JP2024020797A - Muscle atrophy treatment agent - Google Patents

Abstract

To provide a muscle atrophy treatment agent that can effectively prevent muscle atrophy and ameliorate associated symptoms.SOLUTION: A muscle atrophy treatment agent contains IL-33, or an agonist or ligand of the IL-33 receptor.SELECTED DRAWING: Figure 6

Description

The present invention relates to a therapeutic agent for muscular atrophy, which is used for the treatment or prevention of muscular atrophy and the like.

Muscle tissue can undergo muscle damage and muscle-related pathologies due to external factors and overuse. Muscle is originally a tissue with excellent regenerative ability, and after muscle damage or pathological conditions, muscle stem cells and other factors normally cause muscle regeneration and muscle hypertrophy.

However, as the subject ages, has chronic pathological conditions, or is in bed for a long period of time associated with these conditions, muscle regeneration and muscle hypertrophy do not occur sufficiently or normally, resulting in muscle atrophy and muscle degeneration. Muscle weakness due to muscle atrophy and muscle degeneration is associated with a decline in the subject's activity and quality of life. In other words, if muscle atrophy and muscle degeneration can be prevented, it is thought that a decline in QOL can be prevented. In particular, the incidence of disuse muscular atrophy among the elderly has been increasing in recent years, and countermeasures against this disease are an urgent issue in Japan, which has become a super-aging society.

As a means of preventing muscle atrophy, there are many reports of the effectiveness of exercise therapy, but no practical medication for oral administration or injection has yet been established. Furthermore, if we can better understand what happens in muscle atrophy, it may be useful for treatments that prevent or improve muscle atrophy.

Regarding the functions of the main cells within muscles, it is known that satellite cells are responsible for cell regeneration and hypertrophy. Satellite cells are stem cells surrounding muscle fibers, localized in the basement membrane, and are normally dormant, but become activated by muscle damage or exercise, and proliferate and differentiate into myoblasts. On the other hand, muscles undergo fatty degeneration and fibrosis due to injury or disease. As for the cause, mesenchymal progenitor cells called fibro-adipogenic-progenitors (FAPs) have been reported. It has also been reported that supplementing factors secreted by FAPS restores the function of aged satellite cells, and that a model in which FAPS is suppressed shows a decrease in satellite cells and atrophy of muscle fibers.

Here, Patent Document 1 discloses a skeletal muscle damage repair promoter containing a specific peptide or a salt thereof as an active ingredient. In this technique, the skeletal muscle damage repair promoter of the present invention is intended to promote the activation and/or differentiation of muscle satellite cells at the damaged site of skeletal muscle.

International Publication No. 2018/230535

The technique of Patent Document 1 discloses that activation and/or differentiation of muscle satellite cells can be promoted to repair damage to skeletal muscle, that is, muscle tear, muscle atrophy, or muscle degeneration. However, in this technique, a peptide with a specific sequence has an angiogenic effect and a collagen production promoting effect, and these effects activate bone and muscle tissues. This study does not aim to elucidate the mechanism of mesenchymal progenitor cell FAPs, that is, specific factors related to muscle atrophy, or to specifically treat or prevent muscle atrophy.

Elucidating the mechanisms of muscle atrophy, especially the effects of mesenchymal progenitor cells FAPs, will greatly contribute to the prevention and amelioration of muscle atrophy, as well as the establishment of practical therapeutic drugs. There is a strong need for explanations and treatments.

The present invention was made based on this background, and its purpose is to provide a therapeutic agent for muscle atrophy that can effectively prevent muscle atrophy and improve symptoms.

The present invention includes the following aspects.
[1] A therapeutic agent for muscle atrophy containing IL-33 or an agonist or ligand of the IL-33 receptor as an active ingredient.

[2] The therapeutic agent for muscle atrophy according to [1], wherein the IL-33 receptor is ST2.

[3] The therapeutic agent for muscle atrophy according to [1] or [2], wherein the muscle atrophy is caused by an increase in the expression level of the IL-33 receptor in muscle tissue.

[4] The therapeutic agent for muscle atrophy according to any one of [1] to [3], wherein the muscle atrophy is disuse muscular atrophy.

[5] The content of the IL-33 or IL-33 receptor agonist or ligand contained in the active ingredient is 80% by mass or more, 90% by mass or more, or 100% by mass, [1 ] - [4] The therapeutic agent for muscle atrophy according to any one of [4].

According to the present invention, it is possible to provide a therapeutic agent for muscle atrophy that can effectively prevent muscle atrophy and improve symptoms.

It is a graph diagram showing changes in cell number by FACS analysis in this example. FIG. 2 is a graph showing the muscle fiber cross-sectional area (CSA) measured from the cross section of TA (tibialis anterior muscle) in this example. FIG. 2 is a schematic diagram of RNA extraction from FAPs in this example. FIG. 2 is a graph diagram showing the analysis results of expression levels by RNA-seq and qPCR in this example. FIG. 2 is a graph showing the CSA results of the IL-33 and ST2 administration groups of young mice in this example. FIG. 2 is a graph showing the CSA results of IL-33 and ST2 administration groups of aged mice in this example.

Hereinafter, the therapeutic agent for muscle atrophy according to the present invention will be described by showing embodiments. However, the present invention is not limited to the following embodiments.

[Treatment agent for muscle atrophy]
The therapeutic agent for muscle atrophy of the present embodiment contains IL-33 or an agonist or ligand of the IL-33 receptor as an active ingredient.

IL-33 (interleukin 33) is a protein belonging to the interleukin 1 family. Interleukin 1 is a family member known for its role in immune regulation and inflammation. Among these, IL-33 is known to be a specific ligand for ST2, which is a receptor of the IL-1 family. It has been known that NF-κB and MAPK signaling pathways are induced when ST2 is stimulated by IL-33.
IL-33 is a dual-function protein that functions as a nuclear factor and an inflammatory cytokine, and its nuclear localization and association with heterochromatin is mediated by its N-terminal domain, which connects IL-33 to the NF-κB complex. It acts as a novel transcriptional regulator of the p65 subunit. The C-terminal domain is said to bind to the ST2 receptor and activate the production of type 2 cytokines (eg, IL-5 and IL-13) from polarized Th2 cells and ILC2 cells.

Since IL-33 increases inflammation in the airways and arthritis, it is thought that IL-33 is associated with inflammation in these areas. IL-33 is also known to have a protective effect on myocardium.

The active ingredient of the therapeutic agent for muscle atrophy of this embodiment may also be an agonist or a ligand for the IL-33 receptor. Here, the term "IL-33 receptor" broadly refers to protein components that can accept IL-33 in vivo.
The IL-33 receptor is particularly preferably ST2. ST2 is a member of the IL1 receptor family, also known as IL1RL1 (interleukin 1 receptor-like 1).

The IL-33 receptor agonist or ligand includes IL-33 recombinant proteins derived from various animals such as humans, mice, and rats, and any substance that can act as an IL-33 receptor agonist or ligand.

The agonist or ligand for IL-33 or the IL-33 receptor of the present embodiment may be a chemically modified substance, or a protein having homology to these substances. More specifically, it is preferable that the substance has a structure that has the activity of binding to the IL-33 receptor described below, and within this range, changes can be made as appropriate based on the structure of the substance described above.

The present inventors found that muscle atrophy was suppressed by administering IL-33, as shown in the Examples described below. Therefore, IL-33, or an agonist or ligand that similarly binds to the IL-33 receptor, can be used as a therapeutic agent for muscle atrophy in treatments including symptom improvement and prevention.

Muscle atrophy, which is used in therapeutic agents for muscle atrophy, broadly refers to the atrophy of muscle tissue. Muscle atrophy specifically refers to muscle damage caused by injury or disease, or deterioration or degeneration of muscle tissue due to injury, disease, or the course of treatment, followed by recovery through muscle regeneration or muscle hypertrophy. Refers to the atrophy of muscles due to lack or insufficiency. It also refers to various diseases that can cause atrophy of muscle tissue.
Causes of failure or insufficient recovery through muscle regeneration and muscle hypertrophy include genetics, injury to the nervous system, and aging.
Preferably, the muscle atrophy is due to sarcopenia (immobile muscle atrophy). Sarcopenia can refer specifically to the loss of muscle mass due to aging, and in humans it can refer to the loss of muscle mass that occurs after the age of 65.

It is also preferable that the muscle atrophy is a state in which the expression level of the IL-33 receptor is increased in muscle tissue.

It is also preferable that the muscle atrophy is due to muscle atrophy. Muscle atrophy refers to symptoms of muscle atrophy or diseases that exhibit such symptoms. As mentioned above, muscular atrophy includes cases in which muscular atrophy symptoms are exhibited due to genetic factors, injuries to the nervous system, and other factors, and muscular atrophy due to long-term disuse of muscles.

Moreover, it is also preferable that the muscular atrophy is disuse muscular atrophy. Disuse muscular atrophy is atrophy of muscle tissue that results from prolonged disuse of muscles. Reasons for not using muscles for a long period of time include injury or illness, the course of its treatment, and a decline in activity frequency due to aging.

It is also preferable that the content of the IL-33 or the agonist or ligand of the IL-33 receptor contained in the active ingredient is 80% by mass or more, 90% by mass or more, or 100% by mass.
The therapeutic agent for muscle atrophy of the present embodiment may also contain components other than the above-mentioned IL-33 or IL-33 receptor agonist or ligand; however, the above-mentioned IL-33 or IL-33 receptor agonist or Effectiveness can be achieved even when the ligand is the only active ingredient (single agent).

[Pharmaceutical composition]
The therapeutic agent for muscle atrophy of this embodiment is suitably used for the treatment of muscle atrophy. That is, the active ingredient contained in the therapeutic agent for muscle atrophy of this embodiment or the therapeutic agent for muscle atrophy can also be referred to as a pharmaceutical composition used for treating muscle atrophy.
Furthermore, a pharmaceutical composition containing other ingredients in the therapeutic agent for muscle atrophy can also be prepared. The pharmaceutical composition for the treatment of muscle atrophy may appropriately contain various other components contained in conventionally known pharmaceutical compositions.

The form of the pharmaceutical composition of this embodiment is not particularly limited, and may be, for example, a solution, a dispersion such as a sol or gel, or a powder. The pharmaceutical composition can be administered orally, for example, in the form of a tablet, capsule, elixir, etc., or parenterally, in the form of an enema, etc.

As the pharmaceutically acceptable carrier, those commonly used in the formulation of pharmaceutical compositions can be used without particular limitation. More specifically, examples include binders such as gelatin, cornstarch, gum tragacanth, and gum arabic; excipients such as starch and crystalline cellulose; leavening agents such as alginic acid; and solvents such as water, ethanol, and glycerin. .

Pharmaceutical compositions may also contain excipients. Additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavoring agents such as peppermint and red oil; stabilizers such as benzyl alcohol and phenol; phosphoric acid. Buffers such as salts and sodium acetate; solubilizing agents such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives and the like.

Pharmaceutical compositions may be formulated by combining the above-mentioned proteasome inhibitors, the above-mentioned pharmaceutically acceptable carriers and excipients, as appropriate, in a unit dosage form as required by generally accepted pharmaceutical practice. Can be done.

The dosage of the pharmaceutical composition varies depending on the patient's symptoms, body weight, age, sex, etc., and cannot be determined unconditionally, but in the case of subcutaneous administration, for example, 0.000001 mg/kg to 10 mg/kg body weight per day. It may be appropriate to administer the active ingredient at body weight, preferably eg 0.00001 to 1 mg/kg body weight, more preferably 0.0005 to 0.01 mg/kg body weight.

(Effects of this embodiment)
According to the therapeutic agent for muscle atrophy of the present embodiment, treatment such as prevention of muscle atrophy and improvement of symptoms can be effectively performed.

As shown in the Examples, the present inventors attempted to elucidate the molecular mechanism of skeletal muscle atrophy involving FAPs. Specifically, we conducted analyzes using mice in a lower limb immobilization model and an aging model.

FAPs in skeletal muscle were isolated using flow cytometry, and gene expression profiles were analyzed using RNA-seq. As a result, it was revealed that in the lower limb immobilization model, the expression of the intraskeletal IL-33 gene and the ST2 gene located downstream thereof was significantly increased. ST2 protein is a receptor for IL-33 protein. On the other hand, increased expression of the IL-33 gene was suppressed in old mice, suggesting that it contributes to the worsening of disuse muscle atrophy due to aging.
Therefore, we administered IL-33 protein and soluble ST2 protein, which functions as an inhibitor, to mice and examined their effects on muscle atrophy.We found that young and old mice administered soluble ST2 protein While muscle atrophy worsened, muscle atrophy was suppressed in aged mice administered with IL-33 protein.

These results revealed that the IL-33 gene signal in FAPs negatively regulates muscle atrophy (suppresses muscle atrophy). In addition, because this signal is reduced in aged mice, it was thought that muscle atrophy due to disuse progresses excessively. This suggested that the IL-33 gene is a useful therapeutic target for disuse muscular atrophy in the elderly.

[Treatment method for muscle atrophy]
The therapeutic agent for muscle atrophy of this embodiment can be used in a method for treating muscle atrophy that includes administering an effective amount of the active ingredient to a patient.

Methods for treating muscle atrophy include a wide range of methods, including methods for treating the subject of muscle atrophy, methods for preventing muscle atrophy, methods for treating the prognosis of muscle-related diseases, and the like. Patients include a wide range of humans and other animals, including subjects suffering from muscle atrophy as described above, and subjects undergoing preventive or prognostic treatment.

The animal to be treated is preferably a mammal. Examples of mammals include humans, monkeys, marmosets, cows, horses, sheep, pigs, goats, deer, alpacas, dogs, cats, rabbits, hamsters, guinea pigs, rats, and mice. Among these, humans are preferred.

The effective amount of the active ingredient to be administered can be selected, for example, based on the dosage for the above-mentioned pharmaceutical composition. That is, the effective amount varies depending on the patient's symptoms, body weight, age, gender, etc. and cannot be determined unconditionally, but in the case of subcutaneous administration, it is, for example, 0.000001 mg/kg body weight to 10 mg/kg body weight per day, preferably. For example, it is considered appropriate to administer the active ingredient in an amount of 0.00001 to 1 mg/kg body weight, more preferably 0.0005 to 0.01 mg/kg body weight.

[Other aspects of this embodiment]
Another aspect of this embodiment is the active ingredient comprising IL-33, or an agonist or ligand of the IL-33 receptor, for use in the treatment of muscle atrophy.
Another aspect of this embodiment is the use of the active ingredient, including IL-33, or an agonist or ligand of the IL-33 receptor, for producing a therapeutic agent for muscle atrophy.
The composition and method of use of the active ingredient can be selected from those described above.

Examples are shown below. Note that the present invention is not limited to the examples.

[Isolation of single cells from muscle tissue]
A lower limb fixation model mouse was created by fixing the lower limbs of the mouse with wire. Lower limb immobilization model mice were prepared by immobilizing them for 3 days or 2 weeks. Thereafter, lower limb muscles were collected from each lower limb fixed model mouse, tissues such as blood vessels were removed, and the tissue was minced using a cutting base material (manually using scissors, etc.). Enzyme (collagenase Type 2) treatment was performed at 37°C for a total of 70 minutes, red blood cells were removed with red blood cell solubilization buffer, and subsequent washing was performed to obtain a cell sample.

Next, the cell sample was stained with an antibody at a concentration of 1×10 ⁶ to 3×10 ⁶ cells/100 μl of solution. FACS analysis was performed using antibodies FITC-CD31, FITC-CD45, PE-Pdgfra, PE/Cy7-Sca1, and biotin-SM/C2.6 (dye-antigen). In FACS gating, operations for removing dead cells, blood cells/endothelial cells, and doublets were performed sequentially. SM/C. The satellite cells isolated in 2.6 and FAPs stained with Pdgfra and Sca1 were separated, and the FAPs were mainly analyzed.

Figure 1 shows the results of changes in cell number. Figure 1(a) shows cell/weight FAPS (PDGFRα+, Sca-1+), and Figure 1(b) shows cell/weight SCs. Male and female mixture, N=3-6, *=p<0.05, respectively. On the horizontal axis, N indicates a control (non-fixed) mouse, 3D indicates a model mouse with lower limbs fixed for 3 days, and 2W indicates a lower limb fixation model mouse fixed for 2 weeks. Furthermore, 8W, 50W, and 80W are the ages of the mice, respectively; 8 weeks old was a sample of a young mouse, and 50 weeks old and 80 weeks old were samples of an old mouse.

From the results shown in the figure, FAPs increased depending on the week of lower limb immobilization, and there was a significant difference between N and 2W. Since muscle atrophy occurs due to lower limb immobilization, the number of stem cells per weight increases in response to muscle atrophy. In addition, it increased due to muscle atrophy in both young and old age. The results also showed that the number decreased with age.

[Muscle fiber cross-sectional area measurement]
In the same manner as described above, a lower limb fixation model mouse was prepared in which N was a control (non-fixed), 3D was fixed for 3 days, and 2W was fixed for 2 weeks, and TA (tibialis anterior muscle) was collected and frozen. Sections of 10 μm cross section were made 2 mm distal to the proximal end of the TA. The area around the fibers was stained with laminin, photographed using a fluorescence microscope, and the cross-sectional area of the muscle fibers (CSA) included in the cross section was measured using image analysis software (Image J). The measurement range was 100-10000 ^μm2 .

FIG. 2 shows a histogram of the cross-sectional area of muscle fibers measured from a cross section of TA (tibialis anterior). It can be confirmed that the longer the number of days of immobilization, the more the cross-sectional area of muscle fibers decreases. In other words, it was confirmed that muscle atrophy occurs depending on the number of fixed days.

[RNA extraction from FAPs]
RNA was extracted from FAPs using cells extracted from the tissue.
FIG. 3(a) shows an outline of the analysis. Young (8W) and old (80W) normal (unfixed) and 2W lower limb fixation model mice were prepared for 2 weeks, and RNA was extracted from FAPs and analyzed using next-generation RNA sequencing.
FIG. 3(b) shows an outline of the analysis results. When we extracted the RNAs that had changed by 2 or more (i.e., 4 times or more) in Log2 between Normal and 2W, we found that 611 types of RNA had changed in the 8W mouse and 985 types had changed in the 80W mouse. . There were 205 types of RNAs that had overlapping changes in 8W and 80W mice, that is, RNAs whose expression levels increased due to muscle atrophy in both young and old mice.
Among these, the RNA that increased due to muscle atrophy included ST2, which is an IL-33 receptor.

The expression levels of IL-33 and ST2 were analyzed using RNA-seq under TPMlog2 conversion conditions.
FIG. 4(a) shows the analysis results of the expression level of IL-33, and FIG. 4(b) shows the analysis results of the expression level of IL1rL1 (ST2) by RNA-seq. The expression levels of both IL-33 and ST2 are increased in 3D, and are also increased in 2W compared to Normal. Furthermore, when comparing the young (8W) and old (80W) subjects, both ST2 levels increased, but the increase in IL-33 was greater in the young subjects.

Furthermore, the expression levels of IL-33 and ST2 were analyzed using qPCR using an Applied Biosystems 7300 device after RNA was extracted from the collected TA and cDNA was synthesized.
FIG. 4(c) shows the analysis results of the expression levels of IL-33 and ST2 by qPCR. In mice with muscle atrophy that had been immobilized for 3D to 2W, an increase in the expression level was confirmed with a significant difference (N=2).
These results suggested that IL-33 and its receptor ST2 are strongly involved in muscle atrophy.

[Drug administration test to lower limb immobilization model mouse]
IL-33 and ST2 were administered to lower limb immobilization model mice, which is a model of muscle atrophy, and their effects were examined.
IL-33 administration group: recombinant IL-33 2 μg/PBS 100 μl/time sST2 administration group: recombinant sST2 5 μg/PBS 100 μl/time Negative control group: PBS 100 μl
It was administered so that Here, sST2 refers to soluble ST2, which is a protein that acts as an antagonist of IL-33 by trapping IL-33 in the blood.
The above administration was carried out twice a week for each group, as well as young (8W) and old (80W) lower limb immobilization model mice, and sacrificed after 2 weeks (total of 4 administrations).
As an evaluation method, CSA (muscle fiber cross-sectional area) of TA (tibialis anterior muscle) was measured.

FIG. 5(a) shows a graph of the CSA results of the IL-33 administration group of young mice. FIG. 5(b) shows a graph of the CSA results of the ST2 administration group of young mice. The figure is a histogram of the cross-sectional area of muscle fibers measured from a cross section of TA (tibialis anterior). The muscle fiber area is classified into ^100μm2 sections, and the ratio of the number of fibers in each section to the total is expressed (NC: negative control N=3, IL-33, ST2 administration group N=4).
Overall, it can be confirmed that CSA is reduced by lower limb immobilization. Here, regarding the comparison between the control (NC) and the administration group, the results showed that in young mice, no effect was observed due to IL-33 administration, and there was a tendency to decrease after ST2 administration.

FIG. 6(a) shows a graph of the CSA results of the IL-33 administered group of aged mice. FIG. 6(b) shows a graph of the CSA results of the ST2 administration group of old mice. Similarly to the above, the figure is a histogram of the cross-sectional area of muscle fibers measured from a cross section of TA (tibialis anterior muscle). The muscle fiber area is classified into sections of 100 ^μm2 , and the ratio of the number of fibers in each section to the total is expressed (NC: negative control N=3, IL33, ST2 administration group N=4).
Overall, it can be confirmed that CSA is reduced by lower limb immobilization. Here, regarding the comparison between the negative control in which only PBS was administered after 2 weeks of immobilization (2W) and the IL-33 and ST2 administration group, it was found that in old mice, IL-33 administration had a preventive effect on muscle atrophy, whereas ST2 administration had a preventive effect on muscle atrophy. The effect of worsening muscle atrophy was observed.

From these results, no effect of IL-33 administration was observed in young mice, and a tendency towards muscle atrophy was observed in ST2 administration. On the other hand, in old mice, it was suggested that IL-33 administration had a preventive effect on atrophy, while sST2 administration worsened atrophy. ST2 is a receptor for IL-33 and also functions as an inhibitor of IL-33.
These mutual relationships suggested that age-related decline in IL-33 signals may lead to exacerbation of muscle atrophy.
IL-33, or a substance with similar activity (for example, an ST2 ligand or agonist), may be applied to prevent muscle atrophy due to immobility after trauma or in medical diseases.

According to the present invention, a therapeutic agent for muscle atrophy that can effectively prevent muscle atrophy, improve symptoms, etc. can be obtained.

Claims (5)

A therapeutic agent for muscle atrophy containing IL-33 or an agonist or ligand of the IL-33 receptor as an active ingredient. The therapeutic agent for muscle atrophy according to claim 1, wherein the IL-33 receptor is ST2. 2. The therapeutic agent for muscle atrophy according to claim 1, wherein the muscle atrophy is caused by an increase in the expression level of the IL-33 receptor in muscle tissue. The therapeutic agent for muscle atrophy according to claim 1, wherein the muscle atrophy is disuse muscular atrophy. Claim 1, wherein the content of the IL-33 or the agonist or ligand of the IL-33 receptor contained in the active ingredient is 80% by mass or more, 90% by mass or more, or 100% by mass. therapeutic agent.

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