing an isolated antibody or antigen-binding fragment thereof, including a variable region, and a pharmaceutically active moiety conjugated thereto (eg, the amilinomimetic peptide of the invention), the antibody or antigen thereof. The binding fragment does not specifically bind to any human antigen in vivo. In the present disclosure, with respect to an antibody or antigen-binding fragment thereof according to an embodiment of the invention, the phrase "conjugate comprising an antibody or antigen-binding fragment thereof and a pharmaceutically active moiety conjugated thereto" is "pharmaceutically active". It is used interchangeably with "antibodies conjugated to various parts or antigen-binding fragments thereof".

As used herein, the term "antibody binding fragment" refers to, for example, diabodies, Fabs, Fab', F (ab') 2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) ₂ , Bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabody (ds diabody), single chain antibody molecule (scFv), single domain antibody (sdab) scFv dimer (divalent diabody), 1 A multispecific antibody formed from a portion of an antibody containing one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a divalent domain antibody, or any that binds to an antigen but does not contain a complete antibody structure. Refers to antibody fragments such as other antibody fragments. The antigen-binding fragment can bind to the parent antibody or the same antigen to which the parent antibody fragment binds. According to certain embodiments, the antigen binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment (ie, a portion of the heavy chain contained 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" is a conventional single chain in the art that comprises a heavy chain variable chain and a light chain variable region connected by short peptides of about 15 to about 20 amino acids. Refers to an antibody. As used herein, the term "single domain antibody" refers to conventional single domain antibodies in the art that include heavy chain variable and heavy chain constant regions, or contain only heavy chain variable regions. ..

The phrase "isolated antibody or antibody fragment" refers to an antibody or antibody fragment that is substantially free of other antibodies with different antigen specificities (eg, isolated antibodies that specifically bind to the target antigen). Is substantially free of antibodies that do not specifically bind to the target antigen). In addition, isolated antibodies or antibody fragments may be substantially free of other cellular materials and / or chemicals.

The antibody variable region consists of a "framework" region interrupted by three "antigen binding sites". Antigen binding sites are defined using various terms: (i) Complementarity determining regions (CDRs), which are three in VH (HCDR1, HCDR2, HCDR3) and in VL based on sequence specificity. three to (LCDR1, LCDR2, LCDR3) are present (Wu and Kabat J Exp Med 132 : 211-50,1970; Kabat et al Sequences of Proteins of Immunological Interest, 5 th Ed.Public Health Service, National Institutes of Health , Bethesda, Md., 1991). (Ii) "Hypervariable region", "HVR", or "HV", three in VH (H1, H2, H3) and three in VL (L1, L2, L3) are Chuthia and Lesk (Chothia). and Lesk Mol Biol 196: 901-17, 1987) refers to the region of the antibody variable domain that is hypervariable in the structure. Other terms include "IMGT-CDR" (Lefranc et al., Dev Compart Immunol 27: 55-77, 2003) and "Specificity Determining Residue User (SDRU)" (Almagro Mole 4) Is included. The International ImmunoGeneTics (IMGT) database (http://www_mgt_org) provides standard numbering and definitions of antigen binding sites. Correspondence between CDR, HV, and IMGT outline description is described in Lefranc et al. , Dev Comparat Immunol 27: 55-77, 2003.

A "framework" or "framework sequence" is the rest of the sequence of the variable region, excluding those defined as antigen binding sites. Since 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 of the amino acid sequences of SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, respectively. It contains a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of the amino acid sequences of 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 human IgG4 Fc region. The human IgG4 Fc region has a reduced ability to bind FcγR and complement factors compared to other IgG subtypes. Preferably, the Fc region contains a human IgG4 Fc region with a substitution that eliminates effector function. Thus, the isolated antibody further comprises an Fc region having a modified human IgG4 Fc region containing one or more of the following substitutions: using proline instead of glutamate at residue 233, residue. using alanine or valine in place of phenylalanine at 234, and residues 235 (EU numbering, Kabat, E.A.et al., ( 1991) Sequences of Proteins of Immunological Interest, 5 th Ed., U.S. Use alanine or glutamate instead of leucine in Dept. of Health and Human Services, Bethesda, Md., NIH Publication no. 91-3242). Removing the N-linked glycosylation site in the IgG4 Fc region by using Ala instead of Asn at residue 297 (EU numbering) is another way to ensure that residual effector activity is eliminated. Is.

Preferably, the antibodies of the invention can exist as dimers joined together by disulfide bonds and various non-covalent interactions. Thus, Fc moieties useful for the antibodies of the invention stabilize heavy chain dimer formation and prevent the formation of semi-IgG4 Fc chains, such as substitution of serine for proline at position at 228 (EU numbering). It can be a human IgG4 Fc region containing.

In another embodiment, the C-terminal Lys residue of the heavy chain is removed as is commonly found in recombinantly produced monoclonal antibodies.

"Human antibody" refers to an antibody in which both the framework and the antigen binding site have heavy and light chain variable regions derived from sequences of human origin. If the antibody contains a constant region, the constant region is also derived from a sequence of human origin.

Human antibodies are heavy or light chain variable regions that are "derived" from sequences of human origin when the variable region of the antibody is obtained from a system that uses human germline immunoglobulins or rearranged immunoglobulin genes. including. Such systems include transgenic non-human animals, such as the human immunoglobulin gene library presented on phage and mice carrying the human immunoglobulin loci described herein. "Human antibodies" are when compared to human germline or rearranged immunoglobulin sequences, for example by the introduction of naturally occurring somatic mutations or deliberate substitutions within the framework or antigen binding site. Can contain differences in amino acids. Typically, human antibodies are at least about 80%, 81%, 82%, 83%, 84%, 85% of the amino acid sequence in the amino acid sequence as encoded by the human germline or rearranged immunoglobulin gene. , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In some cases, "human antibodies" may be referred to, for example, by Knappik et al. , J Mol Biol 296: 57-86, 2000, consensus framework sequences obtained from human framework sequence analysis, or, for example, Shi et al. , J Mol Biol 397: 385-96, 2010, and Synthetic HCDR3 integrated into the human immunoglobulin gene library presented on phage as described in International Patent Application Publication No. 2009/085462. Antibodies whose antigen binding site is derived from a non-human species are not included in the definition of "human antibody".

The isolated humanized antibody can be synthetic. Human antibodies, which are derived from human immunoglobulin sequences, can be produced using systems such as phage displays incorporating synthetic CDRs and / or synthetic frameworks, or are mutagenesis in vitro. The characteristics of the antibody can be improved, and an antibody that does not naturally exist in the human antibody repertoire repertoire in vivo can be obtained.

As used herein, the term "recombinant antibody" is intended to express an antibody, antibody isolated from a transgenic or chromosome-introduced animal (eg, mouse) of a human immunoglobulin gene or a hybridoma prepared from it. Prepared by antibodies isolated from host cells transformed into, antibodies isolated from recombinant combinatorial antibody libraries, and any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Includes all antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed, created, or isolated, or antibodies produced in vitro using Fab arm exchange.

As used herein, the term "monoclonal antibody" refers to the preparation of antibody molecules of a single molecule composition. The monoclonal antibody of the present invention can be produced by a hybridoma method, a phage display technique, a single lymphocyte gene cloning technique, or a recombinant DNA method. For example, a monoclonal antibody can be produced by a hybridoma containing B cells obtained from a transgenic non-human animal, such as a transgenic mouse or rat, and has a genome containing 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 missing.

As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of an immunoglobulin molecule is derived from more than one species. The variable regions of both the light and heavy chains are often variable antibodies from one species of mammal (eg, mice, rats, rabbits, etc.) with the desired specificity, affinity, and ability. The constant region corresponds to the region, while the constant region corresponds to the sequence of antibodies from another species of mammal (eg, human) in order to avoid inducing an immune response in that species.

As used herein, the term "multispecific antibody" refers to an antibody that comprises a plurality of immunoglobulin variable domain sequences, wherein the plurality of first immunoglobulin variable domain sequences bind to a first epitope. Multiple second immunoglobulin variable domain sequences, including germline sequences that have specificity or lack any known binding specificity, have binding specificity for the second epitope or are optional. Containing a germline sequence lacking the known binding specificity of, this first and / or second immunoglobulin variable domain optionally conjugates a conjugated pharmaceutically active moiety (eg, a therapeutic peptide). include. In certain embodiments, the first and second epitopes are on the same antigen, eg, the same protein (or subunit of a multimeric protein). In certain embodiments, the first and second epitopes overlap or substantially overlap. In certain embodiments, the first and second epitopes do not overlap or are substantially non-overlapping. In certain embodiments, the first and second epitopes are on different antigens, eg, different proteins (or subunits of different multimeric proteins). In certain embodiments, the first and second immunoglobulin variable domains contain the same conjugate, pharmaceutically active moiety. In certain embodiments, the first and second immunoglobulin variable domains contain different pharmaceutically active moieties. In certain embodiments, only the first immunoglobulin variable domain comprises a conjugated pharmaceutically active moiety. In certain embodiments, only the second immunoglobulin variable domain comprises a conjugated pharmaceutically active moiety. In certain embodiments, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In certain embodiments, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody, or a quadrispecific antibody molecule.

As used herein, the term "bispecific antibody" binds to no more than two epitopes or two antigens and / or two conjugated pharmaceutically active moieties (eg, the same). Or a multispecific antibody that comprises a different pharmaceutically active moiety). Bispecific antibodies are a first immunoglobulin variable domain sequence containing a germline sequence that has binding specificity to a first epitope or lacks any known binding specificity, and a second epitope. A second immunoglobulin variable domain sequence comprising a germline sequence having binding specificity to or lacking any known binding specificity, the first and / or second immunoglobulin variable domain. Optionally include conjugated pharmaceutically active moieties. In certain embodiments, the first and second epitopes are on the same antigen, eg, the same protein (or subunit of a multimeric protein). In certain embodiments, the first and second epitopes overlap or substantially overlap. In certain embodiments, the first and second epitopes are on different antigens, eg, different proteins (or subunits of different multimeric proteins). In certain embodiments, the first and second immunoglobulin variable domains contain the same conjugate, pharmaceutically active moiety. In certain embodiments, the first and second immunoglobulin variable domains contain different pharmaceutically active moieties. In certain embodiments, only the first immunoglobulin variable domain comprises a conjugated pharmaceutically active moiety. In certain embodiments, only the second immunoglobulin variable domain comprises a conjugated pharmaceutically active moiety. In certain embodiments, the bispecific antibody comprises a first heavy chain variable domain sequence and a germline sequence that has binding specificity for the first epitope or lacks any known binding specificity. A second heavy chain variable domain sequence and a light chain variable domain sequence containing a light chain variable domain sequence and a germline sequence having binding specificity for a second epitope or lacking any known binding specificity. The first and / or second heavy chain variable domain optionally comprises a conjugated pharmaceutically active moiety. In certain embodiments, the first and second heavy chain variable domains comprise the same conjugate pharmaceutically active moiety. In certain embodiments, the first and second heavy chain variable domains contain different pharmaceutically active moieties. In certain embodiments, only the first heavy chain variable domain comprises a conjugated pharmaceutically active moiety. In certain embodiments, only the second heavy chain variable domain comprises a conjugated pharmaceutically active moiety.

As used herein, "full-length antibody" means an antibody having two full-length antibody heavy chains and two full-length antibody light chains. Full-length antibody heavy chains (HC) consist of well-known heavy chain variable domains and constant domains VH, CH1, CH2, and CH3. Full-length antibody light chains (LCs) consist of well-known light chain variable domains and constant domains VL and CL. Full-length antibodies may lack the C-terminal lysine (K) in either one or both heavy chains.

The term "Fab arm" or "half molecule" means a pair of heavy chain-light chains that specifically bind to an antigen.

Full-length bispecific antibodies prefer heterodimer formation of two antibody half-molecules with different specificities, either in an in vitro cell-free environment or using co-expression. By introducing substitutions at the heavy chain CH3 interface in each half molecule, it can be produced using Fab arm exchange (or half molecule exchange) between two monospecific divalent antibodies. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and the dissociation-association of the CH3 domain. 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 intra-heavy chain disulfide bond with the cysteine residue of the second parent monospecific antibody molecule, while at the same time the CH3 domain of the parent antibody Dissociation-release and reshape by association. The CH3 domain of the Fab arm may be modified to favor heterodimer formation over homodimer formation. The resulting product is a bispecific antibody with two Fab arms or half molecules, each capable of binding to a different epitope.

As used herein with respect to antibodies, "homodimer formation" refers to the interaction of two heavy chains with the same CH3 amino acid sequence. As used herein with respect to an antibody, "homodimer" refers to an antibody having two heavy chains having the same CH3 amino acid sequence.

As used herein with respect to antibodies, "heterodimer formation" refers to the interaction of two heavy chains with non-identical CH3 amino acid sequences. As used herein with respect to an antibody, "heterodimer" refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

A "knob-in-hole" strategy (see, eg, PCT WO 2006/028936) can be used to generate full-length bispecific antibodies. Briefly, the selected amino acids that form the CH3 domain interface in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid (hole) having a small side chain is introduced into the heavy chain of an antibody that specifically binds to the first antigen, and an amino acid (knob) having a large side chain specifically binds to the second antigen. It is introduced into the heavy chain of the antibody to be used. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction between the heavy chain with the "hole" and the heavy chain with the "knob". Exemplary CH3 substitution pairs forming knobs and holes are T366Y / F405A, T366W / F405W, F405 / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_ Represented as a modified position in the first CH3 domain of the first heavy chain / a modified position in the second CH3 domain of the second heavy chain).

Heavy chain heterodimer formation using other strategies, eg, electrostatic interaction by substituting positively charged residues on one CH3 surface and negatively charged residues on a second CH3 surface. The promotion may be used as described in US Patent Application Publication Nos. 2010/0015133, 2009/018212, 2010/028637, or 2011/0123532. In another strategy, heterodimer formation can be facilitated by the following substitutions, as described in US Patent Application Publication No. 2012/0149876 or US Patent Application Publication No. 2013/0195884: 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_T366L_K392L_T394W (the first first modified in a CH3 domain location / second heavy chain heavy chain Represented as a modified position in the second CH3 domain).

In addition to the methods described above, bispecific antibodies are two monospecific homodimers in an in vitro cell-free environment according to the method described in International Patent Application Publication No. 2011/131746. Bispecific heterodimer antibodies are formed from two parent monospecific homodimer antibodies under reducing conditions that introduce asymmetric mutations into the CH3 region of the antibody to isomerize disulfide bonds. Can be generated by In this method, the first unispecific divalent antibody and the second unispecific divalent antibody are modified to have certain substitutions in the CH3 domain that promote the stability of the heterodimer. However, these antibodies are co-incubated under reducing conditions sufficient for cysteine in the hinge region to isomerize disulfide bonds, thereby producing bispecific antibodies by Fab arm exchange. Incubation conditions can be optimally reverted to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithiothreitol (DTE), glutathione, tris (2-carboxyethyl) phosphine (TCEP), L-cysteine. , And beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris (2-carboxyethyl) phosphine. For example, at a temperature of at least 20 ° C., in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol, at pH 5-8, eg pH 7.0 or pH 7.4, for at least 90 minutes. Incubation may be used.

Throughout the specification, the numbering of amino acid residues in the constant region of an antibody is defined by Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), according to the EU index described in.

Conjugate In another general aspect, the invention presents a synthetic therapeutic peptide (eg, a synthetic therapeutic peptide) in a site-specific manner such that the antibody bound to the peptide has an extended / increased half-life compared to the peptide alone. , Amilinomimetic peptide) and the like, the conjugate comprising the antibody of the invention, covalently conjugated to a pharmaceutically active moiety. The present invention also relates to pharmaceutical compositions and methods of use thereof. This conjugate includes, among other things, obesity, type 2 diabetes, metabolic syndrome (ie, syndrome X), insulin resistance, impaired glucose tolerance (eg, glucose tolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, Others such as hypoglycemia due to congenital hyperinsulinism (CHI), impaired glucose tolerance, atherosclerosis, diabetic nephropathy, and hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and / or lipid levels Preventing, treating or ameliorating diseases or disorders such as cardiovascular risk factors, osteoporosis, inflammation, non-alcoholic steatohepatitis (NAFLD), non-alcoholic steatohepatitis (NASH), renal disease, and eczema. It is useful for.

In certain embodiments, the antibodies of the invention are conjugated to a pharmaceutically active moiety with at least one cysteine residue substitution capable of prolonging / increasing the half-life of the pharmaceutically active moiety. Modified to include. In certain embodiments, at least one cysteine residue substitution is included in the complementarity determining regions of the antibody. In certain embodiments, at least one cysteine residue substitution is in the heavy chain complementarity determining regions (HCDRs). In certain embodiments, at least one cysteine residue substitution is in HCDR3, which comprises the amino acid sequence of SEQ ID NO: 49. In certain embodiments, an antibody comprising HCDR3 comprising the amino acid sequence of SEQ ID NO: 49 has at least one additional cysteine substitution that can be conjugated to a pharmaceutically active moiety.

In certain embodiments, the pharmaceutically active moiety may include a linker. The linker can be chemically modified to allow the conjugate of the antibody to the pharmaceutically active moiety. Examples of the linker include a peptide linker, a hydrocarbon linker, a polyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, a polyester linker, a hybrid linker consisting of PEG and an embedded heterocycle, or a hydrocarbon chain. However, it is not limited to these. The PEG linker can contain, for example, 2 to 24 PEG units.

In certain embodiments, the monoclonal antibodies of the invention are one, two, three, four, five, or six pharmaceutically active moieties of interest (eg, therapeutic peptides (s)). ). In a preferred embodiment, the non-targeted monoclonal antibody is conjugated to the two pharmaceutically active moieties of interest. In certain embodiments in which the monoclonal antibody is conjugated to at least two pharmaceutically active moieties of interest, the pharmaceutically active moieties of interest may or may be the same pharmaceutically active moiety. It can be a pharmaceutically active part.

Methods of conjugates of the antibodies of the invention with the pharmaceutically active moieties of the invention are known in the art. Briefly, the antibodies of the invention are reduced with a reducing agent (eg, TCEP (Tris (2-carboxyethyl) phosphine)), purified (eg, by protein A adsorption or gel filtration) and pharmaceutically active. Can be conjugated to a moiety (eg, by providing a cryodried peptide to the reducing antibody under conditions that allow conjugation). After the conjugation reaction, the conjugate is ion exchange chromatography or hydrophobic interaction chromatography (HIC). ) Can be purified by the final purification step of protein A adsorption. In certain embodiments, the antibody of the invention can be purified prior to reduction using the HIC method. For a more detailed description of, see, for example, Example 103 and Denner et al., Antibodies 4: 197-224 (2015).

In certain embodiments, the amilinomimetic peptide is a derivative of the amilinomimetic peptide of formula I modified by one or more processes selected from the group consisting of amidation, lipidation, and PEGylation. , Or its pharmaceutically acceptable salt.

In certain embodiments, the conjugate comprises a monoclonal antibody or fragment thereof conjugated to an amilinomimetic peptide, which amilinomimetic peptide is selected from the group consisting of SEQ ID NOs: 4-28.

In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof is covalently attached to the amilinomimetic peptide at the lysine residue of the amilinomimetic peptide via a linker. The linker may include, for example, a PEG chain of 2 to 24 PEG units, an alkyl chain containing 2 to 10 carbon atoms, or a linker selected from the group consisting of bonds.

In certain embodiments, _{only one of Z 25} , Z ₂₆ , Z ₂₉ , and Z ₃₄ in Formula I is lysine, which is a linker of the monoclonal antibody or antigen-binding fragment thereof. Covalently attached to the modified cysteine residue. In a preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof according to one embodiment of the present invention is conjugated to the amilinomimetic peptide at residue 25 or 26 of the amilinomimetic peptide. In another preferred embodiment, an electromotive agent such as bromoacetamide is introduced onto the amilinomimetic side chain such as the amino side chain of lysine at residue 25 or 26 of the amilinomimetic. , A CDR of a monoclonal antibody or fragment thereof, preferably a sulfhydryl group of a Cys residue modified to HCDR3, thereby covalently binding the amilinomimetic peptide to the monoclonal antibody or fragment thereof. Create a connection by. More preferably, the amilinomimetic peptides are SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: It is selected from the group consisting of 28. In one embodiment, the electrophile is introduced directly onto the amilinomimetic side chain. In another embodiment, the electrophile is introduced over the amilinomimetic side chain via a linker.

Also provided are pharmaceutical compositions comprising the conjugates of the invention and further comprising a pharmaceutically acceptable carrier.

Also provided herein are amilinomimetic peptides that exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with pramlintide or davalintide. As an example of a method of determining sequence identity between two analogs, two peptides, pramlintide (SEQ ID NO: 2) and (SEQ ID NO: 4), are aligned.

プラムリンチド（配列番号２）

Pramlintide (SEQ ID NO: 2)

（配列番号４）

(SEQ ID NO: 4)

The sequence identity of the analog to pramlintide was calculated by subtracting the number of different residues from the total number of aligned residues (ie, the number of identical residues aligned) and dividing by the total number of pramlintide residues. It is a thing. In this example, the different residues are K1 and C2 which are absent. Here, N3 is replaced but retained in its original position. Therefore, in this example, the sequence identity is (37-2) / 37 × 100.

If the compound according to the invention is an amilinomimetic peptide attached to a mAb, it is expected that the mAb will have two copies of the peptide attached to it. Those skilled in the art will appreciate that the reaction product may comprise a partially conjugated reaction product, resulting in a single amilinomimetic peptide bound to mAb. It is understood that such mono-binding compounds, di-binding compounds, and mixtures thereof are all included within the scope of the present invention.

Amilinomimetic peptides Pramlintide and / or davalintide may be effective in the treatment of obesity given its role in suppressing appetite and food intake. However, the therapeutic utility of pramlintide and / or davalintide as a therapeutic agent is limited by its rapid metabolism and short circulating half-life. Thus, the present invention relates, in a broad sense, to modified pramlintides and / or davalintide conjugates that extend the half-life of amilinomimetic peptides and reduce the metabolism of the peptides in vivo.

In certain embodiments of the invention, the modified pramlintide and / or davalintide peptide is an amilinomimetic peptide. The terms "Amilinomimetic Peptide", "Amilinomimetic Analog", and "Amilinomimetic Peptide Analog" can be used interchangeably.

The peptide sequences described herein are described according to conventional practice, with the N-terminal region of the peptide on the left and the C-terminal region on the right. The isomer form of the amino acid is known, but unless explicitly stated otherwise, it is the L form of the amino acid. For convenience in describing the molecules of the invention, conventional and non-conventional abbreviations (both one-letter and three-letter codes) and functional parts of various amino acids are used. These abbreviations are well known to those of skill in the art, but are listed below for clarity: A = Ala = alanine; R = Arg = arginine; N = Asn = aspartic acid; D = Asp = aspartic acid. ΒA = βAla = β-alanine; C = Cys = cysteine; hC = hCys = homocysteine; E = Glu = glutamic acid; Q = Gln = glutamine; G = Gly = glycine; H = His = histidine; I = Ile = isoleucine L = Leu = leucine; K = Lys = lysine; Nle = norleucine; F = Phe = phenylalanine; P = Pro = proline; S = Ser = serine; T = Thr = threonine; W = Trp = tryptophan; Y = Tyr = Tyrosine and V = Val = valine.

Additional amino acid abbreviations used herein include: ACPC = 2-aminocyclopentanecarboxylic acid; β-Aib = β-aminoisobutyric acid = 3-amino-2-methylpropionic acid; β -HA = β-hAla = β-homoalanine = 3-aminobutyric acid; α-MeL = α-MeLeu = α-methylleucine; β ^3- hP = β ^3- hPro = β ³ -homoproline; β-hT = β- hThr = β-homothreonine;

For convenience, the amino acid residue numbering rules used in naming the amilinomimetic peptides of the invention follow those of pramlintide and / or davalintide. The particular amino acid substitution introduced into the peptide for the native residue at the corresponding position of pramlintide and / or davalintide is indicated by the appropriate amino acid code followed by the position of the substitution. Thus, "hC7" in an amilinomimetic peptide refers to a peptide in which the corresponding native Cys7 residue of pramlintide has been replaced with homocysteine. Similarly, for "K (Ac) 26" in _{amilinomic peptides, CH 3} C (O) -substituted lysine on ε-amine replaces the corresponding native Ile26 residue of pramlintide. Refers to a peptide that is present. Further amino acid substitutions occurring within the amilinomimetic peptide will be described according to this rule and will be recognized by those of skill in the art as such.

Also, for convenience, the naming convention used for the amilinomimetic peptides of the invention is to place the amino residues involved in the cycle from the N-terminal residues involved in the cycle, from left to right, between them. Incorporate with the linking group (s). In all cases, the N-terminal amino acid residue of the cycle is linked to an acetyl linking group via its α-amino functional group, which in turn is the side chain residue of the amino acid at position 7 of the amilinomimetic peptide. Connect to. Thus, "cyclo - _(N3-COCH 2 -hC7)" is used to Lys1 and Cys2 residues natural to describe the cycle of Amiri flea main ticks peptides lacking the sequence, Asn3 of α- The amino functional group is acylated with an acetyl residue, and the methyl group is further attached to the side chain of the hCys7 residue via a thioether bond. Similarly, "cyclo - _(S2-COCH 2 -hC7)" is used to Lys1 residues natural to describe the cycle of Amiri flea main ticks peptides lacking, Cys2 residues naturally The α-amino functional group is replaced by Ser2 acylated with an acetyl group, and the above acetyl group is linked to the side chain of the hCys7 residue by a thioether bond.

Lysine residues can be incorporated at various positions in the amilinomimetic sequence to provide a convenient functional handle for further derivatization. The lysine residue can be modified to bind to the monoclonal antibody either directly or indirectly. In indirect binding to a monoclonal antibody, the lysine residue can be modified to include a linker that allows the amilinomimetic peptide to bind to the monoclonal antibody. Those skilled in the art will recognize that the associated orthologous gas can also be used so effectively and is contemplated herein.

The term "K (γ-Glu)" appearing in the peptide sequence refers to a ricinyl residue whose side chain ε-amino group is acylated by the γ-carboxyl group of glutamic acid.

The term "K (Ac)" represents a lysynyl residue whose side chain ε-amino group is substituted with an acetyl group.

The term "K (Allloc)" represents a ricinyl residue whose side chain ε-amino group is substituted with an allyloxycarbonyl group.

The term _"K (OEG 2 -Pal)", the side chain ε- amino group is substituted with 17-amino-10-oxo -3,6,12,15- tetraoxa-9 Azaheputadekan acid, 17 The −amino group represents a lysynyl residue that is further substituted with palmitic acid by an amide bond between the 17-amino group and palmitic acid.

The term "(OEG) ₂ " refers to two consecutively linked together via an amide link (ie, 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid). Represents an OEG unit.

The term "K (OEG) ₂ " is a ricinyl residue whose side chain ε-amino group is acylated with 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid. Represents.

The term "K (OEG) _2- γ-Glu-Pal" has a side chain ε-amino group of (22S) -22-amino-10,19-dioxo-3,6,12,15-tetraoxa-9. , 18-Diazatricosan diic acid is acylated via its 1-carboxylic acid functional group, and its 22-amino group represents a lysynyl residue that is further amidated by palmitinsan.

The term "dPEGx" is a separate oligomer that contains x ethylene glycol units that are linked to propanoic acid at one end and that contain a terminal amino functional group on the distal end and can be further functionalized. Point to.

The term "K (dPEG12)" has a side chain ε-amino group of 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatria. Represents a ricinyl residue that is acylated with a Contan-39-acid via its 39-carboxylic acid functional group.

The term "K (dPEG12-AcBr)" has a side chain εamino group of 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecanoki. A lysynyl acid acylated with a Santrantoria Contan-39-acid via its 39-carboxylic acid functional group, the acid being on its 1-amine group-C (O) CH ₂ Represents a ricinyl acid that is amidated with a Br group.

Half-life extension portion In addition to the antibody of the invention or its antigen-binding fragment, the conjugate of the invention halves a pharmaceutically active moiety (eg, an amilinometric peptide), eg, through covalent interactions. One or more other parts can be incorporated to extend the period. Other exemplary half-life extension portions include, but are not limited to, albumin, albumin variants, albumin-binding proteins and / or domains, transferrin, and fragments and analogs thereof. Additional half-life extension moieties that can be incorporated into the conjugates of the invention for the desired properties include, for example, polyethylene glycol (PEG) molecules such as PEG5000 or PEG20,000, fatty acids of varying chain lengths and Fatty acid esters such as lauric acid ester, myristic acid ester, stearic acid ester, arachiic acid ester, behenic acid ester, oleic acid ester, arachidonic acid ester, octane diic acid, tetradecanoic acid, octadecane diic acid, and docosan diic acid. In addition, polylysine, octane, and fatty acids (dextran, cellulose, oligosaccharides, or polysaccharides) can be mentioned. These moieties may be direct fusions with the protein scaffold coding sequence or may be produced by standard cloning and expression techniques. Alternatively, the moiety can be attached to the recombinantly and chemically produced conjugate of the invention using a well-known chemical bonding method.

A PEGylated moiety may be added, for example, to the peptide molecule of the invention by incorporating a cysteine residue into the C-terminus of the molecule and binding the PEGylated group to cysteine using a well-known method.

Peptide molecules of the invention incorporating additional moieties can be compared for functionality by several well-known assays. For example, the biological or pharmacokinetic activity of a therapeutic peptide of interest may be analyzed, evaluated and compared using known in vitro or in vivo assays, either alone or in a conjugate according to the invention. Can be done.

Pharmaceutical Compositions In another general aspect, the invention relates to pharmaceutical compositions comprising the conjugates and compounds of the invention, as well as pharmaceutically acceptable carriers. As used herein, the term "pharmaceutical composition" means a product comprising a conjugate of the invention along with a pharmaceutically acceptable carrier. The conjugates and compounds of the present invention, as well as compositions containing them, are also useful in the manufacture of agents for therapeutic applications referred to herein.

As used herein, the term "carrier" means any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicles, microspheres, liposome inclusions. Refers to other materials known in the art for use in the body or pharmaceutical formulations. It will be appreciated that the properties of the carrier, excipient or diluent depend on the route of administration for the particular application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the effect of the composition according to the invention or the biological activity of the composition according to the invention. According to certain embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in the pharmaceutical composition of the antibody can be used in the present invention.

Pharmaceutically acceptable acidic / anionic salts for use in the present invention include acetate, benzenesulfonate, benzoate, bicarbonate, hydrogen tartrate, bromide, calcium edetate, cansilicic acid. Salts, carbonates, chlorides, citrates, dihydrochlorides, edetates, edicylates, estruates, esylates, fumarates, glycetates, gluconates, glutamates, glycolylarsa Nylate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandel Acids, mesylates, methyl bromides, methyl nitrates, methyl sulfates, mucoates, napsylates, nitrates, pamoates, pantothenates, phosphates / diphosphates, polygalacturonates, salicylic acids Examples include, but are not limited to, salts, stearate, basic acetate, succinate, sulfate, tannate, tartrate, theocrate, tosylate, and triethiodide. Examples of the organic acid or inorganic acid include hydroiodic acid, perchloric acid, sulfuric acid, phosphoric acid, propionic acid, glycolic acid, methanesulfonic acid, hydroxyethanesulfonic acid, oxalic acid, 2-naphthalenesulfonic acid, p-. Examples include, but are not limited to, toluenesulfonic acid, cyclohexanesulfamic acid, saccharic acid or trifluoroacetic acid.

Pharmaceutically acceptable basic / cationic salts include aluminum, 2-amino-2-hydroxymethyl-propane-1,3-diol (tris (hydroxymethyl) aminomethane, tromethane, or "TRIS". Also known), ammonia, benzathine, t-butylamine, calcium, chloroprocine, choline, cyclohexylamine, diethanolamine, ethylenediamine, lithium, L-lysine, magnesium, meglumin, N-methyl-D-glucamine, piperidine, potassium, Examples include, but are not limited to, prokine, quinine, sodium, triethanolamine, or zinc.

In some embodiments of the invention, the conjugate of the invention is comprising in an amount of about 0.001 to about 100 mg / mL, about 0.01 to about 50 mg / mL, or about 0.1 to about 25 mg / mL. Pharmaceutical formulations are provided. The pharmaceutical formulation may have a pH of about 3.0 to about 10, for example about 3 to about 7, or about 5 to about 9. The formulation is further selected from the group consisting of buffers, preservatives (s), tonicity agents (s), chelating agents (s), stabilizers, and surfactants (s). It may contain at least one component.

Formulations of the pharmaceutically active ingredient having a pharmaceutically acceptable carrier are such as described in, for example, Remington: The Science and Practice of Pharmacy (eg, 21st edition (2005) and any subsequent revisions). Known in the technical field. Non-limiting examples of additional ingredients include buffers, diluents, solvents, tonicity modifiers, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can be used in formulating the pharmaceutical compositions of the present invention.

In one embodiment of the invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, i.e., a formulation containing water. The liquid formulation may include solutions, suspensions, emulsions, microemulsions, gels and the like. Aqueous formulations typically contain at least 50% w / w of water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w / w of water.

In one embodiment, the pharmaceutical composition can be formulated as an injectable agent that can be injected, for example, via an injection device (eg, a syringe or infusion pump). Injections can be delivered, for example, subcutaneously, intramuscularly, intraperitoneally, or intravenously.

In another embodiment, the pharmaceutical composition is a solid formulation, eg, a lyophilized or spray-dried composition that can be used as is or that the physician or patient adds a solvent and / or diluent prior to use. be. Solid dosage forms include tablets such as compressed tablets and / or coated tablets, and capsules (eg, hard or soft gelatin capsules). The pharmaceutical composition may also be in the form of, for example, sachets, dragees, powders, granules, lozenges, or powders for reconstruction.

Dosage forms may be immediate release, in which case they may contain water-soluble or dispersible carriers, or dosage forms may be delayed release, sustained release, or modified release, in which case they. May include a water-insoluble polymer that regulates the rate of dissolution of the dosage form in the gastrointestinal tract.

In other embodiments, the pharmaceutical composition may be delivered intranasally, intraoralally, or sublingually.

The pH of the aqueous product can be pH 3 to pH 10. In one embodiment of the invention, the pH of the pharmaceutical product 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 invention, the pharmaceutical composition comprises a buffer. Non-limiting examples of buffers include arginine, aspartic acid, bicin, citrate, disodium monohydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, carbonate. Included are sodium, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, glycine, and tris (hydroxymethyl) -aminomethane, and mixtures thereof. The buffer may be present individually or in aggregate at a concentration of about 0.01 to about 50 mg / mL, for example about 0.1 to about 20 mg / mL. A pharmaceutical composition comprising each one of these specific buffers constitutes 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, bronopol, butyl4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorohexidine, chlorphenesin, o-cresol, m-cresol, p- Examples thereof include cresol, ethyl 4-hydroxybenzoate, imidourea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl4-hydroxybenzoate, sodium dehydroacetate, thiomerosal, and mixtures thereof. Preservatives may be present individually or in aggregates at concentrations of about 0.01 to about 50 mg / mL, such as about 0.1 to about 20 mg / mL. A pharmaceutical composition comprising each one of these specific preservatives constitutes an alternative embodiment 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), argitol (glycerol, 1,2-propane, etc.). Includes diol propylene glycol, etc.), 1,3-propanediol, and 1,3-butanediol, etc.), polyethylene glycol (eg, PEG400), and mixtures thereof. Another example of an isotonic agent is sugar. Non-limiting examples of sugars include, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta-HPCD, soluble starch, hydroxyethyl starch. , And can be monosaccharides, disaccharides, or polysaccharides, including sodium carboxymethyl cellulose. Another example of an isotonic agent is a sugar alcohol, 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, zulucitol, xylitol, and arabitol. Pharmaceutical compositions containing each isotonic agent listed in this paragraph constitute an alternative embodiment of the present invention. The isotonic agent may be present individually or in the aggregate at a concentration of about 0.01 to about 50 mg / mL, for example about 0.1 to about 20 mg / mL. A pharmaceutical composition comprising each one of these specific isotonic agents constitutes an alternative embodiment of the present invention.

In another embodiment of the invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include citric acid, aspartic acid, salts of ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. The chelating agent may be present individually or in the aggregate at a concentration of about 0.01 to about 50 mg / mL, for example about 0.1 to about 20 mg / mL. A pharmaceutical composition comprising each one of these specific chelating agents constitutes an alternative embodiment of the present invention.

In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and / or one or more protease inhibitors.

In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, which is carboxy- / hydroxycellulose and derivatives thereof (such as HPC, HPC-SL, HPC-L, and HPMC), cyclodextrin, etc. , 2-Methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (such as sodium chloride), sulfur-containing substances such as monothioglycerol), or thioglycolic acid. Stabilizers may be present individually or in aggregates at concentrations of about 0.01 to about 50 mg / mL, such as about 0.1 to about 20 mg / mL. A pharmaceutical composition comprising each one of these specific stabilizers constitutes an alternative embodiment of the present invention.

In a further embodiment of the invention, the pharmaceutical composition comprises one or more surfactants, preferably a surfactant, at least one surfactant, or two different surfactants. The term "surfactant" refers to any molecule or ion consisting of a water-soluble (hydrophilic) and lipophilic (lipophilic) moiety. The surfactant can be selected, for example, from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants, and / or bipolar surfactants. Surfactants may be present individually or in aggregates at a concentration of about 0.1 to about 20 mg / mL. A pharmaceutical composition comprising each one of these specific surfactants constitutes an alternative embodiment of the present invention.

In a further embodiment of the invention, the pharmaceutical composition comprises one or more protease inhibitors such as EDTA and / or benzamidine hydrochloride (HCI). Protease inhibitors may be present individually or in aggregates at concentrations of about 0.1 to about 20 mg / mL. A pharmaceutical composition comprising each one of these specific protease inhibitors constitutes an alternative embodiment of the present invention.

The pharmaceutical compositions of the present invention may contain sufficient amounts of amino acid bases to reduce polypeptide aggregate formation during storage of the composition. The term "amino acid base" refers to one or more amino acids (methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine, etc.) or analogs thereof. Any amino acid may be present in either its free base form or its salt form. Any stereoisomer of the amino acid base (ie, L, D, or a mixture thereof) may be present. Amino acid bases may be present individually or in combination with other amino acid bases at concentrations of about 0.01 to about 50 mg / mL, such as about 0.1 to about 20 mg / mL. A pharmaceutical composition comprising each one of these specific amino acid bases constitutes an alternative embodiment of the present invention.

It will also be apparent to those skilled in the art that the therapeutically effective amount of the conjugate of the invention or a pharmaceutical composition thereof will vary depending on the desired effect. Therefore, the optimal dose can be readily determined by one of ordinary skill in the art and will vary with the specific conjugate used, the method of administration, the strength of the preparation, and the progression of the condition. In addition, factors related to the specific subject being treated, including the subject's age, weight, diet, and duration of administration, raise the need to adjust the dose to the appropriate therapeutic concentration.

For all indications, the conjugates of the invention are preferably in single or divided doses (eg, single doses of 2, 3, 4, 5, 6, 7, 8, 9, or 10 partial doses). At a dose of about 1 μg to about 50 mg per day, or about 0.01 to about 500 μg / kg per dose, more preferably about 0.05 to about 250 μg / kg, most preferably about. It is administered peripherally at less than 50 μg / kg. Dosages within these ranges will, of course, depend on the potency of each agonist and will be readily determined by one of ordinary skill in the art. Therefore, the above dose is an example of the average case. Of course, there may be individual cases in which a higher or lower dose range is effective, and such cases are also within the scope of the present invention.

In certain embodiments, the conjugates of the invention are at a dose of about 1 μg to about 5 mg, or at a dose of about 0.01 to about 500 μg / kg, more preferably from about 0.05 to about 250 μg / kg. In terms of dose, most preferably at doses less than about 50 μg / kg, at doses of about 1 μg to about 5 mg, or more preferably at doses of about 0.01 to about 500 μg / kg, more preferably from about 0.05 to about 250 μg / kg. Of the dose, most preferably administered with a second therapeutic agent (eg, rilaglutide) at a dose of less than about 50 μg / 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 acetates, adipates, benzoates, benzenesulfonates, citrates, cypress salts, dodecyl sulfates, hydrochlorides, hydrobromates, lactates, malees. Included are salts, methanesulfonates, nitrates, oxalates, pivalates, propionates, succinates, sulfates and tartrates. Basic salts include alkali metal salts such as ammonium salt, sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, salts with organic bases such as dicyclohexylamino salt, and amino acids such as arginine. Salt is mentioned. Further, the basic nitrogen-containing group may be quaternized with, for example, an alkyl halide.

The pharmaceutical composition of the present invention can be administered by any means that realizes the intended use. Examples include parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, percutaneous, oral or intraocular administration. It may be administered by the oral route. Formulations suitable for parenteral administration include, for example, aqueous salts of water-soluble salts, acidic solutions, alkaline solutions, dextrose aqueous solutions, isotonic carbohydrate solutions, and aqueous solutions of active conjugates in water-soluble forms such as cyclodextrin inclusion complexes. ..

The invention also includes methods of making pharmaceutical compositions, comprising mixing a pharmaceutically acceptable carrier with any of the conjugates of the invention. In addition, the invention includes pharmaceutical compositions made 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 polymorphic or amorphous forms, and are therefore intended to be included within the scope of the invention. In addition, the conjugate may form, for example, a solvate with water (ie, a hydrate) or a common organic solvent with a solvate. As used herein, the term "solvate" means a physical association of a conjugate of the invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonds, such as hydrogen bonds. In certain cases, for example, when one or more solvent molecules are incorporated into the crystalline lattice of a crystalline solid, the solvate becomes separable. The term "solvate" is intended to include both solution phase solvates and separable solvates. Non-limiting examples of suitable solvates include etanolate, metanolate and the like.

The present invention is intended to include polymorphs and solvates of the conjugates of the present invention within that scope. Therefore, the term "administration" in the therapeutic methods of the invention is a conjugate of the invention, or a polymorph or solvent thereof that would clearly fall within the scope of the invention, even if not specifically disclosed. Means for treating, ameliorating, or preventing the syndromes, disorders, or diseases described herein using Japanese products are included.

In another embodiment, the invention relates to a conjugate of the invention for use as a drug.

The present invention includes the prodrug of the conjugate of the present invention within its scope. In general, such prodrugs are functional derivatives of conjugates that can be easily converted to the required conjugates in vivo. Therefore, in the therapeutic method of the present invention, the term "administration" may or may not be specifically disclosed using a specifically disclosed conjugate, but is specific in vivo after administration to a patient. It shall include the treatment of the various disorders described using conjugates that convert to gates. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs" (Ed. H. Bundgaard, Elsevier, 1985).

Moreover, within the scope of the invention, any element, whether naturally occurring or synthetically produced, and in a natural abundance ratio or isotopic, especially when referred to with respect to the conjugate of the invention. In any of the body-enriched forms, it is intended to contain all isotopes of the element and mixtures of isotopes. For example, references to hydrogen include, within that range, ¹ H, ² H (D), and ³ H (T). Similarly, references to carbon and oxygen include, within their scope, ¹² C, ¹³ C and ¹⁴ C, ^{and 16} O and ¹⁸ O, respectively. Such isotopes may be radioactive isotopes or non-radioactive isotopes. Radiolabeled conjugates of the present ^{invention, 3 H, 11 C, 18} F, 122 I, 123 I, 125 I, 131 I, 75 Br, 76 Br, 77 Br, and radioactive selected from the group consisting of ⁸² Br It may contain isotopes. Preferably, the radioisotope is selected from the group consisting of ³ H, ¹¹ C, and ^{18 F.}

Some conjugates of the invention may exist as atropisomers. Atropisomers are stereoisomers that result from impaired rotation around a single bond, and the interference of conformational strains with respect to rotation is high enough to allow isolation of conformers. It is understood that all such conformers and mixtures thereof are included within the scope of the present invention.

If the conjugates according to the invention have at least one stereocenter, they may as a result exist as enantiomers or diastereomers. It is understood that all such isomers and mixtures thereof are included within the scope of the present invention.

If the process of preparing the conjugate according to the invention yields a mixture of stereoisomers, these isomers can be separated by conventional methods such as preparative chromatography. Conjugates can be prepared as racemates, or individual enantiomers can be prepared either by enantioselective synthesis or degradation. Conjugates can be decomposed into their constituent enantiomers by standard methods, such as (-)-di-p-toroil-D-tartaric acid and / or (+). After forming a diastereomer pair by forming a salt with an optically active acid such as -di-p-toluoil-L-tartaric acid, fractional crystallization and regeneration of a free base are performed. The conjugate can also be degraded by forming an ester or amide of the diastereomer and then performing chromatographic separation to remove the chiral auxiliary. Alternatively, the conjugate may be degraded using high performance liquid chromatography (HPLC) or a chiral column via SFC. In some cases, in the ¹ H NMR spectrum, there may be conjugate rotational isomers observable by ^{1 1} H NMR resulting in complex multiplex and peak integration.

During any of the processes for preparing the conjugates of the invention, it may be necessary and / or desirable to protect sensitive or reactive groups in any of the relevant molecules. This is from Protective Groups in Organic Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973, and T.M. W. Greene & P. G. M. Conventional protecting groups, such as those described in Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, each of which is incorporated herein by reference in its entirety for all purposes. Can be achieved by. The protecting group can be removed at a subsequent convenient stage using methods well known in the art.

Methods of Use The present invention relates to a method of preventing, treating, or ameliorating an amylin receptor-mediated syndrome, disorder, or disease in a subject in need thereof. Including administration of a conjugate, compound, or pharmaceutical composition to a subject in need thereof.

The present invention requires that a disorder, disease, or condition, or any one or more symptoms of the disorder, disease, or condition be prevented, treated, delayed, or ameliorated. The method also provides an effective amount of a conjugate, compound, or pharmaceutical composition of the invention to be administered to a subject in need thereof.

According to certain embodiments, the disorder, or condition, is obesity, type I or type II diabetes, metabolic syndrome (ie, syndrome X), insulin resistance, impaired glucose tolerance (eg, glucose tolerance), hyperglycemia, Related to hyperinsulinemia, hypertriglyceridemia, impaired glucose tolerance (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and uncontrolled cholesterol and / or lipid levels A group consisting of other cardiovascular risk factors such as high blood pressure and cardiovascular risk factors, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty hepatitis (NASH), renal disease, and / or eczema. Is selected from.

According to certain 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 the disease, disorder or condition being treated, or its associated symptoms, (ii) shortening the duration of the disease, disorder or condition being treated, or its associated symptoms. , (Iii) to prevent the progression of the disease, disorder or condition to be treated, or related symptoms, (iv) to cause regression of the disease, disorder or condition to be treated, or related symptoms, (iv). v) Preventing the development or onset of the disease, disorder or condition being treated, or related symptoms, (vi) preventing the recurrence of the disease, disorder or condition being treated, or related symptoms, (vi) vii) Reducing hospitalization for subjects with the disease, disorder or condition being treated, or related symptoms, (viii) Length of stay for subjects with the disease, disorder or condition being treated, or related symptoms To increase the survival rate of subjects with (ix) the disease, disorder or condition being treated, or related symptoms, (xi) the disease, disorder or condition of the subject to be treated, or related to it. Inhibiting or alleviating symptoms and / or (xii) enhancing or ameliorating the preventive or therapeutic effect of another treatment.

The therapeutically effective amount or dose is the disease, disorder or condition to be treated, means of administration, target site, physiological condition of the subject (including, for example, age, weight, health condition), whether the subject is human or animal. It can depend on various factors such as, other agents administered, and whether the treatment is prophylactic or therapeutic. The therapeutic dose is optimally escalated to optimize safety and efficacy.

As used herein, the terms "treat," "treating," and "treatment" are all at least one that is associated with a disease, disorder, or condition. It refers to the improvement or reversal of measurable physical parameters, which are not always recognizable in the subject, but may be recognizable in the subject. The terms "treat," "treat," and "treat" may also refer to causing regression of a disease, disorder, or condition, preventing its progression, or at least slowing its progression. In certain embodiments, "treat," "treat," and "treat" alleviate, prevent the progression or onset of one or more symptoms associated with a disease, disorder, or condition, or shorten its duration. Point to. In certain embodiments, "treat," "treat," and "treat" refer to the prevention of recurrence of a disease, disorder, or condition. In certain embodiments, "treating," "treating," and "treating" refer to improving the survival rate of a subject having a disease, disorder, or condition. In certain embodiments, "treat," "treat," and "treat" refer to the disappearance of a disease, disorder, or condition in a subject.

In one embodiment, the invention is a method of preventing, treating, delaying, or ameliorating obesity, or any one or more symptoms of obesity, in a subject in need thereof. Provided, the method comprises administering an effective amount of a conjugate, compound, or pharmaceutical composition of the invention to a subject in need thereof. In some embodiments, the body weight of the subject is relative to the body weight of the subject prior to administration of any of the conjugates, compounds, pharmaceutical compositions, forms, or agents of the invention described herein, or the present. For example, about 0.01 to about 0.1%, about 0.1, as compared to a control subject that has not received any of the conjugates, compositions, forms, agents, or combinations of the invention described herein. ~ About 0.5%, about 0.5 ~ about 1%, about 1 ~ about 5%, about 2-3%, about 5 ~ about 10%, about 10 ~ about 15%, about 15 ~ 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% reduction.

In some embodiments, weight loss is 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 Maintained for years.

The present invention is intended to prevent, treat, delay or ameliorate a syndrome, disorder, or disease, or any one or more symptoms of the syndrome, disorder, or disease. , The method to be performed on a subject who needs it, and the syndrome, disorder, or disease is obesity, type I or type II diabetes, metabolic syndrome (ie, syndrome X), insulin resistance, impaired glucose tolerance (eg, glucose tolerance). , Glucose tolerance), hyperglycemia, hyperinsulinemia, hypertriglycerideemia, hypoglycemia due to congenital hyperinsulinosis (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and managed Other cardiovascular risk factors such as hypertension and cardiovascular risk factors not associated with cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic steatohepatitis (NAFLD), non-alcoholic steatohepatitis (NASH), kidney Provided is a method selected from the group consisting of diseases, as well as eczema, wherein an effective amount of a conjugate, compound, or pharmaceutical composition of the invention is administered to a subject in need thereof. include.

As used herein, metabolic syndrome is hyperglycemic (eg, high fasting blood glucose), high blood glucose, abnormal cholesterol levels (eg, low HDL levels), abnormal triglyceride levels (eg, high triglyceride), large waistline. (Ie, peri-lumbar), increased fat in the abdominal region, insulin resistance, glucose tolerance, high C-reactive protein levels (ie, preinflammatory conditions), and plasma plasminogen activator inhibitor-1 and fibrinogen levels (ie) , A blood clot state) refers to an object having any one or more.

The present invention provides a method of reducing food intake to a subject in need thereof, which requires administration of an effective amount of a conjugate, compound, or pharmaceutical composition of the present invention. Including what to do with the subject. In some embodiments, the food intake of the subject is relative to the food intake of the subject prior to administration of any of the conjugates, compounds, compositions, forms, agents, or combinations of the invention described herein. Or, for example, about 0.01 to about 0.1% as compared to a control subject that has not received any of the conjugates, compounds, compositions, forms, agents, or combinations of the invention described herein. , About 0.1 to about 0.5%, about 0.5 to about 1%, about 1 to about 5%, about 2 to about 3%, about 5 to about 10%, about 10 to about 15%, about Reduced by 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% NS.

In some embodiments, the reduction in food intake is 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 It will be maintained for 20 years.

The present invention provides a method of reducing glycated hemoglobin (A1C) to a subject in need thereof, the method comprising administering an effective amount of a conjugate, compound or pharmaceutical composition of the invention. , Including doing to the subject who needs it. In some embodiments, the subject A1C is the subject A1C prior to administration of any of the conjugates, compounds, compositions, forms, agents, or combinations of the invention described herein, or. For example, about 0.001-about 0.01%, about, as compared to a control subject that has not received any of the conjugates, compounds, compositions, forms, agents, or combinations of the invention described herein. 0.01 to about 0.1%, about 0.1 to about 0.2%, about 0.2 to about 0.3%, about 0.3 to about 0.4%, about 0.4 to about 0 5.5%, about 0.5 to about 1%, about 1 to about 1.5%, about 1.5 to about 2%, about 2 to about 2.5%, about 2.5 to about 3%, about Reduced by 3 to about 4%, about 4 to about 5%, about 5 to about 6%, about 6 to about 7%, about 7 to about 8%, about 8 to about 9%, or about 9 to about 10% NS.

In another embodiment, a method of reducing fasting blood glucose levels for a subject in need thereof is provided, the method of administration of an effective amount of a conjugate, compound, or pharmaceutical composition of the invention. Includes doing to the subject who needs it. Fasting blood glucose levels are such as to the fasting blood glucose level of the subject prior to administration of any of the conjugates, compounds, compositions, forms, agents, or combinations of the invention described herein, or herein. About 140 to less than about 150 mg / dL, about 140 to about 130 mg / dL, as compared to a control subject not receiving any of the conjugates, compounds, compositions, forms, agents, or combinations of the invention described in. Less than, about 130 to less than 120 mg / dL, about 120 to less than 110 mg / dL, about 110 to less than about 100 mg / dL, about 100 to less than 90 mg / dL, or about 90 to less than about 80 mg / dL obtain.

The present invention provides a method of regulating amyrin receptor activity in a subject in need thereof, the method comprising administering an effective amount of the conjugate, compound, or pharmaceutical composition of the present invention. Includes doing what you need. As used herein, "regulating" refers to increasing or decreasing receptor activity.

In some embodiments, an effective amount of the conjugate or compound of the invention, or form, composition, or agent thereof, is once daily, twice daily, three times daily, four times daily, 1 It is administered to subjects who need it 5 times a day, 6 times a day, 7 times a day, or 8 times a day. In other embodiments, an effective amount of the conjugate or compound of the invention, or form, composition, or agent thereof, is once every two days, once a week, twice a week, three times a week, a week. It is administered to subjects who need it 4 times a week, 5 times a week, 6 times a week, 2 times a month, 3 times a month, or 4 times a month.

Another embodiment of the invention is to prevent, treat, delay or ameliorate a disease, disorder, or syndrome, or any one or more symptoms of the disease, disorder, or syndrome. To a subject who needs it, the method is to administer an effective amount of a conjugate, compound, or pharmaceutical composition of the invention to a subject who needs it in combination therapy. including. In certain embodiments, the combination therapy is a second therapeutic agent. In certain embodiments, the combination therapy is surgical therapy.

As used herein, the term "combined" refers to the use of multiple therapies in the context of administration of two or more therapies to a subject.

As used herein, combination therapy is one or more additional therapeutic agents, or one or more, at the same time as an effective amount of a conjugate or compound of the invention, or form, composition, or agent thereof. Refers to giving surgical treatment to a subject who needs it. In some embodiments, one or more additional therapeutic agents or surgical treatments may be administered on the same day as an effective amount of the conjugate of the invention, whereas in other embodiments, one or more additional therapeutic agents. Alternatively, surgery may be administered in the same week or month as an effective amount of the conjugate or compound of the invention.

In certain embodiments, the disease or disorder is selected from the group consisting of obesity, type II diabetes, metabolic syndrome, insulin resistance, and dyslipidemia, the second therapeutic agent being an antidiabetic agent. In certain embodiments, the anti-diabetic agent can be a glucagon-like peptide-1 (GLP-1) receptor regulator.

The invention also requires the use of combination therapies to prevent, treat, delay or ameliorate any of the diseases, disorders, syndromes, or symptoms described herein. Concomitant therapy requires an effective amount of the conjugate, compound, or pharmaceutical composition of the invention in combination with any one or more of the following therapeutic agents. Includes administration to subjects: dipeptidyl peptidase-4 (DPP-4) inhibitors (eg, sitagliptin, saxagliptin, linagliptin, allogliptin, etc.); GLP-1 receptor agonists (eg, exenatide and lixenatide, etc.) Short-acting GLP-1 receptor agonists; Intermediate-acting GLP-1 receptor agonists (eg, rilaglutide); Long-acting GLP-1 receptor agonists such as long-acting exenatide, albiglutide, and duraglutide; sodium-glucose Cotransporter-2 (SGLT-2) inhibitors (eg, canaglifodin, dapaglifodin, empaglifodin, etc.); bile acid sequestering agents (eg, colleseverum, etc.); dopamine receptor agonists (eg, bromocryptin rapid release); Biguanide (eg, metformin); insulin; oxintomodulin; sulfonylurea (eg, chlorpropamide, glymepyride, glypidide, glybrid, glybenclamid, glybornulide, glycosepid, glycropyramide, trazamide, tolbutamide, acetohexamide, kabutamide, etc.) And thiazolidinedione (eg, pioglitazone, rosiglitazone, robeglitazone, siglitazone, dalglitazone, englycazone, netoglitazone, riboglycazone, troglitazone, etc.). In some embodiments, the dose of the additional therapeutic agent (s) may be. , Reduced when given in combination with the conjugates or compounds of the invention. In some embodiments, additional therapeutic agents (s) may be used in combination with the conjugates or compounds of the invention. , Each may be used at a lower dose than when used alone.

Diseases or disorders include obesity, type I or type II diabetes, metabolic syndrome (ie, syndrome X), insulin resistance, impaired glucose tolerance (eg, glucose tolerance), hyperglycemia, hyperinsulinemia, hypertriglycerideemia , Hypoglycemia due to congenital hyperinsulinosis (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and / or lipid levels, etc. In certain embodiments, selected from the group consisting of other cardiovascular risk factors, osteoporosis, inflammation, non-alcoholic steatohepatitis (NAFLD), non-alcoholic steatohepatitis (NASH), renal disease, and eczema. , The second therapeutic agent can be liraglutide.

The present invention requires the use of combination therapies to prevent, treat, delay or ameliorate any of the diseases, disorders, syndromes, or symptoms described herein. Concomitant therapy comprises administering an effective amount of a conjugate, compound, or pharmaceutical composition of the invention to a subject in need thereof in combination with surgical therapy. In certain embodiments, the surgical treatment is obesity surgery (eg, gastric bypass surgery such as Ruwai's gastric bypass surgery; sleeve gastric resection; adjustable gastric band surgery; biliary gastrointestinal avoidance with duodenal switch; Intragastric balloon; gastric grafting, and combinations thereof).

In embodiments where one or more additional therapeutic agents or surgical treatments are administered on the same day as an effective amount of the conjugate or compound of the invention, the conjugate or compound of the invention is an additional therapeutic agent or compound. It can be administered before, after, or at the same time as surgery. The use of the term "combined" does not limit the order in which the treatment is administered to the subject. For example, a first therapeutic agent (eg, the composition described herein) may be applied to a subject prior to administration of the second therapeutic agent (eg, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1). Hours, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 224 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, Or 12 weeks ago) at the same time or thereafter (eg, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, It can be administered 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later).

Further embodiments of the invention include the incorporation of further proteolytic stabilization modifications of the amyrin agonist peptide, preferably in the peptide region between residues 10 and 17. Such proteolytic stabilization modifications include, but are not limited to, amino acid substitutions with one or more non-proteinogenic amino acids, C-α-alkylated amino acids, homologous amino acids, synthetic amino acids, and the like. A combination of two or more such proteolytic stabilization modifications is contemplated herein.

In a further embodiment of the invention, the thioether cyclized amilinomimetic peptide or derivative thereof contains at least one amino acid residue that is derivatized with an extended half-life moiety. Such half-life extension portions are introduced into suitable (resistant) sites via conjugates on the residues appropriately mutated at the above positions. Examples of half-life extension portions 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.

Further embodiments of the present invention include amino acid substitutions and / or peptide modifications introduced to improve the physicochemical properties of the thioether cyclized amyrin peptide. In certain examples, the native amyrin amino acid residue may be mutated to a residue that reduces the pI of the peptide, whereby it can be formulated for easier administration, while at the same time amyrin receptor potency. Is maintained. In another example, derivatization with water-soluble functional groups (such as, but not limited to, polyethylene glycol) is contemplated as a means of improving the solubility of thioether-cyclized amyrin analogs in suitable blended vehicles.

Embodiment The present invention also provides the following non-limiting embodiments.

Embodiment 1 is a conjugate comprising a monoclonal antibody bound to an amilinomimetic peptide or an antigen-binding fragment thereof, wherein the amilinomimetic peptide is represented by Formula I (SEQ ID NO: 53) or The derivative or pharmaceutically acceptable salt:

（式中、
ｎは、１又は２であり、
Ｚ_２は、直接結合、セリン、又はグリシンであり、
Ｚ_４は、Ｔ又は

(During the ceremony,
n is 1 or 2
Z ₂ is a direct bond, serine, or glycine,
Z ₄ is T or

であり、
Ｚ_５は、Ａ、β−アラニン、

And
Z ₅ is A, β-alanine,

であり、
Ｚ_６は、Ｔ又は

And
Z ₆ is T or

であり、
Ｚ_１０は、Ｑ又はＥであり、
Ｚ_１１は、Ｒ又はＫであり、Ｋのε−アミンが、任意選択で−Ｃ（＝ＮＨ）ＮＨ_２によって置換されたものであり、
Ｚ_１２は、Ｌ又は

And
Z ₁₀ is Q or E,
Z ₁₁ is R or K, in which the ε-amine of K is optionally replaced by _{-C (= NH) NH 2.}
Z ₁₂ is L or

であり、
Ｚ_１６は、Ｌ又は

And
Z ₁₆ is L or

であり、
Ｚ_２５は、Ｐ又はＫであり、
Ｚ_２６は、Ｉ又はＫであり、
Ｘは、ＡＴＺ_１０Ｚ_１１Ｚ_１２ＡＮＦＺ_１６ＶＨＳＳＮＮＦＧＺ_２５Ｚ_２６ＬＰＺ_２９ＴＮＶＧＺ_３４（配列番号５４）、又はＶＬＧＲＬＳＱＥＬＨＲＬＱＴＹＰＲＴＮＴＧＳ（配列番号５５）であり、
Ｚ_２９は、Ｐ又はＫであり、
Ｚ_３４は、Ｓ又はＫである）
その誘導体が、アミド化、グリコシル化、カルバミル化、硫酸化、リン酸化、環化、脂質化、及びＰＥＧ化からなる群から選択される１つ以上のプロセスによって修飾されている式Ｉの化合物である、コンジュゲートである。

And
Z ₂₅ is P or K and
Z ₂₆ is I or K,
X is ATZ ₁₀ Z ₁₁ Z ₁₂ ANFZ ₁₆ VHSSNNFGZ ₂₅ Z ₂₆ LPZ ₂₉ TNVGZ ₃₄ (SEQ ID NO: 54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55).
Z ₂₉ is P or K
Z ₃₄ is S or K)
A compound of formula I in which the derivative is modified by one or more processes selected from the group consisting of amidation, glycosylation, carbamylation, sulfation, phosphorylation, cyclization, lipidation, and PEGylation. There is a conjugate.

In the second embodiment, the amilinomimetic peptide of formula I in which the amilinomimetic peptide is modified by one or more processes selected from the group consisting of amidation, lipidation, and PEGylation. The conjugate according to embodiment 1, which is a derivative or a pharmaceutically acceptable salt thereof.

In the third embodiment, the amilinomimetic peptide is represented by the formula I, or is a derivative thereof or a pharmaceutically acceptable salt, and in the formula,
Z ₂ is a direct bond,
Z ₅ is β-alanine,

であり、
Ｚ_６は、Ｔである、実施形態１に記載のコンジュゲートである。

And
Z ₆ is the conjugate according to embodiment 1, which is T.

In the fourth embodiment, the amilinomimetic peptide is represented by the formula I, or is a derivative thereof or a pharmaceutically acceptable salt, and in the formula,
Z ₁₆ is L,
Z ₁₂ is

である、実施形態１に記載のコンジュゲートである。

The conjugate according to the first embodiment.

In the fifth embodiment, the amilinomimetic peptide is represented by the formula I, or is a derivative thereof or a pharmaceutically acceptable salt, and in the formula,
Z ₁₁ is R,
Z ₁₂ is

である、実施形態１に記載のコンジュゲートである。

The conjugate according to the first embodiment.

Embodiment 6 is the conjugate of Embodiment 1, wherein the amilinomimetic peptide is selected from the group consisting of SEQ ID NOs: 4-28.

Embodiment 7 is any of embodiments 1 to 6, wherein the monoclonal antibody or antigen-binding fragment thereof is covalently bound to the amilinomimetic peptide at the lysine residue of the amilinomimetic peptide via a linker. It is the conjugate described in one.

In Embodiment 8, heavy chain complementarity determining regions 1 (HCDR1), HCDR2, and HCDR3, in which the monoclonal antibody or antigen-binding fragment thereof has the polypeptide sequences of SEQ ID NOs: 47, 48, 49, 50, 51, and 52, respectively. , And the conjugate according to any one of embodiments 1-7, comprising light chain complementarity determining regions 1 (LCDR1), LCDR2, and LCDR3.

In embodiment 9, the isolated monoclonal antibody comprises a heavy chain variable domain (VH) having the polypeptide sequence of SEQ ID NO: 43 and a light chain variable domain (VL) having the polypeptide sequence of SEQ ID NO: 45. , The conjugate according to the eighth embodiment.

Embodiment 10 is the conjugate according to embodiment 9, further comprising an Fc portion.

11th embodiment 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.

In the twelfth embodiment, a monoclonal antibody or an antigen thereof is used as a side chain of an amilinomimetic peptide, preferably an amino acid side chain of a lysine residue of the amilinomimetic peptide, and a bromoacetamide derivatized linker. An embodiment comprising reacting the sulfhydryl group of the cysteine residue of SEQ ID NO: 49 of the binding fragment with a covalent link between the amilinomimetic peptide and the monoclonal antibody or antigen binding fragment thereof. The method for generating a conjugate according to any one of 1 to 11.

Embodiment 13 is a pharmaceutical composition comprising the conjugate according to any one of embodiments 1-11 and a pharmaceutically acceptable carrier.

Embodiment 14 is a method of treating or preventing obesity to a subject in need thereof, wherein an effective amount of the pharmaceutical composition according to Embodiment 13 is administered to the subject in need thereof. It is a method to include.

In the fifteenth embodiment, administration of an effective amount of the pharmaceutical composition to a subject in need thereof is performed by about 5 to about 10%, about 10% of the body weight of the subject before administration of the pharmaceutical composition. The method of embodiment 14, which results in weight loss of ~ about 15%, about 15 ~ about 20%, or about 20 ~ about 25%.

Embodiment 16 is a method of treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, sugar tolerance. Dysfunction, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and uncontrolled cholesterol And / or other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with lipid levels, osteoporosis, inflammation, non-alcoholic steatohepatitis (NAFLD), non-alcoholic steatohepatitis (NASH), renal disease, Also selected from the group consisting of eczema, the method comprises administering an effective amount of the pharmaceutical composition according to embodiment 13 to a subject in need of treatment or prevention.

Embodiment 17 is the method of embodiment 16, wherein the disease or disorder is obese.

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.

20th embodiment is the method according to 16th embodiment, wherein the disease or disorder is metabolic syndrome.

21st embodiment is the method according to 16th embodiment, wherein the disease or disorder is a renal disease.

Embodiment 22 is the method of embodiment 16, wherein the disease or disorder is non-alcoholic steatohepatitis (NASH).

23rd embodiment is the method according to 16th embodiment, wherein the disease or disorder is non-alcoholic fatty liver disease (NAFLD).

Embodiment 24 is a method of reducing food intake to a subject in need thereof, the method comprising administering an effective amount of the pharmaceutical composition according to embodiment 13 to a subject in need thereof. Is.

In the 25th embodiment, administration of an effective amount of the pharmaceutical composition to a subject in need thereof is performed by about 5 to about 10%, about 5 to about 10%, as compared with the food intake of the subject before administration of the pharmaceutical composition. 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 24. The method of embodiment 24, which results in a 45-to 50% reduction in food intake.

Embodiment 26 is a method of regulating amyrin receptor activity to a subject in need thereof, wherein an effective amount of the pharmaceutical composition according to embodiment 13 is administered to the subject in need thereof. It is a method including.

Embodiment 27 is the method according to any one of embodiments 14-26, wherein the pharmaceutical composition is administered by 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 14-28, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.

In embodiment 30, the disease or disorder is selected from the group consisting of obesity, type 2 diabetes, metabolic syndrome, insulin resistance, and dyslipidemia, the second therapeutic agent being at least one antidiabetic agent. The method according to the form 29.

Embodiment 31 is the method of embodiment 30, wherein the antidiabetic agent is a glucagon-like peptide-1 receptor regulator.

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 14-32, wherein the pharmaceutical composition is administered daily, weekly, or monthly to a subject in need thereof.

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 a day.

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 a 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 the conjugate according to any one of embodiments 1-14 or the pharmaceutical composition of embodiment 13, preferably the kit further comprising 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 infusion device.

Embodiment 39 is a method for producing a pharmaceutical composition comprising a compound selected from the group consisting of SEQ ID NOs: 4 to 42 and a pharmaceutically acceptable carrier.

Synthesis The compounds or conjugates of the present invention can be synthesized according to common synthetic methods known to those skilled in the art. The following synthetic description is for illustrative purposes only and is by no means intended to limit the invention.

The thioether-cyclized amilinomimetic peptides or derivatives of the invention can be synthesized by a variety of known conventional procedures for forming continuous peptide linkages between amino acids, automated peptide synthesizers, conventional Bench synthesis, or a combination of both approaches, preferentially solid phase peptide synthesis, as generally described by Merrifield (JAm. Chem. Soc., 1963, 85, 2149-2154). (SPPS). The conventional procedure for peptide synthesis is that the free amino group of one amino acid residue (the other reactive functional groups are well protected) and the free carboxyl group of another amino acid (its reactive functional group is also suitable). Accompanied by condensation with). Examples of condensing agents commonly used for peptide bond formation are diisopropylcarbodiimide (DIC), 2- (1H-) with or without 1-hydroxybenztriazole (HOBT) or ethyl cyano (hydroxyimino) acetate (Oxyma Pure). Benzotriazole-1-yl) -1,1,3,3-tetramethylaminoium hexafluorophosphate (HBTU), 2- (1H-7-azabenztriazole-1-yl) -1,1,3,3 -Tetramethylaminium hexafluorophosphate (HATU), 2- (6-chloro-1H-benztriazole-1-yl) -1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), 1- Cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyOxim), 2- (1H-benzotriazole-1-yl) -1,1,3,3-tetramethylaminium Examples thereof include tetrafluoroborate (TBTU) bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP).

The automated peptide synthesis method was described by Yu (J. Org. Chem., 1992, 57, 4781-4784) and recently improved by Parasek (J. Pept. Sci., 2007, 13, 143-148). As such, it can be done at room temperature (rt) or high temperature, preferably by applying microwave heating.

The compounds of the present invention (C-terminal amides) are N-α-FMOC-protected amino acid carboxy-terminals that are bonded onto conventional solid-phase resins using suitable binders. It can be conveniently prepared using the -α-FMOC protected amino acid method. Suitable conventional commercially available solid phase resins include Rinkamide MBHA resin, Rinkamide AM resin, Tentagel SRAM Resin, FMOC-PAL-PEG PS resin, SpheriTide Rink amide resin, Chemmatrix Rink resin, Seeber amide resin, TG Seeber. Examples include resin. The resin-bound FMOC-amino acid may then be deprotected by exposure to 20% piperidine in either DMF or NMP, the treatment of which serves to selectively remove FMOC protecting groups. The additional FMOC-protected amino acids are then sequentially bound and deprotected, thereby producing the desired resin-bound, protected peptide. In certain cases, it may be necessary to utilize an orthogonally reactive protecting group for another amine in the peptide sequence that will withstand FMOC deprotection conditions. Protecting groups such as 4-methyltrityl (Mtt) or 4-methoxytrityl (Mmt) can all be removed by 1% TFA / DCM treatment, or preferably allyloxycarbonyl (alloc; Pd (PPh ₃ )). _{Can be removed by 4} / PhSiH ₃ treatment), 1- (4,4-dimethyl-2,6-dioxocyclohexa-1-iriden) ethyl (Dde; can be removed by treatment with 2-3% hydrazine / DMF) , And 1- (4,4-dimethyl-2,6-dioxocyclohexa-1-iriden) -3-methylbutyl (ivDde; which can be removed by treatment with 2-3% hydrazine / DMF) are such. Can be used effectively in some cases.

In conventional peptide synthesis methods, the reactive side chains of alpha amino acids are generally protected throughout the synthesis with suitable protecting groups in order to inactivate them against binding and deprotection protocols. Although multiple protecting groups for amino acid side chains are known in the art, the following protecting groups are most preferred herein: tert-butyl for serine, threonine, glutamic acid, aspartic acid, and tyrosine: t-Bu); trityl (Trt) for aspartic acid, glutamine, cysteine, homocysteine, and histidine; tert-butyloxycarbonyl (Boc) for tryptophan and the ε-amino group of lysine; and 2, for arginine. 2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl (Pbf). These protecting groups are removed by treatment with a strong acid such as high concentration trifluoroacetic acid (TFA).

Upon completion of SPPS, the resin-bound side chain protected peptides are deprotected and predominantly (along with various combinations of carbocation sweeping agents such as triisopropylsilane (TIPS), water, phenol, and anisole. A cutting cocktail consisting of TFA) is used to simultaneously cut from the resin. The crude solid peptide is then isolated by precipitating the peptide / cocktail filtrate with cold ether. The crude peptide thus obtained is then dissolved at a low concentration (less than about 5 mg / mL) in a predominantly aqueous solvent system containing an organic co-solvent such as acetonitrile or ethanol. When the pH of the solution is raised above 7, the peptide then undergoes an intramolecular cyclization reaction to form the corresponding crude thioether cyclized amylin analog of the invention. The thioether-cyclized amyrin analog thus formed can be purified using purification techniques generally known in the art. A preferred method of peptide purification as used herein is reverse phase high performance liquid chromatography (HPLC). The purified peptide is then characterized by liquid chromatography / mass spectrometry (LC / MS).

The following examples and embodiments described herein are for purposes of illustration only, and various amendments or modifications in consideration thereof will be presented to those skilled in the art, the purpose and scope of the present application, and the accompanying claims. It is understood that it is included in the range of. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Abbreviations The following abbreviations may be used below and throughout the specification.

Example 1: Synthesis of thioether cyclized amilinomimetic peptide (Scheme 1)
Step A: Synthesis of Resin-Binded C-Termide Amide Peptide The protected peptidyl resin is a low-filled Linkamide resin, preferably on a 0.1 mmol scale, using the FMOC strategy described above, as shown in Scheme 1. Was synthesized on a CEM Liberty Blue Microwave peptide synthesizer using FMOC-PAL-PEG PS resin (approximately 0.16-0.2 meq / g, supplied by Applied Biosystems). Standard FMOC-protected amino acids (supplied by Novabiochem (EMD Millpore), Bachem, Peptides International, Sigma-Aldrich, or Chem-Impex) are used as binders at DIC / Oxyma and a reaction temperature of about 90 ° C. for 4 minutes. It was bound in excess of 5 times the resin filling used. FMOC-Arg (Pdf) -OH was double-bonded at 90 ° C. for 4 minutes each, and FMOC-His (Trt) -OH was double-bonded at room temperature for 4 minutes, followed by 50 ° C. for 8 minutes using a two-step protocol. Combined. One FMOC deprotection was performed at 90 ° C. for 1.5 minutes using 20% piperidine (deprotecting solution) in DMF.

Step B: Procedure for bromoacetylation of resin-bound peptide (Scheme 2)
The FMOC deprotected peptide resin (0.1 mmol) was treated with a solution of bromoacetic anhydride (6-20 equivalents) in DMF (5 mL) in a microwave reactor at 50 ° C. for 5 minutes at that time point. By now, the reaction was largely determined to be complete by the Kaiser ninhydrin test. If the binding was determined to be incomplete, the binding was repeated with the new reagent.

Step C: Peptide cleavage procedure from the resin After SPPS was completed, the resin was thoroughly washed with DMF and then with DCM and dried. The resin is then subjected to TFA / water / TIPS / (95: 2.5: 2.5) (Cleavage Cocktail A), or more preferably TFA / water / phenol / TIPS (88: 5: 5: 2) (Cleavage). It was treated with a cleavage cocktail (10 mL / 0.1 mmol scale) consisting of any of Cocktail B), heated in a microwave reactor at 38 ° C. for 40 minutes and then filtered. The resin was washed with TFA and the combined filtrate was concentrated under a stream of nitrogen to a volume of about 2.5 mL and the peptide was precipitated by adding cold diethyl ether (40 mL). The peptide / ether suspension was centrifuged and the ether layer was decanted. The peptide pellet was resuspended in ether, centrifuged, decanted and the process repeated 3 times. The crude peptide thus obtained was dried under a gentle stream of nitrogen.

Step D: Peptide cyclization (thioether formation) procedure Deoxidize crude cysteine-containing or homocysteine-containing peptide at a concentration of less than 4 mg / mL MeCN / water (50-60% MeCN) or EtOH / water (50-60%) It was dissolved in EtOH). The pH of the peptide solution was then raised to about 7-9 by adding either solid NaHCO ₃ , _{saturated aqueous NaHCO 3 solution, or 1M aqueous solution of Tris buffer (pH 7.5).} The resulting solution was stirred at room temperature for 3-16 hours. Typically, cyclization was completed within 1 hour as determined by analytical LC / MS.

Step E: Peptide Purification Procedure The cyclization reaction mixture is acidified to pH 1.5-3 by adding TFA, the solution is concentrated and most of the organic co-solvent (MeCN or EtOH) is slightly turbid. It was removed to the point where it occurred. Minimal amounts of co-solvent were added back as needed to homogenize the mixture, and the resulting solution was then directly purified by preparative HPLC in multiple injections. Purification was performed on either the Agilent PrepStar HPLC system or the Gilson HPLC 2020 Personal Purification System using a reverse phase C18 or C8 column selected from: Varian Pursuit XR C18 (21 x 250 mm, 100 Å, 5 μm). ); Varian Pursuit XR Diphenyl (30 x 100 mm, 100 Å, 5 μm); Zorbax 300 SB-C8 (21 x 250 mm, 300 Å, 5 μm); Waters Atlantis T3 C18 (19 x 250 mm, 100 Å, 5 μm); Agilent Polaris 5 A (30 x 250 mm, 180 Å, 5 μm). The mobile phase is gradient elution of buffer A (0.1% TFA in water) and buffer B (0.1% TFA in MeCN) with an initial concentration in the range of 10-20% B to a final concentration of 40-90% B. The run time ranged from 36 to 80 minutes. UV detection was monitored at 220 and 254 nm. Product-containing fractions were analyzed by analytical HPLC on an Agilent 1100 HPLC system using the appropriate column types from above (4.6 x 250 mm, 300 Å, 5 μm). The pure fractions were combined, concentrated to remove most of the organic phase and then lyophilized. Subsequent TFA / HCI salt exchange was carried out by Andrushchenko, et al. , (J. Pept. Sci., 2006, 13, 37-43), performed by triple lyophilization from 2 mM HCI.

（式中、ＬＧは、脱離基である）
Ｘは、ＡＴＺ_１０Ｚ_１１Ｚ_１２ＡＡＮＦＬＨＳＳＮＮＦＧＺ_２５Ｚ_２６ＬＰＺ_２９ＴＮＶＧＺ_３４又はＶＬＧＲＬＳＱＥＬＨＲＬＱＴＹＰＲＴＮＴＧＳ
^＊アミノ酸が保護される
スキーム１：チオエーテル環化アミリノミメティックペプチドの合成：アミリン／プラムリンチド／ダバリンチド類似体

(In the formula, LG is a leaving group)
X is ATZ ₁₀ Z ₁₁ Z ₁₂ AANFLHSSNNFGZ ₂₅ Z ₂₆ LPZ ₂₉ TNVGZ ₃₄ or VLGRLSQELHRLQTYPRTNTGS
^* Amino acid protection Scheme 1: Synthesis of thioether cyclized amilinomimetic peptides: amyrin / pramlintide / davalintide analogs

Example 2: Synthesis of lipid-added amilinomimetic peptide (Scheme 2)
Step A: Procedure for introducing derivatized lysine residues into a peptide sequence constructed on a standard Linkamide resin Prepared at a time prior to the desired point of derivatization and described in Example 1A. The resin-bound C-terminal amide peptide prepared as described above is either Dde-Lys (FMOC) -OH or ivDde-Lys (FMOC) -OH, followed by FMOC-OEG-OH (one or two units in tandem). (The ones bound to) are sequentially linked, and then optionally using the DIC / Oxyma coupling method as described in Example 1A under microwave conditions (manually or on a Liberty Blue Peptide Synthesizer). FMOC-Glu-OtBu was connected in. After FMOC deprotection, the resin is microwaved in a solution of lipophilic acid [eg, palmitic acid] (5-10 eq), DIC (5-10 eq), and HOBT or Oxyma (5-10 eq). Under the conditions, it was treated at 90 ° C. for 10 minutes. The reaction was then drained and the resin was washed with DMF. Scheme 2 shows the introduction of lipidated lysine at position Z _25. Those skilled in the art, a similar approach will recognize that results in lipidation lysine _{Z 26, Z 29,} or _{Z 34.}

^＊アミノ酸が保護される
スキーム２：誘導体化されたリジン残基をアミリノミメティックペプチドに導入する手順

^* Amino acid protection Scheme 2: Procedure for introducing derivatized lysine residues into amilinomimetic peptides

Step B: Procedure for deprotecting Dde-protected or ivDde-protected ricinyl peptide Derivatized ricinyl peptide resin is subjected to DMF (6 mL / 0.1 mmol) under microwave conditions at 90 ° C. for 3.5 minutes. Resin) treated with a solution of 3% hydrazine. The reaction was drained and this procedure was repeated two more times. The reaction was drained and the resin was thoroughly washed with DMF and then with DCM.

Step C: Procedure for directly incorporating the FMOC-Lys (Pal-Glu-OtBu) -OH residue If the palmitoylated-γ-Glu-lysinyl residue is incorporated into the sequence, FMOC-Lys (Pal-Glu-OtBu-). OH (available from Peptides International or Active Peptide) can be used directly in the procedure described in Example 1A.

Example 3: Synthesis of BrAc-dPEGx-derivatized amilinomimetic peptide (Scheme 3)
The _{thioether cyclized peptide obtained from Example 1E, wherein one of Z 25} , Z ₂₆ , Z ₂₉ , or Z ₃₄ is K (NH ₂ ) (7 μmol) and BrAc- in DMA (0.3 mL). The solution with dPEGx-OTFP (3 eq) was treated with DIEA (7 eq) and the resulting solution was stirred with rt. As described in Example 1E, when the reaction (about 1 hour) was complete, the mixture was acidified with TFA, diluted with water (0.1% TFA) and purified by reverse phase chromatography. Scheme 3 depicts the introduction of lipidated lysine at position Z _25. Those skilled in the art, a similar approach will recognize that providing BrAc-dPEGx- derivatized lysine _{Z 26, Z 29,} or _{Z 34.}

^＊アミノ酸が保護される
ＬＧは脱離基である
スキーム３：ＢｒＡｃ−ｄＰＥＧｘ−誘導体化アミリノミメティックペプチドの合成手順

^* Amino acid protected LG is a leaving group Scheme 3: Procedure for synthesizing BrAc-dPEGx-derivatized amilinomimetic peptide

Example 4: Synthesis of thioether cyclized amilinomimetic peptide-mAb conjugates Expression and purification of mAbs Fully human monoclonal antibodies (mAbs) are recombinantly expressed in mammalian expression hosts and are standard known in the art. It can be purified from the cell culture supernatant using various methods. For example, the cDNA sequences encoding the light chain (LC) and heavy chain (HC) of mAbs, each containing the appropriate signal peptide that allows secretion, are separated using standard molecular biological methods. It can be cloned into a mammalian expression vector or a single expression vector. The expression vector used can be a commercially available one such as pEE12.4, pcDNA ™ 3.1 (+), or pIRESpuro3, or any custom expression vector with similar functionality. In such vectors, transcription of the heavy and light chains of mAb is driven by one of the known effective promoters, such as the hCMV-MIE promoter, respectively. Transfection grade plasmid DNA is used for separate LC and HC expression constructs, or for a single construct that expresses both LC and HC, using standard methods such as the QIAGEN plasmid Midi Kit. Prepared.

Purified plasmid DNA is prepared for transfection with a lipid-based transfection reagent (following the manufacturer's instructions), such as Freestyle ™ Max Transfection Reagent, followed by CHO-S or HEK 293-F, etc. Transfected into a standard mammalian expression host cell line. If mAb LC and HC are encoded by separate expression constructs, the two constructs are transfected simultaneously. Before and after transfection, mammalian cells are cultured for maintenance or for mAb expression according to standard cell culture methods, whereby cell density is in the range for maintaining the culture medium used. Other cell culture conditions that are present and adhered to are determined by the particular mammalian host cell line utilized. These parameters are typically described in the vendor from which the cell line was obtained, or in the scientific literature. For example, CHO-S cells are maintained suspended in CHO Freestyle ™ medium and shaken at 125 RPM in a humidified incubator set at _{37 ° C. and 8% CO 2, with cell concentrations of 1.5-2.} .0 × 10 ^{Divide when 6} cells / mL.

Cell culture supernatants from transiently transfected mammalian cells expressing mAbs are harvested days after transfection, clarified by centrifugation and filtered. The duration of expression of CHO-S cells is typically 4 days, but can be regulated and can vary for different mammalian host cell lines. Large-scale transfections (> 10 liters) are concentrated 10-fold using a concentrator such as Centramate. Using a protein A affinity column, such as HiTrap MabSelectSure, using standard methods of binding mAbs to protein A resin, washing the resin, and eluting the protein using low pH buffer, Purify the mAb from the clarified supernatant. Immediately neutralize the protein fraction by elution into a tube containing pH 7 buffer, pool and filter the peak fraction, phosphate buffered saline (PBS), pH 7.2. In the evening, dialyze at 4 ° C. After dialysis, mAbs are filtered again (0.2 μ filter) and protein concentration is determined by absorbance at 280 nm. Quality of purified mAb protein is assessed by SDS-polyacrylamide gel electrophoresis (PAGE) and analytical size exclusion HPLC, and endotoxin levels are measured using the Kabuto Crab Blood Cell Extraction Component (LAL) assay. Purified mAbs are stored at 4 ° C.

Expression and Purification of MSCB97 from Transfectally Transfected CHO Cells ExpiCHO-by transiently transfecting MSCB97 cells with purified plasmid DNA of the MSCB97 expression construct according to the manufacturer's recommendations. It was expressed in S ™ cells (ThermoFisher Scientific, plasmid, MA; Catalog No. A29127). Briefly, ExpiCHO-S ™ cells are suspended in ExpiCHO ™ expression medium (Thermo Fisher Scientific, Catalog No. A29100) in a shaking incubator set at _{37 ° C., 8% CO 2 and 125 RPM.} Keeped inside. On the day of transfection, cells are passaged so that they can be diluted to a number of ^{6.0 x 10 6 cells per mL to maintain cell viability> 98%.} Transfections were performed using the ExpiFectamine ™ CHO Transfection Kit (Thermo Fisher Scientific Catalog No. A29131). For every 1 mL of diluted cells to be transfected, 1 microgram of plasmid DNA is used and diluted in OptiPRO ™ SFM complexing medium. ExpiFectamine ™ CHO Reagent is also diluted to OptiPRO ™ using a 1: 3 ratio (v / v, DNA: Reagent). Diluted DNA and transfection reagent were combined for 1 minute to form a DNA / lipid conjugate and then added to cells. After overnight incubation, ExpiCHO ™ feed and ExpiFectamine ™ CHO enhancer were added to the cells. Before collecting the culture supernatant, the cells were cultured at 32 ° C. with shaking for 5 days.

Culture supernatants from transiently transfected ExpiCHO-S ™ cells are clarified through centrifugation (30 minutes, 6000 rpm) and then collected by filtration (0.2 μ PES membrane, Corning). bottom. First, a large-scale transfection (5-20 liters) was concentrated 10-fold using the Pall Centramate Tangential Flow Filtration system. 10 x DPBS (Phosphate Buffered Saline), pH 7.2 was equilibrated using the AKTA FPLC chromatography system at a relative concentration of about 20 mg protein per mL of resin (DPBS, pH 7.2). ) HiTrap MabSelect Pure Protein A column (GE Healthcare; Little Chromat, United Kingdom) was added to the supernatant to a final concentration of 1 x prior to loading. After filling, the column was washed with 10 column volumes of DPBS, pH 7.2. The protein was eluted with a volume of 0.1 M Na-acetic acid (pH 3.5) in 10 columns. The protein fraction was immediately neutralized by eluting into a tube containing 2.0 M Tris (pH 7), which is 20% of the volume of the eluted fraction. The peak fractions were pooled and the pH was adjusted to about 5.5 with additional Tris as needed. The purified protein was filtered (0.2 μ) and the concentration was determined by absorbance at 280 nm on a BioTek Synergy HT ™ spectrophotometer. The quality of the purified protein was evaluated by SDS-PAGE and analytical size exclusion HPLC (Dionex HPLC system). Endotoxin levels were measured using a turbidity titration LAL assay (Pyrotell®-T, Assays of Cape Cod).

Conjugation of MSCB97 to Amilinomic Peptides To the purified mAb (1.2 mL, 19 mg / mL) was added 4 equivalents of TCEP followed by EDTA (100 mM; 12 μL). After 2 hours at room temperature, LCMS analysis showed that the disulfide adduct at position C102 was completely reduced. The reduced mAbs were treated with a Zebra desalting spin column (7 × 10 mL, 7K MWCO, Trisacetic acid 100 mM, pH 5.6 pre-equilibrium) to remove free cysteine / GSH. To this reduced mAB is added a solution of the bromoacetylated amilinomimetic peptide obtained in Example 3 in Milli Q grade water (7 eq vs. mAb, 30-35 mg / mL) and then EDTA. (100 mM; 13.5 μL) was added. 1 M of Tris acetate buffer (pH of about 9) was added dropwise to adjust the pH of the reactants to 7.9. The reaction was allowed to proceed overnight at room temperature with gentle stirring. The reaction is then _{diluted with saturated (NH 4} ) ₂ SO ₄ (10% v / v) and the crude conjugate is purified by hydrophobic interaction chromatography (TOSOH TSKgel Phoenix HIC) with a linear gradient (40-100). % B / A, Solvent A: 5% i-PrOH, 1M (NH ₄ ) ₂ SO ₄ , 100 mM phosphate buffer, pH 6.0; Solvent B: 20% i-PrOH, 100 mM phosphate buffer, It eluted at pH 6.0). Final purification was performed by protein A adsorption (PBS buffer) and elution (NaOAc, pH 3.5). The pH of the product was adjusted to 6 with 2.5 M Tris (pH 7.7; 10 v%) and dialyzed against PBS to give the final sample.

Example 5: Peptide and Peptide-Bioconjugate Analysis and characterization Method A Purified peptide is composed of HP1100 series HPLC using a WATER Atlantics T3 C18 (4.6 x 250 mm, 300 Å, 5 μm) column. Hewlett-Packard Series 1100 MSD system 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) can be applied at a flow rate of 1 mL / min. Used at a column temperature of 35 ° C. [Method A1: 15-60% B over 22 minutes; Method A2: 40-90% B over 22 minutes]. Mass spectrometry of each peptide was provided by electron spray analysis (ES-API, positive ion scan). In all cases, multiple charged species were observed, with 1/3 [M + 3] + and 1/4 [M + 4] ++ ions being the most prominently observed ions. All products resulted in the expected multiple charged ions, to the extent acceptable. The results of mass spectrum analysis of the peptides and the observed LC retention time (RT) are shown in Table 1.

Method B: Purified peptides were analyzed by HPLC on a Shimadzu 10AVP system using a YMC-PACK-ODS-A (4.6 x 250 mm, 200 Å, 5 μm) column. A linear solvent gradient (20-80% B over 30 minutes) was used at a flow rate of 1 mL / 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 EE, cation scan). In all cases, multiple charged species were observed, with 1/3 [M + 3] + and 1/4 [M + 4] + ions being the most prominently observed ions. All products resulted in the expected multiple charged ions, to the extent acceptable. The results of mass spectrum analysis of the peptides and the observed LC retention time (RT) are shown in Table 1.

Method C: Hewlett Packard Series 1100 constructed using a HP 1100 series HPLC using a purified amilinomimetic peptide-mAb conjugate using a MAbPac HIC-10 (4.6 x 1000 mm, 1000 Å, 5 μm) column. Analyzed by hydrophobic interaction chromatography (HIC) on the MSD system. A linear solvent gradient (0-100% B over 30 minutes) was used at a flow rate of 0.5 mL / min (buffer A: 5% i-PrOH, 1.5M (NH _{4 ) 2} SO ₄ ). , 100 mM phosphate buffer, pH 6.0; buffer solution B 20% i-PrOH, 100 mM phosphate buffer, pH 6.0). Mass spectrometry of intact was obtained on a Waters Xevo G2-XS QToF spectrometer (TOF MS ES, cation scan). Table 1 shows the results of analytical characterization of the amilinomimetic peptide-mAb conjugate and conjugation method.

Example 6: Human calcitonin / RAMP3 receptor cAMP assay (AMY3R assay)
The method used to test the efficacy of adenylomymetic analogs in vitro is via modification of the human calcitonin G-protein linking receptor with its receptor activation modified protein 3 (CTR / RAMP3). It was a cell line assay designed to measure the cAMP produced by adenylate cyclase through interaction. Production of cAMP in human AMY3R-transfected 1321N1 astrocytes (DiscoverX) was dose-dependently induced by amilinomimetic analogs and controls and measured by the LANCE FRET-based competitive cAMP immunoassay (PerkinElmer).

Cells were cultured in DMEM, 10% FBS, 2.5 μg / mL puromycin and 800 μg / μL G418. For the assay, cells were harvested by removing the medium, washing with PBS and Basen solution and lifting the cells (Life Technologies). The cells were centrifuged at 450 xg for 5 minutes and the supernatant was aspirated. Cells 0. 1X HBSS (Life Technologies), 5 mM HEPES (Life Technologies), 0.1% BSA (PerkinElmer), 1.0 mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma). Resuspended at 5 × 10 ⁶ cells / mL, 10 μL of suspended cells were added to each well of a 384-well white optiplate (PerkinElmer) to a final cell density of 5000 cells / well. Amyrin analogs and control dilutions were prepared at 1X WBSS, 5 mM HEPES, 0.1% BSA and each sample was added to the designated wells at 10 μL / well. The plates were incubated at room temperature for 30 minutes with shaking. Each assay plate was then added with a 20 μL / well LANCE cAMP detection reagent mixture (PerkinElmer) and incubated at room temperature for 2 to 24 hours with shaking. Plates were read with a PerkinElmer Envision plate reader using a protocol based on the manufacturer's recommendations contained in the LANCE Ultra cAMP Kit. All samples were measured in quadruples. EC50, LogEC50, HillSlope (nH), top and bottom, etc. by analyzing the data using Crucible in-house data analysis software designed by Edward Shaw and plotting the raw cAMP value vs. log compound concentration. I got the parameters of. The data were fitted with a 4-P model using the nonlinear weighted least squares method within the R environment implemented by Janssen R &D's nonclinical statistics and computation department (Open Source http: // crane. us.r-project.org /).

The efficacy of the amilinomimetic analogs of the invention against pramlintide used as a control in the same assay is shown in Table 2 below.

Example 7: Human calcitonin / RAMP1 complex cAMP assay (AMY1R assay)
The efficacy and selectivity of amilinomimetic analogs in vitro was evaluated using a cell line assay designed to measure regulated cAMP production of human CTR or CTR / RAMP1 complex (AMY1R). .. Production of cAMP in COS7 cells transiently transfected into human CTR or AMY1R was induced dose-dependently by amilinomimetic analogs and controls, and the HTRF cAMP kit (CisBio cAMP Dynamic kit, Catalog No .: 62AM4PEC) was used for measurement.

A plasmid encoding the HA-tagged human calcitonin receptor was tagged with its N-terminus immediately after the signal peptide having the signal peptide 3xHA using EcoRV and XhoI (ENST000000426151.5). ) Was subcloned into pcDNA3.1 (+). A plasmid encoding a flag-tagged human RAMP1 was cloned using EcoRV and XhoI into a human RAMP1 ORF (ENST000000254661) tagged at the N-terminus immediately after the signal peptide having the flag tag (DYKDDDDK (SEQ ID NO: 66)). .4) was generated by subcloning into pcDNA3.1 (+).

COS-7 cells are cultured in DMEM (Thermo Fisher Scientific # 11965092) containing 10% FBS (Hyclone # SH30070.03) and 1% penicillin-streptomycin (Thermo Fisher Scientific # 15140122), and cultured in DMEM (Thermo Fisher Scientific # 11965092). E2312) was used for transfection in 384 wells of poly-D-lysine coated white plate (Corning # 356663). For each condition, the mixing ratio of DNA (μg): Fugene HD (μL) was 1: 3, and 10,000 cells / well were added at 40 μL over 10 μL of the DNA: Fugene mixture. The plates were _{incubated in a CO 2} incubator at 37 ° C. for 48 hours. The CTR: RAMP1 cDNA transfection ratio (2: 9) was optimized to suit the formation of the amyrin-1 receptor (AMY1R). The amount of transfected CTR cDNA was optimized so that the expression of calcitonin and amylin receptors did not differ significantly (assessed by enzyme binding immunoadsorption assay for HA tags).

On the day of the assay, the culture medium was replaced with an assay buffer at pH 7.4 containing HBSS with calcium and magnesium, 20 mM HEPES and BSA without 0.1% fatty acids, at 37 ° C. The cells were starved for 1 hour. The assay buffer was then replaced with a new assay buffer containing 500 μM IBMX and the compound was added to the assay buffer (without IBMX). The plates were incubated at room temperature for 30 minutes with shaking. cAMP was detected according to the manufacturer's protocol (CisBio cAMP Dynamic kit, catalog number: 62AM4PEC). Fluorescence was read with a PHERAstar plate reader using excitation at 337 nm and radiation at 260 and 665 nm. The data were normalized with the maximum response of pramlintide. The determination of Emax and EC50 is based on Graphpad Prism 7.0. The agonist response curve analyzed in the curve matching program was performed using the 4-parameter logistic dose response equation in. The data presented represent three independent experiments performed in quadruples for each compound. The data is expressed as an average value. The AMY1R potency of a compound relative to the AMY1R potency of pramlintide (SEQ ID NO: 2) is expressed as a fold change. Their potency in AMY1R over the potency of the compounds in CTR is also presented as a magnification difference (Table 3).

For cell surface receptor expression determination, cells from the same transfection used in the cAMP assay were seeded on 96-well plates, fixed with 4% paraformaldehyde and blocked with PBS + 1% FBS. Rat anti-HA peroxidase (clone 3F10, Roche Bioscience # 12013819001) was applied at 0.5 mg / L for 30 minutes. After washing with blocking buffer and PBS, chemiluminescence was detected using a SuperSignal substrate (Pierce, Rockford, IL, USA) and a PHERAstar plate reader.

Example 8: In vivo Efficacy Test Gastric Excretion: Acetaminophen (AAP) Absorption in Lean C57BI / 6N Mice Male Lean C57BL / 6 mice (6-8 weeks old) were obtained from the Taconic Laboratory. One mouse was housed in each cage with an AlphaDri bedding in a temperature controlled room with a 12 hour light / dark cycle. Mice were allowed to freely ingest water and maintained on a normal diet (Lab Diet Cat: 5K75). Animals were acclimatized to the facility at least one week before the start of the experiment.

The day before dosing, mice were divided into a cohort of 10 animals based on their individual body weight. The next day from 5:00 pm to 6:00 pm, no food was given to the animals, via subcutaneous administration, at a dose of 30 nmol / kg (3 nmol / mL), either vehicle (PBS, pH 7.4) or the test compound. Treated with. After 18 hours, an acetaminophen (AAP) suspension mixture [AAP (10 mg / mL), HPMC (5 mg / mL), gum acacia (50 mg / mL)] was administered to the animals by oral tube feeding (10 mL). / Kg). Whole blood samples (tail, approximately 25 μL) were collected on DMPK-C dried blood spot cards at 5 minutes, 10 minutes, 15 minutes, 30 minutes, and 60 minutes. The blood dot card was completely dried and the desiccant was placed in a separate bag containing the desiccant until LC / MS analysis was performed using standard techniques. A statistical analysis was performed using a one-way ANOVA using Dunnett's post-test in Prism. All data are presented as an average.

Food intake in lean C57BL6N mice that have been deprived of food: Acutely administered male C57BL / 6 mice (6-8 weeks old) were obtained from the Taconic Laboratory. One mouse was housed in each cage with an AlphaDri bedding in a temperature controlled room with a 12 hour light / dark cycle. Mice were allowed to freely ingest water and maintained on a normal diet (Lab Diet Cat: 5K75). Animals were acclimated to BioDAQ cages (Research Diets, Inc., New Brunswick, NJ) more than 72 hours prior to the start of the experiment.

Once acclimatized to the BioDAC cage, mice were divided into a cohort of 10 animals based on individual body weight and individual food intake over the previous 24 hours. From 4:00 to 5:00 pm, weigh the animals and via subcutaneous administration at a dose of 30 nmol / kg (3 nmol / mL), either in vehicle (PBS, pH 7.4) or in test compound. Treated. Changes in food weight in each cage were then recorded by the BioDAQ automatic monitoring system overnight (16-18 hours) without food and for 48 consecutive hours thereafter. Debris was vacuumed daily from the area around the hopper and cage. Food was replenished as needed. The ratio of average cumulative food intake to vehicle was calculated over 12-48 hours after administration. The results are reported in Table 4. A statistical analysis was performed using a bidirectional ANOVA using Dunnett's post-test in Prism. All data are presented as an average.

ＮＤ＝決定されず
^＊ｐ＜０．０５ ^＊＊ｐ＜０．０１ ^＊＊＊ｐ＜０．００１ ^§ｐ＜０．０００１

ND = not decided
^* P <0.05 ^** p <0.01 ^*** p <0.001 ^§ p <0.0001

Example 9: PK study in male C57BL6N mice Male C57BL / 6N mice (6-8 weeks old, Taconic) were placed in a temperature controlled room with a 12 hour light / dark cycle using AlphaDri bedding. They were housed alone and were allowed to freely eat the LabDiet5K75 rodent diet and drink drinking water. Mice were grouped based on body weight after eating (N = 3). The compound was formulated in PBS (1 nmol / mL) and administered subcutaneously at a dose of 10 nmol / kg. Protease inhibitor cocktails (Roche complete protease inhibitor and Millipore DPPIV inhibitor, 2 It was collected in an EDTA-coated Sarsteaded Microvette® tube containing .5 μL) and placed on ice. The final blood draw (7 days later) was a terminal blood draw performed at a target volume of 500 μL (25 μL protease inhibitor cocktail). The sample was then centrifuged at 4 ° C. for 10 minutes (rotational speed 10,000 rpm). Plasma was then transferred to a 96-well plate (approximately 25 μL plasma per well) and stored at −80 ° C. until bioanalysis was performed.

Plasma samples were analyzed using an LC-MS / MS assay for quantifying surrogate peptides. In this assay, immunoaffinity capture was used to extract the analyte from plasma with an anti-human IgG Fc antibody, followed by protease digestion (trypsin or pepsin) and reverse phase LC-MS / MS analysis. Multiple reaction monitoring (MRM) MS analysis was performed on an API 5000 triple quadrupole mass spectrometer operated in positive electron spray mode. Peptides from the N-terminal region of the amilinomimetic sequence were monitored as slogates for quantification of active conjugates, while peptides located on the Fc region of mAbs were monitored as slogates at total levels of mAbs. Standard curves and quality control samples were prepared by spiked reference standards for amilinomimetic conjugates in plasma and processed simultaneously using the same procedure as test samples. The data are shown in Table 5.

^＊除去フェーズ中に採取された、制限された時点でのサンプリングから推定

^* Estimated from sampling at limited time taken during the removal phase

Those skilled in the art will appreciate that modifications can be made to the aforementioned embodiments without departing from the broader conception of the invention. Accordingly, it is understood that the present invention is not limited to the particular embodiments disclosed and is intended to include modifications within the spirit and scope of the invention as defined by this description.

All references cited are incorporated herein by reference.

An exemplary amilinomimetic sequence of the invention or a conjugate thereof includes:

SEQ ID NO: 1
Name: Amyrin (1-37)
Structural formula:

SEQ ID NO: 2
Name: Pramlintide (1-37)
Structural formula:

SEQ ID NO: 3
Name: Davalintide (1-32)
Structural formula:

SEQ ID NO: 4
Name: [cyclo _{- (N3-COCH 2 -C7)} ] - pramlintide 3-37
Structural formula:

SEQ ID NO: 5
Name: [cyclo _{- (N3-COCH 2 -hC7)} , K (Ac) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 6
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5] - pramlintide 3-37
Structural formula:

SEQ ID NO: 7
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, K (Ac) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 8
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, K (Alloc) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 9
Name: [cyclo _{- (N3-COCH 2 -C7)} , β-A5, K (Ac) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 10
Name: [cyclo _{- (N3-COCH 2 -C7)} , β-A5, K (Alloc) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 11
Name: [cyclo _{- (N3-COCH 2 -C7)} , Abu5, K (Ac) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 12
Name: [cyclo _{- (N3-COCH 2 -hC7)} , Abu5, K (Ac) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 13
Name: [cyclo _{- (N3-COCH 2 -hC7)} , (S) -β-Aib5-, K (Ac) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 14
Aka: [cyclo _{- (N3-COCH 2 -hC7)} , (R) -β-Aib5, K (AcA) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 15
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-hA5, K (Ac) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 16
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-hP5, K (Ac) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 17
Name: [cyclo _{- (N3-COCH 2 -hC7)} , { pyrrolidinyl - (3S) - carboxylic} 5, K (Ac) 26 ] - pramlintide 3-37
Structural formula:

SEQ ID NO: 18
Name: [cyclo _{- (N3-COCH 2 -hC7)} , {(1R, 2R) -ACPC} 5, K (Ac) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 19
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-hT4, K (Ac) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 20
Name: [cyclo _{- (N3-COCH 2 -C7)} , β-hT4, K (Ac) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 21
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-hT6, K (Ac) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 22
Name: [cyclo _{- (N3-COCH 2 -C7)} , β-hT6, K (Ac) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 23
Name: [cyclo _{- (S2-COCH 2 -hC7)} , β-A5, K (Ac) 26] - pramlintide 2-37
Structural formula:

SEQ ID NO: 24
Name: [cyclo _{- (S2-COCH 2 -C7)} , β-A5, K (Ac) 26] - pramlintide 2-37
Structural formula:

SEQ ID NO: 25
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, K (Ac) 25] - pramlintide 3-37
Structural formula:

SEQ ID NO: 26
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, K (Ac) 29] - pramlintide 3-37
Structural formula:

SEQ ID NO: 27
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, K (Ac) 34] - pramlintide 3-37
Structural formula:

SEQ ID NO: 28
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5] - Dabarinchido 3-32
Structural formula:

SEQ ID NO: 29
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, K (OEG 2 -Pal) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 30
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, K (OEG 2 -γ-Glu-Pal) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 31
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, α-MeL12, K (dPED12-AcBr) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 32
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, hR11, K (dPEG12-AcBr) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 33
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, E10, K (dPEG12-AcBr) 26] - pramlintide 3-37
Structural formula:

SEQ ID NO: 34
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, α-Mel12, K (dPEG12-AcBr) 25] - pramlintide 3-37
Structural formula:

SEQ ID NO: 35
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, hR11, K (dPEG12-AcBr) 25] - pramlintide 3-37
Structural formula:

SEQ ID NO: 36
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, E10, K (dPEG12-AcBr) 25] - pramlintide 3-37
Structural formula:

SEQ ID NO: 37
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, α-Mel12, K (dPEG12) 26] - pramlintide 3-37 mAb homodimers conjugated structure:

SEQ ID NO: 38
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, hR11, K (dPEG12) 26] - pramlintide 3-37 mAb homodimers conjugated structure:

SEQ ID NO: 39
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, E10, K (dPEG1226] - pramlintide 3-37 mAb homodimer conjugate structural formula:

SEQ ID NO: 40
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, α-Mel12, K (dPEG12) 25] - pramlintide 3-37 mAb homodimers conjugated structure:

SEQ ID NO: 41
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, hR11, K (dPEG25] - pramlintide 3-37 mAb homodimers conjugated structure:

SEQ ID NO: 42
Name: [cyclo _{- (N3-COCH 2 -hC7)} , β-A5, E10, K (dPEG12) 25] - pramlintide 3-37 mAb homodimers conjugated structure:

SEQ ID NO: 43
Name: MSCB97 VH (heavy chain variable region)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSAISGSGGSTYYADSVKGRFTISPDNSKNTLYLQMNSLRAEDTAVYYCAKYDGCYGELDFWGQGTTVSS

SEQ ID NO: 44
Name: MSCB97 HC (heavy chain)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLAQMNSLRAEDTAVYYCAKYDGCYGELDFWGQGTLVTVSSASTKGPSVFSTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO: 45
Name: MSCB97 VL (light chain variable region)
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDDFAVYYCQQRSNWPLTFGQGTKVEIK

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
DANSRAT

SEQ ID NO: 52
Name: MSCB97 LCDR3
QQRSNWPLT

Claims (22)

Formula I (SEQ ID NO: 53):

(During the ceremony,
n is 1 or 2;
Z ₂ is a direct bond, serine, or glycine,
Z ₄ is T or

And
Z ₅ is A, β-alanine,

And
Z ₆ is T or

And
Z ₁₀ is Q or E,
Z ₁₁ is R or K, wherein the ε-amine of K is optionally substituted _{with -C (= NH) NH 2.}
Z ₁₂ is L or

And
Z ₁₆ is L or

And
Z ₂₅ is P or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in two compounds of the same formula I on the mAb. Exists and
Z ₂₆ is I or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in two compounds of the same formula I on the mAb. Exists and
X is ATZ ₁₀ Z ₁₁ Z ₁₂ ANFZ ₁₆ VHSSNNFGZ ₂₅ Z ₂₆ LPZ ₂₉ TNVGZ ₃₄ (SEQ ID NO: 54) or VLGRLSQELHRLQTYPRTNTGS (SEQ ID NO: 55).
Z ₂₉ is P or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in two compounds of the same formula I on the mAb. Exists and
Z ₃₄ is S or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in two compounds of the same formula I on the mAb. Exists. )
Compounds, and pharmaceutically acceptable salts thereof. Z ₂ is a direct bond,
Z ₅ is β-alanine,

And
Z ₆ is T,
The compound according to claim 1 and a pharmaceutically acceptable salt thereof. Z ₁₆ is L,
Z ₂₅ is P or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in two compounds of the same formula I on the mAb. Exists and
Z ₂₆ is I or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in two compounds of the same formula I on the mAb. Exists and
Z ₂₉ is P or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in two compounds of the same formula I on the mAb. Exists and
Z ₃₄ is S or K, and the ε-amine of K is optionally -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

The mAb was optionally substituted via another thioether bond to a second compound of formula I, resulting in two compounds of the same formula I on the mAb. Exists,
The compound according to claim 1 and a pharmaceutically acceptable salt thereof. Z ₂ is a direct bond,
Z ₅ is β-alanine,

And
Z ₆ is T,
The compound according to claim 3 and a pharmaceutically acceptable salt thereof. Z ₁₁ is R or K, and the ε-amine of K is substituted with _{-C (= NH) NH 2.}
Z ₂₅ is P or K, and the ε-amine of K is -C (= O) CH ₃ or

The mAb was substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb.
Z ₂₆ is I or K, and the ε-amine of K is -C (= O) CH ₃ , -C (O) CH ₂ CH = CH ₂ ,

The mAB was substituted via another thioether bond to a second compound of formula I, resulting in the presence of two identical formula I compounds on the mAb.
Z ₂₉ is P or K, and the ε-amine of K is substituted with _{-C (= O) CH 3.}
Z ₃₄ is S or K, and the ε-amine of K is substituted with _{-C (= O) CH 3.}
The compound according to claim 3 and a pharmaceutically acceptable salt thereof. Z ₂ is a direct bond,
Z ₅ is β-alanine,

And
Z ₆ is T,
The compound according to claim 5 and a pharmaceutically acceptable salt thereof. Z ₁₂

The compound according to claim 5 and a pharmaceutically acceptable salt thereof.

The compound according to claim 5 and a pharmaceutically acceptable salt thereof, which are selected from the group consisting of (SEQ ID NOs: 4-42, respectively, from top to bottom). The compound according to claim 1, wherein the compound is selected from the group consisting of SEQ ID NOs: 4-42 or is a pharmaceutically acceptable salt thereof. The compound according to any one of claims 1 to 9, and
A pharmaceutical composition comprising a pharmaceutically acceptable carrier. A method of treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, dyslipidemia, hyperglycemia. Disease, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinism (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and uncontrolled cholesterol and / or lipid levels A group consisting of other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with, osteoporosis, inflammation, non-alcoholic steatohepatitis (NAFLD), non-alcoholic steatohepatitis (NASH), renal disease, and eczema. Selected from
The method comprising administering an effective amount of the pharmaceutical composition of claim 10 to said subject in need thereof. The method of reducing food intake to a subject in need thereof, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition according to claim 10. A method of regulating amyrin receptor activity in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition according to claim 10. Method. 13. The method of claim 13, wherein the amyrin receptor comprises AMY1R and / or AMY2R, and / or AMY3R. The method of claim 14, wherein the amyrin receptor is AMY1R. 14. The method of claim 14, wherein the amyrin receptor is AMY3R. The method according to any one of claims 11 to 14, wherein the pharmaceutical composition is administered by injection. The method according to any one of claims 11 to 16, wherein the pharmaceutical composition is administered in combination with at least one antidiabetes drug. The method of claim 18, wherein the anti-diabetic agent is a glucagon-like peptide-1 receptor regulator. 18. The method of claim 18, wherein the pharmaceutical composition is administered in combination with liraglutide. The kit comprising the conjugate according to any one of claims 1 to 8, preferably further comprising liraglutide and an injectable device. The method for producing a pharmaceutical composition according to claim 10.