CN111533800A

CN111533800A - Novel somatotropin-releasing hormone analogous peptide modification and dimerization preparation and application thereof

Info

Publication number: CN111533800A
Application number: CN202010193221.0A
Authority: CN
Inventors: 唐松山; 张旭东; 杨莉; 谭宏梅; 罗群; 唐婧晅
Original assignee: Zhejiang Huzhou Nafu Biomedical Co Ltd
Current assignee: Tang Lin
Priority date: 2020-03-18
Filing date: 2020-03-18
Publication date: 2020-08-14
Anticipated expiration: 2040-03-18
Also published as: WO2021160188A1; CN113929761A; CN113929761B; CN111533800B

Abstract

The invention provides the application of molecular variant of human growth hormone releasing hormone (hGHRH) peptide and dimer thereof in treating GH deficiency, infertility, senile dementia, diabetes and enhancing immunity. The dimer of the invention is formed by two same hGHRH-like peptide monomers containing single cysteine molecule allosteric through cysteineDisulfide bonds, and forms an H-type structure (single Ser → Cys substitution within the molecule). The invention also carries out fatty acid chain modification on the side chain-amino group of one lysine of the GHRH analog peptide. The GH sustained release time of the GHRH-like peptide monomer or dimer in vivo is prolonged by fatty acid modification, and reaches 17 days at most. The invention discovers that GHRH dimer with long-acting activity has Aib →²Ala substitution,¹⁸C‑¹⁸C disulfide bond formation, C20 fatty acid chain modification, and C-terminal amidation structures.

Description

Novel somatotropin-releasing hormone analogous peptide modification and dimerization preparation and application thereof

Technical Field

The invention belongs to the field of medical biology, and particularly relates to preparation of various human novel growth hormone releasing hormone similar peptide monomers and dimers and application thereof in treatment.

Background

Central growth hormone-releasing hormone-growth hormone-insulin-like growth factor (GHRH-GH-IGFs) endocrine axis, regulate body cell growth and sexual cell reproduction. The human GHRH complete sequence is hGHRH (1-44) NH₂hGHRH (1-29) is a core peptide with 51% bioactivity. The in vitro or in vivo activity of the novel hGHRH-like peptides and dimers thereof can be determined by measuring Growth Hormone (GH) release values by pituitary incubation in vitro or subcutaneous injection in vivo.

New studies have found that the effect of hGHRH molecules on peripheral tissues differs from that on central mechanisms of action, such as in the central, hypothalamic GHRH-pituitary GHRH receptor-pituitary GH with axis-specific effects, in the ovaries or testis of mature animals, although they also contain GHRH, GHRH receptor and GH molecular signals, they do not necessarily produce such axis-specific effects. In vivo injection of some novel GHRH analog peptides or dimeric molecules thereof will produce up-regulation of the GHRH receptor in germ cells to increase conception rates without promoting increased GH in vivo, indicating that reproduction of mature animals in subjects is not directly related to GH. In vitro pituitary incubation of such peptides (which do not produce GH in vivo) induces pituitary GH secretion. Therefore, the reproduction relationship of GH and mature animals is shown by measuring GH in vivo and in vitro pituitary GH secretion and the pregnancy rate of representative GHRH test animals of the test animals, and the new possibility of the peptides in treatment is shown.

GHRH peptide has wide pharmacological activity, such as treating infertility, regulating immunity, senile dementia, promoting wound healing, reconstructing myocardial cell, improving sleep quality, reducing weight or treating diabetes etc.

Although hGHRH analogs have great potential for use in biomedical and agronomic fields, their short half-life or low activity limits their utility. The structure-activity relationship indicates that the N-terminus¹Y is required for pituitary GH release in GHRH.¹P-GHRH shows pituitary GH release in vitro, but no GH release in vivo.¹F-GHRH shows very low GH release in vitro and in vivo. As the sequence extends towards the C-terminus, its activity and half-life increase. When two amino acid residues at the N-terminal of the GHRH molecule are cleaved by aminopeptidase (DPP-4) in the blood, the molecule is inactivated. hGHRH (1-29) NH₂Has a half-life of 13 minutes, natural hGHRH (1-44) NH₂ Half life 17 minutes. Up to now, hGHRH (1-44) NH₂Is the most active of these analogs.

Many protein precursors need to be associated with long fatty chains in the endoplasmic reticulum to increase maturation processing ability, or some protein molecules distributed in the cell membrane, the endoplasmic reticulum membrane or the golgi membrane, etc. will be anchored to these membrane phase systems via long fatty acid chains to increase localization ability. These fatty acylated GHRH molecules significantly increase half-life and at the same time, multiply increase their molecular activity. The invention utilizes the technologies to generate a series of novel long-acting GHRH molecules, and is applied to the treatment of infertility, immunity regulation, senile dementia, wound healing promotion, myocardial cell reconstruction, sleep quality improvement, weight reduction or diabetes.

Disclosure of Invention

It is an object of the present invention to overcome the above-mentioned deficiencies of the prior art and to provide Growth Hormone Releasing Hormone (GHRH) like peptide monomers. The GHRH-like peptide of the present invention has a molecular allosteric conformation to the main peptide chain, on the one hand, a single C → S or homocysteine (Hcy) → T substitution inside the monomeric peptide, and the position of cysteine in the peptide chain has been studied. On the other hand, the side chain-amino group of lysine (K) in the peptide chain is modified with a glutamyl fatty acid or glutamyl fatty diacid or [2 × AEEAC- γ -Glu (N- α -fatty diacid) ] substituent.

In order to achieve the purpose, the invention adopts the technical scheme that: a Growth Hormone Releasing Hormone (GHRH) -like peptide monomer, wherein a serine S of the peptide chain is substituted with a cysteine C, or a threonine T is substituted with a homocysteine (Hcy), and one of the lysines of the main peptide chain is a lysine modified on the side chain-amino group with a glutamyl fatty acid [ γ -Glu (N- α -fatty acid) ] or glutamyl fatty diacid [ γ -Glu (N- α -fatty diacid) ] or [2 × AEEAC- γ -Glu (N- α -fatty diacid) ].

Preferably, the specific sequence of the analogous peptide monomer is any one of the following:

(1)(NH₂)X₁-X₂-DAIFTNCY-X₁₁-X₁₂-VLGQLSA-X₂₀-X₂₁-LLQDIMSR-NH₂or;

(2)(NH₂)X₁-X₂-DAIFTNSY-X₁₁-X₁₂-VLGQLCA-X₂₀-X₂₁-LLQDIMSR-NH₂or;

(3)(NH₂)X₁-X₂-DAIFTNSY-X₁₁-X₁₂-VLGQLSA-X₂₀-X₂₁-LLQDIMCR-NH₂or;

(4)(NH₂)X₁-X₂-DAIF-Hcy-NSY-X₁₁-X₁₂-VLGQLSA-X₂₀-X₂₁-LLQDIMS-X₂₉-NH₂or;

(5)(NH₂)X₁-X₂-DAIFTNCY-X₁₁-X₁₂-VLGQLSA-X₂₀-X₂₁-LLQDIMS-X₂₉-QQGESNQE-X₃₈-GA-X₄₁-A-X₄₃-L-NH₂or;

(6)(NH₂)X₁-X₂-DAIFTNSY-X₁₁-X₁₂-VLGQLCA-X₂₀-X₂₁-LLQDIMS-X₂₉-QQGESNQE-X₃₈-GA-X₄₁-A-X₄₃-L-NH₂or;

(7)(NH₂)X₁-X₂-DAIFTNSY-X₁₁-X₁₂-VLGQLSA-X₂₀-X₂₁-LLQDIMC-X₂₉-QQGESNQE-X₃₈-GA-X₄₁-A-X₄₃-L-NH₂or;

(8)(NH₂)X₁-X₂-DAIFTNSY-X₁₁-X₁₂-VLGQLSA-X₂₀-X₂₁-LLQDIMS-X₂₉-QQGECNQE-X₃₈-GA-X₄₁-A-X₄₃-L-NH₂or;

(9)(NH₂)X₁-X₂-DAIF-Hcy-NSY-X₁₁-X₁₂-VLGQLSA-X₂₀-X₂₁-LLQDIMS-X₂₉-QQGESNQE-X₃₈-GA-X₄₁-A-X₄₃-L-NH₂

wherein, X₁Is Y or P or F; x₂Is A or α -aminoisobutyric acid (α Aib); X₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is K, or is R, or is a side chain-amino glutamyl fatty acid [ gamma-Glu (N- α -fat acid)]Or glutamyl fatty diacid [ gamma-Glu (N- α -fat diacid)](the aliphatic chain is 18 or 20 carbons) or the side chain-amino group [2 × AEEAC-gamma-Glu (N- α -fatty diacid)](the aliphatic diacid is 18 or 20 carbons) modified lysine. Wherein the capital letter is an abbreviation of L-alpha-amino acid or an amino acid substitution symbol, the Arabic numerals are an amino acid residue arrangement sequence, NH₂Represents an N-terminal or C-terminal amide group structure.

Preferably, when X₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is side chain-amino glutamyl fatty acid [ gamma-Glu (N- α -fat acid)]Or glutamyl fatty diacid [ gamma-Glu (N- α -fat diacid)]Modified lysine has a structure shown in chemical formula 1; when X is present₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is a side chain-amino group [ alpha ]2 × AEEAC-gamma-Glu (N- α -fatty diacid)]Modified lysine, the structure of which is shown in chemical formula 2:

the invention also provides a GHRH-like peptide homodimer, which is formed by connecting the same monomers through a disulfide bond formed by cysteine to form an H-type GHRH-like peptide homodimer.

Preferably, the amino acid sequence of the dimer is any one of:

wherein, X₁Is Y or P or F; x₂Is A or α -aminoisobutyric acid (α Aib); X₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is K, or is R, or is a side chain-amino glutamyl fatty acid [ gamma-Glu (N- α -fat acid)]Or glutamyl fatty diacid [ gamma-Glu (N- α -fat diacid)]Modified lysine, or in the side chain-amino group [2 × AEEAC-gamma-Glu (N- α -fatty diacid)]A modified lysine. Wherein the capital letter is an abbreviation of L-alpha-amino acid or an amino acid substitution symbol, the Arabic numerals are an amino acid residue arrangement sequence, NH₂Represents an N-terminal or C-terminal amide group structure.

Preferably, when X₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is side chain-amino glutamyl fatty acid [ gamma-Glu (N- α -fat acid)]Or glutamyl fatty diacid [ gamma-Glu (N- α -fat diacid)]Modified lysine has a structure shown in chemical formula 1; when X is present₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is on the side chain-amino group [2 × AEEAC-gamma-Glu (N- α -fatty diacid)]The structure of modified lysine is shown in chemical formula 2.

The invention also provides the application of the growth hormone releasing hormone similar peptide or the homodimer in preparing medicines for treating GH deficiency (growth hormone deficiency), infertility, senile dementia, immunity enhancement or diabetes.

The invention also provides a medicament for treating GH deficiency, infertility, senile dementia, immune enhancement or diabetes, which is characterized in that the medicament is the growth hormone releasing hormone analog peptide or the pharmaceutically acceptable salt of the homodimer as an active ingredient.

The invention has the beneficial effects that: the ghrelin-like dimer of the present invention can improve the in vitro or/and in vivo ghrelin-releasing activity, and thus can treat a disease of low GH secretion. Through in vitro pituitary GH release activity experiments, the following advantages can be obtained: (1) the monomeric peptides of the invention have an N-terminus compared to standard S1 or S2 peptides¹Y-or¹The P-GHRH-like peptide monomer shows at least 2 times of in vitro GH release activity,¹F-GHRH shows low GH-releasing activity in vitro and in vivo; (2) dimer activity of the present invention: the 2Y series shows high GH release activity in vivo and in vitro, the 2P series only shows in vitro activity, and the 2F series shows very low GH release activity in vivo and in vitro; (3) n-terminal is¹Y-or¹P(1-29)NH₂The activity of the dimeric peptide is only¹Y-or¹P(1-44)NH₂About 50% of the dimeric peptide; (4) lysine side chain-amino modification [ AEEA)₂The activity of the-gamma Glu-fatty diacid peptide is slightly increased compared with that of the [ N-gamma Glu (N- α -fatty acid) ] modification, the longer the modified fatty acid chain is, the higher the activity is, and the fatty acid chain C20 is the best.

Drawings

FIG. 1 is a diagram of the analysis of testis weights of experimental animals.

FIG. 2 is a diagram of serum cholinesterase analysis of Alzheimer's disease mice.

FIG. 3 is a diagram showing the result of serum cholinesterase analysis of diabetic mice.

FIG. 4 is a graph showing statistical analysis of the total number of monocytes in animals on different days before and after administration.

FIG. 5 is a graph of statistical analysis of the percentage of monocytes in animals before and 18 days after dosing.

FIG. 6 is a diagram showing the analysis of the total number of monocytes in experimental animals.

FIG. 7 is a statistical analysis of spleen weights tested.

FIG. 8 is an analysis of H-E staining for treatment with a low immunity model [ FIG. A is a picture of H-E staining (4X 10 fold, 100 μm); and B, analyzing the percentage of spleen red marrow of the experimental animals).

Detailed Description

In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.

Example 1: preparation of monomeric peptides and dimers

Firstly, a monomer peptide synthesis process: manual solid phase peptide synthesis operation steps.

1. Swelling resin: amino resin (amino resin for C-terminal amidated sequence) (available from Nankai Synthesis Technologies, Inc., Tianjin) was placed in a reaction vessel, and 15ml/g of methylene chloride (DCM, Dikma Technologies Inc.) was added thereto and shaken for 30min. SYMPHONY type 12-channel polypeptide synthesizer (SYMPHONY model, software version.201, protein technologies Inc.).

2. Grafting with the first amino acid: the solvent was removed by suction filtration through a sand core, 3-fold molar addition of the first Fmoc-amino acid at the C-terminus (all Fmoc-amino acids supplied by Suzhou Tianma pharmaceutical group Fine Chemicals, Inc.), 10-fold molar addition of 4-Dimethylaminopyridine (DMAP) and N, N' -Dicyclohexylcarbodiimide (DCC), and finally addition of Dimethylformamide (DMF) (purchased from Dikma Technologies Inc.) for dissolution and shaking for 30min. Blocking with acetic anhydride.

3. Deprotection: DMF was removed, 20% piperidine-DMF solution (15ml/g) was added for 5min, the solvent was removed by filtration, and 20% piperidine-DMF solution (15ml/g) was added for 15 min. Piperidine is supplied by Shanghai chemical company, national drug group.

4. And (3) detection: the solvent was removed by suction. Taking dozens of resins, washing the resins with ethanol for three times, adding ninhydrin, KCN and phenol solution one drop each, heating the mixture at the temperature of 105 ℃ and 110 ℃ for 5min, and turning dark blue to be a positive reaction.

5. Resin washing: two washes with DMF (10ml/g), two washes with methanol (10ml/g) and two washes with DMF (10ml/g) were performed in sequence.

6. Condensation: depending on the specific synthesis conditions, the following methods may be used alone or in combination in the polypeptide synthesis:

the method a comprises the following steps: three times of protective amino acid and three times of 2- (7-azobenzotriazol) -tetramethyluronium hexafluorophosphate (HBTU, Suzhou Tianma pharmaceutical group fine chemicals Co., Ltd.) were dissolved in DMF as little as possible and added to the reaction vessel. Ten times of N-methylmorpholine (NMM, Suzhou Tianma pharmaceutical group, Fine chemical Co., Ltd.) was immediately added to react for 30min, and the detection was negative.

The method b: three times of protected amino acid FMOC-amino acid and three times of 1-hydroxybenzotriazole (HOBt, Suzhou Tianma pharmaceutical group fine chemicals Co., Ltd.) are dissolved by DMF as little as possible, added into a reaction tube, immediately added with three times of N, N' -Diisopropylcarbodiimide (DIC) and reacted for 30min, and the detection shows negative.

7. Resin washing: in this order DMF (10ml/g) was washed once, methanol (10ml/g) was washed twice and DMF (10ml/g) was washed twice.

8. The operation of 2 to 6 steps was repeated, and as shown by the amino acids in Table 1, the corresponding amino acids were sequentially linked from right to left. With K fatty acid modifications, synthesized as follows 9.

9. Synthesizing K { N- - [ gamma-Glu- (N-alpha-fatty acid) ] } or K { N- - [ gamma-Glu- (N-alpha-fatty diacid) ] }: adding 10ml 2% hydrazine hydrate to react for 30min to remove the protecting group Dde of Fmoc-Lys (Dde) -OH, exposing the side chain amino group, alternately washing with DMF and methanol for six times, and detecting ninhydrin as blue. 550mg of Fmoc-Glu-OTBU and 250mg of HOBT are weighed, dissolved in DMF, added with 0.3ml of DIC, mixed evenly, added into a reactor to react with lysine side chain amino for 1h, pumped to dry, washed 4 times by DMF, and detected as colorless by ninhydrin. 5ml of 20% piperidine DMF solution was added to the reactor for reaction for 20min to remove the amino protecting group Fmoc of Fmoc-Glu-OTBU, followed by six washes with DMF and methanol alternately, and ninhydrin was detected as blue. Weighing 300mg of fatty acid or fatty diacid and 250mg of HOBT, dissolving with DMF, adding 0.3ml of DIC, mixing uniformly, adding into a reactor, reacting for 1h, draining, washing with DMF for 4 times, detecting the ninhydrin to be colorless, and washing with methanol for 2 times, draining.

Synthesis of K { N- - [2 × AEEAC- γ -Glu- (N- α -fatty diacid) ] }: after the Dde-Lys (Fmoc) group is removed, 2mM of Fmoc-AEEAC-OH and 2mM of benzotriazole-1-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 45mM of HOBt are added, the mixture is dissolved in DMF, 0.375mM of N, N' -Diisopropylethylamine (DIPEA) is added to the mixture under ice water bath for activation for 3min, and the mixture is added into a reaction kettle for reaction for 2h, and the end point of the experiment is judged by the detection of the ninhydrin method. After the reaction was complete, Fmoc was removed from 20% piperidine-DMF solution (15ml/g) and washed 6 times with DMF. Fmoc-AEEAC-OH, Fmoc-Glu-OtBu and the fatty diacid groups were coupled again in the same way. Reacting with 2% hydrazine hydrate for 30min to remove the sequence lysine protecting group Dde, and grafting on the amino group of the lysyl side chain through step 8.

10. The polypeptide after condensation was passed twice through DMF (10ml/g), twice DCM (10ml/g) and twice DMF (10ml/g) and dried by suction for 10 min. Ninhydrin test negative.

11. Removing FMOC protecting group of final N-terminal amino acid of the peptide chain, detecting to be positive, and draining the solution for later use.

12. The resin was washed twice with DMF (10ml/g), twice with methanol (10ml/g), twice with DMF (10ml/g) and twice with DCM (10ml/g) and dried by suction for 10 min.

13. Cleavage of the polypeptide from the resin: preparing cutting fluid (10 ml/g): TFA 94% (j.t. baker chemical company), water 2.5%, ethanedithinol (EDT, Sigma-Aldrich Chemistry) 2.5% and trisisopyrophyllane (TIS, Sigma-Aldrich Chemistry) 1%. Cutting time: and (4) 120 min.

14. Drying and washing: the lysate is blown dry as much as possible with nitrogen, washed six times with ether and then evaporated to dryness at normal temperature.

15. The polypeptides were HPLC purified, identified and stored at-20 ℃ in the dark as follows.

Secondly, the inspection method comprises the following steps:

1. purification of the polypeptide by HPLC: dissolving the crude peptide with pure water or adding a small amount of acetonitrile or other solvents, and purifying according to the following conditions: high performance liquid chromatography (analytical; software Class-VP. service System; manufacturer Japan SHIMADZU) and Venusi MRC-ODS C18 column (30X 250mm, Tianjin Bonna-Agela Technologies). Mobile phase A liquid: 0.1% trifluoroacetic acid aqueous solution, mobile phase B liquid: 0.1% trifluoroacetic acid + 99.9% acetonitrile (purchased from acetonitrile FisherScientific). Flow rate: 1.0ml/min, a loading volume of 30. mu.l, a detection wavelength of 220 nm. Elution procedure: 0-5 min: 90% of solution A and 10% of solution B; 5-30 min: 90% solution A/10% solution B → 20% solution A/80% solution B.

2. Finally, the purified effective solution was lyophilized (Freezone Plus 6, L ABCCONCO) to obtain the final product.

3. And (3) identification: a small amount of finished polypeptide is respectively taken and analyzed by HPLC for purity: high performance liquid chromatography (Shimadzu, Japan) and Venusi MRC-ODS C18 column (4.6X150mm, Tianjin Bonna-Agela Technologies). Mobile phase A liquid: 0.1% trifluoroacetic acid in water, mobile phase B liquid: 99.9% acetonitrile + 0.1% trifluoroacetic acid solution, flow rate: 1.0ml/min, a loading volume of 10. mu.l, a detection wavelength of 220 nm. Elution procedure: 0-5 min: 100% of solution A; 5-30 min: 100% solution A → 20% solution A/80% solution B. The purity required to be determined is greater than 95%. See the patent (Chinese patent ZL201410612382.3) granted by us for a specific method.

MS method for identifying molecular weight of polypeptide: adding water into polypeptide with qualified purity for dissolving, adding 5% acetic acid, 8% acetonitrile and 87 water for dissolving, testing electrospray ionization mass spectrometry to determine molecular weight, and the specific method is disclosed in the patent (Chinese patent ZL 201410612382.3).

4. Sealing and packaging the powdery polypeptide, and storing at-20 deg.C in dark.

And thirdly, forming a dimer: monomeric peptides with a single cysteine inside the peptide chain at a concentration of 1mg/ml were incubated overnight at 37 ℃ in aqueous disodium hydrogen phosphate at a pH of 9.5 to form homodimeric peptides. Many poorly soluble dimeric peptides were centrifuged at 4000 rpm for 20 minutes and the pellet was taken as pure dimeric peptide. The dissolved monomeric or dimeric peptide is separated by Sephadex G-25 chromatography (the dimeric fraction is usually the first peak followed by the monomeric or contaminating fraction with physiological saline or NaCl-PB solution as the flow term in a 2X 60cm G-25 column and natural flow rate). The dimeric peptide can be identified by SDS-PAGE electrophoresis or mass spectrometry of the peptide without a thiol reducing agent, and the specific method is shown in an issued patent (Chinese patent ZL 201410612382.3).

Four, GHRH analog peptide monomers and dimers thereof were synthesized by the present research laboratory or entrusted commercial companies, and the inventors confirmed their structures by HPLC purity, ESI or laser flight mass spectrometry, and cysteine oxidation. The GHRH monomeric peptides synthesized by the present invention are shown in table 1, and the amino acid sequences of the homodimeric peptides are shown in table 2.

Fifthly, peptide species: the amino acid sequences of GHRH monomer-like peptides synthesized according to the above method are shown in Table 1, and their HPLC purities>95% in hGHRH (1-29) NH₂(S1) or hGHRH (1-44) NH₂(S2) as a positive control.

TABLE 1 amino acid sequences of novel GHRH monomeric peptides synthesized according to the present invention and their single injection sustained GH-releasing activity

Note: CFA (carbon fat acid) or CFDA (carbon fat diacid) is carbon fatty acid or carbon fatty diacid; the K [ N- - (gamma-Glu-N-alpha-CFA) ], K [ N- - (2 × AEEAC-gamma-Glu-N-alpha-CFDA) ] represents fatty acyl or fatty diacid glutamyl modification of lysine K side chain-amino, and the specific structure is shown as

formula

1 or 2.

TABLE 2 amino acid sequences of synthetic GHRH dimer peptides of the present invention and their single injection sustained GH-releasing activity

CFA (carbon fat acid) is carbon fatty acid, CFDA (carbon fat diacid) is carbon fatty diacid, K [ N- - (gamma-Glu-N- α -CFA or CFDA)]、K[N--(2×AEEAC-γ-Glu-N-α-CFDA)]The structure of the modification of fatty acyl or fatty diacid glutamyl of lysine K side chain-amino is shown as

formula

1 or 2.

Representing a disulfide bond.

Example 2: growth hormone releasing Activity assay

1. Growth Hormone (GH) release assay in mice: kunming mice (female, 18-22g in weight, purchased from the Experimental animals center of TCM university, Guangzhou, n ═ 6). The care and use regimens for all experimental animals were in accordance with the experimental animal guidelines. In the breeding process, mice are bred alternately in light and shade for 12:12 hours at the breeding temperature of 26 +/-1 ℃. Peptide was injected subcutaneously at the hip of Kunming mice at a dose of 0.0133. mu. mol/kg, and blood was collected from the eyeball 1 hour after administration and from the serum at 6000 rpm/30 min after centrifugation, respectively, for GH assay. In the experiment, all the novel monomeric or dimeric peptides were dissolved with T-NaCl-PB solution (detailed in the administration setting), and the negative control group was injected with T-NaCl-PB solution. The murine GH hormone was tested using an ELISA kit (shanghai, combined mai bioengineering, ltd., china). The results are shown in tables 1 and 2.

2. Setting the administration concentration: because many monomeric or dimeric peptides are poorly soluble, the positive, novel monomeric, or dimeric peptides under this test were formulated with T-NaCl-solution (pH6.5) or T-NaCl-PB (pH8.0) solutions: taking a proper amount of pure peptide powder, dropwise adding pure medicinal Tween 80 (Nanjing Will chemical Co., Ltd., China) with the final concentration of 3%, and stirring with a magnetic stirrer at 900 rpm for 5-10 minutes. After the peptide is dispersed in the tween, slowly dropwise adding normal saline (monomer peptide dissolved) or NaCl-PB solution (normal saline solution pH8.0 buffered by disodium hydrogen phosphate, dimer dissolved), continuously stirring until all the set solution is added, continuously and rapidly stirring twice, each time for 20 minutes, and standing for 15 minutes. Centrifuging at 3000 rpm for 10min, and collecting supernatant for use (precipitating to obtain pure peptide, which can be repeatedly dissolved for use). All peptide concentrations were determined by the LOWRY method, with a blank of T-NaCl-PB or T-NaCl solution (NaCl-PB or NaCl solution containing 3% Tween 80).

Our preliminary experiments show that 0.0133. mu. mol/kg¹The Y-GHRH peptide induced significant GH release in mice or rat pituitary, so all peptides tested were used at a dose of 0.0133. mu. mol/kg in this test. In the measurement of mouse GH release duration, for the novel GHRH peptide with 1-29 amino acid sequence, hGHRH (1-29) NH was used as the positive control group₂(S1); positive drug control hGHRH (1-44) NH was used for novel GHRH peptides of 1-44 amino acid sequence₂(S2)。

3. In vitro rat pituitary growth hormone releasing Activity S-D female rat (body weight 200 + -20 g, purchased from the university of Chinese medicine, university of medicine center.) rat undergoes sudden death by dislocation, pituitary was harvested at 30 minutes, after washing with sterilized lactated Ringer' S buffer (LRB), pituitary was immediately placed in a 1 × 10cm glass tube containing 1ml LRB, incubated at 37 ℃ and all glass slides were incubatedGlass tube total heat preservation 5 h (P)₁,P₂,I₃,I₄And I₅Representing different periods of 5 hour incubation, each period representing 1 hour), while gently shaking every 5 min. After every 1 hour, all buffer was aspirated for pituitary GH hormone testing, while 1ml of fresh LRB with no or with GHRH-like peptide was added to the pituitary for incubation. In the first 2 hours (P)₁And P₂) After the preincubation, the subsequent incubation is carried out for 3-6 hours (I)₃、I₄And I₅) In this experiment, the polypeptide is initially increased and a blank LRB solution is used as a control. The net GH release level of the peptide is [ peptide (I)₃+I₄+I₅) -blank (I)₃+I₄+I₅)]. Rat pituitary rat GH hormone was tested using an ELISA kit (shanghai amalimei bioengineering ltd, china). The results are shown in tables 1 and 2.

4. Novel GHRH-like peptide sustained release GH assay in vivo: kunming mice (female, with the weight of 18-22g, n is 6) are injected with peptide in a single subcutaneous way at the hip of the Kunming mice according to the dose of 0.0133 mu mol/kg, 50 microlitres of blood is collected at the inner canthus at the right time 9 points in the morning on different days before and after administration until GH secretion reaches the normal value, and experimental blank control groups are injected with T-NaCl (monomer peptide) or T-NaCl-PB (dimer) solution. The canthus blood 6000 r/heart separation 30min, taking serum to determine GH. The results are shown in tables 1 and 2.

5. And (4) conclusion: (1) for the Y or 2Y series of GHRH peptides, whether in vivo or in vitro, the secretion of pituitary GH is obviously stimulated, so the peptides can be used for treating GH deficiency; (2) for the P or 2P series of GHRH peptides, the in vitro tests clearly stimulated pituitary GH secretion but no significant GH release in vivo, suggesting that such peptides may not act on the pituitary in vivo; (3) for the F or 2F series of GHRH peptides, both in vivo and in vitro, pituitary GH secretion is stimulated to be very low; (4) according to these results, a significantly long-acting GH sustained release time was observed in vivo for the Y or 2Y series of peptides, but not for the P or 2P series of peptides, or the F or 2F series of peptides showed a shorter GH sustained release time at the observed dose because of their low activity; (5) according to the hydrolysis mechanism of aminodipeptidase (DPP-4), the peptide loses activity after two amino acids at the N-terminal of the novel GHRH are hydrolyzed,therefore, other GHRH peptides having the same sequence should have similar half-lives, such as Y, P and F series having the same sequence, or 2Y, 2P and 2F series having similar half-lives, except that the first amino acid at the N-terminus is different. Although the P and 2P series of peptides or the F and 2F series of peptides show no or very low GH values in vivo, their half-lives should be similar to the corresponding Y and 2Y sequences; (6) GHRH (1-29) contains 51% of activity, and has important application value in science and commerce due to convenient and simple synthesis and low cost, and the GHRH preparation method independently embodies the contents. For peptides containing 1-29 or 1-44, (S1) hGHRH (1-29) NH is used₂Or (S2) hGHRH (1-44) NH₂The standard is made, so that the activity of the peptide is better reflected; (7) the dimers showed at least 2-fold more in vivo duration compared to for the monomeric peptides. According to the position of disulfide bonds, the release time of GH in vivo is Y1 series 2-6 days, Y2 series 5-8 days, Y3 series 2-5 days, Y4 series 3-5 days, Y5 series 2-8 days, Y6 series 3-5 days, Y7 series 3-5 days, Y8 series 2-4 days, 2Y1 series 4-11 days, 2Y2 series 10-15 days, 2Y3 series 4-9 days, 2Y4 series 4-8 days, 2Y5 series 5-17 days, 2Y6 series 6-9 days, 2Y7 series 6-9 days and 2Y8 series 3-7 days, and obviously the necessary long-acting structure comprises Aib →²Ala substitution,¹⁸C-¹⁸C disulfide bond formation, K₂₀Or K₂₁Fatty acid modification and C-terminal amidation structure, the position of disulfide bond is close to C terminal, and the sustained release time of GH is shorter.

Example 3: treatment of infertility models with dimeric peptides

1. The sterile infertility model test method comprises the following steps: the male rats of 130 Zhonghua hamster or the female rats of 65 Zhonghua hamster (five weeks old, 18-22 g/rat, provided by Sichuan Daoho laboratory animal center) were selected, because preliminary experiments showed that the male rats were significantly less sensitive to peptides than the female rats, the male sterile model n ═ 20, while the female infertility model n ═ 10, because the GHRH of Chinese hamster had the highest 93% homology with human, and were divided into blank control groups (male rats 20, female rats 10) and model rats (male rats 110, female rats 55). All model groups were intraperitoneally injected with 20mg/kg cyclophosphamide (Jiangsu Henry pharmaceuticals, Inc., national drug Standard H32020857, lot number 12032925) once a week for a total of five times. After the injection of cyclophosphamide at the fourth week, the model mice were divided into a model control group (Placebo), a human gonadotropin hMG group and a dimer peptide dose group 2Y-47, 2P-104 or 2F-120 according to the non-differential body weight of the hamster, and each group of male mouse model n-20 or each group of female mouse model n-10 (cyclophosphamide injection causes death of animals, so the number of prepared models was increased). The dosage was weighed and calculated before each group was administered, and the dimer peptide was administered intramuscularly at a dose of 0.0133. mu. mol/kg (dissolved in T-NaCl-PB solution), and the positive control hMG group was administered intramuscularly at 200 units/kg (16.65. mu. mol/kg) (dissolved in T-NaCl-PB solution) (urocortin hMG, gauge intramuscular injection of 400U/mg or 12.012U/. mu. mol, manufactured by Zhuhaili bead pharmaceutical industry). The blank control group and Placebo group were injected with T-NaCl-PB solution once a day until the end of the experiment. At 6-8 weeks, model and normal rats were mated with a cage 1:1 heterosexual, after which only therapeutic drugs were given, and the physiological changes (mental, activity, secretions, etc.) during the estrus of the rats were observed and whether the rats were pregnant or not was observed. At the end of the tenth week, mice were killed by dislocation and pregnant mice were counted by dissection. And (3) pregnancy judgment: the red beaded particles can be seen in the oviduct of the female mouse; newborn mice: it was born ten weeks ago.

2. And (3) carrying out statistics on the pregnancy rate of the hamster: organ gravimetric analysis showed that Placebo group showed significant testicular reduction (P <0.05) compared to the blank control group. Compared with Placebo group, the weight of testis in hMG group and 2Y-47 group is significantly increased (P <0.05 or 0.01), showing that hMG and 2Y-47 significantly stimulate testis action. The testis weights were reduced to different degrees in the 2P-104 or 2F-120 groups compared to the hMG group or 2Y-47 group (FIG. 1). Ovarian gravimetric analysis of female models also showed similar changes.

The hMG, 2Y-47 and 2P-104 groups showed a significant increase in total pregnancy compared to the Placebo or the Placebo group, regardless of the female or male mouse model, and they showed statistical differences from the Placebo or the Placebo group, whereas the 2F-120 group showed a decrease in pregnancy rate and showed no difference from the Placebo or the Placebo group (tables 3 and 4), showing that the reproductive capacity of the adult gerbils was not related to GH.

TABLE 3 female mouse model group pregnancy Rate statistics (Mean + -SD)

P <0.05, a, b vs blank control group, Placebo group comparison, chi-square test.

TABLE 4 Male mouse model group pregnancy Rate statistics (Mean SD)

3. And (4) conclusion: (1) an adult 60kg patient is injected with 75-150U of hMG per time per day, the dose ratio of the hMG to the hamster is 9.1 times, the converted hamster dose is averagely 17.05U/kg (1.41968 mu mol/kg), but the hMG has low response to the hamster, 200U/kg (16.65 mu mol/kg) achieves similar pregnancy rate with certain peptides after multiple times of investigation, and the doses of 2Y-47 and 2P-104 are 1/500 of the hMG; (2) in vivo, the central GHRH-GHRH receptor-GH endocrine axis pathway is specific, but testis or ovarian cells present GHRH, GHRH receptors, and GH molecules that do not necessarily induce consistent responses of GH in vivo and in vitro; (3) the high pregnancy rates of 2Y-47 and 2P-104 showed that high GH secretion in vivo was not correlated with pregnancy rates.

Example 4: treatment of model senile dementia with dimer peptides

1. The preparation and treatment method of the senile dementia model comprises the following steps: c57BL/6 mice (SPF grade, male, purchased from Experimental animals center of Guangzhou university of traditional Chinese medicine, 20-25 g/mouse) were light/dark cycle adjusted at 12/12 hours with constant temperature of 23 + -1 deg.C and humidity of 50-60%, and were fed freely with water. After purchase of the animals, the experiment was started 3 days after adaptive feeding.

Mice were randomly divided by body weight into a blank control group, a Placebo group (model control group), a positive control group and a dimer peptide 2Y-47, 2P-104 and 2F 120-administered group, 10 mice per group. The mice of the model control group, the dimer peptide administration group and the positive control group are intragastrically filled with 10mg/kg of aluminum trichloride every day and are simultaneously intraperitoneally injected with 120mg/kg of D-galactose (modeling medicament: aluminum trichloride + D-galactose), the mice of the blank control group are intragastrically filled with distilled water with the same volume every day and are simultaneously intraperitoneally injected with normal saline with the same volume, and the mice of each group are continuously fed with the modeling medicament for 24 weeks. The mice of each group were injected with 0.0133 μmol/kg of dimer peptide (dissolved in T-NaCl-PB solution) subcutaneously on day 1 after the intraperitoneal injection of the model drug in the 13 th week, the blank control group and the model control group were injected with T-NaCl-PB solution subcutaneously, and the positive control group was injected with 38.74mg/kg of donepezil pamoate for injection (diluted with T-NaCl-PB solution) subcutaneously (clinical lot of positive drug 2018L02708, 3mL in specification: 255.4mg, manufactured by Huajiang river, Hai.e., pharmaceutical Co., Ltd.; and the mouse dose was 38.74mg/kg, calculated as 9.1-fold equivalent dose for mice and adults). Each group was administered by subcutaneous injection once every two weeks for 12 weeks, and the molding drugs were administered daily routinely throughout the administration period.

2. Water maze test method: data acquisition and processing of learning and memory of mice was automatically monitored and processed by Morris maze images. The mice are trained in the Morris water maze experiment 3 days before model making and drug administration, each group of mice is trained 3 times per day, the mice are trained to escape from water surface swimming from the starting point of the SMG-2 type square water maze (developed by the institute of medicine of Chinese academy of medical science, China) to reach the terminal stair, and the water maze automatically records the error times and the terminal reaching time (latency) of the mice entering each blind end. Mice were tested for learning and memory the day before and week 12 after treatment, 2 times for each group of mice, Mean error number and Mean latency values were taken, experimental data were Mean ± standard deviation (Mean ± SD), statistical analysis by t-test.

3. As a result: table 5 results show that over the 12 th week of modeling, there was a significant increase in latency and number of errors in each model group (P <0.05) compared to the blank control group, indicating that the model of alzheimer's disease was successful. The Placebo group showed a significant increase (P <0.05 or 0.01) compared to the latency or number of errors in each group before or after dosing. For the latency or number of errors in each group after administration, 2Y-47 and the positive control group showed a significant decrease compared to Placebo, but no significant difference between the 2P-104 and 2F-120 groups. In the above experiments, no experimental phenomena such as weight loss and dysphoria of the mice in the administration group and the mice in the blank control group are found, and the drug has good tolerance to the mice. In conclusion, the 2Y-47 of the invention has better effect, and preliminarily shows that: therapeutic efficacy in Alzheimer's disease is associated with high GH secretion.

Table 5: statistical analysis of memory ability of dementia model mice (Mean + -SD) of each experimental group before and after 12 weeks drug treatment

P <0.05 or 0.01, double greek letter mapping comparative statistical analysis, t-test.

4. Cholinesterase analysis: the cholinesterase assay kit (manufactured by Beijing Jiuqiang Biotechnology corporation) and Hitachi full-automatic biochemical analyzer (model 7180, Japan) were used for assay, and the specifications of the kit and the apparatus were referred to the manufacturer's instructions. The results are shown in FIG. 2: compared with Placebo group, the positive drug and the 2Y-47 group have significant difference (P <0.05), and the animal serum cholinesterase assay shows dose-dependent reduction. Compared with the blank control group, the cholinesterase of Placebo, 2P-104 and 2F-120 groups showed a significant increase. The result shows that 2Y-47 has certain treatment effect on the senile dementia.

Example 5: treatment of diabetes mellitus model II

II, a diabetes model establishing method: c57Bl6/J mice were placed in a standard diet in an environment of SPF rating with free access to water. All experimental operations are conducted according to the ethical and use system guiding principles of experimental animals. After a day of feeding according to the standard diet, 5-week-old C57B16/J male mice were divided into 6 groups: blank control group, Placebo group (T2D model control group), liraglutide group, 2Y-47 low and medium groups (L-2Y-47, M-2Y-47, H-2Y-47). All T2D models were fed 60 kcal% of a high fat diet (D12492, changzhou mouse two biotech limited) until the end of the experiment. The blank control group maintained the standard diet until the end of the experiment. The method for establishing the diabetes model comprises the following steps: after 4 weeks of high-fat feeding, mice were intraperitoneally injected with 75mg/kg streptozotocin (STZ, Sigma chemical Co., USA), 3 days later, re-intraperitoneally injected with a 50mg/kg dose of STZ, and 3 weeks later, diabetic mice had blood glucose equal to or greater than 11 mM. These groups were treated on a high fat diet for an additional 35 days. The liraglutide group was injected subcutaneously with 1.126 nmol/100. mu.L liraglutide, the 2Y-47 group was injected subcutaneously with 3.378, 1.126, 0.375 nmol/100. mu.L (diluted with T-NaCl-PB solution), the blank control and the Placebo group with T-NaCl-PB solution.

2. The results are shown in FIG. 3: after 2Y-47 injection, blood glucose showed no significant change and serum cholinesterase showed a dose-dependent decrease. Compared with the blank control group, the increase of cholinesterase was significant in Placebo, liraglutide and each of the 2Y-47 groups (P < 0.05). Compared with the liraglutide group, the M-and H-2Y-47 groups have no significance, and show that 2Y-47 can reduce cholinesterase and can increase the cognitive function of diabetes.

Example 6: therapeutic effect of dimer of the present invention on hypoimmunity of animals

1. Influence of the drug on the immunity of normal mice: 24 Balb/c male mice were selected, weighing 18-22 g. After 2 days of feeding, the blank control group and the 2Y-47 administration group were randomly divided into 6 groups without weight difference. The 2Y-47 peptide was injected subcutaneously into hip at a single dose of 0.0133 μmol at 12 am, followed by 50 μ l of anticoagulated blood from the intraorbital canthus at 12 pm daily, and blood routine (Sysmex XS-500i and reagents, Japan) was determined until the end of the 18 day experiment. The result of the test in the 2Y-47 group minus the result of the test in the blank control group is the final result.

2. The experimental results are as follows: blood routine analysis shows that after 2Y-47 peptide injection, only the total number and percentage of mononuclear cells are obviously different at different time points, and no influence is shown on other red blood cells, white blood cells or blood platelets and related indexes thereof. On day 1 after administration, each index decreased, probably as a stress response in the animal, and returned to normal the next day. As can be seen in fig. 4, the total number of monocytes was significantly increased at

days

2, 7, 14, 15 (P <0.05) compared to the pre-and post-drug day 1 results. Figure 5 shows a significant increase in monocyte percentage at

days

2, 7, 14, 15 (P <0.05, 0.01, or 0.001) compared to pre-and/or post-drug day 1 results, with peak results at day 15 and a drop to normal at day 18. These results preliminarily show that 2Y-47 peptide promotes monocyte proliferation and enhances immunity.

3. The preparation and treatment method of the low immunity model comprises the following steps: balb/c mice 60, weighing 18-22g, were randomized into 10 groups (male and female halves) without weight difference between the control blank and model groups (Placebo group, positive control group, L-2Y-47, M-2Y-47 and H-2Y-47). Mice in all model groups were injected with cyclophosphamide 80mg/kg intraperitoneally once a day for 3 consecutive days. After 7 days of the experiment, the therapeutic drugs were administered, and each of the positive control groups was subcutaneously injected with 0.2427mg (0.07808. mu. mol) of thymalfasin (trade name: Thai, 1.6mg specification, approved reference H20030407, product batch 16070106, manufactured by Hainan Shuangcheng pharmaceutical Co., Ltd., dissolved in T-NaCl-PB solution), 2Y-47 groups were subcutaneously injected with 0.2342, 0.07808 and 0.02603. mu. mol, and the blank control group and Placebo group were injected with T-NaCl-PB solution. Each group was injected 2 times per week for 4 weeks. One day before treatment and 4 weeks after treatment, 50 microliters of anticoagulated blood was taken from the inner canthus of the mice, blood routine was determined, and spleen was weighed and H-E stained for mice were sacrificed. Counting red pith: H-E stained sections of three animals per group were taken, 5 sections were taken for each animal and photographed, and the percentage of red marrow cells in the entire field was counted using IPP software, which was the red marrow statistical result. The method is described in Liu Changjiang river and other papers [ Liu Changjiang river and other papers, establishment and evaluation of cyclophosphamide induced mice immunologic hypofunction model, Weifang medical college bulletin 37(1), 4-6,2015 ].

4. And (4) evaluating the results: after the experiment, the analysis of the total number of blood mononuclear cells (FIG. 6) showed that the thymalfasin group showed a significant increase in lymphocytes, but no significant increase in monocytes, a dose-dependent increase in the number of monocytes in the 2Y-47 group, and a significant increase in the H-2Y-47 group (P <0.05), as compared to the Placebo group. Comparison of spleen weights (FIG. 7) showed a significant increase in spleen weight (P <0.05) in thymalfasin and H-2Y-47 animals compared to the placebo group. Spleen H-E staining (FIG. 8A) showed a significant increase in white marrow in thymalfasin groups, and in red marrow in 2Y-47 groups. Spleen red marrow was significantly increased in M-and H-2Y-47 groups (P <0.05) compared to the blank control group and Placebo group (FIG. 8B). These preliminary results show that the 2Y-GHRH sequence represented by 2Y-47 enhances immunity by increasing macrophage numbers in the red marrow region.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A Growth Hormone Releasing Hormone (GHRH) -like peptide monomer, wherein serine S on the peptide chain is substituted with cysteine C, or threonine T is substituted with homocysteine (Hcy), and one of the lysines on the main peptide chain is a lysine that is side chain-amino modified with a glutamyl fatty acid [ γ -Glu (N- α -fatty acid) ] or glutamyl fatty diacid [ γ -Glu (N- α -fattydiacide) ] or [2 × AEEAC- γ -Glu (N- α -fatty acid) ].

2. The GHRH analog peptide monomer of claim 1, wherein the analog peptide monomer has a specific sequence selected from any one of:

wherein, X₁Is P or Y or F; x₂Is A or α -aminoisobutyric acid (α Aib); X₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is K, or is R, or is a side chain-amino glutamyl fatty acid [ gamma-Glu (N- α -fat acid)]Or glutamyl fatty diacid gamma-Glu (N- α -fatty diacid) modified lysine, or side chain-amino [2 × AEEAC-gamma-Glu (N- α -fatty diacid)]A modified lysine.

3. The somatotropin releasing hormone analog peptide monomer of claim 2 wherein when X is₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is side chain-amino glutamyl fatty acid [ gamma-Glu (N- α -fat acid)]Or glutamyl fatty diacid gamma-Glu (N- α -fat diacid) modified lysine, the structure is shown in chemical formula 1;when X is present₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is on the side chain-amino group [2 × AEEAC-gamma-Glu (N- α -fatty diacid)]Modified lysine, the structure of which is shown in chemical formula 2:

chemical formula 1:

chemical formula 2:

4. a GHRH-like peptide homodimer formed by the linkage of identical monomers according to any one of claims 1 to 3 via a cysteine-forming disulfide bond, constituting a type H GHRH-like peptide homodimer.

5. The dimer of claim 4, wherein the amino acid sequence is any one of:

6. The somatotropin releasing hormone analog peptide monomer of claim 5 wherein when X is₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is side chain-amino glutamyl fatty acid [ gamma-Glu (N- α -fat acid)]Or glutamyl fatty diacid gamma-Glu (N- α -fatty diacid) modified lysine, the structure is shown in chemical formula 1, when X is₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is on the side chain-amino group [2 × AEEAC-gamma-Glu (N- α -fatty diacid)]The structure of modified lysine is shown in chemical formula 2.

7. Use of a ghrelin-like peptide according to any of claims 1 to 3, or a homodimer according to any of claims 4 to 6, for the manufacture of a medicament for the treatment of GH deficiency, infertility, senile dementia, immune enhancement or diabetes.

8. A medicament for treating GH deficiency, infertility, senile dementia, immune enhancement or diabetes, which comprises the ghrelin-like peptide of any one of claims 1 to 3, or the homodimer of any one of claims 4 to 6 and a pharmaceutically acceptable salt thereof as an active ingredient.