JP2022534228A - Methods for preventing the formation of calcified deposits and inactivating xenoantigens in biological matrices - Google Patents
Methods for preventing the formation of calcified deposits and inactivating xenoantigens in biological matrices Download PDFInfo
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Abstract
本発明は、単離した生物学的マトリックスをフェノール性化合物の混合物を含む溶液と接触させる工程を含む、単離した生物学的マトリックスの内部の石灰化沈着物の形成を防止するための方法を開示する。The present invention provides a method for preventing the formation of calcified deposits within an isolated biological matrix comprising contacting the isolated biological matrix with a solution comprising a mixture of phenolic compounds. Disclose.
Description
本発明は、医学、生物医学または獣医学の分野、特に、ヒトまたは動物の体に埋め込まれる生物学的および生体適合性のマトリックスの製造に応用される。 The invention finds application in the medical, biomedical or veterinary fields, in particular in the production of biological and biocompatible matrices to be implanted in the human or animal body.
バイオプロテーゼ(bioprosthetic substitutes)の生産は、現在、大幅な市場拡大中である。外科的処置の臨床的改善、術後合併症の減少、新しい免疫調節薬の開発および管理が、移植片と宿主の間の相互作用メカニズムのより深い知識と一体となって、すべて、動物または相同組織により構成される生物学的プロテーゼの使用を促進するのに寄与する。この点で、代表的な1つのセクターは、特に心臓弁置換の確立された治療が引き起こし得る社会的および健康的な影響の点で、心血管セクターである。 The production of bioprosthetic substitutes is currently undergoing a significant market expansion. Clinical improvements in surgical procedures, reduction in postoperative complications, development and management of new immunomodulatory agents, all coupled with a deeper knowledge of the interaction mechanisms between graft and host, have led to the development of animal or homologous biomarkers. It contributes to promoting the use of tissue-constructed biological prostheses. In this regard, one sector that is representative is the cardiovascular sector, particularly in terms of the social and health impact that established therapies for heart valve replacement can cause.
生物医学技術は、機能不全の自然弁の開閉機能を模倣できる置換用の人工弁を開発し、外科的に適用することを可能とする。望ましいバイオプロテーゼ心臓弁(bioprosthetic heart valve substitute、BHV)は、類似の本来の健康な弁の流れと重り得る経弁流を可能にして、長寿命を確保し、溶血または血栓形成の影響を生じないようにしなければならない。 Biomedical technology allows the development and surgical application of replacement prosthetic valves that can mimic the opening and closing functions of dysfunctional native valves. Desirable bioprosthetic heart valve substitutes (BHVs) allow transvalvular flow that can overlap with similar native healthy valve flow to ensure longevity and no hemolysis or thrombogenic effects. must be done.
最も頻繁に用いられる弁代替物は、異種組織、特にブタ(pig)の弁、またはウシ、ウマまたはブタ(porcine)の心膜で製造された弁に由来する生物学的プロテーゼである。 The most frequently used valve substitutes are biological prostheses derived from heterologous tissue, especially pig valves or valves made of bovine, equine or porcine pericardium.
このような人工弁および代替物は、それらの臨床応用を著しく制限する多くの重大な問題に悩まされている。 Such prosthetic valves and substitutes suffer from a number of significant problems that severely limit their clinical application.
国際特許出願WO2017/093147は、バイオプロテーゼの製造のための生体組織における異種抗原、特にα-Galの不活化のための方法を開示している。石灰化に関する態様は、その有効性を裏付ける実験的記載がないことにより証明されるとして、付随的に言及されている。 International patent application WO2017/093147 discloses a method for the inactivation of heterologous antigens, in particular α-Gal, in living tissue for the production of bioprostheses. Aspects relating to mineralization are incidentally referred to as evidenced by the lack of experimental descriptions supporting their effectiveness.
米国特許出願US2014/018909は、特に脂質成分の酸化から生じる、糖尿病患者に蓄積する反応の生成物から組織を保護する、心血管分野での適用を意図した方法を開示している。これらの異化生成物は、AGEと名付けられ、糖尿病患者の血管および心臓弁などのバイオプロテーゼの変性メカニズムを促進する原因となる。当該方法は、ペンタガルホイルグルコース(PGG)に関連してのみ開示されており、ブタの心臓弁を脱細胞化することにより得られるコラーゲンマトリックス、および先に脱細胞化されたブタの頸動脈をアルカリで処理することにより得られるエラスチンマトリックスに対して行われる。 US patent application US2014/018909 discloses a method intended for cardiovascular applications to protect tissue from products of reactions that accumulate in diabetic patients, particularly resulting from oxidation of lipid components. These catabolites, termed AGEs, are responsible for promoting degenerative mechanisms of bioprostheses such as blood vessels and heart valves in diabetic patients. The method is disclosed only in connection with pentagalfoyl glucose (PGG), a collagen matrix obtained by decellularizing porcine heart valves, and a previously decellularized porcine carotid artery. It is carried out on elastin matrices obtained by treatment with alkali.
国際特許出願WO01/21228は、心臓弁代替物の製造に用い得る生物学的または合成組織の石灰化を軽減するためのガロタン酸の使用を開示している。 International Patent Application WO01/21228 discloses the use of gallotanic acid to reduce calcification of biological or synthetic tissue that may be used in the manufacture of heart valve replacements.
本特許出願の発明者らは、驚くべきことに、プロテーゼ、特に人工心臓の生体適合性を増加させるための方法を発見した。特に、生物適合性の増加は、石灰化沈着物の形成の防止、生物学的マトリックス中の異種抗原の不活性化、生物学的マトリックス中における血栓形成の防止、生物学的マトリックスへの脂質浸潤の防止、細胞成分が介在する炎症過程の開始の防止、生物学的マトリックスへの生物付着過程の防止の1つ以上の増加を含む。 The inventors of the present patent application have surprisingly discovered a method for increasing the biocompatibility of prostheses, especially artificial hearts. In particular, increased biocompatibility prevents the formation of calcified deposits, inactivates xenoantigens in biological matrices, prevents thrombus formation in biological matrices, and lipid infiltration of biological matrices. prevention of cellular component-mediated initiation of inflammatory processes; prevention of bioadhesion processes to biological matrices.
(本発明の目的)
第1の目的において、本発明は、生物学的マトリックスにおける石灰化沈着物の形成を防止するための方法を開示する。
(Object of the present invention)
In a first object, the present invention discloses a method for preventing the formation of calcified deposits in biological matrices.
好ましい実施形態において、該方法は、生物学的マトリックスをフェノール性化合物の混合物を含む溶液と暗所で35±2℃の温度にて2時間未満の時間接触させる工程を含む。 In preferred embodiments, the method comprises contacting the biological matrix with a solution comprising a mixture of phenolic compounds at a temperature of 35±2° C. in the dark for a period of less than 2 hours.
本発明の一態様によれば、開示する方法はまた、生物学的マトリックス中の異種抗原を不活性化する。 According to one aspect of the invention, the disclosed method also inactivates heterologous antigens in biological matrices.
本発明の別の態様によれば、開示する方法は、生物学的マトリックス上での血栓形成を防止する。 According to another aspect of the invention, the disclosed method prevents thrombus formation on biological matrices.
本発明の更なる態様によれば、開示する方法は、生物学的マトリックスへの脂質浸潤を防止する。 According to a further aspect of the invention, the disclosed methods prevent lipid infiltration into biological matrices.
本発明のさらに別の態様によれば、開示する方法は、細胞成分が介在する炎症過程の開始を防止する。 According to yet another aspect of the invention, the disclosed methods prevent initiation of inflammatory processes mediated by cellular components.
本発明の別の更なる態様によれば、開示の方法は、生物学的マトリックスへの微生物バイオフィルムの形成(生物付着)を防止する。 According to another further aspect of the invention, the disclosed method prevents the formation of microbial biofilms (biofouling) on biological matrices.
好ましい実施形態において、開示する方法は、石灰化沈着物の形成の防止、生物学的マトリックス中の異種抗原の不活性化、生物学的マトリックス上の血栓形成の防止、生物学的マトリックスへの脂質浸潤の防止、細胞成分が介在する炎症過程の開始の防止、生物学的マトリックスへの生物付着過程の防止の1つ以上を提供する。 In preferred embodiments, the disclosed methods prevent the formation of calcified deposits, inactivate xenoantigens in biological matrices, prevent thrombus formation on biological matrices, prevent thrombus formation on biological matrices, It provides one or more of the following: prevention of infiltration, prevention of initiation of inflammatory processes mediated by cellular components, prevention of bioadhesion processes to biological matrices.
より好ましい実施形態において、開示する方法は、石灰化沈着物の形成の防止、生物学的マトリックス中の異種抗原の不活性化、の防止生物学的マトリックス上の血栓形成の防止、の防止生物学的マトリックスへの脂質浸潤の防止、およびの防止細胞成分が介在する炎症過程の開始の防止、生物学的マトリックスへの生物付着過程の防止を提供する。 In a more preferred embodiment, the disclosed methods prevent the formation of calcified deposits, inactivate xenoantigens in biological matrices, prevent thrombus formation on biological matrices, prevent biological prevention of lipid infiltration into biological matrices, and prevention of initiation of inflammatory processes mediated by cellular components, prevention of biofouling processes into biological matrices.
本発明の方法で得られた生物学的マトリックスは、本出願の別の目的である。 A biological matrix obtained by the method of the invention is another object of the present application.
医学、生物医学および/または獣医学の分野における心疾患の処置に使用するための本発明の方法で得られた生物学的マトリックスは、本出願の第2の目的を表す。 A biological matrix obtained by the method of the invention for use in the treatment of heart disease in the medical, biomedical and/or veterinary fields represents a second object of the present application.
本発明の一態様において、本発明の方法に従って得られた生物学的マトリックスの使用を含む、ヒトまたは動物における心疾患の処置のための方法を開示する。 In one aspect of the invention, a method is disclosed for the treatment of heart disease in humans or animals comprising the use of a biological matrix obtained according to the method of the invention.
本特許出願の方法で用いるためのフェノール性化合物またはフェノール性化合物の混合物を含む溶液は、本発明の更なる目的を表す。 A solution comprising a phenolic compound or a mixture of phenolic compounds for use in the method of the present patent application represents a further object of the present invention.
本発明の別の態様によれば、フェノール性化合物の混合物を含む本発明の溶液を調製するための方法を開示する。 According to another aspect of the invention, a method for preparing a solution of the invention comprising a mixture of phenolic compounds is disclosed.
更なる目的において、本発明は、本発明の方法を実施するためのキットを開示する。 In a further object, the invention discloses kits for carrying out the methods of the invention.
(本発明の詳細な説明)
第1の目的によれば、本発明は、生物学的マトリックスにおける石灰化沈着物の形成を防止するための方法を開示する。
(Detailed description of the invention)
According to a first object, the present invention discloses a method for preventing the formation of calcified deposits in biological matrices.
本発明の目的のために、石灰化は、生物学的マトリックスの表面上または生物学的マトリックスの内部で生じ得る。 For purposes of the present invention, mineralization can occur on the surface of the biological matrix or within the biological matrix.
本明細書の目的のために、本発明による方法は、FACTATMと称される。 For the purposes of this specification, the method according to the invention is referred to as FACTA ™ .
特に、該方法は、生物学的マトリックスをフェノール性化合物の混合物を含む溶液と接触させる工程を含む。 In particular, the method includes contacting the biological matrix with a solution containing a mixture of phenolic compounds.
本発明の目的のために、生物学的マトリックスは、異種マトリックスであり、すなわち、ヒト起源のものではない。 For the purposes of the present invention, a biological matrix is a heterologous matrix, ie not of human origin.
特に、それらはウマ、ブタまたはウシを起源とし得る。 In particular, they may be of equine, porcine or bovine origin.
本発明の好ましい実施態様において、生物学的マトリックスは、血管、心臓弁、腱、靭帯、心膜、筋膜、硬膜、鼓膜、腸粘膜下組織、軟骨、脂肪組織および骨組織、骨盤、腹部および乳房の組織として意図されるべきである。 In preferred embodiments of the invention, the biological matrix is blood vessels, heart valves, tendons, ligaments, pericardium, fascia, dura mater, tympanic membrane, intestinal submucosa, cartilage, adipose and bone tissue, pelvis, abdomen. and breast tissue.
より好ましい実施形態において、生物学的マトリックスは、心血管プロテーゼ(心臓弁)および心膜組織パッチを含む群より選択される。 In a more preferred embodiment, the biological matrix is selected from the group comprising cardiovascular prostheses (heart valves) and pericardial tissue patches.
このような心臓弁の例は、TrifectaTM Valve with GlideTM TechnologyおよびEpicTM Mitral Valve(Abbott/St. Jude Medical, St. Paul, MN, USA)によって代表される。 Examples of such heart valves are represented by the Trifecta ™ Valve with Glide ™ Technology and the Epic ™ Mitral Valve (Abbott/St. Jude Medical, St. Paul, MN, USA).
例示のみを目的とする他の例は、Sapien 3、Sapien 3 UltraおよびSapien XT経カテーテル心臓弁(Edwards Lifesciences、Irvine、CA、USA)、Inspiris Resilia、Intuity Valve System、Magna Easy、Perimount RSRおよびPerimount Valve(Edwards Lifesciences、Irvine、CA、USA)、AvalusTM、Contegra Valved Conduit、FreestyleTM、Hancock IITM、MosaicTMおよびMosaicTM Ultra、MelodyTMおよびCoreValve 経カテーテル弁置換プラットフォーム(Medtronic、Minneapolis、MN、USA)、Acurate NeoTMおよびLotu EdgeTM Aortic Valve System(Boston Scientific、Marlborough、MA、USA)、Accufit(登録商標)Transapical Mitral Valve(Sinomed、Tianjin、China)、Xinli(登録商標)(KingstronBio、Jiangsu、China)、BioconduitTM、BiomitralTM、BiopulmonicTM ConduitおよびInjectable BiopulmonicTM(BioIntegral、Mississauga、ON、Canada)であり得る。
Other examples, for illustrative purposes only, are the
さらに好ましい実施形態において、本発明に従って処置できる心臓弁は、経カテーテル的弁留置術(TAVI)により埋め込み可能な心臓弁により表され;該弁は、カテーテルを通して埋め込まれる必要があり、したがって、カテーテル内に収容されるように柔軟である必要がある。 In a further preferred embodiment, the heart valves treatable according to the present invention are represented by heart valves implantable by transcatheter valve placement (TAVI); should be flexible to accommodate
本発明の目的のために、上記のフェノール性化合物混合物内のフェノール性化合物は、単純フェノール、フェノールアルデヒド、フェノール酸、フェニルアミン、フェノール化合物、フラボノイド、フェニルプロパノイドおよびタンニンを含む群から選択されるフェノール性化合物またはポリフェノール性化合物(場合により同義語として本明細書で用いる)として意図される。 For the purposes of the present invention, the phenolic compounds within the above phenolic compound mixture are selected from the group comprising simple phenols, phenolaldehydes, phenolic acids, phenylamines, phenolic compounds, flavonoids, phenylpropanoids and tannins. It is intended as a phenolic compound or a polyphenolic compound (as the terms are sometimes used interchangeably herein).
特に、フェノール性化合物は、バニリン、ケイ皮酸、フェニルアラニン、クマリン、キサントン、カテキン、フラボノニド、フラボン、カルコン、フラバノノール、フラバノール、ロイコアントシアニジン、アントシアニジン、ヒドロキシケイ皮酸、フェニルプロパノイドを含む群より選択される。 In particular, the phenolic compound is selected from the group comprising vanillin, cinnamic acid, phenylalanine, coumarin, xanthones, catechins, flavononides, flavones, chalcones, flavanonols, flavanols, leucoanthocyanidins, anthocyanidins, hydroxycinnamic acids, phenylpropanoids. be.
より具体的には、フェノール性化合物は、レスベラトロール、アロイン、シナリン、エピガロカテキン、タンニン酸、コーヒー酸、クロロゲン酸、ヒドロキシチロソール、ロスマリン酸、ナリゲニン、没食子酸、ヘスペリジン、キナ酸、エレノール酸、ピノレシノール、ルテオリン、アピゲニン、タンゲリチン、イソラムネチン、ケンフェロール、ミリセチン、エリオジクチオール、ヘスペレチン、ナリンゲニン、テアフラビン、テアルビジン、ダイゼイン、ゲニステイン、グリシテイン、プテロスチルベン、デルフィニジン、マルビジン、ペラルゴニジン、ペオニジン、チコリ酸、フェルラ酸、サリチル酸を含む群より選択され得る。 More specifically, the phenolic compounds are resveratrol, aloin, cinnaline, epigallocatechin, tannic acid, caffeic acid, chlorogenic acid, hydroxytyrosol, rosmarinic acid, narigenin, gallic acid, hesperidin, quinic acid, elenol acid, pinoresinol, luteolin, apigenin, tangeritin, isorhamnetin, kaempferol, myricetin, eriodictyol, hesperetin, naringenin, theaflavin, thearubigin, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, pelargonidin, peonidin, chicoric acid, ferulic acid It may be selected from the group comprising acid, salicylic acid.
本発明の一実施形態によれば、クルクミンも用い得る。 Curcumin may also be used according to one embodiment of the present invention.
本発明の目的のために、上記に開示したフェノール性化合物またはポリフェノール性化合物の誘導体も含まれ;例えば、塩またはエステルも用い得る。 Also included for purposes of the present invention are derivatives of the phenolic or polyphenolic compounds disclosed above; for example, salts or esters may also be used.
本発明の一実施態様において、溶液は、上記に開示したフェニルプロパノイドのうちの2つ以上の混合物を含む。 In one embodiment of the invention, the solution comprises a mixture of two or more of the phenylpropanoids disclosed above.
本発明の溶液内で、各フェニルプロパノイドは、約0.2~5mg/ml±0.5mg/ml(w/溶液の総量)の間に含まれる濃度で存在し得る。 Within the solutions of the invention, each phenylpropanoid may be present at a concentration comprised between about 0.2-5 mg/ml±0.5 mg/ml (w/total amount of solution).
以下に、上記の開示に従ったいくつかの溶液を示す:
特に、本発明の方法において、接触工程を2時間未満の時間実施する。 In particular, in the process of the invention the contacting step is carried out for a period of less than 2 hours.
好ましくは、接触工程を約1時間実施する。 Preferably, the contacting step is carried out for about 1 hour.
本発明の好ましい実施態様において、接触工程を約30分間継続する。 In a preferred embodiment of the invention, the contacting step lasts about 30 minutes.
より好ましい実施態様において、第1の接触工程を1回さらに30分間繰り返す。 In a more preferred embodiment, the first contacting step is repeated once for an additional 30 minutes.
所望により、2回の接触工程の間に、すすぎ工程を実施し得る。 Optionally, a rinsing step can be performed between the two contacting steps.
本発明の好ましい実施態様によれば、当該方法は、完全に暗所で、すなわち光への曝露を回避して実施する。 According to a preferred embodiment of the invention, the method is carried out in complete darkness, ie avoiding exposure to light.
接触工程の温度について、約35℃±2℃の温度で実施することが好ましい。 Regarding the temperature of the contacting step, it is preferred to carry out at a temperature of about 35°C ± 2°C.
本発明の好ましい実施態様において、接触工程の後、マトリックスを1回以上の洗浄工程に供し得る。 In preferred embodiments of the invention, the matrix may be subjected to one or more washing steps after the contacting step.
好ましくは、該洗浄工程は、適切な緩衝液を用いて実施し;例えば、適切な緩衝液は、リン酸緩衝液によって代表され得る。 Preferably, the washing step is performed using a suitable buffer; for example, a suitable buffer may be represented by phosphate buffer.
各洗浄工程は、約15分間実施し得る。 Each wash step may be performed for about 15 minutes.
本発明の一実施態様によれば、当該方法は、天然の生物学的マトリックス、ならびに、他の目的のために、例えばタンパク質、脂質および細胞の構造を安定化させるためにおよび宿主の潜在的な抗原作用を低下させるために、以前に処置されている生物学的マトリックスの両方で実施し得る。 According to one embodiment of the present invention, the method uses the natural biological matrix, as well as for other purposes, such as to stabilize proteins, lipids and cellular structures, and to the host's potential It can be performed on both previously treated biological matrices to reduce antigenic effect.
本発明の目的のために、生物学的マトリックスは、脱細胞化された細胞外マトリックス、部分的に消化されたマトリックス、および動物由来のゼラチンを含み得る。 For purposes of the present invention, biological matrices may include decellularized extracellular matrices, partially digested matrices, and animal-derived gelatins.
例えば、本発明の方法に従って生物学的マトリックスを処置する前に、それをグルタルアルデヒド、ホルムアルデヒドおよびケルセチンでの処置に供し得る。 For example, prior to treating a biological matrix according to the methods of the present invention, it may be subjected to treatment with glutaraldehyde, formaldehyde and quercetin.
本発明の一態様によれば、開示する方法はまた、生物学的マトリックス中の異種抗を不活性化する。 According to one aspect of the invention, the disclosed methods also inactivate xenoantibodies in biological matrices.
用語「異種抗原」は、免疫系により認識され、ヒト宿主生物に抗体/免疫介在性/炎症反応を誘発し得る動物起源を意図し;本明細書において、「異種抗原」、「抗原」、「異種抗原」、「エピトープ」および「危機的抗原」なる用語は、同じ意味を有し得て、一緒にまたは互いの代わりに用い得る。 The term "xenoantigen" intends an animal origin that can be recognized by the immune system and elicit an antibody/immune-mediated/inflammatory response in a human host organism; The terms "heterologous antigen", "epitope" and "critical antigen" can have the same meaning and can be used together or in place of each other.
本発明の目的のために、「異種抗原」は、α-Galエピトープを指す。 For the purposes of the present invention, "heterologous antigen" refers to the α-Gal epitope.
本発明の別の態様によれば、開示する方法は、生物学的マトリックス上およびその中の血栓形成および血塊を防止する。 According to another aspect of the invention, the disclosed methods prevent thrombus formation and clots on and within biological matrices.
本発明の更なる態様によれば、開示する方法は、生物学的マトリックスへの脂質浸潤を防止する。 According to a further aspect of the invention, the disclosed methods prevent lipid infiltration into biological matrices.
本発明のさらに別の態様によれば、開示する方法は、細胞成分が介在する炎症過程の開始を防止する。 According to yet another aspect of the invention, the disclosed methods prevent initiation of inflammatory processes mediated by cellular components.
本発明の別の更なる態様によれば、開示の方法は、生物学的マトリックス上での微生物バイオフィルムの形成(生物付着)を防止する。 According to another further aspect of the invention, the disclosed method prevents the formation of microbial biofilms (biofouling) on biological matrices.
特に、本発明の方法は、プロテーゼ表面の接着能力を低下させることにより、スタフィロコッカス・アウレウス(Staphylococcus aureus)により形成されるバイオフィルムの形成を防止することが証明されている。 In particular, the method of the present invention has been shown to prevent the formation of biofilms formed by Staphylococcus aureus by reducing the adhesion capacity of the prosthesis surface.
本発明の方法は、グラム陰性およびグラム陽性の両方の細胞に有効である。 The methods of the invention are effective for both Gram-negative and Gram-positive cells.
本発明の方法で得られた生物学的マトリックスは、本出願の別の目的である。 A biological matrix obtained by the method of the invention is another object of the present application.
特に、該生物学的マトリックスは、開示の方法で得られた心血管バイオプロテーゼによって代表され、これは、石灰沈着物の形成、血栓の構造化、リポタンパク質浸潤、ならびに細菌付着およびその結果の組織コロニー形成に対する有意な耐性を有する。 In particular, said biological matrix is represented by a cardiovascular bioprosthesis obtained by the disclosed method, which is characterized by the formation of calcification deposits, thrombus structuring, lipoprotein infiltration, and bacterial adhesion and resulting tissue Has significant resistance to colonization.
第2の目的によれば、本発明は、医学または獣医学の分野における心疾患の処置に使用するために上記の方法で得られた生物学的マトリックスを開示する。 According to a second object, the present invention discloses a biological matrix obtained by the method described above for use in the treatment of heart disease in the medical or veterinary field.
本発明の更なる目的として、本特許出願の方法で用いるためのフェノール性化合物の混合物を含む溶液が開示する。 As a further object of the present invention, a solution containing a mixture of phenolic compounds for use in the method of the present patent application is disclosed.
特に、該溶液は、2つ以上の上記のフェノール性化合物を混合することにより調製される。 In particular, the solution is prepared by mixing two or more of the above phenolic compounds.
より詳細には、該溶液は、適切な緩衝液中で調製され得る。 More specifically, the solution can be prepared in a suitable buffer.
例えば、リン酸ナトリウム緩衝液を用い得る。 For example, a sodium phosphate buffer may be used.
あるいは、フェノール性化合物の混合物は、NaOHの溶液中で調製され得る。 Alternatively, mixtures of phenolic compounds can be prepared in a solution of NaOH.
本発明の溶液は、オキシダーゼ活性を付与された適切な酵素を加えて;例えば、チロシナーゼ、L-グロノラクトンオキシダーゼ、ラッカーゼなどを用い得る。 Solutions of the present invention may be used with the addition of suitable enzymes endowed with oxidase activity; eg tyrosinase, L-gulonolactone oxidase, laccase and the like.
溶液は、好ましくは、暗所で、すなわち光への曝露を避けて調製する。 Solutions are preferably prepared in the dark, ie avoiding exposure to light.
本発明の第3の目的によれば、上記に開示した方法を実施するためのキットを開示する。 According to a third object of the invention, a kit is disclosed for carrying out the method disclosed above.
特に、該キットは、:
適切な緩衝液を含む容器、
溶解させるべき適量のフェノール性化合物の混合物を含む容器、
洗浄緩衝液を保持する1つ以上の容器、
手順の適用のタイミングおよび様式の説明を含む取扱説明書
を含む。
In particular, the kit comprises:
a container containing a suitable buffer,
a container containing an appropriate amount of the mixture of phenolic compounds to be dissolved;
one or more containers holding wash buffers;
An instruction manual containing instructions on when and how to apply the procedure is included.
本発明の一態様において、フェノール性化合物は、緩衝液に溶解させる粉末形態で存在し得る。 In one aspect of the invention, the phenolic compound may be present in powder form to be dissolved in a buffer.
本発明を、以下の実験セクションに関連してさらに説明する。 The invention is further described in connection with the experimental section below.
(実験セクション)
以下の実験セクションでは、用語「α-Gal抗原のノックアウト動物」は、α-ガラクトシルトランスフェラーゼ酵素をコードする遺伝子がサイレンシングされている動物を指す。このような酵素は、α-Galエピトープの膜糖タンパク質およびリポタンパク質を攻撃する役割を果たす。その非存在は、問題のエピトープを完全に欠き、この点で人体の組織に完全に匹敵する組織の生成を引き起こす。本発明では、α-Gal抗原のノックアウト動物血管組織を絶対陰性対照として用いた。
(experimental section)
In the experimental section below, the term "α-Gal antigen knockout animals" refers to animals in which the gene encoding the α-galactosyltransferase enzyme has been silenced. Such enzymes serve to attack membrane glycoproteins and lipoproteins for α-Gal epitopes. Its absence causes the production of tissue that is completely devoid of the epitope in question and perfectly comparable to that of the human body in this regard. In the present invention, α-Gal antigen knockout animal vascular tissue was used as an absolute negative control.
異種抗原の不活性化
市販のTrifecta GTTMバイオプロテーゼ大動脈心臓弁をパッケージから取り出し、リン酸緩衝液でそれぞれ15分間2回洗浄した。上記で示したフェノール性混合物に基づいて様々な溶液を調製し、0.2μmの滅菌膜でろ過した。Trifecta GTTM弁を、室温にて、それぞれ25±5分間2回、暗所で適度だが一定の撹拌下で、各単一溶液と共にインキュベートした。
Xenoantigen Inactivation Commercially available Trifecta GT ™ bioprosthetic aortic heart valves were removed from packaging and washed twice with phosphate buffer for 15 minutes each. Various solutions were prepared based on the phenolic mixtures shown above and filtered through a 0.2 μm sterile membrane. Trifecta GT ™ valves were incubated with each single solution at room temperature twice for 25±5 minutes each in the dark under moderate but constant agitation.
インキュベートの終わりに、処置したバイオプロテーゼ心臓弁をリン酸緩衝液でそれぞれ15分間2回洗浄した。 At the end of the incubation, the treated bioprosthetic heart valves were washed twice with phosphate buffer for 15 minutes each.
元のTrifecta GTTM弁のセットを、対照として未処置の既製品として採用した。 A set of original Trifecta GT ™ valves was taken as untreated off-the-shelf as a control.
処置試料の表面上でまだ活性であるエピトープの存在の評価は、発明者らが例示した方法の修正に基づくものであり、イタリア特許第0001409783号およびEP2.626.701に記載されている。 Evaluation of the presence of epitopes still active on the surface of treated samples is based on a modification of the method exemplified by the inventors and is described in Italian Patent No. 0001409783 and EP2.626.701.
簡潔には、処置および未処置のTrifectaTM弁からの組織試料を試験管に入れ、リン酸緩衝液を加えて、1000μL~1500μLの最終容量にした。 Briefly, tissue samples from treated and untreated Trifecta ™ valves were placed in test tubes and phosphate buffer was added to a final volume of 1000 μL-1500 μL.
その後、α-Galエピトープに対するモノクローナルマウス抗体(本実施例において、これはM86と呼ばれるIgMクローンである)を、[1:50]v/vの好ましい濃度で加え、全体を、一定であるが適度な撹拌下で37±2℃にて120±10分間インキュベートした。 A monoclonal mouse antibody directed against the α-Gal epitope (in this example, this is an IgM clone called M86) is then added at a preferred concentration of [1:50] v/v, and the whole is given a constant but moderate Incubate for 120±10 minutes at 37±2° C. under moderate agitation.
最後に、試料を周囲温度にて30±5分間、14,750×gで遠心分離した。 Finally, the samples were centrifuged at 14,750×g for 30±5 minutes at ambient temperature.
M86抗体とのインキュベート中に、ウェルの底を、ウェル(cell)当たり100μLのリン酸緩衝液中5μg/mlのα-Gal/血清アルブミンで覆った、96ウェルプレートを調製した。このようにして調製したプレートを、37℃にてすべてを安定化させることが好ましいが、30℃~40℃の温度にて60±10分間インキュベートした。 During incubation with M86 antibody, 96-well plates were prepared in which the bottom of the wells was covered with 100 μL per cell of 5 μg/ml α-Gal/serum albumin in phosphate buffer. Plates prepared in this manner were incubated for 60±10 minutes at a temperature between 30°C and 40°C, although all were preferably stabilized at 37°C.
その後、周囲温度にてリン酸緩衝液を用いて1ウェル当たり300μLで3回の洗浄を行った。第1の洗浄を5分間作用させ、その後2回の洗浄を3分間行った。 This was followed by 3 washes of 300 μL per well with phosphate buffer at ambient temperature. The first wash was allowed to act for 5 minutes, followed by two washes of 3 minutes.
ブロッキングを、血清アルブミンを用いて1ウェル当たり300μLで行い、続いて暗所で周囲温度にて60±10分間インキュベートした。その後、上記のように3回洗浄を行った。 Blocking was performed with serum albumin at 300 μL per well followed by incubation for 60±10 minutes at ambient temperature in the dark. Then three washes were performed as above.
各ウェルに、処置した各試料から遠心分離後に採取した100μLの上清を加えた。 To each well was added 100 μL of supernatant collected after centrifugation from each treated sample.
試料をプレートに負荷し、各タイプの試料がウェルのカラム全体を占有した。その後、プレートを、すべてを37℃にて安定させることが好ましいが、30℃~40℃の温度にて120±10分間インキュベートする。 Samples were loaded onto the plate, each type of sample occupying an entire column of wells. The plates are then incubated for 120±10 minutes at a temperature between 30°C and 40°C, although everything is preferably stabilized at 37°C.
その後、上記のように3回洗浄を行い、ペルオキシダーゼ酵素と結合した二次抗体(ウサギポリクローナル抗マウス)の溶液を1ウェル当たり100μL添加する(このような抗体の理想的な溶液は[1:1000]、[1:500]および[1:100]であることが分かっており、好ましくは中間の[1:500]を採用した)。 This is followed by 3 washes as above and the addition of 100 μL per well of a solution of secondary antibody (rabbit polyclonal anti-mouse) conjugated to peroxidase enzyme (ideal solution of such antibody is [1:1000 ], [1:500] and [1:100], preferably the middle [1:500] was taken).
その後、プレートを、すべてを37℃にて安定させることが好ましいが、30℃~40℃の温度にて60±10分間再度インキュベートする。 The plates are then incubated again for 60±10 minutes at a temperature between 30°C and 40°C, although everything is preferably stabilized at 37°C.
その後、上記のように3回の洗浄を行った。ペルオキシダーゼ酵素の開発溶液を1ウェル当たり100μL加え、続いてプレートを暗所で5±1分間インキュベートする。 This was followed by 3 washes as above. Add 100 μL per well of peroxidase enzyme development solution and then incubate the plate in the dark for 5±1 minutes.
続いて、H2SO4 2Mで構成される停止溶液を1ウェル当たり50μL加え、その後、プレートを450nmにてプレートリーダーで読み取った。 Subsequently, 50 μL per well of stop solution consisting of H 2 SO 4 2M was added, after which the plate was read on a plate reader at 450 nm.
採用した様々なポリフェノール混合物の全体的な分析は、未処置試料と比較して、処置TrifectaTM弁で80±2%~95±0.8%のα-Gal抗原の不活化を得ることが可能であることを示している。 Global analysis of the various polyphenol mixtures employed shows that it is possible to obtain 80±2% to 95±0.8% α-Gal antigen inactivation in treated Trifecta ™ valves compared to untreated samples. It is shown that.
同じタイプの処置弁および未処置弁を、次のインビトロおよびインビボ分析に供した。 Treated and untreated valves of the same type were subjected to the following in vitro and in vivo analyses.
特に、本発明による溶液を用いて処置を行った。 In particular, treatment was carried out with the solution according to the invention.
FACTA TM 処置の経時的安定性の評価
FACTATM処置は、本発明に係る特定のポリフェノールを含有する混合物の作用に基づいている。しかしながら、特定の用量のポリフェノールは毒性があり(LD50 5g/Kg)、肝臓および腎臓に蓄積し得る。FACTATM処置の安定性を確認するために、経時的なフェノール性残留物の放出を評価した。FACTATM処置後、市販BHV弁尖を、製造元から供給されたとおりに保管溶液中に放置した。7および14日ならびに1、3および9か月の種々の時点で、組織から放出されたフェノール性化合物の量を測定した(各時点でn=9の試料)。対照として、未処置の市販BHV弁尖のセットを同じ時点で分析した。溶液中に存在する総ポリフェノール含有量を、アルカリ性pHの条件下で、最終的に保存溶液中に存在するフェノール性化合物とジアゾニウム塩との間の反応に基づく手順を用いて評価した。反応生成物は安定した発色団であり、480nmの波長での吸光度分析により検出および定量化できる。試料中に存在するフェノールの濃度を、カテキン標準を用いた外部キャリブレーション法により定量し、カテキン当量のミリモル濃度(mM)として表す。
Evaluation of stability of FACTA TM treatment over time
The FACTA ™ treatment is based on the action of mixtures containing specific polyphenols according to the invention. However, certain doses of polyphenols are toxic (LD50 5 g/Kg) and can accumulate in liver and kidney. To confirm the stability of the FACTA ™ treatment, the release of phenolic residues over time was assessed. After FACTA ™ treatment, commercial BHV leaflets were left in storage solution as supplied by the manufacturer. The amount of phenolic compound released from the tissue was measured at various time points of 7 and 14 days and 1, 3 and 9 months (n=9 samples at each time point). As a control, a set of untreated commercial BHV leaflets were analyzed at the same time points. The total polyphenol content present in the solution was evaluated using a procedure based on the reaction between phenolic compounds and diazonium salts ultimately present in the stock solution under conditions of alkaline pH. The reaction product is a stable chromophore that can be detected and quantified by absorbance analysis at a wavelength of 480 nm. The concentration of phenol present in the samples was quantified by an external calibration method using catechin standards and expressed as millimolar concentrations (mM) of catechin equivalents.
結果を図1に示す。 The results are shown in Figure 1.
合計で、160ppmのフェノール性化合物が9か月で処置組織から放出された。放出は、処置後最初の14日間にほとんど生じる。毒性の可能性に関して、処置組織からのポリフェノール漏出の総量が健康上のリスクをもたらさないことが確認でき;実際、食事の摂取後のヒト血漿中に存在するポリフェノール濃度より低い。 In total, 160 ppm of phenolic compounds were released from treated tissues in 9 months. Release occurs mostly during the first 14 days after treatment. Regarding potential toxicity, it can be confirmed that the total amount of polyphenol leakage from the treated tissue does not pose a health risk; in fact, it is lower than the polyphenol concentrations present in human plasma after ingestion of a meal.
インビトロ石灰化アッセイ
未処置(B)およびFACTATM処置(BT)の市販BHVからの弁尖、天然(WT)およびFACTATM処置の天然の大動脈ブタ弁尖(WT T)およびα-Galノックアウトブタ大動脈弁尖(KO)を、2%のペニシリンおよびストレプトマイシンを含むプールした正常なヒト血清(Innovative Research、Peary Court Novi、MI)中で37℃にて14日間インキュベートした。対照として、試料のセットを同じ条件にてPBS中でインキュベートした。インキュベート後、試料を10分間、PBS中で2回洗浄し、12時間110℃にてHNO3中で酸加水分解に供した。カルシウムの評価を、EPA6010D法の指示に従って誘導結合プラズマにより加水分解された試料で実施し、μgCa2+/mgの乾燥脱脂重量(d.d.w.)として表す。
In vitro calcification assay Leaflets from untreated (B) and FACTA TM -treated (BT) commercial BHV, native (WT) and FACTA TM -treated native aortic pig leaflets (WT T) and α-Gal knockout porcine aorta Leaflets (KO) were incubated for 14 days at 37° C. in pooled normal human serum containing 2% penicillin and streptomycin (Innovative Research, Peary Court Novi, Mich.). As a control, a set of samples were incubated in PBS under the same conditions. After incubation, the samples were washed twice in PBS for 10 minutes and subjected to acid hydrolysis in HNO3 at 110°C for 12 hours. Calcium evaluation was performed on samples hydrolyzed by inductively coupled plasma according to the instructions of the EPA 6010D method and is expressed as μg Ca 2+ /mg dry defatted weight (ddw).
結果を図2に示す。 The result is shown in figure 2.
FACTATM処置BHV(BT)は、未処置BHV(B)およびα-Galノックアウト組織(KO)と比較してそれぞれ、90%(B対BT、p<0.05)および44.4%(BT対KO、p<0.05)の石灰性傾向が減少する。ノックアウト組織は、石灰化が最も少ない生物学的支持体と見なされ、異種移植誘発性石灰化試験で絶対陰性対照として使用される。FACTATM処置BHVは、KO標準より石灰化が約50%少ないことが示されている。FACTATM法を天然組織(WT T、グルタルアルデヒドで固定されていない)に適用すると、参照組織KOと比較した抗石灰化効果は85%に上昇する(WT T対KO、p<0.05)。 FACTA TM -treated BHV (BT) compared to untreated BHV (B) and α-Gal knockout tissue (KO) by 90% (B vs. BT, p<0.05) and 44.4% (BT vs. KO, p <0.05) decrease in calcareous tendencies. Knockout tissue is considered the least mineralized biological support and is used as an absolute negative control in xenograft-induced mineralization studies. FACTA ™ treated BHV has been shown to have approximately 50% less calcification than the KO standard. Application of the FACTA ™ method to native tissue (WT T, not glutaraldehyde-fixed) increases the anticalcification effect compared to reference tissue KO to 85% (WT T vs. KO, p<0.05).
流体力学的評価
弁の流体力学的性能を、Vivitro(登録商標)Pulse Duplicator System(Vivitro Labs lnc.、Victoria、BC、Canada)において模擬拍動流下で評価した。フローシュミレーターを用いて、心臓の左側をモデル化した。試験弁を、カスタムメイドのシリコンホルダー(図3)に取り付け、拍動流システムの大動脈位置に配置した。25mm Bjork-Shiley傾斜ディスク弁を、参照弁として僧帽弁位置に用いた。試験を、室温にて0.9%(w/v)NaCl中で実施した。弁を、ISO 5840-3に従って、5つの拍動流条件(表1)下で試験した。フロー条件は、2.5~9l/分の心拍出量に対応していた。試験中、拡張期/収縮期の血圧を80/120mm Hgに設定したが、収縮期の持続時間は心周期の35%を占めていた。各フロー条件にて、弁は5分間前処置した後、血圧およびフロー信号を各弁群の各フロー条件にて10サイクルで記録した(FACTATM処置BHV、n=3;未処置BHV、n=2)。
弁の流体力学的性能を、弁全体の平均圧力降下(MPD)とピーク圧力降下(PPD)、弁を通るルート平均二乗前方駆出血流(QRMS)、弁を通るピーク前方駆出血流(Qピーク)、弁有効オリフィス面積(EOA)、弁閉鎖中および弁が完全に閉じている間の弁を通る逆流、および弁エネルギー損失の点で評価した。ISO 5840-3で定義されているように、EOAを、前方駆出血流期の陽圧間隔中の体積流量および弁全体の圧力差のみを用いて測定した。各試験の結果を、記録したサイクル数で平均化した(n=10)。データを、Microsoft Excel(登録商標)およびPrism(登録商標)7 for Windows(v7.03、GraphPad Software lnc.、California)で分析し、平均±標準偏差(SD)として表した。対応のない両側t検定を用いて、0.05の信頼水準にて処置群と未処置群間の有意差を評価した。
The hydrodynamic performance of the valve was measured by mean pressure drop (MPD) and peak pressure drop (PPD) across the valve, root mean square forward ejection blood flow (Q RMS ) through the valve, and peak forward ejection blood flow through the valve. (Q- peak ), valve effective orifice area (EOA), regurgitation through the valve during valve closure and while the valve is fully closed, and valve energy loss. EOA, as defined in ISO 5840-3, was measured using only volumetric flow and pressure difference across the valve during positive pressure intervals during the forward ejection phase. The results of each test were averaged by the number of cycles recorded (n=10). Data were analyzed with Microsoft Excel® and
各フロー条件での10サイクルを平均した未処置弁および処置弁のMPD-QRMSプロファイルを、それぞれ図4Aおよび4Bに示す。両方の弁群は、それらの間で優れた再現性および同様のQRMS-PDプロファイルを示した。 MPD-Q RMS profiles of untreated and treated valves averaged over 10 cycles at each flow condition are shown in Figures 4A and 4B, respectively. Both groups of valves showed excellent reproducibility and similar Q RMS -PD profiles between them.
各フロー条件で計算した処置弁および未処置弁の平均EOAを図5に示す。フロー条件100/80(bpm/sv)を除いて、未処置群と処置群の間でEOAに統計的に有意な差はなく、処置群は未処置群と比較して有意に小さい(p<0.05)EOAを示した。
The average EOA for treated and untreated valves calculated for each flow condition is shown in FIG. Except for the
各条件での10サイクルを平均した未処置弁および処置弁のPPD-Qピークプロファイルは、それぞれ図6Aおよび6Bに示す。両方の群は、同様のPPD-Qピークプロファイルを示した。 PPD-Q peak profiles for untreated and treated valves averaged over 10 cycles for each condition are shown in Figures 6A and 6B, respectively. Both groups showed similar PPD-Q peak profiles.
各フロー条件での未処置群および処置群の平均動的および静的逆流量を図7に示す。両方の群の動的逆流(クロージング量)および静的逆流(クローズド量)は負であり、弁を通る逆流を表している。一般的に、逆流量は、より低いフロー条件でより顕著であり、流速の増加とともに増加した。未処置群と処置群の間で逆流量に統計的に有意な差は見られなかった。 The mean dynamic and static regurgitant flow for the untreated and treated groups for each flow condition are shown in FIG. Both groups of dynamic regurgitation (closing volume) and static regurgitation (closed volume) were negative, representing regurgitation through the valve. In general, backflow was more pronounced at lower flow conditions and increased with increasing flow rate. There was no statistically significant difference in reflux flow between untreated and treated groups.
処置群および未処置群の平均エネルギー損失を図8に示す。フロー条件が増加すると、前方駆出血流とクローズド期でのエネルギー損失について反対のパターンが観察された。前方駆出血流期中の最小のエネルギー損失は、低フロー条件(60/60)で観察され、流速の増加とともに増加した。対照的に、クローズド期中の最大のエネルギー損失は、低フロー条件で観察され、流速の増加とともに減少した。試験したフロー条件のいずれにおいても、未処置群と処置群の間でエネルギー損失に有意差はなかった。 Average energy loss for treated and untreated groups is shown in FIG. As the flow conditions increased, opposite patterns were observed for forward ejection blood flow and energy loss in the closed phase. Minimal energy loss during the forward ejection blood flow phase was observed at low flow conditions (60/60) and increased with increasing flow velocity. In contrast, the greatest energy loss during the closed phase was observed at low flow conditions and decreased with increasing flow velocity. There was no significant difference in energy loss between untreated and treated groups in any of the flow conditions tested.
生体力学的評価
破損までの低ひずみ速度の一軸引張荷重を、100Nロードセルを備えたlnstron(登録商標)引張試験機(5967 Dual Column Series)で実施した。1つの弁尖を各弁から切除し、試験寸法6×3mm(長さ×幅)の試験片を単離するために用いた。試料の厚さを、Mitutoyo(登録商標)厚さ計を用いて試料の長さに沿って3点で測定し、平均した。試験中、各試料に20mm/分のひずみ速度で0.01Nまで予め荷重をかけ、その後同じ速度で破損するまで荷重をかけた。各試料の記録された荷重-伸び応答を、工学的応力-工学的ひずみに変換した。応力-ひずみ曲線を用いて、弾性相およびコラーゲン相の勾配、遷移応力およびひずみ、破損ひずみならびに極限引張強さを計算した。データをExcelで分析し、平均±SDとして表した。対応のない両側t検定を用いて、0.05の信頼水準にて処置群と未処置群間の有意差を評価した。
Biomechanical Evaluation Low strain rate uniaxial tensile loading to failure was performed on an lnstron® tensile tester (5967 Dual Column Series) equipped with a 100N load cell. One leaflet was excised from each valve and used to isolate specimens with test dimensions of 6 x 3 mm (length x width). The sample thickness was measured at three points along the length of the sample using a Mitutoyo® thickness gauge and averaged. During testing, each specimen was preloaded to 0.01 N at a strain rate of 20 mm/min and then loaded at the same rate until failure. The recorded load-elongation response of each sample was converted to engineering stress-engineering strain. Stress-strain curves were used to calculate the elastic and collagen phase gradients, transition stress and strain, failure strain and ultimate tensile strength. Data were analyzed in Excel and expressed as mean±SD. A two-tailed unpaired t-test was used to assess significant differences between treated and untreated groups at a confidence level of 0.05.
FACTATM処置群および未処置群の平均生体力学的パラメーターを図9に示す。破損ひずみの場合、処置群と未処置群の間に有意差が見られた(p<0.05)。FACTATM処置群と未処置群の間で、他のパラメーターのいずれにも有意差は見られなかった。2つの群間の弾性相勾配(図9A)および遷移ひずみ(図9C)の場合、統計的有意性は見られなかったが、これらのパラメーターは、未処置群と比較して処置群で増加(エラスチン相勾配)または減少(遷移ひずみ)することが示された。より高い弾性相およびより低い遷移ひずみは、低ひずみの下で、それぞれより硬く、伸びにくい材料を示す。 Mean biomechanical parameters of FACTA ™ treated and untreated groups are shown in FIG. For strain to failure, a significant difference was found between treated and untreated groups (p<0.05). No significant differences in any of the other parameters were found between the FACTA ™ treated and untreated groups. No statistical significance was found for the elastic phase gradient (Fig. 9A) and transition strain (Fig. 9C) between the two groups, although these parameters were increased in the treated group compared to the untreated group (Fig. 9C). elastin phase gradient) or decreased (transition strain). A higher elastic phase and a lower transition strain indicate a harder and less extensible material under low strain, respectively.
ブタ動物モデルにおけるインビボ研究
FACTATM処置(n=5)および未処置(n=3)の市販のTrifecta GT BHV(St.Jude Medical, USA)を、ブタ動物モデルの肺の位置に埋め込んだ。1か月のフォローアップ後、BHVを検査のために取り除いた。外植されたBHVを、4℃の滅菌PBSで15分ステップで2回洗浄し、巨視的評価のためにすぐに写真が撮った(図10)。続いて、弁尖を各弁から個別に切除した。組織学的および免疫組織化学的試験のための弁尖をすぐにOCT(最適切断温度)コンパウンドに包埋し、残りの弁尖をELISA試験によるα-Gal定量化に供した。非移植Trifecta BHVを参照対照として用いた。
In vivo studies in porcine animal models
FACTA ™ -treated (n=5) and untreated (n=3) commercial Trifecta GT BHV (St. Jude Medical, USA) were implanted at the lung location in porcine animal models. After 1 month follow-up, BHV was removed for examination. Explanted BHVs were washed twice in 15 minute steps with sterile PBS at 4° C. and immediately photographed for macroscopic evaluation (FIG. 10). The leaflets were then excised individually from each valve. Leaflets for histological and immunohistochemical studies were immediately embedded in OCT (optimal cutting temperature) compound and the remaining leaflets were subjected to α-Gal quantification by ELISA test. Non-implanted Trifecta BHV was used as a reference control.
肉眼検査
図10から分かるとおり、未処置の市販BHVは、FACTATMで処置されたものと比較して、心室表面に重大な線維性沈着物および血塊が存在することを示している。未処置BHVのこれらの特有の特性は、大動脈表面の縫合リング周囲と弁尖上の両方での均質で厚いパンヌスの形成とともに、図11でよりよく検出できる。
As can be seen from gross inspection FIG. 10, untreated commercial BHV show significant fibrous deposits and clots on the ventricular surface compared to those treated with FACTA ™ . These unique features of untreated BHV are better detectable in Figure 11, along with the formation of a homogenous thick pannus both around the suture ring on the aortic surface and over the leaflets.
組織学的分析
組織をOCTコンパウンド(Tissue Tek;Sakura Finetek、Tokyo、Japan)に包埋し、液体窒素で凍結冷却(cryocooled)し、6mmの凍結切片に切断した。組織学的分析を、Bio-Optica(Milan、Italy)の市販キットを用いて、製造元から提供される指示に従って実施した。用いた組織学キットを表2に示す。
一般的に、未処置の弁からの弁尖は、図12(ボックスAおよびE)の黒色の矢印で示すように心室表面に均質な線維性パンヌスが存在することを示す。異物反応は、未処置(図12)とFACTATM処置の市販BHV(図13)の両方で、外植されたすべての弁尖にはっきりと見られる。未処置の市販の弁では、細胞成分がマトリックスに浸透できることに留意されたい(図12、ボックスDおよびH)。 In general, leaflets from untreated valves show the presence of homogeneous fibrous pannus on the ventricular surface as indicated by the black arrows in Figure 12 (boxes A and E). A foreign body reaction is evident in all explanted leaflets, both untreated (Figure 12) and FACTA ™ -treated commercial BHV (Figure 13). Note that cellular components are able to penetrate the matrix in untreated commercial valves (Fig. 12, boxes D and H).
FACTATM処置は、細胞外マトリックスのその後の変性の原因となる炎症性細胞成分(図13、ボックスC)の内部間質への浸透を打ち消すバリア効果を発揮できると考えられる。 It is conceivable that FACTA ™ treatment can exert a barrier effect that counteracts the penetration of inflammatory cellular components (FIG. 13, Box C) into the inner stroma responsible for the subsequent degeneration of the extracellular matrix.
未処置の弁では、線維性パンヌスに内在し、細胞層に隣接する領域に、図14(ボックスA、B、CおよびD)に示すとおり、おそらくLDLを処理できない単球の作用のため、豊富な脂質浸潤の存在を観察することが可能である。単球は脂質滴を取り込むが、それらを分解することはできず、泡沫細胞になり、その後カルシウム沈着の除核部位として機能する。FACTATM処置は、この危機的状態からの組織の保存を保証する(図14、ボックスE、F、G、およびH、脂質浸潤は存在しない)。 In untreated valves, areas residing in the fibrous pannus and adjacent to the cell layer have an abundance of It is possible to observe the presence of lipid infiltration. Monocytes take up lipid droplets but are unable to break them down, becoming foam cells and subsequently serving as enucleation sites for calcification. FACTA TM treatment ensures preservation of tissue from this critical condition (Figure 14, boxes E, F, G and H, no lipid infiltration present).
脂質浸潤の大量の存在から予測できるように、ブタにおける1か月のフォローアップ後の未処置の市販BHVは著しく石灰化しており、特に弁尖の外部界面のレベルでの顕著なカルシウム集積が見られる(図15)。しかしながら、カルシウムの大きな沈着が明らかでないところは、間質の内部に影響を及ぼす微小石灰化の広範な広がりを示す(図15、ボックスD、E、GおよびH)。 As expected from the massive presence of lipid infiltrate, untreated commercial BHV after 1-month follow-up in pigs was markedly calcified, with marked calcium accumulation, especially at the level of the external interface of the leaflets. (Fig. 15). However, where large deposits of calcium are not evident, it shows widespread spread of microcalcifications affecting the interior of the stroma (Fig. 15, boxes D, E, G and H).
図16から分かるように、FACTATM処置の市販BHVを、カルシウム沈着から最適に保護する。 As can be seen from Figure 16, FACTA ™ treatment optimally protects commercial BHV from calcification.
図17に示すように、線維性パンヌスの発生に加えて、未処置の市販の弁は、弁尖の心室表面に構造化された血栓が発生する極めて強い傾向を示した。 As shown in FIG. 17, in addition to developing fibrous pannus, untreated commercial valves showed a very strong tendency to develop structured thrombi on the ventricular surface of the leaflets.
特に、図18には、構造化された血栓形成に至る繊維状のパンヌスの存在を示すパネルが示されている。 In particular, FIG. 18 shows a panel showing the presence of fibrous pannus leading to structured thrombus formation.
マロリー3色染色では、FACTATM処置の市販BHVは、パンヌスまたは構造化された血栓などの異常な線維形成の存在を示さない(図19)。弾性繊維に対応する、いくつかの外植された試料(図19、ボックスEおよびF、GおよびH)におけるコラーゲンマトリックス内部のピンク/紫の縞の存在に注意することが重要である。興味深いことに、このような弾性繊維は、未処置の市販BHVの外植片には全く見られない。 By Mallory's trichrome staining, FACTA ™ -treated commercial BHV does not show the presence of abnormal fibrosis such as pannus or structured thrombi (FIG. 19). It is important to note the presence of pink/purple streaks inside the collagen matrix in some of the explanted samples (Fig. 19, boxes E and F, G and H), corresponding to elastic fibers. Interestingly, no such elastic fibers are found in untreated commercial BHV explants.
動物モデルに埋め込まれていない市販BHVを、参照対照として組織学的に処置した。試験から、この組織が異物反応を示さず(図20、ボックスAおよびB)、その結果、線維性パンヌスまたは血栓の発生による影響を受けないことが明らかになった(図20、ボックスEおよびF)。脂質浸潤または石灰化沈着物もない(図20、それぞれボックスCおよびD、GおよびH)。弾性繊維は、FACTATM処置の市販BHV(図19、ボックスEおよびF、GおよびH)に見られるものと同様に、マロリー染色(図20、ボックスEおよびF)によって十分に代表されることに留意されたい。 Commercially available BHV not implanted in animal models were treated histologically as a reference control. Testing revealed that this tissue did not exhibit a foreign body reaction (Figure 20, Boxes A and B) and was consequently unaffected by the development of fibrous pannus or thrombi (Figure 20, Boxes E and F). ). There are also no lipid infiltrates or calcified deposits (Fig. 20, boxes C and D, G and H, respectively). Elastic fibers were found to be well represented by Mallory staining (Fig. 20, boxes E and F), similar to those seen in FACTA TM -treated commercial BHV (Fig. 19, boxes E and F, G and H). Please note.
図21は、FACTATM処置の市販BHVの異なる組織学的染色を示す。このパネルは、FACTATM処置がバイオプロテーゼ組織に提供するすべての改善を要約する。処置により発揮されるバリア効果が明確に示されており;実際、宿主細胞集団はマトリックス内に浸透できない(ボックスBおよびD)。このバリア効果は、ボックスEに示すように、脂質浸潤が存在しないことによりさらに確認される。最後に、組織には石灰沈着がない(ボックスF)。 FIG. 21 shows different histological staining of FACTA ™ -treated commercial BHV. This panel summarizes all the improvements that FACTA ™ treatment provides to bioprosthetic tissue. The barrier effect exerted by the treatment is clearly demonstrated; indeed, the host cell population cannot penetrate into the matrix (boxes B and D). This barrier effect is further confirmed by the absence of lipid infiltration, as shown in Box E. Finally, the tissue is free of calcification (Box F).
細菌付着に対する耐性
FACTATM処置および未処置の市販の弁に対する抗接着性細菌活性を、スタフィロコッカス・アウレウスATCC 6538(グラム陽性)の細菌種で評価した。細菌を、37℃にてトリプシンソイブロス(TSB)中で一晩増殖させた。総細菌量を、TSB中の10倍段階希釈(10-1から10-7)により評価し、適切な選択培地(MSA-マンニトール選択寒天)を含むペトリ皿に播種し、一晩インキュベーターに保持した。インキュベートの最後に、コロニー形成単位(UFC)を計数して、微生物の有効濃度を決定した。さらに、600nmでの光学密度(OD600)の値を、後者とブロスの有効な微生物量との間の直線性を検証するために、各段階(tiled)希釈から決定した。市販のFACTATM処置および未処置のBHV弁尖を、細菌接着に同じ有用な表面を得るために、生検用パンチ(直径3mm)で切断した。このようにして得られた組織パンチをPBSで洗浄し、適度であるが一定の撹拌下で、PBS+ゲンタマイシン(300μg/mL)中で室温にて一晩インキュベートした。
resistance to bacterial adhesion
Anti-adhesive bacterial activity against FACTA ™ -treated and untreated commercial valves was evaluated with the bacterial strain Staphylococcus aureus ATCC 6538 (Gram-positive). Bacteria were grown overnight in tryptic soy broth (TSB) at 37°C. Total bacterial load was assessed by 10-fold serial dilutions (10-1 to 10-7) in TSB, plated on petri dishes containing the appropriate selective medium (MSA-mannitol selective agar) and kept overnight in an incubator. . At the end of the incubation, colony forming units (UFC) were counted to determine the effective concentration of microorganisms. In addition, optical density (OD600) values at 600 nm were determined from each tiled dilution to verify the linearity between the latter and the effective microbial load of the broth. Commercially available FACTA ™ -treated and untreated BHV leaflets were cut with a biopsy punch (3 mm diameter) to obtain the same useful surface for bacterial adhesion. The tissue punches thus obtained were washed with PBS and incubated overnight at room temperature in PBS plus gentamicin (300 μg/mL) under moderate but constant agitation.
一晩インキュベート後、市販BHV組織パンチをPBSで徹底的に洗浄して、残っている抗生物質を除去した。続いて、FACTATM処置試料および未処置試料を、S.アウレウスの細菌懸濁液(細菌負荷1×107CFU/mL)に、室温にて適度であるが一定の撹拌下で90分間曝露した。インキュベートの終わりに、組織試料を3回の適度なボルテックス混合に供して、緩く結合した細菌の分離を促進した。
After overnight incubation, commercial BHV tissue punches were washed extensively with PBS to remove residual antibiotic. FACTA ™ -treated and untreated samples were then exposed to a bacterial suspension of S. aureus (
続いて、異なる組織パンチをUltraturraxによりホモジナイズし、得られたホモジネートを段階希釈し、適切な選択的増殖培地を含むペトリ皿に固定させた。最後に、37℃にて24時間インキュベートした後、試料の各タイプについてコロニー形成単位を係数した。細菌の抗付着活性を、以下の式を用いて計算した:
図22に示すように、FACTATM処置は、組織表面でのS.アウレウスの接着能力を著しく制限する(96±4%の低下)。 As shown in Figure 22, FACTA ™ treatment significantly limits the ability of S. aureus to adhere to tissue surfaces (96±4% reduction).
野生型マウス動物モデルにおける石灰化緩和特性
FACTATM技術の石灰化緩和特性を評価するために、Trifecta GTTMモデル(Abbott/St. Jude)からの処置(F)および未処置(C)の心膜のバイオプロテーゼ心臓弁(BHV)弁尖を、C57Bl/6野生型マウスの背中に皮下埋込みした。合計30匹のマウスが登録され、各動物は1個の試料を受け取った。組織試料を、1、2および4か月のフォローアップ後に外植した。対照試料として、カルシウム定量もまた、既製品の元のTrifecta GTTM弁尖、標識UN(未埋込)で行った。カルシウム含有量を、誘導結合プラズマ(ICP - 0.048μg/mg組織のカルシウム定量閾値を有する特定のタイプの質量分析)により評価し、1mgの乾燥脱脂重量(d.d.w.)当たりのμgとして表した。
Calcification relaxation properties in a wild-type mouse animal model
Treated (F) and untreated (C) pericardial bioprosthetic heart valve (BHV) leaflets from the Trifecta GT TM model (Abbott/St. Jude) to evaluate the calcification relaxation properties of the FACTA TM technique. were implanted subcutaneously into the backs of C57BI/6 wild-type mice. A total of 30 mice were enrolled and each animal received one sample. Tissue samples were explanted after 1, 2 and 4 months follow-up. As a control sample, calcium quantification was also performed on off-the-shelf original Trifecta GT ™ leaflets, labeled UN (unimplanted). Calcium content was assessed by inductively coupled plasma (ICP - a specific type of mass spectrometry with a calcium quantification threshold of 0.048 μg/mg tissue) and expressed as μg per mg dry defatted weight (ddw).
ICP分析により行ったカルシウム定量をさらに確認するために、4か月のフォローアップ後のFACTATM処置試料(F)および未処置試料(C)の組織学的調製を行った。組織学的調製物を、生体組織におけるカルシウム沈着を強調するための特定の技術であるフォン・コッサ染色により分析した。 To further confirm the calcium quantification performed by ICP analysis, histological preparations of FACTA ™ -treated (F) and untreated (C) samples after 4 months follow-up were performed. Histological preparations were analyzed by von Kossa staining, a specific technique for highlighting calcification in living tissue.
ICP分析によるカルシウム定量の結果を図23に示す。 The results of calcium quantification by ICP analysis are shown in FIG.
未処置の元の試料(C)は、埋込のわずか1か月後に石灰化するかなりの傾向を示した。当該傾向は、フォローアップ中に顕著に継続し、埋込の4か月の終わりに定量した1mgのd.d.w.当たり7.17μgのカルシウム沈着をもたらす(表1)。一方、FACTATM処置試料(F)は、4か月のフォローアップ後でもごくわずかな量のカルシウムを示す。注目すべきことに、F試料のカルシウム含有量は、UNで検出された総カルシウムを有意に超えておらず、経時的にカルシウムの取り込みがないことが確認された(表1)。
フォン・コッサ組織染色の結果を図24に示す。 The results of von Kossa tissue staining are shown in FIG.
カルシウム沈着物は黒色の斑点として認識できる(存在する場合は矢印で示される)。ICP分析によると、FACTATM処置試料(F)は、マウスの皮下組織における4か月のフォローアップ後のカルシウム沈着の有意な存在を示さない。一方、元の未処置の対照試料(C)は、様々なカルシウム凝集体の存在を示しており、かなりの寸法のものもある(C4ボックス)。 Calcium deposits are visible as black spots (indicated by arrows if present). By ICP analysis, the FACTA ™ treated sample (F) shows no significant presence of calcification in the subcutaneous tissue of mice after 4 months follow-up. On the other hand, the original untreated control sample (C) shows the presence of various calcium aggregates, some of considerable size (C4 box).
α-Galノックアウトマウス動物モデルにおける石灰化緩和特性
試験の目的は、α-GALノックアウト(KO)マウスの動物モデルに埋め込まれると、市販のTrifecta GTTMのBHVおよびそのFACTATM処置対応物から切除された弁尖中のカルシウム取り込みの評価であった。KOマウスは、α-Gal異種抗原の発現を抑制するように遺伝子改変された、特別に開発されたBCI保有マウスモデルである。α-Gal KOマウスは、α-Gal抗原が特定の抗Gal抗体の産生を刺激する、ヒトと同様の免疫応答メカニズムを有することにより特徴付けられる。弁尖を、C57BI/6α-Gal KOマウスの背中に皮下埋込みした。
Calcification relaxation properties in an α-Gal knockout mouse animal model The aim of the study was to test excised BHV of the commercially available Trifecta GT ™ and its FACTA ™ -treated counterparts when implanted in an α-GAL knockout (KO) mouse animal model. It was an assessment of calcium uptake in the valve cusps. The KO mouse is a specially developed BCI-bearing mouse model genetically modified to suppress expression of the α-Gal xenoantigen. α-Gal KO mice are characterized by having immune response mechanisms similar to humans, in which α-Gal antigen stimulates the production of specific anti-Gal antibodies. Leaflets were implanted subcutaneously into the back of C57BI/6α-Gal KO mice.
カルシウム含有量を、誘導結合プラズマ(ICP - 特定のタイプの質量分析)により評価し、1mgの乾燥脱脂重量(d.d.w.)当たりのμgとして表した。市販のTrifecta GT弁(Abbott/St. Jude)から切除したFACTATM処置弁尖(F)および元の弁尖(C)を、α-Galマウスの野生型(WT)およびノックアウト(KO)の背中に皮下埋込みした。各動物は1個の試料を受け取った。 Calcium content was assessed by inductively coupled plasma (ICP - a specific type of mass spectrometry) and expressed as μg per mg dry defatted weight (ddw). FACTA TM -treated (F) and original (C) leaflets excised from commercial Trifecta GT valves (Abbott/St. Jude) were placed on the backs of wild-type (WT) and knockout (KO) α-Gal mice. was subcutaneously implanted in the Each animal received one sample.
対照試料として、カルシウム定量もまた、既製品の元のTrifecta GTTM弁尖、標識UN(未埋込)で行った。 As a control sample, calcium quantification was also performed on off-the-shelf original Trifecta GT ™ leaflets, labeled UN (unimplanted).
結果を図25に示す。 The results are shown in FIG.
1か月および2か月のフォローアップにてF試料中で決定されたカルシウムの量は、ICP定量閾値(0.048μg/mg)より低いため、ごくわずかであった。F試料中のカルシウム含有量は、UNで検出された総カルシウムを超えておらず、1か月および2か月のフォローアップにてカルシウムの取り込みがないことが確認された。一方、KOに埋め込まれたC標本は、カルシウムの有意かつ均質な存在を示すことにより、石灰沈着促進効果を示す。結果は、ヒトに生じるのと同様に、KOマウスでのカルシウム沈着の促進におけるα-Galの明確な役割を示している。 The amount of calcium determined in the F samples at 1 and 2 months follow-up was negligible as it was below the ICP quantification threshold (0.048 μg/mg). The calcium content in the F samples did not exceed the total calcium detected in the UN, confirming no calcium uptake at 1 and 2 months follow-up. On the other hand, C specimens embedded in KO show a procalcification effect by showing a significant and homogeneous presence of calcium. The results demonstrate a distinct role for α-Gal in promoting calcification in KO mice, similar to what occurs in humans.
FACTATM処置は、α-Gal KOモデルのような重要な生理学的システムでも石灰化を中和するのに効果的であることが証明されている。 FACTA ™ treatment has proven effective in neutralizing calcification also in important physiological systems such as the α-Gal KO model.
上記の開示から、本発明により提供される利点は、当業者には直ちに明らかである。 From the above disclosure, the advantages provided by the present invention are readily apparent to those skilled in the art.
例えば、本発明の方法は、カルシウム塩の沈着を制限でき、それ故に、弁の石灰性ジストロフィーのエピソードの形成を防止できる。 For example, the method of the present invention can limit the deposition of calcium salts and thus prevent the formation of episodes of valvular calcific dystrophy.
また、本発明の方法は、処置された心血管バイオプロテーゼを血餅および構造化血栓の形成に対して保護することを示した。 The methods of the present invention have also been shown to protect treated cardiovascular bioprostheses against the formation of clots and structured thrombi.
本発明の方法は、血液循環からのリポタンパク質の浸潤、およびその結果としての、処置された心血管バイオプロテーゼにおける細胞介在性炎症組織応答の開始を回避することを可能にする。 The method of the invention makes it possible to avoid the infiltration of lipoproteins from the blood circulation and the consequent initiation of a cell-mediated inflammatory tissue response in the treated cardiovascular bioprosthesis.
最後に、本発明により、細菌によるコロニー形成を回避し、心内膜炎の発生から心血管組織を保護する方法が開発された。 Finally, in accordance with the present invention, a method has been developed to avoid bacterial colonization and protect cardiovascular tissue from the development of endocarditis.
本発明で開示した方法は、極めて安定で安全であることが証明された。 The method disclosed in the present invention has proven to be extremely stable and safe.
収集した証拠は、処置弁の流体力学的および生体力学的完全性を有意に変化させなかったことを示している。それにもかかわらず、処置弁に対して得られた流体力学的パラメーターの値は、ISO5840-3規格に示されている値の範囲内である。 Collected evidence indicates that it did not significantly alter the hydrodynamic and biomechanical integrity of the procedural valve. Nevertheless, the hydrodynamic parameter values obtained for the procedural valves are within the range of values given in the ISO 5840-3 standard.
また、当該方法は、インビトロおよびインビボの研究の両方で、石灰化沈着物の形成を阻害できることが証明された。FACTATMで処置されたBHVは、明らかに血栓形成性ではなく、細胞および脂質の浸潤から保護された。 Also, the method has been demonstrated in both in vitro and in vivo studies to be able to inhibit the formation of calcified deposits. BHV treated with FACTA ™ were apparently not thrombogenic and were protected from cellular and lipid infiltration.
最後であるが、重要なことには、本発明により、従来のデバイスおよび機械で実施できる方法が考案された。 Last but not least, the present invention has devised a method that can be implemented with conventional devices and machines.
本発明キットに関して、クリニックおよび病院などの医療施設において有用な、上記の本発明による方法により、すでに調製されたバイオプロテーゼの自律的処置を目的とする。 With respect to the kit of the invention, it is intended for the autonomous treatment of bioprostheses already prepared by the method according to the invention described above, useful in medical facilities such as clinics and hospitals.
本発明は、多くの改変および変更の影響を受けやすく、それらはすべて、特許請求の範囲内にある。 The invention is susceptible to many modifications and variations, all of which fall within the scope of the claims.
さらに、すべての構成要素は、他の技術的に同等の構成要素で置き換え得る。 Moreover, all components may be replaced by other technically equivalent components.
Claims (23)
の1つを含む、請求項1~6のいずれか一項に記載の方法。 The solution is a combination of the following phenylpropanoids:
A method according to any one of claims 1 to 6, comprising one of
の1つを含む、請求項13~17のいずれか一項に記載の溶液。 The solution is a combination of the following phenylpropanoids:
The solution according to any one of claims 13-17, comprising one of
緩衝液に溶解させるべき適量の請求項13~18のいずれか一項に記載の溶液を含む容器
洗浄緩衝液を含む1つ以上の容器、
該方法を実施するためのタイミングおよび様式の説明を含む取扱説明書
を含む、請求項1~10のいずれか一項に記載の方法を実施するためのキット。 a container containing a suitable buffer,
a container containing a suitable amount of the solution according to any one of claims 13 to 18 to be dissolved in a buffer one or more containers containing a wash buffer;
A kit for performing the method according to any one of claims 1 to 10, comprising instructions containing instructions on when and how to perform the method.
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PCT/IB2020/054885 WO2020234845A1 (en) | 2019-05-22 | 2020-05-22 | Method for preventing the formation of calcified deposits and for inactivating xenoantigens in biological matrices |
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CN118105544A (en) * | 2022-11-30 | 2024-05-31 | 上海微创心通医疗科技有限公司 | Biological tissue material and preparation method and application thereof |
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US20040153145A1 (en) * | 2002-11-26 | 2004-08-05 | Clemson University | Fixation method for bioprostheses |
CN100360190C (en) * | 2005-11-30 | 2008-01-09 | 中国科学院上海硅酸盐研究所 | Method for preparing artificial biological valve with biological activity |
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