CN113181364B - Application of CD47 blocker in preparation of medicine for preventing and/or treating acute pancreatitis - Google Patents

Abstract

The invention discloses an application of a CD47 blocker in preparation of a medicine for preventing and/or treating acute pancreatitis, and belongs to the technical field of biological medicines. The invention simulates two clinically relevant acute pancreatitis models: the experimental results prove that the CD47 blocking agent is injected into a mouse body in the abdominal cavity, the generation and development of the two AP models can be remarkably improved, and the two AP models can be used for clinically preventing/treating AP. The medicine for inhibiting acute pancreatitis provided by the invention has high safety, strong pharmacological action and definite curative effect. The invention provides a new medicine source for preventing, diagnosing, detecting, protecting, treating and researching pancreatitis diseases, is easy to popularize and apply clinically, and can generate huge clinical application prospect and social benefit in a short time.

Description

Application of CD47 blocker in preparation of medicine for preventing and/or treating acute pancreatitis

Technical Field

The invention belongs to the technical field of biological medicines, and relates to an application of a CD47 blocking agent in preparation of a medicine for preventing and/or treating acute pancreatitis.

Background

Acute Pancreatitis (AP) is a non-infectious inflammatory disease of the digestive tract caused by excessive activation of pancreatic zymogen, and has the clinical characteristics of Acute disease, rapid onset, extremely high fatality rate and the like. Clinical mild disease patients account for most of AP, can recover pancreatic functions through medical conservative treatment, and have low fatality rate and risk degree. A few patients may have involved peri-pancreatic organs or distant tissues due to poor control, progress to Severe Acute Pancreatitis (SAP), with a high clinical early mortality of SAP, later stage combined with systemic inflammatory infection, progress to systemic inflammatory response syndrome, with mortality rates of up to 40-70% (Chan Y C, leung P S. Acute Pancreas disease: animal models and recent advances in basic research [ J ]. Pancreas,2007Jan 34 (1): 1-14 (Greenberg J A, jonathan H, mohammad B, al clinical practice regulation: management of age Pancreas disease. J. Canadian Journal of Surgery,2016,59 (2): 128-140).

Acinar cell injury/death due to activation and release of trypsinogen within acinar cells is one of the early events that regulate the development of AP. Acinar cells that are excessively injured and not removed in time can sustainably damage the pancreas, involve peripheral organs and distant organs, regulate excessive inflammatory reactions and multi-organ dysfunction. Whether these damaged acinar cells can be recognized by macrophages in early lesions and cleared in time. It is not yet known in the AP study.

CD47 molecules, also known as integrin-associated proteins (IAPs), which are widely expressed on the surface of a variety of cells, are well known "don't eat me" molecules, and belong to the class of membrane surface receptor molecules that can play an important role in a variety of cancer diseases. High expression of CD47 on the surface of many malignant cells can make them resistant to macrophage recognition effects and thus escape programmed clearance of tumor cells (Hayat S, bianoconi V, pirro M, et al. CD47: role in the immune system and application to cancer therapy [ J ]. Cellular Oncology,2019,43 (1)). It has been shown that CD47 is significantly highly expressed in atherosclerotic disease, and the use of anti-CD 47 Blocking Antibodies helps to clear vasculopathy material and improve atherosclerosis in various mouse models and patients (Ryan, john J.CD47-Blocking Antibodies and atherosclerosis [ J ]. Jacc Basic to Translational Science,2016,1 (5): 413-415).

The regulatory role of CD47 in AP is unknown, and early and timely clearance of damaged/dead acinar cells (macrophage cellularity) to inhibit CD47 has yet to be investigated.

Disclosure of Invention

In order to overcome the disadvantages of the prior art described above, it is an object of the present invention to provide the use of a CD47 blocker for the preparation of a medicament for the prevention and/or treatment of acute pancreatitis.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses an application of a CD47 blocking agent in preparation of a medicine for preventing and/or treating acute pancreatitis.

Preferably, the drug is one that reduces the inflammatory response by targeted removal of damaged acinar cells.

Further preferably, the drug is a drug that increases macrophage phagocytosis of damaged acinar cells.

Preferably, the medicament is a medicament for ameliorating pancreatic injury.

Preferably, the drug is a drug that reduces serum amylase and serum lipase levels.

The invention discloses a product, the active component of which is a CD47 blocking agent, and the application of the product at least comprises one of the following applications:

a) Improving inflammatory response of acute pancreatitis;

b) Enhancing the cell burial and elimination effect of the macrophage;

c) Inhibiting disease occurrence/development of acute pancreatitis;

the product is a medicament, additive or active ingredient.

The invention also discloses a medicine for preventing/treating acute pancreatitis, which consists of a CD47 blocking agent and auxiliary materials which can be added in pharmacy.

Preferably, the adjuvant comprises one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier and a lubricant.

Preferably, the drug can be introduced into body tissues by oral, injection, spray, nasal, eye drop, penetration, absorption, and physically or chemically mediated methods; or mixed or coated with other substances and introduced into body.

Further, the CD47 blocker referred to in the present invention is purchased from Bio X Cell, inc. under the trade name BE0283; clone number: the MIAP410.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides an application of a CD47 blocking agent in preparation of a medicine for inhibiting acute pancreatitis, which is obtained by simulating two clinically relevant acute pancreatitis models: (1) performing intraperitoneal injection of ranolanin (100 ug/kg, 1 needle per hour and 7 continuous needles) to induce acute pancreatitis in C57BL/6 mice; (2) bile duct ligation induced severe acute pancreatitis in C57BL/6 mice. Experimental results prove that the CD47 blocking agent is injected into a mouse body in an abdominal cavity, the generation and development of the two AP models can be obviously improved, and the AP blocking agent can be used for clinically preventing/treating AP. Meanwhile, experiments prove that the CD47 blocking agent has the effect of inhibiting acute pancreatitis by targeted removal of damaged acinar cells to achieve the effects of relieving AP inflammatory reaction and reducing AP severity.

The medicine for inhibiting acute pancreatitis provided by the invention has high safety, strong pharmacological action and definite curative effect. The invention provides a new medicine source for preventing, diagnosing, detecting, protecting, treating and researching pancreatitis diseases, is easy to popularize and apply clinically, and can generate huge clinical application prospect and social benefit in a short time.

Drawings

FIG. 1 shows the expression of CD47 in the pancreatic tissue of mice with acute pancreatitis induced by ranunculin in example 1 and its statistics; wherein (a) is the expression of CD47 and GAPDH proteins; (b) is statistics for CD47 and GAPDH proteins; (c) mouse pancreatic tissue CD47 RNA level.

FIG. 2 is a graph of the results of the intraperitoneal injection of the CD47 blocker in example 2 on significantly improving the pancreatic injury of mice with acute pancreatitis induced by ranolog-derived drugs (edema, inflammatory exudation, necrosis); wherein, (a) is a mouse pancreatic tissue HE staining pathological diagram; and (b) is a pathological scoring statistical chart of the pancreatic injury.

FIG. 3 is a graph showing the results of the CD47 blocking agent administered intraperitoneally to reduce the levels of serum amylase and serum lipase in acute pancreatitis in mice induced by ranolanin in example 2; wherein (a) is serum amylase level; and (b) is serum lipase level.

FIG. 4 is a graph showing that intraperitoneal injection of the CD47 blocking agent in example 2 can significantly increase macrophage "cellularity"; as in figure (a), a mouse pancreas histoelectron micrograph, scar bar =2uM; (b) Is a statistical chart of proportion of acinar cells phagocytosed and damaged by macrophages.

FIG. 5 shows that intraperitoneal injection of CD47 blocker can improve pancreatic injury of mice with acute pancreatitis induced by bile duct ligation in example 3; wherein, (a) is a mouse pancreatic tissue HE staining pathological diagram; and (b) is a pathological scoring statistical chart of the pancreatic injury.

FIG. 6 is a graph showing the results of the intraperitoneal injection of the CD47 blocker in example 3, which can reduce the serum amylase and serum lipase levels of acute pancreatitis in mice induced by bile duct ligation; wherein (a) is serum amylase level; and (b) is serum lipase level.

FIG. 7 is the activation of CD47 expression in the cholecystokinin (CCK) -induced acinar cell in vitro acute pancreatitis model of example 4; wherein (a) is mouse pancreatic tissue acinar cell CD47mRNA level; (b) Is the CD47mRNA level of the mouse acinar cell line 266-6 cells.

FIG. 8 is a graph of the results of CD47 blockers upregulating macrophage phagocytosis and not affecting acinar cell death (CCK-induced acute AP model in vitro) in example 5; wherein (a) is the level of phagocytic acinar cells by macrophages, and (b) is the level of cellular LDH release detection.

FIG. 9 is a graph of the results of CD47 blockers upregulating macrophage phagocytosis and not affecting acinar cell death (STS-induced AP damage model in vitro) in example 6; wherein (a) is the level of phagocytic acinar cells of macrophages, and (b) is the level of CCK8 detection of cells.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

the experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified. The reagent materials used in the examples are all available in routine purchase, and the quantitative experiments referred to in the examples are all set up in at least three replicates, and the results are averaged.

The C57/BL6 mice used were purchased from the model animal center of Nanjing university.

The ranophanin (Caerulein) was purchased from MCE under the designation HY-A0190.

Cholecystokinin (CCK) was used as purchased from sigma-aldrich under the designation T6515.

The CD47 blocking agent used was purchased from Bio X Cell, inc. under the catalog number BE0283; clone number: the MIAP410.

Example 1 activation of cd47 in ranolanin-induced acute pancreatitis in mice

Ranulin-induced mouse mild acute pancreatitis model

Experimental animals: male C57BL/6J mice (body weight 20-25 g) at 6-8 weeks.

The experimental animals were divided into a normal control group and a model group at different times (6 h,12h, 24h). The mice in the model group are injected with ranolamin (100 ug/kg, 1h interval and 7 continuous needles) in the abdominal cavity to construct an acute pancreatitis model. Control mice were given a stringent PBS control intraperitoneal injection. And (4) reserving mouse serum for detecting serum enzymology level and inflammatory factor level. Subsequently, mice underwent in vivo heart perfusion to remove circulating blood, rapidly extracted pancreatic tissue for fixed dehydration, made HE-stained paraffin sections and immunohistochemical staining, and the results are shown in fig. 1, and in acute pancreatitis induced by ranulin, pancreatic tissue edema, inflammatory exudation, and acinar cell necrosis are important indicators of the severity of the reaction disease. As shown in fig. 1 (a) and (b), the expression of CD47 in pancreatic tissue was observed by western blot, which showed that CD47 was continuously highly expressed on necrotic acinar cells and was positively correlated with the severity of acute pancreatitis disease, and GPDH was a control protein. As shown in FIG. 1 (c), the results of observing CD47mRNA levels in mouse pancreatic tissues by Q-PCR were consistent with those at protein level.

Example 2 use of anti-CD 47 blocking Agents for prevention/treatment of acute pancreatitis

1. Ranulin-induced mouse acute pancreatitis model

According to the actual operation and model induction conditions of the example 1, a 12h ranophanin induction model is selected as a follow-up study. The molding was carried out in the same manner as in example 1.

2. anti-CD 47 blocking agent intraperitoneal injection

The mice in the model group were randomly divided into a Control group, an AP (oral panicotis) group, and an anti-CD 47 blocking agent (5 mg/kg) treatment group, 7 mice in each group, and were intraperitoneally injected with an anti-CD 47 blocking agent (soluble in PBS solution) 0h before the hamartine molding, and the AP group was given a stringent Control with a stringent IgG antibody. All mice were anesthetized with 5% chloral hydrate by intraperitoneal injection 12h after the first injection of ranophanin, and the serum of the mice was retained for seroenzymology. Subsequently, mice were perfused in vivo heart to remove circulating blood, pancreatic tissue was rapidly extracted for fixed dehydration, paraffin sections for HE staining and immunohistochemical staining were made.

As a result, as shown in FIGS. 2 and 3, it can be seen that 5mg/kg of the anti-CD 47 blocking agent can significantly improve the pancreatic tissue damage (edema, inflammatory exudation and acinar cell necrosis), improve the serum enzymology and the serum amylase level. Furthermore, early clearance of damaged/dead acinar cells by macrophages could improve acute pancreatitis injury in mice and as suggested by the mouse pancreatic histoelectron micrograph (scar bar =2 uM) of fig. 4: the phagocytosis and phagocytic ratio of macrophages (continuous phagocytosis is visible) can be remarkably increased in mice injected with 5mg/kg anti-CD 47 blocking agent in the abdominal cavity. The above results suggest that inhibition of CD47 may improve or ameliorate the development of acute pancreatitis. * Represents a significant difference P < 0.05, represents a significant difference P < 0.01, and represents a significant difference P < 0.001.

Example 3 use of anti-CD 47 blocking agent for prevention/treatment of Severe acute pancreatitis

1. Bile duct ligation (PDL) -induced mouse severe acute pancreatitis model

Experimental animals: 6-8 week C57/BL6 Male mice (weight 20-25 g)

The experimental animals were divided into a sham-operated group, a model group and an anti-CD 47 blocker (5 mg/kg) treatment group (10 animals per group)

1. The experimental animals were anesthetized by intraperitoneal injection of 5% chloral hydrate (injected according to body weight, 8 ul/g).

2. After anaesthetizing all experimental animals, the animals were fixed on the operating table. After the abdominal cavity of the model group is opened, the common bile duct is separated in a blunt manner by using surgical forceps, and a #4 silk thread is applied to the lower end of the common bile duct close to the pancreas for ligation, so that the abdominal cavity is closed layer by layer and the skin is sutured. The sham operation group only performed the operation related to opening the abdomen and closing the abdomen, and did not perform the operation of ligating the bile duct. All mice were returned to the 37 ℃ thermostated table for 30min after surgical procedures.

2. anti-CD 47 blocking agent intraperitoneal injection

The treatment group was treated by intraperitoneal injection of anti-CD 47 blocking agent (5 ug/mouse) at 0h, the mice in the model group were strictly controlled by intraperitoneal injection of IgG control antibody, and the sham-operated mice were each intraperitoneally injected with PBS solution. Serum is kept for 24h,48h and 48h after the start of modeling of all mice, 5% chloral hydrate is injected into the abdominal cavity for anesthesia, and pancreas tissues are kept for preparing paraffin sections stained by HE.

As a result, as shown in (a) and (b) of FIG. 5, 5mg/kg of an anti-CD 47 blocking agent significantly improved the pancreatic tissue damage (pancreatic tissue edema, inflammatory exudation, acinar cell necrosis) in PDL mice, and in addition, as shown in (a) and (b) of FIG. 6, inhibition of CD47 improved the seroenzymology and serum amylase level.

Example 4 expression of CD47 in cholecystokinin (CCK) -induced AP acinar cell injury

Primary acinar cells: pancreatic acinar cells of 6-8 weeks C57/BL6 male mice were extracted, seeded in six-well plates, and stably cultured in a 1M Hepes medium (10% FBS), 37 degrees Celsius, 5% CO2 incubator. CCK was used to establish an in vitro cell injury (0h, 1h,2h, 6h) model.

Mouse pancreatic acinar cell carcinoma strain (266-6 cells): 266-6 cells (purchased from ATCC banks) were seeded at 25cm ² In cell culture flasks, DMEM medium (containing 10% FBS,100U/ML penicillin and 100ug/ML streptomycin) was used, 37 degrees Celsius, 5% CO ₂ Stably culturing and subculturing in an incubator. Cells in good state in logarithmic growth phase are inoculated to a six-hole plate, a new DMEM culture medium is replaced, and CCK is used for establishing an in vitro cell injury (0 h,3h,6h and 9 h) model.

Acinar cell CD47mRNA levels were observed by Q-PCR. As shown in fig. 7 (a) and (b), CD47 was consistently highly expressed on necrotic acinar cells and correlated positively with the severity of acinar cell injury. * Represents a significant difference P < 0.05, represents a significant difference P < 0.01, and represents a significant difference P < 0.001.

Example 5 use of anti-CD 47 blockers to upregulate macrophage phagocytosis without affecting acinar cell death (CCK-induced acinar cell injury model)

The mouse bone marrow-derived macrophages (BMDM) were extracted and plated on 10mm cell plates, and the cells were cultured in 1640 medium (containing 10% FBS,100U/ML penicillin and 100ug/ML streptomycin) together with M-CSF stimulating factor to induce macrophage maturation in vitro and plated on 24-well plates. Lipopolysaccharide (LPS) was used to induce BMDM polarization. 266-6 cells were treated as in example 4. The two cells are co-cultured for 6 hours, and then the cells are collected for subsequent experiments. The specific grouping is as follows:

control group: culturing for 6 hours;

IgG group: igG control antibody, incubated for 6 hours;

CCK + IgG group: 5uM cholecystokinin (CCK) and IgG control antibody were added to each well and incubated for 6 hours;

CCK + anti-CD 47 blocker group: 5uM cholecystokinin (CCK) and 5ug/ml anti-CD 47 blocking agent were added per well for 6 hours of incubation.

Each set of cells was collected and subjected to acinar cell tracer staining and immune cell staining. As shown in fig. 8 (a) - (b): the damaged acinar cells are phagocytized by macrophages, and the phagocytosis ratio of the macrophages can be remarkably increased by applying anti-CD 47 blocking agent intervention.

Example 6 use of anti-CD 47 blockers to upregulate macrophage phagocytosis without affecting acinar cell death (Staurosporine STS-induced acinar cell injury model)

BMDM cells and 266-6 cells were treated as in example 5. The specific grouping is as follows:

control group: culturing for 6 hours;

IgG group: igG control antibody, incubated for 6 hours;

CCK + IgG group: 500nM staurosporine (STS) and IgG control antibody were added to each well and incubated for 6 hours;

CCK + anti-CD 47 blocker group: 5uM staurosporine (CCK) and 5ug/ml anti-CD 47 blocking agent were added to each well and incubated for 6 hours.

Each set of cells was collected and subjected to acinar cell tracer staining and immune cell staining. As shown in fig. 9 (a) - (b): the damaged acinar cells are phagocytized by macrophages, and the phagocytosis ratio of the macrophages can be remarkably increased by applying the anti-CD 47 blocking agent for intervention.

The above contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention should not be limited thereby, and any modification made on the basis of the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims (4)

1. The application of the CD47 blocker in preparing the medicine for preventing and/or treating acute pancreatitis is characterized in that the medicine is a medicine for reducing inflammatory reaction by targeted clearing of damaged acinar cells, and the CD47 blocker is a CD47 antibody.
2. 2. The use of claim 1, wherein the medicament is a medicament that increases phagocytosis of damaged acinar cells by macrophages.
3. 3. The use of claim 1, wherein the medicament is a medicament for ameliorating pancreatic injury.
4. 4. The use of claim 1, wherein the agent is an agent that reduces serum amylase and lipase levels.

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