CN115429870B

CN115429870B - Application of interleukin 40 in preventing and treating neutropenia

Info

Publication number: CN115429870B
Application number: CN202211246205.9A
Authority: CN
Inventors: 肖敏; 刘世明; 伍志华; 楚心唯; 谢玮; 张千兵; 闵志群; 黄泽勇
Original assignee: Guangzhou Medical University
Current assignee: Guangzhou Medical University
Priority date: 2022-10-12
Filing date: 2022-10-12
Publication date: 2023-07-21
Anticipated expiration: 2042-10-12
Also published as: CN115429870A

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of interleukin 40 in preventing and treating neutropenia. In order to develop more effective neutrophilic granulocytopenia therapeutic drugs, the invention discovers that interleukin 40 (IL-40) can generate more effective signals for activating neutrophilic granulocytopenia proliferation and differentiation, not only can obviously increase the quantity of peripheral blood neutrophilic granulocytes, but also can obviously increase the percentage of the neutrophilic granulocytes in bone marrow to the nucleated cells of the bone marrow, and can effectively prevent and treat the occurrence of the neutrophilic granulocytopenia. In addition, the IL-40 has better control effect on the neutropenia induced by various factors (such as radiotherapy, chemotherapy and antibiotics), has better application prospect, and is expected to be used for controlling the neutropenia induced by various factors.

Description

Application of interleukin 40 in preventing and treating neutropenia

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of interleukin 40 in preventing and treating neutropenia.

Background

Interleukin 40 (IL-40) has recently been identified as a B cell-associated cytokine that is involved in the immune response mechanism and B cell homeostasis. IL-40, a small secreted protein (27 kDa) encoded by the C17orf99 gene (chromosome 17 open reading frame 99), was originally discovered by Catan et al in 2017. Based on its unique structural properties, IL-40 belongs to a small number of so-called "orphan" cytokines, because it does not share homology with any established cytokine family. To date, little has been done regarding the C17orf99 gene or its product IL-40 protein. It is currently known that C17orf99 or IL-40 is expressed only in mammals, especially in fetal liver, bone marrow and activated B cells with enhanced expression. IL-40 has been found to be involved in lactation and to affect IgA production by studies in IL-40 knockout mice. Moreover, IL-40 knockout mice exhibit an abnormal B cell population, suggesting that IL-40 plays an important role in B cell development. Under in vitro conditions, IL-40 is expressed by B cells following activation of anti-CD 40, which can be further enhanced by Transforming Growth Factor (TGF) - β. Recent studies have found that C17orf99 expression is down-regulated in human airway epithelial cells co-cultured with IL-38-treated macrophages, suggesting that IL-40 has an important role in inflammation. In addition, there are studies to find that C17orf99 is one of four autoantigens that distinguish autoimmune hepatitis, suggesting that IL-40 is involved in autoimmune inflammation, but IL-40 has many unknown roles in the regulation of immune mechanisms.

Neutrophils are the most abundant cells in the blood circulation, accounting for 60% to 70% of human leukocytes, and the majority of immune cells. The cells are produced by Hematopoietic Stem Cells (HSCs) in Bone Marrow (BM), proliferate and differentiate from HSCs to form mature neutrophils with a high number of neutrophils. These particles contain proteases which can cause neutrophils to kill invading microorganisms (e.g., bacteria and fungi) and form a first line of defense against invading pathogens. Thus, neutrophils play a key role in innate immunity.

Neutropenia is defined as an abnormally low number of circulating neutrophils in the peripheral blood. Since neutrophils account for the majority of peripheral blood leukocytes, they can migrate to the infected area, phagocytize, digest and destroy microorganisms, playing a key role in the host's defense mechanisms against infection. Thus, a decrease in neutrophil count would expose the patient to risk of infection. It has been found that the severity of the risk of neutropenia-related infection is inversely proportional to Absolute Neutrophil Count (ANC) and directly proportional to the duration of neutropenia. Forty years ago, a scholars have published neutral granulesCytopenia reports, defining quantitative risk infections, these principles are still applicable today: generally speaking, ANC<100 cells/mm ³ Will develop a severe infection within 1 to 4 weeks of the onset of neutropenia; while those ANCs<1000 cells/mm ³ With a greatly increased risk of infection with prolonged infection time.

The primary causes of neutropenia (unknown reasons) are rare, and most of them are secondary. Common pathogenesis is: reduced proliferation of granulocytes under the influence of infection, ionizing radiation, anti-tumor or other drugs; granulocyte maturation disorder caused by megaloblastic anemia, myelodysplastic syndrome, etc.; the granulocytic life reduction caused by hyperthyroidism, infection, inflammation or certain drugs; granulocytes are abnormally distributed, granulocytes in the circulation pool migrate to the marginal pool, the granulocyte count is reduced, and after adrenal hormone is injected, the granulocytes enter the circulation pool from the marginal pool, and the count is recovered to be normal, wherein the condition is related to factors such as allergy, viremia, hemolysis, hemodynamic changes and the like.

Chemotherapeutic agents, also known as cytotoxic agents, have been used in antitumor therapy since 1940 and have played an important role in tumor therapy. The mechanism of action of chemotherapeutic agents is complex, including affecting DNA chemistry, inhibiting nucleic acid synthesis, acting on nucleic acid transcription and DNA replication, interfering with mitotic tubulin synthesis, and the like. However, the target of chemotherapeutic agents is non-specific and normal cells are also affected, which can cause unavoidable damage to the body during chemotherapy, such as alopecia, myelosuppression, and gastrointestinal toxicity. Leukopenia is very common in the chemotherapy process, and in clinical work, the smooth progress of chemotherapy is often influenced due to the side effect, so that the curative effect of the chemotherapy is reduced, and the life quality of a patient is directly influenced. Therefore, preventing and relieving the phenomenon of neutrophil inhibition after chemotherapy, promoting the quantity and function recovery of bone marrow and peripheral blood neutrophil, and becoming the key for ensuring smooth completion of chemotherapy and improving the curative effect of chemotherapeutic drugs.

Radiation therapy has been a significant means of clinical treatment of tumors at this stage for over 100 years. Tumor radiotherapy mainly comprises three parts in clinic, namely radiotherapy physics (radiotherapy technology), clinical radiobiology and tumor radiotherapy. Among them, radiation therapy technology is the most rapidly developed at the current stage and is considered as the most important means for the comprehensive treatment of tumors. Although the radiotherapy has ideal treatment effect, the side effect is obvious, because the normal tissues around the tumor cells are irradiated to different degrees while the radioactive rays irradiate the tumor cells in the radiotherapy process. Therefore, neutropenia is one of the common complications after radiotherapy, and how to effectively cope with the disease, ensures that the radiotherapy and chemotherapy of tumor patients can be normally carried out, and becomes one of the difficulties to be solved in the current clinical treatment process of malignant tumors.

During clinical anti-infective therapy, the patient's body temperature may suddenly rise and be accompanied by a decrease in neutrophils after a period of antibiotic treatment. Chloramphenicol is a broad-spectrum antibiotic mainly used for intestinal tract infection, serious bacterial infection and rickettsia diseases, such as typhoid, paratyphoid, bacillary dysentery, bacillus pneumonia, meningitis, typhoid, etc. Chloramphenicol also inhibits bone marrow hematopoietic function when destroying pathogenic microorganisms in the body, causing a decrease in erythrocytes, leukocytes, and platelets. The main link of chloramphenicol action on the bone marrow system is to inhibit protein synthesis in mitochondria of bone marrow hematopoietic cells. If the patient is febrile and has a concomitant neutropenia due to antibiotics rather than infections, simply increasing the anti-infective strength is not effective, but rather the patient's condition is further exacerbated.

In the in vitro studies of hematopoietic cells, it was found that some cytokines stimulated the formation of cell colonies in semi-solid media by different hematopoietic stem cells, and such factors were designated Colony Stimulating Factors (CSF). According to the scope of action of colony stimulating factors, it can be seen that they are named granulocyte CSF (G-CSF), macrophage CSF (M-CSF), granulocyte and macrophage CSF (GM-CSF) and multipotent colony stimulating factor (MultiCSF, also known as IL-3), respectively. These factors play a role in promoting proliferation and differentiation of hematopoietic stem cells at different developmental stages, and are essential stimulators for hematopoiesis. Currently, the CSF used clinically to prevent granulocytopenia is mainly granulocyte and macrophage colony stimulating factor (GM-CSF) or granulocyte colony stimulating factor (G-CSF). Both have obvious prevention and treatment effects on neutropenia caused by chemotherapy, but with the wide clinical application of GM-CSF and G-CSF, problems are gradually revealed.

Currently, there are two main classes of recombinant human neutrophil-stimulating growth factors (rhG-CSF) for therapeutic use, short-acting and long-acting, respectively. The short-acting type needs to be injected for a plurality of times every day or every week, and the long-acting type does not need to be injected for a plurality of times, so that the use of patients is facilitated. However, neither long-acting nor short-acting G-CSF can meet the needs of patients.

Sierra et al designed a randomized, double-blind trial to compare the effects of short-and long-acting G-CSF on prevention of neutropenia following chemotherapy in acute leukemia patients (A single dose of pegfilgrastim compared with daily filgrastim for supporting neutrophil recovery in patients treated for low-to-intermediate risk acute myeloid leukemia: results from a randomized, double-blind, phase 2 trial,BMC Cancer 2008,8:195), starting with recombinant human G-CSF the day before the end of chemotherapy in acute leukemia patients, for three consecutive days. Two groups, one group being short acting G-CSF (n=41) and the other group being long acting G-CSF (n=42), showed that, although all patients were treated with G-CSF immediately before the end of chemotherapy, severe neutropenia symptoms occurred after G-CSF withdrawal and continued for a period of up to 3 weeks, with no significant difference in the efficacy of the two groups. This result suggests that recombinant human G-CSF has not yet achieved satisfactory clinical therapeutic results.

In view of the foregoing, there is a strong need in the art to develop more effective therapies for neutropenia in order to more effectively reduce the incidence of neutropenia and shorten the duration of neutropenia.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention discovers that IL-40 can not only obviously increase the quantity of peripheral blood neutrophils, but also obviously increase the percentage of the neutrophils in bone marrow to bone marrow nucleated cells, and can effectively prevent and treat various neutropenia.

In order to achieve the above purpose, the present invention is realized by the following technical scheme:

the invention provides application of interleukin 40 in preparing a medicament for preventing and treating neutropenia.

Interleukin 40 has been shown to have a significantly better effect on the treatment of neutropenia than existing recombinant proteins in a BALB/C mouse model.

Preferably, the control is to increase the number of peripheral blood neutrophils and/or increase the percentage of neutrophils in bone marrow that are nucleated cells in bone marrow.

Preferably, the neutropenia comprises neutropenia caused by radiotherapy, chemotherapy and antibiotics.

The causes of neutropenia are many, the drugs applicable to the neutropenia with different causes are generally different, and a broader-spectrum neutropenia drug is fresh. For this reason, most of the current drugs can only treat the neutropenia induced by one factor, and the effect on the neutropenia induced by other factors is not ideal. The research shows that interleukin 40 (IL-40) has better prevention and treatment effects on the neutropenia induced by various factors (such as radiotherapy, chemotherapy and antibiotics), can obviously increase the quantity of peripheral blood neutrophils, can obviously increase the percentage of the neutrophils in bone marrow to the bone marrow nucleated cells, has better application prospect, and is expected to be used for preventing and treating the neutropenia induced by various factors.

More preferably, the neutropenia caused by radiotherapy is radiation-induced neutropenia.

More preferably, the chemotherapy-induced neutropenia is cytarabine-induced neutropenia.

According to the invention, compared with recombinant mouse G-CSF, recombinant mouse IL-40 can stimulate proliferation and differentiation of bone marrow neutrophils more quickly and effectively. Therefore, the occurrence rate of neutropenia can be reduced more effectively and the duration of neutropenia can be shortened effectively after the patient starts to use the medicine one day before the chemotherapy is finished, and the medicine has no serious side effect.

More preferably, the antibiotic-induced neutropenia is chloramphenicol-induced neutropenia.

The invention also provides a medicine for preventing and treating neutropenia, which takes interleukin 40 as a main active ingredient.

The recombinant IL-40 of the invention can be used alone or in combination with other compounds. When combined, safe and effective amount of recombinant human IL-40 is applied to a patient in need of treatment, wherein the dosage is pharmaceutically acceptable, and the specific dosage is comprehensively considered by combining the factors of the administration route, the health condition of the patient and the like.

Preferably, the application form of the medicine is enriched, so that the medicine is applicable to different ranges, and the medicine also comprises a pharmaceutically acceptable carrier.

Further, the carrier is a functional pharmaceutical adjuvant acceptable in the pharmaceutical field and comprises a surfactant, a suspending agent, an emulsifier and some novel pharmaceutical polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid-polylactic acid copolymer (PLGA), hyaluronic acid and the like. Excipients such as diluents, binders, lubricants, disintegrants, co-solvents, stabilizers and the like may also be included.

Preferably, to increase the applicable range of the medicine, the dosage forms of the medicine include, but are not limited to, tablets, sachets, granules, dripping pills, liquid agents and injections. Pharmaceutical formulations may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically), and if some drugs are unstable under gastric conditions, they may be formulated as enteric coated tablets.

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

in order to develop more effective neutrophilic granulocytopenia therapeutic drugs, the invention discovers that interleukin 40 (IL-40) can generate more effective signals for activating neutrophilic granulocytopenia proliferation and differentiation, not only can obviously increase the quantity of peripheral blood neutrophilic granulocytes, but also can obviously increase the percentage of the neutrophilic granulocytes in bone marrow to the nucleated cells of the bone marrow, and can effectively prevent and treat the occurrence of the neutrophilic granulocytopenia. In addition, the IL-40 has better control effect on the neutropenia induced by various factors (such as radiotherapy, chemotherapy and antibiotics), has better application prospect, and is expected to be used for controlling the neutropenia induced by various factors.

Drawings

FIG. 1 is a graph showing the results of the counts of peripheral blood neutrophils in BALB/C mice in cytarabine-induced neutropenia of recombinant mouse G-CSF and recombinant mouse IL-40;

FIG. 2 shows the percent change of bone marrow neutrophils to bone marrow nucleated cells in cytarabine-induced neutropenia for recombinant mouse G-CSF and recombinant mouse IL-40;

FIG. 3 is a statistical histogram derived from FIG. 2;

FIG. 4 is a graph showing the results of the counts of peripheral blood neutrophils in BALB/C mice in radiation-induced neutropenia of recombinant mouse G-CSF and recombinant mouse IL-40;

FIG. 5 shows the percentage change of bone marrow neutrophils to bone marrow nucleated cells in radiation induced neutropenia of recombinant mouse G-CSF and recombinant mouse IL-40;

FIG. 6 is a statistical histogram derived from FIG. 5;

FIG. 7 is a graph showing the results of the counts of peripheral blood neutrophils in BALB/C mice in chloramphenicol-induced neutropenia with recombinant mouse G-CSF and recombinant mouse IL-40;

FIG. 8 shows the percent change of bone marrow neutrophils to bone marrow nucleated cells in chloramphenicol-induced neutropenia of recombinant mouse G-CSF and recombinant mouse IL-40;

fig. 9 is a statistical histogram derived from fig. 8.

Detailed Description

The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

The experimental procedures described in the examples below, unless otherwise specified, are generally carried out according to conventional procedures, such as those described in the molecular cloning laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) by Sambrook et al. The test materials used in the examples described below, unless otherwise specified, are commercially available from conventional sources. Interleukin 40 as used in the examples was recombinant mouse IL-40, free of endotoxin, 99% pure or more, purchased from MyBioSource, inc., U.S.A..

EXAMPLE 1 Effect of IL-40 and G-CSF on the Cytosine induced BALB/C mouse model of neutropenia

BALB/C mice, purchased from Shanghai south mode center, 20G, male and female, were randomly assigned, 10 each, and divided into four experimental groups, control group, cytarabine group, cytarabine+G-CSF group, cytarabine+IL-40 group. Control mice were intraperitoneally injected with PBS buffer (100. Mu.L/day) for 10 days; cytarabine group mice were intraperitoneally injected with PBS-dissolved cytarabine (ara-c, 100 mg/kg/day) for 10 days; cytarabine+g-CSF group mice were given an intraperitoneal injection of cytarabine (100 mg/kg/day) on the first day, and recombinant mouse G-CSF (100 μg/kg/day) was given an intraperitoneal injection of cytarabine (100 mg/kg/day) on the second day, both for 9 days; cytarabine + IL-40 group mice were given an intraperitoneal injection of cytarabine (100 mg/kg/day) on the first day, and the recombinant mouse IL-40 (50 μg/kg/day) was given an intraperitoneal injection of cytarabine (100 mg/kg/day) on the second day, followed by a continuous injection of both for 9 days. On day 11, peripheral blood and bone marrow were collected from mice, and neutrophil counts were measured with a whole blood cell analyzer, respectively, and the percentage of bone marrow neutrophil occupied nuclear cells was measured with a flow cytometer.

The results are shown in figures 1-3, with the absolute value of peripheral blood neutrophils significantly decreasing after 10 consecutive days of cytarabine administration in cytarabine group mice. Cytarabine+G-CSF group mice, peripheral blood neutrophil absolute value did not return to the mouse control group level on day 11. Whereas the absolute value of peripheral blood neutrophils in mice of the group consisting of cytarabine + IL-40 exceeded the level of control group, which was 2.08 times that of the group consisting of cytarabine + G-CSF. The percentage of bone marrow neutrophil-occupied nuclear cells of the cytarabine+IL-40 group is 1.58 times that of the cytarabine+G-CSF group, and exceeds the percentage of bone marrow neutrophil-occupied nuclear cells of the control group.

The results show that in a mouse model of cytarabine-induced neutropenia, the treatment effect of IL-40 is obviously better than that of G-CSF, the quantity of peripheral blood neutrophils can be obviously increased, the percentage of the neutrophils in bone marrow to bone marrow nucleated cells can be obviously increased, and the IL-40 is indicated to be capable of effectively preventing the occurrence of chemotherapy drug-induced neutropenia.

Examples 2 IL-40 and G-CSF Effect on radiation (irradialization) induced BALB/C mouse model of neutropenia

BALB/C mice, purchased from Shanghai southwest model center, 20G, each half of male and female, were randomly assigned, and each group was divided into four experimental groups, namely, a control group, an irradiation group, an irradiation+G-CSF group, and an irradiation+IL-40 group. Control mice received no irradiation; mice in the irradiation group receive 10 days of irradiation with total amount of 5 Gy; group of mice irradiated +G-CSF received 0.5Gy of radiation on the first day, starting on the second day, and 0.5Gy of radiation daily and intraperitoneal injection of recombinant mouse G-CSF (100. Mu.g/kg/day) was performed for 9 consecutive days; the mice in the radiation + IL-40 group received 0.5Gy radiation on the first day and 0.5Gy radiation daily and intraperitoneal injection of recombinant mouse IL-40 (50. Mu.g/kg/day) on 9 consecutive days, starting on the second day. On day 11, peripheral blood and bone marrow were collected from mice, and neutrophil counts were measured with a whole blood cell analyzer, respectively, and the percentage of bone marrow neutrophil occupied nuclear cells was measured with a flow cytometer.

The results are shown in fig. 4-6, and the absolute value of peripheral blood neutrophils is significantly reduced after 10 consecutive days of irradiation in the irradiated mice. Mice in the +G-CSF group were irradiated and peripheral blood neutrophil absolute values did not return to the mouse control level on day 11. Whereas the absolute value of peripheral blood neutrophils of mice in the +IL-40 group had exceeded the level of control group, which was 2.16 times that of the +G-CSF group. The percentage of bone marrow neutrophils occupied by nuclear cells in the irradiated + IL-40 group was 1.48 times that in the irradiated + G-CSF group, exceeding the percentage of bone marrow neutrophils occupied by nuclear cells in the control group.

The results show that in the mouse model of the radiation-induced neutropenia, the treatment effect of the IL-40 is obviously better than that of the G-CSF, the quantity of peripheral blood neutrophils can be obviously increased, the percentage of the neutrophils in bone marrow to the bone marrow nucleated cells can be obviously increased, and the IL-40 is indicated to be capable of effectively preventing the radiation-induced neutropenia.

EXAMPLE 3 Effect of IL-40 and G-CSF on chloramphenicol (Chloramphenicol) -induced BALB/C mouse model of neutropenia

BALB/C mice, purchased from Shanghai south mode center, 20G, male and female, randomly distributed, 10 each, divided into four experimental groups, control group, chloramphenicol group, chloramphenicol+G-CSF group, chloramphenicol+IL-40 group. The control mice were intraperitoneally injected with solvent for 21 days; chloramphenicol group mice were intraperitoneally injected with chloramphenicol (200 mg/kg/day) for 21 days; chloramphenicol + G-CSF group mice were intraperitoneally injected with chloramphenicol on the first day, and starting on the second day, intraperitoneally injected with recombinant mouse G-CSF (100 μg/kg/day) and chloramphenicol (200 mg/kg/day), both for 20 days; chloramphenicol + IL-40 group mice were intraperitoneally injected with chloramphenicol (200 mg/kg/day) on the first day, and on the second day, recombinant mice IL-40 (50 μg/kg/day) and chloramphenicol (200 mg/kg/day) were intraperitoneally injected, both for 20 days. On day 21, peripheral blood and bone marrow were collected from mice, and neutrophil counts were measured with a whole blood cell analyzer, respectively, and the percentage of bone marrow neutrophil occupied nuclear cells was measured with a flow cytometer.

As a result, as shown in FIGS. 7 to 9, after continuous intraperitoneal injection of chloramphenicol for 21 days in chloramphenicol mice, the absolute value of peripheral blood neutrophils was significantly decreased. The chloramphenicol + G-CSF group mice did not return to the mouse control group level on day 22 of the absolute value of peripheral blood neutrophils. The absolute value of peripheral blood neutrophil of the mice in the group of chloramphenicol and IL-40 exceeds the level of the control group, which is 2.02 times that of the mice in the group of chloramphenicol and G-CSF. The percentage of bone marrow neutrophils occupied by nuclear cells in the group of chloramphenicol + IL-40 mice was 1.31 times that of the group of chloramphenicol + G-CSF, exceeding the percentage of bone marrow neutrophils occupied by nuclear cells in the control group.

The results show that in a mouse model of chloramphenicol-induced neutropenia, the treatment effect of IL-40 is obviously better than that of G-CSF, so that the quantity of peripheral blood neutrophils can be obviously increased, the percentage of neutrophils in bone marrow to bone marrow nucleated cells can be obviously increased, and the IL-40 is indicated to be capable of effectively preventing the occurrence of chloramphenicol-induced neutropenia.

The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.

Claims

1. The application of interleukin 40 in preparing a medicament for preventing and treating neutropenia is characterized in that the neutropenia is selected from the group consisting of radiotherapy, chemotherapy and neutropenia caused by antibiotics.

2. The use according to claim 1, wherein the control is an increase in the number of peripheral blood neutrophils and/or an increase in the percentage of neutrophils in bone marrow nucleated cells.

3. The use according to claim 1, wherein the radiation therapy induced neutropenia is radiation induced neutropenia.

4. The use according to claim 1, wherein the chemotherapy-induced neutropenia is cytarabine-induced neutropenia.

5. The use according to claim 1, wherein the antibiotic-induced neutropenia is chloramphenicol-induced neutropenia.