Patent Application
20210171978
The present invention relates to a non-human model animal useful as a disease research animals that oxidative stress is involved deeply, in particular, useful as a model animal for aging, kidney disease, cardiovascular disease, high blood pressure, aortic dissection, chronic obstructive pulmonary disease, age-dependent epilepsy, dyslipidemia, anemia, osteoporosis, immune disorder, and particularly relates to a non-human model animal in which a gene encoding thioredoxin is modified.
The living body is equipped with the antioxidant ability by antioxidant enzymes and antioxidants, and there is a system (redox regulation) that maintains homeostasis by adapting to various oxidative stress by controlling the redox state of the living body. However, if this control mechanism cannot sufficiently process the oxidative stress caused by the endogenous or extrinsic factors of the living body, or if the control mechanism fails for some reasons, various obstacles appear in the living body. Excessive oxidative stress damages DNA, proteins, lipids, etc., causing cell dysfunction and cell death, and is therefore considered to be one of the important causes in cancer and aging.
Thioredoxin is a protein with a molecular weight of about 12 kDa consisting of 105 amino acids in human, rat and mouse, and has two cysteine residues in its active site. The dithiol/disulfide exchange reaction between the cysteine residues contributes to the redox reaction of the substrate. The reduced thioredoxin bearing dithiol reduces the disulfide bond of a substrate protein and becomes itself in oxidized form.
Thioredoxin is induced by various factors such as ultraviolet light, radiation, oxidants, viral infection, ischemia-reperfusion injury, administration of anti-cancer agents, etc., which are factors that cause oxidative stress, and it regulates transcription factors that regulate various gene expression and the activity of intracellular signaling molecules (J. Annual Review of Immunology. 15, 351-369 (1997), Annual Review of Biophysics and Biomolecular Structure 30, 421-455 (2001)). Thioredoxin acts defensively against oxidative stress-induced diseases, is resistant to various chemical substances and anticancer agents, and thioredoxin expression is involved in longevity (J Gerontol A Biol Sci Med Sci., 66A: 1286-1299 (2011), The Journal of Clinical Investigation 112 (9), 1395-1406 (2003), Nephrol Dial Transplant; 22: 1547-1557 (2007)). As a conventional technique, a thioredoxin-deficient mouse has been created, but this defective gene homozygous mouse died in the fetal period, and the heterozygote was normal (Non-Patent Document 1). In addition, there is a transgenic animal that expresses a thioredoxin gene having a dominant negative effect in the heart and serves as a hypertrophic cardiomyopathy model (Non-patent Document 2).
The problem to be solved is the absence of non-human animal models of various diseases caused by diminished thioredoxin function. The object of the present invention is to provide a non-human animal model applicable for aging, kidney disease, cardiovascular disease, hypertension, aortic dissection, chronic obstructive pulmonary disease, age-dependent epilepsy, dyslipidemia, anemia, osteoporosis, immune abnormality, etc.
The present invention provides a non-human animal model applicable as a disease model such as aging kidney disease, cardiovascular disease, hypertension, aortic dissection, chronic obstructive pulmonary disease, age-dependent epilepsy, dyslipidemia, anemia, osteoporosis, immune disorder, etc. caused by hypofunction of thioredoxin. In order to solve the above-mentioned problems, the present inventors administered to male rats ethylnitrosourea, a chemical mutagen which randomly induces point mutations on the genome, and produced an offspring rat group by mating with female rats, selected rats with phenotypic abnormalities from the group, and confirmed gene abnormalities to find out for the first time that the causative gene was a mutation in the Txn1 gene. As a result of further studies, the present inventors succeed in the production of a non-human animal having mutations in the Txn1 gene, which can be born even if homozygous, and applicable as a disease model such as aging, kidney disease, cardiovascular disease, hypertension, chronic obstructive pulmonary disease, age-dependent epilepsy, dyslipidemia, anemia, osteoporosis, immune disorder, and the present invention has been completed.
That is, the present invention is, consists of the following.
1. A non-human model animal in which a gene encoding thioredoxin is modified.
2. The non-human model animal according to item 1 above, wherein the modified gene encoding thioredoxin decrease an amount of NADPH (nicotinamide adenine dinucleotide phosphate) consumed per unit time to reduce a disulfide bond of a substrate protein and a decrease rate thereof is in a range of 10% to 90%.
3. The non-human model animal according to item 1 or 2 above, wherein the animal is adapted as a disease model selected from the group consisting of aging, kidney disease, cardiovascular disease, hypertension, aortic dissection, chronic obstructive pulmonary disease, age-dependent epilepsy, dyslipidemia, anemia, osteoporosis and immune disorder.
4. A method for screening a drug, wherein the method uses the non-human model animal according to any one of items 1 to 3 above.
The non-human model animal of the present invention in which the gene encoding the thioredoxin is modified shows disease such as aging, kidney disease, cardiovascular disease, hypertension, aortic dissection, chronic obstructive pulmonary disease, age-dependent epilepsy, dyslipidemia, anemia, osteoporosis, immune disorder, and can be used as a model animal for elucidating the pathological conditions of these diseases and screening therapeutic agents.
The non-human model animal of the present invention is a non-human model animal of a disease that develops due to a decrease in the function of thioredoxin due to a modified gene encoding thioredoxin. Examples of the non-human model animal of the present invention include mammals other than humans, and examples thereof include monkeys, marmosets, cows, pigs, dogs, cats, guinea pigs, rabbits, rats and mice. In particular, rodents such as rats, mice, and guinea pigs are preferable because they are easy to handle, and among them, rats are preferable.
The non-human model animal of the present invention is a non-human model animal obtained by modifying the gene encoding thioredoxin. Thioredoxin is a protein having a molecular weight of about 12 kDa consisting of 105 amino acids in human, rat and mouse, and has two cysteine residues in its active site. It contributes to the redox reaction of the substrate by a dithiol/disulfide exchange reaction between the cysteine residues. The reduced thioredoxin having dithiol reduces disulfide bonds of substrate proteins and becomes itself in oxidized form. The gene encoding thioredoxin is any gene called TXN, TRX, TRX1, TRDX, Txn1, Txn, Trx1 or ADF. For example, human-derived genes are called TXN, TRX, TRX1 or TRDX, rat-derived genes are called Txn1 or Txn, and mouse-derived genes are called Txn1, Txn, Trx1 or ADE As a gene encoding thioredoxin, for example, the rat-derived gene Txn1 includes the gene specified by the nucleotide sequence shown in GenBank Accession No. NM_053800 (SEQ ID NO: 1), and the Txn1 protein is specified by the amino acid sequence shown in SEQ ID NO: 2.
The non-human animal model of the present invention in which the gene encoding thioredoxin is modified in each of homozygous model and heterozygous model shows disease such as aging, kidney disease, cardiovascular disease, hypertension, aortic dissection, chronic obstructive pulmonary disease, age-dependent epilepsy, dyslipidemia, anemia, osteoporosis, and immune disorders. Hereinafter, the modified gene is also simply referred to as “modified thioredoxin gene”. The modified thioredoxin gene according to the present invention may reduce the reducing function of substrate protein on which thioredoxin acts to 10% to 90%, preferably 10% to 70%, more preferably 26.9% to 53.9%. Since the consumption of C35S thioredoxin was reduced to 2.55% to 7.45%, the functionally reduced range of the modified thioredoxin gene in the present invention is a value excluding the range which C35S shows reduction. The modified thioredoxin gene may have a nucleotide sequence capable of expressing thioredoxin in which the 54th amino acid F is replaced with L. Such nucleotide sequences includes, in addition to the nucleotide sequence encoding thioredoxin in which the 54th codon TTC is modified to CTC, any nucleotide sequence encoding a protein having the same amino acid sequence as the modified thioredoxin due to the degeneracy of the gene codon.
The genetically modified non-human model animal of the present invention may be any animal that can express a modified thioredoxin by a nucleotide sequence (modified thioredoxin gene) encoding thioredoxin on the genome of the animal. Such animals can be achieved by placing the modified thioredoxin gene on the genome. For example, it can be produced by artificially changing the genetic information of an individual using genetic engineering. Examples of genetically modified non-human model animals include transgenic animals into which a specific gene has been externally introduced, and knock-in animals in which a specific gene has been replaced. In addition, it can be prepared by genome editing of a model animal by introducing, deleting, replacing a gene in the chromosome thereof further in combination with an effector.
A transgenic non-human animal generally refers to a non-human animal in which an exogenous gene called a transgene is artificially integrated into the host genome and is inherited for generations. It is often produced for the purpose of expressing a mutant protein different from the protein possessed by the host. As a method for producing a transgenic non-human, a method known per se, for example, a microinjection method can be adopted. It is possible to introduce the modified thioredoxin gene of the present invention into the pronucleus of a fertilized egg collected from a donor animal, transplant the fertilized egg into the oviduct of the recipient animal, and spontaneously give birth to produce a transgenic non-human animal. The transgenic non-human animal of the present invention can be prepared by introducing the modified thioredoxin gene of the invention into unfertilized eggs, fertilized eggs, progenitor cells including sperm and its primordial germ cells, etc., preferably at the stage of embryogenesis in the development of non-human animals (more preferably, in the single cell before cleavage of the fertilized egg or at the cleavage stage and generally before the 8-cell stage), using calcium phosphate method, electric pulse method, lipofection method, agglutination method, microinjection method, particle gun method, DEAE-dextran method, and the like. In addition, a transgenic non-human animal can be produce also by that the modified thioredoxin gene of the present invention is introduced into somatic cells, living organs, tissue cells and the like by the gene transfer method to prepare cell cultures, tissue cultures and the like and these cells are fused with the germline precursor cells described above by a cell mixing method known per se.
The fertilized egg into which the gene has been introduced is then transplanted into the oviduct of a foster mother to become a fetus, and DNA is extracted from a part of the body (for example, the tip of the tail for mice and rats) at the age of 3- to 4-weeks after birth and the presence of the transgene can be confirmed by Southern analysis or PCR. If the individual confirmed to have the transgene is the first generation, the transgene is transmitted to its offspring (F1). Furthermore, by crossing this F1 individual with a wild-type animal or another F1 animal, an individual (F2) having a transgene in one (heterozygous) or both (homozygous) of the diploid chromosome can be prepared.
According to another embodiment, the non-human animal of the present invention that expresses thioredoxin in which the 54th amino acid F is replaced by L can be produced also by introducing the modified thioredoxin gene of the present invention described above into ES cells (embryonic stem cells). For example, it can be prepared by introducing the modified thioredoxin gene of the present invention into HPRT-negative (hypoxanthine guanine phosphoribosyl transferase gene-deficient) ES cells derived from normal rat blastocysts. ES cells in which the modified thioredoxin gene of the present invention is integrated on the rat endogenous gene are selected by the HAT selection method. Then, the selected ES cells are microinjected into a fertilized egg (blastocyst) obtained from another normal non-human animal. The blastocyst is transplanted into the uterus of another normal non-human animal as a foster mother. Then, a chimeric transgenic non-human animal is born from the foster parent non-human animal. A heterozygous transgenic non-human animal can be obtained by mating the chimeric transgenic non-human animal with a normal non-human animal. By mating the heterozygous transgenic non-human animals with each other, homozygous transgenic non-human animals can be obtained. It can be said that these transgenic non-human animals are model animals in which the gene encoding thioredoxin of the present invention is modified.
According to another embodiment, a knock-in non-human animal expressing thioredoxin in which the 54th amino acid F is replaced with L can be prepared by introducing the modified thioredoxin gene of the present invention into ES cells. For example, it can be prepared by introducing the modified thioredoxin gene of the present invention into ES cells of a normal non-human animal such as rat. ES cells in which the modified thioredoxin gene of the present invention is substituted on the endogenous gene of a non-human animal are selected by the positive selection method using G418 and the negative selection method using the herpes thymidine kinase gene. Then, the selected ES cells are microinjected into a fertilized egg (blastocyst) obtained from another normal non-human animal. The blastocyst is transplanted into the uterus of another normal non-human animal as a foster mother. Then, a chimeric knock-in non-human animal is born from the foster mother non-human animal. A heterozygous knock-in rat can be obtained by mating the chimeric knock-in non-human animal with a normal non-human animal. A homo knock-in non-human animal can be obtained by mating the heterozygous knock-in rats. It can be said that these knock-in non-human animals are non-human model animals in which the gene encoding thioredoxin of the present invention is modified.
In yet another aspect, the non-human model animal of the present invention can also be produced by using the technique of genome editing. As a method of genome editing, a method known per se or any method developed in the future can be applied. For example, gene-specific disruption or knock-in of a target gene in a model animal is performed by a technique such as CRISPR/Cas system, Transcription Activator-Like Effector Nucleases (TALEN), Zinc Finger Nuclease (ZFN), and the like to produce non-human model animals.
Hereinafter, for a better understanding of the present invention, details of the contents of the present invention will be described by examples and experimental examples.
In this example, rats with phenotypic abnormalities are analyzed, and the production of the Txn1 gene-modified rats based on the analysis results is described.
1) Confirmation of Rat with Phenotypic Abnormalities
Ethyl nitrosourea (N-ethyl-N-Nitorosourea; ENU), a chemical mutagen which induces random point mutations in the genome (40 mg/kg) was intraperitoneally administered to male rats F344/NSlc twice (at the time of 9- or 10-week-old), and the rats were mated with female rats F344/NSlc to produce offspring rats. Rats with phenotypic abnormalities were selected from the offspring rats, and genes involved in phenotypic abnormalities were searched. As a gene analysis, a backcross group was produced by mating with a wild type, and linkage analysis with the disease type was performed using a DNA polymorphism marker on all chromosomes. This narrowed down the relevant loci involved in phenotypic abnormalities, and identified that the causative gene was a mutation of Txn1 gene by genome sequence analysis using a next-generation sequencer.
The amino acids in the vicinity of thioredoxin mutation site of the rat (54th F) were aligned and analyzed with other animal species. The 54th amino acid F was conserved across animal species (
2) Production of Txn1 Gene-Modified Rat
Rats in which the 54th codon TTC of Txn1 gene was changed to CTC confirmed in 1) above were backcrossed with wild type (WT) rats of the same strain for more than ten generations to match the genetic background with WT, to produce Txn1 gene-modified rats (heterozygotes: Hetero). By crossing the heterozygous Txn1 gene-modified rats with each other, homozygous Txn1 gene-modified rats, heterozygous Txn1 gene-modified rats and wild-type rats (WT) were obtained. In the following experiments, this sibling WT was used as a control. Hereinafter, the homozygous mutant rat is referred to as “Homo” and the heterozygous mutant rat is referred to as “Hetero”. Wild-type rat (WT) is referred to as “WT”. The same applies to the following experimental examples.
Txn1 protein expression was confirmed in Txn1 gene-modified rats prepared in Example 1. Kidney, heart, liver and brain tissues were excised from One-month-old and 4-month-old WT, Hetero and Homo, and 12-month-old WT and Hetero, frozen in liquid nitrogen and stored in a deep freezer. Protein was extracted from each tissue using Lysis Buffer, and subjected to ultrasonication. After removing the precipitate with a cooling centrifuge and measuring the protein concentration, Western blotting was carried out using an anti-Txn1 antibody and an anti-GAPDH antibody as an internal control. It was revealed that there was no significant difference in the expression level of Txn1 protein in each tissue of Hetero and Homo rats compared with WT. From this, it was considered that the mutant Txn1 protein was also stably expressed without degradation.
In this experimental example, Txn1 recombinant protein was prepared using the Txn1 gene cDNA of the Homo rat and the WT rat prepared in Example 1, and the Txn1 activity was measured. RNA was extracted from WT and Homo rats, normal type (WT) and mutant type (F54L) cDNA were prepared by RT-PCT, and the cDNA were inserted into pCMV-Tag2B vector having FLAG-tag. Further, a mutant C35S in which the 35th cysteine, the active center of Txn1, was mutated to serine was prepared and introduced into pCMV-Tag2B vector. The sequences of all the prepared plasmids were confirmed by sequence analysis. Each vector (WT, F54L, C35S, Empty vector) was introduced into human normal cultured cells HEK293 to cause gene expression, and then FLAG-fused thioredoxin protein was purified using anti-FLAG antibody beads (Sigma; ANTI-FLAG M2 Agarose Affinity Gel, A2220). After quantifying the protein concentration and comparing and confirming the amount of each protein by Western blot using anti-Txn1 antibody (Cell Signaling Technology, #2298) and anti-FLAG antibody (Sigma, F3165), Thioredoxin Activity Assay Kit (Redoxica, TR-7011K) and a trace spectrophotometer (De Novix, DS-11+) were used to measure the activity of Txn1.
In the present experimental example, proteins were extracted from the tissues of Homo, Hetero, and WT rats prepared in Example 1 and Txn1 activity was measured. Tissues of 4-month-old Homo, Hetero, and WT were extracted, frozen in liquid nitrogen, and stored in a deep freezer. After protein was extracted from kidney tissue using Lysis Buffer and subjected to ultrasonication, the precipitate was removed by a cooling centrifuge and the protein concentration was quantified. After confirming the amount of protein by Western blot using anti-Txn1 antibody (Cell Signaling Technology, #2298), thioredoxin Activity Assay Kit (Redoxica, TR-7011K) and a trace spectrophotometer (DeNovix, DS-11+) was used to measure the activity of Txn1.
In this experimental example, the lifespan of the Homo, Hetero and WT rats produced in Example 1 were confirmed. Compared with the lifespan of WT rats of 2 to 3 years, Homo rats died in about 3-4 months, Hetero tended to die from about 9 months, and it was confirmed that Txn1 gene-modified rats have a short lifespan, and male Txn1 gene-modified rats had a shorter lifespan than females due to sex difference (
In this experimental example, the body weight of the Homo, Hetero and WT rats produced in Example 1 was measured over the age of 0 to 12 months, and the health conditions were observed.
In this experimental example, the pathological states of progressive renal dysfunction, hypercholesterolemia, and anemia in the Homo, Hetero and WT rats produced in Example 1 were confirmed. Blood and urine were collected from 0 to 12 month-old rats and subjected to biochemical examination. In
Kidney tissues of WT, Hetero and Homo rats aged 1-month and 3-month, and WT and Hetero rats aged 10 to 12-month were excised and subjected to histopathological analysis.
The number of dialysis patients due to end-stage renal failure is increasing worldwide, which is a big problem in the medical economy. The number of dialysis patients in Japan was about 300,000 in 2011 (1 out of 450 in the total population). The causes are various, but it is estimated that the number of patients with chronic kidney disease (CKD), a reserve arm of end-stage renal failure, is 12.9% of Japanese adults and 13.3 million people (1 out of 8 adults) is CKD patients requiring intervention. CKD is said to have 5 billion patients worldwide. Background factors of CKD are many lifestyle-related diseases such as diabetes and hypertension, and due to the aging of society, it is expected that the number of patients will increase further in the next 10 years. Since the Txn1 gene-modified rats have a rapid progression of renal dysfunction, it is possible to determine the effect of a therapeutic agent in a short period of time, and in particular, to evaluate the prolongation of life prognosis, which is a clinical problem. Thus it can be used as an excellent model rat for chronic kidney disease. There are many experimentally induced kidney disease models, but the present invention is a hereditary chronic kidney disease model, the chronic kidney disease model rat according to the present invention is superior in that symptoms occur due to spontaneous occurrence without pretreatment of animals unlike other kidney disease models, and that the chronic kidney disease model rat according to the present invention exhibit many renal disease-related complications while the other kidney disease models exhibit only specific symptoms. The rat of the present invention, the cell isolated from the rat of the present invention, and the cell in which the NADPH consumption per unit time of thioredoxin is reduced to 10% to 90% of the normal level are considered to be used not only for studying the elucidation of the mechanism of the pathological condition, but also for the search for biomarkers reflecting the pathological condition, the screening of therapeutic agents and the analysis of drug efficacy.
In this experimental example, the pathology of heart disease in the Homo, Hetero and WT rats produced in Example 1 was confirmed.
In this experimental example, lesions of chronic obstructive pulmonary disease in Homo, Hetero and WT rats produced in Example 1 were confirmed.
In this experimental example, thymus atrophy in Homo, Hetero and WT rats produced in Example 1 was confirmed. The thymus is a primary lymphoid organ involved in the immune system such as T cell differentiation and maturation, and consists of two lobes called thoracic lobules. Immature lymphocytes are present in the cortex, which is the outer part of the mature thymus, and the inner medulla contains lymphocytes as well as antigen-presenting cells such as macrophages and dendritic cells.
In the present experimental example, ovarian atrophy in Homo and WT rats produced in Example 1 was confirmed. Ovarian atrophy and a decrease in mature follicles were observed in Homo rats compared to WT rats (
In this experimental example, blood was collected from the Homo, Hetero and WT rats produced in Example 1 at 0- to 12-month-old, subjected to biochemical examination for calcium, inorganic phosphorus, and alkaline phosphatase. In addition, Computed Tomography (CT) was performed on 4-month-old Homo, Hetero and WT rats.
In this experimental example, epileptic seizures in Homo, Hetero and WT rats produced in Example 1 were confirmed. Epileptic seizures concentrated in 4- to 5-week-old rats and epileptic seizures gradually decreased and disappeared after 6 weeks. More specifically, we confirmed that epileptic seizures changed in age-dependent manner.
Txn1 gene-modified rats exhibited partial seizures, repeated bouncing seizures, and generalized tonic seizures from the age of 4 weeks.
Brain lesions from 3-week-old Txn1 gene-modified rats, before the observation of epileptic seizures, to 4-week-old, 5-week-old, and 7-week-old rats were subjected to pathological examination by HE staining. Three- to five-week-old Hetero rats showed vacuolar degeneration in inferior colliculus, thalamus and hypothalamus (
Age-dependent epilepsy is an epilepsy syndrome found in childhood. There is a characteristic that the disease type changes with the development of the brain, and onset and age-dependent changes in the disease type are observed at a specific age. This age-dependent change also includes spontaneous healing. Age-dependent epilepsy includes West syndrome, Lennox-Gastaut syndrome, Doose syndrome, childhood absence epilepsy, and benign childhood epilepsy with centrotemporal spikes.
Recently, various causative genes of age-dependent epilepsy have been identified by genetic analysis. Along with this, attempts have been made to produce model animals in epilepsy research facilities around the world, but there are no model animals that reflect the pathology and lesions of age-dependent epilepsy in genetically modified mice and rats. It is considered that the Txn1 gene-modified rat can be used as an excellent age-dependent epilepsy model rat. The rat of the present invention, neurons isolated from the rat of the present invention, and cells in which NADPH consumption of thioredoxin per unit time is reduced to 10% to 90% of the normal level are considered to be useful not only for studying the elucidation of the mechanism of the pathology but also for preventing the onset, screening of therapeutic agents, and drug efficacy analysis.
Txn1 gene-modified mice were created using the CRISPR/Cas9 system. First, a guide RNA containing a sequence near the 54th codon of the mouse Txn1 gene was prepared, and introduced into fertilized eggs of C57BL/6J mice along with Cas9 protein and a single-stranded DNA fragment having a mutant sequence of the 54th codon (including 2 silent mutations). The 136 fertilized eggs that were introduced were transplanted into the uterus of temporary mother. The number of pups F0 born was 9, but 5 died. The remaining 4 animals (F0-#1, F0-#2, F0-#3, F0-#4) were excised at the tail tips, and using the obtained DNA, 363 bases including the 54th codon of the Txn1 gene was amplified by PCR and was subjected to sequence analysis. The above gene-modified mouse F0 was crossed with a C57BL/6J wild type mouse to obtain offspring mouse F1.
The upper part of
As described in detail above, the non-human model animal of the present invention in which the gene encoding thioredoxin is modified is useful as a disease research animal deeply associated with oxidative stress, particularly, as model animals for aging, kidney disease, cardiovascular disease, chronic obstructive lung, age-dependent epilepsy, dyslipidemia, anemia, osteoporosis, immune disorders, etc. It is also useful for evaluating the effectiveness of health foods and nutritional foods.
The non-human model animal of the present invention in which the gene encoding thioredoxin is modified is useful for the pathological condition analysis of the linkage of a plurality of organ diseases, because the plurality of diseases described above appear. For example, cardiorenal linkage, cardiopulmonary linkage, renal-brain linkage, and the like. In addition, it is useful for evaluating the effects of pharmaceuticals, health foods, etc. on multiple coexisting diseases. It is useful for developments for pharmaceuticals and health foods for human multi-organ disease patients, for example, how pharmaceuticals for heart disease etc. influence/effect co-existing kidney and lung diseases, and how drugs pharmaceuticals for kidney disease etc. influence/effect co-existing heart and brain diseases.
The non-human model animal of the present invention in which the gene encoding thioredoxin is modified can provide each organ, primary cultured cells such as fetal fibroblasts and its cell lines or stem cells, iPS cells derived from the non-human model animal of the present invention. Each cell can be applied to studies and screening of therapeutic agents in aging, kidney disease, cardiovascular disease, chronic obstructive pulmonary disease, age-dependent epilepsy, dyslipidemia, anemia, osteoporosis and the like.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017-238612 | Dec 2017 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2018/045008 | 12/6/2018 | WO | 00 |
