CN111514301A

CN111514301A - Application of TGF (transforming growth factor) signal inhibitor in preventing and treating cancerous anemia

Info

Publication number: CN111514301A
Application number: CN202010435961.0A
Authority: CN
Inventors: 项鹏; 汪建成; 王博彦
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2020-05-21
Filing date: 2020-05-21
Publication date: 2020-08-11

Abstract

The invention discloses application of a TGF (transforming growth factor) signal inhibitor in preparing a medicament for treating and/or preventing cancerous anemia. The research of the invention shows that TGF signal inhibitors have obvious prevention and treatment effects on cancerous anemia, and the TGF signal inhibitors are mainly beneficial to maintaining bone homeostasis, improving hematopoietic microenvironment, delaying the occurrence of cancerous anemia, relieving or alleviating symptoms of cancerous anemia of patients and improving the treatment effect of tumors by reducing bone absorption. Therefore, the TGF signal inhibitor has good application value in the aspects of treating and/or preventing cancerous anemia and improving the treatment effect of tumor.

Description

Application of TGF (transforming growth factor) signal inhibitor in preventing and treating cancerous anemia

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a TGF (transforming growth factor) signal inhibitor in preparation of a medicine for treating and/or preventing cancer anemia and a medicine for treating and/or preventing cancer anemia.

Background

Cancer anemia is a common concomitant condition in patients with tumors, manifested by a progressive decrease in hemoglobin levels and a reduction in the number of red blood cells. Anemia occurs in at least 30% of cancer patients and symptoms gradually worsen as the tumor progresses. Patients with cancerous anemia are often associated with fatigue, lethargy, dyspnea, anorexia and a progressive deterioration of cognitive functions which significantly affect the quality of life of the patient and lead to a poor tolerance of the patient in the treatment of tumors, impairing the therapeutic efficacy of the tumor. Therefore, the treatment of cancer anemia plays an important role in prolonging the life span of tumor patients and improving the life quality of the patients.

With the development of a great deal of research, the mechanism of occurrence of cancerous anemia is also well understood. The pathological and physiological characteristics of cancer anemia are various, wherein the destruction of hematopoietic microenvironment plays an important role, and the negative balance is caused by the comprehensive factors such as tumor secretion and metabolic disorder of the body. Despite the numerous advances currently made in preclinical studies in tumor-bearing animal models, no drugs have yet been clinically approved for the prevention and treatment of cancerous anemia. Clinically, cancer anemia is mainly treated by means of red blood cell transplantation, erythropoietin injection and the like, but the method can generate a series of adverse reactions such as immune reaction, blood vessel embolism and the like. Therefore, there is a need to understand the etiology and pathological processes of cancer anemia and to perform targeted intervention in the early stage of the occurrence of cancer anemia to achieve better therapeutic effects.

Erythrocytes are produced by the process of stepwise erythropoietic differentiation of hematopoietic stem cells. The earliest erythroid progenitors formed were burst-forming unit erythroid cells, which were mostly dormant and able to differentiate into colony-forming unit erythroid cells. The colony forming unit erythroid cells then produce proerythroid cells under the action of erythropoietin. The protoerythrocytes undergo several stages of maturation, including basophilic, polychromatic and orthochromatic erythrocytes, then reticulocytes, and finally mature erythrocytes. In recent years, work by many researchers has shown that hematopoiesis is a complex process that is tightly controlled by the microenvironment, including the surrounding endosteum and bone marrow. The endosteal microenvironment is composed primarily of osteoblasts and osteoclasts, and its maintenance of homeostasis depends on a dynamic balance between bone formation and resorption, a process known as skeletal remodeling. Skeletal remodeling activity is closely related to hematopoiesis, and it can regulate the proliferation, differentiation and long-term hematopoiesis of hematopoietic stem cells by direct or indirect means. In addition, the bone marrow microenvironment, as part of the hematopoietic microenvironment, also plays an important role in homeostatic maintenance of erythroid progenitor cells and regulation of stress-induced proliferation and differentiation.

Transforming growth factor beta (TGF- β) belongs to a cytokine superfamily that promotes cell growth and transformation in hematopoietic microenvironments, is an important regulator of proliferation and differentiation of different cell types, and has been shown to be involved in the pathogenesis of a variety of myeloid diseases. TGF β in bone marrow is mainly derived from bone matrix. During bone matrix formation, osteoblasts produce non-functional TGF β and store it in the bone matrix. During bone resorption, this stored TGF β is released from the bone matrix and is further cleaved by osteoclasts to become activated TGF β. In this process, TGF plays a dual role: activated TGF β can promote migration of bone marrow stromal cells to the site of bone resorption and induce bone formation, thereby regulating bone balance. However, excessive release of TGF β in the bone matrix is a pathological cause and a key regulatory factor for a variety of diseases. Several researchers have demonstrated that muscle weakness occurring in cancer is associated with the osteolytic process of certain invasive tumors that over-release TGF during tumor-induced bone destruction. For example, in patients with Camurati-Engelmann's disease, abnormal osteogenesis results in the release of TGF β causing severe bone hypertrophy and osteoarthritis. However, the role of TGF β in the pathogenesis, development and progression of cancer anemia has not been clearly studied.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing medicines for preventing and treating the cancerous anemia and provides a new idea and a new medicine selection for preventing and treating the cancerous anemia. Through a great deal of creative work, the inventor discovers for the first time that the TGF signal inhibitor can effectively treat and/or prevent the cancerous anemia, especially provides a new idea for treating the tumor-induced anemia, is favorable for delaying or relieving the anemia symptoms of tumor patients and improves the tumor treatment effect.

The invention aims to provide application of a TGF (transforming growth factor) signal inhibitor in preparing a medicament for treating and/or preventing cancerous anemia.

It is another object of the present invention to provide a medicament for treating and/or preventing cancer anemia, said medicament comprising a TGF signaling inhibitor.

Another object of the present invention is to provide the use of a TGF signaling inhibitor for the manufacture of a medicament for improving the therapeutic effect of an anticancer drug.

Another object of the present invention is to provide a therapeutic agent for cancer accompanied by cancerous anemia, which comprises a TGF signaling inhibitor and an anticancer agent having a therapeutic effect on the cancer.

The above object of the present invention is achieved by the following technical solutions:

SB505124, chemical name: 2- [4- (1, 3-benzodioxol-5-yl) -2- (tert-butyl) -1H-imidazol-5-yl ] -6-methylpyridine, having the following structural formula:

SB431542, chemical name: 4- [4- (3, 4-methylenedioxyphenyl) -5- (2-pyridyl) -1H-imidazol-2-yl ] benzamide having the formula:

SB505124 is synthesized for the first time in 2002, is a selective small molecule inhibitor of TGF-beta I type receptors ALK4, ALK5 and ALK7, has potential anticancer activity, and has potential treatment potential including ischemic brain injury, solid tumor, cartilage injury, renal insufficiency, diabetic nephropathy, stem cell mobilization, inflammatory diseases and pulmonary fibrosis; SB431542 was first synthesized in 2002, is a selective inhibitor of TGF-beta type I receptor ALK5, also inhibits ALK4 and ALK7, and also has certain functions of immunoregulation and tumor growth and metastasis inhibition.

The research of the invention shows that TGF signal inhibitors such as SB505124, SB431542 and the like have important application prospects in the aspects of preventing and treating cancerous anemia.

Accordingly, the present invention provides the use of a TGF signalling inhibitor in the manufacture of a medicament for the treatment and/or prevention of cancerous anemia.

Preferably, the TGF signaling inhibitor is a TGF- β type I receptor inhibitor.

The TGF-. beta.type I receptor inhibitor is preferably a structural analogue of SB 505124.

More preferably, the TGF- β type I receptor inhibitor is SB505124 or a pharmaceutically acceptable salt thereof, or SB431542 or a pharmaceutically acceptable salt thereof.

Herein, the SB505124 is the compound 2- [4- (1, 3-benzodioxol-5-yl) -2- (tert-butyl) -1H-imidazol-5-yl ] -6-methylpyridine.

Herein, the SB431542 is the compound 4- [4- (3, 4-methylenedioxyphenyl) -5- (2-pyridyl) -1H-imidazol-2-yl ] benzamide.

Herein, the cancerous anemia is tumor-induced anemia.

The cancerous anemia of the present invention includes, but is not limited to, syndromes that are marked by a decrease in peripheral red blood cell count, a decrease in hemoglobin concentration, and erythropoiesis impairment during tumorigenesis and tumor treatment.

The invention also provides a medicament for the treatment and/or prevention of cancer anemia, the medicament comprising a TGF signalling inhibitor.

The TGF signal inhibitor is an effective component in the medicament.

Preferably, the TGF signaling inhibitor is a TGF- β type I receptor inhibitor.

Based on the above results, the present invention also provides the use of a TGF signaling inhibitor for the manufacture of a medicament for improving the therapeutic effect of an anticancer agent.

The improvement of the therapeutic effect of the anticancer drug means improvement of the therapeutic effect on cancer accompanied by cancerous anemia.

Preferably, the TGF signaling inhibitor is a TGF- β type I receptor inhibitor.

The invention also provides a therapeutic drug for cancers accompanied by cancerous anemia, which comprises a TGF (transforming growth factor) signal inhibitor and an anti-cancer drug having a therapeutic effect on the cancers, and the combination of the TGF signal inhibitor and the anti-cancer drug improves the anti-cancer effect.

Preferably, the TGF signaling inhibitor is a TGF- β type I receptor inhibitor.

The medicaments of the invention are useful as a treatment and/or prevention of cancerous anemia in a mammal (e.g., human, mouse, rat, rabbit, dog, cat, cow, horse, pig, monkey, etc.).

The term "treating" as used herein refers to alleviating or ameliorating a disorder, i.e., delaying or inhibiting the development of the disorder or at least one clinical symptom.

The term "prevention" as used herein refers to the administration of a medicament according to the invention to a subject prior to the onset of symptoms of the disorder.

The subject of the present invention can refer to any animal, including but not limited to mammals such as human, mouse, rat, rabbit, dog, cat, cow, horse, pig, monkey, etc.

The medicament of the present invention may be administered via any physiologically acceptable route, for example, oral, injection, etc.

The medicine can be prepared into pharmaceutically acceptable dosage forms. Generally, the amount of active ingredient in the formulation is 1-95% of the total weight of the formulation.

The dosage form of the drug is adapted to different modes of administration. For oral administration, the drug may be formulated into solid preparations such as tablets, powders, granules, capsules or liquid preparations such as solutions or suspensions, etc. For injection, the drug may be formulated as an injectable solution or suspension, or as an injectable dry powder; the injectable dry powder may be dissolved or dispersed in sterile water or other sterile injectable medium immediately prior to use, and may be used immediately. The preparation of various preparations can adopt the conventional production method in the pharmaceutical field.

In a preferred embodiment of the invention, the medicament is in the form of injection or oral preparation.

The medicament can also contain pharmaceutically acceptable auxiliary materials or carriers, and the pharmaceutically acceptable auxiliary materials or carriers can be optionally used according to requirements. Such adjuvants or carriers are widely known and include: binders for oral formulations (such as starch, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone), diluents (such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and/or glycerol), lubricants (such as silicon dioxide, talc, stearic acid or salts thereof, usually magnesium or calcium stearate, and/or polyethylene glycol), and, if desired: disintegrating agents (such as starch, agar, alginic acid or a salt thereof, usually sodium alginate) and/or effervescent mixtures, solubilising agents, stabilisers, suspending agents, colours, flavouring agents etc.; preservatives, solubilizers, stabilizers and the like for injectable formulations; bases for topical formulations, diluents, lubricants, preservatives, and the like.

The term "pharmaceutically acceptable" as used herein means that the excipient or carrier is compatible with the active ingredient, preferably, it stabilizes the active ingredient and is not harmful to the individual being treated.

The phrase "pharmaceutically acceptable salts" as used herein refers to certain salts that retain their original biological activity and are suitable for pharmaceutical use. The compounds SB505124 and SB431542 are capable of forming many different salts with different inorganic and organic acids. The acids from which the salts can be prepared are those that form non-toxic acid addition salts including, but not limited to, citrate, maleate, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acetate, lactate, salicylate, citrate, succinate, maleate, gentisate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate.

When the medicament is to be used for the treatment and/or prevention, or other treatment and/or prevention, of the cancerous anemia, the total daily amount of the compound of the present invention must be determined by the attending physician within the scope of sound medical judgment. For any particular patient, the specific therapeutically effective dose level will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the particular compound employed; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound employed; the duration of treatment; and similar factors known in the medical arts. For example, it is common in the art to start doses of the compound at levels below those required to achieve the desired therapeutic effect and to gradually increase the dose until the desired effect is achieved.

A subject is in need of such treatment if the subject would benefit biologically, medically or in quality of life from the treatment.

The invention has the following beneficial effects:

cell level and in vivo animal experiments show that TGF signal inhibitors such as SB505124, SB431542 and the like are mainly beneficial to maintaining bone homeostasis, improving hematopoietic microenvironment, delaying the occurrence of cancerous anemia and relieving or alleviating the symptoms of cancerous anemia of patients by reducing bone absorption, so that cancerous anemia is effectively treated and/or prevented, and a new thought is provided for preventing and treating cancerous anemia and improving the treatment effect of tumors.

Drawings

In fig. 1 to 6, Control: control group, C57BL/6 mice without any treatment, LLC: c57BL/6 mice subcutaneously implanted with LLC tumor cells (i.e., tumor-bearing); in fig. 3-5: LLC + SB 505124: c57BL/6 mice transplanted with LLC tumor cells subcutaneously and administered with SB505124 in vivo; in fig. 6: LLC + DMSO: c57BL/6 mice (corresponding to LLC + SB505124) implanted with LLC tumor cells subcutaneously and administered DMSO in vivo, LLC + PEG: c57BL/6 mice (corresponding to LLC + SB431542) implanted subcutaneously with LLC tumor cells, administered in vivo with 2% DMSO + 30% PEG300+ 68% PBS, LLC + SB 431542: c57BL/6 mice subcutaneously transplanted with LLC tumor cells and administered with SB431542 in vivo;

FIG. 1 shows the results of conventional blood tests and erythroid progenitor cell differentiation tests of control mice and tumor-bearing mice; a: peripheral red blood cell number; b: a hemoglobin level; c: mean hemoglobin concentration of red blood cells; d: the number of cells in which erythroid progenitor cells differentiate into different stages (I, II, III, IV, V); e: statistical analysis of the number percentage of different stages of erythroid progenitor cell differentiation;

FIG. 2 is a bone marrow microenvironment analysis of control and tumor-bearing mice; a: detecting the result of TGF beta by DAPI fluorescence labeling; b: indicating a level of phosphorylation of Smad 2/3; p-Smad2 and p-Smad3 in the figure refer to phosphorylated Smad2 and Smad3, respectively; GAPDH is glyceraldehyde-3-phosphate dehydrogenase, used as an internal control;

FIG. 3 shows the results of the measurement of the numbers of cells in different stages of the differentiation of erythroid progenitor cells in control mice, tumor-bearing mice and mice administered with SB505124 tumor-bearing mice; a: the number of cells in the erythroid progenitor cells at different stages of differentiation; b: statistical analysis of the number percentage of erythroid progenitor cell clusters at stage III of differentiation in ter119+ cells; c: statistical analysis of the number percentage of the erythroid progenitor cell clusters at stage V of differentiation in ter119+ cells;

FIG. 4 shows the results of bone detection in control mice, tumor-bearing mice and tumor-bearing mice administered with SB 505124; a: femoral scan, wherein Transverse: cross-cut, corona: coronal, Sagittal: sagittal; b: a three-dimensional structure chart taking the whole subchondral bone inner cavity as a reconstruction area; c: the ratio of bone surface area to bone volume; d: bone volume to total volume ratio; e: number of trabeculae; f: cortical bone thickness;

FIG. 5 shows the results of the detection of the expression of the myelofibrosis factor in the control mouse, the tumor-bearing mouse and the tumor-bearing mouse administered with SB 505124;

FIG. 6 is a graph of the therapeutic effect of different TGF- β type I receptor inhibitors on cancer anemia; a: the number of red blood cells in the peripheral blood; b: a hemoglobin level; c: myelofibrosis factor expression level.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

SB505124 is purchased from Selleck, USA, under the drug Cat No. S2186.

SB431542 is purchased from Selleck, USA, and has a drug catalog number S1067.

Example 1 culture of Lewis Lung cancer LLC cells and construction of mouse tumor-bearing model

1.1 LLC cell culture: LLC cells (ATCC # CRL-1642) were cultured in DMEM basal medium (containing 10% fetal bovine serum) for use.

1.2 mouse breeding method: feeding mice with conventional mouse feed (protein content of 20-25 wt%, fat content of 5-10 wt%, and crude fiber content of 3-5 wt%).

1.3 construction of mouse tumor-bearing model

Male C57BL/6 mice (purchased from the model animal research institute of Nanjing university) with a weight difference of not more than 2g at 8 weeks of age were randomly divided into a control group and an LLC cell transplantation group, 6 mice per group, and approximately 4 × 10 subcutaneous injections were given to the groin of each LLC group⁶LLC cells, the experimental process in the clean bench, attention to sterile operation. Control mice were not treated at all. LLC cell transplantation (i.e., tumor-bearing) was counted as day 0, then 1 day, 2 days, and so on.

Example 2 evaluation of hematopoietic function in tumor-bearing mice

2.1 routine measurement of mouse blood

Tumor-bearing mice of 21 days and control mice were anesthetized with 10% chloral hydrate, and blood samples of the inferior vena cava of the mice were extracted. The blood sample is immediately tested for red blood cell count, hemoglobin level, and mean hemoglobin concentration of red blood cells using a conventional blood analyzer. As shown in the graphs A-C in FIG. 1, the number of red blood cells, the hemoglobin level and the mean hemoglobin concentration of red blood cells in the tumor-bearing mice were significantly reduced compared to the control group, indicating that the tumor-bearing mice had anemia symptoms.

2.2 mouse erythroid progenitor cell differentiation assay

Tumor-bearing 21-day mice and control mice were anesthetized with 10% chloral hydrate, and the femur and bone marrow of the mice were isolated. Cell suspensions were centrifuged and flow analyzed using flow antibody labeled Ter119, CD44 to give Panel D in FIG. 1, and panel E was obtained by statistical analysis. As shown in figure E, significant retardation of erythroid progenitor cell differentiation between stage III (i.e., polychromatic erythroblasts) and stage V (i.e., mature erythrocytes) and a significant impairment of hematopoietic function was observed in tumor-bearing mice compared to the control group.

Example 3 detection of TGF signaling pathway in tumor-bearing mice

Tumor-bearing 21-day mice and control mice were anesthetized with 10% chloral hydrate, and the femurs of the mice were isolated. After fixation with 4% paraformaldehyde overnight, decalcification was performed with 10% EDTA for 2 weeks, and cryosectioning was performed. By fluorescence labeling of TGF β with DAPI, an increase in TGF β levels in the tumor-bearing mouse bone marrow microenvironment was observed, as shown in panel a in figure 2.

Extracting protein from the cell suspension obtained in the step 2.2 by using RIPA lysate, performing SDS-PAGE electrophoresis, transferring a membrane, incubating Smad2/3 and p-Smad2/3 antibodies, and developing to obtain a graph B in the graph 2, wherein the level of the p-Smad2/3 microenvironment of the bone marrow of the tumor-bearing mouse is increased, which indicates that a TGF signal path in the bone marrow microenvironment of the tumor-bearing mouse is activated.

Example 4 Effect of SB505124 on hematopoietic function of tumor-bearing mice

Male C57BL/6 mice with a weight difference of not more than 2g at 8 weeks of age were selected as Control group (Control), tumor-bearing group (LLC) and SB505124 administration group (LLC + SB 505124).

Control group: no treatment was performed.

Tumor-bearing group 4 × 10⁶Individual LLC cells were transplanted subcutaneously into the groin of mice. After 7 days, 100 μ l of LDMSO was intraperitoneally injected daily.

SB505124 administration group: after 7 days of LLC cell transplantation (i.e., tumor-bearing), SB505124 (dissolved in pure DMSO) was intraperitoneally injected daily for 2 weeks. The amount of SB505124 administered was 5mg/kg body weight/day.

21 days after tumor bearing, 10% chloral hydrate is used for anesthesia, the thighbone of the mouse is separated, and the bone marrow of the mouse is taken. After digestion and centrifugation, cell suspensions were obtained and flow analysis was performed using flow antibody labeled Ter119, CD44 to obtain panel A in FIG. 3, and statistical analysis to obtain panels B and C, it was observed that the blockade of erythroid progenitor cell differentiation between stage III (i.e., multi-colored erythroblasts) and stage V (i.e., mature erythrocytes) was alleviated in tumor-bearing mice administered SB505124 compared to LLC group mice.

From the experimental results, it can be seen that the hematopoietic function of tumor-bearing mice was significantly restored after in vivo administration of SB 505124.

Example 5 Effect of SB505124 on bone Mass in tumor-bearing mice

Male C57BL/6 mice with a weight difference of not more than 2g at 8 weeks of age were selected as Control group (Control), tumor-bearing group (LLC), and SB505124 administration group (LLC + SB 505124).

Control group: no treatment was performed.

SB505124 administration group: after 7 days of LLC cell transplantation (i.e., tumor-bearing), SB505124 was intraperitoneally injected daily for 2 weeks. The amount of SB505124 administered was 5mg/kg body weight/day.

21 days after tumor loading, the femur was isolated by anesthesia with 10% chloral hydrate, fixed in 4% paraformaldehyde for 48 hours, and the entire femur was scanned using an Inveon PET/CT scanner (Siemens), resulting in panel A in FIG. 4. The image was reconstructed and analyzed using Inveon research Workplace 4.1 software, and three-dimensional structural analysis was performed with the entire subchondral bone cavity as the reconstruction region, to obtain Panel B in FIG. 4.

As shown in fig. 4, panels C-F, the ratio of bone surface area to bone volume was increased, the ratio of bone mass to total volume, the number of trabeculae and the thickness of cortical bone decreased in tumor-bearing mice compared to control mice, indicating increased bone resorption in tumor-bearing mice. However, the symptoms of the tumor-bearing mice administered with SB505124 are improved, which indicates that the administration of SB505124 can slow down bone resorption of the tumor-bearing mice.

From the experimental results, the bone remodeling ability of tumor-bearing mice was significantly restored after in vivo administration of SB 505124.

Example 6 Effect of SB505124 on myelofibrosis in tumor-bearing mice

Control group: no treatment was performed.

Bone marrow cell suspension was obtained by the method mentioned in 2.2, cells were lysed by TRIzol kit, single-stranded cDNA was obtained by reverse transcription of mRNA, and then the expression status of each of the fibrosis genes Acta2, Col3a1 and Fn was detected by fluorescent quantitative PCR using LC480 system of Roche.

The primers used for the fluorescent quantitative PCR were as follows:

(1) mouse Acta2 primers were:

5'-CTGACAGAGGCACCACTGAA-3' (F); and

5’-CATCTCCAGAGTCCAGCACA-3’(R)；

(2) the mouse Col3a1 primers are:

5'-ACGTAGATGAATTGGGATGCAG-3' (F); and

5’-GGGTTGGGGCAGTCTAGTG-3’(R)，

(3) mouse Fn primers were:

5'-ATGTGGACCCCTCCTGATAGT-3' (F); and

5’-GCCCAGTGATTTCAGCAAAGG-3’(R)，

as shown in fig. 5, the expression of the fibrosis genes Acta2, Col3a1 and Fn was significantly increased in tumor-bearing mice compared to the control group mice, whereas the expression of the genes was significantly down-regulated in tumor-bearing mice administered SB 505124.

From the experimental results, the hematopoietic microenvironment of tumor-bearing mice was improved after in vivo administration of SB 505124.

The invention confirms that the incidence of cancerous anemia is related to a TGF (transforming growth factor) signaling pathway through the above examples, and evaluates the activation condition of the TGF beta-Smad 2/3 signaling pathway in a tumor-bearing mouse hematopoietic microenvironment. By sectioning and immunofluorescent staining of mouse bone marrow and counting TGF β levels, it was found that tumor-bearing mice had more TGF β released from the bone matrix than normal mice. Meanwhile, the Western Blot result also indicates that the phosphorylation level of Smad2/3 of tumor-bearing mice is increased. These results suggest that activation of the TGF β signaling pathway is accompanied after the mice develop cancer anemia.

The inventor firstly utilizes the micCT to scan the thighbone of a tumor-bearing mouse, and finds that the bone quality is damaged and the bone balance is damaged in the process of the cancer anemia of the mouse; at the same time, myelofibrosis increases and the microenvironment of the bone marrow deteriorates. While SB505124 successfully reduced bone destruction, improved bone balance and bone marrow microenvironment by in vivo administration. The above results demonstrate that administration of SB505124 improves the hematopoietic microenvironment in the case of cancerous anemia.

The inventor of the application finds that the tumor-bearing mice applied with SB505124 have lighter anemia symptoms than the tumor-bearing mice not applied with SB505124 on the same days of tumor bearing by the conventional and erythroid progenitor cell differentiation detection of the mouse blood.

The experimental results in the above examples show that SB505124 has an obvious curative effect on the prevention and treatment of cancerous anemia, suggesting that SB505124 administered to a patient with a tumor can block the destruction of the tumor to the homeostatic balance of various tissues of the whole body such as bone marrow, and is beneficial to delaying the occurrence of cancerous anemia, alleviating the anemia symptoms of the patient with a tumor, and improving the tumor treatment effect.

Example 7 therapeutic Effect of other TGF-. beta.type I receptor inhibitors on cancer anemia

Male C57BL/6 mice with body weight difference not more than 2g at 8 weeks of age were selected as tumor-bearing group #1(LLC + DMSO), tumor-bearing group #2(LLC + PEG), and tumor-bearing group # 431542 (LLC + SB 431542).

Tumor-bearing group # 1: 4 × 10⁶Individual LLC cells were transplanted subcutaneously into the groin of mice. After 7 days, 100. mu.L of DMSO was intraperitoneally injected daily, which was the same as the tumor-bearing group (LLC) in examples 4-6.

SB505124 administration group: after 7 days of LLC cell transplantation (i.e., tumor-bearing), SB505124 was intraperitoneally injected daily for 2 weeks. The amount of SB505124 administered was 5mg/kg body weight/day. The SB505124 administration group in this example is the same as the SB505124 administration group in examples 4-6.

Tumor-bearing group # 2: 4 × 10⁶Individual LLC cells were transplanted subcutaneously into the groin of mice. After 7 days, 100 μ L of 2% DMSO + 30% PEG300+ 68% PBS was intraperitoneally injected daily.

SB431542 administration group: after 7 days of LLC cell transplantation (i.e., tumor bearing), SB431542 (dissolved in 2% DMSO + 30% PEG300+ 68% PBS) was intraperitoneally injected daily for 2 weeks. SB431542 was administered at 5mg/kg body weight/day.

Tumor-bearing 21-day mice were divided into four groups, two groups of which were administered two TGF- β type I receptor inhibitors of SB505124 and SB431542, respectively, while the other two groups were administered two formulated solvents of the two drugs, respectively, in order to exclude the difference in the formulation method of the drugs.

The mice were anesthetized with 10% chloral hydrate and the mice's inferior vena cava blood samples were extracted. The blood sample is immediately tested for red blood cell count and hemoglobin levels using a conventional blood analyzer. As shown in fig. 6, panels a-B, the numbers of red blood cells and hemoglobin levels in tumor-bearing mice administered SB505124 or SB431542 were significantly increased compared to the tumor-bearing group alone, suggesting that both TGF- β type I receptor inhibitors have a certain therapeutic effect on cancer anemia.

In order to preliminarily search the treatment mechanism of the two drugs, the method in the same example 6 is adopted, and the expression conditions of the fibrosis genes Acta2, Col3a1 and Fn in bone marrow are detected by using fluorescent quantitative PCR. As shown in panel C of FIG. 6, the expression levels of the myelofibrosis-associated genes Acta2, Col3a1 and Fn were significantly reduced after administration of SB505124 or SB431542, suggesting that both TGF- β type I receptor inhibitors may alleviate the symptoms of cancer anemia by alleviating the degree of myelofibrosis.

The results indicate that the occurrence and development of the cancer anemia are closely related to the activation of the TGF-beta pathway, and the treatment effect of the TGF-beta type I receptor inhibitor on the cancer anemia has certain universality.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

Use of a TGF signalling inhibitor in the manufacture of a medicament for the treatment and/or prevention of cancerous anemia.
2. The use according to claim 1, wherein the TGF signalling inhibitor is a TGF- β type I receptor inhibitor.
3. The use according to claim 2 wherein the TGF- β type I receptor inhibitor is SB505124 or a pharmaceutically acceptable salt thereof, or SB431542 or a pharmaceutically acceptable salt thereof.
4. The use of any one of claims 1-3, wherein the cancer anemia is tumor-induced anemia.
5. A medicament for the treatment and/or prevention of cancerous anemia, comprising an inhibitor of TGF signalling.
6. The medicament of claim 5, wherein the cancer anemia is tumor-induced anemia.
Use of a TGF signaling inhibitor in the manufacture of a medicament for improving the therapeutic effect of an anti-cancer agent.
8. The use according to claim 7, wherein the improvement in the therapeutic effect of the anticancer agent is an improvement in the therapeutic effect on cancer accompanied by cancer anemia.
9. The use according to claim 7, wherein the TGF signalling inhibitor is a TGF- β type I receptor inhibitor.
10. A therapeutic agent for cancer accompanied by cancerous anemia, comprising a TGF signaling inhibitor and an anticancer agent having a therapeutic effect on said cancer, wherein said TGF signaling inhibitor is a TGF-beta type I receptor inhibitor.