Long chain modified ghrh agonists and uses thereof

Abstract

The application relates to the field of biological medicine, and discloses a long-chain modified GHRH agonist and application thereof. The application provides several GHRH agonists which are modified by long-chain fatty acylation at C terminals. Compared with natural GHRH and existing GHRH agonists, the GHRH agonists modified by long-chain fatty acylation at C terminals have greatly improved biological activity, are not prone to be enzymolyzed in vivo, have better stability, have a serum half-life of more than 24 hours, can realize long-acting GHRH receptor agonism, and can play multiple therapeutic effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a long-chain modified GHRH agonist and its application. BACKGROUND

[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, prevent ischemia-reperfusion injury, and therefore have wide clinical application prospects in cardiovascular diseases, diabetes, regenerative medicine, etc.

[0003] The GH secretion-promoting effect of GHRH makes it a promising drug, but natural GHRH is easily degraded in the body and loses its biological activity. Chromatographic analysis of rat pituitary glands showed that the half-life of GHRH was only about 22 minutes, so natural GHRH is not ideal as a candidate drug. Currently, two GHRH polypeptide analogues, Tesamorelin and Sermorelin, are on the market, but the latter has been withdrawn. Tesamorelin is a substitute for the addition of trans-3-hexenoyl to the first tyrosine of hGHRH(1-44)NH2. This modification enables it to resist the degradation of dipeptidyl aminopeptidase-IV (CD26), thereby 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 fat metabolism disorder in HIV patients, improve immune function and sleep quality of patients, and help reduce plasma cholesterol levels in patients with type II diabetes. Currently, the Tesamorelin injection produced by Theratechnologies, a Canadian pharmaceutical company, has been approved for marketing in the United States and Canada for the treatment of fat metabolism disorder in HIV patients. Schally AV et al. replaced the 12th and 21st amino acids of hGHRH with ornithine to design and synthesize the early GHRH agonist JI series. However, compared with natural GHRH, the new polypeptide does not exhibit very significant serum stability, for example, the serum stability of its most effective agonist MR409 is about 100 minutes, and its biological half-life in animals will be further reduced, and no further research on its late drug properties and other systems has been reported.

[0004] In summary, the stability and biological activity of the currently developed GHRH agonists are generally not ideal, so it is necessary to develop more GHRH agonists with high stability and high biological activity. SUMMARY

[0005] To solve the above technical problems, the present application provides a long-chain modified GHRH agonist and its application. The present application synthesizes a series of GHRH agonists modified by long-chain fatty acylation at the C-terminal. Compared with natural GHRH and existing GHRH agonists, the GHRH agonists modified by long-chain fatty acylation at the C-terminal have significantly improved biological activity, and are not easily enzymatically degraded in vivo, so their stability is greatly improved, and the serum half-life is more than 24 hours, which can achieve long-acting GHRH receptor agonism and play multiple therapeutic effects.

[0006] The specific technical solutions of the present application are as follows:

[0007] In the first aspect, the present application provides a long-chain modified GHRH agonist, whose chemical structural formula is:

[0008]

[0009] The present application synthesizes the above-mentioned GHRH agonist modified by long-chain fatty acylation at the C-terminal. Compared with natural GHRH and existing GHRH agonists, the GHRH agonist modified by long-chain fatty acylation at the C-terminal has significantly improved biological activity, and is not easily enzymatically degraded in vivo, so its stability is better, and the serum half-life is more than 24 hours, which can achieve long-acting GHRH receptor agonism and play multiple therapeutic effects.

[0010] The present application finds that the modification site and the length of the carbon chain of long-chain fatty acylation have a significant impact on the stability and activity of the GHRH agonist. Specifically:

[0011] For the modification site: based on the electron microscope structure prediction of the binding of GHRH to GHRH receptor, the N-terminal of GHRH (1-29) has a close binding with GHRH receptor. Therefore, the modification of the N-terminal may significantly affect its activity. And the modification at the middle site may destroy its secondary structure, thereby greatly affecting its activity (the present application team previously tried to select 16 and 21 as the modification site of long-chain, and found that no activity was detected). In contrast, the amino acid side chain near the C-terminal has less binding with the target, and contains more hydrophobic groups, so it is easier to modify, and at the same time, it will not cause great damage to its secondary structure. In addition, the modification at the C-terminal can be more conveniently synthesized by solid phase; if the modification is performed at other sites, the synthesis cost is also higher.

[0012] 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.

[0013] Preferably, the chemical structural formula of the GHRH agonist is:

[0014]

[0015] This invention has found that compounds modified at the C-terminus with the aforementioned specific chain length exhibit better stability and activity.

[0016] 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.

[0017] Preferably, the drug comprises the following (a) and / or (b):

[0018] (a) GHRH agonists;

[0019] (b) Pharmaceutically acceptable salts and / or esters of GHRH agonists.

[0020] Further preferably, the drug also includes one or more of pharmaceutically acceptable dressings, excipients, solvents, and buffer solutions.

[0021] 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.

[0022] 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.

[0023] Thirdly, the present invention provides a disease treatment medicament based on the above-mentioned GHRH agonist, comprising the following (a) and / or (b):

[0024] (a) GHRH agonists;

[0025] (b) Pharmaceutically acceptable salts and / or esters of GHRH agonists.

[0026] 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.

[0027] Further preferably, the disease treatment drug also includes one or more of pharmaceutically acceptable dressings, excipients, solvents, and buffer solutions.

[0028] 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.

[0029] 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.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] This 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 exceeding 24 hours. They can achieve long-acting GHRH receptor activation and exert a variety of therapeutic effects. Attached Figure Description

[0032] Figure 1 A comparison of serum stability of different GHRH agonists.

[0033] Figure 2 A comparison of the GH release capacity of different GHRH agonists.

[0034] Figure 3 A comparison of the levels of CREB phosphorylation promoted by different GHRH agonists.

[0035] Figure 4 A comparison of the levels of CREB phosphorylation promoted by different GHRH agonists.

[0036] Figure 5 A comparison of the levels of CREB phosphorylation promoted by different GHRH agonists.

[0037] Figure 6 A comparison of the levels of CREB phosphorylation promoted by different GHRH agonists.

[0038] Figure 7 A comparison of the levels of CREB phosphorylation promoted by different GHRH agonists.

[0039] 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.

[0040] 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

[0041] The present invention will be further described below with reference to embodiments.

[0042] General Implementation Examples

[0043] In a first aspect, the present invention provides a long-chain modified GHRH agonist, the chemical structural formula of which is:

[0044]

[0045]

[0046] In some preferred embodiments, the chemical structural formula of the GHRH agonist is as follows:

[0047]

[0048] 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.

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

[0050] (a) GHRH agonists;

[0051] (b) Pharmaceutically acceptable salts and / or esters of GHRH agonists.

[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 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 medicament based on the above-mentioned GHRH agonist, comprising the following (a) and / or (b):

[0056] (a) GHRH agonists;

[0057] (b) Pharmaceutically acceptable salts and / or esters of GHRH agonists.

[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 GHRH agonist of this invention comprises one or more of the following non-natural amino acid compounds in its structural formula:

[0064]

[0065] This invention synthesizes several new 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.,

[0071] 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).

[0072] The amino acid sequence of MR409 is as follows:

[0073] 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.

[0074] 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.

[0075] Table 2-1: Design of γ-AA peptide substitution for GHRH agonists

[0076]

[0077]

[0078] Table 2-2: Rational design of GHRH agonists using Aib and γ-AA peptide together

[0079]

[0080] Table 2-3: Rational design of GHRH agonists with good activity using long aliphatic chains

[0081]

[0082]

[0083] The amino acid sequences of the GHRH agonists provided by this invention are as follows:

[0084] HD-1A:

[0085] 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

[0086] 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.

[0087] 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.

[0088] HD-1B:

[0089] 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

[0090] 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.

[0091] 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.

[0092] HD-2A:

[0093] 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

[0094] 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.

[0095] 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.

[0096] HD-2B:

[0097] 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

[0098] 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.

[0099] 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.

[0100] HD-3A:

[0101] 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

[0102] 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.

[0103] 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.

[0104] HD-3B:

[0105] 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

[0106] 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.

[0107] 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.

[0108] HD-4A:

[0109] 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

[0110] 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.

[0111] 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.

[0112] HD-4B:

[0113] 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

[0114] 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.

[0115] 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.

[0116] HD-5A:

[0117] 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

[0118] 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.

[0119] 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.

[0120] HD-5B:

[0121] 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

[0122] 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.

[0123] 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.

[0124] HD-6A:

[0125] 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

[0126] 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-12-aminododecanoic acid.

[0127] 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.

[0128] HD-6B:

[0129] 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

[0130] 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-12-aminododecanoic acid.

[0131] 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.

[0132] HD-7A:

[0133] 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

[0134] 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.

[0135] 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.

[0136] HD-7B:

[0137] 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

[0138] 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.

[0139] 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.

[0140] HD-8A:

[0141] 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-I le-γAA2-Arg-NH2

[0142] 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.

[0143] 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.

[0144] HD-8B:

[0145] AcTyr-Aib-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Orn-Le u-Leu-Gln-Asp-Ile-γAA2-Arg-NH2

[0146] 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.

[0147] 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.

[0148] HD-9A:

[0149] 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

[0150] 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.

[0151] 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.

[0152] HD-9B:

[0153] AcTyr-Aib-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Om-Leu-Leu-γAA3-Ile-γAA2-Arg-NH2

[0154] 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.

[0155] 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.

[0156] HD-10A:

[0157] 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

[0158] 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.

[0159] 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.

[0160] HD-10B:

[0161] 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

[0162] 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.

[0163] 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.

[0164] HD-1 1A:

[0165] 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

[0166] 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.

[0167] 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.

[0168] HD-11B:

[0169] 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

[0170] 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.

[0171] 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.

[0172] HD-12A:

[0173] 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

[0174] 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.

[0175] 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.

[0176] HD-12B:

[0177] 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

[0178] 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.

[0179] 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.

[0180] (1) Stability testing: A major problem limiting the use of GHRH analogues 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.

[0181] (2) GH release test

[0182] 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. 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 2 As shown in the figure. 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 of HD-6A and HD-11A and their serum stability.

[0183] (3) Cell viability test

[0184] 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.

[0185] like Figures 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-6A, HD-9A, and HD-11A show better activity.

[0186] (4) Animal models validated that GHRH agonists promote blood flow recovery in ischemic lower limbs.

[0187] 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 the difference was not statistically significant. On postoperative day 14, the blood perfusion in groups 6A and 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).

[0188] (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. - 5After culturing the culture 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. The results are as follows: Figure 9 As shown in the figure. The results showed that, compared with the NC group, MR409, 6A, and 11A could all promote HUVEC cell migration, with 6A and 11A showing more significant promoting effects.

[0189] 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.

[0190] 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 of claim 1, characterized in that: The chemical structural formula is:

3. Use of a 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 cardiovascular disease, diabetes and related complications, 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 comprises the following (a) and / or (b): (a) GHRH agonist; (b) pharmaceutically acceptable salt and / or ester of GHRH agonist.

5. Use according to claim 4, characterized in that, The drug further comprises one or more of pharmaceutically acceptable dressings, excipients and solvents.

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 cream / 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 or swallowed.

8. A medicament for the treatment of a disease based on the GHRH agonist according to claim 1 or 2, characterized in that The drug comprises the following (a) and / or (b): (a) GHRH agonist; (b) pharmaceutically acceptable salt and / or ester of GHRH agonist. The disease is cardiovascular disease, diabetes and related complications, 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 further comprises one or more of pharmaceutically acceptable dressings, excipients and solvents.

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 cream / liquid / gel or a micro-needle.

Images