TW201141510A - Pharmaceutical compositions for the stimulation of stem cells - Google Patents

Pharmaceutical compositions for the stimulation of stem cells Download PDF

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TW201141510A
TW201141510A TW099141986A TW99141986A TW201141510A TW 201141510 A TW201141510 A TW 201141510A TW 099141986 A TW099141986 A TW 099141986A TW 99141986 A TW99141986 A TW 99141986A TW 201141510 A TW201141510 A TW 201141510A
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pharmaceutical composition
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pharmaceutical
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TW099141986A
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Chinese (zh)
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Roland Gordon-Beresford
Vinciane Gaussin
Christian Homsy
Dwan Isa Jean-Pierre Latere
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Cardio3 Biosciences Sa
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/529—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim forming part of bridged ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1833—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Endocrinology (AREA)
  • Cardiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Diabetes (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Vascular Medicine (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Hospice & Palliative Care (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to a human or veterinary pharmaceutical composition (B) for the stimulation of stem cells, comprising at least two stem-cells-stimulating-agents and at least one pharmaceutically acceptable excipient.

Description

201141510 ·' 六、發明說明： ， 【發明所屬之技術領域】 [0001] 本發明係一般關於適合針對組織及/或器官之醫藥組成物 。特別是，其係相關於對需要心臟組織再生之個體投予 幹細胞刺激物至其心臟，而治療心臟疾病。特別是本 發明揭露使用該幹細胞刺激物，進而在活體内以心臟幹 細胞增進心臟組織的再生。 【先前技術】 [0002] 心肌梗塞（MI)導致心肌喪失、疤痕產生、心室重新建構 Ο ，而最終心臟衰竭。藥理方面、基於導管方面及外科方 面之介入已改善冠狀動脈疾病（CAD)病人之存活率，雖然 他們不能再生已死之心肌。結果，減少之死仓率（mortal ity ) 因為缺血性心臟衰竭 而伴隨著增加之病態 (morbidity)。近幾年來，以幹細胞為基礎之治療已興 起’其對心臟修復為一具潛力之策略（Dimmeler S. et al·’ J Clin Invest 2005，11，572-583)。對於修 復損壞的心肌之最理想細胞來源為密集研究之主題。能201141510 · ' VI. Description of the invention: , [Technical field to which the invention pertains] [0001] The present invention relates generally to pharmaceutical compositions suitable for tissues and/or organs. In particular, it relates to the treatment of heart diseases by administering stem cell stimuli to the heart of an individual in need of cardiac tissue regeneration. In particular, the present invention discloses the use of the stem cell stimulant, thereby enhancing cardiac tissue regeneration by cardiac stem cells in vivo. [Prior Art] [0002] Myocardial infarction (MI) leads to myocardial loss, scarring, ventricular remodeling, and ultimately heart failure. Pharmacological, catheter-based, and surgical interventions have improved the survival rate of patients with coronary artery disease (CAD), although they are unable to regenerate dead myocardium. As a result, the reduced mortality is accompanied by increased morbidity due to ischemic heart failure. In recent years, stem cell-based therapies have emerged as a potential strategy for cardiac repair (Dimmeler S. et al.' J Clin Invest 2005, 11, 572-583). The most ideal source of cells for repairing damaged myocardium is the subject of intensive research. can

Q 使心肌再生的幹細胞’其重要特徵包括自我更新、複製 產生（clonogenicity)以及分化成心肌細胞 '内皮細胞 及血管平滑肌細胞之能力。 [0003] 過去十年來，研究者已在動物研究中施予多種骨趙（BM) 來源幹細胞/前驅（progen i tor )細胞進行心臟修復治療 ，例如負系（1 ineage negative, 1 Iiineg)c-kit 陽性 (c-kit positive，c-kit pos)骨髓幹細胞（Orlic et al., Nature 2001; 410： 701-705; Kajstura 099141986 表單編號A0101 第3頁/共34頁 1003114781-0 201141510 et al., Circ. Res. 2005; 96: 127-137; Rota et al., Proc Natl Acad Sci USA 2007; 104: 17783-1 7788)、骨髓來源間葉幹細胞（BM-derived mesenchymal stem cells, MSCs) (Min et al.,The important features of Q stem cells that regenerate the heart muscle include self-renewal, clonogenicity, and the ability to differentiate into cardiomyocytes 'endothelial cells and vascular smooth muscle cells. [0003] Over the past decade, researchers have administered a variety of bone-derived (BM)-derived stem cells/progen i tor cells for cardiac repair in animal studies, such as the negative (1 ineage negative, 1 Iiineg) c- C-kit positive (c-kit pos) bone marrow stem cells (Orlic et al., Nature 2001; 410: 701-705; Kajstura 099141986 Form No. A0101 Page 3 of 34 1003114781-0 201141510 et al., Circ. Res. 2005; 96: 127-137; Rota et al., Proc Natl Acad Sci USA 2007; 104: 17783-1 7788), BM-derived mesenchymal stem cells (MSCs) (Min et Al.,

Ann Thor Surg 2002; 74: 1568-1575; Amado et al., Proc Natl Acad Sci USA 2005; 102: 1 1474-1 1479)以及内皮前驅細胞（endothelial pro -genitor cells, EPCs) (Cho et al., J Exp Med 2007; 204: 3257-3269; Schuh et al., Basic Res Cardio2008; 103:60-77)。儘管這些研究顯示骨 髓來源幹細胞/前驅細胞分化為具有心肌細胞及血管細胞 標記特徵之細胞，但其他研究則指出移植之骨髓幹細胞 在受損之心臟中不獲有心臟之表現型。（Balsam et al., Nature 2004; 428: 668-673; Murry et al., Nature 2004; 428: 664-668; Nygren et al·， Nat Med 2004; 10: 494-501)。 [0004]因此’增進移植後骨髓來源幹細胞/前驅細胞的分化進而 有效地利用這些細胞於心臟再生治療，對於研究者而言 仍然是個巨大挑戰。除了骨髓來源幹細胞/前驅細胞外， 其他幹細胞來源也可能被用於心臟再生治療。尤其，常 駐心臟幹細胞（resident cardiac stem cells， CSCs)被發現存在於心臟之本身。（Beitramj et ai.， Cell 2003； 114: 763-776; Uranek et al., ProcAnn Thor Surg 2002; 74: 1568-1575; Amado et al., Proc Natl Acad Sci USA 2005; 102: 1 1474-1 1479) and endothelial pro-genitor cells (EPCs) (Cho et al., J Exp Med 2007; 204: 3257-3269; Schuh et al., Basic Res Cardio 2008; 103: 60-77). Although these studies have shown that bone marrow-derived stem/progenitor cells differentiate into cells with cardiomyocyte and vascular cell marker characteristics, other studies have indicated that transplanted bone marrow stem cells do not have a cardiac phenotype in the damaged heart. (Balsam et al., Nature 2004; 428: 668-673; Murry et al., Nature 2004; 428: 664-668; Nygren et al., Nat Med 2004; 10: 494-501). [0004] Therefore, it is still a great challenge for researchers to improve the differentiation of bone marrow-derived stem cells/precursor cells after transplantation and to effectively utilize these cells for cardiac regeneration therapy. In addition to bone marrow-derived stem/precursor cells, other stem cell sources may also be used for cardiac regeneration therapy. In particular, resident cardiac stem cells (CSCs) are found in the heart itself. (Beitramj et ai., Cell 2003; 114: 763-776; Uranek et al., Proc

Natl Sci USA 2006; 103: 9226-9231)。 因為常駐心臟幹細胞已位於心臟内並且被設計去產生心 099141986 表單編號A0101 第4頁/共34頁 1003114781-0 201141510 [0005] 臟組織，所以當發生心臟組織大量喪失時，它們代表著 對心臟再生治療能有所功效之合理來源。由於常駐心臟 幹細胞具有獨特特徵，辨認出常駐心臟幹細胞對於利用 來自心臟本身且天生被設計來重建心臟組織之細胞之心 肌再生帶來了無比興奮以及如火花般強烈之希望。 〜肌修復需要新的肌細胞（my〇Cyte)及冠狀血管之生成 ’且其不能由已經完全確定將分化為肌肉細胞系 (myocyte lineage)之細胞來達成。當出現梗塞時，單 ❹ 獨生成肌肉細胞無法恢復不能運動（akinetic)區域之 了收縮性此’肌肉細胞在沒有企管生成下，將無法生長 及存活。小動脈（arteriolies)對於血液供應是緊要的 ’而氧氣的遞送由微血管網界f支:配。相同填，單獨血管 新生（neovasculogenesis)也不能恢復死亡的心肌或 重建心室壁梗塞部份之收縮力（contractile activity) 。 在動物梗塞的心肌局部注射常駐心臟幹細胞 （ CSCs) ’觀察到修復了壞死（necr〇tic)組織並改善心室 〇 功能（Beltrami et a 1.，Cell 2003; 114: 763-776; Bearzi et al., Proc Natl Sci USA 2007; 1 04: 1 4068-14073) ’其對於常駐心臟幹細胞參 與肌細胞（myocytes)、内皮細胞、平滑肌細胞以及纖 維母細胞之正常再生’形成了新的範例基礎。對於企圖 發展關於未來病人治療的策略，新的假說必須被提出， 以朝向確立常駐心臟幹細胞之以個人為基礎之臨床治療 領域方向邁進。 [0006] 關於新發現之常駐心臟幹細胞之臨床應用已有各種不同 099141986 表單編號A0101 第5頁/共34頁 1003114781-0 201141510 的嘗試。第一步是常駐心臟幹細胞之分離、培養、複製 以及擴張。此類細胞將被注射回梗塞的心臟以企圖再生 具功能之心肌。然而，常駐心臟幹細胞族群之匱乏，結 合著嚴苛之細胞培養條件以及貧乏之產量，為使用此方 法之限制因素。描述於本技術領域之其他替代方法為利 用外生（exogenous)之物質去蒐集並分化内生（endogenous) 的常駐心臟幹細胞 。然而 ，沒有 關於此 方法之 功效之清楚證據被描述，因而對於在活體内有效地蒐集 並分化常駐心臟幹細胞之能力投下了不確定性。 【發明内容】 [0007] 本發明提供關於刺激活體内常駐心臟幹細胞之新方法， 在一方面’使它們走向心臟系（cardiac lineage)，尤 其是從它們得到有心肌細胞標記特徵且有令人滿意之功 能，及顯著數量之細胞。 [0008] 本發明關於可作為刺激幹細胞之人類或獸醫上使用之醫 藥組成物（B)，其包括至少兩種幹細胞刺激物以及至少一 種醫藥可接受之賦形劑。 [0009] 該至少兩種幹細胞刺激物可從下列物質所組成之群組中 選出：TGFH、BMP-4、FGF-2、IGF-1、活化蛋白 A(Activin-A)，alpha-凝血蛋白酶 、alpha-thrombin) 、 Cardiotrophin 1 、 CardiogenolC或上述各物之混合物。特別是，該至少兩 種幹細胞刺激物可從下列物質所組成之群組中選出：TGF 石-1、BMP-4、FGF-2、IGF-1、活化蛋白 A(Activin-A) 、Cardiotrophin 1 、Cardi〇genolC或 099141986 表單編號A0101 第6頁/共34頁 1003114781-0 201141510 [0010] D [0011] [0012] [0013] ❹ [0014] 上述各物之混合物。 本發明也關於一種醫藥混合物（c 〇 c k t a i 1)，其包括根據 本發明之醫藥組成物（B)及包括至少一種醫藥有效物質 (active substance)之組成物（A)。本發明之組成物或 混合物能提供刺激物導引（st imu 1 at i ng agent-guided) 之幹細胞 ，其表示當 常駐心臟幹細胞被放 置去接 觸這些組成物或混合物時，會被刺激而進行分化。因此 ，這些被刺激的幹細胞可能會分化進一種入心臟系 (cardiac lineage)且可能變為心肌細胞。 在本文件之架構内，除非有相反的指示，在引號之間所 標出的術語具有以下之定義。 如於此之所用，術語『刺激或刺激的』在此處是指幹細 胞之蒐集（recruitment)、增殖（proliferation)，存 活及/或分化。 如於此之所用，術語『心臟組織』或『心肌』意指肌肉 細胞、企管及纖維母細胞。 名詞『心臟幹細胞（CSCs)』、『心臟前驅細胞』、『常 駐心臟幹細胞』或『常駐心臟前驅細胞』在此處是指在 心肌内呈現之幹細胞。它們為自我更新的、複製產生的 (clonogenic)、多功能的，且可能產生心肌。 名詞『幹細胞刺激物』在此處是指一種增進幹細胞能力 之物質，使其被蒐集至再生之處，進行增殖並分化為心 臟組織。 099141986 表單編號A0101 第7頁/共34頁 1003114781-0 [0015] 201141510 [0016] 名詞『幹細胞刺激物組成物』在此處是指一種包括至少 兩種幹細胞刺激物之組成物。 [0017] 名詞『刺激物導引之幹細胞』在此處是指一種接觸如上 所述之幹細胞刺激物組成物之幹細胞，並進一步進行分 化，及朝向心臟系（cardiac lineage)分化。 [0018] 名詞『分化』在此處是指較不專業化（specialized) 之細胞變為較專業化之細胞之過程。 [〇〇19] 除非另行定義’於此所使用之技術上或科學上之術語具 有本發明所屬技術領域通常知識者所一般了解之相同竟 義。雖然與於此所描述之相似或相當之方法與材料可被 使用而實施本發明’但適當之方法與材料將敘述於下。 所有於此所提及之出版、專利申請、專利及其他參考文 獻以全文方式併入作為參考。若有衝突，本發明說明奎 ’包括定義’將作為本發明術語意義之標準。此外，材 料、方法及實施例僅為例示而非企圖加以限制。 【實施方式】 [0020] 在一較佳之具體實施例，醫藥組成物（…可能包含至少五 種幹細胞刺激物，其選自下列物質所組成之組群：TGF万一 1、BMP-4、FGF-2、IGF-1、活化蛋sA(Activin A)、Natl Sci USA 2006; 103: 9226-9231). Because the resident cardiac stem cells are already in the heart and are designed to produce heart 099141986 Form No. A0101 Page 4 / Total 34 Page 1003114781-0 201141510 [0005] Dirty tissue, so when cardiac tissue loss occurs, they represent the regeneration of the heart A reasonable source of treatment can be effective. Because of the unique characteristics of resident cardiac stem cells, the identification of resident cardiac stem cells has created a tremendous excitement and sparking hope for the use of cardiac muscle regeneration from cells that are naturally designed to reconstruct heart tissue. ~ Muscle repair requires new myocytes (my〇Cyte) and the formation of coronary blood vessels' and it cannot be achieved by cells that have been completely determined to differentiate into myocyte lineage. When an infarction occurs, the muscle cells alone cannot recover the contractility of the akinetic region. The muscle cells will not grow and survive without being managed. Arteriolies are critical to the blood supply' while oxygen is delivered by the microvascular network. With the same filling, neovascularization (neovasculogenesis) also failed to restore the dead heart muscle or reconstruct the contractile activity of the ventricular wall infarction. Local injection of resident cardiac stem cells (CSCs) in the infarcted myocardium was observed to repair necrotic tissue and improve ventricular spasm function (Beltrami et al 1., Cell 2003; 114: 763-776; Bearzi et al. , Proc Natl Sci USA 2007; 1 04: 1 4068-14073) 'It forms a new paradigm for the involvement of resident cardiac stem cells in the normal regeneration of myocytes, endothelial cells, smooth muscle cells, and fibroblasts. For the attempt to develop strategies for future patient care, new hypotheses must be proposed to move towards the establishment of a personal-based clinical treatment area for resident cardiac stem cells. [0006] There are various clinical applications for newly discovered resident cardiac stem cells. 099141986 Form No. A0101 Page 5 of 34 attempts at 1003114781-0 201141510. The first step is the isolation, culture, replication, and expansion of resident cardiac stem cells. Such cells will be injected back into the infarcted heart in an attempt to regenerate a functional myocardium. However, the lack of resident cardiac stem cell populations, combined with harsh cell culture conditions and poor yields, are limiting factors in the use of this method. An alternative method described in the art is to use exogenous substances to collect and differentiate endogenous resident cardiac stem cells. However, no clear evidence of the efficacy of this method has been described, and thus uncertainty has been placed on the ability to efficiently collect and differentiate resident cardiac stem cells in vivo. SUMMARY OF THE INVENTION [0007] The present invention provides a novel method for stimulating the resident cardiac stem cells in vivo, on the one hand 'making them toward a cardiac lineage, especially from which they have cardiomyocyte marker characteristics and are satisfactory The function, and a significant number of cells. The present invention relates to a pharmaceutical composition (B) useful as a human or veterinarian for stimulating stem cells, comprising at least two stem cell stimulators and at least one pharmaceutically acceptable excipient. [0009] The at least two stem cell stimulators can be selected from the group consisting of TGFH, BMP-4, FGF-2, IGF-1, Activin A (Activin-A), alpha-coagulinase, Alpha-thrombin), Cardiotrophin 1, CardiogenolC or a mixture of the above. In particular, the at least two stem cell stimulators can be selected from the group consisting of TGF stone-1, BMP-4, FGF-2, IGF-1, Activin A (Activin-A), Cardiotrophin 1 , Cardi〇genolC or 099141986 Form No. A0101 Page 6 / Total 34 Page 1003114781-0 201141510 [0010] [0012] [0013] [0014] [0014] A mixture of the above. The invention also relates to a pharmaceutical mixture (c 〇 c k t a i 1) comprising a pharmaceutical composition (B) according to the invention and a composition (A) comprising at least one pharmaceutically active substance. The composition or mixture of the present invention is capable of providing a stem cell of a stimulant guide, which means that when resident cardiac stem cells are placed in contact with these compositions or mixtures, they are stimulated to differentiate. . Therefore, these stimulated stem cells may differentiate into a cardiac lineage and may become cardiomyocytes. Within the framework of this document, unless otherwise indicated, the terms indicated between the quotation marks have the following definitions. As used herein, the term "stimulated or stimulating" as used herein refers to the recruitment, proliferation, survival and/or differentiation of stem cells. As used herein, the term "heart tissue" or "myocardium" means muscle cells, arteries, and fibroblasts. The terms "heart stem cells (CSCs)", "cardiac precursor cells", "resident cardiac stem cells" or "resident cardiac precursor cells" herein refer to stem cells present in the myocardium. They are self-renewing, clonogenic, multifunctional, and may produce myocardium. The term "stem cell stimulator" as used herein refers to a substance that enhances the ability of stem cells to be collected and regenerated to differentiate and differentiate into heart tissue. 099141986 Form No. A0101 Page 7 of 34 1003114781-0 [0015] The term "stem cell stimulant composition" as used herein refers to a composition comprising at least two stem cell stimuli. [0017] The term "stimulator-directed stem cells" as used herein refers to a stem cell that contacts a stem cell stimulant composition as described above, further differentiated, and differentiated toward a cardiac lineage. [0018] The term "differentiation" as used herein refers to the process by which less specialized cells become more specialized cells. [0019] Unless otherwise defined, the technical or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, the appropriate methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are hereby incorporated by reference in their entirety. In the event of a conflict, the present invention illustrates that Qué' includes definitions as a standard for the meaning of the terms of the present invention. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. [Embodiment] [0020] In a preferred embodiment, the pharmaceutical composition (... may comprise at least five stem cell stimulators selected from the group consisting of TGF-1, BMP-4, FGF -2, IGF-1, activated egg sA (Activin A),

Cardiotrophin l、Cardi〇genol C以及上述各物之混 合物。 [0021] 特別是，醫藥組成物（B)可能包含丁讣万^、BMp_4、 FGF-2、IGF-1、活化蛋白A(Activin_A)、Cardi〇 —Cardiotrophin l, Cardi〇genol C and mixtures of the above. [0021] In particular, the pharmaceutical composition (B) may comprise Ding Wan Wan, BMp_4, FGF-2, IGF-1, Activin A (Activin_A), Cardi〇 —

trophin 1 以及 Cardiogenol C 099141986 表單編號A0101 第8頁/共34頁 1003114781-0 201141510 - [0022]再者，該醫藥組成物（B)可能進一步選擇性地包含al_Trophin 1 and Cardiogenol C 099141986 Form No. A0101 Page 8 of 34 1003114781-0 201141510 - [0022] Furthermore, the pharmaceutical composition (B) may further optionally contain al_

Pha-凝血蛋白酶.該醫藥組成物（B)可能進一步包含凝血 蛋白酶抑制劑（thrombin inhibitors)，如hirudin, bivalirudin, lepirudin， deirudin， ar- gatroban, melagatran, ximelagatran, dabigat-ran，及肝素（heparin). Alpha-凝血蛋白酶為一種凝 金劑。或者，在某些情況下，該醫藥組成物（B)可能選擇 性地不含alpha-凝血蛋白酶。 [0023] o 本發明之醫藥組成物（B)可能進一步包含至少一種物質， 其選自由生長因子、細胞激素（cytokines )、賀爾蒙 .... ….. 及其混合物所組成之組群。議至少一種物寶可能選自由 +.... 下列所組成之組群： _ 骨形態發生蛋白（Bone morphogenetic proteins， BMP) ’ 如BMP-1 、 BMP-2 、 BMP-5 、 BMP-6; -表皮生長因子（Epidermal growth factor，EGF) -紅血球生成素（Erythropoietin, .EPO); _纖維母細胞生長因子（Fibroblast growth factors, FGF)，如FGF-1、FGF 4、FGF 5、FGF 12、FGF 13、FGF 15、FGF 20; -顆粒細胞-群落刺激因子（Granulocyte-colony stimulating factor, G-CSF); -生長分化因子9 (Growth differentiation factor-9, GDF-9); -肝細胞生長因子（Hepatocyte growth factor, HGF); 099141986 表單編號A0101 第9頁/共34頁 1003114781-0 201141510 -類胰島素生長因子（IGF)，如IGF-2; -肌肉生長抑制素（Myostatin，GDF-8); -神經營養素（Neurotrophins)，如 NT-3、NT-4、 NT-1以及神經生長因子（Nerve growth factor, NGF)； -血小板來源生長因子（Platelet-derived growth factor， PDGF)，如PDGF-beta 、 PDGF-AA 、 PDGF-BB; -血小板生成素（Thrombopoietin，TPO) _ 轉化生長因子（Transforming growth factor alpha) -轉化生長因子 yS (Transforming growth factors召 ，TGF-卢）如TGF 冷1、TGF β2 ' TGF-/S3; -血管内皮生長因子（Vascular endothelial growth factor’VEGF)，如VEGF A、VEGF C; -TNF a:、白血病抑制因子（Leukemi a i nhib i tory factor，LIF)、介白素6(interleukin 6，IL-6) 、視黃酸（retinoic acid)、C SDF-1 (基質細胞來源 因子（stromal cell—derived factor-1)) 'BDNF ( 腦來源神經營養因子（brain-derived neurotrophic factor))、Periostin、jk 管收縮素 II (Angiotensin 11)、FI t3 配體（FI t3 1 igand)、神 經膠來源神經營養因子（Glial- Derived Neurotrophic Factor) 、 肝素 （Heparin) 、 類胰島素生長因子結合 蛋白 3( Insulin-Like Growth Factor Binding Protein-3)、類胰島素生長因子結合蛋白5(Insulin -Like Growth Factor Binding Protein-5)、介白素 099141986 表單編號A0101 第10頁/共34頁 1003114781-0 201141510 3(interleukin-3)、介白素8(interleukin-8)、中 期因子（Midkine)、黃體激素（Progesterone)、腐胺 (Putrescine)、幹細胞因子、TGF-alpha、Wntl、Pha-coagulation protease. The pharmaceutical composition (B) may further comprise thrombin inhibitors such as hirudin, bivalirudin, lepirudin, deirudin, ar-gatroban, melagatran, ximelagatran, dabigat-ran, and heparin Alpha-coagulation protease is a gelling agent. Alternatively, in some cases, the pharmaceutical composition (B) may alternatively be free of alpha-coagulation protease. [0023] The pharmaceutical composition (B) of the present invention may further comprise at least one substance selected from the group consisting of growth factors, cytokines, hormones, ..., and mixtures thereof . It is suggested that at least one kind of treasure may be selected from the group consisting of: _ _ Bone morphogenetic proteins (BMP) ' such as BMP-1, BMP-2, BMP-5, BMP-6; - Epidermal growth factor (EGF) - Erythropoietin (.EPO); Fibroblast growth factors (FGF), such as FGF-1, FGF 4, FGF 5, FGF 12, FGF 13, FGF 15, FGF 20; - Granulocyte-colony stimulating factor (G-CSF); - Growth differentiation factor-9 (GDF-9); - Hepatocyte growth factor (Hepatocyte growth factor, HGF); 099141986 Form No. A0101 Page 9 of 34 1003114781-0 201141510 - Insulin-like growth factor (IGF), such as IGF-2; - Myostatin (GDF-8); - Neurotrophins such as NT-3, NT-4, NT-1 and Nerve growth factor (NGF); - Platelet-derived growth factor (PDGF), such as PDGF-beta , PDGF-AA, PDGF-BB; - platelets Thrombopoietin (TPO) _ Transforming growth factor alpha - Transforming growth factor yS (Transforming growth factors, TGF-Lu) such as TGF cold 1, TGF β2 ' TGF-/S3; - vascular endothelial growth factor (Vascular endothelial growth factor 'VEGF), such as VEGF A, VEGF C; -TNF a:, leukemia inhibitory factor (Leukemi ai nhib i tory factor, LIF), interleukin 6, IL-6, yellow Retinoic acid, C SDF-1 (stromal cell-derived factor-1) 'BDNF (brain-derived neurotrophic factor), Periostin, jk tube contractile hormone II ( Angiotensin 11), FI t3 1 igand, Glial-Derived Neurotrophic Factor, Heparin, Insulin-Like Growth Factor Binding Protein- 3) Insulin-Like Growth Factor Binding Protein-5, Interleukin 099141986 Form No. A0101 Page 10 of 34 1003114781-0 2011 41510 3 (interleukin-3), interleukin-8, midkine, progesterone, putrescine, stem cell factor, TGF-alpha, Wntl,

Wnt3a、Wnt5a、半胱胺酸-天門冬胺酸特殊蛋白酶 4(caspase-4)、趨化素配體l(chemokine ligand 1)、趨化素配體2、趨化素配體5、趨化素配體7、趨化 素配體11、趨化素配體20、結合球蛋白（haptoglobin) Ο 、凝集素（lectin)、類固醇-25羥化酶（cholesterol. 25-hydroxylase) 、 syntaxin-8 、 syntaxin-11 、藍 胞漿素（ceruloplasmin)、補體成份l(complement component 1)、補體成份f、插入素alpha 6(integrin alpha 6)、溶梅體酸脂酶l:(’lysosomal acid lipase l)、J-2 微球奎白（J-2 micro-globulin) 、 泛素 （ubiquitin)， 巨禮細胞移動抑制因 子（macrophage migration inhibitory factor) 、絲切蛋白（cofilin)、親環素 it(xyclophillin A) Ο 、FKBP12、NDPK、前纖維蛋白 l(pro:filin 1)、半胱 胺酸蛋白酶抑制劑C (c y s t a t i n C ) 、#5環素 (calcyclin) 、stanniocalcin-l、前列線素E-2 ( PGE-2)、mpCCL2、ID0、誘發性一氧化氮合酶 （iNOS )、HLA-G5、M-CSF、血管生成素（angiopoietin)、 PIGF、MCP-1、胞外間質分子（extracellular matrix molecules)、CCL2 (MCP-1)、CCL3 (MIP-1)、CCL4 (MIP-1)>CCL5 (RANTES) ' CCL7 (MCP-3) ' CCL20 (MIP-3) ' CCL26 (eotaxin-3) ' CX3CL1 (fractalkine) 、 CXCL5 (ENA-78) 、 CXCL11 099141986 表單編號A0101 第11頁/共34頁 1003114781-0 201141510 (i-TAC) ' CXCLl (GRO) > CXCL2 (GRO) ' CXCL8 (IL-8)、CCL10 (IP-10)以及上述各物之混合物。 [0024] 被刺激的幹細胞可能為常駐心臟幹細胞（CSCs)或循環幹 細胞（circulating stem cells)或注射之幹細胞。 [0025] 該醫藥組成物可能含有一級粒子（pr imary part i c 1 es) 。該一級粒子可能選自下列所組成之組群：海藻酸鹽（ alginates)、幾丁聚醣（chitosan)、葡萄聚糖（ dextran)、纖維素、脂質體（liposomes)、聚酯微 米球體或奈米球體（microspheres or nanospheres ΐ of polyesters)，如PLGA、聚己内醋（p〇lycapro-lactone)或共聚醋（copo.lyesters)。例如，該一級 粒子可包覆該醫藥組成物（B)之該至少兩種幹細胞刺激物 。因此，該一級粒子可包覆包含於醫藥組成物（B)中的幹 細胞刺激物。術語『一級』代表該醫藥組成物可能被包 覆於如前述所定義之第一型式粒子。 [0026] 有利者為’該醫藥組成物（B)可與包含至少一種醫藥有效 物質之組成物（A)結合’而形成醫藥之混合物。在一具體 i 實施例，該至少一種醫藥有效物質可能選自下列物質所 組成之組群：類胰島素生長因子dGF — 丨）、肝細胞生長因 子（HGF)及/或其變化形如NK1、1K1、1K2、HP 11或HP21 ，及其上述各物之組合物。在另一具體實施例，該組成 物（A)可進一步包含SCF-1 ’該組成物（A)可進一步包含 二級粒子’其選自下列所組成之組群：海藻酸鹽（a 1 _ ginates)、幾丁聚醣（chitosan)、葡萄聚糖（dex — 099141986 tran )、纖維素、脂質體（ΐ i p0S0mes )、聚酷微米球 表單編號删1 第12頁/共34頁 1003114781-0 201141510 - 體或奈米球體（microsPheres 〇r nanospheres of … polyesters) ’ 如pLGA、聚己内㉝（p〇iycapr〇iac_ tone)或共聚酯（c〇p〇lyesters)。該二級粒子可包 覆該至少-種醫藥有效物質。術語ρ二級』在此處是指 該組成物（Α)可能被包覆於如前述所定義之第二型式粒子 。此外，該—級粒子群（sec〇ndary 被設 計成可使包含於其中之物質先遞送之後，包含於一級粒 子群（primary particies)内之物質再行遞送之。 q [0027]該醫藥之混合物可能包含該組成物（B)之樣品及該組成物 (A)之樣品。或者’組成物U)及（B)兩者可混合為一樣 品。然而，當混合在一起時，組成物（A)及（B)可施加或 遞送於一區域或區域之周圍、而分別治療之。 [0028] 本發明所述之該醫藥組成物或該醫藥混合物可作為藥品 之使用。或者’本發明所述之該醫藥組成物或該醫藥混 合物可作為心臟組織再生之使用。或者，本發明所述之 該醫藥組成物或該醫藥混合物可作為心臟疾病治療之使 0 用’包括心臟衰竭、心臟缺血或心肌梗塞。 [0029] 本發明之另一方面，提供了人類或動物之常駐心臟幹細 胞作用於體内或體外之程序。該程序包含施加本發明所 述之該醫藥組成物（B)或該醫藥混合物於該人類或 步驟。 [0030] 該醫藥組成物（B)之施加，可能在首先初步施加包含至少 一種醫藥有效物質之組成物（A)之後。 [〇〇3丨]該施加可藉由連續注射組成物（A)、（B)或混合物而完成 099141986 表單編號A0101 第13頁/共34頁 1〇〇3114781~〇 201141510 [0032] [0033] [0034] [0035] [0036] 099141986 再者’本發明所述之該醫藥組成物或醫藥混合物之連續 兩次施加之間隔可從1小時至180天。每次施加可重覆。 或者’該組成物（A)之每次或某些次之施加可為選擇性者 〇 因此，該醫藥組成物（B)或醫藥混合物可以非腸胃之方式 施加。再者’該醫藥組成物（B)或醫藥混合物可被施加於 人類或動物之循環系統。該醫藥組成物（B)或醫藥混合物 可被施加於靜脈及/或動脈。 本發明之該醫藥組成物（B)或醫藥之混合物可被施加於心 臟組織。在較佳之具體實施例中，該醫藥組成物（B)可在 冠狀血管内施加，組成物（A)之該初步施加可在靜脈内。 在此處所用之TGF/S代表TGF-1、TGF-2或TGF-3且可為 任何具有TGF/3活性之多肽類（polypeptide) ’如人類 TGf'召。例如，TGFyS可為重組TGF点。在一具體實施例 中’ TGF/S可為TGF冷-1。可使用任何適當濃度之TGF召 。例如，可使用每毫升〇. 1至1〇〇奈克（ng)之TGF/3C例如 ’大約每毫升33奈克之TGFyS 1)。 BMP可為任何具有BMP活性之多肽類，如人類BMP。例如 ’ BMP可為重組bmp。在一具體實施例中，BMP可為BMP4 。可使用任何濃度之BMP。例如，可使用每毫升0. 1至 2〇〇奈克（ng)之BMP(例如，大約每毫升65奈克之BMP4) 〇 FGF 2可為任何具有FGF 2活性之多肽類，如人類FGF 表單編號A0101 第14頁/共34頁 100311478卜0 [0037] 201141510 2。例如，FGF 2可為重組FGF 2。可使用任何濃度 之FGF 2。例如，可使用每毫升〇· 1至200奈克（ng)之 FGF 2 (例如，大約每毫升65奈克之FGF 2)。 [0038] IGF-1可為任何具有igF-Ι活性之多肽類，如人類IGF-1 。例如，IGF-1可為重組IGF-1。可使用任何濃度之igf-1。例如，可使用每毫升1至1000奈克（ng)之IGF_1 (例 如’大約每毫升650奈克之IGF-1)。 [0039] Ο 活化蛋白A (Activin-A)可為任何具有活化蛋白a活性 之多肽類’如人類活化蛋白A。例如，活化蛋白a可為重 組活化蛋白A。可使用任何濃度之活化蛋白a。例如，可 使用每毫升0. 1至500奈克（ng)之活化蛋白A(例如，大約 每毫升130奈克之活化蛋白A)。 [0040] Ο [0041] α-凝血蛋白酶（a-Thrombin)可為任何具有凝血 蛋白酶活性之多肽類，如人類凝血蛋白酶。例如，α -凝血蛋白酶可為重組α-建血蛋白酶或合成α—凝血蛋白 酶。可使用任何濃度之α -凝血蛋舀酶&例如，可使用每 毫升0· 05至100單位之α -凝血蛋白酶。Wnt3a, Wnt5a, caspase-4, chemokine ligand 1, chemotactic ligand 2, chemokine ligand 5, chemotaxis Ligand 7, chemokine ligand 11, chemokine ligand 20, haptoglobin Ο, lectin, cholesterol-25 hydroxylase (cholesterol. 25-hydroxylase), syntaxin-8 , syntaxin-11, ceruloplasmin, complement component 1, complement component f, integrin alpha 6, lysing lipase l: ('lysosomal acid lipase l ), J-2 micro-globulin, ubiquitin, macrophage migration inhibitory factor, cofilin, xyclophillin A) Ο, FKBP12, NDPK, profibrin 1 (pro: filin 1), cysteine C (cystatin C), #5 cyclin (calcyclin), stanniocalcin-l, prostaglandin E-2 (PGE-2), mpCCL2, ID0, induced nitric oxide synthase (iNOS), HLA-G5, M-CSF, angiogenesis (angiopoietin), PIGF, MCP-1, extracellular matrix molecules, CCL2 (MCP-1), CCL3 (MIP-1), CCL4 (MIP-1)>CCL5 (RANTES) 'CCL7 ( MCP-3) ' CCL20 (MIP-3) ' CCL26 (eotaxin-3) ' CX3CL1 (fractalkine) , CXCL5 (ENA-78) , CXCL11 099141986 Form No. A0101 Page 11 of 34 1003114781-0 201141510 (i- TAC) 'CXCLl (GRO) > CXCL2 (GRO) 'CXCL8 (IL-8), CCL10 (IP-10) and mixtures of the above. [0024] The stimulated stem cells may be resident cardiac stem cells (CSCs) or circulating stem cells or injected stem cells. [0025] The pharmaceutical composition may contain a first order particle (pr imary part i c 1 es). The first stage particle may be selected from the group consisting of alginates, chitosan, dextran, cellulose, liposomes, polyester microspheres or nai Microspheres or nanospheres ΐ of polyesters, such as PLGA, p〇lycapro-lactone or copo.lyesters. For example, the primary particle can coat the at least two stem cell stimulators of the pharmaceutical composition (B). Therefore, the primary particle can coat the stem cell stimulating substance contained in the pharmaceutical composition (B). The term "level" means that the pharmaceutical composition may be coated with a first type of particle as defined above. [0026] It is advantageous that the pharmaceutical composition (B) can be combined with the composition (A) comprising at least one pharmaceutically effective substance to form a mixture of medicines. In a specific embodiment, the at least one pharmaceutically effective substance may be selected from the group consisting of insulin-like growth factor dGF (丨), hepatocyte growth factor (HGF), and/or variants thereof such as NK1, 1K1 , 1K2, HP 11 or HP21, and combinations thereof. In another embodiment, the composition (A) may further comprise SCF-1 'the composition (A) may further comprise secondary particles 'which are selected from the group consisting of alginate (a 1 _ Ginates), chitosan, dextran (dex - 099141986 tran ), cellulose, liposome (ΐ i p0S0mes ), poly microsphere form number deletion 1 page 12 / total 34 pages 1003114781-0 201141510 - microsPheres 〇r nanospheres of ... polyesters' such as pLGA, p〇iycapr〇iac_ tone or copolyester (c〇p〇lyesters). The secondary particles may comprise the at least one pharmaceutically effective substance. The term "secondary" as used herein means that the composition (Α) may be coated with a second type of particle as defined above. In addition, the sigma particle group (sec〇ndary is designed such that the substance contained in the primary particies is delivered after the substance contained therein is delivered first. q [0027] The mixture of medicines A sample containing the composition (B) and a sample of the composition (A) may be contained, or both 'composition U' and (B) may be mixed into one sample. However, when mixed together, the compositions (A) and (B) can be applied or delivered around a region or region and treated separately. [0028] The pharmaceutical composition or the pharmaceutical mixture of the present invention can be used as a medicine. Alternatively, the pharmaceutical composition or the pharmaceutical mixture of the present invention can be used as a cardiac tissue regeneration. Alternatively, the pharmaceutical composition or the pharmaceutical mixture of the present invention may be used as a treatment for heart disease, including heart failure, cardiac ischemia or myocardial infarction. In another aspect of the invention, there is provided a procedure for the resident cardiac or human brain of a human or animal to act in vivo or in vitro. The procedure comprises applying the pharmaceutical composition (B) or the pharmaceutical mixture described herein to the human or step. [0030] The application of the pharmaceutical composition (B) may be after the initial application of the composition (A) comprising at least one pharmaceutically effective substance. [〇〇3丨] The application can be completed by continuously injecting the composition (A), (B) or a mixture. 099141986 Form No. A0101 Page 13 of 34 1〇〇 3114871~〇201141510 [0033] [0036] [0036] 099141986 Further, the interval between two consecutive application of the pharmaceutical composition or pharmaceutical mixture of the present invention may range from 1 hour to 180 days. Repeat each time it is applied. Alternatively, each or some of the application of the composition (A) may be selective. Therefore, the pharmaceutical composition (B) or the pharmaceutical mixture may be applied parenterally. Further, the pharmaceutical composition (B) or the pharmaceutical mixture can be applied to a human or animal circulatory system. The pharmaceutical composition (B) or pharmaceutical mixture can be applied to veins and/or arteries. The pharmaceutical composition (B) or a mixture of medicines of the present invention can be applied to heart tissue. In a preferred embodiment, the pharmaceutical composition (B) can be applied in a coronary vessel, and the initial application of the composition (A) can be in the vein. As used herein, TGF/S stands for TGF-1, TGF-2 or TGF-3 and can be any polypeptide having TGF/3 activity, such as human TGf. For example, TGFyS can be a recombinant TGF spot. In one embodiment, 'TGF/S can be TGF cold-1. Any appropriate concentration of TGF can be used. For example, TGF/3C per ml of 〇1 to 1 〇〇Ng (ng) can be used, for example, 'about 36 ng per liter of TGFyS 1 per ml. The BMP can be any polypeptide having BMP activity, such as human BMP. For example, 'BMP can be a recombinant bmp. In a specific embodiment, the BMP can be BMP4. Any concentration of BMP can be used. For example, a BMP of 0.1 to 2 ng per gram (ng) may be used (for example, about 65 ng per ml of BMP4) 〇FGF 2 may be any polypeptide having FGF 2 activity, such as a human FGF form number. A0101 Page 14 of 34 100311478 Bu 0 [0037] 201141510 2. For example, FGF 2 can be recombinant FGF 2 . Any concentration of FGF 2 can be used. For example, FGF 2 (e.g., about 65 ng of FGF 2 per ml) may be used in an amount of from 1 to 200 nanograms (ng) per ml. [0038] IGF-1 can be any polypeptide having igF-Ι activity, such as human IGF-1. For example, IGF-1 can be recombinant IGF-1. Any concentration of igf-1 can be used. For example, 1 to 1000 ng per gram of IGF_1 (e.g., about 650 ng per gram of IGF-1) can be used. [0039] 活化 Activin A (Activin-A) can be any polypeptide having activated protein a activity, such as human activating protein A. For example, activated protein a can be a recombinant activated protein A. Any concentration of activated protein a can be used. For example, activated protein A per ml of 0.1 to 500 ng (e.g., about 130 ng per ml of activated protein A) may be used. [0041] The a-Thrombin may be any polypeptide having thromboplastin activity, such as human coagulation protease. For example, the alpha-coagulation protease can be a recombinant alpha-casein or a synthetic alpha-coagulase. Any concentration of α-coagulase & can be used, for example, from 0.05 to 100 units of α-coagulin per ml.

Cardiotrophin可為任何具有Cardiotrophin活性之多 肽類，如人類Cardiotrophin-1。例如，Cardiotrophin can be any peptide having Cardiotrophin activity, such as human Cardiotrophin-1. E.g,

Cardiotrophin可為重組Cardiotrophin。可使用任何 ί辰度之Cardi〇trophiη。例如，可使用每毫升〇 〇5至 100奈克（ng)之Cardiotrophin (例如，大約每毫升13 奈克之Cardiotrophin-Ι)。 IL 6可為任何具有IL 6活性之多肽類，如人類丨[6 099141986 表單編號A0101 第15頁/共34頁 1003114781-0 201141510 [0042] [0043] [0044] [0045] [0046] 。例如’IL 6可為重組IL 6。可使用任何濃度之 6。例如’可使用每毫升1〇至4〇〇奈克（ng)之6。 可使用任何濃度之Cardi ogenol C或一種醫藥上可接受 之鹽類（例如’鹽酸鹽Cardiogenol C (Cardiogen〇l C hydrochloride))。例如，可使用每毫升1至looo奈 克（ng)之Cardiogenol C (例如，大約每毫升350奈克 之Cardiogenol C)。 視黃酸可為任何具有視黃酸活性之分子，如合成視黃酸 、天然視黃酸、一種維生素A代謝物、一種維生素a之天 然衍生物，或一種維生素A之合成衍生物。可使用任何濃 度之視黃酸。例如，可使用1 x 1Q-7至4 χ 微莫耳 濃度（/zM)之視黃酸。 在某些情況下’可使用添加有了〇?0-1(例如，2.5奈 克/毫升）、BMP4 (例如’ 5奈克/毫升）、FGF 2 (例 如’ 5奈克/毫升）、IGF-1 (例如，50奈克/毫升）、活 化蛋白A (例如’ 50奈克/毫升）、Cardiotrophin (例 如’ 1奈克/毫升）、〇:-凝血蛋白酶（例如，1單位/毫升） 及Cardiogenol C [例如1〇〇奈莫耳濃度（nM)]之含血清 或不含血清之培養基（media)。在某些情況下，培養基 (例如’含血清或不含企清之培養基）可包含企小板溶解 產物（lysate)，（例如，人類血小板溶解產物）。 在某些情況下’用來刺激常駐心臟幹細胞之组成物可額 外包含選擇性之因子，如TNF a、LIF及VEGF-A。 TNF «可為任何具有TNF α活性之多肽類，如人類 099141986 表單編號Α0101 第16頁/共34頁 1003114781-0 201141510 [0047] [0048] Ο [0049] Ο TNF α。例如，tnf α可為重組TNF α:。可使用任 何濃度之TNF α。例如，可使用每毫升〇. 5至100奈克 之TNF α 。 LIF可為任何具有l I f活性之多肽類，如人類LIF。例如 ，LIF可為重組LIF。可使用任何濃度之LIF。例如，可 使用每毫升0. 25至200奈克之LIF。 VEGF-Α可為任何具有veGF-A活性之多肽類，如人類VE-GF-A。例如’ VEGF-A可為重組VEGF-A。可使用任何濃 度之VEGF-A。例如，可使用每毫升〇. 5至4〇〇奈克之VE-GF-A。 在此處所提供之組成物可使用任何適當之方法製備。例 如，在此處所提供之組成物寸使用商業上可取得之刺激 物製備。在某些情況下，在此處所提供之組成物之製備 可包含細胞溶解產物（如血小板溶解產物）或來自細胞， 例如心肌細胞或經TNF «刺激之内皮細胞培養基。例如 ’在此處所提供之組成物可由血小板溶解產物並添加商 業上可取得之因子而製備。在某些情況下，在此處所提 供之組成物可使用分離自適當培養基之因子而製備。在 某些情況下’這些因子可溶解於培養基，例如含有或不 含有血清之細胞培養之培養基。 實施例 試驗在急性梗塞之豬隻中執行。建立以下之流程。梗塞 在Τ0時以90分鐘之左前降支閉塞（left anteri〇r descending (LAD) occlusi〇n)， 隨後以 3〇 分鐘之再灌流 099141986 表單編號A0101 第17頁/共34頁 1003114781-0 [0050] 201141510 (reperfusion)來執行。再灌流結束時（T1)，一級組成 物以非腸胃之方式施加於動物，藉由在閉塞區末端之遠 處以冠狀企管内遞送之。裝載BrdU之滲透性幫浦也於τι 時植入皮下。14天後（T2) ’二級組成物以非腸胃之方式 施加於動物。兩種組成物之施加，可以不同之施加方式 來達成’例如靜脈内注射、肌肉内注射或冠狀血管内注 射。最後’在第42天（T3)，豬隻被施以安樂死。 [0051] 成份之濃度被表示於括弧内。兩種組成物以各別或組合 之方式被測試： -組成物A由IGF-1[8微克（ug)於15毫升之磷酸鹽緩衝溶 液（PBS)]以及HGF [ 2微克（pg.)於.1 5毫:升之鱗酸鹽緩衝溶 液（PBS)]所組成，以及 -組成物B由TGF々-1 [0. 5微克（mg)於15毫升之磷酸鹽緩 衝溶液（PBS)]、BMP4[1微克（Ug)於15毫升之磷酸鹽緩 衝溶液（PBS)]、FGF 2[1微克（叫）於15毫升之磷酸鹽 緩衝溶液（PBS)] ' IGF-1[10微克。2)於15毫升之磷酸 鹽緩衝溶液（PBS)]、活性蛋白A[2微克（以2)於15毫升之 碟酸鹽緩衝溶液（PBS)]、Cardiotrophin 1 [0.2微克 (Mg)於15毫升之磷酸鹽緩衝溶液（PBS)]以及Cardio-genol C [5.2微克（ug)於15毫升之磷酸鹽緩衝溶液 (PBS)]所組成。 兩種組成物皆在一種醫藥上可接受之賦形劑内。該醫藥 上可接受之賦形劑可為磷酸鹽緩衝溶液（PBS)、哈特曼氏 液（Hartmann’ s solut i on)、林格氏乳酸液（ringer· ’ s lactate)、有/或沒有添加白蛋白（aibumin)或 099141986 表單編號A0101 第18頁/共34頁 1003114781-0 201141510 任何適當之蛋白質穩定組成物之生理食鹽水（〇 g %Cardiotrophin can be a recombinant Cardiotrophin. Any Cardi〇trophiη can be used. For example, 5 to 100 nanograms (ng) of Cardiotrophin per milliliter (e.g., about 13 ng per liter of Cardiotrophin-Ι) can be used. IL 6 can be any polypeptide having IL 6 activity, such as human 丨 [6 099141986 Form No. A0101 Page 15 / Total 34 Page 1003114781-0 201141510 [0044] [0044] [0046] [0046] For example, 'IL 6 can be recombinant IL 6 . Any concentration of 6 can be used. For example, '6 per ml to 4 〇〇 Ng (ng) per ml can be used. Any concentration of Cardiogenol C or a pharmaceutically acceptable salt (e.g., 'Cardiogenol C (Cardiogenol C Hydrochloride)) can be used. For example, Cardiogenol C (e.g., approximately 350 ng per ml of Cardiogenol C) per ml can be used. The retinoic acid can be any molecule having retinoic acid activity, such as synthetic retinoic acid, natural retinoic acid, a vitamin A metabolite, a natural derivative of vitamin A, or a synthetic derivative of vitamin A. Any concentration of retinoic acid can be used. For example, 1 x 1Q-7 to 4 χ micromolar (/zM) retinoic acid can be used. In some cases, 'can be used with 〇?0-1 (for example, 2.5 ng/ml), BMP4 (eg '5 ng/ml), FGF 2 (eg '5 ng/ml), IGF -1 (for example, 50 ng/ml), activated protein A (eg '50 ng/ml), Cardiotrophin (eg '1 ng/ml), 〇:-coagulinase (eg 1 unit/ml) and Cardiogenol C [eg 1 〇〇Nemo concentration (nM)] serum-containing or serum-free medium (media). In some cases, the culture medium (e.g., serum-containing or culture-free medium) may comprise a lysate (e.g., human platelet lysate). In some cases, the composition used to stimulate resident cardiac stem cells may additionally contain selective factors such as TNF a, LIF and VEGF-A. TNF « can be any polypeptide having TNFα activity, such as human 099141986 Form No. Α0101 Page 16 of 34 1003114781-0 201141510 [0048] 00 [0049] TNF TNF α. For example, tnf α can be recombinant TNF α:. Any concentration of TNF alpha can be used. For example, TNF α of from 0.5 to 100 ng per ml may be used. The LIF can be any polypeptide having l I f activity, such as human LIF. For example, the LIF can be a recombinant LIF. Any concentration of LIF can be used. For example, a LIF of 0.25 to 200 Ng per ml can be used. VEGF-Α can be any polypeptide having veGF-A activity, such as human VE-GF-A. For example, 'VEGF-A can be recombinant VEGF-A. Any concentration of VEGF-A can be used. For example, VE-GF-A per liter of 0.5 to 4 〇〇Nike can be used. The compositions provided herein can be prepared using any suitable method. For example, the compositions provided herein are prepared using commercially available irritants. In some cases, the compositions provided herein may be prepared from cell lysates (e. g., platelet lysates) or from cells, such as cardiomyocytes or TNF-stimulated endothelial cell culture media. For example, the compositions provided herein can be prepared from platelet lysates and the addition of commercially available factors. In some cases, the compositions provided herein can be prepared using factors isolated from an appropriate medium. In some cases, these factors can be dissolved in a medium, such as a medium cultured with or without serum. EXAMPLES Tests were performed in pigs with acute infarction. Establish the following process. The infarction was occluded at 90° left anteri〇r descending (LAD) occlusi〇n, followed by reperfusion for 3 minutes. 099141986 Form No. A0101 Page 17 of 34 1003114781-0 [0050 ] 201141510 (reperfusion) to execute. At the end of reperfusion (T1), the primary composition is applied to the animal in a parenteral manner and delivered intracoronically at the distal end of the occlusion zone. The osmotic pump loaded with BrdU is also implanted subcutaneously at τι. After 14 days (T2), the secondary composition was applied to the animals in a non-gastrointestinal manner. The application of the two compositions can be achieved in different ways, e.g., intravenous, intramuscular, or intracoronary injection. Finally, on day 42 (T3), the pigs were euthanized. [0051] The concentration of the ingredients is indicated in parentheses. The two compositions were tested individually or in combination: - Composition A consisted of IGF-1 [8 micrograms (ug) in 15 milliliters of phosphate buffered saline (PBS)] and HGF [2 micrograms (pg.) .1 5 milliliters: literate buffer solution (PBS)], and - composition B from TGF々-1 [0.5 micrograms (mg) in 15 ml of phosphate buffer solution (PBS)], BMP4 [1 μg (Ug) in 15 ml of phosphate buffered saline (PBS)], FGF 2 [1 μg (called) in 15 ml of phosphate buffered saline (PBS)] IGF-1 [10 μg. 2) in 15 ml of phosphate buffered saline (PBS)], active protein A [2 μg (in 2) in 15 ml of discate buffer solution (PBS)], Cardiotrophin 1 [0.2 μg (Mg) in 15 ml Phosphate buffer solution (PBS)] and Cardio-genol C [5.2 micrograms (ug) in 15 ml phosphate buffer solution (PBS)]. Both compositions are in a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be phosphate buffered saline (PBS), Hartmann's solut i on, Ringer's lactate, with or without Add albumin (aibumin) or 099141986 Form No. A0101 Page 18 of 34 1003114781-0 201141510 Any suitable protein stable composition of physiological saline (〇g %

NaCl) 〇 [0052] 評估每組5隻動物之五組處理組。 處理組1為控制組，在Τ1及Τ2僅接受鹽水溶液。 處理組2在Τ1接受包含組成物Α之溶液及在Τ2接受鹽水溶 液，記錄為混合物A。 處理組3在T1接受包含組成物a之溶液及在T2接受包含組 成物B之溶液，記錄為混合物A + B。 Ο 處理組4在T1及T2接受包含組成物b之溶液，記錄為混合 物 B+B。 處理組5在T1接受包含組成物B之溶液及在τ 2接受鹽水溶 液，記錄為混合物B。 該實驗流程（protocal)總結於表一。 ο 表一 處理組1 處理組2 處理組3 處理組4 處理組5 Τ0:梗塞 — T1: 30分鐘 鹽水 A A B B T2: 2 週 鹽水 鹽水 B B 鹽水 T3: 6 週 安樂死 安樂死 安樂死 安樂死 安樂死 [0053] 在不同時間間隔執行血液分析（Bl0〇d anaiyses)。血 液樣本收集自每處理組兩隻豬，經由頸靜脈（jugular vein)之冠狀靜脈竇（coronary sinus)以及經由耳 靜脈之靜脈血。在第一次施加後，樣品在T1+5分鐘、 Τ1 + 1小時以及Τ1 + 6小時之時間被收集。在第二次施加後 099141986 表單編號Α0101 第19頁/共34頁 1003114781-0 201141510 ，樣品在T2 + 5分鐘' T2 + 1小時、T2 + 6小日夸以及T2 + 24小 時之時間被收集。樣品以酵素連結免疫吸附分析（ELISA) 之免疫分析法加以分析以定量IGF-1以及cardiotroph -i η 1 〇 [0054] 核磁共振造影（MRI)也在Τ1 + 3天、Τ2以及Τ3時在所有動 物身上執行，以研究疤痕區域、整體左心室功能、局部 功能（壁之運動及增厚）以及心室之局部灌流。核磁共振 造影允許偵測及確認改善心室功能之新血管、組織及細 胞之呈現。 [0055] 組織病理學也被執行以決定疤痕區域以及辨識及定量c-k i t 陽性心臟幹細胞（c-k i t positive cardiac stem cells)。組織病理學也提供新金管及心肌細胞之分佈、 大小以及密度之數據。組織病理學允許紀錄證明組織及 細胞層級之修復過程。 [0056] 在心臟修復分析中，四項關鍵之變數被考慮：（1)重建之 組織之數量或心肌質量及冠狀脈管系統；（2 )恢復之肌肉 細胞及血管之數量及大小；（3)新生成之肌肉細胞及血管 與周圍心肌之整合；及/或（4)再生之心肌結構之來源。 [0057] 梗塞結果 [0058] 核磁共振造影之影像被用來評估梗塞大小、梗塞重量以 及梗塞區域。結果列於下表二。 1003Π4781-0 099141986 表單編號A0101 第20頁/共34頁 201141510 : [0059] 表二 梗塞區域（百分比） 梗塞重量（克） 梗塞體積（立 方公分） 組1 (控制组） 19.8 15.6 22.0 組2 (混合物A) 18.7 15.8 20.7 組5 (混合物B) 13.7 13.2 15.6 組4 (混合物B+B) 18.8 22.2 25.2 組3 (混合物A+B) 9.6 10.3 10.3 [0060] 實驗顯示在控制組之梗塞區域大約為19. 8%，在分別使用 混合物A及混合物B + B之组2及4大約為19%。令人驚訝地 ，使用根據本發明之組成物(B)，在組5中（混合物B)之梗 塞區域限縮至13. 7%。因此，根據本發明之組成物（B)與 其他組成物相比，對於治療梗塞，例如心肌梗塞，非常 有效。 [0061] 此外，令人驚訝地觀察到，當初步注射混合物A後，接著 注射混合物B，梗塞區域更進一步限縮至大約9. 6%。這是 一個根據其他組所觀察之結果所不能加以預期之結果。 確實，單獨使用於組2之混合物A幾乎無效。使用根據本 發明之醫藥混合物可觀察到發生協同（synergistic ) 效應。此結果以組織病理學及免疫組織化學測試被證實 [0062] 組織病理學 [0063] 結果係以取自梗塞之周邊區或中心區内之部分採分別彙 整。各組之結果列於下表三。表三所顯示之資料為分析 099141986 表單編號 A0101 第 21 頁/共 34 頁 1003114781-0 201141510 表三 周邊區 梗塞中心區 梗塞/疤 痕（百分 比） 透壁性 (Transmurality) (百分比） 梗塞/疤 痕（百分 比） 透壁性 (Transmurality) (百分比） 組1 (控制組） 33.0 4. 0 73. 3 13.3 組2 (混合物 A) 36. 0 6. 0 56.7 6.7 組5 (混合物B) 26.0 6.0 30.0 0.0 組4 (混合物 B+B) 31.4 12.9 70.0 10.0 組3 (混合物 A+B) 20. 0 0. 0 25. 0 0.0 [0064] 梗塞與疤痕大小之間之比率代表梗塞大小，而透壁性（ transmural ity)則為一參數，其表明梗塞是否位於心 肌之外表面或其延伸遍及心肌之内表面。透壁性數值越 高，梗塞強度越大。 [0065] 關於梗塞之周邊區，表三顯示混合物A、混合物B + B或控 制組對於梗塞大小幾乎沒有影響。在那些例子，梗塞與 疤痕大小之間之比率從31. 4百分比至36. 0百分比。相反 地，當使用根據本發明之組成物（即混合物B)時，梗塞與 疤痕大小之間之比率令人驚訝地降至26百分比。當使用 根據本發明之醫藥混合物（即混合物A + B )時，此值可進一 步降低至20百分比。本實驗呈現出本發明之該醫藥組成 物及醫藥混合物對治療心臟疾病是有效的。當實驗在梗 塞中心區執行時，該效果也能被證實。 [00661 再者，單獨施以混合物B或混合物A + B時，令人驚訝地展 099141986 表單編號 A0101 第 22 頁/共 34 頁 1003114781-0 201141510 . 現出透壁性之減低。這表示本發明之組成物（B)本身或當 ' 其與組成物（A)結合使用，能限制梗塞擴張至心肌之外表 面。此為根據本發明之醫藥組成物及醫藥混合物為作為 治療心臟疾病或問題之強而有力之組成物之另一證據。 [0067]因此，由上述實驗可清楚得知，根據本發明之醫藥組成 物及醫藥混合物兩者皆適合治療續發自心肌缺血（secondary to myocardial ischemia) 、 缺血或心肌梗 塞而引起之心臟衰竭。 Q [0068] 免疫組織化學 [0〇69]在梗塞區段内’執行試驗以評佑微細與管密度（vWF-陽性 血管/平方毫米）（microvessel density, vWF- •丄. positive vessels/mm2)、BtdU陽性細胞（BrdU positive cells) 以及 c-kit 陽性細胞 （c-kit positive cells)。使用 von Willebrand 因子（von Willebrand factor，vWF)對微細血管密度之定量，能測定在梗塞區 域内新生血管之數量。BrdU陽性細胞試驗代表包括心臟NaCl) 〇 [0052] Five treatment groups of 5 animals per group were evaluated. Treatment group 1 was a control group, and only saline solution was accepted at Τ1 and Τ2. Treatment group 2 received a solution containing the composition Τ at Τ1 and a saline solution at Τ2, and recorded as mixture A. Treatment group 3 received a solution containing composition a at T1 and a solution containing composition B at T2, and recorded as mixture A + B. Ο Treatment group 4 received a solution containing composition b at T1 and T2 and recorded as mixture B+B. Treatment group 5 received a solution containing composition B at T1 and a saline solution at τ 2 and recorded as mixture B. The experimental procedure (protocal) is summarized in Table 1. ο Table 1 Treatment Group 1 Treatment Group 2 Treatment Group 3 Treatment Group 4 Treatment Group 5 Τ 0: Infarction - T1: 30 minutes saline AABB T2: 2 weeks saline saline BB saline T3: 6 weeks of euthanasia euthanasia euthanasia [0053] in different Blood analysis (Bl0〇d anaiyses) was performed at time intervals. Blood samples were collected from two pigs per treatment group, via coronary sinus of the jugular vein and venous blood via the auricular vein. After the first application, the samples were collected at T1 + 5 minutes, Τ 1 + 1 hour, and Τ 1 + 6 hours. After the second application 099141986 Form No. Α0101 Page 19 of 34 1003114781-0 201141510, samples were collected at T2 + 5 minutes 'T2 + 1 hour, T2 + 6 small days and T2 + 24 hours. The samples were analyzed by enzyme-linked immunosorbent assay (ELISA) immunoassay to quantify IGF-1 and cardiotroph-i η 1 〇 [0054] Magnetic resonance imaging (MRI) was also performed at Τ1 + 3 days, Τ2, and Τ3 Animals were performed to study scar areas, overall left ventricular function, local function (wall movement and thickening), and local perfusion of the ventricles. Magnetic resonance imaging allows the detection and confirmation of new blood vessels, tissues and cells that improve ventricular function. [0055] Histopathology is also performed to determine scar regions and to identify and quantify c-k i t positive cardiac stem cells. Histopathology also provides data on the distribution, size, and density of new gold tubes and cardiomyocytes. Histopathology allows for the recording of tissue and cell-level repair processes. [0056] In cardiac repair analysis, four key variables are considered: (1) the number of reconstructed tissues or myocardial mass and coronary vasculature; (2) the number and size of muscle cells and blood vessels recovered; (3) The newly formed muscle cells and blood vessels are integrated with the surrounding myocardium; and/or (4) the source of the regenerated myocardial structure. Infarct Results [0058] Images of nuclear magnetic resonance imaging were used to assess infarct size, infarct weight, and infarct area. The results are shown in Table 2 below. 1003Π4781-0 099141986 Form No. A0101 Page 20 of 34 201141510: [0059] Table 2 Infarct area (percentage) Infarct weight (g) Infarct volume (cubic centimeters) Group 1 (control group) 19.8 15.6 22.0 Group 2 (mixture A) 18.7 15.8 20.7 Group 5 (mixture B) 13.7 13.2 15.6 Group 4 (mixture B+B) 18.8 22.2 25.2 Group 3 (mixture A+B) 9.6 10.3 10.3 [0060] The experiment shows that the infarct area in the control group is approximately 19 8%, in groups 2 and 4 using Mixture A and Mix B + B, respectively, approximately 19%. 7%。 In the stalk area of the group 5 (mixture B) was limited to 13.7%. Therefore, the composition (B) according to the present invention is very effective for treating infarction such as myocardial infarction as compared with other compositions. 6%。 [0061] In addition, it was surprisingly observed that after the initial injection of the mixture A, followed by the injection of the mixture B, the infarct area was further reduced to about 9.6%. This is a result that cannot be expected based on the results observed by other groups. Indeed, the mixture A used alone in Group 2 was almost ineffective. A synergistic effect can be observed using the pharmaceutical mixture according to the invention. This result was confirmed by histopathological and immunohistochemical tests. [0062] Histopathology [0063] The results were collected separately from the peripheral or central regions of the infarct. The results of each group are listed in Table 3 below. The information shown in Table 3 is the analysis 099141986 Form No. A0101 Page 21 of 34 1003114781-0 201141510 Table 3 Infarcts/scars in the central area of the infarct area (percentage) Transmurality (percentage) Infarction/scarring (percentage Transmurality (percent) Group 1 (control group) 33.0 4. 0 73. 3 13.3 Group 2 (mixture A) 36. 0 6. 0 56.7 6.7 Group 5 (mixture B) 26.0 6.0 30.0 0.0 Group 4 (mixture B+B) 31.4 12.9 70.0 10.0 Group 3 (mixture A+B) 20. 0 0. 0 25. 0 0.0 [0064] The ratio between infarct size and scar size represents infarct size, and transmural ity ) is a parameter indicating whether the infarct is located on the outer surface of the myocardium or extends over the inner surface of the myocardium. The higher the transmural value, the greater the infarct intensity. Regarding the peripheral zone of the infarct, Table 3 shows that the mixture A, the mixture B + B or the control group had little effect on the infarct size. In those cases, the ratio between infarction and scar size is from 31.4% to 36.0%. Conversely, when the composition according to the invention (i.e., mixture B) was used, the ratio between infarct size and scar size was surprisingly reduced to 26 percent. When the pharmaceutical mixture according to the invention (i.e., mixture A + B ) is used, this value can be further reduced to 20%. This experiment demonstrates that the pharmaceutical composition and pharmaceutical mixture of the present invention are effective for treating heart diseases. This effect can also be confirmed when the experiment is performed in the central region of the infarct. [00661 Furthermore, when mixture B or mixture A + B is applied alone, it is surprisingly exhibited 099141986 Form No. A0101 Page 22 of 34 1003114781-0 201141510 . The reduction in transmissivity is observed. This means that the composition (B) of the present invention itself or when it is used in combination with the composition (A) can restrict the expansion of the infarct to the surface of the myocardium. This is another evidence that the pharmaceutical composition and the pharmaceutical mixture according to the present invention are strong and powerful components for treating heart diseases or problems. [0067] Therefore, it is clear from the above experiments that both the pharmaceutical composition and the pharmaceutical mixture according to the present invention are suitable for treating a heart caused by secondary to myocardial ischemia, ischemia or myocardial infarction. Depletion. Q [0068] Immunohistochemistry [0〇69] Perform an experiment in the infarct segment to evaluate micro-vessel density (vWF- • 血管. positive vessels/mm2) , BtdU positive cells and c-kit positive cells. The quantification of microvessel density using the von Willebrand factor (vWF) allows the determination of the number of neovascularizations in the infarcted area. BrdU positive cell test representative including heart

Q 細胞之細胞增殖。c-kit陽性細胞試驗顯示出在所選擇之 梗塞區段内之心臟幹細胞之數量。結果列於表4。這些試 驗只針對組1(控制組）、組3(混合物a + B)以及組5(混合 物B)執行。 099141986 表單編號A0101 第23頁/共34頁 1003114781-0 201141510 表四 組1 (控制組） 組3 (混合物A+B) 組5 (混合物B) 微細血管密度 (vWF-陽性血管/毫米平方） 27.9 34.3 34.3 BrdU陽性細胞（％) 22. 1 52.7 36.0 c-kit陽性細胞（％) 1.9 1.2 1.8 [0070] 結果顯示，當根據本發明，結合組成物（A)及（B)或組成 物（B)單獨注射入心臟，它們對於心臟幹細胞之刺激或心 臟細胞之增殖具有重大之影響。的確，在本發明之組成 物或本發明之混合物刺激下，微細it管密度增加且新血 管增生。從組3或5獲得之結果，相對於控制組之2 7. 9， 分別達到34. 2及34. 3。此結果於BrdU陽性細胞試驗中得 到證實，其顯示出本發明之組成物增加了細胞增殖，且 觀察到強大之細胞活動力。當混合物B被注射時，觀察到 36. 0%之BrdU陽性細胞，相較之下，控制組僅有22. 1%。 此清楚地突顯出，根據本發明之醫藥組成物促進細胞增 殖，因此在心肌周圍形成新的心肌細胞及血·管。當使用 根據本發明之醫藥混合物時，此效果能更進一步增強。 施以該混合物能使該值達到52. 7%。因此，根據本發明之 醫藥組成物及醫藥混合物兩者皆適合改善心臟組織之再 生。 該醫藥混合物對於誘發及促進心臟幹細胞活化及增殖之 能力，以c-kit陽性細胞試驗得到證實。c-kit陽性細胞 試驗能展現出常駐心臟幹細胞被消耗，此乃由於當使用 混合物A + B時，與控制組相較，其數量顯著降低。因此， 099141986 表單編號A0101 第24頁/共34頁 1003114781-0 201141510 再生之心肌結構源自於常駐心臟幹細胞。本發明之組成 物及/或混合物對於在活體内刺激常駐心臟幹細胞為有效 者。 [0071] 於此所使用之術語及描述僅為列舉之用，而不意指作為 其限制。精於該技術領域者將認知到，如包含於下述之 申請專利範圍之定義，本發明之精神與範疇内有許多可 能之變化，其中所有術語將以它們最為廣泛可能之意思 來作理解。因此，所有修改及變更將於其他人閱讀及理 解本發明之先前敘述時發生。特別是，前述之數量、材 料及其他參數可能依照應用之需要而有所改變。 【圖式簡單說明】 [0072] 無 【主要元件符號說明】 [0073] 無 099141986 表單編號A0101 第25頁/共34頁 1003114781-0Cell proliferation of Q cells. The c-kit positive cell assay showed the number of cardiac stem cells in the selected infarct segment. The results are shown in Table 4. These tests were performed only for Group 1 (control group), Group 3 (mixture a + B), and Group 5 (mixture B). 099141986 Form No. A0101 Page 23 of 34 1003114781-0 201141510 Table Four Group 1 (Control Group) Group 3 (Mixture A+B) Group 5 (Mixture B) Microvessel Density (vWF-positive vessel/mm square) 27.9 34.3 34.3 BrdU-positive cells (%) 22. 1 52.7 36.0 c-kit positive cells (%) 1.9 1.2 1.8 [0070] The results show that when combined with the composition (A) and (B) or composition (B) according to the present invention Injecting into the heart separately, they have a major impact on the stimulation of cardiac stem cells or the proliferation of cardiac cells. Indeed, under the stimulation of the composition of the invention or the mixture of the invention, the density of the micro-it tube increases and the new blood vessels proliferate. The results obtained from the group 3 or 5 are 34. 2 and 34.3, respectively, relative to the control group. This result was confirmed in the BrdU positive cell assay, which showed that the composition of the present invention increased cell proliferation, and strong cell motility was observed. When the mixture B was injected, 36.0% of BrdU-positive cells were observed, compared with 22.1% in the control group. This clearly highlights that the pharmaceutical composition according to the present invention promotes cell proliferation, thereby forming new cardiomyocytes and blood vessels around the myocardium. This effect can be further enhanced when the pharmaceutical mixture according to the invention is used. 7%。 By this mixture can achieve a value of 52.7%. Therefore, both the pharmaceutical composition and the pharmaceutical mixture according to the present invention are suitable for improving the regeneration of heart tissue. The ability of the pharmaceutical mixture to induce and promote cardiac stem cell activation and proliferation was confirmed by c-kit positive cell assays. The c-kit positive cell assay showed that the resident cardiac stem cells were consumed because the amount of the mixture was significantly lower when compared to the control group when the mixture A + B was used. Therefore, 099141986 Form No. A0101 Page 24 of 34 1003114781-0 201141510 The regenerated myocardial structure is derived from resident cardiac stem cells. The compositions and/or mixtures of the present invention are effective for stimulating resident cardiac stem cells in vivo. The terms and descriptions used herein are for illustrative purposes only and are not intended to be limiting. It will be appreciated by those skilled in the art that many variations are possible in the spirit and scope of the invention as defined in the scope of the claims. Accordingly, all modifications and changes will occur to others upon reading and understanding the invention. In particular, the quantities, materials and other parameters described above may vary depending on the needs of the application. [Simple diagram description] [0072] None [Main component symbol description] [0073] None 099141986 Form No. A0101 Page 25 of 34 1003114781-0

Claims (1)

201141510 七、申請專利範圍： 1 . 一種人類或獸醫用之幹細胞刺激醫藥組成物（B)，其特徵 為’其包括至少兩種幹細胞刺激物以及至少一種醫藥上可 接受之賦形劑（pharmaceutically acceptable excipient) ° 2.根據申請專利範圍第1項所述之醫藥組成物，其特徵為， 該至少兩種幹細胞刺激物係選自於下列所組成之組群： TGF 点-1、BMP-4 ' FGF-2、IGF-1、活化蛋白 A (Activin-A) 、 Cardiotrophin 1 、 CardiogenolC及 上述各物之組合物。 3 ·根據申請專利範圍第2項所述之醫藥組成物，其特徵為， 該幹細胞刺激物為TGFyS-1、BMP-4、FGF-2、IGF-1、 活化蛋白A 'Cardiotrophin 1 以及Cardiogenol C。 4 ·根據前述各項申請專利範圍中任一項所述之醫藥組成物， 其特徵為’該組成物進一步至少包括凝血蛋白酶抑制劑（ thrombin inhibitors) ’而後者係選自於下列所組成 之組群.hirudin、bivalirudin、lepirudin、 deirudin ' argatroban ' melagatran ' ximelagatran'dabigatran以及肝素（heparin)。 5 .根據前述各項申請專利範圍中任一項所述之醫藥組成物， 其特徵為’其進一步包括至少一種物質，而該物質係選自 於下列所組成之組群：生長因子、細胞激素、賀爾蒙及上 述各物之組合物。 6 ·根據申請專利範圍第5項所述之醫藥組成物，其特徵為， 該至少一種之物質係選自於下列所組成之組群： 099141986 表單編號A0101 第26頁/共34頁 1003114781-0 201141510 • _ 骨形態發生蛋白（Bone morphogenet i c protei ns, o BMP)，如BMP-l、BMP-2、BMP-5以及BMP-6 ; -表皮生長因子（Epidermal growth factor, EGF); -紅jk球生成素（Erythropoietin, EPO); -纖維母細胞生長因子（Fibroblast growth factors，FGF)，如FGF-1、FGF 4、FGF 5、FGF 12、FGF 13、FGF 15 以及 FGF 20; -顆粒細胞--群落刺激因子（Granulocyte-colony stimulating factor, G-CSF)； -顆粒細胞--巨噬細胞群落刺激因子 (Granulocyte-macrophage cilony stimulating factor, GM-CSF)； . :. ...... . -生長分化因子9 (Growth' differentiation factor-9, GDF-9)； -肝細胞生長因子（Hepatocyte growth factor, HGF); 〇 -類胰島素生長因子（IGF)，如JGF-2; _肌肉生長抑制素（財〇5七3七111，、::.:61^-8); -神經營養素（Neurotrophins)，如 NT-3、NT-4、 NT-1以及神經生長因子（Nerve growth factor, NGF); _血小板來源生長因子（Platelet-derived growth factor，PDGF)，如 PDGF-beta、PDGF-AA、PDGF-BB 099141986 _ 血小板生成素（Thrombopoietin，TPO) -轉化生長因子（Transforming growth factor 表單編號A0101 第27頁/共34頁 1003114781-0 201141510 alpha, TGF) -轉化生長因子 ^^Transforming growth factors^ ，TGF-/S)如TGF /5 1 ' TGF β2 ' TGF-^3 ； —灰管内皮生長因子（Vascular endothelial growth factor, VEGF)，如 VEGF A、VEGF C; TNF a、白企病抑制因子（Leukemia inhibi tory factor，LIF)、介白素6(interleukin 6，IL-6)、視 頁酸（retinoic acid)、基質細胞來源因子（stromal cell-derived factor-1，C SDF-1)、腦來源神經營 養因子(brain-derived neurotrophic factor, BDNF)、Periostin、血管收縮素II (Angiotensin 11)、F113配體、神經膠來源神經營養因子 (Glial-Derived Neurotrophic Factor)、類胰島素 生長因子結合蛋白 3(Insulin-Like Growth Factor Binding Protein-3)、類胰島素生長因子結合蛋白 5(Insulin- Like Growth Factor Binding Protein-5)、介白素3、介白素8、中期因子（Midkine) 、黃體激素、腐胺（Putrescine)、幹細胞因子、 TGF-alpha、Wntl、Wnt3a、Wnt5a、半脱胺酸-天門冬 胺酸特殊蛋白酶4(caspase-4)、趨化素配體 l(chemokine ligand 1)、趨化素配體2、趨化素配體5 、趨化素配體7、趨化素配體11、趨化素配體2 〇、結合球 蛋白（haptoglobin)、凝集素（lectin)、類固醇-25經 化酶（cholesterol 25-hydroxylase) ' syntaxin-8 、syntaxin-11、藍胞漿素（cerui〇piasmin)、補體成 份l(complement component 1)、補體成份3、插入 099141986 表單編號A0101 第28頁/共34頁 1003114781Ό 201141510 Ο201141510 VII. Patent application scope: 1. A stem cell stimulating pharmaceutical composition (B) for human or veterinary use, characterized in that it comprises at least two stem cell stimulants and at least one pharmaceutically acceptable excipient (pharmaceutically acceptable) The pharmaceutical composition according to claim 1, wherein the at least two stem cell stimulating substances are selected from the group consisting of TGF point-1, BMP-4' FGF-2, IGF-1, Activin A (Activin-A), Cardiotrophin 1, Cardiogenol C, and combinations thereof. 3. The pharmaceutical composition according to claim 2, wherein the stem cell stimulator is TGFyS-1, BMP-4, FGF-2, IGF-1, activated protein A 'Cardiotrophin 1 and Cardiogenol C . The pharmaceutical composition according to any one of the preceding claims, characterized in that the composition further comprises at least a thrombin inhibitors and the latter is selected from the group consisting of Group. Hirudin, bivalirudin, lepirudin, deirudin 'argatroban ' melagatran ' ximelagatran 'dabigatran and heparin. 5. The pharmaceutical composition according to any one of the preceding claims, characterized in that it further comprises at least one substance selected from the group consisting of growth factors, cytokines , hormones and combinations of the above. 6. The pharmaceutical composition according to claim 5, wherein the at least one substance is selected from the group consisting of: 099141986 Form No. A0101 Page 26 of 34 1003114781-0 201141510 • _ Bone morphogenet ic protei ns (o BMP), such as BMP-1, BMP-2, BMP-5 and BMP-6; Epidermal growth factor (EGF); -Red jk Erythropoietin (EPO); - Fibroblast growth factors (FGF), such as FGF-1, FGF 4, FGF 5, FGF 12, FGF 13, FGF 15 and FGF 20; -Granulocyte-colony stimulating factor (G-CSF); - Granulocyte-macrophage cilony stimulating factor (GM-CSF); . :. - Growth differentiation factor-9 (GDF-9); - Hepatocyte growth factor (HGF); 〇-like insulin growth factor (IGF), such as JGF-2; _ muscle growth inhibition Prime (Financial 5 7 3 7 111,, :::: 61^-8); - Neurotrophins, such as NT-3, NT-4, NT-1, and Nerve growth factor (NGF); Platelet-derived growth factor (PDGF), such as PDGF-beta, PDGF-AA, PDGF-BB 099141986 _ thrombopoietin (TPO) - transforming growth factor (Transforming growth factor Form No. A0101 Page 27 of 34 1003114781-0 201141510 alpha, TGF) - Transforming Growth Factor ^^Transforming Growth factors^, TGF-/S) such as TGF /5 1 'TGF β2 ' TGF-^3 ; - Vascular endothelial growth factor (VEGF), such as VEGF A, VEGF C; TNF a, white Leukemia inhibiry factor (LIF), interleukin 6, IL-6, retinoic acid, stromal cell-derived factor-1 (C SDF-1) ), brain-derived neurotrophic factor (BDNF), Periostin, angiotensin II, F113 ligand, Glial-Derived Neurotrophic Factor, Insulin-Like Growth Factor Binding Protein-3, Insulin-Like Growth Factor Binding Protein-5, Interleukin 3, Interleukin 8, Midkine (Midkine), progesterone, putrescine, stem cell factor, TGF-alpha, Wntl, Wnt3a, Wnt5a, heptamic acid-aspartate specific protease 4 (caspase-4), chemokine ligand l (chemokine ligand 1), chemokine ligand 2, chemokine ligand 5, chemokine ligand 7, chemokine ligand 11, chemokine ligand 2 结合, haptoglobin, agglutination Lectin, cholesterol 25-hydroxylase 'syntaxin-8, syntaxin-11, cerui〇piasmin, complement component 1 , complement component 3, insertion 099141986 Form No. A0101 Page 28/Total 34 Page 1003114781Ό 201141510 Ο 素alpha 6(integrin alpha 6)、溶酶體酸脂酶 l(lysosomal acid lipase l)、J-2微球蛋白（J-2 microglobulin)、泛素（ubiquitin)、巨喔細胞移動抑 制因子（macrophage migration inhibitory factor)、絲切蛋白（cofilin)、親環素 A(cyclophillin A)、FKBP12、NDPK、前纖維蛋白 l(profilin 1)、半胱胺酸蛋白酶抑制劑C(cystatin C)、妈環素（calcyclin)、stanniocalcin-1、前列腺 素E-2 (PGE-2)、mpCCL2、IDO、誘發性一氧化氮合酶 (iNOS)、HLA-G5、M-CSF、jk管生成素 (angiopoietin)、PIGF、MCPH、胞#間質分子 .. . ..... (extracellular matrix molecules) 、 CCL2 (MCP-1)、CCL3 (MIP-1)、CCL4 (MlP-1)、 CCL5(RANTES) 、 CCL7(MCP-3) 、 CCL20(MIP-3)、 CCL26 (eotaxin-3) ' CX3CL1(fractalkine) ' CXCL5(ENA-78)、CXCL11( i-TAO ' CXCLUGRO)、 CXCL2(GR0)、CXCL8(IL-8)、CCUO(IP-IO)以及上述 各物之組合物。 7 .根據前述各項申請專利範圍中任一項所述之醫藥組成物， 其特徵為，該被刺激的幹細胞為常駐心臟幹細胞或循環幹 細胞或由注射注入之幹細胞。 8 .根據各項前述申請專利範圍中任一項所述之醫藥組成物， 其至少包括一級粒子群（primary particies)。 .根據申請專利範圍第8項所述之醫藥組成物，其特徵為， 該一級粒子群（PrimaiT particles)係選自於下列所 組成之組群：海藻酸鹽（alginates)、幾丁聚糖 099141986 表單編號A0101 第29頁/_共34頁 1003114781-0 201141510 (chitosan)、葡萄聚糖（dextran)、纖維素、脂質體（ liposome)以及聚酯微米球體或奈米球體（ microspheres or nanospheres of polyesters) 5 如PLGA '聚己内醋（polycaprolactone)或共聚醋 (copolyesters) ° 1〇 .根據申請專利範圍第8或第9項所述之醫藥組成物，其特徵 為’該一級粒子群（primary particles)包覆於該醫 藥組成物之幹細胞刺激物上。 11 . 一種醫藥混合物，其特徵為，其包括前述申請專利範圍任 一項之醫藥組成物（B)及包括至少一種醫藥有效 (pharmaceutically acti.ve)物質之組咸物（A)。 12 .根據申請專利範圍第u項所述之醫藥混合物，其特徵為， 該至少一種醫藥有效物質係選自於下列所組成之組群：類 騰島素生長因子（IGF-1)、肝細胞生長因子（HGF)及/或 其變形體如NK1、1K1、1K2、HP11或HP21，及上述各物 之組合物。 13 ·根據申請專利範圍第丨丨或第12項所述之醫藥混合物，其 特徵為’該至少一種醫藥有效物質進一步至少包括SCF一 1 〇 14 . 根據鈾述申請專利範圍第11至第1 3項中任一項所述之醫 藥混合物，其特徵為，該組成物進一步包括二級粒子 群（secondary particles)，其係選自於下列所組成 之組群：海藻酸鹽、幾丁聚醣、葡萄聚糖、纖維素、脂質 體或聚酯微米球體或奈米球體’如PLga、聚己内酯或共 聚醋（polycaprolactone or cop〇lyesters)。 根據申請專利範圍第1 4項所述之醫藥混合物，其特徵為， 099141986 1003114781-0 表單編號A0101 第3〇頁/共34頁 15 . 201141510 . 違一級粒子群（secondary part ic 1 es )包覆該至少一 . 種醫藥有效物質（pharmaceut i ca 11 y act i ve substance )。 16 .根據申請專利範圍第14或第15項所述之醫藥混合物其 特徵為，該二級粒子群（secondary particies)被設 計成可使包含於其中之物質先遞送之後，包含於一級粒子 群（primary particles)内之物質再行遞送者。 17 . —種根據申請專利範圍第1至第10項中任一項所述之醫藥Insegrin alpha 6, lysosomal acid lipase l, J-2 microglobulin, ubiquitin, macrophage cell migration inhibitor (macrophage) Migration inhibitory factor), cofilin, cyclophilin A, FKBP12, NDPK, profilin 1 , cystatin C, cystein (calcyclin), stanniocalcin-1, prostaglandin E-2 (PGE-2), mpCCL2, IDO, induced nitric oxide synthase (iNOS), HLA-G5, M-CSF, jk tubeogen (angiopoietin), PIGF, MCPH, cell #interstitial molecules ..... (extracellular matrix molecules), CCL2 (MCP-1), CCL3 (MIP-1), CCL4 (MlP-1), CCL5 (RANTES), CCL7 (MCP-3), CCL20 (MIP-3), CCL26 (eotaxin-3) 'CX3CL1(fractalkine) 'CXCL5(ENA-78), CXCL11( i-TAO ' CXCLUGRO), CXCL2(GR0), CXCL8(IL- 8), CCUO (IP-IO) and combinations of the above. The pharmaceutical composition according to any one of the preceding claims, wherein the stimulated stem cells are resident cardiac stem cells or circulating stem cells or stem cells injected by injection. 8. The pharmaceutical composition according to any of the preceding claims, comprising at least a primary fractional. The pharmaceutical composition according to claim 8, wherein the primary particle group is selected from the group consisting of alginate, chitosan 099141986 Form No. A0101 Page 29 / _ Total 34 pages 1003114781-0 201141510 (chitosan), dextran, cellulose, liposome and microspheres or nanospheres of polyesters 5, such as PLGA 'polycaprolactone or PEG (copolyesters) ° 1 〇. The pharmaceutical composition according to claim 8 or 9 of the patent application, characterized by 'the primary particles of the primary particles The stem cell stimulator is coated on the pharmaceutical composition. A pharmaceutical mixture characterized by comprising the pharmaceutical composition (B) according to any one of the preceding claims, and a group salt (A) comprising at least one pharmaceutically acti.ve. 12. The pharmaceutical mixture according to claim 5, wherein the at least one pharmaceutically effective substance is selected from the group consisting of: an allergen-like growth factor (IGF-1), hepatocytes Growth factors (HGF) and/or variants thereof such as NK1, 1K1, 1K2, HP11 or HP21, and combinations of the above. 13. The pharmaceutical mixture according to claim </ RTI> or claim 12, characterized in that the at least one pharmaceutically effective substance further comprises at least SCF-1 〇14. According to the uranium claim patent range 11th to 13th The pharmaceutical mixture according to any one of the preceding claims, wherein the composition further comprises secondary particles selected from the group consisting of alginate, chitosan, Glucan, cellulose, liposome or polyester microspheres or nanospheres such as PLga, polycaprolactone or cop〇lyesters. Pharmaceutical mixture according to item 14 of the patent application, characterized by 099141986 1003114781-0 Form No. A0101 Page 3 of 34. 201141510 . Secondary part ic 1 es coating The at least one pharmaceutically effective substance (pharmaceut i ca 11 y act i ve substance ). The medical mixture according to claim 14 or 15, wherein the secondary particies are designed to be included in the first-order particle group after the substance contained therein is delivered first ( The substance in the primary particles is re-delivered. 17 . The medicine according to any one of claims 1 to 10 組成物或申請專利範圍第11至第16項中任一項所述之醫 藥混合物’其作為藥物之使用者。 18 · —種根據申請專利範圍第1至第1〇項中任一項所述之醫藥 組成物或一種根據申請專利範圍第U至第16項中任一項 所述之醫藥混合物，其作為心臟組織再生之使用者。 19 . 一種根據申請專利範圍第1至第1〇項中任一項所述之醫藥 組成物或一種根據申請專利範圍第11至第16項中任一項 所述之醫藥混合物，其作為治療心臟組織退化之使用者。 2〇 . —種根據申請專利範圍第1至第10項中任一項所述之醫藥 組成物或一種根據申請專利範圍第U至第16項中任一項 之醫藥混合物’其作為治療包括心臟衰竭、心臟缺血或心 肌梗塞等心臟疾病之使用者。 21 . —種於活體内或活體外作用於人類或動物之心臟幹細胞之 方法’其特徵為，將一種根據申請專利範圍第1至第10項 中任一項所述之醫藥組成物（B)施加於該人類或該動物者 22 .根據申請專利範圍第21項所述之方法，其特徵為，該醫藥 組成物（B)之施加係於首先初步施加包含至少一種醫藥有 表單編號A0101 第31頁/共34頁 1003114781-0 201141510 效物質之組成物（A)之後者。 23 .根據申請專利範圍第21或第22項所述之方法，其特徵為 ，該施加係藉由注射方式來執行者。 24 ·根據前述申請專利範圍第21至第23項中任一項所述之方 法，其特徵為，該施加係以循連續注射方式（ sequential injection)來執行者。 25 ·根據前述申請專利範圍第21至第24項中任一項所述之方 法，其特徵為，根據申請專利範圍第1至第1〇項中任一項 所述之fe藥組成物（B)之連續兩次施加之間隔係從丨小時 至180天者。 26 .根據前述申請專利範圍第21至第25項中任一項所述之方 法，其特徵為，其每次施加係以重複之方式作施加者。 27 ·根據前述申請專利範圍第21至第26項中任一項所述之方 法’其特徵為’該醫藥組成物（A)或（B)係以腸胃外之方 式作施加者。 28 .根據前述申請專利範圍第21至第27項中任一項所述之方 法’其特徵為’該醫藥組成物（A)或（B)係施加於人類或 動物之循環系統中者。 29 .根據前述申請專利範圍第21至第28項中任一項所述之方 法’其特徵為，該醫藥組成物（A)或（B)係施加於靜脈及/ 或動脈中者。 30 ♦根據前述申請專利範圍第21至第29項中任一項所述之方 法’其特徵為，該醫藥組成物（A)或（B)係施加於一個心 臟組織内者。 31 099141986 根據前述申請專利範圍第21至第30項中任一項所述之方 法’其特徵為，該醫藥組成物（。係在冠狀血管内施加， 表單編號A0101 第32頁/共34頁 1003114781-0 201141510 而該組成物（A)之初步施加係在靜脈内者。 32 .根據前述申請專利範圍第21至第26項中任一項所述之方 法，其特徵為，該組成物（A)之每次施加與否係為選擇性 者。The composition or the pharmaceutical mixture described in any one of the claims 11 to 16 is used as a user of the drug. The pharmaceutical composition according to any one of claims 1 to 1 or the pharmaceutical mixture according to any one of the claims U to 16 of the patent application, as a heart The user who organizes the regeneration. A pharmaceutical composition according to any one of claims 1 to 1 or a pharmaceutical mixture according to any one of claims 11 to 16, which is used as a therapeutic heart. A user whose organization is degraded. The pharmaceutical composition according to any one of claims 1 to 10, or the pharmaceutical mixture according to any one of claims U to 16 of the patent application, which comprises a heart as a treatment A user of heart disease such as failure, cardiac ischemia or myocardial infarction. A method of cultivating a cardiac stem cell which is applied to a human or an animal in vivo or in vitro, wherein the pharmaceutical composition according to any one of claims 1 to 10 (B) The method of claim 21, wherein the application of the pharmaceutical composition (B) is first applied initially comprising at least one medicine having a form number A0101. Page / Total 34 pages 1003114781-0 201141510 The composition of the effect substance (A). A method according to claim 21 or 22, wherein the application is performed by an injection method. The method according to any one of the preceding claims, wherein the application is performed in a sequential injection. The method according to any one of the preceding claims, wherein the composition of the fe-feet according to any one of claims 1 to 1 (B) The interval between two consecutive applications is from 丨 hours to 180 days. The method according to any one of the preceding claims, wherein each application is applied in a repeated manner. The method according to any one of the preceding claims, wherein the pharmaceutical composition (A) or (B) is administered parenterally. The method according to any one of the preceding claims, wherein the pharmaceutical composition (A) or (B) is applied to a human or animal circulatory system. The method according to any one of the preceding claims, wherein the pharmaceutical composition (A) or (B) is applied to a vein and/or an artery. The method according to any one of the preceding claims, wherein the pharmaceutical composition (A) or (B) is applied to a heart tissue. The method of any one of the preceding claims, wherein the pharmaceutical composition is applied in a coronary blood vessel, Form No. A0101, Page 32 of 34, 1003114781 And the method of any one of the preceding claims, wherein the composition (A) is in the vein. The method according to any one of the preceding claims, wherein the composition (A) Each time it is applied, it is a selective one. 099141986 表單編號A0101 第33頁/共34頁 1003114781-0099141986 Form No. A0101 Page 33 of 34 1003114781-0
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