JP7562066B2 - Preventive or therapeutic agent for osteoarthritis, and pharmaceutical composition for preventing or treating osteoarthritis - Google Patents
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Description
特許法第３０条第２項適用 ２０１９年３月２５日に日本大学リポジトリのウェブサイト＜ＵＲＬ：ｈｔｔｐ：／／ｒｅｐｏｓｉｔｏｒｙ．ｎｉｈｏｎ－ｕ．ａｃ．ｊｐ／ｘｍｌｕｉ／ｈａｎｄｌｅ／１１２６３／１５３０＞に公表Article 30, paragraph 2 of the Patent Act applied. Published on the Nihon University Repository website on March 25, 2019 at URL: http://repository.nihon-u.ac.jp/xmlui/handle/11263/1530.
本発明は、変形性関節症の予防又は治療剤、及び変形性関節症の予防又は治療用医薬組成物に関する。 The present invention relates to a preventive or therapeutic agent for osteoarthritis, and a pharmaceutical composition for preventing or treating osteoarthritis.
変形性関節症（Ｏｓｔｅｏａｒｔｈｒｉｔｉｓ；ＯＡ）は、軟骨が変性及び摩耗する病気である。ＯＡの中でも変形性膝関節症（以下、「膝ＯＡ」と略記する場合がある）は、膝関節の軟骨の摩耗及び消失を特徴とし、主に加齢に伴って発症する疾患であり、日本には２４００万人の患者がいると推定されている。軟骨変性が重度な症例では手術（人工膝関節置換術等）が行われるが、発症初期から中期では、消炎鎮痛剤やヒアルロン酸注射等の対症療法が主となっている。近年、発症初期から中期の膝ＯＡに対して、患者の血液から調製される多血小板血漿（ＰＲＰ）や脂肪組織等から調製される間葉系幹細胞（ＭＳＣ）を関節内注射する治療法（例えば、非特許文献１参照）が保険外診療として行われており、一定の有効性が示されている。 Osteoarthritis (OA) is a disease in which cartilage degenerates and wears away. Among OA, knee osteoarthritis (hereinafter sometimes abbreviated as "knee OA") is characterized by wear and loss of cartilage in the knee joint, and is a disease that mainly develops with aging, with an estimated 24 million patients in Japan. In cases where cartilage degeneration is severe, surgery (such as total knee replacement surgery) is performed, but in the early to mid-stages of onset, symptomatic treatments such as anti-inflammatory analgesics and hyaluronic acid injections are the norm. In recent years, for early to mid-stage knee OA, a treatment method in which platelet-rich plasma (PRP) prepared from the patient's blood or mesenchymal stem cells (MSC) prepared from adipose tissue, etc. are injected into the joint (see, for example, Non-Patent Document 1) has been performed as an out-of-pocket medical treatment, and a certain degree of effectiveness has been shown.
一方、発明者らは、これまで、脂肪細胞から脱分化脂肪細胞（ＤＦＡＴ）を簡便且つ大量に作製する方法を開発している（例えば、特許文献１参照）。得られたＤＦＡＴが歯周組織の再生（例えば、特許文献２参照）や、真皮の再建（例えば、特許文献３参照）に有用であることが確かめられている。 Meanwhile, the inventors have developed a method for easily and mass-producing dedifferentiated fat cells (DFAT) from adipocytes (see, for example, Patent Document 1). The obtained DFAT has been confirmed to be useful for the regeneration of periodontal tissue (see, for example, Patent Document 2) and reconstruction of the dermis (see, for example, Patent Document 3).
上述したとおり、膝ＯＡの治療法は対症療法が主となっている。ＰＲＰを用いた治療法では、異なった採取方法による調製キットが多数存在し、成分も雑多であり、治療効果も安定していない。ＭＳＣを用いる治療法では、採取時の侵襲が大きく、増殖速度が遅く、さらに採取後の細胞には、間葉系幹細胞以外の細胞も多く混在している。そのため、短期間で純度の高い細胞を大量に調製することが困難である。
さらに、膝ＯＡのような慢性疾患では、長期に亘り継続的な治療により効果を維持することが求められる。しかしながら、ＰＲＰやＭＳＣは１回の採取で、通常１回の治療しかできない。そのため、長期に亘り継続的な治療により効果を維持できる新規の治療方法が求められている。
As mentioned above, the treatment of knee OA is mainly symptomatic treatment. In the treatment using PRP, there are many preparation kits with different collection methods, the components are diverse, and the treatment effect is not stable. In the treatment using MSC, the collection is highly invasive, the proliferation rate is slow, and many cells other than mesenchymal stem cells are mixed in the cells after collection. Therefore, it is difficult to prepare a large amount of highly pure cells in a short period of time.
Furthermore, in chronic diseases such as knee OA, it is necessary to maintain the effect by continuous treatment over a long period of time. However, PRP and MSCs can only be collected once and usually only one treatment can be performed. Therefore, a new treatment method that can maintain the effect by continuous treatment over a long period of time is required.
本発明は、上記事情に鑑みてなされたものであって、新規の変形性関節症の予防又は治療剤、及び変形性関節症の予防又は治療用医薬組成物を提供する。 The present invention has been made in consideration of the above circumstances, and provides a novel agent for preventing or treating osteoarthritis, and a pharmaceutical composition for preventing or treating osteoarthritis.
本発明者らは、上記目的を達成すべく鋭意研究を重ねた結果、ＤＦＡＴ細胞を膝ＯＡモデルラットに投与することで、優れた治療効果を発揮することを見出し、本発明を完成するに至った。 As a result of intensive research conducted by the inventors to achieve the above-mentioned objective, they discovered that administering DFAT cells to a rat model of knee OA exhibited excellent therapeutic effects, leading to the completion of the present invention.
すなわち、本発明は、以下の態様を含む。
本発明の第１態様に係る変形性関節症の予防又は治療剤は、脱分化脂肪細胞又はその培養上清を有効成分として含有する。
前記脱分化脂肪細胞が変形性関節症患者又は患畜由来の自家細胞であってもよい。
変形性関節症が変形性膝関節症であってもよい。
That is, the present invention includes the following aspects.
The preventive or therapeutic agent for osteoarthritis according to the first aspect of the present invention contains dedifferentiated adipocytes or a culture supernatant thereof as an active ingredient.
The dedifferentiated adipocytes may be autologous cells derived from a patient or animal suffering from osteoarthritis.
The osteoarthritis may be knee osteoarthritis.
本発明の第２態様に係る変形性関節症の予防又は治療用医薬組成物は、上記第１態様に係る変形性関節症の予防又は治療剤、及び薬学的に許容可能な担体を含む。 The pharmaceutical composition for preventing or treating osteoarthritis according to the second aspect of the present invention comprises the agent for preventing or treating osteoarthritis according to the first aspect and a pharma- ceutical acceptable carrier.
上記態様の変形性関節症の予防又は治療剤、及び変形性関節症の予防又は治療用医薬組成物によれば、ＯＡの進行を抑制することができる。 The above-mentioned preventive or therapeutic agent for osteoarthritis and pharmaceutical composition for preventing or treating osteoarthritis can inhibit the progression of OA.
≪変形性関節症≫
一般に、ＯＡ（Ｏｓｔｅｏａｒｔｈｒｉｔｉｓ；変形性関節症）は、全身のあらゆる関節に起こり得る疾患である。ＯＡを引き起こす関節として具体的には、膝関節、股関節、脊椎等が挙げられ、それぞれ、変形性膝関節症（膝ＯＡ）、変形性股関節症、変形性脊椎症と呼ばれる。中でも、本実施形態のＯＡの予防又は治療剤は、変形性膝関節症（膝ＯＡ）に対して特に好適に用いられる。
<Osteoarthritis>
In general, OA (Osteoarthritis) is a disease that can occur in any joint in the body. Specific examples of joints that cause OA include the knee joint, hip joint, and spine, which are called knee osteoarthritis (knee OA), hip osteoarthritis, and spinal osteoarthritis, respectively. Among them, the preventive or therapeutic agent for OA of the present embodiment is particularly suitable for use in knee osteoarthritis (knee OA).
≪変形性関節症の予防又は治療剤≫
本実施形態の変形性関節症（ＯＡ）の予防又は治療剤は、ＤＦＡＴ細胞又はその培養上清を有効成分として含有する。
なお、ここでいう「ＤＦＡＴ細胞を有効成分として含有する」とは、ＤＦＡＴ細胞又はその培養上清をＯＡの予防又は治療効果を奏する成分として含有することを意味する。
<Preventive or therapeutic agent for osteoarthritis>
The preventive or therapeutic agent for osteoarthritis (OA) of this embodiment contains DFAT cells or a culture supernatant thereof as an active ingredient.
As used herein, "containing DFAT cells as an active ingredient" means containing DFAT cells or their culture supernatant as an ingredient that exerts a preventive or therapeutic effect on OA.
現在、ＯＡの治療への臨床応用が検討されているＭＳＣは、ＥＳ細胞と比べて、腫瘍形成能が低いと考えられており、骨髄、羊水、臍帯血及び脂肪組織等から採取することができる。しかしながら、骨髄からのＭＳＣの採取は侵襲性が高く、ＭＳＣは骨髄中の全有核細胞数に対して０．００１％以上０．０１％以下程度の極微量しか含まれず、加齢とともに増殖力が低下するため、採取対象となるドナーの年齢に増殖力は依存する。羊水からのＭＳＣの採取は、侵襲性は低いが、ＭＳＣは羊水中の全有核細胞数に対して１％以上２%以下程度の微量しか含まれず、増殖が遅い。脂肪組織からのＭＳＣの採取は、侵襲性は低いが、ＭＳＣは間質血管分画中の全有核細胞数に対して０．３％以上４％以下程度の微量しか含まれないため、細胞治療に必要な１０８個程度の細胞数を得るためには、通常５０ｍＬ以上３００ｍＬ以下の脂肪組織の採取が必要となる。また、脂肪組織のＭＳＣは、骨髄及び羊水中のＭＳＣに対して増殖力は高いが、加齢とともに増殖力が低下するため、採取対象となるドナーの年齢に増殖力は依存する。さらに、細胞組織からＭＳＣを採取する際に、ＭＳＣ以外の細胞を多く含むため、継代培養を複数回繰り返して純化する必要があり、純化に時間を要する。
これに対して、ＤＦＡＴ細胞は、脂肪組織の約３０％を占める成熟脂肪細胞を原料としているため、１０ｍＬ程度の少量の脂肪組織から細胞数１０８個程度の細胞治療に十分なＤＦＡＴ細胞が得られる。従って、採取に伴う侵襲性は、脂肪組織からＭＳＣを採取するより低い。また、脂肪組織から成熟脂肪細胞を単離する際に、その他の細胞が混入しにくいため、培養早期より純度の高いＤＦＡＴ細胞が得られる。さらに、ＤＦＡＴ細胞の増殖力はＭＳＣと同等であるが、その増殖力や多分化能はＭＳＣと異なり採取対象となるドナーの年齢や基礎疾患の影響を受けずに保たれる。
また、後述する実施例に記載のとおり、ＤＦＡＴ細胞を膝ＯＡモデルラットに関節内投与することで、軟骨変性抑制効果が示されており、ＤＦＡＴ細胞の投与はＯＡの治療効果を期待できる。
MSCs, which are currently being considered for clinical application in the treatment of OA, are considered to have a lower tumorigenicity than ES cells, and can be collected from bone marrow, amniotic fluid, umbilical cord blood, adipose tissue, etc. However, collection of MSCs from bone marrow is highly invasive, and MSCs are contained in extremely small amounts of about 0.001% to 0.01% of the total number of nucleated cells in bone marrow, and their proliferation ability decreases with age, so their proliferation ability depends on the age of the donor from whom they are collected. Collection of MSCs from amniotic fluid is less invasive, but MSCs are contained in only trace amounts of about 1% to 2% of the total number of nucleated cells in amniotic fluid, and they proliferate slowly. Collection of MSCs from adipose tissue is less invasive, but MSCs are contained in only trace amounts of about 0.3% to 4% of the total number of nucleated cells in the interstitial vascular fraction, so in order to obtain about 10 8 cells required for cell therapy, it is usually necessary to collect 50 mL to 300 mL of adipose tissue. In addition, MSCs in adipose tissue have a higher proliferation rate than MSCs in bone marrow and amniotic fluid, but the proliferation rate decreases with age, so the proliferation rate depends on the age of the donor from whom the MSCs are collected. Furthermore, when MSCs are collected from cell tissue, they contain many cells other than MSCs, so they need to be purified by repeating subculture multiple times, which takes time.
In contrast, DFAT cells are made from mature adipocytes, which account for approximately 30% of adipose tissue, and therefore, DFAT cells sufficient for cell therapy, with a cell number of approximately 108 , can be obtained from a small amount of adipose tissue, approximately 10 mL. Therefore, the invasiveness associated with collection is lower than that of MSC collection from adipose tissue. In addition, when isolating mature adipocytes from adipose tissue, other cells are less likely to be mixed in, so that highly pure DFAT cells can be obtained from the early stage of culture. Furthermore, the proliferation ability of DFAT cells is equivalent to that of MSCs, but unlike MSCs, their proliferation ability and multipotency are maintained without being affected by the age or underlying disease of the donor from whom they are collected.
Furthermore, as described in the Examples below, intra-articular administration of DFAT cells into knee OA model rats has been shown to have an inhibitory effect on cartilage degeneration, and administration of DFAT cells is expected to be effective in treating OA.
ＤＦＡＴ細胞は、ＯＡ患者又は患畜由来の自家細胞であってもよく、当該患者又は患畜以外のドナー由来の他家細胞であってもよい。当該患者又は患畜以外のドナーとしては、ＯＡ患者又は患畜と同種の動物であればよく、年齢及び性別は問わない。
ＯＡの治療にＯＡ患者又は患畜由来の自家細胞を用いる場合、ＤＦＡＴ細胞は、上述のとおり増殖力が高いことから、少量の成熟脂肪細胞から大量に自家細胞のＤＦＡＴ細胞を調製することができ、１回の組織採取から複数回（例えば、５回程度）の治療に必要な細胞数を得ることができる。また、自家細胞であるため、免疫拒絶を受けるリスクがない。
また、当該患者又は患畜以外のドナー由来の他家細胞である場合、免疫拒絶により速やかに排除される可能性があるが、他家細胞であるＤＦＡＴ細胞が炎症部において抗炎症作用を発現することができるため、十分に治療効果が期待できる。また、他家細胞である場合、外科手術時に廃棄される脂肪組織から採取された成熟脂肪細胞を用いて調製することができ、侵襲度が低い。さらに、これら廃棄される脂肪組織から採取された成熟脂肪細胞を用いて調製されたＤＦＡＴ細胞からなるバンキングシステムを容易に構築することができ、このバンキングシステムを利用することで、患者又は患畜に適したＤＦＡＴ細胞を適宜選択して用いることもできる。
The DFAT cells may be autologous cells derived from an OA patient or animal patient, or allogeneic cells derived from a donor other than the patient or animal patient. The donor other than the patient or animal patient may be an animal of the same species as the OA patient or animal patient, regardless of age or sex.
When autologous cells derived from OA patients or animals are used for the treatment of OA, since DFAT cells have high proliferation ability as described above, a large amount of autologous DFAT cells can be prepared from a small amount of mature adipocytes, and the number of cells required for multiple treatments (for example, about 5 times) can be obtained from a single tissue collection. In addition, since the cells are autologous, there is no risk of immune rejection.
In addition, allogeneic cells derived from donors other than the patient or animal may be quickly eliminated due to immune rejection, but DFAT cells, which are allogeneic cells, can exhibit anti-inflammatory effects at the site of inflammation, so sufficient therapeutic effects can be expected. In addition, allogeneic cells can be prepared using mature adipocytes collected from adipose tissue discarded during surgery, and are less invasive. Furthermore, a banking system consisting of DFAT cells prepared using mature adipocytes collected from such discarded adipose tissue can be easily constructed, and by using this banking system, DFAT cells suitable for the patient or animal can be appropriately selected and used.
ＤＦＡＴ細胞は、公知の方法、例えば、特開２０００－８３６５６号公報（参考文献１）に記載の方法を用いて、動物の脂肪組織から採取された成熟脂肪細胞から調製することができる。具体的には、まず、脂肪組織からコラゲナーゼ処理等により成熟脂肪細胞を単離する。次いで、単離された成熟脂肪細胞を天井培養法によって培養することでＤＦＡＴ細胞が得られる。調製されたＤＦＡＴ細胞は、継代培養を少なくとも１回した後、すぐに使用することができる。 DFAT cells can be prepared from mature adipocytes collected from animal adipose tissue using known methods, for example, the method described in JP 2000-83656 A (Reference 1). Specifically, mature adipocytes are first isolated from the adipose tissue by collagenase treatment or the like. DFAT cells are then obtained by culturing the isolated mature adipocytes using the ceiling culture method. The prepared DFAT cells can be used immediately after at least one subculture.
ＤＦＡＴ細胞は、ＣＤ３１陰性、ＣＤ４５陰性及びＨＬＡ－ＤＲ陰性である。ＣＤ３１、ＣＤ４５及びＨＬＡ－ＤＲは、細胞表面抗原であり、例えばフローサイトメトリー、細胞染色等により検出することができる。そのため、これら細胞表面抗原を検出することで、ＤＦＡＴ細胞の純度を確認することができる。具体的には、例えば、蛍光標識抗体を用いるフローサイトメトリーでは、ネガティブコントロール（アイソタイプコントロール）と比較してより強い蛍光を発する細胞が検出された場合、当該細胞は当該細胞表面抗原について「陽性」と判定される。蛍光標識抗体は、当該技術分野において公知の任意の抗体を使用することができ、例えば、イソチオシアン酸フルオレセイン（ＦＩＴＣ）、フィコエリスリン（ＰＥ）、アロフィコシアニン（ＡＰＣ）等により標識された抗体が挙げられるが、これらに限定されない。細胞染色において、着色する又は蛍光を発する細胞が顕微鏡下にて観察された場合、当該細胞は当該細胞表面抗原について「陽性」と判定される。細胞染色は、抗体を使用する免疫細胞染色であってもよく、抗体を使用しない非免疫細胞染色であってもよい。
上記細胞表面抗原を検出するタイミングとしては、特別な限定はなく、例えば、成熟脂肪細胞からＤＦＡＴ細胞が調製された直後、継代培養中、製剤化する前等が挙げられる。
DFAT cells are CD31 negative, CD45 negative and HLA-DR negative. CD31, CD45 and HLA-DR are cell surface antigens and can be detected by, for example, flow cytometry, cell staining and the like. Therefore, the purity of DFAT cells can be confirmed by detecting these cell surface antigens. Specifically, for example, in flow cytometry using a fluorescently labeled antibody, when a cell is detected that emits stronger fluorescence compared to a negative control (isotype control), the cell is judged to be "positive" for the cell surface antigen. The fluorescently labeled antibody can be any antibody known in the art, and examples include, but are not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC) and the like. In cell staining, when a colored or fluorescent cell is observed under a microscope, the cell is judged to be "positive" for the cell surface antigen. Cell staining may be immune cell staining using an antibody, or may be non-immune cell staining using no antibody.
The timing for detecting the cell surface antigen is not particularly limited, and examples include immediately after DFAT cells are prepared from mature adipocytes, during subculture, and before formulation.
本実施形態のＯＡの予防又は治療剤は、ＤＦＡＴ細胞の代わりに、ＤＦＡＴ細胞の培養上清を含んでいてもよい。後述する実施例に示すように、軟骨変性抑制効果は、炎症性サイトカイン存在下でＤＦＡＴ細胞から産生される因子によって、引き起こされるものであると考えられる。そのため、例えば、炎症性サイトカイン（例えば、ＴＮＦ－α、ＩＦＮ－γ等）存在下で培養したＤＦＡＴ細胞の培養上清を、本実施形態のＯＡの予防又は治療剤の有効成分として使用することができる。医療製剤として用いる観点から、ＤＦＡＴ細胞の培養上清は、培養液成分を実質的に含まないことが好ましい。 The preventive or therapeutic agent for OA of this embodiment may contain a culture supernatant of DFAT cells instead of DFAT cells. As shown in the Examples described below, it is believed that the cartilage degeneration inhibitory effect is caused by factors produced by DFAT cells in the presence of inflammatory cytokines. Therefore, for example, a culture supernatant of DFAT cells cultured in the presence of inflammatory cytokines (e.g., TNF-α, IFN-γ, etc.) can be used as an active ingredient of the preventive or therapeutic agent for OA of this embodiment. From the viewpoint of use as a medical preparation, it is preferable that the culture supernatant of DFAT cells does not substantially contain culture medium components.
≪ＯＡの予防又は治療用医薬組成物≫
本実施形態のＯＡの予防又は治療用医薬組成物（以下、「本実施形態の医薬組成物」と略記する場合がある）は、上記ＯＡの予防又は治療剤、及び薬学的に許容可能な担体を含む。本実施形態の医薬組成物を投与することにより、ＯＡを予防又は治療することができる。
<Pharmaceutical composition for preventing or treating OA>
The pharmaceutical composition for preventing or treating OA of this embodiment (hereinafter sometimes abbreviated as "pharmaceutical composition of this embodiment") contains the above-mentioned agent for preventing or treating OA and a pharma- ceutical acceptable carrier. OA can be prevented or treated by administering the pharmaceutical composition of this embodiment.
本実施形態の医薬組成物は、公知の方法を用いて適宜製剤とすることができ、一般的な細胞製剤が含む成分を配合することができる。具体的には、本実施形態の医薬組成物の製剤においては、製剤上の必要に応じて、適宜の薬学的に許容される担体、例えば、賦形剤、結合剤、溶剤、溶解補助剤、懸濁化剤、乳化剤、等張化剤、緩衝剤、安定化剤、無痛化剤、防腐剤、抗酸化剤、着色剤、滑沢剤、崩壊剤、湿潤剤、吸着剤、甘味剤、希釈剤等の任意成分を配合することができる。
投与経路は、皮下投与、経皮投与、筋肉内投与、血管内投与、関節内投与等の非経口投与経路が好ましく、関節内投与が特に好ましい。
The pharmaceutical composition of the present embodiment can be appropriately formulated using a known method, and can be formulated with components contained in general cell preparations. Specifically, in the formulation of the pharmaceutical composition of the present embodiment, any optional components such as appropriate pharma- ceutical acceptable carriers, for example, excipients, binders, solvents, solubilizers, suspending agents, emulsifiers, isotonicity agents, buffers, stabilizers, soothing agents, preservatives, antioxidants, colorants, lubricants, disintegrants, wetting agents, adsorbents, sweeteners, diluents, etc., can be formulated according to the needs of the formulation.
The administration route is preferably a parenteral route such as subcutaneous administration, transdermal administration, intramuscular administration, intravascular administration, or intraarticular administration, with intraarticular administration being particularly preferred.
本実施形態の医薬組成物の投与量としては、ＯＡの重症度や、剤型、投与対象の体重等によって変わり得るが、ＤＦＡＴ細胞を、例えば、１回当たり、１．０×１０4個／ｋｇ体重以上１．０×１０9個／ｋｇ体重以下の範囲で投与することができる。また、本実施形態の医薬組成物の投与は、単回投与でもよく、複数回投与であってもよい。複数回投与である場合は、例えば、２時間以上１２時間以下の期間毎、毎日、又は２日、１週間、数週間、１か月若しくは数か月に１回等の頻度で投与することができる。 The dosage of the pharmaceutical composition of this embodiment may vary depending on the severity of OA, the dosage form, the body weight of the subject, etc., but the DFAT cells can be administered in a range of 1.0 x 104 cells/kg body weight or more and 1.0 x 109 cells/kg body weight or less per administration. The pharmaceutical composition of this embodiment may be administered once or multiple times. When administered multiple times, it can be administered, for example, every 2 hours to 12 hours, every day, or once every 2 days, 1 week, several weeks, 1 month, or several months.
本実施形態の医薬組成物の投与対象としては、哺乳動物であることが好ましい。哺乳動物としては、特別な限定はないが、例えば、ヒト、サル、イヌ、ネコ、ニワトリ、ウサギ、ブタ、ウシ、ヤギ、ヒツジ、マウス、ラット、モルモット、ハムスター、ウマ等が挙げられる。中でも、哺乳動物としては、ヒトが好ましい。 The subject of administration of the pharmaceutical composition of this embodiment is preferably a mammal. There are no particular limitations on the mammal, but examples include humans, monkeys, dogs, cats, chickens, rabbits, pigs, cows, goats, sheep, mice, rats, guinea pigs, hamsters, and horses. Of these, humans are preferred as mammals.
また、一実施形態において、本発明は、ＯＡの予防又は治療用医薬組成物の製造のための、ＤＦＡＴ細胞又はその培養上清の使用を提供することができる。
また、一実施形態において、本発明は、ＯＡの予防又は治療のための、ＤＦＡＴ細胞又はその培養上清の使用を提供することができる。
また、一実施形態において、本発明は、ＯＡ患者又は患畜に投与して、軟骨変性の抑制のために使用される、ＤＦＡＴ細胞又はその培養上清を提供することができる。
また、一実施形態において、本発明は、ＤＦＡＴ細胞又はその培養上清の治療有効量をＯＡ患者又は患畜に投与する、ＯＡの治療方法を提供することができる。
In addition, in one embodiment, the present invention can provide use of DFAT cells or a culture supernatant thereof for the manufacture of a pharmaceutical composition for the prevention or treatment of OA.
In addition, in one embodiment, the present invention can provide use of DFAT cells or a culture supernatant thereof for the prevention or treatment of OA.
In addition, in one embodiment, the present invention can provide DFAT cells or a culture supernatant thereof, which is administered to an OA patient or animal patient to suppress cartilage degeneration.
In addition, in one embodiment, the present invention can provide a method for treating OA, comprising administering a therapeutically effective amount of DFAT cells or a culture supernatant thereof to an OA patient or animal patient.
以下、実施例により本発明を説明するが、本発明は以下の実施例に限定されるものではない。 The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.
［実施例１］
１．ラットＤＦＡＴ細胞の調製
ラットＤＦＡＴ細胞は、特開２０００－８３６５６号公報（参考文献１）に記載の方法を用いて、ラット脂肪組織から採取された成熟脂肪細胞から、予め調製した。
[Example 1]
1. Preparation of Rat DFAT Cells Rat DFAT cells were prepared in advance from mature adipocytes collected from rat adipose tissue, using the method described in JP-A-2000-83656 (Reference 1).
２．膝ＯＡモデルラットの作製及びＤＦＡＴ細胞の投与
膝ＯＡモデルラットは、１０～１２週齢のＷｉｓｔａｒラット（雄性）に前十字靭帯（ＡＣＬ）切離及び内側半月板（ＭＭ）切除（以下、「ＡＣＬＴ＋ＭＭｘ処置」と略記する場合がある。）により作製した（図１参照）。ＡＣＬＴ＋ＭＭｘ処置1週間後に、ラットＤＦＡＴ細胞（ＤＦＡＴ投与群）又はＰＢＳ（Ｃｏｎｔｒｏｌ群）を１週間毎に４回関節内投与した（各群ｎ＝１０、図２及び３参照）。処置５週間後に、両群の膝関節軟骨の変性を肉眼的及び組織学的に評価した。
2. Creation of knee OA model rats and administration of DFAT cells Knee OA model rats were created by anterior cruciate ligament (ACL) transection and medial meniscus (MM) resection (hereinafter sometimes abbreviated as "ACLT + MMx treatment") in 10-12 week old Wistar rats (male) (see Figure 1). One week after ACLT + MMx treatment, rat DFAT cells (DFAT administration group) or PBS (control group) were administered intra-articularly four times every week (n = 10 per group, see Figures 2 and 3). Five weeks after treatment, degeneration of knee articular cartilage in both groups was evaluated macroscopically and histologically.
３．肉眼的評価
肉眼的評価では、両群の膝関節をＩｎｄｉａ Ｉｎｋ染色により評価した。結果を図４に示す。図４において、破線部分は、軟骨変性範囲を示す。
図４に示すように、ＤＦＡＴ群のほうがＣｏｎｔｒｏｌ群に比べて、大腿骨顆部及び脛骨近位部の関節軟骨の変性範囲が少ない傾向にあった。
3. Macroscopic Evaluation In the macroscopic evaluation, the knee joints of both groups were evaluated by India Ink staining. The results are shown in Figure 4. In Figure 4, the dashed line indicates the area of cartilage degeneration.
As shown in FIG. 4, the DFAT group tended to have a smaller range of degeneration of the articular cartilage in the femoral condyle and proximal tibia than the control group.
４．組織学的評価
組織学的評価では、両群の膝関節に対してサフラニンＯ染色を行い、Ｍａｎｋｉｎ’ｓ ｓｃｏｒｅ及びＯＡＲＳＩ ｓｃｏｒｅを用いて定量評価を行なった。Ｍａｎｋｉｎ’ｓ ｓｃｏｒｅにおける評価基準を表１に、ＯＡＲＳＩ ｓｃｏｒｅにおける評価基準を表２に示す。なお、ＯＡＲＳＩ ｓｃｏｒｅは「Ｓｃｏｒｅ＝Ｇｒａｄｅ×Ｓｔａｇｅ」で示され、Ｇｒａｄｅ及びＳｔａｇｅの各評価基準を表３に示す。両群の大腿骨遠位関節面の染色像を図５に、両群の大腿骨遠位関節面の組織学的スコアを図６Ａ及び図６Ｂに示す。また、両群の脛骨近位関節面の染色像を図７に、両群の脛骨近位関節面の組織学的スコアを図８Ａ及び図８Ｂに示す。
4. Histological evaluation In the histological evaluation, the knee joints of both groups were stained with Safranin O, and quantitative evaluation was performed using Mankin's score and OARSI score. The evaluation criteria for Mankin's score are shown in Table 1, and the evaluation criteria for OARSI score are shown in Table 2. The OARSI score is shown as "Score = Grade x Stage", and the evaluation criteria for Grade and Stage are shown in Table 3. The stained images of the distal femoral articular surface of both groups are shown in Figure 5, and the histological scores of the distal femoral articular surface of both groups are shown in Figures 6A and 6B. In addition, the stained images of the proximal tibial articular surface of both groups are shown in Figure 7, and the histological scores of the proximal tibial articular surface of both groups are shown in Figures 8A and 8B.
図５～図６Ｂに示すように、大腿骨遠位関節面の組織学検討から、Ｃｏｎｔｒｏｌ群では、硝子軟骨変性による菲薄化と軟骨表面の不整及び裂隙が認められた。一方、ＤＦＡＴ群では、関節軟骨の変性の程度が軽い傾向にあった。軟骨変性の組織学的スコアであるＭａｎｋｉｎ’ｓ ｓｃｏｒｅ及びＯＡＲＳＩ ｓｃｏｒｅは、Ｃｏｎｔｒｏｌ群に比べてＤＦＡＴ群で有意に低値を示した。 As shown in Figures 5 to 6B, histological examination of the distal femoral articular surface revealed thinning due to hyaline cartilage degeneration, as well as irregularities and cracks in the cartilage surface in the control group. On the other hand, the degree of degeneration of the articular cartilage tended to be milder in the DFAT group. The Mankin's score and OARSI score, which are histological scores for cartilage degeneration, were significantly lower in the DFAT group compared to the control group.
図７～図８Ｂに示すように、脛骨近位関節面の組織学検討から、Ｃｏｎｔｒｏｌ群では、硝子軟骨変性による菲薄化と軟骨細胞の減少が認められた。一方、ＤＦＡＴ群では、関節硝子軟骨変性による菲薄化と軟骨細胞の減少が抑制されていた。軟骨変性の組織学的スコアであるＭａｎｋｉｎ’ｓ ｓｃｏｒｅ及びＯＡＲＳＩ ｓｃｏｒｅは、Ｃｏｎｔｒｏｌ群に比べてＤＦＡＴ群で有意に低値を示した。 As shown in Figures 7 to 8B, histological examination of the proximal tibial articular surface revealed thinning due to hyaline cartilage degeneration and a decrease in chondrocytes in the control group. On the other hand, thinning due to hyaline cartilage degeneration and a decrease in chondrocytes were inhibited in the DFAT group. Mankin's score and OARSI score, which are histological scores for cartilage degeneration, were significantly lower in the DFAT group than in the control group.
［実施例２］
１．ＤＦＡＴ細胞の調製
ヒト膝ＯＡ患者（ｎ＝３）の皮下脂肪組織と膝蓋下脂肪体から単離した成熟脂肪細胞から、上記参考文献１に記載の方法を用いて、ヒトＤＦＡＴ細胞を予め調製した。
[Example 2]
1. Preparation of DFAT cells Human DFAT cells were prepared in advance from mature adipocytes isolated from subcutaneous adipose tissue and infrapatellar fat pads of human knee OA patients (n=3) using the method described in Reference 1 above.
２．滑膜線維芽細胞との共培養
滑膜線維芽細胞（ＳＦ）をＴＮＦαで刺激するとアグリカン分解酵素であるＡＤＡＭＴＳ４の遺伝子発現が亢進することが知られている。健常者由来滑膜線維芽細胞（Ｎ－ＳＦ）１×１０６ｃｅｌｌｓ、又は膝ＯＡ患者由来滑膜線維芽細胞（ＯＡ－ＳＦ）１×１０６ｃｅｌｌｓを、１×１０６ｃｅｌｌｓとＴＮＦ－α存在下（終濃度１０ｎｇ／ｍＬ）又は非存在下で１２時間間接的に共培養した（図９参照）。その後、各条件下で培養した滑膜線維芽細胞から全ＲＮＡを抽出し、ＡＤＡＭＴＳ４の発現をリアルタイムＲＴ－ＰＣＲ法にて定量評価した。結果を図１０に示す。
2. Co-culture with synovial fibroblasts It is known that stimulation of synovial fibroblasts (SF) with TNFα enhances gene expression of ADAMTS4, an aggrecan degrading enzyme. 1×10 6 cells of healthy subject-derived synovial fibroblasts (N-SF) or 1×10 6 cells of knee OA patient-derived synovial fibroblasts (OA-SF) were indirectly co-cultured with 1×10 6 cells in the presence or absence of TNF-α (
図１０に示すように、滑膜線維芽細胞によるＡＤＡＭＴＳ４の発現は、ＦＰ－ＤＦＡＴ、又はＳＣ－ＤＦＡＴと共培養することにより、有意に抑制された。この作用は、健常者由来滑膜線維芽細胞（Ｎ－ＳＦ）でも、膝ＯＡ患者由来滑膜線維芽細胞（ＯＡ－ＳＦ）でも認められた。 As shown in Figure 10, the expression of ADAMTS4 by synovial fibroblasts was significantly suppressed by co-culturing with FP-DFAT or SC-DFAT. This effect was observed in both synovial fibroblasts derived from healthy subjects (N-SF) and synovial fibroblasts derived from patients with knee OA (OA-SF).
３．抗炎症及び免疫制御に関与する遺伝子群の発現
「１．」で調製された膝蓋下脂肪体由来ＤＦＡＴ（ＦＰ－ＤＦＡＴ）、及び皮下脂肪組織由来ＤＦＡＴ（ＳＣ－ＤＦＡＴ）をＴＮＦ－α又はＩＦＮ－γで刺激し、抗炎症及び免疫制御に関与する遺伝子群（ＰＴＧＳ２、ＴＮＦＡＩＰ６、ＰＲＧ４、及びＢＭＰ２）の発現をリアルタイムＲＴ－ＰＣＲ法にて定量評価した。ＰＴＧＳ２の発現量の結果を図１１に、ＴＮＦＡＩＰ６の発現量の結果を図１２に、ＰＲＧ４の発現量の結果を図１３に、及びＢＭＰ２の発現量の結果を図１４に示す。
3. Expression of genes involved in anti-inflammation and immune regulation The DFAT (FP-DFAT) derived from the infrapatellar fat pad prepared in "1." and the DFAT (SC-DFAT) derived from the subcutaneous adipose tissue were stimulated with TNF-α or IFN-γ, and the expression of genes involved in anti-inflammation and immune regulation (PTGS2, TNFAIP6, PRG4, and BMP2) was quantitatively evaluated by real-time RT-PCR. The results of the expression level of PTGS2 are shown in FIG. 11, the results of the expression level of TNFAIP6 in FIG. 12, the results of the expression level of PRG4 in FIG. 13, and the results of the expression level of BMP2 in FIG. 14.
図１１及び図１２に示すように、ＦＰ－ＤＦＡＴ又はＳＣ－ＤＦＡＴを炎症性サイトカイン（ＴＮＦ－αやＩＦＮ－γ）で刺激することで抗炎症因子であるＰＴＧＳ２及びＴＮＦＡＩＰ６の発現が顕著に増加した。 As shown in Figures 11 and 12, stimulating FP-DFAT or SC-DFAT with inflammatory cytokines (TNF-α and IFN-γ) significantly increased the expression of anti-inflammatory factors PTGS2 and TNFAIP6.
図１３及び図１４に示すように、ＦＰ－ＤＦＡＴは、軟骨形成促進因子であるＰＲＧ４を定常状態で発現し、この発現は炎症性サイトカイン（ＴＮＦ－αやＩＦＮ－γ）で刺激することで抑制された。また、ＤＦＡＴ細胞は、ＴＮＦ－αで刺激することで、軟骨形成促進因子であるＢＭＰ２を発現した。ＰＲＧ４及びＢＭＰ２の発現は、ＳＣ－ＤＦＡＴに比べて、ＦＰ－ＤＦＡＴで高い傾向にあった。 As shown in Figures 13 and 14, FP-DFAT expressed PRG4, a chondrogenesis-promoting factor, in a steady state, and this expression was suppressed by stimulation with inflammatory cytokines (TNF-α and IFN-γ). Furthermore, DFAT cells expressed BMP2, a chondrogenesis-promoting factor, when stimulated with TNF-α. The expression of PRG4 and BMP2 tended to be higher in FP-DFAT than in SC-DFAT.
以上のことから、膝ＯＡに対するＤＦＡＴ細胞の軟骨変性抑制効果が明らかになった。また、ＤＦＡＴ細胞の軟骨変性抑制メカニズムは、炎症下におけるＤＦＡＴ細胞による抗炎症因子の誘導によるものであることが明らかになった。 These findings demonstrated the inhibitory effect of DFAT cells on cartilage degeneration in knee OA. Furthermore, it was revealed that the mechanism by which DFAT cells inhibit cartilage degeneration is due to the induction of anti-inflammatory factors by DFAT cells under inflammation.
本実施形態の変形性関節症の予防又は治療剤、及び変形性関節症の予防又は治療用医薬組成物によれば、ＯＡの進行を抑制することができる。 The preventive or therapeutic agent for osteoarthritis and the pharmaceutical composition for preventing or treating osteoarthritis of this embodiment can inhibit the progression of OA.
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