JP6429401B2 - Agent for normalizing excessive accumulation of mutated type I collagen in the endoplasmic reticulum - Google Patents

Description

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

Osteogenesis Imperfecta (OI) is a disease that is prone to fractures, mainly due to mutations in type I collagen, a component of bone. Normal type I collagen is formed by two α1 chains translated by the COL1A1 gene and one α2 chain translated by the COL1A2 gene forming a helical structure. There are two types of osteogenesis imperfecta: one type in which type I collagen with a distorted helical structure is produced due to mutations in the COL1A1 or COL1A2 gene (qualitative abnormality), and one type in which the number of type I collagen is reduced due to nonsense mutations (quantitative abnormality). In the qualitative abnormality type, the normal helical structure cannot be constructed, resulting in delayed secretion of type I collagen from the endoplasmic reticulum, the site of protein translation. Due to the delayed secretion from the endoplasmic reticulum, excessive post-translational modification occurs in the endoplasmic reticulum, and type I collagen is overmodified with glycans. In addition, delayed secretion from the endoplasmic reticulum increases the volume of the endoplasmic reticulum. Currently, type I collagen that cannot form a normal helical structure and type I collagen with overmodified sugar chains are thought to be the main causes of osteogenesis imperfecta, and it is believed that these lead to the formation of poor-quality bones that are easily broken and brittle.

It is known that when defective proteins that do not have the correct higher-order structure accumulate in the endoplasmic reticulum, a state known as endoplasmic reticulum stress occurs. It is also known that cells that sense this unbalanced state (endoplasmic reticulum stress) respond to maintain homeostasis by suppressing the translation of new proteins, folding defective proteins into the correct higher-order structure, and degrading defective proteins that cannot be folded into the correct higher-order structure. The responses that sense this state of endoplasmic reticulum stress and maintain homeostasis are collectively known as the UPR (unfolded protein response).

Recently, Mirigian et al. reported that in an experiment using an osteogenesis imperfecta model mouse with an amino acid substitution mutation of Gly610Cys in the procollagen α2 chain, type I collagen with an irregular helical structure accumulated in the endoplasmic reticulum and expanded the endoplasmic reticulum, but did not observe upregulation of immunoglobulin-binding protein, an indicator of UPR, or activation of NFκB, an indicator of endoplasmic reticulum overload (ER overload) (Non-Patent Document 1). In other words, it was shown that the accumulation of type I collagen with an irregular helical structure that causes osteogenesis imperfecta in the endoplasmic reticulum does not cause the conventionally known endoplasmic reticulum stress state or endoplasmic reticulum overload state, but rather causes a different cellular stress state.

Currently, the only medication used to treat osteogenesis imperfecta is a bisphosphonate, which is administered to reduce the risk of fracture. This treatment does not improve the poor bone quality of osteogenesis imperfecta, but rather increases bone mass and strengthens the bones, but does not prevent fractures. There is currently no cure for osteogenesis imperfecta.

Chemical chaperones are low molecular weight compounds involved in the formation and stabilization of protein higher-order structures, and examples of such compounds include 4-phenylbutyric acid (PBA), trimethylamine-N-oxide (TMAO), tauroursodeoxycholic acid (TUDCA), betaine, taurine, sarcosine, and lutein. Sodium 4-phenylbutyrate has been approved as a treatment for urea cycle disorders and is in clinical use. Sodium 4-phenylbutyrate is also known to have the effect 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 reduces endoplasmic reticulum stress in human neuroblastoma SK-N-MC cells.

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 objective of the present invention is to discover a substance that has the effect of normalizing the excessive accumulation of mutated type I collagen in the endoplasmic reticulum, and to provide a medicine that is useful for treating diseases caused by mutations in type I collagen, such as osteogenesis imperfecta.

In order to solve the above problems, the present invention includes the following inventions.
[1] An agent for normalizing excessive accumulation of mutated type I collagen in the endoplasmic reticulum, the agent comprising 4-phenylbutyric acid or a pharma- ceutical acceptable salt thereof as an active ingredient.
[2] The agent according to the above [1], wherein the pharma- ceutically acceptable salt of 4-phenylbutyric acid is sodium 4-phenylbutyrate.
[3] A pharmaceutical for treating a disease caused by a mutation in type I collagen, comprising the agent according to [1] or [2] above.
[4] The pharmaceutical according to the above [3], wherein the disease caused by a mutation in type I collagen is osteogenesis imperfecta, Caffey disease, or Ehlers-Danlos syndrome.
[5] The pharmaceutical according to the above [3], wherein the disease associated with excessive accumulation of type I collagen in the endoplasmic reticulum is osteogenesis imperfecta.

The present invention can provide an agent for normalizing excessive accumulation of mutated type I collagen in the endoplasmic reticulum. The present invention can also provide a medicine useful for treating diseases caused by mutations in type I collagen, such as osteogenesis imperfecta.

FIG. 1 shows the amount of type I collagen accumulated in the endoplasmic reticulum in skin fibroblasts from a patient with osteogenesis imperfecta having a missense mutation in the COL1A1 gene, and the effect of chemical chaperones on this amount, compared with normal skin fibroblasts.

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

4-Phenylbutyric acid ( ^{chemical formula: C6H5(CH2)3COOH} _{, CAS number} : 1821-12-1) can be purchased as a commercial product from, for example, Sigma-Aldrich (product number: P21005) or Merck (catalog number: 820986). 4-Phenylbutyric acid can also be synthesized by a known method.

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

As the active ingredient of ^the endoplasmic reticulum excessive accumulation normalizing agent of the present invention, it is preferable to use sodium 4-phenylbutyrate. Sodium 4-phenylbutyrate (chemical formula: _C6H5 ( _CH2 ) _3COONa , CAS number: 1716-12-7) is approved as a therapeutic drug for urea cycle disorders and is in clinical use (trade name: Buphenyl). As the endoplasmic reticulum excessive accumulation normalizing agent of the present invention, sodium 4-phenylbutyrate sold as a pharmaceutical for treating said urea cycle disorders can be suitably used. In addition, commercially available products can be purchased and used. Sodium 4-phenylbutyrate synthesized by a known method can also be used.

Type I collagen is composed of two α1 chains and one α2 chain, and has a triple helix structure. Mutations in type I collagen may be those in at least one of the COL1A1 gene encoding the α1 chain and the COL1A2 gene encoding the α2 chain, but preferably have mutations at the protein level. Examples of mutations at the protein level include missense mutations (amino acid substitution mutations), amino acid deletion mutations, amino acid insertion mutations, amino acid duplication mutations, and frameshift mutations. It is believed that type I collagen with such mutations at the protein level accumulates in the endoplasmic reticulum because it cannot form a normal helical structure. The presence of a mutation in type I collagen can be confirmed by analyzing the base sequence of the COL1A1 gene or COL1A2 gene of the target to which the endoplasmic reticulum excessive accumulation normalizing agent of the present invention is to be applied, using a known method.

Type I collagen is mainly composed of three repeated amino acids, glycine-X-Y (X and Y are different amino acids). Glycine is located 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 replaced by another amino acid, it is thought that the normal helical structure cannot be maintained and the strength of collagen is weakened. Type I collagen in which glycine is replaced by another amino acid is thought to accumulate in the endoplasmic reticulum because the helical structure becomes distorted. Therefore, it is preferable that type I collagen having a mutation has a mutation in which glycine is replaced by 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 a state in which the amount of accumulated type I collagen (abundance) in cells expressing type I collagen having a mutation (e.g., skin fibroblasts, osteoblasts, etc. of a patient with osteogenesis imperfecta) is increased compared to the amount of accumulated type I collagen (abundance) in the endoplasmic reticulum in normal type I collagen-expressing cells (e.g., skin fibroblasts, osteoblasts, etc. of a healthy person). In addition, normalization of the excessive accumulation of type I collagen having a mutation in the endoplasmic reticulum means reducing the amount of mutant type I collagen excessively accumulated in the endoplasmic reticulum to approach the amount of type I collagen in the endoplasmic reticulum of normal type I collagen-expressing cells. The amount of type I collagen in the endoplasmic reticulum can be confirmed, for example, as described in Example 1, by preparing a specimen of type I collagen-expressing cells, performing fluorescent immunostaining using an anti-type I collagen antibody and an antibody against an endoplasmic reticulum marker, and measuring the fluorescence intensity of type I collagen that colocalizes with the endoplasmic reticulum marker.

The endoplasmic reticulum excessive accumulation normalizing agent of the present invention can be used as a medicine for treating a disease caused by a mutation in type I collagen. That is, the present invention provides a medicine for treating a disease caused by a mutation in type I collagen, which contains the endoplasmic reticulum excessive accumulation normalizing agent of the present invention. In other words, the present invention provides a medicine for treating a disease caused by a mutation in type I collagen, which contains 4-phenylbutyric acid or a pharma- ceutical acceptable salt thereof as an active ingredient.

Diseases caused by mutations in type I collagen include osteogenesis imperfecta (OI), Caffey disease, and Ehlers-Danlos syndrome (EDS). Osteogenesis imperfecta is a congenital disease that shows various degrees of connective tissue symptoms in addition to easy fractures and progressive bone deformation due to bone fragility throughout the body. More than 90% of cases of osteogenesis imperfecta are caused by genetic mutations in type I collagen. Caffey disease is also called juvenile cortical osteophytosis. Caffey disease is an infantile bone disorder characterized by acute inflammation of the soft tissues and localized thickening of the underlying bone cortex due to amino acid substitutions in the α1 chain of type I collagen, and is often self-limited. Ehlers-Danlos syndrome is a hereditary connective tissue disorder characterized by hyperextensibility of the skin and joints, and fragility of various tissues. Ehlers-Danlos syndrome is classified into several types, and the causative gene for the multiple joint laxity type is the type I collagen gene (COL1A1, COL1A2).

The pharmaceutical of the present invention contains 4-phenylbutyric acid or a pharma- ceutical acceptable salt thereof as the active ingredient, and can be formulated by appropriately blending pharma- ceutical acceptable carriers or additives according to a known method for producing pharmaceutical preparations (e.g., the method described in the Japanese Pharmacopoeia). 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), troches, syrups, liquids, emulsions, suspensions, controlled release preparations (e.g., immediate release preparations, sustained release preparations, sustained release microcapsules, etc.), aerosols, films (e.g., orally disintegrating films, oral mucosal patch films, etc.), injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.), drip infusions, transdermal preparations, ointments, lotions, patches, suppositories (e.g., rectal suppositories, vaginal suppositories, etc.), pellets, nasal preparations, pulmonary preparations (inhalants), eye drops, and other oral or parenteral preparations. The blending ratio of the carrier or additive can be appropriately set based on the range normally adopted in the pharmaceutical field. There are no particular limitations on the carriers or additives that can be added, but examples include various carriers such as water, physiological saline, other aqueous solvents, and aqueous or oily bases; and various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavorings, and fragrances.

Examples of additives that can be mixed into tablets, capsules, etc. include binders such as gelatin, corn starch, tragacanth, and gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, and alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose, or saccharin, and flavors such as peppermint, saffron oil, or cherry. When the dosage unit form is a capsule, the above-mentioned materials may further contain liquid carriers such as oils and fats. Sterile compositions for injection can be prepared according to normal formulation procedures (e.g., dissolving or suspending the active ingredient in a solvent such as water for injection or natural vegetable oil). Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose, and other auxiliary drugs (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), and may be used in combination with appropriate solubilizing agents such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50, etc.). As the oily liquid, for example, sesame oil, soybean oil, etc. are used, and may be used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol, etc. In addition, it may be mixed with buffering agents (e.g., phosphate buffer, sodium acetate buffer, etc.), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives (e.g., benzyl alcohol, phenol, etc.), antioxidants, etc.

The active ingredient of the pharmaceutical of the present invention can be added in a proportion of usually 0.01 to 100% (w/w), preferably 0.1 to 95% (w/w), based on the total amount of the formulation, although this varies depending on the dosage form, administration method, carrier, etc.

Sodium 4-phenylbutyrate, the active ingredient of the pharmaceutical of the present invention, has been administered to humans as a therapeutic agent for urea cycle disorders, and therefore can be safely administered to humans and other mammals (e.g., rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.).

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

The pharmaceutical agent of the present invention can be used in combination with other drugs. Examples of other drugs include bisphosphonate preparations that are administered to patients with osteogenesis imperfecta to reduce the risk of fracture.

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

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

Example 1: Effect of chemical chaperones on accumulation of mutated type I collagen in the endoplasmic reticulum
1. Materials and Methods (1) Cells Dermal fibroblasts collected during surgery from a patient with osteogenesis imperfecta (having a c.2461G>A (p.G821S) mutation in the COL1A1 gene) were used. Primary human dermal fibroblasts, neonatal (Human Dermal Fibroblasts, neonatal, Gibco, Cat.# C0045C) were used as normal cells. Experiments were performed using cells at passage numbers 5 to 6. These cells were seeded at 6 to 8 x ¹⁰³ cells/well in a 96-well plate and cultured for 2 days in a normal culture medium for dermal fibroblasts, DMEM (Wako), 10% FBS (Equitech Bio), and 0.5% penicillin-streptomycin (Gibco), with wells containing and without chemical chaperones.

(2) 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") were used as chemical chaperones. 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) was used as the primary antibody for type I collagen, and anti-PDI (protein disulfide isomerase) monoclonal antibody (Enzo, Cat.# ADI-SPA-891) was used as the primary antibody for the endoplasmic reticulum marker. 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 then incubated with blocking buffer (PBS, 1% BSA, 10% FBS, 0.3 M glycine, 0.1% Tween 20) for 1 hour at room temperature. Then, anti-collagen type I antibody and anti-PDI clonal antibody were incubated overnight at 4°C in primary antibody solutions diluted 1:100 and 1:200 in blocking buffer, respectively. Then, each secondary antibody was incubated at room temperature for 1 hour in secondary antibody solutions diluted 1:500 in blocking buffer, respectively.

(4) Measurement of Fluorescence Intensity Fluorescently stained cells were photographed using an In Cell Analyzer 6000 (GE Healthcare), and the fluorescence intensity of type I collagen, which colocalized with the endoplasmic reticulum marker, was measured using an In Cell Developer Toolbox (GE Healthcare).

2. Results The results are shown in FIG. 1. The amount of type I collagen accumulated in the endoplasmic reticulum was shown 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 normal cell (normal) group not added with chemical chaperone set to 1. In the osteogenesis imperfecta patient cell (OI) group not added with chemical chaperone, more than 1.6 times the amount of type I collagen accumulated in the endoplasmic reticulum compared to normal cells. In the osteogenesis imperfecta patient cell added with 4PBA, the amount of type I collagen accumulated in the endoplasmic reticulum was reduced in a concentration-dependent manner, and in the 5 mM group, it was reduced to the same level as in normal cells. In addition, 4PBA had little effect on normal cells. On the other hand, in the group added with TMAO to the osteogenesis imperfecta patient cell, a reduction in the amount of type I collagen accumulated in the endoplasmic reticulum was observed at concentrations of 50 mM and 5 mM, but no significant effect was observed as with 4PBA. These results suggest that the reduction (normalization) of type I collagen accumulation in the endoplasmic reticulum in cells from patients with osteogenesis imperfecta is not merely a result of the restoration of higher-order structure by chemical chaperones, but that some other action of 4PBA also contributes.

The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. In addition, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims (5)

An agent for normalizing excessive accumulation of mutated type I collagen in the endoplasmic reticulum, characterized in that the active ingredient is 4-phenylbutyric acid or a pharma- ceutical acceptable salt thereof. The agent according to claim 1, wherein the pharma- ceutically acceptable salt of 4-phenylbutyric acid is sodium 4-phenylbutyrate. 3. A pharmaceutical for treating osteogenesis imperfecta having a mutation in which glycine present at a site constituting the triple helix structure of type I collagen is replaced with another amino acid, comprising the agent according to claim 1 or 2. The pharmaceutical composition according to claim 3, wherein the glycine is glycine of the α1 chain of type I collagen . The pharmaceutical composition according to claim 3, wherein the glycine is the 821st glycine in the α1 chain of type I collagen .