JP2014231476A - IκB KINASE α ACTIVATION INHIBITOR - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a castration-resistant relapsed prostate cancer therapeutic agent, a male contraceptive agent and a myasthenia gravis therapeutic agent comprising IκB kinase α activation inhibitor (IKKα activation inhibitor) as an active ingredient.SOLUTION: The noraristeromycin (NAM) of an adenosine analog represented by the following formula and having the selective inhibitory action of IκB kinase α is used as a castration-resistant relapsed prostate cancer therapeutic agent, a male contraceptive agent and a myasthenia gravis therapeutic agent.

Description

  The present invention relates to a castration resistant relapsed prostate cancer therapeutic agent, a male contraceptive agent, and a myasthenia gravis therapeutic agent containing an IκB kinase α activation inhibitor as an active ingredient.

NF-κB (nuclear factor-kappa B) is a transcription factor discovered by David Baltimore et al. In 1986, and has been reported to be involved in the expression of a number of genes, and plays a central role in the immune response. It is known as one of transcription factors.
In addition, NF-κB has been recognized to be constantly activated in malignant tumors, and further, it has been reported to be involved in the proliferation of HIV.
The inactive NF-κB forms a complex with the inhibitory protein IκB, and the phosphorylation of IκB by the IKK complex releases NF-κB, and then NF-κB It is transferred to the nucleus and activated. The IKK complex is composed of three types of proteins: IκB kinase α (IKKα), IκB kinase β (IKKβ), and NEMO (IKKγ).

  Okamoto et al. In Patent Document 1

  It has been reported that the adenosine analog, noraristeromycin (NAM), having a selective inhibitory action on IκB kinase α, is useful for the treatment of AIDS and the like.

(1) Castration-resistant relapsed prostate cancer treatment In recent years, the incidence of prostate cancer patients in Japan has been increasing. The number of deaths due to prostate cancer followed an increase, reaching 883 in 1970, but approximately 11 times as many as 9,989 in 2008. In addition, the number of affected persons was 10,940 in 1994, but a significant increase is expected to exceed 80,000 in 2020.
So far, estrasite, ifomide, cisplatin, peplomycin, and UFT have been used in Japan as chemotherapeutic agents with insurance indication. However, although these can be expected to temporarily reduce PSA and improve symptoms, no effect of extending the overall survival rate has been observed (Non-patent Document 1). In addition, many of the target cases are elderly, and there is a problem in patient tolerance.
Docetaxel is a taxane anticancer agent. In both reports on Randomized controlled study (RCT) with mitoxantrone + prednisolone as the control group, castration-resistant prostate cancer (castration) was obtained by anticancer chemotherapy using the same drug. -Life prognostic extension effect (18.9 months and 18 months) on resist prosthetic cancer (CRPC) was observed (Non-patent Document 2). Based on these results, docetaxel was expanded to cover prostate cancer in May 2004 in the US and in Japan in August 2008, and is now a standard treatment for anticancer chemotherapy for CRPC. It's getting on. However, even in various reports in Japan, the progression-free period is 3 to 11 months, which is not always satisfactory (Non-Patent Document 3), and anticancer chemotherapy using the same agent has become the standard treatment for CRPC. The current situation is not. Overseas, side-effects centered on the cardiovascular system, digestive system, and blood system are observed in 45-54%, and treatment-related death is recognized in 0.3-2% (Non-patent Document 2). Even in reports in Japan (Non-Patent Document 3, Non-Patent Document 4 and Non-Patent Document 5), the incidence of blood system side effects is particularly high at 75-80%, and the reality is that there are many elderly people as target patients. In view of this, development of a treatment method that is lower risk and high benefit is desired.
It is a known fact that NF-kB is activated in various cancers, and research and development of various molecular targeted drugs targeting its pathway and I-κB kinase β (IKKβ) are actively conducted. However, small molecules have not been successful, except for a few cases such as the proteosome inhibitor Velcade.

  On the other hand, M.M. Karin et al. Reported from a study of Maspin in cancer that the I-κB kinase α (IKKα) regulates metastasis of prostate cancer in 2007 using a TRAMP mouse with a mutation in IKKα. (Non-Patent Document 6). Furthermore, although there are reports on cancer using an IKKβ-specific inhibitor, there has been no report on studies on cancer using an IKKα-specific inhibitor (Non-patent Document 7). In 2010, K.K. Cheng et al. Reported on PC-3 and DU145, which are castration-resistant relapsed prostate cancer strains, using IKKα-specific siRNA, and reported that they suppressed migration but not growth. (Non-patent Document 8).

2 Contraceptives for men The selection of contraceptive methods in Japan is characterized by the overwhelming majority of condoms, removal methods and rhythm methods (Ogino method) even after the low-dose pill was approved in 1999. (Non-patent document 9). The only contraceptive methods that men can actively select are condoms, removal methods, and sterilization. Conventionally, low reversible surgical infertility, vas deferens without using a scalpel, direct injection into the vas deferens to reduce fertility, polyurethane or silicone plugs to close the vas deferens, new polymers Improvement techniques such as a method of lowering the pH in the vas deferens by injecting sperm and a fertilization ability by reducing testicular overheating have been devised. In addition, as a male contraceptive, which is under study, the University of Edinburgh research team, together with the World Health Organization (WHO), conducts clinical trials of contraceptive injections to men once every two months. It started in 2009 with 400 couples at 10 centers in Europe, Asia, South America and Australia. This contraceptive contains testosterone, progesterone, and hormones, and has the effect of rapidly reducing the number of sperm during the early hours of administration. The University of Washington in the United States is conducting research and development on oral contraceptives that perform contraception by administering "testosterone", a type of male hormone, but only 90% of contraceptive success rates have been shown. At Bar-Ilan University in Israel, we have succeeded in developing a synthetic compound that suppresses the fertility of mouse sperm for a month at a low dose. Rho Kinase Inhibitors (Non-Patent Document 10) reports that it has a contraceptive effect by a mechanism that prevents the sperm from reaching the testicular to the enormous part of the vas deferens by inhibiting the contraction of the smooth muscle that constitutes the vas deferens. Yes. Various other male contraceptive approaches have been reported. An oral contraceptive for men that is reversible and has a high contraceptive success rate as used by women has been developed in Indonesia, 2011. Only "Gandarsa" scheduled for practical use in the year. Although approval in Western countries has yet to be determined, a survey conducted by a German company in 2005 revealed that the international demand for male contraceptives is very high. In Spain, Germany, Mexico, and Brazil in particular, 60% of the men surveyed want to use new contraceptives, indicating a high market demand. In addition, the expansion of options for easy and effective contraceptive methods for men avoids “unwanted pregnancies” and leads to men's active action to reduce women's economic costs and physical risks. It is considered to have social significance.

  To the knowledge of the inventors, there is no document describing the relationship between IKKα and male contraceptives.

3. About the treatment for myasthenia gravis Myasthenia gravis (MG) is specifically sensitized to the neuromuscular synaptic muscle side receptor (acetylcholine receptor; AChR) that uses acetylcholine (ACh) as a transmitter. It develops when autoantibodies (anti-AChR antibodies) are produced depending on helper T cells. However, there are about 24% of MG patients for whom anti-AChR antibodies have not been proven, and the etiology is not clear. From the onset mechanism of MG, it was considered that the thymus (an organ having the following important ability) is important as a target organ for MG treatment.
(1) Source of antibody-producing B cells (2) Source of helper T cells (3) Source of antigen-presenting cells (4) Expression of MHC class II protein (5) Expression of antigen protein (AChR) (6) Enhanced cytokine expression (7) Positive / negative selection of immune cells Therefore, there is no clear evidence that thymectomy is effective as a treatment for non-thymoma myasthenia gravis, but thymectomy depends on the presence or absence of thymoma. Regardless, it is positioned as a basic treatment for systemic MG (except children who develop 11 years of age and younger and elderly people aged 50-70 and older). As a result of thymectomy, remission rate was 15.9% at 6 months, 22.4% at 1 year, 36.9% at 3 years, 45.8% at 5 years, 55.7% at 10 years , It was 67.2% in 15 years and 50% in 20 years (Non-patent Document 11). On the other hand, complications related to thymectomy were also 33% overall (acute respiratory failure associated with crisis (6%), infection (11%), nerve damage such as recurrent nerve / phrenic nerve (2%) Etc.) (Non-patent Document 12).
Anticholinesterase drugs are also used for all types of MG as a symptomatic treatment, not as a radical treatment, but thymectomy is not performed, and about 1/4 cases of MG are treated with anticholinesterase alone. Therefore, there is a report that it will die within 3 years of onset (Non-patent Document 13). In addition, there are steroid drugs that are administered in the hope of an immunosuppressive effect, but their effectiveness has been described in combination with other therapies such as thymectomy (improvement rate 63.4-100%). However, there are many reports that consider side effects (38-66.7%) occurring at a high rate and transient initial exacerbations at the time of introduction as problems. Steroids are the first choice as immunosuppressive drugs, but only Prograf is insured in Japan when it cannot be used due to steroid resistance or side effects of steroids. Other treatment methods include blood purification therapy and high-dose immunoglobulin therapy. By combining them, the above-mentioned mortality rate is reduced to 1% or less even in cases where the control of MG symptoms is insufficient. In addition, the mortality related to thymectomy is also reported to be 1% or less (Non-patent Document 14).
On the other hand, there are the following cases for which thymectomy cannot be recommended as the first choice. In these cases, the above-described medical treatment is the first choice, but recent studies have reported improvement in symptoms and improvement in remission rate by performing thymectomy.
(1) Children under 11 years old who are worried about growth inhibition and immune function abnormalities (2) Older adults who need a certain period of time until surgical invasion, patient's life expectancy and the effect of surgery appear (3) Good response to drug therapy (4) Anti-AChR antibody negative (about 50% of ocular muscle type; about 80% develops to general type within 2 years after onset, about 20% of systemic type)

  To the knowledge of the present inventors, there is no document describing the relationship between IKKα and a drug for myasthenia gravis.

JP2007-99702

NEJM 2004 351: p15134 NEJM 2004 351: p1502 & p1513 Urological Surgery 2009 22: p551 Jpn J Clin Oncol 2007 37: p603 Urological Surgery 2008 21: p1091 Nature 2007 446: p690 Expert Opin Ther Target 2008 12: p1109 Pharm Res 2010 United Nations, World Contraceptive Use 2009 Auton Autocod Pharmacol 2006 26: p169 Ann Thorac Surg 1996 62: p853 Ann Surg 1997 32: p324 J Neurol Neurosurg Psychiatry 1989 52: p1121 Ann Surg 1997 32: p324

  An object of the present invention is to provide a castration resistant relapsed prostate cancer therapeutic agent, a male contraceptive agent, and a myasthenia gravis therapeutic agent containing an IκB kinase α activation inhibitor as an active ingredient.

That is, the present invention relates to a castration resistant relapsed prostate cancer therapeutic agent containing an IκB kinase α activation inhibitor as an active ingredient.
The present invention also relates to a male contraceptive containing an IκB kinase α activation inhibitor as an active ingredient.
Furthermore, the present invention relates to a therapeutic agent for myasthenia gravis comprising an IκB kinase α activation inhibitor as an active ingredient.

FIG. 1 is a diagram showing evaluation of cell growth inhibitory ability using a PC-3 cell line. FIG. 2 is a diagram showing cell growth inhibitory capacity evaluation using the DU145 cell line. FIG. 3 is a diagram showing cell growth inhibitory capacity evaluation using the LNCaP cell line. FIG. 4 is a view showing evaluation of cell migration inhibitory ability of Control using PC-3 cell line. FIG. 5 is a diagram showing an evaluation of the cell migration inhibition ability of the NAM addition group using the PC-3 cell line. FIG. 6 is a diagram showing an evaluation of the cell migration inhibitory activity of the MLN-120B addition group using the PC-3 cell line. FIG. 7 is a diagram showing evaluation of cell migration inhibitory ability of Control using DU145 cell line. FIG. 8 is a diagram showing an evaluation of the cell migration inhibition ability of the NAM addition group using the DU145 cell line. FIG. 9 is a diagram showing an evaluation of the ability to inhibit cell migration in the MLN-120B addition group using the DU145 cell line. FIG. 10 is a view showing evaluation of cell migration inhibitory ability of Control using the LNCaP cell line. FIG. 11 is a diagram showing an evaluation of the cell migration inhibitory activity of the NAM addition group using the LNCaP cell line. FIG. 12 is a diagram showing an evaluation of cell migration inhibition ability of the MLN-120B addition group using the LNCaP cell line. FIG. 13 is a diagram showing evaluation of apoptosis induction ability using DU145 cell line. FIG. 14 shows HE-stained images of testicular seminiferous stages VII and VIII after repeated administration of 0.3 mg / kg 42 days of NAM to 6-week-old DBA / 1JNCrlj male mice used in a collagen arthritis model. FIG. 15 shows HE stained images of testicular seminiferous stages VII and VIII after repeated administration of 0.1 mg / kg for 42 days to 6 week old DBA / 1JNCrlj male mice used in a collagen arthritis model. FIG. 16 shows HE stained images of seminiferous tubule stages VII and VIII after repeated administration of MC for 42 days to 6-week-old DBA / 1JNCrlj male mice used in a collagen arthritis model. It is. FIG. 17 shows HE staining of testicular seminiferous stage VII and VIII after repeated administration of 0.3 mg / kg for 42 days to DBA / 1JNCrlj male mice of 6 weeks old used in a collagen arthritis model and resting for 84 days. FIG. FIG. 18 shows HE-stained images of seminiferous tubule stages VII and VIII of testis after repeated administration of 0.1 mg / kg for 42 days to DBA / 1JNCrlj male mice of 6 weeks old used in a collagen arthritis model and resting for 84 days. It is a figure which shows FIG. 19 shows HE-stained images of testicular seminiferous stages VII and VIII after 10-week-old DBA / 1JNCrlj male mice were repeatedly administered NAM at 1 mg / kg for 7 days. FIG. 20 is a diagram showing HE stained images of testicular seminiferous stages VII and VIII after repeated administration of MC for 7 days to 10-week-old DBA / 1JNCrlj male mice. FIG. 21 is a diagram showing HE-stained images of testicular seminiferous stages VII and VIII after 10-week-old administration of MLN-120B to 10-week-old DBA / 1JNCrlj male mice at 10 mg / kg for 14 days. FIG. 22 is a diagram showing HE-stained images of testicular seminiferous tubule stages VII and VIII after 10-week-old DBA / 1JNCrlj male mice were repeatedly administered NAM at 1 mg / kg for 14 days. FIG. 23 shows HE stained images of testicular seminiferous stages VII and VIII after repeated administration of HPMC for 14 days to 10-week-old DBA / 1JNCrlj male mice. FIG. 24 is a diagram showing HE-stained images in the thymus after repeated administration of 1 mg / kg for 7 days to 10-week-old DBA / 1JNCrlj male mice. FIG. 25 is a view showing an HE-stained image in the thymus after repeated administration of MC for 7 days to 10-week-old DBA / 1JNCrlj male mice. FIG. 26 is a diagram showing HE-stained images in the thymus after repeated administration of MLN-120B at 10 mg / kg for 7 days to 10-week-old DBA / 1JNCrlj male mice. FIG. 27 shows HE-stained images in the thymus after repeated administration of HPMC for 7 days to 10-week-old DBA / 1JNCrlj male mice. FIG. 28 is a diagram showing an enlarged cortical image of HE staining in the thymus after repeated administration of 1 mg / kg 7 days of NAM to 10-week-old DBA / 1JNCrlj male mice. FIG. 29 is a diagram showing an enlarged cortical image of HE staining in the thymus after repeated administration of MC to 10-week-old DBA / 1JNCrlj male mice for 7 days. FIG. 30 shows the relative weight in the thymus after repeated administration of NAM 1 mg / kg, MLN-120B 10 mg / kg, HPMC and MC for 7 days in 10 week old DBA / 1JNCrlj male mice. FIG. 31 is a diagram showing HE-stained images in the thymus after 14-day withdrawal of NAM repeatedly administered to DBA / 1JNCrlj male mice at 10 weeks of age at 1 mg / kg for 14 days.

Next, the present invention will be described in detail.
The present invention relates to the following therapeutic agents and contraceptives.
1) A castration resistant relapsed prostate cancer therapeutic agent comprising an IκB kinase α activation inhibitor as an active ingredient.
2) IKK- [alpha] have the following _{IC 50} 1 [mu] M with respect to activity, castration-resistant recurrent prostate cancer therapeutic agent comprising as an active ingredient the IκB kinase α activator inhibitors with more _{IC 50} 10 [mu] M to IKKβ activity.
3) The castration resistant relapsed prostate cancer therapeutic agent according to 1), wherein the IκB kinase α activation inhibitor is noraristeromycin.
4) A male contraceptive containing an IκB kinase α activation inhibitor as an active ingredient.
5) IKK- [alpha] has the following _{IC 50} 1 [mu] M with respect to activity, male contraceptives containing as an active ingredient the IκB kinase α activator inhibitors with more _{IC 50} 10 [mu] M to IKKβ activity.
6) The male contraceptive according to 4), wherein the IκB kinase α activation inhibitor is noraristeromycin.
7) A therapeutic agent for myasthenia gravis comprising an IκB kinase α activation inhibitor as an active ingredient.
8) IKK- [alpha] has the following _{IC 50} 1 [mu] M with respect to activity, myasthenia gravis therapeutic agent containing as an active ingredient the IκB kinase α activator inhibitors with more _{IC 50} 10 [mu] M to IKKβ activity.
9) The therapeutic agent for myasthenia gravis according to 7), wherein the inhibitor of IκB kinase α activation is noraristeromycin.

The IκB kinase α activation inhibitor which is an active ingredient of the present invention includes NAM and 18a described in Table 1 of Bioorg med chem lett 17 1233-1237 2007; Table 1 of Bioorg Med Chem Lett 17 3972-3777 2007 8p, 8q described in Table 12, 12a, 12d described in Table 2, NPI-1387 described in US2008 / 0064753, and compounds described in WO2008 / 32121 and WO2008 / 10508.

First, a pharmacological test for castration resistant relapsed prostate cancer of an IκB kinase α activation inhibitor will be described.
Casting resistant relapsed prostate cancer lines PC-3 and DU145, and castration non-resistant prostate cancer line LNCaP using noraristeromycin (NAM) having specific inhibitory effect on IKKα activation Proliferation, mobility, and apoptosis induction effect were examined in vitro. As a result, the metastasis inhibitory effect was confirmed, and it was newly confirmed that there was a growth inhibitory effect and an apoptosis induction effect. Moreover, it confirmed that these effects were notably shown in PC-3 and DU145 which are a castration resistant relapsed prostate cancer strain | stump | stock compared with MLN-120B which is the preceding IKK (beta) specific inhibitor. (Example 1 described later, FIGS. 1 to 13)
Therefore, the IκB kinase α activation inhibitor is useful as a castration resistant relapsed prostate cancer therapeutic agent.

Next, the test results on the effect of an IκB kinase α activation inhibitor as a male contraceptive will be described.
Noralisteromycin (NAM), which has a specific inhibitory effect on IKKα activation, is a type II collagen arthritis model mouse, and semen from twice-daily administration of 0.1 mg / kg, which is an ED50 of arthritis inhibitory effect. A tendency to suppress formation was observed, and administration of 0.3 mg / kg twice a day to prevent arthritis 100% completely suppressed spermatogenesis (insufficient spermatogenesis due to loss of early spermatocytes). On the other hand, such a change was not recognized by administration of MLN-120B which is an IKKβ specific inhibitor. Moreover, it was confirmed by histologically reversible change that the spermatogenesis disorder observed by oral administration of NAM was analyzed by the analysis after the drug withdrawal period. (Example 2 described later, FIGS. 14 to 23)
Therefore, IκB kinase α activation inhibitors are useful as male contraceptives.

Next, a pharmacological test for myasthenia gravis by an inhibitor of IκB kinase α activation is described.
As described in Example 3 below, the following effects were discovered by repeated oral administration of NAM to a type II collagen arthritis model mouse by administering 0.3 mg / kg twice a day to prevent arthritis 100%. On the other hand, administration of MLN-120B, an IKKβ specific inhibitor, did not show the following changes.
(1) From the early stage of NAM administration, atrophy of the thymus was observed (when 10 week-old DBA / 1JNCrlj male mice were administered 1 mg / kg twice daily for 1 week, the relative weight was The value was about 38.5% compared to the control group).
(2) From the histological analysis, a decrease in lymphocytes was confirmed.
(3) It was suggested that the thymus weight and lymphocyte count were recovered by the withdrawal of NAM, and that this was a reversible change (approximately 8 weeks after the withdrawal, 100% of the onset was suppressed by administration of NAM. Arthritis developed in 2/3 cases of type II collagen arthritis model mice).
Based on the above results, it is possible to treat myasthenia gravis by controlling the function of the thymus medically in cases where thymectomy cannot be recommended or in cases resistant to anticholinesterase or immunosuppressive drugs. It was suggested. (Example 3 described later, FIGS. 24-31)
Therefore, IκB kinase α activation inhibitors are useful as therapeutic agents for myasthenia gravis.

The IκB kinase α activation inhibitor which is an active ingredient of the present invention can be administered to humans by an appropriate administration method such as general oral administration or parenteral administration.
For formulation, it can be produced into a dosage form such as a tablet, granule, powder, capsule, suspension, injection, suppository and the like by a conventional method in the technical field of formulation.
For these preparations, usual excipients, disintegrants, binders, lubricants, dyes, diluents and the like are used. Here, as the excipient, lactose, D-mannitol, crystalline cellulose, glucose and the like, as the disintegrant, starch, carboxymethylcellulose calcium (CMC-Ca), etc., as the lubricant, magnesium stearate, Examples of binders include talc and the like, and hydroxypropylcellulose (HPC), gelatin, polyvinylpyrrolidone (PVP), and the like.

The dose is usually about 0.01 mg to 100 mg of the compound of the present invention, which is an active ingredient in an injection, and 0.1 mg to 500 mg per day by oral administration in adults, but may be increased or decreased depending on age, symptoms, etc. Can do.

EXAMPLES Next, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.

Castration-resistant relapsed prostate cancer drug 1) Test substance Noristosterycin (NAM)
MLN-120B
2) Cells used (purchased from ATCC)
PC-3 (ATCC No. CRL-1435), castration resistant DU145 (ATCC No. HTB-81), castration resistant LNCaP (ATCC No. CRL-1740), castration non-resistant 3) Preculture conditions Each cell line Was incubated under conditions of 37 ° C. and 5% CO 2 (HERACELL 150i, Thermo SCIENTIFIC). The culture conditions are as follows.

PC-3;
F12K medium (GIBCO, 21127-022) + 10% fetal bovine serum (GIBCO, 12483, Lot No. 356833)

DU145;
MEM medium (GIBCO, 10370-021) + Sodium Pyruvate 100 mM (100 ×) (GIBCO, 11360-070) + GlutaMax I (GIBCO, 35050061) + 10% fat bovine serum (GIBCO, 12483, Lot No. 3383)

LNCaP;
RPMI-1640 medium (GIBCO, A10491-01) + 10% fetal bovine serum (GIBCO, 12483, Lot No. 356833)

4) Experimental method 1 Solution preparation 100% Dimethylsulfoxide (DMSO, Wako Pure Chemicals, Lot No. STL3279) dissolved compounds (30, 10, 3, 1, 0.3, 0.1, 0.03 and 0. 3). (01 mmol / L) was prepared as a stock solution and stored at 4 ° C. until use (SANYO, MPR-411FRS).

2 Determination of optimal cell concentration / culture days Table 1 shows the results.

The culture equipment used is as follows.
Cell growth inhibitory ability evaluation; 96 well flat bottom plate (grainer bio-one, 655180)

Cell migration inhibitory evaluation: 35 mmΦ tissue culture dish (grainer bio-one, 627160)

Apoptosis induction ability evaluation; Chamber Slide II (8) (IWAKI, 5732-008)

3 Evaluation of cell growth inhibition ability; Use Premix WST-1 Cell Proliferation Assay System (MK400, Takara Bio Inc.) Add 260 μL of sterile PBS (-) to all the wells outside 96 well plate to reduce evaporation of medium It was. Cells were seeded at the cell concentrations shown in Table 1 and cultured for 24 hours. The stock solution was diluted 100-fold with each medium and added to each well (each concentration; n = 6) at 10 μL / well. The negative control group (n = 6) was a final concentration 0.1% DMSO medium. In the WST-1 assay medium control group (cell non-seeded), 100 μL / well of 0.1% DMSO medium (n = 6) was also prepared according to the Premix WST-1 Cell Proliferation Assay System manual. After culturing for 72 hours, 10 μL / well of WST-1 was added, incubated for 1 hour at 37 ° C. under 5% CO ₂ , using Plate Reader (ImmunoReader NJ-2000), measurement wavelength (450 nm) and control Measured at wavelength (620 nm).

4 Cell migration inhibition ability evaluation; Wound-healing assay
After seeding at the number of cells shown in Table 1 and culturing for 24 hours, the stock solution was added to the dish so that the final concentrations were 10 μmol / L NAM, MLN-120B and 0.1% DMSO (negative control). After culturing for 72 hours and confirming that the number of cells had reached 90% confluence under a microscope, Wounding was performed using a 200 μL sterile chip. Thereafter, the cells were cultured for a maximum of 72 hours, and healing was observed.

5 Apoptosis induction ability evaluation; Use of TUNEL assay (Promega, G3250 DeadEnd ^™ Fluorometric TUNEL System) was used for seeding with the number of cells shown in Table 1. However, since only the adhesion of LNCaP was weak, 10 μg / mL poly-L-lysine hydride (Sigma, P9155-MG) was coated and the cells were seeded. After culturing for 24 hours, the stock solution was added to the well so that the final concentration was 10 μmol / L NAM, MLN-120B and 0.1% DMSO (negative control). Then, it culture | cultivated for the period shown in Table 1, and performed TUNEL assay according to the manual.

5) Results 1 Cell concentration and number of culture days Table 1 shows the seeded cell concentration and the maximum number of culture days (after compound addition) for each cell line.

2 Cell growth inhibitory ability evaluation The results are shown in FIGS. NAM showed a significant growth inhibitory effect in all cell lines. On the other hand, MLK-120B, which is an IKKβ inhibitor, showed a significant inhibitory effect only at a concentration of 10 μmol / L in PC-3 and LNCaP cell lines.

3 Cell migration inhibitory ability evaluation The results are shown in FIGS. NAM was a PC-3 and DU145 cell line, and showed a remarkable cell migration inhibitory effect compared with the control. LNCaP showed the possibility that all cells were deformed and died. On the other hand, MLN-120B did not show a cell migration inhibitory effect in all cell lines compared to the control.

4 Apoptosis induction ability evaluation The results are shown in FIG. The addition of NAM showed TUNEL-positive only in the DU145 cell line (PC-3 and LNCaP; data not shown). On the other hand, MLN-120B was TUNEL-negative in all cell lines (data not shown).

6) Discussion NAM, an IKKα activation inhibitor, has been shown to exhibit high cell growth inhibitory ability, cell migration inhibitory ability, and apoptosis induction ability in vitro against prostate cancer cell lines. In particular, the high efficacy alone based on a novel mechanism for castration resistant PC-3 and DU145 suggested the possibility of leading to the development of new therapeutic agents and treatment methods.

Contraceptives for men 1) Test substance Noraristeromycin (NAM)
MLN-120B

2) Animals used (purchased from Nihon Charles River Co., Ltd.)
Male DBA / 1JNCrlj mouse 6 weeks old; for type II collagen arthritis model, as a sex immature mouse (5 weeks old at the time of purchase)
10 weeks of age; as a naive, sexually mature mouse (9 weeks old at the time of purchase)

3) Breeding environment Animals were raised individually by placing a floor covering in a polycarbonate cage with a cross partition (KN-606: Natsume Seisakusho Co., Ltd.). Tap water and solids in an environment with a temperature of 23 ° C (acceptable range: 20-26 ° C), humidity of 55% (acceptable range: 35-75%), and illumination time of 7 am-7 pm (light / dark cycle 12 hours) Feed (CRF-1: Oriental Yeast Co., Ltd.) is freely ingested. After about one week of habituation, only healthy animals were subjected to the test.

4) Grouping Based on the body weight measured on the day before administration, grouping was performed.

5) Identification method of test system The animals were marked with tails using oil-based felt pens to identify individuals.

6) Experimental method Experiment 1: Examination in a mouse model of type II collagen arthritis NAM was prepared once a day using an aqueous solution (MC) of 1% methylcellulose (Metroses SM-15, Shin-Etsu Chemical Co., Ltd.) (0.3 And 0.1 mg / 5 mL), 2 times a day (9: 00-10: 00 and 16: 00-17: 00) using a gastric sonde under non-fasting conditions to be 5 mL / kg / time In addition, 6-week-old male DBA / 1JNCrlj mice for the preparation of a type II collagen arthritis model were repeatedly forcibly administered orally for 42 days from the first type II collagen immunization to the ridge (period of spermatogenesis in the mouse testis; 1 cycle). did. Thereafter, a resting period of 84 days (2 cycles) was set, autopsy was performed after repeated administration for 42 days and resting for 84 days, and histological examination of the testis was performed. Individuals who confirmed high inflammation in the ridge were excluded from this study because of their effects on testicular tissue.

Experiment 2: Examination in Naive mice NAM was prepared once a day (1 mg / 5 mL) using MC, and twice a day (9: 00-10: 00 and 16) using a gastric sonde under non-fasting conditions. : 07: 00 to 17:00), repeated oral gavage was performed at 5 mL / kg / dose. MLK-120B, an IKKβ inhibitor, was prepared once a day using 0.5% hydroxypropylmethylcellulose (METOLOSE 60SH-50, Shin-Etsu Chemical Co., Ltd.) / 0.2% tween80 (WAKO) (HPMC) ( 1 mg / mL) and repeated oral gavage to 10-week-old male DBA / 1JNCrlj mice (Naive) under the same conditions as NAM at 10 mL / kg / dose. Necropsy was performed 7 and 14 days after the start of oral administration, and histological examination of the testis was performed.

7) Result experiment 1
FIGS. 14 to 16 show HE stained images of seminiferous tubule stages VII and VIII after repeated administration of NAM for 42 days to 6 week old DBA / 1JNCrlj male mice. With 0.3 mg / kg NAM (FIG. 14), spermatogenesis was completely suppressed. In 0.1 mg / kg NAM (FIG. 15), a decrease in sperm cells and the like was observed.
FIGS. 17 and 18 show HE-stained images of seminiferous tubule stages VII and VIII of the testis after repeated administration of NAM for 42 days to 6-week-old DBA / 1JNCrlj male mice. Spermatogenesis that was not observed at all after administration of 0.3 mg / kg NAM for 42 days (FIG. 14) resumed after 84 days of withdrawal (FIG. 17). In addition, the number of sperm cells, which had decreased after 42 days of administration with 0.1 mg / kg NAM (see FIG. 15), recovered after the withdrawal (FIG. 18).
Experiment 2
19 and 20 show HE-stained images of testicular seminiferous tubule stages VII and VIII after repeated administration of 1 mg / kg NAM for 7 days to sex-matured 10-week-old DBA / 1JNCrlj male mice. The initial spermatocytes (preleptene phase spermatocytes; arrows) observed in the MC administration group (FIG. 20) were not observed at all in the NAM administration group.
FIGS. 21 to 23 show HE-stained images of testicular seminiferous stages VII and VIII after 10-day repeated administration of 10 mg / kg MLN-120B and 1 mg / kg NAM to sexually mature 10-week-old DBA / 1JNCrlj male mice. Indicated. At 10 mg / kg MLN-120B (FIG. 21), the detachment of spermatocytes as shown by NAM (FIG. 21) was not observed. In 1 mg / kg NAM (FIG. 22), in addition to the preleptoten spermatocytes shown in FIG. 19, no pachytene spermatocytes (see FIG. 20) were observed.

8) Discussion The suppression of spermatogenesis observed with NAM administration was caused by the loss of early spermatocytes, and was observed regardless of the age at the start of administration (presence of sexual maturity) or the presence or absence of antigen. Further, this change was not observed even after 14 days of repeated administration of MLK-120B, an IKKβ inhibitor, suggesting the possibility of an action based on suppression of IKKα activation. This spermatogenesis disorder caused by NAM is histologically reversible, indicating the possibility as a male contraceptive having a high contraceptive effect similar to that of female oral contraceptives.

Myasthenia gravis treatment

1) Test substance Noraristeromycin (NAM)
MLN-120B

2) Animals used (purchased from Nihon Charles River Co., Ltd.)
Male DBA / 1JNCrlj mouse 10 weeks old (9 weeks old at the time of purchase)

3) Breeding environment Animals were raised individually by placing a floor covering in a polycarbonate cage with a cross partition (KN-606: Natsume Seisakusho Co., Ltd.). Tap water and solids in an environment with a temperature of 23 ° C (acceptable range: 20-26 ° C), humidity of 55% (acceptable range: 35-75%), and illumination time of 7 am-7 pm (light / dark cycle 12 hours) Feed (CRF-1: Oriental Yeast Co., Ltd.) is freely ingested. After about one week of habituation, only healthy animals were subjected to the test.

4) Grouping Based on the body weight measured on the day before administration, grouping was performed.

5) Identification method of test system The animals were marked with tails using oil-based felt pens to identify individuals.

6) Experimental method NAM was prepared (1 mg / 5 mL) once a day using a 1% methylcellulose (Metroses SM-15, Shin-Etsu Chemical Co., Ltd.) aqueous solution (MC), and using a stomach tube under non-fasting conditions. Twice a day (9: 00-10: 00 and 16: 00-17: 00), repeated oral gavage was performed on 10-week-old male DBA / 1JNCrlj mice at 5 mL / kg / dose.
MLK-120B, an IKKβ inhibitor, is prepared once a day using 0.5% hydroxypropyl methylcellulose (METOLOSE 60SH-50, Shin-Etsu Chemical Co., Ltd.) / 0.2% tween 80 (WAKO) (HPMC). (1 mg / mL). Administration is 10-week-old males at 10 mL / kg / dose twice a day (9: 00-10: 00 and 16: 00-17: 00) using a gastric sonde under non-fasting conditions. DBA / 1JNCrlj mice were administered by repeated gavage.
Necropsy was performed 7 and 14 days after administration, and thymus weight measurement and histological examination were performed.

7) Results FIGS. 24 to 30 show HE stained images and relative weights (each group; n = 4) in the thymus after repeated administration of NAM and MLN-120B for 7 days. As shown in FIG. 24 and FIG. 28, a decrease in lymphocytes centered on the cortex (range of arrows) was observed by repeated oral administration of NAM, but in MLN-120B (FIG. 26), a decrease in lymphocytes was observed. Was not observed. The relative thymus weight of the NAM administration group (FIG. 30) was significantly lower (about 38.5%, p <0.001, t-test) compared to the MC group. On the other hand, the MLN-120B administration group showed no significant difference compared to the HPMC group.
Furthermore, even when repeated oral administration (total 14 days) was performed for 7 days, in the MLN-120B administration group, the thymus tissue and weight change particularly noted were not observed as compared with the HPMC group. In NAM, thymus tissue and weight changes were similar to repeated administration for 7 days (data not shown).
When a 14-day withdrawal was performed on a 14-day administration group of NAM that showed changes in thymus tissue and weight, the number of thymic cortical lymphocytes recovered dramatically (FIG. 31), and the weight was similar to that of the control group. Recovered (data not shown).
8) Discussion It was suggested that thymic atrophy accompanied by the decrease in lymphocytes observed by repeated oral administration of NAM may be an action based on suppression of IKKα activation. In addition, since it is reversibly histologically, it has been shown that in myasthenia gravis, various adverse effects due to thymectomy can be avoided and the therapeutic effect equivalent to that of thymectomy can be exhibited.

In FIGS. 14 to 16, − represents 50 μm.
In FIGS. 19 and 20, − represents 50 μm.
The arrowheads in FIGS. 19 and 20 represent pakiten phase spermatocytes.
The arrow in FIG. 20 represents the preleptoten phase spermatocytes.
-Of FIGS. 21-23 represents 50 micrometers.
-Of FIGS. 24-27 represents 100 micrometers.
The arrow range in FIG. 24 represents the cortex.
-Of FIG. 28, 29 represents 50 micrometers.
31 in FIG. 31 represents 100 μm.
The arrow range in FIG. 31 represents the cortex.

Claims (9)

  A castration resistant relapsed prostate cancer therapeutic agent comprising an IκB kinase α activation inhibitor as an active ingredient. Have the following _{IC 50} 1 [mu] M to IKKα activity, castration-resistant recurrent prostate cancer therapeutic agent comprising as an active ingredient the IκB kinase α activator inhibitors with more _{IC 50} 10 [mu] M to IKKβ activity.   The therapeutic agent for castration-resistant relapsed prostate cancer according to claim 1, wherein the IκB kinase α activation inhibitor is noralisteromycin.   A male contraceptive containing an IκB kinase α activation inhibitor as an active ingredient. Have the following _{IC 50} 1 [mu] M to IKKα activity, male contraceptives containing as an active ingredient the IκB kinase α activator inhibitors with more _{IC 50} 10 [mu] M to IKKβ activity.   5. The male contraceptive agent according to claim 4, wherein the IκB kinase α activation inhibitor is noralisteromycin.   A therapeutic agent for myasthenia gravis comprising an IκB kinase α activation inhibitor as an active ingredient. Have the following _{IC 50} 1 [mu] M to IKKα activity, myasthenia gravis therapeutic agent containing as an active ingredient the IκB kinase α activator inhibitors with more _{IC 50} 10 [mu] M to IKKβ activity.   The therapeutic agent for myasthenia gravis according to claim 7, wherein the inhibitor of IκB kinase α activation is noralisteromycin.

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