Long-chain modified GHRH agonist and use thereof

Abstract

The present invention provides a GHRH agonist modified with long-chain fatty acylation at the C-terminus. Compared with natural GHRH and existing GHRH agonists, the GHRH agonist modified with long-chain fatty acylation at the C-terminus exhibits significantly improved biological activity and is less susceptible to enzymatic degradation in vivo, thereby showing better stability. The serum half-life exceeds 24 hours, enabling long-acting agonism of GHRH receptors and exerting various therapeutic effects.

Description

A long-chain modified GHRH agonist and its application Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to a long-chain modified GHRH agonist and its application. Background Technology

[0002] Growth hormone-releasing hormone (GHRH) is a polypeptide composed of 44 amino acids, mainly synthesized and secreted by the hypothalamus. Studies have shown that GHRH agonists (GHRH-A) can promote angiogenesis and tissue repair, and prevent ischemia-reperfusion injury, thus showing broad clinical application prospects in cardiovascular diseases, diabetes, regenerative medicine, and other fields.

[0003] GHRH's GH-secreting effect makes it a valuable drug, but natural GHRH is easily enzymatically degraded in vivo, losing its biological activity. Chromatographic analysis of rat pituitary glands shows that GHRH has a half-life of only about 22 minutes, making natural GHRH an unsuitable candidate drug. Currently, two GHRH peptide analogs, tesamorelin and sermorelin, are marketed, but the latter has been withdrawn from the market. Tesamorelin is a derivative of hGHRH (1-44)NH2 with the addition of a trans-3-hexenoyl group. This modification makes it resistant to degradation by dipeptidyl aminopeptidase-IV (CD26), thus prolonging its plasma half-life to about 60 minutes. The effect of hGHRH is limited to about 2–3 hours, while tesamorelin can induce GH release for up to 8 hours. Tesamorelin can improve lipid metabolism disorders in HIV patients, enhance immune function and sleep quality, and help lower plasma cholesterol levels in patients with type II diabetes. Currently, temorelin injection, manufactured by the Canadian pharmaceutical company Theratechnologies, has been approved for marketing in the United States and Canada for the treatment of lipid metabolism disorders in HIV patients. Schally AV et al. designed and synthesized the early GHRH agonist JI series by replacing the 12th and 21st amino acids of hGHRH with ornithine. However, compared to natural GHRH, the new peptides did not exhibit very significant serum stability. For example, the most effective agonist, MR409, has a serum stability of approximately 100 minutes, and its biological half-life in animals is further reduced. No in-depth systematic studies on its later drug-like properties have been reported.

[0004] In summary, the stability and bioactivity of currently developed GHRH agonists are generally not ideal, therefore it is necessary to develop more GHRH agonist products with high stability and high bioactivity. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a long-chain modified GHRH agonist and its applications. This invention synthesizes a series of GHRH agonists modified with C-terminal long-chain fatty acylation. Compared with natural GHRH and existing GHRH agonists, the C-terminal long-chain fatty acylated GHRH agonists exhibit significantly enhanced biological activity and are less susceptible to enzymatic degradation in vivo, thus greatly improving their stability. With a serum half-life exceeding 24 hours, they can achieve long-acting GHRH receptor activation and exert various therapeutic effects.

[0006] The specific technical solution of the present invention is as follows: Firstly, the present invention provides a long-chain modified GHRH agonist, the chemical structural formula of which is: This invention synthesizes the above-mentioned GHRH agonist modified with C-terminal long-chain fatty acid acylation. Compared with natural GHRH and existing GHRH agonists, the biological activity of this C-terminal long-chain fatty acid acylation modified GHRH agonist is greatly improved, and it is not easily enzymatically hydrolyzed in vivo, thus it has better stability and a serum half-life of more than 24 hours, which can achieve long-acting GHRH receptor agonism and exert a variety of therapeutic effects.

[0007] This invention reveals that the sites of long-chain fatty acylation modifications and the length of the carbon chain significantly affect the stability and activity of GHRH agonists. Specifically: Regarding modification sites: Based on electron microscopy predictions of GHRH binding to the GHRH receptor, the N-terminus of GHRH(1-29) binds tightly to the GHRH receptor. Therefore, modification of the N-terminus may significantly affect its activity. Modification at the middle site may disrupt its secondary structure, thus greatly affecting its activity (the team previously attempted to select positions 16 and 21 as modification sites for long chains, but no activity was detected). In contrast, the amino acid side chains near the C-terminus bind less to the target site and contain more hydrophobic groups, making them easier to modify without significantly disrupting their secondary structure. Furthermore, C-terminal modification allows for more convenient solid-phase synthesis of the target peptide; modification at other sites increases synthesis costs.

[0008] Regarding carbon chain length: Our team discovered that fatty acid chains of different lengths have different effects on improving drug stability and function. Specifically: Short-chain fatty acids (e.g., C8 to C10): Short-chain fatty acids can improve the water solubility of peptides, but their membrane permeability and binding ability to plasma proteins are weaker, so their effect on prolonging half-life may not be as significant as that of long-chain fatty acids. Long-chain fatty acids (e.g., C16 and above): Long-chain fatty acids such as palmitic acid can more effectively increase the binding of peptides to albumin, prolong half-life, and enhance membrane permeability, but excessively long chains may affect water solubility, making it difficult for the drug to distribute evenly in the body. Therefore, the selection of fatty acid chain length is usually a balancing process, and the chain length should be determined according to the specific needs of the drug. An appropriate chain length can maximize the improvement of half-life, membrane permeability, and metabolic stability, while avoiding solubility and distribution problems caused by excessively long chains.

[0009] Preferably, the chemical structural formula of the GHRH agonist is: This invention has found that compounds modified at the C-terminus with the aforementioned specific chain length exhibit better stability and activity.

[0010] Secondly, this invention provides the application of GHRH agonists having the above-described structural formula in the preparation of therapeutic drugs for diseases. The diseases include cardiovascular diseases, diabetes and related complications, non-alcoholic fatty liver disease, metabolic diseases such as obesity, tumors, spinal muscular atrophy, optic nerve injury, ischemic stroke, colitis, growth hormone deficiency, or infertility.

[0011] Preferably, the drug comprises the following (a) and / or (b): (a) a GHRH agonist; (b) a pharmaceutically acceptable salt and / or ester of a GHRH agonist.

[0012] Further preferably, the drug also includes one or more of pharmaceutically acceptable dressings, excipients, solvents, and buffer solutions.

[0013] Preferably, the drug is an injectable preparation, an oral preparation, a patch, a spray, an implantable micropump, eye drops, a topical lotion / liquid / gel, or a microneedle.

[0014] Further preferably, the drug is administered via subcutaneous injection, intravenous injection, intramuscular injection, micropump implantation, oral / nasal inhalation, eye drops, oral administration, or topical application.

[0015] Thirdly, the present invention provides a disease treatment medicament based on the above-mentioned GHRH agonist, comprising the following (a) and / or (b): (a) a GHRH agonist; (b) a pharmaceutically acceptable salt and / or ester of the GHRH agonist.

[0016] The diseases mentioned include cardiovascular diseases, diabetes and related complications, non-alcoholic fatty liver disease, metabolic diseases such as obesity, tumors, spinal muscular atrophy, optic nerve injury, ischemic stroke, colitis, growth hormone deficiency, or infertility.

[0017] Further preferably, the disease treatment drug also includes one or more of pharmaceutically acceptable dressings, excipients, solvents, and buffer solutions.

[0018] Preferably, the disease treatment drug is an injectable preparation, an oral preparation, a patch, a spray, an implantable micropump, eye drops, a topical lotion / liquid / gel, or a microneedle.

[0019] Further preferably, the drug is administered via subcutaneous injection, intravenous injection, intramuscular injection, micropump implantation, oral / nasal inhalation, eye drops, oral administration, or topical application.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides several GHRH agonists modified with long-chain fatty acid acylation at the C-terminus. Compared with natural GHRH and existing GHRH agonists, these GHRH agonists have higher biological activity and are not easily enzymatically hydrolyzed in vivo, thus exhibiting better stability and a serum half-life of more than 24 hours. They can achieve long-acting GHRH receptor activation and exert a variety of therapeutic effects. Attached Figure Description

[0021] Figure 1 shows a comparison of serum stability of different GHRH agonists.

[0022] Figure 2 is a comparison of the GH release capacity of different GHRH agonists.

[0023] Figure 3 is a comparison of the phosphorylation levels of CREB promoted by different GHRH agonists.

[0024] Figure 4 is a comparison of the phosphorylation levels of CREB promoted by different GHRH agonists.

[0025] Figure 5 is a comparison of the phosphorylation levels of CREB promoted by different GHRH agonists.

[0026] Figure 6 is a comparison of the phosphorylation levels of CREB promoted by different GHRH agonists.

[0027] Figure 7 is a comparison of the phosphorylation levels of CREB promoted by different GHRH agonists.

[0028] Figure 8 is a comparison of the effects of different GHRH agonists on the restoration of blood flow in ischemic lower limbs in animal models.

[0029] Figure 9 shows the effect of different GHRH agonist treatments on the migration ability of HUVECs as detected by transwell assay. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments.

[0031] In a first aspect, the present invention provides a long-chain modified GHRH agonist, the chemical structural formula of which is: In some preferred embodiments, the chemical structural formula of the GHRH agonist is as follows: Secondly, this invention provides the application of GHRH agonists having the above-described structural formula in the preparation of therapeutic drugs for diseases. The diseases include cardiovascular diseases, diabetes and related complications, non-alcoholic fatty liver disease, metabolic diseases such as obesity, tumors, spinal muscular atrophy, optic nerve injury, ischemic stroke, colitis, growth hormone deficiency, or infertility.

[0032] In some preferred embodiments, the medicament comprises the following (a) and / or (b): (a) a GHRH agonist; (b) a pharmaceutically acceptable salt and / or ester of a GHRH agonist.

[0033] In some more preferred embodiments, the medicament also includes one or more of pharmaceutically acceptable dressings, excipients, solvents, and buffer solutions.

[0034] In some preferred embodiments, the drug is an injectable formulation, an oral formulation, a patch, a spray, an implantable micropump, eye drops, a topical lotion / liquid / gel, or a microneedle.

[0035] In some more preferred embodiments, the drug is administered via subcutaneous injection, intravenous injection, intramuscular injection, micropump implantation, oral / nasal inhalation, eye drops, oral administration, or topical application.

[0036] Thirdly, the present invention provides a disease treatment medicament based on the above-mentioned GHRH agonist, comprising the following (a) and / or (b): (a) a GHRH agonist; (b) a pharmaceutically acceptable salt and / or ester of the GHRH agonist.

[0037] The diseases mentioned include cardiovascular diseases, diabetes and related complications, non-alcoholic fatty liver disease, metabolic diseases such as obesity, tumors, spinal muscular atrophy, optic nerve injury, ischemic stroke, colitis, growth hormone deficiency, or infertility.

[0038] In some preferred embodiments, the disease treatment drug also includes one or more of pharmaceutically acceptable dressings, excipients, solvents, and buffer solutions.

[0039] In some preferred embodiments, the disease treatment drug is an injectable formulation, an oral formulation, a patch, a spray, an implantable micropump, eye drops, a topical lotion / liquid / gel, or a microneedle.

[0040] In some more preferred embodiments, the drug is administered via subcutaneous injection, intravenous injection, intramuscular injection, micropump implantation, oral / nasal inhalation, eye drops, oral administration, or topical application.

[0041] Specific embodiments and comparative examples.

[0042] The GHRH agonist of this invention comprises one or more of the following non-natural amino acid compounds in its structural formula: This invention synthesizes several new GHRH agonists with different chemical structures, as shown in Table 1: Table 1 The amino acid sequences of the GHRH agonists listed in the table above are designed based on natural GHRH agonists and the known synthetic GHRH agonist MR409.

[0043] The differentiating amino acid sites of the newly designed GHRH agonists of this invention from their natural GHRH agonists and the GHRH agonist MR409 are shown in Tables 2-1 to 2-3, respectively. Note: In Tables 2-1 to 2-3, in the amino acid sequences of HD-1A to HD-12B, "-" indicates that they are the same as MR409.

[0044] Table 2-1: Design of γ-AA peptide substitution for GHRH agonists Table 2-2: Rational design of GHRH agonists using Aib and γ-AA peptide together Table 2-3: Rational design of GHRH agonists with good activity using long aliphatic chains The specific composition of each numbered GHRH agonist provided by this invention is as follows: HD-1A: NMeTyr-DAla-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-Nle-Asp-Arg-γAA1-NH2, i.e.: N-methyltyrosine-D-alanine-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-glutamine-aspartic acid-isoleucine-ornithine-aspartic acid-arginine-γAA peptide 1.

[0045] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 521.77 [M+7H] 7+, 608.48 [M+6H] 6+ 729.91 [M+5H] 5+ 912.10[M+4H] 4+ MS theoretical value: 3645.29.

[0046] HD-1B: AcTyr-DAla-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-Nle-Asp-Arg-γAA1-NH2, which is: Acetyltyrosine-D-alanine-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-ornithine-leucine-glutamine-aspartic acid-isoleucine-ornithine-aspartic acid-arginine-γAA peptide 1.

[0047] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 613.15 [M+6H] 6+ 735.56 [M+5H] 5+ 919.08 [M+4H] 4+ MS theoretical value: 3673.30.

[0048] HD-2A: NMeTyr-DAla-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-γAA2-Arg-NH2, which is: N-methyltyrosine-D-alanine-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-glutamine-aspartic acid-isoleucine-γAA peptide 2-arginine.

[0049] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 567.95 [M+6H] 6+ 681.35 [M+5H] 5+ 851.35[M+4H] 4+ MS theoretical value: 3402.03.

[0050] HD-2B: AcTyr-DAla-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-γAA2-Arg-NH2, which is: Acetyltyrosine-D-alanine-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-glutamine-aspartic acid-isoleucine-γAA peptide 2-arginine.

[0051] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 686.90 [M+5H] 5+ 858.40[M+4H] 4+ MS theoretical value: 3430.04.

[0052] HD-3A: NMeTyr-DAla-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-γAA3-Ile-γAA2-Arg-NH2, which is: N-methyltyrosine-D-alanine-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-γAA peptide3-isoleucine-γAA peptide2-arginine.

[0053] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 493.90 [M+7H] 7+ , 576.05 [M+6H] 6+ 690.95 [M+5H] 5+ 863.45 [M+4H] 4+ MS theoretical value: 3450.22.

[0054] HD-3B: AcTyr-DAla-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-γAA3-Ile-γAA2-Arg-NH2, which is: Acetyltyrosine-D-alanine-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-γAA peptide3-isoleucine-γAA peptide2-arginine.

[0055] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 580.69 [M+6H] 6+ 696.58 [M+5H] 5+ 870.36 [M+4H] 4+ MS theoretical value: 3478.23.

[0056] HD-4A: NMeTyr-DAla-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-γAA2-Arg-γAA1-NH2, which is: N-methyltyrosine-D-alanine-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-glutamine-aspartic acid-isoleucine-γAA peptide2-arginine-γAA peptide1.

[0057] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 524.60 [M+7H] 7+ , 611.84 [M+6H] 6+ 734.02[M+5H] 5+ 917.09 [M+4H] 4+ MS theoretical value: 3665.38.

[0058] HD-4B: AcTyr-DAla-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-γAA2-Arg-γAA1-NH2, which is: Acetyltyrosine-D-alanine-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-glutamine-aspartic acid-isoleucine-γAA peptide2-arginine-γAA peptide1.

[0059] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 616.56 [M+6H] 6+ 739.57 [M+5H] 5+ 924.14[M+4H] 4+ MS theoretical value: 3693.39.

[0060] HD-5A: NMeTyr-DAla-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-γAA3-Ile-γAA2-Arg-γAA1-NH2, which is: N-methyltyrosine-D-alanine-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-γAA peptide3-isoleucine-γAA peptide2-arginine-γAA peptide1.

[0061] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 531.55 [M+7H] 7+ , 619.85 [M+6H] 6+ 743.55 [M+5H] 5+ 929.15 [M+4H] 4+ MS theoretical value: 3713.57.

[0062] HD-5B: AcTyr-DAla-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-γAA3-Ile-γAA2-Arg-γAA1-NH2, which is: Acetyltyrosine-D-alanine-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-γAA peptide3-isoleucine-γAA peptide2-arginine-γAA peptide1.

[0063] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 535.50 [M+7H] 7+ , 624.60[M+6H] 6+ 749.20 [M+5H] 5+ 936.00 [M+4H] 4+ MS theoretical value: 3741.58.

[0064] HD-6A: NMeTyr-DAla-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-Nle-Asp-Arg-Ada-NH2, which is: N-methyltyrosine-D-alanine-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-ornithine-leucine-glutamine-aspartic acid-isoleucine-ornithine-aspartic acid-arginine-12-aminododecanoic acid.

[0065] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 597.46 [M+6H] 6+ 716.72[M+5H] 5+ 895.55 [M+4H] 4+ MS theoretical value: 3579.26.

[0066] HD-6B: AcTyr-DAla-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-Nle-Asp-Arg-Ada-NH2, which is: Acetyltyrosine-D-alanine-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-ornithine-leucine-glutamine-aspartic acid-isoleucine-ornithine-aspartic acid-arginine-12-aminododecanoic acid.

[0067] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 722.32 [M+5H] 5+ 902.58 [M+4H] 4+ MS theoretical value: 3607.27.

[0068] HD-7A: Tyr-Aib-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-Nle-Asp-Arg-NH2, which is: tyrosine-α-aminoisobutyric acid-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-glutamine-aspartic acid-isoleucine-ornithine-aspartic acid-arginine.

[0069] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 564.62 [M+6H] 6+ 677.26 [M+5H] 5+ 846.28 [M+4H] 4+ MS theoretical value: 3381.93.

[0070] HD-7B: AcTyr-Aib-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-Nle-Asp-Arg-NH2, which is: Acetyltyrosine-α-aminoisobutyric acid-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-glutamine-aspartic acid-isoleucine-ornithine-aspartic acid-arginine.

[0071] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 685.68 [M+5H] 5+ 856.79 [M+4H] 4+ MS theoretical value: 3423.97.

[0072] HD-8A: Tyr-Aib-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-γAA2-Arg-NH2, which is: tyrosine-α-aminoisobutyric acid-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-glutamine-aspartic acid-isoleucine-γAA peptide 2-arginine.

[0073] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 567.97 [M+6H] 6+ 681.29 [M+5H] 5+ 851.30[M+4H] 4+ MS theoretical value: 3402.03.

[0074] HD-8B: AcTyr-Aib-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-γAA2-Arg-NH2, which is: Acetyltyrosine-α-aminoisobutyric acid-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-glutamine-aspartic acid-isoleucine-γAA peptide 2-arginine.

[0075] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 689.70 [M+5H] 5+ 861.80[M+4H] 4+ MS theoretical value: 3444.06.

[0076] HD-9A: Tyr-Aib-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-γAA3-Ile-γAA2-Arg-NH2, which is: tyrosine-α-aminoisobutyric acid-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-γAA peptide3-isoleucine-γAA peptide2-arginine.

[0077] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 493.87 [M+7H] 7+ , 575.97 [M+6H] 6+ 690.92[M+5H] 5+ 863.34[M+4H] 4+ MS theoretical value: 3450.22.

[0078] HD-9B: AcTyr-Aib-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-γAA3-Ile-γAA2-Arg-NH2, which is: Acetyltyrosine-α-aminoisobutyric acid-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-γAA peptide3-isoleucine-γAA peptide2-arginine.

[0079] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 583.00 [M+6H] 6+ 699.35 [M+5H] 5+ 873.87[M+4H] 4+ MS theoretical value: 3492.25.

[0080] HD-10A: Tyr-Aib-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-γAA2-Arg-γAA1-NH2, which is: tyrosine-α-aminoisobutyric acid-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-glutamine-aspartic acid-isoleucine-γAA peptide2-arginine-γAA peptide1.

[0081] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 524.64 [M+7H] 7+ , 611.89 [M+6H] 6+ 734.02[M+5H] 5+ 917.14[M+4H] 4+ MS theoretical value: 3665.39.

[0082] HD-10B: NMeTyr-Aib-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-γAA2-Arg-γAA1-NH2, which is: N-methyltyrosine-α-aminoisobutyric acid-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-glutamine-aspartic acid-isoleucine-γAA peptide2-arginine-γAA peptide1.

[0083] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 526.61 [M+7H] 7+ , 614.20[M+6H] 6+ 736.82 [M+5H] 5+ 920.67 [M+4H] 4+ MS theoretical value: 3679.41.

[0084] HD-11A: Tyr-Aib-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-γAA2-Arg-γAA1-Ada-NH2, which is: tyrosine-α-aminoisobutyric acid-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-glutamine-aspartic acid-isoleucine-γAA peptide2-arginine-γAA peptide1-12-aminododecanoic acid.

[0085] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 552.80 [M+7H] 7+ , 644.76 [M+6H] 6+ 773.46 [M+5H] 5+ 966.45 [M+4H] 4+ MS theoretical value: 3862.71.

[0086] HD-11B: NMeTyr-Aib-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-γAA2-Arg-γAA1-Ada-NH2, which is: N-methyltyrosine-α-aminoisobutyric acid-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-glutamine-aspartic acid-isoleucine-γAA peptide2-arginine-γAA peptide1-12-aminododecanoic acid.

[0087] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 554.58 [M+7H] 7+ , 647.12[M+6H] 6+ 776.26 [M+5H] 5+ 970.02[M+4H] 4+ MS theoretical value: 3876.74.

[0088] HD-12A: Tyr-Aib-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-γAA3-Ile-γAA2-Arg-Ada-NH2, which is: tyrosine-α-aminoisobutyric acid-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-γAA peptide3-isoleucine-γAA peptide2-arginine-12-aminododecanoic acid.

[0089] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 522.10 [M+7H] 7+ , 608.85 [M+6H] 6+ 730.42[M+5H] 5+ 912.65 [M+4H] 4+ MS theoretical value: 3647.54.

[0090] HD-12B: NMeTyr-Aib-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-γAA3-Ile-γAA2-Arg-Ada-NH2, which is: N-methyltyrosine-α-aminoisobutyric acid-aspartic acid-alanine-isoleucine-phenylalanine-threonine-asparagine-serine-tyrosine-arginine-ornithine-valine-leucine-2-aminobutyric acid-glutamine-leucine-serine-alanine-arginine-ornithine-leucine-γAA peptide3-isoleucine-γAA peptide2-arginine-12-aminododecanoic acid.

[0091] The purity of the target peptide was >95%, and its purity was determined using RP-HPLC. MS result: 524.10 [M+7H] 7+ , 611.25 [M+6H] 6+ 733.24[M+5H] 5+ 916.20[M+4H] 4+ MS theoretical value: 3661.57.

[0092] (1) Stability testing: A major problem limiting the use of GHRH analogs is their susceptibility to rapid degradation by proteases, making them unstable. Improving the resistance of GHRH agonists to proteolytic degradation is one of the main objectives of this invention. This invention tested the stability of GHRH agonists HD-2A, HD-3A, HD-4A, HD-5A, HD-6A, HD-8A, HD-9A, HD-11A, HD-11B, HD-12A, and HD-12B in serum. By using 10... -4 MR-409 (control GHRH agonist) and HD-2A, HD-3A, HD-4A, HD-5A, HD-6A, HD-8A, HD-9A, HD-11A, HD-11B, HD-12A, and HD-12B were incubated with human serum at 30°C for 24 hours. Samples were collected at different time points during the reaction. The content of intact GHRH agonists that had not been degraded was analyzed by high-performance liquid chromatography (HPLC). The relative residual intact GHRH agonist content (%) was compared with the amount of peptides before the addition of serum (time 0 hours), which was used to obtain the stability. The results showed that the serum stability of GHRH agonists HD-6A, HD-11A, HD-11B, HD-12A, and HD-12B was significantly improved compared with MR-409.

[0093] (2) GH Release Assay: The most basic function of GHRH is to promote GH release. Based on this, using GHRH (1-29) and MR-409 as positive controls, this invention tested the ability of GHRH agonists HD-4A, HD-6A, HD-9A, HD-11A / B, and HD-12A / B to promote GH release. The detection method is briefly described as follows: Pituitary glands of C57 mice were extracted, pituitary cells were isolated, resuspended in F12-K medium, 100 μL of medium was extracted, and GH concentration was detected using an ELISA Kit. The result was recorded as 0 min, and a final concentration of 2 × 10⁻⁶ was added. -5 After administration of the GHRH agonist M, 100 μL of culture medium was extracted from the detection system at 15 min, 30 min, and 60 min to detect GH concentration. The Primm assay results are shown in Figure 2. The results showed that, compared with GHRH and MR-409, the GHRH agonists HD-6A and HD-11A had a significantly stronger ability to promote GH release. This result was determined by the combined effect of the target agonistic activity and serum stability of HD-6A and HD-11A.

[0094] (3) Cell viability assay: In cells containing GHRH-R, after GHRH or a GHRH agonist binds to GHRH-R, the activated second messengers include not only adenylate cyclase-cAMP-PKA and Ca2+, but also... 2+ Calmodulin includes phosphoinositol-diacylglycerol-protein kinase C (PKC), L-type calcium channels, and the arachidonic acid-eicosanoic acid pathway. Increased cAMP levels stimulate PKA to activate cAMP response element-binding protein (CREB), leading to its phosphorylation and stimulating GHRH gene transcription. Therefore, the activity of different GHRH agonists can be determined by detecting P-CREB. In this cell experiment, MCF7 cells carrying GHRH receptors were used as the experimental subjects to preliminarily screen the activity of various GHRH agonists.

[0095] As shown in Figures 3-7, the GHRH agonists of the present invention exhibit significantly higher activity / efficacy in promoting CREB phosphorylation levels than most known GHRH agonists such as natural GHGH and MR409. Among them, HD-4A, HD-6A, HD-9A, and HD-11A show better activity.

[0096] (4) Animal Model Validation: GHRH Agonist Promotes Lower Limb Blood Flow Recovery. A mouse lower limb ischemia model was established by ligating the femoral artery, and the recovery of lower limb blood flow after GHRH agonist treatment was observed. The GHRH agonist concentration was 10 μg / mouse / day. Doppler ultrasound was performed on the first, third, seventh, and 14th days after model establishment. The results are shown in Figure 8-A. After the lower limb ischemia model was established, the blood perfusion of the affected limb was significantly lower than that of the control side. On the seventh day after surgery, the blood flow recovery of the three groups treated with GHRH agonist was better than that of the control group, but there was no statistical difference. On the 14th day after surgery, the blood perfusion of the 6A and 11A groups was significantly improved compared with the NC and MR409 groups (Figure 8-B). At the same time, the gangrene of the toes of the mice was also significantly improved 14 days after GHRH agonist administration (Figure 8-C, D).

[0097] (5) Cellular experiments to verify the effect of GHRH agonist on the migration ability of human umbilical vein endothelial cells (HUVECs) HUVECs were seeded in the upper chamber of a Transwell culture medium containing serum-free medium, and the lower chamber was supplemented with medium containing 15% fetal bovine serum and 10% HUVECs. -5 After culturing in the medium containing MGHRH agonists for 24 hours, the upper chamber was fixed with paraformaldehyde, stained with crystal violet, and the distribution of cells in the lower layer of the upper chamber was observed under a microscope, as shown in Figure 9. The results showed that, compared with the NC group, MR409, 6A, and 11A all promoted HUVEC cell migration, with 6A and 11A showing more significant promoting effects.

[0098] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0099] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A long-chain modified GHRH agonist, characterized in that: The chemical structural formula is:

2. The GHRH agonist according to claim 1, characterized in that: The chemical structural formula is:

3. The use of the GHRH agonist according to claim 1 or 2 in the preparation of a disease treatment drug, characterized in that: The diseases mentioned include cardiovascular disease, diabetes and related complications, non-alcoholic fatty liver disease, obesity, spinal muscular atrophy, optic nerve injury, ischemic stroke, colitis, growth hormone deficiency, or infertility.

4. The application according to claim 3, characterized in that: The drug includes the following (a) and / or (b): (a) GHRH agonists; (b) Pharmaceutically acceptable salts and / or esters of GHRH agonists.

5. The application according to claim 4, characterized in that, The drug also includes one or more of pharmaceutically acceptable dressings, excipients, and solvents.

6. The application according to claim 5, characterized in that, The drug can be an injectable preparation, an oral preparation, a patch, a spray, an implantable micropump, an eye drop, a topical lotion / liquid / gel, or a microneedle.

7. The application according to claim 6, characterized in that, The drug can be administered via subcutaneous injection, intravenous injection, intramuscular injection, micropump implantation, oral / nasal inhalation, eye drops, or oral ingestion.

8. A disease treatment medicament based on the GHRH agonist of claim 1 or 2, characterized in that... Including the following (a) and / or (b): (a) GHRH agonists; (b) Pharmaceutically acceptable salts and / or esters of GHRH agonists; The diseases mentioned include cardiovascular disease, diabetes and related complications, non-alcoholic fatty liver disease, obesity, spinal muscular atrophy, optic nerve injury, ischemic stroke, colitis, growth hormone deficiency, or infertility.

9. The disease treatment drug as described in claim 8, characterized in that, It also includes one or more of pharmaceutically acceptable dressings, excipients, and solvents.

10. The disease treatment drug as described in claim 9, characterized in that, The disease treatment drugs are injectable preparations, oral preparations, patches, sprays, implantable micropumps, eye drops, topical lotions / liquids / gels, or microneedles.

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