CN117229384A - Novel somatostatin analogue peptide monomer, dimer or tetramer and preparation and application thereof - Google Patents

Novel somatostatin analogue peptide monomer, dimer or tetramer and preparation and application thereof Download PDF

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CN117229384A
CN117229384A CN202310853772.9A CN202310853772A CN117229384A CN 117229384 A CN117229384 A CN 117229384A CN 202310853772 A CN202310853772 A CN 202310853772A CN 117229384 A CN117229384 A CN 117229384A
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terminal
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amino acid
nacl
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唐松山
罗群
余立城
杜艳
王新瑞
王剑云
周雅曼
吴睿卿
胡雨橦
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Guangdong Pharmaceutical University
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Guangdong Pharmaceutical University
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Abstract

The invention discloses a novel growth hormone releasing hormone similar peptide monomer, dimer or tetramer, preparation and application thereof, which promote the expression of human organ autocrine GHRH receptor and KI67 signal, promote the proliferation and/or activation of mesenchymal stem cells of organs, and are used for preparing medicaments for treating organ injury protection such as infertility, senile dementia or anticancer; the dimer sequence is one of the following:(N-terminal) X 1 ‑X 2 ‑DAIFTNSYR‑X 12 ‑VLGQLSAR‑X 21 ‑LLQDIMSRQQGESNQERGARARL‑C 45 (C-terminal) -C 45 (C-terminal) -LRARAGREQNSEGQQRSMIDQLL-X 21 ‑RASLQGLV‑X 12 ‑RYSNTFIAD‑X 2 ‑X 1 (N-terminal);the tetramer sequence is one of the following:

Description

Novel somatostatin analogue peptide monomer, dimer or tetramer and preparation and application thereof
technical field:
the invention relates to the technical field of biological medicines, in particular to a novel growth hormone releasing hormone analogue peptide monomer, dimer or tetramer, and preparation and application thereof.
The background technology is as follows:
in the last 30 years, hypothalamic Growth Hormone Releasing Hormone (GHRH) -pituitary Growth Hormone (GH) -peripheral insulin-like growth factor 1 (IGF 1) endocrine axis pathway, has endocrine axis effects, and is involved in growth and fertility regulation. Over the last 15 years, autocrine GHRH, GHRH receptors and GH signals were found in many organ cells, acting through autocrine but without axicorresponding effects. For a long time, different GHRH and GH molecules have been considered homohormones, ignoring their functional differences, because of the final GH-releasing activity. It has now been found that low GH secretion 1 P-GHRH peptide is secreted higher than GH 1 The Y-GHRH peptide can promote proliferation and/or activity of mesenchymal stem cells of organismBy melting, organ protection is increased, while GH does not.
The invention comprises the following steps:
the invention aims to provide a GHRH analogue peptide monomer, dimer or tetramer, and preparation and application thereof.
The invention is realized by the following technical scheme:
a GHRH-like peptide monomer having an amino acid sequence of one of:
1.1 (N-terminal) X 1 -X 2 -DAIFTNSYR-X 12 -VLGQLSAR-X21-LLQDIMSRQQGESNQERGARARL-C 45 -NH 2 (C-terminal)
1.2 (N-terminal) X 1 -X 2 -DAIFTNSYRRVLGQLSAR-X 21 -LLQDIMSRQQGESNQERGARARL 44 -X 45 -NH 2 (C-terminal)
1.3 (N-terminal) X 1 -X 2 -DAIFTNSYRRVLG-X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -LLQDIMSRQQGESNQERGARARL 44 -NH 2 (C-terminal)
The capital letter in the sequence is abbreviation or amino acid substitution symbol of L-alpha-amino acid, arabic numerals are amino acid residue arrangement sequence, NH 2 Represents the N-terminal or C-terminal amide structure, cysteine residue C 45 C-terminal CONH of (C-terminal) 2 The method comprises the steps of carrying out a first treatment on the surface of the Wherein X is 1 Is Y or P, X 12 Is K, X 45 Is a diamino-amino acid such as diaminopropionic acid Dap or ornithine; x is X 16 -X 20 Only one of which is a diamino-amino acid and is structurally similar to the corresponding sequence Q 16 Or L 17 、S 18 、A 19 、R 20 If diaminopropionic acid Dap is analogous to A or S, ornithine is analogous to Q, L or R, the remainder of the sequence is still corresponding to Q 16 、L 17 、S 18 、A 19 、R 20 The amino acid sequence is unchanged; when X is 21 Is modified by epsilon amino [ gamma-Glu (N-alpha-alkanoic acid) with K side chain, and has a structural formula shown as formula 1, wherein n=16-18; when X is 21 For side-chain epsilon-amino [2 XAEEAC-gamma-Glu (N-alpha-fatty diacid)]Modified riceIn the case of amino acid, the structure is shown as formula 2:
a growth hormone releasing hormone analog peptide dimer, the amino acid sequence of said dimer being one of five sequences:
(N-terminal) X 1 -X 2 -DAIFTNSYR-X 12 -VLGQLSAR-X 21 -LLQDIMSRQQGESNQERGARARL-C 45 (C-terminal) -C 45 (C-terminal) -LRARAGREQNSEGQQRSMIDQLL-X 21 -RASLQGLV-X 12 -RYSNTFIAD-X 2 -X 1 (N-terminal);
wherein the first three sequences are U-type dimers, and the second two sequences are H-type dimers. X is X 46 Are sulfhydryl-containing amino acids such as cysteine (C) or threonine (T), homocysteine (HCY).
Wherein (N-terminal) X 1 -X 2 -DAIFTNSYR-X 12 -VLGQLSAR-X 21 -LLQDIMSRQQGESNQERGARARL-C 45 (N-terminal) -C 45 (N-terminal) -LRARAGREQNSEGQQRSMIDQLL-X 21 -RASLQGLV-X 12 -RYSNTFIAD-X 2 -X 1 The (N end) is formed by connecting monomer sequences 1.1 through disulfide bonds;
is formed by extension of monomer sequence 1.2;
dimerization was achieved from monomer sequence 1.2 by using oxalic acid as linker.
Dimer was formed from monomer sequence 1.3 via oxalic acid as linker.
Monomer sequence 1.3 extends to dimer.
A GHRH-like peptide tetramer formed by disulfide-linking the identical dimers via cysteine, comprising a U or H-type homotetramer as follows; the amino acid sequence is one of the following structures:
u-type tetramer:
type H tetramer:
Wherein the inter-amino acid '-' linkage is a peptide bond, and the'-S-S-' linkage is a disulfide bond.
The invention also protects the application of the GHRH similar peptide monomer, dimer or tetramer in preparing medicines for treating infertility, senile dementia or anticancer.
The invention also provides a medicine for treating infertility, senile dementia or anticancer, which takes the monomer, dimer or tetramer of the somatostatin analogue peptide as an active ingredient.
The beneficial effects of the invention are as follows: (1) The somatostatin similar peptide monomer, dimer or tetramer of the invention can improve or extremely reduce GH release activity, promote proliferation or/and activation of mesenchymal stem cells of human organs, and can be used for preparingA medicament for treating organ injury protection diseases, such as infertility, senile dementia or anticancer. (2) Polymer Activity of the invention: 1 the Y-series U and H-type polymers show high GH release activity in vitro and in vivo, 1 the P-series polymer has low GH activity; k side chain epsilon-amino modification [ (AEEA) 2 -gamma Glu-fatty diacid]The peptide activity is higher than that of [ N-epsilon-gamma Glu (N-alpha-fatty acid)]The modification is slightly increased, and the longer the modified fatty acid chain is, the higher the activity is, and the fatty acid chain C18-20 is the best; (3) 1 The H or U-shaped polymer of Y-GHRH can enter the blood brain barrier to promote the expression of brain GHSR and KI67, proliferate and activate the brain mesenchymal stem cells. 1 The U or H-type polymer of the P-GHRH has no obvious promotion effect on the release of pituitary GH, and can promote the expression of a peripheral tissue testis/ovary autocrine GHRH receptor and KI67, proliferate and activate peripheral mesenchymal stem cells; (4) Single administration, the modified H-type peptide single/double/quadruplex maintains the plasma stability for 7/21/35 days, and the modified U-type single/double/quadruplex maintains the plasma stability for 9/30/45 days;
(5) The monomer is not readily soluble in NaCl-PB solution at pH8 (disodium hydrogen phosphate buffered saline pH 8), but the di/tetramer is completely dissolved in this solution. The monomers were dissolved in a pH 4 solution, but the di/tetramers were not dissolved in this solution, which has a reference meaning in pharmaceutical formulations, showing that different polymers produced different spatial conformations.
Description of the drawings:
fig. 1 is a statistical plot of female model birth rate. a, b, c, d, e, f, g, H, i, j, k, L correspond to the NaCl-PB, model Control, hGH, hMG,2Y-L/M/H,2P-L/M/H,4P-M,4Y-M groups. * (x) corresponds to P<0.05,0.01,0.001，X 2 And (5) chi square inspection.
FIG. 2 is a chart of female model follicular number analysis. a, b, c, d, e, f, g, H, i, j, k, L correspond to the NaCl-PB, model Control, hGH, hMG,2Y-L/M/H,2P-L/M/H,4P-M,4Y-M groups. * P <0.05,0.01,0.001, t-test.
Figure 3 is a female model ovarian GHRH receptor immunofluorescence assay. a, b, c, d, e, f, g, H, i, j, k, L correspond to the NaCl-PB, model Control, hGH, hMG,2Y-L/M/H,2P-L/M/H,4P-M,4Y-M groups. * P <0.05,0.01,0.001, t-test.
Figure 4 is an ovarian GH immunofluorescence assay. a, b, c, d, e, f, g, H, i, j, k, L correspond to the NaCl-PB, model Control, hGH, hMG,2Y-L/M/H,2P-L/M/H,4P-M,4Y-M groups. * P <0.05,0.01,0.001, t-test.
FIG. 5 is a graph of GH protein analysis from female model blood. a, b, c, d, e, f, g, H, i, j, k, L correspond to the NaCl-PB, model Control, hGH, hMG,2Y-L/M/H,2P-L/M/H,4P-M,4Y-M groups. * P <0.05,0.01,0.001, t-test.
FIG. 6 is a graph of analysis of KI67 protein expression in female model ovarian tissue. a, b, c, d, e, f, g, H, i, j, k, L correspond to the NaCl-PB, model Control, hGH, hMG,2Y-L/M/H,2P-L/M/H,4P-M,4Y-M groups. * P <0.05,0.01,0.001, t-test.
Fig. 7 is a pattern of analysis of TUNEL staining of female model ovarian tissue. a, b, c, d, e, f, g, H, i, j, k, L correspond to the NaCl-PB, model Control, hGH, hMG,2Y-L/M/H,2P-L/M/H,4P-M,4Y-M groups. * P <0.05,0.01,0.001, t-test.
Fig. 8 is a graph of estrogen analysis in female model blood. a, b, c, d, e, f, g, H, i, j, k, L correspond to the NaCl-PB, model Control, hGH, hMG,2Y-L/M/H,2P-L/M/H,4P-M,4Y-M groups. * P <0.05,0.01,0.001, t-test.
Fig. 9 is a graph of analysis of blood testosterone levels in a female model. a, b, c, d, e, f, g, H, i, j, k, L correspond to the NaCl-PB, model Control, hGH, hMG,2Y-L/M/H,2P-L/M/H,4P-M,4Y-M groups. * P <0.05,0.01,0.001, t-test.
Fig. 10 is male model reproductive rate. A, b, c, d, e, f, g correspond to the NaCl-PB, model Control, hMG,2P-L/M/H,4P-M group. * (x) corresponds to P<0.05,0.01,0.001，X 2 And (5) chi square inspection.
Fig. 11 is a graph of male model testis tissue GHRH receptor assay. A, b, c, d, e, f, g correspond to the NaCl-PB, model Control, hMG,2P-L/M/H,4P-M group. * P <0.05,0.01, t-test.
Fig. 12 is a drawing of male model blood GH analysis. A, b, c, d, e, f, g correspond to the NaCl-PB, model Control, hMG,2P-L/M/H,4P-M group. * P <0.05,0.01, t-test.
FIG. 13 is a graph of male testis tissue KI67 protein analysis. A, b, c, d, e, f, g correspond to the NaCl-PB, model Control, hMG,2P-L/M/H,4P-M group. * P <0.05,0.01, t-test.
Fig. 14 is a pattern of analysis of TUNEL staining of male model testis tissue. A, b, c, d, e, f, g correspond to the NaCl-PB, model Control, hMG,2P-L/M/H,4P-M group. * P <0.05,0.01, t-test.
FIG. 15 is a graph showing epididymal sperm count (A), motility (B) and deformity (C). A, b, c, d, e, f, g correspond to the NaCl-PB, model Control, hMG,2P-L/M/H,4P-M group. * P <0.05,0.01, t-test.
Fig. 16 is a male model blood testosterone assay. A, b, c, d, e, f, g correspond to the NaCl-PB, model Control, hMG,2P-L/M/H,4P-M group. * P <0.05,0.01, t-test.
FIG. 17 is a water maze test analysis of APP/PS1 transgenic mice. The groups a, b, c, d, e, f, g, H correspond to the NaCl-PB, model Control, galanthine (galanthamine), 2Y-L/M/H,4P1-M, 4Y-M. * P <0.05,0.01, t-test.
FIG. 18 is a graph of APP/PS1 transgenic mice, hippocampus KI67 (A) and TUNEL (B) analysis. a, b, c, d, e, f, g, H correspond to the NaCl-PB, model Control, galanthine, 2Y-L/M/H,4P1-M,4Y-M groups. * P <0.05,0.01, t-test.
FIG. 19 is a graph of APP/PS1 transgenic mice analysis of cortical KI67 (A) and TUNEL (B). a, b, c, d, e, f, g, H correspond to the NaCl-PB, model Control, galanthine, 2Y-L/M/H,4P1-M,4Y-M groups. * P <0.05,0.01, t-test.
FIG. 20 is a graph of analysis of hippocampal Tau (A) and cortical Tau (B) of APP/PS1 transgenic mice. a, b, c, d, e, f, g, H correspond to the NaCl-PB, model Control, galanthine, 2Y-L/M/H,4P1-M,4Y-M groups. * P <0.05,0.01, t-test.
FIG. 21 is a graph of analysis of hippocampal GFAP (A) and cortical GFAP (B) in APP/PS1 transgenic mice. a, b, c, d, e, f, g, H correspond to the NaCl-PB, model Control, galanthine, 2Y-L/M/H,4P1-M,4Y-M groups. * P <0.05,0.01, t-test.
FIG. 22 is a graph of analysis of hippocampal Abeta (A) and cortical Abeta (B) from APP/PS1 transgenic mice. a, b, c, d, e, f, g, H correspond to the NaCl-PB, model Control, galanthine, 2Y-L/M/H,4P1-M,4Y-M groups. * P <0.05,0.01, t-test.
FIG. 23 is a graph of analysis of hippocampal synaptophysin SYN (A) and cortical SYN (B) from APP/PS1 transgenic mice. a, b, c, d, e, f, g, H correspond to the NaCl-PB, model Control, galanthine, 2Y-L/M/H,4P1-M,4Y-M groups. * P <0.05,0.01, t-test.
FIG. 24 is a graph of hippocampal GHSR (A) and cortical GHSR (B) analysis of APP/PS1 transgenic mice. a, b, c, d, e, f, g, H correspond to the NaCl-PB, model Control, galanthine, 2Y-L/M/H,4P1-M,4Y-M groups. * P <0.05,0.01, t-test.
FIG. 25 shows the inhibition of proliferation of cancer cells by the U-type peptide (A) and the H-type peptide (B). HepG2, A549 and MDA-MB-231 are human hepatoma cells, human lung carcinoma cells and human breast carcinoma cell lines, respectively. * P <0.05,0.01, t-test.
The specific embodiment is as follows:
the following is a further illustration of the invention and is not a limitation of the invention.
Example 1: GHRH analog peptide monomer, dimer or tetramer and preparation thereof
1. Preparation of growth hormone analogue peptide dimer:
1. 2g of Rink Amide Am Resin (AM resin, hereinafter, where the C-terminal is an amide group CONH) was weighed 2 or-NH 2 Is synthesized by AM resin) and 2-chlorine resin (2-Cl-Trt resin, the following is the peptide with carboxyl COOH or-OH at the C end, which is synthesized by 2-chlorine resin), the peptide is soaked by Dichloromethane (DCM) for 15min, pumped down, 20% piperidine is added to stir and react for 20min, the N, N-Dimethylformamide (DMF) is washed for 2 times, methanol is washed for 2 times, and the DMF is used for 2 times.
2. 164mg of Fmoc-L-Dap (ALLOC) -OH or Fmoc-L-Orn (ALLOC) -OH,3 times the amount of 2- (7-azobenzotriazole) -tetramethylurea Hexafluorophosphate (HBTU) was weighed into a 20ml solid phase reactor, dissolved in DCM, and 0.1ml DIC (N, N-diisopropylcarbodiimide) was added for 2 hours.
3. 10ml of pyridine and acetic anhydride solution was added to carry out a blocking reaction for 0.5h, followed by washing with DMF 1 times, methanol 1 time, and DMF 2 times, and then draining.
4. A20% piperidine DMF solution was added to the reactor and reacted for 20min, washed 2 times with DMF, 2 times with methanol and 2 times with DMF, and ninhydrin was detected as blue.
5. 424mg of Fmoc-Leu-OH, HOBT 81mg were weighed, dissolved in DMF and 0.1ml of DIC was added, mixed well, added to the reactor for reaction for 2h, drained, washed 1 time with DMF, 1 time with methanol, 2 times with DMF, and ninhydrin was detected as colorless.
6. 20% piperidine in DMF was added and reacted for 20min, washed 2 times with DMF, 2 times with methanol and 2 times with DMF, and ninhydrin was detected as blue.
7. Repeating the steps 5 and 6 until the sequence monomer is long-chain: [ X ] 1 -X 2 -DAIFTNSYRRVLGQLSAR-X 21 -LLQDI-MSRQQGESNQERGARARL-X 45 -resin]Fmoc-Pro-OH or Fmoc-Tyr-OH, and ninhydrin was detected as colorless.
8. Adding 2% hydrazine hydrate to react for 30min to remove a protective group Dde of Fmoc-Lys (Dde) -OH, exposing side chain amino, washing 2 times with DMF, washing 2 times with methanol, washing 2 times with DMF, and detecting ninhydrin as blue;
9. Fmoc-Glu-Otbu 255mg, HOBT 81mg, were weighed, dissolved in DMF, and 0.1ml of DIC was added, mixed well, added to the reactor and reacted with lysine side chain amino groups for 2h, drained, washed 1 time with DMF, washed 1 time with methanol, and then 2 times with DMF, and ninhydrin was detected as colorless.
10. Adding 20% piperidine DMF solution into the reactor for reaction for 20min, removing amino protecting group Fmoc of Fmoc-Glu-Otbu, washing 2 times with DMF, washing 2 times with methanol, washing 2 times with DMF, and detecting ninhydrin as blue;
11. weighing the electric acid mono-tert-butyl ester (239 mg) of eicosanedioic acid, dissolving HOBT (81 mg) in DMF, adding 0.1ml of DIC, uniformly mixing, adding into a reactor for reaction for 1h, pumping, washing with DMF for 1 time, washing with methanol for 1 time, washing with DMF for 2 times, and detecting ninhydrin as colorless;
12. Removing X 45 Fmoc-Dap (ALLOC) -OH or Fmoc-Orn (ALLOC) -OH side chain amino ALLOC protecting group, specifically: washing 3 times with DCM, adding 400mmol of phenylsilane, reacting for 5 minutes, adding 2mmol of Pd (PPh 3) 4, reacting for 120 minutes, pumping, washing 1 time with DMF, washing 1 time with methanol, washing 2 times with DMF, and detecting the ninhydrin as colorless.
13. Adding X 46 Boc-Cys (phacm) -OH, which isAlpha carboxyl and X 45 Side chain amino condensation. Repeating steps 5-11 until the second strand X 1 -X 2 -DAIFTNSYRRVLGQLSAR-X 21 -LLQDIMSRQQGESNQERGARARL 47 -X 46 And ninhydrin detected as colorless, completing a U peptide containing two GHRH copy sequences:
14. similarly, repeating steps 5-11 completes the synthesis of the following monomeric peptide chain:
(NH 2 )X 1 -X 2 -DAIFTNSYR-X 12 -VLGQLSAR-X 21 -LLQDIMSRQQGESNQERGARARL-C 45 -NH 2 (C-terminal)
15. Dimer synthesis with oxalic acid as linker: cleaving the monomer from the 2-chloro resin obtained in step 7, and removing X from the collected polypeptide 45 The side chain amino protecting group ALLOC was added with 5 times of oxalic acid and reacted for 5 hours to form the following dimer:
16. synthesis of GHRH sequence-containing type H peptide: the first chain monomer long chain was synthesized with AM resin according to the following sequence: (N-terminal) X 1 -X 2 -DAIFTNSYRRVLG 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -LLQDIMSRQQGESNQERGARARL-NH 2 (C-terminal).
17. Continuing to covalently extend the 16-step peptide chain: carrying out the steps 8-11 to obtain a composition containing X 21 And the protecting group Dde of the side chain amino is hydrolyzed and combined with the gamma-carboxyl of Glu, and then fatty acid is added to form a fatty acid modified side chain.
18. X containing diamino groups 16 Or X 17 、X 18 、X 19 、X 20 One of which is a side chain amino protecting group ALLOC, the side chain amino group of which is hydrolyzed with X 46 Alpha-carboxyl reaction of Boc-Cys (phacm) -OH with X 46 Alpha-amino as a reactive group to sequentially prolong Q of a second chain 16 -X 1 Amino acid sequence, completing the following "J" type structure:
19. synthesis of sequence (N-terminal) LSAR-X on 2-chloro resin 21 LLQDIMSRQQGESNQERGARAR-OH (C-terminus), cleavage of the polypeptide from the resin. In the fully protected active group state, the polypeptide was collected and mixed with 5-fold mol of leucinamide, reacted in methylene chloride for 24 hours, and the DCM phase solution was separated by repeated washing with a separating funnel 3 times and collected.
20. Adding the DCM phase solution obtained in the step 19 into the AM resin solid phase obtained in the step 18 to react for 6 hours, and then carrying out Q with the second chain 16 Alpha carboxyl reaction, combining the polypeptide in the step 19 on the right side of the J structure to complete the 17 th-44 th amino acid sequence of the second chain, wherein the sequence is as follows:
21. and finally, adding 15ml of 95% lysate to react for 2 hours at room temperature, completely hydrolyzing a side chain protecting group, settling diethyl ether, centrifuging to obtain a crude product, and identifying by mass spectrum.
22. The crude peptide is dissolved in water, the aqueous phase is repeatedly extracted by warm water with the temperature of 50 ℃, the aqueous phase is purified by Sephadex G-25 (1 x 60cm, eluting peak 1) and SP-Sepharose chromatographic column (3 x 10 cm), the pH of the mobile phase of the aqueous phase is 11 to 7.8, and the effective peak is collected.
23. Hydrolysis X 46 Sodium dihydrogen phosphate with pH=9.5 is added and incubated overnight at 37 ℃ to form tetramer (see the following method: preparation of peptide tetramer similar to three and growth hormone releasing hormone).
24. H-type dimer synthesis with oxalic acid as linker: the first fully protected monomer long chain was synthesized with 2-chloro resin according to the following sequence: (N-terminal) X 1 -X 2 -DAIFTNSYRRVLG 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 LLQDIMSRQQGESNQERGA-RARL-OH (C-terminal) completing X 21 Side chain amino fatty acid modification, followed by removal of X 16 Or X 17 、X 18 -、X 19 -、X 20 A side chain amino protecting group ALLOC. The crude peptide was dissolved in DCM, the DCM phase was repeatedly extracted with 50℃warm water and purified by Sephadex G-25 chromatography (1X 60cm, 1 st elution peak) and the effective peak was collected. 5 times the amount of oxalic acid was added to the effective peak and reacted for 5 hours to form the following dimer:
the dimeric somatostatin-like peptides synthesized according to the above method are shown in table 1.
2. Purification by HPLC or normal pressure chromatography
1. And (3) placing the prepared peptide monomer and dimer crude product into a vessel. 2-5ml of 50% acetonitrile aqueous solution is used for dissolving and preparing 1 microgram/microliter, and can slightly ultrasonic for 2 minutes.
2. The solution was filtered through a 0.45 μm membrane.
3. Sample analysis: the peptide samples prepared above were loaded at 10 μl and analyzed by analytical grade HPLC. The mobile phase is water and acetonitrile, the time is 30min, gradient elution is carried out, HPLC is firstly balanced for 5min by using an initial gradient, then sample injection is carried out, the initial gradient is 95% of water, the acetonitrile is 5%, the end proportion is 5% of water and the acetonitrile is 95%.
4. Sample preparation: and (5) preparing the dissolved sample for sample injection. The preparative HPLC was equilibrated for 10min, starting gradient water 95%, acetonitrile 5%, ending gradient water 25%, acetonitrile 75% gradient time 40min. The sample coming out of the detector is collected.
5. Sample identification: the collected samples were sampled for purity and Mass Spectrometry (MS) identification.
6. And finally, freeze-drying the purified solution to obtain a finished product.
3. Preparation of a growth hormone releasing hormone analogue peptide tetramer: the sulfur-containing dimer peptide was prepared at a concentration of 1mg/ml, and incubated overnight at 37℃in an aqueous solution of disodium hydrogen phosphate at pH=9.5 to form a homotetrameric peptide. The poorly soluble tetrameric peptide was centrifuged at 4000 rpm for 20 min and the pellet was taken as the pure tetrameric peptide. The supernatant was separated by Sephadex G-25 chromatography (at 1X 60cm G-25 column and natural flow rate, using NaCl-PB solution as flow term, the polymer fraction was usually peak 1). Mixing the precipitate with the effective components obtained by column chromatography, and injecting experimental animals with NaCl-PB solution for maximum dissolution. Tetrameric peptides can be identified by peptide-SDS-PAGE electrophoresis without thiol reducing agents, see our issued patent (China patent ZL 201410612382.3).
The growth hormone releasing hormone analogue peptide tetramer synthesized according to the above method is shown in table 1:
table 1 amino acid sequences of novel GHRH mono/di/tetra chain peptides synthesized and peak effect time and plasma stability of GH release in vivo in a single injection (day)
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Note that: CFA (carbon fatty acid) is a carbon fatty acid, CFDA (carbon fatty diacid) is a carbon fatty diacid; the K [ N-epsilon- (gamma-Glu-N-alpha-CFA or CFDA) ], K [ N-epsilon- (AEEA) 2-gamma-Glu-N-alpha-CFDA) ] represents fatty acyl or fatty diacid glutamyl modification of lysine K side chain epsilon-amino, and the specific structure is shown in formula 1 or 2. "-S-S-" means disulfide bonds. Ornithine ORN and diaminopropionic acid DAP are diamino amino acids and the other single letters are amino acid abbreviations. AIB is alpha or beta aminoisobutyric acid.
Example 2 stability of somatostatin analogue peptide monomer, dimer or tetramer in human plasma:
hGHRH ELISA kit (CEA 438Hu, wuhan cloud cloning technologies Co., ltd., china) was designed to detect the N-terminal amino acid of hGHRH. N-terminal dipeptide as hGHRH molecule 1 Tyr- 2 When Ala is degraded by dipeptidyl aminopeptidase 4 (DPP-4), the remaining part loses the binding activity to the GHRH antibody. The hGHRH kit is more sensitive to detection of the entire hGHRH polymer. Mu.g of mono-or di-tetra-chain peptide was incubated in 1.7ml of normal human fresh plasma (purchased from the blood department of the army hospital, guangdong province) at 37.+ -. 0.5 ℃ for 0, 5, 10, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 45 days. At each incubation time point, 100 μl of each peptide-plasma mixed solution was sampled and stored at-80 ℃. After incubation, all samples were measured together by hGHRH ELISA. The experiments were designed for blank plasma control and multiplex experiments at each incubation point. The standard concentration of hGHRH is on the y-axis with the reading data at 450nm wavelength on the x-axis, according to the equation y=ax 2 +bx+c the final peptide amount was calculated. The duration of the polymer activity was obtained by comparison with the 0-day data of the mixture and blank plasma. The results showed that the U-type peptide single/double/quadruplex maintained 9/30/45 day plasma stability and the H-type peptide single/double/quadruplex maintained 7/21/35 day plasma stability (see Table 1).
Example 3 GHRH-like peptide GH release Activity assay in vivo growth hormone release: male Kunming mice were 4 weeks old (n=6) [ SCXK (Guangdong) 2016-0041, purchased from southern university of medical science ], and each mouse was subcutaneously injected with a single 9 μg dose of peptide, and 25 microliters of inner canthus blood was taken every two days for measurement of blood GH values at 0 time before and 1-21 days after the drug. Because GH in animals fluctuates obviously at different times every day, excessive blood taking points can cause blood loss stress, the maximum blood taking time is set to 21 days, and the absorption time of polymer peptide is also obviously different, the secretion kinetics of modified peptide GH is greatly different from that of natural peptide, so that a NaCl-PB control group is set up at each blood taking point, and the peak effect time of GH release is observed. The results showed that after a single subcutaneous injection of 9 μg peptide, the H-type peptide group reached a peak effect at 15 days, after which GH release gradually decreased, while the U-type peptide group continued to increase from 13 days, reaching a peak at 21 days, indicating that the U-type polymer of the application had greater stability than the H-type polymer (see Table 1).
Example 4 GHRH-like peptide di/tetramers have efficacy in female infertility, male sterility, alzheimer's disease and anti-cancer treatments.
1. Raising and breeding experimental animals: golden hamsters were purchased from experimental animal technologies limited [ SCXK (jing) 2016-0011 ] of beijing velutinin, china, and reproductive capacity measurements were performed; APP/PS1 transgenic mice and gene negative C57BL/6 mice were supplied by Sedum laboratory animal Co., ltd. [ SCXK (Su) 2016-0010 ], and senile dementia experiments were performed. Hamsters or transgenic mice were bred and bred in the Guangdong police hospital animal house according to the corporate guidelines. The propagation process comprises the following steps: puberty 8-week-old female and male golden mice, or APP/PS1 transgenic male mice and C57 mice, 1:1 mating with the cage for 2 weeks, and feeding female mice for 2 weeks respectively, and waiting for the baby of the mice to be born. The mice and the mother mice are kept in the same cage for two weeks and are kept in separate cages. All mice in the experiment were bred and propagated in the animal house, and the transgenic mice were subjected to PCR technique in the manner provided by the manufacturer to identify the gene mice. Golden hamsters or transgenic mice were subjected to modeling and treatment according to the guidelines for laboratory animal care and use. The experimental animals were kept under constant temperature (25 ℃) 14 hours light/10 hours darkness, and feed was freely provided. The study has been approved by the university of guangdong medicine and the cantonese army hospital for animal ethics.
2. Dose design: the clinical dose conversion rate of 80g of golden hamster to 60kg of adult human was 7.4:1.hMG dose: 1mg hMG contains 400U (molecular weight 22672.9D, FSH: LH=1:1, no.200902, lizhu group Lizhu pharmaceutical Co., china). The clinical dose was 75U per day of intramuscular injection for one course of treatment, and hamster dose was converted to 0.74U per hamster daily of leg muscle (0.082 nmol). hGH dose: 1mg hGH contains 3 units (molecular weight 22125D, injected with recombinant human growth hormone, no.202101009, unknown sea-Ji biomedical Co., ltd. Of Zhongshan, china) and has a clinical dose of 0.1U/kg for subcutaneous injection (sc), and hamster dose was converted into 0.0179U (0.27 nmol) for back subcutaneous injection per hamster per day. Dimer and tetramer peptides: this experiment was performed with reference to the simiglutide dose (semaglutinide, a fatty acid modified similar diabetes drug, which was injected once every 7 days of clinical treatment), and each hamster was injected subcutaneously with the dimer or tetramer peptide once every 1 week. The low, medium and high dose groups 0.09, 0.27, 0.81nmol,2P1-M (molecular weight 11220.5D), 4P-M (molecular weight 22838.4D), 4P1-M (molecular weight 22438.9D) and 4Y-M (molecular weight 22200.6D) were each set to 0.27nmol for 2Y (molecular weight 11101.3D) or 2P (molecular weight 11420.2D), respectively.
3. Modeling and experiments are performed from four aspects of female infertility, male sterility, senile dementia model and anticancer effect.
1. Female infertility: female infertility models and mating tests were designed based on the normal golden hamster breeding process and our previously published article [ Zhang JH et al 2018 ]. 156 female hamsters (4 weeks old) were randomized into 12 groups (NaCl-PB group and 11 model groups) with no significant difference in body weight (n=13). NaCl-PB was subcutaneously injected with NaCl-PB solution (disodium hydrogen phosphate adjusted to pH 8.0) and 11 animals from the model group were respectively intraperitoneally injected (ip) with 200mg/kg Cyclophosphamide (CPA) (batch: 0G390A,Baxter Oncology GmbH, germany) 1 time a week for 4 weeks. After the third CPA injection, hamster models were randomly divided into 9 groups (Model Control, hGH, hMG, 2P-L/M/H, 2Y-L/M/H, 4P-M, 4Y-M) with no statistical difference in body weight (n=13), and changes in body weight during treatment were observed. NaCl-PB group was used as a normal control, and NaCl-PB solution was injected. The next day after regrouping, each female model mice was subcutaneously injected with 2P-L/M/H or 2Y-L/M/H0.09/0.27/0.81 nmol doses, and groups 4P-M and 4Y-M were subcutaneously injected with 0.27nmol of 4P and 4Y on the back, once a week. hMG group served as positive control, and each animal was injected with 0.74U (0.082 nmol) per day in the hind leg muscle. hGH group was a positive control and each animal injected subcutaneously back daily with 0.0179U (0.27 nmol). hMG group on day 8 after the 4 th CPA injection, animals of all groups were each assigned a 1:1 were randomly mated for 2 weeks. Subsequently, each model hamster was kept separately for two weeks, waiting for the baby hamster to born. During the experiment, the hair color, activity and feeding condition of the model group were observed, and the body weight was measured once a week. During birth of the hamsters, the date and number of birth of the model mice were recorded and the birth rate was calculated. After 8 weeks, model mice were sacrificed by cervical dislocation, blood with or without anticoagulant was collected from the eyes, serum or plasma was obtained by 6000 rpm/30 min separation, and stored at-70 ℃. Animals liver, ovary, spleen, heart and lung were taken and weighed. Ovaries were rapidly excised and stored at-70 ℃ for immunoblotting (n=5-10) or Immunohistochemical (IHC) evaluation (n=3) in PBS solution fixed in 10% formaldehyde. Pathological changes in ovary and blood were evaluated by staining with H-E, TUNEL, KI67, GHRH receptor and GH. Three animal tissues were stained for each group, 5-18 per section were fluorescent photographed, and the fluorescence intensities were counted using IPP software.
The results were as follows: fur color and activity: normal golden hamsters have bright golden fur. As the CPA injection amount increased, mao Pise of the golden hamster model became grey, and its activity was weakened. After CPA is deactivated, coat color and activity are progressively improved. With increasing number of peptide injections, the activity of the hamster in the 2Y model was stronger than that of the NaCl-PB and 2P and 4P-M groups, since 2Y had more GH released.
Female model body weight change: the differences in body weight between model groups prior to CPA injection were not statistically significant. Four weeks after CPA injection, hamster model body weight loss was a maximum of 21.4%. In peptide injection 1-3 th The weight of each model group was significantly reduced compared to the NaCl-PB group (P<0.01 or 0.001). After the third drug injection, the body weight of group 2Y gradually increased, while the body weights of groups 2P and 4P showed a dose-dependent decrease (see table 2). The results show that CPA weight loss is due to CPA toxicity, leading to systemic stem cell depletion. The greater increase in body weight of the 2Y group was due to the release of more GH.
Table 2 change in body weight of golden mice model (x±sd, n=13)
P <0.05, or 0.01, 0.001 vs a, b, c, d, e, f, g, H, i, j, k, L represent NaCl-PB, model Control, hGH, hMG,2Y-L/M/H,2P-L/M/H,4P-M,4Y-M groups, respectively
Female model organ weight change: the heart weights were significantly reduced (P <0.01 or 0.001) in the other model groups except for the 2Y-H group compared to the corresponding NaCl-PB group (see Table 3). 2P induces a dose-dependent increase in model liver, lung, left or right kidney weight, or 2Y induces a dose-dependent increase in model heart, liver, lung weight. The 2P-L/H, 4P-M and 4Y-M groups were lighter (P <0.05 or 0.01) than the model control group or hGH, hMG group. The 2P-L/M and 4P groups induced livers lighter than the model control group, or lungs lighter than the hGH or hMG groups (P <0.05 or 0.01). The 2P-H group had a heavier left kidney (P < 0.05) than the 2P-L or 4P group. The results show that 2P induces an increase in organ weight dependence because it increases proliferation and activation of stem cells of the organ, and has a good protective effect on the corresponding organ. 2Y induces an organ weight-dependent increase because it promotes GH secretion.
Table 3 change in organ weight of golden mice model (x±sd, n=13)
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P <0.05, or 0.01, 0.001 vs a, b, c, d, e, f, g, H, i, j, k, L represent NaCl-PB, model Control group Model Control, growth hormone hGH, urotropin hMG,2Y-L/M/H,2P-L/M/H,4P-M,4Y-M groups, respectively.
Female model birth rate: the group 2P-L/M/H or 2Y-L/M/H, 4P-M,4Y-M has 75/69/46% or 45/13/50%, 61%, 39% birth rate respectively. NaCl-PB, model control The birth rate of hGH or hMG groups was 100%, 20% or 25% (FIG. 1). Compared with NaCl-PB group, the birth rate of each model group has significant difference (P<0.05, 0.01 or 0.001). The 4P, 4Y and 2P-L/M groups showed higher yields than the model control, hGH or hMG, 2Y-L/M/H groups (P<0.05 or 0.01). Birth rates of the 2Y-M group or the 2Y-H group were lower or higher than those of the model control group (P<0.05 or 0.001). And (3) with 1 The comparison of the P-GHRH, 1 Y-GHRH has stronger animal specificity.
Female model ovarian H-E staining: morphological changes in female hamster ovary were observed and follicle count was performed. The ovarian cortex of the NaCl-PB group is very large and even the whole ovary consists of cortex. The follicles and the larger corpus luteum of each stage are orderly distributed in the interstitial cell mass, and the dense granular cells are obviously layered. The model control group had sparse early follicles and more closed follicles, and no corpus luteum was found. The medullary area has messy cells, broken small vessel walls, increased cell gaps and increased follicles at all levels. The 2P and 4P groups had decreased cortical mesenchymal cells, increased medullary cells, no connective tissue found in the whole ovarian tissue, disordered medullary area cells and rare mesenchymal cells, and visible small vessel wall rupture. The hMG group had follicles and corpus luteum at each level. The hGH group had little connective tissue in the medulla and less cortical follicular distribution. Classification counting and statistics of ovarian tissue follicles (fig. 2) found that 2Y induced either the corpus luteum or primary follicles to decrease in dose-dependent manner, or the occlusion follicles to increase in dose-dependent manner. 2P and 4P cause dose-dependent increases in mature follicles, corpus luteum, atresia follicles, or dose-dependent decreases in secondary follicles, but there is no significant meaning between the two. The secondary follicles in group 2Y-L were significantly more than in the model control group, the mature follicles in group 2P-L were significantly less than in group 2Y-H, or the mature follicles in group 2P-H were more than in NaCl-PB or group 2Y-L (P < 0.05).
Female model ovarian GHRH receptor: fluorescent staining showed that GHRH receptors are widely distributed in mesenchymal cells, luteal and basement membrane cells exhibit stronger GHRH receptor staining, and granular cells in follicles are less expressed. Group 2P showed an increase in dose-dependent GHRH receptor expression, higher expression of GHRH receptor in group 2P-M than in the model control group or group 2Y-L/M (P <0.05 or 0.01) and a significant increase in GHRH receptor in group 2P-H than in the model control group or group 2Y-LM/H (P <0.05 or 0.01, 0.001) (fig. 3). The results show that the increase in GHRH receptor promotes birth rate.
Female model ovarian growth hormone expression: GH is widely distributed in mesenchymal cells, and luteal and basement membrane cells are strongly stained. In the granulosa cells within the follicle, GH expression is significantly reduced. Group 2Y-M ovarian GH expression was higher than that of NaCl-PB or hGH, 2Y-L/H, 2P-M/H, 4P-M, 4Y-M (P <0.05 or 0.01), and group 2Y-H ovarian GH expression was lower than that of the model control group (P < 0.05) (FIG. 4). The results show that GH expression is increased, inhibiting birth rate.
Female model blood growth hormone analysis: the blood GH level of group 2P was increased in a dose-dependent manner, and the blood growth hormone level of group 2P-H was significantly higher than that of NaCl-PB, model control, hGH, 2Y-M/H or 2P-L/M groups (P <0.05, 0.01 or 0.001). The hGH group had blood GH levels lower than those of NaCl-PB, model control, hMG, 2Y-M/H or 2P-L/M/H, 4P-M, 4Y-M groups (P < 0.01 or 0.001) (FIG. 5). The results show that GH expression is increased, inhibiting birth rate.
Female model ovarian KI67 protein expression: the outer cortical mesenchymal cells of the NaCl-PB group were seen as dense KI67 positive cells, while the inner cortex and medullary areas were seen as sparse KI67 positive cells. Intrafollicular granulosa cells are rarely KI67 positive cells. hGH groups showed scattered KI67 positive stromal cells in basement membrane, cortical and medullary areas. In hMG or 2Y group, KI67 protein is mainly distributed in follicular stromal cells and basement membrane cells, which have a higher staining capacity than stromal cells. KI67 protein expression was significantly reduced in the model control, hGH, hMG and 2P-L groups compared to the NaCl-PB group (P <0.05,0.01 or 0.001). The KI67 proteins of groups 2Y and 2P showed a dose-dependent increase (P <0.01 or 0.001) and the expression levels were higher than those of group 2P-L (P <0.05,0.01 or 0.001). There were significant differences (P <0.01 or 0.001) between the 2Y-M/H group and the model control group or hGH, hMG, 2Y-L group. The expression level of KI67 was higher in the 2P group than in the model control or hGH, hMG group (P < 0.001). The 4P and 4Y groups were significantly higher than the model control group or hGH, hMG, 2P-L groups, or lower than 2Y-M or 2P-M/H (P <0.05,0.01 or 0.001) (FIG. 6). The results showed that KI67 reflects the proliferation capacity of the mesenchymal stem cells of the organs, all GHRH peptides were shown to promote KI67 protein expression, and 2Y and 2P showed better proliferation capacity. 4P and 4Y take longer to take blood due to peptide absorption, slightly less effective than dimer. hGH has no proliferative effect.
Female model ovary TUNEL staining: in the NaCl-PB group, TUNEL positive stromal cells were found mainly in the cortical areas, and granulosa cells and medullary stromal cells were also stained positively. Model control and hMG groups of follicular stromal cells and basement membrane cells had stronger TUNEL staining. The model control group had more staining of the medullary cells TUNEL than the cortical mesenchymal cells, while the hMG group had more staining of the cortical cells TUNEL than the cortical mesenchymal cells. TUNEL staining was significantly increased (P < 0.001) in model control, hGH, hMG groups compared to NaCl-PB groups, or hGH was significantly higher than in model control groups (P < 0.01). Dose-dependent reduction occurred in group 2Y compared to model control, hGH or hMG group (P <0.05, 0.01 or 0.001). The TUNEL staining intensity was significantly less for the 2P or 4P, 4Y group than for the model control, hGH, hMG, Y-L/M/H group (P <0.05, 0.01 or 0.001) (fig. 7).
Female model blood hormone: estrogen (fig. 8) or testosterone levels (fig. 9) in hamster blood were determined using E2 or T ELISA kits. 2P-induced estrogen levels increased in a dose-dependent manner (P < 0.01), with groups 2P-H significantly higher than NaCl-PB,2P-L or 2Y-H (P <0.05 or 0.01). The estrogen content of group 2Y-H or group 2P-L is lower than that of hGH and group 2Y-L/M (P <0.05 or 0.01, 0.001). hGH induced estrogen levels higher than those of NaCl-PB (P < 0.05). The estrogen levels were lower in the 4P-M group than in the hGH group and higher in the 4Y-M group than in the 2Y-H group (FIG. 8). The blood testosterone levels were lower in all hamster models than in the NaCl-PB group, the 2P-induced testosterone was increased in a dose-dependent manner, whereas the blood testosterone levels were significantly lower in the 2P-L group than in the NaCl-PB, model control, hGH, hMG or 2Y-H group (P <0.05 or 0.01), and the blood testosterone levels were significantly lower in the 4P-M group than in the NaCl-PB group (P < 0.05).
2. Male sterility model and experiment: there were no statistical differences in body weight for 112 male hamsters (4 weeks old) and were randomly assigned to 7 groups (NaCl-PB group and 6 model groups) (n=16). NaCl-PB group was intraperitoneally injected with NaCl-PB solution, and model animals were intraperitoneally injected with 200mg/kg CPA 1 time per week for 4 weeks. After the third CPA injection, hamster models were re-randomized into 6 groups (Model Control, hMG, 2P-L/M/H, 4P-M group) with no statistical differences in body weight (n=16). The NaCl-PB group is a normal control group, and NaCl-PB solution is injected. After model regrouping, each model mouse was injected subcutaneously at the back with 0.09, 0.27, 0.81nmol dose of 2P (L/M/H), and the 4P-M group with 0.27nmol of 4P once a week. hMG group was positive control, and each animal was injected with 0.74U (0.082 nmol) per day in the hind leg muscle. On the following day after CPA injection, male models were each assigned a 1:1 were randomly mated for 2 weeks. Subsequently, pairs of hamsters were kept separately for two weeks, waiting for the baby hamsters to born. Model coat color, activity, feeding were observed and body weight was measured once a week. During birth of the hamsters, the date and number of birth of the model mice were recorded and the birth rate was calculated. After the whole experiment is finished for 8 weeks, a male model mouse is killed by cervical dislocation, blood with anticoagulant or not is collected by picking an eyeball, serum or plasma is obtained after 6000 revolutions/30 min of heart separation, and the blood is preserved at the temperature of minus 70 ℃. Testes were excised rapidly, stored at-70 ℃ for immunoblotting detection (n=7-13) or fixed in 10% formaldehyde in PBS for Immunohistochemical (IHC) evaluation (n=3). The liver, testis, spleen, heart and lung of the male model were removed and weighed. Staining with H-E, TUNEL, KI, GHRH receptor, GH. Epididymal sperm count, morphological analysis, and the like. Three animal tissue sections per group, 5-20 per section were fluorescent photographed and counted by IPP software.
Model observations and weight and organ weight statistics: the changes in coat color and activity, body weight and organ weight of the male model are similar to those of the female model, as described in the female model.
Male model reproduction rate: the birth rates of the 2P-M and 4P-M, model control or hMG groups were significantly reduced (P <0.05, 0.01 or 0.001) compared to the NaCl-PB group. The 4P-M and 2P-M groups were more productive than the model control or hMG groups (P <0.01 or 0.001). The 2P-L/H group was significantly higher than the model control group or hMG group (P < 0.05) (fig. 10).
Male model testicle GHRH receptor assay: fluorescent staining showed that GHRH receptors are distributed in the seminiferous, seminal vesicles, sperm cells and mature sperm heads. Group 2P showed an increase in dose-dependent GHRH receptor expression (P <0.05 or 0.01), while an increase in GHRH receptor expression was observed with cell maturation. The model control group showed a significant decrease (P < 0.01) compared to the NaCl-PB group. Compared to the model control groups, significant increases occurred in the 2P-M/H and 4P-M groups (P <0.05 or 0.01) (FIG. 11). It was shown that 2P induces GHRH receptor expression, activating mesenchymal stem cells.
Male model blood GH analysis: the model control, 2P-M/H and 4P-M groups showed significant increases (P <0.05 or 0.01) compared to the NaCl-PB group. Compared to the model control group, hMG showed a significant decrease (P < 0.05). Compared to hMG, significant increases occurred in the 2P-M/H and 4P-M groups (P < 0.05) (fig. 12).
Male model testis KI67 protein expression: the KI67 protein is mainly distributed in seminoma, spermatids and spermatids of seminiferous tubules of testis. The expression of KI67 protein in group 2P was dose-dependent increased. . The model control group showed significantly reduced KI67 protein expression (P < 0.05) compared to the NaCl-PB group. KI67 protein expression was significantly increased in the 2P and 4P-M groups compared to the model control group (P <0.05 or 0.01). It was shown that 2P induced KI67 expression, promoting mesenchymal stem cell proliferation (fig. 13).
Model testis TUNEL staining: TUNEL staining is mainly distributed in seminoma cells of seminiferous tubules of testes, with weak staining of seminoma and sperm cells, and no staining of sperm. TUNEL was significantly increased (P < 0.05) in the model control group compared to the NaCl-PB group. TUNEL staining was significantly reduced in the 2P and 4P groups compared to the model control group (P <0.05 or 0.01). TUNEL was significantly increased in the 4P-M group compared to the 2P-H group (P <0.05 (fig. 14).
Epididymal sperm count and sperm morphology analysis: compared to the NaCl-PB group, 200mg/kg CPA intraperitoneal injection of model animals significantly resulted in reduced sperm count (fig. 15A) and motility (fig. 15B) and increased sperm malformation rate (fig. 15C) (P <0.05, 0.01, or 0.001). Compared with the model control group, the 2P-M group significantly increases the number of sperms (P < 0.05), and the hMG, 2P and 4P groups significantly reduce the sperm malformation (P <0.05, 0.01 or 0.001). The hMG, 2P and 4P groups showed significantly increased sperm malformation (P <0.01 or 0.001) compared to the NaCl-PB group or model control group.
Model blood testosterone: the 2P group increased with dose-dependent testosterone. The 2P-L group showed significant testosterone reduction (P < 0.05) compared to the NaCl-PB group. The 2P-H group significantly increased testosterone concentration (P < 0.05) compared to the 2P-L group (fig. 16).
And (3) result evaluation: 1 the P-GHRH peptide proliferates and activates mesenchymal stem cells by increasing the autocrine KI67 and GHRH receptor signals, and repairs cell damage.
3. APP/PS1 transgenic model and experiment: 80 transgenic mice (4 weeks old) were randomly divided into 8 groups (NaCl-PB group and 7 model groups) according to no statistical difference in body weight (n=10). NaCl-PB group was used as a normal control, and NaCl-PB solution was injected. Each model mouse was subcutaneously injected back with 0.09, 0.27, 0.81nmol dose of 2Y (L/M/H) groups, and 0.27nmol doses of 4P1-M and 4Y-M groups, once every 9 days. The galanthamine (Galanthaline) group was injected with a dose of 0.177mg/kg (0.112. Mu. Mol/20 g) as a positive control, once daily into the hind leg muscle of each animal, and all animals were injected in a volume of 350. Mu.l. After 12 weeks, the experiment was ended, and the animal cerebral cortex and hippocampus were removed and stained with H-E, TUNEL, KI, GHSR, GFAP, A. Beta., TAU and SYN fluorescence, respectively. Liver, ovary, spleen, heart and lung were weighed. Blood GH and biochemical analysis, etc. to evaluate brain function change. Three animal tissues were stained for each group, 5-40 per section were fluorescent photographed, and the fluorescence intensities were counted using IPP software.
Mouse Morris water maze test: the APP/PS1 transgenic mice are subjected to Morris water maze experimental training after 12 weeks of administration, the mice are trained for 7 days, water maze starts from different directions of a circular water maze to avoid swimming on the water surface and reach an end stair, the water maze automatically records the error times of the mice entering each blind end and the arrival end time (latency), average error times and average latency values are taken, experimental data are average +/-standard deviation (Mean +/-SD), and t-test statistical analysis is carried out. The results showed that escape latency decreased to varying degrees with the number of training days. Starting on day 3, the average incubation period of transgenic mice in NaCl-PB and 2Y groups was progressively shorter. On days 4 and 5, the average incubation period of transgenic mice in groups 2Y-L or 2Y-H was significantly shorter (P < 0.05) compared to the model control group. On day 5, the average incubation period of transgenic mice in group 2Y-H was significantly shorter (P < 0.05) compared to galantamine or 2Y-L. On day 6, the average incubation period of transgenic mice was significantly shorter in the 2Y-L group (P < 0.05) compared to NaCl-PB, model control and Galanthine groups (FIG. 17).
Hippocampus KI67 and TUNEL were used to detect the proliferation and apoptosis of mesenchymal stem cells of hippocampus. The results showed a significant decrease in model control KI67 (P < 0.05) compared to NaCl-PB group. Compared to the model control group, KI76 in groups of galantamine, 2Y-L/M/H, 4P1-M and 4Y-M showed a significant increase (P <0.05 or 0.01) (FIG. 18A).
The model control, galantamine, 2Y-L/H, 4P1-M and 4Y-M groups showed a significant increase in TUNEL index (P <0.01 or 0.001) compared to the NaCl-PB group. The 2Y-M group showed a decrease in significance (P <0.01 or 0.001) compared to the model control, galantamine, 2Y-L group, 4P1-M and 4Y-M groups showed a decrease in the significance of TUNEL index (P <0.05 or 0.01 or 0.001) compared to the model control and 2Y-H group, and 2Y-M showed the greatest decrease (FIG. 18B).
Cortex KI67 and TUNEL: the galanthamine, 2Y-H, 4P1-M and 4Y-M groups showed a significant increase in KI67 index (P <0.05 or 0.01) compared to the NaCl-PB group. The 2Y-L group showed a significant decrease in KI67 index (P < 0.05) compared to the model control group. The 4P1-M and 4Y-M groups showed a significant increase in KI67 index (P < 0.05) compared to the 2Y-L group (FIG. 19A).
Model control, galantamine, 2Y-L group TUNEL index showed increased significance (P < 0.001), or 2Y-M and 4P1-M groups showed decreased significance (P <0.01 or 0.001) compared to NaCl-PB group. Compared with the model control group, the TUNEL indexes of the groups 2Y-L/M/H, 4P1-M and 4Y-M are remarkably reduced (P <0.05,0.01 or 0.001). Compared with galantamine and Y-L, the TUNEL index of groups 2Y-M/H, 4P1-M and 4Y-M was significantly reduced (P <0.05,0.01 or 0.001). The TUNEL index significance was increased in the 2Y-H and 4Y-M groups compared to the 2Y-M groups (P <0.05 or 0.01), the TUNEL index significance was decreased in the 4P1-M groups compared to the 2Y-H groups (P < 0.01), and the TUNEL index significance was increased in the 4Y-M groups compared to the 4P1-M groups (P < 0.05) (fig. 19B).
Hippocampus and cortex TAU, TAU protein is microtubule-associated protein, and the TAU protein molecule in normal mature brain contains 2-3 phosphate groups, whereas brain TAU protein in Alzheimer's disease patients is abnormally hyperphosphorylated, and per molecule TAU protein can contain 5-9 phosphate groups and lose normal biological functions. Abnormal accumulation of Tau protein is a real source of cognitive decline and memory loss in patients with Alzheimer's Disease (AD). The results showed that the model control group showed an increase in hippocampal Tau significance or a decrease in hippocampal Tau index significance in the groups of galanthamine, 2Y-M/H, 4P1-M, 4P-M (P <0.05 or 0.01, 0.001) compared to the NaCl-PB group. The hippocampal Tau significance of galantamine or groups 2P-M/H, 4P1-M, 4Y-M was reduced by P <0.05 or 0.01,0.001 compared to model control or 2P-L/H. The hippocampal Tau index was significantly increased in group 2Y-L compared to the galanthamine group (P < 0.05) (fig. 20A).
The model controls showed an increase in cortical Tau significance (P <0, 01 or 0.001) in the galantamine and 2P-L groups compared to the NaCl-PB group. The cortical Tau significance of the 2P-M/H, 4P1-M, 4Y-M groups was reduced by P <0.05 or 0.001 compared to NaCl-PB, model control, galanthamine and 2P-L groups (FIG. 20B).
Hippocampus and cortex GFAP, glial Fibrillary Acidic Protein (GFAP), is a biomarker of reactive astrocyte proliferation. GFAP is a sensitive biomarker for detection and tracking of reactive astrocytopathy and aβ pathology. The results showed a significant decrease in hippocampal GFAP index in the 2Y-M/H, 4P1-M, 4Y-M groups (P <0.05 or 0.01, 0.001) compared to NaCl-PB, model control or galanthamine group (fig. 21A).
The model control or galanthamine group had increased GFAP significance (P < 0.001) compared to NaCl-PB. The 2Y-M/H or 4P1-M, 4Y-M groups showed significantly reduced cortical GFAP index (P <0.05 or 0.01, 0.001) compared to the NaCl-PB or model control, galantamine, 2Y-L/M/H, 4P1-M groups (FIG. 21B).
Hippocampus and cortex aβ amyloid precursor protein-APP is concentrated in neuronal synapses. APP can be decomposed by alpha-, beta-, gamma-protease, and continuous action of beta-protease and gamma-protease can cause APP to be decomposed to produce Abeta. Aβ1-42 is more toxic and readily aggregates to form the core of Aβ precipitation. The results showed a significant increase in hippocampal aβ (P < 0.05) compared to the NaCl-PB group. Compared to the model control group or the galanthamine group, the galanthamine or the 2Y-L/M/H, 4P1-M, 4Y-M groups had a reduced hippocampal Abeta significance (P <0.05 or 0.01, 0.001) (FIG. 22A).
Cortical aβ was significantly increased in the model control group compared to the NaCl-PB group (P < 0.01). Compared with the model control group, the cortical Abeta of the galanthamine or the 2Y-L/M/H, 4P1-M and 4Y-M groups is obviously reduced (P <0.05 or 0.01 and 0.001). Cortical aβ was significantly less pronounced in the P1-M, 4Y-M groups than in the galantamine and 2Y-L groups (P <0.05 or 0.01, 0.001) (fig. 22B).
Hippocampal and cortical SYN synaptosin SYN is a presynaptic marker commonly used to detect the density and distribution of synapses. Synaptic loss is an early event in AD and is considered an important marker of synaptic changes. Synaptic plasticity is considered to be the basis of brain learning and memory capacity. The results showed a reduction in hippocampal SYN significance (P < 0.001) in the model control and 2P-L/M groups compared to the NaCl-PB group. The galanthamine group significantly increased hippocampal SYN (P <0.05 or 0.01, 0.001) over NaCl-PB, model control group or 2P-L/M group. Group 2Y showed a dose-dependent significant increase in hippocampal SYN (P <0.01 or 0.001). The hippocampal SYN of groups 4P1-M and 4Y-M were significantly increased (P <0.01 or 0.001) (fig. 23A).
Cortical SYN was significantly reduced (P <0.01 or 0.001) in the model control and 2P-L groups compared to NaCl-PB groups. The significance of the galanthamine group is increased by SYN (P < 0.05) compared with the model control group or the 2P-M group, and the significance of the 2P-L group is reduced by SYN (P < 0.01) compared with the galanthamine group. 2P-L/M/H was dose-dependent (P <0.05 or 0.001). The cortical SYN of the 2Y-H, 4P1-M or 4Y-M groups showed a significant increase (P < 0.05) over the model control or galantamine, group 2P-L/M (fig. 23B).
Hippocampus and cortex GHSR: GHSR is a receptor of brain acetylated GHRH, and exogenous GHRH can activate GHSR and has a protective effect on brain nerve cells. The results showed a decrease in hippocampal GHRH significance (P < 0.001) in all model groups compared to the NaCl-PB group. Compared to model control groups, the hippocampal GHRH of groups galanthamine, 2Y-H and 4Y-M were significantly increased (P <0.05 or 0.001). Compared to the galanthamine group, the 2Y-M and 4P1-M groups showed a significant decrease in hippocampal GHRH (P <0.05 or 0.01). The 4Y-M group had a significant increase in hippocampal GHSR over the 4P1-M group (fig. 24A).
The 2P-M/H or 4P1-M, 4Y-M groups showed a significant increase in cortical GHRH (P <0.05 or 0.01, 0.001) compared to the NaCl-PB group, model control group, galantamine and 2P-L group. The 4P1-M group had a significant GHSR reduction (P < 0.05) compared to 2P-H (fig. 24B).
4. Evaluation of anticancer Effect:
culturing human liver cancer cell HepPG2, or human lung cancer cell A549, human breast cancer cell MDA-MB231 with DMEM fetal bovine serum culture medium to 80% abundance, adding 2P or 2Y 0, 2.4, 4.7, 9.4, 18.9, 37.8 μg/ml into the compound wells, continuously culturing for 24 hours, staining with CCK8, and measuring absorbance value with 415nm enzyme marker instrument. The inhibition rate calculation formula: 100 (measurement tube A) 415 Blank tube A 415 ) Blank tube A 415 . Concentration-inhibition was plotted as shown in fig. 25.
For 2P, a concentration of 9.4 μg/ml plays a significant inhibitory role on cancer cell lines HePG2 or A549 (FIG. 25A). For 2Y, a concentration of 2.4 μg/ml plays a significant role in inhibiting cancer cell lines HePG2 or A549. Both have weak inhibition on B123 (fig. 25B).

Claims (6)

1. A somatostatin analogue peptide monomer characterized in that the amino acid sequence of said monomer is one of the following:
(N-terminal) X 1 -X 2 -DAIFTNSYR-X 12 -VLGQLSAR-X 21 -LLQDIMSRQQGESNQERGARARL-C 45 -NH 2 (C-terminal);
(N-terminal) X 1 -X 2 -DAIFTNSYRRVLGQLSAR-X 21 -LLQDIMSRQQGESNQERGARARL 44 -X 45 -NH 2 (C-terminal);
(N-terminal) X 1 -X 2 -DAIFTNSYRRVLG-X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -LLQDIMSRQQGESNQERGARARL 44 -NH 2 (C-terminal);
the capital letter in the sequence is abbreviation or amino acid substitution symbol of L-alpha-amino acid, arabic numerals are amino acid residue arrangement sequence, NH 2 Represents the N-terminal or C-terminal amide structure, cysteine residue C 45 C-terminal of (C) is CONH 2 The method comprises the steps of carrying out a first treatment on the surface of the Wherein X is 1 Is Y or P, X 12 Is K, X 45 Is a diamino-amino acid; x is X 16 -X 20 Only one of the amino acids is a diamino-amino acid and is boundIs similar to the corresponding sequence Q 16 Or L 17 、S 18 、A 19 、R 20 The remaining sequence is still corresponding to Q 16 Or L 17 、S 18 、A 19 、R 20 The amino acid sequence is unchanged; when X is 21 Is modified by epsilon amino [ gamma-Glu (N-alpha-alkanoic acid) with K side chain, and has a structural formula shown as formula 1, wherein n=16-18; when X is 21 For side-chain epsilon-amino [2 XAEEAC-gamma-Glu (N-alpha-fatty diacid)]When modified, the structure of the modified lysine is shown in formula 2:
2. a growth hormone releasing hormone analog peptide dimer, wherein the amino acid sequence of said dimer is one of the following:
(N-terminal) X 1 -X 2 -DAIFTNSYR-X 12 -VLGQLSAR-X 21 -LLQDIMSRQQGESNQERGARARL-C 45 (C-terminal) -C 45 (C-terminal) -LRARAGREQNSEGQQRSMIDQLL-X 21 -RASLQGLV-X 12 -RYSNTFIAD-X 2 -X 1 (N-terminal);
wherein X is 46 Is an amino acid containing sulfhydryl group.
3. A ghrelin-like dimer as claimed in claim 2, wherein X is 46 Is cysteine C, threonine T or homocysteine HCY.
4. A GHRH-like peptide tetramer, characterized in that said tetramer is formed by disulfide bonds formed by cysteines between identical dimers as defined in claim 2, constituting a U or H-type homotetramer; the amino acid sequence is one of the following structures:
5. Use of a GHRH-like peptide monomer of claim 1, a dimer of claim 2 or a tetramer of claim 4 in the manufacture of a medicament for the treatment of infertility, senile dementia or anticancer.
6. A medicament for treating infertility, senile dementia or cancer, which comprises the somatostatin-like peptide monomer as claimed in claim 1, the dimer as claimed in claim 2 or the tetramer as claimed in claim 4 as an active ingredient in pharmaceutically acceptable salt thereof.
CN202310853772.9A 2023-07-12 2023-07-12 Novel somatostatin analogue peptide monomer, dimer or tetramer and preparation and application thereof Pending CN117229384A (en)

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CN109180800A (en) * 2018-08-01 2019-01-11 广东药科大学 Novel growth hormone releasing hormone is similar to peptide dimer and its application
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