CN115429870B - Application of interleukin 40 in prevention and treatment of neutropenia - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and in particular relates to the application of interleukin 40 in preventing and treating neutropenia. In order to develop a more effective drug for treating neutropenia, the present invention has found through research that interleukin 40 (IL-40) can produce more effective signals for activating the proliferation and differentiation of neutrophils, can not only significantly increase the number of neutrophils in peripheral blood, but also significantly increase the percentage of neutrophils in bone marrow, which can effectively prevent the occurrence of neutropenia. Moreover, IL-40 has a good preventive effect on neutropenia induced by various factors (such as radiotherapy, chemotherapy, and antibiotics), showing a good application prospect, and is expected to be used to prevent and treat neutropenia induced by various factors.

Description

Application of interleukin 40 in prevention and treatment of neutropenia

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to the application of interleukin 40 in preventing and treating neutropenia.

Background technique

Interleukin 40 (IL-40) was recently identified as a B cell-associated cytokine involved in immune response mechanisms and B cell homeostasis. IL-40, as a small secreted protein (27 kDa) encoded by the C17orf99 gene (chromosome 17 open reading frame 99), was originally discovered by Catalan et al. in 2017. Based on its unique structural properties, IL-40 belongs to a small number of so-called "orphan" cytokines because it has no homology to any established cytokine family. So far, there are few studies on C17orf99 gene or its product IL-40 protein. It is currently known that C17orf99 or IL-40 is only expressed in mammals, especially in fetal liver, bone marrow and activated B cells. Studies have found that IL-40 is related to lactation and affects IgA production by studying IL-40 knockout mice. Moreover, IL-40 knockout mice exhibit abnormal B cell populations, suggesting that IL-40 plays an important role in B cell development. In vitro, IL-40 is expressed by B cells following anti-CD40 activation, which can be further enhanced by transforming growth factor (TGF)-β. A recent study found that the expression of C17orf99 was downregulated in human airway epithelial cells co-cultured with IL-38-treated macrophages, suggesting that IL-40 plays an important role in inflammation. In addition, studies have found that C17orf99 is also one of the four autoantigens that distinguish autoimmune hepatitis, indicating that IL-40 is related to autoimmune inflammation, but IL-40 still has many unknown roles in the regulation of immune mechanisms.

Neutrophils are the most abundant cells in the blood circulation, comprising 60 to 70 percent of the body's white blood cells, and the majority of immune cells. The cells are generated from hematopoietic stem cells (HSCs) in the bone marrow (BM), from which they proliferate and differentiate to form mature neutrophils with numerous neutrophil granules. These granules contain proteases, which enable neutrophils to deliver a lethal blow to invading microbes, such as bacteria and fungi, and form the first line of defense against invading pathogens. Therefore, neutrophils play a key role in innate immunity.

Neutropenia is defined as abnormally low numbers of circulating neutrophils in the peripheral blood. Since neutrophils account for the majority of peripheral blood leukocytes, they can migrate to infected areas, engulf, digest, and destroy microorganisms, playing a key role in the host's defense mechanism against infection. Therefore, a decrease in the number of neutrophils puts the patient at risk of infection. The severity of neutropenia-associated infection risk was found to be inversely proportional to absolute neutrophil count (ANC) and directly proportional to the duration of neutropenia. Forty years ago, some scholars published a report on neutropenia, which determined the quantitative risk of infection, and these principles still apply today: generally speaking, patients with ANC<100 cells/ ^mm3 will develop severe infection within 1 to 4 weeks of the onset of neutropenia; while those patients with ANC<1000 cells/ ^mm3 , the risk of infection increases greatly with the prolongation of infection time.

Primary neutropenia (with unknown cause) is rare, mostly secondary. The common pathogenesis is: decreased granulocyte hyperplasia caused by infection, ionizing radiation, anti-tumor or other drugs; granulocyte maturation disorder caused by megaloblastic anemia, myelodysplastic syndrome, etc.; hypersplenism, infection, inflammation or certain drugs The shortened granulocyte lifespan; factors such as hemodynamic changes.

Chemotherapy drugs, also known as cytotoxic drugs, have been used in antitumor therapy since 1940 and have played an important role in tumor treatment. The mechanism of action of chemotherapeutic drugs is complex, including affecting the chemical structure of DNA, inhibiting nucleic acid synthesis, acting on nucleic acid transcription and DNA replication, and interfering with mitotic tubulin synthesis. However, the targets of chemotherapy drugs are non-specific, and normal cells will also be affected, causing inevitable damage to the body during chemotherapy, such as hair loss, bone marrow suppression, and gastrointestinal toxicity. Leukopenia is very common during chemotherapy. In clinical work, this side effect often affects the smooth progress of chemotherapy, which leads to a decrease in the efficacy of chemotherapy and directly affects the quality of life of patients. Therefore, preventing and alleviating neutrophil suppression after chemotherapy and promoting the recovery of the number and function of neutrophils in bone marrow and peripheral blood have become the key to ensure the smooth completion of chemotherapy and improve the efficacy of chemotherapy drugs.

Radiation therapy has a history of more than 100 years and is an important means of clinical treatment of tumors at this stage. Oncology radiotherapy mainly includes three parts clinically, namely radiation therapy physics (radiotherapy technology), clinical radiobiology and tumor radiation therapy. Among them, radiotherapy technology is the most rapidly developed at the current stage and is considered to be the most important means of comprehensive treatment of tumors. Although the therapeutic effect of radiotherapy is ideal, the side effects are also very obvious, because in the process of radiotherapy, while the radiation is irradiating tumor cells, the normal tissues around the tumor cells are also irradiated to varying degrees. Therefore, neutropenia is one of the common complications after radiotherapy. How to effectively deal with this disease and ensure the normal progress of radiotherapy and chemotherapy in cancer patients has become one of the problems to be solved in the current clinical treatment of malignant tumors.

In the course of clinical anti-infection treatment, after a period of antibiotic treatment, the patient's body temperature will suddenly rise and be accompanied by a decrease in neutrophils. Chloramphenicol is a broad-spectrum antibiotic, mainly used for intestinal infections, severe bacterial infections and rickettsial diseases, such as typhoid, paratyphoid, bacillary dysentery, bacillary pneumonia, meningitis, typhus, etc. When chloramphenicol eliminates pathogenic microorganisms in the body, it will also inhibit the hematopoietic function of the bone marrow, causing red blood cells, white blood cells, and platelets to decrease. The main link of chloramphenicol's effect on the bone marrow system is to inhibit the protein synthesis in the mitochondria of bone marrow hematopoietic cells. If the patient has fever and neutropenia at this time, the cause is antibiotics rather than infection, then simply increasing the anti-infection efforts will not only be ineffective, but will further aggravate the patient's condition.

In the in vitro study of hematopoietic cells, it was found that some cytokines could stimulate different hematopoietic stem cells to form cell colonies in semi-solid medium, and these factors were named colony-stimulating factors (CSF). According to the scope of action of colony-stimulating factor, it can be seen that they are respectively named as granulocyte CSF (G-CSF), macrophage CSF (M-CSF), granulocyte and macrophage CSF (GM-CSF) and multipotent colony-stimulating factor (multi-CSF, also known as IL-3). These factors play a role in promoting proliferation and differentiation of hematopoietic stem cells at different developmental stages, and are essential stimulating factors for hematopoiesis. At present, the CSF used clinically to prevent and treat neutropenia 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 application of GM-CSF and G-CSF in clinical practice, some problems have gradually emerged.

At present, there are mainly two types of recombinant human neutrophil-stimulating growth factor (rhG-CSF) for treatment, which are short-acting and long-acting. Among them, the short-acting type requires multiple injections every day or every week, while the long-acting type does not require multiple injections, which is convenient for patients to use. 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 effect of short-acting and long-acting G-CSF in preventing neutropenia after chemotherapy in patients with acute leukemia (A single dose of pegfilgrastim compared with daily filgrastim for supporting neutrophil recovery in patient treated for low-to-intermediate risk acute myeloid leukemia:res ults from arandomized, double-blind, phase 2 trial, BMC Cancer 2008, 8:195), patients with acute myeloid leukemia started to use recombinant human G-CSF one day before the end of chemotherapy, and used it for three consecutive days. They were divided into two groups, one was the short-acting G-CSF group (n=41) and the other was the long-acting G-CSF group (n=42). It was found that although all patients were treated with G-CSF before the end of chemotherapy, severe neutropenia still occurred after G-CSF was stopped and lasted for up to 3 weeks. There was no significant difference in efficacy between the two groups. This result suggests that recombinant human G-CSF has not yet achieved a satisfactory clinical therapeutic effect.

To sum up, it can be seen that there is an urgent need in this field to develop more effective drugs for the treatment of neutropenia in order to more effectively reduce the incidence of neutropenia and shorten the duration of neutropenia.

Contents of the invention

In order to overcome the above-mentioned deficiencies in the prior art, the present invention has found through research that IL-40 can not only significantly increase the number of neutrophils in peripheral blood, but also significantly increase the percentage of neutrophils in bone marrow to bone marrow nucleated cells, and can effectively prevent and treat the occurrence of various neutropenias.

To achieve the above object, the present invention is achieved through the following technical solutions:

The invention provides the application of interleukin 40 in the preparation of medicine for preventing and treating neutropenia.

In the BALB/C mouse model, interleukin 40 has been confirmed to have a significantly better effect on the treatment of neutropenia than existing recombinant proteins.

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

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

There are many inducing factors for neutropenia, and the drugs applicable to different causes of neutropenia are generally different, and there are few broad-spectrum neutropenia drugs. It is also for this reason that most current drugs can only target neutropenia induced by a certain factor, and the effect on neutropenia induced by other factors is not ideal. However, the present invention has found through research that interleukin 40 (IL-40) has a good preventive effect on neutropenia induced by various factors (such as radiotherapy, chemotherapy, and antibiotics). It can not only significantly increase the number of neutrophils in peripheral blood, but also significantly increase the percentage of neutrophils in bone marrow.

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

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

The present invention finds through research that, compared with recombinant mouse G-CSF, recombinant mouse IL-40 can stimulate the proliferation and differentiation of bone marrow neutrophils faster and more effectively. Therefore, it can more effectively reduce the incidence of neutropenia and shorten the duration of neutropenia, without serious side effects, when it is used one day before the end of chemotherapy.

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

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

The recombinant IL-40 of the present invention can be used alone or in combination with other compounds. In the case of combined medication, a safe and effective amount of recombinant human IL-40 is applied to patients in need of treatment, and the dosage is a pharmaceutically approved dosage, and the specific dosage should be comprehensively considered in combination with factors such as the route of administration and the health status of the patient.

Preferably, in order to enrich the application form of the drug so that it can be used in different ranges, the drug also includes a pharmaceutically acceptable carrier.

Further, the carrier is an acceptable functional pharmaceutical excipient in the pharmaceutical field, including surfactants, suspending agents, emulsifiers, and some new pharmaceutical polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid polylactic acid copolymer (PLGA), hyaluronic acid, etc. Excipients such as diluents, binders, lubricants, disintegrants, solubilizers, and stabilizers may also be included.

Preferably, in order to improve the scope of application of the medicine, the dosage form of the medicine includes but not limited to tablet, capsule, granule, drop pill, liquid and injection. Pharmaceutical formulations can be administered orally or parenterally (eg intravenously, subcutaneously, intraperitoneally or topically), and if certain drugs are unstable under gastric conditions, they can be prepared as enteric-coated tablets.

Compared with prior art, the beneficial effect of the present invention is:

In order to develop more effective medicines for treating neutropenia, the present invention has found through research that interleukin 40 (IL-40) can produce more effective signals for activating the proliferation and differentiation of neutrophils, which can not only significantly increase the number of neutrophils in peripheral blood, but also significantly increase the percentage of neutrophils in bone marrow, which can effectively prevent and treat the occurrence of neutropenia. Moreover, IL-40 has a good preventive effect on neutropenia induced by various factors (such as radiotherapy, chemotherapy, antibiotics), shows a good application prospect, and is expected to be used to prevent and treat neutropenia induced by various factors.

Description of drawings

Fig. 1 is recombinant mouse G-CSF and recombinant mouse IL-40 in cytarabine-induced neutropenia, BALB/C mouse peripheral blood neutrophil count result;

Figure 2 shows the changes in the percentage of bone marrow neutrophils accounting for bone marrow nucleated cells in cytarabine-induced neutropenia with recombinant mouse G-CSF and recombinant mouse IL-40;

Fig. 3 is the statistical histogram obtained by Fig. 2;

Fig. 4 is recombinant mouse G-CSF and recombinant mouse IL-40 in radiation-induced neutropenia, the counting result of peripheral blood neutrophils of BALB/C mouse;

Figure 5 shows the changes in the percentage of bone marrow neutrophils accounting for bone marrow nucleated cells in irradiation-induced neutropenia with recombinant mouse G-CSF and recombinant mouse IL-40;

Fig. 6 is the statistical histogram obtained by Fig. 5;

Fig. 7 is recombinant mouse G-CSF and recombinant mouse IL-40 in the neutropenia induced by chloramphenicol, the enumeration result of neutrophils in the peripheral blood of BALB/C mouse;

Figure 8 shows the change in the percentage of bone marrow neutrophils accounting for bone marrow nucleated cells in chloramphenicol-induced neutropenia with recombinant mouse G-CSF and recombinant mouse IL-40;

FIG. 9 is a statistical histogram obtained from FIG. 8 .

Detailed ways

Specific embodiments of the present invention will be further described below. It should be noted here that the descriptions of these embodiments are used to help understand the present invention, but are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not constitute a conflict with each other.

The experimental methods in the following examples, unless otherwise specified, are generally implemented according to conventional operations, such as the method described in the Molecular Cloning Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) written by Sambrook et al. The test materials used in the following examples, unless otherwise specified, can be purchased through conventional commercial channels. The interleukin 40 used in the examples is recombinant mouse IL-40, free of endotoxin, with a purity of over 99%, purchased from MyBioSource, USA.

Example 1 Effect of IL-40 and G-CSF on Cytarabine-Induced BALB/C Mouse Neutropenia Model

BALB/C mice were purchased from Shanghai Nanfang Modeling Center. One 20 g mouse was half male and half female. They were randomly assigned, 10 mice in each group, and divided into four experimental groups, namely the control group, the cytarabine group, the cytarabine+G-CSF group, and the cytarabine+IL-40 group. The mice in the control group were intraperitoneally injected with PBS buffer (100 μL/day) for a total of 10 days; the mice in the cytarabine group were injected intraperitoneally with cytarabine (ara-c, 100 mg/kg/day) dissolved in PBS for a total of 10 days; 100mg/kg/day), for 9 consecutive days; the mice in the cytarabine+IL-40 group were injected with cytarabine (100mg/kg/day) intraperitoneally on the first day, and from the second day, they were intraperitoneally injected with recombinant mouse IL-40 (50μg/kg/day) and cytarabine (100mg/kg/day) for 9 consecutive days. On the 11th day, the peripheral blood and bone marrow of the mice were collected, and the neutrophil count was detected by a whole blood analyzer, and the percentage of nucleated cells occupied by neutrophils in the bone marrow was detected by a flow cytometer.

The results are shown in Figures 1-3, after administration of cytarabine to the mice in the cytarabine group for 10 consecutive days, the absolute value of peripheral blood neutrophils decreased significantly. In the cytarabine+G-CSF group, the absolute value of peripheral blood neutrophils did not return to the level of the control group on day 11. However, the absolute value of neutrophils in the peripheral blood of the mice in the cytarabine+IL-40 group has exceeded the level of the control group, which is 2.08 times that of the cytarabine+G-CSF group. The percentage of nuclear cells occupied by bone marrow neutrophils in the cytarabine+IL-40 group was 1.58 times that of the cytarabine+G-CSF group, exceeding the percentage of bone marrow neutrophils in the control group.

The above results show that in the mouse model of cytarabine-induced neutropenia, the therapeutic effect of IL-40 is significantly better than that of G-CSF, which can not only significantly increase the number of peripheral blood neutrophils, but also significantly increase the percentage of neutrophils in bone marrow nucleated cells, suggesting that IL-40 can effectively prevent the occurrence of chemotherapy-induced neutropenia.

Example 2 Effects of IL-40 and G-CSF on BALB/C Mouse Neutropenia Model Induced by Irradiation

BALB/C mice were purchased from Shanghai Nanfang Modeling Center. One 20 g mouse, half male and half male, were randomly allocated, 10 mice in each group, and divided into four experimental groups, namely control group, irradiation group, irradiation+G-CSF group, and irradiation+IL-40 group. The mice in the control group did not receive irradiation; the mice in the irradiation group received a total of 5 Gy irradiation for 10 days; the mice in the irradiation + G-CSF group received 0.5 Gy irradiation on the first day, and then 0.5 Gy irradiation per day and intraperitoneal injection of recombinant mouse G-CSF (100 μg/kg/day) for 9 consecutive days; Inject recombinant mouse IL-40 (50 μg/kg/day). On the 11th day, the peripheral blood and bone marrow of the mice were collected, and the neutrophil count was detected by a whole blood analyzer, and the percentage of nucleated cells occupied by neutrophils in the bone marrow was detected by a flow cytometer.

The results are shown in Figures 4-6, after 10 consecutive days of irradiation in the irradiation group, the absolute value of peripheral blood neutrophils decreased significantly. The absolute value of neutrophils in the peripheral blood of the mice in the irradiation+G-CSF group did not return to the level of the control group on day 11. However, the absolute value of peripheral blood neutrophils in the irradiation+IL-40 group was 2.16 times higher than that in the irradiation+G-CSF group. The percentage of nucleated cells occupied by bone marrow neutrophils in the irradiation + IL-40 group was 1.48 times that of the irradiation + G-CSF group, exceeding the percentage of bone marrow neutrophils in the control group.

The above results show that in the mouse model of radiation-induced neutropenia, the therapeutic effect of IL-40 is significantly better than that of G-CSF. It can not only significantly increase the number of peripheral blood neutrophils, but also significantly increase the percentage of neutrophils in bone marrow nucleated cells, suggesting that IL-40 can effectively prevent the occurrence of radiation-induced neutropenia.

Example 3 Effects of IL-40 and G-CSF on the Neutropenia Model of BALB/C Mice Induced by Chloramphenicol

BALB/C mice were purchased from Shanghai Nanfang Modeling Center. One 20 g mouse, half male and half male, were randomly assigned, 10 mice in each group, and divided into four experimental groups, namely control group, chloramphenicol group, chloramphenicol+G-CSF group, and chloramphenicol+IL-40 group. Mice in the control group were intraperitoneally injected with solvent for a total of 21 days; mice in the chloramphenicol group were injected intraperitoneally with chloramphenicol (200 mg/kg/day) for a total of 21 days; mice in the chloramphenicol+G-CSF group were injected intraperitoneally with chloramphenicol on the first day, and from the next day, injected intraperitoneally with recombinant mouse G-CSF (100 μg/kg/day) and chloramphenicol (200 mg/kg/day) for 20 consecutive days; mice in the chloramphenicol+IL-40 group were injected intraperitoneally with chloramphenicol (200 mg/kg/day) on the first day /day), and from the next day, intraperitoneal injection of recombinant mouse IL-40 (50 μg/kg/day) and chloramphenicol (200 mg/kg/day), and continuous injection of both for 20 days. On the 21st day, the peripheral blood and bone marrow of the mice were collected, and the neutrophil count was detected with a whole blood analyzer, and the percentage of nucleated cells occupied by neutrophils in the bone marrow was detected with a flow cytometer.

The results are shown in Figures 7-9, after chloramphenicol mice were intraperitoneally injected with chloramphenicol for 21 consecutive days, the absolute value of peripheral blood neutrophils decreased significantly. In the mice of the chloramphenicol+G-CSF group, the absolute value of peripheral blood neutrophils did not return to the level of the mouse control group on day 22. However, the absolute value of peripheral blood neutrophils in the chloramphenicol+IL-40 group was 2.02 times higher than that in the chloramphenicol+G-CSF group. The percentage of nucleated cells occupied by bone marrow neutrophils in the chloramphenicol+IL-40 group was 1.31 times that of the chloramphenicol+G-CSF group, exceeding the percentage of bone marrow neutrophils occupied by the control group.

The above results show that in the mouse model of chloramphenicol-induced neutropenia, the therapeutic effect of IL-40 is significantly better than that of G-CSF, which can not only significantly increase the number of peripheral blood neutrophils, but also significantly increase the percentage of neutrophils in bone marrow nucleated cells, suggesting that IL-40 can effectively prevent 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. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions and modifications to these embodiments still fall within the protection scope of the present invention.

Claims (5)

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.