A pseudo-polypeptide ghrh agonist and uses thereof

Abstract

The present application relates to the field of biological medicine, and discloses a kind of polypeptide GHRH agonist and its application.The present application provides several non-natural amino acid substitution polypeptide GHRH agonist, compared with natural GHRH and existing GHRH agonist, the biological activity of the polypeptide GHRH agonist is greatly improved, and it is not easy to be enzymolysis in vivo, so its stability is better, serum half-life is more than 24 hours, can realize long-acting agonist GHRH receptor and play a variety of therapeutic effects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a polypeptide-like GHRH agonist and its application. Background Technology

[0002] Growth hormone-releasing hormone (GHRH) is a 44-amino acid polypeptide primarily synthesized and secreted by the hypothalamus. As a member of the hypothalamus's regulatory peptides, GHRH acts on the GHRH receptor (GHRH-R) on the anterior pituitary cell membrane, stimulating the pituitary gland to synthesize and secrete growth hormone (GH). GH reaches the liver via the bloodstream, inducing hepatocytes to secrete insulin-like growth factor 1 (IGF-1). IGF-1, by binding to its receptor, activates the PI3K and MAPK signaling pathways in target tissues, thereby promoting cell division and growth. The initial GHRH transcript consists of 108 amino acids, including the first 20 amino acid signal peptide and the propeptides at the beginning and end. The active form of GHRH in the human body consists of 40 and 44 amino acids, but the first 29 amino acids of GHRH possess complete biological activity (GHRH(1-29)NH2). Among these, the 25 amino acids at positions 3-27 participate in the formation of the α-helix. It is generally believed that maintaining and regulating the hypothalamic-pituitary GHRH / GH / IGF-1 neuroendocrine axis is the main function of GHRH. For this reason, GHRH-R was initially thought to be expressed only in pituitary cells. However, subsequent studies have shown that GHRH-R is expressed in various peripheral tissues and organs, including the placenta, male and female gonads, and lymphocytes, in addition to the hypothalamus. Besides being an important member of the neuroendocrine system, GHRH's anti-apoptotic, inflammatory regulation, and tissue healing-promoting effects have been confirmed in various tissues and cells. Furthermore, increasing research indicates 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, and regenerative medicine.

[0003] However, natural GHRH is easily enzymatically broken down in vivo, losing its biological activity. Chromatographic analysis of the rat pituitary gland shows that the half-life of GHRH is only about 22 minutes, making natural GHRH an undesirable candidate drug. Sermorelin, a marketed GHRH agonist, consists of the first 29 amino acids of GHRH and possesses complete GHRH biological activity. It was first patented by Carlsberg Biotech in 1979 and has since been used for the diagnosis and treatment of growth hormone deficiency. Studies in GH-deficient rats have shown that sermorelin increases testosterone secretion, suggesting its potential therapeutic effect on male hypogonadism. Recently, Yuanhao Chang et al., using high-throughput drug screening, discovered that sermorelin can inhibit the transcription and translation of tumor cells by regulating cell cycle and nuclear division, showing potential therapeutic value for glioblastoma. Nevertheless, the biological activity of GHRH(1-29) and later-developed GHRH agonists remains unsatisfactory, with relatively short biological half-lives. For example, the serum stability of GHRH(1-29) is approximately 30 minutes.

[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 peptide-like GHRH agonist and its applications. This invention synthesizes a series of non-natural amino acid-substituted peptide-like GHRH agonists. Compared to natural GHRH and existing GHRH agonists, these agonists contain non-natural amino acids in their structural formulas, resulting in significantly enhanced biological activity. Furthermore, they 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 agonism and exert various therapeutic effects.

[0006] The specific technical solution of this invention is as follows:

[0007] In a first aspect, the present invention provides a pseudo-peptide GHRH agonist having one of the following chemical structural formulas:

[0008]

[0009]

[0010]

[0011] This invention synthesizes the aforementioned peptide-like GHRH agonist. Compared to natural GHRH and existing GHRH agonists, this peptide-like GHRH agonist replaces some of the original natural amino acids with non-natural amino acids in its structural formula. The strategy of rationally designing and replacing non-natural amino acids and peptide-like backbones is the most effective solution. This invention provides several novel non-natural amino acids as replacements, possessing backbones different from natural amino acids, yet capable of mimicking α-helices in their natural state. The non-natural amino acids of this invention exhibit unusual folding stability through a series of helical structures with well-defined hydrogen bond patterns. Ultimately, the resulting peptide-like GHRH agonist exhibits significantly enhanced biological activity and is less susceptible to enzymatic degradation in vivo, thus demonstrating better stability. With a serum half-life exceeding 24 hours, it can achieve long-acting GHRH receptor agonism and exert various therapeutic effects.

[0012] Furthermore, based on replacing some natural amino acids with non-natural amino acids in the GHRH agonist structure, this invention discovered that long-chain fatty acylation modification at the C-terminus of the structure can further enhance stability and activity (HD-11A, HD-11B, HD-12A, HD-12B). Moreover, the site of the long-chain fatty acylation modification and the carbon chain length have a significant impact on the stability and activity of the GHRH agonist. Specifically:

[0013] Regarding modification sites: Based on electron microscopy predictions of GHRH binding to the GHRH receptor, our team determined that 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 (our team previously attempted to modify long chains at positions 16 and 21, but found no activity 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.

[0014] 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.

[0015] Preferably, the chemical structural formula of the pseudo-peptide GHRH agonist is one of the following:

[0016]

[0017] This invention has found that compounds modified at the C-terminus with the aforementioned specific chain length exhibit better stability and activity.

[0018] Secondly, this invention provides the application of a peptide-like GHRH agonist having the above-described structural formula in the preparation of a drug for treating 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.

[0019] Preferably, the drug comprises the following (a) and / or (b):

[0020] (a) Peptide-like GHRH agonists;

[0021] (b) Pharmaceutically acceptable salts and / or esters of the pseudo-peptide GHRH agonist.

[0022] Further preferably, the drug also includes one or more of pharmaceutically acceptable dressings, excipients, solvents, and buffer solutions.

[0023] 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.

[0024] 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.

[0025] Thirdly, the present invention provides a disease treatment drug based on the above-mentioned pseudo-peptide GHRH agonist, comprising the following (a) and / or (b):

[0026] (a) Peptide-like GHRH agonists;

[0027] (b) Pharmaceutically acceptable salts and / or esters of the pseudo-peptide GHRH agonist.

[0028] The diseases mentioned include cardiovascular disease, diabetes and related complications, non-alcoholic fatty liver disease, metabolic disease, tumors, spinal muscular atrophy, optic nerve injury, ischemic stroke, colitis, growth hormone deficiency, or infertility.

[0029] Further preferably, the disease treatment drug also includes one or more of pharmaceutically acceptable dressings, excipients, solvents, and buffer solutions.

[0030] 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.

[0031] 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.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides several non-natural amino acid-substituted peptide-like GHRH agonists. Compared with natural GHRH and existing GHRH agonists, these peptide-like GHRH agonists have higher biological activity and are not easily enzymatically hydrolyzed in vivo, thus exhibiting better stability. Their serum half-life exceeds 24 hours, enabling long-term GHRH receptor activation and various therapeutic effects. Attached Figure Description

[0033] Figure 1 A comparison of serum stability of different GHRH agonists.

[0034] Figure 2 A comparison of the GH release capacity of different GHRH agonists.

[0035] Figure 3 A comparison of the levels of CREB phosphorylation promoted by different GHRH agonists.

[0036] Figure 4 A comparison of the levels of CREB phosphorylation promoted by different GHRH agonists.

[0037] Figure 5 A comparison of the levels of CREB phosphorylation promoted by different GHRH agonists.

[0038] Figure 6A comparison of the levels of CREB phosphorylation promoted by different GHRH agonists.

[0039] Figure 7 A comparison of the levels of CREB phosphorylation promoted by different GHRH agonists.

[0040] Figure 8 A comparative diagram to verify the effect of different GHRH agonists on the restoration of blood flow in ischemic lower limbs in animal models.

[0041] Figure 9 The graph shows the effect of different GHRH agonist treatments on the migration ability of HUVECs as detected by transwell assay. Detailed Implementation

[0042] The present invention will be further described below with reference to embodiments.

[0043] General Implementation Examples

[0044] In a first aspect, the present invention provides a pseudo-peptide GHRH agonist having one of the following chemical structural formulas:

[0045]

[0046]

[0047]

[0048] Secondly, this invention provides the application of a peptide-like GHRH agonist having the above-described structural formula in the preparation of a drug for treating 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.

[0049] In some preferred embodiments, the drug comprises the following (a) and / or (b):

[0050] (a) Peptide-like GHRH agonists;

[0051] (b) Pharmaceutically acceptable salts and / or esters of the pseudo-peptide GHRH agonist.

[0052] In some more preferred embodiments, the medicament also includes one or more of pharmaceutically acceptable dressings, excipients, solvents, and buffer solutions.

[0053] 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.

[0054] 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.

[0055] Thirdly, the present invention provides a disease treatment drug based on the above-mentioned pseudo-peptide GHRH agonist, comprising the following (a) and / or (b):

[0056] (a) Peptide-like GHRH agonists;

[0057] (b) Pharmaceutically acceptable salts and / or esters of the pseudo-peptide GHRH agonist.

[0058] 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.

[0059] In some preferred embodiments, the disease treatment drug also includes one or more of pharmaceutically acceptable dressings, excipients, solvents, and buffer solutions.

[0060] 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.

[0061] 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.

[0062] Specific embodiments and comparative examples

[0063] The structural formula of the pseudo-peptide GHRH agonist of this invention includes one or more of the following non-natural amino acid compounds:

[0064]

[0065] This invention synthesizes several novel peptide-like GHRH agonists with different chemical structures, as shown in Table 1:

[0066] Table 1

[0067]

[0068]

[0069]

[0070] 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. The amino acid sequence of the natural GHRH agonist is shown in SEQ ID NO: 1: YADAIFTNSYRKVLGQLSARKLLQDIMSR (i.e., Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2).

[0071] The amino acid sequence of MR409 is as follows:

[0072] 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-NHCH3.

[0073] The differentiating amino acid sites of the newly designed pseudo-peptides 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.

[0074] Table 2-1: Design of γ-AA peptide substitution for GHRH agonists

[0075]

[0076]

[0077] Table 2-2: Rational design of GHRH agonists using Aib and γ-AA peptide together

[0078]

[0079] Table 2-3: Rational design of GHRH agonists with good activity using long aliphatic chains

[0080]

[0081]

[0082] The amino acid sequences of the numbered pseudo-peptides GHRH agonists provided by this invention are as follows:

[0083] HD-1A:

[0084] 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

[0085] That 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-ornithine-aspartic acid-arginine-gamma-acid-1.

[0086] 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.

[0087] HD-1B:

[0088] 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

[0089] That 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-ornithine-aspartic acid-arginine-gamma-acid-1.

[0090] 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.

[0091] HD-2A:

[0092] 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

[0093] That 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.

[0094] 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.

[0095] HD-2B:

[0096] 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

[0097] That 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.

[0098] 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.

[0099] HD-3A:

[0100] 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

[0101] That 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 peptide 3-isoleucine-γAA peptide 2-arginine.

[0102] 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.

[0103] HD-3B:

[0104] 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

[0105] That 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 peptide 3-isoleucine-γAA peptide 2-arginine.

[0106] 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.

[0107] HD-4A:

[0108] 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

[0109] That 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.

[0110] 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.

[0111] HD-4B:

[0112] 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

[0113] That 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.

[0114] 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.

[0115] HD-5A:

[0116] 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

[0117] That 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 peptide 3-isoleucine-γAA peptide 2-arginine-γAA peptide 1.

[0118] 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.

[0119] HD-5B:

[0120] 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

[0121] That 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.

[0122] 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.

[0123] HD-7A:

[0124] 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

[0125] That 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-ortholeucine-aspartic acid-arginine.

[0126] 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.

[0127] HD-7B:

[0128] 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

[0129] That 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-ornithine-leucine-glutamine-aspartic acid-isoleucine-ornithine-aspartic acid-arginine.

[0130] 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.

[0131] HD-8A:

[0132] 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

[0133] That 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.

[0134] 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.

[0135] HD-8B:

[0136] 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

[0137] That 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.

[0138] 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.

[0139] HD-9A:

[0140] 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

[0141] That 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 peptide 3-isoleucine-γAA peptide 2-arginine.

[0142] 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.

[0143] HD-9B:

[0144] 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

[0145] That 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 peptide 3-isoleucine-γAA peptide 2-arginine.

[0146] The purity of the target peptide is >95%, and its purity was determined using RP-HPLC. The MS value was 583.00 [M+6H]. 6+ 699.35 [M+5H] 5+ 873.87[M+4H] 4+ MS theoretical value: 3492.25.

[0147] HD-10A:

[0148] 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

[0149] That 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,

[0150] 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.

[0151] HD-10B:

[0152] 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

[0153] That 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.

[0154] 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.

[0155] HD-11A:

[0156] 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

[0157] That 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-γAA peptide 1-12-aminododecanoic acid.

[0158] 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.

[0159] HD-11B:

[0160] NMeTyr-Aib-Asp-Ala-Ile-Phe-ThT-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-γAA2-Arg-γAA1-Ada-NH2

[0161] That 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 peptide 2-arginine-γAA peptide 1-12-aminododecanoic acid.

[0162] 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.

[0163] HD-12A:

[0164] 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

[0165] That 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 peptide 3-isoleucine-γAA peptide 2-arginine-12-aminododecanoic acid.

[0166] 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.

[0167] HD-12B:

[0168] 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

[0169] That 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 peptide 3-isoleucine-γAA peptide 2-arginine-12-aminododecanoic acid.

[0170] 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.

[0171] (1) Stability testing: A major problem limiting the use of GHRH analogues is their susceptibility to rapid protease degradation and instability. Improving the resistance of GHRH agonists to protease 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-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-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-11A, HD-11B, HD-12A, and HD-12B was significantly improved compared with MR-409.

[0172] (2) GH release test

[0173] 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-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 drawn, and GH concentration was detected using an ELISA Kit. The result was recorded as 0 min. Finally, a final concentration of 2 × 10⁻⁶ GH was added. -5 After M was administered the GHRH agonist, 100 μL of culture medium was extracted from the detection system at 15 min, 30 min, and 60 min to detect GH concentration. The Prim's analysis results are as follows: Figure 2As shown in the figure. The results show that, compared with GHRH and MR-409, the GHRH agonist HD-11A has a significantly stronger ability to promote GH release. This result is determined by the combination of HD-11A's target agonistic activity and its serum stability.

[0174] (3) Cell viability test

[0175] In cells containing GHRH-R, upon binding of GHRH or a GHRH agonist 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.

[0176] like Figure 3-7 As shown, 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-9A, and HD-11A show better activity.

[0177] (4) Animal models validated that GHRH agonists promote blood flow recovery in ischemic lower limbs.

[0178] A mouse model of lower limb ischemia was established by ligating the femoral artery, and the recovery of blood flow in the ischemic lower limbs was observed after treatment with a GHRH agonist. The GHRH agonist was administered at a concentration of 10 μg / mouse / day. Doppler ultrasound examinations of the mouse lower limbs were performed on the day of model establishment, day 3, day 7, and day 14. The results are as follows: Figure 8 As shown in -A, after the establishment of the lower limb ischemia model, the blood perfusion of the affected limb was significantly lower than that of the control side in all groups of mice. On postoperative day 7, the blood flow recovery in the three groups treated with GHRH agonists was better than that in the control group, but there was no statistically significant difference. On postoperative day 14, the blood perfusion in group 11A was significantly improved compared to the NC and MR409 groups. Figure 8 -B). Simultaneously, 14 days after administration of the GHRH agonist, the paw gangrene in mice was significantly improved ( Figure 8 -C, D).

[0179] (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- 5 After culturing the culture medium containing M GHRH 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. The results are as follows: Figure 9 As shown in the figure. The results showed that, compared with the NC group, both MR409 and 11A could promote HUVEC cell migration, with 11A showing a more significant promoting effect.

[0180] 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.

[0181] 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 peptidomimetic GHRH agonist, characterized in that: HD-1A: HD-1B: HD-2A: HD-2B: HD-3A: HD-3B: HD-4A: HD-4B: HD-5A: HD-5B: HD-8A: HD-8B: HD-9A: HD-9B: HD-10A: HD-10B: HD-11A: HD-11B: HD-12A: HD-12B:

2. The peptidomimetic GHRH agonist of claim 1, characterized in that: HD-4A: HD-11A: HD-11B: HD-12A: HD-12B:

3. Use of a peptidomimetic GHRH agonist according to claim 1 or 2 for the preparation of a medicament for the treatment of a disease, characterized in that: The disease is diabetes, non-alcoholic fatty liver, obesity, spinal muscular atrophy, optic nerve injury, ischemic stroke, colitis, growth hormone deficiency, or infertility.

4. Use according to claim 3, characterized in that: The drug includes the following (a) and / or (b): (a) a polypeptide GHRH agonist; (b) a pharmaceutically acceptable salt and / or ester of the polypeptide GHRH agonist.

5. Use according to claim 4, characterized in that, The drug also includes one or more of a pharmaceutically acceptable dressing, excipient, and solvent.

6. Use according to claim 5, characterized in that, The drug is an injection preparation, an oral preparation, a patch, a spray, an implanted micro-pump, eye drops, a smeared milk / liquid / gel, or a micro-needle.

7. Use according to claim 6, characterized in that, The drug is administered subcutaneously, intravenously, intramuscularly, implanted with a micro-pump, inhaled through the mouth and nose, dropped into the eyes, swallowed, or applied topically.

8. A medicament for the treatment of a disease based on the polypeptide GHRH agonist according to claim 1 or 2, characterized in that The drug includes the following (a) and / or (b): (a) a polypeptide GHRH agonist; (b) a pharmaceutically acceptable salt and / or ester of the polypeptide GHRH agonist. The disease is diabetes, non-alcoholic fatty liver, obesity, spinal muscular atrophy, optic nerve injury, ischemic stroke, colitis, growth hormone deficiency, or infertility.

9. The medicament for treating disease according to Claim 8, wherein The drug also includes one or more of a pharmaceutically acceptable dressing, excipient, and solvent.

10. The medicament for treating disease according to Claim 9, wherein The drug is an injection preparation, an oral preparation, a patch, a spray, an implanted micro-pump, eye drops, a smeared milk / liquid / gel, or a micro-needle.

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