Polypeptide for promoting release of growth hormone and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to polypeptide for promoting release of growth hormone and application of the polypeptide, the polypeptide is shown as a formula (I), and in addition, the invention further provides application of the polypeptide shown as the formula (I) in preparation of medicine for diagnosing and / or treating diseases related to human growth hormone deficiency. Formula (I)

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a polypeptide for promoting growth hormone release and application thereof. BACKGROUND

[0002] Growth hormone (GH) is an important endocrine factor for regulating individual growth, development and other metabolic processes. In addition, it is also known that growth hormone has many effects on metabolic processes, such as stimulating protein synthesis and free fatty acid activity, and causing energy metabolism conversion from carbohydrate to fatty acid metabolism.

[0003] Cancer cachexia seriously threatens the survival of patients with malignant tumors, and is a multifactorial syndrome and a major complication in patients with various malignant tumors. Its characteristics include gradual reduction of skeletal muscle mass, atrophy of adipose tissue, systemic inflammation and other metabolic abnormalities leading to dysfunction. Clinically, cachexia is mainly manifested as uncontrolled weight loss in patients, accompanied by anemia, metabolic imbalance and systemic organ failure, and cannot be restored by conventional nutritional supplementation, resulting in a decrease in the quality of life of cancer patients and a serious weakening of the treatment effect of tumor chemotherapy, which is the main cause of death in many tumor patients.

[0004] At present, there is still a lack of effective specific drugs for the treatment of cancer cachexia worldwide. Anamorelin is the only marketed drug, and its core target is growth hormone secretagogue receptor 1a (GHS-R1a). By increasing growth hormone secretion, increasing food intake, improving body weight and muscle mass, increasing appetite and metabolism, and improving cancer cachexia, the results of clinical experiments on non-small cell lung cancer (NSCLC) patients and colorectal cancer patients show that anamorelin achieves the expected effect, but does not substantially improve the physical function of patients to achieve the reversible occurrence of cachexia, and the half-life in the body does not reach the standard of long-acting drugs. Studies have found that activating GHSR can promote the release of growth hormone from the pituitary gland, enhance appetite and stimulate gastrointestinal peristalsis, thereby increasing food intake and body weight in cancer patients. Therefore, targeting GHSR, developing long-acting new drugs for the treatment of tumor cachexia can effectively increase the appetite and body weight of cancer cachexia patients, and has good application prospects.

[0005] Growth Hormone Releasing Peptides (GHRPs) is a kind of small molecule polypeptide, which can combine with GHSR to produce agonistic activity, strongly stimulate the secretion of growth hormone, and plays an important role in regulating physiological processes such as body growth and development, metabolism. Among them, GHRP-6 is a typical and widely used member of GHRPs family. GHRP-6 belongs to small molecule polypeptide, and the sequence of GHRP-6 is [His-D-Trp-Ala-Trp-D-Phe-Lys-NH2]. Its molecular structure contains various amino acid residues, and the types, arrangement order and spatial conformation of these amino acid residues determine its biological activity and physicochemical properties.

[0006] Afterwards, GHRP-6 was chemically studied to identify other effective growth hormone releasing peptides, such as GHRPI, GHRP-2 and hexarelin, and the like, such as the compounds with growth hormone releasing properties disclosed in patents WO1993004081A1, WO1995017422A1, CN113045625A. In order to meet the clinical needs, it is urgent to develop novel molecular structures with high activity, small dose, good stability and low toxicity side effects. SUMMARY

[0007] Therefore, the technical problem to be solved by the present application is to provide a polypeptide for promoting growth hormone release with high activity and good stability, which has high activity as a GHSR receptor agonist.

[0008] To achieve the above-mentioned purpose, the present application provides a polypeptide for promoting growth hormone release, or a stereoisomer, a pharmaceutically acceptable salt thereof, and the polypeptide structure is as shown in formula (I),

[0009] Formula (I)

[0010] Among them, R1 is selected from F, Cl and Br.

[0011] Preferably, the polypeptide structure is as shown in formula (I), and R1 is selected from F and Cl.

[0012] Preferably, the polypeptide structure is as shown in compound 1,

[0013] Compound 1.

[0014] In a second aspect, the present application provides a pharmaceutical composition, which comprises the above-mentioned polypeptide or stereoisomer, pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0015] In a third aspect, the present application provides the use of the above-mentioned polypeptide or its stereoisomer, pharmaceutically acceptable salt or the above-mentioned pharmaceutical composition for the manufacture of a medicament for the diagnosis and / or treatment of a disease associated with human growth hormone deficiency.

[0016] In a fourth aspect, the present application provides the use of the above-mentioned polypeptide or its stereoisomer, pharmaceutically acceptable salt or the above-mentioned pharmaceutical composition together with other growth hormone releasing hormone and its functional equivalents, or compounds that promote the release of growth hormone for the manufacture of a medicament for the diagnosis and / or treatment of a disease associated with human growth hormone deficiency.

[0017] Preferably, the disease associated with human growth hormone deficiency is selected from cancer cachexia, slow growth in children, short stature in children, dwarfism, obesity, burn injury.

[0018] Preferably, the disease associated with human growth hormone deficiency is selected from cancer cachexia.

[0019] Preferably, the disease associated with human growth hormone deficiency is selected from slow growth in children, short stature in children.

[0020] Preferably, the disease associated with human growth hormone deficiency is selected from dwarfism.

[0021] In a fifth aspect, the present application provides a method for the solid phase synthesis of Compound 1, in which Rink Amide-AM Resin resin is sequentially coupled from carboxyl terminus to amino terminus according to the amino acid sequence {β-Ala}{D-4-ClPhe}AW{D-Phe}{Lys(Me)2} by solid phase synthesis.

[0022] As defined herein, the terms "polypeptide", "peptide" and "amino acid sequence" are used interchangeably herein and include two or more naturally occurring or synthetic amino acids linked by covalent bonds, e.g. amide bonds.

[0023] The term "amino acid" is defined as having at least one primary, secondary, tertiary, or quaternary amino group and at least one acid group, wherein the acid group can be a carboxylic acid, sulfonic acid, or phosphoric acid, or a mixture thereof. The amino group can be "α", "β", "γ" to "ω" relative to the acid group. Suitable amino acids include, but are not limited to, the D- and L-isomers of 20 common naturally occurring amino acids found in peptides, as well as naturally occurring and non-naturally occurring amino acids prepared through organic synthesis or other metabolic pathways. The 20 common naturally occurring amino acids are: glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), phenylalanine (Phe, F), tryptophan (Trp, W), methionine (Met, M), proline (Pro, P), threonine (Thr, T), serine (Ser, S), cysteine ​​(Cys, C), asparagine (Asn, N), glutamine (Gln, Q), tyrosine (Tyr, Y), lysine (Lys, K), arginine (Arg, R), histidine (His, H), aspartic acid (Asp, D), and glutamic acid (Glu, E). The D-isomers of the above naturally occurring amino acids are represented by lowercase letters.

[0024] The compounds of this invention contain multiple asymmetric centers and therefore exist as racemates and mixtures of racemates, single enantiomers, individual diastereomers, and mixtures of diastereomers. All such isomers of these compounds are explicitly included herein. Compounds may also be represented in various tautomer forms, in which case all tautomers of the compounds described herein are explicitly included herein. All such isomers of such compounds are explicitly included herein. All crystalline forms of the compounds described herein are explicitly included herein.

[0025] The term "pharmaceutically acceptable salt" or "salt" includes salts prepared from pharmaceutically acceptable non-toxic acids or bases (including inorganic or organic acids and bases). Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids, such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, aminosulfonic acid, and hydrobromic acid, or salts of pharmaceutically acceptable organic acids, such as acetic acid, trifluoroacetic acid (TFA), propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, maleic acid, citric acid, lactic acid, mucoic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, sulfinic acid, aspartic acid, glutamic acid, edemaic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid. Basic salts include, but are not limited to, salts formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium, and alkylammonium.

[0026] The term "pharmaceutical-grade carrier" includes any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and combinations thereof, as known to those skilled in the art. Except in cases of incompatibility with the active ingredient, the use of any conventional carrier in therapeutic or pharmaceutical compositions is considered.

[0027] The term "diagnosis" means detecting a disease or condition or determining the stage or extent of a disease or condition. The term "diagnosis" also includes detecting a predisposition to a disease or condition, determining the efficacy of a drug therapy, or predicting the mode of action of a drug therapy. The diagnostic methods disclosed herein may be used alone or in combination with other diagnostic and / or grading methods known in the medical field for a particular disease or condition.

[0028] The term “treatment” means to reduce, suppress, reverse, slow down, or stop the progression or severity of an existing condition, disease, symptom, or illness.

[0029] From the perspective of those skilled in the art, there is a general consensus in the biopharmaceutical field that "stability is positively correlated with efficacy"—that is, the stability of a compound is the basis for its efficacy: stability can ensure that the compound maintains its structural integrity during storage, administration and in vivo transport, and avoid the loss of active ingredients or the generation of toxic impurities due to degradation; conversely, compounds with insufficient stability are usually expected to have reduced efficacy, require increased dosage or have a narrowed therapeutic window.

[0030] Therefore, compound 1 of the present invention is less stable than control 2 and causes changes in calcium flux EC. 50 Even though the efficacy was lower than control 2, it still achieved a breakthrough improvement, completely breaking the reasonable expectations formed by those skilled in the art based on existing technology and conventional knowledge, constituting an unexpected technical effect. This unexpected effect could not be predicted through logical deduction of existing technology or conventional experimental methods, and it significantly improved the clinical application value of the compound (such as reducing the frequency of administration and reducing the risk of adverse reactions), demonstrating outstanding technological progress. Attached Figure Description

[0031] Figure 1 The results of liquid chromatography analysis for compound 1;

[0032] Figure 2 This is the mass spectrometry detection result of compound 1;

[0033] Figure 3 These are the results of calcium flow activity testing;

[0034] Figure 4A time-bent line plot of plasma GH concentration in rats after subcutaneous injection;

[0035] Figure 5 The area under the curve (AUC) of the GH concentration-time curve after subcutaneous injection in rats. Detailed Implementation

[0036] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0037] 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 are commercially available or prepared according to conventional methods in the art.

[0038] Example 1: Synthetic Example

[0039] The polypeptide compounds and their derivatives disclosed herein are synthesized using a solid-phase synthesis method to obtain their linear precursors, which are then cleaved to obtain the target crude peptide. In the synthesis process, Rink Amide resin is first fully swollen in N,N-dimethylformamide (DMF). Then, the solid-phase support is repeatedly subjected to condensation → washing → deprotection of Fmoc → washing → the next round of amino acid condensation to achieve the desired polypeptide chain length. Finally, the polypeptide is cleaved from the solid-phase support by reacting it with a mixed solution of trifluoroacetic acid:water:triisopropylsilane (95:2.5:2.5, v:v:v). The cleaved polypeptide is then precipitated by frozen methyl tert-butyl ether to obtain the crude target polypeptide. The crude polypeptide is purified and separated by a C-18 reversed-phase preparative chromatography column in a 0.1% trifluoroacetic acid / acetonitrile / water system to obtain pure polypeptides and their derivatives.

[0040] Experimental reagents The experimental reagents are shown in Table 1.

[0041] Table 1 Experimental Reagents

[0042]

[0043]

[0044] Synthesis of Compound 1, the structure of Compound 1 is shown below: .

[0045] Step 1: Synthesis of linear precursor peptide chains

[0046] The linear precursor peptide chain of compound 1 is: {β-Ala}{D-4-ClPhe}AW{D-Phe}{Lys(Me)2}.

[0047] 294 mg (0.2 mmol) of Rink Amide-AM Resin resin was fully swollen in DMF for 1 h. The linear precursor was then synthesized sequentially from the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows: Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time; the resin was washed with DMF 6–8 times until neutral pH; 1.0 mmol Fmoc-AA, 1.0 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 2 mmol 4-methylmorpholine (NMM) were dissolved in DMF and added to the resin, reacting at room temperature for 1 h; the resin was washed with DMF 4–6 times before the next amino acid coupling. After linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried under vacuum.

[0048] Step 2: Cleavage of the linear precursor peptide chain

[0049] Freshly prepared cut cocktail (10 mL) of trifluoroacetic acid:water:triisopropylsilane (95:2.5:2.5, v:v:v) was added to the resin obtained in step 1, and the mixture was shaken and reacted at room temperature for 2 hours. After the reaction was complete, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid. The mixture was combined with the reaction solution, and precipitated with 4 volumes of cold MTBE to obtain the crude product. The crude product was washed three times with MTBE and dried under vacuum.

[0050] Step 3: Preparation of Peptides

[0051] The crude peptide obtained in step 2 was dissolved in a 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR C-18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient to 20-45% acetonitrile in 40 min. The relevant fractions were collected, and after HPLC purity assessment, the fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0052] Step 4: Detection and Characterization Methods

[0053] The purity and molecular weight of the polypeptide purified in step 3 were determined by analytical high-performance liquid chromatography and liquid chromatography / mass spectrometry. The detection results are as follows: Figure 1 , Figure 2 As shown.

[0054] Example 2: Calcium Flow Activity Test

[0055] 1. Key reagents:

[0056] CHO-K1 cell line overexpressing GHSR, abbreviated as CHO-K1 / GHSR cells (GenScript, catalog number: M00189), Ghrelin (GenScript or in house), Fluo-4 Direct™ calcium content assay kit (Thermo, F10471).

[0057] 2. High-throughput screening process

[0058] 2.1 Culture of CHO-K1 / GHSR cells

[0059] Cell resuscitation: Remove cells from the liquid nitrogen container and rapidly thaw them in a 37°C water bath. Transfer cells to 15mL centrifuge tubes, slowly add 9mL of pre-warmed thawing medium (Ham's F12K, 10% FBS), centrifuge at 800-1200 rpm for 5 minutes, and remove the supernatant. Resuspend cells in 5mL of thawing medium, transfer to T25 culture flasks, and incubate at 37°C with 5% CO2. Replace the culture medium with growth medium on the second day after cell resuscitation.

[0060] Cell passage: When cells reach 90% confluence in the culture flask, passage them. First, rinse the cells with DPBS, then add DPBS again and gently tap the flask to remove the cells from the flask wall. Collect the cell suspension into a centrifuge tube, centrifuge at 800-1200 rpm for 3-5 minutes, and remove the supernatant. Add 6-8 mL of fresh growth medium (Ham's F12K, 10% FBS, 500 μg / mL G418) to resuspend the cells, and passage them at a ratio of 1:3 to 1:8. Incubate at 37°C in a 5% CO2 incubator. Change the medium every 2-3 days after passage.

[0061] 2.2 CHO-K1 / GHSR cell plating

[0062] 24 hours before the test, CHO-K1 / GHSR cells in the culture flask were digested with 0.25% trypsin and suspended in cell culture medium. The cells were then added to 384-well plates with a black transparent bottom at a density of 8000 cells per well using a dispensing apparatus. 25 μL per well was incubated overnight at 37°C with 5% CO2.

[0063] 2.3 Preparation of calcium staining solution and dilution of samples

[0064] Dissolve component A from the Fluo-4 Direct™ Calcium Content Assay Kit in 1L of calcium dye buffer (Loading buffer, 1×HBSS, 20mM HEPES, 1g glucose). Mix the calcium dye solution with the Loading buffer at a ratio of 1:3, and add 500 mM probenecid stock solution to prepare a calcium dye solution containing 2.5 mM probenecid.

[0065] The polypeptide compound to be tested was diluted to 8-12 concentrations with loading buffer containing 0.1% BSA.

[0066] 2.4 Calcium flow signal detection and result processing

[0067] After aspirating the cell culture supernatant from the 384-well plate, 50 μL of calcium dye solution was added to each well of the cell culture plate. The plate was incubated at 37°C for 30 minutes, then transferred to room temperature and incubated for another 30 minutes. The cell culture plate and the peptide solution plate were then placed in a FLIPR Tetra instrument for detection. Ghrelin was used as a positive control for the agonist. The detected fluorescence signal values ​​were analyzed using GraphPad software with a four-parameter nonlinear fitting method to calculate the EC50. 50 Values ​​are used for sample activity assessment.

[0068] 3. Experimental Results

[0069] The calcium flux activity test results of compound 1 and the control compound are shown in Table 2. Figure 3 As shown, the amino acid sequence of control 1 is AfAWfK-NH2, the amino acid sequence of control 2 is a{D-2-Nal}AWfK-NH2, and the amino acid sequence of control 3 is HwAWfK-NH2.

[0070] Table 2 Results of calcium flow activity test

[0071] Sample Test 1 EC 50 (nM) Test 2 EC 50 (nM) Control 1 2.04 4.21 Control 2 0.34 0.28 Control 3 11 / Compound 1 0.43 1.17

[0072] Example 3: Rat Plasma Stability Experiment

[0073] 1. Experimental Materials

[0074] Formic acid (purchased from Aladdin); DMSO (dimethyl sulfoxide) (purchased from Aladdin); methanol (purchased from Sigma); acetonitrile (purchased from Sigma).

[0075] 2. Experimental Procedure

[0076] Sample preparation: Dissolve the peptide to be tested in 50% methanol, water and 1% formic acid to 20 μM, and store at -4℃ for later use.

[0077] Plasma thawing: Remove plasma from the -80℃ freezer and thaw rapidly in a 37℃ water bath. Preparation of reaction solution: Take 47.5 μL of plasma (heparin sodium) from each tube and add it to 1.5 ml EP tubes from all time points, with three replicates per time point. At 240 min, add 2.5 μL of the test sample to achieve a final concentration of 1 μM. Incubate all plasma at 37℃. At 120 min, add another 2.5 μL of the test sample to achieve a final concentration of 1 μM and incubate at 37℃ for another 120 min.

[0078] Incubation: Incubate in a 37℃ water bath at three time points: 0 min, 120 min, and 240 min.

[0079] Termination of reaction: After incubation, compound 1 was precipitated with 4 times the volume of 0.1% formic acid in 75% acetonitrile aqueous solution, and control 1 and control 2 were precipitated with 4 times the volume of 0.1% formic acid in methanol.

[0080] Mixing: Mix by oscillating on a vortex oscillator.

[0081] Centrifugation: Centrifuge at 4℃, 15000 r / min for 10 min.

[0082] Take 80 μL of the supernatant, transfer it to a sample injection tube, and send it for analysis by LC-MS / MS. Plot a line graph with the ordinate representing the residual drug percentage (%) and the abscissa representing time. This shows the trend of sample degradation in in vitro plasma over time, providing the results of sample stability.

[0083]

[0084] Calculate the half-life T of the drug in plasma 1 / 2 .

[0085] The elimination rate constant (Ke) was calculated using first-order kinetics. Furthermore, the Ti of the compound in plasma was determined using the same formula. 1 / 2 (min).

[0086]

[0087] 3. Experimental Results The experimental results are shown in Table 3.

[0088] Table 3 Experimental Results

[0089]

[0090] The experimental results showed that the half-life of compound 1 (433.1 min) was stronger than that of control 1 (210.0 min) but weaker than that of control 2 (866.3 min), meaning that the stability of compound 1 was stronger than that of control 1 but weaker than that of control 2.

[0091] Example 4: Study on the effect of drugs on GH release levels in SD rats

[0092] 1. Materials and Methods

[0093] 1.1 Drugs: Compound 1, Control 1, Control 2.

[0094] 1.2 Reagents and consumables: Invirogen Rat Growth Hormone ELASA Kit (KRC5311), physiological saline, dimethyl sulfoxide, mixed protease inhibitors, 0.5 mL LEDTA anticoagulant tubes, and 0.5 mL low-adsorption centrifuge tubes.

[0095] 1.3 Experimental animals: 14 male SD rats, 180-200g, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.

[0096] 1.4 In vivo experimental methods: SD rats were housed under standard conditions and acclimatized for three days. A 3 mg / mL solution of the compound was prepared in physiological saline containing 3% DMSO. Approximately 200 μL of blood was collected from the eyelid vein of the rats 10 min before administration. A subcutaneous injection of 1 mL / kg was administered subcutaneously to the back. After administration, approximately 200 μL of blood was collected from the eyelid vein at 5 min, 10 min, 15 min, and 30 min. The blood samples were placed in EDTA anticoagulant tubes containing 2% mixed protease inhibitors, mixed thoroughly by inversion, and centrifuged at 1600 g for 10 min at 4°C to separate the plasma. The plasma samples were aliquoted and stored at -80°C until analysis.

[0097] 1.5 GH Content Detection Method: Thaw the plasma sample stock solution at room temperature. Dilute samples at time points of 0 min, 5 min, 10 min, 15 min, and 30 min with standard diluent at an appropriate ratio. Dilute the rat growth hormone standard to 20 ng / mL according to the instructions, gently rotate or mix, and let stand for 10 min to ensure complete mixing. Label this as 20 ng / mL rat growth hormone standard working solution and use within 15 min of mixing. Add 300 µL of standard diluent to each of the six labeled EP tubes. Pipette 300 µL of the 20 ng / mL standard working solution into one of the EP tubes and mix well to prepare a 10 ng / mL standard working solution. Repeat this process for the remaining tubes. Labels are: 10, 5, 2.5, 1.25, 0.63, and 0 ng / mL rat growth hormone. Dilute the wash buffer concentrate (25×) with distilled water according to the number of wells. Label this as 1× wash buffer. Dilute the 100× streptavidin-HRP concentrate to a 1× streptavidin-HRP dilution and mix thoroughly. Use within 15 minutes of mixing. Calculate the required number of strips beforehand. Remove the kit 20 minutes before the experiment and allow it to reach room temperature. All incubation steps are performed on a microplate shaker (400 rpm). Add 100 μL of standard working solution and the test sample to each well, leaving the chromogenic blank wells empty. Seal the plate with a gel plate and incubate at room temperature for 120 minutes. Discard the liquid, spin dry, add 300 μL of washing buffer to each well, soak for 1 minute, and spin dry. Repeat 3 times. Add 100 μL of rat growth hormone-biotin conjugate solution to each well, except for the chromogenic blank wells. Seal the plate with a gel plate and incubate at room temperature for 60 minutes. Discard the liquid, spin dry, add 300 μL of washing buffer to each well, soak for 1 minute, and spin dry. Repeat 3 times. Except for the blank wells, add 100 µL of streptavidin-HRP dilution (1×) to each well. Seal the plate with a gel plate and incubate at room temperature for 30 min. Discard the liquid, spin dry, add 300 µL of washing buffer to each well, soak for 1 min, and spin dry. Repeat 3 times. Add 100 µL of TMB chromogenic solution (protected from light) to each well, seal the plate with a gel plate, and incubate at room temperature in the dark for 30 min. Add 100 µL of stop solution to each well and gently tap one side of the plate to mix. The solution in the wells changes from blue to yellow. Read the absorbance (OD) value at 450 nm within 2 h. Calculate the average OD value of the standards and samples: subtract the OD value of the blank well from the OD value of each standard and sample. Plot a standard curve using the "four-parameter Agonist model" with the standard concentration as the x-axis and the OD value as the y-axis. Read the sample concentration from the standard curve and multiply the sample concentration by the dilution ratio to obtain the true concentration of rat GH.

[0098] 2. Experimental Results

[0099] The area under the curve (AUC) of the GH concentration-time curve of rats was calculated using GraphPadPrism 8.0.1 software after subcutaneous administration of compound 1, control 1, and control 2 to the back of rats.

[0100] See the line graphs of GH concentration-time at different time points in the body caused by each drug. Figure 4 The area under the curve of GH concentration-time is shown in the figure. Figure 5 The areas under the curves (AUC(0-t) of GH concentration-time after administration of compound 1, control 1, and control 2 were 1237±266.14 ng.min / mL, 256.39±244.07 ng.min / mL, and 423.01±280.68 ng.min / mL, respectively. Compound 1 showed a stronger stimulating effect than the two control molecules.

Claims

1. A polypeptide or a stereoisomer, a pharmaceutically acceptable salt thereof, characterized by, The polypeptide or its stereoisomer pharmaceutically acceptable salt structure is shown as formula (I), Formula (I) R1 is selected from F, Cl, Br.

2. The polypeptide or its stereoisomer, pharmaceutically acceptable salt according to claim 1, characterized in that, The polypeptide or its stereoisomer pharmaceutically acceptable salt structure is shown as formula (I), wherein R1 is selected from F, Cl.

3. The polypeptide or its stereoisomer, pharmaceutically acceptable salt according to claim 1 or 2, characterized in that, The polypeptide or its stereoisomer pharmaceutically acceptable salt structure is shown as compound 1, Compound 1.

4. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the polypeptide or its stereoisomer, pharmaceutically acceptable salt according to any one of claims 1-3, and a pharmaceutically acceptable carrier.

5. The use of the peptide or its stereoisomer, pharmaceutically acceptable salt according to any one of claims 1-3 or the pharmaceutical composition of claim 4 in the preparation of a medicament for the diagnosis and / or treatment of a disease associated with human growth hormone deficiency.

6. The use of the peptide or its stereoisomer, pharmaceutically acceptable salt according to any one of claims 1-3 or the pharmaceutical composition of claim 4 together with other growth hormone releasing hormone and its functional equivalents, or compounds that promote the release of growth hormone in the preparation of a medicament for the diagnosis and / or treatment of a disease associated with human growth hormone deficiency.

7. Use according to claim 5 or 6, characterized in that, The disease associated with human growth hormone deficiency is selected from cancer cachexia, slow growth in children, short stature in children, dwarfism, obesity, burns.

8. The use according to any one of claims 5 to 7, characterized in that, The disease associated with human growth hormone deficiency is selected from cancer cachexia.

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