WO2024254754A1 - Polypeptide for preventing and/or relieving hepatic ischemia-reperfusion injury, and use thereof - Google Patents

Abstract

The present invention provides a use of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or an inverse peptide thereof in the preparation of a drug for preventing and/or relieving hepatic ischemia-reperfusion injury, wherein the polypeptide can be modified.

Description

Polypeptide for preventing and/or alleviating liver ischemia-reperfusion injury and its application Technical Field

The present invention belongs to the field of biomedicine, and in particular, relates to a polypeptide for preventing or alleviating liver ischemia-reperfusion injury and an application thereof.

Background Art

Ischemia-reperfusion injury (IRI) refers to organ damage caused when blood supply is temporarily blocked and then restored. Hepatic ischemia-reperfusion injury usually occurs during liver transplantation, liver surgery, or in cases of liver trauma, shock, or ischemic hepatitis. The pathological process of ischemia-reperfusion injury is complex and the exact mechanism is not fully understood. During ischemia-reperfusion, factors such as cellular adenosine triphosphate (ATP) depletion, intracellular calcium overload, increased oxygen free radicals, enhanced inflammatory response, mitochondrial dysfunction, and endoplasmic reticulum stress response lead to tissue damage (Rampes, S. and Ma, D., Hepatic ischemia-reperfusion injury in liver transplant setting: mechanisms and protective strategies. J Biomed Res. 2019 Jul 28; 33(4): 221-234.). On the contrary, improvement of the above processes can help prevent or alleviate ischemia-reperfusion injury by reducing the time of organ hypoxia during surgery, optimizing blood flow restoration techniques, inhibiting harmful pro-inflammatory responses or promoting beneficial anti-inflammatory responses, alleviating oxidative stress, reducing ATP depletion, and reducing calcium overload.

Pannexin-1 channel protein (PANX1) is a transmembrane protein involved in the release of adenosine triphosphate (ATP) to the extracellular space. PANX1 can be activated by a variety of factors, such as mechanical stimulation, membrane depolarization, increased intracellular calcium levels, increased extracellular potassium levels, cleavage of the C-terminal region of PANX1 protein, receptor protein activation, Src family kinase (SFK)-mediated phosphorylation, proinflammatory cytokine TNFα, etc. Activated PANX1 releases ATP to the extracellular space and participates in inflammation and injury responses (Koval, M., et al. Pannexin 1 as a driver of inflammation and ischemia-reperfusion injury. Purinergic Signal. 2021 Dec; 17(4): 521-531.). Activation of PANX1 by the proinflammatory cytokine TNFα induces increased intracellular calcium levels (Yang Y. et al. Endothelial Pannexin 1 Channels Control Inflammation by Regulating Intracellular Calcium. J Immunol. 2020 Jun 1; 204(11): 2995-3007.).

Studies have shown that genetic deletion of endothelial cell PANX1 can reduce the size of ischemic stroke infarction (Good, ME, et al. Endothelial cell Pannexin1 modulates severity of ischemic stroke stroke by regulating cerebral inflammation and myogenic tone. JCI Insight. 2018 Mar 22; 3(6): e96272.). The PANX1 inhibitor carbenoxolone (CBX) has a protective effect on renal ischemia-reperfusion injury in mice (Jankowski, J. et al. Epithelial and Endothelial Pannexin1 Channels Mediate AKI. J Am Soc Nephrol. 2018 Jul; 29(7): 1887-1899.). Clinical trial results show that patients taking the PANX1 inhibitor probenecid have significantly reduced vascular diseases, especially myocardial infarction and stroke (Kim, SC et al. Cardiovascular Risks of Probenecid Versus Allopurinol in Older Patients With Gout. J Am Coll Cardiol. 2018 Mar 6; 71(9): 994-1004.). PANX1 inhibitors carbenoxolone and probenecid significantly reduced lung dysfunction, edema, cytokine production and neutrophil infiltration in mice with lung ischemia-reperfusion injury (Sharma, AK et al. Pannexin-1 channels on endothelial cells mediate vascular inflammation during lung ischemia-reperfusion injury. Am J Physiol Lung Cell Mol Physiol. 2018 Aug 1; 315(2): L301-L312.). These data provide strong evidence for preventing or alleviating ischemia-reperfusion injury by intervening in PANX1.

Although there are many small molecule compounds that can inhibit PANX1, such as probenecid, carbenoxolone, brilliant blue FCF, etc., most PANX1 antagonists also bind to unrelated channel proteins, transport proteins, and non-membrane proteins. ¹⁰ panx is a specific peptide antagonist of PANX1, but its inhibitory effect is not as good as that of small molecule antagonists (Navis, KE, et al., Pannexin 1 Channels as a Therapeutic Target: Structure, Inhibition, and Outlook. ACS Chem Neurosci. 2020 Aug 5; 11(15): 2163-2172.). Therefore, it is very necessary to develop efficient and specific therapeutic drugs for PANX1.

Summary of the invention

With reference to the structure of PANX1 protein, two new mimetic peptides were invented (Chinese patent application number 202211496945.8, PCT application number PCT/CN2022/134256). Cell experiments have shown that the new mimetic peptides are better than carbenoxolone and ¹⁰ panx in inhibiting PANX1. The inventors used one of the mimetic peptides, QE20, in a mouse experiment to prove that the peptide has the effect of preventing and alleviating liver ischemia-reperfusion injury.

The present invention provides a use of a polypeptide in preparing a medicament for preventing and/or alleviating liver ischemia-reperfusion injury, wherein the polypeptide is a polypeptide fragment consisting of an amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence of SEQ ID NO: 1 is as follows:

In a specific embodiment, one or more amino acids in the polypeptide of the present invention may be in the form of the D-enantiomer. More preferably, all amino acids in the polypeptide are in the form of the D-enantiomer.

In a specific embodiment, the polypeptide of the present invention can be further modified. The polypeptide of the present invention can be modified by various well-known technical means. Polypeptide modification includes but is not limited to amino acid replacement, N-terminal modification, C-terminal modification, side chain modification, amino acid modification, peptide backbone modification, binding to other polypeptides or proteins, etc. The polypeptide modification means can optimize the physicochemical properties of the polypeptide, improve its water solubility, prolong the in vivo action time, change its in vivo distribution, eliminate immunogenicity, and reduce toxic side effects.

In a specific embodiment, the polypeptide of the present invention is linked to the N-terminus of the polypeptide via stearic acid, and the C-terminus of the polypeptide is modified by amination.

In a specific embodiment, the polypeptide of the present invention may be a fragment of a polypeptide in the opposite direction (i.e., a reverse polypeptide) composed of the amino acid sequence shown in SEQ ID NO:1.

Beneficial Effects

This application has developed a new pharmaceutical application for the polypeptide QE20. This application first confirms that the polypeptide QE20 has a good effect in preventing and inhibiting ischemia-reperfusion injury. Therefore, it has potential application value in preventing and alleviating ischemia-reperfusion injury in the fields of liver transplantation, heart transplantation, kidney transplantation, lung transplantation, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing that the polypeptide of the present invention has no toxicity to normal human liver cells L-02 at a concentration of 50 μM.

Figure 2 shows the degree of liver damage at different times after liver ischemia-reperfusion in mice, A: effect on ALT; B: effect on AST.

FIG3 is a graph showing that the polypeptide of the present invention prevents and alleviates liver ischemia-reperfusion injury in mice by intraperitoneal injection, A: effect on ALT; B: effect on AST.

FIG4 is a graph showing that the polypeptide of the present invention prevents and alleviates liver ischemia-reperfusion injury in mice by intravenous injection, A: effect on ALT; B: effect on AST.

DETAILED DESCRIPTION

Hereinafter, the present invention will be described in detail by way of examples. However, the examples provided herein are only for illustrative purposes and are not intended to limit the present invention.

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

Example

Peptide synthesis

The peptide was synthesized by a peptide synthesis company (Zhejiang Paipeptide Biological Co., Ltd., No. 291, Fucheng Road, Xiasha Street, Jianggan District, Hangzhou) through a solid phase method, and its amino acid sequence is as follows:

Stearic acid is conjugated to the N-terminus of the polypeptide SEQ ID NO: 1 (FKHLWLPLNGSESQLSNKQE), and the C-terminus is aminated to become the polypeptide of the present application (named QE20). The steps are as follows: First, the aminated polypeptide is synthesized by a solid phase method, and then stearic acid is conjugated to the N-terminus of the polypeptide by a chemical reaction. The stearic acid polypeptide is freed from the solid phase by a chemical reaction. The synthesized polypeptide is purified by reverse phase chromatography to a purity of >95%. The purity of the polypeptide is determined by high performance liquid chromatography (HPLC), and the molecular weight of the polypeptide is determined by mass spectrometry (MALDI-TOF).

Preparation of peptides for experiments

First, the peptide QE20 prepared above was dissolved in 2.5% volume DMSO (D2650, Sigma-Aldrich), and then 1×PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , pH 7.2-7.4) (absin, abs962, Shanghai, China) buffer was added to a final concentration of 1 mM.

Experimental steps and results

(1) Cell hypoxia and reoxygenation experiment

Normal human hepatocytes L- ₀₂ (ATCC, HL-7702, BFN608006124, BLUEFBIO, Shanghai, China) were cultured in DMEM medium (Gibco, C11995500BT) (hereinafter referred to as complete medium) containing 10% FBS (Gibco, 10270-106) and 1% penicillin-streptomycin (Gibco, 15140-122) at 37°C in a humidified incubator (20% O ₂ , 5% CO ₂ and 94% N ₂ , hereinafter referred to as normal incubator) containing 5% CO 2 until the logarithmic growth phase, and the cell growth density reached more than 80%. After separation, the cells were seeded into 3.5 cm culture dishes at a density of 5×10 ⁵ cells/mL per well.

Culture the cells for 24 to 48 hours until the cell growth density reaches more than 80%. Gently remove the culture medium and wash the cells once with 1×PBS preheated at 37°C. In the hypoxia-reoxygenation experiment group, add 1mL of sugar-free and serum-free DMEM culture medium (Gibco, 11966025) to each culture dish, and then add 50μL of 1×PBS without or with the following test samples and the final concentrations are: 150μM carbenoxolone (Sigma-Aldrich, C4790), 200μM ¹⁰ Panx (Tocris Bioscience, Cat#3348), 50μM QE20. Incubate in a normal incubator for 30 minutes, then transfer to a hypoxic incubator at 37°C, containing 1.0% O ₂ , 5% CO ₂ and 94% N ₂ and culture for 12 hours (this is the hypoxia stage). After the culture was completed, the cell culture medium in the hypoxic incubator was removed, and then washed once with 1×PBS incubated at 37°C, replaced with 1 mL of fresh complete culture medium incubated at 37°C, and 1×PBS, 150 μM carbenoxolone, 200 μM ¹⁰ Panx, and 50 μM QE20 were added to the corresponding wells, and the cells were transferred to a normal incubator and continued to be cultured for 4 hours (this was the reoxygenation stage).

After the cell culture was completed, the culture medium was discarded, the cells were washed once with 1×PBS, and then incubated at 37°C for 2 minutes with 0.25% trypsin solution (Beyotime, C0205) without EDTA. The reaction was then neutralized by adding complete culture medium. The cells were collected and centrifuged (1000 rpm) for 3 minutes, the supernatant was discarded, and the cells were washed twice with 1×PBS precooled at 4°C. Antibody staining was performed according to the steps of the cell apoptosis detection kit (BD Pharmingen ^™ FITC Annexin V Apoptosis Detection Kit I, 556547). After staining, the cells were detected by flow cytometry (BD LSRFortessa ^™ ).

The results are shown in FIG1 . Compared with the control group (PBS), 150 μM carbenoxolone, 200 μM ¹⁰ Panx, and 50 μM QE20 did not cause significant toxicity to the cells.

(2) Detection of liver damage at different time points after liver ischemia-reperfusion in mice

Eight-week-old male C57BL/6J mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and fed with SPF rat growth and breeding feed (Beijing Keao Xieli Feed Co., Ltd.) under pathogen-free conditions and fasted for 12 hours before surgery.

The mice in the experimental group were anesthetized by intraperitoneal injection of 1.25% avertin. Then the mice's limbs were fixed, the abdomen was disinfected with alcohol, and the abdomen was opened layer by layer along the midline of the abdomen to expose the liver hilum. The mouse hilum was ligated with 4-0 non-absorbable surgical sutures (Jiangsu Yangzhou Fuda Medical Equipment Co., Ltd.) to construct a 70% liver (left lobe and middle lobe) blood flow occlusion model (the liver color turned grayish white), and then the incision was covered with wet gauze and placed under a warm lamp. After 1 hour, the ligature was loosened, and the ligature was gently rubbed with a cotton swab. After the blood flow to the liver was restored, the abdominal cavity was closed, and the mice were placed under a warm lamp to wake up. The sham operation group (Sham, 4 mice) underwent the same surgical operation, but the hilum was not ligated. Eyeballs were examined 3 hours (4 mice), 6 hours (4 mice), 12 hours (4 mice), and 24 hours (5 mice) after surgery. Blood was collected and placed in a 1.5 mL Eppendorf tube, allowed to stand at room temperature for 30 min, then centrifuged at 4°C, 5000 rpm, and serum was transferred to another 1.5 mL Eppendorf tube. Alanine aminotransferase (ALT) (Jiangsu Kangshang Biomedical Technology Co., Ltd.) and aspartate aminotransferase (AST) (Jiangsu Kangshang Biomedical Technology Co., Ltd.) were detected according to the steps of the kit.

Mouse liver function (ALT, AST) tests showed that the liver damage was most severe after 1 hour of ischemia and 6 hours of reperfusion in mice, as shown in Figure 2.

(3) Mouse liver ischemia-reperfusion injury experiment (intraperitoneal injection)

According to the above experimental results, the mouse liver was ischemic for 1 hour and then reperfused for 6 hours to construct the mouse ischemia-reperfusion model. The experimental groups were divided into NC (Normal Control) group (5 mice), Panx1 ^-/- group [5 8-week-old Panx1 gene-deficient male mice, Saiye (Suzhou, Jiangsu) Biotechnology Co., Ltd.)] and QE20 group (6 mice). Two hours before surgery, the NC group and Panx1 ^-/- group were intraperitoneally injected with an equal volume (200 μl) of sterile 1× PBS, and the QE20 group was intraperitoneally injected with 50 mg/kg QE20. The subsequent experimental steps were the same as above (2).

The sham-operated groups (4 mice in the NC Sham group and 5 mice in the Panx1 ^-/- Sham group) underwent the same surgical procedure, but without ligating the hepatic hilum.

Figure 3 shows that Panx1 gene deletion can reduce liver ischemia-reperfusion injury (reduced ALT by 76%, reduced AST by 76%), and after intraperitoneal injection of peptide QE20, the degree of liver ischemia-reperfusion injury is even lower than that of Panx1 gene deletion (reduced ALT by 91%, reduced AST by 91%).

(4) Mouse liver ischemia-reperfusion injury experiment (intravenous injection)

The average weight of 8-week-old mice was 22 grams. The mouse liver ischemia-reperfusion model was constructed as above (3), that is, the mouse liver was ischemic for 1 hour and then reperfused for 6 hours. The experimental groups were divided into NC (Normal Control) group (3 mice) and QE20 group. One hour before surgery, the NC group was intravenously injected with 100 μl of sterile 1× PBS, and the QE20 group was intravenously injected with the same volume containing 34 μg of peptide (3 mice, the final concentration of peptide in blood was about 10 μM) and 169 μg of peptide (4 mice, the final concentration of peptide in blood was about 50 μM). The subsequent experimental steps were the same as above (2).

The results in Figure 4 show that the effect of peptide QE20 in preventing and inhibiting ischemia-reperfusion injury in mouse liver depends on the concentration of the peptide in the blood. Low concentration of peptide (10 μM) can effectively prevent and inhibit ischemia-reperfusion injury in mouse liver (reducing ALT by 42% and AST by 34%), while high concentration of peptide (50 μM) has an increasing effect (reducing ALT by 83% and AST by 85%).

The above results indicate that the polypeptide QE20 of the present application has the effect of inhibiting and alleviating liver ischemia-reperfusion injury. Therefore, the polypeptide of the present application has potential application value in preventing and alleviating ischemia-reperfusion injury in the fields of liver transplantation, heart transplantation, kidney transplantation, lung transplantation, etc.

Claims (6)

Use of a polypeptide in the preparation of a drug for preventing and/or alleviating liver ischemia-reperfusion injury, wherein the polypeptide is a polypeptide fragment consisting of the amino acid sequence shown in SEQ ID NO: 1, or the polypeptide is a reverse peptide fragment of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. The use according to claim 1, wherein one or more amino acids in the polypeptide are in the D-enantiomer form. The use according to claim 1, wherein all the amino acids of the polypeptide are in the D-enantiomer form. The use according to claim 1, wherein the polypeptide is modified. The use according to claim 4, wherein the modification comprises amino acid replacement, N-terminal modification, C-terminal modification, side chain modification, amino acid modification, peptide backbone modification, and binding to other polypeptides or proteins. The use according to claim 1, wherein the polypeptide is linked to the N-terminus of the polypeptide via stearic acid, and the C-terminus of the polypeptide is modified by amination.