JP5974409B2 - False joint treatment - Google Patents
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- JP5974409B2 JP5974409B2 JP2015228687A JP2015228687A JP5974409B2 JP 5974409 B2 JP5974409 B2 JP 5974409B2 JP 2015228687 A JP2015228687 A JP 2015228687A JP 2015228687 A JP2015228687 A JP 2015228687A JP 5974409 B2 JP5974409 B2 JP 5974409B2
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Description
本発明は、偽関節の治療剤に関する。より詳細には、骨形成タンパク質-2(BMP-2)のN末端にアラニン（Ala）残基が付加したAla-BMP-2とβ-リン酸三カルシウム（β-TCP）とからなる偽関節治療剤に関する。 The present invention relates to a therapeutic agent for pseudo joints. More specifically, a pseudo-joint consisting of Ala-BMP-2 with an alanine (Ala) residue added to the N-terminus of bone morphogenetic protein-2 (BMP-2) and β-tricalcium phosphate (β-TCP) It relates to a therapeutic agent.
偽関節とは骨折部の骨癒合プロセスが完全に停止したものをいい、骨折の重篤な後遺症のひとつであり、骨癒合不全ともいう。骨折整復後に骨折部に血行不良や不安定性があると癒合が遷延し、ついには修復作用が停止した難治性の偽関節となるケースはしばしば発生する。偽関節が疑われるとまず超音波治療などが行われるが、それでも癒合が得られない場合は外科的な処置を行う。ロッキングプレートや髄内釘による確実な固定または再固定を行い、偽関節部の不良組織を除去してギャップがある場合はそこに骨移植が行われる。 Pseudo-joint refers to a bone fracture process that has completely stopped, and is one of the serious aftereffects of fractures. If there is poor circulation or instability in the fracture after fracture reduction, the fusion is prolonged, and in some cases, it becomes a refractory pseudo-joint that finally stops repairing. If a false joint is suspected, ultrasonic therapy is performed first. If fusion is still not possible, surgical treatment is performed. Secure fixation or re-fixation with a locking plate or intramedullary nail is performed, and if there is a gap by removing the defective tissue in the false joint, bone grafting is performed there.
骨移植では、患者自身の腸骨から採骨した自家骨が、移植材としてはゴールドスタンダードである。しかし、移植用の自家骨を採骨するのに健常部に侵襲を加えることの問題があり、採骨部痛、感染など様々な合併症が生じる可能性がある。そのため自家骨に変わる代替材料の開発が進められている。難治性偽関節に用いる骨移植材は、周囲からの修復作用に乏しい母床に移植するので、移植材自体に高い骨形成能が必要である。例えばハイドロキシアパタイト（HAP）やβ-TCPなど骨の無機成分であるリン酸カルシウムからなる人工骨の一般使用の普及は進んでいるものの、これらの材料は骨形成の足場とはなるが自ら骨形成する性質はなく、難治性偽関節のような疾患には適応にならない。 In bone grafting, autologous bone taken from the patient's own iliac bone is the gold standard for grafting. However, there is a problem of invading the healthy part in order to extract autologous bone for transplantation, and various complications such as bone pain and infection may occur. Therefore, the development of alternative materials that can be used as substitutes for autologous bones is underway. Since a bone graft material used for an intractable pseudo-joint is transplanted to a mother bed having poor repair action from the surroundings, the graft material itself needs to have a high bone forming ability. For example, although hydroxyapatite (HAP) and β-TCP are commonly used as artificial bones made of calcium phosphate, which is an inorganic component of bone, these materials provide a bone-forming scaffold but have the ability to form bone themselves. It is not indicated for diseases such as refractory false joints.
高い骨形成能をもたらすものとして、BMPなどの骨形成促進タンパクや骨髄細胞を培養したものを担体とともに骨移植材として応用する骨再生の研究が行われている。骨髄細胞を用いる手法は、培養のために数週間から数ヶ月の時間が必要であること、自家細胞を用いるが故に1ロットが1ユニットとなり、培養のコストとともに通常はロット単位で行われるような安全性や性能確認も製品ごとになって、それらのコストは膨大なものになるという欠点があった。 Bone regeneration research is being carried out in which bone formation-promoting proteins such as BMP and bone marrow cells are cultured together with a carrier as a bone grafting material to provide high bone forming ability. The method using bone marrow cells requires several weeks to several months for culturing, and because of the use of autologous cells, one lot becomes one unit. Safety and performance confirmation are also product-specific, and their costs are enormous.
BMPは未分化細胞に作用して骨芽細胞への分化を強力に促進し、骨を誘導するタンパクである。TGF-βスーパーファミリーに属し、BMPの中でもBMP-2、BMP-4、BMP-7などが骨形成には効果があるとされており、近年は遺伝子組み換えによって製造されたBMP-2、BMP-7が欧米では製品化されている。 BMP is a protein that acts on undifferentiated cells to strongly promote differentiation into osteoblasts and induce bone. BMP-2, BMP-4, BMP-7, etc. belong to the TGF-β superfamily, and BMP-2, BMP-7, etc. are effective for bone formation. Recently, BMP-2, BMP- 7 has been commercialized in the West.
BMPの担体として、欧米で実用化されているBMP製品にはタイプＩコラーゲンが用いられている。BMP-2とコラーゲンスポンジとを組み合わせた製品（製品名：Infuse メドトロニック社）は、急性開放脛骨骨幹部骨折、椎間板変性症における脊椎固定術、抜歯窩治癒過程に伴う骨欠損に対する上顎洞底挙上術および局所的な歯槽堤形成術で米国では認可されている。さらに、自家骨移植の代替をするためにBMP製品の偽関節へのオフラベル使用も行われている。しかし、コラーゲンは生体吸収性ではあるものの、骨を修復するということでは骨伝導能などの性質はなく、外力に対してつぶれてしまうなどの力学的な欠点もある。偽関節への適応においても、自家骨移植に比較して、必ずしも満足な結果は得られていない。 As a BMP carrier, type I collagen is used in BMP products that have been put to practical use in Europe and the United States. The combination of BMP-2 and collagen sponge (Product name: Infuse Medtronic Co., Ltd.) is used for acute open tibial shaft fractures, spinal fusion in intervertebral disc degeneration, and maxillary sinus floor elevation for bone defects associated with the extraction socket healing process Approved in the United States for surgical and local alveolar plasty. In addition, BMP products are being used off-label in the false joints to replace autologous bone grafts. However, although collagen is bioresorbable, repairing bone does not have properties such as osteoconductivity, and has mechanical disadvantages such as collapsing against external forces. Even in the adaptation to the pseudo-joint, satisfactory results are not always obtained as compared with autologous bone transplantation.
また、大腸菌発現系由来の、BMP-2のN末端にアラニンが付加したAla-BMP-2と、その担体として、連通気孔を有するβ-TCP多孔体とを組み合わせた移植材についても研究されている。原田らは、この大腸菌発現系由来Ala-BMP-2と連通気孔を有する多孔質β-TCP顆粒とを複合した移植材をイヌ尺骨全周性欠損モデルにて評価し、このモデルでは良好な結果を得ている（非特許文献１）。これにより、大腸菌発現系由来Ala-BMP-2と連通気孔を有するβ-TCP多孔体は高い機能を有する骨移植材であることは判明した。 In addition, transplantation materials that combine Ala-BMP-2 with an alanine added to the N-terminus of BMP-2 derived from the E. coli expression system and a β-TCP porous body with continuous air holes as its carrier have been studied. Yes. Harada et al. Evaluated the graft material composed of this Escherichia coli expression system-derived Ala-BMP-2 and porous β-TCP granules with open air vents in a canine ulna peripheral defect model. (Non-Patent Document 1). As a result, it was found that the β-TCP porous body having Ela coli expression system-derived Ala-BMP-2 and communicating pores is a bone graft material having a high function.
しかしながら、上記モデルのような実験的に作製した骨欠損では、骨癒合プロセスが機能しているため、移植母床としての環境がよく、骨癒合プロセスが完全に停止した難治性偽関節に上記複合移植材を適応する場合には、自家骨の使用が必須であると考えられていた。自家骨は、骨形成に寄与する細胞や因子をナチュラルに含んでおり、基質、ミネラルは骨形成の良い環境をもたらすものである。それゆえ、このような自家骨とBMPの骨誘導能との併用は、実際の難治性の症例では効果が高く、移植材を構成するものとして自家骨は必要なものであった。特に、ネコやイヌなどのヒト以外の動物は、患部を安静に保つことは難しく、より骨誘導能の高い自家骨の使用は必須であると考えられていた。実際、原田らは、ネコおよびイヌの重症度の高い骨癒合不全の症例の治療の際には、大腸菌発現系由来Ala-BMP-2と、連通気孔を有する多孔質β-TCP顆粒とを複合し、これにさらに自家骨を混合して調製した製剤を使用している（非特許文献２及び３）。 However, in bone defects created experimentally as in the above model, the bone fusion process is functioning, so the environment as a transplant mother bed is good, and the above complex is added to the intractable pseudojoint where the bone fusion process has completely stopped. The use of autologous bone was considered essential when applying the graft. Autologous bones naturally contain cells and factors that contribute to bone formation, and the matrix and minerals provide a good environment for bone formation. Therefore, the combined use of the autologous bone and the osteoinductive ability of BMP is highly effective in actual intractable cases, and autologous bone is necessary as a component of the transplant. In particular, it was difficult for animals other than humans such as cats and dogs to keep the affected area at rest, and it was considered essential to use autologous bone with higher osteoinductive ability. In fact, Harada et al. Combined Ala-BMP-2 derived from the E. coli expression system with porous β-TCP granules with open air vents in the treatment of cats and dogs with severe bone union. However, a preparation prepared by further mixing autologous bone with this is used (Non-patent Documents 2 and 3).
上述のとおり、移植材への自家骨の使用は、患者の健常部に侵襲を加えることによる採骨部痛、感染などの合併症リスクを不可避的に伴う。しかしながら、骨形成促進タンパクと人工骨素材担体との複合体のみでは、実際の難治性偽関節、特に安静を保つことが困難なヒト以外の動物における難治性偽関節への適応には不十分であり、良好な治療効果を得るには自家骨の使用が不可欠であるというジレンマがあった。
従って、本発明の目的は、自家骨を用いることなく、自家骨配合移植材と同等の効果を奏する、難治性の骨癒合不全に適応可能な新規移植材を提供することである。
As described above, the use of autologous bone as a transplant material inevitably involves the risk of complications such as bone picking pain and infection caused by invading the healthy part of a patient. However, the complex of bone formation-promoting protein and artificial bone material carrier alone is not sufficient for adaptation to actual refractory pseudo-joints, especially refractory pseudo-joints in non-human animals that are difficult to maintain rest. There was a dilemma that the use of autologous bone was indispensable to obtain a good therapeutic effect.
Accordingly, an object of the present invention is to provide a novel graft material that can be applied to intractable bone union failure without using autologous bone and that exhibits the same effect as the autologous bone-blown graft material.
本発明者らは、大腸菌発現系由来Ala-BMP-2（Ec Ala-BMP-2）と、従来から販売されている哺乳類由来のCHO細胞で発現されたBMP-2（CHO BMP-2）との間で、コラーゲンスポンジまたは連通気孔を有するβ-TCP多孔体をそれぞれ担体として用いた場合のin vivoでの骨誘導能を比較した。その結果、コラーゲンスポンジを担体として用いた場合は、Ec Ala-BMP-2とCHO BMP-2との間で骨誘導能に有意な差はなかったのに対し、β-TCP多孔体を担体として用いた場合、Ec Ala-BMP-2の骨誘導能はCHO BMP-2のそれよりも著しくすぐれていた。さらに、Ec Ala-BMP-2とβ-TCP多孔体とからなる移植材を、自家骨を用いることなく難治性の骨癒合不全であるイヌ偽関節の治療に用いたところ、驚くべきことに、自家骨を用いないにもかかわらず、自家骨を用いるのと同等の骨再生効果を示した。
以上のように、本発明者らは、Ec Ala-BMP-2とβ-TCP多孔体とからなる移植材は、自家骨を用いることなく自家骨配合移植材と同等の偽関節治療効果を示すという、従来のBMP-2製剤では達成できない効果を奏することを見出し、本発明を完成した。
The present inventors include E. coli expression system-derived Ala-BMP-2 (Ec Ala-BMP-2), and BMP-2 (CHO BMP-2) expressed in CHO cells derived from mammals that have been conventionally sold. The in vivo osteoinductive ability when using a collagen sponge or a β-TCP porous body having continuous vents as a carrier was compared. As a result, when collagen sponge was used as a carrier, there was no significant difference in osteoinductivity between Ec Ala-BMP-2 and CHO BMP-2, whereas β-TCP porous material was used as a carrier. When used, the osteoinductive capacity of Ec Ala-BMP-2 was significantly better than that of CHO BMP-2. Furthermore, surprisingly, when a graft material composed of Ec Ala-BMP-2 and β-TCP porous material was used for the treatment of canine pseudo-joint that is intractable bone union failure without using autologous bone, Despite not using autologous bone, it showed the same bone regeneration effect as using autologous bone.
As described above, the present inventors show that the graft material composed of Ec Ala-BMP-2 and β-TCP porous body exhibits the same pseudo-joint treatment effect as that of a bone graft material without using bone. The present inventors have found that the conventional BMP-2 preparation has an effect that cannot be achieved, and thus completed the present invention.
すなわち、本発明は、以下のとおりである。
（１）活性成分としてAla-BMP-2とβ-TCP多孔体のみを含む偽関節治療用製剤。
（２）治療対象がヒトを除く哺乳動物である、（１）記載の製剤。
（３）ヒトを除く哺乳動物がペット動物である、（２）記載の製剤。
（４）ヒトを除く哺乳動物がイヌまたはネコである、（２）記載の製剤。
（５）β-TCP多孔体が、気孔経1μm前後のミクロ気孔と、気孔径数百μmのマクロ気孔とを含み、かつ気孔率が60%以上である、（１）から（４）のいずれかに記載の製剤。
（６）活性成分としてAla-BMP-2とβ-TCP多孔体のみを用いる、ヒトを除く哺乳動物の偽関節治療方法。
That is, the present invention is as follows.
(1) A pseudo-joint treatment preparation containing only Ala-BMP-2 and β-TCP porous material as active ingredients.
(2) The preparation according to (1), wherein the treatment target is a mammal other than a human.
(3) The preparation according to (2), wherein the mammals other than humans are pet animals.
(4) The preparation according to (2), wherein the mammals other than humans are dogs or cats.
(5) Any of (1) to (4), wherein the β-TCP porous material includes micropores having a pore diameter of about 1 μm and macropores having a pore diameter of several hundreds of μm, and the porosity is 60% or more. A preparation according to the above.
(6) A method for treating pseudo-joints in mammals other than humans, using only Ala-BMP-2 and β-TCP porous material as active ingredients.
本発明の偽関節治療用製剤は、自家骨を用いないため、自家骨採取のために健常部への侵襲を伴わずに治療でき、かつ自家骨を使用した場合と同等の高い効果が得られる。 Since the preparation for treating pseudo-joints of the present invention does not use autologous bone, it can be treated without invading the healthy part for autologous bone collection, and the same high effect as when autologous bone is used can be obtained. .
以下、本発明を詳細に説明する。
（Ｉ）骨形成タンパク質-2 (BMP-2)
「BMP-2」は、BMPファミリー又はGDFファミリーに属し、BMP-2のI型およびII型受容体への結合活性を有し、かつ骨形成を誘導する能力、或いは骨の再生および修復能力を有する（これらをまとめて、以下「BMP-2活性」と記載する）。
Hereinafter, the present invention will be described in detail.
(I) Bone morphogenetic protein-2 (BMP-2)
“BMP-2” belongs to the BMP family or GDF family, has the ability to bind BMP-2 to type I and type II receptors, and to induce bone formation, or to regenerate and repair bone. (These are collectively referred to as “BMP-2 activity” hereinafter).
本発明に用いられるBMP-2が由来する動物種は特に限定されないが、例えば、治療対象である哺乳動物由来のBMP-2が挙げられる。好ましくはヒトである。イヌ、ネコのBMP-2は、ヒトと同一のアミノ酸配列を有する。 The animal species from which BMP-2 used in the present invention is derived is not particularly limited, and examples thereof include BMP-2 derived from a mammal to be treated. Preferably it is a human. Dog and cat BMP-2 has the same amino acid sequence as human.
本発明で使用されるBMP-2は、Ala-BMP-2が好ましい。「Ala-BMP-2」は、N末端にアラニン残基が付加された活性型ヒトBMP-2ホモダイマーである。Ala-BMP-2は、BMP-2と同様に、システインノットと呼ばれる3個のジスルフィド結合と、1個の分子間ジスルフィド結合とを形成する、高度に保存されたシステイン残基をもつ。Ala-BMP-2は、1分子あたり1個のシステインノット、1個のα-ヘリックス、少なくとも4個のβ-シートで構成され、これによってモノマーはダイマーを形成することができる。Ala-BMP-2はBMP-2と同様にホモダイマーを形成し、活性型となる。 The BMP-2 used in the present invention is preferably Ala-BMP-2. “Ala-BMP-2” is an active human BMP-2 homodimer with an alanine residue added to the N-terminus. Ala-BMP-2, like BMP-2, has a highly conserved cysteine residue that forms three disulfide bonds called cysteine knots and one intermolecular disulfide bond. Ala-BMP-2 is composed of one cysteine knot, one α-helix, and at least four β-sheets per molecule, which allows monomers to form dimers. Ala-BMP-2 forms a homodimer like BMP-2 and becomes active.
BMP-2は哺乳動物細胞内では糖鎖修飾を受けた不活性型ホモダイマーの前駆体BMP-2として合成されたのち、細胞外に分泌されてからプロテアーゼによってプロセシングされて活性型ホモダイマーの成熟タンパク質となる。 BMP-2 is synthesized as a BMP-2 precursor of an inactive homodimer that has undergone glycosylation in mammalian cells, and then secreted outside the cell and then processed by a protease to produce a mature protein of the active homodimer. Become.
ヒトBMP-2のヌクレオチド配列およびアミノ酸配列は、GenBank（NCBI、米国）に登録されており、登録番号（Accession Number）はNM_001200である。 The nucleotide sequence and amino acid sequence of human BMP-2 are registered in GenBank (NCBI, USA), and the registration number (Accession Number) is NM_001200.
特に本発明で使用されるAla-BMP-2は、BMP-2ホモダイマーのアミノ酸配列のN末端にそれぞれアラニン残基が付加された、糖鎖が付加されていないタンパク質である(WO 2012/029148)。Ala-BMP-2は、大腸菌をはじめとする原核細胞発現系を用いて組換え生産することができる。あるいは、自体公知の無細胞翻訳系を用いてin vitro合成することもできる。 In particular, Ala-BMP-2 used in the present invention is a protein in which an alanine residue is added to the N-terminus of the amino acid sequence of a BMP-2 homodimer and no sugar chain is added (WO 2012/029148). . Ala-BMP-2 can be produced recombinantly using prokaryotic expression systems such as E. coli. Alternatively, it can be synthesized in vitro using a cell-free translation system known per se.
欧米のBMP-2製品は哺乳類細胞の発現系にて製造されており、これらのアミノ酸配列はヒト内在性のものと同一であるが、糖鎖修飾のパターンは異なっている。大腸菌で生産されたAla-BMP-2は糖鎖修飾がないことにより溶解性が低下しているため、局所因子であるBMPの局所での停留性が高まり、それによって作用効率が向上するメリットがある。 Western BMP-2 products are produced in mammalian cell expression systems, and these amino acid sequences are identical to those endogenous to humans, but the glycosylation pattern is different. Since Ala-BMP-2 produced in E. coli has reduced solubility due to the absence of sugar chain modification, the local retention of BMP, a local factor, is enhanced, thereby improving the efficiency of action. is there.
（ＩＩ）β-リン酸三カルシウム（β-TCP）
BMP-2の担体としては、材料単独でも人工骨として応用されている骨伝導能を有するリン酸カルシウム系の多孔体が望ましく、特に生体吸収性で自家骨置換の性質を有するβ-TCP多孔体が好ましい。リン酸カルシウムはタンパクもよく吸着しBMPの保持性も高い。多孔体の気孔構造は、良好な気孔連通性が確保されていることが必要であり、気孔経は1μm前後のミクロ気孔と数百μmのマクロ気孔が含まれていること、気孔率は60％以上であることが望ましい。
(II) β-tricalcium phosphate (β-TCP)
As a carrier for BMP-2, a calcium phosphate based porous material having osteoconductivity that is applied as an artificial bone even with the material alone is desirable, and a β-TCP porous material having bioresorbable and self-bone replacement properties is particularly preferable. . Calcium phosphate adsorbs proteins well and has high BMP retention. The pore structure of the porous body needs to ensure good pore connectivity, the pore diameter includes micropores around 1 μm and macropores of several hundred μm, and the porosity is 60% The above is desirable.
β-TCPの具体的な例としては、オスフェリオン（登録商標；オリンパス社製）、スーパーポア（登録商標；HOYA Technosurgical社製）等を用いることができる。 As specific examples of β-TCP, Osferion (registered trademark; manufactured by Olympus), Superpore (registered trademark; manufactured by HOYA Technosurgical), and the like can be used.
（ＩＩＩ）BMP-2とβ-TCP多孔体とからなる偽関節治療用製剤
本発明の偽関節治療用製剤（以下、本発明製剤ともいう。）は、活性成分として、上記のAla-BMP-2及びβ-TCP多孔体のみを含有する。ここで「のみを含有する」とは、Ala-BMP-2及びβ-TCP多孔体と組み合わせた場合に、該2成分のみの場合と比較して、難治性偽関節の治療上区別できない程度で、骨再生作用に関与し得る添加剤を含むことを許容することを意味する。
本発明製剤は、骨再生作用自体に直接的に関与しない限り、他の医薬上許容される添加剤を含有してもよい。該添加剤としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液（例えば、D-ソルビトール、D-マンニトール、塩化ナトリウムなど）などの水性液、緩衝剤（例えば、リン酸塩緩衝液、酢酸ナトリウム緩衝液）、無痛化剤（例えば、塩化ベンザルコニウム、塩酸プロカインなど）、安定剤（例えば、ヒト血清アルブミン、ポリエチレングリコールなど）、保存剤、酸化防止剤などが挙げられる。
(III) Formulation for treating pseudo-joint comprising BMP-2 and β-TCP porous body The formulation for treating pseudo-joint of the present invention (hereinafter also referred to as the present formulation) is the above-mentioned Ala-BMP- as an active ingredient. Contains only 2 and β-TCP porous material. Here, “contains only” means that when combined with Ala-BMP-2 and β-TCP porous material, it is indistinguishable from the treatment of refractory false joints compared to the case of only the two components. It means to allow the inclusion of additives that can be involved in the bone regeneration action.
The preparation of the present invention may contain other pharmaceutically acceptable additives as long as they are not directly involved in the bone regeneration action itself. Examples of such additives include aqueous solutions such as physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.), buffers (eg, phosphate) Salt buffer, sodium acetate buffer), soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizer (eg, human serum albumin, polyethylene glycol, etc.), preservative, antioxidant, etc. .
本発明製剤は、例えば、Ala-BMP-2、好ましくは大腸菌発現系由来のAla-BMP-2の凍結乾燥物(調製法については、例えばWO 2012/029148を参照)を、適当な溶媒（例、注射用水、希塩酸等の水性溶媒）に0.1〜10mg/mlの濃度となるように溶解し、得られたAla-BMP-2溶液をβ-TCP多孔体に、β-TCPのg体積あたり0.1〜10mgのBMP-2が含まれるように含浸させることにより調製することができる。 The preparation of the present invention comprises, for example, a freeze-dried product of Ala-BMP-2, preferably Ala-BMP-2 derived from an E. coli expression system (for the preparation method, see, for example, WO 2012/029148) and an appropriate solvent (eg, , Water for injection, aqueous solvent such as dilute hydrochloric acid) to a concentration of 0.1 to 10 mg / ml, and the resulting Ala-BMP-2 solution was added to the β-TCP porous body to 0.1 β per g volume of β-TCP. It can be prepared by impregnation to contain ˜10 mg of BMP-2.
得られた本発明製剤は、移植対象である哺乳動物の骨欠損部に、十分な骨再生が得られる程度のサイズ及び形状に調整して、移植することができる。哺乳動物として、例えば、イヌ、ネコ、ウサギなどのペット動物、ウシ、ウマ、ヒツジ、ヤギ、ブタなどの家畜動物、マウス、ラット、モルモットなどの実験動物、及びヒト、サル、オランウータン、チンパンジーなどの霊長類が挙げられるが、これらに限定されない。安静を保つことの困難なヒト以外の哺乳動物では、偽関節の治療はより困難となるので、好ましい一実施態様において、本発明製剤の移植対象として、ヒト以外の哺乳動物が挙げられる。より好ましくは、ペット動物、特に好ましくは、イヌまたはネコである。 The obtained preparation of the present invention can be transplanted to a bone defect part of a mammal to be transplanted, adjusted to a size and shape sufficient to obtain sufficient bone regeneration. Examples of mammals include pet animals such as dogs, cats and rabbits, domestic animals such as cattle, horses, sheep, goats and pigs, laboratory animals such as mice, rats and guinea pigs, and humans, monkeys, orangutans and chimpanzees. Examples include, but are not limited to, primates. In mammals other than humans that are difficult to maintain rest, treatment of pseudo-joints is more difficult, and in a preferred embodiment, non-human mammals are mentioned as transplant targets of the preparation of the present invention. More preferred are pet animals, particularly preferred are dogs and cats.
以下に実施例を挙げて本発明をより具体的に説明するが、本発明はこれら実施例に何ら限定されるものではない。 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
実施例１ イヌ橈骨尺骨骨折後の骨癒合不全症例に対する本発明製剤の移植
大腸菌で製造されたAla-BMP-2と連通気孔を有するβ-TCP多孔体の組合せ移植材を、自家骨の併用なしでイヌ橈骨尺骨の骨折後の骨癒合不全症例に適用した。
動物種はチワワ、年齢1歳、メス、体重は1.35kgで、経緯として落下事故により受傷、ギプス固定による保存的治療を行ったが治癒せず、癒合不全となった。3ヵ月後に手術治療を実施した。
手術は、プレートおよびスクリューにより整復固定、骨再建術を実施した。デブリードマンによって生じた骨欠損部に、大腸菌で製造されたAla-BMP-2の溶液（WO 2012/029148に記載の凍結乾燥物を0.5mM塩酸溶媒に再溶解したもの）を、連通気孔を有する多孔質β-TCP顆粒（オスフェリオン（登録商標；オリンパス社製）；気孔率75％、気孔経100〜400μm（1μm程度の細孔を含む）気孔性状で、顆粒径1〜3mmのもの）にしみ込ませた移植材を補填した。欠損の長さは4mmで、これは橈骨長に対して8.9％、また手術側橈骨は、健常側の橈骨長に対して83%の脚短縮となった。移植材の調製は、連通気孔を有する多孔質β-TCP顆粒0.25gをディッシュにとり、大腸菌発現系由来Ala-BMP-2溶液（濃度1.25mg/ml）0.2mlを、β-TCP顆粒にまんべんなく滴下してしみ込ませることにより行った。すなわち、連通気孔を有する多孔質β-TCP顆粒0.25gに、大腸菌発現系由来Ala-BMP-2が0.25mgを複合したものを移植材として使用した。
Example 1 Combination of Ala-BMP-2 produced by Escherichia coli transplanted with Escherichia coli transplanted in the present invention for a case of bone union failure after canine ulna fracture without a combination of autologous bone Therefore, it was applied to the case of bone union failure after fracture of the canine ulna.
The animal species was Chihuahua, age 1 year, female, weight 1.35kg. As a background, she was injured by a fall accident and conservative treatment was performed by fixing the cast. Surgical treatment was performed 3 months later.
The operation was reduced and fixed with a plate and screw, and bone reconstruction was performed. In the bone defect produced by Debridement, a solution of Ala-BMP-2 produced in Escherichia coli (the lyophilized product described in WO 2012/029148 was redissolved in 0.5 mM hydrochloric acid solvent) Porous β-TCP granules (Osferion (registered trademark; manufactured by Olympus)); Porosity 75%, Porosity 100 to 400 μm (including pores of about 1 μm), Porosity, granules with a diameter of 1 to 3 mm The soaked transplant was supplemented. The length of the defect was 4 mm, which was 8.9% of the rib length, and the surgical rib had a leg shortening of 83% of the healthy rib length. To prepare the graft material, take 0.25 g of porous β-TCP granules with continuous air holes in a dish, and add 0.2 ml of Ala-BMP-2 solution (concentration 1.25 mg / ml) derived from E. coli expression system evenly onto β-TCP granules. It was done by letting it soak. Specifically, 0.25 g of porous β-TCP granules having continuous air holes and 0.25 mg of E. coli expression system-derived Ala-BMP-2 were used as a transplant material.
術後は、X線撮影により移植部の骨再生の状態を観察した。移植したβ-TCP顆粒は経時的に不明瞭となり、骨形成の進行と母床骨との癒合の進行を伺わせた。画像上では移植部と母床との連続性が完全となり、骨再生が進行した術後18週で、荷重伝達を戻していくために、プレート両端のスクリュー各1本を残してその他のスクリューを抜去、この時点で良好に橈骨尺骨は再建されていた。さらに術後42週で、すべてのスクリューとプレートを抜去し、以降は再骨折などなく運動機能は保たれていた。 After the operation, the state of bone regeneration at the transplanted part was observed by X-ray photography. The transplanted β-TCP granules became unclear over time, suggesting the progress of bone formation and the fusion with the mother bone. On the image, the continuity between the transplanted part and the mother bed is complete, and 18 weeks after the operation when bone regeneration has progressed, in order to return the load transmission, leave one screw at each end of the plate and the other screws. The ribs were successfully reconstructed at this point. Furthermore, 42 weeks after the operation, all screws and plates were removed, and after that, the motor function was maintained without re-fracture.
実施例２ ラット異所性骨化モデルにおけるBMP-2の骨誘導能の比較
大腸菌で製造されたAla-BMP-2（OB1）と市販されている哺乳動物細胞で製造した、BMP-2（製品名：Infuse メドトロニック社）について、担体をコラーゲンスポンジとした場合とβ-TCPとした場合とで比較を行った。
１．コラーゲンスポンジでの比較
７週齢のラットの背部の皮膚を切開、剥離し、皮下にOB1またはInfuseを含ませたコラーゲンスポンジ（テルダーミス真皮欠損用グラフト：コラーゲン単層タイプ、オリンパステルモバイオマテリアル社製）または希釈溶媒（グリシンバッファー（組成：スクロース50 mg、グリシン250 mg、L-グルタミン酸37 mg、塩化ナトリウム 1 mg、ポリソルベート80 1 mg（滅菌水10 mL中））又は0.5mM HCl）を含ませたコラーゲンスポンジを埋植した。埋植後、空気が入らないように縫合した。埋植は1回投与とした。埋植日を0日とし、3週間観察した。担体コラーゲンは、片側あたり5×5mm（厚みは3mm）になるように無菌的に切ったものを使用した。これに被験物質を所定量含む濃度に調製した溶液20μlを滴下して15分放置し移植材とした。具体的には、表１の通りの試験群で埋植を行った。
各ラットへの埋植については、麻酔は塩酸ケタミン（ケタラール、第一三共（株））及びキシラジン（セラクタール2 %注射液、バイエルメディカル（株））の併用麻酔（混合比3：1）を行った。術部の広い範囲を電気バリカンにて剃毛し、ヒビテンアルコール（0.5%グルコン酸クロルヘキシジン−エタノール溶液、大日本住友製薬（株））で消毒後、背部4箇所(左右2箇所ずつ)に縦皮切を加え、皮下組織に移植材を埋植した。左右とも同様に埋植し、ナイロン糸もしくは絹糸で縫合した。抜糸は行わなかった。
Example 2 Comparison of osteoinductive ability of BMP-2 in rat ectopic ossification model Ala-BMP-2 (OB1) produced in E. coli and BMP-2 (product) produced in commercially available mammalian cells Name: Infuse Medtronic) was compared between the case where the carrier was collagen sponge and the case where β-TCP was used.
1. Comparison with collagen sponge Collagen sponge with incision and peeling of the skin of the back of 7-week-old rats and OB1 or Infuse under the skin (graft for derdermis dermal defect: collagen single layer type, manufactured by Olympus Terumo Biomaterials) Or collagen containing dilute solvent (glycine buffer (composition: sucrose 50 mg, glycine 250 mg, L-glutamic acid 37 mg, sodium chloride 1 mg, polysorbate 80 1 mg in sterile water 10 mL) or 0.5 mM HCl) A sponge was implanted. After implantation, it was sutured so that air did not enter. Implantation was performed once. The day of implantation was set to day 0 and observed for 3 weeks. The carrier collagen used was aseptically cut to 5 × 5 mm (thickness 3 mm) per side. To this, 20 μl of a solution prepared to a concentration containing a predetermined amount of the test substance was dropped and left for 15 minutes to obtain a transplant material. Specifically, implantation was performed in the test group as shown in Table 1.
For implantation in each rat, anesthesia should be combined with ketamine hydrochloride (Ketalar, Daiichi Sankyo Co., Ltd.) and xylazine (Seractal 2% Injection, Bayer Medical Co., Ltd.) (mixing ratio 3: 1). went. A wide area of the surgical site is shaved with an electric clipper, disinfected with Hibiten Alcohol (0.5% chlorhexidine gluconate-ethanol solution, Dainippon Sumitomo Pharma Co., Ltd.), then vertically peeled on 4 backs (2 on each side) A cut was made and the graft material was implanted in the subcutaneous tissue. The left and right were implanted in the same manner and sutured with nylon or silk thread. No yarn removal was performed.
３週間後、ケタセラ3:1の過麻酔下でラットを安楽死させ採血後、剖検を行い、異常の有無を確認したが異常は認められなかった。背筋膜下埋植担体を採取し、DEXA装置(DCS-600EX, ALOKA)にて埋植後の骨塩量を測定した。OB1で骨塩量が高い傾向が見られるものの、有意差は見られなかった(図1)。 Three weeks later, rats were euthanized under Ketacera 3: 1 overanesthesia, and after blood collection, necropsy was performed to confirm the presence or absence of abnormalities, but no abnormalities were observed. The subscapular implant carrier was collected and the bone mineral content after implantation was measured with a DEXA device (DCS-600EX, ALOKA). Although there was a tendency for OB1 to have a high bone mineral content, no significant difference was observed (FIG. 1).
２．β-TCPでの比較
７週齢のラットの背部の皮膚を切開、剥離し、皮下にOB1またはInfuseを含ませたβ-TCP（スーパーポア：HOYA社製）または希釈溶媒（グリシンバッファー（組成：スクロース50 mg、グリシン250 mg、L-グルタミン酸37 mg、塩化ナトリウム 1 mg、ポリソルベート80 1 mg（滅菌水10 mL中））又は0.5mM HCl）を含ませたβ-TCPを埋植した。埋植後、空気が入らないように縫合した。埋植は1回投与とした。埋植日を0日とし、3週間観察した。担体β-TCPは、片側あたり50mgになるように秤量したものを使用した。これに被験物質を所定量含む濃度に調製した溶液40μlを滴下して15分放置し移植材とした。具体的には、表２の通りの試験群で埋植を行った。
各ラットへの埋植については、麻酔は塩酸ケタミン（ケタラール、第一三共（株））及びキシラジン（セラクタール2 %注射液、バイエルメディカル（株））の併用麻酔（混合比3：1）を行った。術部の広い範囲を電気バリカンにて剃毛し、ヒビテンアルコール（0.5%グルコン酸クロルヘキシジン−エタノール溶液、大日本住友製薬（株））で消毒後、背部4箇所(左右2箇所ずつ)に縦皮切を加え、皮下組織に移植材を埋植した。左右とも同様に埋植し、ナイロン糸もしくは絹糸で縫合した。抜糸は行わなかった。
2. Comparison with β-TCP Incision and detachment of the skin of the back of 7-week-old rats, β-TCP (Superpore: manufactured by HOYA) containing OB1 or Infuse subcutaneously, or a diluent solvent (glycine buffer (composition: Β-TCP containing 50 mg of sucrose, 250 mg of glycine, 37 mg of L-glutamic acid, 1 mg of sodium chloride, 1 mg of polysorbate 80 (in 10 mL of sterilized water)) or 0.5 mM HCl) was implanted. After implantation, it was sutured so that air did not enter. Implantation was performed once. The day of implantation was set to day 0 and observed for 3 weeks. The carrier β-TCP used was weighed so as to be 50 mg per side. To this, 40 μl of a solution prepared to a concentration containing a predetermined amount of the test substance was dropped and left for 15 minutes to obtain a transplant material. Specifically, implantation was performed in the test groups as shown in Table 2.
For implantation in each rat, anesthesia should be combined with ketamine hydrochloride (Ketalar, Daiichi Sankyo Co., Ltd.) and xylazine (Seractal 2% Injection, Bayer Medical Co., Ltd.) (mixing ratio 3: 1). went. A wide area of the surgical site is shaved with an electric clipper, disinfected with Hibiten Alcohol (0.5% chlorhexidine gluconate-ethanol solution, Dainippon Sumitomo Pharma Co., Ltd.), then vertically peeled on 4 backs (2 on each side) A cut was made and the graft material was implanted in the subcutaneous tissue. The left and right were implanted in the same manner and sutured with nylon or silk thread. No yarn removal was performed.
３週間後、ケタセラ3:1の過麻酔下でラットを安楽死させ採血後、剖検を行い、異常の有無を確認したが異常は認められなかった。背筋膜下埋植担体を採取し、DEXA装置(DCS-600EX, ALOKA)にて埋植後の骨塩量を測定した。OB1で骨塩量が著しく高く、強い骨誘導能があることが分かった(図２)。 Three weeks later, rats were euthanized under Ketacera 3: 1 overanesthesia, and after blood collection, necropsy was performed to confirm the presence or absence of abnormalities, but no abnormalities were observed. The subscapular implant carrier was collected and the bone mineral content after implantation was measured with a DEXA device (DCS-600EX, ALOKA). It was found that OB1 has a significantly high bone mineral content and strong osteoinductive ability (FIG. 2).
以上のように、大腸菌発現系由来Ala-BMP-2と連通気孔を有する多孔質β-TCP顆粒の組合せによる移植材において、その骨誘導能は動物細胞発現系由来BMP-2の連通気孔を有する多孔質β-TCP顆粒の組合せによる移植材に比べ著しく骨誘導能が高く、自家骨を用いることなく難治性の骨癒合不全を治癒し得ることが確認できた。 As described above, in the transplantation material by the combination of Ala-BMP-2 derived from E. coli expression system and porous β-TCP granule having open pores, the osteoinductive ability has open pores of BMP-2 derived from animal cell expression system It was confirmed that the osteoinductive ability was remarkably higher than that of the transplanted material with the combination of porous β-TCP granules, and it was possible to cure refractory bone union failure without using autologous bone.
実験的な骨欠損の修復ではなく、実際の難治性症例において有効性が判明したことから、ヒト、並びにヒト以外の哺乳動物（例えば、イヌ、ネコ等のペット動物）における難治性骨癒合不全、偽関節の治療材料として、Ala-BMP-2とβ-TCP多孔体との組合せによる骨移植材は、臨床応用し得るものといえる。 Since it was found effective in actual refractory cases rather than experimental bone defect repair, refractory osteounion failure in humans and non-human mammals (eg, pet animals such as dogs, cats, etc.) As a material for the treatment of pseudo-joints, bone graft materials using a combination of Ala-BMP-2 and β-TCP porous material can be said to be clinically applicable.
Claims (5)
み、かつ気孔率が60%以上である、請求項１又は２に記載の製剤。 The preparation according to claim 1 or 2, wherein the β-TCP porous material comprises micropores having a pore size of about 1 µm and macropores having a pore size of several hundreds µm, and the porosity is 60% or more.
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