Medicinal composition for treating fibrosis
阅读说明：本技术 纤维化治疗用医药组合物 (Medicinal composition for treating fibrosis ) 是由 水岛彻 田中健一郎 于 2019-09-26 设计创作，主要内容包括：本发明提供一种对肺泡上皮细胞不显示损伤性、诱导肺成纤维细胞的选择性细胞死亡并且抑制肺的纤维化的新型纤维化治疗药。纤维化治疗用医药组合物含有式(I)或式(II)所示的化合物、该化合物的作为医药能够允许的盐或它们的溶剂合物。(式(I)中,R～1表示可以被卤原子取代的C-(1－4)烷基,l表示3～6的整数。式(II)中,n表示8～12的整数。)(The present invention provides a novel therapeutic agent for fibrosis, which does not cause damage to alveolar epithelial cells, induces selective cell death of lung fibroblasts, and inhibits fibrosis of the lung. A pharmaceutical composition for the treatment of fibrosis, which comprises a compound represented by the formula (I) or (II), a pharmaceutically acceptable salt of the compound, or a solvate of the compound and the salt. (in the formula (I), R 1 Represents C which may be substituted by halogen atoms 1－4 Alkyl, l represents an integer of 3 to 6. In the formula (II), n represents an integer of 8 to 12. ))
1. A pharmaceutical composition for the treatment of fibrosis, comprising:
comprising a compound represented by the formula (I) or the formula (II), a pharmaceutically acceptable salt of the compound, or a solvate of the compound and the salt,
in the formula (I), R1To representC which may be substituted by halogen atoms1－4An alkyl group, l represents an integer of 3 to 6,
in the formula (II), n represents an integer of 8 to 12.
2. The pharmaceutical composition of claim 1, wherein:
comprising a compound represented by the formula (I), a pharmaceutically acceptable salt of the compound, or a solvate of the compound and the salt.
3. The pharmaceutical composition of claim 2, wherein:
in the formula (I), l is 4 or 5, R1Is methyl, ethyl or trifluoromethyl.
4. The pharmaceutical composition of claim 2 or 3, wherein:
in the formula (I), l is 5, R1Is methyl or ethyl.
5. The pharmaceutical composition of claim 1, wherein:
comprising a compound represented by the formula (II), a pharmaceutically acceptable salt of the compound, or a solvate of the compound and the salt.
6. The pharmaceutical composition of claim 5, wherein:
in formula (II), n is 10.
7. The pharmaceutical composition according to any one of claims 1 to 6, wherein:
the fibrosis is pulmonary fibrosis.
8. The pharmaceutical composition of claim 7, wherein:
the pulmonary fibrosis is idiopathic pulmonary fibrosis.
9. The pharmaceutical composition according to any one of claims 1 to 8, wherein:
it is formulated for administration via the respiratory tract.
10. The pharmaceutical composition according to any one of claims 1 to 8, wherein:
it is formulated for oral administration.
11. The pharmaceutical composition according to any one of claims 1 to 8, wherein:
it is formulated for intravenous administration.
12. A compound represented by the formula (I) or the formula (II), a pharmaceutically acceptable salt thereof, or a solvate of either:
can be used for treating fibrosis, and can be used for treating fibrosis,
in the formula (I), R1Represents C which may be substituted by halogen atoms1－4An alkyl group, l represents an integer of 3 to 6,
in the formula (II), n represents an integer of 8 to 12.
13. Use of a compound represented by the formula (I) or the formula (II), a pharmaceutically acceptable salt thereof or a solvate thereof for producing a pharmaceutical composition for the treatment of fibrosis,
in the formula (I), R1Represents C which may be substituted by halogen atoms1－4An alkyl group, l represents an integer of 3 to 6,
in the formula (II), n represents an integer of 8 to 12.
14. A method of treating fibrosis, comprising:
administering an effective amount of a compound represented by formula (I) or formula (II), a pharmaceutically acceptable salt of the compound, or a solvate of the compound,
in the formula (I), R1Represents C which may be substituted by halogen atoms1－4An alkyl group, l represents an integer of 3 to 6,
in the formula (II), n represents an integer of 8 to 12.
Technical Field
The present invention relates to a pharmaceutical composition for the treatment of fibrosis.
Background
Idiopathic Pulmonary Fibrosis (IPF) is a persistent disease with a poor prognosis and a median survival time after diagnosis is very short, ranging from 2.8 to 4.2 years. Heretofore, steroids or immunosuppressive agents have been used as therapeutic agents for IPF, but no effect has been reported in large-scale clinical trials. In addition, 2 new drugs such as pirfenidone and nintedanib have been marketed as anti-fibrotic drugs in recent years. These drugs have been shown to suppress a decline in Forced Vital Capacity (FVC) in patients with IPF in clinical trials (non-patent documents 1 to 3), but their effectiveness in long-term use has not been clarified yet. In addition, both drugs cause high-frequency and serious side effects, particularly gastrointestinal disorders, which have been problematic (non-patent documents 2 and 3). Therefore, it is very important to develop a new IPF therapeutic agent that is safer and more effective.
The mechanism of onset and exacerbation of IPF is not clearly understood, and it is considered that pulmonary fibroblasts abnormally proliferate and activate because alveolar epithelial cells are damaged by various stimuli (non-patent documents 4 to 6). Specifically, it has been reported that myofibroblasts increase and accumulate in lung tissues of IPF patients as a result of activation of fibroblasts (non-patent document 7). In addition, it has been reported that myofibroblasts increase and accumulate in an animal IPF model as in human (non-patent document 8). As a result, it is considered that the lung is fibrosed due to abnormal production and accumulation of extracellular matrix such as collagen, and abnormal repair and remodeling are performed.
The sources of myofibroblasts have been reported in several ways, and are roughly classified into cells differentiated from fibroblasts and cells differentiated from epithelial-mesenchymal transition (EMT). Specifically, it has been reported that fibroblast cells are differentiated into myofibroblasts by the action of a stimulus such as Transforming Growth Factor (TGF)) - β 1 (non-patent document 9). On the other hand, it was also found that when TGF- β 1 acts on alveolar epithelial cells, epithelial mesenchymal transition is induced, and the alveolar epithelial cells are transformed into myofibroblasts (non-patent documents 10 and 11). Therefore, a compound that inhibits the induction into myofibroblasts without damaging alveolar epithelial cells or a compound that inhibits collagen production by fibroblasts is considered to be a good therapeutic agent for IPF. Actually, it has been reported that pirfenidone and nintedanib, which have been marketed in recent years, inhibit the differentiation from fibroblasts to myofibroblasts, the EMT of alveolar epithelial cells, and the production of collagen by activated fibroblasts (non-patent documents 12 to 14).
Documents of the prior art
Patent document
Non-patent document 1: am.J.Respir.Crit.Care Med.192, e 3-e 19(2015)
Non-patent document 2: lancet 377, 1760-1769 (2011)
Non-patent document 3: N.Engl.J.Med.370, 2071-2082 (2014)
Non-patent document 4: am.J.Pathol.170, 1807-1816 (2007)
Non-patent document 5: chest 122,286S-289S (2002)
Non-patent document 6: chest 132, 1311-1321 (2007)
Non-patent document 7: int.J.mol.Med.34, 1219-1224 (2014)
Non-patent document 8: physiol. Rep.2, (2014)
Non-patent document 9: proc.am.thorac.Soc.5, 338-342 (2008)
Non-patent document 10: am.J.Physiol.Lung Cell mol.Physiol 293, L525-534 (2007)
Non-patent document 11: respir Res.6,56(2005)
Non-patent document 12: Eur.J.Pharm.Sci.58, 13-19 (2014)
Non-patent document 13: BMC palm Med.12,24(2012)
Non-patent document 14: J.Pharmacol.exp.Ther.349, 209-220 (2014)
Disclosure of Invention
Technical problem to be solved by the invention
The technical problem to be solved by the invention is as follows: disclosed is a novel therapeutic agent for fibrosis, which does not cause damage to alveolar epithelial cells, induces selective cell death of lung fibroblasts, and inhibits fibrosis of the lung.
Technical solution for solving technical problem
Accordingly, the present inventors have conducted various studies to find a drug having the above-mentioned action, using an approved drug whose safety to humans is sufficiently confirmed, and as a result, have found that a compound represented by the following formula (I) or (II) selectively acts on pulmonary fibroblasts without damaging alveolar epithelial cells, and has an excellent in vivo (in vivo) pulmonary fibrosis inhibitory action, thereby completing the present invention.
That is, the present invention provides the following inventions [1 ] to [14 ].
[1 ] A pharmaceutical composition for the treatment of fibrosis, which comprises a compound represented by the formula (I) or (II), a pharmaceutically acceptable salt of the compound, or a solvate of the compound and the pharmaceutically acceptable salt.
(in the formula (I), R1Represents C which may be substituted by halogen atoms1－4Alkyl, l represents an integer of 3 to 6.
In the formula (II), n represents an integer of 8 to 12. )
[ 2] A pharmaceutical composition according to [1 ], which comprises a compound represented by the formula (I), a pharmaceutically acceptable salt of the compound, or a solvate of the compound and the pharmaceutically acceptable salt.
[ 3] the pharmaceutical composition according to [ 2], wherein, in the formula (I), l is 4 or 5, R1Is methyl, ethyl or trifluoromethyl.
[ 4] the pharmaceutical composition according to [ 2] or [ 3], wherein, in the formula (I), l is 5, R1Is methyl or ethyl.
[ 5 ] A pharmaceutical composition according to [1 ], which comprises a compound represented by the formula (II), a pharmaceutically acceptable salt of the compound, or a solvate of the compound and the pharmaceutically acceptable salt.
[ 6 ] the pharmaceutical composition according to [ 5 ], wherein, in the formula (II), n is 10.
The pharmaceutical composition according to any one of [1 ] to [ 6 ], wherein the fibrosis is pulmonary fibrosis.
The pharmaceutical composition according to [ 8 ] or [ 7 ], wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis.
The pharmaceutical composition according to any one of [1 ] to [ 8 ] which is formulated for administration via the respiratory tract.
[ 10 ] the pharmaceutical composition according to any one of [1 ] to [ 8 ], which is formulated for oral administration.
The pharmaceutical composition according to any one of [1 ] to [ 8 ], which is formulated for intravenous administration.
[12] A compound represented by formula (I) or formula (II), a pharmaceutically acceptable salt thereof, or a solvate of either thereof, which is useful for treating fibrosis.
(in the formula (I), R1Represents C which may be substituted by halogen atoms1－4Alkyl, l represents an integer of 3 to 6.
In the formula (II), n represents an integer of 8 to 12. )
[13] Use of a compound represented by formula (I) or formula (II), a pharmaceutically acceptable salt thereof, or a solvate of either thereof for the manufacture of a pharmaceutical composition for the treatment of fibrosis.
(in the formula (I), R1Represents C which may be substituted by halogen atoms1－4Alkyl, l represents an integer of 3 to 6.
In the formula (II), n represents an integer of 8 to 12. )
[14] A method of treating fibrosis, comprising: administering an effective amount of a compound represented by formula (I) or formula (II), a pharmaceutically acceptable salt of the compound, or a solvate of the compound and the pharmaceutically acceptable salt.
(in the formula (I), R1Represents C which may be substituted by halogen atoms1－4Alkyl, l represents an integer of 3 to 6.
In the formula (II), n represents an integer of 8 to 12. )
Effects of the invention
The compounds represented by the formula (I) or the formula (II) are compounds which have been used as medicines and have been confirmed for their stability to humans. Further, these compounds exhibit an action completely different from the currently known pharmacological actions, namely, a selective cell death-inducing action on lung fibroblasts in an amount that does not cause damage to alveolar epithelial cells, and a pulmonary fibrosis-inhibiting action, and are useful as a therapeutic agent for fibrosis, particularly a therapeutic agent for idiopathic pulmonary fibrosis.
Drawings
FIG. 1A shows the effect of idebenone (idebenone) on the viable cell number (%) of LL29 cells and A549 cells.
FIG. 1B shows the effect of idebenone on LDH activity of LL29 cells and A549 cells.
FIG. 1C shows the effect of idebenone on cell death of LL29 cells.
FIG. 1D shows the effect of idebenone on cell death of A549 cells.
FIG. 2 shows the effect of pirfenidone and nintedanib on the viable cell number (%) of LL29 cells and A549 cells.
Figure 3A shows the effect of idebenone (Ide) on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 3B shows the effect of idebenone (Ide) on BLM-dependent pulmonary fibrosis (Ashcroft score).
Figure 3C shows the effect of idebenone (Ide) on BLM-dependent pulmonary fibrosis (hydroxyproline levels).
Figure 3D shows the effect of idebenone (Ide) on BLM-dependent pulmonary fibrosis (elastic resistance of the whole and alveoli, FVC).
Figure 4A shows the therapeutic effect of idebenone (Ide) on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 4B shows the therapeutic effect of idebenone (Ide) on BLM-dependent pulmonary fibrosis (ashcroft score).
Figure 4C shows the therapeutic effect of idebenone (Ide) on BLM-dependent pulmonary fibrosis (hydroxyproline levels).
Figure 4D shows the therapeutic effect of idebenone (Ide) on BLM-dependent pulmonary fibrosis (elastic resistance of the whole and alveoli, FVC).
Figure 5A shows the effect of idebenone (Ide) and CoQ10 on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 5B shows the effect of idebenone (Ide) and CoQ10 on BLM-dependent pulmonary fibrosis (ashcroft score).
Figure 5C shows the effect of idebenone (Ide) and CoQ10 on BLM-dependent pulmonary fibrosis (hydroxyproline levels).
Figure 6A shows the effect of idebenone (Ide) and CoQ10 on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 6B shows the effect of idebenone (Ide) and CoQ10 on BLM-dependent pulmonary fibrosis (ashcroft score).
Figure 6C shows the effect of idebenone (Ide) and CoQ10 on BLM-dependent pulmonary fibrosis (hydroxyproline levels).
FIG. 7A shows the effect of idebenone (Ide) and CoQ10 on myofibroblasts (α -SMA cell staining).
FIG. 7B shows the effect of idebenone (Ide) and CoQ10 on myofibroblasts (. alpha. -SMA positive cell number).
FIG. 8A shows the effect of idebenone (Ide) and CoQ10 on collagen.
FIG. 8B shows the effect of idebenone (Ide) and CoQ10 on α -SMA, COL1A 1.
FIG. 9A shows the effect of tolperisone (tolperisone) on the viable cell number (%) of LL29 cells and A549 cells.
Fig. 9B shows the effect of pirfenidone and nintedanib on viable cell number (%) of LL29 cells and a549 cells.
Figure 10A shows the effect of tolperisone (Tol) on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 10B shows the effect of tolperisone (Tol) on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 10C shows the effect of tolperisone (Tol) on BLM-dependent pulmonary fibrosis (hydroxyproline levels).
Figure 10D shows the effect of tolperisone (Tol) on BLM-dependent pulmonary fibrosis (elastic resistance of the whole and alveolar, FVC).
Figure 11A shows the effect of tolperisone (Tol) on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 11B shows the effect of tolperisone (Tol) on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 11C shows the effect of tolperisone (Tol) on BLM-dependent pulmonary fibrosis (hydroxyproline levels).
Figure 11D shows the effect of tolperisone (Tol) on BLM-dependent pulmonary fibrosis (elastic resistance of the whole and alveolar, FVC).
Figure 12A shows the therapeutic effect of tolperisone (Tol) on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 12B shows the therapeutic effect of tolperisone (Tol) on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 12C shows the therapeutic effect of tolperisone (Tol) on BLM-dependent pulmonary fibrosis (hydroxyproline levels).
Figure 12D shows the therapeutic effect of tolperisone (Tol) on BLM-dependent pulmonary fibrosis (elastic resistance of the whole and alveolar, FVC).
FIG. 13A shows the effect of the same isoactive agents (eperisone, tizanidine) on the viable cell number (%) of LL29 cells and A549 cells.
Fig. 13B shows the effect of the same isoactive agents (lanpirone, etanerone) on the viable cell numbers (%) of LL29 cells and a549 cells.
Figure 14A shows the effect of eperisone (Epe) and tizanidine (Tiza) on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 14B shows the effect of eperisone (Epe) and tizanidine (Tiza) on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 14C shows the effect of eperisone (Epe) and tizanidine (Tiza) on BLM-dependent pulmonary fibrosis (hydroxyproline levels).
Figure 14D shows the effect of eperisone (Epe) and tizanidine (Tiza) on BLM-dependent pulmonary fibrosis (elastic resistance of the whole and alveoli, FVC).
FIG. 15 shows the effect of tolperisone (Tol) and eperisone (Epe) on α -SMA, COL1A 1.
Figure 16A shows the therapeutic effect of tolperisone (Tol), pirfenidone (Pir), and nintedanib (Nin) on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 16B shows the therapeutic effect of tolperisone (Tol), pirfenidone (Pir), and nintedanib (Nin) on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 16C shows the therapeutic effect of tolperisone (Tol), pirfenidone (Pir), and nintedanib (Nin) on BLM-dependent pulmonary fibrosis (elastic resistance of the whole lung and alveoli, FVC).
Figure 17A shows the therapeutic effect of eperisone (Epe) on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 17B shows the therapeutic effect of eperisone (Epe) on BLM-dependent pulmonary fibrosis (collagen staining).
Figure 17C shows the therapeutic effect of eperisone (Epe) on BLM-dependent pulmonary fibrosis (elastic resistance of the whole lung and alveoli, FVC).
Detailed Description
The active ingredient contained in the pharmaceutical composition for the treatment of fibrosis of the present invention is a compound represented by formula (I) or formula (II), a pharmaceutically acceptable salt of the compound, or a solvate of the compound and the pharmaceutically acceptable salt.
(in the formula (I), R1Represents C which may be substituted by halogen atoms1－4Alkyl, l represents an integer of 3 to 6.
In the formula (II), n represents an integer of 8 to 12. )
It is known that the compound represented by the formula (I) is one of antispasmodics, acts on both central nervous system and vascular smooth muscle, improves muscular tone by alleviating pain of skeletal muscle, improving ischemia, and relieving tension, and has an action of eliminating hard mass and spasm of muscle. However, the effect on fibrosis is completely unknown. In particular, the compound represented by formula (I) has an effect of specifically inducing cell death in fibroblasts, more preferably lung fibroblasts. The compound represented by the formula (I) has an effect of specifically inhibiting the activation of fibroblasts, more preferably lung fibroblasts. The compound represented by the formula (I) has an effect of inhibiting tissue fibrosis, particularly lung tissue fibrosis. In addition, the compound represented by the formula (I) has an effect of inhibiting a decrease in respiratory function. The compound represented by the formula (I) has an effect of inhibiting tissue damage, particularly lung tissue damage.
In the formula (I), as R1C which may be substituted by halogen atoms1－4Examples of the alkyl group include C which may be substituted with 1 to 3 chlorine, fluorine, bromine or iodine atoms1－4An alkyl group. Specific examples thereof include methyl, ethyl, trifluoromethyl, n-propyl, n-butyl, isopropyl, sec-butyl and the like, more preferred are methyl, ethyl and trifluoromethyl, and still more preferred are methyl and ethyl.
In the formula (I), l represents an integer of 3-6. Among them, an integer of 3 to 5 is more preferable, 4 or 5 is further preferable, and 5 is further preferable. As the compound represented by the formula (I), more preferred is a compound wherein l is 4 or 5 and R is1A compound which is methyl, ethyl or trifluoromethyl, further preferably l is 5 and R is1A compound that is methyl or ethyl. Wherein l is 5 and R1The compound that is methyl is tolperisone. l is 5 and R1The compound that is ethyl is eperisone. l is 4 and R1The compound that is ethyl is etanerithronate. l is 4 and R1The compound that is trifluoromethyl is lanpirimol.
The compound represented by the formula (II) is known as a therapeutic agent for cognitive disorders or cognitive impairment of the alzheimer type.
The effect of these compounds of formula (II) on fibrosis is completely unknown. In particular, the compound represented by formula (II) has an effect of specifically inducing cell death in fibroblasts, more preferably lung fibroblasts. The compound represented by the formula (II) has an effect of specifically inhibiting the activation of fibroblasts, more preferably lung fibroblasts. The compound represented by the formula (II) has an effect of inhibiting tissue fibrosis, particularly lung tissue fibrosis. In addition, the compound represented by the formula (II) has an effect of inhibiting a decrease in respiratory function. In addition, the compound of formula (II) has an effect of inhibiting tissue damage, particularly lung tissue damage.
In the formula (II), n is an integer of 8 to 12, more preferably 9 or 10, and still more preferably 10. Wherein the compound with n-10 is idebenone.
The salt of the compound represented by the formula (I) or (II) is not particularly limited as long as it is a pharmaceutically acceptable salt, and examples thereof include inorganic acid salts such as hydrochloride, sulfate and nitrate, and organic acid salts such as acetate, oxalate, citrate and tartrate. Among them, hydrochloride of the compound represented by the formula (I) is more preferable.
As solvates of the compound represented by the formula (I) or the formula (II) or a pharmaceutically acceptable salt of the compound, hydrates, alcoholates and the like can be mentioned, with hydrates being more preferred.
The compound represented by the formula (I) or the formula (II) is a known compound as described above, and can be produced by a known production method.
As shown in examples described below, the compound represented by the formula (I) or the formula (II), a pharmaceutically acceptable salt thereof, or a solvate thereof exhibits a selective cell death-inducing effect on pulmonary fibroblasts and an excellent pulmonary fibrosis-inhibiting effect in an amount that does not cause damage to alveolar epithelial cells. Therefore, these compounds are useful as therapeutic agents for various fibrosis. Among them, the fibrosis includes pulmonary fibrosis, idiopathic pulmonary fibrosis, scleroderma, renal fibrosis, hepatic fibrosis, cardiac fibrosis and fibrosis of other organs or tissues, and is preferably used for pulmonary fibrosis and idiopathic pulmonary fibrosis, and particularly preferably used for idiopathic pulmonary fibrosis.
Therefore, a pharmaceutical composition containing a compound represented by the formula (I) or the formula (II), a pharmaceutically acceptable salt of the compound, or a solvate of the compound or the salt is useful as a pharmaceutical composition for the treatment of fibrosis, more preferably a pharmaceutical composition for the treatment of pulmonary fibrosis, and still more preferably a pharmaceutical composition for the treatment of idiopathic pulmonary fibrosis.
Examples of the form of the pharmaceutical composition of the present invention include oral preparations (tablets, coated tablets, powders, granules, capsules, solutions, etc.), respiratory tract-through preparations, intraperitoneal preparations, intravenous preparations, injections, suppositories, patches, ointments, etc., and preferably oral preparations, respiratory tract-through preparations, or intravenous preparations. Among them, since intraperitoneal administration to animals is considered to be an administration route equivalent to intravenous administration to humans by those skilled in the art, the results of intraperitoneal administration to animals can be considered as the results of intravenous administration to humans.
These administration forms can be prepared by a generally known method using a pharmaceutically acceptable carrier in addition to the above-mentioned active ingredients. Examples of such carriers include various substances commonly used in ordinary medicines, such as excipients, binders, disintegrants, glidants, diluents, dissolution aids, suspending agents, isotonic agents, pH adjusters, buffers, stabilizers, colorants, flavors, and flavoring agents.
The dosage of the pharmaceutical composition of the present invention varies depending on the administration route, sex, body weight, age, symptom, etc., and as the amount of the above-mentioned active ingredient, the amount administered per 1 day for an adult is usually preferably 0.5mg to 3000mg, more preferably 1mg to 300 mg. The dose for 1 day may be administered in a single dose or in multiple doses.
Examples
The present invention will be described in more detail below with reference to examples.
Example 1
a. Experimental methods
(1) Administration of Bleomycin (BLM), idebenone, tolperisone and eperisone
For male ICR mice under normal rearing, BLM was administered via the respiratory tract for the first day to prepare BLM lung injury model mice.
Pre-administration: idebenone was administered to the respiratory tract 1 time a day from the first day until day 7. Idebenone was administered via the respiratory tract 1 hour prior to BLM administration the first day.
Post-administration: idebenone was administered to the respiratory tract 1 time on day 1 from day 10to day 18. Both drugs were suspended in 0.9% NaCl and used.
Post-administration: tolperisone was administered 1 time a day from day 10to day 19. Both drugs were suspended in 0.9% NaCl and used.
Post-administration: eperisone was administered 1 time a day from day 10to day 19. Both drugs were suspended in 0.9% NaCl and used.
Among them, since intraperitoneal administration to animals is considered to be an administration route equivalent to intravenous administration to humans by those skilled in the art, the results of intraperitoneal administration to animals can be considered as the results of intravenous administration to humans.
(2) Real-time (Real-time) RT-PCR
Total RNA (total RNA) from cells was extracted using RNeasy kit (Qiagen). For Total RNA 2.5. mu.g, reverse transcription reaction was carried out using first-strand cDNA synthesis kit (TAKARA) according to the protocol of TAKARA. For the synthesized cDNA, SsoFast EvaGreen Supermix was used, utilizing CFX96TMReal time system (Real time system) for analysis. In order to make the total RNA amount comparable in each reaction, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal standard.
(3) Cell culture
DMEM Medium (Dulbecco's Modified Eagle's Medium) containing 10% FBS, Ham's F-12K (Kaighn's) containing 15% FBS was used at 37 ℃ with 5% CO2A549 cells (human alveolar epithelial cells) and LL29 cells (lung fibroblasts from IPF patients) were cultured under the conditions of (1).
For the determination of the number of living cells, MTT method or Countess was usedTMTrypan blue staining was performed with an Automated Cell Counter (Invitrogen, Carlsbad, CA). For the evaluation of cell death, LDH activity in the medium was determined based on the protocol of the test kit.
(4) Tissue staining method and tissue immunostaining method
The extracted lung tissue was fixed in 10% formalin (formalin) for 24 hours, and then embedded in paraffin to prepare a paraffin section having a thickness of 4 μm.
For H & E staining, first, staining was performed with Mayer's hematoxyline (Mayer's hematoxyline), and then staining was performed with a 1% eosin (eosin) solution. After staining, the cells were enclosed in a malino and histological analysis was performed using a Nanozomer-XR digital slide scanner (Hamamatsu Photonics, Shizuoka, Japan) or Olympus BX51 microscope (Tokyo, Japan).
Masson's trichrome staining was performed using a first mordant (5 w/v% potassium dichromate (potassium dichromate), 5 w/v% trichloroacetic acid (trichloroacetic acid)), Wegener's iron hematoxylin (Weigart's iron methoxylin), a second mordant (1.25 w/v% phosphotungstic acid, 1.25 w/v% phosphomolybdic acid (phosphomolydic acid)), 0.75 w/v% orange G (orange G), ponceau/xylidine mixture (0.12 w/v% xylidine poseau, 0.04 w/v% acid fuchsin, 0.02 w/v% azored (azophloxin)), aniline blue (aniline blue). After staining, the cells were enclosed in malinol and histology was performed using a Nanozomer-XR digital slide scanner (digital slide scanner). For pulmonary fibrosis, the scores were scored using an ashcroft score. In addition, the Ashcroft score is referenced J.Clin.Pathol.41, 467-470 (1988).
For the immunostaining of α -SMA, 2.5% goat serum (goat serum) was blocked for 10min and then antibody treatment was performed once (against α -SMA, 1: 100 dilution (dilution)). After 12h, sections were cultured for 2h using Alexa Fluor 594 goat anti-rabbit immunoglobulin (goat anti-rabbit immunoglobulin) G and DAPI. Thereafter, VECTASHIELD are enclosed. The sections were photographed using a microscope (Olympus DP 71).
The alpha-SMA positive areas were quantified using Image J software (software) (National Institutes of Health, Bethesda, Md.). In addition, the number of 8-OhdG positive cells was determined by using Definiens Tissue StudioTMsoftware (CTC Life Science Corporation, Tokyo, Japan) was used for quantification.
(5) Measurement of Lung function and forced Lung volume
Using computer controlled small animal exhalingLung function was measured using a suction apparatus (FlexiVent; SCIRES, Montreal, Canada) according to the literature. A8 mm metal tube (1.27 mm in outer diameter and 0.84mm in inner diameter, respectively) was inserted into the trachea of a mouse under anesthesia with chloral hydrate (500mg/kg), and a tidal volume of 8.7ml/kg and a tidal volume of 2-3 cm H were used2Positive end expiratory pressure ventilation of O mechanically breathes it at a rate of 150 breaths/min.
The total respiratory elastic resistance (total respiratory elasticity resistance) and tissue elastic resistance (tissue elasticity resistance) were measured using fast shot (snap shot) and forced oscillation (forced oscillation) techniques, respectively. Furthermore, forced vital capacity was measured according to Chest142, 1011-1019 (2012) using the computer-controlled small animal ventilator and negative pressure vessel (SCIRQ, Montreal, Canada) described above. All data were analyzed using FlexiVent software (version 5.3; SCIReq, Montreal, Canada).
(6) Quantification of Hydroxyproline (Hydroxyproline)
The upper left lung lobe of the mouse thus extracted was homogenized in 0.5mL of 5% TCA, and 0.5mL of concentrated hydrochloric acid was added to the precipitate obtained by centrifugation, followed by heating at 110 ℃ for 16 hours. Thus, the precipitate is hydrolyzed to extract hydroxyproline (hydroxyproline). To the extracted hydroxyproline (hydroxyproline), 1.4 w/v% chloramine (chloramine) T was added and left to stand for 20 minutes, and then heated at 65 ℃ for 10 minutes together with Ehrich's reagent (1M DMBA, 70 v/v% isopropyl alcohol (isoproapanol) and 30 v/v% perchloric acid (perchloric acid)) to develop red to violet. Thereafter, the absorbance (550nm) was measured.
(7) Statistical analysis
All values are expressed as mean. + -. standard error of the mean (SEM). The significance difference test was performed by One-way ANOVA, with Dunnett's test (Dunnett's test) between groups and Student's t test (Student's t-test) between groups. In addition, SPSS22 software was used for the test. When the value of P is less than 0.05, it is judged that there is a significant difference.
It is: p <0.05 (relative to the blank),. ANG.: p <0.01 (relative to blank), #: p <0.05 (alone relative to BLM), # #: p <0.01 (alone with respect to BLM)
b-1. Results
(1) Effect of idebenone on cell death and cell proliferation
In the fibrotic mechanism of IPF, activation of lung fibroblasts is considered important. To date, it has been reported that fibroblasts abnormally proliferate and activate in the lungs of IPF patients, and that collagen is abnormally produced from activated lung fibroblasts. Therefore, the present inventors screened for an approved drug that selectively acts on lung fibroblasts without showing damaging properties to alveolar epithelial cells. In the screening, the decrease in cell survival rate of lung fibroblasts by the compound was used as a simple index for inhibiting the activity of lung fibroblasts.
As for a specific method, lung fibroblasts (LL29 cells) and human alveolar epithelial cells (a549 cells) from IPF patients were treated with approved drugs, respectively, and cell survival rate after 24 hours was evaluated by the MTT method. As a result, IC from LL29 cells50Of the compounds having a value (concentration required to reduce cell viability by 50%) lower than that of A549 cells, compounds were selected in which IC was particularly significantly seen between the two cells50A compound with a difference in value-idebenone.
Also as a reproducibility test of screening, the cell death inducing effect of idebenone was studied in a smaller amount in the screening. As a result, the cell survival rate of LL29 cells (lung fibroblasts) was decreased at a lower concentration of idebenone than that of a549 cells (alveolar epithelial cells) was observed (fig. 1A). Next, cell death was evaluated by quantifying LDH released from the cells, and in LL29 cells, LDH was released in the medium by idebenone (150 μ M), but LDH release was hardly seen in a549 cells (fig. 1B). From these results, it was shown that idebenone selectively induced cell death for LL29 cells compared to a549 cells.
From FIGS. 1A and 1B, idebenone is considered to have a possibility of inhibiting the cell growth of LL29 cells at 75 to 125. mu.M. Thus, the present inventors determined the number of live cells and the number of dead cells using trypan blue staining. As a result, idebenone inhibited the proliferation of LL29 cells from 75 μ M and induced cell death of LL29 cells from 175 μ M (fig. 1C). On the other hand, idebenone inhibited cell proliferation in epithelial cells from 175 μ M, and no induction of cell death was seen at the concentrations studied (fig. 1D).
In addition, neither of pirfenidone and nintedanib, which are conventional IPF therapeutic agents, selectively induces cell death of fibroblasts after studies (fig. 2).
(2) Effect of idebenone on Bleomycin (BLM) -dependent pulmonary fibrosis
Considering the development of idebenone as a therapeutic agent for IPF, it is also important to show an effect in an animal model of IPF. As an animal model of IPF, a Bleomycin (BLM) lung injury model is commonly used, and it has been reported that the BLM lung injury model reproduces typical characteristics of IPF symptoms, and it has been reported that alveolar epithelial cells are injured, lung fibroblasts proliferate and accumulate, and respiratory function is decreased by administering BLM through the respiratory tract. Therefore, the prevention effect and the treatment effect of idebenone on pulmonary fibrosis were studied using the BLM lung injury model.
While BLM-dependent lung injury (hypertrophy, alveolar wall or interstitial edema) and collagen accumulation were confirmed by H & E staining and masson trichrome staining when BLM was administered to mice via the respiratory tract, these injury and collagen accumulation were significantly suppressed by idebenone-administered via the respiratory tract (fig. 3A). Further, the effect of idebenone on BLM-dependent pulmonary fibrosis was evaluated using the ashcroft score for quantitative fibrosis and the hydroxyproline amount, which is an amino acid having a large content in lung collagen, as indicators based on tissue images. The ashcroft score and hydroxyproline amount increased dependently on BLM, but their increase was significantly suppressed by the administration of idebenone (fig. 3B, fig. 3C).
Considering the clinical application of idebenone, it is important to show the effect not only on histological indexes but also on indexes such as respiratory function. It has been reported that in IPF patients, the lung becomes hard due to fibrosis, and therefore the lung elastic resistance increases, and the Forced Vital Capacity (FVC) decreases. Therefore, the present inventors measured these indices using a mouse ventilator. The increase in total respiratory system elastic resistance (elastic resistance of the whole lung including bronchi, bronchioles, and the whole alveoli) and tissue elastic resistance (elastic resistance of the alveoli) was observed by administering BLM, but the increase was suppressed by administering idebenone via the respiratory tract (fig. 3D). In addition, although FVC decreased dependently on BLM, the decrease tended to be suppressed by respiratory tract administration of idebenone (fig. 3D). From the above results, it was revealed that by administering idebenone via the respiratory tract, BLM-dependent pulmonary fibrosis and respiratory function decrease were suppressed. In addition, idebenone (18.8mg/kg) administered via the respiratory tract alone did not have an effect on pulmonary fibrosis and respiratory function (FIGS. 3A-D).
(3) Therapeutic effect of idebenone on BLM-dependent pulmonary fibrosis
Next, the present inventors studied the therapeutic effect by administering idebenone to mice in which fibrosis was induced by the previous administration of BLM. To date, the development of pulmonary fibrosis has been reported when BLM is administered over about 10 days. Therefore, the present inventors started transrespiratory administration of idebenone 10 days after the administration of BLM, and evaluated pulmonary fibrosis and respiratory function 20 days after the administration of BLM. Lung injury or fibrosis was significantly inhibited by idebenone administration via the respiratory tract after 20 days from the start of BLM administration (fig. 4A-C). In addition, BLM-dependent reduction of respiratory function was also significantly suppressed by respiratory tract administration of idebenone (fig. 4D). From the above results, it was revealed that idebenone improved BLM-dependent pulmonary fibrosis and respiratory function reduction even in mice induced fibrosis by the previous administration of BLM.
(4) Comparison of idebenone with CoQ10
To understand the mechanism that determines the effect of idebenone, the inventors compared the effect of idebenone and CoQ10 on bleomycin pulmonary fibrosis. As shown in FIGS. 5A-C, lung injury and fibrosis caused by bleomycin were inhibited by respiratory administration of idebenone or CoQ 10.
Next, the present inventors compared the therapeutic effects of idebenone and CoQ10 on BLM pulmonary fibrosis. As shown in fig. 6A-C, idebenone inhibited lung injury and fibrosis caused by bleomycin, but CoQ10 did not.
As described above, myofibroblasts play an important role in pulmonary fibrosis and bleomycin-induced pulmonary fibrosis in IPF patients. Therefore, the effects of idebenone and COQ10 were studied focusing on myofibroblasts. As shown in fig. 7A, B, the number of α -SMA positive cells (myofibroblasts) in the lung increased due to bleomycin administration, but this increase was significantly suppressed by idebenone. On the other hand, the inhibitory effect was not seen in CoQ 10. Therefore, the effects on TGF- β 1-dependent lung fibroblast activation (differentiation into myofibroblasts) were compared using idebenone and CoQ 10. LL29 cells were treated with idebenone or CoQ10, then treated with TGF-. beta.1, and the amount of collagen in the medium was measured. As shown in FIG. 8A, the treatment with TGF-. beta.1 increased the amount of collagen, but the increase was suppressed by idebenone. On the other hand, CoQ10 was not inhibited. Next, the effect of idebenone was examined using the mRNA amounts of α -SMA and collagen as indices. As shown in FIG. 8B, the treatment of LL29 cells with TGF-. beta.1 induced the expression of α -SMA and Col1a1 mRNA, but the treatment with idebenone inhibited the induction. On the other hand, CoQ10 was not inhibited. These results show that idebenone acts on fibroblasts, inhibiting the activation of lung fibroblasts.
b-2. Results
(1) Effect of tolperisone on cell death
In the same manner as in (b-1) above, lung fibroblasts (LL29 cells) and human alveolar epithelial cells (a549 cells) derived from IPF patients were treated with approved drugs, respectively, and the cell survival rate after 24 hours was evaluated by the MTT method. As a result, IC from LL29 cells50(refiningConcentration required for a 50% reduction in cell viability) values lower than those of a549 cells, compounds were selected in which IC was particularly significantly seen between the two cells50A compound with a poor value of (a), tolperisone.
Also used as a reproducibility test for screening, the cell death inducing effect of tolperisone was investigated at a smaller dose. As a result, tolperisone at a lower concentration than the concentration at which a549 cell (alveolar epithelial cell) cell survival was seen decreased cell survival of LL29 cells (lung fibroblasts) (fig. 9A). Next, fibroblast-selective cell death was investigated using pirfenidone and nintedanib, which are existing IPF therapeutic agents, and both of these drugs hardly induced fibroblast-selective cell death (fig. 9B).
(2) Effect of tolperisone administered via respiratory tract on BLM-dependent pulmonary fibrosis
The therapeutic effect of tolperisone on pulmonary fibrosis was studied using the BLM lung injury model.
BLM was administered to mice via the respiratory tract, and collagen was stained by H & E staining and masson trichrome staining, confirming BLM-dependent lung injury (hypertrophy, alveolar wall or interstitial edema) and collagen accumulation, but tolperisone administered via the respiratory tract inhibited these injury and collagen accumulation, although less (fig. 10A, B). Further, the effect of tolperisone on BLM-dependent pulmonary fibrosis was evaluated using the amount of hydroxyproline, which is an amino acid having a large content in collagen in the lung, as an index. The amount of hydroxyproline increased dependently on BLM, and this increase was hardly inhibited even by tolperisone administered via the respiratory tract (fig. 10C). Subsequently, the respiratory function was measured using a mouse ventilator. By administering BLM, increases in total respiratory system elastic resistance (elastic resistance of the whole lung including bronchi, bronchioles, and the whole alveoli) and tissue elastic resistance (elastic resistance of the alveoli) were observed, but by administering tolperisone, a tendency was observed that these increases were suppressed (fig. 10D). In addition, although FVC decreased dependently on BLM, the decrease was suppressed by tolperisone administration (fig. 10D). From the above results, it was shown that by administering tolperisone via the respiratory tract, BLM-dependent pulmonary fibrosis and a decrease in respiratory function were suppressed.
(3) Effect of orally administered tolperisone on BLM-dependent pulmonary fibrosis
BLM was administered to mice via the respiratory tract, and collagen was stained by H & E staining and masson trichrome staining, confirming BLM-dependent lung injury (hypertrophy, alveolar wall or interstitial edema) and collagen accumulation, but tolperisone administered orally inhibited these injuries and collagen accumulation (fig. 11A, B). The amount of hydroxyproline increased dependently on BLM, but this increase was significantly inhibited if tolperisone was administered orally (fig. 11C). Subsequently, the respiratory function was measured using a mouse ventilator. By administering BLM, increases in total respiratory system elastic resistance (elastic resistance of the whole lung including bronchi, bronchioles, and the whole alveoli) and tissue elastic resistance (elastic resistance of the alveoli) were seen, but these increases were suppressed by administering tolperisone (fig. 11D). In addition, although FVC decreased dependently on BLM, this decrease was suppressed by tolperisone administration (fig. 11D). From the above results, it was shown that by oral administration of tolperisone, BLM-dependent pulmonary fibrosis and a decrease in respiratory function were suppressed.
(4) Effect of intraperitoneal tolperisone on BLM-dependent pulmonary fibrosis
BLM was administered to mice via the respiratory tract and collagen was stained by H & E staining and masson trichrome staining, confirming BLM-dependent lung injury (hypertrophy, alveolar wall or interstitial edema) and collagen accumulation, but tolperisone administered intraperitoneally inhibited these injuries and collagen accumulation (fig. 12A, B). The amount of hydroxyproline increased dependently on BLM, but this increase was significantly inhibited if tolperisone was administered intraperitoneally (fig. 12C).
Subsequently, the respiratory function was measured using a mouse ventilator. By administering BLM, increases in total respiratory system elastic resistance (elastic resistance of the whole lung including bronchi, bronchioles, and the whole alveoli) and tissue elastic resistance (elastic resistance of the alveoli) were seen, but these increases were suppressed by administering tolperisone (fig. 12D). In addition, although FVC decreased dependently on BLM, this decrease was suppressed by tolperisone administration (fig. 12D). From the above results, it was shown that the administration of tolperisone intraperitoneally inhibited BLM-dependent pulmonary fibrosis and respiratory function decrease.
(5) Effect of tolperisone on the same drug (selectivity for fibroblast)
To elucidate the mechanism by which tolperisone acts selectively on fibroblasts, it was analyzed in fig. 13 whether fibroblast-selective cell death could be seen with the same isoactive. As a result, eperisone at a concentration lower than the concentration at which the cell survival rate of a549 cells (alveolar epithelial cells) was decreased reduced decreased the cell survival rate of LL29 cells (lung fibroblasts), but such an effect was not seen in tizanidine (fig. 13A). Eperisone is a compound that is very similar in chemical structure to tolperisone. Therefore, fibroblast selectivity was analyzed using compounds with a similar chemical structure to tolperisone. As a result, selective cell death of fibroblasts similar to tolperisone was observed in etanereisone and lanpiritone, which are chemical structural analogues of tolperisone (fig. 13B).
(6) Effect of intraperitoneal administration of eperisone and tizanidine on BLM-dependent pulmonary fibrosis
BLM administration to mice via the respiratory tract, collagen staining by H & E staining and masson trichrome staining, and eperisone administration intraperitoneally inhibited BLM-dependent lung injury and collagen accumulation, but not in tizanidine (fig. 14A, B). A propensity towards inhibition of BLM-dependent hydroxyproline levels was observed by intraperitoneal administration of eperisone, but not in tizanidine (figure 14C). Subsequently, the respiratory function was measured using a mouse ventilator. By administering BLM, an increase in total respiratory system elastic resistance (elastic resistance of the whole lung including bronchi, bronchioles, and the whole alveoli) and a decrease in FVC were observed. On the other hand, eperisone inhibited these responses, but tizanidine did not (fig. 14D). From the above results, it was revealed that eperisone inhibits BLM-dependent pulmonary fibrosis and respiratory function degradation in the same manner as tolperisone.
(7) Inhibition of fibroblast activation by tolperisone and eperisone
In FIG. 15, the effect of tolperisone and eperisone on the activation of fibroblasts (increase in the amount of mRNA for. alpha. -SMA and collagen) when treated with TGF-. beta.1 was examined. If LL29 cells were treated with TGF-. beta.1, the expression of α -SMA and Col1a1 mRNA was increased, but if tolperisone, eperisone treatment was performed, the induction was inhibited. These results show that tolperisone and eperisone act on fibroblasts and inhibit the activation of lung fibroblasts.
(8) Effect of tolperisone and existing IPF therapeutics on BLM-dependent pulmonary fibrosis
Pirfenidone and nintedanib are used in clinical settings as IPF therapeutics. Thus, the effectiveness of tolperisone and these drugs was compared using the BLM lung injury model.
Mice were dosed on day0 (day0) with bleomycin (BLM, 1mg/kg) or vehicle (vehicle). 1 day of the mice was orally administered pirfenidone (200mg/kg), nintedanib (30mg/kg), and tolperisone (5mg/kg) 1 time for 9 days (from day 10to day 18). After 20 days, lung tissue sections were prepared and collagen was stained by H & E staining and masson trichrome staining.
As a result, as shown in fig. 16A and 16B, it was confirmed that BLM-dependent lung injury (hypertrophy, alveolar wall or interstitial edema) and accumulation of collagen were observed, and pirfenidone and nintedanib hardly inhibited BLM-dependent lung injury. On the other hand, by administering tolperisone, a significant improvement in BLM lung injury was seen.
Subsequently, the respiratory function was measured using a mouse ventilator. As a result, as shown in fig. 16C, the increase of total respiratory system elastic resistance (elastic resistance of the whole lung including bronchi, bronchioles and the whole alveoli) and the decrease of FVC were observed by administering BLM, but tolperisone improved these changes. On the other hand, two drugs other than tolperisone did not ameliorate these changes. From the above results, the effect of tolperisone is expected to be higher than that of the conventional IPF therapeutic agent.
(9) Effect of orally administered eperisone on BLM-dependent pulmonary fibrosis
Eperisone is used in clinical settings as an oral muscle tone-improving drug. Therefore, in addition to the aforementioned intraperitoneal administration, the effect of oral administration was also analyzed.
Mice were dosed on day0 (day0) with bleomycin (BLM, 1mg/kg) or vehicle (vehicle). Eperisone (15 or 50mg/kg) was orally administered 1 time a day to the mice for 9 days (from day 10to day 18). After 20 days, lung tissue sections were prepared and subjected to histological analysis. Collagen was stained by H & E staining and masson trichrome staining. As a result, as shown in fig. 17A and 17B, by orally administering eperisone, BLM-dependent lung injury and collagen accumulation were concentration-dependently inhibited. In addition, as shown in fig. 17C, an increase in total respiratory elastic resistance (total respiratory system elasticity) and alveolar elastic resistance (tissue elasticity) and a decrease in FVC were seen by administering BLM, but these changes were improved concentration-dependently by oral administration of eperisone. From the above results, eperisone also showed effectiveness in oral administration as a method of administration in clinical use.
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