JP6429401B2 - Normalizing agent for excessive accumulation of endoplasmic reticulum in type I collagen with mutation - Google Patents

Description

  The present invention relates to a normalizing agent for excessive accumulation of type I collagen having a mutation in the endoplasmic reticulum and a medicine containing the same.

  Osteogenesis Imperfecta (OI) is a disease that is easily fractured by mutations in type I collagen, which is a component of bone. Normal type I collagen is formed by taking a helical structure of two α1 chains translated by the COL1A1 gene and one α2 chain translated by the COL1A2 gene. In osteogenesis imperfecta, these COL1A1 gene and COL1A2 gene have mutations, a type in which type I collagen having an irregular helical structure is produced (qualitative abnormality), and a type in which the number of type I collagen is reduced by nonsense mutation The existence of (quantitative abnormalities) is known. Since the normal helical structure cannot be constructed in the type of qualitative abnormality, the secretion of type I collagen from the endoplasmic reticulum, which is the site of protein translation, is delayed. Due to delayed secretion from the endoplasmic reticulum, post-translational modification occurs excessively in the endoplasmic reticulum, and type I collagen undergoes sugar chain hypermodification. In addition, delayed secretion from the endoplasmic reticulum increases the capacity of the endoplasmic reticulum. Currently, type I collagen that cannot build such a normal helical structure and type I collagen that has undergone sugar chain overmodification are considered to be the main causes of osteogenesis imperfecta. It is thought that a brittle bone that is easily broken is formed.

  It is known that a state of endoplasmic reticulum stress occurs when defective proteins that do not have the correct higher-order structure accumulate in the endoplasmic reticulum. Cells that perceive such an unbalanced state (endoplasmic reticulum stress) suppress translation of new proteins to maintain homeostasis, fold defective proteins into the correct higher order structure, It is also known to perform reactions such as degrading defective proteins that cannot be folded. Such reactions for sensing endoplasmic reticulum stress states and maintaining homeostasis are collectively referred to as UPR (unfolded protein response).

  Recently, Mirigian et al., In an experiment using an osteogenesis imperfecta model mouse having an amino acid substitution mutation of Gly610Cys in the procollagen α2 chain, type I collagen with an irregular helical structure accumulates in the endoplasmic reticulum and expands the endoplasmic reticulum. However, it was reported that up-regulation of immunoglobulin-binding protein serving as an index of UPR and activation of NFκB serving as an index of endoplasmic reticulum overload (ER overload) were not observed (Non-patent Document 1). In other words, the accumulation of type I collagen having a distorted helical structure that causes bone dysplasia in the endoplasmic reticulum is not a known endoplasmic reticulum stress state or endoplasmic reticulum overload state. It has been shown to cause a condition.

  At present, the only therapeutic agents used for osteogenesis imperfecta are bisphosphonates administered to reduce the risk of fracture. This treatment does not improve the bone quality of osteogenesis imperfecta, but increases the amount of bone and reinforces the bone, and does not prevent fracture. There is currently no fundamental treatment for osteogenesis imperfecta.

  A chemical chaperone is a low-molecular compound involved in the formation and stabilization of protein higher-order structures, such as 4-phenylbutyric acid (PBA), trimethylamine-N-oxide (TMAO), tauroursodeoxycholic acid (TUDCA), betaine, and taurine. Sarcosine, lutein, etc. are known. Sodium 4-phenylbutyrate is approved as a therapeutic agent for urea cycle disorders and is in clinical use. Further, sodium 4-phenylbutyrate is known to have an action of reducing endoplasmic reticulum stress by functioning as an endoplasmic reticulum chaperone. For example, Non-Patent Document 2 discloses that sodium 4-phenylbutyrate acts as a chaperone in vitro and reduced endoplasmic reticulum stress in human neuroblastoma SK-N-MC cells. Yes.

Mirigian LS et al., J Bone Miner Res. 2016 Aug; 31 (8): 1608-16.doi: 10.1002 / jbmr.2824.Epub 2016 Apr 13. Kubota K et al., Journal of Neurochemistry, 2006, 97, 1259-1268.DOI: 10.1111 / j.1471-4159.2006.03782.x

  The present invention finds a substance having an action to normalize excessive accumulation of type I collagen having a mutation in the endoplasmic reticulum, and provides a medicament useful for the treatment of diseases caused by type I collagen mutation such as osteogenesis imperfecta. The task is to do.

The present invention includes the following inventions in order to solve the above problems.
[1] A normalizing agent for excessive accumulation of type I collagen having a mutation, comprising 4-phenylbutyric acid or a pharmaceutically acceptable salt thereof as an active ingredient.
[2] The agent according to [1], wherein the pharmaceutically acceptable salt of 4-phenylbutyric acid is sodium 4-phenylbutyrate.
[3] A medicament for treating a disease caused by a mutation in type I collagen, comprising the agent according to [1] or [2].
[4] The medicament according to [3], wherein the disease caused by a mutation in type I collagen is osteogenesis imperfecta, Kaffey disease, or Eras-Danlos syndrome.
[5] The medicament according to [3], wherein the disease accompanied by excessive accumulation of type I collagen in the endoplasmic reticulum is osteogenesis imperfecta.

  The present invention can provide a normalizing agent for excessive accumulation of type I collagen having a mutation in the endoplasmic reticulum. In addition, according to the present invention, it is possible to provide a medicament useful for the treatment of diseases caused by mutations in type I collagen such as osteogenesis imperfecta.

The figure which shows the result which compared with the normal skin fibroblast the amount of vesicle accumulation of type I collagen in the skin fibroblast of the osteogenesis imperfecta patient who has a missense mutation in COL1A1 gene, and the effect of the chemical chaperone on it is there.

  The present invention provides a normalizing agent for excessive accumulation in the endoplasmic reticulum of type I collagen having a mutation, comprising 4-phenylbutyric acid or a pharmaceutically acceptable salt thereof as an active ingredient.

4-Phenylbutyric acid (chemical formula: C ₆ H ₅ (CH ₂ ) ³ COOH, CAS number: 1821-12-1) is, for example, Sigma-Aldrich (product number: P21005), Merck (catalog number) : 820986) can be purchased and used. 4-Phenylbutyric acid can be synthesized by a known method.

  In the present invention, the pharmaceutically acceptable salt is not particularly limited as long as it maintains the efficacy of 4-phenylbutyric acid and does not adversely affect the human body. A pharmaceutically acceptable salt of 4-phenylbutyric acid can be obtained by treating 4-phenylbutyric acid with an appropriate cation. Examples of pharmaceutically acceptable salts of 4-phenylbutyric acid include those formed with metal cations such as aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, benzathine, chloroprocaine, choline, diethanolamine, and ethylenediamine. , And those formed with organic cations such as meglumine and procaine.

It is preferable to use sodium 4-phenylbutyrate as an active ingredient of the endoplasmic reticulum excessive accumulation normalizing agent of the present invention. Sodium 4-phenylbutyrate (Chemical Formula: C ₆ H ₅ (CH ₂ ) ³ COONa, CAS No. 1716-12-7) is approved and clinically used as a treatment for urea cycle disorders (Brand name: Buphenyl). As the normalizing agent for excessive accumulation in the endoplasmic reticulum of the present invention, sodium 4-phenylbutyrate sold as a pharmaceutical for treating the urea cycle abnormality can be suitably used. Moreover, a commercial item can be purchased and used. Sodium 4-phenylbutyrate synthesized by a known method may be used.

  Type I collagen is composed of two α1 chains and one α2 chain, and has a triple helical structure. The type I collagen having a mutation is not particularly limited as long as it has a mutation in at least one of the COL1A1 gene encoding the α1 chain and the COL1A2 gene encoding the α2 chain, but preferably has a mutation at the protein level. Examples of the mutation at the protein level include missense mutation (amino acid substitution mutation), amino acid deletion mutation, amino acid insertion mutation, amino acid duplication mutation, and frame shift mutation. It is considered that type I collagen having such a mutation at the protein level accumulates in the endoplasmic reticulum because a normal helical structure cannot be constructed. It can be confirmed that the type I collagen has a mutation by analyzing the base sequence of the COL1A1 gene or COL1A2 gene to which the endoplasmic reticulum hyperaccumulation normalizing agent of the present invention is applied by a known method. it can.

  Type I collagen is mainly composed of three amino acid repeats of glycine-XY (X and Y are different amino acids). Glycine is at the center of the triple helix structure of collagen and is thought to play an important role in maintaining the structure. Therefore, when glycine is substituted with another amino acid, the normal helical structure cannot be maintained and the strength of collagen is considered to be weakened. And it is thought that type I collagen in which glycine is substituted with another amino acid accumulates in the endoplasmic reticulum because the helical structure becomes distorted. Therefore, the type I collagen having a mutation preferably has a mutation in which glycine is substituted with another amino acid in at least one of the α1 chain and the α2 chain.

  The excessive accumulation of type I collagen having a mutation in the endoplasmic reticulum means the amount of type I collagen accumulated in the endoplasmic reticulum in normal type I collagen-expressing cells (for example, healthy human skin fibroblasts, osteoblasts, etc.) The amount of accumulated type I collagen (abundance) in type I collagen-expressing cells (for example, skin fibroblasts, osteoblasts, etc. of osteogenesis imperfecta patients) is increased compared to Means the state. In addition, normalization of excessive accumulation of type I collagen having a mutation in the endoplasmic reticulum means that the amount of mutant type I collagen that is excessively accumulated in the endoplasmic reticulum is decreased, and normal type I collagen-expressing cells have an endoplasmic reticulum. It means close to the amount of type I collagen. For the amount of type I collagen in the endoplasmic reticulum, for example, as described in Example 1, a sample of type I collagen-expressing cells is prepared, and fluorescent immunostaining is performed using an anti-type I collagen antibody and an antibody against the endoplasmic reticulum marker. This can be confirmed by measuring the fluorescence intensity of type I collagen that co-localizes with the endoplasmic reticulum marker.

  The endoplasmic reticulum hyperaccumulation normalizing agent of the present invention can be implemented as a medicament for treating diseases caused by type I collagen mutation. That is, the present invention provides a medicament for treating a disease caused by a mutation in type I collagen, which contains the above-mentioned normalizing agent for excessive accumulation in the endoplasmic reticulum of the present invention. In other words, the present invention provides a medicament for treating diseases caused by mutations in type I collagen, comprising 4-phenylbutyric acid or a pharmaceutically acceptable salt thereof as an active ingredient.

  Examples of diseases caused by mutations in type I collagen include osteogenesis imperfecta (OI), Caffey disease, Ehlers-Danlos syndrome (EDS), and the like. Osteogenesis dysfunction is a congenital disease that shows various degrees of connective tissue symptoms in addition to easy fracture and progressive bone deformation due to bone fragility throughout the body. Over 90% of cases of osteogenesis imperfecta are due to genetic mutations in type I collagen. Cuffy's disease is also called pediatric cortical bone growth. Cuffy's disease is an infantile bone disorder characterized by acute inflammation of soft tissue caused by amino acid substitution of α1 chain of type I collagen and local thickening of the underlying bone cortex, many of which are spontaneously healing. Eras-Danros syndrome is a hereditary connective tissue disease characterized by hyperextension of the skin and joints and fragility of various tissues. Eras-Danlos syndrome is classified into several disease types. Among them, the causative gene of the multiple joint relaxation type is type I collagen gene (COL1A1, COL1A2).

  The medicament of the present invention contains 4-phenylbutyric acid or a pharmaceutically acceptable salt thereof as an active ingredient, and is pharmacologically according to a known method (for example, a method described in the Japanese Pharmacopoeia) as a method for producing a pharmaceutical preparation. An acceptable carrier or additive can be appropriately blended to prepare a preparation. Specifically, for example, tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (including soft capsules and microcapsules) , Lozenges, syrups, solutions, emulsions, suspensions, controlled release formulations (eg, immediate release formulations, sustained release formulations, sustained release microcapsules, etc.), aerosols, film agents (eg, orally disintegrating films) , Oral mucosa adhesive film, etc.), injection (eg, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.), drip, transdermal preparation, ointment, lotion, patch Oral or parenteral preparations such as suppositories (for example, rectal suppositories, vaginal suppositories), pellets, nasal preparations, pulmonary preparations (inhalants), eye drops and the like. The blending ratio of the carrier or additive can be appropriately set based on the range usually employed in the pharmaceutical field. Carriers or additives that can be blended are not particularly limited, but various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases; excipients, binders, pH adjusters, disintegrants, absorption enhancers Various additives such as an agent, a lubricant, a coloring agent, a corrigent, and a fragrance are included.

  Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like A swelling agent, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, red oil, or cherry are used. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. A sterile composition for injection can be prepared according to a usual formulation procedure (for example, an active ingredient is dissolved or suspended in a solvent such as water for injection or natural vegetable oil). As an aqueous solution for injection, for example, isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants are used, and appropriate solubilizers such as You may use together with alcohol (ethanol etc.), polyalcohol (propylene glycol, polyethyleneglycol, etc.), nonionic surfactants (polysorbate 80, HCO-50, etc.). As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. In addition, buffers (eg phosphate buffer, sodium acetate buffer etc.), soothing agents (eg benzalkonium chloride, procaine hydrochloride etc.), stabilizers (eg human serum albumin, polyethylene glycol etc.), preservatives (eg For example, you may mix | blend with benzyl alcohol, phenol, etc.), antioxidant, etc.

  The medicament of the present invention varies depending on the dosage form, administration method, carrier and the like, but the active ingredient is usually 0.01 to 100% (w / w), preferably 0.1 to 95% (w / w) based on the total amount of the preparation. It can be added at a ratio of w).

  Since sodium 4-phenylbutyrate, which is an active ingredient of the medicament of the present invention, has been administered to humans as a therapeutic agent for urea cycle abnormalities, humans and other mammals (for example, rats, mice, rabbits, sheep) , Pigs, cows, cats, dogs, monkeys, etc.).

  The dose of the pharmaceutical agent of the present invention varies depending on the subject of administration, symptoms, administration route, etc., but in the case of oral administration, for example, generally about 0.01 mg to 20 g per day in a human body weight of about 60 kg, Preferably it is about 0.1 mg to 15 g, more preferably about 0.5 mg to 10 g. For parenteral administration, the single dose varies depending on the patient's condition, symptoms, administration method, etc. For example, in the case of injection, it is usually about 0.01 to 500 mg per kg body weight, preferably about 0.01 to 300 mg, more preferably about 0.01 to 200 mg. The total daily dose may be a single dose or divided doses.

  The medicament of the present invention can be used in combination with other drugs. Examples of other drugs include bisphosphonate preparations administered for the purpose of reducing fracture risk for osteogenesis imperfecta.

The present invention includes the following inventions.
A method for normalizing the excessive accumulation of type I collagen having a mutation in the endoplasmic reticulum, comprising administering 4-phenylbutyric acid or a pharmaceutically acceptable salt thereof to a mammal.
4-phenylbutyric acid or a pharmaceutically acceptable salt thereof for use in normalizing endoplasmic reticulum hyperaccumulation of type I collagen having a mutation.
Use of 4-phenylbutyric acid or a pharmaceutically acceptable salt thereof for producing a normalizing agent for excessive accumulation of endoplasmic reticulum of type I collagen having a mutation.
A method for treating a disease caused by a mutation in type I collagen, comprising administering 4-phenylbutyric acid or a pharmaceutically acceptable salt thereof to a mammal.
4-phenylbutyric acid or a pharmaceutically acceptable salt thereof for use in the treatment of diseases caused by mutations in type I collagen.
Use of 4-phenylbutyric acid or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of diseases caused by mutations in type I collagen.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these.

[Example 1: Effect of chemical chaperone on intravesicular accumulation of type I collagen with mutations]
1. Materials and Methods (1) Cells Skin fibroblasts collected at the time of surgery from patients with osteogenesis imperfecta (COL1A1 gene has c.2461G> A (p.G821S) mutation) were used. As normal cells, newborn human dermal fibroblasts (Human Dermal Fibroblasts, neonatal, Gibco, Cat. # C0045C) were used. Experiments were performed using cells of passage number 5-6. These cells were seeded in a 96-well plate at 6-8 × 10 ³ cells / well, and DMEM (Wako), 10% FBS (Equitech Bio), 0.5% penicillin, which is a culture medium for normal skin fibroblasts. -Wells to which a chemical chaperone was added and not added to streptomycin (Gibco) were cultured for 2 days.

(2) Chemical chaperone As chemical chaperones, sodium 4-phenylbutyrate (Sigma-Aldrich, Cat. # SML0309, hereinafter abbreviated as “4PBA”) and trimethylamine-N-oxide dihydrate (Sigma-Aldrich, Cat. # 317594, hereinafter abbreviated as “TMAO”). 4PBA was added to the medium at concentrations of 5 mM, 1 mM, and 0.2 mM, and TMAO was added to the medium at concentrations of 100 mM, 50 mM, and 5 mM.

(3) Quantification of type I collagen in the endoplasmic reticulum Fluorescent immunostaining was performed to quantify the accumulation of type I collagen in the endoplasmic reticulum. Anti-type I collagen antibody (Abcam, Cat. # Ab34710) is used as the primary antibody against type I collagen, and anti-PDI (protein disulfide isomerase) monoclonal antibody (Enzo, Cat. # ADI-SPA-891) is used as the primary antibody against the endoplasmic reticulum marker. ) Was used. Anti-Rabbit IgG (Jackson Immunoresearch; 711-485-152) and Anti-Mouse IgG (Jackson Immunoresearch; 715-505-151) were used as secondary antibodies.
After culturing the cells for 2 days, the cells were fixed by incubating with 4% paraformaldehyde for 10 minutes followed by cold methanol for 1 minute and blocking buffer (PBS, 1% BSA, 10% FBS, 0.3 M glycine, 0.1% Tween 20) was added and incubated at room temperature for 1 hour. Thereafter, anti-collagen type I antibody and anti-PDI clonal antibody were incubated overnight at 4 ° C. with a primary antibody solution diluted 1: 100 and 1: 200 in blocking buffer, respectively. Subsequently, each secondary antibody was incubated with a secondary antibody solution diluted 1: 500 with a blocking buffer for 1 hour at room temperature.

(4) Measurement of fluorescence intensity Fluorescence of type I collagen showing fluorescence-stained cells with In Cell Analyzer 6000 (GE Healthcare) and colocalizing with endoplasmic reticulum markers using In Cell Developer Toolbox (GE Healthcare) The strength was measured.

2. Results The results are shown in FIG. The amount of accumulation of type I collagen in the endoplasmic reticulum was expressed as a relative value of the fluorescence intensity of type I collagen in each group, with the fluorescence intensity of type I collagen in the group without normal chemical chaperone added as 1. In the group without added chemical chaperone of osteogenesis imperfecta patient cells (OI), type I collagen more than 1.6 times that of normal cells was accumulated in the endoplasmic reticulum. In patients with osteogenesis imperfecta added with 4PBA, the amount of type I collagen accumulated in the endoplasmic reticulum decreased in a concentration-dependent manner, and decreased to the same level as normal cells in the 5 mM group. In addition, the influence of 4PBA on normal cells was small. On the other hand, even in the group in which TMAO was added to cells with osteogenesis imperfecta, the amount of type I collagen accumulated in the endoplasmic reticulum was decreased at concentrations of 50 mM and 5 mM, but no significant effect was observed as in 4PBA. It was. As a result, the decrease (normalization) in the endoplasmic reticulum type I collagen accumulation in cells with osteogenesis imperfecta is not simply a restoration of higher-order structure by chemical chaperone, and some other action by 4PBA contributes. It is thought that.

  The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

Claims (5)

  A normalizing agent for excessive accumulation of type I collagen having a mutation, comprising 4-phenylbutyric acid or a pharmaceutically acceptable salt thereof as an active ingredient.   The agent according to claim 1, wherein the pharmaceutically acceptable salt of 4-phenylbutyric acid is sodium 4-phenylbutyrate. A medicament for the treatment of osteogenesis imperfecta having a mutation in which a glycine present in the site constituting the triple helical structure of type I collagen is substituted with another amino acid, containing the agent according to claim 1 or 2. . The medicament according to claim 3, wherein the glycine is α1 chain glycine of type I collagen . The medicament according to claim 3, wherein the glycine is the 821st glycine of α1 chain of type I collagen .