Pseudopolypeptide GHRH agonist and application thereof

Abstract

The invention relates to the field of biological medicine, and discloses a quasi-polypeptide GHRH agonist and application thereof. The invention provides a plurality of non-natural amino acid substituted quasi-polypeptide GHRH agonists, and compared with natural GHRH and existing GHRH agonists, the quasi-polypeptide GHRH agonists have the advantages that the biological activity is greatly improved, and the quasi-polypeptide GHRH agonists are not easily subjected to enzymolysis in vivo, so that the stability is better, the serum half-life period exceeds 24 hours, and long-acting excitation of GHRH receptors can be realized and various treatment effects can be exerted.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly relates to a pseudo-peptide GHRH agonist and its application. Background Art

[0002] Growth hormone-releasing hormone (GHRH) is a polypeptide composed of 44 amino acids, which is mainly synthesized and secreted by the hypothalamus. As a member of the hypothalamic regulatory peptides, GHRH acts on the GHRH receptor (GHRH-R) on the cell membrane of the anterior pituitary gland, stimulating the pituitary gland to synthesize and secrete growth hormone (GH). GH reaches the liver through the blood, inducing hepatocytes to secrete Insulin-like growth factors 1 (IGF-1). The latter binds to its receptor, activates the PI3K and MAPK signaling pathways in target tissues, and plays a role in stimulating cell division, thereby promoting human growth and development. The initial transcript of GHRH consists of 108 amino acids, including the first 20 amino acid signal peptide and the propeptides at both ends. The active form of GHRH in humans is composed of 40 and 44 amino acids, but the first 29 amino acids of GHRH have complete biological activity (GHRH(1-29)NH2). Among them, 25 amino acids at positions 3-27 participate in the formation of the α helix. It is generally believed that maintaining and regulating the GHRH / GH / IGF-1 neuroendocrine axis of the hypothalamus-pituitary is the main function of GHRH. For this reason, GHRH-R was initially considered to be expressed only in pituitary cells. However, subsequent studies have shown that in addition to the hypothalamus, GHRH-R is expressed in various peripheral tissue organs such as the placenta, male and female gonads, and lymphocytes. In addition to being an important member of the neuroendocrine system, the anti-apoptotic, inflammation-regulating, and tissue-healing-promoting effects of GHRH have also been confirmed in various tissues or cells. In addition, more and more studies have shown that GHRH agonists (GHRH-A) can play a role in promoting angiogenesis and tissue repair and preventing ischemia-reperfusion injury, and thus have broad clinical application prospects in the fields of cardiovascular diseases, diabetes, and regenerative medicine.

[0003] However, natural GHRH is easily enzymatically degraded in vivo and loses its biological activity. Chromatographic analysis of the rat pituitary gland shows that the half-life of GHRH is only about 22 minutes. Therefore, natural GHRH is not an ideal candidate drug. Sermorelin is an already marketed GHRH agonist, which consists of the first 29 amino acids of GHRH and has the complete biological activity of GHRH. Its drug patent was first applied for by Carlsberg Bioscience in 1979 and has since been used for the diagnosis and treatment of growth hormone deficiency. Studies on rats with GH deficiency have shown that sermorelin can cause an increase in testosterone secretion, suggesting its potential therapeutic effect on male hypogonadism. Recently, Yuanhao Chang et al. used high-throughput drug screening and found that sermorelin can inhibit the transcription and translation of tumor cells by regulating the cell cycle and nuclear division, and has potential therapeutic value for glioblastoma. Nevertheless, the biological activities of GHRH(1-29) and later developed GHRH agonists are still not ideal, and their biological half-lives are still short. For example, the serum stability of GHRH(1-29) is about 30 minutes.

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

[0005] To solve the above technical problems, the present invention provides a peptidomimetic GHRH agonist and its application. The present invention synthesizes a series of peptidomimetic GHRH agonists substituted with non-natural amino acids. Compared with natural GHRH and existing GHRH agonists, their structural formulas contain non-natural amino acids, their biological activities are significantly improved, and they are not easily enzymatically degraded in vivo. Therefore, their stability is greatly improved, the serum half-life exceeds 24 hours, and long-term activation of the GHRH receptor and the exertion of various therapeutic effects can be achieved.

[0006] The specific technical solutions of the present invention are as follows: In the first aspect, the present invention provides a peptidomimetic GHRH agonist, and its chemical structural formula is one of the following: The present invention synthesizes the above-mentioned peptidomimetic GHRH agonist. Compared with natural GHRH and existing GHRH agonists, in the structural formula of this peptidomimetic GHRH agonist, some of the original natural amino acids are replaced by unnatural amino acids. The strategy of rational design and replacement using unnatural amino acids and peptidomimetic backbones is the most effective solution. Several unnatural amino acids provided by the present invention are novel unnatural amino acids, which have a backbone different from that of natural amino acids, but can mimic the α-helix in the natural state. The unnatural amino acids of the present invention exhibit unusual folding stability by adopting a series of helical structures with clear hydrogen bond patterns. Finally, the resulting peptidomimetic GHRH agonist has greatly improved biological activity and is not easily enzymatically degraded in vivo. Therefore, it has better stability, a serum half-life exceeding 24 hours, and can achieve long-term activation of the GHRH receptor and exert various therapeutic effects.

[0007] In addition, on the basis of replacing some natural amino acids with unnatural amino acids in the structural formula of the GHRH agonist, the present invention discovers that long-chain fatty acylation modification at the C-terminus of the structural formula can further improve stability and activity (HD-11A, HD-11B, HD-12A, HD-12B). Moreover, the modification site and the carbon chain length of the long-chain fatty acylation have a significant impact on the stability and activity of the GHRH agonist. Specifically: Regarding the modification site: The research team of the present invention predicts based on the electron microscopy structure of the binding of GHRH to the GHRH receptor that the N-terminus of GHRH (1-29) has a tight binding with the GHRH receptor. Therefore, modification of the N-terminus may significantly affect its activity. Modification at its middle site may disrupt its secondary structure, thus greatly affecting its activity (the research team of the present invention previously tried to select the 16th and 21st positions as the sites for modifying the long chain, and it was found that no activity was detected). In contrast, the amino acid side chains near the C-terminus have less binding to the target and contain more hydrophobic groups, so they are easier to modify and will not cause great damage to its secondary structure. In addition, modification at the C-terminus can more conveniently synthesize the target polypeptide by solid phase; if modification is carried out at other sites, the synthesis cost is also higher.

[0008] For the carbon chain length: The research team of the present invention found that fatty chains of different lengths have different effects on enhancing drug stability and function. Specifically: Short-chain fatty acids (such as C8 to C10): Short-chain fatty acids can improve the water solubility of polypeptides, but have weak membrane permeability and binding ability to plasma proteins, so the effect of prolonging the half-life may not be as significant as that of long-chain fatty acids. Long-chain fatty acids (such as C16 and above): Long-chain fatty acids such as palmitic acid can more effectively increase the binding of polypeptides to albumin, prolong the half-life, and enhance membrane permeability, but overly long chains may affect water solubility, resulting in uneven distribution of the drug in the body. Therefore, the selection of the fatty acid chain length is usually a balancing process, and the chain length needs to be determined according to the specific requirements of the drug. An appropriate chain length can maximize the half-life, membrane permeability, and metabolic stability, while avoiding solubility and distribution problems caused by overly long chains.

[0009] Preferably, the chemical structural formula of the pseudo-polypeptide GHRH agonist is one of the following: The present invention found that compounds with the above specific chain lengths modified at the C-terminus have better stability and activity.

[0010] In a second aspect, the present invention provides the use of a pseudo-polypeptide GHRH agonist having the above structural formula in the preparation of a drug for treating diseases. Among them, the diseases are cardiovascular diseases, diabetes and related complications, non-alcoholic fatty liver, obesity and other metabolic diseases, 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 pseudo-polypeptide GHRH agonist; (b) Pharmaceutically acceptable salts and / or esters of the pseudo-polypeptide GHRH agonist.

[0012] More preferably, the drug further comprises one or more of pharmaceutically acceptable dressings, excipients, solvents and buffers.

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

[0014] More preferably, the administration method of the drug is subcutaneous injection, intravenous injection, intramuscular injection, micropump implantation, nasal and oral inhalation, eye drop instillation, swallowing or topical application.

[0015] In a third aspect, the present invention provides a drug for treating diseases based on the above pseudo-polypeptide GHRH agonist, comprising the following (a) and / or (b): (a) A pseudo-polypeptide GHRH agonist; (b) Pharmaceutically acceptable salts and / or esters of peptidomimetic GHRH agonists.

[0016] Wherein, the diseases are cardiovascular diseases, diabetes and related complications, non-alcoholic fatty liver, metabolic diseases, tumors, spinal muscular atrophy, optic nerve injury, ischemic stroke, colitis, growth hormone deficiency or infertility.

[0017] Further preferably, the drug for treating the diseases further comprises one or more of pharmaceutically acceptable dressings, excipients, solvents and buffers.

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

[0019] Further preferably, the administration mode of the drug is subcutaneous injection, intravenous injection, intramuscular injection, micropump implantation, nasal and oral inhalation, eye drop instillation, swallowing or topical application.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides several peptidomimetic GHRH agonists substituted with unnatural amino acids. Compared with natural GHRH and existing GHRH agonists, the peptidomimetic GHRH agonist has higher biological activity and is not easily enzymatically degraded in vivo. Therefore, it has better stability, a serum half-life exceeding 24 hours, and can achieve long-term activation of the GHRH receptor and exert various therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a comparison chart of serum stabilities of different GHRH agonists.

[0022] Figure 2 It is a comparison chart of GH release capabilities of different GHRH agonists.

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

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

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

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

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

[0028] Figure 8 Comparison chart for validating the promotion of blood flow recovery in ischemic lower limbs by different GHRH agonists in an animal model.

[0029] Figure 9 Chart showing the effect of treatment with different GHRH agonists on the migration ability of HUVECs detected by transwell. Detailed implementation methods

[0030] The present invention will be further described below in conjunction with the embodiments.

[0031] General embodiment In a first aspect, the present invention provides a peptidomimetic GHRH agonist, the chemical structural formula of which is one of the following: In a second aspect, the present invention provides the use of a peptidomimetic GHRH agonist having the above structural formula in the preparation of a drug for treating diseases. Among them, the diseases are cardiovascular diseases, diabetes and related complications, non-alcoholic fatty liver, obesity and other metabolic diseases, tumors, spinal muscular atrophy, optic nerve injury, ischemic stroke, colitis, growth hormone deficiency or infertility.

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

[0033] In some more preferred embodiments, the drug further comprises one or more of a pharmaceutically acceptable dressing, excipient, solvent and buffer.

[0034] In some preferred embodiments, the drug for treating diseases is an injection preparation, an oral preparation, a patch, a spray, an implanted micropump, eye drops, a topical lotion / cream / gel or a microneedle.

[0035] In some more preferred embodiments, the administration method of the drug is subcutaneous injection, intravenous injection, intramuscular injection, micropump implantation, nasal and oral inhalation, eye drop instillation, swallowing or topical application.

[0036] In a third aspect, the present invention provides a drug for treating diseases based on the above peptidomimetic GHRH agonist, comprising the following (a) and / or (b): (a) A peptidomimetic GHRH agonist; (b) The pharmaceutically acceptable salts and / or esters of peptidomimetic GHRH agonists.

[0037] Wherein, the diseases are cardiovascular diseases, diabetes and related complications, non-alcoholic fatty liver, obesity and other metabolic diseases, tumors, spinal muscular atrophy, optic nerve injury, ischemic stroke, colitis, growth hormone deficiency or infertility.

[0038] In some preferred embodiments, the medicament for treating the diseases further comprises one or more of pharmaceutically acceptable dressings, excipients, solvents and buffers.

[0039] In some preferred embodiments, the medicament for treating the diseases is an injection preparation, an oral preparation, a patch, a spray, an implantable micropump, eye drops, a smear lotion / gel or a microneedle.

[0040] In some more preferred embodiments, the administration mode of the medicament is subcutaneous injection, intravenous injection, intramuscular injection, micropump implantation, nasal and oral inhalation, eye drop instillation, swallowing or topical application.

[0041] Specific examples and comparative examples The structural formula of the peptidomimetic GHRH agonist in the embodiment of the present invention includes one or more of the following unnatural amino acid compounds: The present invention newly synthesizes a variety of peptidomimetic GHRH agonists with different chemical structural formulas, as specifically shown in Table 1: Table 1 Wherein: the amino acid sequences of each numbered GHRH agonist in the above table are designed based on the natural GHRH agonist 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).

[0042] The amino acid sequence of MR409 is: 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。

[0043] The amino acid sites that distinguish each of the newly designed peptidomimetic GHRH agonists of the present invention from its natural GHRH agonist 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 the same as MR409.

[0044] Table 2-1: Design of substitutions of γ-AA peptides for GHRH agonists Table 2-2: Rational design of GHRH agonists using Aib and γ-AA peptides Table 2-3: Rational design of GHRH agonists with better activity using long fatty chains The amino acid sequences of the peptidomimetic GHRH agonists with each number provided by the present invention are specifically 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 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-leucine-glutamine-aspartic acid-isoleucine-norleucine-aspartic acid-arginine-γAA peptide 1.

[0045] The purity of the target peptide > 95%, and its purity was determined using RP-HPLC. MS measured value: 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 i.e.: 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-leucine-glutamine-aspartic acid-isoleucine-norleucine-aspartic acid-arginine-γAA peptide 1.

[0047] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 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 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-leucine-glutamine-aspartic acid-isoleucine-γAA peptide 2-arginine.

[0049] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 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 That is: Acetyl-Tyrosine-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-Leucine-Glutamine-Aspartic acid-Isoleucine-γAA peptide 2-Arginine.

[0051] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 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 That is: N-Methyl-Tyrosine-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-Leucine-γAA peptide 3-Isoleucine-γAA peptide 2-Arginine.

[0053] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 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 That is: Acetyl-Tyr-D-Ala-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

[0055] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 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 That is: N-Methyl-Tyr-D-Ala-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

[0057] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 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 Namely: Acetyl-Tyr-D-Ala-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

[0059] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 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 Namely: N-Methyl-Tyr-D-Ala-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

[0061] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 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 Namely: Acetyl-Tyrosine-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-Leucine-γAA peptide 3-Isoleucine-γAA peptide 2-Arginine-γAA peptide 1

[0063] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 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-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 Namely: 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-Leucine-Glutamine-Aspartic Acid-Isoleucine-Norleucine-Aspartic Acid-Arginine

[0065] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 564.62 [M+6H] 6+ 677.26 [M+5H] 5+ 846.28 [M+4H] 4+ ; MS theoretical value: 3381.93

[0066] 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 Namely: 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-leucine-glutamine-aspartic acid-isoleucine-norleucine-aspartic acid-arginine

[0067] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 685.68 [M+5H] 5+ 856.79 [M+4H] 4+ ; MS theoretical value: 3423.97

[0068] 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 Namely: 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-leucine-glutamine-aspartic acid-isoleucine-γAA peptide 2-arginine

[0069] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 567.97 [M+6H] 6+ 681.29 [M+5H] 5+ 851.30 [M+4H] 4+ ; MS theoretical value: 3402.03

[0070] 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 Namely: Acetyl-Tyr-α-aminoisobutyric acid-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-2-aminobutyric acid-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-γAA peptide 2-Arg-NH2

[0071] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 689.70 [M+5H] 5+ 861.80 [M+4H] 4+ ; MS theoretical value: 3444.06

[0072] 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 Namely: Tyr-α-aminoisobutyric acid-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-2-aminobutyric acid-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-γAA peptide 3-Ile-γAA peptide 2-Arg-NH2

[0073] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 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

[0074] 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 Namely: Acetyl-Tyr-α-aminoisobutyric acid-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-2-aminobutyric acid-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-γAA peptide 3-Ile-γAA peptide 2-Arg-NH2

[0075] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 583.00 [M+6H] 6+ 699.35 [M+5H] 5+ 873.87 [M+4H] 4+ ; MS theoretical value: 3492.25

[0076] 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 Namely: Tyr-α-aminoisobutyric acid-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-2-aminobutyric acid-Gln-Leu-Ser-Ala-Arg-Orn-Leu-Leu-Gln-Asp-Ile-γAA peptide 2-Arg-γAA peptide 1

[0077] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 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

[0078] 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 That is: N-methyl 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-leucine-glutamine-aspartic acid-isoleucine-γAA peptide 2-arginine-γAA peptide 1.

[0079] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 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.

[0080] 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 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-leucine-glutamine-aspartic acid-isoleucine-γAA peptide 2-arginine-γAA peptide 1-12-aminododecanoic acid.

[0081] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 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.

[0082] HD-11B: 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 That is: N-methyl 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-leucine-glutamine-aspartic acid-isoleucine-γAA peptide 2-arginine-γAA peptide 1-12-aminododecanoic acid.

[0083] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 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.

[0084] 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 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-leucine-γAA peptide 3-isoleucine-γAA peptide 2-arginine-12-aminododecanoic acid.

[0085] The purity of the target peptide > 95%, and its purity was determined by RP-HPLC. MS measured value: 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.

[0086] 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 That is: N - methyl - tyrosine - α - amino - isobutyric acid - aspartic acid - alanine - isoleucine - phenylalanine - threonine - asparagine - serine - tyrosine - arginine - ornithine - valine - leucine - 2 - aminobutyric acid - glutamine - leucine - serine - alanine - arginine - ornithine - leucine - leucine - γAA peptide 3 - isoleucine - γAA peptide 2 - arginine - 12 - aminododecanoic acid.

[0087] The purity of the target peptide is > 95%, and its purity is determined by RP - HPLC. MS measured value: 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.

[0088] (1) Stability experiments One of the main problems restricting the use of GHRH analogs is that they are easily and rapidly degraded by proteases and are unstable. Improving the ability of GHRH agonists to resist proteolysis is one of the main objectives of the present invention. The present invention detected the stability of GHRH agonists HD - 2A, HD - 3A, HD - 4A, HD - 5A, HD - 8A, HD - 9A, HD - 11A, HD - 11B, HD - 12A, HD - 12B in serum. By incubating 10 -4 μM MR - 409 (control GHRH agonist) and HD - 2A, HD - 3A, HD - 4A, HD - 5A, HD - 8A, HD - 9A, HD - 11A, HD - 11B, HD - 12A, HD - 12B with human serum at 30 °C for 24 hours, samples of the reaction were collected at different times in the middle, and the content of the intact GHRH agonist that was not degraded was analyzed using high - performance liquid chromatography (HPLC). Compared with the amount of the polypeptide before adding serum (time 0 hour), the relative residual intact GHRH agonist content % was obtained, which is the stability. The results showed that the serum stability of GHRH agonists HD - 11A, HD - 11B, HD - 12A, HD - 12B was significantly improved compared to MR - 409.

[0089] (2) GH release test The most basic function of GHRH is to promote GH release. Based on this, using GHRH (1-29) and MR-409 as positive controls, the present invention detected 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: Extract the pituitary glands of C57 mice, isolate pituitary cells, resuspend them in F12-K medium, withdraw 100 μl of the medium, and detect the GH concentration using an ELISA kit, which is recorded as 0 min. After adding GHRH agonists at a final concentration of 2×10 -5 M, 100 μl of the medium was withdrawn from the detection system at 15 min, 30 min, and 60 min respectively for detecting the GH concentration. The results analyzed by Prism are as Figure 2 shown. The results showed that compared with GHRH and MR-409, the ability of the GHRH agonist HD-11A to promote GH release was significantly stronger. This result is comprehensively determined by the agonist activity of HD-11A on the target and its serum stability.

[0090] (3) Cell viability test In cells containing GHRH-R, after GHRH or GHRH agonists bind to GHRH-R, the activated second messengers include not only adenylate cyclase-cAMP-PKA and Ca 2+ calmodulin, but also phosphoinositide-diacylglycerol-protein kinase C (PKC), L-type calcium channels, and arachidonic acid-eicosanoid pathway. The increase in cAMP also stimulates PKA to activate cAMP response element-binding protein (CREB), phosphorylate it, and thus stimulate the transcription of the GHRHR gene. That is to say, the activity of different GHRH agonists can be judged by detecting P-CREB. In this cell experiment, MCF7 cells with GHRH receptors were selected as the experimental subjects to preliminarily screen the activities of various GHRH agonists.

[0091] As Figures 3 - 7 shown, the activities / efficacies of the GHRH agonists of the present invention to promote the phosphorylation level of CREB are mostly significantly higher than those of known GHRH agonists such as natural GHRH and MR409. Among them, HD-4A, HD-9A, and HD-11A have good activities.

[0092] (4) Animal model verification of the promotion of blood flow recovery in ischemic lower limbs by GHRH agonists A mouse model of lower limb ischemia was constructed by ligating the femoral artery, and the blood flow recovery of the ischemic lower limbs of mice after treatment with GHRH agonists was observed. The administration concentration of GHRH agonists was 10 μg / mouse / day, and Doppler ultrasound of the lower limbs of mice was performed on the day, the 3rd day, the 7th day, and the 14th day after modeling. The results are as Figure 8As shown in Figure -A, after the establishment of the lower limb ischemia model in mice of each group, the blood perfusion of the affected limb was significantly lower than that of the control side. On the 7th day after surgery, the blood flow recovery of the three groups of mice treated with the GHRH agonist was better than that of the control group mice, but there was no statistical difference. On the 14th day after surgery, compared with the NC group and the MR409 group, the blood perfusion of the mice in the 11A group had been significantly improved ( Figure 8 -B). At the same time, after 14 days of administration of the GHRH agonist, the toe gangrene situation of the mice was also significantly improved ( Figure 8 -C, D).

[0093] (5) Cell experiment to verify the effect of the GHRH agonist on the migration ability of human umbilical vein endothelial cells (HUVECs) HUVECs were seeded on the upper chamber of Transwell, and the culture medium used was serum-free medium. The lower chamber was added with a culture medium containing 15% fetal bovine serum and 10- 5 M GHRH agonist. After culturing for 24 h, the upper chamber was fixed with paraformaldehyde and stained with crystal violet, and then the distribution of cells in the lower layer of the upper chamber was observed under a microscope. The results are as Figure 9 shown. The results showed that compared with the NC group, both MR409 and 11A could promote the migration of HUVECs, and the promoting effect of 11A was more obvious.

[0094] The raw materials and equipment used in the present invention are all common raw materials and equipment in the art without special instructions; the methods used in the present invention are all conventional methods in the art without special instructions.

[0095] The above are only the preferred embodiments of the present invention, and do not limit the present invention in any way. Any simple modifications, changes, and equivalent transformations made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A polypeptide mimetic GHRH agonist, characterized in that: The chemical structure is one of the following: HD-1A: HD-1B: HD-2A: HD-2B: HD-3A: HD-3B: HD-4A: HD-4B: HD-5A: HD-5B: HD-7A: HD-7B: HD-8A: HD-8B: HD-9A: HD-9B: HD-10A: HD-10B: HD-11A: HD-11B: HD-12A: HD-12B:

2. The polypeptide mimetic GHRH agonist according to claim 1, characterized in that: The chemical structure is one of the following: HD-4A: HD-11A: HD-11B: HD-12A: HD-12B:

3. Use of the mimetic polypeptide GHRH agonist according to claim 1 or 2 in the preparation of a drug for treating a disease, characterized in that: The diseases are cardiovascular disease, diabetes and related complications, non-alcoholic fatty liver disease, metabolic diseases, tumors, spinal muscular atrophy, optic nerve damage, ischemic stroke, colitis, growth hormone deficiency or infertility.

4. The use according to claim 3, characterized in that: The drug comprises the following (a) and / or (b): (a) Peptide-like GHRH agonists; (b) a pharmaceutically acceptable salt and / or ester of the polypeptide mimetic GHRH agonist.

5. The use according to claim 4, characterized in that: The medicament further comprises one or more of a pharmaceutically acceptable dressing, an excipient, a solvent and a buffer.

6. The use according to claim 5, characterized in that: The drug is an injectable preparation, an oral preparation, a patch, a spray, an implantable micropump, eye drops, an smear emulsion / liquid / gel or a microneedle.

7. The use according to claim 6, characterized in that: The drug can be administered by subcutaneous injection, intravenous injection, intramuscular injection, micropump implantation, oral and nasal inhalation, eye drops, swallowing or topical application.

8. A disease treatment drug based on the mimetic polypeptide GHRH agonist according to claim 1 or 2, characterized in that Includes (a) and / or (b) below: (a) Peptide-like GHRH agonists; (b) a pharmaceutically acceptable salt and / or ester of a polypeptide mimetic GHRH agonist; The diseases are cardiovascular disease, diabetes and related complications, non-alcoholic fatty liver disease, metabolic diseases, tumors, spinal muscular atrophy, optic nerve damage, ischemic stroke, colitis, growth hormone deficiency or infertility.

9. The disease treating drug according to claim 8, characterized in that: Also included is one or more of a pharmaceutically acceptable dressing, excipient, solvent and buffer.

10. The disease treating drug according to claim 9, characterized in that: The drug is an injectable preparation, an oral preparation, a patch, a spray, an implantable micropump, eye drops, an smear emulsion / liquid / gel or a microneedle.

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