CN117209587A - Application of growth hormone release related polypeptide in preventing and treating immune checkpoint inhibitor related myocarditis - Google Patents
Application of growth hormone release related polypeptide in preventing and treating immune checkpoint inhibitor related myocarditis Download PDFInfo
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- CN117209587A CN117209587A CN202311305815.6A CN202311305815A CN117209587A CN 117209587 A CN117209587 A CN 117209587A CN 202311305815 A CN202311305815 A CN 202311305815A CN 117209587 A CN117209587 A CN 117209587A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses application of a growth hormone release related polypeptide in preventing and treating myocarditis related to immune checkpoint inhibitor. The growth hormone release related peptide is derived from an endogenous ligand of a human growth hormone secretion receptor, and the amino acid sequence of the endogenous ligand is shown as SEQ ID NO: 1. The growth hormone release related peptide can reduce myocardial injury markers such as CK, CKBB, LDH-1, cTnI and cTnT release in serum of mice with myocarditis related to immune checkpoint inhibitor, reduce inflammatory cell infiltration and fibrosis degree of myocardial tissue, improve cardiac function of the mice with myocarditis, and inhibit apoptosis and autophagy of myocardial cells. When the growth hormone releasing related peptide is used as a medicine component, the invention has the advantages of small molecular weight, low immunogenicity, high specificity, safe action mode, high patentability and the like. The invention provides a new drug choice for treating ICIS-related myocarditis, and has good application prospect.
Description
Technical Field
The invention relates to application of a growth hormone release related polypeptide in preventing and treating immune checkpoint inhibitor related myocarditis, and belongs to the technical field of biological medicines.
Background
Since 2011, the use of immune checkpoint inhibitors (immune checkpoint inhibitors, ICIs) has brought a new choice for advanced malignancy treatment. Clinical data indicate that 1.14% of patients suffer from ICIs-related myocarditis with mortality rates approaching 50%. ICIs-related myocarditis is clinically manifested as chest pain, shortness of breath, pulmonary edema and even cardiogenic shock, and arrhythmia may also occur, leading to syncope and sudden death. Unlike traditional cardiomyopathy, the main pathogenesis of myocarditis associated with immune checkpoint inhibitors is thought to be that immune cells residing around the myocardium are activated to attack normal cardiomyocytes for injury, and pathology is primarily manifested by inflammatory cell infiltration. Whereas traditional ischemic cardiomyopathy is a late manifestation of coronary heart disease, due to loss of cardiomyocytes, fibrosis or scar hyperplasia caused by coronary atherosclerosis. The pathogenesis of the two is different, and the treatment means are also different. Ischemic cardiomyopathy is mainly treated by traditional cardiovascular therapeutic drugs such as digitalis and aminophylline and vasodilators. However, ici s-related myocarditis is mainly treated by high-dose hormone impact, and traditional cardiovascular medicines are almost ineffective for treating the ici s-related myocarditis. However, in clinical practice it has been found that a proportion of patients have reduced clinical benefit due to the occurrence of hormonal resistance, which may eventually progress to MACE (Major Adverse Cardiovascular Events, major cardiovascular adverse events). For this reason, it is urgent to find highly sensitive and specific therapeutic means for ICIs-related myocarditis.
Polypeptides are produced by proteasome degradation and can play an important role as endogenous ligands or receptors mediating a variety of signaling pathways. There are studies showing that the circulating antimicrobial peptide LL-37 has become a novel biomarker in cardiovascular disease in addition to BNP and ANP. Polypeptides also occupy a unique place in the field of drug development, and from early on, to date, hundreds of polypeptide drugs have been approved for sale worldwide. Wherein eptifibatide has been clinically used to treat acute coronary syndrome. Provides a brand new choice for the research and development of polypeptide drugs in the field of cardiovascular diseases.
Growth hormone releasing peptide (ghrelin) is an endogenous ligand for the growth hormone secretagogue receptor (Growth hormone secretagogue receptor, GHSR). ghrelin is found by Kojima et al in mouse and human gastric endocrine cells and hypothalamic arciform nuclei, and is the only natural ligand of GHSR found so far, which contains 28 amino acid residues and has a molecular weight of 33kDa. The ghrelin precursor protein of human and rat consists of 117 amino acids, and the N-terminal pre-23 peptide is characterized by secretion signal peptide; the first 4 amino acid fragments at the N-terminus of ghrelin are the smallest active centers and the P-R structure at the C-terminus (proline-arginine) is the recognition site. Ghrelin differs from ghrelin in humans and rats by only 2 amino acid residues, and the coding gene sequence has 82.9% homology. ghrelin has two secreted forms in vivo: one is that the serine at position 3 of the N-terminal of ghrelin is octanoylated, the other is that this site is not octanoylated. The 3 rd serine of ghrelin is a substantial part of ghrelin that exerts biological functions. Initial studies suggest that the de-octanoylated ghrelin (des-acyl ghrelin) is not biologically active, whereas recent studies have found that both the de-octanoylated and the octanoylated ghrelin are capable of promoting proliferation of spinal nerve epithelium; the de-octanoylated ghrelin has endocrine function, can promote cell proliferation, and has anti-apoptosis effect.
The pulse release of Growth Hormone (GH) at the pituitary is regulated mainly by three factors, hypothalamic growth hormone releasing hormone (growth hormone releasing hormone, GHRH), somatostatin (SS) and ghrelin. Promoting GH secretion, GHRH, SS inhibiting, ghrelin can act synergistically with GHRH to promote the formation of a local neuroendocrine regulatory feedback loop in the hypothalamus. In a system that regulates GH secretion, GHRH binds to its receptor, increasing intracellular levels of cyclic adenosine monophosphate; ghrelin binds to its receptor, K + Depolarization and inhibition of channels, leading to an increase in intracellular IP3 concentration, intracellular Ca 2+ The concentration increases, eventually stimulating GH secretion. The release of GH from pituitary somatotrophic hormone cells may also be controlled by Growth Hormone Releasing Polypeptide (GHRP). A hexapeptide, H-His-D-Trp-Ala-Trp-D-Phe-Lys-CONH, has been found 2 (GHRP-6) regulates the release of growth hormone in somatotrophic hormone cells in a dose dependent manner in several species including humans (bonerset al, endocrinology 1984,114,1537-1545). A subsequent chemical study of GHRP-6 identified other potent growth hormone releasing elements such as GHRP-1, GHRP-2 and Haxarelin (hexarelin) and the like:
GHRP-1：Ala-His-D-(2’)-Nal-Ala-Trp-D-Phe-Lys-NH 2 ；
GHRP-2：D-Ala-D-(2’)-Nal-Ala-Trp-D-Nal-Lys-NH 2 ；
sea sand relin: his-D-2-MeTrp-Ala-Trp-D-Phe-Lys-NH 2 。
GHRP-1, GHRP-2, GHRP-6 and Haixorelin are synthetic growth hormone releasing elements (GHS's) which stimulate the secretion of growth hormone by a different mechanism than growth hormone releasing elements.
The present patent (CN 112955165 a) discloses the use of GHRP-6 as a delayed cardioprotector and restorative agent, GHRP-6 being advantageous for reversing cytotoxic events derived from ventricular dyskinesia and diastolic dysfunction in order to reconstruct myocardial function as such and improve perfusion of coronary arteries, myocardium and the rest of the tissues and organs of animals and humans. However, no report is currently available on ghrelin-derived growth hormone release-related polypeptides and their use in the prevention and treatment of immune checkpoint inhibitor-related myocarditis.
Disclosure of Invention
The purpose of the invention is that: aiming at the technical problem of poor curative effect of hormone for treating the ICIs related myocarditis, the invention provides a ghrelin-derived growth hormone release related polypeptide and application thereof in preventing and treating immune checkpoint inhibitor related myocarditis.
In order to achieve the above object, the present invention provides a growth hormone releasing related polypeptide, which is characterized in that the amino acid sequence of the polypeptide is: X-P-Y, wherein P is as shown in SEQ ID NO:1, and X is: NH (NH) 2 Or any one amino acid or any two amino acid combination, Y is: OH or any one amino acid or a combination of any two amino acids.
Preferably, the polypeptide is GHRL-12 having the amino acid sequence as set forth in SEQ ID NO:1 (i.e. X is NH) 2 Y is OH), and the amino acid sequence of the polypeptide is derived from ghrelin protein.
The invention also provides a gene for encoding the growth hormone releasing related polypeptide GHRL-12, which has the nucleotide sequence shown in SEQ ID NO:2, and a nucleotide sequence shown in the following formula.
The invention also provides application of the growth hormone release related polypeptide or pharmaceutically acceptable salt form thereof in preparing medicines for preventing and/or treating immune checkpoint inhibitor related myocarditis.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for preparing a medicament for preventing and treating ICIs-related myocarditis by applying ghrelin-derived growth hormone release-related polypeptide GHRL-12 for the first time, and the method is verified on an animal model; in particular, the growth hormone release related polypeptide can reduce the content of CK, CKBB, LDH-1, cTnI and cTnT in mice in the myocarditis group, improve the level of LVEF and FS in mice in the myocarditis group, reduce LVEDs and LVEDd, reduce the infiltration and fibrosis degree of inflammatory cells in myocardial tissues of the mice in the myocarditis group, and obviously inhibit the activation of apoptosis and autophagy key proteins; in addition, the growth hormone release related polypeptide is derived from natural protein, and has the advantages of small molecular weight, small immunogenicity, safe action mode, high patentability and the like when being used as a pharmaceutical component.
Drawings
FIG. 1 is a schematic representation of GHRL-12 in mice with improved ICis-associated myocarditis; A. the mice in each group had a body weight increase every 7 days; B. the heart-to-body ratio of each group of mice; C. the heart-to-shin ratio of each group of mice; * P <0.05, < P <0.01, < P <0.001, one-way ANOVA, n=6.
FIG. 2 shows that GHRL-12 improves cardiac function in mice with ICis-associated myocarditis; a: m-type cardiac ultrasonic images of mice in each experimental group; b: left Ventricular Ejection Fraction (LVEF) statistics for each group of mice; c: left ventricular short axis reduction (FS) statistics for each group of mice; d: end-systole inner diameters (LVEDs) statistics for each group of mice; e: end-diastole inner diameter (LVEDd) statistics for each group of mice; * P <0.05, < P <0.01, < P <0.001, one-way ANOVA, n=6.
FIG. 3 shows that GHRL-12 improves inflammatory cell infiltration and fibrosis in myocardial tissue of mice with ICis-associated myocarditis; a: HE staining results for each group of mice; b: statistics of inflammatory cell infiltration in HE in A with ImageJ; c: masson staining results for each group of mice; d: performing ImageJ statistical analysis on the dyeing result in step C; * P <0.05, < P <0.01, < P <0.001, one-way ANOVA, n=6.
FIG. 4 shows GHRL-12 reduces the release of markers of myocardial injury from mice with ICis-related myocarditis; a: detecting and analyzing results of various groups of mouse Creatine Kinase (CK); b: detection and analysis results of creatine kinase isozymes (Creatine kinase Isoenzyme, CKBB) in serum of each group of mice; c: detection and analysis results of each group of mouse lactate dehydrogenase (Lactate dehydrogenase, LDH) -1; d: detection and analysis results of the troponin (cardiac TroponinI) I of each group of mice; e: detection and analysis results of troponin (cardiac TroponinI) T of each group of mice; * P <0.05, < P <0.01, < P <0.001, one-way ANOVA, n=6.
FIG. 5 shows that GHRL-12 reduces inflammatory factor release in mice with ICis-associated myocarditis; a: results of detection and analysis of interleukin-1β (IL-1β) in serum of each group of mice by ELISA; b: results of detection and analysis of interleukin-6 (IL-6) in serum of each group of mice by ELISA method; c: detection and analysis of the results of tumor necrosis factor-alpha (TNF-alpha) of each group of mice by ELISA; * P <0.05, < P <0.01, < P <0.001, one-way ANOVA, n=6.
FIG. 6 shows that GHRL-12 significantly inhibits apoptosis and activation of autophagy-related proteins in mice with ICis-related myocarditis; A. HMGB1, ATG5, beclin-1 and LC3B protein expression pictures in myocardial tissue of each group of mice; B. statistics of ATG5 protein expression in myocardial tissue of each group of mice; C. the expression statistics of the Beclin-1 protein in the myocardial tissue of each group of mice; D. HMGB1 protein expression statistics in myocardial tissue of each group of mice; E. LC3B protein expression statistics in myocardial tissue of each group of mice; F. myocardial tissue TUNEL staining pictures of each group of mice; G. quantitative results of TUNEL staining of myocardial tissue of each group of mice; H. PARP and caspase-3 protein expression pictures of myocardial tissues of mice in each group; I. the myocardial tissue clear-caspase-3 and caspase-3 protein expression statistics of each group of mice; J. calculating the expression ratio statistics of the clear-PAPRP and PARP proteins of myocardial tissues of each group of mice; * P <0.05, < P <0.01, < P <0.001, one-way ANOVA, n=3.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.
The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1: GHRL-12 polypeptide (i.e. "growth hormone releasing related polypeptide") sequence and preparation thereof
The GHRL-12 polypeptide is synthesized by Shanghai peptide biotechnology company through solid phase chemistry, the purity is more than 95%, the GHRL-12 polypeptide has 12 amino acids, the GHRL-12 polypeptide is positioned at the 12 th-23 th amino acid of human Ghrelin protein, and the amino acid sequence is as follows: val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Pro-Pro-Ala (VQQRKESKKPPA, SEQ ID NO: 1), a nucleotide sequence (SEQ ID NO: 2) of 5'-gtg cag cag aga aag gag agc aag aag ccc cct gcc-3', a molecular Mass (MW) of 1395.62, an isoelectric Point (PI) of 10.29, and an in vivo half-life of more than 20h, indicates that the polypeptide has good drug properties.
Example 2: animal experiment
1. Animal origin:
balb/c mice of 6-8 weeks old, weight 20-25g, supplied by Nanjing Jixiaokang laboratory animal Limited liability company, are raised in the laboratory animal center of the auxiliary Zhongshan hospital at the double denier university.
2. Experimental grouping:
control group (CON group), myocarditis group (ICIs group), GHRL-12group (GHRL-12 group) and GHRL-12+ myocarditis group (GHRL-12+ ICIs group) were given alone. Each group of 6 GHRL-12 polypeptides is administrated according to 10mg/kg, and is prepared into injection by taking physiological saline as solvent and is administrated by tail vein injection.
Establishment of a mice model for myocarditis associated with administration of ghrl-12 and immune checkpoint inhibitor:
mice in the GHRL-12group and the GHRL+myocarditis group are continuously injected with 10mg/kg GHRL-12 for one week by tail vein, and meanwhile, mice in the control group and the myocarditis group have the same volume of nonfunctional scramble peptide by tail vein. Mice in the myocarditis group and the GHRL-12+icis group were given 250 μg cTnI on day 7 and day 14, respectively, and 5mg/kg of anti-PD-1 was intraperitoneally injected 5 times every other day from day 14.
The mice of each group were weighed every 7 days and the general status was recorded.
After one week of model construction, mice were placed in an induction chamber containing 2% isoflurane (Hebei Yi Kagaku, shanghai, china) and continuously supplied with oxygen for 2L/min. After the mice were anesthetized completely, they were lying on an isothermal bench. The heart rate of the mice was maintained at 350-550bpm and M-mode images were recorded.
1mL of orbital vein blood was collected, and the supernatant was collected by conventional centrifugation to determine serum Creatine Kinase (CK), creatine kinase isozyme (CKBB), lactate dehydrogenase (LDH-1), troponin I (cTnI) and troponin T (cTnT) levels; ELISA (enzyme-linked immunosorbent assay) detects the levels of serum inflammatory factors TNF-alpha, IL-1 beta and IL-6.
Heart tissue was sectioned and stained to observe the level of myocarditis cell infiltration and the extent of fibrosis. TUNEL staining is carried out on myocardial tissue, myocardial fibers are stained red by alpha-actinin marks, DAPI staining nuclei are blue, and apoptotic myocardial cells are stained green by TUNEL.
Adding the myocardial tissue blocks into the protein lysate prepared according to the proportion, and extracting tissue proteins. Identification of apoptosis-critical proteins Caspase-3 and PARP and autophagy-critical proteins HMGB1, ATG5, beclin-1 and LC3B expression.
4. Experimental results
(1) GHRL-12 improves the general condition of mice with ICIS-related myocarditis
FIG. 1 shows that the body weight of the mice in the administration group of GHRL-12 alone was not significantly different from the body weight of the mice in the group of gastric lavage physiological saline. While myocarditis mice had insignificant weight gain, showing statistical differences from day 19 (p= 0.0248). On day 31, myocarditis mice with GHRL-12-dry prognosis had more pronounced weight gain, with statistical differences (p=0.0008) from mice in the myocarditis alone group (fig. 1A). After the modeling was completed, the results of measuring the mice' heart rate and heart-to-shin rate revealed that the ICIs group heart rate and heart-to-shin rate were significantly increased and the GHRL-12+ myocarditis group heart rate and heart-to-radius-to-shin rate were significantly decreased compared to the control group and GHRL-12group (fig. 1B and 1C). The results show that GHRL-12 improves the general condition of mice with ICis related myocarditis.
(2) GHRL-12 improves cardiac function of mice with ICIs-related myocarditis
Mice administered with GHRL-12 alone had no significant change in cardiac function, and myocarditis mice had decreased Left Ventricular Ejection Fraction (LVEF) and left ventricular short axis reduction (FS), while LVEF and FS increased to some extent after GHRL-12 intervention (fig. 2B and 2C); meanwhile, myocarditis mice had both elevated left ventricular end-Systole (LVEDs) and left ventricular end-diastole (LVEDd) (p < 0.05), and both had decreased after GHRL-12 intervention (figures 2D and 2E). The results show that GHRL-12 improves the cardiac function of mice with ICis-related myocarditis.
(3) GHRL-12 improves inflammatory cell infiltration and fibrosis of myocardial tissue of mice with ICis-related myocarditis
HE staining results show that the myocardial cells of the mice in the control group and the mice in the GHRL-12group are orderly arranged, the cell morphology is normal, and inflammatory cell infiltration is avoided. Whereas the myocarditis group mice had partially ruptured myocardial tissue, the cell arrangement was disordered, there was significant inflammatory cell infiltration, the myocarditis group mice had reduced myocardial tissue rupture, the myocardial cell arrangement was partially disturbed, and the inflammatory cell infiltration was reduced (fig. 3A). Statistics of inflammatory cell infiltration in HE were performed with ImageJ, and found that there was a statistical difference in the degree of inflammatory cell infiltration in mice with myocarditis compared to the control group (P < 0.05), and in mice with GHRL-12+ myocarditis compared to the myocarditis group (P < 0.05) (fig. 3B). Masson staining showed that mice with myocarditis group had a disturbed myocardial fiber arrangement, and the collagen content was significantly increased compared to the control group and GHRL-12group, while mice with GHRL-12+ myocarditis group had a relatively regular myocardial fiber arrangement, and the myocardial collagen content was significantly reduced (FIG. 3C). The ImageJ analysis showed that there was a statistical difference in the degree of myocardial fiber alignment disorder in mice with myocarditis compared to the collagen content (P < 0.05) and in GHRL-12+ myocarditis compared to the myocarditis (P < 0.05) (fig. 3D). The result shows that GHRL-12 can obviously reduce the inflammatory cell infiltration and the fibrosis degree of the myocardial tissue of mice with the ICis related myocarditis.
(4) GHRL-12 reduces ICIS-related myocarditis mouse myocardial injury zymogram marker release
Creatine Kinase (CK) results showed no significant difference in serum CK values for the GHRL-12 mice compared to the control group, whereas the serum CK values for the myocarditis mice were significantly increased, whereas the serum CK values for the GHRL-12+ myocarditis mice were significantly decreased (fig. 4A). Meanwhile, after a single GHRL-12 dry prognosis, creatine kinase isozymes (Creatine kinase Isoenzyme, CKMB) in the serum of mice were not significantly changed, and CKMB was significantly increased in mice of the myocarditis group, while CKMB was significantly decreased in mice of the GHRL-12+ myocarditis group (fig. 4B). Likewise, GHRL-12 can reduce the release of lactate dehydrogenase (Lactate dehydrogenase, LDH) -1 (fig. 4C). Both murine troponin (cardiac TroponinI) I and T were significantly elevated in the myocarditis group, and GHRL-1 reduced release of murine troponin I and T (fig. 4D and 4E). The result shows that GHRL-12 can obviously reduce the release of myocardial zymogram markers and reduce the damage of ICI to cardiac function.
(5) GHRL-12 reduces inflammatory factor release in mice with ICIs-related myocarditis
ELISA results show that interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha in the serum of the myocarditis mice are obviously increased, and have statistical difference (p < 0.05); serum IL-1β, IL-6 and TNF- α were significantly reduced in mice from the GHRL-12+ myocarditis group and were statistically significant (P < 0.05) compared to the myocarditis group (FIGS. 5A-C). The result shows that GHRL-12 can relieve the damage of ICIs to cardiac muscle, inhibit the overactivation of immune cells and inhibit the release of inflammatory factors.
(6) GHRL-12 improving ICIS-associated myocarditis mice significantly inhibit activation of apoptosis-related proteins and autophagy-related genes
Autophagy key proteins Beclin-1, hmgb1, atg5 and LC3B were all significantly elevated in myocardial tissue of mice in the myocarditis group, but GHRL-12 intervention alone did not affect autophagy. Mice in the GHRL-12+ myocarditis group, beclin-1, HMGB1, ATG5 and LC3B were significantly reduced and had statistical significance (FIGS. 6A-E). TUNEL staining of myocardial tissue showed no significant change in apoptotic cardiomyocytes in the control and GHRL-12 groups. The group of ICIs significantly increased apoptotic cardiomyocytes, while the group of GHRL-12+icis significantly decreased apoptotic cardiomyocytes (fig. 6F-G). Western blotting technique further detects apoptosis-related protein changes, and found that clear-caspase-3 and PARP are significantly activated in myocardial tissue of mice in myocarditis group, while GHRL-12+ myocarditis group is inhibited to a certain extent and has statistical difference (P < 0.05) (FIG. 6E-F). The results indicate that GHRL-12 can inhibit activation of apoptosis and autophagy key proteins.
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (4)
1. A growth hormone releasing related polypeptide, wherein the amino acid sequence of the polypeptide is: X-P-Y, wherein P is as shown in SEQ ID NO:1, and X is: NH (NH) 2 Or any one amino acid or any two amino acid combination, Y is: OH or any one or any two of amino acidsA combination of amino acids.
2. The growth hormone releasing related polypeptide of claim 1 wherein said polypeptide is GHRL-12 having the amino acid sequence as set forth in SEQ ID NO:1, the amino acid sequence of which is derived from ghrelin protein.
3. A gene encoding the growth hormone releasing related polypeptide of claim 2, wherein said gene has the amino acid sequence as set forth in SEQ ID NO:2, and a nucleotide sequence shown in the following formula.
4. Use of a growth hormone release related polypeptide according to claim 1 or 2, or a pharmaceutically acceptable salt form thereof, for the manufacture of a medicament for the prevention and/or treatment of immune checkpoint inhibitor related myocarditis.
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