WO2024213022A1 - Incretin analogues and preparation method therefor, and application - Google Patents

Abstract

Provided are incretin analogues and a preparation method therefor, and an application. The incretin analogues include polypeptides and substituents. Also provided are a pharmaceutical composition including the incretin analogues, a preparation method for the incretin analogues, and a use of the incretin analogues in treating diseases. The provided incretin analogues have GIPR and GLP-1R agonistic activity, are dual-receptor agonists, and have significant effects in terms of treating diabetes, decreasing blood sugar and reducing body weight.

Description

Incretin analogs and preparation methods and applications thereof Technical Field

The present disclosure relates to the field of therapeutic polypeptides, and in particular to incretin analogs and preparation methods and uses thereof.

Background Art

Glucagon-like peptide-1 (GLP-1) is a hormone secreted by the L cells of the small intestine and belongs to the incretin hormone. GLP-1 acts on pancreatic β cells to promote the synthesis and secretion of insulin. It can also stimulate the proliferation and differentiation of pancreatic β cells and inhibit the apoptosis of pancreatic β cells, thus protecting the function of the pancreas. It also has the effects of suppressing appetite and delaying gastric emptying, achieving the effect of lowering blood sugar through these effects.

Glucose-dependent insulinotropic polypeptide (GIP) is the first incretin hormone discovered. It is composed of 42 amino acids and has 68% homology with GLP-1. It is mainly produced and secreted by K cells located in the proximal small intestine and has multiple physiological functions. In terms of blood sugar regulation, GIP activates its glucose-dependent insulinotropic hormone receptor (GIPR) in pancreatic β cells to enhance insulin secretion in a glucose-dependent manner, while increasing glucagon secretion during hypoglycemia and inhibiting glucagon secretion during hyperglycemia; the interaction with GIPR on the surface of fat cells can promote fatty acid synthesis, thereby achieving the effect of regulating blood sugar and fat metabolism.

GLP-1 analogs have dose-dependent side effects such as nausea and vomiting in clinical practice, which reduces patients' compliance with medication. The incretin effect of GIP is weakened in patients with type 2 diabetes (T2DM), which may be related to the downregulation of receptors. Studies have shown that improving blood sugar control through anti-diabetic treatment can restore the pancreatic function of GIP in patients with T2DM, indicating that co-stimulation of GLP-1R and GIPR can play a synergistic hypoglycemic effect.

Currently, there is still a clinical need in this field to develop GIPR/glucagon-like peptide-1 receptor (GLP-1R) dual receptor agonists with better efficacy and higher safety.

SUMMARY OF THE INVENTION

The present disclosure provides an incretin analog, and also provides a pharmaceutical composition comprising the incretin analog, a preparation method of the incretin analog and an application thereof. The incretin analog disclosed in the present disclosure is a single molecule dual receptor agonist, which can simultaneously stimulate GLP-1R and GIPR.

In one aspect, the present disclosure provides a polypeptide or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS (SEQ ID NO: 17), wherein:

_X2 is selected from Aib or Ala,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr, Ile, Ala or Aib,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence; and the polypeptide optionally has a C-terminal amidation modification.

In one aspect, the present disclosure provides an incretin analog or a pharmaceutically acceptable salt thereof, comprising a polypeptide and a substituent, wherein the polypeptide comprises an amino acid sequence as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS (SEQ ID NO: 17), wherein:

_X2 is selected from Aib or Ala,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr, Ile, Ala or Aib,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence; and

The substituent is linked to the polypeptide via Cys in the polypeptide; the polypeptide optionally has a C-terminal amidation modification.

In some embodiments, the C-terminus of the polypeptide has a -COOH group, i.e., the polypeptide has a C-terminus with a free carboxyl group. In some embodiments, the C-terminus of the polypeptide has a -CONH ₂ group, i.e., the polypeptide has a C-terminus amidation modification, i.e., the polypeptide has an amidation-modified C-terminus.

In another aspect, the present disclosure provides a pharmaceutical composition comprising the incretin analog of the present disclosure or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

On the other hand, the present disclosure provides a method for preparing the incretin analog or a pharmaceutically acceptable salt thereof, comprising preparing the polypeptide of the present disclosure by chemical synthesis and/or recombinant expression, preparing the modifier of the present disclosure by chemical synthesis, and linking the modifier to the polypeptide via Cys in the polypeptide.

In another aspect, the present disclosure provides use of the incretin analog or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the present disclosure in preparing a medicament.

In another aspect, the present disclosure provides a method for treating a disease in a subject in need thereof, comprising administering to the subject an incretin analog or a pharmaceutically acceptable salt thereof of the present disclosure, or a pharmaceutical composition of the present disclosure.

In another aspect, the present disclosure provides a method for treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an incretin analog or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 and 2 are the weight change rate curves of mice after drug administration;

FIG3 is a blood glucose level curve of mice in a type 2 diabetes mouse model after administration;

Figure 4 shows the body weight change rate curve of DIO mice after drug administration.

DETAILED DESCRIPTION OF THE INVENTION

Peptides

In one aspect, the present disclosure provides a polypeptide or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS (SEQ ID NO: 17), wherein:

_X2 is selected from Aib or Ala,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr, Ile, Ala or Aib,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence; and the polypeptide optionally has a C-terminal amidation modification.

In some embodiments, the polypeptide or a pharmaceutically acceptable salt thereof comprises an amino acid sequence as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS (SEQ ID NO: 16), wherein:

_X2 is selected from Aib or Ala,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr or Ile,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence; and the polypeptide optionally has a C-terminal amidation modification.

In some embodiments, said _X2 is Aib.

In some embodiments, _X12 is Tyr, _X13 is selected from Cys or Tyr, _X16 is selected from Cys or Lys, _X20 is selected from Cys or Lys, _X21 is selected from Cys or Leu, _X24 is selected from Cys or Gln, and _X27 is selected from Cys or Leu. In some embodiments, _X2 is Aib, _X12 is Tyr, _X13 is selected from Cys or Tyr, _X16 is selected from Cys or Lys, _X20 is selected from Cys or Lys, _X21 is selected from Cys or Leu, _X24 is selected from Cys or Gln, and _X27 is selected from Cys or Leu.

In some embodiments, _X20 is Cys.

In some embodiments, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val. In some embodiments, _X2 is Aib, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val.

In some embodiments, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val. In some embodiments, _X2 is Aib, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val.

In some embodiments, _X12 is selected from Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu. In some embodiments, _X2 is Aib, _X12 is selected from Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu.

In some embodiments, _X12 is Lys, _X13 is selected from Tyr, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu. In some embodiments, _X2 is Aib, _X12 is Lys, _X13 is selected from Tyr, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu.

In some embodiments, _X12 is Tyr, _X13 is selected from Tyr or Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu. In some embodiments, _X2 is Aib, _X12 is Tyr, _X13 is selected from Tyr or Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu.

In some embodiments, the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS (SEQ ID NO: 17), wherein:

_X2 is selected from Aib or Ala,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr, Ile, Ala or Aib,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence.

In some embodiments, the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS (SEQ ID NO: 16), wherein:

_X2 is selected from Aib or Ala,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr or Ile,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence.

In some embodiments, in the polypeptide or a pharmaceutically acceptable salt thereof, _X2 is Aib.

In some embodiments, the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr, Ile, Ala or Aib,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence.

In some embodiments, the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr or Ile,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence.

In some embodiments, the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib, _X12 is Tyr, _X13 is selected from Cys or Tyr, _X16 is selected from Cys or Lys, _X20 is selected from Cys or Lys, _X21 is selected from Cys or Leu, _X24 is selected from Cys or Gln, _X27 is selected from Cys or Leu, and there is only one Cys in the amino acid sequence.

In some embodiments, in the polypeptide or a pharmaceutically acceptable salt thereof, _X20 is Cys.

In some embodiments, the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gin, and _X27 is selected from Leu, Ile or Val.

In some embodiments, the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gin, and _X27 is selected from Leu, Ile or Val.

In some embodiments, the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib, _X12 is selected from Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is GIn, and _X27 is Leu.

In some embodiments, the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib, _X12 is Lys, _X13 is selected from Tyr, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is GIn, and _X27 is Leu.

In some embodiments, the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib, _X12 is Tyr, _X13 is selected from Tyr or Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is GIn, and _X27 is Leu.

In some embodiments, the polypeptide or a pharmaceutically acceptable salt thereof comprises any one of the following amino acid sequences:

(1)YAibEGTFTSDYSYCLEKQAAKLFVQWLLAGGPSSGAPPPS;

(2)YAibEGTFTSDYSYYLECQAAKLFVQWLLAGGPSSGAPPPS;

(3)YAibEGTFTSDYSYYLEKQAACLFVQWLLAGGPSSGAPPPS;

(4)YAibEGTFTSDYSYYLEKQAAKCFVQWLLAGGPSSGAPPPS;

(5)YAibEGTFTSDYSYYLEKQAAKLFVCWLLAGGPSSGAPPPS;

(6)YAibEGTFTSDYSYYLEKQAAKLFVQWLCAGGPSSGAPPPS;

(7)YAibEGTFTSDYSYILEKQAACLFVQWLLAGGPSSGAPPPS;

(8)YAibEGTFTSDYSYILEKQAACLFVQWLIAGGPSSGAPPPS;

(9)YAibEGTFTSDYSYILEKQAACLFVQWLVAGGPSSGAPPPS;

(10)YAibEGTFTSDYSSILEKQAACLFVQWLLAGGPSSGAPPPS;

(11)YAibEGTFTSDYSKILEKQAACLFVQWLLAGGPSSGAPPPS;

(12)YAibEGTFTSDYSIILEKQAACLFVQWLLAGGPSSGAPPPS;

(13)YAibEGTFTSDYSKYLEKQAACLFVQWLLAGGPSSGAPPPS;

(14)YAibEGTFTSDYSKALEKQAACLFVQWLLAGGPSSGAPPPS; or

(15)YAibEGTFTSDYSKAiBLEKQAACLFVQWLLAGGPSSGAPPPS.

In some embodiments, the amino acid sequence of the polypeptide is selected from any one of the following:

(1)YAibEGTFTSDYSYCLEKQAAKLFVQWLLAGGPSSGAPPPS;

(2)YAibEGTFTSDYSYYLECQAAKLFVQWLLAGGPSSGAPPPS;

(3)YAibEGTFTSDYSYYLEKQAACLFVQWLLAGGPSSGAPPPS;

(4)YAibEGTFTSDYSYYLEKQAAKCFVQWLLAGGPSSGAPPPS;

(5)YAibEGTFTSDYSYYLEKQAAKLFVCWLLAGGPSSGAPPPS;

(6)YAibEGTFTSDYSYYLEKQAAKLFVQWLCAGGPSSGAPPPS;

(7)YAibEGTFTSDYSYILEKQAACLFVQWLLAGGPSSGAPPPS;

(8)YAibEGTFTSDYSYILEKQAACLFVQWLIAGGPSSGAPPPS;

(9)YAibEGTFTSDYSYILEKQAACLFVQWLVAGGPSSGAPPPS;

(10)YAibEGTFTSDYSSILEKQAACLFVQWLLAGGPSSGAPPPS;

(11)YAibEGTFTSDYSKILEKQAACLFVQWLLAGGPSSGAPPPS;

(12)YAibEGTFTSDYSIILEKQAACLFVQWLLAGGPSSGAPPPS;

(13)YAibEGTFTSDYSKYLEKQAACLFVQWLLAGGPSSGAPPPS;

(14)YAibEGTFTSDYSKALEKQAACLFVQWLLAGGPSSGAPPPS; or

(15)YAibEGTFTSDYSKAiBLEKQAACLFVQWLLAGGPSSGAPPPS.

In some embodiments, the polypeptide or a pharmaceutically acceptable salt thereof comprises an amino acid sequence as shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In some embodiments, the polypeptide or a pharmaceutically acceptable salt thereof comprises an amino acid sequence as shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some embodiments, the polypeptide or a pharmaceutically acceptable salt thereof comprises an amino acid sequence as shown in SEQ ID NO:1, 3, 5 or 7. In some embodiments, the polypeptide or a pharmaceutically acceptable salt thereof comprises an amino acid sequence as shown in SEQ ID NO:3. In some embodiments, the polypeptide or a pharmaceutically acceptable salt thereof comprises an amino acid sequence as shown in SEQ ID NO:7. In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In some embodiments, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some embodiments, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1, 3, 5 or 7. In some embodiments, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 3. In some embodiments, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 7.

In some embodiments, in the above polypeptide or its pharmaceutically acceptable salt, the polypeptide has a C-terminus with a free carboxyl group. In other embodiments, in the above polypeptide or its pharmaceutically acceptable salt, the polypeptide has a C-terminus amidation modification.

The polypeptide or its pharmaceutically acceptable salt of the present invention is linked to the substituent of the present invention via Cys in the polypeptide to obtain the incretin analog of the present invention. Specifically, the polypeptide or its pharmaceutically acceptable salt is linked to the substituent of the present invention via the thiol group of Cys in the polypeptide.

Incretin analogs

In one aspect, the present disclosure provides an incretin analog or a pharmaceutically acceptable salt thereof, which comprises the above polypeptide and a substituent disclosed herein.

In some embodiments, the pancreatic analogs or pharmaceutically acceptable salts thereof of the present disclosure comprise the above-mentioned polypeptide, and a substituent, wherein the substituent is linked to the polypeptide via Cys in the polypeptide.

In some embodiments, the present disclosure provides an incretin analog or a pharmaceutically acceptable salt thereof, comprising a polypeptide and a substituent, wherein the polypeptide comprises an amino acid sequence as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS (SEQ ID NO: 17), wherein,

_X2 is selected from Aib or Ala,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr, Ile, Ala or Aib,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence; and

The substituent is linked to the polypeptide via Cys in the polypeptide; the polypeptide optionally has a C-terminal amidation modification.

In some embodiments, the present disclosure provides an incretin analog or a pharmaceutically acceptable salt thereof, comprising a polypeptide and a substituent, wherein the polypeptide comprises an amino acid sequence as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS (SEQ ID NO: 16), wherein,

_X2 is selected from Aib or Ala,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr or Ile,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence; and

The substituent is linked to the polypeptide via Cys in the polypeptide; the polypeptide optionally has a C-terminal amidation modification.

In some embodiments, said _X2 is Aib.

In some embodiments, _X12 is Tyr, _X13 is selected from Cys or Tyr, _X16 is selected from Cys or Lys, _X20 is selected from Cys or Lys, _X21 is selected from Cys or Leu, _X24 is selected from Cys or Gln, and _X27 is selected from Cys or Leu. In some embodiments, _X2 is Aib, _X12 is Tyr, _X13 is selected from Cys or Tyr, _X16 is selected from Cys or Lys, _X20 is selected from Cys or Lys, _X21 is selected from Cys or Leu, _X24 is selected from Cys or Gln, and _X27 is selected from Cys or Leu.

In some embodiments, _X20 is Cys.

In some embodiments, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val. In some embodiments, _X2 is Aib, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val.

In some embodiments, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val. In some embodiments, _X2 is Aib, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val.

In some embodiments, _X12 is selected from Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu. In some embodiments, _X2 is Aib, _X12 is selected from Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu.

In some embodiments, _X12 is Lys, _X13 is selected from Tyr, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu. In some embodiments, _X2 is Aib, _X12 is Lys, _X13 is selected from Tyr, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu.

In some embodiments, _X12 is Tyr, _X13 is selected from Tyr or Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu. In some embodiments, _X2 is Aib, _X12 is Tyr, _X13 is selected from Tyr or Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu.

In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the polypeptide comprises an amino acid sequence as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS (SEQ ID NO: 17), wherein:

_X2 is selected from Aib or Ala,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr, Ile, Ala or Aib,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence; and

The substituent is linked to the polypeptide via Cys in the polypeptide; the polypeptide optionally has a C-terminal amidation modification.

In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the polypeptide comprises an amino acid sequence as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS (SEQ ID NO: 16), wherein:

_X2 is selected from Aib or Ala,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr or Ile,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence; and

The substituent is linked to the polypeptide via Cys in the polypeptide; the polypeptide optionally has a C-terminal amidation modification.

In some embodiments, said _X2 is Aib.

In some embodiments, _X12 is Tyr, _X13 is selected from Cys or Tyr, _X16 is selected from Cys or Lys, _X20 is selected from Cys or Lys, _X21 is selected from Cys or Leu, _X24 is selected from Cys or Gln, and _X27 is selected from Cys or Leu. In some embodiments, _X2 is Aib, _X12 is Tyr, _X13 is selected from Cys or Tyr, _X16 is selected from Cys or Lys, _X20 is selected from Cys or Lys, _X21 is selected from Cys or Leu, _X24 is selected from Cys or Gln, and _X27 is selected from Cys or Leu.

In some embodiments, _X20 is Cys.

In some embodiments, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val. In some embodiments, _X2 is Aib, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val.

In some embodiments, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val. In some embodiments, _X2 is Aib, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val.

In some embodiments, _X12 is selected from Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu. In some embodiments, _X2 is Aib, _X12 is selected from Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu.

In some embodiments, _X12 is Lys, _X13 is selected from Tyr, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu. In some embodiments, _X2 is Aib, _X12 is Lys, _X13 is selected from Tyr, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu.

In some embodiments, _X12 is Tyr, _X13 is selected from Tyr or Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu. In some embodiments, _X2 is Aib, _X12 is Tyr, _X13 is selected from Tyr or Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu.

In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS (SEQ ID NO: 17), wherein:

_X2 is selected from Aib or Ala,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr, Ile, Ala or Aib,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence; and

The substituent is linked to the polypeptide via Cys in the polypeptide; the polypeptide optionally has a C-terminal amidation modification.

In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS (SEQ ID NO: 16), wherein:

_X2 is selected from Aib or Ala,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr or Ile,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence; and

The substituent is linked to the polypeptide via Cys in the polypeptide; the polypeptide optionally has a C-terminal amidation modification.

In some embodiments, in the incretin analog or a pharmaceutically acceptable salt thereof, _X2 is Aib.

In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr, Ile, Ala or Aib,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence; and

The substituent is linked to the polypeptide via Cys in the polypeptide.

In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr or Ile,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

_X27 is selected from Cys, Leu, Ile or Val, and

There is one and only one Cys in the amino acid sequence; and

The substituent is linked to the polypeptide via Cys in the polypeptide.

In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib, _X12 is Tyr, _X13 is selected from Cys or Tyr, _X16 is selected from Cys or Lys, _X20 is selected from Cys or Lys, _X21 is selected from Cys or Leu, _X24 is selected from Cys or Gln, _X27 is selected from Cys or Leu, and there is one and only one Cys in the amino acid sequence; and the substituent is linked to the polypeptide through the Cys in the polypeptide.

In some embodiments, in the incretin analog or a pharmaceutically acceptable salt thereof, _X20 is Cys.

In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val; and the substituent is linked to the polypeptide through the thiol group of Cys in the polypeptide.

In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val; and the substituent is linked to the polypeptide through the thiol group of Cys in the polypeptide.

In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib, _X12 is selected from Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu; and the substituent is linked to the polypeptide through the thiol group of Cys in the polypeptide.

In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib, _X12 is Lys, _X13 is selected from Tyr, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu; and the substituent is linked to the polypeptide through the thiol group of Cys in the polypeptide.

In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is Aib, _X12 is Tyr, _X13 is selected from Tyr or Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu; and the substituent is linked to the polypeptide through the Cys in the polypeptide.

In some embodiments, the incretin analog or a pharmaceutically acceptable salt thereof comprises a polypeptide and a substituent, wherein the polypeptide comprises any one selected from the following amino acid sequences:

(1)YAibEGTFTSDYSYCLEKQAAKLFVQWLLAGGPSSGAPPPS;

(2)YAibEGTFTSDYSYYLECQAAKLFVQWLLAGGPSSGAPPPS;

(3)YAibEGTFTSDYSYYLEKQAACLFVQWLLAGGPSSGAPPPS;

(4)YAibEGTFTSDYSYYLEKQAAKCFVQWLLAGGPSSGAPPPS;

(5)YAibEGTFTSDYSYYLEKQAAKLFVCWLLAGGPSSGAPPPS;

(6)YAibEGTFTSDYSYYLEKQAAKLFVQWLCAGGPSSGAPPPS;

(7)YAibEGTFTSDYSYILEKQAACLFVQWLLAGGPSSGAPPPS;

(8)YAibEGTFTSDYSYILEKQAACLFVQWLIAGGPSSGAPPPS;

(9)YAibEGTFTSDYSYILEKQAACLFVQWLVAGGPSSGAPPPS;

(10)YAibEGTFTSDYSSILEKQAACLFVQWLLAGGPSSGAPPPS;

(11)YAibEGTFTSDYSKILEKQAACLFVQWLLAGGPSSGAPPPS;

(12)YAibEGTFTSDYSIILEKQAACLFVQWLLAGGPSSGAPPPS;

(13)YAibEGTFTSDYSKYLEKQAACLFVQWLLAGGPSSGAPPPS;

(14)YAibEGTFTSDYSKALEKQAACLFVQWLLAGGPSSGAPPPS; or

(15)YAibEGTFTSDYSKAiBLEKQAACLFVQWLLAGGPSSGAPPPS;

Furthermore, the substituent is linked to the polypeptide via Cys in the polypeptide.

In some embodiments, the incretin analog consists of a polypeptide and a substituent, wherein the polypeptide comprises any one selected from the following amino acid sequences:

(1)YAibEGTFTSDYSYCLEKQAAKLFVQWLLAGGPSSGAPPPS;

(2)YAibEGTFTSDYSYYLECQAAKLFVQWLLAGGPSSGAPPPS;

(3)YAibEGTFTSDYSYYLEKQAACLFVQWLLAGGPSSGAPPPS;

(4)YAibEGTFTSDYSYYLEKQAAKCFVQWLLAGGPSSGAPPPS;

(5)YAibEGTFTSDYSYYLEKQAAKLFVCWLLAGGPSSGAPPPS;

(6)YAibEGTFTSDYSYYLEKQAAKLFVQWLCAGGPSSGAPPPS;

(7)YAibEGTFTSDYSYILEKQAACLFVQWLLAGGPSSGAPPPS;

(8)YAibEGTFTSDYSYILEKQAACLFVQWLIAGGPSSGAPPPS;

(9)YAibEGTFTSDYSYILEKQAACLFVQWLVAGGPSSGAPPPS;

(10)YAibEGTFTSDYSSILEKQAACLFVQWLLAGGPSSGAPPPS;

(11)YAibEGTFTSDYSKILEKQAACLFVQWLLAGGPSSGAPPPS;

(12)YAibEGTFTSDYSIILEKQAACLFVQWLLAGGPSSGAPPPS;

(13)YAibEGTFTSDYSKYLEKQAACLFVQWLLAGGPSSGAPPPS;

(14)YAibEGTFTSDYSKALEKQAACLFVQWLLAGGPSSGAPPPS; or

(15)YAibEGTFTSDYSKAiBLEKQAACLFVQWLLAGGPSSGAPPPS;

Furthermore, the substituent is linked to the polypeptide via Cys in the polypeptide.

In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is selected from any one of the following:

(1)YAibEGTFTSDYSYCLEKQAAKLFVQWLLAGGPSSGAPPPS;

(2)YAibEGTFTSDYSYYLECQAAKLFVQWLLAGGPSSGAPPPS;

(3)YAibEGTFTSDYSYYLEKQAACLFVQWLLAGGPSSGAPPPS;

(4)YAibEGTFTSDYSYYLEKQAAKCFVQWLLAGGPSSGAPPPS;

(5)YAibEGTFTSDYSYYLEKQAAKLFVCWLLAGGPSSGAPPPS;

(6)YAibEGTFTSDYSYYLEKQAAKLFVQWLCAGGPSSGAPPPS;

(7)YAibEGTFTSDYSYILEKQAACLFVQWLLAGGPSSGAPPPS;

(8)YAibEGTFTSDYSYILEKQAACLFVQWLIAGGPSSGAPPPS;

(9)YAibEGTFTSDYSYILEKQAACLFVQWLVAGGPSSGAPPPS;

(10)YAibEGTFTSDYSSILEKQAACLFVQWLLAGGPSSGAPPPS;

(11)YAibEGTFTSDYSKILEKQAACLFVQWLLAGGPSSGAPPPS;

(12)YAibEGTFTSDYSIILEKQAACLFVQWLLAGGPSSGAPPPS;

(13)YAibEGTFTSDYSKYLEKQAACLFVQWLLAGGPSSGAPPPS;

(14)YAibEGTFTSDYSKALEKQAACLFVQWLLAGGPSSGAPPPS; or

(15)YAibEGTFTSDYSKAiBLEKQAACLFVQWLLAGGPSSGAPPPS;

Furthermore, the substituent is linked to the polypeptide via Cys in the polypeptide.

In some embodiments, the incretin analog or a pharmaceutically acceptable salt thereof comprises a polypeptide and a substituent, wherein the polypeptide comprises an amino acid sequence as shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; and the substituent is linked to the polypeptide via Cys in the polypeptide. In some embodiments, the incretin analog or a pharmaceutically acceptable salt thereof comprises a polypeptide and a substituent, wherein the polypeptide comprises an amino acid sequence as shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; and the substituent is linked to the polypeptide via Cys in the polypeptide. In some such embodiments, the polypeptide comprises an amino acid sequence as shown in SEQ ID NO:1, 3, 5 or 7. In some such embodiments, the polypeptide comprises an amino acid sequence as shown in SEQ ID NO:3. In some such embodiments, the polypeptide comprises an amino acid sequence as shown in SEQ ID NO:7.

In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the polypeptide comprises an amino acid sequence as shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; and the substituent is linked to the polypeptide through Cys in the polypeptide. In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the polypeptide comprises an amino acid sequence as shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; and the substituent is linked to the polypeptide through Cys in the polypeptide. In some such embodiments, the polypeptide comprises an amino acid sequence as shown in SEQ ID NO:1, 3, 5 or 7. In some such embodiments, the polypeptide comprises an amino acid sequence as shown in SEQ ID NO:3. In some such embodiments, the polypeptide comprises an amino acid sequence as shown in SEQ ID NO:7.

In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; and the substituent is linked to the polypeptide through Cys in the polypeptide. In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; and the substituent is linked to the polypeptide through Cys in the polypeptide. In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1, 3, 5 or 7; and the substituent is linked to the polypeptide through Cys in the polypeptide. In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO:3; and the substituent is linked to the polypeptide via Cys in the polypeptide. In some embodiments, the incretin analogue consists of a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO:7; and the substituent is linked to the polypeptide via Cys in the polypeptide.

In some embodiments, the substituent is linked to the polypeptide via Cys at position 13, 16, 20, 21, 24, or 27 of the polypeptide. In some embodiments, the substituent is linked to the polypeptide via Cys at position 20 of the polypeptide. In some embodiments, the substituent is linked to the polypeptide via Cys at position 20 of the polypeptide whose amino acid sequence is shown in SEQ ID NO:7.

In some embodiments, the substituent is linked to the polypeptide via the thiol group of Cys at position 13, 16, 20, 21, 24 or 27 of the polypeptide. In some embodiments, the substituent is linked to the polypeptide via the thiol group of Cys at position 20 of the polypeptide. In some embodiments, the substituent is linked to the polypeptide via the thiol group of Cys at position 20 of the polypeptide having an amino acid sequence as shown in SEQ ID NO:7.

In some embodiments, the substituent is connected to the polypeptide by forming a thioether bond with the thiol group of Cys at position 13, 16, 20, 21, 24 or 27 of the polypeptide. In some embodiments, the substituent is connected to the polypeptide by forming a thioether bond with the thiol group of Cys at position 20 of the polypeptide. In some embodiments, the substituent is connected to the polypeptide by forming a thioether bond with the thiol group of Cys at position 20 of the polypeptide whose amino acid sequence is shown in SEQ ID NO:7.

In some embodiments, the substituent comprises a fatty acid group.

In some embodiments, the fatty acid group comprises a carbon chain comprising at least 8 consecutive -CH ₂ -structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising at least 10 consecutive -CH ₂ -structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising at least 12 consecutive -CH ₂ -structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising at least 14 consecutive -CH ₂ -structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising at least 16 consecutive -CH ₂ -structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising at least 18 consecutive -CH ₂ -structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising at least 20 consecutive -CH ₂ -structural units.

In some embodiments, the fatty acid group comprises a carbon chain comprising 8-20 consecutive -CH ₂ - structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising 10-20 consecutive -CH ₂ - structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising 12-20 consecutive _{-CH 2 - structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising 14-20 consecutive -CH 2} - structural units.

In some embodiments, the fatty acid group comprises a carbon chain comprising 8, 10, 12, 14, 16, 18, or 20 consecutive _-CH2- structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising 16 or 18 consecutive _-CH2- structural units.

In some embodiments, the structure of the fatty acid group is as follows:

Where n is an integer between 8 and 20.

The fatty acid groups may be bound to albumin.

In some embodiments, the substituent further comprises a linker. The linker is connected to the fatty acid group via an amide bond. In some such embodiments, the structure of the fatty acid group is as shown in Formula I, wherein n is an integer between 8 and 20, and the wavy line In some embodiments, n is an integer between 10 and 20. In some embodiments, n is an integer between 12 and 20. In some embodiments, n is an integer between 14 and 20. In some embodiments, n is 8, 10, 12, 14, 16 or 18. In some embodiments, n is 16 or 18.

In some embodiments, the substituent has the following structure:

in,

Lk represents a linker, _R1 is a fatty acid group of the structure shown in Formula I, n is an integer from 8 to 20, the wavy line in Formula I represents the connection position between _R1 and Lk, and the wavy line in Formula II represents the connection position between the substituent and the polypeptide of the present disclosure.

In some embodiments, n is an integer between 10 and 20. In some embodiments, n is an integer between 12 and 20. In some embodiments, n is an integer between 14 and 20. In some embodiments, n is 8, 10, 12, 14, 16, or 18. In some embodiments, n is 16 or 18.

In some embodiments, the Lk is selected from one or more combinations of Ala, D-Ala, Arg, Asp, Asn, Glu, D-Glu, γ-Glu, Gln, Gly, Lys, ε-Lys, Pro, Phe, Ser or AEEA, or is missing; preferably one or more combinations of Asp, Glu, γ-Glu, Gly, Ser, AEEA, Lys or ε-Lys; more preferably one or more combinations of Glu, γ-Glu, AEEA, Lys or ε-Lys. In some embodiments, the Lk is selected from one or more combinations of γ-Glu, AEEA or ε-Lys. In some embodiments, the -Lk- is -AEEA-γ-Glu-γ-Glu-, and the substituent structure is: In some embodiments, the -Lk- is -γ-Glu-AEEA-AEEA-, and the substituent structure is: In some embodiments, the -Lk- is -γ-Glu-ε-Lys-ε-Lys-, and the structure of the substituent is:

In some embodiments, the structure of the substituent is as shown in Formula II, wherein the Lk represents a linker and is selected from one or a combination of γ-Glu, AEEA or ε-Lys, _R1 is a fatty acid group of the structure shown in Formula I, n is an integer from 14 to 20, the wavy line in Formula I represents the connection position between _R1 and Lk, and the wavy line in Formula II represents the connection position between the substituent and the polypeptide of the present disclosure; preferably, n is 8, 10, 12, 14, 16 or 18; more preferably, n is 16 or 18.

In some embodiments, the substituent is selected from the following structures: Wherein, the wavy lines in Formulae II-1, II-2, II-3, II-4, II-5 and II-6 indicate the connection position of the substituent to the polypeptide of the present disclosure.

In some embodiments, the incretin analog or a pharmaceutically acceptable salt thereof comprises a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is YAibEGTFTSDYSYYLEKQAACLFVQWLLAGGPSSGAPPPS, and the polypeptide optionally has a C-terminal amidation modification; the substituent is connected to the polypeptide via the thiol group of Cys in the polypeptide, and the structure of the substituent is as shown in Formula II-1, II-2, II-3, II-4, II-5 or II-6. In some embodiments, the structure of the substituent is as shown in Formula II-1. In some embodiments, the structure of the substituent is as shown in Formula II-2. In some embodiments, the structure of the substituent is as shown in Formula II-3. In some embodiments, the structure of the substituent is as shown in Formula II-4. In some embodiments, the structure of the substituent is as shown in Formula II-5. In some embodiments, the structure of the substituent is as shown in Formula II-6.

In some embodiments, the incretin analog or a pharmaceutically acceptable salt thereof comprises a polypeptide and a substituent, wherein the amino acid sequence of the polypeptide is YAibEGTFTSDYSYILEKQAACLFVQWLLAGGPSSGAPPPS, and the polypeptide optionally has a C-terminal amidation modification; the substituent is connected to the polypeptide via the thiol group of Cys in the polypeptide, and the structure of the substituent is as shown in Formula II-1, II-2, II-3, II-4, II-5 or II-6. In some embodiments, the structure of the substituent is as shown in Formula II-1. In some embodiments, the structure of the substituent is as shown in Formula II-2. In some embodiments, the structure of the substituent is as shown in Formula II-3. In some embodiments, the structure of the substituent is as shown in Formula II-4. In some embodiments, the structure of the substituent is as shown in Formula II-5. In some embodiments, the structure of the substituent is as shown in Formula II-6.

The substituents attached to the polypeptides of the present disclosure can non-covalently bind to albumin via the fatty acid group, thereby extending the half-life of the incretin analog in vivo.

The structures of exemplary incretin analogs provided by the present disclosure are as follows:

Incretin analogs 1:

Incretin analog 2:

Incretin analog 3:

Incretin analog 4:

Incretin analog 5:

Incretin analog 6:

Incretin analog 7:

Incretin analogs 8:

Incretin analogs 9:

Incretin analog 10:

Incretin analogue 11:

Incretin analog 12:

Incretin analog 13:

Incretin analog 14:

Incretin analog 15:

Incretin analog 16:

Incretin analog 17:

Incretin analog 18:

Incretin analog 19:

Incretin analog 20:

Incretin analog 21:

Incretin analog 22:

Incretin analog 23:

Incretin analog 24:

Incretin analog 25:

Incretin analog 26:

Incretin analog 27:

Incretin analog 28:

Incretin analog 29:

Incretin analog 30:

Incretin analog 31:

Incretin analog 32:

Incretin analog 33:

Incretin analog 34:

Incretin analog 35:

Incretin analog 36:

Incretin analog 37:

Incretin analog 38:

Incretin analog 39:

Incretin analog 40:

Incretin analog 41:

Incretin analog 42:

Incretin analog 43:

Incretin analog 44:

Incretin analog 45:

Incretin analog 46:

Incretin analog 47:

Incretin analog 48:

Incretin analog 49:

Incretin analog 50:

Incretin analog 51:

Incretin analog 52:

Incretin analog 53:

Incretin analog 54:

Incretin analog 55:

Incretin analog 56:

Incretin analog 57:

Incretin analog 58:

Incretin analog 59:

Incretin analog 60:

Incretin analog 61:

Incretin analog 62:

Incretin analog 63:

Incretin analog 64:

Incretin analog 65:

Incretin analog 66:

Incretin analog 67:

Incretin analog 68:

Incretin analog 69:

Incretin analog 70:

Incretin analog 71:

Incretin analog 72:

Incretin analog 73:

Incretin analog 74:

Incretin analog 75:

Incretin analog 76:

Incretin analog 77:

Incretin analog 78:

Incretin analog 79:

Incretin analog 80:

Incretin analog 81:

Incretin analog 82:

Incretin analog 83:

Incretin analog 84:

In some embodiments, in the above-mentioned incretin analogs or pharmaceutically acceptable salts thereof, the polypeptide has a C-terminus with a free carboxyl group. In other embodiments, in the above-mentioned incretin analogs or pharmaceutically acceptable salts thereof, the polypeptide has a C-terminus amidation modification.

The incretin analogs provided by the present disclosure have agonist activity of GIPR and GLP-1R, are dual receptor agonists, and are significantly effective in treating diabetes, lowering blood sugar, and reducing body weight.

Modifiers

The present disclosure provides a modifier comprising a fatty acid group. The modifier is used to modify the polypeptide of the present disclosure to obtain an incretin analog. The polypeptide connected with the modifier can non-covalently bind to albumin through the fatty acid group, thereby extending the half-life of the incretin analog in vivo.

In some embodiments, the fatty acid group comprises a carbon chain comprising at least 8 consecutive -CH ₂ -structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising at least 10 consecutive -CH ₂ -structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising at least 12 consecutive -CH ₂ -structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising at least 14 consecutive -CH ₂ -structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising at least 16 consecutive -CH ₂ -structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising at least 18 consecutive -CH ₂ -structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising at least 20 consecutive -CH ₂ -structural units.

In some embodiments, the fatty acid group comprises a carbon chain comprising 8-20 consecutive -CH ₂ - structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising 10-20 consecutive -CH ₂ - structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising 12-20 consecutive _{-CH 2 - structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising 14-20 consecutive -CH 2} - structural units.

In some embodiments, the fatty acid group comprises a carbon chain comprising 8, 10, 12, 14, 16, 18, or 20 consecutive _-CH2- structural units. In some embodiments, the fatty acid group comprises a carbon chain comprising 16 or 18 consecutive _-CH2- structural units.

In some embodiments, the structure of the fatty acid group is as shown in Formula I, wherein n is an integer between 8-20.

In some embodiments, the modifier further comprises a linker. The linker is connected to the fatty acid group via an amide bond. In some such embodiments, the structure of the fatty acid group is as shown in Formula I, wherein n is an integer between 8 and 20, and the wavy line In some embodiments, n is an integer between 10 and 20. In some embodiments, n is an integer between 12 and 20. In some embodiments, n is an integer between 14 and 20. In some embodiments, n is 8, 10, 12, 14, 16 or 18. In some embodiments, n is 16 or 18.

In some embodiments, the structure of the modifier is as follows:

in,

Lk represents a linker, _R1 is a fatty acid group of the structure shown in Formula I, _R2 is an iodoacetyl group or a bromoacetyl group, n is an integer from 8 to 20, and the wavy line in Formula I represents the connection position between _R1 and Lk. In some embodiments, _R2 is a bromoacetyl group.

In some embodiments, n is an integer between 10 and 20. In some embodiments, n is an integer between 12 and 20. In some embodiments, n is an integer between 14 and 20. In some embodiments, n is 8, 10, 12, 14, 16, or 18. In some embodiments, n is 16 or 18.

In some embodiments, the Lk is selected from one or more combinations of Ala, D-Ala, Arg, Asp, Asn, Glu, D-Glu, γ-Glu, Gln, Gly, Lys, ε-Lys, Pro, Phe, Ser or AEEA, or is missing; preferably one or more combinations of Asp, Glu, γ-Glu, Gly, Ser, AEEA, Lys or ε-Lys; more preferably one or more combinations of Glu, γ-Glu, AEEA, Lys or ε-Lys. In some embodiments, the Lk is selected from one or more combinations of γ-Glu, AEEA or ε-Lys. In some embodiments, the -Lk- is -AEEA-γ-Glu-γ-Glu-, and the modifier structure is: In some embodiments, the -Lk- is -γ-Glu-AEEA-AEEA-, and the modifier structure is: In some embodiments, the -Lk- is -γ-Glu-ε-Lys-ε-Lys-, and the structure of the modifier is:

In some embodiments, the structure of the modifier is as shown in Formula III, wherein the Lk represents a linker and is selected from one or a combination of γ-Glu, AEEA or ε-Lys, _R1 is a fatty acid group of the structure shown in Formula I, n is an integer between 14 and 20, and the wavy line in Formula I represents the connection position between _R1 and Lk; preferably, n is 8, 10, 12, 14, 16 or 18; more preferably, n is 16 or 18.

In some embodiments, the modifier is selected from the following structures:

Pharmaceutical composition

In another aspect, the present disclosure provides a pharmaceutical composition comprising the incretin analogs of the present disclosure or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition comprises the incretin analogs of the present disclosure or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

In one embodiment, the pharmaceutical composition can be a solid preparation, such as a lyophilized preparation. In another embodiment, the pharmaceutical composition can be a liquid preparation, such as an aqueous preparation.

Preparation method

In another aspect, the present disclosure provides a method for preparing an incretin analog or a pharmaceutically acceptable salt thereof, comprising preparing a polypeptide of the present disclosure by chemical synthesis and/or recombinant expression, preparing a modifier of the present disclosure by chemical synthesis, and linking the modifier to the polypeptide via Cys in the polypeptide. In some embodiments, the modifier is linked to the polypeptide via the thiol group of Cys in the polypeptide.

The polypeptides of the present disclosure can be prepared by chemical synthesis, for example, using solid phase synthesis to prepare the polypeptides of the present disclosure. Alternatively, the polypeptides of the present disclosure can be prepared by recombinant expression methods, for example, by culturing a host cell containing a DNA sequence encoding the polypeptide in a suitable culture medium under conditions that allow polypeptide expression. Alternatively, the polypeptides of the present disclosure can be prepared by a combination of recombinant expression and chemical synthesis. Non-limiting examples of host cells suitable for expressing these polypeptides include Escherichia coli or CHO cells.

In some embodiments, the method for preparing the polypeptide of the present disclosure comprises the following steps:

(1) selecting a suitable resin and synthesizing the polypeptide on the resin using a solid phase synthesis method to obtain a resin peptide;

(2) Add a side chain protecting group scavenger containing a strong acid to the resin peptide, filter, add an appropriate amount of organic solvent to precipitate, centrifuge, wash the precipitate with an organic solvent, and dry to obtain a crude polypeptide.

In the method for preparing the polypeptide of the present disclosure, after step (2), a step of purifying the crude polypeptide may be further included. The purification method includes but is not limited to reverse phase chromatography or ion exchange chromatography, preferably reverse phase chromatography.

In some embodiments, the resin is Wang resin or MBHA resin.

In some embodiments, the side chain protecting group scavenger is selected from one, two or more combinations of thioanisole, triisopropylsilane, phenol, water, 1,2-ethanedithiol or meta-cresol. In some embodiments, the strong acid is trifluoroacetic acid (TFA). In some specific embodiments, the side chain protecting group scavenger containing a strong acid is an aqueous solution containing TFA and TIS, wherein TFA:TIS:water=95:2.5:2.5 (v:v:v).

In some embodiments, step (1) comprises:

(a) selecting a suitable resin for swelling, removing the amino protecting group with a removing agent, and washing the resin with a solvent;

(b) adding a protected amino acid and a condensation reagent to couple the amino acid, removing the amino protecting group with a removing agent, and washing the resin with a solvent;

(c) Repeat step (b) to sequentially couple the remaining amino acids. After the last amino acid is coupled, the amino protecting group is removed with a removing agent, and the resin is washed with a solvent to obtain a resin peptide.

The sequential coupling of the remaining amino acids is to connect the amino acids one by one from the C-terminus to the N-terminus according to the amino acid sequence of the polypeptide.

In some embodiments, the amino protecting group is selected from tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z) or 9-fluorenyl-methoxycarbonyl (Fmoc), preferably 9-fluorenyl-methoxycarbonyl (Fmoc).

In some embodiments, the solvent in step (1) is selected from N,N-dimethylformamide (DMF), dichloromethane (DCM) or N-methylpyrrolidone (NMP), preferably DMF or DCM.

In some embodiments, the removing agent in step (1) is selected from a DMF solution of piperidine (v/v) at a concentration of 10%-40%, preferably a DMF solution of piperidine (v/v) at a concentration of 20-25%. In some embodiments, the time required for removing the amino protecting group can be selected from 20-50 min, preferably 25-35 min.

In some embodiments, the condensation reagent is selected from one or a combination of carbodiimide-type reagents, benzotriazolium salt-type reagents or 1-hydroxybenzotriazole (HOBt). In some embodiments, the carbodiimide-type reagent is selected from one of dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide (DIC) or 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC). In some embodiments, the benzotriazolium salt-type reagent is selected from one of 2-(1H-benzotriazolyl L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), hexafluorophosphate benzotriazole-1-oxy tris (dimethylamino) phosphorus (BOP) or hexafluorophosphate benzotriazole-1-yl-oxy tripyrrolidinophosphorus (PyBOP). In some specific embodiments, the condensation reagents are DIC and HOBt, or TBTU and HOBt; preferably DIC and HOBt.

use

In another aspect, the present disclosure provides the use of the incretin analogs of the present disclosure or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the present disclosure in the preparation of a medicament. In some embodiments, the medicament is used to treat diseases including but not limited to hyperglycemia, diabetes, impaired glucose tolerance or obesity.

On the other hand, the present disclosure provides a method for treating a disease in a subject in need thereof, comprising administering to the subject an incretin analog of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. On the other hand, the present disclosure provides a method for treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an incretin analog of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In some embodiments, the disease includes, but is not limited to, hyperglycemia, diabetes, impaired glucose tolerance, or obesity.

In some embodiments, non-limiting examples of diabetes include type 1 diabetes, type 2 diabetes, maturity-onset diabetes of the young (MODY), or gestational diabetes.

In one embodiment, the present disclosure provides the use of the incretin analog or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diabetes. In one embodiment, the present disclosure provides the use of the incretin analog or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating type 2 diabetes. In one embodiment, the present disclosure provides the use of the incretin analog or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating obesity.

In one embodiment, the present disclosure provides the use of the pharmaceutical composition in the preparation of a medicament for treating diabetes. In one embodiment, the present disclosure provides the use of the pharmaceutical composition in the preparation of a medicament for treating type 2 diabetes. In one embodiment, the present disclosure provides the use of the pharmaceutical composition in the preparation of a medicament for treating obesity.

In one embodiment, the present disclosure provides a method for treating diabetes in a subject in need, comprising administering to the subject an incretin analog of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In one embodiment, the present disclosure provides a method for treating type 2 diabetes in a subject in need, comprising administering to the subject an incretin analog of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In one embodiment, the present disclosure provides a method for treating obesity in a subject in need, comprising administering to the subject an incretin analog of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In one embodiment, the present disclosure provides a method for treating diabetes in a subject in need, comprising administering to the subject a therapeutically effective amount of an incretin analog of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In one embodiment, the present disclosure provides a method for treating type 2 diabetes in a subject in need, comprising administering to the subject a therapeutically effective amount of an incretin analog of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In one embodiment, the present disclosure provides a method for treating obesity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an incretin analog or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure.

The present disclosure also provides the following specific implementation schemes, but the protection scope of the present disclosure is not limited thereto:

Embodiment 1. A polypeptide or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein:

_X2 is selected from Aib or Ala,

_X12 is selected from Tyr, Lys, Ile or Ser,

_X13 is selected from Cys, Tyr, Ile, Ala or Aib,

X ₁₆ is selected from Cys or Lys,

X ₂₀ is selected from Cys or Lys,

X ₂₁ is selected from Cys or Leu,

X ₂₄ is selected from Cys or Gln,

X ₂₇ is selected from Cys, Leu, Ile or Val, and there is one and only one Cys in the amino acid sequence; and the polypeptide optionally has a C-terminal amidation modification.

Embodiment 2. The polypeptide or pharmaceutically acceptable salt thereof according to Embodiment 1, wherein _X2 is Aib.

Embodiment 3. The polypeptide or pharmaceutically acceptable salt thereof according to embodiment 1 or 2, wherein X ₁₃ is selected from Cys, Tyr or Ile.

Embodiment 4. The polypeptide or pharmaceutically acceptable salt thereof according to embodiment 1 or 2, wherein _X12 is Tyr, _X13 is selected from Cys or Tyr, _X16 is selected from Cys or Lys, _X20 is selected from Cys or Lys, _X21 is selected from Cys or Leu, _X24 is selected from Cys or Gln, and _X27 is selected from Cys or Leu.

Embodiment 5. The polypeptide or pharmaceutically acceptable salt thereof according to any one of Embodiments 1 to 4, wherein X ₂₀ is Cys.

Embodiment 6. The polypeptide or pharmaceutically acceptable salt thereof according to embodiment 1 or 2, wherein _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val.

Embodiment 7. The polypeptide or pharmaceutically acceptable salt thereof according to embodiment 1 or 2, wherein X ₁₂ is selected from Lys, Ile or Ser, X ₁₃ is selected from Tyr, Ile, Ala or Aib, X ₁₆ is Lys, X ₂₀ is Cys, X ₂₁ is Leu, X ₂₄ is Gln, and X ₂₇ is Leu.

Embodiment 8. The polypeptide or pharmaceutically acceptable salt thereof according to embodiment 1 or 2, wherein _X12 is Lys, _X13 is selected from Tyr, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu.

Embodiment 9. The polypeptide or pharmaceutically acceptable salt thereof according to embodiment 1 or 2, wherein X ₁₂ is Tyr, X ₁₃ is selected from Tyr or Ile, X ₁₆ is Lys, X ₂₀ is Cys, X ₂₁ is Leu, X ₂₄ is Gln, and X ₂₇ is Leu.

Embodiment 10. A polypeptide or a pharmaceutically acceptable salt thereof according to Embodiment 1, wherein the polypeptide or a pharmaceutically acceptable salt thereof comprises an amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

Embodiment 11. The polypeptide or pharmaceutically acceptable salt thereof according to any one of Embodiments 1-10, wherein the polypeptide has a C-terminal amidation modification.

Embodiment 12. An incretin analog or a pharmaceutically acceptable salt thereof, comprising the polypeptide according to any one of embodiments 1-11, and a substituent, wherein the substituent is linked to the polypeptide via Cys in the polypeptide.

Embodiment 13. An incretin analog or a pharmaceutically acceptable salt thereof according to Embodiment 12, wherein the substituent is linked to the polypeptide via Cys at position 13, 16, 20, 21, 24 or 27 of the polypeptide; preferably, it is linked to the polypeptide via Cys at position 20 of the polypeptide.

Embodiment 14. The incretin analog or a pharmaceutically acceptable salt thereof according to Embodiment 12 or 13, wherein the substituent comprises a fatty acid group.

Embodiment 15. An incretin analog or a pharmaceutically acceptable salt thereof according to Embodiment 14, wherein the fatty acid group comprises a carbon chain comprising at least 8 consecutive -CH ₂ -structural units; preferably, the carbon chain comprises 8-20 consecutive -CH ₂ -structural units; more preferably, the carbon chain comprises 8, 10, 12, 14, 16, 18 or 20 consecutive -CH ₂ -structural units; most preferably, the carbon chain comprises 16 or 18 consecutive -CH ₂ -structural units.

Embodiment 16. The incretin analog or a pharmaceutically acceptable salt thereof according to Embodiment 14, wherein the structure of the fatty acid group is as follows:

wherein n is an integer between 8 and 20; preferably, n is 8, 10, 12, 14, 16 or 18; more preferably, n is 16 or 18.

Embodiment 17. The incretin analog or a pharmaceutically acceptable salt thereof according to any one of Embodiments 12-16, wherein the substituent further comprises a linker.

Embodiment 18. The incretin analog or a pharmaceutically acceptable salt thereof according to Embodiment 12 or 13, wherein the structure of the substituent is as follows:

in,

Lk represents a linker, R1 _is The fatty acid group of the structure shown, n is an integer between 8 and 20, the wavy line in formula I represents the connection position between _R1 and Lk, and the wavy line in formula II represents the connection position between the substituent and the polypeptide.

Embodiment 19. An incretin analog or a pharmaceutically acceptable salt thereof according to Embodiment 18, wherein Lk is selected from one or a combination of Ala, D-Ala, Arg, Asp, Asn, Glu, D-Glu, γ-Glu, Gln, Gly, Lys, ε-Lys, Pro, Phe, Ser or AEEA, or is absent; preferably one or a combination of Asp, Glu, γ-Glu, Gly, Ser, AEEA, Lys or ε-Lys; more preferably one or a combination of Glu, γ-Glu, AEEA, Lys or ε-Lys.

Embodiment 20. The incretin analog or pharmaceutically acceptable salt thereof according to Embodiment 18 or 19, wherein the Lk is selected from one or a combination of γ-Glu, AEEA or ε-Lys.

Embodiment 21. The incretin analog or a pharmaceutically acceptable salt thereof according to Embodiment 18, wherein:

(1) The Lk is -AEEA-γ-Glu-γ-Glu-, and the substituent structure is:

(2) The Lk is -γ-Glu-AEEA-AEEA-, and the substituent structure is: or,

(3) The Lk is -γ-Glu-ε-Lys-ε-Lys-, and the substituent structure is:

Embodiment 22. The incretin analog or a pharmaceutically acceptable salt thereof according to Embodiment 12 or 13, wherein the substituent is selected from the following structures: The wavy lines in formulae II-1, II-2, II-3, II-4, II-5 and II-6 indicate the connection position between the substituent and the polypeptide.

Embodiment 23. The incretin analog or a pharmaceutically acceptable salt thereof according to Embodiment 12, wherein the incretin analog is selected from:

Incretin analog 1, its structure is:

Incretin analog 2, its structure is:

Incretin analog 3, its structure is:

Incretin analog 4, its structure is:

Incretin analog 5, its structure is:

Incretin analog 6, its structure is:

Incretin analog 7, its structure is:

Incretin analog 8, its structure is:

Incretin analog 9, its structure is:

Incretin analog 10, the structure of which is:

Incretin analog 11, its structure is:

Incretin analog 12, the structure of which is:

Incretin analog 13, its structure is:

Incretin analog 14, the structure of which is:

Incretin analog 15, its structure is:

Incretin analog 16, its structure is:

Incretin analog 17, its structure is:

Incretin analog 18, its structure is:

Incretin analog 19, its structure is:

Incretin analog 20, the structure of which is:

Incretin analog 21, its structure is:

Incretin analog 22, its structure is:

Incretin analog 23, its structure is:

Incretin analog 24, the structure of which is:

Incretin analog 25, its structure is:

Incretin analog 26, its structure is:

Incretin analog 27, its structure is:

Incretin analog 28, its structure is:

Incretin analog 29, its structure is:

Incretin analog 30, the structure of which is:

Incretin analog 31, its structure is:

Incretin analog 32, its structure is:

Incretin analog 33, its structure is:

Incretin analog 34, the structure of which is:

Incretin analog 35, its structure is:

Incretin analog 36, its structure is:

Incretin analog 37, its structure is:

Incretin analog 38, its structure is:

Incretin analog 39, its structure is:

Incretin analog 40, the structure of which is:

Incretin analog 41, its structure is:

Incretin analog 42, the structure of which is:

Incretin analog 43, its structure is:

Incretin analog 44, its structure is:

Incretin analog 45, its structure is:

Incretin analog 46, its structure is:

Incretin analog 47, its structure is:

Incretin analog 48, its structure is:

Incretin analog 49, its structure is:

Incretin analog 50, the structure of which is:

Incretin analog 51, its structure is:

Incretin analog 52, the structure of which is:

Incretin analog 53, its structure is:

Incretin analog 54, the structure of which is:

Incretin analog 55, its structure is:

Incretin analog 56, its structure is:

Incretin analog 57, its structure is:

Incretin analog 58, its structure is:

Incretin analog 59, its structure is:

Incretin analog 60, its structure is:

Incretin analog 61, its structure is:

Incretin analog 62, its structure is:

Incretin analog 63, its structure is:

Incretin analog 64, its structure is:

Incretin analog 65, its structure is:

Incretin analog 66, its structure is:

Incretin analog 67, its structure is:

Incretin analog 68, its structure is:

Incretin analog 69, its structure is:

Incretin analog 70, the structure of which is:

Incretin analog 71, its structure is:

Incretin analog 72, its structure is:

Incretin analog 73, its structure is:

Incretin analog 74, its structure is:

Incretin analog 75, its structure is:

Incretin analog 76, its structure is:

Incretin analog 77, its structure is:

Incretin analog 78, its structure is:

Incretin analog 79, its structure is:

Incretin analog 80, its structure is:

Incretin analog 81, its structure is:

Incretin analog 82, its structure is:

Incretin analog 83, its structure is:

Or incretin analog 84, whose structure is:

Embodiment 24. A pharmaceutical composition comprising the incretin analog or a pharmaceutically acceptable salt thereof according to any one of Embodiments 12-23, and one or more pharmaceutically acceptable excipients.

Embodiment 25. Use of the incretin analogue or a pharmaceutically acceptable salt thereof according to any one of Embodiments 12-23, or the pharmaceutical composition according to Embodiment 24 in the preparation of a drug.

Embodiment 26. The use according to embodiment 25, wherein the disease for which the medicament is used to treat is hyperglycemia, diabetes, impaired glucose tolerance and/or obesity.

Embodiment 27. The use according to embodiment 26, wherein the diabetes is type 1 diabetes, type 2 diabetes, maturity-onset diabetes of the juvenile and/or gestational diabetes.

Embodiment 28. A method of treating a disease in a subject in need thereof, comprising administering to the subject the incretin analog or a pharmaceutically acceptable salt thereof according to any one of Embodiments 12-23, or the pharmaceutical composition according to Embodiment 24.

Embodiment 29. The method according to embodiment 28, wherein the disease is hyperglycemia, diabetes, impaired glucose tolerance and/or obesity.

Embodiment 30. The method according to embodiment 29, wherein the diabetes is type 1 diabetes, type 2 diabetes, maturity-onset diabetes of the juvenile and/or gestational diabetes.

Embodiment 31. A modifier having the following structure:

in,

Lk represents a linker, R1 _is The fatty acid group of the structure shown, R ₂ is iodoacetyl or bromoacetyl, n is an integer from 8 to 20, and the wavy line in Formula I indicates the connection position between R ₁ and Lk; wherein,

Lk is selected from one or more combinations of Ala, D-Ala, Arg, Asp, Asn, Glu, D-Glu, γ-Glu, Gln, Gly, Lys, ε-Lys, Pro, Phe, Ser or AEEA, or is absent; preferably, it is one or more combinations of Asp, Glu, γ-Glu, Gly, Ser, AEEA, Lys or ε-Lys; more preferably, it is one or more combinations of Glu, γ-Glu, AEEA, Lys or ε-Lys.

Embodiment 32. The modifier according to embodiment 31, wherein the Lk is selected from one or a combination of γ-Glu, AEEA or ε-Lys.

Embodiment 33. The modifier according to embodiment 31, wherein

(1) The Lk is -AEEA-γ-Glu-γ-Glu-, and the modifier structure is:

(2) The Lk is -γ-Glu-AEEA-AEEA-, and the modifier structure is: or,

(3) The Lk is -γ-Glu-ε-Lys-ε-Lys-, and the modifier structure is:

Embodiment 34. The modifier according to embodiment 31, wherein the modifier is selected from the following structures:

Embodiment 35. A method for preparing the incretin analog or a pharmaceutically acceptable salt thereof according to any one of embodiments 12-23, comprising preparing the polypeptide according to any one of embodiments 1-11 by chemical synthesis and/or recombinant expression, preparing the modifier according to any one of embodiments 31-34 by chemical synthesis, and linking the modifier to the polypeptide via Cys in the polypeptide.

Definition and Description

Unless otherwise indicated, the following terms used in this disclosure have the following meanings. A particular term should not be considered to be indefinite or unclear in the absence of a special definition, but should be understood according to the common meaning in the art. When a trade name appears in this disclosure, it is intended to refer to the corresponding commercial product or its active ingredient.

The term "mercapto" refers to a -SH group.

The structure of the term "iodoacetyl" is as follows: The structure of the term "bromoacetyl" is as follows:

Unless otherwise specified, the key is a solid wedge. and dotted wedge key Indicates the absolute configuration of a stereocenter.

Unless otherwise specified, when a group has a bondable site, the bond from that site to another group is represented by a wavy line. express.

The compounds of the present disclosure (e.g., incretin analogs or modifications) may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomeric forms of the compounds of the present disclosure (including but not limited to diastereomers, enantiomers and atropisomers and mixtures thereof such as racemic mixtures) are constituents of the present disclosure. The compounds of the present disclosure containing asymmetric carbon atoms may be isolated in optically pure form or in racemic form. Optically pure forms may be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

The term "treatment" generally refers to an operation to obtain a desired pharmacological and/or physiological effect. The effect may be preventive, in terms of completely or partially preventing a disease or its symptoms; and/or may be therapeutic, in terms of partially or completely stabilizing or curing a disease and/or side effects produced by the disease. As used herein, "treatment" encompasses any treatment of a patient's disease, including but not limited to preventing the occurrence or recurrence of a disease, inhibiting a disease or disease state, alleviating symptoms of a disease, reducing any direct or indirect pathological consequences of a disease, preventing the metastasis of a disease, slowing the progression of a disease, improving or alleviating the state of a disease, extending the frequency and duration of symptom-free periods, and resolving or improving the prognosis of a disease.

A "therapeutically effective amount" is any amount of a drug that protects a subject from the onset of disease or promotes disease regression, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of impairment or disability resulting from disease affliction. The ability of a drug to promote disease regression can be evaluated using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in in vitro assays.

The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "excipient" refers to any ingredient other than the active ingredient (e.g., the incretin analogs of the present disclosure). The choice of excipient will depend to a large extent on factors such as the specific mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipients" include, but are not limited to, excipients, diluents, fillers, binders, disintegrants, solubilizers, stabilizers, colorants, flavorings, surfactants, emulsifiers, buffers or encapsulating materials. The amount of each excipient may vary within the conventional range in the art.

As used herein, "pharmaceutically acceptable salts" may be, for example, metal salts, ammonium salts, salts formed with organic bases, salts formed with inorganic acids, salts formed with organic acids, salts formed with basic or acidic amino acids, and the like. Non-limiting examples of metal salts include, but are not limited to, salts of alkali metals, such as sodium salts, potassium salts, and the like; salts of alkaline earth metals, such as calcium salts, magnesium salts, barium salts, and the like; aluminum salts, and the like. Non-limiting examples of salts formed with organic bases include, but are not limited to, salts formed with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and the like. Non-limiting examples of salts formed with inorganic acids include, but are not limited to, salts formed with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like. Non-limiting examples of salts formed with organic acids include, but are not limited to, salts formed with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, malic acid, maleic acid, tartaric acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Non-limiting examples of salts formed with basic amino acids include, but are not limited to, salts formed with arginine, lysine, ornithine, etc. Non-limiting examples of salts formed with acidic amino acids include, but are not limited to, salts formed with aspartic acid, glutamic acid, etc.

As used herein, "amino protecting group" refers to a chemical group introduced to protect the amino group participating in the condensation reaction, which can be selected from tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z) or 9-fluorenyl-methoxycarbonyl (Fmoc), preferably 9-fluorenyl-methoxycarbonyl (Fmoc).

Herein, the terms "pharmaceutical composition" and "formulation" have the same meaning and are used interchangeably.

As used herein, the terms "subject," "patient," or "subject" are used interchangeably. "Subject," "patient," or "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some embodiments, the terms "subject," "patient," or "subject" are mammals. In some embodiments, the subject, patient, or subject is a mouse. In some embodiments, the subject, patient, or subject is a human.

When describing the position of amino acids in the polypeptides disclosed herein, it refers to sequential numbering based on the N-terminal starting amino acid of the corresponding amino acid sequence as the first position. For example, the 20th Cys in the polypeptide with an amino acid sequence such as SEQ ID NO:7 refers to the 20th Cys in the sequence, starting with the first amino acid at the N-terminus of SEQ ID NO:7 as position 1.

The words “comprise”, “comprise” or “comprises” and their English variations such as comprises or comprising should be construed in an open, non-exclusive sense, ie, “including but not limited to”.

Herein, singular terms include plural referents and vice versa unless the context clearly dictates otherwise.

As used herein, "about" means within the acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation as practiced in the art. Alternatively, "about" may mean a range of up to ±5%, such as fluctuations within ±2%, within ±1%, or within ±0.5% of a given specific numerical range. When a specific value is given in the present disclosure or claims, unless otherwise indicated, the meaning of "about" should be considered to be within the acceptable error range for that specific value. In this document, unless otherwise indicated, all values of drug doses, times, step parameters, or conditions are modified by "about" by default.

The abbreviations of natural amino acid residues in polypeptides are as follows: Phe or F for phenylalanine, Leu or L for leucine, Ile or I for isoleucine, Met or M for methionine, Val or V for valine, Ser or S for serine, Pro or P for proline, Thr or T for threonine, Ala or A for alanine, Tyr or Y for tyrosine, His or H for histidine, Gln or Q for glutamine, Asn or N for asparagine, Lys or K for lysine, Asp or D for aspartic acid, Glu or E for glutamic acid, Cysteine is Cys or C, Trp or W for tryptophan, Arg or R for arginine, and Gly or G for glycine. The abbreviations of non-natural amino acid residues in polypeptides are as follows: D-alanine is D-Ala, D-glutamic acid is D-Glu, and 2-aminoisobutyric acid is Aib.

The structure of Aib in the polypeptide is as follows:

In the polypeptide amino acid sequence disclosed herein, when _X2 is Aib, the α-amino group of Aib is connected to the α-carboxyl group of Y, and the α-carboxyl group of Aib is connected to the α-amino group of E.

The term "AEEA" means 2-[2-(2-aminoethoxy)ethoxy]acetic acid.

The glutamic acid residue in which the γ-carboxyl group participates in the formation of the peptide bond and the α-carboxyl group is a free carboxyl group is γ-Glu (also written as gGlu or γGlu), which has the following structure:

The lysine residue in which the ε-amino group participates in the formation of the peptide bond and the α-amino group is a free amino group is ε-Lys (also written as eLys or εLys), which has the following structure:

For the purpose of description and disclosure, all patents, patent applications and other identified publications are expressly incorporated herein by reference. These publications are provided only because their disclosure precedes the filing date of the present disclosure. All statements regarding the dates of these documents or the representations of the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates of these documents or the contents of these documents. Furthermore, any reference to these publications herein does not constitute an admission that the publications are part of the common general knowledge in the art in any country.

Example

For the sake of clarity, the present disclosure is further described with examples, but the examples are not intended to limit the scope of the present disclosure. Similarly, the present disclosure is not limited to any specific preferred embodiments described herein. It should be understood by those skilled in the art that equivalent substitutions made to the technical features of the present disclosure, or corresponding improvements, still fall within the scope of protection of the present disclosure. Unless otherwise specified, the reagents used in the following examples are all commercially available products, and the preparation of the solutions can adopt conventional techniques in the art.

Unless otherwise specified, the operations disclosed herein will be performed using conventional techniques of organic synthesis, biochemistry, protein purification, etc. within the skill of the art, or according to product instructions, which techniques are fully explained in the literature. In the following examples, efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account.

The detection, purification and characterization methods used in the examples are as follows: Tirzepatide used in the examples is from Eli Lilly and Company, and Semaglutide is from Novo Nordisk.

The list of abbreviations is as follows:

1. RP-HPLC analysis method:

The chromatographic column was Kromasil 100-3.5-C4 4.6×150 mm, the flow rate was 1 mL/min, the detection wavelength was 215 nm, the mobile phase A was 0.05% TFA (v/v) in water, and the mobile phase B was 0.05% TFA (v/v) in acetonitrile. The gradient elution parameters are shown in Table 1.

Table 1. Gradient elution parameters

2. RP-HPLC purification method A:

The chromatographic column was Kromasil 300-10-C4 10×250 mm, the flow rate was 5 mL/min, the detection wavelengths were 215 nm and 280 nm, the mobile phase A was 50 mM PBS (pH 8.0) containing 5 mM TCEP, and the mobile phase B was acetonitrile. The gradient elution parameters are shown in Table 2.

Table 2. Gradient elution parameters

3. RP-HPLC purification method B:

The chromatographic column was Kromasil 300-10-C4 10×250 mm, the flow rate was 5 mL/min, the detection wavelengths were 215 nm and 280 nm, the mobile phase A was 50 mM PBS (pH 8.0), and the mobile phase B was acetonitrile. The gradient elution parameters are shown in Table 3.

Table 3. Gradient elution parameters

Example 1: Solid phase synthesis and purification of polypeptides

Polypeptide 1, polypeptide 2, polypeptide 3, polypeptide 4, polypeptide 5, polypeptide 6, polypeptide 7, polypeptide 8, polypeptide 9, polypeptide 10, polypeptide 11, polypeptide 12, polypeptide 13, polypeptide 14 and polypeptide 15 were prepared according to the following method. The amino acid sequences of polypeptide 1 to polypeptide 15 are shown in SEQ ID NO: 1-15, respectively.

1.1 Materials and reagents

MBHA resin (Xi’an Lanxiao Technology New Materials Co., Ltd., catalog number: 09-191010), with a substitution value (SD) of 0.29 mmol/g.

The protected amino acids are: Fmoc-Ser(tBu)-OH (CAS: 71989-33-8), Fmoc-Pro-OH (CAS: 71989-31-6), Fmoc-Ala-OH (CAS: 35661-39-3 ), Fmoc-Gly-OH (CAS: 29022-11-5), Fmoc-Leu-OH (CAS: 35661-60-0), Fmoc-Trp (Boc)-OH (CAS: 143824-78-6), Fmoc-Gln(Trt)-OH(CAS: 132327-80-1), Fmoc-Val-OH (CAS: 68858-20-8), Fmoc -Phe-OH(CAS: 35661-40-6), Fmoc-Lys(Boc)-OH(CAS: 71989-26-9), Fmoc-Glu(OtBu)-OH(CAS: 71989-18-9), Fmoc-Cys(Trt)-OH(CAS: 103213-32-7), Fmoc-Tyr(tBu)-OH(CAS: 71989-38-3), Fmoc-Asp(OtBu)-OH(CAS: 71989-14 -5), Fmoc-Thr(tBu)-OH (CAS: 71989-35-0), Fmoc-Aib-OH (CAS: 94744-50-0).

Synthesis reagents: HOBt, DIC, DMF, DCM, piperidine.

1.2 Instruments

CS-BIO peptide synthesizer, Waters 600 semi-preparative HPLC, Beckman centrifuge, Buchi rotary evaporator

1.3 Operation steps

a. Solid Phase Synthesis of Peptides

Weigh 1g of MBHA resin, place it in the reactor of CS-BIO peptide synthesizer, add 10mL DCM, soak for 2h, add 15mL of 20% PIP (v/v) DMF solution, mix for 25min to remove the amino protecting group, and wash the resin with DCM 6 times; weigh Fmoc-Ser (tbu) -OH, DIC and HOBt equivalent to three times the amount of resin material, add 10mL DMF to dissolve, add to the reactor for reaction, the reaction temperature is room temperature, and the reaction progress is monitored by ninhydrin reaction. If it is monitored as blue-purple, it means that the condensation reaction is incomplete, and if it is colorless, the reaction is complete. After the reaction is completed, wash the resin with DCM 6 times; then add 15mL of 20% PIP (v/v) DMF solution, mix for 20min to remove the amino protecting group, and wash the resin with DMF 6 times. At this time, ninhydrin is detected as blue-purple.

The coupling reaction of the next amino acid was continued according to the above method, in the order of C-terminus to N-terminus, and this cycle was repeated until all the amino acids were coupled. The last amino acid was deprotected and washed, and the resin was washed 6 times with DCM to obtain a resin peptide.

b. Lysis and precipitation

The resin peptide was dried under vacuum and weighed. The cleavage reagent was prepared according to TFA:TIS:water=95:2.5:2.5 (v:v:v), and the cleavage reagent was added to the resin peptide at a ratio of 10 mL of cleavage reagent per 1 g of resin peptide, stirred at room temperature for 3 hours, filtered, and part of the TFA was removed by rotary evaporation, and then 10 times the volume of icy ether was added to precipitate the polypeptide, centrifuged, and the precipitate was repeatedly washed with icy ether for 4-5 times, vacuum dried, and the crude polypeptide was obtained and weighed.

c. Purification

The crude polypeptide was purified according to RP-HPLC purification method A, and samples with a purity of more than 90% were combined and verified by LC-MS mass spectrometry. The theoretical value m/z and the measured value m/z are shown in Table 4.

Table 4. Theoretical and measured m/z values of peptides

Example 2: Synthesis of Modifiers

Example 2.1: Synthesis of C18 Diacid-AEEA-gGlu-gGlu-Lys(ε-bromoacetyl)-COOH

The structure of C18 diacid-AEEA-gGlu-gGlu-Lys(ε-bromoacetyl)-COOH is shown in Formula III-1.

(1) Materials and reagents

2-Chlorotrityl chloride (2-CTC) resin (Xi’an Lanxiao Technology New Materials Co., Ltd., catalog number: 03-210310), with a substitution value of 1.15 mmol/g.

The synthetic materials are: Fmoc-Lys(Alloc)-OH (CAS: 146982-27-6), Fmoc-AEEA-OH (CAS: 166108-71-0), Fmoc-Glu-OtBu (CAS: 84793-07-7), and octadecanediolatoic acid mono-tert-butyl ester (CAS: 843666-40-0).

Synthesis reagents: HOBt, DIC, DMF, DCM, PIP, DIEA.

(2) Instruments

CS-BIO peptide synthesizer, Waters 600 semi-preparative high performance liquid chromatograph, Beckman centrifuge, BUCHI rotary evaporator.

(3) Synthetic modifiers

a. Solid Phase Synthesis

Weigh 1.00 g of 2-CTC resin, place it in a CS-BIO peptide synthesizer reactor, add 10 mL of DCM, swell for 1 hour, weigh 2-3 times the amount of Fmoc-Lys(Alloc)-OH and 4-6 times the amount of DIEA, add 10 mL of DMF to dissolve, put it into the reactor, react at room temperature for 2 hours, that is, the first amino acid is coupled to the resin, then wash the resin with DCM 6 times, and the substitution value (SD) of the resin at this time is measured to be 0.57 mmol/g; then add 10 mL of 20% PIP (v/v) DMF solution, mix for 10 minutes to remove the amino protecting group Fmoc, repeat this process once, and then wash the resin with DCM 6 times. To couple the second amino acid, weigh three times the amount of Fmoc-Glu-otBu, HOBt and DIC as much as the amount of resin material, add 10mL DMF/DCM (v:v＝1:1) mixed solvent to dissolve, react at room temperature, monitor the reaction progress with ninhydrin, and the reaction is complete when it is colorless. Wash the resin with DCM 6 times. Then, continue the coupling reaction of γ-Glu, AEEA and octadecane dioic acid according to the above coupling method, and repeat this cycle until the coupling is completed to obtain the resin peptide.

Weigh tetrakis(triphenylphosphine)palladium (equivalent to 0.1 times the amount of resin substance) and phenylsilane (equivalent to 10 times the amount of resin substance), add 15 mL DCM to dissolve, and then add to the reactor to react with the resin peptide for 25 minutes. This process must be carried out under light-proof and nitrogen protection conditions. After the reaction is completed, wash the resin peptide with DCM (15 mL × 6 times, 2 minutes each time), 0.02M N,N-diethyldithiocarbamic acid DMF solution (15 mL × 3 times, 2 minutes each time) and DMF (15 mL × 6 times, 2 minutes each time), and take a small amount of resin peptide for ninhydrin detection. The purple-black color of the resin particles indicates that the deprotection is complete.

Add bromoacetyl bromide twice the amount of the resin material and 10 mL of DMF. React at room temperature for 1 h. Monitor the progress of the reaction with ninhydrin. The reaction is complete when it turns colorless. Wash the resin peptide 6 times with DCM, dry the resin peptide in vacuum and set aside.

b. Lysis and precipitation

The cleavage reagent was prepared according to TFA:TIS: _H2O =95:2.5:2.5 (v:v:v), and added to the resin peptide at a ratio of 10 mL of cleavage reagent per 1 g of resin peptide. The reaction was carried out at room temperature for 1 hour, and the resin was removed by filtration. The filtrate was rotary evaporated at 40°C to remove TFA as much as possible, and the modifier was precipitated with 7-10 times the volume of ice ether relative to the cleavage reagent, placed in a -20°C refrigerator for 20 minutes, centrifuged, and vacuum dried to obtain the modifier.

LC-MS determined that m/z = 968.43 [M+H] ⁺ , consistent with the theoretical molecular weight of 966.96 Da.

Example 2.2: Synthesis of C20 Diacid-AEEA-gGlu-gGlu-Lys(ε-bromoacetyl)-COOH

C20 diacid-AEEA-gGlu-gGlu-Lys(ε-bromoacetyl)-COOH was synthesized by referring to the method of Example 2.1, and its structure is shown in Formula III-2.

LC-MS determined that m/z = 996.55 [M+H] ⁺ , consistent with the theoretical molecular weight of 995.02 Da.

Example 2.3: Synthesis of C18 Diacid-gGlu-AEEA-AEEA-Lys(ε-bromoacetyl)-COOH

C18 diacid-gGlu-AEEA-AEEA-Lys(ε-bromoacetyl)-COOH was synthesized by referring to the method of Example 2.1, and its structure is shown in Formula III-3.

LC-MS determined that m/z = 984.49 [M+H] ⁺ , consistent with the theoretical molecular weight of 983.01 Da.

Example 2.4: Synthesis of C20 Diacid-gGlu-AEEA-AEEA-Lys(ε-bromoacetyl)-COOH

C20 diacid-gGlu-AEEA-AEEA-Lys(ε-bromoacetyl)-COOH was synthesized by referring to the method of Example 2.1, and its structure is shown in Formula III-4.

LC-MS determined that m/z = 1012.51 [M+H] ⁺ , consistent with the theoretical molecular weight of 1011.06 Da.

Example 2.5: Synthesis of C18 Diacid-gGlu-eLys-eLys-Lys(ε-bromoacetyl)-COOH

C18 diacid-gGlu-eLys-eLys-Lys(ε-bromoacetyl)-COOH was synthesized by referring to the method of Example 2.1, and its structure is shown in Formula III-5.

LC-MS determined that m/z = 950.53 [M+H] ⁺ , consistent with the theoretical molecular weight of 949.04 Da.

Example 2.6: Synthesis of C20 Diacid-gGlu-eLys-eLys-Lys(ε-bromoacetyl)-COOH

C20 diacid-gGlu-eLys-eLys-Lys(ε-bromoacetyl)-COOH was synthesized by referring to the method of Example 2.1, and its structure is shown in Formula III-6.

LC-MS determined that m/z = 978.56 [M+H] ⁺ , consistent with the theoretical molecular weight of 977.09 Da.

Example 3: Synthesis of incretin analogs

Synthesis of incretin analog 1:

(1) Modification of Peptide 1

Dissolve polypeptide 1 in water, adjust the pH to 7-8 with saturated ammonium bicarbonate (the concentration of polypeptide 1 is 2 mg/ml), dissolve C18 diacid-AEEA-gGlu-gGlu-Lys(ε-bromoacetyl)-COOH with saturated ammonium bicarbonate to make the concentration of C18 diacid-AEEA-gGlu-gGlu-Lys(ε-bromoacetyl)-COOH be 10 mg/mL, add C18 diacid-AEEA-gGlu-gGlu-Lys(ε-bromoacetyl)-COOH solution to polypeptide 1 solution at a molar ratio of 3:1 between C18 diacid-AEEA-gGlu-gGlu-Lys(ε-bromoacetyl)-COOH and polypeptide 1, react at room temperature for 0.5-1h, monitor according to RP-HPLC analysis method, and obtain a reaction solution.

(2) Purification and characterization of incretin analog 1

The reaction solution was purified according to RP-HPLC purification method B, and samples with a purity of more than 90% were combined and freeze-dried. LC-MS mass spectrometry confirmed that m/z = 1259.8760 [M+4H] ⁴⁺ was measured, which was consistent with the theoretical value m/z = 1260.37 [M+4H] ⁴⁺ .

Synthesis of other incretin analogs:

Referring to the synthesis method of incretin analog 1, incretin analogs 2 to 42, incretin analog 59, incretin analog 60, incretin analog 65, incretin analog 66, incretin analog 72 to incretin analog 84 were prepared and characterized. The theoretical value m/z and the measured value m/z of each incretin analog are shown in Table 5.

Incretin analogs 1:

Incretin analog 2:

Incretin analog 3:

Incretin analog 4:

Incretin analog 5:

Incretin analog 6:

Incretin analog 7:

Incretin analogs 8:

Incretin analogs 9:

Incretin analog 10:

Incretin analogue 11:

Incretin analog 12:

Incretin analog 13:

Incretin analog 14:

Incretin analog 15:

Incretin analog 16:

Incretin analog 17:

Incretin analog 18:

Incretin analog 19:

Incretin analog 20:

Incretin analog 21:

Incretin analog 22:

Incretin analog 23:

Incretin analog 24:

Incretin analog 25:

Incretin analog 26:

Incretin analog 27:

Incretin analog 28:

Incretin analog 29:

Incretin analog 30:

Incretin analog 31:

Incretin analog 32:

Incretin analog 33:

Incretin analog 34:

Incretin analog 35:

Incretin analog 36:

Incretin analog 37:

Incretin analog 38:

Incretin analog 39:

Incretin analog 40:

Incretin analog 41:

Incretin analog 42:

Incretin analog 59:

Incretin analog 60:

Incretin analog 65:

Incretin analog 66:

Incretin analog 72:

Incretin analog 73:

Incretin analog 74:

Incretin analog 75:

Incretin analog 76:

Incretin analog 77:

Incretin analog 78:

Incretin analog 79:

Incretin analog 80:

Incretin analog 81:

Incretin analog 82:

Incretin analog 83:

Incretin analog 84:

Table 5. Theoretical and Measured m/z Values of Incretin Analogs

Example 4: Determination of in vitro activity of incretin analogs by time-resolved fluorescence energy transfer (TR-FRET)

cAMP Hunter ^TM CHO-K1 GIPR Gs Cell Line (eurofins DiscoverX, catalog number: 95-0146C2) and GLP-1R-CRE-bla CHO-K1 (Life Technologies, catalog number: K1783) cell lines were used, and Tirzepatide was used as a positive control to measure the in vitro cell activity of the samples to be tested (peptides, incretin analogs). Tirzepatide can specifically bind to GIPR and GLP-1R on the cell surface, leading to the activation of membrane adenylate cyclase and the formation of cyclic adenosine monophosphate (cAMP) in the cell. The amount of cAMP generated is positively correlated with the concentration of Tirzepatide. The amount of intracellular cAMP generated can be accurately determined by time-resolved fluorescence resonance energy transfer immunoassay (TR-FRET). The detection kit is the LANCE UltracAMP Kit of PerkinElmer. The experimental data were analyzed using Prism5 software, with sample concentration as the X-axis and the corresponding fluorescence ratio as the Y-axis. A four-parameter equation was used for fitting, and the dose-response curves of the positive control and the sample to be tested were drawn to calculate the EC ₅₀ value. The EC ₅₀ ratios of the samples and the positive control are shown in Table 6.

Table 6. In vitro activities of peptides and incretin analogs

“/” indicates inactive.

Example 5: In vivo pharmacodynamic evaluation in mice

64 SPF normal ICR mice (Shanghai Lingchang Biotechnology Co., Ltd.) were raised in an animal laboratory at 20-26°C with 12-hour light and dark alternations, with free access to water and food, and were randomly divided into 8 groups before the experiment, with 8 mice in each group. The specific grouping and dosing regimen are shown in Table 7.

Table 7. Grouping and dosing regimen

The mice were fasted 2h before administration. Tirzepatide or incretin analogs were injected subcutaneously in the abdomen of each administration group, with an injection volume of 10mL/kg. The normal control group was injected with an equal volume of normal saline. The day of administration was D0. The mice were weighed and recorded every day, and the weight change rate was calculated. Starch solution (10.6g/kg, calculated according to the average weight of mice) was given 71h after administration. Blood was collected from the tail tip of the mice, and blood sugar was measured about 4.5h before administration and 72h after administration with a blood glucose meter, and the blood sugar change rate of each group of mice was calculated.

The formula for calculating the weight change rate is as follows:

Body weight change rate (%) = (Dt body weight - D0 body weight) / D0 body weight × 100%, wherein Dt body weight is the body weight on Dt, and D0 body weight is the body weight on D0. The body weight change rate of mice is shown in Figures 1 and 2.

The formula for calculating blood sugar change rate is as follows:

Blood sugar change rate (%) = (blood sugar increase value of normal control group - blood sugar increase value of each drug administration group) / blood sugar increase value of normal control group × 100%.

The blood glucose change rates of mice are shown in Table 8.

Table 8. Changes in blood glucose in ICR mice 72 hours after administration

Example 6: In vivo pharmacokinetic evaluation in beagle dogs

Beagle dogs (Nanjing Yadong Experimental Animal Research Center) (weight 10-13kg) were randomly divided into groups, 3 in each group, and administered with a dose of 10nmol/kg by subcutaneous injection. Blood was collected from the eye socket within 30min before administration and 0.5, 1, 2, 4, 8, 10, 24, 48, 72, 96, 120, 144, and 168h after administration to prepare the plasma samples to be tested. 100μL of the plasma sample to be tested and the standard curve sample were drawn, and the N ¹⁵ -labeled semaglutide (prepared in-house) internal standard solution (solvent: acetonitrile: methanol = 3:2 (v:v) solution containing 0.1% (v/v) glacial acetic acid) was added, vortexed to mix, and the supernatant was diluted for LC-MS analysis. The non-compartmental model was used to fit and calculate the pharmacokinetic parameters in each group of beagle dogs, including peak time (T _max ), peak drug concentration (C _max ), area under the concentration-time curve (AUC _(0-t) ), and elimination half-life (t _1/2 ), which are shown in Table 9-1 and Table 9-2.

Table 9-1. Pharmacokinetic parameters in beagle dogs

Table 9-2. Pharmacokinetic parameters in beagle dogs

Example 7: Pharmacodynamic evaluation in a type 2 diabetes mouse model

25 SPF db/db mice (from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were housed in an animal laboratory at 20-26°C with alternating light and dark for 12 hours each, with free access to water and food, and were randomly divided into 4 groups before the experiment, with 5 mice in each group. The specific grouping and dosing regimen are shown in Table 10.

Table 10. Grouping and dosing regimen

Each treatment group received a subcutaneous injection of Semaglutide or incretin analogs in the abdomen with an injection volume of 2 mL/kg, and the normal control group received an injection of the same volume of PBS. The day of administration was D1, and the drug was administered once every 3 days for 60 consecutive days. Blood sugar was measured before the first and second administrations, 48h and 72h after administration, and blood sugar was measured 72h after each subsequent administration, and blood sugar was measured 48h after the last administration (i.e. D60).

[Corrected 25.04.2024 in accordance with Article 91]
The blood glucose levels in db/db mice are shown in FIG3 , wherein “↓” indicates drug administration on the same day.

Example 8: In vivo pharmacodynamic evaluation in diet-induced obese (DIO) mice

Twenty SPF DIO mice (from Shanghai Model Organisms Science Co., Ltd.) were housed in an animal laboratory at 20-26°C with alternating light and dark for 12 hours each, with free access to water and food, and were randomly divided into 4 groups before the experiment, with 4 mice in each group. The specific grouping and dosing regimen are shown in Table 11.

Table 11. Grouping and dosing regimen

Each treatment group was injected subcutaneously with Semaglutide or incretin analogs at a volume of 2 mL/kg, and the normal control group was injected with the same volume of PBS. The drug was administered once every 3 days, with the day of administration being D1, for 46 consecutive days. During the administration process, body weight was measured every 3 days. The weight change rate was calculated as follows:

Body weight change rate (%) = (Dt body weight - D0 body weight) / D0 body weight × 100%, wherein Dt body weight is the body weight on Dt, and D0 body weight is the body weight on D0.

[Corrected 25.04.2024 in accordance with Article 91]
The rate of body weight change in DIO mice is shown in FIG4 .

The sequence information of the present disclosure is summarized in Table S1 below.

Table S1. Summary of sequence information

Although the present disclosure has been described in detail above with general descriptions and specific implementation schemes, it is obvious to those skilled in the art that some modifications or improvements may be made to the present disclosure. Therefore, these modifications or improvements made without departing from the spirit of the present disclosure are within the scope of protection claimed by the present disclosure.

Claims (17)

A polypeptide or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence as shown in YX ₂ EGTFTSDYSX ₁₂ X ₁₃ LEX ₁₆ QAAX ₂₀ X ₂₁ FVX ₂₄ WLX ₂₇ AGGPSSGAPPPS, wherein: _X2 is selected from Aib or Ala, _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is selected from Cys, Tyr, Ile, Ala or Aib, X ₁₆ is selected from Cys or Lys, X ₂₀ is selected from Cys or Lys, X ₂₁ is selected from Cys or Leu, X ₂₄ is selected from Cys or Gln, _X27 is selected from Cys, Leu, Ile or Val, and There is one and only one Cys in the amino acid sequence; and the polypeptide optionally has a C-terminal amidation modification. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, wherein _X13 is selected from Cys, Tyr or Ile. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein _X2 is Aib. The polypeptide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein _X12 is Tyr, _X13 is selected from Cys or Tyr, _X16 is selected from Cys or Lys, _X20 is selected from Cys or Lys, _X21 is selected from Cys or Leu, _X24 is selected from Cys or Gln, and _X27 is selected from Cys or Leu. The polypeptide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein _X20 is Cys. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1 or 3, wherein: (a) _X12 is selected from Tyr, Lys, Ile or Ser, _X13 is Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is selected from Leu, Ile or Val; (b) _X12 is selected from Lys, Ile or Ser, _X13 is selected from Tyr, Ile, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu; (c) _X12 is Lys, _X13 is selected from Tyr, Ala or Aib, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu; or (d) _X12 is Tyr, _X13 is selected from Tyr or Ile, _X16 is Lys, _X20 is Cys, _X21 is Leu, _X24 is Gln, and _X27 is Leu. The polypeptide or a pharmaceutically acceptable salt thereof according to claim 1, wherein the polypeptide or a pharmaceutically acceptable salt thereof comprises any one selected from the following amino acid sequences: (1)YAibEGTFTSDYSYCLEKQAAKLFVQWLLAGGPSSGAPPPS; (2)YAibEGTFTSDYSYYLECQAAKLFVQWLLAGGPSSGAPPPS; (3)YAibEGTFTSDYSYYLEKQAACLFVQWLLAGGPSSGAPPPS; (4)YAibEGTFTSDYSYYLEKQAAKCFVQWLLAGGPSSGAPPPS; (5)YAibEGTFTSDYSYYLEKQAAKLFVCWLLAGGPSSGAPPPS; (6)YAibEGTFTSDYSYYLEKQAAKLFVQWLCAGGPSSGAPPPS; (7)YAibEGTFTSDYSYILEKQAACLFVQWLLAGGPSSGAPPPS; (8)YAibEGTFTSDYSYILEKQAACLFVQWLIAGGPSSGAPPPS; (9)YAibEGTFTSDYSYILEKQAACLFVQWLVAGGPSSGAPPPS; (10)YAibEGTFTSDYSSILEKQAACLFVQWLLAGGPSSGAPPPS; (11)YAibEGTFTSDYSKILEKQAACLFVQWLLAGGPSSGAPPPS; (12)YAibEGTFTSDYSIILEKQAACLFVQWLLAGGPSSGAPPPS; (13)YAibEGTFTSDYSKYLEKQAACLFVQWLLAGGPSSGAPPPS; (14)YAibEGTFTSDYSKALEKQAACLFVQWLLAGGPSSGAPPPS; or (15)YAibEGTFTSDYSKAiBLEKQAACLFVQWLLAGGPSSGAPPPS. An incretin analog or a pharmaceutically acceptable salt thereof, comprising the polypeptide according to any one of claims 1 to 7, and a substituent, The substituent is linked to the polypeptide via Cys in the polypeptide; Preferably, the substituent is linked to the polypeptide via Cys at position 13, 16, 20, 21, 24 or 27 of the polypeptide; preferably, it is linked to the polypeptide via Cys at position 20 of the polypeptide. The incretin analog or a pharmaceutically acceptable salt thereof according to claim 8, wherein the substituent comprises a fatty acid group; preferably, the fatty acid group comprises a carbon chain, and the carbon chain comprises at least 8 consecutive -CH ₂ - structural units; preferably, the carbon chain comprises 8-20 consecutive -CH ₂ - structural units; more preferably, the carbon chain comprises 8, 10, 12, 14, 16, 18 or 20 consecutive -CH ₂ - structural units; most preferably, the carbon chain comprises 16 or 18 consecutive -CH ₂ - structural units; Preferably, the structure of the fatty acid group is as follows: wherein n is an integer between 8 and 20; preferably, n is 8, 10, 12, 14, 16 or 18; More preferably, n is 16 or 18. According to the incretin analog or a pharmaceutically acceptable salt thereof according to claim 8, the structure of the substituent is as follows: in, Lk represents a linker, R1 _is The fatty acid group of the structure shown, n is an integer from 8 to 20, the wavy line in formula I represents the connection position of R ₁ and Lk, and the wavy line in formula II represents the connection position of the substituent and the polypeptide; Optionally, the Lk is selected from one or more combinations of Ala, D-Ala, Arg, Asp, Asn, Glu, D-Glu, γ-Glu, Gln, Gly, Lys, ε-Lys, Pro, Phe, Ser or AEEA, or is absent; preferably one or more combinations of Asp, Glu, γ-Glu, Gly, Ser, AEEA, Lys or ε-Lys; more preferably one or more combinations of Glu, γ-Glu, AEEA, Lys or ε-Lys; more preferably one or more combinations of γ-Glu, AEEA or ε-Lys; Preferably, wherein: (1) The Lk is -AEEA-γ-Glu-γ-Glu-, and the substituent structure is: (2) The Lk is -γ-Glu-AEEA-AEEA-, and the substituent structure is: or, (3) The Lk is -γ-Glu-ε-Lys-ε-Lys-, and the substituent structure is: The incretin analog or a pharmaceutically acceptable salt thereof according to claim 8, wherein the substituent is selected from the following structures:

The wavy lines in formulas II-1, II-2, II-3, II-4, II-5 and II-6 indicate the position at which the substituent is attached to the polypeptide. According to claim 8, the incretin analog or a pharmaceutically acceptable salt thereof, the incretin analog is selected from: incretin analog 1, whose structure is:
Incretin analog 2, its structure is:
Incretin analog 3, its structure is:
Incretin analog 4, its structure is:
Incretin analog 5, its structure is:
Incretin analog 6, its structure is:
Incretin analog 7, its structure is:
Incretin analog 8, its structure is:
Incretin analog 9, its structure is:
Incretin analog 10, the structure of which is:
Incretin analog 11, its structure is:
Incretin analog 12, the structure of which is:
Incretin analog 13, its structure is:
Incretin analog 14, the structure of which is:
Incretin analog 15, its structure is:
Incretin analog 16, its structure is:
Incretin analog 17, its structure is:
Incretin analog 18, its structure is:
Incretin analog 19, its structure is:
Incretin analog 20, the structure of which is:
Incretin analog 21, its structure is:
Incretin analog 22, the structure of which is:
Incretin analog 23, its structure is:
Incretin analog 24, the structure of which is:
Incretin analog 25, its structure is:
Incretin analog 26, its structure is:
Incretin analog 27, its structure is:
Incretin analog 28, its structure is:
Incretin analog 29, its structure is:
Incretin analog 30, the structure of which is:
Incretin analog 31, its structure is:
Incretin analog 32, its structure is:
Incretin analog 33, its structure is:
Incretin analog 34, the structure of which is:
Incretin analog 35, its structure is:
Incretin analog 36, its structure is:
Incretin analog 37, its structure is:
Incretin analog 38, its structure is:
Incretin analog 39, its structure is:
Incretin analog 40, the structure of which is:
Incretin analog 41, its structure is:
Incretin analog 42, the structure of which is:
Incretin analog 43, its structure is:
Incretin analog 44, its structure is:
Incretin analog 45, its structure is:
Incretin analog 46, its structure is:
Incretin analog 47, its structure is:
Incretin analog 48, its structure is:
Incretin analog 49, its structure is:
Incretin analog 50, the structure of which is:
Incretin analog 51, its structure is:
Incretin analog 52, the structure of which is:
Incretin analog 53, its structure is:
Incretin analog 54, the structure of which is:
Incretin analog 55, its structure is:
Incretin analog 56, its structure is:
Incretin analog 57, its structure is:
Incretin analog 58, its structure is:
Incretin analog 59, its structure is:
Incretin analog 60, its structure is:
Incretin analog 61, its structure is:
Incretin analog 62, its structure is:
Incretin analog 63, its structure is:
Incretin analog 64, its structure is:
Incretin analog 65, its structure is:
Incretin analog 66, its structure is:
Incretin analog 67, its structure is:
Incretin analog 68, its structure is:
Incretin analog 69, its structure is:
Incretin analog 70, the structure of which is:
Incretin analog 71, its structure is:
Incretin analog 72, its structure is:
Incretin analog 73, its structure is:
Incretin analog 74, its structure is:
Incretin analog 75, its structure is:
Incretin analog 76, its structure is:
Incretin analog 77, its structure is:
Incretin analog 78, its structure is:
Incretin analog 79, its structure is:
Incretin analog 80, its structure is:
Incretin analog 81, its structure is:
Incretin analog 82, its structure is:
Incretin analog 83, its structure is: or Incretin analog 84, its structure is:
A pharmaceutical composition comprising the incretin analogue or a pharmaceutically acceptable salt thereof according to any one of claims 8 to 12, and one or more pharmaceutically acceptable excipients. A method for treating a disease in a subject in need thereof, comprising administering to the subject the incretin analog or a pharmaceutically acceptable salt thereof according to any one of claims 8 to 12, or the pharmaceutical composition according to claim 13; Preferably, the disease is hyperglycemia, diabetes, impaired glucose tolerance and/or obesity; Preferably, the diabetes is type 1 diabetes, type 2 diabetes, maturity-onset diabetes of the juvenile and/or gestational diabetes. A modifier having the following structure: in, Lk represents a linker, R1 _is The fatty acid group of the structure shown, R ₂ is iodoacetyl or bromoacetyl, n is an integer from 8 to 20, and the wavy line in Formula I indicates the connection position between R ₁ and Lk; wherein, Lk is selected from one or more combinations of Ala, D-Ala, Arg, Asp, Asn, Glu, D-Glu, γ-Glu, Gln, Gly, Lys, ε-Lys, Pro, Phe, Ser or AEEA, or is absent; preferably one or more combinations of Asp, Glu, γ-Glu, Gly, Ser, AEEA, Lys or ε-Lys; more preferably one or more combinations of Glu, γ-Glu, AEEA, Lys or ε-Lys; more preferably one or more combinations of γ-Glu, AEEA or ε-Lys; Preferably, wherein (1) The Lk is -AEEA-γ-Glu-γ-Glu-, and the modifier structure is: (2) The Lk is -γ-Glu-AEEA-AEEA-, and the modifier structure is: or, (3) The Lk is -γ-Glu-ε-Lys-ε-Lys-, and the modifier structure is: The modifier according to claim 15, wherein the modifier is selected from the following structures:

A method for preparing an incretin analogue or a pharmaceutically acceptable salt thereof according to any one of claims 8 to 12, comprising preparing the polypeptide according to any one of claims 1 to 7 by chemical synthesis and/or recombinant expression, preparing the modifier according to claim 15 or 16 by chemical synthesis, and linking the modifier to the polypeptide via Cys in the polypeptide.