WO2025162423A1 - Triagonist for glp-1r, gipr, and gcgr - Google Patents

Abstract

A triagonist for GLP-1R, GIPR, and GCGR, which can be used for preventing and/or treating metabolic disorders.

Description

Triple agonist targeting GLP-1R, GIPR, and GCGR Technical Field

The present invention belongs to the field of biomedicine and relates to a triple agonist targeting GLP-1R, GIPR and GCGR.

Background Art

Glucagon-like peptide 1 (GLP-1) acts on multiple tissues and plays a key regulatory role in the body's blood sugar balance, fat metabolism, food intake and other processes, exerting blood sugar lowering, weight loss and even cardioprotective effects. In addition, glucagon (GCG), glucose-dependent insulinotropic polypeptide (GIP), fibroblast growth factor 21 (FGF21) and others also play an important role in these metabolic processes. Glucagon-like peptide 1 receptor (GLP-1R), glucose-dependent insulinotropic polypeptide receptor (GIPR) and glucagon receptor (GCGR) are all present in pancreatic β cells. In the process of regulating blood sugar balance, β cell function can be improved by activating GLP-1R and/or GIPR and/or GCGR, protecting the pancreatic islets while increasing insulin secretion and controlling blood sugar.

Activation of the GLP-1 receptor (GLP-1R) can affect the central nervous system, producing anorexia and slowing gastric emptying, thereby reducing food intake. The weight loss effects of GLP-1 receptor agonists have been well-established in clinical practice. Newer-generation weight loss molecules leverage GLP-1 activity by incorporating other complementary gastrointestinal hormones. For example, activation of the GCGR can reduce lipogenesis and increase fat conversion to heat energy. Therefore, GLP-1R/GCGR dual agonists can reduce food intake while increasing energy consumption, thereby achieving enhanced weight loss. Innovent Biologics' Mazdutide is a GLP-1R/GCGR dual agonist. The role of GIP in weight loss remains controversial, but its glucose-lowering effects are well established, stimulating insulin secretion directly or indirectly to maintain blood sugar stability. While the weight loss effects of GIP remain controversial, Eli Lilly's GLP-1R/GIPR dual-targeting Tirzepatide is already on the market.

Currently, a key area of multi-target synergy based on GLP-1 receptor agonists is the GLP-1R/GIPR/GCGR tri-agonist. Eli Lilly's Retatrutide is the most advanced in development and has already initiated Phase III clinical trials. Furthermore, Hanmi's triple-target efocipegtrutide is in Phase II clinical trials, primarily targeting non-alcoholic steatohepatitis (NASH). The triple-target NN9423, jointly developed by Novo and Roche, and Sanofi's triple-target SAR441255 were both terminated after Phase I clinical trials. GGG tri-agonists developed by other companies are still in the preclinical stage.

The market potential in areas such as weight loss and NASH is enormous, and there is a persistent demand for better drugs. The downstream signaling of GLP-1R, GIPR, and GCGR receptors is complex, potentially with positive and negative feedback regulation on glucose and lipid metabolism. This presents opportunities for comprehensive regulation of glucose and lipid metabolism, but also increases the difficulty of designing an ideal multi-active peptide.

Summary of the Invention

The purpose of the present disclosure is to provide a polypeptide or its derivative having a novel polypeptide sequence and a pharmaceutically acceptable salt molecule thereof, which has triple agonist activity against GLP-1R, GIPR and GCGR simultaneously, and has the characteristics of strong biological activity and high stability, and can meet the clinical treatment needs of various metabolic diseases related to GLP-1R, GIPR and GCGR. Compared with Eli Lilly's (GCGR/GIPR/GLP-1R) triple agonist Retatrutide, the GLP-1R, GIPR and GCGR disclosed in the present disclosure have good performance in terms of agonist activity, stability, and animal weight loss. The triple agonist disclosed in the present disclosure provides improved blood sugar control and enhanced weight loss, and can be used as a therapeutic agent for diabetes, obesity and related diseases.

In a first aspect, the present disclosure provides a triple agonist compound of GLP-1R, GIPR and GCGR or a pharmaceutically acceptable salt thereof, comprising a compound having a sequence shown in Formula I:
Xaa1-Aib-Gln-Gly-Thr-Xaa6-Thr-Ser-Xaa9-Xaa10-Ser-Ile-Xaa13-Xaa14-Asp-Xaa16-
Xaa17-Lys-Xaa19-Xaa20-Xaa21-Phe-Ile-Glu-Xaa25-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Ser-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-R ₁ (general formula I)

in,

Xaa1 is Tyr or His;

Xaa6 is αMeF(2F), Phe, αMeF, αMeF(2,4F), F(2F) or F(2,4F);

Xaa9 is Asp or αMeD;

Xaa10 is Tyr, αMeY, or Leu;

Xaa13 is Leu, Tyr, or αMeL;

Xaa14 is Leu or αMeL;

Xaa16 is Lys, Har, 3Pal, Arg, or NεMeK;

Xaa17 is Har, Arg, or Gln;

Xaa19 is Gln or Ala;

Xaa20 is 4Pal, 3Pal, Aib, Gln, Ala or NεMeK;

Xaa21 is Asp, Glu, or Ala;

Xaa25 is Tyr, Trp, 3Pal, or αMeY;

Xaa28 is Ala or Glu;

Xaa29 is Gly, dAla, or Aib;

Xaa30 is Gly or Lys;

Xaa31 is Pro, dPro, or trans-Hyp;

Xaa32 is Pro or Ser;

Xaa34 is Aib or Gly;

Xaa35 is Lys or Ala;

Xaa36 is Pro or trans-Hyp;

Xaa37 is Pro or trans-Hyp;

Xaa38 is Pro or trans-Hyp; and/or

Xaa39 is Lys or Ser;

and at least one amino acid Lys residue is connected to a fatty acid side chain; R ₁ is a carboxyl terminal (C-terminal) modification of the compound sequence, and R ₁ is selected from -NH ₂ or -OH.

In some embodiments, the above-mentioned compound or a pharmaceutically acceptable salt thereof has a sequence shown in Formula I and at least one amino acid Lys residue is linked to a fatty acid side chain; _R1 is a carboxyl terminal (C-terminal) modification of the compound sequence, _{and R1} is selected from _-NH2 or -OH.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, the connection can be direct or indirect (eg, via a linker).

In some embodiments, any of the above compounds or pharmaceutically acceptable salts thereof, in general formula I, contain any one or more of the following amino acids: Xaa1 is Tyr, Xaa9 is Asp, Xaa10 is Tyr, Xaa13 is Leu, Xaa14 is Leu, Xaa16 is Lys, Xaa19 is Ala, Xaa21 is Glu, Xaa29 is Gly, Xaa30 is Gly, Xaa31 is Pro, Xaa34 is Gly, Xaa35 is Ala, Xaa36 is Pro, Xaa37 is Pro, Xaa38 is Pro, and Xaa39 is Ser.

In some embodiments, in the above compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa1 is Tyr.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa9 is Asp.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa10 is Tyr.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa13 is Leu.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa14 is Leu.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa16 is Lys.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa19 is Ala.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa21 is Glu.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa29 is Gly.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa30 is Gly.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa31 is Pro.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa34 is Gly.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa35 is Ala.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa36 is Pro.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa37 is Pro.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa38 is Pro.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, in Formula I, Xaa39 is Ser.

In some embodiments, in any of the above compounds or pharmaceutically acceptable salts thereof, in Formula I, the compound is linked to the fatty acid side chain via the epsilon amino group on the Lys residue at position 18.

In some embodiments, in any of the above compounds or pharmaceutically acceptable salts thereof, the fatty acid side chain is selected from

One or more of, wherein x is any integer from 4 to 38;

Preferably, the fatty acid side chain is selected from:

HOOC(CH ₂ ) ₁₄ CO-, HOOC(CH ₂ ) ₁₅ CO-, HOOC(CH ₂ ) ₁₆ CO-, HOOC(CH ₂ ) ₁₇ CO-, HOOC(CH ₂ ) ₁₈ CO-, HOOC(CH ₂ ) ₁₉ CO-, HOOC(CH ₂ ) ₂₀ CO-, HOOC(CH ₂ ) ₂₁ CO- and HOOC(CH ₂ ) ₂₂ One or more of CO-.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, the fatty acid side chain is linked to the amino acid Lys residue via a linker.

In some embodiments, in any of the above compounds or pharmaceutically acceptable salts thereof, the linker is selected from

One or more of, wherein m is 0, 1, 2 or 3; n is 1 or 2; p is any integer from 1 to 5;

Preferably, the connector is:

wherein m is 0, 1, 2 or 3, and n is 1; more preferably, wherein m is 1, and n is 1.

In some embodiments, the compounds are M1-M42 and M45-M69 shown in Table 2 herein.

In some embodiments, any of the above compounds or pharmaceutically acceptable salts thereof have the general formula II:
Tyr-Aib-Gln-Gly-Thr-Xaa6-Thr-Ser-Asp-Tyr-Ser-Ile-Leu-Leu-Asp-Lys-Xaa17-Lys-Ala-
Xaa20-Glu-Phe-Ile-Glu-Xaa25-Leu-Leu-Xaa28-Gly-Gly-Pro-Xaa32-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R ₁ (General formula II)

in,

Xaa6 is αMeF(2F), αMeF, or αMeF(2,4F);

Xaa17 is Har or Arg;

Xaa20 is 4Pal, 3Pal, Aib or Ala;

Xaa25 is Tyr or Trp;

Xaa28 is Ala or Glu; and/or

Xaa32 is Pro or Ser.

In some embodiments, in any of the aforementioned compounds or pharmaceutically acceptable salts thereof, R ₁ is "NH ₂ ".

In some embodiments, the compound is M36, M37, M40, M52, M53, and M68 as shown in Table 2 herein.

In a second aspect, the present disclosure provides a polypeptide, the amino acid sequence of which is shown in the above-mentioned general formula I or the above-mentioned general formula II.

In a third aspect, the present disclosure provides nucleic acid molecules encoding the above-mentioned polypeptides.

In a fourth aspect, the present disclosure provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

In a fifth aspect, the present disclosure provides a recombinant cell comprising the aforementioned nucleic acid molecule or the aforementioned recombinant vector and capable of expressing and optionally secreting the polypeptide.

In a sixth aspect, the present disclosure provides a method for preparing any of the aforementioned compounds or pharmaceutically acceptable salts thereof or the aforementioned polypeptides;

Preferably, the preparation method comprises the steps of preparing the compound or the polypeptide using chemical methods and/or biological methods;

Preferably, the chemical method includes liquid phase or solid phase polypeptide synthesis; the biological method includes molecular biology method and/or cell biology method.

In a seventh aspect, the present disclosure provides a pharmaceutical composition comprising any one of the aforementioned compounds or pharmaceutically acceptable salts thereof or the aforementioned polypeptides, and pharmaceutically acceptable excipients.

The pharmaceutical compositions of the present disclosure may be administered by any suitable route known in the art, including, but not limited to, oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, and/or cerebrospinal;

Preferably, the pharmaceutical composition is in the form of solid, liquid or semi-solid.

In some embodiments, the pharmaceutical compositions of the present disclosure are formulated as liquids suitable for administration by injection or infusion.

In some embodiments, the pharmaceutical compositions of the present disclosure further include one or more additional pharmaceutically active ingredients;

The pharmaceutically active ingredient may have a beneficial effect on preventing and/or treating metabolic disorders;

Preferably, the metabolic disorder is diabetes or a diabetes-related disorder, or obesity or an obesity-related disorder;

More preferably, the diabetes-related disorder is insulin resistance, glucose intolerance, elevated fasting blood glucose, prediabetes, type 1 diabetes, type 2 diabetes, gestational diabetes hypertension, dyslipidemia, bone-related disorder, or a combination thereof;

The obesity-related disorder is obesity-related inflammation, obesity-related gallbladder disease, or obesity-induced sleep apnea; or may be associated with a disorder selected from atherogenic dyslipidemia, dyslipidemia, elevated blood pressure, hypertension, a prothrombotic state, and a proinflammatory state, or a combination thereof;

The active ingredient is, for example, a pharmaceutical active ingredient for treating diabetes, obesity, dyslipidemia or hypertension, such as an insulin receptor agonist, a GIP receptor agonist, a glucagon receptor agonist, amylin receptor agonist, an FGF21 receptor agonist, a GDF15 receptor agonist, a PCSK9 inhibitor, and the like.

In some embodiments, the pharmaceutical compositions of the present disclosure can be used in combination with one or more other pharmaceutical compositions. In some embodiments, different pharmaceutical compositions can be administered to patients in need simultaneously, sequentially, or separately. In some embodiments, different pharmaceutical compositions are administered to patients in need in a temporal order, for example, in 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month, 2 months, 3 months or longer, administering each pharmaceutical composition once, twice, three times or more per day simultaneously, sequentially, or separately;

The one or more other pharmaceutical compositions are, for example, insulin receptor agonists, GIP receptor agonists, glucagon receptor agonists, amylin receptor agonists, FGF21 receptor agonists, GDF15 receptor agonists, PCSK9 inhibitors, metformin, α-glucosidase inhibitors, SGLT2 inhibitors, thiazolidinediones, sulfonylureas, gliflozins, etc.

In an eighth aspect, the present disclosure provides any of the aforementioned compounds or pharmaceutically acceptable salts thereof, the aforementioned polypeptides, and any of the aforementioned pharmaceutical compositions for use in treatment.

In a ninth aspect, the present disclosure provides use of any of the aforementioned compounds or pharmaceutically acceptable salts thereof, the aforementioned polypeptides, or any of the aforementioned pharmaceutical compositions in the preparation of a medicament for preventing and/or treating metabolic disorders;

Preferably, the metabolic disorder is diabetes or a diabetes-related disorder, or obesity or an obesity-related disorder;

More preferably, the diabetes-related disorder is insulin resistance, glucose intolerance, elevated fasting blood glucose, prediabetes, type 1 diabetes, type 2 diabetes, gestational diabetes hypertension, dyslipidemia, bone-related disorder, or a combination thereof;

The obesity-related disorder is obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH); or may be associated with a disorder selected from atherogenic dyslipidemia, dyslipidemia, increased blood pressure, hypertension, a prothrombotic state, and a proinflammatory state, or a combination thereof.

In a tenth aspect, the present disclosure provides a method for preventing and/or treating metabolic disorders, comprising the step of administering a therapeutically effective amount of any one of the aforementioned compounds or pharmaceutically acceptable salts thereof, the aforementioned polypeptides, or any one of the aforementioned pharmaceutical compositions to a patient in need thereof;

Preferably, the metabolic disorder is diabetes or a diabetes-related disorder, or obesity or an obesity-related disorder;

More preferably, the diabetes-related disorder is insulin resistance, glucose intolerance, elevated fasting blood glucose, prediabetes, type 1 diabetes, type 2 diabetes, gestational diabetes hypertension, dyslipidemia, bone-related disorder, or a combination thereof;

The obesity-related disorder is obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH); or may be associated with a disorder selected from atherogenic dyslipidemia, dyslipidemia, increased blood pressure, hypertension, a prothrombotic state, and a proinflammatory state, or a combination thereof.

The medicaments of the present disclosure may be administered to the patient by any suitable route known in the art, including but not limited to oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic and/or cerebrospinal.

In some embodiments, in any of the methods described above, the administration cycle of any of the compounds described above or their pharmaceutically acceptable salts, the polypeptides described above or the pharmaceutical compositions described above is one or more times daily, weekly, every two weeks, every three weeks, every 1 month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or every 12 months, for example, daily, weekly, every two weeks, every three weeks, every 1 month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or every 12 months. 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111

In some embodiments, in any of the above methods, the total number of times any of the above compounds or pharmaceutically acceptable salts thereof, the above polypeptides or any of the above pharmaceutical compositions are administered can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

The present invention also provides a kit comprising any of the above-mentioned compounds or pharmaceutically acceptable salts thereof, the above-mentioned polypeptides or any of the above-mentioned pharmaceutical compositions and optionally instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A shows the changes in body weight of mice in each drug-treated group over time in the C57BL/6J DIO mouse model.

Figure 1B shows the changes in cumulative food intake over time in each drug-treated group of mice in the C57BL/6J DIO mouse model.

DETAILED DESCRIPTION

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

The present invention will be further described below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

definition

Unless otherwise stated, the terms used herein have the following definitions.

Herein, the conventional one-letter and three-letter codes for the natural amino acids are used, as well as the commonly recognized three-letter codes for other amino acids, such as homoarginine (Har).All amino acid residues in the peptides of the present disclosure are in the L-configuration unless otherwise specified.

In the present disclosure, Aib represents α-aminoisobutyric acid, Har represents homoarginine, αMeF(2F) represents α-methyl-2-fluoro-L-phenylalanine, 4Pal represents 3-(4-pyridyl)-L-alanine, 3Pal represents 3-(3-pyridyl)-L-alanine, dAla represents D-alanine, dPro represents D-proline, trans-Hyp represents trans-hydroxyproline, F(2F) represents 2-fluoro-L-phenylalanine, F(2,4F) represents 2,4-difluoro-L-phenylalanine, NεMeK represents Nε-methyl-L-lysine, αMeL represents α-methyl-L-leucine, αMeY represents α-methyl-L-tyrosine, αMeF represents α-methyl-L-phenylalanine, αMeF(2,4F) represents α-methyl-2,4-difluoro-L-phenylalanine, and αMeD represents α-methyl-L-aspartic acid. The structural formulas of these amino acids are as follows:

The sequences disclosed herein are sequences in which an "NH ₂ " moiety is incorporated at the carboxyl terminus (C-terminus) of the sequence, and the "NH ₂ " moiety represents an amide group (-CONH ₂ ) at the C-terminus.

The terms "polypeptide" or "peptide" or "protein" are used interchangeably. A "polypeptide" or "peptide" or "protein" is any chain of two or more amino acids, including naturally occurring or non-naturally occurring (e.g., synthetic) amino acids or amino acid analogs, regardless of post-translational modification (e.g., glycosylation or phosphorylation), wherein the amino acids in any chain are covalently linked by peptide bonds.

The terms “comprising,” “including,” “having,” or “containing” should be understood to imply the inclusion of specified components but not the exclusion of any other components.

The term "agonist" refers to a substance (ligand) that activates the receptor.

The terms "compound," "triagonist compound," "derivative," or "polypeptide derivative" are used interchangeably herein to refer to a polypeptide having a fatty acid side chain modification.

The terms "patient," "subject," and "individual" are used interchangeably and include humans and non-human animals, including mammals such as monkeys, rats, mice, cows, pigs, goats, sheep, dogs, and cats.

When used in reference to an animal, human, subject, cell, tissue, organ, or biological fluid, "administering" and "treating" refer to contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering" and "treating" can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating a cell includes contacting an agent with a cell and contacting an agent with a fluid, wherein the fluid is contacted with the cell. "Administering" and "treating" also mean the in vitro and ex vivo treatment of a cell, for example, by an agent, diagnostic agent, binding composition, or by other cells.

As used herein, "preventing" or "treating" includes delaying the development of symptoms associated with a disease and/or lessening the severity of symptoms that will or are expected to develop due to the disease. The terms also encompass alleviating existing symptoms, preventing additional symptoms, and alleviating or preventing the underlying causes of the symptoms. Thus, the terms indicate that a beneficial result has been conferred on a vertebrate subject, such as a human, suffering from a disease.

As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of a GLP-1R, GIPR and GCGR triagonist compound, or a pharmaceutically acceptable salt thereof, or a polypeptide thereof, which, when administered alone or in combination with another therapeutic agent to a cell, tissue or subject, is effective in preventing or alleviating the disease or condition being treated. A therapeutically effective dose further refers to an amount of the GLP-1R, GIPR and GCGR triagonist compound, or a pharmaceutically acceptable salt thereof, or a polypeptide thereof, sufficient to cause a reduction in symptoms, such as treating, curing, preventing or alleviating the associated medical condition, or increasing the rate of treatment, cure, prevention or alleviating the symptoms of the condition. The effective amount for a particular subject may vary depending on a variety of factors, such as the disease being treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects. The effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. When administered to an individual, the therapeutically effective amount refers to the active ingredient alone. When a combination is administered, a therapeutically effective amount refers to the combined amount of the active ingredients that produces the therapeutic effect, regardless of whether they are administered in combination, serially or simultaneously. A therapeutically effective amount will alleviate symptoms by typically at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40% and most preferably at least 50%.

A triple agonist compound of GLP-1R, GIPR and GCGR or a pharmaceutically acceptable salt thereof

The compound disclosed herein is a triple agonist of GLP-1R, GIPR and GCGR, and has agonist activity on the three receptors of GLP-1R, GIPR and GCGR.

GLP-1R agonists bind to GLP-1R and activate signaling through the receptor, for example, by generating cAMP or inducing Ca ²⁺ release. The activity of GLP-1R agonists is assessed by measuring the signal generated by luciferase driven by a cAMP response element.

Similarly, GIPR agonists can bind to GIPR and activate signaling through the receptor, for example by generating cAMP or inducing Ca2 ⁺ release. The activity of GIPR agonists is assessed herein by measuring the signal generated by luciferase driven by a cAMP response element.

Similarly, GCGR agonists can bind to GCGR and activate signaling through the receptor, for example by generating cAMP or inducing Ca ²⁺ release. The activity of GCGR agonists is assessed herein by measuring the signal generated by luciferase driven by a cAMP response element.

The present disclosure obtains a triple agonist compound with good activity and stability for GLP-1R, GIPR, and GCGR through polypeptide design. Compared with the GCGR/GIPR/GLP-1R triple agonist Retatrutide, the triple agonist compound of the present disclosure shows significantly better drug effects and demonstrates good GCGR/GIPR/GLP-1R activation synergy.

The triple agonist compounds of GLP-1R, GIPR and GCGR designed in the present disclosure are shown in Table 2.

In particular, the amino acid sequence of the polypeptide derivative M36 of the present disclosure is shown in SEQ ID NO: 36, and the epsilon amino group at the amino acid residue K at position 18 is linked to 2*AEEA+γGlu+C20 diacid via an amide bond;

Its structural formula is shown in formula (III):

The amino acid sequence of the polypeptide derivative M37 of the present disclosure is shown in SEQ ID NO: 37, and the epsilon amino group at the amino acid residue K at position 18 is linked to the 2*AEEA+γGlu+C20 diacid via an amide bond;

Its structural formula is shown in formula (IV):

The amino acid sequence of the polypeptide derivative M40 of the present disclosure is shown in SEQ ID NO: 40, and the epsilon amino group at the amino acid residue K at position 18 is linked to the 2*AEEA+γGlu+C20 diacid via an amide bond;

Its structural formula is shown in formula (V):

The amino acid sequence of the polypeptide derivative M52 of the present disclosure is shown in SEQ ID NO: 52, and the epsilon amino group at the amino acid residue K at position 18 is linked to the 2*AEEA+γGlu+C20 diacid via an amide bond;

Its structural formula is shown in formula (VI):

The amino acid sequence of the polypeptide derivative M53 of the present disclosure is shown in SEQ ID NO: 53, and the epsilon amino group at the amino acid residue K at position 18 is linked to the 2*AEEA+γGlu+C20 diacid via an amide bond;

Its structural formula is shown in formula (VII):

The amino acid sequence of the polypeptide derivative M68 of the present disclosure is shown in SEQ ID NO: 68, and the epsilon amino group at the amino acid residue K at position 18 is linked to the 2*AEEA+γGlu+C20 diacid via an amide bond;

Its structural formula is shown in formula (VIII):

It should be understood that the compounds of the present disclosure can also be provided in the form of salts. Pharmaceutically acceptable salts include salts in the form of anions and salts in the form of cations. Some examples of salts in the form of anions include hydrochlorides, citrates, chloride salts, and acetates. Preferably, the salt is acetate. Some examples of salts in the form of cations include salts in which the cation is selected from the group consisting of alkali metals (e.g., sodium and potassium), alkaline earth metals (e.g., calcium), and the like.

In addition, the compounds of the present invention can also form coordination complexes with metal ions (such as Mn2 ⁺ and Zn2 ⁺ ), thereby existing in the form of complexes. Due to the presence of hydroxyl or carboxylic acid in the compounds of the present invention, the derivatives can also react with suitable carboxylic acids or alcohols to form esters, thereby existing in the form of esters. The compounds of the present invention can also exist in the form of prodrugs, and prodrugs can be converted into one of the parent compounds in vivo or in vitro. Generally, at least one biological activity of the compound will be reduced in the prodrug form, and can be activated by the conversion of the prodrug to release the compound or its metabolites. Some examples of prodrugs include the use of protecting groups, which can be removed in situ to release the active compound or to suppress the removal of drugs in the body.

Synthetic triagonist compounds or peptides

The triagonist compounds or polypeptides disclosed herein can be prepared using chemical and/or biological methods. Chemical methods are preferred, for example, liquid or solid phase peptide synthesis methods can be used to synthesize the triagonist compounds or polypeptides disclosed herein. Biological methods include molecular biology methods and cell biology methods.

The method for preparing the triagonist compound or polypeptide of the present disclosure may comprise the following steps:

Synthesize the disclosed triagonist compounds or polypeptides stepwise or by fragment assembly according to the polypeptide sequence by liquid or solid phase polypeptide synthesis; or

A nucleic acid construct encoding a polypeptide sequence of a tri-agonist compound is transferred into a host cell, and then after culturing under certain conditions for a period of time, a tri-agonist compound polypeptide product is obtained from the host cell culture, for example, the polypeptide (which may or may not contain non-natural amino acids) is expressed from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants, or animals) using recombinant technology; and the polypeptide is optionally modified (e.g., by introduction of non-natural amino acids, C-terminal modification, and/or fatty acid side chain modification) to obtain the tri-agonist compound; or

A nucleic acid construct encoding the polypeptide sequence of a tri-agonist compound is used to express a polypeptide of the tri-agonist compound (which may or may not contain unnatural amino acids) in a cell-free system, and the polypeptide is optionally modified (e.g., by introducing unnatural amino acids, C-terminal modifications, and/or fatty acid side chain modifications) to obtain a tri-agonist compound.

In some embodiments, the triagonist compounds of the present disclosure are prepared by solid phase peptide synthesis on a suitable resin. The solid phase peptide synthesis steps are well known in the art, for example, by attaching an N-terminally protected amino acid and its carboxyl terminus to an inert solid support carrying a cleavable linker to initiate solid phase synthesis. The solid support can be any polymer that allows initial amino acid coupling, such as MBHA resin. In some embodiments, Fmoc-Linker-MBHA-Resin is used as the starting material, and according to the solid phase synthesis method, amino acids with Fmoc-protecting groups are sequentially connected to obtain a protected linear polypeptide resin, during which the Fmoc-protecting groups are sequentially removed, and TBTU is used as a condensing agent for peptide grafting reaction. After obtaining a protected linear polypeptide resin, the peptide is cut from the resin, and then the side chain protecting groups and coupling building blocks are removed simultaneously to obtain a fatty acid-modified polypeptide resin. The peptide is cut from the resin and separated and purified by a chromatographic column, and then freeze-dried to obtain a powdered refined polypeptide derivative.

In some embodiments, recombinant technology is used to prepare the polypeptide sequence of the triagonist compound of the present disclosure. In this case, the present disclosure also provides a nucleic acid molecule encoding the polypeptide sequence of the triagonist compound of the present disclosure, the nucleotide sequence of which can be a codon-optimized sequence according to the host to be transferred; the nucleic acid molecule can be a DNA fragment or an RNA fragment, which can usually be obtained by amplification using a PCR instrument or artificial synthesis.

In some embodiments, the polypeptide sequence of the three agonist compounds of the present disclosure is prepared using recombinant technology. In this case, the present disclosure also provides a recombinant vector comprising the above-mentioned nucleic acid molecule; the recombinant vector includes a cloning vector and an expression vector, the cloning vector is used to replicate the relevant sequence, and the expression vector is used to express the relevant gene. The vector can be any vector commonly used in the art, such as a plasmid, a phage, a cosmid, a minichromosome or a virus. In addition to the nucleic acid encoding the polypeptide sequence of the above three agonist compounds, the expression vector can include not only a promoter for initiating transcription of the gene encoding the polypeptide sequence, but also a signal peptide sequence, a terminator for terminating transcription of the gene encoding the polypeptide sequence, and an enhancer sequence.

The method for constructing a recombinant expression vector can be any known method. The promoter described above, the nucleic acid encoding the polypeptide sequence, and other DNA segments (e.g., terminators, enhancers) if present, can be introduced into a suitable selected vector as a basis in a predetermined order. For example, a recombinant vector can be constructed by using restriction endonucleases and ligases, etc.

In some embodiments, the polypeptide sequence of the tri-agonist compound of the present invention is prepared using recombinant technology. In this case, the present disclosure also provides a recombinant cell comprising the above-mentioned recombinant vector, wherein the recombinant cell expresses the polypeptide sequence of the tri-agonist compound of the present invention with or without induction. In some embodiments, the method for constructing the recombinant cell comprises the following: transforming the recombinant expression vector into an expression host cell, culturing and adding an inducer to induce expression (if necessary) to obtain the polypeptide sequence of the tri-agonist compound. Further, the expression host cell is a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, plant cell, animal cell, etc.

More specifically, the method for constructing the above-mentioned recombinant cell comprises the following steps:

(1) Amplification of the gene encoding the polypeptide sequence of the tri-agonist compound;

(2) Construction of recombinant expression vector;

(3) The recombinant expression vector is transformed or transfected into the expression host cell;

(4) Optionally screen to obtain positive clones.

The polypeptides of the present disclosure may be secreted outside the cell or expressed on the cell surface or inside the cell.

In some embodiments, using recombinant technology to prepare the polypeptide sequences of the tri-agonist compounds of the present disclosure comprises the following steps:

(1) culturing the above-mentioned recombinant cells and adding an inducer to induce (if necessary) the expression of the polypeptide sequence of the triagonist compound of the present disclosure to obtain a cell culture;

(2) Optionally, isolating and purifying the polypeptide sequence of the tri-agonist compound of the present disclosure from the cell culture (eg, cells, cell culture supernatant).

Biological activity

In this disclosure, _EC50 values are used as a numerical measure of the potency of an agonist for a given receptor (i.e., GLP-1R, GIPR, and GCGR). The _EC50 value refers to the concentration that elicits 50% of the maximal effect. In the same assay for a specific receptor, a compound with a low _EC50 value can be considered to have a higher potency at the receptor.

The present invention tested the cellular activity of the triple agonist compound and found that the triple agonist compound of the present invention has good agonist activity for GLP-1R, GIPR and GCGR receptors, and can effectively activate the corresponding downstream pathways of GLP-1R, GIPR and GCGR receptors, and exert appropriate agonist effects.

Furthermore, the thermally accelerated stability test confirmed that the compound of the present disclosure has a stability comparable to or better than that of Eli Lilly's Retatrutide (M0).

Furthermore, the weight loss effect was evaluated in a mouse model, and the results showed that the various tri-agonist compounds disclosed herein exhibited significantly higher weight loss effects and food intake inhibition effects than Retatrutide.

Pharmaceutical composition

The triagonist compound or pharmaceutically acceptable salt or polypeptide of the present disclosure can be formulated into a pharmaceutical composition, wherein the triagonist compound or pharmaceutically acceptable salt or polypeptide thereof is present in a therapeutically effective amount.

The pharmaceutical compositions described herein contain, in addition to the active ingredient tri-agonist compound or its pharmaceutically acceptable salt or polypeptide, a pharmaceutically acceptable excipient. Those skilled in the art are familiar with pharmaceutically acceptable excipients, such as non-toxic fillers, stabilizers, diluents, carriers, solvents or other formulation excipients. For example, diluents, excipients, such as microcrystalline cellulose, mannitol, etc.; fillers, such as starch, sucrose, etc.; binders, such as starch, cellulose derivatives, alginates, gelatin and/or polyvinyl pyrrolidone; disintegrants, such as calcium carbonate and/or sodium bicarbonate; absorption enhancers, such as quaternary ammonium compounds; surfactants, such as cetyl alcohol; carriers, solvents, such as water, saline, kaolin, bentonite, etc.; lubricants, such as talc, calcium/magnesium stearate, polyethylene glycol, etc.

In some embodiments, the triagonist compounds of the present disclosure, or pharmaceutically acceptable salts or polypeptides thereof, are formulated into a liquid suitable for administration by injection or infusion.

The tri-agonist compounds or pharmaceutically acceptable salts or polypeptides disclosed herein can be used alone or in combination with any compound that is beneficial for preventing and/or treating metabolic disorders in a pharmaceutical composition, which is expected to enhance the beneficial therapeutic effects of the tri-agonist compounds or pharmaceutically acceptable salts or polypeptides disclosed herein. The active ingredients are, for example, active pharmaceutical ingredients for treating diabetes, obesity, dyslipidemia, or hypertension, such as insulin receptor agonists, GIP receptor agonists, glucagon receptor agonists, amylin receptor agonists, FGF21 receptor agonists, GDF15 receptor agonists, PCSK9 inhibitors, and the like.

The pharmaceutical composition of the present disclosure can also be used in combination with one or more other drugs, and such combination is expected to have a synergistic effect in preventing and/or treating metabolic disorders. The one or more other drugs are, for example, insulin receptor agonists, GIP receptor agonists, glucagon receptor agonists, amylin receptor agonists, FGF21 receptor agonists, GDF15 receptor agonists, PCSK9 inhibitors, metformin, α-glucosidase inhibitors, SGLT2 inhibitors, thiazolidinediones, sulfonylureas, glimepirides, etc.

Medical conditions

The formulations of the present disclosure can be used to prevent or treat metabolic disorders by administering an effective amount of a triagonist compound as described herein, or a pharmaceutically acceptable salt or polypeptide thereof.

The metabolic disorder herein may be diabetes or a diabetes-related disorder, or obesity or an obesity-related disorder.

The diabetes-related disorder is insulin resistance, glucose intolerance, elevated fasting blood glucose, prediabetes, type 1 diabetes, type 2 diabetes, gestational diabetes, hypertension, dyslipidemia, a bone-related disorder, or a combination thereof.

The obesity-related disorder is obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH); or may be associated with a disorder selected from atherogenic dyslipidemia, dyslipidemia, elevated blood pressure, hypertension, a prothrombotic state, and a proinflammatory state, or a combination thereof.

Example 1: Preparation of polypeptide derivatives

Using solid-phase organic synthesis, Fmoc-protected amino acid strategy, and solid-phase peptide synthesis (SPPS) technology, peptide derivatives were synthesized, cleaved, and purified to obtain the target product. Taking compound M0 (Retatrutide) in Table 2 as an example, the preparation process is as follows:

1. Solid phase synthesis:

Use Fmoc-Linker MBHA Resin (substitution degree = 0.32 mmol/g) and Fmoc/tBu technology to condense the amino acids from the C-terminus to the N-terminus (from right to left) according to the above peptide sequence and the method in Table 1.

Table 1 Synthesis procedures

The following amino acids were coupled sequentially:
A-01Fmoc-Ser(tBu)-OH,A-02Fmoc-Pro-OH,A-03Fmoc-Pro-OH,A-04Fmoc-Pro-OH,A-05Fmoc-
Ala-OH,A-06Fmoc-Gly-OH,A-07Fmoc-Ser(tBu)-OH,A-08Fmoc-Ser(tBu)-OH,A-09Fmoc-Pro-OH ,A-10Fmoc-Gly-OH,A-11Fmoc-Gly-OH,A-12Fmoc-Glu(OtBu)-OH,A-13Fmoc-Leu-OH,A-14Fmoc-L eu-OH,A-15Fmoc-Tyr(tBu)-OH,A-16Fmoc-Glu(OtBu)-OH,A-17Fmoc-Ile-OH,A-18Fmoc-Phe-OH ,A-19Fmoc-Ala-OH,A-20Fmoc-Aib-OH,A-21Fmoc-Gln(Trt)-OH,A-22Fmoc-Ala-OH,A-23Fmoc-Ly s(Dde)-OH,A-24Fmoc-Lys(Boc)-OH,A-25Fmoc-Asp(OtBu)-OH,A-26Fmoc-Leu-OH,A-27Fmoc-a- Me-Leu-OH,A-28Fmoc-Ile-OH,A-29Fmoc-Ser(tBu)-OH,A-30Fmoc-Tyr(tBu)-OH,A-31Fmoc-Asp( OtBu)-OH,A-32Fmoc-Ser(tBu)-OH,A-33Fmoc-Thr(tBu)-OH,A-34Fmoc-Phe-OH,A-35Fmoc-Thr( tBu)-OH,A-36Fmoc-Gly-OH,A-37Fmoc-Gln(Trt)-OH,A-38Fmoc-Aib-OH,A-39Boc-Tyr(tBu)-OH.

Finally, a linear polypeptide resin is formed. After the linear polypeptide resin is synthesized, the Dde protection is removed by the hydrazine hydrate method, and the following building blocks are coupled in sequence:
B01Fmoc-AEEA-OH,B02Fmoc-γ-Glu-OtBu,B03 C20 diacid-OtBu

The obtained polypeptide derivative resin is washed, transferred out, dried to a constant weight, and then cracked.

2. Peptide derivative resin cleavage:

Preparation of cleavage reagent: Calculate the amount of cleavage reagent (TFA: _H2O :EDT:TIS = 95:1:2:2 (volume ratio)) based on 1g of polypeptide derivative resin to 10ml±2ml of cleavage reagent. Place the required cleavage reagents _H2O , TFA, EDT, and TIS into the cleavage reaction bottle in sequence. Control the cleavage reagent temperature at 0-10°C.

The cleavage reagent was added to the peptide derivative resin with stirring. After the system temperature stabilized, the reaction was stirred at 25-30°C for 2.5 hours. The lysate was filtered and precipitated with 5 times the liquid volume of glacial ether. The precipitate was filtered and washed three times with 3 times the liquid volume of glacial ether. After drying under reduced pressure at room temperature, a crude solid product was obtained.

3. Purification and freeze-drying:

The crude product was ground finely, purified water was prepared, and the ground crude product was slowly added to the solution under stirring. Simultaneously, an acetonitrile aqueous solution was added dropwise. After the crude product was completely added and dissolved, it was filtered through a 0.45 μm microporous filter membrane. The crude product was purified using a C-18 preparative column with mobile phases A: 0.1% TFA/ _H2O , B: 0.1% TFA/ACN. Separation and purification were performed at room temperature using an appropriate gradient. The target product was collected, analyzed, tested, and classified. The impurity purity was required to be ≥90%. Unqualified target products were collected and separated and purified again using an appropriate gradient. The qualified main peak was freeze-dried under reduced pressure to obtain a powdered, refined polypeptide derivative lyophilized powder.

The remaining polypeptide derivatives in Table 2 were prepared in a similar manner.

Table 2 Peptide derivatives

In Table 2, M1-M69 represent the derivatives obtained by modifying the amino acid sequence with a fatty acid-containing side chain in the present application; the modification and fatty acid columns represent the position of the fatty acid side chain modification and the specific side chain connected to the position, respectively. Taking M0 as an example, the modification 17K and the fatty acid AEEA+γGlu+C20 diacid in the table refer to the fatty acid side chain modification on the 17th lysine of the polypeptide sequence of M0 (SEQ ID NO: 70), specifically, the modification is achieved by connecting the ε amino group on the lysine to the AEEA+γGlu+C20 diacid through an amide bond; taking M1 as an example, the modification 18K and the fatty acid 2*AEEA+γGlu+C20 diacid in the table refer to the fatty acid side chain modification on the 18th lysine of the polypeptide sequence of M1 (SEQ ID NO: 1), specifically, the modification is achieved by connecting the ε amino group on the lysine to the 2*AEEA+γGlu+C20 diacid through an amide bond. The amino acid at the C-terminus of the polypeptide derivative has an " _NH2 " modification.

The structural formula of AEEA+γGlu+C20 diacid is shown in formula (I):

The structural formula of 2*AEEA+γGlu+C20 diacid is shown in formula (II):

In formula (II), Part is the linker 2*AEEA+γGlu,

Part of it is the fatty acid side chain C20 diacid,

In the connector Part of it is γGlu.

The abbreviations used in the above preparation process have the following meanings:
AA：Amino Acid
AEEA: 2-(2-(2-Aminoethoxy)ethoxy)acetic acid
Boc：t-Butyloxy carbonyl, tert-Butyloxycarbonyl
ACN: Acetonitrile, acetonitrile
DMF：N,N-Dimethyl formamide
DIEA：N,N-Diisopropylethylamine
Dde：2-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, 2-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl
EDT: 1,2-Ethanedithiol
Fmoc：9-fluorenylmethyloxycarbonyl，9-fluorenylmethyloxycarbonyl
OtBu：O-tert-butyl ester, tert-butyl ester
Pip：Piperidine, piperidine
TBTU: O-(Benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium Tetrafluoroborate
tBu：tertiary butyl
TFA: Trifluoroacetic acid
TIS: Triisopropylsilane, triisopropylsilane
Trt：Triphenylmethyl，triphenylmethyl γGlu：γ-Glutamic Acid，γ-glutamic acid
C20 diacid: eicosanedioic acid

Example 2: Cellular activity detection of polypeptide derivatives

The purpose of this experiment is to detect the activity or efficacy of the polypeptide derivatives of the present disclosure as shown in Table 2 on human GLP-1, GIP and GCG receptors in vitro using luciferase assay.

Using standard methods, CHO-K1/Ga15/GCGR cells (GenScript Biotech, catalog number M00345) were transfected with the plasmid pGL4.29 [luc2P/CRE/Hygro] (Promega, catalog number E8471, in which the hygromycin selection marker was replaced with puromycin) containing multiple copies of a cAMP response element (CRE)-driven luciferase expression cassette. The cells were cultured in F12K + 10% FBS medium supplemented with puromycin (6 μg/mL), hygromycin (100 μg/mL), and G418 (400 μg/mL) to generate a CHO cell line stably transfected with GCGR/CRE-luc. Monoclonal cells were isolated by limiting dilution for subsequent viability testing.

The lyophilized powder of the polypeptide derivative obtained in Example 1 was dissolved in 20 mM phosphate buffer at pH 7.0 and diluted with growth medium (GLP-1R cells: DMEM medium containing 10% FBS, GIPR cells: DMEM medium containing 10% FBS, GCGR cells: F12K medium containing 10% FBS) to obtain polypeptide derivative samples with initial concentrations of 10 nM (GLP-1R), 20 nM (GIPR), and 200 nM (GCGR). The polypeptide derivative samples were then gradiently diluted with growth medium to obtain seven concentrations of samples with concentrations differing by 4 times (GLP-1R), 5 times (GIPR), and 8 times (GCGR). 50 μL of the sample assay solution of the corresponding concentration was added to each well of a white 96-well plate.

HEK293/CRE-Luc/GLP1R cell lines (GenScript Biotech Co., Ltd., catalog number M00562), HEK293T/GIPR/CRE-LUC (Xining Biotech, catalog number GIPRSCF001), and the above-constructed stably transfected CHO cells with GCGR/CRE-luc were resuspended in growth medium at 6 × 10 ⁵ cells/mL. 50 μL of the cell suspension was added to a white 96-well plate containing the sample assay solution. After incubation at 37°C, 5% CO ₂ for 24 hours, 100 μL of Luciferase substrate (Promega, catalog number E2650) was added to each well and incubated for 3 minutes. Luminescence was measured on a SPARK (TECAN) microplate reader using SparkControl Magellan 3.0 software. A standard curve was plotted based on the fluorescence values, and the EC ₅₀ was calculated.

The results are shown in Table 3.

Table 3 GLP-1R, GIPR, and GCGR activity results

Table 3 shows that the triple agonist compound disclosed herein has good agonist activity for GLP-1R, GIPR and GCGR receptors, and can effectively activate the corresponding downstream pathways of GLP-1R, GIPR and GCGR receptors, exerting a suitable agonist effect.

Example 3: Thermal Accelerated Stability Test

The lyophilized powder of the polypeptide derivative obtained in Example 1 was mixed with sodium dihydrogen phosphate and dissolved in ultrapure water to a final concentration of 1 mg/ml and 1.42 mg/ml, respectively. The solution was first adjusted to a pH of approximately 10.5 with a 0.5 M aqueous sodium hydroxide solution, and then adjusted to a pH of approximately 7.4 with a 10% by volume aqueous hydrochloric acid solution. The solution was then filtered through a 0.22 μm sterile filter in a clean bench and placed into a sterilized vial. The vial was capped and placed in a 40°C stability test chamber. The day of the experiment was designated as day 0, and testing was performed on the 7th day (7d) and the 30th day (30d). During the experiment, changes in the properties of the polypeptide derivative were observed and recorded. The sample was then centrifuged at 10,000 rpm and 4°C for 3 min. The supernatant was transferred to a liquid phase injection vial, and the concentration and purity of the polypeptide derivative were determined using the following liquid chromatography method.

Reverse phase chromatography conditions: Agilent 1260 high performance liquid chromatograph (reverse phase chromatography column: Sepax Bio-C18 4.6*250mm 3μm ); automatic sampler temperature: 15°C; column temperature: 25°C; detection wavelength: 280 nm; mobile phase A: 100% H ₂ O + 0.05% TFA, mobile phase B: 100% ACN; flow rate: 1.0 ml/min; elution gradient see Table 4.

Table 4 Liquid chromatography elution gradient list

The concentration changes of the polypeptide derivatives are shown in Table 5, and the purity changes are shown in Table 6.

Table 5 Concentration changes

Table 5 shows that the tri-agonist compounds of the present disclosure exhibited very low concentration changes in the 7-day or 30-day heat-accelerated stability test compared to when formulated, and their stability was comparable to or even better than that of Retatrutide within a reasonable error range.

Table 6 Purity changes detected by reverse phase chromatography

Similarly, Table 6 shows that the tri-agonist compounds of the present disclosure exhibited very low changes in molecular purity in the 7-day or 30-day thermal accelerated stability test compared to when formulated. Compared with Retatrutide, the purity changes were basically equivalent within a reasonable error range, and some molecules were even better.

In summary, from the perspective of changes in concentration and purification, the three agonist compounds disclosed herein all exhibited good stability, which was comparable to or better than the stability of Retatrutide.

Example 4: Animal Experiment

C57BL/6J DIO male mice (Beijing Huafukang Biotechnology Co., Ltd.) were used for this study. Mice averaged 40-60 g, were 31 weeks old (26 weeks into the high-fat modeling process), and were fed a high-fat diet, D12492. Mice were allowed to acclimate to the experimental environment for at least 10-14 days prior to the start of the experiment. The experimental animals were housed at a room temperature of 20°C to 23°C and a relative humidity of 40% to 50%. Purified water was provided from drinking bottles, with free access to water.

Each peptide derivative test group consisted of five mice. The experimental animals were subcutaneously injected once daily (QD) for 17 consecutive days with either a peptide derivative or vehicle (the vehicle was 20 mM PB, pH 7.4, and the peptide derivative was administered in a volume of 5 mL/kg). The day of the first dose was designated Day 1 (D1). The initial body weight and initial feed addition were recorded on Day 0. From Day 1 to Day 18, the body weight and remaining food were recorded daily. The body weight change rate and food intake were calculated, and any abnormalities were recorded and reported. The mice remained in good condition throughout the experiment, and no abnormalities were observed. Body weight change rate = (BWDn - _BWD1 )/ _BWD1 * 100%, where BW represents body weight, and _D1 and Dn represent Day 1 and Day N, respectively. Graphs represent mean ± standard error of the mean (SEM). Cumulative food intake is the total amount of food consumed by each group of animals up to a specific time point after dosing. The results are shown in Figures 1A and 1B. Compared with the vehicle group, the mice in each experimental group showed significant weight loss and food intake inhibition. For example, on day 18, even the group with the lowest weight loss effect still lost nearly 26% of its weight. Even more surprising is that after 2 weeks of administration, the three agonist compounds disclosed herein all showed significantly higher weight loss effects (except M53) and food intake inhibition effects than Retatrutide.

Claims (18)

A triple agonist compound of GLP-1R, GIPR and GCGR or a pharmaceutically acceptable salt thereof, comprising a sequence represented by general formula I:
Xaa1-Aib-Gln-Gly-Thr-Xaa6-Thr-Ser-Xaa9-Xaa10-Ser-Ile-Xaa13-Xaa14-Asp-Xaa16-
Xaa17-Lys-Xaa19-Xaa20-Xaa21-Phe-Ile-Glu-Xaa25-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Ser-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-R ₁ (general formula I) in, Xaa1 is Tyr or His; Xaa6 is αMeF(2F), Phe, αMeF, αMeF(2,4F), F(2F) or F(2,4F); Xaa9 is Asp or αMeD; Xaa10 is Tyr, αMeY, or Leu; Xaa13 is Leu, Tyr, or αMeL; Xaa14 is Leu or αMeL; Xaa16 is Lys, Har, 3Pal, Arg, or NεMeK; Xaa17 is Har, Arg, or Gln; Xaa19 is Gln or Ala; Xaa20 is 4Pal, 3Pal, Aib, Gln, Ala or NεMeK; Xaa21 is Asp, Glu, or Ala; Xaa25 is Tyr, Trp, 3Pal, or αMeY; Xaa28 is Ala or Glu; Xaa29 is Gly, dAla, or Aib; Xaa30 is Gly or Lys; Xaa31 is Pro, dPro, or trans-Hyp; Xaa32 is Pro or Ser; Xaa34 is Aib or Gly; Xaa35 is Lys or Ala; Xaa36 is Pro or trans-Hyp; Xaa37 is Pro or trans-Hyp; Xaa38 is Pro or trans-Hyp; and/or Xaa39 is Lys or Ser; At least one amino acid Lys residue is connected to a fatty acid side chain, _R1 is a carboxyl terminal (C-terminal) modification of the compound sequence, and _R1 is selected from _-NH2 or -OH. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: in the general formula I, it contains any one or more of the following amino acids: Xaa1 is Tyr, Xaa9 is Asp, Xaa10 is Tyr, Xaa13 is Leu, Xaa14 is Leu, Xaa16 is Lys, Xaa19 is Ala, Xaa21 is Glu, Xaa29 is Gly, Xaa30 is Gly, Xaa31 is Pro, Xaa34 is Gly, Xaa35 is Ala, Xaa36 is Pro, Xaa37 is Pro, Xaa38 is Pro, and Xaa39 is Ser. The compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: in the general formula I, the compound is connected to the fatty acid side chain through the epsilon amino group on the Lys residue at position 18. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, characterized in that: the fatty acid side chain is selected from
One or more of, wherein x is any integer from 4 to 38; Preferably, the fatty acid side chain is selected from:
HOOC(CH ₂ ) ₁₄ CO-, HOOC(CH ₂ ) ₁₅ CO-, HOOC(CH ₂ ) ₁₆ CO-, HOOC(CH ₂ ) ₁₇ CO-,
One or more of HOOC(CH ₂ ) ₁₈ CO-, HOOC(CH ₂ ) ₁₉ CO-, HOOC(CH ₂ ) ₂₀ CO-, HOOC(CH ₂ ) ₂₁ CO-, and HOOC(CH ₂ ) ₂₂ CO-. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that the fatty acid side chain is connected to the amino acid Lys residue via a linker. The compound or pharmaceutically acceptable salt thereof according to claim 5, characterized in that: the linker is selected from
One or more of, wherein m is 0, 1, 2 or 3; n is 1 or 2; p is any integer from 1 to 5; Preferably, the connector is:
Where m is 0, 1, 2 or 3, n
is 1; more preferably, wherein m is 1 and n is 1. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, characterized in that the compound is M1-M42 and M45-M69 shown in Table 2 herein. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, characterized in that the compound has the general formula II:
Tyr-Aib-Gln-Gly-Thr-Xaa6-Thr-Ser-Asp-Tyr-Ser-Ile-Leu-Leu-Asp-Lys-Xaa17-Lys-Ala-
Xaa20-Glu-Phe-Ile-Glu-Xaa25-Leu-Leu-Xaa28-Gly-Gly-Pro-Xaa32-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R ₁ (General formula II) in, Xaa6 is αMeF(2F), αMeF, or αMeF(2,4F); Xaa17 is Har or Arg; Xaa20 is 4Pal, 3Pal, Aib or Ala; Xaa25 is Tyr or Trp; Xaa28 is Ala or Glu; and/or Xaa32 is Pro or Ser. The compound or pharmaceutically acceptable salt thereof according to claim 8, characterized in that the compound is M36, M37, M40, M52, M53 and M68 shown in Table 2 herein. A polypeptide, the amino acid sequence of which is shown as the general formula I in claim 1 or 2 or the general formula II in claim 8. A nucleic acid molecule encoding the polypeptide according to claim 10. A recombinant vector comprising the nucleic acid molecule according to claim 11. A recombinant cell comprising the nucleic acid molecule of claim 11 or the recombinant vector of claim 12 and capable of expressing and optionally secreting the polypeptide of claim 10. A method for preparing the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, or the polypeptide according to claim 10; Preferably, the preparation method comprises the steps of preparing the compound or the polypeptide using chemical methods and/or biological methods; Preferably, the chemical method comprises liquid phase or solid phase polypeptide synthesis. A pharmaceutical composition comprising the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, or the polypeptide according to claim 10, and a pharmaceutically acceptable excipient. The compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, the polypeptide according to claim 10 or the pharmaceutical composition according to claim 15, for use in treatment. Use of the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, the polypeptide according to claim 10 or the pharmaceutical composition according to claim 15 in the preparation of a medicament for preventing and/or treating metabolic disorders; Preferably, the metabolic disorder is diabetes or a diabetes-related disorder, or obesity or an obesity-related disorder; More preferably, the diabetes-related disorder is insulin resistance, glucose intolerance, elevated fasting blood glucose, prediabetes, type 1 diabetes, type 2 diabetes, gestational diabetes hypertension, dyslipidemia, bone-related disorder, or a combination thereof; The obesity-related disorder is obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH); or may be associated with a disorder selected from atherogenic dyslipidemia, dyslipidemia, increased blood pressure, hypertension, a prothrombotic state, and a proinflammatory state, or a combination thereof. A method for preventing and/or treating metabolic disorders, comprising the step of administering to a patient in need thereof a therapeutically effective amount of the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, the polypeptide according to claim 10, or the pharmaceutical composition according to claim 15; Preferably, the metabolic disorder is diabetes or a diabetes-related disorder, or obesity or an obesity-related disorder; More preferably, the diabetes-related disorder is insulin resistance, glucose intolerance, elevated fasting blood glucose, prediabetes, type 1 diabetes, type 2 diabetes, gestational diabetes hypertension, dyslipidemia, bone-related disorder, or a combination thereof; The obesity-related disorder is obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH); or may be associated with a disorder selected from atherogenic dyslipidemia, dyslipidemia, increased blood pressure, hypertension, a prothrombotic state, and a proinflammatory state, or a combination thereof.