TW201302207A - Composition for use in treating and preventing mucosa related disease - Google Patents
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本發明係一種玻尿酸混合物用於製造治療及預防黏膜相關病變或疾病之組成物的用途，其中黏膜相關病變或疾病之症狀包含潰瘍、發炎、過敏反應及流血。 The present invention is a use of a hyaluronic acid mixture for the manufacture of a composition for treating and preventing mucosal-related diseases or diseases, wherein the symptoms of mucosal-related diseases or diseases include ulcers, inflammation, allergic reactions, and bleeding.
玻尿酸具有不同之英文名詞(hyaluronic acid、hyaluronan、hyaluronate及sodium hyaluronate)，可簡稱為HA，係一種天然的糖胺聚多醣(glycosaminoglycan)，其係由交替的N-乙醯-D-葡萄糖胺(N-acetyl-D-glucosamine)及D-葡萄糖醛酸(glucuronic acid)組成，此雙糖由交替之β-1,3及β-1,4糖苷鍵鏈結。天然的玻尿酸通常具有高黏滯度，其分子量介於5萬道爾頓(Dalton，Da)至數百萬道爾頓之間。 Hyaluronic acid has different English nouns (hyaluronic acid, hyaluronan, hyaluronate and sodium hyaluronate), which can be referred to as HA, is a natural glycosaminoglycan, which is composed of alternating N-acetyl-D-glucosamine ( N-acetyl-D-glucosamine) and D-glucuronic acid, which are linked by alternating β-1,3 and β-1,4 glycosidic linkages. Natural hyaluronic acid typically has a high viscosity with a molecular weight between 50,000 Daltons (Daton) and millions of Daltons.
天然之玻尿酸為黏彈性流體，填充於細胞與膠原蛋白纖維空隙之中且覆蓋在某些表皮組織上。主要用來保護及潤滑細胞，提供一平台來運輸調節性T細胞，穩定膠原蛋白網狀結構，以及保護它免於受到機械性的破壞。玻尿酸在肌腱、肌腱鞘及黏滑膜表面也是一種主要的潤滑劑，此係藉由其潤滑特性及高吸震能力。玻尿酸也有助於組織流變力學(rheological mechanics)，運動和細胞增殖(參照Delpech,B.,Girard,N.,Bertrand,P.,Courel,M.-N.,Chauzy,C.,Delpech,A.,1997.J.Intern.Med.242,41-48)。並參與一些細胞表面的受體交互作用(receptor interaction)，特別指主要的受體-CD44。CD44已被廣泛接受為一種淋巴球 細胞活化之標記(參照Teder P,Vandivier RW,Jiang D,Liang J,Cohn L,Pure E,Henson PM,Noble PW.Science 2002；296：155-158)。 Natural hyaluronic acid is a viscoelastic fluid that fills the gaps between cells and collagen fibers and covers certain epidermal tissues. Primarily used to protect and lubricate cells, providing a platform to transport regulatory T cells, stabilize the collagen network, and protect it from mechanical damage. Hyaluronic acid is also a major lubricant on the surface of tendons, tendon sheaths and sticky synovial membranes, which are characterized by their lubricating properties and high shock absorption. Hyaluronic acid also contributes to the organization of rheological mechanisms, motion and cell proliferation (see Delpech, B., Girard, N., Bertrand, P., Courel, M.-N., Chauzy, C., Delpech, A). , 1997. J. Intern. Med . 242, 41-48). And participate in some cell surface receptor interactions, especially the main receptor - CD44. CD44 has been widely accepted as a marker for lymphocyte activation (see Teder P, Vandivier RW, Jiang D, Liang J, Cohn L, Pure E, Henson PM, Noble PW. Science 2002; 296: 155-158).
近來玻尿酸常以其鈉鹽形態應用於臨床治療中，主要用於眼科、皮膚科、整形外科、一般外科、關節炎、動脈治療及化粧品中。含鹼金屬離子、鹼土金屬離子(例如鎂)、鋁離子、銨離子及經取代銨離子的鹽類之玻尿酸可充作輔助藥物吸收之載體(參照比利時專利第904,547號)。在玻尿酸之重金屬鹽類中，銀鹽被用為殺黴菌劑；而金鹽可用以治療類風濕性關節炎(參照PCT專利WO 87/05517)。 Recently, hyaluronic acid is often used in clinical treatment in the form of its sodium salt, mainly used in ophthalmology, dermatology, orthopedics, general surgery, arthritis, arterial therapy and cosmetics. Hyaluronic acid containing an alkali metal ion, an alkaline earth metal ion (for example, magnesium), an aluminum ion, an ammonium ion, and a salt of a substituted ammonium ion can be used as a carrier for assisting drug absorption (refer to Belgian Patent No. 904,547). Among the heavy metal salts of hyaluronic acid, silver salts are used as fungicides; and gold salts can be used to treat rheumatoid arthritis (refer to PCT Patent WO 87/05517).
美國專利第5,888,986號揭露使用有效濃度玻尿酸治療膀胱炎之方法及相關結構，其中玻尿酸的重量平均分子量(Mw)超過20萬道爾頓。在實施例中僅運用特定重量平均分子量之單一玻尿酸。例如，利用重量平均分子量65萬道爾頓或190萬道爾頓之玻尿酸來治療膀胱炎，但特定重量平均分子量之單一玻尿酸，無法兼顧治療速效及持久的效果。 U.S. Patent No. 5,888,986 discloses the use of an effective concentration of hyaluronic acid for the treatment of cystitis and related structures wherein the weight average molecular weight (Mw) of hyaluronic acid exceeds 200,000 Daltons. In the examples, only a single hyaluronic acid of a specific weight average molecular weight is used. For example, hyaluronic acid having a weight average molecular weight of 650,000 Daltons or 1.9 million Daltons is used to treat cystitis, but a single hyaluronic acid having a specific weight average molecular weight cannot balance the therapeutic effect with long-lasting effects.
美國專利申請案第2005/0080037號揭露玻尿酸治療急性和過度扭傷和應變，其中玻尿酸之重量平均分子量僅介於90萬道爾頓至120萬道爾頓間，特定重量平均分子量之單一玻尿酸無法兼顧治療速效及持久的效果。 U.S. Patent Application No. 2005/0080037 discloses the treatment of acute and excessive sprains and strains by hyaluronic acid, wherein the weight average molecular weight of hyaluronic acid is only between 900,000 Daltons and 1.2 million Daltons, and the specific hyaluronic acid of a specific weight average molecular weight cannot be balanced. Treatment for quick and long lasting results.
美國專利第7,354,910號揭露重量平均分子量介於60萬道爾頓至120萬道爾頓間之玻尿酸能用來治療發炎性腸疾病(inflammatory bowel disease,IBD)。然而，僅單一重量平均分子量的玻尿酸，在其注射入病人的體內後，無法兼顧速效及持久的效果，因此在臨床上對病人而言非常不 方便。 U.S. Patent No. 7,354,910 discloses that hyaluronic acid having a weight average molecular weight of between 600,000 Daltons and 1.2 million Daltons can be used to treat inflammatory bowel disease (IBD). However, only a single weight average molecular weight hyaluronic acid cannot be combined with a quick-acting and long-lasting effect after it is injected into a patient's body, so it is clinically very uncomfortable for the patient. Convenience.
歐洲專利第1369119號揭露了重量平均分子量介於60萬道爾頓至300萬道爾頓間之玻尿酸能用來治療自體免疫疾病。然而，該專利僅利用具特定重量平均分子量之單一玻尿酸，而無法兼顧治療速效及持久的效果。 European Patent No. 1369119 discloses that hyaluronic acid having a weight average molecular weight of between 600,000 Daltons and 3 million Daltons can be used to treat autoimmune diseases. However, this patent utilizes only a single hyaluronic acid having a specific weight average molecular weight, and cannot achieve both a fast-acting and long-lasting effect.
黏膜(mucous membrane,mucosa)覆蓋在大部分的內皮表面，表皮表面，並具有吸收(於腸胃道)及分泌(於腸胃道及呼吸道)功能。黏膜覆蓋在對外部環境曝露及內部器官的凹陷表面，及伴隨著皮膚覆蓋在身體的一部份：鼻孔、嘴、嘴唇、眼瞼、耳朵、生殖道及肛門。由黏膜及腺體分泌具有黏性的濃厚液體稱為黏液(mucus)，而依據黏膜在身體的不同部份有不同的稱呼，同時黏膜未必皆會分泌黏液。 The mucous membrane (mucosa) covers most of the surface of the endothelium, the surface of the epidermis, and has functions of absorption (in the gastrointestinal tract) and secretion (in the gastrointestinal tract and respiratory tract). The mucous membrane covers the exposed surface of the external environment and the concave surface of the internal organs, and is accompanied by skin covering a part of the body: nostrils, mouth, lips, eyelids, ears, reproductive tract and anus. The thick liquid that is secreted by mucous membranes and glands is called mucus, and depending on the mucosa, different parts of the body are called, and mucus does not necessarily secrete mucus.
眼睛：結膜炎(conjunctivitis)係結膜發炎，(結膜是眼睛的最外層及眼瞼的內部表面)最常起因於感染或過敏反應(參照Richards A,May 2010.「結膜炎("Conjunctivitis")」.Pediatr Rev 31(5)：196-208)。 Eyes: Conjunctivitis is inflammation of the conjunctiva (the conjunctiva is the outermost layer of the eye and the inner surface of the eyelid) most often caused by infection or allergic reactions (see Richards A, May 2010. "Conjunctivitis"). Pediatr Rev 31(5): 196-208).
耳朵：耳炎(otitis)是人類及其他動物耳朵發炎或感染的一般名詞。可分為：外耳炎、中耳炎及內耳炎。 Ear: Otitis is a general term for inflammation or infection of the ear of humans and other animals. Can be divided into: otitis externa, otitis media and otitis externa.
過敏性鼻炎：鼻炎是鼻黏膜發炎，其特徵為複合性症狀包括以下任一種之結合：打噴嚏、鼻塞、發癢及流鼻水。眼睛、耳朵、鼻竇及喉嚨皆包含在內。過敏性鼻炎是鼻炎的最常見起因，同時影響了20%的族群。過敏性鼻炎並非是威脅性命的疾病，然而會產生併發症且會明顯影響生活品質，間接導致出許多生活花費(參照Bousquet J et al.「過 敏性鼻炎管理手冊(Allergic rhinitis management pocket reference)」2008.Allergy 2008 Aug；63(8)：990-996)。美國專利申請案第2005/0107330號揭露一種用來局部治療鼻炎的醫藥組合物，其包含至少一個酸性糖胺聚多糖。該發明亦包含至少一個適宜局部施用的擬交感藥物或生理上可接受之鹽類或衍生物，其具有對黏膜之血管收縮或消腫的作用，且其並未揭露或教導結合兩種不同重量平均分子量之玻尿酸。 Allergic rhinitis: Rhinitis is inflammation of the nasal mucosa characterized by a combination of any of the following: sneezing, stuffy nose, itching, and runny nose. Eyes, ears, sinuses and throat are included. Allergic rhinitis is the most common cause of rhinitis and affects 20% of the population. Allergic rhinitis is not a life-threatening disease, but it can cause complications and can significantly affect the quality of life, indirectly leading to a lot of cost of living (see Bousquet J et al . "Allergic rhinitis management pocket reference" 2008 Allergy 2008 Aug; 63(8): 990-996). U.S. Patent Application Serial No. 2005/0107330 discloses a pharmaceutical composition for topical treatment of rhinitis comprising at least one acidic glycosaminoglycan. The invention also encompasses at least one sympathomimetic or physiologically acceptable salt or derivative suitable for topical administration, which has an effect on vasoconstriction or swelling of the mucosa, and which does not disclose or teach the incorporation of two different weights The average molecular weight of hyaluronic acid.
嘴巴：口腔黏膜位於口腔的表皮。口腔潰瘍是口腔內部的開放性潰瘍，或在少數例子中，會造成嘴唇上或口腔周圍的黏膜或表皮的裂開。潰瘍的類型歧異，具有多重相關的原因：物理性的磨損、酸性水果、感染、其他醫學狀態、給藥方法以及癌症的和非專一性進程(cancerous and nonspecific processes)。一旦形成，潰瘍可能因發炎及/或第二次感染而持續。兩種常見形態為口腔潰瘍及嘴唇皰疹(cold sores)。嘴唇附近的嘴唇皰疹係由病毒所導致(參照J.M.Casiglia,et al.,October 2006.「口腔潰瘍(Aphthous stomatitis)」.Emedecine)。 Mouth: The oral mucosa is located in the epidermis of the mouth. Oral ulcers are open ulcers inside the mouth, or in a few cases, cracks in the mucous membranes or epidermis around the lips or around the mouth. The type of ulcer is heterogeneous and has multiple related causes: physical wear, acidic fruit, infection, other medical conditions, methods of administration, and cancerous and nonspecific processes. Once formed, the ulcer may persist due to inflammation and/or a second infection. Two common forms are oral ulcers and cold sores. Lip herpes near the lips are caused by viruses (see JM Casiglia, et al ., October 2006. "Aphthous stomatitis". Emedecine ).
支氣管炎：支氣管係用於將氣流從氣管輸送到肺臟之呼吸道。支氣管炎為支氣管的黏膜發炎，可分為兩種：急性及慢性。每一種都有獨特的病因、病徵及療法。急性支氣管炎經常在如感冒或流感的急性病毒疾病進程中發生。慢性支氣管炎最常發展於吸入性刺激所導致之氣管的再發性損傷。吸煙是最常見的起因，接著為空氣污染及職業性暴露刺激源(occupational exposure to irritants)(Cohen, Jonathan and William Powderly.「感染性疾病(Infectious Diseases.)」2nd ed.Mosby(Elsevier),2004.「第三章氣管、支氣管以及囊性纖維化(Chapter 33：Bronchitis,Bronchiectasis,and Cystic Fibrosis)」)。美國專利申請案第2003/0171332號揭露一種藉由能結合CD44的多醣(polysaccharide)來治療呼吸道疾病的方法，但只利用單一種玻尿酸。 Bronchitis: The bronchial system is used to transport airflow from the trachea to the respiratory tract of the lungs. Bronchitis is inflammation of the mucosa of the bronchi and can be divided into two types: acute and chronic. Each has its own unique cause, symptoms and treatments. Acute bronchitis often occurs in the course of acute viral diseases such as colds or flu. Chronic bronchitis is most often developed as a recurrent injury to the trachea caused by inhalation stimulation. Smoking is the most common cause, followed by exposure to irritants (Cohen, Jonathan and William Powderly. "Infectious Diseases." 2nd ed. Mosby (Elsevier), 2004 " Chapter 3: Trachea , Bronchial, and Cystic Fibrosis " ( Chapter 33: Bronchitis , Bronchiectasis, and Cystic Fibrosis ). U.S. Patent Application Serial No. 2003/0171332 discloses a method for treating respiratory diseases by binding to a polysaccharide of CD44, but using only a single hyaluronic acid.
腸胃道黏膜：包覆消化腔或管道內開放空間之腸胃壁的最內層是黏膜。此層係直接與食物團接觸，並負責吸收、消化及分泌等消化作用的重要過程。 Gastrointestinal mucosa: The innermost layer of the gastrointestinal wall that coats the open space in the digestive tract or duct is the mucosa. This layer is in direct contact with the food group and is responsible for the important processes of digestion, digestion and secretion.
黏膜在每個消化道器官是高度特殊化的，其在胃中面對低pH、在小腸中吸收多種不同的物質，而同樣在大腸中吸收一定份量的水。為反映出這些器官的不同需求，黏膜的結構能由分泌腺體的套疊(如胃小凹，gastric pits)所組成，或為了增加表面積呈折疊狀(Abraham L.Kierszenbaum,2002，「組織學以及細胞生物學：從導論至病理學(Histology and Cell Biology：an introduction to pathology)」。 The mucosa is highly specialized in each digestive tract organ, which faces a low pH in the stomach, absorbs a variety of different substances in the small intestine, and also absorbs a certain amount of water in the large intestine. To reflect the different needs of these organs, the structure of the mucosa can be composed of nests that secrete glands (such as gastric pits) or folds to increase surface area (Abraham L. Kierszenbaum, 2002, Histology And Histology and Cell Biology: an introduction to pathology.
消化道黏膜在身體與同時含有營養物及潛在致病性微生物及毒素的消化腔之間形成一道屏障。黏膜的挑戰在於當需要將進入體內的有害分子及有機體(organisms)強力排開時，同時允許有效的傳輸營養物穿透表皮入人體內。胃及腸黏膜的排除特性稱之為「腸胃道屏障(gastrointestinal barrier)」。一般而言，毒素及微生物皆能突破單一的表皮細胞層而毫無阻礙的進入體循環。 The digestive tract mucosa forms a barrier between the body and the digestive tract that contains both nutrients and potentially pathogenic microorganisms and toxins. The challenge of mucous membranes is to allow efficient delivery of nutrients through the epidermis into the body when it is necessary to force the harmful molecules and organisms into the body. The exclusion characteristic of the stomach and intestinal mucosa is called the "gastrointestinal barrier". In general, toxins and microorganisms can break through a single epidermal cell layer without any hindrance into the systemic circulation.
胃食道逆流症(gastroesophageal reflux disease,GERD) 是一種胃酸從胃逆流至食道所致的慢性症狀或黏膜損傷，典型的症狀是心口灼熱。胃食道逆流症通常是導因於胃與食道間屏障的變化，其包括：下食道括約肌(lower esophageal sphincter)的不正常放鬆，在正常情況下，該下食道括約肌會保持胃的頂部呈封閉狀；食道受損始於胃逆流之驅逐作用；或者橫隔膜疝氣(hiatal hernia)。該等變化可能是永久的或暫時的(參照DeVault KR,1999.「胃食道逆流症之診斷與治療之最新指引-美國胃腸病學會之執業參數委員會(Updated guidelines for the diagnosis and treatment of gastroesophageal reflux disease.The Practice Parameters Committee of the American College of Gastroenterology)」.Am J Gastroenterol 94(6)：1434-42)。 Gastroesophageal reflux disease (GERD) is a chronic symptom or mucosal damage caused by gastric acid reflux from the stomach to the esophagus. The typical symptom is heartburn. Gastroesophageal reflux disease is usually caused by changes in the barrier between the stomach and the esophagus. It includes: abnormal relaxation of the lower esophageal sphincter. Under normal circumstances, the lower esophageal sphincter keeps the top of the stomach closed. Esophageal damage begins with the expulsion of the stomach countercurrent; or hiatal hernia. These changes may be permanent or temporary (see DeVault KR, 1999. Updated guidelines for the diagnosis and treatment of gastroesophageal reflux disease) .The Practice Parameters Committee of the American College of Gastroenterology)". Am J Gastroenterol 94(6): 1434-42).
消化性潰瘍(peptic ulcer)：消化性潰瘍係一種腸胃道之區域的潰瘍，其定義為黏膜糜爛等於或大於0.5公分，該區域通常是酸性的，而因此導致特別疼痛。幾乎70-90%的潰瘍與幽門螺旋桿菌(Helicobacter pylori)有關，該菌呈螺旋狀生活在胃的酸性環境中。潰瘍也會因藥物而產生或惡化，例如阿司匹靈(aspirin)、保栓通錠(Plavix)、異丁苯丙酸(ibuprofen)及其他的非類固醇消炎劑(non-steroid anti-inflammatory drugs，NSAIDs)。 Peptic ulcer: A peptic ulcer is an ulcer in the area of the gastrointestinal tract, defined as mucosal erosion equal to or greater than 0.5 cm, which is usually acidic and thus causes particularly pain. Almost 70-90% of the ulcers are associated with Helicobacter pylori , which lives in the acidic environment of the stomach in a spiral shape. Ulcers can also be caused or worsened by drugs such as aspirin, Plavix, ibuprofen, and other non-steroidal anti-inflammatory drugs. , NSAIDs).
與一般咸信相反，消化性潰瘍在十二指腸比在胃裡更易發生。大約4%的胃潰瘍是起因於惡性腫瘤，且需要多種活組織檢驗來排除其係癌症。十二指腸潰瘍(duodenal ulcer)一般而言是良性的。消化性潰瘍是藉由發作部位予以分類：胃(稱為胃潰瘍)、十二指腸(稱為十二指腸潰 瘍)、食道(稱為食道潰瘍)、梅克爾氏憩室(Meckel's Diverticulum，稱為梅克爾氏憩室潰瘍)。 In contrast to general salty, peptic ulcers are more likely to occur in the duodenum than in the stomach. Approximately 4% of gastric ulcers are caused by malignant tumors and require multiple biopsy tests to rule out cancer. Duodenal ulcers are generally benign. Peptic ulcers are classified by the site of the attack: stomach (called gastric ulcer), duodenum (called duodenal ulcer) Ulcer), esophagus (called esophageal ulcer), Meckel's Diverticulum (called Meckel's diverticulum ulcer).
上述實施例皆代表黏膜相關病變或疾病但不受限於上述實施例。所揭露的病變或疾病至今可能用一些不同藥物或治療方法來治療，但這些治療手段大部分是藉由高度特別製造方法所製成的複雜成份藥物來達成，而非屬簡單及安全的治療手段。除了本發明之發明人外，並未有先前技術教導或預測結合兩個具有不同重量平均分子量之玻尿酸(LMWHA，低重量平均分子量玻尿酸；HMWHA，高重量平均分子量玻尿酸)，而該兩個玻尿酸之分子量係以150萬道爾頓來做界限區分。此代表在本技術領域具有通常知識者無法藉由結合任何其他前案導出本發明之混合物應用的概念。 The above examples all represent mucosa-related lesions or diseases but are not limited to the above examples. The disclosed lesions or diseases may have been treated with a number of different drugs or treatments to date, but most of these treatments are achieved by complex ingredients made from highly specialized manufacturing methods, rather than simple and safe treatments. . In addition to the inventors of the present invention, there is no prior art teaching or prediction to combine two hyaluronic acids (LMWHA, low weight average molecular weight hyaluronic acid; HMWHA, high weight average molecular weight hyaluronic acid) having different weight average molecular weights, and the two hyaluronic acid The molecular weight is distinguished by a limit of 1.5 million Daltons. This represents a concept that would not be able to derive the application of the mixture of the present invention by any of the prior art in connection with any of the prior art.
為了克服先前技術之缺點，本發明提供一種玻尿酸混合物用於製造治療及預防黏膜相關病變或疾病之組成物的用途，其中所述的組成物包括治療上有效劑量之玻尿酸混合物，而該混合物係包含至少兩個不同重量平均分子量之玻尿酸，用以緩和或排除先前提到之問題。 In order to overcome the disadvantages of the prior art, the present invention provides a use of a hyaluronic acid mixture for the manufacture of a composition for treating and preventing a mucosal-related disease or disease, wherein the composition comprises a therapeutically effective amount of a hyaluronic acid mixture, and the mixture comprises At least two different weight average molecular weight hyaluronic acids are used to alleviate or eliminate the previously mentioned problems.
本發明之整體概念首先需理解到玻尿酸會被玻尿酸水解酶(hyaluronidase,HAase)分解。而線狀玻尿酸(linear HA)(LMWHA)及球狀玻尿酸(globular HA)(HMWHA)會被分解成小片段，惟其中球狀玻尿酸因空間立體結構，而能在降解後補充線狀玻尿酸，因此有助於治療效用，同時能延長作用時間。減少需不斷補充線狀玻尿酸的困擾。已知 玻尿酸水解酶的活性能被用來作為發炎的標記。簡述如下：低及高重量平均分子量的玻尿酸的混合，可結合低重量平均分子量玻尿酸容易附著在傷口及快速治療的效果，及高重量平均分子量玻尿酸可因降解而不斷補充低重量平均分子量玻尿酸的概念，因此該混合物同時可用於治療並具長效。因此，較短的玻尿酸減少後，可不斷地被補充用於持續的傷口治療或提供一種軟性障壁使組織免於損傷或進一步損傷。以下將詳細描述本發明之概念。 The overall concept of the present invention first understands that hyaluronic acid is decomposed by hyaluronidase (HAase). Linear hyaluronic acid (linear HA) (LMWHA) and globular HA (HMWHA) are broken down into small fragments, but globular hyaluronic acid can replenish linear hyaluronic acid after degradation due to spatial stereostructure. It helps the therapeutic effect while prolonging the duration of action. Reduce the need to constantly replenish linear hyaluronic acid. A known The activity of hyaluronic acid hydrolase can be used as a marker of inflammation. Briefly described as follows: the combination of low and high weight average molecular weight hyaluronic acid can be combined with the low weight average molecular weight hyaluronic acid to adhere to the wound and rapid treatment effect, and the high weight average molecular weight hyaluronic acid can continuously supplement the low weight average molecular weight hyaluronic acid due to degradation. Concept, so the mixture is both therapeutic and long-lasting. Thus, after a decrease in hyaluronic acid, it can be continuously replenished for sustained wound treatment or provide a soft barrier to protect the tissue from damage or further damage. The concept of the present invention will be described in detail below.
本發明之用於治療及預防黏膜相關病變或疾病之組合物係包含一有效治療劑量之玻尿酸混合物，其包括一低重量平均分子量玻尿酸(LMWHA)及一高重量平均分子量玻尿酸(HMWHA)。如圖1所示，低重量平均分子量玻尿酸係重複的雙醣單元而形成線狀結構。然而，高重量平均分子量玻尿酸，一般咸信線狀玻尿酸巨分子的羧基(carboxylic group)藉由與二/三/多胺反應形成醯胺鍵結(amide linkage)，並同時形成分子間鍵橋(intramolecular bridge)。基於此反應，開始纏繞之玻尿酸結構會轉形成球狀奈米粒子(spherical nanoparticle)(參照美國專利第7,879,818號)。不同的分子量有不同的流變、黏附、組織支架的功能及在水溶液中的降解性，因此玻尿酸混合物能平衡藥物功效及降解速率，以利於治療及預防黏膜相關病變或疾病，同時達成適宜的及延長的治療效果。 The composition for treating and preventing mucosal related diseases or diseases of the present invention comprises a therapeutically effective amount of a hyaluronic acid mixture comprising a low weight average molecular weight hyaluronic acid (LMWHA) and a high weight average molecular weight hyaluronic acid (HMWHA). As shown in Fig. 1, the low weight average molecular weight hyaluronic acid is a repeating disaccharide unit to form a linear structure. However, the high weight average molecular weight hyaluronic acid, the carboxylic group of the generalized linear hyaluronic acid macromolecule, forms an amide linkage by reaction with a di/tri/polyamine, and simultaneously forms an intermolecular bond bridge ( Intramolecular bridge). Based on this reaction, the hyaluronic acid structure that begins to wrap is transformed into spherical nanoparticle (refer to U.S. Patent No. 7,879,818). Different molecular weights have different rheology, adhesion, tissue scaffold function and degradability in aqueous solution, so hyaluronic acid mixture can balance drug efficacy and degradation rate to facilitate the treatment and prevention of mucosal related diseases or diseases, and at the same time achieve appropriate Prolonged treatment effect.
Tranchepain F.等人揭露了玻尿酸之所以會有不同的生物功能係基於其分子量。從物理化學的觀點，藉由小牛睪丸玻尿酸水解酶催化玻尿酸水解的研究指出，動力學參數 係依賴玻尿酸鏈的長度。獲得不同分子量的玻尿酸的新製程係藉由玻尿酸水解酶催化玻尿酸水解(參照Tranchepain F et al.,「藉由玻尿酸水解酶水解之一完整系列玻尿酸片段：藉由簡單高性能液相層析儀研究玻尿酸質量之應用(Acomplete set of hyaluronan fragments obtained from hydrolysis catalyzed by hyaluronidase： Application to studies of hyaluronan mass distribution by simple HPLC devices.)」Anal Biochem.2006 Jan 15；348(2)：232-42.)。相似的，同一批研究團隊亦揭露了玻尿酸之所以具有不同的生物功能係強烈依賴其鏈長。一般認為玻尿酸水解酶催化玻尿酸水解係參與控制較長鏈與較短鏈玻尿酸間的平衡。較短鏈玻尿酸似乎因太短而無法形成一個穩定的複合物，而較長鏈玻尿酸也在形成複合物上遭遇困難，這可能是因為立體構造上的阻礙(steric hindrance)。 Tranchepain F. et al. revealed that hyaluronic acid has different biological functions based on its molecular weight. From a physicochemical point of view, studies on the hydrolysis of hyaluronic acid by calf hydrazide hyaluronan hydrolase indicate that the kinetic parameters depend on the length of the hyaluronic acid chain. A new process for obtaining hyaluronic acid of different molecular weights catalyzes the hydrolysis of hyaluronic acid by hyaluronan hydrolase (cf. Tranchepain F et al ., "A complete series of hyaluronic acid fragments hydrolyzed by hyaluronic acid hydrolase: by simple high performance liquid chromatography A complete set of hyaluronan fragments obtained from hydrolysis catalyzed by hyaluronidase: Application to studies of hyaluronan mass distribution by simple HPLC devices.) Anal Biochem .2006 Jan 15;348(2):232-42.). Similarly, the same team of researchers also revealed that hyaluronic acid has different biological functions depending on its chain length. It is generally believed that hyaluronic acid hydrolase catalyzes the hydrolysis of hyaluronic acid to control the balance between longer chain and shorter chain hyaluronic acid. The shorter chain hyaluronic acid appears to be too short to form a stable complex, while the longer chain hyaluronic acid also encounters difficulties in forming complexes, possibly due to steric hindrance.
數篇報告闡述玻尿酸水解酶參與了發炎反應。滑液(Synovial fluid,SF)玻尿酸水解酶的活性能用來作為滑膜炎症(Synovial inflammation)的標記(參照Nagaya H et al.,「評估風濕性關節炎及骨關節炎病人作為關節標記分子之滑液及血清玻尿酸水解酶活性(Examination of synovial fluid and serum HAase activity as a joint marker in rheumatoid arthritis and osteoarthritis patients.)」Ann Rheum Dis.,1999,58(3)：186-8)。玻尿酸是牙周韌帶(periodontal ligament,PDL)之細胞外基質(extracellular matrix)的主要成份，能促進其結構及功能的完整。玻尿酸有助於咀嚼期間牙周韌帶之緩衝效用，同時在發炎及傷口 癒合上亦占有重要地位。研究指出牙周韌帶纖維母細胞能釋出玻尿酸水解酶，並於生理狀態及發炎狀態下，能產生細胞外玻尿酸代謝所需的玻尿酸水解酶的活性(Ohno S et al.,「玻尿酸水解酶於人類牙周韌帶纖維母細胞之表現及活性(Expression and activity of hyaluronidase in human periodontal ligament fibroblasts.)」J Periodontol.,2002,,73(11)：1331-7)。玻尿酸水解酶作用後產生的較短玻尿酸片段參與了發炎反應及血管生成(angiogenesis)，然而原生的(native)玻尿酸卻不會(Tranchepain F et al.,同上述)。 Several reports have stated that hyaluronan hydrolase is involved in the inflammatory response. The activity of synovial fluid (SF) hyaluronic acid hydrolase can be used as a marker for Synovial inflammation (see Nagaya H et al ., "Assessing rheumatoid arthritis and osteoarthritis patients as joint marker molecules""Examination of synovial fluid and serum HAase activity as a joint marker in rheumatoid arthritis and osteoarthritis patients." Ann Rheum Dis ., 1999, 58(3): 186-8). Hyaluronic acid is the main component of the extracellular matrix of the periodontal ligament (PDL), which promotes the integrity of its structure and function. Hyaluronic acid contributes to the cushioning effect of the periodontal ligament during chewing, and also plays an important role in inflammation and wound healing. Studies have shown that periodontal ligament fibroblasts can release hyaluronan hydrolase, and under physiological and inflammatory conditions, can produce the activity of hyaluronan hydrolase required for extracellular hyaluronic acid metabolism (Ohno S et al ., "Hyaluronic acid hydrolase Expression and activity of hyaluronidase in human periodontal ligament fibroblasts.) J Periodontol ., 2002, 73(11): 1331-7). The shorter hyaluronic acid fragment produced by the action of hyaluronic acid hydrolase is involved in the inflammatory response and angiogenesis, whereas the native hyaluronic acid does not (Tranchepain F et al ., supra).
依據本發明之定義，具有重量平均分子量低於150萬道爾頓的玻尿酸被歸類為低重量平均分子量玻尿酸(LMWHA)。較佳的，所述之低重量平均分子量玻尿酸平均分子量範圍係介於5萬至150萬道爾頓之間。更佳的，所述之低重量平均分子量玻尿酸平均分子量範圍係介於10萬至150萬道爾頓之間。最佳的，所述之低重量平均分子量玻尿酸平均分子量範圍係介於10萬至50萬道爾頓之間。具有重量平均分子量高於150萬道爾頓的玻尿酸被歸類為高重量平均分子量玻尿酸(HMWHA)。較佳的，所述之高重量平均分子量玻尿酸平均分子量範圍係介於150萬至500萬道爾頓之間。，最佳的，所述之高重量平均分子量玻尿酸平均分子量範圍係介於150萬至250萬道爾頓之間。將組合低重量平均分子量玻尿酸及高重量平均分子量玻尿酸的混合物投予至一需要治療之標的時，低重量平均分子量玻尿酸可快速地覆蓋發炎部位來治療黏膜相關病變或疾病，且高重量平均分子量玻尿酸可延長治療效果。因 此，治療可迅速且長效地進行。玻尿酸的一般化學結構式如圖1所示。 According to the definition of the present invention, hyaluronic acid having a weight average molecular weight of less than 1.5 million Daltons is classified as low weight average molecular weight hyaluronic acid (LMWHA). Preferably, the low weight average molecular weight hyaluronic acid has an average molecular weight ranging from 50,000 to 1.5 million Daltons. More preferably, the low weight average molecular weight hyaluronic acid has an average molecular weight ranging from 100,000 to 1.5 million Daltons. Most preferably, the low weight average molecular weight hyaluronic acid has an average molecular weight ranging from 100,000 to 500,000 Daltons. Hyaluronic acid having a weight average molecular weight of more than 1.5 million Daltons is classified as a high weight average molecular weight hyaluronic acid (HMWHA). Preferably, the high weight average molecular weight hyaluronic acid has an average molecular weight ranging from 1.5 million to 5 million Daltons. Preferably, the high weight average molecular weight hyaluronic acid has an average molecular weight ranging from 1.5 million to 2.5 million Daltons. When a mixture of low weight average molecular weight hyaluronic acid and high weight average molecular weight hyaluronic acid is administered to a therapeutic target, the low weight average molecular weight hyaluronic acid can rapidly cover the inflamed site to treat mucosal related diseases or diseases, and the high weight average molecular weight hyaluronic acid Can extend the treatment effect. because Thus, the treatment can be carried out quickly and in a long-term manner. The general chemical structure of hyaluronic acid is shown in Figure 1.
玻尿酸的量化並非僅得以用分子量表示，也可以利用特性黏度(intrinsic viscosity,η)來表示。此係與聚合物之分子量直接相關。藉由Mark-Houwink-Sakurada(MHS)方程式表達：[η]=KMα。對玻尿酸而言，在0.15 M氯化鈉(NaCl)磷酸鹽緩衝液(phosphate buffcr)、pH 7.5、20℃中，K是0.00057，指數α為0.75(「用以作為生物醫學及組織工程醫藥產品應用之起始物的玻尿酸鑑定及測試標準指南(Standard Guide for Characterization and Testing of Hyaluronan as Starting Materials Intended for Use in Biomedical and Tissue Engineered Medical Product Applications)」,ASTM Designation：F 2347-03)。 The quantification of hyaluronic acid is not only expressed by molecular weight, but also by intrinsic viscosity (η). This is directly related to the molecular weight of the polymer. Expressed by the Mark-Houwink-Sakurada (MHS) equation: [η] = KM α . For hyaluronic acid, in 0.15 M sodium chloride (NaCl) phosphate buffer (phosphate buffcr), pH 7.5, 20 ° C, K is 0.00057, index α is 0.75 ("for biomedical and tissue engineering pharmaceutical products "Standard Guide for Characterization and Testing of Hyaluronan as Starting Materials Intended for Use in Biomedical and Tissue Engineered Medical Product Applications", ASTM Designation : F 2347-03).
本發明之結果顯示無論在玻尿酸水解酶或人造胃液中，高重量平均分子量玻尿酸皆能持續補充低重量平均分子量玻尿酸以保持其長效。如圖2所示，在玻尿酸水解酶存在的環境中，所有三種玻尿酸滯留時間(retention time)會隨著降解的增加而明顯增加。如圖3所示，在酸性環境中，所有三種玻尿酸的滯留時間會隨著降解的增加而緩慢增加。實驗參數係模擬生理環境，但真實的生理環境卻可能因相當複雜而導致與實驗結果間有些許的差異。惟玻尿酸降解的趨勢卻不會因此改變，因為其天然分子特徵係高重量平均分子量玻尿酸能補充低重量平均分子量玻尿酸。 The results of the present invention show that the high weight average molecular weight hyaluronic acid can continuously replenish the low weight average molecular weight hyaluronic acid to maintain its long-lasting effect in hyaluronic acid hydrolase or artificial gastric juice. As shown in Figure 2, in the presence of hyaluronic acid hydrolase, all three hyaluronic acid retention times increased significantly with increasing degradation. As shown in Figure 3, in an acidic environment, the residence time of all three hyaluronic acids increases slowly as the degradation increases. The experimental parameters simulate the physiological environment, but the real physiological environment may be slightly different from the experimental results. However, the tendency of hyaluronic acid degradation does not change, because its natural molecular characteristics are high weight average molecular weight hyaluronic acid can supplement low weight average molecular weight hyaluronic acid.
本發明混合物之另一較佳的實施例包含，但不限於：依重量比1：1(w/w)混合高、低重量平均分子量玻尿酸， 混合比例的較佳實施例可以依據臨床需求調整為重量比20：80至80：20之間。具有較高比例低重量平均分子量的玻尿酸混合物對於加速治療更有幫助。相反地，較高比例高重量平均分子量的玻尿酸混合物能提供較長的降解時間而因此延長治療效果。 Another preferred embodiment of the mixture of the present invention comprises, but is not limited to, mixing high and low weight average molecular weight hyaluronic acid according to a weight ratio of 1:1 (w/w), The preferred embodiment of the mixing ratio can be adjusted to a weight ratio of between 20:80 and 80:20 depending on clinical requirements. Hyaluronic acid mixtures with a higher proportion of low weight average molecular weight are more helpful for accelerating treatment. Conversely, a higher proportion of high weight average molecular weight hyaluronic acid mixture can provide longer degradation times and thus prolong the therapeutic effect.
如本文所使用之用語黏膜包含，但不限於：眼睛的黏膜、耳朵的黏膜、鼻子的黏膜、口腔的黏膜、呼吸道的黏膜、腸胃道的黏膜、尿道的黏膜及生殖道的黏膜。本發明之黏膜相關病變或疾病的症狀係包含潰瘍、發炎反應、過敏反應或流血。黏膜相關病變或疾病的較佳實施例包括但不限於：結膜炎、耳炎、過敏性鼻炎、牙齦炎、口腔潰瘍、支氣管炎、胃食管逆流症(gastroesophageal reflux disease,GERD)、食道炎、胃炎、腸炎、消化性潰瘍(peptic ulcer,PU)、發炎性腸疾(inflammatory bowel disease,IBD)、刺激性腸症候群(irritable bowel syndrome,IBS)、尿道炎、膀胱炎和***炎。腸炎的更佳實施例包括但不限於：急性腸炎、慢性腸炎、感染性腸炎、缺血性腸炎(ischemic enteritis)、放射性腸炎(radioenteritis)及藥物誘發之腸炎。消化性潰瘍的更佳實施例包括但不限於：胃潰瘍、十二指腸潰瘍、食道潰瘍及梅爾氏憩室潰瘍(Meckel's Diverticulum ulcer)。發炎性腸疾(IBD)的更佳實施例包括但不限於：克隆氏症(Crohn's disease)及潰瘍性結腸炎(ulcerative colitis)。刺激性腸症候群(IBS)的更佳實施例包括但不限於：腹腔疾病(coeliac disease)、果糖吸收不良(fructose malabsorption)、溫和感染、諸如梨形鞭毛 蟲症(giardiasis)之寄生蟲感染、慢性官能性便秘及慢性官能性腹痛。 As used herein, the mucosa includes, but is not limited to, the mucosa of the eye, the mucosa of the ear, the mucosa of the nose, the mucosa of the mouth, the mucosa of the respiratory tract, the mucosa of the gastrointestinal tract, the mucosa of the urethra, and the mucosa of the genital tract. The symptoms of the mucosa-related disease or disease of the present invention include ulcers, inflammatory reactions, allergic reactions, or bleeding. Preferred examples of mucosal related lesions or diseases include, but are not limited to, conjunctivitis, otitis, allergic rhinitis, gingivitis, oral ulcers, bronchitis, gastroesophageal reflux disease (GERD), esophagitis, gastritis, Enteritis, peptic ulcer (PU), inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), urethritis, cystitis and vaginitis. More preferred embodiments of enteritis include, but are not limited to, acute enteritis, chronic enteritis, infectious enteritis, ischemic enteritis, radioactive enteritis, and drug-induced enteritis. More preferred embodiments of peptic ulcer include, but are not limited to, gastric ulcer, duodenal ulcer, esophageal ulcer, and Meckel's Diverticulum ulcer. More preferred examples of inflammatory bowel disease (IBD) include, but are not limited to, Crohn's disease and ulcerative colitis. More preferred examples of stimulating bowel syndrome (IBS) include, but are not limited to, coeliac disease, fructose malabsorption, mild infection, such as pear-shaped flagella. Parasitic infection of giardiasis, chronic functional constipation and chronic functional abdominal pain.
本發明結果顯示，如圖4所示，相同重量平均分子量之玻尿酸在受傷的結腸組織中被吸收的程度明顯高於在正常的結腸組織(P<0.01)。比較三種不同重量平均分子量之玻尿酸在受傷的結腸組織中被吸收的程度，其中由螢光指數顯示的35萬道爾頓玻尿酸被受傷結腸組織所吸收的量，明顯高於另外二個重量平均分子量玻尿酸(100萬及200萬道爾頓)。進一步言，100萬道爾頓玻尿酸被正常或受傷結腸組織所吸收的螢光指數高於200萬道爾頓玻尿酸。此結果解釋了低重量平均分子量玻尿酸快速散佈及覆蓋的效果，其係提供快速的傷口癒合及保護組織免於進一步受傷。結合上述數據，本發明係提供一種快速及持續治療或預防黏膜相關病變或疾病的方法。 The results of the present invention show that, as shown in Fig. 4, the degree of hyaluronic acid of the same weight average molecular weight was significantly absorbed in the injured colon tissue than in the normal colon tissue (P < 0.01). Comparison of the extent to which three different weight average molecular weight hyaluronic acids were absorbed in injured colon tissue, where the amount of 350,000 dalton hyaluronic acid shown by the fluorescence index was absorbed by the injured colon tissue, significantly higher than the other two weight average molecular weights. Hyaluronic acid (1 million and 2 million Daltons). Furthermore, the fluorescence index of 1 million Dalton hyaluronic acid absorbed by normal or injured colon tissue is higher than 2 million Dalton hyaluronic acid. This result explains the effect of rapid dispersion and coverage of low weight average molecular weight hyaluronic acid, which provides rapid wound healing and protects tissue from further injury. In conjunction with the above data, the present invention provides a rapid and sustained method of treating or preventing mucosal related diseases or diseases.
本發明組合物之另一較佳實施例包括加入賦形劑(excipient)使之形成下列適用於各部位或方式的劑型：眼睛、耳朵、口腔、鼻子、呼吸道、腸胃道或局部使用。本發明口服劑型之更佳實施例係選自下列由固態劑型、溶液包括但不限於懸浮液、錠劑包括但不限於緩釋型錠劑、及膠囊包括但不限於腸溶性膠囊(enteric-coated capsule)所組成之群組。口服固態劑型之最佳實施例係每日服用50毫克至1000毫克之間的劑量。腸胃道劑型之更佳實施例係選自下列由固態劑型、灌注液(perfusion)、灌腸劑(enema)、栓劑及包括懸浮液之溶液所組成之群組。局部使用劑型之更佳實施例係選自下列由灌注液、灌腸劑、噴劑、吸入劑 及點滴所組成之群組。 Another preferred embodiment of the composition of the present invention comprises the addition of an excipient to form the following dosage forms suitable for use in various parts or forms: eye, ear, mouth, nose, respiratory, gastrointestinal or topical use. More preferred embodiments of the oral dosage form of the present invention are selected from the following solid dosage forms, solutions including, but not limited to, suspensions, lozenges including, but not limited to, sustained release tablets, and capsules including, but not limited to, enteric-coated capsules (enteric-coated) Group of capsules. The preferred embodiment of the oral solid dosage form is a daily dose of between 50 mg and 1000 mg. A more preferred embodiment of the gastrointestinal dosage form is selected from the group consisting of solid dosage forms, perfusions, enemas, suppositories, and solutions including suspensions. A more preferred embodiment of the topical dosage form is selected from the group consisting of a perfusate, an enema, a spray, and an inhalant. And the group consisting of drip.
本發明之用於治療過敏性鼻炎或支氣管炎之較佳實施例係使用本發明之組成物作為結合藥物的佐劑(adjuvant)，其中藥物包括抗組織胺藥、抗過敏藥、抗充血藥、類固醇或抗氣喘藥。本發明用於治療腸炎之較佳實施例係使用本發明之組成物作為結合藥物的佐劑，其中藥物包括抗生素或鎮痙劑(antispasmodic)。本發明之用於治療消化性潰瘍之較佳實施例係使用本發明之組成物作為結合藥物的佐劑，其中藥物包括凝血劑(coagulant)、抗生素、制酸劑(antacid)、H2阻斷劑(H2 blocker)、鉀氫離子幫浦阻斷劑(potassium hydrogen ion pump blocker,PPI)、細胞或黏膜保護劑。本發明用於治療發炎性腸疾之較佳實施例係使用本發明之組成物作為結合藥物的佐劑，其中藥物包括類固醇、免疫抑制劑(immunosuppressive agent)、抗生素、5-胺柳酸(5-aminosalicylic acid,5-ASA)及其衍生物或抗發炎藥。 A preferred embodiment of the present invention for treating allergic rhinitis or bronchitis is the use of the composition of the present invention as a drug-binding adjuvant, wherein the drug comprises an antihistamine, an antiallergic agent, an anti-congestant, Steroids or anti-asthmatic drugs. A preferred embodiment of the present invention for treating enteritis is the use of the composition of the present invention as a drug-binding adjuvant, wherein the drug comprises an antibiotic or an antispasmodic. A preferred embodiment of the present invention for treating peptic ulcers uses the composition of the present invention as a drug-binding adjuvant, wherein the drug includes a coagulant, an antibiotic, an antacid, and an H2 blocker. (H2 blocker), potassium hydrogen ion pump blocker (PPI), cell or mucosal protective agent. A preferred embodiment of the present invention for treating inflammatory bowel disease is the use of the composition of the present invention as a drug-binding adjuvant, wherein the drug comprises a steroid, an immunosuppressive agent, an antibiotic, 5-amine salicylic acid (5) -aminosalicylic acid, 5-ASA) and its derivatives or anti-inflammatory drugs.
本發明闡述了藉由不同重量混合比例的玻尿酸來治療發炎性腸疾，其係藉由大鼠的平均體重變化來作指標。如圖5A及5B所示，A組代表高重量平均分子量(HMWHA)：低重量平均分子量(LMWHA)之重量混合比例為8：2。B組代表HMWHA：LMWHA之重量混合比例為1：1。C組代表控制組，其中之玻尿酸由磷酸鹽緩衝液(phosphate buffer saline,PBS)代替。雖然統計顯著性僅在第5及第6天發生，但本圖式的重點是在於每一組從第1天至第14天的趨勢變化。隨著實驗天數增加，實驗趨勢顯示A組及B組皆具和 緩結腸炎(屬於炎症性腸疾病之一種)的效應。如圖5A所示，相較控制組與A、B組之大鼠平均體重變化，經過整個實驗流程直到第14天，可看出玻尿酸治療結腸炎之投用會導致A、B組之大鼠平均體重持續高於比控制組。有意義的是，該趨勢亦顯示B組一般而言比A組有較佳的治療效應，代表較高比例的低重量平均分子量玻尿酸有較佳的治療效果。此結果支持本發明利用高重量平均分子量玻尿酸來補充低重量平均分子量玻尿酸以延長治療效應之概念。 The present invention describes the treatment of inflammatory bowel disease by hyaluronic acid in different weight mixing ratios, which is measured by the average body weight change of the rat. As shown in Figures 5A and 5B, Group A represents a high weight average molecular weight (HMWHA): low weight average molecular weight (LMWHA) weight mixing ratio of 8:2. Group B represents HMWHA: LMWHA has a weight mixing ratio of 1:1. Group C represents the control group in which hyaluronic acid is replaced by phosphate buffer saline (PBS). Although statistical significance occurs only on days 5 and 6, the focus of this diagram is on the trend change from day 1 to day 14 for each group. As the number of experimental days increases, the experimental trend shows that both groups A and B have The effect of colitis (a type of inflammatory bowel disease). As shown in Fig. 5A, compared with the control group and the average body weight of the rats in groups A and B, after the whole experimental procedure until the 14th day, it can be seen that the administration of hyaluronic acid to colitis causes the rats of group A and B. The average weight continued to be higher than the control group. Significantly, this trend also shows that group B generally has a better therapeutic effect than group A, and represents a higher proportion of low-weight average molecular weight hyaluronic acid with better therapeutic effect. This result supports the concept of the present invention utilizing a high weight average molecular weight hyaluronic acid to supplement the low weight average molecular weight hyaluronic acid to prolong the therapeutic effect.
本發明利用大鼠磨擦鼻子來作為過敏症狀的指標及利用磨擦鼻子次數的減少來作為改善過敏的指標。如圖6所示，結果顯示在第28天時，控制組(僅投予PBS)與玻尿酸組之結果具有顯著差異，代表過敏症狀已被成功的誘發。雖然第30天時，結果並未有統計上顯著差異，但過敏症狀在控制組與A組之間仍然有明顯的減少。因此，改善過敏症狀的趨勢可藉由投予本發明玻尿酸混合物至標的之實驗結果高度證實。 The present invention utilizes a rat rubbing nose as an indicator of allergy symptoms and a reduction in the number of rubbing noses as an indicator for improving allergy. As shown in Fig. 6, the results showed that on the 28th day, the results of the control group (only PBS administration) and the hyaluronic acid group were significantly different, indicating that the allergy symptoms have been successfully induced. Although there was no statistically significant difference on the 30th day, there was still a significant reduction in allergy symptoms between the control group and the A group. Therefore, the tendency to improve allergy symptoms can be highly confirmed by the experimental results of administering the hyaluronic acid mixture of the present invention to the target.
或許本發明的各個實驗結果有些許不同，然而需留意的是由本發明的概觀趨勢仍能推導出本發明的混合物具有改善包括過敏之黏膜相關病變或疾病的能力。本發明藉由數個相關實驗予以證實，而為了適當的顯示結果，本發明採取較短的實驗時程。因此在實際運用本發明概念至動物體或人體時，應會需要比實驗更長的時程。基於理論及本發明的結果，高重量平均分子量玻尿酸應足以補充長期的輔助，及/或補償體內低重量平均分子量玻尿酸的快速治療效應。 Perhaps the results of the various experiments of the present invention are somewhat different, however, it is to be noted that the appendix of the present invention can still deduce that the mixture of the present invention has the ability to improve mucosal-related lesions or diseases including allergies. The present invention has been confirmed by several related experiments, and the present invention takes a shorter experimental time course for proper display of results. Therefore, when the concept of the present invention is actually applied to an animal or a human body, a longer time course than the experiment should be required. Based on the theory and the results of the present invention, the high weight average molecular weight hyaluronic acid should be sufficient to supplement long-term assistance and/or compensate for the rapid therapeutic effects of low weight average molecular weight hyaluronic acid in the body.
本發明用於治療刺激性腸症候群(IBS)之較佳實施例係使用本發明之組成物作為結合藥物的佐劑，其中藥物包括抗過敏藥、鎮痙劑、痢抑制劑(antidiarrheal)、神經組織崩解劑(neurolytic)、鎮定劑(transquilizer)、麻醉鎮痛劑(narcotic analgesic)、抗憂鬱劑(antidepressant)或血清素拮抗劑(serotonin antagonist)。 A preferred embodiment of the present invention for treating irritation of intestinal tract syndrome (IBS) is the use of a composition of the present invention as a drug-binding adjuvant, wherein the drug comprises an antiallergic agent, an antispasmodic agent, an antidiarrheal inhibitor, and a nervous tissue. A neurolytic, a transquilizer, an narcotic analgesic, an antidepressant or a serotonin antagonist.
口服劑型(例如腸溶衣錠，enteric coating tablet)之較佳實施例中，腸溶衣在胃裡提供較佳的抗溶離及抗消化的能力。直到抵達腸道及結腸時，腸溶包衣才會溶解使得玻尿酸混合物釋放出來形成結腸發炎部位(位於升結腸或橫結腸的部位)的保護膜。此目的係為了加速治癒發炎部位，並藉由長降解速率(長時間的長鏈玻尿酸降解成短鏈玻尿酸)來延長治療時間。 In a preferred embodiment of an oral dosage form (e.g., enteric coating tablet), the enteric coating provides better resistance to dissolution and digestion in the stomach. Until the intestines and colon are reached, the enteric coating will dissolve and the hyaluronic acid mixture will be released to form a protective film on the site of the colonic inflammation (where the ascending colon or transverse colon is located). The aim is to speed up the healing of the site of inflammation and to extend the treatment time by a long rate of degradation (long-term degradation of long-chain hyaluronic acid into short-chain hyaluronic acid).
栓劑的較佳實施例中，栓劑內含玻尿酸混合物而可投予至肛門，使得混合物將可在直腸溶解並散佈至結腸的其他部位(例如降結腸)，以俾在結腸發炎部位形成保護膜，而能達到加速治療及持續釋放的效果。 In a preferred embodiment of the suppository, the suppository contains a hyaluronic acid mixture which can be administered to the anus such that the mixture will dissolve in the rectum and spread to other parts of the colon (e.g., descending colon) to form a protective film on the inflamed site of the colon. It can achieve accelerated treatment and sustained release.
灌注液(例如灌腸劑)的較佳實施例中，玻尿酸混合物在此係為主要的活性成份並與賦形劑混合(例如PBS緩衝液)，可直接使用或將上述玻尿酸混合物置於軟管中並注射於結腸。玻尿酸混合物將會灌注入結腸並擴散至結腸的其他部位(例如降結腸)，以便形成一於結腸發炎部位之保護膜，進而加速治療發炎部位，並達到持續釋放的效果。 In a preferred embodiment of the perfusate (e.g., enema), the hyaluronic acid mixture is herein the primary active ingredient and is mixed with excipients (e.g., PBS buffer), either directly or in a hose. And injected into the colon. The hyaluronic acid mixture will be infused into the colon and spread to other parts of the colon (such as the descending colon) to form a protective film on the inflamed area of the colon, which in turn accelerates the treatment of the inflamed area and achieves a sustained release effect.
本發明接受治療標的之更佳的實施例為哺乳動物，最 佳的實施例之標的為人類。 A more preferred embodiment of the invention for receiving a therapeutic target is a mammal, most The preferred embodiment is labeled as human.
依照以上描述，混合至少兩種藥學上可接受的不同重量平均分子量的玻尿酸鹽類後，可達到快速覆蓋受傷或發炎表面，同時縮短治療周期及拉長降解的時間以便延長覆蓋的周期。因此，快速的治療及持續的釋放效應將可同時達成。本發明提供一種較簡單及安全的方法來治療前述病變或疾病，並可降成本以提供更佳的選擇來幫助相關病人。 According to the above description, after mixing at least two pharmaceutically acceptable hyaluronic acid salts of different weight average molecular weights, rapid coverage of the injured or inflamed surface can be achieved while shortening the treatment period and lengthening the degradation time to prolong the coverage period. Therefore, rapid treatment and sustained release effects can be achieved simultaneously. The present invention provides a relatively simple and safe method of treating the aforementioned lesions or diseases and can be cost effective to provide a better choice to assist the patient in question.
本發明所提供者僅為最佳實施例，本技術領域具有通常知識者一般而言皆可運用本發明之構想對本發明做出轉換、修飾及變化。然這些動作之核心概念應已由本發明所揭露。因此，這些轉換、修飾及變化接落入本案申請專利範圍之範疇。本文所述以及伴隨圖式揭示者皆用作例示說明而非限制 The present invention is intended to be only a preferred embodiment, and it is obvious to those skilled in the art that the present invention can be modified, modified, and changed. However, the core concepts of these actions should have been disclosed by the present invention. Therefore, these conversions, modifications and variations fall within the scope of the patent application scope of this application. The descriptions of the present invention and accompanying drawings are used as illustrations and not limitation.
步驟： step:
1.將0.25克高重量平均分子量玻尿酸鈉粉末(標示為HHA，高重量平均分子量，分子量：2百萬道爾頓，購自Freda)及0.25克低重量平均分子量玻尿酸鈉粉末(標示為LHA，低重量平均分子量，分子量：35萬道爾頓，購自Freda)分別加入50毫升磷酸鹽緩衝液中，以形成濃度為0.5%的溶液，接著攪拌6小時直到粉末完全溶解。 1. 0.25 g of high weight average molecular weight sodium hyaluronate powder (labeled HHA, high weight average molecular weight, molecular weight: 2 million Daltons, available from Freda) and 0.25 grams of low weight average molecular weight sodium hyaluronate powder (labeled LHA, The low weight average molecular weight, molecular weight: 350,000 Daltons, purchased from Freda, was separately added to 50 ml of phosphate buffer to form a 0.5% solution, followed by stirring for 6 hours until the powder was completely dissolved.
2.將0.05克LHA粉末及0.2克HHA粉末(比例為2：8，中間重量平均分子量，MHA)加進50毫升PBS緩衝液 中，接著攪拌6小時直到粉末完全溶解。 2. Add 0.05 g of LHA powder and 0.2 g of HHA powder (ratio 2:8, intermediate weight average molecular weight, MHA) to 50 ml PBS buffer The mixture was stirred for 6 hours until the powder was completely dissolved.
3.凝膠透過層析儀(gel permeation chromatograph,GPC)系統的移動相溶液由下列方式製備：(1)將35.49克之磷酸氫二鈉(Na2HPO4)粉末加入450毫升之去離子蒸餾水(deionized-distilled water,dd water)，在室溫中攪拌30分鐘後以形成0.5體積莫耳濃度(M)Na2HPO4溶液；(2)將18克NaH2PO4粉末加入250毫升之去離子蒸餾水，在室溫中攪拌30分鐘後以形成0.5 M NaH2PO4溶液。 3. The mobile phase solution of the gel permeation chromatograph (GPC) system is prepared by (1) adding 35.49 grams of disodium hydrogen phosphate (Na 2 HPO 4 ) powder to 450 ml of deionized distilled water ( Deionized-distilled water, dd water), stirred at room temperature for 30 minutes to form a 0.5 volume molar concentration (M) Na 2 HPO 4 solution; (2) 18 grams of NaH 2 PO 4 powder was added to 250 ml of deionized The water was distilled and stirred at room temperature for 30 minutes to form a 0.5 M NaH 2 PO 4 solution.
4.將玻尿酸水解酶粉末於4℃加入磷酸鹽緩衝液中，以製備1 U/ml玻尿酸水解酶。 4. The hyaluronic acid hydrolase powder was added to the phosphate buffer at 4 ° C to prepare a 1 U/ml hyaluronic acid hydrolase.
5.將2毫升玻尿酸樣本、1毫升10 U/ml玻尿酸水解酶及7毫升磷酸鹽緩衝液混合加入於15毫升玻璃管中，劇烈振盪3分鐘。 5. 2 ml of hyaluronic acid sample, 1 ml of 10 U/ml hyaluronic acid hydrolase and 7 ml of phosphate buffer were mixed and added to a 15 ml glass tube, and shaken vigorously for 3 minutes.
6.將該玻璃管放在水浴槽中以50 rpm的轉速搖晃。分別在15、30、45、60、75、90、105及120分鐘時各取1毫升的溶液，然後在上述時間點分別加入1毫升的玻尿酸水解酶。所述的每一個1毫升溶液係以0.45微米(μm)孔徑之濾膜過濾。取20微升(μl)溶液注射入GPC系統中，然後記錄圖譜。 6. Place the glass tube in a water bath and shake at 50 rpm. 1 ml of each solution was taken at 15, 30, 45, 60, 75, 90, 105 and 120 minutes, respectively, and then 1 ml of hyaluronic acid hydrolase was added at the above time. Each of the 1 ml solutions was filtered through a 0.45 micron (μm) pore size filter. A 20 microliter (μl) solution was injected into the GPC system and the map was recorded.
7. GPC系統的狀態設定為：(1)管柱：2X GMPWx1(TSK-gel)；(2)移動相流動速率：每分鐘1毫升(ml/min)；(3)溫度：30℃。 7. The state of the GPC system is set to: (1) column: 2X GMPWx1 (TSK-gel); (2) mobile phase flow rate: 1 ml per minute (ml/min); (3) temperature: 30 °C.
8.所有顯示的數值係指觀察的平均數。組織學指數(histological index)是利用Student’s t-test分析。 8. All values shown refer to the average observed. The histological index was analyzed using Student's t-test.
結果： result:
圖2顯示GPC圖譜的玻尿酸滯留時間。縱軸代表在GPC中的滯留時間，水平軸代表在含有玻尿酸水解酶溶液中的玻尿酸之滯留時間。水平虛線從上到下分別代表200萬、100萬、35萬及1.7萬道爾頓分子量的玻尿酸滯留時間。隨著三種玻尿酸降解的增加，滯留時間也明顯地隨之增加。歷經15分鐘的降解後，HHA及MHA的重量平均分子量降解至大約100萬道爾頓。歷經37分鐘的降解後，HHA及MHA的重量平均分子量降解至大約35萬道爾頓。歷經90分鐘的降解後，HHA、MHA及LHA的重量平均分子量降解至三者之間沒有明顯差異存在，且皆大於1.7萬道爾頓。 Figure 2 shows the hyaluronic acid retention time of the GPC map. The vertical axis represents the residence time in GPC, and the horizontal axis represents the residence time of hyaluronic acid in the solution containing hyaluronic acid hydrolase. The horizontal dashed line represents the hyaluronic acid retention time of the molecular weights of 2 million, 1 million, 350,000 and 17,000 Daltons from top to bottom. As the three hyaluronic acid degradation increases, the residence time also increases significantly. After 15 minutes of degradation, the weight average molecular weight of HHA and MHA degraded to approximately 1 million Daltons. After 37 minutes of degradation, the weight average molecular weight of HHA and MHA degraded to approximately 350,000 Daltons. After 90 minutes of degradation, the weight average molecular weight of HHA, MHA and LHA degraded to no significant difference between the three, and both were greater than 17,000 Daltons.
步驟： step:
1. LHA、MHA及HHA之製備皆同於實施例1。 1. The preparation of LHA, MHA and HHA is the same as in Example 1.
2. GPC系統的移動相溶液之製備同於實施例1。 2. The mobile phase solution of the GPC system was prepared in the same manner as in Example 1.
3.將5.72毫升的17.5N HCl及90毫升的去離子蒸餾水混合並攪拌10分鐘以製備人造胃液(0.1 N HCl)作為儲備溶液。 3. 5.72 ml of 17.5 N HCl and 90 ml of deionized distilled water were mixed and stirred for 10 minutes to prepare an artificial gastric juice (0.1 N HCl) as a stock solution.
4.在15毫升玻璃管中，將2毫升之HHA、MHA及LHA分別與8毫升人造胃液混合並劇烈攪拌3分鐘。 4. In a 15 ml glass tube, mix 2 ml of HHA, MHA and LHA with 8 ml of artificial gastric juice and vigorously stir for 3 minutes.
5.將該玻璃管放在水浴槽中，在37℃下以50rpm的轉速搖晃。分別在6、12、24及48小時各取1毫升的溶液，然後在上述時間點分別加入1毫升的人造胃液。以0.45微米孔徑之濾膜過濾每一個1毫升溶液。取20微升溶液注射入GPC系統中，然後記錄圖譜。 5. The glass tube was placed in a water bath and shaken at 37 ° C at 50 rpm. 1 ml of each solution was taken at 6, 12, 24 and 48 hours, respectively, and then 1 ml of artificial gastric juice was added at the above time points. Each 1 ml solution was filtered through a 0.45 micron pore size filter. A 20 microliter solution was injected into the GPC system and the map was recorded.
6.所有顯示的數值係指觀察的平均數。組織學指數(histological index)是利用Student’s t-test分析。 6. All values shown refer to the average observed. The histological index was analyzed using Student's t-test.
結果： result:
圖3顯示GPC圖譜的玻尿酸滯留時間。縱軸代表在GPC中的滯留時間，水平軸代表在含有人造胃液中的玻尿酸的滯留時間。水平虛線從上到下分別代表200萬、100萬、35萬及17萬道爾頓分子量的玻尿酸滯留時間。隨著三種玻尿酸降解的增加，滯留時間也緩慢地隨之增加。歷經7小時的降解後，HHA及MHA的重量平均分子量降解至大約100萬道爾頓。歷經31小時的降解後，MHA的重量平均分子量降解至大約35萬道爾頓。歷經35小時的降解後，HHA的重量平均分子量也降解至大約35萬道爾頓。上述數據皆指出玻尿酸在人造胃液中是緩慢地降解，而HHA及MHA的重量平均分子量歷經6小時的降解後皆大於100萬道爾頓，而HHA及MHA的重量平均分子量歷經24小時的降解後皆大於35萬道爾頓。 Figure 3 shows the hyaluronic acid retention time of the GPC map. The vertical axis represents the residence time in GPC, and the horizontal axis represents the residence time of hyaluronic acid in the artificial gastric juice. The horizontal dashed line represents the hyaluronic acid retention time of the molecular weights of 2 million, 1 million, 350,000 and 170,000 Daltons from top to bottom. As the three hyaluronic acid degradation increases, the residence time also slowly increases. After 7 hours of degradation, the weight average molecular weight of HHA and MHA degraded to approximately 1 million Daltons. After 31 hours of degradation, the weight average molecular weight of the MHA degraded to approximately 350,000 Daltons. After 35 hours of degradation, the weight average molecular weight of HHA also degraded to approximately 350,000 Daltons. The above data indicate that hyaluronic acid is slowly degraded in artificial gastric juice, and the weight average molecular weight of HHA and MHA is greater than 1 million Dalton after 6 hours of degradation, while the weight average molecular weight of HHA and MHA is degraded after 24 hours. Both are more than 350,000 Daltons.
步驟： step:
1. LHA及HHA之製備如同實施例1。將MHA(分子量：100萬道爾頓，購自Freda)加入50毫升的磷酸鹽緩衝液，然後攪拌6小時直到粉末完全溶解，以俾接下來步驟之使用。 1. Preparation of LHA and HHA is as in Example 1. MHA (molecular weight: 1 million Daltons, purchased from Freda) was added to 50 ml of phosphate buffer, and then stirred for 6 hours until the powder was completely dissolved for use in the next step.
2.螢光玻尿酸(HA-f)由下列方式製備：(1)將0.39克MES游離酸[2-(N-嗎啉)-乙磺酸(2-(N-morpholino) ethanesulfonic acid，購自Calbiochem)]溶解於100毫升之去離子蒸餾水中。(2)溶液A：將65毫克的胺基螢光(fluoresceinamine)粉末(異構物I，購自Fluka公司)溶於9毫升95%酒精溶液並避光攪拌10分鐘。(3)溶液B：將359毫克[N-(3-二甲腔基丙基)-N-乙基碳二醯亞胺氯化氫][N-(3-Dimethylamino propyl)-N-ethyl carbodiimide hydrochloride,EDC](購自Sigma)粉末溶於9毫升MES緩衝液，並攪拌10分鐘。(4)溶液C：216毫克N-羥基丁二醯亞胺(N-Hydroxysuccinimde,NHS)粉末(購自Sigma)溶於9毫升MES緩衝液，並攪拌10分鐘。(5)將3毫升溶液A緩慢地滴入50毫升0.5%的玻尿酸溶液，並於避光下攪拌10分鐘。(6)將3毫升B溶液及5毫升C溶液分別滴入步驟(5)之溶液，並於避光下攪拌10分鐘。(7)將0.02 M MES緩衝液緩慢地滴入步驟(6)的溶液直到體積達到100毫升，並於室溫並避光下攪拌24小時。(8)將反應後的產物倒入透析管中(分子量：12000-14000)，以5公升的去離子蒸餾水作為透析液，並於4℃下避光攪拌5天，期間每12小時換一次透析液，直到透析液中沒有螢光為止。(9)透析後的液體以50毫升塑膠離心管分裝，接著保存在-20℃冰箱隔夜。之後在避光下以冷凍乾燥機乾燥。(10)乾燥的HA-f粉末保存在-20℃冰箱。(11)將50毫克HA-f粉末緩慢地加入10毫升磷酸鹽緩衝液中，並攪拌6小時，直到粉末完全溶解。 2. Fluorescent hyaluronic acid (HA-f) was prepared in the following manner: (1) 0.39 g of MES free acid [2-(N-morpholine)-ethanesulfonic acid (2-(N-morpholino)) Ethylsulfonic acid (purchased from Calbiochem)] was dissolved in 100 ml of deionized distilled water. (2) Solution A: 65 mg of fluoresceinamine powder (isomer I, available from Fluka) was dissolved in 9 ml of a 95% alcohol solution and stirred for 10 minutes in the dark. (3) Solution B: 359 mg of [N-(3-dimethylpropylpropyl)-N-ethylcarbodiimide hydrochloride] [N-(3-Dimethylamino propyl)-N-ethyl carbodiimide hydrochloride, EDC] (purchased from Sigma) powder was dissolved in 9 ml of MES buffer and stirred for 10 minutes. (4) Solution C: 216 mg of N-Hydroxysuccinimde (NHS) powder (purchased from Sigma) was dissolved in 9 ml of MES buffer and stirred for 10 minutes. (5) 3 ml of the solution A was slowly dropped into 50 ml of a 0.5% hyaluronic acid solution, and stirred for 10 minutes in the dark. (6) 3 ml of the B solution and 5 ml of the C solution were respectively dropped into the solution of the step (5), and stirred for 10 minutes in the dark. (7) The 0.02 M MES buffer was slowly dropped into the solution of the step (6) until the volume reached 100 ml, and stirred at room temperature for 24 hours in the dark. (8) Pour the reacted product into a dialysis tube (molecular weight: 12000-14000), use 5 liters of deionized distilled water as dialysate, and stir at 4 ° C for 5 days in the dark, during which dialysis is changed every 12 hours. Liquid until there is no fluorescence in the dialysate. (9) The dialysis liquid was dispensed in a 50 ml plastic centrifuge tube and then stored in a -20 ° C refrigerator overnight. It was then dried in a freeze dryer under light. (10) The dried HA-f powder was stored in a refrigerator at -20 °C. (11) 50 mg of HA-f powder was slowly added to 10 ml of phosphate buffer and stirred for 6 hours until the powder was completely dissolved.
3.將7-8周大的史-道二氏大鼠(Sprague-Dawley,SD)的結腸組織切下後，以磷酸鹽緩衝液沖洗，再切成3-4公分 長，最後浸泡在磷酸鹽緩衝液中。 3. Cut the colon tissue of 7-8 weeks old Shih Tzu's rats (Sprague-Dawley, SD), rinse with phosphate buffer, and cut into 3-4 cm. Long, finally soaked in phosphate buffer.
4.受傷的結腸組織是以牙刷縱向刷20次製備，之後浸泡在磷酸鹽緩衝液中。 4. The injured colon tissue was prepared by brushing 20 times in the longitudinal direction of the toothbrush and then immersed in phosphate buffer.
5.將正常與受傷的結腸組織放入12孔培養盤中，每孔分別加入1毫升0.5% HA-f溶液，然後在室溫下搖晃2小時。過多的HA-f溶液在2小時後以微吸管吸除，再浸泡至磷酸鹽緩衝液10分鐘後，移除磷酸鹽緩衝液。重複上述步驟三次。 5. Place normal and injured colon tissue into a 12-well culture dish, add 1 ml of 0.5% HA-f solution to each well, and then shake at room temperature for 2 hours. Excessive HA-f solution was aspirated by a micropipette after 2 hours, and after soaking in phosphate buffer for 10 minutes, the phosphate buffer was removed. Repeat the above steps three times.
6.將乾淨的結腸組織之襯裡組織(lining tissue)朝上放置在12孔培養盤中，然後放在IVIS(活體分子影像系統，in vivo image system,XENOGEN公司)的平台(dock)上。出廠參數是設定為綠螢光蛋白(green fluorescent protein,GFP)，激發(excitation)波長設定為465奈米(nm)，而發射(emission)波長設定為500奈米，影像再由軟體捕捉。 6. Place the clean colon tissue lining tissue upside in a 12-well culture plate and place it on a dock of IVIS ( in vivo image system, in vivo image system, XENOGEN). The factory parameters were set to green fluorescent protein (GFP), the excitation wavelength was set to 465 nm (nm), and the emission wavelength was set to 500 nm, and the image was captured by the software.
7.所有顯示的數值係指觀察的平均數。組織學指數(histological index)是利用Student’s t-test分析。 7. All values shown refer to the average observed. The histological index was analyzed using Student's t-test.
結果： result:
圖4顯示螢光指數的定量及排列結果。正常結腸組織的螢光指數定義為1。其他結腸組織再依據定義的數值作校準。結果顯示相同重量平均分子量的玻尿酸，黏附在受傷的結腸組織明顯比在正常結腸組織多(P<0.01)。比較三種不同重量平均分子量玻尿酸黏附在受傷結腸組織的結果後，被吸收的35萬道爾頓玻尿酸之螢光指數明顯高過其他二種玻尿酸(200萬與100萬道爾頓)。甚者，不論在正常 或受傷結腸組織中，被吸收的100萬道爾頓玻尿酸之螢光指數高過200萬道爾頓玻尿酸。 Figure 4 shows the quantification and arrangement results of the fluorescence index. The fluorescence index of normal colon tissue is defined as 1. Other colon tissues are then calibrated against defined values. The results showed that hyaluronic acid of the same weight average molecular weight was significantly more adherent to the injured colon tissue than in normal colon tissue (P < 0.01). Comparing the results of three different weight average molecular weight hyaluronic acid adhesion to injured colon tissue, the fluorescence index of the absorbed 350,000 Dalton hyaluronic acid was significantly higher than the other two hyaluronic acids (2 million and 1 million Daltons). Even if it is normal In the injured colon tissue, the absorbed 1 million Dalton hyaluronic acid has a fluorescence index higher than 2 million Dalton hyaluronic acid.
步驟： step:
1.測試目的：為了評估以重量比例混合兩種不同重量平均分子量玻尿酸來治療或預防結腸炎的效果，以三硝基苯磺酸(trinitrobenzenesulfonic acid,TNBS)在無特定病原(specific-pathogen-free,SPF)等級的史-道二氏大鼠大鼠中誘發出結腸炎。 1. Test purpose: In order to evaluate the effect of mixing two different weight average molecular weight hyaluronic acid to treat or prevent colitis by weight ratio, trinitrobenzenesulfonic acid (TNBS) in specific pathogen-free Colitis was induced in the history of the SPF class of rats.
2.測試樣本：IBD98，其包含低重量平均分子量(LMWHA)及高重量平均分子量(HMWHA)，其中HMWHA是200萬道爾頓玻尿酸，LMWHA是35萬道爾頓玻尿酸，混合比例為8：2(歸類為A組)及1：1(歸類為B組)。溶於磷酸鹽緩衝液中產生0.125%(w/v)濃度的玻尿酸溶液。 2. Test sample: IBD98, which contains low weight average molecular weight (LMWHA) and high weight average molecular weight (HMWHA), of which HMWHA is 2 million Dalton hyaluronic acid, LMWHA is 350,000 Dalton hyaluronic acid, the mixing ratio is 8:2 (Classified as Group A) and 1:1 (Classified as Group B). Dissolved in phosphate buffer to produce a 0.125% (w/v) concentration of hyaluronic acid solution.
3.方法：(1)測試標的：挑選8周大的大鼠，並將其分成三組：A組代表玻尿酸混合比例8：2；B組代表玻尿酸混合比例1：1；C組代表以磷酸鹽緩衝液代替玻尿酸的控制組。(2)動物實驗：所有實驗組(A、B組)的大鼠先使其空腹兩天。在實驗第1天，大鼠麻醉後經由直腸投予濃度為每毫升50毫克之1毫升三硝基苯磺酸。從第4至第14天，每天將A與B組的大鼠，經由直腸分別投予1毫升之兩種IBD98。在第九天，觀察A與B組一半大鼠的體重。在第14天觀察另一半大鼠的體重變化。所有控制組的大鼠先使其空腹兩天。在實驗第1天，大鼠麻醉後經由直 腸投予濃度為每毫升50毫克之1毫升三硝基苯磺酸。從第4至第14天，每天經由直腸投予1毫升磷酸鹽緩衝液。在第九天，觀察C組一半大鼠的體重。在第14天觀察另一半大鼠的體重變化。 3. Methods: (1) Test target: Eight-week-old rats were selected and divided into three groups: group A represents hyaluronic acid mixing ratio of 8:2; group B represents hyaluronic acid mixing ratio of 1:1; group C represents phosphoric acid Salt buffer instead of hyaluronic acid control group. (2) Animal experiments: Rats in all experimental groups (groups A and B) were given an empty stomach for two days. On the first day of the experiment, rats were anesthetized with a concentration of 50 mg of 1 ml of trinitrobenzenesulfonic acid per ml via the rectum. From day 4 to day 14, rats of groups A and B were administered 1 ml of two IBD98 via the rectum. On the ninth day, the body weight of half of the rats in group A and group B was observed. The change in body weight of the other half of the rats was observed on the 14th day. Rats in all control groups were given an empty stomach for two days. On the first day of the experiment, the rats were anesthetized via straight Intestine is administered at a concentration of 50 mg of 1 ml of trinitrobenzenesulfonic acid per ml. From day 4 to day 14, 1 ml of phosphate buffer was administered via the rectum daily. On the ninth day, the body weight of half of the rats in group C was observed. The change in body weight of the other half of the rats was observed on the 14th day.
結果： result:
1.發炎指標：本發明利用平均體重的變化來觀察結腸炎(IBD)改善的結果。對於確認治療結果而言，體重的變化是一種較方便且直接之指標。 1. Inflammatory index: The present invention utilizes changes in mean body weight to observe the results of improved colitis (IBD). Changes in body weight are a convenient and straightforward indicator of the outcome of a treatment.
2.觀察A組與B組的趨勢可發現，如圖5及圖5B所示，從第1天至第14天代表發炎症腸疾(IBD)的結腸炎，在兩組皆有緩和的結果。觀察控制組與實驗組的平均體重變化可發現，如圖5B所示，透過投用玻尿酸結腸炎的治療效果會使實驗組的平均體重，在全部實驗期間即使直到第14天，皆保持的比控制組的平均體重來得高。 2. Observing the trend of group A and group B, it can be found that, as shown in Fig. 5 and Fig. 5B, colitis which represents inflammatory bowel disease (IBD) from day 1 to day 14 has a mitigating result in both groups. . Observing the average body weight change between the control group and the experimental group, as shown in Fig. 5B, the therapeutic effect of the administration of hyaluronic acid colitis caused the average body weight of the experimental group to be maintained even during the whole experiment period until the 14th day. The average weight of the control group is high.
步驟： step:
1.測試目的：在無特定病原等級的史-道二氏大鼠中誘導出過敏性疾病，在此為過敏性鼻炎。 1. Test purpose: Allergic diseases were induced in a history-specific T. sinensis-free rat, which is allergic rhinitis.
2.測試樣本：IBD98，其包含低重量平均分子量及高重量平均分子量，其中HMWHA是200萬道爾頓玻尿酸，LMWHA是35萬道爾頓玻尿酸，混合比例為1：1，溶於磷酸鹽緩衝液中產生0.125%(w/v)濃度的玻尿酸溶液。 2. Test sample: IBD98, which contains low weight average molecular weight and high weight average molecular weight, wherein HMWHA is 2 million Dalton hyaluronic acid, LMWHA is 350,000 Dalton hyaluronic acid, the mixing ratio is 1:1, soluble in phosphate buffer A 0.125% (w/v) concentration of hyaluronic acid solution was produced in the solution.
3.方法：(1)測試標的：挑選8周大的大鼠9隻，隨機分類為二組：由4隻大鼠組成的控制組(磷酸鹽緩衝液)及由5隻大鼠組成的實驗組(玻尿酸)。(2)動物實驗： 從實驗第1天至第7天，每天透過腹腔內注射(intra-peritoneally,IP)內含1毫克卵白蛋白(ovalbumin)及10毫克明礬(alum)的生理食鹽水。從第8天至第13天只做觀察。從第14天至第27天，每天將內含卵白蛋白的食鹽水(10 μg/10 μl/每個鼻孔)利用微量吸移管(micropipette)滴入大鼠的兩側鼻孔。經過誘發過敏之抗原反應後，玻尿酸對鼻子上症狀的治療效果分別在第28天及第30天做評估。在滴入卵白蛋白之1小時前，大鼠先滴入玻尿酸溶液或磷酸鹽緩衝液(25 μl/每個鼻孔)。在內含卵白蛋白的食鹽水(10 μg/10 μl/每個鼻孔)滴入兩側的鼻孔後，大鼠被放置在觀察籠裡(1隻大鼠/每籠)，且其30分鐘過程中磨擦鼻子的次數被計數。磨擦鼻子的頻率係以每30分鐘磨擦鼻子的次數計算。 3. Methods: (1) Test criteria: 9 rats of 8 weeks old were randomly divided into two groups: a control group consisting of 4 rats (phosphate buffer) and an experiment consisting of 5 rats. Group (hyaluronic acid). (2) Animal experiment: From the first day to the seventh day of the experiment, intraperitoneally (IP) contained 1 mg of ovalbumin and 10 mg of alum physiological saline per day. Only observe from day 8 to day 13. From day 14 to day 27, egg albumin-containing saline (10 μg/10 μl per peritone) was instilled into the nostrils on both sides of the rat using a micropipette. After the allergic antigen reaction, the therapeutic effect of hyaluronic acid on the symptoms on the nose was evaluated on the 28th and 30th day, respectively. One hour before the instillation of ovalbumin, the rats were first dropped into hyaluronic acid solution or phosphate buffer (25 μl per nostril). After instillation of ovalbumin-containing saline (10 μg/10 μl per nostril) into the nostrils on both sides, the rats were placed in an observation cage (1 rat per cage) and their 30 minute course The number of times the nose is rubbed is counted. The frequency of rubbing the nose is calculated as the number of times the nose is rubbed every 30 minutes.
結果： result:
1.過敏改善指標：本發明利用大鼠磨擦鼻子的次數來做為過敏症狀改善的指標。 1. Allergy improvement index: The present invention uses the number of times the rat rubs the nose as an indicator for improving allergy symptoms.
2.如圖6所示，玻尿酸樣本對於抗原誘發的鼻子症狀的治療效果。因卵白蛋白所誘發的平均磨擦鼻子頻率，在第28天是56.25±16.1次/30分鐘，在第30天是70.0±41.3次/30分鐘。與控制組比較起來，實驗組磨擦鼻子的頻率在第28天有統計意義的顯著減少，然而在第30天，仍然有明顯的減少。 2. As shown in Figure 6, the hyaluronic acid sample has a therapeutic effect on antigen-induced nasal symptoms. The average friction nose frequency induced by ovalbumin was 56.25 ± 16.1 times / 30 minutes on day 28 and 70.0 ± 41.3 times / 30 minutes on day 30. Compared with the control group, the frequency of the experimental group rubbing the nose was statistically significantly reduced on the 28th day, but on the 30th day, there was still a significant decrease.
圖1係玻尿酸的一般化學結構式。 Figure 1 is a general chemical structural formula of hyaluronic acid.
圖2為藉由凝膠透過層析儀顯示的玻尿酸滯留時間； 其中縱軸代表在GPC裡的滯留時間，橫軸代表玻尿酸在玻尿酸水解酶(HAase)溶液中的降解時間。 Figure 2 is a hyaluronic acid retention time displayed by a gel permeation chromatograph; The vertical axis represents the residence time in GPC, and the horizontal axis represents the degradation time of hyaluronic acid in hyaluronic acid hydrolase (HAase) solution.
圖3係藉由GPC顯示的玻尿酸滯留時間，其中縱軸代表在GPC裡的滯留時間，橫軸代表玻尿酸在人造胃酸(artificial gastric juice)溶液中的降解時間。 Figure 3 shows the hyaluronic acid retention time by GPC, where the vertical axis represents the residence time in GPC and the horizontal axis represents the degradation time of hyaluronic acid in an artificial gastric juice solution.
圖4係藉由螢光係數展現的玻尿酸在正常及受傷結腸組織的附著性。 Figure 4 shows the adhesion of hyaluronic acid to normal and injured colon tissue by fluorescence coefficient.
圖5A係玻尿酸混合物在治療結腸炎的效果，其係在實驗第1至9天由大鼠的平均體重來展示。 Figure 5A is an effect of a hyaluronic acid mixture in the treatment of colitis, which is shown by the average body weight of the rats on days 1 to 9 of the experiment.
圖5B係玻尿酸混合物在治療結腸炎的效果，其係在實驗第1至14天由大鼠的平均體重來展示。 Figure 5B is an effect of a hyaluronic acid mixture in the treatment of colitis, which is shown by the average body weight of the rats on days 1 to 14 of the experiment.
圖6係藉由卵白蛋白(10 μg/10 μl/鼻孔)誘發大鼠磨擦鼻子以觀察玻尿酸減少大鼠鼻子過敏的效果，第28天時，玻尿酸組及控制組間具有顯著差異(＊ p<0.05) Figure 6 shows the effect of hyaluronic acid on rat nasal allergy induced by ovalbumin (10 μg/10 μl/nostril). On day 28, there was a significant difference between hyaluronic acid group and control group ( * p < 0.05)
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