JP6508670B2 - Cartilage degeneration inhibitor - Google Patents

Description

The present invention relates to a cartilage degeneration inhibitors.

  Osteoarthritis (OA), a typical disease that causes joint destruction, results in profound pain and motor dysfunction. In Japan, the total number of patients with OA is 12 million out of a total population of 1250 million, and it is said that 7 million people need treatment. However, the cause of OA has not been elucidated, and there is no drug that prevents the progression of OA. For this reason, at present, surgical treatment such as artificial joint replacement is performed on advanced cases of OA. In addition, as conservative treatment for OA, rehabilitation such as strength training and range of motion training, physical therapy such as thermal treatment, orthosis, intraarticular administration of hyaluronic acid, and administration of various anti-inflammatory and analgesic agents are performed. In addition to primary OA, OA also occurs after meniscal injury or resection, or after trauma. Furthermore, considering the future aging society, the number of patients targeted will be considerable.

  Articular cartilage can not self-repair once it is damaged. Therefore, until now, many studies have focused on cartilage. On the other hand, the importance of subchondral bone supporting cartilage has been recognized. Subchondral bone plays an important role in cartilage homeostasis. There are also reports that changes in subchondral bone affect cartilage degeneration. In actual OA patients, changes such as osteosclerosis and osteophyte formation of subchondral bone are seen as progression of OA. In the early stage of OA, bone hardening of the subchondral bone is observed first.

  In recent years, it has become clear that sensory nerves control bone metabolism. The neuropeptide calcitonin gene-related peptide (calcitonin gene-related peptide; CGRP) has been shown to play an important role in promoting osteoblast differentiation / proliferation and suppressing osteoclast differentiation. CGRP expression is increased in pain and has been shown to play an important role in OA pain.

  Non-Patent Document 1 discloses a study on pain in OA using an OA model rat administered a CGRP antibody. In addition, attempts have been reported to control the progression of OA by controlling the changes in subchondral bone in OA. For example, Non-Patent Document 2 discloses the results of a clinical test in which bisphosphonate, which is an osteoporosis treatment, was actually administered to a patient with OA.

Benschop RJ, 16 others, "Development of a novel antibody to calcitonin gene-related peptide for the treatment of osteoarthritis-related pain.", Osteoarthritis Cartilage, 2014, 22 (4), 578-585 Nishii T, 4 others, "Alendronate treatment for hip osteoarthritis: prospective randomized 2-year trial.", Clinical Rheumatology, 2013, 32 (12), 1759-1766

  However, in the above Non-Patent Document 1, the effect of CGRP antibody on pain in OA is evaluated, and the effect of CGRP antibody on bone metabolism and cartilage degeneration in OA is not evaluated. Therefore, a drug that can suppress cartilage degeneration of OA has not been found yet. Moreover, according to the above-mentioned nonpatent literature 2, as a result of a clinical test, bisphosphonate was not able to control progress of OA.

  If the progressive disease OA can be controlled by drugs at an early stage, surgical treatment is reduced, and not only benefits for patients but also medical economic effects are great. Therefore, there is an urgent need to develop a drug that can be administered safely and with low invasiveness to OA.

This invention is made in view of the said situation, and it aims at providing the cartilage degeneration inhibitor which can suppress joint destruction safely and with low invasion.

The inventor considered that the progress of cartilage degeneration and eventually joint degeneration could be suppressed if it was possible to prevent osteosclerosis in the early stage of OA, in particular the hardening of the subchondral bone, and conducted intensive studies. As a result, the present invention is completed. That is,
Cartilage degeneration inhibitor according to the perspective of the present invention,
Including Orsege Pant .

  According to the present invention, joint destruction can be suppressed safely and with low invasion.

It is a figure which shows the image of the safranin-O stained knee joint section of OA model mouse. It is a figure which shows the osteoarthritis score of OA model mouse. It is a figure which shows the ratio of the area of the subchondral bone of OA model mouse. FIG. 8 shows images of osteocalcin-stained knee joint sections of OA model mice and the percentage of osteocalcin-positive cells in bone marrow cells. It is a figure which shows the image of the knee joint section which stained tartaric acid resistant acid phosphatase (TRAP) of OA model mouse, and the number of TRAP positive cells per unit area. The ratio of the CGRP positive area to the image of the knee joint section labeled with CGRP of the OA model mouse and the total area of the visual field is shown. It is a figure which shows the image of the safranin-O-stained knee joint section of OA model mouse | mouth which administered BIBN4096. It is a figure which shows the osteoarthritis score of OA model mouse | mouth which administered BIBN4096. It is a figure which shows the image of the knee joint section which stained matrix metalloproteinase (MMP) 13 of OA model mouse | mouth which administered BIBN4096. It is a figure which shows the ratio of the MMP13 positive cell in the chondrocytes in OA model mouse | mouth which administered BIBN4096. It is a figure which shows the ratio of the area of the subchondral bone of OA model mouse | mouth which administered BIBN4096. FIG. 2 shows an image obtained by micro-computed tomography (μCT) and the ratio of the length of the medial end (A) to the length of the lateral end (B). FIG. 6 shows images of osteocalcin-stained knee joint sections of OA model mice administered BIBN4096 and the percentage of osteocalcin-positive cells in bone marrow cells. It is a figure which shows the image of the knee joint section which stained TRAP of OA model mouse which administered BIBN4096, and the number of TRAP positive cells per unit area. The expression level and ALP activity of osteocalcin in a cultured cell are shown. It is a figure which shows the image of the cultured cell which stained TRAP. It is a figure which shows the number of TRAP positive cells in a cultured cell. It is a figure which shows the image of the safranin-O-stained knee joint section of OA model mouse | mouth which administered risedronate.

An embodiment according to the present invention will be described.
Embodiment
The embodiment will be described in detail. The cartilage degeneration inhibitor according to the present embodiment contains a CGRP receptor antagonist. The CGRP receptor is a heterotrimeric protein composed of GPCR calcitonin receptor-like receptor (CLR), receptor activity modifying protein (RAMP) and intracellular receptor component protein (RCP). When CGRP, which is a 37-amino acid peptide, binds to the CGRP receptor, intracellular cAMP is increased, resulting in actions such as vasodilation.

  A CGRP receptor antagonist is any substance that antagonizes CGRP, inhibits the interaction between CGRP and the CGRP receptor, and blocks the signal from the CGRP receptor. CGRP receptor antagonists include any substance that directly or indirectly inhibits the interaction between CGRP and CGRP receptor, for example, nucleic acids such as synthetic products, natural products, peptides, antibodies and aptamers. As a CGRP receptor antagonist, for example, Orsegepanto (BIBN4096), Telkage panto (MK-0974), MS-694153 and the like can be mentioned. Alternatively, benzodiazepine CGRP receptor antagonists (see, for example, WO 2005/013894) may be used as a CGRP receptor antagonist.

  The CGRP receptor antagonist may be synthesized by a known method, or a commercially available one may be used, or a compound included in the compound library is selected as a compound having CGRP receptor antagonistic activity. It may be a compound. For selection of compounds having CGRP receptor antagonistic activity, known methods such as receptor binding assay using cells expressing human CGRP receptor can be used. Cells expressing the CGRP receptor can be produced by genetic engineering techniques. For example, after the membrane is isolated from the cells by a known method, and radioactive iodine labeled human CGRP and a test substance are incubated with the isolated membrane, human CGRP contained in the membrane fraction is quantified based on the radioactive iodine. Thus, compounds having CGRP receptor antagonistic activity can be selected.

  The CGRP receptor antagonist may be a substance that inhibits the interaction between CGRP and CGRP receptor by interacting with CGRP. Thus, a CGRP receptor antagonist may be, for example, a CGRP antibody such as ALD403 and LY2951742. An antibody against CGRP may be produced by a known antibody production method.

  The cartilage degeneration inhibitor according to the present embodiment contains the above CGRP receptor antagonist as an active ingredient. The said cartilage degeneration inhibitor is manufactured by a well-known method. The dosage form of the above cartilage degeneration inhibitor is not limited, but tablets, capsules, injections, solutions, rectal suppositories, vaginal suppositories, nasal absorbents, transdermal absorbents, pulmonary absorbents and buccal absorption Agents. The cartilage degeneration inhibitor may be, for example, a combination compounded with a pharmacologically acceptable carrier. The pharmacologically acceptable carrier is various organic carrier substances or inorganic carrier substances used as pharmaceutical materials. Further, the cartilage degeneration inhibitor may contain pharmacologically acceptable additives such as sucrose and a buffer. Additives such as preservatives, antioxidants, coloring agents, and sweeteners can also be used, if necessary.

  The cartilage degeneration inhibitor according to the present embodiment suppresses bone hardening and cartilage degeneration of subchondral bone as shown in Example 1 below. Therefore, the cartilage degeneration inhibitor is preferably administered to a patient with a cartilage degeneration disease. Although the administration method of the cartilage degeneration inhibitor is optional, it is preferably oral administration or intravenous injection. The dose of the cartilage degeneration inhibitor is appropriately adjusted according to the patient's body weight, condition of the patient such as the condition, etc., and the dose of the active ingredient CGRP receptor antagonist per day is 0. It is 1 mg to 1 g, or 1 to 100 mg, preferably 2.5 to 10 mg. Of course, the administration method such as the administration frequency and the administration interval can be appropriately adjusted while observing the condition of the patient.

  As described above in detail, the cartilage degeneration inhibitor according to the present embodiment suppresses bone hardening and cartilage degeneration of subchondral bone. There are no blood vessels in the cartilage, and efficient transport of the cartilage degeneration inhibitor to the affected area may be a problem. However, since there is a blood vessel in the subchondral bone, the cartilage degeneration inhibitor is effective in treating the subchondral bone via blood circulation. It can be reached and medicinal effects can be obtained efficiently. Therefore, the cartilage degeneration inhibitor is safe and can suppress joint destruction with low invasion.

  Further, the above-mentioned cartilage degeneration inhibitor may contain orsegepant as the above-mentioned CGRP receptor antagonist. It can be safely administered to patients because it is used in clinical trials as a treatment for migraine headaches.

  In another embodiment, provided is a CGRP receptor antagonist for use as a cartilage degeneration inhibitor. Another embodiment is the use of a CGRP receptor antagonist for the manufacture of a cartilage degeneration inhibitor. In yet another embodiment, a method of suppressing osteosclerosis of the subchondral bone comprising administering a CGRP receptor antagonist, or a method of inhibiting cartilage degeneration, comprising administering a CGRP receptor antagonist Is provided. In yet another embodiment, there is provided a method of treating a cartilage disease, comprising the step of administering a CGRP receptor antagonist to a patient with a cartilage disease.

  As shown in Example 1 below, a CGRP receptor antagonist inhibits bone hardening of subchondral bone. Subchondral bone osteosclerosis is seen with the progression of OA in actual OA patients, as described above. Therefore, the CGRP receptor antagonist may be contained as an active ingredient in the osteoarthritis inhibitor.

  Another embodiment provides a CGRP receptor antagonist for use as an osteoarthritis inhibitor. Another embodiment is the use of a CGRP receptor antagonist for the manufacture of an osteoarthritis inhibitor. In yet another embodiment, there is provided a method of treating osteoarthritis comprising administering a CGRP receptor antagonist to a patient with osteoarthritis.

  The present invention will be described more specifically by the following examples, but the present invention is not limited by the examples. In addition, all the results of this example are shown by mean value ± standard deviation. Comparison of 3 groups or 4 groups was performed by the Turkey-Kramer post hoc test. The Mann Whitney U test was used to detect differences between the two groups. A P value less than 0.05 was considered statistically significant.

Example 1 Examination of Cartilage Degeneration Inhibitory Effect of CGRP Receptor Antagonist
An OA model mouse was produced by the following procedure. First, OA surgery was performed in which the medial collateral ligament of the right knee joint of a 10-week-old C57 / BL6 mouse was dissected. To assess early changes in subchondral bone, mice undergoing OA surgery were euthanized after 2 and 7 days of excess anesthesia (n = 6 at each time point). Mice subjected to sham operation (sham) were similarly euthanized (n = 6).

  To investigate the effect of CGRP receptor antagonist on the development of OA, mice were injected intraperitoneally with orsegepant (BIBN4096, Tocris Bioscience) at a dose of 0.6 mg / kg / 100 μl after OA surgery. Given to The control group received phosphate buffered saline (PBS) at the same dose.

  Knee joints were collected one week after injection, four weeks and eight weeks later, and after fixation with 4% paraformaldehyde (PFA), μCT and histologic evaluation were performed. In μCT, after fixing for 24 hours in 4% PFA, the sample was analyzed using a high resolution μCT apparatus (SkyScan 1176, manufactured by Toyo Technica Co., Ltd.) according to the manual. The obtained images were reconstructed, and the lengths of the medial end and the lateral end were calculated for sham, the control group and the BIBN4096 administration group.

  The knee joint fixed with 4% PFA was decalcified with 20% ethylenediaminetetraacetic acid (EDTA) for 2 weeks and embedded in paraffin. A 4 μm thick sagittal section taken from the load bearing area in the middle of the medial compartment of the knee joint was prepared for histological evaluation. The sections were stained with the Safranin-O Fast Green method and pathological changes in the medial femoral condyle and tibial plateau were assessed based on the OARSI (Osteo Arthritis Research Society International) recommendation for the evaluation of knee cartilage in joint degeneration in mice .

  For each section, the subchondral bone area / tissue area (BT / TV,%) at the epiphysis of the inner plateau was measured using an image analyzer (BZ-9000, manufactured by Keyence Corporation). Other sections were used for immunohistochemistry and TRAP staining. For TRAP staining, a kit manufactured by Wako Pure Chemical Industries, Ltd. was used according to the manual. TRAP positive cells in the epiphysis of the subchondral bone were counted for each section.

  For immunohistochemical evaluation, first, paraffin-embedded knee joint sections were deparaffinized with xylene-substituted ro-Par Clearant (Anatech) and returned with serially diluted ethanol and water. For antigen unmasking, sections in 10 mM sodium citrate buffer (pH 6.0) were microwaved and held at 80-85 ° C. for 1.5 minutes. After antigen unmasking, the slides were cooled at room temperature for 20 minutes. After washing with PBS, sections were blocked for 30 minutes at room temperature with 5% serum.

  Subsequently, anti-CGRP antibody (50-fold dilution, manufactured by Abcam), anti-osteocalcin antibody (2000-fold dilution, manufactured by Takara Bio) and anti-MMP 13 antibody (50-fold dilution, manufactured by BD Bioscience Pharmingen) are prepared at 4 ° C. Incubate with sections overnight. After washing with PBS, sections were incubated with biotinylated goat anti-rabbit secondary antibody for 30 minutes at room temperature. As a secondary antibody against CGRP, Alexa Fluor 488-conjugated anti-rabbit IgG (Molecular Probes; Invitrogen) was used.

  Next, the sections were incubated for 30 minutes using Vectastain ABC-AP alkaline phosphatase (Vector Laboratories). Slides were washed and sections were incubated with alkaline phosphatase substrate for 20-30 minutes.

  Immunohistochemical signals were quantified as follows. For bone formation analysis, the total number of bone marrow cells in the epiphysis of subchondral bone was counted to calculate the percentage of osteocalcin positive cells. For CGRP expression, 5 fields of the epiphysis of the subchondral bone were randomly selected, and each CGRP positive area was measured under a 200-fold magnification using ImageJ software (National Institutes of Health). Here, the ratio of CGRP positive area to the total area of each visual field was calculated. For the expression of MMP13, four photographs of the section were taken under 400 × magnification, and for each photograph, the number of MMP13 positive cells in total chondrocytes found in articular cartilage of the section was counted.

(result)
FIG. 1 shows an image of a section prepared from the knee joint of an OA model mouse. In the OA model mice in which the medial collateral ligament was dissected, bone formation of the subchondral bone proceeded with the passage of 2 days and 7 days, and the decrease of the bone marrow cavity was observed. In addition, in the OA model mouse, cartilage degeneration progressed over time. In addition, the bar in the figure demonstrated below shows 100 micrometers in length.

  An OARSI score indicating the degree of OA is shown in FIG. 2 for the tibia and femur of the OA model mouse. In OA model mice, OA progressed statistically significantly in both tibia and femur over time. FIG. 3 shows the ratio of the area of subchondral bone to the area of tissue in OA model mice. The subchondral bone area was increased at day 7 as compared to the normal knee sham.

  FIG. 4 shows images of sections of knee joints of OA model mice stained with osteocalcin and ratios of osteocalcin positive cells to bone marrow cells in immunohistochemical evaluation. As OA progressed, the subchondral bone marrow cavity decreased. In addition, the proportion of osteocalcin positive cells, ie, osteoblasts, was significantly increased. FIG. 5 shows an image of a knee joint section of a TRAP-stained OA model mouse and the number of TRAP-positive cells per unit area in an immunohistochemical evaluation. On the second day, the number of TRAP-positive cells, ie osteoclasts, increased but decreased on the seventh day. FIG. 6 shows images of knee joint sections labeled with CGRP in subchondral bone and the ratio of CGRP positive area to the total area of each visual field. In the subchondral bone of OA model mice, the expression of CGRP increased with time.

  Images of knee joint sections when BIBN4096 is administered to OA model mice are shown in FIG. In the control group, bone hardening and cartilage degeneration of the subchondral bone progressed with 1, 4, 8 weeks over time. On the other hand, in the BIBN4096 administration group, the osteochondral bone was hardened in a mild degree and the progress of cartilage degeneration was also light. FIG. 8 shows the OARSI scores of the control group and the BIBN4096 administration group. In the BIBN4096 administration group, the OARSI score showed a significantly low value.

  The expression of the cartilage degeneration marker MMP13 in knee joint sections of OA model mice is shown in FIG. There was less expression of MMP13 in the BIBN4096 administration group as compared to the control group. As shown in FIG. 10, the percentage of MMP13 positive cells in chondrocytes in the BIBN4096 administration group was significantly smaller than in the control group. FIG. 11 shows the ratio of the area of subchondral bone to the area of tissue in the BIBN4096 administration group. The bone mass of the subchondral bone was shown to be significantly smaller at 1, 4 and 8 weeks as compared to the control group.

  The image obtained by analysis by μCT and the ratio of the length of the medial end to the length of the lateral end are shown in FIG. In the control group, bone hardening of the medial tibial condyle was observed, and the length of the medial epiphysis separated was short. On the other hand, in the BIBN4096 administration group, no bone hardening of the medial tibial condyle was observed, and the length of the medial end was equal to that of sham.

  FIG. 13 shows an image of a section of a knee joint stained with osteocalcin and the ratio of osteocalcin-positive cells to bone marrow cells in the BIBN4096-administered group. The percentage of osteoblasts in the BIBN4096 administration group was significantly smaller than that in the control group. FIG. 14 shows an image of the knee joint section of the BIBN4096-administered group stained with TRAP and the number of TRAP-positive cells per unit area. The number of osteoclasts in the BIBN4096 administration group was significantly increased as compared to the control group.

Example 2 Influence of BIBN 4096 on Bone Formation
Second and third passage human bone marrow mesenchymal stem cells (MSC) (manufactured by Life Technologies), MSC growth medium (StemPro MSC SFM) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Manufactured by Life Technologies). Passage 4 MSCs were seeded in 12-well plates to induce bone formation. An osteogenesis induction medium (StemPro osteogenesis differentiation kit, manufactured by Life Technologies) containing 10% FBS and 1% penicillin / streptomycin was changed twice a week and incubated for 21 days. In addition, bone formation induction medium was similarly induced by the bone formation induction medium further containing 100 nM CGRP (manufactured by Peptide Laboratories) and the bone formation induction medium further containing 100 nM CGRP and BIBN4096.

  After 21 days of incubation, RNA was isolated using Trizol (Life Technologies) for real-time polymerase chain reaction (PCR) analysis. Then, proteins were extracted for evaluation of alkaline phosphatase activity.

  For real-time PCR analysis, first, using a Superscript VLIO kit (manufactured by Invitrogen) according to the manual, complementary DNA was synthesized from 1 μg of total RNA. As a probe for real-time PCR, osteocalcin (Hs01587814_g1) and GADPH (Hs02758991_m1) of Taqman Gene Expression Assays probes (manufactured by Life Technologies) were used. The expression of each gene was assessed against the expression of GADPH. The ΔΔCt method was used for data analysis of real-time PCR.

  For evaluation of alkaline phosphatase (ALP) activity, a bone formation marker, cell lysates were collected by treatment with 0.1% Triton X, and centrifuged at 14,000 rpm for 15 minutes. The supernatant was evaluated using ALP assay kit (manufactured by Wako Pure Chemical Industries, Ltd.) according to the manual. The absorbance at 405 nm was measured for each sample on a microplate spectrometer.

(result)
FIG. 15 shows the expression level and ALP activity of osteocalcin. In the presence of CGRP, osteocalcin expression was increased by induction of bone formation. On the other hand, the addition of BIBN4096 significantly reduced the expression level of osteocalcin. Similarly, ALP activity increased in the presence of CGRP. On the other hand, addition of BIBN4096 significantly reduced ALP activity.

Example 3 Effect of BIBN 4096 on Osteoclastogenesis
Raw 264.7 cells were cultured in α-minimal essential medium containing 10% FBS and 1% penicillin / streptomycin in 12-well plates. In order to evaluate osteoclastogenesis, the medium is changed every 3 days to a medium containing 50 ng / mL M-CSF (manufactured by Wako Pure Chemical Industries, Ltd.) and 50 ng / mL RANKL (manufactured by Wako Pure Chemical Industries), Culture was performed for one week. The medium was also cultured in the same manner as in the medium further containing 100 nM CGRP, and the medium further containing 100 nM CGRP and BIBN4096. On the seventh day, TRAP of cells was stained using the above kit manufactured by Wako Pure Chemical Industries, Ltd. TRAP positive multinucleated cells having 4 or more nuclei were identified as osteoclasts. Osteoclasts in 10 fields of the microscope were counted in each well under microscopic observation at 200 × magnification. All experiments were performed at least three times in triplicate.

(result)
An image of TRAP-stained cells is shown in FIG. The TRAP positive cells confirmed by the induction of differentiation to osteoclasts were reduced by adding CGRP. On the other hand, addition of BIBN4096 increased TRAP-positive cells. FIG. 17 shows the number of TRAP-positive cells (osteoclasts). Addition of BIBN4096 significantly increased the number of TRAP-positive cells.

  From the above results, in early OA, it was shown that hardening of subchondral bone associated with cartilage degeneration and increased expression of CGRP. Furthermore, it has been demonstrated that CGRP receptor antagonists inhibit this subchondral bone osteosclerosis and cartilage degeneration and inhibit OA.

(Comparative example)
In the above OA model mice, risedronate, which is one of bisphosphonates, was intravenously administered at a dose of 0.06 mg / kg / 100 μl after OA surgery. One week and four weeks after administration, the knee joint was recovered and histologically evaluated in the same manner as in Example 1 above.

(result)
FIG. 18 shows images of sections of a knee joint stained with Safranin-O Fast Green 1 week and 4 weeks after risedronate administration. Progression of osteosclerosis and cartilage degeneration of the subchondral bone is observed as compared with 1 week and 4 weeks of the BIBN4096 administration group of FIG. 7. Bisphosphonates are considered to be incapable of suppressing osteosclerosis and cartilage degeneration of the subchondral bone because they suppress the function of osteoclasts.

  The embodiments described above are for explaining the present invention, and do not limit the scope of the present invention. That is, the scope of the present invention is indicated not by the embodiments but by the claims. And, various modifications made within the scope of the claims and the meaning of the invention are considered to be within the scope of the present invention.

The present invention is suitable for pharmaceuticals for inhibiting cartilage degeneration. Cartilage degeneration inhibitor according to the present invention reduces the surgical therapy for advanced cases of OA, suppression of motor dysfunction, which contributes to the improvement of improvement and quality of life of patients motor function.

Claims (1)

Including Orsege Pant ,
Cartilage degeneration inhibitor.