JP2005281235A - Chronic rejection inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chronic rejection inhibitor which is safe without adverse drug reaction and can effectively inhibit chronic rejection after organ transplantation or tissue transplantation, particularly after organ transplantation. <P>SOLUTION: The chronic rejection inhibitor after organ transplantation or tissue transplantation, particularly after organ transplantation contains a compound represented by formula (I), its pharmacologically acceptable salt or a solvate thereof as an active ingredient. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an agent for suppressing chronic rejection after organ transplantation or tissue transplantation, comprising as an active ingredient pirfenidone represented by formula (I).

  At present, the surgical technique for organ transplantation has improved significantly, and organ transplantation has been established as one of the treatments for serious diseases. Kidney, heart and liver transplantation has already become established as general medicine, and lung, pancreas and small intestine transplantation It is becoming increasingly popular (Non-Patent Document 1). However, the success of organ transplant surgery depends on how the transplant rejection after surgery can be controlled. Rejection occurs when a living body recognizes a graft as a foreign body and a series of immune reactions are induced to eliminate it. In normal transplantation, some incompatibility of histocompatibility antigens can be avoided. First, effective suppression of rejection is required in the clinical setting.

In general, rejection of transplanted organs can be classified into hyperacute rejection, acute rejection, and chronic rejection (Non-patent Document 2).
Hyperacute rejection is a response of an existing antibody to a transplant antigen and occurs immediately after transplantation. Clinically, it includes cases of acute facilitation, blood group incompatible transplantation and the like.

  Acute rejection is a biological response that occurs in the transplanted organ, and generally a cellular response has a major role. Although CD4 positive helper T cells recognize class II antigens, an immune reaction occurs, but there are a variety of immunocompetent cells involved in rejection. Helper T cells (Th0) are induced to differentiate into Th1 and Th2 by cytokines, with the former regulating cellular immunity and the latter regulating humoral immunity. Acute rejection occurs clinically within 3 months after transplantation, and is more common within 5 months after the 5th day.

  Chronic rejection is a vascular-stromal lesion mainly caused by immunological mechanisms. Clinically, the function of the transplanted organ gradually decreases, and histologically, it is characterized by fibrosis and parenchymal cell loss and disappearance. It is a clinicopathological disease. Immunologically does not indicate a specific biological phenomenon. It is an irreversible progressive lesion caused as a result of repeated acute rejection, and ischemia plays an important role as a consequence of transplant arteritis. It usually develops after 3 months after transplantation.

  Conventionally, rejection inhibitors that have been used clinically include steroids (such as prednisolone and methylprednisolone), antimetabolites (such as azathioprine and mizoribine), and immunosuppressants (such as cyclosporine and tacrolimus (FK506)). It is done. However, these rejection inhibitors are those that are mainly used to suppress acute rejection, and are also maintained in the chronic phase. I can't say that. Thus, with some exceptions, transplant patients must continue to take immunosuppressants throughout their lives after transplantation, resulting in serious side effects such as nephrotoxicity, hepatotoxicity, central nervous system disorders, decreased white blood cells and platelets. These were frequently observed, and there was a problem with the safety of long-term administration after organ transplantation, and this drug was insufficient for use in suppressing chronic rejection.

  In fact, there is no therapeutic drug that focuses on chronic rejection after organ transplantation or tissue transplantation as a standard treatment method in actual medical practice. Basically, the basic treatment policy is to reduce the initial high-dose treatment for suppressing acute rejection. Therefore, for treatment of such chronic rejection, a method that basically uses a combined immunosuppressant treatment centered on cyclosporine, tacrolimus, steroids, etc., and increases or decreases the drug according to the symptoms Has been adopted.

  Recently, it was revealed that pirfenidone (5-methyl-1-phenyl-2 (H) -pyridone) having antipyretic, analgesic and anti-inflammatory actions, which is a known compound, has an antifibrotic action and fibrosis. It has been reported that it is useful as an agent for repairing lesion tissues and inhibiting fibrotic lesions (Patent Document 1). Patent Document 1 describes that it is effective for suppression of myocardial infarction, myocardial fibrosis, fibrous vascular degeneration (atherosclerosis), vascular stenosis, etc., but suppression of rejection for organ transplantation or tissue transplantation There is no description about the effect.

There have also been reports on antifibrosis and TGF-β1 production inhibitory effects when pirfenidone and cyclosporine are administered in combination to tracheal transplanted rats. However, this report does not relate to organ transplantation such as kidney, pancreas, small intestine, lung, liver or heart, nor does it describe or even suggest any suppression of chronic rejection after transplantation (non-patented) Reference 3).
Furthermore, a pharmaceutical composition effective for suppression of transplant rejection using a pyrimidine derivative as an active ingredient, which may contain an immunosuppressive agent (such as cyclosporine) and an antifibrotic agent (such as pirfenidone) has been reported ( Patent Document 2). However, this Patent Document 2 does not describe a specific example of a pharmaceutical composition containing pirfenidone, and does not describe anything about chronic rejection of a transplanted organ or transplanted tissue.
Furthermore, in recent years, the presence of transplanted inflammatory factor-1 (AIF-1) has been reported as one factor of chronic rejection in transplantation (Non-patent Documents 4 to 5 and Patent Document 3). Therefore, the effectiveness of the chronic rejection inhibitor after transplantation can be determined using the inhibitory effect on AIF-1 as an index.
These prior documents described above do not describe that pirfenidone is useful for suppressing chronic rejection after organ transplantation or tissue transplantation.

JP-A-2-215719 WO03 / 094904 US6077948 Today's transplant vol. 11 No. 1 53 1998) Histopathological diagnosis criteria for human transplant organ rejection November 1998 [1st edition], Japanese Society for Transplantation Japanese Society for Pathology / Editor, Kanehara Publishing Co., Ltd. Pulmonary Pharmacology & Therapeutics (2002), 15 (5), 433-437 Journal of Biomedical Materials Research, Part A (2003), 64A (1), 12-19 Immunology 104: 307, 2001 12-19

  An object of the present invention is to provide a safe chronic rejection inhibitor that can effectively suppress chronic rejection after transplantation and has no side effects.

  As a result of intensive research on various substances in order to find a drug that effectively suppresses rejection that occurs during organ transplantation, particularly chronic rejection, the present inventors have found that pirfenidone exhibits chronic rejection after organ transplantation. The present invention was completed by discovering that it is a drug that is effectively suppressed and has no side effects.

（２）臓器移植が腎移植、膵移植、小腸移植、肺移植、肝移植または心移植であることを特徴とする上記（１）に記載の慢性拒絶反応抑制剤。
（３）臓器移植が心移植であることを特徴とする上記（１）に記載の慢性拒絶反応抑制剤。
（４）組織移植が皮膚移植、骨移植または心臓弁移植であることを特徴とする上記（１）に記載の慢性拒絶反応抑制剤。
（５）免疫抑制剤および上記（１）に記載の慢性拒絶反応抑制剤からなる合剤。 That is, the present invention
(1) Chronic rejection after organ transplantation or tissue transplantation, comprising a compound represented by formula (I), a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient Inhibitor.

(2) The chronic rejection inhibitor as described in (1) above, wherein the organ transplantation is kidney transplantation, pancreatic transplantation, small intestine transplantation, lung transplantation, liver transplantation or heart transplantation.
(3) The chronic rejection inhibitor as described in (1) above, wherein the organ transplant is a heart transplant.
(4) The chronic rejection inhibitor as described in (1) above, wherein the tissue transplant is a skin transplant, a bone transplant or a heart valve transplant.
(5) A mixture comprising an immunosuppressant and the chronic rejection inhibitor described in (1) above.

（８）心機能低下が心拍動強度の低下であることを特徴とする上記（７）に記載の心機能低下抑制剤。
（９）免疫抑制剤および上記（７）に記載の心機能低下抑制剤からなる合剤。
（１０）免疫抑制剤がシクロスポリン、タクロリムス、ステロイド剤、アザチオプリン、ミゾリビン、ミコフェノール酸モフェチル、抗Ｔ細胞抗体、ラパマイシンおよび１５−デオキシスパガリンから選ばれる１種または２種以上であることを特徴とする上記（９）に記載の合剤。 (6) The immunosuppressant is one or more selected from cyclosporine, tacrolimus, steroids, azathioprine, mizoribine, mycophenolate mofetil, anti-T cell antibody, rapamycin and 15-deoxyspagarin. The combination according to (5) above.
(7) Decreased cardiac function after organ transplantation or tissue transplantation, comprising a compound represented by formula (I), a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient Inhibitor.

(8) The cardiac function lowering inhibitor as described in (7) above, wherein the decrease in cardiac function is a decrease in heartbeat intensity.
(9) A combination comprising an immunosuppressive agent and the cardiac function lowering inhibitor described in (7) above.
(10) The immunosuppressant is one or more selected from cyclosporine, tacrolimus, steroids, azathioprine, mizoribine, mycophenolate mofetil, anti-T cell antibody, rapamycin and 15-deoxyspagarin. The combination according to (9) above.

(11) A transplanted inflammatory factor after organ transplantation or tissue transplantation, comprising a compound represented by formula (I), a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient -1 production inhibitor.

  The chronic rejection inhibitor of the present invention can effectively suppress chronic rejection after organ transplantation or tissue transplantation, particularly after organ transplantation, and is extremely useful in the field of treatment of organ transplantation or tissue transplantation, particularly organ transplantation. Useful for. Further, it can be used for prevention and treatment of chronic rejection for a long period of time without any side effects seen in known immunosuppressive agents used in the medical field as acute rejection inhibitors. Furthermore, since the chronic rejection inhibitor of the present invention suppresses the production of transplanted inflammatory factor-1, cerebral infarction, brain trauma, glioma, skeletal muscle trauma, inflammatory disease, Kawasaki, to which this factor is related, It is also effective for treating diseases, SARS, and the like.

  The chronic rejection inhibitor of the present invention is a human or non-human animal that has undergone organ transplantation such as kidney, pancreas, small intestine, lung, liver or heart, or tissue transplantation such as skin, bone, heart valve or the like (for example, Can be administered to mammals such as dogs, cats, cows, horses, etc., but preferably can be administered to humans or non-human animals transplanted by heart. It is also effective for treating patients suffering from cerebral infarction, brain trauma, glioma, skeletal muscle trauma, inflammatory disease, Kawasaki disease, SARS, etc. involving transplanted inflammatory factor-1.

  Patients after organ transplantation develop symptoms of acute rejection (fever, general malaise, swelling, reduced function, etc.), which are treated and alleviated by administration of immunosuppressants. However, after about 3 months after organ transplantation, a chronic rejection reaction that often causes a decrease in the function of the transplanted organ often occurs several years later. Until now, no effective treatment or treatment for chronic rejection has been known, and the fundamental treatment had to rely on retransplantation. The phenotypes of chronic rejection of these organs vary depending on the organ. Is done. The cause is said to be both an immunological cause (cell infiltration that cannot be suppressed by immunosuppressive therapy) and an inflammatory cause, but the cause is not clear, but obstruction of the feeding artery to the organ tissue Probably the main cause.

  The chronic rejection inhibitor of the present invention is particularly preferably applied to a heart transplant patient in view of its therapeutic / preventive effect. Heart transplant rejection includes acute rejection that occurs up to 3 months after transplantation and chronic rejection that gradually weakens the coronary artery several months after transplantation, for example, after about 3 months. Symptoms.

  As mentioned above, coronary arteriosclerosis is known as a chronic rejection reaction in the transplanted heart, and the cardiac function of the transplanted heart is lowered due to the chronic rejection reaction, and in the worst case, the grafted heart is removed and attached, resulting in death. .

  In the present invention, the effectiveness against chronic rejection after heart transplantation was measured using heartbeat intensity as one of the cardiac functions as shown in Test Example 1 in the Examples described later. As described above, using as an index the inhibitory effect on transplanted inflammatory factor-1 (AIF-1) (see Non-patent Documents 4 to 5 and Patent Document 5) reported as one factor of chronic rejection in transplantation, organs are used. The effectiveness of the chronic rejection inhibitor after transplantation was determined (see: FIG. 1, Test Example 2).

  The pirfenidone, pharmacologically acceptable salt or solvate represented by the above formula (I) used as an active ingredient of the chronic rejection inhibitor of the present invention is a known compound, and is described in Patent Document 1 or 2 can be produced. Pharmacologically acceptable salts of pirfenidone include salts with amino acids, inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, etc.) or organic acids (acetic acid, citric acid, maleic acid, fumaric acid, benzene) Salts with sulfonic acid, p-toluenesulfonic acid and the like). These salts can be formed by a commonly performed method. Of these, salts with inorganic acids and organic acids are preferred. Examples of the solvate of pirfenidone include solvates with organic solvents, hydrates, etc., but hydrates are preferred, and when forming hydrates, any number of water molecules and It may be coordinated.

The chronic rejection inhibitor of the present invention is generally administered to humans or other animals that have undergone organ transplantation, but can be administered prophylactically in advance to patients or animals prior to organ transplantation, or acute rejection. While it is possible to administer to a patient who is experiencing a response, it is preferably administered to a patient who is experiencing chronic rejection.
The active ingredient pirfenidone, pharmacologically acceptable salt or solvate can be formulated by a method known per se, for example, a preparation can be produced by the method described in WO01 / 58448. it can. The dosage form is not particularly limited as long as it can suppress chronic rejection, but generally, orally in a dosage form such as tablets, capsules, granules, powders, powders, powders, pills, liquids, syrups, Alternatively, it can be administered parenterally in dosage forms such as injections (subcutaneous injection, intramuscular injection, intravenous infusion), creams, ointments, suppositories, transdermal absorption agents, nasal agents, inhalants and the like. Preferred dosage forms are oral preparations such as tablets and capsules.
In the preparation of the above preparation, additives used in the field for formulation can be used as appropriate. Carriers, solvents, excipients, humectants, dispersants, suspensions, sweeteners, flavors Agents, flavoring agents, preservatives or other additives can be appropriately selected and contained in the chronic rejection inhibitor.

  The dosage of the active ingredient pirfenidone, pharmacologically acceptable salt or solvate is, in the case of oral preparations, the patient's age, weight, sex, severity of the disease and the efficacy, toxicity, excretion rate of the active ingredient, Although it can be determined in consideration of the absorption rate and the like, it is usually about 80 to 2000 mg / kg / day for adults, and about 80 to 2000 mg / kg / day for parenteral administration.

  In the suppression of chronic transplant rejection, pirfenidone can be used in combination with other immunosuppressive agents. Pirfenidone and other immunosuppressive agents may be contained in the same preparation. A preparation containing pirfenidone and a preparation containing another immunosuppressant may be used as a combination. Such immunosuppressants include cyclosporine, tacrolimus (FK-506), steroidal agents (eg, prednisolone, methylprednisolone), azathioprine, mizoribine, mycophenolate mofetil, anti-T cell antibody (OKT3, ATG), rapamycin, 15-deoxyspagarin and the like are exemplified, and one or more of these immunosuppressive agents can be used. Preferred combination forms include (1) pirfenidone and steroid, (2) pirfenidone and cyclosporine, (3) pirfenidone and tacrolimus, (4) pirfenidone and steroid and cyclosporine, (5) pirfenidone and steroid and tacrolimus, (6 And a combination of pirfenidone, cyclosporine, and azathioprine, and (7) pirfenidone, a steroid, cyclosporine, and azathioprine. In addition, it is desirable to avoid simultaneous administration of cyclosporine and tacrolimus since contraindications have been reported.

  The actual administration timing of drugs for treating rejection can be generally divided into 1) the timing of single administration of immunosuppressive agents, 2) the timing of combined use of immunosuppressants and pirfenidone, and 3) the timing of single administration of pirfenidone. . That is, at the time of combination, pirfenidone and other immunosuppressive agents can be administered at the same time, or the administration time of both drugs can be shifted. In addition, when an immunosuppressive agent is administered orally at the time of combined use, it is desirable to administer pirfenidone orally. Similarly, when the immunosuppressive agent is administered parenterally, it is desirable to administer pirfenidone parenterally.

  Here, the dose of the immunosuppressive agent can be appropriately determined in consideration of the patient's age, weight, sex, degree of disease, efficacy of the active ingredient, toxicity, excretion rate, absorption rate, dosage form, and the like. Regarding the dosage and dosage form of each immunosuppressant, if it conforms to what is described in “2004 Medicinal Products Japan Pharmaceuticals Collection, Japan Pharmaceutical Information Center (JAPIC), Jiho Co., Ltd.” Good. The range of dosage may vary depending on the organ and the number of days before and after transplantation. For example, as a cyclosporine (internal use solution, capsule), take 2-16 mg / kg once or twice a day and start intravenous infusion. When oral administration is possible, it can be switched to oral administration. In the case of tacrolimus (liquid for internal use, capsule), 0.06-0.30 mg / kg is taken once or twice a day, It can be switched to oral administration when it becomes possible to administer from intravenous infusion. Examples of prednisolone preparations include creams, powders, powders, tablets, ointments, and eye ointments. Usually, 5 to 60 mg of prednisolone is taken 1 to 4 times a day. Examples of the preparation of methylprednisolone include tablets. Usually, 4 to 48 mg of methylprednisolone is taken 1 to 4 times a day. As a preparation of mycophenolate mofetil, there is a capsule. Usually, 1 to 3 mg of mycophenolate mofetil is taken twice a day for 12 hours. The preparation of azathioprine includes tablets, and usually 0.5 to 3 mg / kg is taken as azathioprine per day. As a preparation of mizoribine, there are tablets, and 1 to 3 mg / kg of mizoribine is usually taken 1 to 3 times a day.

  Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

Formulation Example 1
A granule containing the following ingredients was produced.
Ingredients Pirfenidone 10 mg
Lactose 700 mg
Corn starch 274 mg
HPC-L 16 mg
1000 mg
Pirfenidone and lactose were passed through a 60 mesh sieve and corn starch was passed through a 120 mesh sieve. These were mixed with a V-type mixer. An HPC-L (low-viscosity hydroxypropylcellulose) aqueous solution was added to the mixed powder, kneaded and granulated (extruded granulated pore diameter 0.5 to 1 mm), and then dried. The obtained dried granules were combed with a vibration sieve (12/60 mesh) to obtain granules.

Formulation Example 2
A powder for capsule filling containing the following components was produced.
Ingredients Pirfenidone 10 mg
Lactose 79 mg
Corn starch 10 mg
Magnesium stearate 1 mg
100 mg
Pirfenidone and lactose were passed through a 60 mesh sieve, and corn starch was passed through a 120 mesh sieve. These and magnesium stearate were mixed in a V-type mixer. 100 mg of the 10-fold powder produced in this way was filled into a No. 5 hard gelatin capsule.

Formulation Example 3
A capsule filling granule containing the following components was produced.
Ingredients Pirfenidone 15 mg
Lactose 90 mg
Corn starch 42 mg
HPC-L 3 mg
150 mg
Pirfenidone and lactose were passed through a 60 mesh screen and corn starch was passed through a 120 mesh screen. These were mixed, the HPC-L solution was added to the mixed powder, kneaded, granulated and dried. After the obtained dried granules were sized, 150 mg thereof was filled into a No. 4 hard gelatin capsule.

Formulation Example 4
A tablet containing the following ingredients was prepared.
Ingredients Pirfenidone 10 mg
Lactose 90 mg
Microcrystalline cellulose 30 mg
CMC-Na 15 mg
Magnesium stearate 5 mg
150 mg
Pirfenidone, lactose, microcrystalline cellulose and CMC-Na (carboxymethylcellulose sodium salt) were passed through a 60 mesh sieve and mixed. The mixed powder was mixed with magnesium stearate to obtain a mixed powder for tableting. This mixed powder was directly hit to obtain 150 mg tablets.

Formulation Example 5
An intravenous formulation containing the following ingredients was prepared.
Pirfenidone 100 mg
Saturated fatty acid glyceride 1000 ml
Solutions of the above components are usually administered intravenously to the patient at a rate of 1 ml per minute.

Formulation Example 6 Production Method of Pirfenidone Tablets To 2,000 g of pirfenidone, 560 g of lactose and 50 g of carmellose calcium are added to prepare a mixed powder. Then, fluidized bed granulation was performed to obtain granules. 5.6% by weight of carmellose calcium and 1.1% by weight of magnesium stearate were added to and mixed with the obtained granules, tableted (13 kN), and pirfenidone uncoated tablets (12.0 × 6.0 mm, 285 mg / tablet containing 200 mg of pirfenidone per tablet).
A 10 wt% aqueous coating solution containing 66.7 g of hydroxypropylmethylcellulose, 6.7 g of triethyl citrate and 26.6 g of titanium oxide was coated with 10 mg / tablet on the above pirfenidone uncoated tablet using a high coater. To obtain pirfenidone tablets.
The composition per tablet is shown in Table 1 below.

Test Example 1 Measurement of Heartbeat Intensity Transplantation experiments were performed between PVG and LEW, which are inbred rats (manufacturer: SEAC Yoshitake Co., Ltd.) with completely different major histocompatibility antigens (MHC). 6-8 week old (130-180 g) male PVG rat hearts were transplanted (cervical ectopic heart transplantation) into the neck of 8-10 week old (230-300 g) male LEW rats. Cervical ectopic heart transplantation was performed by Heron's method in which the recipient's carotid artery and jugular vein were anastomosed to the aorta and pulmonary artery of the donor heart (Heron I. Atechnical accession transplantation in rabbits and rats. 79: 366, 1971). For suppression of acute rejection, cyclosporin A (10 mg / ml, manufactured by Novarteis Pharma, Miglyol 812 solution (manufactured by Dynamit Nobel)) was administered into the rat hind limb muscle daily for 10 days after transplantation.
Pirfenidone (PFD) represented by the formula (I) is suspended in a vehicle (Carboxymethyl cellulose sodium salt, 039-0133335, manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 30 mg / ml, The treatment group was orally administered daily at 400 mg / kg from the day after transplantation, and the same amount of vehicle (0.5% carboxymethylcellulose sodium salt aqueous solution) was orally administered to the control group every day.
The pulsation of the heart transplanted into the rat was observed daily (120 days) by palpation, and the strength of the pulsation was recorded in 15 stages. Table 2 shows the average values obtained by statistical processing (statistically significant differences were determined by Fisher's PLSD analysis after analysis of variance and p <0.05 was determined to be significant). The 15 levels of the heartbeat intensity are based on the following criteria.
0: Cardiac arrest, 0% of normal heartbeat intensity
1: Slightly stronger than 0% of normal heartbeat intensity 2: Slightly weaker than 20% of normal heartbeat intensity 3: 20% of normal heartbeat intensity
6: 40% of normal heartbeat intensity
7: Slightly stronger than 40% of normal heartbeat intensity 8: Slightly weaker than 60% of normal heartbeat intensity 9: 60% of normal heartbeat intensity
10: Slightly stronger than 60% of normal heartbeat intensity 11: Slightly weaker than 80% of normal heartbeat intensity 12: 80% of normal heartbeat intensity
13: Slightly stronger than 80% of normal heartbeat intensity 14: Slightly weaker than 100% of normal heartbeat intensity 15: Normal heartbeat intensity, 100%

  As is apparent from Table 2, when pirfenidone (PFD: 400 mg) was administered to a heart transplanted rat, the decrease in heart rate tended to be suppressed compared to the control group to which pirfenidone was not administered, resulting in decreased cardiac function. It turns out that the inhibitory effect is shown. In particular, the pulsation intensity between 90 days and 120 days after heart transplantation, in which chronic rejection occurs, tends to be superior to the control group.

Test Example 2 Inhibition of Gene Expression of Transplanted Inflammatory Factor-1 (AIF-1) Factor Pirfenidone (PFD) is a vehicle (0.5% carboxymethylcellulose sodium salt solution, 039-0133335, at a concentration of 30 mg / ml, Wako Pure Chemical Industries, Ltd.) was orally administered daily at 400 mg / kg from the day after transplantation to form a treatment group, and the same amount of vehicle (0.5% carboxymethylcellulose sodium salt aqueous solution) was orally administered every day. It was.
Messenger RNA (mRNA) was extracted from rat transplanted hearts 60 days and 120 days after transplantation obtained in the same manner as in Test Example 1 above, and the Taqman probe for quantitative PCR shown below for transplanted inflammatory factor-1 (AIF-1) RT-PCR reaction was performed using (Manufacturer: Applied Biosystems).
Primer sequence
Forward 5'-TGG GAT CAA CAA GCA CTT CCT-3 '; Sequence Listing 1
Reverse 5'-TTG AAG GCC TCC AGT TTG GA-3 '; Sequence Listing 2
Probe sequence
FAM-CCA AGT ACA GCA GTG ATG-MGB; Sequence Listing 3
In the probe sequence, FAM is a fluorescent light emitting molecule (Reporter) 6-carbofluorescein, and MGB is a light reducing molecule (Quencher).
After completion of the RT-PCR reaction, Prism 7700 (manufactured by Applied Biosystems) was used, and Heid CA, Stevens J, Livek KJ, Williams PM. Real time quantitative PCR. Genome Research 6 (10): 986-994, 1996, and the degree of gene expression of AIF-1 in the heart tissue of the treatment group and the heart tissue of the control group was compared by the amount of mRNA. Here, the ratio of the expression relative to the expression of the glyceraldehyde 3-phosphate dehydrogenase gene as an internal standard was determined, and the expression level of AIF-1 was examined with the value of normal heart tissue set to 1. 1 gene expression intensity. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is one of the most basic glycolytic enzymes as a cell energy production system, and is almost constant in any cell and under any conditions. The amount is known to be contained and is often used in the art as an internal standard molecule. Table 3 shows the results of statistically processing the gene expression intensity of the obtained AIF-1. Each period was examined using 5 normal rats and heart transplant rats.

上記数値をグラフ化したのを図１に示す。表３および図１から明らかなように、ピルフェニドン４００ｍｇ／ｋｇ／ｄａｙを６０日間、１２０日間投与した場合、対照群に比較して、移植心におけるＡＩＦ−１の発現量はピルフェニドン投与により有意に抑制されているのが分かる。なお、別途実験によれば、心移植後７日、６０日、１２０日、１８０日のラットにおいて、移植心内ＡＩＦ−１遺伝子発現は、移植後に漸次増大し、移植後１２０日にはピークに達していることを確認した。このことは慢性拒絶反応のピークが１２０日後に生起していることを示す。

FIG. 1 shows a graph of the above numerical values. As is apparent from Table 3 and FIG. 1, when pirfenidone 400 mg / kg / day was administered for 60 days for 120 days, the expression level of AIF-1 in the transplanted heart was significantly suppressed by pirfenidone administration compared to the control group You can see that. In addition, according to a separate experiment, in rats at 7 days, 60 days, 120 days, and 180 days after heart transplantation, the intracardiac AIF-1 gene expression gradually increased after transplantation, and peaked at 120 days after transplantation. Confirmed that it has reached. This indicates that the peak of chronic rejection occurs after 120 days.

  The chronic rejection inhibitor of the present invention has an excellent effect in suppressing and preventing chronic rejection after organ transplantation or tissue transplantation, particularly after organ transplantation, and can be used safely for a long time without side effects. It is a medicine.

2 shows the transplanted intracardiac AIF-1 gene expression intensity (ratio to normal heart tissue) of the pirfenidone administration group (400 mg / kg / day, 60 days, 120 days) and the control group of heart transplanted rats. Data after 120 days after transplantation are significantly different by the Mann-Whitney U test (p <0.05).

Claims (11)

An inhibitor of chronic rejection after organ transplantation or tissue transplantation, comprising a compound represented by formula (I), a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient.

  2. The chronic rejection inhibitor according to claim 1, wherein the organ transplantation is kidney transplantation, pancreatic transplantation, small intestine transplantation, lung transplantation, liver transplantation or heart transplantation.   2. The chronic rejection inhibitor according to claim 1, wherein the organ transplantation is a heart transplantation.   The chronic rejection inhibitor according to claim 1, wherein the tissue transplant is a skin transplant, a bone transplant or a heart valve transplant.   A combination comprising an immunosuppressant and the chronic rejection inhibitor according to claim 1.   The immunosuppressive agent is one or more selected from cyclosporine, tacrolimus, steroids, azathioprine, mizoribine, mycophenolate mofetil, anti-T cell antibody, rapamycin and 15-deoxyspagarin. 5. A mixture according to 5. An inhibitor of cardiac function deterioration after organ transplantation or tissue transplantation, comprising a compound represented by formula (I), a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient.

  The cardiac function lowering inhibitor according to claim 7, wherein the cardiac function deterioration is a reduction in heartbeat intensity.   A combination comprising an immunosuppressive agent and the cardiac function lowering inhibitor according to claim 7.   The immunosuppressive agent is one or more selected from cyclosporine, tacrolimus, steroids, azathioprine, mizoribine, mycophenolate mofetil, anti-T cell antibody, rapamycin and 15-deoxyspagarin. 9. A combination according to 9. Transplantation inflammatory factor-1 production after organ transplantation or tissue transplantation, comprising a compound represented by formula (I), a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient Inhibitor.

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