WO2025087344A1

WO2025087344A1 - Glp1-r/gipr dual peptide agonists and use thereof

Info

Publication number: WO2025087344A1
Application number: PCT/CN2024/127119
Authority: WO
Inventors: Chongqian ZHANG; Hongxia Li; Aiping Zhang; Chao ZONG; Nan Zhang; Zuefeng LV; Yuanhang CHEN; Xuwen WANG; Chun Tang; Jirong Peng; Don Zhang
Original assignee: Beta Pharma Inc
Current assignee: Beta Pharma Inc
Priority date: 2023-10-25
Filing date: 2024-10-24
Publication date: 2025-05-01
Anticipated expiration: 2026-04-25

Abstract

Provided is dual target agonist peptides, and more specifically to a series of dual target agonists that can simultaneously activate the human Glucagon like peptide-1 (GLP-1) receptor and the human glucose dependent insulinotropic polypeptide (GIP) receptor, as well as its pharmaceutical salts and pharmaceutical compositions. The peptides have dual agonist effect and can be used for the treatment of diabetes, obesity and other related diseases.

Description

GLP1-R/GIPR Dual Peptide Agonists and Use Thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/592, 940 filed October 25, 2023 which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is directed to dual target agonist peptides, and more specifically to a series of dual target agonists that can simultaneously activate the human Glucagon like peptide-1 (GLP-1) receptor and the human glucose dependent insulinotropic polypeptide (GIP) receptor, as well as its pharmaceutical salts and pharmaceutical compositions. These peptides have dual agonist effect and can be used for the treatment of diabetes, obesity and other related diseases.

Brief Description of the Related Art

Diabetes is a metabolic disease characterized by hyperglycemia. Hyperglycemia is caused by defects in insulin secretion or impaired biological effects, or both. Long term high blood sugar leads to chronic damage and dysfunction of various tissues, especially the eyes, kidneys, heart, blood vessels, and nerves. There is obvious genetic heterogeneity in type 1 or type 2 diabetes. Diabetes has a familial tendency, and 1/4 to 1/2 of patients have a family history of diabetes. There are at least 60 kinds of genetic syndromes associated with diabetes clinically. Type 1 diabetes has multiple DNA sites involved in the pathogenesis, of which DQ site polymorphism in HLA antigen gene is the most closely related. Many specific gene mutations have been found in type 2 diabetes, such as insulin gene, insulin receptor gene, glucokinase gene, mitochondrial gene, etc. Obesity caused by overeating and reduced physical activity is the main environmental factor of type 2 diabetes, which makes individuals with genetic susceptibility to type 2-diabetes prone to disease. The patients with Type 1 diabetes have abnormal immune system, which will lead to autoimmune reaction and destroy insulin after infection of some viruses such as coxsackie virus, rubella virus, and parotid virus. 90%of diabetes patients worldwide are type 2 non-insulin dependent diabetes. The main reason is the pancreatic islets β Long term chronic Metabolic disorder caused by impaired cell function and long-term insulin resistance. The main clinical manifestations are a lack of insulin levels in the body and an increase in blood glucose concentration in the plasma. It has been confirmed that type 2 diabetes is associated with a variety of high-risk diseases of patients, and it will often lead to cardiovascular disease, renal failure, blindness, Diabetic foot, Nervous system disease, amputation and other diseases of patients' target organs.

Obesity is closely related to type 2 diabetes. Type 2 diabetes is caused by lifestyle, while Type 1 diabetes is often caused by autoimmune disease or virus infection, and has nothing to do with lifestyle (such as obesity) . Obesity and type 2 diabetes are global medical problems, and their incidence rate is gradually rising. According to the World Health Organization, there are more than 500 million obese patients worldwide, mainly distributed in developed and developing countries. Obesity not only brings diabetes, hyperlipidemia, hypertension, coronary heart disease, stroke, gallbladder disease, gout and other diseases, but also shortens life expectancy and reduces the 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 diabetes adults are obese. Obesity has been recognized as the most important risk factor that can be changed in the development of type 2 diabetes. According to the person's Body mass index BMI, obesity can also be defined as when the person's BMI is greater than or equal to 30. Obesity will not only affect the patient's behavior and spirit, but also significantly increase the risk of cardiovascular disease, diabetes, hypertension, musculoskeletal diseases and some cancers.

The number of patients worldwide has increased year by year due to diabetes and its related complications, which has caused a huge burden to patients' health, families and society. The development of diabetes drugs has been one of the core topics of many scientific research institutions and pharmaceutical enterprises for decades. The anti-diabetes related drugs on the market now include various small molecule oral and drug and peptide injection drugs. There are fewer drugs for obesity related indications than diabetes drugs. There is still a huge gap in clinical and market practices.

Glucagon-like peptide-1 (GLP-1) is mainly secreted by intestinal L cells, and when the intestine is stimulated by nutrients, it will secrete a large amount of GLP-1. The function of GLP-1 depends on its receptor, the glucagon like peptide-1 receptor (GLP-1R) . GLP-1R is widely distributed in various tissues of the body. GLP-1 plays a role in various parts of the human body by targeting GLP-1R. GLP-1 can reduce appetite and reduce addictive behavior towards certain foods, which can be used for obesity treatment. This is also why GLP-1R has become an important target for obesity treatment. In the pancreas αGLP-1 can reduce the secretion of Glucagon in the pancreas β In cells, GLP-1 can promote insulin secretion, and both of them can reduce blood sugar, which makes GLP-1R one of the important targets for the treatment of diabetes.

Gastric inhibitory polypeptide receptor (GIPR) is the receptor of GIP and a G protein coupled receptor with seven transmembrane domains. GIPR is expressed in pancreatic cells, stomach, small intestine, adipose tissue, Adrenal cortex, heart, pituitary, bone, lung and spleen. In the pancreatic islets, only α and β Cells express GIPR. GIPR can activate G protein, and the activated G protein can increase the level of cAMP and Ca2+in cells, and regulate the expression of downstream genes through PI3K, PAK, PKB and other signal pathways. When the human body ingests food, GIPR signal can promote the secretion of insulin, thus reducing the blood sugar level. At the same time, GIPR can also affect the storage and metabolism of adipocytes, as well as glucose synthesis and release in the liver. Numerous studies have revealed that GIPR plays an important role in regulating insulin secretion, blood sugar, and lipid metabolism, and is crucial for maintaining normal metabolic status. This makes GIPR an important target for the treatment of diabetes and obesity.

A single drug that can simultaneously reduce blood sugar and weight loss is needed clinically to address the growing population of diabetes and obesity, and to solve the inconvenience of combination therapy. The purpose of the present invention is to provide a peptide derivative with dual agonistic effects on human GLP-1 receptors and human GIP receptors. Compounds of the present invention have better stimulating effects compared to GLP-1 receptor agonists in the art, and can support a longer metabolic half-life.

DETAILED DESCRIPTION OF THE FIGURES

Fig. 1a-Fig. 1m illustrate HPLC purity identification charts of BPP-A-001-BPPS-A-013 respectively.

Fig. 2a-Fig. 2m illustrate mass spectroscopy (MS) identification spectra of BPP-A-001-BPPS-A-013 respectively.

Figure 3 illustrates in vitro stability results of Tirzepatide, BPP-A-001-BPP-A-011, and BPPA-013 in human plasma.

Fig. 4a-Fig. 4n illustrate the metabolic curves of Tirzepatide and BPP-A-001-BPP-A-013 respectively.

Fig. 5a-Fig. 5f illustrate the hypoglycemic effects of Tirzepatide and BPP-A-001-BPP-A-013.

Fig. 6a-Fig. 6d illustrate the weight loss effects of Tirzepatide, BPP-A-001-BPP-A-013.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to peptide analogs having the sequence of SEQ ID NO: 1, or available salts thereof:

Tyr-Aib-Glu-X4-Thr-X6-Thr-Ser-Asp-Tyr-Ser-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-Gly-Gly-Pro-Ser-Ser-Gly-X35-Pro-Pro-Pro-Ser-X40-NH2; (SEQ ID NO: 1)

wherein 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 peptide segments composed of them; Alternatively, independently selected from modified L-type natural amino acids, modified D-type natural amino acids, modified non-natural amino acids, or peptide segments composed of them.

In another aspect, the present invention is directed to a pharmaceutical composition, comprising the peptide analog of SEQ ID NO: 1 and a pharmaceutically acceptable carrier.

In yet another aspect, the present invention is directed to a method of treating obesity and/or diabetes in a patient comprising the step of administering the pharmaceutical composition comprising the peptide analog of SEQ ID NO: 1 and a pharmaceutically acceptable carrier to a patient in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

As indicated above, the present invention is directed to peptide analogs having the sequence of SEQ ID NO: 1, or available salts thereof:

wherein 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 peptide segments composed of them; Alternatively, independently selected from modified L-type natural amino acids, modified D-type natural amino acids, modified non-natural amino acids, or peptide segments composed of them.

In some embodiments, the variable positions of the peptide analogs of SEQ ID NO: 1 may be selected from the following:

X4 is selected from Aib, Ser, Ala, or Gly amino acid residues;

X6 is selected from Tyr, Trp, or Phe amino acid residues;

X12 is selected from Val, Ala, Leu, Ile, Aib, Met, α-Me-Leu, D-Leu amino acid residues;

X13 is selected from Ala, Aib, Ser, Val amino acid residues;

X14 is selected from Ile, Val, Leu, Met, α-Me-Leu, α-Me-Val amino acid residues;

X15 is selected from Thr, Ser, or Asp amino acid residues;

X16 is selected from Ala, Aib, Lys, Met amino acid residues;

X17 is selected from Leu, Val, Met, Ile, α-Me-Leu, α-Me-Val amino acid residues;

X18 is selected from Aib, Val, Ala amino acid residues;

X19 is selected from Glu, Gln, Asn amino acid residues;

X20 is selected from Asp, Lys, Glu, Arg, or modified amino acid residues B amino acid residues;

X21 is selected from Aib, Ala, Val, D-Ala amino acid residues;

X22 is selected from Trp, Phe, Tyr amino acid residues;

X23 is selected from Aib, Ile, Leu, α-Me-Leu, α-Me-Val, Val amino acid residues;

X24 is selected from Gln, Asn, Ala, Met, Leu, Aib, α-Me-Leu, D-Gln amino acid residues;

X25 is selected from Tyr, Phe, Trp amino acid residues;

X26 is selected from Ile, Leu, Aib, α-Me-Leu, α-Me-Val amino acid residues;

X27 is selected from Ile, Val, Leu, Aib, α-Me-Leu, α-Me-Val amino acid residues;

X28 is selected from Aib, Val, Ala, α-Me-Val, D-Ala amino acid residues;

X35 is selected from Aib, Val, Ala, α-Me-Val amino acid residues;

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

wherein m is selected from integers 2-6, n is selected from integers 10-26, z is selected from integers 1-6, Y is selected from Asp, D-Asp, -Asp, α-Me-Asp, GABA residues or Y is absent.

The peptide analogs of the invention may be made using standard biochemical techniques that are known in the art, for example, solid-phase peptide synthesis. In addition, GLP-1R, GIPR reporter gene cell lines that are known in the art may be used to evaluate the activation effect of the peptide analogs of the invention.

The peptide analogs disclosed herein can be administered to a patient as free peptides, but are preferably administered as a pharmaceutical composition. Accordingly, the invention encompasses pharmaceutical compositions comprising a peptide analog or a salt (including a pharmaceutically acceptable salt) of a peptide, such as the peptide of SEQ ID NO: 1, together with at least one pharmaceutically acceptable carrier. The pharmaceutical composition may contain a compound or salt of SEQ ID NO: 1 as the only active agent, but preferably contains at least one additional active agent. In certain embodiments the pharmaceutical composition is in a dosage form that contains from about 0.1 mg to about 2000 mg, from about 10 mg to about 1000 mg, from about 100 mg to about 800 mg, or from about 200 mg to about 600 mg of a compound comprising the peptide of SEQ. ID NO: 1 (Formula I) and optionally from about 0.1 mg to about 2000 mg, from about 10 mg to about 1000 mg, from about 100 mg to about 800 mg, or from about 200 mg to about 600 mg of an additional active agent in a unit dosage form. The pharmaceutical composition may also include a molar ratio of a peptide, such as SEQ ID NO: 1, and an additional active agent. For example, the pharmaceutical composition may contain a molar ratio of about 0.5: 1, about 1: 1, about 2: 1, about 3: 1 or from about 1.5: 1 to about 4: 1 of an additional active agent to peptide.

The peptides or pharmaceutical compositions disclosed herein may be administered orally, topically, parenterally, by inhalation or spray, sublingually, transdermally, via buccal administration, rectally, as an ophthalmic solution, or by other means, in dosage unit formulations containing conventional pharmaceutically acceptable carriers. The pharmaceutical composition may be formulated as any pharmaceutically useful form, e.g., as an aerosol, a cream, a gel, a pill, a capsule, a tablet, a syrup, a transdermal patch, or an ophthalmic solution. Some dosage forms, such as tablets and capsules, are subdivided into suitably sized unit doses containing appropriate quantities of the active components, e.g., an effective amount to achieve the desired purpose.

Carriers include excipients and diluents and must be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the patient being treated. The carrier can be inert or it can possess pharmaceutical benefits of its own. The amount of carrier employed in conjunction with the compound is sufficient to provide a practical quantity of material for administration per unit dose of the compound.

Classes of carriers include, but are not limited to binders, buffering agents, coloring agents, diluents, disintegrants, emulsifiers, flavorants, glidants, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. Some carriers may be listed in more than one class, for example vegetable oil may be used as a lubricant in some formulations and a diluent in others. Exemplary pharmaceutically acceptable carriers include sugars, starches, celluloses, powdered tragacanth, malt, gelatin, talc, and vegetable oils. Optional active agents may be included in a pharmaceutical composition, which do not substantially interfere with the activity of the compound of the present invention.

The pharmaceutical compositions /combinations can be formulated for oral administration. These compositions contain between 0.1 and 99 weight % (wt%) of a compound of Formula I and usually at least about 5 wt%of a compound of Formula I. Some embodiments contain from about 25 wt%to about 50 wt%or from about 5 wt%to about 75 wt%of the compound of Formula I.

The peptides of SEQ ID NO: 1, as well as pharmaceutical compositions comprising the compounds, are useful for treating obesity and/or diabetes in a patient.

According to the present invention, the method of treating obesity and/or diabetes in a patient comprises the step of administering the pharmaceutical composition containing the peptides of SEQ ID NO: 1 to a patient in need of such treatment. In one embodiment, the patient is a mammal, and more specifically a human. As will be understood by one skilled in the art, the invention also encompasses methods of treating non-human patients such as companion animals, e.g. cats, dogs, and livestock animals.

A therapeutically effective amount of a pharmaceutical composition is preferably an amount sufficient to reduce or ameliorate the symptoms of a disease or condition. A therapeutically effective amount of a compound or pharmaceutical composition described herein will also provide a sufficient concentration of a compound of SEQ ID NO: 1 when administered to a patient. A sufficient concentration is preferably a concentration of the compound in the patient’s body necessary to prevent or combat the disorder. Such an amount may be ascertained experimentally, for example by assaying blood concentration of the compound, or theoretically, by calculating bioavailability.

According to the invention, the methods of treatment disclosed herein include providing certain dosage amounts of a compound of SEQ ID NO: 1 to a patient. Dosage levels of each compound of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day) . The amount of compound that may be combined with the carrier materials to produce a single dosage form will vary depending upon the patient treated and the particular mode of administration. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of each active compound. In certain embodiments 25 mg to 500 mg, or 25 mg to 200 mg of a compound of Formula I are provided daily to a patient. Frequency of dosage may also vary depending on the compound used and the particular disease treated. However, for treatment of most obesity or diabetes disorders, a dosage regimen of 4 times daily or less can be used and in certain embodiments a dosage regimen of 1 or 2 times daily is used.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

The peptides of SEQ ID NO: 1 may be administered singularly (i.e., sole therapeutic agent of a regime) to treat obesity and/or diabetes, or may be administered in combination with another active agent. One or more peptides of SEQ ID NO: 1 may be administered in coordination with a regime of one or more other active agents known in the art.

As will be appreciated by one skilled in the art, the methods of treatment provided herein are also useful for treatment of mammals other than humans, including for veterinary applications such as to treat horses and livestock, e.g. cattle, sheep, cows, goats, swine and the like, and pets (companion animals) such as dogs and cats.

For diagnostic or research applications, a wide variety of mammals will be suitable subjects including rodents (e.g. mice, rats, hamsters) , rabbits, primates, and swine such as inbred pigs and the like. Additionally, for in vitro applications, such as in vitro diagnostic and research applications, body fluids (e.g., blood, plasma, serum, cellular interstitial fluid, saliva, feces, and urine) and cell and tissue samples of the above subjects will be suitable for use.

All publications and patent applications cited in the specification are herein incorporated by reference in their entirety. It will be apparent to those of ordinary skill in the art that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

EXAMPLES

Example 1: Synthesis, preparation and identification of polypeptide analogs The reagents and amino acids used during the experiment are as follows:

The peptides corresponding to the sequences SEQ ID NO: 2-SEQ ID NO: 14 were synthesized according to the following common scheme. The peptide names correspond to BPP-A-001-BPPS-A-013 in order.

1. Weigh AM resin, swell with DCM for 1 hour, and wash with DMF twice;

2. Remove the Fmoc protecting group of the resin with 20%piperidine in DMF solution, 15min*2 times, wash with DMF 6 times, wash with methanol 1 time, and wash with DMF 2 times;

3. Add the first amino acid + 1.5eq of HOBT + 1.5eq of DIC, use DMF as solvent, react for 1 hour, and wash with DMF 5 times;

4. Take a small amount of resin and use ninhydrin to detect the efficiency of amino reaction;

5. Remove Fmoc protecting group with 20%piperidine in DMF solution, 15min*2 times, wash with DMF 6 times, wash with methanol 1 time, and wash with DMF 2 times;

6. Add 3eq of the second amino acid + 3eq of HOBT + 3eq of DIC, use DMF as solvent, react for 1h, and wash with DMF 5 times;

7. Repeat steps 4-6 and connect the main chain sequence in order;

8. Remove Fmoc at the N-terminus with 20%piperidine in DMF solution, then add 3eq of Boc anhydride + 3eq of DIEA, react for 45min, and wash with DMF 6 times;

9. Remove DDE protecting group of lysine side chain at position 20 with 2%hydrazine hydrate in DMF solution;

10. Repeat steps 6-7 and connect side chain modifications in order;

11. After cleaning the resin with DMF, wash the resin with DCM 3 times and drain;

12. 95%TFA + 2%Tis + 2%EDT + 1%H2O, crack the resin for 2h, and remove all protective groups in the sequence;

13. Wash the precipitated peptide 3 times with ether 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 peak of the target;

15. Dissolve the crude product and separate the crude product with a liquid column;

16. Find the target by mass spectrometry, and detect the purity of the target by HPLC;

17. Collect the qualified samples, 30° rotary evaporation, and remove acetonitrile;

18. Add sodium chloride + sodium diphosphate heptahydrate to the peptide aqueous solution without acetonitrile (peptide aqueous solution: sodium chloride: sodium diphosphate heptahydrate = 1L: 41mg: 7mg) , and adjust the pH value with sodium hydroxide solution;

19. Freeze-dry, review the mass spectrum and purity of the freeze-dried sample to ensure that the sample is qualified.

The HPLC identification purity of the prepared BPP-A-001-BPPS-A-013 peptides reached more than 98%, and their HPLC purity spectra are shown in Figs. 1a-Fig 1m, and their mass spectra are shown in Figs. 2a-Fig 2m.

Example 2: In vitro stability test of peptide analogs:

1. Take 50ul 1mg/ml (0.952mg/ml) Tirzepatide, BPP-A-001-BPP-A-011, BPPA-013 and add them to 200ul Human plasma, mix well and take 50ul, store at -20℃. The remaining 200ul is placed at 37℃ for reaction and incubated for 24h. Then take 50μL to the next step.

2. Add 200ul methanol: acetonitrile (3: 1) solution to 50ul of -20℃ storage solution and 37℃ reaction solution, centrifuge at 3200rpm/min for 15min, and take the supernatant for use.

3. Negative control: Add 120ul methanol: acetonitrile (3: 1) solution to 30ul Human plasma, centrifuge at 3200rpm/min for 15min, and take the supernatant for use.

4. Take 100ul of supernatant to measure HPLC, and compare the peak area of -20℃ storage solution and 37℃ reaction solution.

As shown in Figure 3, BPP-A-001-BPP-A-002, BPP-A-004-BPPA-013 are more stable than Tirzepatide in human plasma. Surprisingly, after 24 hours of human plasma treatment at 37℃, only 83%of Tirzepatide remains. In other groups, except for BPP-A-008, which retains 89%, the others retain more than 90%. This may indicate that BPP-A-001-BPP-A-011 and BPPA-013 may also have a longer metabolic cycle than Tirzepatide in vivo metabolism.

Example 3: In vivo metabolism experiment of peptide drugs

SD rats were grouped according to body weight to test the in vivo metabolic parameters of Tirzepatide and BPP-A-001-BPP-A-013. The method is as follows:

Administration: 3mg/kg 0.5ml/rat, iv injection

Blood collection: Cut the tail and collect blood at 0.167h, 0.5h, 1h, 2h, 4h, 6h, 8h, 24h, and 48h after administration, 200ul/time, placed in EDTA. K2 anticoagulant tube (pre-cooled on ice) .

Centrifugation: 3200g 10min 4℃ centrifugation to obtain plasma, stored at -80℃for HPLC detection after sample processing.

HPLC sample processing: Take a certain amount of the above plasma samples, add 3 times the volume of plasma methanol: acetonitrile (3: 1 v/v) solution, vortex mix, water bath at 37℃ for 30min, centrifuge at 3200g/min for 10min at 4℃, and take the supernatant. Add 3 times volume of methanol: acetonitrile (3: 1 v/v) solution to the supernatant, vortex mix, incubate at 37℃ in a water bath for 5min, centrifuge at 3200g/min for 10min at 4℃, and take the supernatant for testing.

Fig. 4a-Fig. 4n show the metabolic curves of Tirzepatide and BPP-A-001-BPP-A-013, respectively. The results show that BPP-A-001-BPP-A-013 has a similar metabolic curve to Tirzepatide, and except for BPP-A-005, whose half-life is shorter than that of Tirzepatide, the other peptide groups all show a longer half-life than Tirzepatide.

Example 4: Hypoglycemic experiment of peptide drugs

6-8 week old c57BL/6N female mice were used to test the blood glucose of mice using the Aike Lingrui 2 blood glucose tester.

The experimental process is as follows:

1. Grouping, 3 mice in each group, clipping ears for marking.

2. Subcutaneous injection of vehicle solvent, Tirzepatide, BPP-A-001-BPP-A-013 peptide solution, injection volume: 10nmol/kg, 0.2mL/mouse.

3. After fasting for 15-18 hours after peptide injection, blood was collected by tail cutting to measure the initial blood glucose concentration, and then glucose solution was intraperitoneally injected, with an injection volume of 0.2g/mL, 0.2mL/mouse.

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

The results shown in Fig. 5a-Fig. 5f show that BPP-A-001-BPP-A-013 all exhibit good hypoglycemic effects, some of which are significantly better than Tirzepatide, and some are comparable to Tirzepatide. In particular, BPP-A-001, BPP-A-002, BPP-A-003, and BPP-A-004 have unexpected hypoglycemic effects.

Example 5: Weight loss experiment of peptide drugs

6-8 week old c57BL/6N female mice were fed with XTHF60 high-fat diet, and the weight loss effect of Tirzepatide and BPP-A-001-BPP-A-013 was detected by weighing the body weight.

Process:

1. Grouping: Weigh the body weight before grouping, 4 mice per group, and clip the ears for marking.

2. Subcutaneous injection (s. c. ) of vehicle solvent, Tirzepatide, BPP-A-001-BPP-A-013 peptide solution: 10nmol/Kg, 0.2mL/mouse, once every two days, weigh the body weight before administration.

It is worth noting that the Tirzepatide group suffered from severe dehydration on the second and fourth days after administration in the experiment, resulting in a sharp drop in body weight. Unexpectedly, despite this, BPP-A-001-BPP-A-013 all showed good and relatively safe weight loss effects, and the weight loss effects of some peptides were better than Tirzepatide, such as BPP-A-001, BPP-A-002, BPP-A-003, BPP-A-004, BPP-A-005, BPP-A-007, BPP-A-008, etc., as shown in Fig. 6a-6d.

Example 6: Toxicity experiment of peptide drugs in rats

Acute toxicity experiment was conducted using 180-200g SD-Rat-female rats.

Experimental process:

1. Weighing: Baseline body weight was measured before administration.

2. Administration: Vehicle solvent, Tirzepatide, BPP-A-001-BPP-A-013 peptide solution, 3mg/kg 0.5ml/rat, iv injection.

3. Weight monitoring: Weigh every working day for 14 days.

4. Urine collection: Use metabolic cages to collect rat urine for testing one day before blood collection. Before collection, fasting but not water.

5. Blood coagulation, serum biochemistry, and blood routine tests: After 14 days, use disposable venous blood collection needles and disposable vacuum blood collection tubes to collect appropriate amounts of blood, and send for testing after corresponding treatment.

5.1. Hemagglutination test:

1) Collect 2mL of whole blood in a hemagglutination test tube (sodium citrate 1: 9) , and immediately invert it 5-8 times

2) Transfer to a centrifuge tube and centrifuge at 1800G at room temperature for 10 minutes

3) Take yellow plasma and store it in another centrifuge tube;

5.2. Serum biochemical test:

1) Collect 2mL of whole blood in a tube without additives (coagulant/separation gel) , and immediately invert it 5 to 8 times

2) Let stand at room temperature for 20 minutes

3) Transfer to a centrifuge tube and centrifuge at 1800G at room temperature for 10 minutes

4) Take yellow serum and store it in another centrifuge tube;

5.3. Routine blood test (hematology) :

1) Collect 1mL of whole blood in a routine blood test tube (EDTA-K2) , and immediately invert it 5-8 times without centrifugation or other treatments.

6. Clinical observation: Observe the physiological and pathological conditions of all mice.

The results showed that in the clinical observation, mice in the Tirzepatide and BPP-A-001-BPP-A-013 groups showed a decrease in food intake. There were no abnormalities in urine, blood coagulation, serum biochemistry, and blood routine in each experimental group. Tirzepatide caused severe diarrhea, while other experimental groups had no abnormalities. Therefore, BPP-A-001-BPP-A-013 is superior to the control group Tirzepatide in terms of safety.

SEQUENCE LISTING

SEQ ID NO: 1:

Tyr-Aib-Glu-X4-Thr-X6-Thr-Ser-Asp-Tyr-Ser-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-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- {diacid20-gamma-Glu- (AEEA)2-Lys} -AFVQWLIAGGPSSGAPPPS-NH2

SEQ ID NO: 3:

Y- {Aib} -EGTFTSDYSI- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA)2-Lys} -VFVQWLIAGGPSSGAPPPS-NH2

SEQ ID NO: 4:

Y- {Aib} -EGTFTSDYSI- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA)2-Lys} -AFVQWLIVGGPSSGAPPPS-NH2

SEQ ID NO: 5:

Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA)2-Lys} -AFVQWLIVGGPSSGAPPPS-NH2

SEQ ID NO: 6:

Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA)2-Lys} -VFVQWLIAGGPSSGAPPPS-NH2

SEQ ID NO: 7:

Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA)2-Lys} -VFVQWLIVGGPSSGAPPPS-NH2

SEQ ID NO: 8:

Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA)2-Lys} -AFVQWLI- {Aib} -GGPSSGAPPPS-NH2

SEQ ID NO: 9:

Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA)2-Lys} - {Aib} -FVQWLIAGGPSSGAPPPS-NH2 SEQ ID NO: 10:

Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA)2-Lys} - {Aib} -FVQWLI- {Aib} -GGPSSGAPPPS-NH2

SEQ ID NO: 11:

Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu-Asp- (AEEA)2-Lys} -VFVQWLIVGGPSSGAPPPS-NH2

SEQ ID NO: 12:

Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu-Asp- (AEEA)2-Lys} - {Aib} -FVQWLI- {Aib} -GGPSSGAPPPS-NH2

SEQ ID NO: 13:

Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid22-gamma-Glu-Asp- (AEEA)2-Lys} -VFVQWLIVGGPSSGAPPPS-NH2

SEQ ID NO: 14:

Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid22-gamma-Glu-Asp- (AEEA)2-Lys} - {Aib} -FVQWLI- {Aib} -GGPSSGAPPPS-NH2

Claims

A peptide analog having the sequence of SEQ ID NO: 1, or available salts thereof:

Tyr-Aib-Glu-X4-Thr-X6-Thr-Ser-Asp-Tyr-Ser-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-Gly-Gly-Pro-Ser-Ser-Gly-X35-Pro-Pro-Pro-Ser-X40-NH2; (SEQ ID NO: 1)

wherein 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 peptide segments composed of them; Alternatively, independently selected from modified L-type natural amino acids, modified D-type natural amino acids, modified non-natural amino acids, or peptide segments composed of them.
The peptide analog or available salt according to claim 1, wherein:

X4 is selected from Aib, Ser, Ala, or Gly amino acid residues;

X6 is selected from Tyr, Trp, or Phe amino acid residues;

X12 is selected from Val, Ala, Leu, Ile, Aib, Met, α-Me-Leu, D-Leu amino acid residues;

X13 is selected from Ala, Aib, Ser, Val amino acid residues;

X14 is selected from Ile, Val, Leu, Met, α-Me-Leu, α-Me-Val amino acid residues;

X15 is selected from Thr, Ser, or Asp amino acid residues;

X16 is selected from Ala, Aib, Lys, Met amino acid residues;

X17 is selected from Leu, Val, Met, Ile, α-Me-Leu, α-Me-Val amino acid residues;

X18 is selected from Aib, Val, Ala amino acid residues;

X19 is selected from Glu, Gln, Asn amino acid residues;

X20 is selected from Asp, Lys, Glu, Arg, or modified amino acid residues B amino acid residues;

X21 is selected from Aib, Ala, Val, D-Ala amino acid residues;

X22 is selected from Trp, Phe, Tyr amino acid residues;

X23 is selected from Aib, Ile, Leu, α-Me-Leu, α-Me-Val, Val amino acid residues;

X24 is selected from Gln, Asn, Ala, Met, Leu, Aib, α-Me-Leu, D-Gln amino acid residues;

X25 is selected from Tyr, Phe, Trp amino acid residues;

X26 is selected from Ile, Leu, Aib, α-Me-Leu, α-Me-Val amino acid residues;

X27 is selected from Ile, Val, Leu, Aib, α-Me-Leu, α-Me-Val amino acid residues;

X28 is selected from Aib, Val, Ala, α-Me-Val, D-Ala amino acid residues;

X35 is selected from Aib, Val, Ala, α-Me-Val amino acid residues;

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- (CH) n-COOH} ; and

wherein m is selected from integers 2-6, n is selected from integers 10-26, z is selected from integers 1-6, Y is selected from Asp, D-Asp, β-Asp, α-Me-Asp, GABA residues or Y is absent.
A pharmaceutical composition, comprising the peptide analog of SEQ ID NO: 1 and a pharmaceutically acceptable carrier.
The pharmaceutical composition according to claim 3, further comprising an additional active agent.
A method of treating obesity and/or diabetes in a patient comprising the step of administering the pharmaceutical composition of claim 3 to a patient in need of treatment.
The method according to claim 5 wherein the pharmaceutical composition comprises the peptide analog of claims 1or 2 (SEQ ID NO: 1-SEQ ID NO: 14) , or available salts thereof.
The method according to claim 5, further comprising the step of administering to said patient the pharmaceutical composition in a dosage from that contains about 0.1 mg to about 2000mg.
A peptide analog having the sequence of SEQ ID NO: 2-14, or available salts thereof:

SEQ ID NO: 2 : Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA) 2-Lys} -AFVQWLIAGGPSSGAPPPS-NH2;

SEQ ID NO: 3 : Y- {Aib} -EGTFTSDYSI- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA) 2-Lys} -VFVQWLIAGGPSSGAPPPS-NH2;

SEQ ID NO: 4 : Y- {Aib} -EGTFTSDYSI- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA) 2-Lys} -AFVQWLIVGGPSSGAPPPS-NH2;

SEQ ID NO: 5 : Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA) 2-Lys} -AFVQWLIVGGPSSGAPPPS-NH2;

SEQ ID NO: 6 : Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA) 2-Lys} -VFVQWLIAGGPSSGAPPPS-NH2;

SEQ ID NO: 7 : Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA) 2-Lys} -VFVQWLIVGGPSSGAPPPS-NH2;

SEQ ID NO: 8 : Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA) 2-Lys} -AFVQWLI- {Aib} -GGPSSGAPPPS-NH2;

SEQ ID NO: 9 : Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA) 2-Lys} - {Aib} -FVQWLIAGGPSSGAPPPS-NH2;

SEQ ID NO: 10 : Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu- (AEEA) 2-Lys} - {Aib} -FVQWLI- {Aib} -GGPSSGAPPPS-NH2;

SEQ ID NO: 11 : Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu-Asp- (AEEA) 2-Lys} -VFVQWLIVGGPSSGAPPPS-NH2;

SEQ ID NO: 12 : Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid20-gamma-Glu-Asp- (AEEA) 2-Lys} - {Aib} -FVQWLI- {Aib} -GGPSSGAPPPS-NH2;

SEQ ID NO: 13 : Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid22-gamma-Glu-Asp- (AEEA) 2-Lys} -VFVQWLIVGGPSSGAPPPS-NH2; and

SEQ ID NO: 14 : Y- {Aib} -EGTFTSDYSL- {Aib} -LDKIAQ- {diacid22-gamma-Glu-Asp- (AEEA) 2-Lys} - {Aib} -FVQWLI- {Aib}-GGPSSGAPPPS-NH2.