Glp1-r / gipr dual peptide agonists and uses thereof

Abstract

Provided are dual-target agonist peptides, and more specifically a series of dual-target agonists capable of simultaneously activating the human glucagon-like peptide-1 (GLP-1) receptor and the human glucose-dependent insulinotropic polypeptide (GIP) receptor, as well as pharmaceutical salts and pharmaceutical compositions thereof. These peptides have dual agonist effects and can be used to treat diabetes, obesity, and other related diseases.

Description

[0001] Citations of relevant applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 592,940, filed October 25, 2023, the entire contents of which are incorporated herein by reference. Background of the Invention Technical Field

[0003] This invention relates to dual-target agonist peptides, and more specifically to a series of dual-target agonists capable of simultaneously activating human glucagon-like peptide-1 (GLP-1) receptor and human glucose-dependent insulinotropic peptide (GIP) receptor, as well as pharmaceutically acceptable salts and pharmaceutical compositions thereof. These peptides have dual agonist activity and can be used to treat diabetes, obesity, and other related diseases.

[0004] Brief description of related technologies

[0005] Diabetes mellitus is a metabolic disease characterized by hyperglycemia. Hyperglycemia is caused by defects in insulin secretion or impaired biological effects, or both. Long-term hyperglycemia leads to chronic damage and dysfunction in multiple tissues, particularly the eyes, kidneys, heart, blood vessels, and nerves. Significant genetic heterogeneity exists in both type 1 and type 2 diabetes. Diabetes has a familial tendency, with one-quarter to one-half of patients having a family history of the disease. At least 60 genetic syndromes are clinically associated with diabetes. Type 1 diabetes involves multiple DNA loci involved in its pathogenesis, with the DQ locus polymorphism in the HLA antigen gene being the most closely related. Many specific gene mutations have been identified in type 2 diabetes, such as those in the insulin gene, insulin receptor gene, glucokinase gene, and mitochondrial genes. Obesity, caused by overeating and reduced physical activity, is a major environmental factor in type 2 diabetes, making individuals with a genetic predisposition to the disease more susceptible. People with type 1 diabetes have an abnormal immune system. Infection with viruses such as Coxsackievirus, rubella virus, and mumps virus can trigger an autoimmune response that destroys insulin. Globally, 90% of diabetes cases are type 2, non-insulin-dependent diabetes. The primary cause is chronic metabolic disorder of pancreatic beta cells due to impaired function and long-term insulin resistance. The main clinical manifestations are low insulin levels and elevated blood glucose levels. Type 2 diabetes is confirmed to be associated with a number of high-risk diseases and commonly leads to cardiovascular disease, kidney failure, blindness, diabetic foot, neurological disorders, amputation, and other target organ diseases.

[0006] Obesity is closely linked to type 2 diabetes. Type 2 diabetes is caused by lifestyle factors, while type 1 diabetes is usually caused by autoimmune diseases or viral infections and is not related to lifestyle factors such as obesity. Obesity and type 2 diabetes are global medical problems, and their incidence is gradually increasing. According to the World Health Organization, there are more than 500 million obese people worldwide, mainly in developed and developing countries. Obesity not only leads to diseases such as diabetes, hyperlipidemia, hypertension, coronary heart disease, stroke, gallbladder disease, and gout, but also shortens life expectancy and reduces quality of life. In the United States, the prevalence of diabetes among adults is as high as 8%, and more than 90% of newly diagnosed adults with diabetes are obese. Obesity has been identified as the most important modifiable risk factor in the development of type 2 diabetes. Obesity can also be defined as a person with a BMI greater than or equal to 30, based on their body mass index (BMI). Obesity not only affects a person's behavior and mental health, but also significantly increases the risk of cardiovascular disease, diabetes, hypertension, musculoskeletal disorders, and certain cancers.

[0007] The number of people suffering from diabetes and its related complications is increasing annually worldwide, placing a significant burden on patients' health, families, and society. For decades, the development of diabetes drugs has been a core focus for many research institutions and pharmaceutical companies. Currently available anti-diabetic drugs include various small-molecule oral medications and peptide injectables. Fewer drugs are available for obesity-related indications than for diabetes. A significant gap remains between clinical practice and market practice.

[0008] Glucagon-like peptide-1 (GLP-1) is primarily secreted by intestinal L cells, and large amounts are secreted when the intestine is stimulated by nutrients. The function of GLP-1 depends on its receptor, the glucagon-like peptide-1 receptor (GLP-1R). GLP-1R is widely distributed throughout various tissues of the body. GLP-1 exerts its effects in various parts of the body by targeting GLP-1R. GLP-1 can reduce appetite and decrease addictive behaviors towards certain foods, making it useful in obesity treatment. This is why GLP-1R is an important target for obesity therapy. In pancreatic α cells, GLP-1 can reduce glucagon secretion; in pancreatic β cells, GLP-1 can promote insulin secretion, and both of these can lower blood glucose levels, making GLP-1R one of the important targets for diabetes treatment.

[0009] The gastric inhibitory peptide receptor (GIPR) is the receptor for GIPs and a G protein-coupled receptor with seven transmembrane domains. GIPR is expressed in pancreatic cells, the stomach, small intestine, adipose tissue, adrenal cortex, heart, pituitary gland, bone, lung, and spleen. In the pancreatic islets, only α-cells and β-cells express GIPR. GIPR activates G proteins, and activated G proteins increase intracellular cAMP and Ca2+ levels and regulate the expression of downstream genes through PI3K, PAK, PKB, and other signaling pathways. When food is ingested, GIPR signaling promotes insulin secretion, thereby lowering blood glucose levels. GIPR also affects adipocyte storage and metabolism, as well as glucose synthesis and release in the liver. Numerous studies have revealed that GIPR plays a crucial role in regulating insulin secretion, blood glucose, and lipid metabolism, and is essential for maintaining normal metabolic states. This makes GIPR an important target for the treatment of diabetes and obesity.

[0010] Clinically, there is a need for single-drug agents that can simultaneously lower blood sugar and reduce weight to address the growing prevalence of diabetes and obesity, and to overcome the inconvenience of combination therapies. The purpose of this invention is to provide a peptide derivative with dual agonistic activity against both human GLP-1 and human GIP receptors. The compounds of this invention exhibit better stimulatory activity compared to existing GLP-1 receptor agonists and support a longer metabolic half-life. Attached Figure Description

[0011] Figures 1a to 1m The HPLC purity identification chromatograms for BPP-A-001 to BPPS-A-013 are shown respectively.

[0012] Figures 2a to 2m The mass spectrometry (MS) identification spectra of BPP-A-001 to BPPS-A-013 are shown respectively.

[0013] Figure 3 The in vitro stability results of tirzepatide, BPP-A-001 to BPP-A-011 and BPPA-013 in human plasma are presented.

[0014] Figures 4a to 4n The metabolic curves of tesipatide and BPP-A-001 to BPP-A-013 are shown respectively.

[0015] Figures 5a to 5f The hypoglycemic effects of tesipatide and BPP-A-001 to BPP-A-013 were demonstrated.

[0016] Figures 6a to 6d The weight loss effects of tesipatide, BPP-A-001 to BPP-A-013 were demonstrated. Summary of the Invention

[0017] On one hand, the present invention relates to peptide analogs having the sequence SEQ ID NO:1 or their available salts: Tyr-Aib-Glu-X4-Thr-X6-Thr-Ser-Asp-Tyr-Ser-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X 22-X23-X24-X25-X26-X27-X28-Gly-Gly-Pro-Ser-Ser-Gly-X35-Pro-Pro-Pro-Ser-X40-NH2; (SEQ ID NO:1) X4, X6, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X35, and X40 are independently selected from L-type natural amino acids, D-type natural amino acids, non-natural amino acids, or peptides composed of them; alternatively, they are independently selected from modified L-type natural amino acids, modified D-type natural amino acids, modified non-natural amino acids, or peptides composed of them.

[0018] On the other hand, the present invention relates to a pharmaceutical composition comprising a peptide analog of SEQ ID NO:1 and a pharmaceutically acceptable carrier.

[0019] In another aspect, the present invention relates to a method for treating obesity and / or diabetes in patients, the method comprising the steps of administering to a patient requiring treatment a pharmaceutical composition comprising a peptide analog of SEQ ID NO:1 and a pharmaceutically acceptable carrier. Detailed Implementation

[0020] As described above, the present invention relates to peptide analogs having the sequence SEQ ID NO:1 or their available salts: Tyr-Aib-Glu-X4-Thr-X6-Thr-Ser-Asp-Tyr-Ser-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X 22-X23-X24-X25-X26-X27-X28-Gly-Gly-Pro-Ser-Ser-Gly-X35-Pro-Pro-Pro-Ser-X40-NH2; (SEQ ID NO:1) X4, X6, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X35, and X40 are independently selected from L-type natural amino acids, D-type natural amino acids, non-natural amino acids, or peptides composed of them; alternatively, they are independently selected from modified L-type natural amino acids, modified D-type natural amino acids, modified non-natural amino acids, or peptides composed of them.

[0021] In some embodiments, the variable position of the peptide analog of SEQ ID NO:1 can be selected from the following: X4 is selected from the amino acid residues Aib, Ser, Ala, or Gly; X6 is selected from Tyr, Trp, or Phe amino acid residues; X12 is selected from the amino acid residues Val, Ala, Leu, Ile, Aib, Met, α-Me-Leu, and D-Leu. X13 is selected from the amino acid residues Ala, Aib, Ser, and Val; X14 is selected from the amino acid residues Ile, Val, Leu, Met, α-Me-Leu, and α-Me-Val; X15 is selected from Thr, Ser, or Asp amino acid residues. X16 is selected from amino acid residues of Ala, Aib, Lys, and Met; X17 is selected from the amino acid residues Leu, Val, Met, Ile, α-Me-Leu, and α-Me-Val; X18 is selected from the amino acid residues Aib, Val, and Ala. X19 is selected from amino acid residues of Glu, Gln, and Asn. X20 is selected from Asp, Lys, Glu, Arg, or modified amino acid residues B amino acid residues; X21 is selected from the amino acid residues Aib, Ala, Val, and D-Ala; X22 is selected from amino acid residues of Trp, Phe, and Tyr. X23 is selected from the amino acid residues Aib, Ile, Leu, α-Me-Leu, α-Me-Val, and Val. X24 is selected from amino acid residues of Gln, Asn, Ala, Met, Leu, Aib, α-Me-Leu, and D-Gln; X25 is selected from the amino acid residues Tyr, Phe, and Trp. X26 is selected from the amino acid residues Ile, Leu, Aib, α-Me-Leu, and α-Me-Val; X27 is selected from the amino acid residues Ile, Val, Leu, Aib, α-Me-Leu, and α-Me-Val; X28 is selected from amino acid residues of Aib, Val, Ala, α-Me-Val, and D-Ala; X35 is selected from the amino acid residues Aib, Val, Ala, and α-Me-Val; X40 is selected from Asp, Lys, Glu, Arg, or modified amino acid residue B; or X40 is absent; and Wherein, B is selected from Lys{(-(AEEA)m-(Y-γ Glu) z-CO-(CH2)n-COOH}; and Where m is selected from integers 2 to 6, n is selected from integers 10 to 26, z is selected from integers 1 to 6, and Y is selected from Asp, D-Asp, -Asp, α-Me-Asp, GABA residues or Y does not exist.

[0022] The peptide analogs of the present invention can be prepared using standard biochemical techniques known in the art, such as solid-phase peptide synthesis. Furthermore, the activation activity of the peptide analogs of the present invention can be evaluated using GLP-1R and GIPR reporter gene cell lines known in the art.

[0023] The peptide analogs disclosed herein can be administered to patients in the form of free peptides, but are preferably administered in the form of pharmaceutical compositions. Therefore, the present invention covers pharmaceutical compositions comprising a peptide analog or a salt of a peptide (including pharmaceutically acceptable salts), such as the peptide of SEQ ID NO:1, and at least one pharmaceutically acceptable carrier. The pharmaceutical composition may contain the compound or salt of SEQ ID NO:1 as the sole active agent, but preferably contains at least one additional (other, additional) active agent. In some embodiments, the pharmaceutical composition is in dosage forms containing, per unit dosage form, about 0.1 mg to about 2000 mg, about 10 mg to about 1000 mg, about 100 mg to about 800 mg, or about 200 mg to about 600 mg of a compound comprising the peptide of SEQ ID NO:1 (Formula I) and optionally about 0.1 mg to about 2000 mg, about 10 mg to about 1000 mg, about 100 mg to about 800 mg, or about 200 mg to about 600 mg of additional active agent. The pharmaceutical composition may also contain a molar ratio between a peptide (such as SEQ ID NO:1) and an additional active agent. For example, the pharmaceutical composition may contain a molar ratio of the additional active agent to the peptide of about 0.5:1, about 1:1, about 2:1, about 3:1, or about 1.5:1 to about 4:1.

[0024] The peptides or pharmaceutical compositions disclosed herein may be administered orally, topically, parenterally, by inhalation or spray, sublingually, transdermally, via buccal application, rectal administration, as an ophthalmic solution, or otherwise, in dosage units containing conventionally pharmaceutically acceptable carriers. The pharmaceutical compositions may be formulated into any pharmaceutically useful form, such as aerosols, creams, gels, pills, capsules, tablets, syrups, transdermal patches, or ophthalmic solutions. Some dosage forms, such as tablets and capsules, are subdivided into appropriately sized unit doses containing suitable amounts of the active ingredient, e.g., an effective amount to achieve the desired purpose.

[0025] The carrier comprises excipients and diluents and must possess sufficiently high purity and low toxicity to be suitable for administration to patients undergoing treatment. The carrier may be inert or it may itself provide pharmaceutical benefits. The amount of carrier used with the compound is sufficient to provide the actual amount of material administered per unit dose of the compound.

[0026] Carriers include, but are not limited to, binders, buffers, colorants, diluents, disintegrants, emulsifiers, flavoring agents, glidants, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. Some carriers may be listed in more than one category; for example, vegetable oils can be used as lubricants in some formulations and as diluents in others. Exemplary pharmaceutically acceptable carriers include sugars, starches, cellulose, powdered tragacanth, malt, gelatin, talc, and vegetable oils. Optional active agents may be included in the pharmaceutical composition that do not materially interfere with the activity of the compounds of the present invention.

[0027] Pharmaceutical compositions / combinations can be formulated for oral administration. These compositions contain 0.1 to 99 wt% of a compound of formula I, and typically contain at least about 5 wt% of a compound of formula I. Some embodiments contain about 25 wt% to about 50 wt% or about 5 wt% to about 75 wt% of a compound of formula I.

[0028] The peptide of SEQ ID NO:1, and pharmaceutical compositions comprising the compound, may be used to treat obesity and / or diabetes in patients.

[0029] According to the present invention, a method for treating a patient with obesity and / or diabetes includes the step of administering a pharmaceutical composition containing the peptide of SEQ ID NO:1 to a patient requiring such treatment. In one embodiment, the patient is a mammal, and more specifically a human. As will be understood by those skilled in the art, the present invention also covers methods for treating non-human patients such as companion animals, such as cats, dogs, and livestock.

[0030] The therapeutically effective amount of the pharmaceutical composition is preferably an amount sufficient to alleviate or improve the symptoms of the disease or condition. When a therapeutically effective amount of the compound or pharmaceutical composition described herein is administered to a patient, it will also provide a sufficient concentration of the compound of SEQ ID NO: 1. Preferably, the sufficient concentration is the concentration of the compound required in the patient to prevent or combat the disease. This amount can be determined experimentally, for example, by measuring the blood concentration of the compound, or theoretically by calculating bioavailability.

[0031] According to the present invention, the treatment methods disclosed herein include: providing a patient with a specific dose of the compound of SEQ ID NO:1. In the treatment of the above-described conditions, a dose level of about 0.1 mg to about 140 mg per kilogram of body weight per day (about 0.5 mg to about 7 g per patient per day) for each compound is useful. The amount of compound that can be combined with a carrier material to produce a single dosage form will vary depending on the patient being treated and the specific method of administration. Dosage units will generally contain about 1 mg to about 500 mg of each active compound. In some embodiments, the patient is provided with 25 mg to 500 mg, or 25 mg to 200 mg of the compound of formula I, per day. The frequency of administration may also vary depending on the compound used and the specific disease being treated. However, for the treatment of most obesity or diabetes conditions, a dosing regimen of four times or less per day may be used, and in some embodiments a dosing regimen of once or twice per day may be used.

[0032] However, it should be understood that the specific dose level for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, weight, general health status, sex, diet, time of administration, route of administration and excretion rate, drug combination, and the severity of the specific disease being treated.

[0033] The peptide of SEQ ID NO:1 can be administered alone (i.e., as the sole therapeutic agent in a treatment regimen) to treat obesity and / or diabetes, or it can be administered in combination with another active agent. One or more peptides of SEQ ID NO:1 can be administered in coordination (coordination) with one or more other active agents known in the art.

[0034] As those skilled in the art will understand, the treatment methods described herein can also be used to treat mammals other than humans, including for veterinary applications such as treating horses and livestock (e.g., cattle, sheep, dairy cows, goats, pigs, etc.), as well as pets (companion animals such as dogs and cats).

[0035] For diagnostic or research applications, a variety of mammals would be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs (e.g., inbred pigs). Furthermore, for in vitro applications, such as in vitro diagnostics and research, bodily fluids (e.g., blood, plasma, serum, intercellular fluid, saliva, feces, and urine) as well as cell and tissue samples from the above subjects would be suitable.

[0036] All publications and patent applications referenced in this specification are incorporated herein by reference in their entirety. It will be apparent to those skilled in the art that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0037] Example

[0038] Example 1: Synthesis, preparation and identification of peptide analogs

[0039] The reagents and amino acids used during the experiment are as follows:

[0040] The peptides corresponding to sequences SEQ ID NO:2 to SEQ ID NO:14 were synthesized according to the following general protocol. The peptide names correspond sequentially to BPP-A-001 to BPPS-A-013.

[0041] 1. Weigh the AM resin, swell it with DCM for 1 hour, and wash it twice with DMF; 2. Remove the Fmoc protecting groups on the resin with a 20% piperidine DMF solution, repeating twice for 15 min each time, washing 6 times with DMF, washing once with methanol, and then washing twice with DMF. 3. Add the first amino acid + 1.5 equivalents of HOBT + 1.5 equivalents of DIC, use DMF as solvent, react for 1 hour, and wash 5 times with DMF; 4. Take a small amount of resin and use ninhydrin to test the amino reaction efficiency; 5. Remove the Fmoc protecting group with a 20% piperidine DMF solution, repeat 15 min × 2 times, wash 6 times with DMF, wash once with methanol, and then wash twice with DMF. 6. Add 3 equivalents of the second amino acid + 3 equivalents of HOBT + 3 equivalents of DIC, use DMF as solvent, react for 1 hour, and wash 5 times with DMF; 7. Repeat steps 4 through 6 and connect the main chain sequences in order; 8. Remove the Fmoc at the N-terminus with a 20% piperidine DMF solution, then add 3 equivalents of Boc anhydride + 3 equivalents of DIEA, react for 45 min, and wash 6 times with DMF. 9. Remove the DDE protecting group of the lysine side chain at position 20 using a 2% hydrazine hydrate DMF solution; 10. Repeat steps 6 and 7 and connect the sidechain trimmers in sequence; 11. After cleaning the resin with DMF, wash the resin three times with DCM and drain. 12. Pyrolyze the resin for 2 hours using 95% TFA + 2% Tis + 2% EDT + 1% H2O, and remove all protecting groups from the sequence. 13. Wash the precipitated peptides three times with ether by centrifugation, and drain the crude product. 14. Take a small amount of crude product and dissolve it for mass spectrometry and HPLC detection to determine the target peak; 15. Dissolve the crude product and separate it using a liquid chromatography column chromatography method; 16. Locate the target analyte using mass spectrometry and determine its purity using HPLC; 17. Collect qualified samples, rotary evaporate at 30°, and remove acetonitrile; 18. Add sodium chloride and sodium diphosphate heptahydrate to an acetonitrile-free peptide aqueous solution (peptide aqueous solution: sodium chloride: sodium diphosphate heptahydrate = 1L: 41mg: 7mg), and adjust the pH value with sodium hydroxide solution. 19. Freeze-dry the sample and check its mass spectrometry and purity to ensure it is qualified.

[0042] The prepared BPP-A-001 to BPPS-A-013 peptides achieved a purity of over 98% according to HPLC identification, and their HPLC purity chromatograms are shown below. Figures 1a to 1m As shown, and its mass spectrum is as follows. Figures 2a to 2m As shown.

[0043] Example 2: In vitro stability test of peptide analogs: 1. Take 50 μl of 1 mg / ml (0.952 mg / ml) tesipatide, BPP-A-001 to BPP-A-011, and BPPA-013 and add them to 200 μl of human plasma. Mix well and take 50 μl, storing at -20°C. Incubate the remaining 200 μl at 37°C for 24 h. Then take 50 μL for the next step.

[0044] 2. Add 200 μl of methanol:acetonitrile (3:1) solution to 50 μl of the -20°C storage solution and the 37°C reaction solution, centrifuge at 3200 rpm / min for 15 min, and collect the supernatant for later use.

[0045] 3. Negative control: Add 120 μl of methanol:acetonitrile (3:1) solution to 30 μl of human plasma, centrifuge at 3200 rpm / min for 15 min, and collect the supernatant for later use.

[0046] 4. Take 100 μl of the supernatant to measure the HPLC and compare the peak areas of the -20°C storage solution and the 37°C reaction solution.

[0047] like Figure 3 As shown, BPP-A-001 to BPP-A-002, and BPP-A-004 to BPPA-013 are more stable than tesipatide in human plasma. Surprisingly, after 24 hours of human plasma treatment at 37°C, only 83% of tesipatide was retained. In the other groups, except for BPP-A-008 which retained 89%, all others retained more than 90%. This may indicate that BPP-A-001 to BPP-A-011 and BPPA-013 may also have a longer metabolic cycle than tesipatide in vivo.

[0048] Example 3: In vivo metabolism experiment of peptide drugs

[0049] SD rats were grouped according to body weight to test in vivo metabolic parameters of tesipatide and BPP-A-001 to BPP-A-013. The method is as follows: Administration: 3 mg / kg 0.5 ml / rat, intravenously. Blood collection: The tail was clipped and blood was collected at 0.167 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h and 48 h after administration, 200 μl each time, and placed in EDTA.K2 anticoagulant tubes (pre-cooled on ice).

[0050] Centrifugation: The plasma was obtained by centrifugation at 3200 g for 10 min at 4°C and stored at -80°C for HPLC detection after sample processing.

[0051] HPLC Sample Preparation: Take a certain amount of the above plasma sample, add 3 times the plasma volume of methanol:acetonitrile (3:1 v / v) solution, vortex mix, incubate in a 37°C water bath for 30 min, centrifuge at 3200 g / min for 10 min at 4°C, and collect the supernatant. Add 3 times the volume of methanol:acetonitrile (3:1 v / v) solution to the supernatant, vortex mix, incubate in a 37°C water bath for 5 min, centrifuge at 3200 g / min for 10 min at 4°C, and collect the supernatant for testing.

[0052] Figures 4a to 4n Metabolic curves for tesipatide and BPP-A-001 to BPP-A-013 are shown separately. The results show that BPP-A-001 to BPP-A-013 have similar metabolic curves to tesipatide, and except for BPP-A-005, which has a shorter half-life than tesipatide, the other peptide groups show longer half-lives than tesipatide.

[0053] Example 4: Hypoglycemic Experiment with Peptide Drugs

[0054] Blood glucose levels in mice were tested using the Aike Lingrui 2 blood glucose tester in 6- to 8-week-old female c57BL / 6N mice.

[0055] The experimental procedure is as follows: 1. Divide the mice into groups of 3 and clip their ears for labeling.

[0056] 2. Subcutaneous injection of carrier solvent, tesipatide, and BPP-A-001 to BPP-A-013 peptide solutions, injection volume: 10 nmol / kg, 0.2 mL / mouse.

[0057] 3. After fasting for 15 to 18 hours following peptide injection, blood was collected by tail clipping to measure initial blood glucose concentration. Then, a glucose solution was injected intraperitoneally (0.2 g / mL) per mouse.

[0058] 4. Tail clipping was performed at 15 min, 30 min, 60 min, 120 min, 180 min and 240 min after glucose injection to measure blood glucose concentration.

[0059] Figures 5a to 5f The results show that BPP-A-001 to BPP-A-013 all exhibited good hypoglycemic effects, with some significantly superior to tesipatide and others comparable to tesipatide. Specifically, BPP-A-001, BPP-A-002, BPP-A-003, and BPP-A-004 showed unexpected hypoglycemic effects.

[0060] Example 5: Weight Loss Experiment with Peptide Drugs

[0061] Female c57BL / 6N mice aged 6 to 8 weeks were fed a high-fat diet of XTHF60, and the weight loss effects of tesipatide and BPP-A-001 to BPP-A-013 were assessed by weighing.

[0062] method: 1. Grouping: Before grouping, weigh the mice, with 4 mice in each group, and clip their ears for labeling.

[0063] 2. Subcutaneous injection (sc) of carrier solvent, tesipatide, BPP-A-001 to BPP-A-013 peptide solutions: 10 nmol / Kg, 0.2 mL / mouse, once every two days, weighing the mouse before administration.

[0064] Notably, in the experiment, the tesipatide group experienced severe dehydration on the second and fourth days after administration, leading to a rapid decrease in weight. Surprisingly, despite this, BPP-A-001 to BPP-A-013 all showed good and relatively safe weight loss effects, and some peptides, such as BPP-A-001, BPP-A-002, BPP-A-003, BPP-A-004, BPP-A-005, BPP-A-007, and BPP-A-008, showed better weight loss effects than tesipatide. Figures 6a to 6d As shown.

[0065] Example 6: Toxicity test of peptide drugs in rats

[0066] Acute toxicity experiments were conducted using 180-200 g SD-Rat-female rats.

[0067] Experimental procedure: 1. Weighing: Measure baseline body weight before application.

[0068] 2. Administration: Solvent, tesipatide, peptide solutions of BPP-A-001 to BPP-A-013, 3 mg / kg, 0.5 ml / rat, intravenously.

[0069] 3. Weight monitoring: Weighing every working day for 14 consecutive days.

[0070] 4. Urine collection: Urine from rats was collected using a metabolic cage the day before blood collection for testing. Rats were fasted before collection, but water intake was not prohibited.

[0071] 5. Coagulation function (blood coagulation), serum biochemistry and routine blood tests: 14 days later, collect an appropriate amount of blood using a disposable venous blood collection needle and a disposable vacuum blood collection tube, and send it for testing after appropriate processing.

[0072] 5.1. Hemagglutination test: 1) Collect 2 mL of whole blood in a blood coagulation test tube (sodium citrate 1:9) and immediately invert it 5 to 8 times; 2) Transfer to centrifuge tubes and centrifuge at 1800 G for 10 minutes at room temperature. 3) Collect the yellow plasma and store it in another centrifuge tube; 5.2. Serum biochemical tests: 1) Collect 2 mL of whole blood into a tube without additives (coagulant / separating gel) and immediately invert it 5 to 8 times; 2) Let stand at room temperature for 20 minutes 3) Transfer to centrifuge tubes and centrifuge at 1800 G for 10 minutes at room temperature. 4) Collect the yellow serum and store it in another centrifuge tube; 5.3. Routine blood tests (hematology): 1) Collect 1 mL of whole blood into a standard blood collection tube (EDTA-K2), and immediately invert it 5 to 8 times. No centrifugation or other treatment is required.

[0073] 6. Clinical observation: The physiological and pathological conditions of all mice were observed.

[0074] The results showed that, in clinical observation, mice in the tesipatide group and the BPP-A-001 to BPP-A-013 groups exhibited reduced food intake. No abnormalities were observed in urine, coagulation function, serum biochemistry, or complete blood count in any of the experimental groups. Tesipatide induced severe diarrhea, while no abnormalities were observed in the other experimental groups. Therefore, the BPP-A-001 to BPP-A-013 groups were superior to the control group tesipatide in terms of safety.

[0075] sequence list

[0076] SEQ ID NO:1: Tyr-Aib-Glu-X4-Thr-X6-Thr-Ser-Asp-Tyr-Ser-X12-X13-X14-X15-X16-X17-X18-X19-X20-X2 1-X22-X23-X24-X25-X26-X27-X28-Gly-Gly-Pro-Ser-Ser-Gly-X35-Pro-Pro-Pro-Ser-X40-NH2 SEQ ID NO:2: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{diacid 20 -γ-Glu-(AEEA)2-Lys}-AFVQWLIAGGPSSGAPPPS-NH2 SEQ ID NO:3: Y-{Aib}-EGTFTSDYSI-{Aib}-LDKIAQ-{dicarboxylic acid 20 -γ-Glu-(AEEA)2-Lys}VFVQWLIAGGPSSGAPPPS-NH2 SEQ ID NO:4: Y-{Aib}-EGTFTSDYSI-{Aib}-LDKIAQ-{dicarboxylic acid 20 -γ-Glu-(AEEA)2-Lys}-AFVQWLIVGGPSSGAPPPS-NH2 SEQ ID NO:5: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{dicarboxylic acid 20 -γ-Glu-(AEEA)2-Lys}-AFVQWLIVGGPSSGAPPPS-NH2 SEQ ID NO:6: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{dicarboxylic acid 20 -γ-Glu-(AEEA)2-Lys}-VFVQWLIAGGPSSGAPPPS-NH2 SEQ ID NO:7: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{dicarboxylic acid 20 -γ-Glu-(AEEA)2-Lys}-VFVQWLIVGGPSSGAPPPS-NH2 SEQ ID NO:8: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{dicarboxylic acid 20 -γ-Glu-(AEEA)2-Lys}-AFVQWLI-{Aib}-GGPSSGAPPPS-NH2 SEQ ID NO:9: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{dicarboxylic acid 20 -γ-Glu-(AEEA)2-Lys}-{Aib}-FVQWLIAGGPSSGAPPPS-NH2 SEQ ID NO:10: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{dicarboxylic acid 20 -γ-Glu-(AEEA)2-Lys}-{Aib}-FVQWLI-{Aib}-GGPSSGAPPPS-NH2 SEQ ID NO:11: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{dicarboxylic acid 20 -γ-Glu-Asp-(AEEA)2-Lys}-VFVQWLIVGGPSSGAPPPS-NH2 SEQ ID NO:12: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{dicarboxylic acid 20 -γ-Glu-Asp-(AEEA)2-Lys}-{Aib}-FVQWLI-{Aib}-GGPSSGAPPPS-NH2 SEQ ID NO:13: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{dicarboxylic acid 22 -γ-Glu-Asp-(AEEA)2-Lys}-VFVQWLIVGGPSSGAPPPS-NH2 SEQ ID NO:14: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{dicarboxylic acid 22 -γ-Glu-Asp-(AEEA)2-Lys}-{Aib}-FVQWLI-{Aib}-GGPSSGAPPPS-NH2。

Claims

1. A peptide analog having the sequence SEQ ID NO:1, or a salt thereof: Tyr-Aib-Glu-X4-Thr-X6-Thr-Ser-Asp-Tyr-Ser-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X 22-X23-X24-X25-X26-X27-X28-Gly-Gly-Pro-Ser-Ser-Gly-X35-Pro-Pro-Pro-Ser-X40-NH2; (SEQ ID NO:1) X4, X6, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X35, and X40 are independently selected from L-type natural amino acids, D-type natural amino acids, non-natural amino acids, or peptides composed of them; or, independently selected from modified L-type natural amino acids, modified D-type natural amino acids, modified non-natural amino acids, or peptides composed of them.

2. The peptide analog or available salt according to claim 1, wherein: X4 is selected from the amino acid residues Aib, Ser, Ala, or Gly; X6 is selected from Tyr, Trp, or Phe amino acid residues; X12 is selected from the amino acid residues Val, Ala, Leu, Ile, Aib, Met, α-Me-Leu, and D-Leu. X13 is selected from the amino acid residues Ala, Aib, Ser, and Val; X14 is selected from the amino acid residues Ile, Val, Leu, Met, α-Me-Leu, and α-Me-Val; X15 is selected from Thr, Ser, or Asp amino acid residues. X16 is selected from amino acid residues of Ala, Aib, Lys, and Met; X17 is selected from the amino acid residues Leu, Val, Met, Ile, α-Me-Leu, and α-Me-Val; X18 is selected from the amino acid residues Aib, Val, and Ala. X19 is selected from amino acid residues of Glu, Gln, and Asn. X20 is selected from Asp, Lys, Glu, Arg, or modified amino acid residues B amino acid residues; X21 is selected from the amino acid residues Aib, Ala, Val, and D-Ala; X22 is selected from amino acid residues of Trp, Phe, and Tyr. X23 is selected from the amino acid residues Aib, Ile, Leu, α-Me-Leu, α-Me-Val, and Val. X24 is selected from amino acid residues of Gln, Asn, Ala, Met, Leu, Aib, α-Me-Leu, and D-Gln; X25 is selected from the amino acid residues Tyr, Phe, and Trp. X26 is selected from the amino acid residues Ile, Leu, Aib, α-Me-Leu, and α-Me-Val; X27 is selected from the amino acid residues Ile, Val, Leu, Aib, α-Me-Leu, and α-Me-Val; X28 is selected from amino acid residues of Aib, Val, Ala, α-Me-Val, and D-Ala; X35 is selected from the amino acid residues Aib, Val, Ala, and α-Me-Val; X40 is selected from Asp, Lys, Glu, Arg, or modified amino acid residue B; or X40 is absent; and Where B is selected from Lys {(-(AEEA)m-(Y-γ Glu)z-CO-(CH)n-COOH}; and Where m is selected from integers 2 to 6, n is selected from integers 10 to 26, z is selected from integers 1 to 6, and Y is selected from Asp, D-Asp, β-Asp, α-Me-Asp, GABA residues or Y is not present.

3. A pharmaceutical composition comprising a peptide analog of SEQ ID NO:1 and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, further comprising an additional active agent.

5. A method for treating obesity and / or diabetes in patients, comprising the following steps: The pharmaceutical composition of claim 3 is administered to a patient who requires treatment.

6. The method of claim 5, wherein the pharmaceutical composition comprises the peptide analogues (SEQ ID NO:1 to SEQ ID NO:14) of claim 1 or 2, or a salt thereof.

7. The method according to claim 5, further comprising the following step: The pharmaceutical composition was administered to the patient in a dosage form containing about 0.1 mg to about 2000 mg.

8. A peptide analog having the sequence SEQ ID NO:2 to SEQ ID NO:14, or a salt thereof: SEQ ID NO:2: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{diacid 20-γ-Glu-(AEEA)2-Lys}-AFVQWLIAGGPSSGAPPPS-NH2; SEQ ID NO:3: Y-{Aib}-EGTFTSDYSI-{Aib}-LDKIAQ-{diacid 20-γ-Glu-(AEEA)2-Lys}-VFVQWLIAGGPSSGAPPPS-NH2; SEQ ID NO:4: Y-{Aib}-EGTFTSDYSI-{Aib}-LDKIAQ-{diacid 20-γ-Glu-(AEEA)2-Lys}-AFVQWLIVGGPSSGAPPPS-NH2; SEQ ID NO:5: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{diacid 20-γ-Glu-(AEEA)2-Lys}-AFVQWLIVGGPSSGAPPPS-NH2; SEQ ID NO:6: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{diacid 20-γ-Glu-(AEEA)2-Lys}-VFVQWLIAGGPSSGAPPPS-NH2; SEQ ID NO:7: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{Diacid 20-γ-Glu-(AEEA)2-Lys}-VFVQWLIVGGPSSGAPPPS-NH2; SEQ ID NO:8: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{Diacid 20-γ-Glu-(AEEA)2-Lys}-AFVQWLI-{Aib}-GGPSSGAPPPS-NH2; SEQ ID NO:9: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{Diacid 20-γ-Glu-(AEEA)2-Lys}-{Aib}-FVQWLIAGGPSSGAPPPS-NH2; SEQ ID NO:10: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{Diacid 20-γ-Glu-(AEEA)2-Lys}-{Aib}-FVQWLI-{Aib}-GGPSSGAPPPS-NH2; SEQ ID NO:11: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{Diacid 20-γ-Glu-Asp-(AEEA)2-Lys}-VFVQWLIVGGPSSGAPPPS-NH2; SEQ ID NO:12: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{Diacid 20-γ-Glu-Asp-(AEEA)2-Lys}-{Aib}-FVQWLI-{Aib}-GGPSSGAPPPS-NH2; SEQ ID NO:13: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{Diacid 22-γ-Glu-Asp-(AEEA)2-Lys}-VFVQWLIVGGPSSGAPPPS-NH2; and SEQ ID NO:14: Y-{Aib}-EGTFTSDYSL-{Aib}-LDKIAQ-{Diacid 22-γ-Glu-Asp-(AEEA)2-Lys}-{Aib}-FVQWLI-{Aib}-GGPSSGAPPPS-NH2.

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