TW492866B

TW492866B - A pharmaceutical composition for treatment or diagnosis of ischemia reperfusion injury

Info

Publication number: TW492866B
Application number: TW84103130A
Authority: TW
Inventors: Jen-Chang Hsia
Original assignee: Jen-Chang Hsia
Priority date: 1995-03-31
Filing date: 1995-03-31
Publication date: 2002-07-01

Abstract

This invention provides to a pharmaceutical composition for treatment or diagnosis of ischemia reperfusion injury, comprising human serum albumin covalently labelled with nitroxide. This invention also provides a pharmaceutical composition for treatment or diagnosis of ischemia reperfusion injury, comprising a membrane permeable nitroxide and a polynitroxide-labelled biocompatible macromolecule such as dextran, hydroxyethy starch, albumin or hemoglobin.

Description

492866 經濟部中央標準局員工消費合作社印製 A7 B7 i、發明説明（) 發明領域本發明係有關使用氮氧化物-標識的巨分子，包括血紅素、白蛋白、免疫球蛋白與脂質體，來減輕一活生物體内與氧有關的物種之毒性作用，以及有關某些生理病狀的診斷與治療。本發明亦有關氮氧化物-標識的巨分子與低分子量氮氧化物之合併使用以維持氮氧化物之活體内作用。本發明中所掲露的新穎化合物與方法，其持徵在於合併使用氮氧化物與生理上可相容的無細胞血紅素溶液及包囊化血紅素溶液，以供用作為一紅血球代用品。另外，本發明敘述將上述氮氧化物與其他的生理活性化合物合併使用以防止由自由基所引起的病理性損害以及氣化壓力，以及敘述其等在疾病的診斷與治療上之用途。發明背景雖然對氧代謝的生理機轉之知曉已有多年，氧化壓力在生理學與醫學中所扮演之一角色仍未被全然地暸解。由氧衍生的自由基對生理學與疾病之衝擊，在醫學與生物學中偽為一具提高的重要性之客題。已知疾病與傷害會導致自由基的位準遠遠超過身體的天然抗氧化能力--其結果是氧化壓力。氧化壓力是失控的毒性自由基（特別是與氣有關的毒性物種）之生理性表徵。毒性自由基被認為是許多病理狀態的導因，這包括了局部缺血-再灌流損害、休克、禿髮症、敗血症、若干藥物毒性、由治療肺部疾病之氧治療所引起的毒性以及離子化放射之臨床或偶發的暴露，亦被認為是老化過程之導因。 (請先閱讀背面之注意事項再填寫本頁) •裝· •本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -4 -492866 A7 B7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs i. Description of the Invention Field of the Invention The present invention relates to the use of nitrogen oxide-labeled macromolecules, including heme, albumin, immunoglobulins and liposomes Reducing the toxic effects of oxygen-related species in a living organism, and the diagnosis and treatment of certain physiological conditions. The present invention also relates to the combined use of nitrogen oxide-labeled macromolecules and low molecular weight nitrogen oxides to maintain the in vivo effects of nitrogen oxides. The novel compounds and methods disclosed in the present invention are characterized by a combined use of nitrogen oxides and a physiologically compatible cell-free heme solution and an encapsulated heme solution for use as a red blood cell substitute. In addition, the present invention describes the use of the above-mentioned nitrogen oxides in combination with other physiologically active compounds to prevent pathological damage and gasification pressure caused by free radicals, and describes their use in the diagnosis and treatment of diseases. BACKGROUND OF THE INVENTION Although the physiological mechanisms of oxygen metabolism have been known for many years, one of the roles of oxidative stress in physiology and medicine has not been fully understood. The impact of oxygen-derived free radicals on physiology and disease is a subject of increasing importance in medicine and biology. Illness and injury are known to cause free radicals to far exceed the body's natural antioxidant capacity-the result is oxidative stress. Oxidative stress is a physiological characterization of uncontrolled toxic free radicals, especially toxic species associated with gas. Toxic free radicals are thought to be the cause of many pathological conditions, including ischemia-reperfusion damage, shock, alopecia, sepsis, toxicity of several drugs, toxicity caused by oxygen therapy for lung diseases, and ion Clinical or occasional exposure to chemical radiation is also considered to be the cause of the aging process. (Please read the precautions on the reverse side before filling out this page) • Loading · • This paper size applies to China National Standard (CNS) A4 (210X297 mm) -4-

A7 B7 經濟部中夬標準局員工消費合作社印製 i、發明説明（）因此，對於其能將自由基與相關的毒性物種解毒，且其在體内具有充分的活性與持續性而能避免被快速地消耗以致自由基之濃度又提高的組成物與方法，實為所需者。更甚者，已有的證據顯示出自由基有助於發展癌症、潰瘍、白内障、封閉型頭部損害以及心臓血管疾病等等。由於其等之高反應力，自由基可氧化核酸、生物膜以及其他的細胞組份，這造成嚴重的或致命的細胞性損害、突變形成或癌症形成。抗癌放射治療法以及許多的抗癌藥物藉由産生對癌細胞有毒的自由基來發揮作用，但該等自由基對在細胞***期間受暴露的正常細胞亦是有毒的，因而引起在作癌症治療時非所欲的副作用。確實地，一般相信許多病理過程會有自由基的生成以為其共同的最終通路，而此為所觀察到的病理學之直接病因。當漸漸認知到活生物条統内氣化壓力之重要性時，對於其能發揮抗氧化劑之功能，且能被設計成可與從氧衍生的自由基相互反應以減輕該等自由基之毒性的化合物與方法，存有一持續的需要。由於離子化放射對一生物體導致生理性損害之機轉至少部分地涉及到一自由基與細胞之相互作用，其帶有自由基或能與自由基相互作用的化合物對被暴露於放射之組織展現出一局部化作用。安全且有效的抗氣化劑會增加病人的抗氧化能力並幫助遏止許多處於自由基生成之階段的病理過程。除了任一臨床上顯著的功能之外，由於自由基物種内未配對的電子可藉由光譜學來偵測，自由基反應可於活體内被監測之，而其能與自由基相互反應的化合物可藉由光A7 B7 Printed by the Consumer Cooperatives of the China Standards Bureau of the Ministry of Economic Affairs i. Description of invention () Therefore, it can detoxify free radicals and related toxic species, and it has sufficient activity and persistence in the body to avoid being Compositions and methods that are rapidly consumed such that the concentration of free radicals are increased are desirable. What's more, the existing evidence shows that free radicals can contribute to the development of cancer, ulcers, cataracts, closed head damage, and heart disease. Due to their high reactivity, free radicals can oxidize nucleic acids, biofilms, and other cellular components, which can cause severe or fatal cellular damage, mutation formation, or cancer formation. Anti-cancer radiotherapy and many anti-cancer drugs work by generating free radicals that are toxic to cancer cells, but these free radicals are also toxic to normal cells exposed during cell division, and cause cancer Unwanted side effects during treatment. Indeed, it is generally believed that many pathological processes will generate free radicals as their common ultimate pathway, and this is the direct cause of the observed pathology. When the importance of gasification pressure in living organisms is gradually recognized, it can be used as an antioxidant and can be designed to interact with free radicals derived from oxygen to reduce the toxicity of these free radicals. There is a continuing need for compounds and methods. The mechanism of physiological damage caused by ionizing radiation to an organism at least in part involves the interaction of a free radical with a cell. The free radical or the compound capable of interacting with the free radical exhibits to the tissue exposed to the radiation. A localization effect. Safe and effective anti-gasification agents increase the patient's antioxidant capacity and help stop many of the pathological processes at the stage of free radical generation. In addition to any clinically significant function, since unpaired electrons in free radical species can be detected by spectroscopy, free radical reactions can be monitored in vivo, and compounds that can interact with free radicals Available by light

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Hi. 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） - 5 - 經濟部中央標準局員工消費合作社印製 492866 Α7 Β7 五、發明説明（）譜學技術來作觀察。已有許多治療方法被提出以用來降低自由基之病理性位準。但是，在發展其能克服氧化壓力或與氧-相關的物種有關聯之毒性化合物及方法上，成功有限。許多抗氧化劑之有用性受限於其在活體内之作用期短，其在有效的劑量位準之毒性，其不能跨越胞膜，以及其不能抗衡高位準的自由基之作用。例如，超氧化物歧化酶（SOD)或觸酶之投藥可促進有毒的自由基相關物種轉化成一無毒形式。但是，這些酵素在細胞間隙内無法有效地發揮作用。作為一 G S Η前驅物之前半胱胺酸（p r 〇 c y s t e i η )，以及維生素和其他抗氯化化學品，可以增進身體的天然抗氧化能力，但是無法處理在傷害與疾病時所碰到的較高位準之自由基，且會被身體快速地消耗掉。自由基具顯著的反應力且存在期間短。該反應力在生物条統内傺為一特別嚴重的危害，因為一自由基與身體組織之間的有害化學反應，偽發生在非常鄰近於生成該自由基之位置處。因此，其固有地發揮降低自由基濃度功能之化合物具有某種有利之作用，雖然該作用在臨床上可能是不顯著的。在生成一適供大量靜脈投藥之血液代用品上所遭遇之困難是，在防止或減輕由與氧有關的物種所引起之全身性毒性上的困難之一重要實例。近幾十年來，科學家及醫師們致力於製造一種可被安全地輸血至人體内之红血球代用品。由於經常性的血荒以及血型不符、血型必需相配和疾本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 6 (請先閲讀背面之注意事項再填寫本頁) 裝---.---^ ——訂 J---7--Α 線----------i------- 經濟部中央標準局員工消費合作社印製 492866 A7 ____ B7___ 五、發明説明（）病傳染等問題之存在，導致工業界、學術界及官方共同努力來發展一可供大量安全的輸血而不會有顯箸的生理上副作之血液代用品。目前，一些公司正在對實驗血液代用品從事臨床測試。但是，沒有預想到的有害生理反應以及研發過程之内在的複雜性，阻礙其走向通過使用許可階段以及成為一臨床上有用的血液代用品。美國海軍之一研究咨詢委員會在1 9 9 2年8月發表一份報告，其中概述了數組從事製造血液代用品所做之努力，評估該等努力的情況，並概略地敘述所碰到的毒性問題。該美國海軍研究咨詢委員會報告反映出現今科學界一致的看法：即令現有之血液代用品産品[一般稱為"以血紅素為主的氧載體（HBOC) 〃]已被證實在氯氣傳輸上是有效的，某些毒性問題仍未克解決。在現有的以血紅素為主的氧載體（HBOC)之臨床研究中所看到的有害輸血反應包括了全身性高血壓與血管收縮。這些有害反應迫使許多製藥公司必需在非常低的劑量位準下進行第一期臨床試驗。現有的以血紅素為主的氣載體（HBOC)之毒性問題已被美國政府列為應優先解決之一事項。該美國海軍研究咨詢委員的建議已為美國國家衛生院（National Institute of H e a 11 h )所採納，並以☆以血红素為Φ的氣jg鵲：羞袢：> 機為主題，公開邀請研究計劃（PA-93-23)來加以推動。因此，醫界與科學界可說是苦於尋覓一種可供輸血且不具有在現有的以血紅素為主的氣載體中所看到的副作用之血液代用品。本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） m. II·: ml ml m in HI 士*9^ —^ϋ n I -^1 m、一：心~Inn ϋϋ - - - —Is in ϋϋ m I t^n j— I: I fn ·ν·— m «^ϋ m m ml —-111 (請先閲讀背面之注意事項再填寫本頁) ^866 ^866 經濟部中夬標準局員工消費合作社印製 A7 B7 i、發明説明（) 紅血球是血液之主要組份，並具有身體的氧氣傳輸条統。已知一血液代用品的最重要特徵是其攜氧的能力。紅血球能攜氧偽因其主要組份是能如同氧載體般作用的血紅素。大多數正在進行作為血液代用品的臨床試驗之産品含有血紅素，該血紅素從紅血球的細胞膜及其餘細胞組份中被分離出，並予以純化以去除實質上所有的雜質。但是，當血紅素從紅血球中被移出並以其天然形式被置放於溶液内時，其很不穩定且會迅速地分解成其組份次單元。因此，在以血紅素為主的氧載體中所用的血紅素必須予以穩定化以防止其在溶液内分解。要發展其在溶液是穩定的且其被穩定化之方式不會令其氣傳輸功能受損的血红素産物，科學人力及資本之實質投注，是不可或缺的。現有的以血紅素為主的血液代用品之輸氣能力，已被充分地證明（參照美國專利第 3,925,344; 4,001,200; 4,001,401; 4,053, 590; 4,061,736； 4,136,093； 4,301,144； 4,336,248； 4,376,095； 4,377,512； 4,401,652； 4,473,494； 4,473, 496 ； 4,600,531 ； 4,584,130 ； 4,857,636 ； 4,826,811; 4,91 1, 929及 5,061,688號）。在身體内，當血液流經肺臓時，位在紅血球内的血紅素與氧分子結合，並將氣分子傳送至身體各部位以契合身體的正常代謝功能所需。但是，大部分活生物必須呼吸以維生的大氣氧氣是一種科學及醫學上的矛盾。在一方面，幾乎所有的活生物體需要氧以維生。在另一方面，各種不同的與氣有關的化學毒性物質偽在正常氧代謝過程中被生本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 8 (請先閱讀背面之注意事項再填寫本頁)Hi. This paper size is in accordance with Chinese National Standard (CNS) A4 (210X297 mm)-5-Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 492866 Α7 Β7 V. Description of Invention () Spectroscopy technology for observation Many treatments have been proposed to reduce the pathological level of free radicals. However, success has been limited in developing toxic compounds and methods that can overcome oxidative stress or are associated with oxygen-related species. The usefulness of many antioxidants is limited by their short duration of action in vivo, their toxicity at effective dosage levels, their inability to cross the cell membrane, and their inability to counteract the effects of high levels of free radicals. For example, administration of superoxide dismutase (SOD) or catalase can promote the conversion of toxic free radical-related species into a non-toxic form. However, these enzymes cannot function effectively in the intercellular space. As a GS Η precursor, cysteine (pr cystei η), as well as vitamins and other anti-chlorinated chemicals, can enhance the body's natural antioxidant capacity, but it cannot deal with the problems encountered in injury and disease. High-level free radicals, which are quickly consumed by the body. Free radicals have a significant reactivity and have a short duration. This reaction force is a particularly serious hazard in the biological system, because the harmful chemical reaction between a free radical and body tissues pseudo-occurs very close to the position where the free radical is generated. Therefore, compounds that inherently function to reduce the concentration of free radicals have some beneficial effect, although the effect may not be clinically significant. The difficulty encountered in generating a blood substitute suitable for large intravenous administration is an important example of the difficulty in preventing or mitigating systemic toxicity caused by oxygen-related species. In recent decades, scientists and physicians have worked to create a red blood cell substitute that can be safely transfused into the body. Due to the frequent blood shortage and blood type inconsistency, the blood type must be matched and the paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 6 (Please read the precautions on the back before filling this page) .--- ^ ——Order J --- 7--Α line ---------- i ------- Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 492866 A7 ____ B7___ 5 Explanation of the invention () The existence of problems such as disease transmission has led to industry, academia, and the government to work together to develop a blood substitute that can be used for a large number of safe blood transfusions without significant physiological side effects. Currently, some companies are conducting clinical tests on experimental blood substitutes. However, unanticipated harmful physiological reactions and the inherent complexity of the research and development process have prevented it from moving through the licensing phase and becoming a clinically useful blood substitute. One of the US Navy's Research Advisory Committee published a report in August 1992 outlining the efforts of the array to make blood substitutes, assessing those efforts, and briefly describing the toxicity encountered problem. The report of the U.S. Naval Research Advisory Committee reflects the consensus of today ’s scientific community that even existing blood substitute products [commonly referred to as " heme-based oxygen carriers (HBOC) 〃] have been shown to be Effectively, certain toxicity issues remain unresolved. The harmful transfusion reactions seen in existing clinical studies of heme-based oxygen carriers (HBOC) include generalized hypertension and vasoconstriction. These adverse reactions have forced many pharmaceutical companies to conduct Phase 1 clinical trials at very low dose levels. The toxicity of the existing heme-based gas carrier (HBOC) has been listed by the US government as a priority. The recommendations of the U.S. Naval Research Advisory Board have been adopted by the National Institute of Health (11 h), and the theme is ☆ with hemoglobin Φ jg 鹊: shame: > machine, open invitation Research Project (PA-93-23). Therefore, the medical and scientific communities are struggling to find a blood substitute that can be used for blood transfusion without the side effects seen in the existing heme-based air carriers. This paper size is in accordance with Chinese National Standard (CNS) A4 (210X297 mm) m. II ·: ml ml m in HI person * 9 ^ — ^ ϋ n I-^ 1 m, one: heart ~ Inn ϋϋ--- —Is in ϋϋ m I t ^ nj— I: I fn · ν · — m «^ ϋ mm ml —-111 (Please read the precautions on the back before filling this page) ^ 866 ^ 866 Ministry of Economic Affairs Zhongli Standard Bureau Printed by employees' consumer cooperatives A7 B7 i. Description of invention () Red blood cells are the main component of blood and have the oxygen transmission system of the body. It is known that the most important feature of a blood substitute is its ability to carry oxygen. Red blood cells can carry oxygen because its main component is heme, which acts like an oxygen carrier. Most of the products undergoing clinical trials as blood substitutes contain heme, which is separated from the red blood cell's cell membrane and the remaining cellular components and purified to remove substantially all impurities. However, when heme is removed from red blood cells and placed in solution in its natural form, it is unstable and rapidly breaks down into its component subunits. Therefore, the hemoglobin used in the heme-based oxygen carrier must be stabilized to prevent its decomposition in solution. In order to develop a heme product that is stable in solution and that it is stabilized in a way that does not impair its gas transmission function, the physical betting of scientific manpower and capital is indispensable. The ability of existing heme-based blood substitutes to be transfused has been fully proven (refer to US Patent Nos. 3,925,344; 4,001,200; 4,001,401; 4,053,590; 4,061,736; 4,136,093; 4,301,144; 4,336,248; 4,376,095; 4,377,512; 4,401,652; 4,473,494; 4,473, 496; 4,600,531; 4,584,130; 4,857,636; 4,826,811; 4,91 1,929 and 5,061,688). In the body, when blood flows through the lungs, the hemoglobin in the red blood cells combines with oxygen molecules and transmits air molecules to various parts of the body to meet the body's normal metabolic functions. However, most living organisms must breathe in order to sustain atmospheric oxygen, which is a scientific and medical contradiction. On the one hand, almost all living organisms need oxygen to survive. On the other hand, various kinds of gas-related chemically toxic substances are faked during normal oxygen metabolism. The paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 8 (Please read the notes on the back first (Fill in this page again)

經濟部中央標準局員工消費合作社印製 492866 A7 __^ __Β7_ 五、發明説明（) 成之。關於在血紅素輸送氧氣的過程中所形成的氧化壓力，已知道在輸送氧氣的過程中，血紅素（Hb)分子本身可被其所攜帶的氧氣（0 2)所氧化。此自我氧化反應産生二種非所欲的産物：氧化血紅素（met-Hb)與超氧化物陰離子（·〇2_) 。其化學反應如下所示： H b + 4 0 2 ----分 me t - H b + 4 · 0 2 ' [1] 該超氣化物陰離子（· 〇2 _)傺為一帶有一額外的電子及一負電荷的氧分子。該超氧化物陰離子（♦ 0厂）具高度反應性且且為有毒的。如此處所述的，諸如超氧化物陰離子之自由基，在一廣範圍的病理過程中，被暗示是為細胞損害之因子。就血紅素輸送氧氣之情況而言，具有潛在破壞性的氧化壓力，傺從血紅素之自我氧化所生成的超氧化物陰離子而生，並在酵素超氧化物歧化酶（S0D)之存在下，藉由下列反應而造成超氧化物陰離子之隨後轉化成有毒的過氧化氫：Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 492866 A7 __ ^ __Β7_ V. Description of the invention (). Regarding the oxidative pressure formed during the transport of oxygen by heme, it is known that during the transport of oxygen, the heme (Hb) molecule itself can be oxidized by the oxygen (02) carried by it. This self-oxidation reaction produces two undesired products: oxidized heme (met-Hb) and superoxide anion (· 〇2_). The chemical reaction is as follows: H b + 4 0 2 ---- divided into me t-H b + 4 · 0 2 '[1] The supergassing anion (· 〇 2 _) 傺 is a band with an additional electron And a negatively charged oxygen molecule. This superoxide anion (Plant 0) is highly reactive and toxic. As described herein, free radicals such as superoxide anions have been implicated as a factor of cell damage in a wide range of pathological processes. In the case of hemoglobin transporting oxygen, the potentially damaging oxidative stress arises from the superoxide anion produced by the self-oxidation of heme, and in the presence of the enzyme superoxide dismutase (SOD), The subsequent conversion of superoxide anions to toxic hydrogen peroxide is caused by the following reactions:

S0D 2 * 0 2 +2H+ ----—少〇2 + Η 2 0 2 [2] 在其中氧化壓力傺為一因子的病理狀態中，可一再地觀察到一種反應，藉此一自由基物種得以在活體内生成毒性化本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） 9 (請先閱讀背面之注意事項再填寫本頁) 裝- -5'，經濟部中央標準局員工消費合作社印製 492866 A7 _________ 五、發明説明（）學物種或導致細胞性損害。紅血球内的超氣化物陰離子及過氧化氫之存在被認為是紅血球的氧化壓力之主要來源。除了利用其内所含血紅素來輸送氧氣外，紅血球之一較少被認知到的待徵是，其含有一組特殊的酵素，該等酵素能將與氧有關的化學物種加以解毒，該等化學物質是在氧氣代謝中所産生的副産物。沒有此等持殊的酵素条統之保護，血紅素的自我氧化會造成红血球的惡化及毀滅。但是在身體内，位在紅血球内的該等酵素条統之保存能力，藉由將在正常代謝期間中所産生的超氧化物陰離子轉變成無毒物質，可使身體免於氧中毒，並_此控制氣化壓力之程度。但是，若此酵素条統瓦解，紅血球的完整性就會受損。一種會産生為在紅血球細胞内的保護条統之一種酵素的基因之損害會導致一可觀察到的病理狀況。例如，葡萄糖-6-磷酸去氫酶缺乏症，一種紅血球的遣傳性障礙，是過氧化氫誘發的溶血貧血症之主因。此障礙是由於被影響的細胞不能維持足供被氧化的縠胱甘肽之還原的NAD (P) Η 位準，造成過氧化氫透過榖胱甘肽過氧化酶之不當的解毒 [P. Hochestein, Free Radical Biology & Medicine, 5_:387 (198δ)]。紅血球細胞的保護性酵素条統可經由一種二步驟化學途徑將有毒的超氧化物陰離子轉變成一種無毒的型式°該途徑的第一個步驟是經由酵素榖胱甘肽過氧化酶將超氧化物陰離子轉變成為過氧化氫（參見式[2] ) °由於過氧化S 亦為有毒的，紅血球含有另一種酵素，觸酶，其如該途徑本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1Τ (請先閲讀背面之注意事項再填寫本頁) 裝· • τ— I J— . 經濟部中央標準局員工消費合作社印製 492866 A7 ___B7_ 五、發明説明（）之第二步驟所示，將過氧化氫轉變成水（參見式[3 ])。觸酶 2 H2〇2 -------今 2 HsO + 〇2 [3] 红血球亦可使用酵素榖胱甘肽過氧化酶而將過氧化氫以及其它的毒性有機過氧化物予以解毒，該榖胱甘呔過氧化酶與榖胱甘呔反應而將過氧化氫及有機過氧化物轉變成水。红血球亦含有一種酵素來防止由血紅素之自我氧化所産生的氧化血红素之生成。該酵素，氧化血红素還原酶可將氣化血紅素轉變回血紅素的天然型式。因此，在身體内，血紅素的自我氧化之毒性作用，被以酵素為主之持殊的反應途徑所防止，該途徑可消除非所欲的氧氣代謝副産物。該等在正常的氧氣輸送過程中可使紅血球免於氣中毒的超氧化物歧化酶、觸酶及榖胱甘肽過氧化酶之酵素性氧氣解毒功能，並不存在於迄今所發展的以血紅素為主的氧載體（hboc)内。若無氣氣解毒功能，現有的HBOC溶液之安全性會由於與氧有關的毒性物種之存在而受損。用來製備現有的HBOC溶液之基本方法是藉由從紅血球中除出血红素，而後將之純化以去除可能在輸血時引起不利反應的所有非-血红素蛋白質及其它雜質（參見美國專利第 4,8 78,210、4,831,012 及 4,925, 57 4號）。氧氣解毒酵素条統之實質破壞或移除，是現有的用以産生可用於HBOCs 中的純化血紅素之分離及純化方法之一不可避免的結果。本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）-1 1 - (請先閲讀背面之注意事項再填寫本頁)S0D 2 * 0 2 + 2H + ----— less 〇2 + Η 2 0 2 [2] In pathological states where oxidative stress 傺 is a factor, a reaction can be repeatedly observed, whereby a radical species Ability to generate toxicity in vivo This paper is sized to the Chinese National Standard (CNS) A4 (210 × 297 mm) 9 (Please read the precautions on the back before filling this page) Pack--5 ', employee of the Central Bureau of Standards, Ministry of Economic Affairs Printed by the Consumer Cooperative 492866 A7 _________ V. Description of the invention () Study species or cause cellular damage. The presence of superoxide anions and hydrogen peroxide in red blood cells is considered to be the main source of oxidative stress in red blood cells. In addition to the use of hemoglobin contained in it to transport oxygen, one of the lesser-known symptoms of red blood cells is that it contains a special group of enzymes that can detoxify chemical species related to oxygen. Substances are by-products produced in the metabolism of oxygen. Without the protection of these special enzyme systems, the self-oxidation of heme can cause the deterioration and destruction of red blood cells. However, in the body, the storage capacity of these enzymes located in red blood cells can protect the body from oxygen poisoning by converting the superoxide anions produced during normal metabolism into non-toxic substances. Control the degree of gasification pressure. However, if this enzyme disintegrates, the integrity of the red blood cells will be compromised. Damage to a gene that produces an enzyme that is a protective system within red blood cells results in an observable pathological condition. For example, glucose-6-phosphate dehydrogenase deficiency, a transmission disorder of red blood cells, is a major cause of hydrogen peroxide-induced hemolytic anemia. This disorder is due to the improper detoxification of hydrogen peroxide through cystathione peroxidase due to the inability of the affected cells to maintain the NAD (P) Η level sufficient for the reduction of oxidized cystathione [P. Hochestein , Free Radical Biology & Medicine, 5_: 387 (198δ)]. The protective enzyme system of red blood cells can convert a toxic superoxide anion into a non-toxic form through a two-step chemical pathway.The first step of this pathway is to convert superoxide via the enzyme cystathione peroxidase. The anion is converted into hydrogen peroxide (see formula [2]) ° Since peroxide S is also toxic, red blood cells contain another enzyme, which touches enzymes. In this way, the paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) 1T (Please read the notes on the back before filling out this page) Install · • τ— IJ—. Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 492866 A7 ___B7_ V. The second step of the invention description () It is shown that hydrogen peroxide is converted into water (see formula [3]). Contact enzyme 2 H2〇2 ------- today 2 HsO + 〇2 [3] Red blood cells can also use the enzyme 榖 glutathione peroxidase to detoxify hydrogen peroxide and other toxic organic peroxides, The cystathionine peroxidase reacts with cystathione to convert hydrogen peroxide and organic peroxides into water. Red blood cells also contain an enzyme to prevent the production of oxidized heme, which is produced by the self-oxidation of heme. This enzyme, oxidized heme reductase, converts vaporized heme back to the natural form of heme. Therefore, in the body, the toxic effects of heme self-oxidation are prevented by the enzyme-based, dedicated response pathway that eliminates unwanted oxygen metabolism by-products. The enzyme oxygen detoxifying functions of superoxide dismutase, catalase, and cystathione peroxidase that can prevent red blood cells from gas poisoning during normal oxygen transport do not exist in hemoglobin developed so far. The main oxygen carrier (hboc). Without qi and gas detoxification, the safety of existing HBOC solutions would be impaired by the presence of oxygen-related toxic species. The basic method used to prepare existing HBOC solutions is to remove hemoglobin from red blood cells and then purify it to remove all non-heme proteins and other impurities that may cause adverse reactions during blood transfusion (see U.S. Patent No. 4 , 8 78,210, 4,831,012 and 4,925, 57 4). The substantial destruction or removal of the oxygen detoxifying enzyme system is an inevitable result of the existing separation and purification methods used to produce purified heme that can be used in HBOCs. This paper size applies to Chinese National Standard (CNS) A4 specification (210X297 mm) -1 1-(Please read the precautions on the back before filling this page)

經濟部中央標準局員工消費合作社印製 492866 A7 B7__ 五、發明説明（）任擇地，不從紅血球中來分離及純化血紅素，已可使用重組技術來製備血紅素。但是，重組型人類血紅素亦為高度純化的，且其不含於紅血球中發現到的氧氣解毒条統。因此，用來産生一高度純化的血紅素溶液之精密技術的發展，是好是壞未可知，因為純化方法會去除有害的雜質以及正常存在於紅血球内之有利的氧氣解毒酵素，而最終會助益於與氧有關的毒性。由現有的HBOCs之靜脈内投藥所引起之一觀察到的毒性副作用是血管收縮或高血壓。已知超氯化物歧化酶（SOD) 在活體外會快速的去除超氣化物陰離子，並且增長氧化氮 (NO)之血管舒張效用。氧化氮是一種其在最近被發現偽為先前僅知的*内皮-衍生之鬆馳因子（EDRF) 〃物質之分子。氧化氮的血管鬆馳效用藉由SOD而得以延長，被認為是該SOD用以防止超氧化物陰離子及氧化氮之間的反應之能力戸万致者[Μ·Ε· Murphy e t a 1 . , P r q c . Natl. _Acad> Sci· USA, 88 ： 10860 (1991)； Ignarro et al., J. PharmacoL! R x d. T h θ r …244:81 (19 8 8) ； Rub a n y i Am. J , Physiol· 2 5 0: H822 (1 986) ； Gry 1 ewski et al., Nature 320: 454 (1986)]。但是，在活體中，由超氣化物陰離子所造成的EDRF之去活化，不曾被觀察到，且一般不被認為是可能的。然而，某些會損害S0D的病理生理上狀況會引起由超氣化物陰離子所導致的毒性作用[Ignarro et al·, J. Phramaco 1 > Toy icol . 2立：535 (1990)]。在現有的HBOC溶液之臨床前本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -12- (請先閱讀背面之注意事項再填寫本頁)Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 492866 A7 B7__ V. Description of the Invention () Optionally, instead of separating and purifying heme from red blood cells, recombination technology can be used to prepare heme. However, recombinant human heme is also highly purified and does not contain the oxygen detoxification system found in red blood cells. Therefore, the development of precision technology to produce a highly purified heme solution is not known for good or bad, because the purification method will remove harmful impurities and the beneficial oxygen detoxifying enzymes normally present in red blood cells, which will eventually help Benefits from oxygen-related toxicity. One of the toxic side effects observed with intravenous administration of existing HBOCs is vasoconstriction or hypertension. It is known that superchloride dismutase (SOD) rapidly removes supergaside anions in vitro and increases the vasodilating effect of nitrogen oxide (NO). Nitric oxide is a molecule that has recently been found to be pseudo-previously known only as * endothelium-derived relaxation factor (EDRF) plutonium. The vasorelaxation effect of nitric oxide is prolonged by SOD, which is considered to be the ability of this SOD to prevent the reaction between superoxide anion and nitric oxide [M · E · Murphy eta 1., P rqc. Natl. _Acad > Sci · USA, 88: 10860 (1991); Ignarro et al., J. PharmacoL! R x d. T h θ r… 244: 81 (19 8 8); Rub anyi Am. J, Physiol. 2 50: H822 (1 986); Gry 1 ewski et al., Nature 320: 454 (1986)]. However, in vivo, the deactivation of EDRF caused by supergassing anions has not been observed and is generally not considered possible. However, certain pathophysiological conditions that can impair SOD can cause toxic effects caused by supergassing anions [Ignarro et al., J. Phramaco 1 > Toy icol. 2 Li: 535 (1990)]. Before the clinical application of the existing HBOC solution, the paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) -12- (Please read the precautions on the back before filling this page)

492866 A7 B7__ 五、發明説明（）動物研究中所觀察到的高血壓作用暗示了在現有的HBOCs 之大量輸血中，超氧化物陰離子的濃度是EDRF的破壞及所觀察到的血管收縮及全身性高血壓的原因。因此，區隔由氧化物陰離子與氧化氮（NO)之反應所導致的高血壓與由血管外滲作用所導致的高血壓以及血紅素對NO之結合，是十分重要的。在一 HBOCs之輸血時，血紅素亦可藉由其與氧化氮反應而産生對應的亞硝醯基-原血红素（NO-heme)加合物來壓低氧化氮的血管舒張作用。更待別的是，已知去氧-血紅素對氧化氮之結合親和力，較其對一*氧化細局的結合親和力要局上好幾値級數° 這些血紅素-M0相互反應已被用於分析氧化氮及研究氧化氮的生物活性。例如，血红素對氧化氮的血管舒張作用之拮抗作用似乎是依賴血紅素的細胞膜通透性而定。在完整的血小板中，血紅素不會抵消L-精胺酸（氧化氮的前驅物）之作用。相對的，在被分解的血小板之胞液中，血紅素係為由氧化氮所調節之L-精胺酸誘發的環GMP形成之一最有效的抑制劑。這些實驗證實血紅素無法有效地穿透血小板膜[R a d 〇 m s k i e t a 1 · , B r . J . Pharmacol . 101: 325 經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) (1990)]。因此，所想要的HBOCs之特徽之一是消除氧化氮與血紅素之相互反應。亦已知血紅素可拮抗内皮-依賴性血管舒張作用[Martin W· et al., J , Pharmaco]. Exp. Th^r . 122:708 (1985)]以及NO-激發的血管平滑肌的鬆馳作用[Grueter C . A . , e t a 1 . , J . Cyclic N u 〇 1 a 〇 t： i d e Res· 5_: 21 1 (1979)]。已有嘗試藉由化學地穩定化、聚本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 492866 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（）合化、包囊化或共軛化位在HBOCs中的血紅素來限制血紅素的血管外滲作用及高血壓作用俾以延長循環時間。因此，雖然現今的HBOCs是為相當地膜不可通透者，並可輸送氧氣，但該等HBOCs溶液在輸血時，不具有防止超氧化物陰離子與氧化氮間反應之能力。上述之例證明在克服氧氣毒性/壓力問題上之困難，即令對氧氣輸送之反應機轉已有合理之瞭解，且數十年來之研究亦為了要改善血紅素生成及形成亦然。現有的血液代用品之毒性問題的一個理想解決方案是一種以血红素為主的組成物，該組成物組合了現有的HBOCs 之氧氣-輸送功能與紅血球的氧氣解毒功能。但是，酵素超氧化物歧化酶（SOD)至現有的HBOC溶液内之一簡單的添加不會是所想要者，蓋因藉由降低超氧化物陰離子的濃度，會助益將血紅素氧化成氧化血紅素的反應，這導致非所欲的氧化血紅素之建立（參見式[1])。又，亦不希望鼓勵一血红素溶液中的超氧化物陰離子至過氧化氫之轉化，因為過氧化氫有毒且具有反應性，並會在儲存期間分解成有毒的羥基團或形成其它有毒的有機過氧化物。由於合成的血液代用品理想地是能供大量輸血的，其會與自由基相互反應之化合物必須在活體内能雒持其功能並且為安定及無毒的。依據本發明，氮氧化物與氮氧化物 -標識的巨分子（包括血紅素、白蛋白以及他者）被用來緩解一活生物體内的自由基物種之毒性作用。依據本發明，要與生物性巨分子合併使用俾以於活體 (請先閲讀背面之注意事項再填寫本頁) -裝_ 訂 k 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 492866 經濟部中央標準局員工消費合作社印製 A7 _B7____ 五、發明説明（）内控制由自由基所引起之損害的氣化氮之能力，産生了可用來設計供治療與診斷用之含氧化氮配方與其一廣範圍的使用方法之可行性。許多其中存在有氧氣-衍生的自由基之生理狀態及過程，可藉由使用本文中所述之化合物予以治療或診斷。膜可通透的低分子量氮氣化物與生物可相容的巨分子（例如血紅素、白蛋白以及他者）之合併使用，容許研究人員可以就其所感興趣之待殊環境來裁製含氮氧化物配方。一多重組份之以氮氧化物為主的条統亦發揮有如一放射保護劑之功能以供用於癌症治療以及放射暴露之治療。在臨床應用上，放射治療法之有效性，會因容許較高幅射劑量被安全地使用，而被增進之。許久以來，殷切地契求其能防止醫學放射治療法之期間的離子輻射或由於環境的幅射暴露所遭遇到的有害作用之試劑。該等試劑亦為研究放射毒性之機轉的有用工具。半胱胺（一種含硫化合物）偽為最早被鑑定出的放射保護劑之一者。它的發現促成美國國防部來贊助超過4,000種的化合物之合成與条統性篩析以嘗試發現更有效的試劑。此極為龐大的計劃造成一些放射保護劑（例如被稱之為WR -272 1的胺基硫醇化合物）之發現。近來，超氧化物技化酶、間白素I以及穎粒白血球-巨噬細胞細胞群剌激因子已被顯示出具有放射保護活性。在這些試劑之一比較中，WR-2721對正常組織顯示出最具體及選擇性保護作用。但是，當被用於正在進行癌症放射治療的病人身上時，對固有的毒性之考慮以及腫瘤的非專丨丨1F (請先閱讀背面之注意事項再填寫本頁) •裝· 、訂 ·線本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）經濟部中央標準局員工消費合作社印製 492866 A7 B7 五、發明説明（）一性保護，削弱了對於WR-2721之使用之熱衷。若干穩定的氮氧化物已被發現具有抗氯化劑及放射保護劑活性。但是，膜可通透的氮氯化物在活體内被快速地減少，且在升高的位準下可能是有毒的。依據本發明，膜可通透的氮氧化物之投藥的實用性，可被增進之。發明概要説明本發明掲露氮氧化物與生物可相容的巨分子之合併使用。特別地，本發明描述低分子量且為膜可通透的氮氯化物與其以一高莫耳比被結合至諸如白蛋白育血紅素的生物可相容的巨分子之氮氧化物的合併使用。本發明亦涵括穩定的氮氧化物自由基（此下稱之為0氮氣化物之使用，俾以對以血红素為主的血液代用品提供红血球的氧氣解毒功能並緩解氧化壓力以及避免與自由基毒性有關聯的生物性損害（包括發炎、熱傷害.、後-局部缺血再灌流傷害、離子幅射、白内障、敗血症、老化等等在内）。在某些實施例中，穩定的氮氧化物被用來製造數種有關於一具有紅血球的氧氣解毒功能之血液代用品的配方。這些配方在此處可能被敘述為以血红素為主的紅血球代用品（HRCS)，因為以血紅素為主的氧載體（HBOC)之氧氣輸送能力可藉由提供身體紅血球之氧氣解毒功能而被增強之。欲克服僅使用氮氧化物時的缺點，在本發明之較佳實施例中，一種聚氮氧化物-標識的巨分子（諸如Tempe-標識 . / 的人類血清白蛋白），與一自由的且膜可通透的氮氧化物一起被輸血俾以於活體内提供氮氧化物之延長的活性。該本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -! 6 - ---------裝-- (請先閲讀背面之注意事項再填寫本頁) 訂* 經濟部中央標準局員工消費合作社印製 492866 A7 _B7__ 五、發明説明（）一配方之一優點是，其為一種可被用於診斷與治療此二種醫療應用的改良型放射保護劑，俾以防止暴露於來自任一來源的放射。在治療性醫療應用中，增加的放射劑量可以被施用，因而改善放射治療會成功的可能性。在某些腫瘤 (例如那些位於腦中者，以及加上本文中所述之顯影，那些含有缺氧之區域者）中，此能力是特別顯著的。又，氮氧化物可利用電子旋轉共振光譜法以及核磁共振光譜法來偵測。在現代的顯影儀器設備之發展下，依據一氮氧化物的自由基之一測定可以進行整體的生物組織與器官之顯影。依據本發明，身體内有活性的氮氧化物位準可被維持一段延長的時間而容許較僅使用低分子量且膜可通透的氮氧化物時，要為改善的顯影對比以及較長的信號持續性。更甚者*不像某些現有的試劑，本發明之組成物能夠橫跨過血液-腦障壁。另外，由於它們的抗氧化活性，本發明所掲露之組成物具有治療用價值，加上其診斷用價值，而容許本發明之新穎組成物與方法可被有利地應用在一廣範圍之應用上。本發明所敘述之材料與方法可供用於呈數種形式之穩定的氮氧化物之製備及投藥，以及其在活體内會被其他於本發明中所述的化合物轉化成有生物活性的氮氧化物或抗氣化酵素模擬物之無活性的且相當無毒的膜可通透的氮氧，化物前驅物之製備及投藥。在任一情況中，在其於解毒有害的自由基之過程後已被化學性地還原後，可藉由氮氣化物-標識的巨分子物種，將被還原的（無活性的）氮氧化物本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) -裝. 訂 492866 經濟部中央標準局員工消費合作社印製 A7 ___—_ B7 __ 五、發明説明（）再活化之。由於再生作用之結果，本發明之氮氧化物相較於低分子量且膜可通透的氮氧化物本身，於活體内具有較長的半衰期。因此，本發明所提供之組成物與方法可以增進其中氮氧化物像為有效的任一種應用之效果。使用本發明之多重組份条統，在低分子量且膜可通透的氮氧化物以及具不同穩定性之含有膜可通透的氮氧化物的物種之間可建立起一動力平衡。待別地，一種膜不可通透的巨分子-結合的氮氧化物會使膜可通透的氮氧化物之活性功能再生之。依據本發明，維持一有活性的氮氧化物在活體内之濃度的能力，對於任一種其中氮氧化物之投藥像為有利的，但由於在活體内的快速減少，或其中一膜可通透的氮氣化物之最佳有效劑量傺為有毒的，而使其利用性受限的應用而言，最終是提供了有利之結果。其因為一穩定不成對的電子而為順磁性的氮氧化物，應可作為核磁共振顯影和電子旋轉共振顯影之顯影劑。但是，由於氮氧化物之快速減少至一光譜法偵測不到的物種，典型地大多為羥基胺，該等試劑之實用性即受到限制。由於自由基物種被包含於再灌流傷害内，且已知其與氧氣代謝有關，使用本發明之化合物作為顯影劑，可以觀察到局部缺血性組織傷害以及缺氧症。以多重組份氮氣化物為主的条統之一明顯的優點是，其能輸送抗氧化性、放射保護性、抗局部缺血性、顯影- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -18- —Hr (請先閱讀背面之注意事項再填寫本頁) 裝· 訂線 492866 A7 B7 五、發明説明（增進性、酵素-模擬物，並於身體的數個區域（例如血管隔室、間質間隙以及細胞内區域）發揮功能之能力。研究人員或臨床撿驗員可依應用所需，來裁用本發明所述之多重組份条統。例如，本發明中所述之不同的配方具有不同的血管舒張作用之位準。基本上，一氮氧化物（包括前驅物及代謝基質），依據其被選擇要進行所欲之之功能（亦即放射保護、顯影、酵素-模擬物等等），而被提供之，並提供另一種以氮氣化物為主的物種以作為一活性之貯庫。前者物種可被認為是一個接受者〃氮氣化物，而後者物種是一個a供給者"氮氧化物。供給者與接受者於活體内應保持實質物質性分隔，且於其自由基部分應具有不同的穩定性。在一較佳的實施例中*該供給者氮氧化物傺為一種聚氮氧化物白蛋白，其主要分佈在血管間隙並發揮有如一活性貯庫之作用。該接受者氮氧化物典型地偽為一低分子量膜可通透的物種（例如TLP或TPH)。經濟部中央標準局員工消費合作社印製 0\\492866 A7 B7__ 5. Description of the invention () The hypertension effect observed in animal studies suggests that the concentration of superoxide anion in the massive transfusion of existing HBOCs is the destruction of EDRF and the observed vasoconstriction and systemicity. Causes of hypertension. Therefore, it is important to distinguish the combination of hypertension caused by the reaction of oxide anions with nitric oxide (NO), hypertension caused by extravasation of blood vessels, and heme to NO. During the transfusion of a HBOCs, heme can also reduce the vasodilation effect of nitric oxide by reacting with nitric oxide to produce the corresponding nitrosino-protoheme (NO-heme) adduct. What's more, it is known that the binding affinity of deoxy-heme to nitrogen oxide is several orders of magnitude higher than its binding affinity to a single oxidation detail. These heme-M0 interactions have been used for Analyze NO and study the biological activity of NO. For example, the antagonistic effect of heme on the vasodilation of nitric oxide appears to depend on the membrane permeability of heme. In intact platelets, heme does not counteract the effects of L-arginine, a precursor of nitric oxide. In contrast, in the cytosol of the decomposed platelets, heme is one of the most effective inhibitors of cyclic GMP formation induced by L-arginine acid regulated by nitric oxide. These experiments confirm that heme cannot effectively penetrate the platelet membrane [R ad omskita 1 ·, B r. J. Pharmacol. 101: 325 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out (This page) (1990)]. Therefore, one of the special characteristics of the desired HBOCs is to eliminate the interaction between nitric oxide and heme. Heme is also known to antagonize endothelial-dependent vasodilation [Martin W. et al., J, Pharmaco]. Exp. Th ^ r. 122: 708 (1985)] and NO-stimulated relaxation of vascular smooth muscle Action [Grueter C. A., eta 1., J. Cyclic Nu 〇1a 〇t: ide Res · 5_: 21 1 (1979)]. Attempts have been made to chemically stabilize the paper size to apply Chinese National Standard (CNS) A4 specifications (210X297 mm) 492866 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs Encapsulation or conjugation of heme in HBOCs can limit the vascular extravasation and hypertensive effect of heme to extend circulation time. Therefore, although today's HBOCs are relatively impermeable membranes and can transport oxygen, these HBOCs solutions do not have the ability to prevent the reaction between superoxide anions and nitrogen oxides during blood transfusion. The above examples demonstrate the difficulty in overcoming the issue of oxygen toxicity / stress, that is, to have a reasonable understanding of the reaction mechanism of oxygen transport, and decades of research to improve heme production and formation. An ideal solution to the toxicity problem of existing blood substitutes is a heme-based composition that combines the oxygen-transport function of existing HBOCs with the oxygen detoxification function of red blood cells. However, a simple addition of the enzyme superoxide dismutase (SOD) to the existing HBOC solution will not be what you want. Gein will help to oxidize heme by reducing the concentration of superoxide anions. The reaction of oxidized heme, which leads to the establishment of undesired oxidized heme (see formula [1]). Also, it is not desirable to encourage the conversion of superoxide anions in a heme solution to hydrogen peroxide, because hydrogen peroxide is toxic and reactive, and will decompose into toxic hydroxyl groups or form other toxic organic compounds during storage. peroxide. Since synthetic blood substitutes are ideal for large blood transfusions, compounds that interact with free radicals must retain their function in vivo and be stable and non-toxic. According to the present invention, nitrogen oxides and nitrogen oxide-labeled macromolecules (including heme, albumin, and others) are used to alleviate the toxic effects of free radical species in a living organism. According to the present invention, it is to be used in combination with biological macromolecules for living organisms (please read the precautions on the back before filling this page)-binding_ order k This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) ) 492866 Printed by A7 _B7____ of the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention () The ability to internally control the gasification of nitrogen caused by free radicals has produced nitrogen oxides that can be used to design for treatment and diagnosis The feasibility of the formulation and its wide range of uses. Many physiological states and processes in which oxygen-derived free radicals are present can be treated or diagnosed by using the compounds described herein. The combination of membrane-permeable low-molecular-weight nitrogen compounds and biocompatible macromolecules (such as heme, albumin, and others) allows researchers to tailor nitrogen-containing oxidation to the specific environment of interest物组合物。 Formula. More than one reorganized nitrogen oxide-based system also functions as a radioprotective agent for cancer treatment and radiation exposure treatment. In clinical applications, the effectiveness of radiation therapy is enhanced by allowing higher radiation doses to be used safely. For a long time, there has been an earnest demand for reagents that can prevent the harmful effects of ionizing radiation during medical radiotherapy or exposure to the environment. These reagents are also useful tools for studying the mechanisms of radiotoxicity. Cysteine, a sulfur-containing compound, was one of the first radioprotectants identified. Its discovery has led the U.S. Department of Defense to sponsor the synthesis and systematic screening of more than 4,000 compounds in an attempt to discover more effective reagents. This extremely ambitious project has led to the discovery of some radioprotective agents, such as the aminothiol compound known as WR-272 1. Recently, superoxide chemokines, melatonin I, and granulocyte-macrophage population stress factors have been shown to have radioprotective activity. Among the comparisons of these reagents, WR-2721 showed the most specific and selective protective effect on normal tissues. However, when used on patients undergoing radiotherapy for cancer, consideration of inherent toxicity and non-speciality of tumors 丨 1F (please read the precautions on the back before filling this page) This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 492866 A7 B7 V. Description of the invention () Uniform protection, weakening the enthusiasm for the use of WR-2721 . Several stable nitrogen oxides have been found to have anti-chlorination and radioprotective activity. However, membrane permeable nitrogen chloride is rapidly reduced in vivo and may be toxic at elevated levels. According to the present invention, the practicality of the administration of the membrane-permeable nitrogen oxides can be enhanced. Summary of the invention The combined use of dew nitrogen oxides of the present invention with biocompatible macromolecules. In particular, the present invention describes the combined use of low molecular weight and membrane permeable nitrogen chlorides with nitrogen oxides which are bound to a biocompatible macromolecule such as albumin to hemoglobin at a high mole ratio. The present invention also includes the use of stable nitrogen oxide free radicals (hereafter referred to as zero nitrogen compounds). It provides oxygen detoxification function of red blood cells and relieves oxidative stress and avoids freedom from the heme-based blood substitutes. Biological damage associated with basic toxicity (including inflammation, thermal injury, post-ischemic reperfusion injury, ion radiation, cataract, sepsis, aging, etc.). In some embodiments, stable nitrogen Oxides are used to make several formulations of blood substitutes with oxygen detoxification of red blood cells. These formulations may be described here as heme-based red blood cell substitutes (HRCS), because heme The oxygen transport capacity of the main oxygen carrier (HBOC) can be enhanced by providing oxygen detoxification function of the body's red blood cells. To overcome the disadvantages when using only nitrogen oxides, in a preferred embodiment of the present invention, a polymer Nitrogen oxide-labeled macromolecules (such as Tempe-labeled human serum albumin) are transfused with a free and membrane-permeable nitrogen oxide 氧化物Provide extended activity of nitrogen oxides in the living body. This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm)-! 6---------- install-(Please read first Note on the back, please fill out this page) Order * Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 492866 A7 _B7__ V. Description of the invention () One of the advantages of a formula is that it is a type that can be used for diagnosis and treatment. Improved radioprotective agents for medical applications to prevent exposure to radiation from any source. In therapeutic medical applications, increased radiation doses can be administered, thus improving the likelihood that radiation therapy will be successful. In some tumors (Such as those located in the brain, plus those described in this article, those with hypoxic areas), this ability is particularly significant. In addition, nitrogen oxides can use electron resonance resonance spectroscopy and nuclear magnetic resonance Spectroscopy to detect. Under the development of modern imaging equipment, the overall biological tissues and organs can be developed based on the determination of one of the nitrogen oxide free radicals. According to the present invention It is clear that the active nitrogen oxide level in the body can be maintained for an extended period of time, which allows for improved development contrast and longer signal persistence than when only low molecular weight and membrane permeable nitrogen oxides are used. What's more, * unlike some existing agents, the composition of the present invention can cross the blood-brain barrier. In addition, due to their antioxidant activity, the composition disclosed by the present invention has therapeutic value. The diagnostic value of this invention allows the novel compositions and methods of the present invention to be advantageously applied to a wide range of applications. The materials and methods described in the present invention can be used to stabilize nitrogen oxides in several forms Preparation and administration, as well as inactive and fairly non-toxic membranes that are converted in vivo by other compounds described in this invention into biologically active nitrogen oxides or anti-gasification enzyme mimics Preparation and administration of nitrogen oxides and precursors. In either case, after it has been chemically reduced after the detoxification of harmful free radicals, the reduced (inactive) nitrogen oxides can be reduced by the nitrogenate-identified macromolecular species Standards are applicable to China National Standard (CNS) A4 specifications (210X297 mm) (Please read the precautions on the back before filling out this page)-Packing. Order 492866 Printed by the Consumers Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 ___ B7 __ 5 2. Description of the invention () Reactivate it. As a result of the regeneration effect, the nitrogen oxide of the present invention has a longer half-life in vivo than the nitrogen oxide itself, which has a low molecular weight and is permeable to the membrane. Therefore, the composition and method provided by the present invention can enhance the effect of any application in which the nitrogen oxide image is effective. Using the multi-component system of the present invention, a dynamic balance can be established between low molecular weight, membrane permeable nitrogen oxides and species with different stability containing membrane permeable nitrogen oxides. Elsewhere, a membrane-impermeable macromolecule-bound nitrogen oxide will regenerate the active function of the membrane-permeable nitrogen oxide. According to the present invention, the ability to maintain a concentration of active nitrogen oxides in vivo is advantageous for any kind of administration image of nitrogen oxides, but due to rapid reduction in vivo, or one of the membranes is transparent The optimal effective dose of nitrogen compounds is toxic, and in applications where its availability is limited, it ultimately provides favorable results. It is a paramagnetic nitrogen oxide because of a stable unpaired electron and should be used as a developer for nuclear magnetic resonance imaging and electron rotation resonance imaging. However, due to the rapid reduction of nitrogen oxides to species not detected by a spectroscopic method, most of them are hydroxylamines, and the usefulness of these reagents is limited. Since free radical species are included in reperfusion injury and are known to be involved in oxygen metabolism, using the compound of the present invention as a developer, ischemic tissue injury and hypoxia can be observed. One of the obvious advantages of the system with multiple reconstituted nitrogen compounds is that it can transport oxidation resistance, radiation protection, ischemia resistance, and development-this paper size applies Chinese National Standard (CNS) A4 specifications ( 210X297 mm) -18- —Hr (Please read the precautions on the back before filling out this page) Binding · 492866 A7 B7 5. Description of the invention (promoter, enzyme-mimic, and in several areas of the body ( (Such as vascular compartments, interstitial spaces, and intracellular areas). Researchers or clinical examiners can use the multiple reorganization systems described in the present invention as required by the application. For example, in the present invention The different formulations described have different levels of vasodilation. Basically, a nitrogen oxide (including precursors and metabolic matrices) is selected to perform the desired function (ie, radiation protection, imaging) , Enzymes-mimetics, etc.), and is provided, and provides another nitrogenate-based species as an active reservoir. The former species can be considered as a recipient 〃 nitrogenate, The latter species is a supplier " nitrogen oxide. The supplier and the recipient should maintain a substantial physical separation in the living body and should have different stability in their free radical moiety. In a preferred embodiment * The donor nitrogen oxide is a kind of polynitrogen oxide albumin, which is mainly distributed in the vascular space and functions as an active depot. The recipient nitrogen oxide is typically faked as a low molecular weight membrane. Species (such as TLP or TPH). Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs.

OilOil

HTPU 丁 (請先閱讀背面之注意事項再填寫本頁)HTPU Ding (Please read the notes on the back before filling this page)

本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）經濟部中央標準局員工消費合作社印製 492866 A7 ___ B7____ 五、發明説明（）在實施上，該接受者氮氧化物（即TPL)變成被快速地還原成TPH直至被供給者氮氧化物物種（即聚氮氧化物白蛋白）氧化成TPL。那些熟習此藝者會暸解到，各別的供給者物種可以變化，只其維能持要實質的物質性分隔以及達致差別性穩定性。例如，相同的氮氧化物可作為接受者與供給者兩者之用。在這樣的例子中，被標識在位於一諸如白蛋白的巨分子物種上之許多胺基基團處的TPL，提供了一實質膜不可通透的供給者氮氧化物。藉由在胺基基團處之標識提供了巨分子-結合的TPL之差別性穩定性，因而使得剩下之羧基基團産生一種酸性微環境，這於該白蛋白-結合的TPL内形成一較不穩定的自由基狀態。任擇地，可以提供不同的未結合的氮氧化物，由於其等固有的化學及電子結構之故，提供所需的分隔及差別性穩定性。使用氮氧化物與生物可相容的巨分子合併使用之較佳的組成物可予以變化之；例如，對於一供輸血之用的生理上可相容的溶液（諸如一以血紅素為主的氧載體），該等組成物包含有：1)被加至一儲存容器内或被包含在一過濾器内之含氮氧化物化合物；氮氧化物可以數種形式被化學地連結至一過濾器内所使用的基質上或被包含在内以作為一有利的投藥方式；2)被共價地連接至一藉由化學或重組交聯而被穩定化的血红素上之氮氧化物；3)被共價地連接至《一聚合化血紅素的氮氧化物，持別是呈2、4與8莫耳當量的氮氧化物之形式者；4)與血紅素被共包囊化在一脂質體本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁)This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 492866 A7 ___ B7____ 5. Description of the invention () In practice, the recipient ’s nitrogen oxides (ie TPL ) Becomes rapidly reduced to TPH until it is oxidized to TPL by the donor's nitrogen oxide species (ie, polynitrogen oxide albumin). Those skilled in the art will understand that individual donor species can change, as long as they maintain a substantial physical separation and achieve differential stability. For example, the same nitrogen oxides can be used for both recipients and suppliers. In such an example, TPL identified at a number of amine groups on a macromolecular species such as albumin provides a supplier of nitrogen oxides that are impermeable to the substantial membrane. The differential stability of the macromolecule-bound TPL is provided by the identification at the amine group, so that the remaining carboxyl group creates an acidic microenvironment, which forms an albumin-bound TPL. More unstable free radical state. Optionally, different unbound nitrogen oxides can be provided which, due to their inherent chemical and electronic structure, provide the required separation and differential stability. The preferred composition using nitrogen oxides in combination with biocompatible macromolecules can be varied; for example, for a physiologically compatible solution (such as a heme-based Oxygen carrier), these compositions include: 1) nitrogen oxide compounds added to a storage container or contained in a filter; nitrogen oxides can be chemically linked to a filter in several forms The matrix used in or is included as an advantageous method of administration; 2) the nitrogen oxides are covalently linked to a heme stabilized by chemical or recombinant cross-linking; 3) It is covalently linked to "a polymerized heme nitrogen oxide, which is in the form of 2, 4 and 8 mole equivalents of nitrogen oxide; 4) co-encapsulated with heme in a lipid The paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page)

經濟部中央標隼局員工消費合作社印製 492866 A7 B7 ____ 五、發明説明（）内或是被嵌入於一脂質體膜上的氮氧化物；5)被共價地結合至一共化血紅素上的氮氧化物；6)以較局的莫耳比被共價地結合至數種形式的白蛋白上的氮氧化物；及7)被共價地結合至免疫球蛋白上的氮氧化物，以及其等在一多重組份条統内的任一組合。上述組成物可被分別地使用，或視應用之需而與低分子量膜可通透的氮氧化物合併使用。更甚者，上述組成物可加上其它的化合物被待別地配方之，俾以改變其等在活體内的反應性及穩定性。持別是，可使用環糊精來增強以血紅素為主的溶液之穩定性。已知道必要的營養物硒會産生超氧化物，而可與一聚氮氧化物巨分子一起被使用以促進其氧化。這些配方之使用亦可加有其他已知的會防止氧化壓力之化合物（其會增進顯影或增加/降低對放射的敏感性），以及其他已知的具有臨床與診斷實用性之化合物。此處提供實驗結果以證實低分子量氮氯化物可藉由與本發明之氮氧化物-標識的巨分子，從一經還原的無活性形式被再生成其活性形式。以下的實驗結果及操作方法顯示出氮氧化物可被連接至生物可相容的巨分子（包括白蛋白以及穩定化、聚合化、共軛化和包囊化血红素）上，俾供用於診斷治療以及與氧化壓力有關的生理狀況之評估。氮氧化物-標識的血紅素與氮氧化物-標識的白蛋白之相互作用，無論是為單獨或與一低分子量氮氣化物之併用，暗示了其他具有一實質血漿半衰期之生物可相容的巨分子可依據本發明被標識以氮氣化物並予以使用，俾以有利地提本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）Printed by the Consumers' Cooperative of the Central Bureau of Standards, Ministry of Economic Affairs 492866 A7 B7 ____ V. Description of the invention () Nitrogen oxides embedded in a liposome membrane; 5) Covalently bound to a co-heme 6) nitrogen oxides that are covalently bound to several forms of albumin at a more local molar ratio; and 7) nitrogen oxides that are covalently bound to immunoglobulins, And any combination of them within a multiple reorganization. The above-mentioned composition can be used separately, or combined with the nitrogen oxides permeable to the low-molecular-weight membrane, as required by the application. What's more, the above composition can be formulated separately with other compounds to change its reactivity and stability in vivo. In other words, cyclodextrin can be used to enhance the stability of a heme-based solution. The necessary nutrient selenium is known to produce superoxide and can be used with a polynitrogen oxide macromolecule to promote its oxidation. The use of these formulations can also be supplemented with other known compounds that prevent oxidation stress (which enhances development or increases / decreases sensitivity to radiation), and other known compounds with clinical and diagnostic utility. The experimental results are provided here to confirm that low molecular weight nitrogen chlorides can be regenerated from their reduced inactive forms into their active forms by using the nitrogen oxide-labeled macromolecules of the present invention. The following experimental results and procedures show that nitrogen oxides can be linked to biocompatible macromolecules (including albumin and stabilized, polymerized, conjugated, and encapsulated heme) for diagnostic purposes Treatment and assessment of physiological conditions related to oxidative stress. The interaction of nitrogen oxide-labeled heme with nitrogen oxide-labeled albumin, either alone or in combination with a low molecular weight nitrogen compound, implies that other biologically compatible giants with a substantial plasma half-life Molecules can be labeled with nitrogen compounds and used in accordance with the present invention, so that the paper size is advantageously applied to the Chinese National Standard (CNS) A4 specification (210X297 mm)

2J (請先閱讀背面之注意事項再填寫本頁)2J (Please read the notes on the back before filling this page)

^866 ^866 經濟部中央標準局員工消費合作社印製 A7 s___—_B7__ 五、發明説明（）供對由自由基化學物種所引起的氧化壓力之坑性或防護。此處亦提供實驗結果以證實本發明的組成物與方法，當與一 HBOC—起被輸血時，係為抗高血壓的，因而可使一 HBOC溶液之輸血變成血管中性的。利用將細胞培養物以及小白鼠暴露於致命的放射劑量而證實了放射保護。大白鼠心臓的EPR顯影顯示出能夠監測局部缺血及再灌流損害的進行，這證實了本發明所掲露之組成物除了顯影-增強性之外，還能使局部缺血免於再灌流損害。圖式夕概要説昍本案圖式含有至少一張彩色相片/顯影圖。本案之彩色圖的拷貝本，會在被要求及付必要費用之後，由專利商標局予以提供之。第1A及1B圖顯示第1天（A)及第30天（B)所記錄的，4-胺基-TEMPO (TEMPO: 2,2,6,6-四甲基六氫吡啶-卜烴氧基）標識的〇 -棉子糖聚合化血红素之電子旋轉共振光譜。第1C 圖是在第1天所記錄的，以同體積未標識的血红素稀釋第 1 A圖中的樣品而得之光譜。第1D圖是第1C圖中的樣品在第 3 0天時之記錄。第2A及2B圖像為電子旋轉共振光譜，其分別顯示出4-(2-溴乙醯胺基）-TEMPO至ω -胺己基-瓊脂糖的共價接合及 4-胺基- TEMPO至1,4-雙（2: 3-璟氧丙氧基）丁烷-活化的瓊 I 脂膠之共價接合。第3A及3B圖像為電子旋轉共振光譜，其分別顯示出4-(2-溴乙醯胺基）-了£[^0及3-馬來醯亞胺-?1?(^几至3,5-雙一本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) -裝· k h^866 經濟部中央標準局員工消費合作社印製 A7 B7 i'發明説明（）溴甲矽基-雙反式丁烯二酸（DBBF)交聯的血紅素或二阿斯匹靈交聯的血紅素之成功的共價接合。第4A圖偽為4 - (2-溴乙醯胺基）- TEMPO之一 ESR光譜。第4B圖偽為4- (2-溴乙醯胺基）-TEMPO標識的HBOC之一 ESR 光譜。第4C圖偽為在室溫下記錄的，配於乳酸化林格氏液 (Lactated Ringer’s solution)内之 15NDi?-TEMPOL 之一 ESR光譜。第5圖傺為帶有不同的氮氧化物對Hb莫耳比之4- (2-溴乙醯胺基）-TEMPO標識的HBOC之一 ESR光譜。第5A圔2:1 ; 第5B圖4:1;以及第5C圖8:1。由第5A圖至第5B圖以至第 5C圖，儀器敏感度予以成比例地降低俾以記錄光譜值，因而其中央波峰會顯示出具有類似的波峰高度。第6圖像為由4- (2-溴乙醯胺基）-TEMPO標識的HBOC與 HND^-TEMPO所構成的混合物之一 ESR光譜，其中前者之 •中央波峰（參見向下箭號）以及後者之高視野波峰（參見向上箭號）會被調整至相似的強度。此為源自第4B圖與第4C 圖的ESR光譜之一重疊影像。第7圔偽為4 - (2-溴乙醯胺基）- TEMPO標識的HBOC在小白鼠體内之一血漿半衰期.。第7A圖是在靜脈輸入〇.5ml的於第6圖中所示樣品後大約10分鐘，於小白鼠尾部所記錄的氮氣化物信號之ESR光譜。第7B圖是在10分鐘的儀器敏感性下所記錄的，第7A圖之中央波峰（Mo)信號強度相對於時間的降低（掃描時間30分鐘）。第7C圖像為第7B圖在其掃描結束後之一延續。本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公釐） -------—裝—^^丨訂—：—'_線 (請先閱讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製 492866 A7 B7___ 五、發明説明（) 第8圖偽為一由4- (2-溴乙醯胺基）-TEMPO標識的HBOC (8g/dl的 Hb 以及 TEMP0:Hb，8:1)與 15NDw-TEMPOL所構成的混合物（在一為32g重的小白鼠，0. 5ml)，使用一套管以供立即記錄被輸入的氮氧化物，從小白鼠尾部所記錄之一血漿半衰期。樣品在注射前之ESR光譜示於第6圖中。第8A 圔傺為一条列的間隔5分鐘而記錄的5個ESR光譜，在各次掃描之間磁場強度以2Ganss之數予以增加俾以顯現出信號強度傺有如時間之一函數而被降低之。第8B圖是第8A圖之延續，其以相同的時間間隔重複記錄一条列的6個ESR光譜，惟在各次掃描之間磁場強度以2GaUSS之數予以降低之。第9A及9B圖僳為電子旋轉共振光譜，其分別顯示出4-(2-溴乙醯胺基）-TEMPO標識的、〇-棉子糖交聯的並且聚合化的人體血紅素以及與戊二醛聚合的3-馬來醯亞胺-PROXYL 標識的DBBF-血紅素。第10 A及10B圔傺為電子旋轉共振光譜，其分別顯示出含有下列物質之脂質體包囊化的人體血紅素：U)3-D0XYL-膽甾烷，（B)16-D0XYL-硬脂酸。第10C圖像為3-D0XYL-膽甾烷及16-D0XYL-硬脂酸此二者之電子旋轉共振光譜。第11圔#為以4-胺基-TEMPO予以標識並以葡聚糖予以共軛化的氮氧化物-標識的血紅素之一電子旋轉共振光譜。第12圔為一過濾器室之一實施例，該過濾器室包含一其上結合有一氮氯化物之固體基質，且一含血紅素溶液可流經其中。第13圔顯示出一大白鼠體内平均動脈壓（MAP)對於單本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁)^ 866 ^ 866 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 s ___—_ B7__ V. Description of the invention () Provides pitting or protection against oxidative stress caused by free radical chemical species. Experimental results are also provided here to confirm that the composition and method of the present invention are antihypertensive when transfused with a HBOC, so that the blood transfusion of a HBOC solution can be made vascular neutral. Radioprotection was demonstrated by exposing cell cultures and mice to lethal radiation doses. EPR imaging of heart palpitations in rats has been shown to be able to monitor the progress of ischemia and reperfusion injury, which confirms that the composition disclosed by the present invention can protect the ischemia from reperfusion injury in addition to development-enhancing properties. . Schematic overview: This scheme contains at least one color photo / development image. Copies of the color maps in this case will be provided by the Patent and Trademark Office upon request and payment of necessary fees. Figures 1A and 1B show 4-amino-TEMPO (TEMPO: 2,2,6,6-tetramethylhexahydropyridine-hydrocarbyloxy) recorded on days 1 (A) and 30 (B). Electron Rotational Resonance Spectrum of the O-raffinose polymerized heme identified. Figure 1C is the spectrum recorded on day 1 when the sample in Figure 1 A was diluted with the same volume of unlabeled heme. Figure 1D is the record of the sample in Figure 1C on day 30. The 2A and 2B images are electron rotation resonance spectra, which show the covalent bonding of 4- (2-bromoacetamido) -TEMPO to omega-aminohexyl-sepharose and 4-amino-TEMPO to 1 Covalent attachment of 4-bis (2: 3-trioxopropoxy) butane-activated agar I liposome. The 3A and 3B images are electron rotation resonance spectra, which respectively show 4- (2-bromoacetamido)-? £ [^ 0 and 3-maleimidine-? 1? (^ Several to 3 , 5-Double-one paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page)-Installed · kh ^ 866 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 i 'Invention Description () Successful covalent bonding of bromosilyl-bis-trans-butenedioic acid (DBBF) cross-linked heme or diaspirin cross-linked heme. Figure 4A Pseudo-ESR spectrum of 4-(2-bromoacetamido) -TEMPO. Figure 4B Pseudo-ESR spectrum of HBOC labeled 4- (2-bromoacetamido) -TEMPO. Figure 4C An ESR spectrum of 15NDi? -TEMPOL in Lactated Ringer's solution, recorded at room temperature. Figure 5: Molar ratio of Hb with different nitrogen oxides ESR spectrum of 4- (2-bromoacetamido) -TEMPO-labeled HBOC. 5A 第 2: 1; 5B Fig. 4: 1; and 5C Fig. 8: 1. From Fig. 5A to 5B Figure to Figure 5C, the sensitivity of the instrument is proportional Lower the tritium to record the spectral value, so its central peak will show a similar peak height. The sixth image is composed of HBOC and HND ^ -TEMPO identified by 4- (2-bromoacetamido) -TEMPO. One of the mixtures of ESR spectra, the central peak of the former (see downward arrow) and the high-field peak of the latter (see upward arrow) will be adjusted to similar intensities. This is derived from Figure 4B and 4C One of the ESR spectra of the figure is an overlay image. The 7th pseudo pseudo- 4- (2-bromoacetamido) -TEMPO-labeled HBOC has a plasma half-life in mice. Figure 7A is entered intravenously. The ESR spectrum of the nitrogen compound signal recorded on the tail of a mouse about 5 minutes after a sample of 5 ml in the sample shown in Figure 6. Figure 7B is recorded with the instrument sensitivity at 10 minutes, and the center of Figure 7A Decrease of the signal intensity of the peak (Mo) with respect to time (scanning time 30 minutes). The 7C image is a continuation of the 7B image after the end of the scan. This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -------— install — ^^ 丨 Order—: —'_ line (Please read the precautions on the back before filling this page) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 492866 A7 B7___ V. Description of the invention () Figure 8 is forged by 4- (2-bromoacetamido) -TEMPO labeled HBOC (8g / dl Hb and TEMP0: Hb, 8: 1) and a mixture of 15NDw-TEMPOL (a 32g mouse, 0.5ml), using a set of tubes for immediate The input nitrogen oxide was recorded, and one of the plasma half-lives was recorded from the tail of the mouse. The ESR spectrum of the sample before injection is shown in Figure 6. The 8A is the 5 ESR spectra recorded for a 5 minute interval in a column. The magnetic field strength is increased by 2Ganss between scans to show that the signal strength is reduced as a function of time. Figure 8B is a continuation of Figure 8A. It repeatedly records a series of 6 ESR spectra at the same time interval, but the magnetic field intensity is reduced by 2GaUSS between each scan. Figures 9A and 9B are electron rotation resonance spectra, which show 4- (2-bromoacetamido) -TEMPO-labeled, 0-raffinose-linked and polymerized human heme, and Dialdehyde polymerized 3-maleimide-PROXYL-labeled DBBF-heme. 10A and 10B10 are electron rotation resonance spectra, which respectively show liposome-encapsulated human heme containing the following substances: U) 3-D0XYL-cholestane, (B) 16-D0XYL-stearin acid. Image 10C is an electron rotation resonance spectrum of both 3-D0XYL-cholestane and 16-D0XYL-stearic acid. Eleventh # is an electron rotation resonance spectrum of one of the nitrogen oxide-labeled heme labeled with 4-amino-TEMPO and conjugated with dextran. Item 12) is an example of a filter chamber that includes a solid matrix having a nitrogen chloride bonded thereto, and a heme-containing solution can flow therethrough. Article 13 shows that the mean arterial pressure (MAP) in a rat is based on the Chinese paper standard (CNS) A4 (210X297 mm) for a single paper size (please read the precautions on the back before filling this page)

經濟部中央標準局員工消費合作社印製 492866 A7 ______B7___ 五、發明説明（）獨配於林格氏乳酸化液内的7.8g/dl l(Uv/v DBBF-Hb (虛線）之靜脈輸血的反應，以及在意識清醒的大白鼠髏内，以7,8g/dl l(Uv/v DBBF-Hb +聚氮氧化物白蛋白5g/dl + TPL lOOmM lOU/v (實線）處理之結果。容許該等大白鼠從手術及麻醉恢復，大約7後才予以進行研究賓驗。第14圖顯示靜脈注射後，大白鼠的TPL血漿濃度與時間之一相關性圖。血漿樣品係獲自於第13圖中所述的大白鼠。利用EPR旋轉密度測定來偵測TPL濃度。第15A、15B與15C圖偽為電子旋轉共振光譜，其分別顯示出·· 15A，配於50mM磷酸鈉緩衝液（pH 7. 6)内的TPL (2mM) ; 15B，配於相同的緩衝液内的TPH (2mM:);以及15C ，聚氮氧化物白蛋白（PNA)。EPR光譜計設定條件如下：微波電源，8mW ;接收器增益：1 . 00e + 03 ;調幅：0. 5G ;調頻·· ΙΟΟΚΗζ ;微波頻率：9.43GHz ;曲線寬度：200G。第16圖之柱狀圖顯示出中國倉鼠V79細胞在12 Gray幅射之下的存活部分。該等V79細胞在X-光放射之前予以預處理10分鐘。在僅使用TP Η或PN A時，未觀察到有放射保護 (其對應之柱顯示出2¾的存活率，同於未經處理的對照組者）。其對應於含有TP Η與PN A之組合的樣品之柱顯示出提高的放射保護（8¾存活率）。第17圖顯示出TPH被PNA轉化成TPL (配方指定批號為 SZ102994)。在25mM之固定濃度下的TPH被混合以配於50mM 磷酸鈉緩衝液（pH 7.6)内之增加濃度的PNA。TPL/TPΗ之比例，以相對於25roM PNA，而予以繪成圖。此比例表示轉化本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -25 - (請先閱讀背面之注意事項再填寫本頁)Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 492866 A7 ______B7___ V. Description of the invention () Response to intravenous transfusion of 7.8g / dl l (Uv / v DBBF-Hb (dashed line)) exclusively in Ringer's lactic acid solution And in a conscious rat's skull, treated with 7,8g / dl l (Uv / v DBBF-Hb + polynitrogen oxide albumin 5g / dl + TPL lOOmM lOU / v (solid line). Allowed These rats recovered from surgery and anesthesia, and were tested after about 7 days. Figure 14 shows the correlation between TPL plasma concentration and time in rats after intravenous injection. Plasma samples were obtained from No. 13 The rats described in the figure. The EPR rotation density measurement was used to detect the TPL concentration. Figures 15A, 15B, and 15C are pseudo-electron resonance resonance spectra, which respectively show 15A, formulated in 50 mM sodium phosphate buffer solution (pH 7.6) TPL (2mM); 15B, TPH (2mM :) in the same buffer; and 15C, polynitrogen oxide albumin (PNA). EPR spectrometer setting conditions are as follows: microwave power, 8mW; Receiver gain: 1. 00e + 03; AM: 0.5G; FM ·· ΙΟΟΚΗζ; Microwave frequency Rate: 9.43GHz; curve width: 200G. The histogram in Figure 16 shows the survival part of Chinese hamster V79 cells under 12 Gray radiation. These V79 cells were pretreated for 10 minutes before X-ray radiation. No radiation protection was observed when only TP Η or PN A was used (the corresponding column showed a survival rate of 2¾, as in the untreated control group). It corresponds to the combination containing TP Η and PN A The column of the sample showed increased radioprotection (8¾ survival rate). Figure 17 shows that TPH was converted by PNA to TPL (the formula specified batch number is SZ102994). TPH at a fixed concentration of 25mM was mixed to make up with 50mM phosphoric acid Increased concentration of PNA in sodium buffer solution (pH 7.6). The ratio of TPL / TPΗ is plotted relative to 25roM PNA. This ratio indicates that the Chinese paper standard (CNS) A4 specification (210X297) is used to convert the paper size. Mm) -25-(Please read the notes on the back before filling this page)

492866 A7 B7 五、發明説明（）效率。TPL濃度之測定像藉由在室溫下培育25 的TP Η以及 7種不同濃度的ΡΝΑ歷時30分鐘，隨後使用10KD膜centrocon 在50 00xg下進行分離歴時1小時。TPL在濾液内之高視野EPR 波峰強度，如所示使用TPL標準曲線來校正並予以作圖。第18圔偽為藉由在各個掃描間以大約1G之量來手動地增加視野強度，於小白鼠尾部記錄的15Μ RPR光譜之一連續記錄。在1 5N TPL的高視野波峰（Μ-1/2)上予以標示出掃描數目。該小白鼠先前被注射以〇.51111的4〇1^15^1丁？1，經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) 該TPL以一為2分鐘之半衰期被還原成ΤΡΗ (結果未示出）。一由ΡΝΑ與1 5N TPL所構成的混合物之第二次注射顯示一類似的1 5N TPL消失速率（參照在30秒之間隔，於一約為2分鐘的半衰期下，所記錄的波峰1-5)，隨後為一同樣快速的波峰強度之回復（參照波峰5-7)。TPL的波峰強度以一為13分鐘之半衰期而衰減（參照在1分鐘之間隔下所記錄的波峰7-15)。在位於1 TPL波峰的兩個分別簇群Β與Β1 (Μ +1/2以及Μ-1/2)之間所顯示出的聚氮氧化物白蛋白之中央廣共振波峰（Α)亦顯示出以一為13分鐘之半衰期而衰減。因此，1 5N TPL之使用清楚地證實了活體内從ΤΡΗ至ΡΝΑ 的旋轉轉移。第19圔顯示出於C57小白鼠體内，在有或無ΡΝ Α之存在下，TPL與TPH之藥理動力學。TPL的血漿位準[其以藉由監測EPR高視野波峰強度而測定的假定單位（arbitrary unit) 來表示]，以有如時間（分鐘）之一函數而予以作圔之。（□) 藉由靜脈内投藥的TPL (本身2mM)，（◊)藉由腹腔内投藥本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） t^«66 經濟部中央標隼局員工消費合作社印製 A7 ------ B7 ^________---- --------- I . ---------------------------------------------------------五、發明説明（）的TPL 275mg/kg，以及（〇）TPH 100mg/kg + PNA 1ml (配方指定批號為SZ102994)。第2 0圖顯示出在以下列處理後，被暴露於10 Gray幅射的C57小白鼠（每組10隻小白鼠，N = 10)之30-天存活率研究：藉由靜脈内投藥的PNA (配方指定批號為SZ102994) (0.5ml/小白鼠），接而10分鐘後給予PBS緩衝液（);0· 5 mi PBS，接而10分鐘後藉由給予腹腔内（ip)投藥200mg/kg 的TPL (·);藉由靜脈内投藥的聚氮氧化物白蛋白（0. 5ml/ 小白鼠），接而10分鐘後給予200mg/kg的TPL (♦)。第21圖顯示出在以下列處理後，被暴露於10 Gray幅射的C57小白鼠之30-天存活率研究：藉由靜脈内投藥的PNA (配方指定批號為SZ102994)(0.5rol/小白鼠），接而10分鐘後給予PBS緩衝液（ ) ; 0.5ml PBS，接而10分鐘後藉由給予腹腔内投藥200mg/kg的TPL (♦);藉由靜脈内投藥的聚氮氧化物白蛋白（0.5ml/小白鼠），接而10分鐘後給予50mg /kg的 TPL (♦卜第22A圖傺為被加入有TPL與聚氮氧化物白蛋白的大白鼠心臓的一個三維空間（25x25x25mm3) EPR顯影（全視圖）。使用在2 · 5小時的局部缺血後所得之144個突出物再建立顯影。第22B圖像為同一顯影之截斷面圖。用以獲得數據之參數如下：光譜視窗：7· 0G ;空間視窗·· 25mm ;最大梯度·· 49.3 G/cm。第23圖像為被加入有TPL與PNA的大白鼠心臓的一個EPR 顯影（25x25mm2)，其獲自於一個3-D空間顯影並出示時間本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公釐） -------丨丨叫 (請先閲讀背面之注意事項再填寫本頁) •裝. 、言 k 經濟部中央標準局員工消費合作社印製 492866 A7 B7 五、發明説明（）以為局部缺血期間之一測定（156分鐘）。用以獲得數據參數如下：10分鐘；光譜視窗：7. 0G ;空間視窗：25mm ;最大梯度：49.3 G/cm。第24圖顯示兩個局部缺血的心臓内之15N TEMPOL EPR 信號之強度相對於局部缺血間期的時間。上行顯示一個以 2mM TPL + PNA處理的心臓（豢），下行顯示一個以2roM TEMP0L 處理的心臓（)。實線傜為對所觀察到的強度數據之兩级指數適合值（double-exponential fittings) Q 半衰期分別為0 · 4分鐘、2 · 9分鐘（)和3 · 3分鐘、3 0 · 1分鐘（·）° 第25圖像為被加人有TPL + PNA (配方指定批號SZ102994) 的大白鼠心臓之橫向切片的一個2-D橫截面顯影（25x25mm2) ，以為在連續的顯影中，局部缺血期間對以數字表示的時間（分鐘：秒）之一函數。該等顯影傜獲自於3-D空間顯影。用以獲得數據之參數與第23圖者相同。第26圖像為對下列各組之冠狀動脈流的回復之一測定 ••未處理的對照組心臓（〇），以TPL 2mM處理的心臓（·），以及以PNA (4g/dl)+TPL (批號102994) 2mM處理的心臟 (▲)。將心臓引至30分鐘的整體性局部缺血，隨後再予45 分鐘的再流動。第27圖偽為在下列各組中之速率壓力産物（RPP)的回復之測定：未處理的對照組心臓（〇 )，以TPL 2mM處理的心臓（鲁），以及以 PNA (4g/dl)+TPL (批號 102994) 2mM處理的心臓（▲)。將心臓引至30分鐘的整體性局部缺血，隨後再予45分鐘的再流動。本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 1 I I I I I I I--I —J n ；--—I -Aw (請先閱讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製 492866 A7 B7 五、發明説明（）第28圔顯示一獲自於第23圖與第24圖所示的顯影，以 15^?[^岬“處理的大白鼠心臓内的了£}^01^之£?以言號強度相對於局部缺血的間期之圖。在對應的3-D空間顯影之特定位置處獲得之強度數值如下：LAD (·)，主動脈（)， LV-尖（▼)以及組織（▲)。第29圖顯示在意識清醒的大白鼠體内，對於靜脈輸入的 7.8g/dl 10%v/v DBBF-Hb + PNA 7.5g/dl + TPL lOOmM 10%v/v (n = 4)之平均動脈壓（MAP)反應。容許該等大白鼠從手術及麻醉恢復，大約7天後才予以進行研究實驗。發明的詳細説明氮氧化物係穩定的自由基，其被顯示出具有模擬超氣化物歧化酶（S0D)的抗氧化催化活性，且其存在於活體内時可與其他的物質相互作用以進行觸酶-模擬物活性。以前，氮氧化物己被用於電子旋轉共振光譜學以作為研究生物巨分子的結構及移動的特性之 ''旋轉標識〃。氮氯化物已被用來撿測反應性自由基中間産物，因其等之化學結構. 提供一具有明確界定的超精細相互反應之穩定不成對的電子。此外，氮氧化物亦被觀察出能産生酵素模仿物之作用 ;某些低分子量氮氧化物已被鑑定出可模擬超氧化物歧化酶（S0D)[A. Samuni et al·, Biol. Chem. 263:17921 (1 988 )]及觸酶[R · J · Meh 1 horn et a 1 · , Free Rad.492866 A7 B7 V. Description of the invention () Efficiency. The TPL concentration was determined by incubating 25 TP at room temperature and 7 different concentrations of PNA for 30 minutes, followed by separation using a 10KD membrane centrocon at 50 00xg for 1 hour. The high field EPR peak intensity of the TPL in the filtrate was corrected and plotted using the TPL standard curve as shown. The eighteenth pseudo is to continuously record one of the 15M RPR spectra recorded in the tail of the mouse by manually increasing the visual field intensity by approximately 1G between each scan. The number of scans is marked on the 15N TPL high field peak (M-1 / 2). The mouse was previously injected with 4.01 ^ 15 ^ 1 Ding of 0.511111? 1. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) The TPL is reduced to TPZ with a half-life of 2 minutes (results not shown). A second injection of a mixture of PNA and 15N TPL showed a similar disappearance rate of 15N TPL (refer to recorded peaks 1-5 at 30 second intervals and a half-life of about 2 minutes. ), Followed by a similarly fast peak intensity recovery (cf. peaks 5-7). The peak intensity of the TPL decays with a half-life of 13 minutes (see peaks 7-15 recorded at 1 minute intervals). The central broad resonance peak (A) of the polynitrogen oxide albumin shown between the two clusters B and B1 (M +1/2 and M-1 / 2) at the 1 TPL peak is also shown Decay with a half-life of 13 minutes. Therefore, the use of 1 5N TPL clearly confirms the rotational transfer from TPH to PNA in vivo. Figure 19 shows the pharmacokinetics of TPL and TPH in C57 mice in the presence or absence of PN A. The plasma level of TPL [represented by an arbitrary unit measured by monitoring the EPR high field peak intensity] is given as a function of time (minutes). (□) TPL by intravenous administration (itself 2mM), (◊) By intraperitoneal administration The paper size is applicable to Chinese National Standard (CNS) A4 (210X297 mm) t ^ «66 Central Bureau of Standards, Ministry of Economic Affairs Printed by employee consumer cooperative A7 ------ B7 ^ ________---- --------- I. -------------------- ------------------------------------- V. Description of the invention () TPL 275mg / kg, and (〇) TPH 100mg / kg + PNA 1ml (the formula specified batch number is SZ102994). Figure 20 shows the 30-day survival study of C57 mice exposed to 10 Gray radiation (10 mice per group, N = 10) after the following treatments: PNA administered intravenously (The designated batch number of the formula is SZ102994) (0.5ml / mouse), then PBS buffer solution () was given after 10 minutes; 0.5 mi PBS, and then 10 minutes later, 200 mg / kg was administered by intraperitoneal (ip) administration. TPL (·); Polynitrogen oxide albumin (0.5 ml / mouse) was administered intravenously, followed by administration of 200 mg / kg of TPL 10 minutes later (♦). Figure 21 shows the 30-day survival rate of C57 mice exposed to 10 Gray radiation after the following treatments: PNA (in the formulation designated batch number SZ102994) (0.5rol / mouse) by intravenous administration ), Followed by 10 minutes with PBS buffer (); 0.5ml PBS, followed by 10 minutes by intraperitoneal administration of 200 mg / kg of TPL (♦); intravenous administration of polynitrogen oxide albumin (0.5ml / mice), and then given 50mg / kg of TPL 10 minutes later (♦ Figure 22A is a three-dimensional space (25x25x25mm3) EPR of the heart of a rat with TPL and polynitrogen oxide albumin Development (full view). 144 projections obtained after 2.5 hours of ischemia were used to establish development. Image 22B is a cross-sectional view of the same development. The parameters used to obtain the data are as follows: Spectral window: 7.0G; space window 25mm; maximum gradient 49.3 G / cm. The 23rd image is an EPR development (25x25mm2) of the heart palpitations of a rat with TPL and PNA added, obtained from a 3-D Space development and presentation of time This paper size applies to Chinese National Standards (CNS) A 4 specifications (210 X 297 mm) ------- 丨丨 call (please read the precautions on the back before filling out this page) • equipment., Printed by the Consumer Cooperatives of the Central Standards Bureau, Ministry of Economic Affairs, printed 492866 A7 B7 V. Description of the invention () It is considered to be one of the ischemic periods (156 minutes). The parameters used to obtain the data are as follows: 10 minutes; spectral window: 7.0G; spatial window: 25mm; maximum gradient: 49.3 G / cm. Figure 24 shows the intensity of the 15N TEMPOL EPR signal relative to the time of the ischemic interval in two ischemic heart palpitations. The top line shows a palpitate (豢) treated with 2mM TPL + PNA, and the bottom line shows a 2roM TEMP0L Processed palpitations (). The solid lines 傜 are double-exponential fittings to the observed intensity data. Q half-lives are 0 · 4 minutes, 2 · 9 minutes (), and 3 · 3 minutes, respectively. 3 0 · 1 minute (·) ° The 25th image is a 2-D cross-section development (25x25mm2) of a transverse slice of a rat heart palpitation with TPL + PNA (recipe designated batch number SZ102994). During development, ischemia A function of the time (minutes: seconds) indicated. The developments were obtained from 3-D space development. The parameters used to obtain the data are the same as those in Figure 23. The 26th image is the coronary arteries for the following groups One of the responses to the flow was determined. • Untreated control group palpitations (0), palpitations (·) treated with TPL 2mM, and hearts treated with PNA (4g / dl) + TPL (lot 102994) 2mM (▲). Cardiac palpitations were induced to global ischemia for 30 minutes, followed by reflow for 45 minutes. Figure 27 is a measurement of the recovery of rate pressure product (RPP) in the following groups: untreated control palpitations (0), palpitations (Lu) treated with TPL 2mM, and PNA (4g / dl) + TPL (batch number 102994) 2 mM treated palpitations (▲). Cardiac palpitations were induced to global ischemia for 30 minutes, and then reflowed for another 45 minutes. This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) 1 IIIIII I--I —J n; --- I -Aw (Please read the notes on the back before filling this page) Central Ministry of Economic Affairs Printed by the Standards Bureau Consumer Cooperative 492866 A7 B7 V. Description of the Invention () 28th shows a development obtained from the developments shown in Figures 23 and 24 in the heart of a white rat treated with 15 ^? [^ Cape " The figure of the intensity of the word versus the ischemia interval. The intensity value obtained at the specific position of the corresponding 3-D spatial development is as follows: LAD (·), aorta (), LV-tip (▼) and tissue (▲). Figure 29 shows that in a conscious rat, 7.8 g / dl 10% v / v DBBF-Hb + PNA 7.5 g / dl for intravenous input + TPL 100 mM 10% v / v (n = 4) mean arterial pressure (MAP) response. The rats were allowed to recover from surgery and anesthesia, and the research experiment was carried out after about 7 days. Detailed description of the invention NOx Is a stable free radical, which has been shown to have antioxidant catalytic activity that mimics superoxide dismutase (S0D), and it exists in It can interact with other substances in the body to perform enzyme-mimetic activity. In the past, nitrogen oxides have been used in electron rotation resonance spectroscopy as a `` rotation mark '' to study the structure and movement characteristics of biological macromolecules. 〃. Nitrogen chlorides have been used to detect reactive free radical intermediates due to their chemical structure. Provide a stable, unpaired electron with a well-defined ultrafine interaction. In addition, nitrogen oxides have also been observed It can produce enzyme mimics; some low molecular weight nitrogen oxides have been identified to mimic superoxide dismutase (S0D) [A. Samuni et al ·, Biol. Chem. 263: 17921 (1 988)] Contact enzyme [R · J · Meh 1 horn et a 1 ·, Free Rad.

[_· , U_: 1 57 (1 992 )]的活性。許多的研究亦顯示可通透胞膜的氮氯化物可短期地保護哺乳類細胞對抗由次黃嘌昤/黃嘌昤氧化酶所産生超氣化物陰離子以及於過氧化氫本紙張尺度適用中國國家標準（CNS ) A4規格（210 X297公釐） (請先閱讀背面之注意事項再填寫本頁)[_ ·, U_: 1 57 (1 992)]. Many studies have also shown that nitrogen chloride, which can permeate the cell membrane, can protect mammalian cells against supergaside anions produced by hypoxanthine / xanthine oxidase in the short term, and for hydrogen peroxide, this paper applies Chinese National Standard (CNS) A4 Specifications (210 X297 mm) (Please read the notes on the back before filling in this page)

492866 A7 B7 五、發明説明（）暴露所引起之細胞中毒。在此所使用的 ''氮氧化物〃一詞是描述穩定的氮氣化物自由基、其前驅物（例如N-H形式）及其衍生物[包含其相應的羥基胺衍生物（N-OH)，其中該氧原子被代之以一羥基，且以一鹵化氫形式而存在]。對本發明的目的而言，該等羥基胺衍生物之氯鹽形式是較為適宜的。在此所述的氮氧化物中.，該不成對的電子偽部分地安定的，因為其氮原子核被連接至二痼磺原子，該等磺原子被強烈的電子供給者所取代的。對位於N -0鍵結的氧原子上之部分負電荷而言，該二値鄰近的碩原子一起將該不成對的電子定位在該氮原子核上。氮氧化物通常可具有一雜環或是直鐽結構。其基礎標準是一個穩定的自由基。在結構上，具有下列結構的氮氧化物是較佳的，其中Ri-R 4為電子供給者，而A為一雜環之基餘成員。 (請先閱讀背面之注意事項再填寫本頁) -裝--- 經濟部中央標準局員工消費合作社印製 ό 1 ·492866 A7 B7 V. Description of the invention () Cell poisoning caused by exposure. The term `` nitrogen oxide '' is used herein to describe stable nitrate radicals, their precursors (such as the NH form), and their derivatives [including their corresponding hydroxylamine derivatives (N-OH), where This oxygen atom is replaced by a hydroxyl group and is present as a hydrogen halide]. For the purposes of the present invention, the chloride salt form of these hydroxylamine derivatives is preferred. In the nitrogen oxides described herein, the unpaired electrons are pseudo-stable because their nitrogen nuclei are connected to disulfonium atoms, which are replaced by strong electron donors. For a portion of the negative charge on the oxygen atom at the N-0 bond, the two adjacent mega atoms together locate the unpaired electron on the nitrogen nucleus. The oxynitride may generally have a heterocyclic or straight-chain structure. The basic criterion is a stable free radical. Structurally, a nitrogen oxide having the following structure is preferable, wherein Ri-R 4 is an electron donor and A is a residual member of a heterocyclic ring. (Please read the precautions on the back before filling out this page)-Equipment --- Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs ό 1 ·

在這些雜環結構中，”Α”代表一痼5-員璟（0¾咯烷基或具一雙鍵的PROXY，亦即吡咯啉）或是6-員的六氫吡啶基或 1.1—訂 J--.------------------- 本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公釐） 492866 A7 B7 五、發明説明（ TEMPO)雜環結構之其餘的磺原子，且其中一磺原子可被取代以一個氧原子唑啉基或DO XYL)以及某些氫可為至多二個溴原子所取代。在該等雜環結構中，可使用穩定的同位素（例如1 5 N及氘）。位在ct碳上的取代必須使得該不成對的電子大體上被維持在一 τι P軌域結構上。R 1以至R 4為烷基（直鏈或支鏈）或芳基，但其較佳者為甲基或乙基。在任一種氮氧化物中，位在其α碳上的取代基必須為強烈的電子供給者以增加安定性，因此甲基（CH 3)或乙基（C 2 Η 5) 是較為適宜的，雖然其它較長的碳鏈物種亦可被使用之。依據本發明，當被連接至生物可相容的巨分子時，氮氧化物的反應力會因微環境之故而被改變之。此反應力可藉由所使用的標識計劃以及與其他已知會改變自由基穩定性或反應力的化合物（例如硒）之反應，而被裁製之。在實施上，立體結構上的考量可能會限制實用且經濟的氮氧化物化合物的範圍。供本發明之使用的較佳氮氧化物包含具有下列結構的氮氧化物： ----------裝------—訂 J—； —線 (請先閲讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製 CH. -〇 0 CH3v^\CH3 Ch/T2 β ΓΟΗ. ch3 CH, 0 I· 2 β CHa 、CH3 3 4 5 一 31 — 本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公釐） 492866 A7 B7 五、發明説明（） DOXPL (4,4-二甲基-3 - _ 唑烷基氧基-) (4,4-二甲基噁唑烷烴氧基） PROXYL (2,2,5,5-四甲基-1_ 吡咯烷基氧基-) (2,2,5,5-四甲基吡咯院-N-煙氧基） TEMPO (2,2,6,6-四甲基-1-六氫吡啶基氧基-) ί2,2,6,6-四甲基六氫 Pit淀-您氧基） (請先閲讀背面之注意事經濟部中央標準局員工消費合作社印製由上述可明顯得知，大多數適合的氮氧化物化合物基本上可以用下列化學式來代表之： R N - 0 設若該R基係擇自於可維持該自由基的安定性之結構中。已有各種不同的技術描述將一氮氧化物共價接合至一生物巨分子（包含血红素、白蛋白、免疫球蛋白以及脂質體）上。參照 M c C ο η n e 1 1 e t a I. , Quart. Rev* Biophvs. 3_：91 (1979)； Hamilton et al., "Structural Chemistry and Molecular Biology, ,T A. Rich et a 1 . , eds. W.H. Freeman, San Francisco, ρ·115 (1968); Griffith et a 1 · , Acc · Chem · Res . 2_: 1 7 (1968) ； Smith I · C · P . ’’Biological Application of Electron Spin Resonance Spectroscopy, TT Swartz, Η·Μ· et a 1 . , eds., Vi ley/ Interscience, New York P.483 (1972)。選用的氮氧化物己被共價地接合至血紅素分子以供研究血紅素的互助式氧氣結合機制之用。關於此處所述之巨分子，至少有兩種可行的方法，即通常被稱之為vv標識計策〃者，可將氮氧化物結合至一巨 4 ，項再填. 裝-- 寫本頁) 本紙張尺度適用中國國家標準（CNS ) A4規格（210x297公羡 ) -32 - 492866 (請先閲讀背面之注意事項再填寫本頁)In these heterocyclic structures, "A" represents a 5-membered fluorene (0¾rolidinyl or PROXY with a double bond, that is, pyrroline) or a 6-membered hexahydropyridyl or 1.1- --.------------------- This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm) 492866 A7 B7 V. Description of the invention (TEMPO ) The remaining sulfonic atom of the heterocyclic structure, and one of the sulfonic atoms may be substituted with one oxygen atom oxazoline group or DO XYL) and some hydrogens may be substituted with up to two bromine atoms. In such heterocyclic structures, stable isotopes (such as 15 N and deuterium) can be used. The substitution at the ct carbon must be such that the unpaired electrons are generally maintained in a τι P orbital structure. R 1 to R 4 are alkyl (straight or branched) or aryl, but preferred is methyl or ethyl. In any kind of nitrogen oxide, the substituent on the α carbon must be a strong electron donor to increase stability, so methyl (CH 3) or ethyl (C 2 Η 5) is more suitable, although Other longer carbon chain species can also be used. According to the present invention, when connected to a biocompatible macromolecule, the reactivity of nitrogen oxides is changed due to the microenvironment. This reactivity can be tailored by the labeling scheme used and the reaction with other compounds (such as selenium) known to alter the stability or reactivity of free radicals. In practice, structural considerations may limit the range of practical and economical nitrogen oxide compounds. Preferred nitrogen oxides for use in the present invention include nitrogen oxides having the following structure: ---------- install -------- order J--; line (please read the first Note: Please fill in this page again.) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs. -〇0 CH3v ^ \ CH3 Ch / T2 β ΓΟΗ. Paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) 492866 A7 B7 V. Description of the invention () DOXPL (4,4-dimethyl-3-_ azolidinyloxy-) (4, 4-dimethyloxazolidinyloxy) PROXYL (2,2,5,5-tetramethyl-1_pyrrolidinyloxy-) (2,2,5,5-tetramethylpyrrole-N- Nicotinyloxy) TEMPO (2,2,6,6-tetramethyl-1-hexahydropyridyloxy-) ί2,2,6,6-tetramethylhexahydro Pit-Yoxy) (please First read the notice on the back. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. From the above, it is obvious that most suitable nitrogen oxide compounds can be basically represented by the following chemical formula: RN-0 From the structure that can maintain the stability of this radical. The same technical description covalently attaches a nitrogen oxide to a biological macromolecule (including heme, albumin, immunoglobulin, and liposomes). Refer to Mc C ο η ne 1 1 eta I., Quart. Rev * Biophvs. 3_: 91 (1979); Hamilton et al., &Quot; Structural Chemistry and Molecular Biology,, T A. Rich et a 1., Eds. WH Freeman, San Francisco, ρ · 115 (1968); Griffith et a 1 ·, Acc · Chem · Res. 2_: 1 7 (1968); Smith I · C · P. '' Biological Application of Electron Spin Resonance Spectroscopy, TT Swartz, Η · Μ · et a 1., eds., Vi ley / Interscience, New York P. 483 (1972). The selected nitrogen oxides have been covalently bonded to the heme molecule for the purpose of studying the mutual oxygen-binding mechanism of heme. Regarding the macromolecules described here, there are at least two feasible methods, commonly known as the vv identification strategy, which can combine nitrogen oxides with a giant 4, and then fill in the items. Loading-write this page ) This paper size applies to Chinese National Standard (CNS) A4 (210x297) -32-492866 (Please read the precautions on the back before filling this page)

A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明 ( ) 分子上〇特定的標識法之重要性係視氮氣化物所要結合的巨分子中之微環境以及氮氧化物由此而形成的催化活性。位在 —· 特定的配位子結合位處之特殊的標 m -會産生一具有一更一致的結合微璟境之均質産物以及因此該氮氧化物的催化專一性及活性更為穩定的化合物〇除非在本文之描述中另有待別提出 9 在此所用的N'血紅素 // 一詞通常是指氧基 -' 、羧基- 一氧化 m 基 -及去氯_ 血红素〇本發明中所使用的血红素可為人類的重組型或為動物來源者並係藉由已知的技術而獲得及純化者。該血紅素可耢由醛基與交聯的血红素衍生物反應，而被共價地接合至吡哆 -5 ? _ 磷酸酯中的吡哆 m 基團或開璟的腺嘌昤三磷酸 (0 -ATP)上( 3該等交聯的衍生物可包含多官能基的雜雙官能基的及同雙功能基的交聯試劑 > 如二醛、聚 m 、二璟氧化物、聚環氣化物、活化的聚羧基基團與二羧基基團 > 例如 3, 5- 雙 -溴甲硅基-雙反式丁烯二酸及 TEMPO 丁二酸或 TOPS (參見美國專利第4,240 ,797號 )， 1環糊精及其陰離子 (例如硫酸）交聯的血紅素及聚合化血紅素。所有在此描述的用於本發明之血红素溶液是生理上可相容的。該血紅素溶液是不含細胞的俾以去除埶原内毒素及其它污染物〇合併使用氮氣化物與人類血清白蛋白動物或重組型白蛋白的較佳組成物包括有： 1) 白蛋白與氮氧化物之非-專- -性標識[例如，在高氮氧化物對白蛋白比下之 4- (2 -溴乙醯胺基） -TEMPO]；本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公釐） 492866 經濟部中央標準局員工消費合作社印製 A7 B7五、發明説明（） 2) 白蛋白在特殊的配位子結合位置處之專一性標識； 3) 白蛋白藉由雙硫鍵的還原與烷化之增進的專一性標識。因此而被使用的白蛋白可予以溫度或化學處理，俾以增加可用的標識位置。另外，白蛋白可如同一單元體、二元物、聚合物而存在，或可被包在微球體内。一用以藉由增加身體的抗氣化能力來減輕氧化壓力之較佳的技術是，使用多重組份之以氮氧化物為主的条統。一第一組份是一種膜可通透的氮氧化物，例如TEMPOL。藉由其電荷特徵以及小型分子尺寸，低分子量之未結合氮氧化物可容易地穿透細胞膜並進入細胞内間隙。一第二組份是另一種含氮氧化物物種，例如一被標識以高莫耳比例的氮氧化物（聚氮氧化物）之生物可相容的巨分子，例如被標識以一為30:1莫耳比例的TEMPOL之人類血清白蛋白。本發明之一多重組份氮氧化物条統的使用，會幫助減輕由一低分子量膜可通透的氮氧化物之大量、濃縮的或重複劑量所引起之毒性。由於氮氧化物的羥基胺形式在作為一抗氧化劑是無活性的，且因為高劑量下之氮氧化物毒性被認為主要是由於抗氧化活性造成細胞的氯化還原狀態之混亂所致，羥基胺狀態展現出遠較其相應的未還原的氮氧化物為低的毒性。因此本發明之一實施例敘述一膜可通透的氮氧化物，合併以一巨分子聚氮氣化物，之一無毒性劑量的使用，俾以於活體内活化一已被還原成一無活性形式之氮氣化物。以類似的方式，該羥基胺可如同一在活體内會被轉化 (請先閲讀背面之注意事 4 項再填· 裝— 寫本頁) 訂A7 B7 Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of the invention () The importance of the specific labeling method on molecules is based on the microenvironment in the macromolecules to which nitrogen compounds are to be bound and the nitrogen oxides formed by them Catalytic activity. A special standard m located at a particular ligand binding site will produce a homogeneous product with a more consistent binding microenvironment and therefore a more stable catalytic specificity and activity of the nitrogen oxide 〇 Unless otherwise mentioned in the description herein 9 The term N'heme // as used herein generally refers to oxy- ', carboxyl-moxide- and dechloro-heme. The heme used can be a recombinant human or an animal origin and is obtained and purified by known techniques. The heme can be reacted covalently to a pyridine m group in pyrido-5? -Phosphate or an open adenine triphosphate by reacting an aldehyde group with a cross-linked heme derivative ( 0-ATP) (3 These cross-linked derivatives may contain polyfunctional heterobifunctional and homobifunctional crosslinking reagents> such as dialdehydes, polym, dioxane, polycyclic rings Gases, activated polycarboxyl groups and dicarboxyl groups > such as 3, 5-bis-bromosilyl-bis-trans-butenedioic acid and TEMPO succinic acid or TOPS (see U.S. Patent No. 4,240,797 No.), 1 cyclodextrin and its anions (such as sulfuric acid) cross-linked heme and polymerized heme. All the heme solutions described herein for use in the present invention are physiologically compatible. The heme solution It is cell-free, which removes protoendotoxin and other pollutants. Combined use of nitrogen compounds and human serum albumin Preferred compositions of animal or recombinant albumin include: 1) Non-specific --- identical identification of albumin and nitrogen oxides [eg, 4- (2-bromoethyl at high nitrogen oxide to albumin ratio) (Amine-based) -TEMPO]; This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm) 492866 A7 B7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention () 2) Albumin Specific identification at specific ligand binding positions; 3) Specific identification of albumin through the reduction and alkylation of disulfide bonds. The albumin that is used can be temperature or chemically treated to increase the number of available markers. In addition, albumin may exist as the same unit, binary, polymer, or may be encapsulated in a microsphere. A better technique to reduce oxidative stress by increasing the body's ability to resist gasification is to use a system consisting mainly of nitrogen oxides. A first component is a membrane that is permeable to nitrogen oxides, such as TEMPOL. With its charge characteristics and small molecular size, low molecular weight unbound nitrogen oxides can easily penetrate the cell membrane and enter the intracellular space. A second component is another nitrogen oxide species, such as a biocompatible macromolecule that is labeled with a high mole ratio of nitrogen oxides (polynitrogen oxides), such as labeled with a 30: 1 mol of TEMPOL in human serum albumin. The use of multiple nitrogen oxide strips in one of the inventions will help alleviate the toxicity caused by large, concentrated or repeated doses of nitrogen oxides that are permeable to a low molecular weight membrane. Because the hydroxylamine form of nitrogen oxides is inactive as an antioxidant, and because the toxicity of nitrogen oxides at high doses is considered to be mainly due to the chaos of the chloride reduction state of cells caused by antioxidant activity, hydroxylamines The state exhibits much lower toxicity than its corresponding unreduced nitrogen oxide. Therefore, one embodiment of the present invention describes a membrane that is permeable to nitrogen oxides, combined with a macromolecular polynitrogen compound, and used at a non-toxic dose, which is activated in vivo-which has been reduced to an inactive form Nitrogen compounds. In a similar way, the hydroxylamine can be transformed in vivo as the same (Please read the note on the back 4 items before filling and packing — write this page)

II 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）經濟部中央標準局員工消費合作社印製 492866 A7 B7 五、發明説明（）成一有活性的抗氧化劑之無毒的氛氧化物前驅物，而被投藥之。其結果是身體内形成一有力的ί/Ι氧化劑之一安全且恆續的治療位準。關於活體内的安全性，其可依據本發明而被投藥的氮氧化物之位準在動物體内耐受良好，且被預期在人體内亦是耐受良好，因為已知道氮氧化物是相當安全的。例如， TEMPO在小白鼠體内之最高耐受腹腔内劑量傺為275mg/kg ，而LD5Q像為341mg/kg。更甚者，一巨分子-結合的氮氧化物會比其活性形式的自由的氮氧化物要安全得多。與自由的氮氧化物合併使用的本發明之氮氣化物-標識的會減少其或有必要被投藥以達到一抗氧化作用的氮氧化物之總量。在抗氧化劑内配方内使用的氮氧化物-標識的巨分子之一增加的優點在於有能力逹到高活性位準的呈其具有改善的安全性之有活性的抗氯化劑形式之氮氧化物。現今於活生物體内所研究的大多數氮氧化物傺為相當低分子量的化合物，其等可容易地穿透過細胞膜進入身體組織内。本發明之巨分子-結合的氮氧化物可被靜脈内輸入，並可因該巨分子物種的膜可通透性而維持在血管區域内。在該一實施例中，一被共價地接合至一巨分子之氮氧化物會産生減輕自由基毒性的作用，並同時將該氮氧化物限定在一位置（亦即血管區域）處，於該處其實用性達最佳化〇 * 當TEMP0L被注入時，其快速地擴散至細胞内間隙内，於該處其在解毒（氯化）自由基之過程中被還原成羥基胺形本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） (請先閲讀背面之注意事項再填寫本頁)II This paper size applies to China National Standard (CNS) A4 (210X297 mm) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 492866 A7 B7 V. Description of the invention () A non-toxic precursor of toxic oxide with active antioxidant Material, and was administered. The result is a safe and consistent treatment level in the body, which is one of the powerful I / I oxidants. Regarding safety in vivo, the level of nitrogen oxides that can be administered according to the present invention is well tolerated in animals, and is expected to be well tolerated in humans, as it is known that nitrogen oxides are equivalent safe. For example, the highest tolerable intraperitoneal dose of TEMPO in mice is 275 mg / kg, while the LD5Q image is 341 mg / kg. What's more, a macromolecule-bound nitrogen oxide is much safer than its active form, free nitrogen oxide. The nitrogenate-labeled compounds of the present invention used in combination with free nitrogen oxides will reduce the total amount of nitrogen oxides that they may or need to be administered to achieve an antioxidant effect. One of the added advantages of nitrogen oxide-labeled macromolecules used in antioxidant-based formulations is their ability to reach high levels of nitrogen oxidation in the form of an active anti-chlorination agent with improved safety Thing. Most of the nitrogen oxides that are studied today in living organisms are relatively low molecular weight compounds that can easily penetrate through cell membranes and enter body tissues. The macromolecule-bound nitrogen oxide of the present invention can be input intravenously and can be maintained in the vascular region due to the membrane permeability of the macromolecular species. In this embodiment, a nitrogen oxide that is covalently bonded to a macromolecule will have the effect of reducing the toxicity of free radicals, and at the same time, the nitrogen oxide is limited to a position (that is, a blood vessel region), at Its practicability is optimized there. * When TEMP0L is injected, it quickly diffuses into the intercellular space, where it is reduced to hydroxylamine-shaped paper in the process of detoxifying (chlorinated) free radicals. Standards are applicable to China National Standard (CNS) A4 specifications (210X 297 mm) (Please read the precautions on the back before filling this page)

492866 經濟部中央標隼局員工消費合作社印製 A7 B7 i、發明説明（）式。藉由其高膜可通透性與惰性化學主幹，該羥基胺亦可自由地分佈於細胞内間隙及細胞外間隙中，並在身體内唯持一段相當長的時間。但是，一旦該氮氧化物被還原成羥基胺即喪施其抗氧化活性。 TEMPOL 4-羥基-2,2,6,6-四甲基六氫吡啶-1}-羥氧基在解毒自由基之過程中被快速地消耗掉；其被還原成一氧代鉸中間産物，該中間産物可被氣化回氮氯化物或進一步被還原成一羥基胺。因此，氮氧化物的生物轉化（在自由基之解毒過程中）形成一種羥基胺。該羥基胺基不是順磁性的（其在ESR光譜學中是不活動的），並且缺乏氮氧化物的抗氧化催化活性。在治療上僅使用TEMPOL不被偏好，因為其會被快速地轉化成羥基胺，且能在要達到一有意義的抗氧化作用之所需的劑量位準下僳為有毒的。但是，羥基胺在化學上是穩定的，且在身體内相當地持久（其氮氧化物分子之主幹相當地無活性），且依據本發明之教示，可被化學地轉化回氮氧化物之活性形式。此活體内轉化使得氮氧化物之安全的臨床使用以提供一維續的抗氧化活性，成為可能。由於其高分子量及低膜可通透性之故，一巨分子聚氮氧化物有被分佈於細胞細外間隙之傾向，切不容易在生化璟境内被還原之。但是，已發現到，巨分子聚氮氧化物能夠將一個電子轉移至羥基胺，造成活體内轉化回一帶有活性抗氧化能力的氮氣化物。此方法有效地將抗氣化能力從細胞外之一抗氧化活性之高能力巨分子儲庫，轉移至其可本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） ---------裝— (請先閲讀背面之注意事項再填寫本頁) 訂經濟部中央標隼局員工消費合作社印製 A7 B7 i'發明説明（）橫跨細胞膜之高移動性膜可通透的氮氯化物上，俾以於細胞内提供抗氧化活性。一旦位於細胞内，該氮氧化物藉由氧化有毒的自由基被還原成羥基胺，並接而循環回至細胞外間隙，於該處被巨分子聚氮氧化物再活化之°更甚者’ 該巨分子聚氮氧化物之反應力可藉由增加其他諸如硒之化合物而被增強之。 _ . 次又，當一高莫耳比之一氮氧化物被結合至一巨分子 (聚氮氧化物），且被容許於活體内與未結合的低分子量氮氧化物接觸時，可以製備一持別有利的含氮氧化物配方。此二種物種之相互作用在周圍組織内提供一維續的抗氧化力。事實上，該巨分子物種像為一種抗氧化活性之貯庫，其能對可穿透胞膜的低分子量物種再充予活性。此處所掲露的這個共生方策提供了有利的投藥方法，諸如一巨分子氮氧化物之局部投藥與一低分子量氮氧化物之口服投藥的組合，俾以提供局部化的、維續的抗氧化活性。依據此處所展現的實驗數據，本發明的含氮氧化物物種之抗氧化活性，已在活髏外及活體内觀察到。依據這些結果，讓氮氧化物-標識的HBOC可參預自由基的氧化/還原反應之反應機轉，證實了配製用於一廣範圍的生理狀況之診斷與治療之新穎HBOC化合物以及其他的氮氧化物-標識的巨分子之可能性。 _ 此外，本發明描述被締合至一容器以供用於諸如靜脈流體、局部試劑等等之藥物的儲藏及投藥之氮氧化物。含氮氣化物化合物可呈固體或液體形式被添加至一容器之内本紙張尺度適用中國國家標隼（CNS ) A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁)492866 A7 B7 printed by the Consumer Cooperatives of the Central Bureau of Standards, Ministry of Economic Affairs i. Invention Description (). With its high membrane permeability and inert chemical backbone, the hydroxylamine can also be freely distributed in the intracellular and extracellular spaces, and only for a considerable period of time in the body. However, once the nitrogen oxide is reduced to hydroxylamine, its antioxidant activity is lost. TEMPOL 4-hydroxy-2,2,6,6-tetramethylhexahydropyridine-1} -hydroxyoxy group is quickly consumed in the process of detoxifying free radicals; it is reduced to a monooxo hinge intermediate product, which Intermediate products can be gasified back to nitrogen chlorides or further reduced to monohydroxyamines. Therefore, the biological transformation of nitrogen oxides (in the process of detoxification of free radicals) forms a hydroxylamine. The hydroxylamine group is not paramagnetic (it is inactive in ESR spectroscopy) and lacks the antioxidant catalytic activity of nitrogen oxides. The use of TEMPOL alone in therapy is not preferred because it is rapidly converted to hydroxylamine and can be toxic at the dose levels necessary to achieve a meaningful antioxidant effect. However, hydroxylamine is chemically stable and relatively durable in the body (its backbone of nitrogen oxide molecules is quite inactive), and according to the teachings of the present invention, it can be chemically converted back to nitrogen oxide activity form. This in vivo conversion makes it possible for safe clinical use of nitrogen oxides to provide one-dimensional continuous antioxidant activity. Due to its high molecular weight and low membrane permeability, a macromolecular polynitrogen oxide tends to be distributed in the fine outer space of the cell, and it is not easy to be reduced in the biochemical region. However, it has been found that macromolecular polynitrogen oxides can transfer an electron to hydroxylamine, causing the living body to convert back to a nitrogen compound with active antioxidant capacity. This method effectively transfers the anti-gasification ability from a high-capacity macromolecular depot with an antioxidant activity outside the cell to its paper size applicable to the Chinese National Standard (CNS) A4 specification (210X 297 mm) --- ------ Equipment— (Please read the precautions on the back before filling in this page) Order the A7 B7 i 'invention description printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs () Permeable nitrogen chloride is used to provide antioxidant activity in cells. Once in the cell, the nitrogen oxide is reduced to hydroxylamine by oxidizing toxic free radicals, and then circulates back to the extracellular space, where it is reactivated by the macromolecular polynitrogen oxide. The reactivity of the macromolecular polynitrogen oxide can be enhanced by adding other compounds such as selenium. _. Once again, when a high mole ratio of nitrogen oxide is bound to a macromolecule (polynitrogen oxide) and allowed to contact in vivo with unbound low molecular weight nitrogen oxide, a Hold other favorable nitrogen oxide formula. The interaction of these two species provides one-dimensional continuous antioxidant power in the surrounding tissue. In fact, the macromolecular species acts as a reservoir of antioxidant activity, which recharges low molecular weight species that can penetrate the cell membrane. The symbiotic strategy disclosed here provides advantageous methods of administration, such as a combination of topical administration of a macromolecular nitrogen oxide and oral administration of a low molecular weight nitrogen oxide to provide a localized, sustained antioxidant active. According to the experimental data presented here, the antioxidant activity of the nitrogen oxide-containing species of the present invention has been observed outside of living skulls and in vivo. Based on these results, the nitrogen oxide-labeled HBOC can participate in the reaction mechanism of free radical oxidation / reduction reactions, confirming novel HBOC compounds formulated for the diagnosis and treatment of a wide range of physiological conditions and other nitrogen oxidation -The possibility of identifying giant molecules. In addition, the present invention describes nitrogen oxides that are associated to a container for storage and administration of drugs such as intravenous fluids, topical agents, and the like. Nitrogen compounds can be added to a container in solid or liquid form. This paper is sized for China National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back before filling this page)

492866 A7 B7__ 五、發明説明（）部或可被共價地接合至一容器之内部。一有關投藥之有利的技術是，在有或無自由的氮氣化物下，將含氮氧化物化合物添加至一用於流體之投藥的在管線中過濾器。例如，一聚氮氧化物白蛋白可以，加上自由的氮氧化物，被併入至一過濾器内，或被接合至一被安裝在一過濾器内之不溶性基質上，該過濾器被用於諸如現有的HBOC之靜脈内流體投藥，俾以在流體被輸入一病人體内之前，清除掉該流體内存有之與氧有關的毒性化合物。此處所述之HRCS配方與氮氧化物-標識的巨分子可減缓與氧有關的毒性物種之形成，此像藉由令一氮氧化物發揮有如一 $超氧化物歧化酶〃之功能，一已知不會發生红血球細胞内的酵素般反應。在這些HRCS配方中，氮氧化物防止由血紅素的自我氧化所産生的非所欲之超氯化物陰離子的蓄積（參照式[1 ])。該等氮氧化物-標識的巨分子，諸如白蛋白與免疫球蛋白，同樣地發揮有如抗氧化酵素模擬物之功能，其等之功能被維持定位在血管及間隙區域内，且其等可與膜可通透的氮氧化物反應以提供細胞内保護。經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 合併使用氮氧化物與免疫球蛋白之較佳的組成物包括一被結合至一對一半抗原或抗原具專一性的免疫球蛋白之氮氧化物-標識的半抗原或抗原。更甚者，依據本發明，氮氣化物化合物之有利的治療作用可被控制及維續之。例如，氮氧化物2 , 2 , 6 , 6 -四甲基-1 -羥氣基-4-哌啶叉琥珀酸（TOPS)可被結合至人類血清白蛋白之主要膽紅素結合位。在活體内，此結合延長了血清本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公釐） 492866 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（）半衰期並顯示出氮氣化物至細胞内間隙之擴散，這降低了所需之劑量（而因此減低潛在的毒性）並延長生物作用。因此，雖然僅有氮氧化物本身（諸如TOPS)，在無一巨分子聚氮氣化物之下，其可具有作為一抗氧化試劑之用途。依據本發明，選擇或設計其可輸送氮氧化物至身體内之特定位置處的載體，俾以作為一用以定位治療性抗氧化活性之工具，是可行的。有鑒於氮氧化物之穩定的化學性質，此處所掲露的組成物可藉由各種不同的途徑被投藥之。該膜可通透的氮氧化物可被腸道外地或口服地投藥之。在其還原形式，羥基胺可在G I条統内局部地發揮作用，或被攝入血液内。因此，在身體所有區域裡，維續的抗氧化活性能被提供之。該巨分子聚氮氧化物可被腸道外地投藥，於該處其留在細胞外間隙内以便再活性被還原的自由基，或可被口服地或局部地/經皮地投藥，於該處其發揮活化循環性、被還原的氮氧化物之作用，因而提供一局部化的抗氧化作用。一以蛋白質為主的巨分子聚氮氧化物與一膜可通透的氮氧化物之待別的反應力，似乎可藉由加熱該巨分子以及於其多胜呔鏈之一级胺基處的標識，而被增進之。已知加熱會改變巨分子之空間結構，拉伸介於胺基與羧基基團之間的氫鍵，並造成該巨分子的四維結構被改變。隨後在胺基基團處的氮氣化物之共價標識，可能於該蛋白質其因加熱而被暴露出的相當内部之位置處發生。在所形成的氮氧化物-標識的巨分子中，這些氮氣化物部分更可免於與溶本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) • 士 n^i 11-1 I 1 I ,〆^im in In - - - - i ! ml i ·· i-ιϋ mu ml ^^^1 . 492866 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（）劑起反應。又，當氮氧化物被接合至該蛋白質上的許多胺基時，所餘的羧基基圍之優勢産生一環繞該結合的氮氣化物之酸性微環境。一酸性環境藉由將位在N -0鍵内之不成對的電子拉向該氧原子，而增加了該氮氧化物之反應力。在本發明針對一紅血球代用品之實施例中，該等氮氧化物之必要性質是，其藉由模擬超氧化物歧化酶以及觸酶活性（其在該過程中大體上不會被消耗掉），來影響HRCS内的超氧化物陰離子串聯（cascade)的過程之能力。在 ''超氧化物氧化酶〃反應中，超氧化物陰離子被氧化變回氣分子，而不會走向過氣化氫的形成。此部分地伴隨著産生一儲存狀況，其中氮氣化物之濃度遠超過於氣的濃度。使用在此所掲露的方式，氮氧化物可防止非所欲之氯化反應的串聯，該等氧化反應造成超氧化物陰離子之開始形成。再者，在此所述之生理上可相容的HRCS溶液可提供比現有的HBOC溶液更佳的優點，因此處所述之該等配方在被輸入病人體内後，該氣氧化物會模擬超氧化物歧化酶及觸酶酵素活性。 ‘ 雖然一廣範圍種類的氮氧化物可被用於本發明，該氮氧化物在一減輕氧氣毒性所需之最低濃度下，應為生理上可接受的。在評估一可實施的物種時，值得注意的是，氮氧化物之相當低的毒性有助於其被發展成為NMR顯影中的對比劑（參見美國專利第4,834,964、4,863,727、5,104, 641號）。熟於此項技藝人士已知道許多用來分離及純化其為生本紙張尺度適用中國國家標準（CNS ) Α4規格（210X297公釐） 1-------裝---_---.丨訂 J--Γ--線 (請先閲讀背面之注意事項再填寫本頁) 492866 經濟部中央標準局員工消费合作社印製 A7 B7 五、發明説明（）理上可相容的血紅素溶液之方法。典型地，經純化的血紅素組成物含有至少99¾血紅素（以總蛋白質量計），一低於約3/ig/ml的磷脂質含量，低於Ug/ml的磷脂醯絲胺酸或磷脂醯乙醇胺，以及一低於6¾的無活性血紅素色素。其可用於本發明之純化的血红素溶液，可使用各種傳統技術來製備，這包含但不限於，C h e u n g e t a 1. , Anal. Boichem. 13.7 -48 1 -484 (1984)、De Venuto et al·, J . Clin, f I p d 509-516 (1977)及 Lee et al., Vith International Symposium on Blood Substitutes, San Diego, CA Mar. 17-20 Abstract H51 (1993) ° 使用於本發明之純化的血紅素溶液，基本上應是不含氧的。溶液中的血紅素可藉由與一化可造成令血紅素釋出氧並使之保持在一實質去氧化狀態下的化學還原劑摻合而將之去氧化。將血紅素溶液去氧化之一較佳方法是藉由將該血紅素溶液暴露在一惰性的、且大體上不含氧氣的氣體 (例如氮氣或一氧化碳）中，而予以進行的，俾以造成從血红素上移除掉結合的氧，同時將位在該溶液中的血红素轉變成一呈無氧之形式者，諸如去氧-血红素或一氯化碩基-血紅素。任擇地，血紅素可經由一對氧具通透性但對血紅素則否之膜，被暴露在一真空或是氣體中。例如，可令一血红素溶液流經一具有一膜壁的擴散室，藉此血紅素沿著該膜流動’而氧分子可通過該膜但血紅素則否。惰性氣體沿著相對於血紅素溶液的膜壁之側邊循環，而造成氧的去除以及溶液内的血紅素成為去氧化狀態之轉化。較佳地，本紙張尺度適用中國國家標準（CNS ) A4規格（21〇X297公釐） 41 ^—ϋ··- mm^nm ϋΒϋ —ί I ml —ϋ ϋ_11 ·ϋϋ mu immmamj Βιϋ-*J, τϋΜ— In. mi ·ϋι - * _ 备 (請先閱讀背面之注意事項再填寫本頁) 492866 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（）在氮氧化物標識化、交聯化、聚合化或共軛化之期間，該去氧-血紅素被維持在一大體上無氧的環境下。在任何結合的氧之移除後，一氮氧化物被共價地接合至該血紅素上。通常是至少一個，且較佳的是一個以上的氮氧化物被共價地接合至一單個血紅素分子上。該氮氧化物可於該血紅素分子的數個位置處被共價接合至血紅素，該等位置包含： (a) 在一或多個自由的巯基（-SH)處，例如在-93 位置處。 (b) 在任一具反應性的胺基（-NH2)處，例如在/3鏈的 Val-Ι和/或/S鏈的離胺酸-82和/或α鐽的離胺酸-99處之DPG位置處； (c) 在任一非-專一性的表面胺基（NH2)或羧基（COOH) 處0 一氮氧化物亦可被結合至在血紅素之交聯與聚合時所涉及之任一種具有一個二價-或三價-聯結子之殘餘的醛、環氧基或是活化的羧基基團，或是被用於共軛化血红素之一試劑[如葡聚糖（Dx)或羥乙基澱粉（HES)或是聚氧化乙烯 (POE)]上的殘餘的具有活性之基團。如前述式[1]中所述的，在儲存期間内，HBOC溶液内的血紅素被氧氣緩慢地自我氧化而形成氧化-血紅素以及超氧化物陰離子。但是，在HRCS (其為本發明之標的）的儲存期間，由此所形成的超氧化物陰離子會將氮氧化物還原成一羥基胺衍生物，且該超氧化物陰離子會經由下式反本紙張尺度適用中國國家標準（CNS ) Α4規格（210X297公釐） ^n- m_i —ϋ im —ϋ- ϋϋ nw mi ϋϋ ι_^ϋ ϋι« ilaj mi TJ τϋ· ian mi 0ml (請先閲讀背面之注意事項再填寫本頁) A7 B7 i、發明説明（）應而被氧化以形成氧分子： H+ + R N-0 + *〇2- -----> R N-OH + 〇2 [4] 式[4]所述的超氧化物陰離子至氧分子之轉化可防止超氧化物陰離子的聚集以及其後的過氣化物之形成。當該組合物内之最初氣含量被維持在一最低值時，在此被敘述為 ''超氧化物氧化酶〃活性的這値活性會是最有效的，該組合物被維保持在一大體上無氧的環境内，且該氮氧化物的濃度足夠防止超氧化物陰離子及過氧化物的形成。因此，H RCS在一大體上無氧的容器内之儲存，是較為適宜的。可容許一溶液被儲存在一無氧的環境内之容品条統，偽為已詳知者，因為許多非-血红素為主的靜脈溶液對氧氣是敏感的。例如，可使用一種在瑱充及密封過程中被淨除掉氧氣的玻璃容器。又，亦可使用其可被封裝於一用以作抗氧密封的外包裝紙（overwrap)内之可撓性塑膠容器。基本上，任何可防止氧與溶液中的血紅素反應之容器，皆可被使用之。為證實一氮氧化物之 > 超氧化物氧化酶〃的活性，將配於溶液内的氮氧化物標識的血紅素之樣品維持在一加速的氧化儲存狀況内，並藉由電子旋轉共振光譜學來研究該氮氧化物的氧化還原狀態歷時一段時間。例如，一個被標識以4-胺基-TEMPO的棉子糖聚合化血紅素溶液，呈其氧化狀態被儲存於一密封的玻璃容品内（第1 A圖）。在該一狀本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公董） (請先閲讀背面之注意事項再填寫本頁) -裝 ---- 經濟部中央標準局員工消費合作社印製 ——1T//,------------------- 經濟部中央標準局員工消費合作社印製 492866 A7 _B7__ 五、發明説明（）態下，溶液内的超氧化物陰離子及氧化-血紅素的形成速率速率有夠快而使得氮氧化物至其羥基胺衍生物的轉化可被方便地監測之（參見式[4]及比較第1A與1B圔）。式[4]表示氮氧化物至其抗磁性羥基衍生物之轉化，被偶合以該超氧化物陰離子變回氧分子之轉化。支持該一轉化的實驗證據被示於第1A及1B圔中。被共價地接合至血紅素之TEMPO 的電子旋轉共振光譜（第1 A圖）被轉化成其抗磁性的衍生物，這造成該樣品在4°C下儲存30天後，其共振峰消失完全的（第1B圖）。當其在血红素之存在下被轉化成其羥基胺衍生物時，該氮氯化物被認為已執行一有如 '"超氧化物氣化酶〃活性。一 HBOC溶液内的氮氣化物之 ''超氧化物歧化酶〃活性，是藉由顯示出該羥基胺衍生物回至一氮氧化物之再轉化而予以證實的（參見式[4]及[5])。當知曉在第1A及1B圖所述的實驗條件下，氮氧化物被完全地轉化成羥基胺（參見式[4])時，，該氮氧化物可如式[5]所示的，經由僅提供更多的超氧化物陰離子而被再生之。為證實此反應機制，血紅素（及由此所形成的超氧化物陰離子）相對於氮氧化物的濃度，藉由以等體積之未標識的血红素來稀釋第1 A圖中的樣品，而被增加之。第1A及1C圖之一比較顯示出，因該稀釋效用而造成該氮氧化物的信號強度降低了約50%。另一方面，在30天的4¾低溫儲存後，如式[5]所預期的，該氮氣化物被部分地再生之（參見第1D圖）。此觀察符合下列二反應之偶合：該羥基胺衍生物回至氮氯化物之再轉化以本紙張尺度適用中國國家標準（CNS)A4規格（ 210X297公釐）-44 - ----—-------^---^ 訂 --丨 — -線 (請先閱讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製 492866 A7 B7_______ 五、發明説明（) 及源自超氧化物陰離子的過氧化氫之形成。 Η + + * 0 2 ~ + R Ν ~ Ο Η----今 Η 2 0 2 + R N_0 [5] 式[4]及[5]相加得到 2 ·〇2 - + 2Η+ ------今 0 2 + Η2〇2 這證實該氮氧化物在”HBOC”溶液内産生有如一低分子量、無金屬的SOD模擬物之作用。氮氧化物的電子旋轉共振光譜之檢測結果，與超氣化物陰離子和羥基胺（R N-0H)的反應相吻合，該反應造成氮氧化物（R P0)及過氯化氫（H2〇2) 的形成。近來，氧代銨陽離子被推論其在氮氧化物催化的超氧化物之技化作用中，有如一中間産物而涉及其中° [參見 Krishna e t a 1 . , P r o c > Natl· Acad · Sc i · USA 8_2_· 5537-5541 (1992)]- 此處所述的HRCS配方可減輕於現有的HOBC溶液内超氧化物陰離子的生成所造成的氧化壓力，且在輸血時’可減少一氧化氮，内皮-衍生的鬆弛因子（E D R F )，的破壞。若能防止EDRF的破壞，當現有的HBOC溶液被輸入至一病人體内所觀察到的血管收縮及全身性高血壓之問題，即可被實質地減輕之。每一血红素分子之氮氧化物的數目可在大約1至40之範圍内，而對專一性標識而言，以大約為2是最為適宜者。但是，由於藥物動力學、毒性學以及免疫學上的考量，本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） —- m -ϋ^— -- - - - l - - τϋ ...... - ----- (請先閱讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製 492866 A7 B7 五、發明説明（）氮氧化物-血紅素比應予以保持在最低值。例如，一諸如卜馬來醯亞胺-PROXYL的氮氧化物，藉由首先製備一個 mM的配於乙醇中的氮氧化物溶液以作為載體溶液，而被共價地接合至溶液内血紅素上。將2莫耳當量的氮氧化物對血紅素直接予以混合添加至一配於乳酸化林格氏液中的 DCL-Hb (8g/dl)内。容許該混合物在22 °C及攪拌下反應，直至高於90¾的氮氧化物被共價地接合至該DCL-Hb，這通常需時1小時。接而使用一具有分子量截留值為3000道爾頓的橫流（cross-f Ιον)膜過濾条統，藉由以10倍體積的乳酸化林格氏緩衝溶液清洗三次，而去除掉未反應的氮氣化物。將滯留物血紅素濃度調整為7-14g/dl，予以無菌過濾並保存在4°C下。在輸血後，當HRSC被完全地氧化，該氮氧化物被預期係發揮有如一個S0D模擬物及其次，一觸酶模擬物之作用。作為一値S0D模擬物，其將該超氧化物陰離子歧化成為過氧化氫（參見式[2])，且其後可保護對抗内皮中的一氧化氮的破壞以預防血管收縮。作為一個觸酶模擬物，其可經由將過氧化氫轉化成為無害的水而防止過氧化氫毒性。（參見式[3])。如上所述，氮氧化物已被共價地結合至血紅素以研究血紅素分子本身的互肋型氣氣結合待性。但是，氮氯化物並未被用於其為生理上可相容的穩定化（也就是交聯的）或聚合化、包囊化或共軛化血紅素溶液。已知的血紅素及氮氧化物之化學暗示，可以藉由選擇一位在血紅素上且未被用來穩定化、聚合化、或共軛化血紅素分子之特殊化合物本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公釐） (請先閲讀背面之注意事項再填寫本頁) 裝· J. 經濟部中央標準局員工消費合作社印製 492866 A7 B7__ 1、發明説明（）所堵塞的可用位置，來進行其已被化學地交聯或經由重組技術而交聯的血紅素之類似的氮氧化物標識。由於某些下列所述的穩定化及聚合化形式之血紅素，目前已涉及臨床試驗，下面詳細說明氮氧化物至此等穩定化及聚合化之以血紅素為主的氧載體之接合，俾以驗證本發明的氧氣解毒功能適用於現有的血紅素溶液者。此處所例示的氮氧化物-標識技術，在氮氣化物-HBOC 之例中，被容易地引用於製造其他具有有用的抗氣化及酵素模擬物活性之氮氧化物-標識的巨分子，例如，氮氯化物-標識的血清白蛋白以及氮氧化物-標識的免疫球蛋白。藉由此技術，其可被氮氧化物容易地標識之血清白蛋白的形式偽為單體型（正常的）白蛋白以及白蛋白同質二聚物、寡聚物以及聚集物（微球體）以及各者之多胜呔片段。此處所述之一氮氧化物-標識的血紅素或其他適當的巨分子之抗氧化酵素模擬物作用，對任一需要一穩定的自由基的應用中，具有實用性。由於應用上的差異，此處所述之配方可以被分別地或合在一起地使用之。例如，在心臓再灌損害的治療與診斷中，希望能夠利用本發明的數個方面之優點，亦卽氧氣輸送、免於氧化壓力之全身性保護、免於氣化壓力之局部化保護以及增強的顯影。在這樣的例子中，此處所述的配方之一組合可被用來作為諸如一現有的HBOC、氮氣化物-標識的白蛋白以及一低分子量氮氣化物，其等可視治療或診斷目的所需，而被同時地或依序地投藥之。因此，下列詳本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -· {· ·ϋϋ —ϋ ffm —In ϋϋ ϋϋΜ —ι_ι ϋϋ mi —IJ TJ rn ϋϋ —a^i (請先閱讀背面之注意事項再填寫本頁) 492866 A7 B7 五、發明説明（）細掲露數種可以任一種組合來應用之配方。實施例1 一含有氮氧化物與氮氧化物-標識的化合物之容器及過濾器本發明在不化學地修飾現有的配方下’可對現有的靜脈内溶液（諸如HBOC溶液）提供氧氣解毒功能。同時在該組成中不需作該血紅素之化學修飾。藉由包含一可與一自由的氮氧化物合併使用之聚氮氧化物，或藉由將氮氧化物共價地接合至一内儲有HBOC的容器之内部表面’在現有的配方中所觀察到的，由氧氣毒性所引起的不利生理作用可被減缓之。經濟部中央標準局員工消費合作社印製 -装---Γ-- (請先閱讀背面之注意事項再填寫本頁) 與含血红素溶液（其為本發明之標的）一起使用的容器必需是生理上可相容的，且具有類似於靜脈流體所使用的容器之性質。典型地，玻璃或一生物可相容的塑膠是合適的。關於本發明的實施例，其中一靜脈溶液被置放於一容器内歴時一段時間，該容器必須是無氯的且可被密封以排除氧。對一玻璃容器而言，一傳統的塞子或封閉裝置卽足夠了。但是，一些現今可用的可撓性塑膠容器像為氧可通透的。在此情況下，一鋁箔外包裝紙或其他的密封機構可被使用以防止氣與溶液中的血紅素接觸° 為將氮氣化物施用至一容器的内部表面上，將一氮氯化物聚合物或一添加有氮氣化物的共聚物之一未瀝濾層直接塗覆於該内部表面。含氮氧化物聚合物可藉由許多依據一般已知的原理或聚合反應之技術，來進行製造，只要該自由基的穩定性在該聚合反應程序中可被保持之。本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）經濟部中央標準局員工消費合作社印製 492866 A7 _ _B7__ 五、發明説明（）又，一個HBOC容器的内部可被修飾成含有一可與一氮氧化物結合的物質之一塗層，諸如其可與一氮氧化物之酮或醛基起反應而形成穩定的腙衍生物之親水性醯肼基團。該塗覆反應是直接進行的。例如，配於ί>Η 5 · 0之乙酸緩衝液中的氮氧化物（1〇〇fflM)被加入至一醯肼活化的塑膠容器内以促進一腙鍵結之形成。當製備好該容器後，予以加入一生理上可相容的溶液。此溶液可為一穩定化及純化的HBOC或此處所掲露的HRCS ，但亦包含任一想要與血红素共輸血的靜脈膠態或晶體溶液。該溶液而後被保持在一大體上無氣的璟境内。除了處理一容器的内部表面外，一具有一活塞（luer) 鎖入口及出口且含有一可使氮氧化物固定於其上的膠質或基質之過濾器型藥室，當血紅素溶液流經該藥室時，可被使用以去除具有活性之氯氣衍生的反應性物種。對於此一應用而言，可將一聚氮氧化物巨分子添加至該過濾器之殼罩内，於是一溶液可在通經其間以直接輸入至病人體内之前，先與該聚氮氧化物起反應—低分子量氮氧化物亦可被包含之。在這些應用中，於輸送之前，氮氧化物可被結合至一軟性或硬性凝膠基質上，藉此發揮大體上有如一無菌的管線内過濾器之作用。各種將小型配位子（諸如氮氣化物）接合至一固體基質上之方法，偽為親和力層析技術中已詳知者，就如用以化學地修飾玻璃及塑膠表面之該等技術一樣。其與無菌溶液傺為可相容的數種型式的基質，已知包含有瓊脂糖凝膠、以聚礙為主的物質、乳膠等等。本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁)492866 A7 B7__ 5. The description of the invention () may be covalently joined to the inside of a container. An advantageous technique for dosing is to add a nitrogen-containing oxide compound to a in-line filter for dosing of fluids with or without free nitrogen. For example, a polynitrogen oxide albumin, plus free nitrogen oxides, can be incorporated into a filter, or joined to an insoluble matrix mounted in a filter, the filter being used Administration of intravenous fluids, such as existing HBOCs, removes oxygen-related toxic compounds present in the fluid before it is introduced into a patient. The HRCS formulation and nitrogen oxide-labeled macromolecules described here can slow the formation of toxic species related to oxygen. This effect is to make a nitrogen oxide function like a superoxide dismutase, A known enzyme-like reaction in red blood cells does not occur. In these HRCS formulations, nitrogen oxides prevent the accumulation of undesired superchloride anions produced by the self-oxidation of heme (see formula [1]). The nitrogen oxide-labeled macromolecules, such as albumin and immunoglobulin, also function as antioxidant enzyme mimetics, and their functions are maintained in the blood vessels and interstitial regions, and they can interact with The membrane is permeable to nitrogen oxides to provide intracellular protection. Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page). A better composition that combines nitrogen oxides and immunoglobulins includes a pair of antigens or antigens Nitrogen oxide-labeled haptens or antigens of specific immunoglobulins. Furthermore, according to the present invention, the beneficial therapeutic effects of the nitrogen compound can be controlled and maintained. For example, nitrogen oxides 2, 2, 6, 6-tetramethyl-1 -hydroxyl-4-piperidineidenesuccinic acid (TOPS) can be bound to the main bilirubin binding site of human serum albumin. In vivo, this combination extends the serum size of this paper to the applicable Chinese National Standard (CNS) A4 specifications (210 X 297 mm) 492866 A7 B7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention () Half-life and display Diffusion of nitrogen compounds into the intercellular space reduces the required dose (and therefore reduces potential toxicity) and prolongs biological effects. Therefore, although there is only nitrogen oxide itself (such as TOPS), it can be used as an antioxidant without a macromolecular polynitride. In accordance with the present invention, it is feasible to select or design a carrier that can deliver nitrogen oxides to specific locations in the body as a tool for locating therapeutic antioxidant activity. In view of the stable chemical nature of nitrogen oxides, the composition disclosed here can be administered in a variety of ways. The membrane's permeable nitrogen oxide can be administered parenterally or orally. In its reduced form, hydroxylamine can act locally in the G I system or be taken into the blood. Therefore, continuous antioxidant activity can be provided in all areas of the body. The macromolecular polynitrogen oxide can be administered outside the intestine, where it remains in the extracellular space for reactivation of reduced free radicals, or it can be administered orally or topically / percutaneously, where It exerts the effect of activating cyclic, reduced nitrogen oxides, thus providing a localized antioxidant effect. It seems that the reaction between a protein-based macromolecular polynitrogen oxide and a membrane permeable nitrogen oxide can be heated by heating the macromolecule and its first-order amine group The logo was promoted. It is known that heating will change the spatial structure of the macromolecule, stretch the hydrogen bond between the amine group and the carboxyl group, and cause the four-dimensional structure of the macromolecule to be changed. Subsequent covalent identification of the nitrogen compound at the amine group may occur at a relatively internal position of the protein which is exposed by heating. Among the formed nitrogen oxide-labeled macromolecules, these nitrogen compounds are more exempt from the dissolution of the paper. The Chinese National Standard (CNS) A4 specification (210X297 mm) is applied (please read the precautions on the back first) (Fill in this page) • Taxi n ^ i 11-1 I 1 I, 〆 ^ im in In----i! Ml i ·· i-ιϋ mu ml ^^^ 1. 492866 Staff Consumer Cooperatives, Central Standards Bureau, Ministry of Economic Affairs Printing A7 B7 5. Description of the invention () The agent reacts. Also, when nitrogen oxides are attached to many amine groups on the protein, the remaining advantages of the carboxyl group create an acidic microenvironment surrounding the bound nitrogen compound. An acidic environment increases the reactivity of the nitrogen oxide by pulling unpaired electrons located within the N-0 bond toward the oxygen atom. In an embodiment of the present invention directed to a red blood cell substitute, the necessary properties of the nitrogen oxides are that they mimic superoxide dismutase and catalase activity (which will not be substantially consumed in the process) To affect the ability of cascades of superoxide anions in HRCS. In the "superoxide oxidase" reaction, the superoxide anion is oxidized back to gas molecules without moving towards the formation of hydrogen gas. This part is accompanied by a storage condition, in which the concentration of nitrogen compounds far exceeds the concentration of gas. Using the method disclosed here, nitrogen oxides can prevent undesired chlorination reactions in series, which cause the formation of superoxide anions. Furthermore, the physiologically compatible HRCS solutions described herein can provide better advantages than existing HBOC solutions. Therefore, after the formulations described herein are introduced into a patient, the aerosol will mimic Superoxide dismutase and catalase enzyme activity. ‘Although a wide variety of nitrogen oxides can be used in the present invention, the nitrogen oxides should be physiologically acceptable at the minimum concentration required to reduce the toxicity of oxygen. When evaluating a viable species, it is worth noting that the relatively low toxicity of nitrogen oxides has helped it develop into a contrast agent in NMR development (see U.S. Patent Nos. 4,834,964, 4,863,727, 5,104,641). Those skilled in this art have already known that many of the paper sizes used to separate and purify their living papers are applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 1 ------- install ---_--- . 丨 Order J--Γ-- line (Please read the notes on the back before filling this page) 492866 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention Solution method. Typically, the purified heme composition contains at least 99¾ heme (based on total protein mass), a phospholipid content below about 3 / ig / ml, and a phospholipid serine or phospholipid below Ug / ml.醯 ethanolamine, and an inactive heme pigment below 6¾. It can be used in the purified heme solution of the present invention and can be prepared using a variety of conventional techniques, including but not limited to C heungeta 1., Anal. Boichem. 13.7 -48 1 -484 (1984), De Venuto et al · , J. Clin, f I pd 509-516 (1977) and Lee et al., Vith International Symposium on Blood Substitutes, San Diego, CA Mar. 17-20 Abstract H51 (1993) ° Purified red blood for use in the present invention The solution should be essentially oxygen-free. Heme in solution can be deoxidized by blending it with a chemical reducing agent that causes it to release oxygen and maintain it in a substantially deoxidized state. One of the preferred methods for deoxidizing a heme solution is by exposing the heme solution to an inert and substantially oxygen-free gas (such as nitrogen or carbon monoxide), thereby causing The bound oxygen is removed from the heme, and at the same time the heme in the solution is converted into an anaerobic form, such as deoxy-heme or mesyl chloride-heme. Alternatively, heme may be exposed to a vacuum or a gas through a membrane that is permeable to oxygen but not to heme. For example, a heme solution can be caused to flow through a diffusion chamber with a membrane wall, whereby heme flows along the membrane 'and oxygen molecules can pass through the membrane but heme is not. The inert gas circulates along the side of the membrane wall opposite to the heme solution, causing the removal of oxygen and the conversion of the heme in the solution into a deoxidized state. Preferably, the paper size is in accordance with Chinese National Standard (CNS) A4 (21 × 297mm) 41 ^ —ϋ ··-mm ^ nm ϋΒϋ —ί I ml —ϋ ϋ_11 · ϋϋ mu immmamj Βιϋ- * J, τϋΜ— In. mi · ϋι-* _ Preparation (Please read the precautions on the back before filling out this page) 492866 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention () During cross-linking, polymerization, or conjugated, the deoxy-heme is maintained in a substantially anaerobic environment. After the removal of any bound oxygen, a nitrogen oxide is covalently attached to the heme. Usually at least one, and preferably more than one nitrogen oxide is covalently attached to a single heme molecule. The nitrogen oxide may be covalently bonded to the heme at several positions on the heme molecule, the positions comprising: (a) at one or more free thiol (-SH) positions, such as at the -93 position Office. (b) At any of the reactive amine groups (-NH2), for example, at / 3-chain Val-1 and / or / S-chain lysine-82 and / or α 鐽 lysine-99 At the DPG position; (c) at any non-specific surface amine (NH2) or carboxyl group (COOH). 0-nitrogen oxides can also be bound to any of the involved in the cross-linking and polymerization of heme A residual aldehyde, epoxy, or activated carboxyl group with a divalent or trivalent linker, or a reagent used to conjugate heme [such as dextran (Dx) or Residual active groups on hydroxyethyl starch (HES) or polyethylene oxide (POE)]. As described in the aforementioned formula [1], during storage, the heme in the HBOC solution is slowly and automatically oxidized by oxygen to form oxidized-heme and superoxide anion. However, during storage of HRCS (which is the subject of the present invention), the superoxide anion thus formed will reduce the nitrogen oxide to a monohydroxyamine derivative, and the superoxide anion will pass through the paper through the following formula The scale applies to the Chinese National Standard (CNS) Α4 specification (210X297 mm) ^ n- m_i —ϋ im —ϋ- ϋϋ nw mi ϋϋ ι_ ^ ϋ ϋι «ilaj mi TJ τϋ · ian mi 0ml (Please read the precautions on the back first Fill out this page again) A7 B7 i. Description of the invention () It should be oxidized to form oxygen molecules: H + + R N-0 + * 〇2- ----- > R N-OH + 〇2 [4] The conversion of the superoxide anion to an oxygen molecule as described in the formula [4] can prevent the aggregation of the superoxide anion and the subsequent formation of an overgas. When the initial gas content in the composition is maintained at a minimum value, the activity described herein as `` superoxide oxidase activity '' will be most effective, and the composition is maintained at a substantial level. In an oxygen-free environment, the concentration of the nitrogen oxide is sufficient to prevent the formation of superoxide anions and peroxides. Therefore, it is more suitable for H RCS to be stored in a substantially oxygen-free container. A container system that allows a solution to be stored in an anaerobic environment is assumed to be well known because many non-heme-based intravenous solutions are sensitive to oxygen. For example, a glass container may be used in which oxygen is purged out during filling and sealing. Also, a flexible plastic container which can be enclosed in an overwrap for oxygen-resistant sealing can also be used. Basically, any container that prevents the reaction of oxygen with the heme in solution can be used. In order to confirm the activity of superoxide oxidase 氮, a sample of heme labeled with nitrogen oxide in the solution was maintained in an accelerated oxidative storage condition, and the electron rotation resonance spectrum was used. It took a while to study the redox state of this nitrogen oxide. For example, a raffinose polymerized heme solution labeled 4-amino-TEMPO is stored in a sealed glass container in its oxidized state (Figure 1A). In this case, the paper size applies the Chinese National Standard (CNS) A4 specification (210X297) (please read the precautions on the back before filling this page)-installed ---- printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs ——1T //, ------------------- Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 492866 A7 _B7__ V. Description of the invention () The formation rates of superoxide anions and oxidized heme are fast enough that the conversion of nitrogen oxides to their hydroxylamine derivatives can be easily monitored (see formula [4] and compare 1A and 1B 圔). Formula [4] shows the conversion of nitrogen oxide to its diamagnetic hydroxy derivative, which is coupled with the conversion of the superoxide anion back to the oxygen molecule. Evidence supporting this transformation is shown in Sections 1A and 1B. The electronic rotational resonance spectrum (Figure 1A) of TEMPO covalently bonded to heme was converted to its diamagnetic derivative, which caused the sample to completely disappear after its storage at 4 ° C for 30 days. (Figure 1B). When it is converted into its hydroxylamine derivative in the presence of heme, the nitrogen chloride is considered to have performed as "" superoxide gasification enzyme 〃 activity. The "superoxide dismutase" activity of nitrogen compounds in a HBOC solution was confirmed by showing the hydroxylamine derivative to retransform to a nitrogen oxide (see formulas [4] and [5 ]). When it is known that under the experimental conditions shown in Figures 1A and 1B, the nitrogen oxides are completely converted into hydroxylamine (see formula [4]), the nitrogen oxides can be expressed by formula [5] via Only more superoxide anions are provided and regenerated. To confirm this reaction mechanism, the concentration of heme (and the superoxide anion formed thereby) with respect to nitrogen oxides was diluted by diluting the sample in Figure 1 A with an equal volume of unlabeled heme. Increase it. A comparison of one of Figures 1A and 1C shows that the signal strength of the nitrogen oxide is reduced by about 50% due to the dilution effect. On the other hand, after 30 days of low-temperature storage at 4¾, as expected by formula [5], the nitrogen compound was partially regenerated (see Figure 1D). This observation is in accordance with the following two reaction coupling: the hydroxylamine derivative is converted back to nitrogen chloride, and the Chinese national standard (CNS) A4 specification (210X297 mm) is applied to this paper scale -44------------- ----- ^ --- ^ Order-丨--line (please read the notes on the back before filling out this page) Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 492866 A7 B7_______ V. Description of Invention () and Formation of hydrogen peroxide from superoxide anions. Η + + * 0 2 ~ + R Ν ~ Ο Η ---- present Η 2 0 2 + R N_0 [5] Adding formulas [4] and [5] gives 2 · 〇2-+ 2Η + --- --- Today 0 2 + Η202. This confirms that the nitrogen oxides in the "HBOC" solution act like a low molecular weight, metal-free SOD mimic. The detection results of the electronic rotating resonance spectrum of nitrogen oxides are in agreement with the reaction of the superoxide anion and hydroxylamine (R N-0H), which causes the nitrogen oxide (R P0) and hydrogen perchloride (H2O2) to react. form. Recently, oxoammonium cations have been deduced to be involved in the technical effects of nitrogen oxide-catalyzed superoxides as an intermediate product [see Krishna eta 1., Proc > Natl · Acad · Sc i · USA 8_2_ · 5537-5541 (1992)]-The HRCS formulation described here can reduce the oxidative stress caused by the generation of superoxide anions in the existing HOBC solution, and can reduce nitric oxide and endothelium during blood transfusion. -Destruction of derived relaxation factor (EDRF). If the destruction of EDRF can be prevented, the problems of vasoconstriction and systemic hypertension observed when an existing HBOC solution is input into a patient's body can be substantially reduced. The number of nitrogen oxides per heme molecule can be in the range of about 1 to 40, and for specific identification, about 2 is the most suitable. However, due to pharmacokinetic, toxicological, and immunological considerations, this paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) —- m -ϋ ^ —----l--τϋ. .....------ (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 492866 A7 B7 V. Description of the invention () Nitrogen oxide-heme ratio It should be kept to a minimum. For example, a nitrogen oxide such as bulimalaimine-PROXYL is covalently bonded to the hemoglobin in the solution by first preparing a mM nitrogen oxide solution in ethanol as a carrier solution. . Heme was directly mixed with 2 mole equivalents of nitrogen oxide and added to a DCL-Hb (8g / dl) formulated in lactated Ringer's solution. The mixture is allowed to react at 22 ° C with stirring until nitrogen oxides above 90¾ are covalently bonded to the DCL-Hb, which usually takes 1 hour. A cross-f membrane membrane system with a molecular weight cutoff of 3000 Daltons was subsequently used. The unreacted nitrogen was removed by washing three times with a 10-fold volume of lactated Ringer's buffer solution. Compound. The retentate hemoglobin concentration was adjusted to 7-14 g / dl, sterile filtered and stored at 4 ° C. After HRSC is completely oxidized after blood transfusion, the nitrogen oxide is expected to function as a SOD mimetic and secondarily, an enzyme mimetic. As a SOD mimetic, it disproportionates this superoxide anion into hydrogen peroxide (see formula [2]), and thereafter protects against the destruction of nitric oxide in the endothelium to prevent vasoconstriction. As a catalase mimetic, it prevents hydrogen peroxide toxicity by converting hydrogen peroxide into harmless water. (See formula [3]). As mentioned above, nitrogen oxides have been covalently bound to heme to study the interribbed gas-binding properties of the heme molecule itself. However, nitrogen chlorides have not been used for physiologically compatible stabilization (i.e., cross-linked) or polymerized, encapsulated, or conjugated heme solutions. Known chemical meanings of heme and nitrogen oxides can be obtained by selecting a special compound on heme that is not used to stabilize, polymerize, or conjugate the heme molecule. Standard (CNS) A4 size (210 X 297 mm) (Please read the precautions on the back before filling out this page). J. Printed by the Consumer Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 492866 A7 B7__ 1. Description of Invention () The clogging is available for similar nitrogen oxide identification of heme that has been chemically crosslinked or crosslinked via recombinant technology. Due to some of the stabilized and polymerized forms of heme described below, clinical trials are currently involved. The following describes in detail the bonding of nitrogen oxides to such stabilized and polymerized heme-based oxygen carriers. It is verified that the oxygen detoxification function of the present invention is applicable to existing heme solutions. The nitrogen oxide-labeling technology exemplified here is easily cited in the example of the nitrogen compound-HBOC to make other nitrogen oxide-labeled macromolecules with useful anti-gasification and enzyme mimic activity, for example, Nitrogen chloride-labeled serum albumin and nitrogen oxide-labeled immunoglobulin. With this technique, the form of serum albumin, which can be easily identified by nitrogen oxides, is pseudo-haplotype (normal) albumin and albumin homodimers, oligomers and aggregates (microspheres), and Each one has more wins. One of the nitrogen oxide-labeled heme or other appropriate macromolecular antioxidant enzyme mimics described herein is useful in any application that requires a stable free radical. Due to application differences, the formulations described herein can be used separately or together. For example, in the treatment and diagnosis of palpitations and reperfusion damage, it is desirable to be able to take advantage of several aspects of the present invention, as well as oxygen delivery, systemic protection from oxidative stress, localized protection from vaporized pressure, and enhancement. Development. In such an example, a combination of the formulations described herein can be used such as an existing HBOC, nitrate-labeled albumin, and a low molecular weight nitrate, which can be required for therapeutic or diagnostic purposes, They are administered simultaneously or sequentially. Therefore, the following detailed paper sizes apply to the Chinese National Standard (CNS) A4 specification (210X297 mm)-· {· · ϋϋ —ϋ ffm —In ϋϋ ϋϋΜ —ι_ι ϋϋ mi —IJ TJ rn ϋϋ —a ^ i (please Read the notes on the back and fill in this page) 492866 A7 B7 V. Description of the invention () There are several kinds of formulas that can be applied in any combination. Example 1 A container and a filter containing nitrogen oxides and nitrogen oxide-labeled compounds The present invention can provide an oxygen detoxification function to an existing intravenous solution (such as HBOC solution) without chemically modifying the existing formula. At the same time, no chemical modification of the heme is required in the composition. Observed in existing formulations by including a polynitrogen oxide that can be used in combination with a free nitrogen oxide, or by covalently bonding the nitrogen oxide to the internal surface of a container containing HBOC Thus, the adverse physiological effects caused by oxygen toxicity can be mitigated. Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs-Packing --- Γ-- (Please read the notes on the back before filling this page) The container used with the hemoglobin solution (which is the subject of the present invention) must be Physiologically compatible and similar in nature to containers used for intravenous fluids. Typically, glass or a biocompatible plastic is suitable. With regard to embodiments of the present invention, where an intravenous solution is placed in a container for a period of time, the container must be chlorine-free and can be sealed to remove oxygen. For a glass container, a conventional stopper or closure is sufficient. However, some of the flexible plastic containers available today appear to be oxygen permeable. In this case, an aluminum foil wrapper or other sealing mechanism can be used to prevent gas from coming into contact with the heme in the solution. To apply a nitrogen compound to the inner surface of a container, a nitrogen chloride polymer or An unleached layer of a nitrogenated copolymer was applied directly to the interior surface. Nitrogen-containing oxide polymers can be manufactured by a number of techniques based on generally known principles or polymerization, as long as the stability of the free radicals can be maintained during the polymerization process. This paper size is in accordance with Chinese National Standard (CNS) A4 (210X297 mm). Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. A coating of a substance that can bind to a nitrogen oxide, such as a hydrophilic hydrazine group that can react with a ketone or aldehyde group of a nitrogen oxide to form a stable hydrazone derivative. The coating reaction proceeds directly. For example, nitrogen oxides (100 fflM) in an acetic acid buffer solution of &> Η5.0 are added to a hydrazine-activated plastic container to promote the formation of a hydrazone bond. When the container is prepared, a physiologically compatible solution is added. This solution can be a stabilized and purified HBOC or HRCS as disclosed herein, but it also contains any intravenous colloidal or crystal solution that is intended to be co-transfused with heme. The solution was then held in a substantially airless plutonium. In addition to treating the inner surface of a container, a filter-type pharmacy with a luer lock inlet and outlet and containing a gelatinous or matrix to which nitrogen oxides can be fixed, when the heme solution flows through the In the pharmacy, it can be used to remove reactive chlorine-derived reactive species. For this application, a polynitrogen oxide macromolecule can be added to the housing of the filter, so a solution can be contacted with the polynitrogen oxide before passing through it for direct input into the patient. React—Low molecular weight nitrogen oxides can also be included. In these applications, nitrogen oxides can be bound to a soft or hard gel matrix before delivery, thereby acting as a bacteria-free in-line filter. Various methods for joining small ligands (such as nitrogen compounds) to a solid substrate are assumed to be well-known in affinity chromatography techniques, as are those techniques for chemically modifying the surfaces of glass and plastic. It is compatible with several types of matrices of sterile solutions. It is known to contain agarose gels, substances that are mainly polymerized, latex, and the like. This paper size applies to China National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page)

I五經濟部中央標準局員工消費合作社印製發明説明（ A7 B7 在該過濾器藥室方法中，該固體基質被共價地連接以一氮氧化物，且被包含在一過濾器殼罩或是其它類似裝置中，俾使一溶液（諸如血紅素）可流經該裝置，並與氮氧化物接觸，並同時被輸入至一病人體内。一實用的方法是使用一般可得的活化瓊脂糖凝膠作為基質，並將該膠質裝入一無菌具活塞鎖藥室内。該藥室接而只要在一含有血紅素的溶液之輸血期間，被***至在流體投藥線上即可。在實施上，可利用各種已知的結構，來提供其包含該過濾器殼罩之結構，其中該氮氧化物及血红素流經該殼罩。例如，參照USP 5, 149,425。例如參照第12圖，殼罩1含有一氮氧化物-標識的瓊脂糖凝膠。例如，一 4-溴-乙醯胺基-TEMPO 標識的ω胺基已基-瓊脂糖（參見第2A圖），一被偶合以4-胺基- TEMPO之1,4-雙（2, 3-環氧基氧丙基）丁烷瓊脂糖（參見第2 B圖）。其他的化合物可被包含在該過濾器殼罩内。在輸送的過程中，含有該溶液的靜脈輸送線會被連接至該活塞入口 2，以使其進入該殼罩1内，於其中該血紅素溶液會碰到被包含在殼罩内或結合至基質4上的含氮氧化物化合物，於此過程中，該含氮氧化物組成物會被輸入並可能起反應，俾以去除該溶液内與氧有關的毒性物種°該血紅素溶液而後經由活塞出口 3流出該藥室，且會被直接輸入病人體内。該4-胺基-TEMPO標識的璟氧基-瓊脂糖之電子旋轉共振被示於第2A圖中。此外，一 ω -胺基己基-瓊脂糖可與4 - (2-溴乙醯胺基）-TEMPO起反應而形成TEMPO標識的瓊脂。其電子旋轉共振光譜示於第2B圖中。另一選擇本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事- -項再填裝—--；--- :寫本頁) 訂"-- 492866 A7 B7 1、發明説明（經濟部中央標準局員工消費合作社印製是經由一羰醯胺鍵結將4-羧基-TEMPO接合至具有磺化二亞胺的胺基-瓊脂糖上。相反的，該4-胺基-TEMPO可容易地經由使用碩化二亞胺，例如卜乙基-3-(3-二甲基胺基-丙基）磺化二亞胺，而被偶合至位在瓊脂糖上的羧基基圍° 標識有4-胺基-TEMPO的藥室之製備如下：在室溫下令配於乳酸林格氏液内的l〇〇mM 4-胺基-TEMPO (Sigma Chem. Co.)循環流經一醒 Avid Chrom Cartridge (Unisyn Tech· I n c .)歴時1小時，而後以氰基氫化硼鈉予以還原歴時6小時。該藥室殼罩的内部以乳酸林格氏液予以徹底地清洗。標識有3-胺基-PR0XYL的藥室，亦可依據上述方法’ 藉由以3-胺基-PR0XYL取代4-胺基-TEMPO，被類似地製備之。 · 實施例2 —氮氧化物-標識的穩定化血紅素為防止血紅素分解成其組份次單元，血红素分子經由其次單元之化學或是重組交聯而被分子内穩定化。在此所稱、'穩定化"血紅素是描述其經由化學或重組交聯而被穩定化的血紅素單體，且亦描述二聚體、三聚體或是較大的寡聚物，其等之組份血紅素分子藉由使用環糊精及其硫酸化衍生物之交聯而被穩定化。將氮氧化物接合至穩定化血紅素之一較佳技術是，藉由將該氮氯化物共價地接合至穩定化血红素的兩値鏈的 /3 -93巯基上。雖然在-93位置處之專一性標識，已在天然的人類血红素上就空間結構的研究而被證實了 [參見 McConnell等人之回顧論文，Quart· Rev. Biophys. 3:91 (請先閲讀背面之注意事項再填寫本頁) -裝--- Ί丨訂_ 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） 51 492866 經濟部中央標準局員工消費合作社印製 A7 _____B7__五、發明説明（） (1970)]，此種交聯的血紅蛋白之專一性標識未曾被報導過。如上所提的，已發展出數種形式之以血紅素為主的氧載體，其經由與DBBF、雙阿斯匹靈交聯的血紅素以及被〇-棉子糖穩定化或寡聚化的血紅素之化學交聯而被穩定之。在本案發明人之美國專利第4,857,626號中所描述的開環糖産生多價醛，該多價醛係衍生自雙糖、寡糖或，較佳地，諸如〇 -棉子糖之三糖。這些化合物之功能為可提供分子内的穩定化（交聯）及分子間的聚合化。藉由控制掲露於本案發明人之先前專利中的反應*該多價醛可被用來産生如上所界定的 ''穩定化〃血紅素而不需要聚合化。在另一例中，一氮氣化物可被共價地接合至穩定化血紅素或聚合化血紅素。因此*其使用多價醛而被穩定化之以血紅素為主的溶液，在本發明的實施例中被認為是一 v穩定化〃血紅素，並在其後的實施例被以為是一聚合化血紅素。為證實經化學修飾的血紅素之/3 -93位置對於氮氧化物的接合，並未造成其立體上的不可接近性，展現出的結果確認了一氮氧化物可被共價地接合至DBBF-Hb的-93位置處。在此實施例中，DBBF-Hb與二種形式的氮氯化物（TEMPO 及PR0XYL)反應，該二種氮氧化物含有兩種形式的巯基專一性功能基團並且具有下列結構式：本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 52 (請先閱讀背面之注意事 •項再填· 裝！ I寫本頁) 訂 ¾ 492866 A7 B7 i'發明説明（）I5 The printed description of the invention published by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (A7 B7 In the filter pharmacy method, the solid matrix is covalently connected with a nitrogen oxide and is contained in a filter housing or In other similar devices, a solution (such as heme) is allowed to flow through the device, contact with nitrogen oxides, and be simultaneously input into a patient. A practical method is to use generally available activated agar. Glucose gel is used as a matrix, and the colloid is filled into a sterile piston lock medicine chamber. The medicine chamber can be inserted into the fluid administration line during the blood transfusion of a solution containing heme. Various known structures can be utilized to provide a structure including the filter housing, wherein the nitrogen oxides and heme flow through the housing. For example, refer to USP 5, 149,425. For example, refer to FIG. 12, the housing Hood 1 contains a nitrogen oxide-labeled agarose gel. For example, a 4-bromo-acetamido-TEMPO labeled omega aminohexyl-sepharose (see Figure 2A), one is coupled with 4 -Amine-Tempo 1,4-bis ( 2,3-epoxyoxypropyl) butane agarose (see Figure 2B). Other compounds can be included in the filter housing. During delivery, an intravenous line containing the solution Will be connected to the piston inlet 2 so that it enters the casing 1 where the heme solution will encounter the nitrogen-containing oxide compound contained in the casing or bound to the substrate 4, during this process In this case, the nitrogen oxide-containing composition will be input and may react to remove oxygen-related toxic species in the solution. The heme solution then flows out of the pharmacy via the piston outlet 3, and will be directly input into the patient. In vivo. The electronic rotation resonance of the 4-amino-TEMPO-labeled methoxy-agarose is shown in Figure 2A. In addition, an omega-aminohexyl-agarose can interact with 4- (2-bromoethyl)醯 Amino group) -TEMPO reacts to form TEMPO-labeled agar. Its electronic rotational resonance spectrum is shown in Figure 2B. Another option This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) (please first Read the note on the back--item refill ---; ---: write Page) Order-492866 A7 B7 1. Description of the invention (Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs, the 4-carboxy-TEMPO is bonded to the amine group with a sulfodiimide via a monocarbonylamide bond. -On agarose. Conversely, the 4-amino-TEMPO can easily be obtained by using a sulfonized diimide, such as ethyl ethyl 3- (3-dimethylamino-propyl) sulfonimide, and The pharmacy, labeled with 4-amino-TEMPO, was coupled to the carboxyl group on the agarose as follows: 100 mM 4-amino- TEMPO (Sigma Chem. Co.) was circulated through Avid Chrom Cartridge (Unisyn Tech · Inc.) For one hour, and then reduced with sodium cyanoborohydride for six hours. The inside of the pharmacy shell was thoroughly washed with lactated Ringer's solution. A pharmacy labeled with 3-amino-PR0XYL can be similarly prepared according to the method described above by replacing 4-amino-TEMPO with 3-amino-PROXXYL. Example 2-Nitrogen oxide-labeled stabilized heme In order to prevent the decomposition of heme into its component subunits, the heme molecules are stabilized intramolecularly by chemical or recombination cross-linking of the next unit. As used herein, 'stabilized' heme describes a heme monomer that is stabilized by chemical or recombinant cross-linking, and also describes a dimer, trimer, or larger oligomer, These component heme molecules are stabilized by crosslinking using cyclodextrin and its sulfated derivatives. One of the preferred techniques for joining nitrogen oxides to stabilized heme is by covalently joining the nitrogen chloride to the / 3 -93 mercapto group of the two fluorene chains of the stabilized heme. Although the specific identification at the -93 position has been confirmed on the spatial structure of natural human heme [see the retrospective paper of McConnell et al., Quart · Rev. Biophys. 3:91 (Please read first Note on the back, please fill in this page again) -Installation --- Ί 丨 Order_ This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 × 297 mm) 51 492866 Printed by A7 of the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention () (1970)], the unique identification of such crosslinked hemoglobin has not been reported. As mentioned above, several forms of heme-based oxygen carriers have been developed, via heme crosslinked with DBBF, double aspirin, and Heme is chemically cross-linked and stabilized. The ring-opening sugars described in U.S. Patent No. 4,857,626 to the inventors of the present case generate polyvalent aldehydes which are derived from disaccharides, oligosaccharides, or, preferably, trisaccharides such as 0-raffinose. The function of these compounds is to provide intramolecular stabilization (crosslinking) and intermolecular polymerization. By controlling the reactions disclosed in the previous patents of the inventor of the present case, * the polyvalent aldehyde can be used to produce '' stabilized osmium hemoglobin as defined above without the need for polymerization. In another example, a nitrogen compound may be covalently bonded to a stabilized heme or a polymerized heme. Therefore * heme-based solutions that are stabilized using polyvalent aldehydes are considered to be a v-stabilized heme in the examples of the present invention, and the following examples are considered to be a polymerization Heme. In order to confirm that the chemically modified heme's / 3 -93 position is bound to nitrogen oxides, it did not cause its inaccessibility in three dimensions. The results showed that a nitrogen oxide can be covalently bonded to DBBF -Hb at -93. In this embodiment, DBBF-Hb reacts with two forms of nitrogen chlorides (TEMPO and PR0XYL). The two nitrogen oxides contain two forms of thiol-specific functional groups and have the following structural formula: Applicable to Chinese National Standard (CNS) A4 specification (210X297 mm) 52 (Please read the notes on the back • items before filling • loading! I write this page) Order ¾ 492866 A7 B7 i 'invention description ()

經濟部中央標準局員工消費合作社印製 4-(2_溴乙醯胺基）-了£丨彳？0 3-馬來醯亞胺基- PROXYL D B B F - H b之製備傺藉由已知的技術’將溶液内的純化的去氣化血红素交聯以雙（3,5-二溴甲矽基）反式丁烯二酸，且所得的産物藉由管柱層析法予以純化之。3 -馬來醯亞胺基（2,2, 5,5-四甲基吡咯烷烴氧基）[3-馬來醯亞胺基 -PROXYL]之共價接合，藉由使用甲醇作為載體溶劑，在一濃度為約lOOmM 3-馬來醯亞胺基-PROXYL之下，將2莫耳當量的此氮氧化物加入至配於乳酸林格氏液内之1 m 1的濃度為約8g/dl之DBBF-Hb中，而予以完成。容許DBBF-Hb在22-23 °C下反應30分鐘，並予以攪拌之。該交聯的程度是可由未反應的氮氧化物之電子旋轉共振信號強度的消失而加以估計。為去除未反應的氮氧化物，該反應混合物使用一具一 30Kd截留值表稱分子量限制（NMWL)聚乙撑基楓（PES)膜 (Filtron Technology Co.)的 Filtron stire室，以 10倍過量體積的乳酸林格氏液予以清洗3次。該氮氧化物-標識的血紅素之電子旋轉共振測量是以一 Broker ESR光譜儀予以記錄之。第3A圖顯示4-(2-溴乙醯胺基）-TEMPO標識的 (請先閱讀背面之注意事項再填寫本頁) -裝. 、τ 線本紙張尺度適用中國國家標準（CNS > A4規格（210X297公釐）經濟部中央標準局員工消費合作杜印製 492866 A7 B7 i、發明説明（) DBBF-Hb之電子旋轉共振光譜。其被3-馬來醯亞胺-PROXYL 類似地標識之DBBF-Hb的電子旋轉共振光譜顯示於第3B圖中。在本實施例中，氮氣化物被共價地鍵結至位在血紅素的二値/3鏈上之單獨的巯基處。因此，在99.00¾去氧_血紅素下，該氮氧化物對血紅素-結合的氧氣比為大約200 : 1 ，蓋因有二個氮氧化物被接合至該血紅素的二個鐽上。但是，在輸血後，該去氧化的HRCS收集位在肺中的氯，而在100¾的氧氣化下，該氮氧化物對血紅素-結合氧氣比變成約1 : 2，因為有四個氧分子被結合至該血紅素的四個球蛋白鐽上，而該二個氮氧化物留在該/3球蛋白鐽上ϋ 使用一為1 :2的血红素對氮氣化物比，高於99¾的氮氧化物會被共價地接合至該DBBF-Hb上。DBBF-Hb亦可被標識以一個位於3-馬來醯亞胺與PR0XYL部分之間的間隔子基圍 (例如一額外的甲基）（亦即3-馬來醯亞胺甲基-PR0XYL)，其會展現出一類似於第3B圖的共振光譜。值得注意的是，其他的氮氧化物可被共價地接合至DPG結合位中的待定胺基（例如 /3 -Val-1、/3 -Lys-82及 oc -Lys-99)，或可被接合至血紅素上其餘的40-pi us表面離胺酸e -胺基。該TEMPO 及PR0XYL氮氣化物的異氡酸衍生物亦可與胺基起反應。例如，4-異氡酸-TEMPI可在一莫耳比約為10:1下被加入至血紅素中。在其它位置處被標識以該氮氣化物的血紅素之共振光譜（未示出）亦是類似於第3A圖至中所示者。將氮氧化物接合至DBBF-Hb的數個位置上之能力暗示本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）袭---^—— (請先閲讀背面之注意事項再填寫本頁) J — 訂--- 線 492866 經濟部中央標準局員工消費合作社印製 A7 B7五、發明説明（）了使用球蛋白二聚體被穩定化的重組型血紅素[D.Loker et al., Native 356:258 (1992)]，可被類似地標識以氮氣化物。製備一氮氧化物-標識的交聯劑（例如TEMPO標識的琥珀酸）之一 DBBF類似物，亦是可能的（參見美國專利第 4,240,797號）。第4圔傺為下列之ESR光譜：（A) 4-(2-溴乙醯胺基）-TEMPO，（B) 4-(2-溴乙醯胺基卜TEMPO標識的HBOC以及（C) 配於乳酸化林格氏液内且在室溫下記錄的1 5NDEi ^TEMPOL (TEMP0L: 4-羥基-2,2,6,6-四甲基六氫吡啶-卜烴氣基）。第4A圖與第4B圖之差異顯示出一小型分子量氮氣化物對一被共價地接合至一巨分子（諸如血紅素）的氮氯化物之移動性。第4C圖顯示出一具有一為1/2的核旋轉之穩定的同位素氮1 5 N生成兩個共振波峰，而具有一為1的核旋轉之天然的-同位素1 4N生成三個共振波峰（比較4A至4C)。在此處所述之實驗組中，這些共振波峰之分離被用來證明小型及巨分子量的氮氧化物之酵素模擬性以及活體内與活體外之氧化/還原反應。依下述來製備具有不同的氮氣化物對血紅素之莫耳比的氮氧化物-標識的H0BC。於一乾淨的通風櫥中，將2、4 與δ莫耳當量的4-(2-溴乙醯胺基卜TEMPO，如同固體粉末，予以直接添加至3値分開的15ml Vacutainer内。在換過橡膠隔板後，接而將4-(2-溴乙醯胺基）_了£卩卩0溶於200从1 氯仿内。接而經由一個2 7號針頭並通過該橡膠隔板，將該 Vacutainer接至高真空（5mmHg)，並移除掉氯仿，而在該 (請先閱讀背面之注意事項再填寫本頁) •裝--- Γ · -ϋϋ^ tMmMmmt ^ ^ ·ϋκ IB·.— · m-ια 線本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐）經濟部中央標準局員工消費合作社印製 492866 A 7 B7 五、發明説明（）Printed by the Consumers Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 4- (2_Bromoacetamido) -Is it £ 丨彳? 0 3-Maleimide-Preparation of imino-PROXYL DBBF-H b. The purified degassed heme in the solution was cross-linked with bis (3,5-dibromosilyl) by known techniques. ) Trans-butenedioic acid, and the resulting product was purified by column chromatography. Covalent bonding of 3-maleimidoimino (2,2,5,5-tetramethylpyrrolidinyloxy) [3-maleimidoimido-PROXYL], by using methanol as a carrier solvent, Under a concentration of about 100 mM 3-maleimidoimino-PROXYL, 2 mol equivalent of this nitrogen oxide was added to 1 m 1 of lactated Ringer's solution at a concentration of about 8 g / dl. DBBF-Hb. Allow DBBF-Hb to react at 22-23 ° C for 30 minutes and stir it. The degree of this crosslinking can be estimated from the disappearance of the intensity of the electron rotation resonance signal of the unreacted nitrogen oxide. To remove unreacted nitrogen oxides, the reaction mixture used a 30Kd cut-off table called a Filtron stire chamber with a molecular weight limited (NMWL) polyethylene ethylene maple (PES) membrane (Filtron Technology Co.) in a 10-fold excess. The volume of lactated Ringer's solution was washed 3 times. The electronic rotation resonance measurement of the nitrogen oxide-labeled heme was recorded with a Broker ESR spectrometer. Figure 3A shows the 4- (2-bromoacetamido) -TEMPO logo (please read the precautions on the back before filling out this page)-installed., Τ Thread paper size applies Chinese national standards (CNS > A4 Specifications (210X297 mm) Consumer cooperation of the Central Bureau of Standards, Ministry of Economic Affairs, printed 492866 A7 B7 i. Description of invention () Electronic rotating resonance spectrum of DBBF-Hb. It is similarly identified by 3-maleimide-PROXYL The electron rotation resonance spectrum of DBBF-Hb is shown in Figure 3B. In this example, the nitrogen compound is covalently bonded to a separate sulfhydryl group on the difluorene / 3 chain of heme. Therefore, in At 99.00¾ deoxy_heme, the ratio of nitrogen oxide to heme-bound oxygen is about 200: 1, and two nitrogen oxides are conjugated to the two hydrazones of the heme. However, in After transfusion, the deoxidized HRCS collects chlorine in the lungs, and under 100¾ oxygenation, the nitrogen oxide to heme-bound oxygen ratio becomes about 1: 2 because four oxygen molecules are bound to On the four globins of the heme, while the two nitrogen oxides remain on the / 3 globulin鐽上 ϋ Using a 1: 2 heme-to-nitrogen ratio, nitrogen oxides above 99¾ will be covalently bonded to the DBBF-Hb. DBBF-Hb can also be identified as a 3-ma The spacer group (for example, an additional methyl group) between the lyme imine and the PR0XYL moiety (ie, 3-maleimide methyl-PR0XYL) will exhibit a resonance similar to that shown in Figure 3B Spectrum. It is worth noting that other nitrogen oxides can be covalently bonded to the pending amine group in the DPG binding site (for example, / 3-Val-1, / 3-Lys-82, and oc-Lys-99), Or it can be attached to the remaining 40-pi us surface lysine e-amine group on heme. The TEMPO and PROXYL nitrogen compounds also react with amine groups. For example, 4-isocyanate -TEMPI can be added to heme at a molar ratio of about 10: 1. The resonance spectrum (not shown) of the heme labeled elsewhere with this nitrogen compound is also similar to Figure 3A to The ability to join nitrogen oxides to several positions of DBBF-Hb implies that this paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) ----- ^ —— (Please read the notes on the back before filling this page) J — Order --- Line 492866 Printed by the Consumers Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Invention Description () Use of globulin Dimer-stabilized recombinant heme [D. Loker et al., Native 356: 258 (1992)] can be similarly identified as a nitrogen compound. It is also possible to prepare a DBBF analogue, a nitroxide-labeled cross-linking agent (such as TEMPO-labeled succinic acid) (see US Patent No. 4,240,797). Section 4 is the following ESR spectrum: (A) 4- (2-bromoacetamido) -TEMPO, (B) 4- (2-bromoacetamido TEMPO-labeled HBOC and (C) compound 15NDEi ^ TEMPOL (TEMP0L: 4-hydroxy-2,2,6,6-tetramethylhexahydropyridine-hydrocarbyl) recorded in lactated Ringer's solution at room temperature. Figure 4A The difference from Figure 4B shows the mobility of a small molecular weight nitrogen compound to a nitrogen chloride that is covalently bonded to a macromolecule such as heme. Figure 4C shows a sample with The stable nuclear isotope nitrogen 1 5 N generates two resonance peaks, while the natural-isotope 1 4N with a nuclear rotation of 1 generates three resonance peaks (compare 4A to 4C). The experiments described here In the group, the separation of these resonance peaks was used to demonstrate the enzyme mimicry of small and large molecular weight nitrogen oxides and the oxidation / reduction reaction in vivo and in vitro. The following have been prepared with different nitrogen compounds to heme. Mole ratio of nitrogen oxides-labeled HOBC. In a clean fume hood, mix 2, 4 and δ Mol equivalents of 4- (2-bromoacetamido T EMPO, like a solid powder, was directly added to a separate 15 ml Vacutainer. After changing the rubber separator, 4- (2-bromoacetamido) was dissolved in 200 μl. 1 inside of chloroform. Connect a Vacutainer to a high vacuum (5mmHg) through a 27 gauge needle and pass through the rubber septum, and remove the chloroform. (Please read the precautions on the back before filling this page ) • Equipment --- Γ · -ϋϋ ^ tMmMmmt ^ ^ · ϋκ IB · .— · · m-ια thread paper size applies to China National Standard (CNS) Α4 specification (210 × 297 mm) Employees' Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs Printing 492866 A 7 B7 V. Description of the invention ()

Vacutainer之下半部處留下一薄層的4-(2-溴乙醒胺基）-TEMPO塗層。經由無菌轉移並通過該橡膠隔板，將適當量的 HBOC引入後，容許該等溶液在室溫下反應，並每隔5分鐘予以間歇地渦镟振盪混合歴時半小時（不是所有的固體會被溶於4與8莫耳比之氮氧化物對血紅素内），該Vacuta iner 接而被置於一値4 °C的冷溫箱内過夜。隔天早上揮復在室溫下之渦谧振盪混合歷時又半小時直至所有的4- (2-溴乙醯胺基）-TEMPO固體最後從該Vacutainer的表面處消失。接而將反應混合物以及對照組轉移至一値無菌透析管，並對乳酸化林格氏液進行透析直至測不到未標識之自由的4-(2-溴乙醯胺基）~TEMP0電子旋轉共振（ESR)信號。在 2、4與δ莫耳當量的4-(2-溴乙醯胺基）-TEMP0對Hb下，4-(2-溴乙醯胺基）-TEMPO-標識的HBOC之ESR光譜，分別示於第5A-5C圖中。在2莫耳當量的4-(2 -溴乙醒胺基）-TEMP〇對 Hb下，其ESR光譜在有或無進行透析者偽大體上相同的，這表示共價標識繫為定量的。在/3球蛋白鏈上的兩痼巯基基團，在H0BC之例中，似乎是共價接合之位置處（這可由利用N-乙基馬來醯亞胺的4 - (2-溴乙醯胺基）-TEMPO標識之選擇性堵塞或藉由逆相H PLC的球蛋白鏈分析而予以證實）。必須注意的是，對於2、4與8的ESR信號強度（波峰Mo)比，在儀器敏感度予以成比例地降低以記錄其光譜值時，會大致呈相同的比例。更甚者，可預期到在甚而更高的莫耳比下，更多的 (2-溴乙醯胺基）-TEMPO可被接合至Hb以作為，例如活髏内本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） , #1 (請先閲讀背面之注意事項再填寫本頁) 裝一---^---j —訂 *--- 經濟部中央標準局員工消費合作社印製 492866 A7 B7 五、發明説明（）的放射保護劑。如下所敘述的，在紅血球代用品配方中，較佳的氮氧化物對血紅素之莫耳比傜為8 :1。參照第6圖，一種由4-(2-溴乙醯胺基）-TEMPO-檫識的 HBOC與15ND15-TEMP0L所構成的混合物之一 ESR光譜，其中 4- (2-溴乙醯胺基）-TEMPO之中央波峰（以向下箭號表示）以及後者之高視野波峰（向上箭號表示）被調整至相似的強度〇共振波峰之分離，容許了涉及相同的分子量氮氧化物 (TEMP0L)及其巨分子共軛物在活體外或活體内（小白鼠）反應之自由基或酵素模擬物活性之同時的監測。例如，該二種氮氧化物之活體内血漿半衰期，藉由參照該等不同的氮氧化物之獨待的光譜恃徵，而被比較之。待別地，以血红素為主的溶液之活體内ESR研究，使用一為8 : 1的氮氧化物對血紅素比（參照第5 C圖），在小白鼠身上進行之，俾以利用其高ESR信號強度之優點。首先，一小型低分子量氮氧化物（1 ID! 7-TEMP0L，參照第4C圖）與一大型分子量4 - (2-溴乙醯胺基）-TEMPO-標識的HBOC (參照第4B圖）之適當的血漿半衰期之測定傺藉由製備該二者之一混合物並將其ESR 強度調整至大約相同（參照第6圔）。在麻醉下，將0.5ml的該混合物注射至一在一熱燈下被擴張的小白鼠尾部靜脈内。將該小白鼠尾部***至一 ESR室内，並在注射後於10分 ’ 鐘内記錄其光譜（參照第7A圔）。參照第7A、7B與7C圖，15ND17-TEMP0L信號無法偵測本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） -5 7 - , 裝—丨丨訂 00 (請先閲讀背面之注意事項再填寫本頁) 492866 經濟部中央標準局員工消費合作社印製 A7 B7_____五、發明説明（）得到，但是4-(2-溴乙醯胺基）-TEMPO-標識的HBOC被清楚地解析之（參照第7B與7C圖有關血漿半衰期之研究，其中第7C圖是第7B圖之一延續）。由於HBOC的血管收縮作用被報導，在大白鼠體内於一 H0BC之大九藥注射之初期5-15分鐘被完全地發展出，氮氧化物-標識的HBOC在輸血至小白鼠體内後，立即偵測其在自由基氧化還原反應中的參與。雌性CH3小白鼠的尾部靜脈，利用80¾的一氧化二氮、20¾ 的氣氣以及3¾的異氣院（isofluorane)，在麻醉之下被裝上套管。在一熱燈之下，該小白鼠的尾部靜脈變成可視觀地擴張，一由一値30號皮下針所構成且連接至一個1英尺長的聚乙烯導管之套管被***至該尾部靜脈内並利用氰基丙烯酸酯固定膠將之定位。關於活體内的ESR測量，被裝有套管之小白鼠在麻醉之下被轉移至一値50m 1的錐形離心管内，該離心管被修改以容許該尾部可由該離心管之端部突出並容許源自該管之開口端的麻醉氣體之持續的流動。該尾部被***至一塑膠管内，該塑膠管接而被固定於一個 TE 102室内。該套管被周期性地沖洗以肝素（100單位/ml) 以確保血管開放性。該套管位在接近於尾部之根部處，並被維持在該ESR室之外，藉此一源自尾部之純信號可在大九藥注射之後被立即地偵測之。0.5ml的樣品（參照第8圔）經由該套管被注射，而光譜計被設定成一為0.5分鐘間隔之重複掃描模式（參照第8A與8B圖）。在第8A圖中*磁場強度以2Gauss之數予以增加而在第8B圔中，磁場強度以2Gauss 之數予以減低，俾以重疊該等共振光譜。該1 5NDi 7-TEMP0 (請先閱讀背面之注意事項再填寫本頁) .裝--- I j 訂 ---線本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） π竭6 1^__I____ 經濟部中央標準局員工消費合作社印製 A7 B7 發明説明（） L信號在注射後2 · 5分鐘内即消失。在相同的時間期間裡’ 該4- (2-溴乙醯胺基）-TEMPOL-標識的HBOC亦以一相似的速率消失。但是，氮氧化物-HBOC信號在血漿内被顯示出係為穩定的（第δΒ圖）。因此，第8B圔加上第7圖之結果顯示出被檫識至巨分子的氮氯化物，相較於小塱低分子量氮氣化物 (例如1 5NDi 7-TEMP0L)具有要長得多的血漿半衰期。所觀察到的自由基反應之性質涉及兩痼途徑： 1.快速相似乎是涉及氮氧化物的自由基（例如超氧化物）氧化成其氧代銨陽離子中間産物，隨後為該氧代銨陽離子還原成該氮氣化物其穩定的羥基胺衍生物。該還原涉及存在於血管區域内的一或兩個還原當量（例如NADH)之參與。氮氧化物至其羥基胺的還原會導致ESR信號強度之一快速的降低，就8:1莫耳比的4 - (2_溴乙醯胺基）- TEMPO檫識的HBOC之例而言，此代表大約25¾的位在HBOC上之4- (2-溴乙醯胺基） -TEMPO。此相涉及小分子及巨分子的氮氧化物。 2 ·依據反應機制，緩慢相（參照第8B圖）似乎表示剩下的75¾的位在HBOC上之4- (2-溴乙醯胺基）-TEMPO之抗氧化酵素模擬物活性，其中該氮氧化物涉及了環形-自由基反應（例如SOD-模擬反應）。當氮氧化物自由基大體上未如一個SOD-模擬物被消耗時，ESR 信號強度之降低速率主要地可歸因於上面所述的反應機制以及次要地HBOC濃度之降低，這是因其在血本紙張尺度適用中國國家標準（CNS ) Μ規格（210X297公釐） ---------裝---^---11 訂，-----線 (請先閱讀背面之注意事項再填寫本頁) 492866 A7 B7 五、發明説明（）管區域内如同其血漿半衰期濃度之一函數而被緩慢地消除所致。此結果證實了聚氮氧化物巨分子之實用性，於此例中是TEMPO-標識的HBOC於活體内將自由基解毒。此實用性被界定藉由氮氧化物於活體内發揮有如酵素模擬物之作用而提供短期的（以分鐘計）自由基之清除以及持續的（以小時計）保護對抗氧化反應。在這個以及各個與含血紅素溶液有關的實施例中，必須瞭解到，未結合的低分子量氮氧化物可被添加至配方内以增加可跨越血管膜至間質間隙内以及周圍的細胞環境之活性氮氧化物的濃度。此處所展現之結果因而將一低分子量氮氧化物至一藥學組成物内之簡單的添加之作用，和本發明之聚氮氧化物巨分子的作用，予以明顯地區別之。本發明使用一聚氮氧化物巨分子及低分子量氮氧化物之一多重組份条統的特殊優點，將在後文更清楚地彰顯之。經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 依據第8圖之光譜分析，氧化/還原循環反應涉及了大約73¾的留在其自由基狀態之4-(2-溴乙醯胺基）-TEMP0標識的HBOC。這表示TEMP0L參與位血管間隙之區域内的活體内氧化/還原反應。為研究以血紅素為主的溶液之血管收縮作用，由經修飾的人類血红素所構成之溶液被用來測試其在完整的速率下之作用。在任一有使用動物之研究中，一律使用人道的處理程序。在研究之前 2-3天，以 Ketamine (40 mg/kg ί·πι·)以本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐）經濟部中央標準局員工消費合作社印製 492866 A7 B7 五、發明説明（）及乙醯丙U蒙（acetylpromazine) (0.75mg)或者戊基巴比妥鈉（40 mg/kgi.P·) ’ 來麻醉雄性 Sprague-Dawley大白鼠。將醫藥级的 Tyson microbore (ID 為 0·05 ’ 0D 為0.03)*** 至股動脈及靜脈内e套管會被賦於外並充填以肝素化的» 萄糖，並被密封以不銹鋼針。在一為2-3天之恢復期後’ 將有意識的動物置於一塑膠拘籠内。從外科手術之2-3天是需要的，俾以確保切口會在交換輸血之前癒合°由於外科手術可能造成小出血，容許復原是很重要的，這樣與外科手術有關的小出血才不會與血液置換之一副作用相混淆。使甩一輸入泵來進行50¾交換輸血，俾以同時分別地輸入及油出測試溶液及血液°移出的血液容積（依據總血液容積為12-15ml)被置換以測試溶液有大約20-30分鐘之久。在交喚輸血後5小時，使用一連接至一圖形記錄器之壓力信號轉移器來監測及記錄動脈血壓。平均動脈血壓偽以脈衝壓力的1 /3來計算。心臓速率傺以血壓痕量予以測定之。實施例3 —穩定化血紅素的氮氧化物標識聚合物雖然從交聯的單體來製造穩定化血紅素之二聚物是可行的，由穩定化或自然的血红素産生血紅素聚合物亦是可能的。血紅素聚合物的溶液含有由單體、二聚物、三聚物、四聚物或其它寡聚體所構成之一混合物。被用來作為一 HBOC之含有聚合化血紅素的溶液通常具有較長的血漿循環時間，且與穩定化單體血紅素相較，具有較高的攜氯能力。該聚合化血紅素是可利用許多的途徑使用不同的聚合化本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）一 61 一 I--------ml-裝—--：---：—訂 '·------•線 (請先閱讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製 492866 A7 B7 五、發明説明（）試劑予以製備而得（參見美國專利第4,001,200、4,857,636 及4, 8 26, 8 1 1號）。將氮氧化物引入至一聚合化血紅素溶液中之較佳方法偽再次地藉由將一氮氧化物共價接合至該血紅素的二個冷球蛋白鏈之/3 -93巯基上。該等頸基尚未知是否涉及穩定化或是聚合過程。是故，最好是在血紅素單體的穩定化及聚合化之前，將氮氧化物共價地接合至血紅素。例如，依據上述在第二實施例所述的步驟，氮氧化物被共價地接合至DBBF-Hb上，而後依據授予Sehgal等人之美國專利第4, 826, 811號中所述的步驟，利用戊二醛將之聚合化。第4B圖為以3-馬來醯亞胺-PR0XYL標識且以戊二醛聚合化的DBBF-Hb之電子旋轉共振光譜。同樣地，其被戊二醛聚合化的DBBF-Hb亦可以相同方式被標識以4- (2-溴乙醯胺基卜TEMPO作標識。使用一相同的方法，一含有未反應的醛基之聚合化血紅素中間産物，例如一種戊二醛-聚合化或是〇 -棉子糖-聚合化的血紅素中間産物，可經由還原性胺化反應被用來與 4-胺基-TEMPO或3-胺基-PR0XYL共價接合，俾以産生氮氧化物-標識的血红素聚合物。關於該還原性胺化反應，該反應的順序以及時間是十分重要的。該4>胺基-TEMPO是在聚合化反應完全後，但在可造成該氮氧化物共價接合至聚合化血紅素的還原反應之前，被加入至戊二醛-聚合化血紅素中。同樣的，一〇 -棉子糖聚合化血紅素的氮氧化物標識可在還原性胺化反應之前，藉由加入4-胺基-TEMPO或是本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -62 - I·· -----------^---_---τ—訂^-----1^11^ 線 (請先閱讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製 492866 A7 -------- B7_ 五、發明説明（） 3-胺基- PROXYL而予以完成。例如，4-胺基- TEMPO標識的〇-棉子糖聚合化血紅素是依據本案發明人之美國專利第4 , 857 ,636號所述的步驟而予以製備的，只除了在聚合化反應完成後，並在使用20奠耳過量的二甲基胺硼複合體之還原反應之前，將6莫耳當量的4-胺基-TEMPO加入之。如此處所述的，血紅素可藉由從雙糖或是開環的糖（包含寡糖，或以諸如〇 -棉子糖的三醣為佳）所衍生的多價醛基予以交聯化及聚合化。同樣地，單醣亦可被用來穩定化及聚合化血紅素’雖然較高分子量的糖是較佳的。一經透稀及清洗過之被檫識以4-胺基-TEMPO的〇-棉子糖聚合化血紅素之共振光譜是顯示於第9A圖。為了增加聚合化血紅素之血紅素寡聚物（Hb„ ,其中n = 2-4)之産率，希望能使用α -璟糊精、0 -環糊精以及7 -環糊精，以及其等表示6-、7-與8-璟化®萄糖分子之硫酸鹽衍生物，來增加交聯劑的聚醛之價數◦該等開環的α -環糊精、/3 -璟糊精以及7 -璟糊精分別具有12、14與16個反應性醛基。這些開環的交聯劑可被用來交聯及聚合化血紅素以製造其富含寡聚物的聚合化血紅素。如前所述的，未反應的醛可被利用以共價地接合至一胺基-気氧化物（例如4-胺基TEMPO或3-胺基- PR0XYL。更甚者，開環的硫酸鹽衍生物（例如硫酸化α -環糊精），因為兩個額外原因，可為一有效的交聯劑：（1)在降低，交聯的血紅素之氣氣親和力上，硫酸基團會模擬DPG之活性，因而改善氧氣奢輸送性質；以及（2)該等硫酸基團會本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） ---------裝---^---^訂 j------線 (請先閱讀背面之注意事項再填寫本頁) 492866 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（）提供作為其可複合多重型（例如n = 2-4)血紅素之親和力標識俾以初始地形成一、、簇〃。一旦該、、簇〃複合物被形成 ’位在環糊精上的該等醛基圍會被帶至接近該等DPG位置内之胺基基團處，因而促進血紅素的次單位之内的與分子之間的共價***聯，這形成血紅素寡聚物之一增高的産率。除了抗氧化酵素模擬物活性之外，該開環的璟糊精聚合化及氮氧化物-標識的血紅素，相較於〇-棉子糖與戊二醛聚合化血紅素*亦會具有改善的産率與組成。實施例4 一氮氧化物-標識的脂質體-包囊化血紅素脂質體偽為由兩性分子之聚集以形成一呈中空球體形式的雙層結構而被形成的顆粒體，以其極性處對面一内部水區域以及外側大量的水。數種用以形成脂質體之可接受的方法像為此技藝中已知者。典型地，像二棕櫚醯磷脂醯膽鹼之分子在水溶液中形成脂質體。脂質體可用膽固醇來配製以增加穩定性，且可包含其它物質，如中性脂質以及表面修飾劑（例如正電荷或是負電荷化合物）。較佳的脂質體是小型單薄板式雙層（u n i 1 a m e 1 1 a r - b Π a y e r e d )球形殼體。亦已知一種用以將血紅素包囊化在一脂質體内的方法 (Farmer et al.,美國專利第4,911,921號）。為達本發明之目的，許多方法可被用來將以氮氧化物為主的氯氣解毒功能引入一脂質體-包囊化血紅素溶液中。例如，可以使 " 用氮氧化物-標識的天然血紅素或一如上所述的氮氧化物-標識的穩定化血紅素作為起始物質，而後藉由習知技術來本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1· ml §am§M§ —a— ernnkwrEB I— ml ϋϋ an·-— —^ϋ_4 Γ y ^i-n·— ^ i (請先閱讀背面之注意事項再填寫本頁) 線 492866 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（) 進行該氮氧化物-標識的血紅素之脂質體包囊化。在本實施例中，純化的血紅素亦可與一膜不可通透的氮氧化物，如在Hsi a等人之美國專利第4, 235,792號中對一旋轉膜免疫分析所掲露之TEMPO-膽鹼氯化物，一起被包囊化。又，任一純化的血紅素可以一由氮氧化物-標識的脂肪酸（例如7-D0XPL-硬脂酸、12-D0XYL-硬脂酸及16-D0XYL-硬脂酸酯）、膽甾烷、一膽固醇類似物（例如3-D0XYL-膽甾烷）或礎脂質（例如12-D0XYL-硬脂酸-標識的磷脂醯膽鹼）所構成的脂質體予以包囊化。可利用一類似於在Tabushiet 等人[L· Am· Soc，106:219 (1984)]所述之方法，來製備被一由3-D0XYL-膽甾烷標識物所構成的脂質體所包囊化的血紅素。如下所載明的，一為5m 1的含有脂質組成物（包含 D0XYL標識的硬脂酸和/或膽甾烷）的氛仿溶液，首先在氮氣流中將之乾燥以去除溶劑。而後，該殘餘物予以真空乾燥，並將所形成的薄膜再散浮於2ml的配於乳酸化林格氏液内之血红素（24g/dl)中。該分散液内的脂質濃度傺為100 mM。該脂質體包囊化血红素而後被旋轉且，較佳地，被培育於37 °C下直至所有的脂質被分散以形成多重薄板式載體。而後，所形成的含有多薄板式脂質體包囊化血红素及自由的未包囊化血紅素之溶液，依據Cook等人的程序（參見美國專利第4,533,254號），迫使其流經一微流動床以形成 0.2徼米脂質體。位在該脂質體内的二棕櫚醯磷脂醯膽鹼： ^ 膽固醇：二棕櫊醯磷脂酸：3-D0XYL-膽甾烷的比例係為0.5: 0,4:0.02:0.07。所形成的3-D0XYL-膽甾烷標識的脂質體- (請先閱讀背面之注意事 ▼項再填. 裝—--^---：" I 訂 :寫本頁) --線本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） ^«66A thin layer of 4- (2-bromoethenyl) -TEMPO coating was left in the lower half of Vacutainer. After aseptic transfer and through the rubber separator, after introducing an appropriate amount of HBOC, the solutions are allowed to react at room temperature, and intermittently vortexed and mixed every 5 minutes for half an hour (not all solids will Was dissolved in 4 and 8 mole ratio of nitrogen oxides to heme), the Vacuta iner was then placed in a cold incubator at 4 ° C overnight. The next morning, the mixture was vortexed and mixed at room temperature for another half an hour until all the 4- (2-bromoacetamido) -TEMPO solid finally disappeared from the surface of the Vacutainer. The reaction mixture and control group were then transferred to a stack of sterile dialysis tubes, and the lactated Ringer's solution was dialyzed until unmarked free 4- (2-bromoacetamido) ~ TEMP0 electron rotation was not detected. Resonance (ESR) signal. The ESR spectra of 4- (2-bromoacetamido) -TEMPO-labeled HBOC at 2, 4 and δ Mol equivalents of 4- (2-bromoacetamido) -TEMP0 versus Hb are shown separately. In Figures 5A-5C. At 2 molar equivalents of 4- (2-bromoethenylamino) -TEMPO to Hb, their ESR spectra are virtually identical with or without dialysis, indicating that the covalent identification is quantitative. The two sulfhydryl groups on the / 3 globin chain appear to be covalently bonded in the HOBC example (this can be determined by the 4- (2-bromoacetamidine) using N-ethylmaleimide (Amine) -Selective blockage of TEMPO labelling or confirmation by globulin chain analysis of reverse-phase H PLC). It must be noted that for the ESR signal intensity (peak Mo) ratios of 2, 4, and 8, the instrument sensitivity is reduced proportionally to record its spectral value, which will be approximately the same proportion. What's more, it can be expected that at even higher mole ratios, more (2-bromoacetamido) -TEMPO can be bonded to Hb as, for example, the paper standard in China is applicable to Chinese national standards (CNS) A4 specification (210X297mm), # 1 (Please read the precautions on the back before filling out this page) Pack one --- ^ --- j —Order * --- Consumer Cooperatives of Central Standards Bureau, Ministry of Economic Affairs Printed 492866 A7 B7 V. The radiation protection agent of the invention (). As described below, in the red blood cell substitute formulation, the preferred molar ratio of nitrogen oxide to heme is 8: 1. Referring to Figure 6, an ESR spectrum of a mixture of 4- (2-bromoacetamido) -TEMPO-coated HBOC and 15ND15-TEMP0L, where 4- (2-bromoacetamido) The central peak of the -TEMPO (indicated by the downward arrow) and the latter's high-field peak (indicated by the upward arrow) are adjusted to similar intensities. The separation of the resonance peaks allows the same molecular weight nitrogen oxides (TEMP0L) and The macromolecular conjugates are monitored simultaneously for free radical or enzyme mimics in vitro or in vivo (mouse). For example, the in vivo plasma half-life of the two nitrogen oxides is compared by referring to the unique spectral characteristics of the different nitrogen oxides. Elsewhere, in vivo ESR studies of heme-based solutions, using an 8: 1 ratio of nitrogen oxides to heme (see Figure 5C), were performed on mice to use them. Advantages of high ESR signal strength. First, a small low molecular weight nitrogen oxide (1 ID! 7-TEMP0L, see Figure 4C) and a large molecular weight 4-(2-bromoacetamido) -TEMPO-labeled HBOC (see Figure 4B) Determination of appropriate plasma half-life is performed by preparing one of the two mixtures and adjusting their ESR intensity to approximately the same (see Section 6). Under anesthesia, 0.5 ml of the mixture was injected into a tail vein of a mouse that was dilated under a heat lamp. The tail of the mouse was inserted into an ESR chamber, and its spectrum was recorded within 10 minutes after injection (refer to Section 7A 圔). Referring to Figures 7A, 7B, and 7C, the 15ND17-TEMP0L signal cannot detect the paper size. Applicable to China National Standard (CNS) A4 specification (210X 297 mm) -5 7-, installed — 丨丨 Order 00 (Please read the back first Please pay attention to this page and fill in this page) 492866 A7 B7_____ printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. The invention description () was obtained, but the 4- (2-bromoacetamido) -TEMPO-labeled HBOC was clearly Ground analysis (refer to Figures 7B and 7C for plasma half-life studies, where Figure 7C is a continuation of Figure 7B). Due to the vasoconstrictive effect of HBOC, it was reported that it was fully developed in the early 5-15 minutes of the injection of a H0BC big nine drug in rats. Nitrogen oxide-labeled HBOC was transfused into mice, Immediately detect its involvement in free radical redox reactions. The tail veins of female CH3 mice were cannulated under anesthesia using 80¾ nitrous oxide, 20¾ gas, and 3¾ isofluorane. Under a heat lamp, the tail vein of the mouse became visually dilated, and a cannula made of a 30-gauge hypodermic needle connected to a 1-foot polyethylene catheter was inserted into the tail vein. It is positioned with a cyanoacrylate fixing glue. For in vivo ESR measurements, the cannulated mice were transferred under anaesthesia to a 50m 1 conical centrifuge tube, which was modified to allow the tail to protrude from the end of the centrifuge tube and A continuous flow of anesthetic gas from the open end of the tube is allowed. The tail is inserted into a plastic tube, and the plastic tube is connected and fixed in a TE 102 room. The cannula was periodically flushed with heparin (100 units / ml) to ensure vascular openness. The cannula is located near the root of the tail and is maintained outside the ESR chamber, whereby a pure signal from the tail can be detected immediately after the injection of the nine drugs. A 0.5ml sample (see Section 8) is injected through the cannula, and the spectrometer is set to a repeat scan mode at 0.5 minute intervals (see Figures 8A and 8B). In Fig. 8A * the magnetic field strength is increased by 2Gauss, and in 8B8, the magnetic field strength is decreased by 2Gauss, so as to overlap the resonance spectra. The 1 5NDi 7-TEMP0 (please read the precautions on the back before filling this page). Installation --- I j Order --- The paper size of the paper applies the Chinese National Standard (CNS) A4 specification (210X297 mm) π exhaust 6 1 ^ __ I____ Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs A7 B7 Description of Invention () The L signal disappeared within 2.5 minutes after the injection. During the same period of time, the 4- (2-bromoacetamido) -TEMPOL-labeled HBOC also disappeared at a similar rate. However, the nitrogen oxide-HBOC signal was shown to be stable in plasma (Figure δB). Therefore, the results of Figure 8B and Figure 7 show that the nitrogen chlorides recognized as macromolecules have a much longer plasma half-life than low molecular weight nitrogen compounds (eg, 15NDi 7-TEMP0L). . The nature of the observed radical reactions involves two pathways: 1. The fast phase appears to be the oxidation of free radicals (such as superoxide) involving nitrogen oxides to their oxoammonium cation intermediates, followed by the oxoammonium cation It is reduced to this nitrogenate with its stable hydroxylamine derivative. This reduction involves the participation of one or two reducing equivalents (e.g. NADH) present in the vascular region. The reduction of nitrogen oxides to their hydroxylamines results in a rapid decrease in one of the ESR signal intensities, as in the case of 8: 1 molar ratio of 4-(2-bromoacetamido) -TEMPO-recognized HBOC, This represents approximately 25¾ of 4- (2-bromoacetamido) -TEMPO on HBOC. This phase involves small and macromolecular nitrogen oxides. 2 · According to the reaction mechanism, the slow phase (refer to Figure 8B) seems to indicate the remaining 75¾ of 4- (2-bromoacetamido) -TEMPO's antioxidant enzyme mimic activity, where the nitrogen Oxides involve cyclic-radical reactions (eg, SOD-mimetic reactions). When nitrogen oxide radicals are not substantially consumed as a SOD-mimetic, the rate of decrease in ESR signal strength can be mainly attributed to the reaction mechanism described above and the decrease in HBOC concentration secondary, which is due to its Chinese national standard (CNS) M specifications (210X297 mm) are applied to the paper size of blood paper. --------- install --- ^ --- 11 order, ----- line (please read the first Note: Please fill in this page again) 492866 A7 B7 V. Description of the invention () The tube area is slowly eliminated as a function of its plasma half-life concentration. This result confirms the utility of polynitrogen oxide macromolecules, in this case TEMPO-labeled HBOC will detoxify free radicals in vivo. This practicality is defined as providing short-term (in minutes) free radical removal and continuous (in hours) protection against oxidation reactions by acting as an enzyme mimetic in nitrogen in the living body. In this and various embodiments related to heme-containing solutions, it must be understood that unbound low molecular weight nitrogen oxides can be added to the formulation to increase the amount of cells that can cross the vascular membrane to the interstitial space and the surrounding cellular environment. Concentration of reactive nitrogen oxides. The results presented here therefore clearly distinguish the effect of the simple addition of a low molecular weight nitrogen oxide into a pharmaceutical composition from the effect of the polynitrogen oxide macromolecules of the present invention. The special advantages of the present invention using a polynitrogen oxide macromolecule and a low molecular weight nitrogen oxide multi-recombination system will be more clearly demonstrated later. Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) According to the spectral analysis in Figure 8, the oxidation / reduction cycle reaction involves about 73¾ of the free radicals left in 4- (2-Bromoacetamido) -TEMP0 identified HBOC. This indicates that TEMP0L participates in the in vivo oxidation / reduction reaction in the area of the vascular space. In order to study the vasoconstriction effect of a heme-based solution, a solution composed of modified human heme was used to test its effect at a complete rate. In any study that uses animals, humane procedures are used. 2-3 days before the study, Ketamine (40 mg / kg ί · π ·) was applied to the Chinese paper standard (CNS) Α4 size (210 × 297 mm) at this paper standard, printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. B7 V. Description of the invention () and acetylpromazine (0.75 mg) or sodium pentyl barbiturate (40 mg / kgi.P.) 'To anaesthetize male Sprague-Dawley rats. Medical-grade Tyson microbore (ID 0 · 05 ’0D is 0.03) inserted into the femoral artery and intravenous e-cannula will be filled and filled with heparinized» glucose, and sealed with a stainless steel needle. After a recovery period of 2-3 days, conscious animals are placed in a plastic cage. It is needed from 2-3 days of surgery, to ensure that the incision will heal before the blood transfusion. As the surgery may cause minor bleeding, it is important to allow recovery so that minor bleeding related to surgery will not be associated with One of the side effects of blood replacement is confused. Make an input pump to perform 50¾ exchange blood transfusion, and simultaneously input and oil out the test solution and blood respectively. The blood volume removed (based on the total blood volume is 12-15ml) is replaced with the test solution for about 20-30 minutes. as long as. Five hours after the transfusion, a pressure signal transfer device connected to a graphic recorder was used to monitor and record arterial blood pressure. The mean arterial blood pressure is calculated as 1/3 of the pulse pressure. Cardiac palpitations were measured as blood pressure traces. Example 3-Stabilized Heme Nitrogen Oxide Labelled Polymers Although it is feasible to make stabilized heme dimers from crosslinked monomers, it is also possible to produce heme polymers from stabilized or natural heme. It is possible. The solution of the heme polymer contains a mixture of monomers, dimers, trimers, tetramers or other oligomers. Polymerized heme-containing solutions that are used as a HBOC usually have a longer plasma circulation time and have a higher chlorine-carrying capacity than the stabilized monomeric heme. The polymerized heme can be used in many ways using different polymerizations. The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm)-61-I ----- ml-pack—- -: ---: — Order '· ------ • Line (Please read the notes on the back before filling out this page) Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 492866 A7 B7 V. Description of Invention () Reagents were prepared (see US Patent Nos. 4,001,200, 4,857,636 and 4, 8 26, 8 1 1). The preferred method of introducing nitrogen oxides into a polymerized heme solution is again by covalently attaching a nitrogen oxide to the / 3 -93 mercapto group of the two cold globulin chains of the heme. It is not known whether these neck groups are involved in stabilization or polymerization. Therefore, it is desirable to covalently bond nitrogen oxides to heme before stabilizing and polymerizing the heme monomer. For example, according to the steps described in the second embodiment above, nitrogen oxides are covalently bonded to DBBF-Hb, and then according to the steps described in U.S. Patent No. 4,826,811 to Sehgal et al. It was polymerized with glutaraldehyde. Fig. 4B is an electron rotation resonance spectrum of DBBF-Hb labeled with 3-maleimide-PR0XYL and polymerized with glutaraldehyde. Similarly, DBBF-Hb polymerized by glutaraldehyde can be labeled in the same way with 4- (2-bromoacetamido TEMPO). Using the same method, one containing unreacted aldehyde groups Polymerized heme intermediates, such as a glutaraldehyde-polymerized or 0-raffinose-polymerized heme intermediate, can be used with 4-amino-TEMPO or 3 via a reductive amination reaction. -Amine-PR0XYL is covalently bonded to produce a nitrogen oxide-labeled heme polymer. Regarding the reductive amination reaction, the order and time of the reaction are very important. The 4 > amino-TEMPO is After the polymerization reaction is completed, but before the reduction reaction that can cause the covalent attachment of the nitrogen oxide to the polymerized heme, it is added to the glutaraldehyde-polymerized heme. Similarly, 10-raffinose The nitrogen oxide label of polymerized heme can be added before the reductive amination reaction by adding 4-amino-TEMPO or this paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) -62-I ----------- ^ ---_--- τ--order ^ ----- 1 ^ 11 ^ line (please first Read the notes on the back and fill in this page) The Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs printed 492866 A7 -------- B7_ V. Invention Description () 3-Amino-PROXYL and completed. For example, 4 -Amine-TEMPO-labeled 0-raffinose polymerized heme is prepared according to the procedures described in the inventor's U.S. Patent No. 4,857,636, except that after the polymerization reaction is completed, and 6 mol equivalent of 4-amino-TEMPO was added prior to the reduction reaction using a 20 mol excess of dimethylamine boron complex. As described herein, heme can be obtained from either a disaccharide or Polyvalent aldehyde groups derived from ring-opening sugars (including oligosaccharides or trisaccharides such as 0-raffinose) are cross-linked and polymerized. Similarly, monosaccharides can also be used for stabilization And polymerized heme 'Although higher molecular weight sugars are preferred. Once diluted and washed, the resonance spectrum of the polymerized heme, which is known as 4-amino-TEMPO's 0-raffinose, is shown in Figure 9A. To increase the amount of polymerized heme oligomers (Hb „, where n = 2-4) Rate, we hope to use α-cyclodextrin, 0-cyclodextrin, and 7-cyclodextrin, and their sulfate derivatives representing 6-, 7-, and 8-fluorinated ® glucose molecules to increase the cross Valence of polyaldehyde of the crosslinking agent. The ring-opening α-cyclodextrin, / 3-pyridoxin and 7-pyridoxin have 12, 14 and 16 reactive aldehyde groups, respectively. Crosslinkers can be used to cross-link and polymerize heme to make its polymerized heme rich in oligomers. As previously mentioned, unreacted aldehydes can be utilized to covalently bond to monoamine- Samarium oxide (eg 4-amino TEMPO or 3-amino-PR0XYL. What's more, ring-opened sulfate derivatives (such as sulfated α-cyclodextrin) can be an effective cross-linking agent for two additional reasons: (1) the reduced, cross-linked heme gas In terms of gas affinity, the sulfuric acid group will simulate the activity of DPG, thus improving the oxygen transport properties; and (2) these sulfuric acid groups will be in accordance with the Chinese National Standard (CNS) A4 specification (210 × 297 mm) on this paper scale --- ------ install --- ^ --- ^ order j ------ line (please read the precautions on the back before filling this page) 492866 Printed by the Consumers Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention () Provide the affinity tag of heme which can be compounded multiple types (for example, n = 2-4) to initially form a cluster. Once the and cluster complexes are formed, the aldehyde groups on the cyclodextrin will be brought close to the amine groups in the DPG positions, thus promoting the subunits of heme. Covalent cross-linking with molecules, which results in an increased yield of one of the heme oligomers. In addition to the activity of antioxidant enzyme mimetics, the ring-opening gelatin polymerization and nitrogen-labeled heme will also have improved properties compared to 0-raffinose and glutaraldehyde polymerized heme * Yield and composition. Example 4 A nitroxide-labeled liposome-encapsulated heme liposome is pseudo-particles formed by the aggregation of amphiphilic molecules to form a double-layered structure in the form of a hollow sphere, opposite to its polarity An internal water area and a large amount of water on the outside. Several acceptable methods for forming liposomes are known to those skilled in the art. Molecules like dipalmitin phospholipids and choline typically form liposomes in aqueous solution. Liposomes can be formulated with cholesterol for added stability, and can include other substances such as neutral lipids and surface modifiers (such as positively or negatively charged compounds). The preferred liposome is a small single thin plate bilayer (u n i 1 a m e 1 1 a r-b Π a y e r e d) spherical shell. A method for encapsulating heme in a liposome is also known (Farmer et al., U.S. Patent No. 4,911,921). For the purpose of the present invention, a number of methods can be used to introduce the detoxification function of chlorine gas, mainly nitrogen oxides, into a liposome-encapsulated heme solution. For example, it is possible to use " nitrogen oxide-labeled natural heme or a nitrogen oxide-labeled stabilized heme as described above as the starting material, and then use conventional techniques to apply this paper standard to the Chinese country Standard (CNS) A4 specification (210X297 mm) 1 · ml §am§M§ —a— ernnkwrEB I— ml ϋϋ an · -— — ^ ϋ_4 Γ y ^ in · — ^ i (Please read the notes on the back first Fill out this page again) Line 492866 A7 B7 Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of the invention () Liposome encapsulation of the nitrogen oxide-labeled heme. In this embodiment, the purified heme can also be contacted with a membrane that is impermeable to nitrogen oxides, such as the TEMPO- Choline chloride is encapsulated together. In addition, any purified heme can be a nitrogen oxide-labeled fatty acid (such as 7-DOXPL-stearic acid, 12-DOXYL-stearic acid and 16-DOXYL-stearate), cholestane, Liposomes composed of a cholesterol analog (such as 3-DOXYL-cholestane) or a base lipid (such as 12-DOXYL-stearic acid-labeled phosphatidylcholine) are encapsulated. A method similar to that described in Tabushiet et al. [L. Am. Soc, 106: 219 (1984)] can be used to prepare a liposome encapsulated with a 3-DOXYL-cholestane marker Heme. As described below, a 5 ml 1 ammonium-containing solution containing a lipid composition (including stearic acid and / or cholestane labeled with DOXYL) was first dried in a nitrogen stream to remove the solvent. Then, the residue was vacuum-dried, and the formed film was re-suspended in 2 ml of heme (24 g / dl) in lactated Ringer's solution. The lipid concentration in this dispersion was 100 mM. The liposome encapsulates the heme and is then rotated and, preferably, incubated at 37 ° C until all the lipids are dispersed to form a multiple thin plate carrier. Then, the resulting solution containing multi-lamellar liposome-encapsulated heme and free unencapsulated heme was forced to flow through a microflow according to the procedure of Cook et al. (See US Patent No. 4,533,254). Bed to form 0.2 mm rice liposomes. The ratio of the dipalmitinphospholipid and choline in the liposome: ^ cholesterol: dibromophospholipid acid: 3-D0XYL-cholestane is 0.5: 0, 4: 0.02: 0.07. The formed 3-D0XYL-cholestane-labeled liposomes-(Please read the note on the back ▼ item before filling. Packing --- ^ ---: " I Order: Write this page)-Threadbook Paper size applies to China National Standard (CNS) A4 (210X297 mm) ^ «66

經濟部中央標準局員工消費合作社印製 i、發明説明（）包囊化血紅素之共振光譜被示於第10 A圖。在此構形中，該氮氣化物被***在該脂質體膜上，且在該脂質體的雙層水交界之内表面及外表面處皆可發現之。以16-DOXYL硬脂酸來取代第10A圖中所示的脂質組成物内之3-D0XYL-膽g 烷，會形成於第10B圔所示的電子共振光譜。由共振光譜所反映出的氮氧化物之移動性，與該硬脂酸的DOXYL-部份主要是位在該脂質雙層物的疏水性内部之解釋相符。以相同的莫耳比例將3-D0XYL膽甾烷及16-D0XYL硬脂酸加入該脂質組成物中，該雙重氮氧化物-標識的脂質體包囊化血红素之共振光譜被示於第10C圖中。第10C圖的共振光譜是第10A及10B圖的複合體，因為在此實施例中的氮氧化物是位在該膜-水交界處及其疏水性脂質雙層内部。藉由將氮氧化物置於該二位置.，此實施例在該脂質雙層疏水性内部與膜-水交界處提供氧氣解毒功能，因此對該包囊化血紅素提供氧氣解毒能力之一額外保留的附加利益。實施例5 --氮氧化物-標識的共鈪化血紅素一由共軛化血红素所構成之生理上可相容的溶液之製備像藉由形成一由血紅素及一被用作為血漿膨脹劑之生物可相容的巨分子之共軛物。血漿膨脹劑，例如葡聚糖（D X) 、聚璟氧乙烷（Ρ0Ε)、羥乙基澱粉（HES)，被用來增加血红素在身體内的循璟半衰期。在此狀態中，血红素分子和該生物可相容的巨分子一起被共同地稱之為血紅素共軛物。有許多方便的方法可將一氮氧化物併入至一血紅素共軛物内。例如，有一値方法是簡單地以一氮氧化物-標識的血本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事_ 蜂項再填· 裝-- 寫本頁) 訂線 492866 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（）红素（例如如TEMPO-PROXYL標識DBBF-Hb)來取代欲予以共軛化的血紅素。這可藉由在共軛化血紅素之製備中，以3-馬來醯亞胺-PR0XYレDBBF-Hb或4-(2-溴乙醯胺基）-TEMP0-DBBF-Hb來取代血紅素或吡哆醛基化血红素，而被完成之。 4-胺基-TEMPO標識的》聚糖共軛化血紅素像依據Tam et a 1. , Proc. Natl . head. Sci.· U: 2 1 28 (1 976))所述的步驟而予以製備的。初始時，一配於0. 15M NaCl及5mM 磷酸緩衝液（pH 7.4)内之8%血紅素溶液被共軛化至過碘酸-氣化的葡聚糖以形成一席夫鹼（Sch iff-base)中間産物。 20莫耳當量的4-胺基-TEMPO被加入至血紅素中以便在氮氧化物及位在該«聚糖上之其餘的反應性醛基圍之間形成席夫鹼。在4°C下培育30分鐘後，一為50莫耳當量且配於水内之二甲基胺硼烷被加入之。該溶液在4°C下再予以培育另2値小時。此後，該溶液被透稀，以乳酸化林格氏緩衝液予以重建，並以Filtron膜過濾單位（Filtron Technology Co.)與予以無菌過濾處理◦該4-胺基-TEMPO標識的Μ聚糖-共軛化血紅素的電子旋轉共振光譜為非常不對稱的三條，這反映其具有一高度的移動自由度（參見第11圖）。被共價地接合至葡聚糖的該TEMPO之增加的移動度，相較於一緊密摺疊的血红素分子，之移動度相比較，相符於被鍵結至一可撓性聚_«聚糖鏈之氮氣化物（參見第3 A及3B圖）。因此，第11圖之共振光譜證實一新穎的氮氧化物-標識的《聚糖共軛化血紅素已被製備出。實施例6 --氮氧化物-標識的白蛋白 (請先閱讀背面之注意事項再填寫本頁) 裝---^---^—訂.------線本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）- 67 - — 492866 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（）本發明之一較佳的實施例是氮氧化物-標識的生物可相容的巨分子與低分子量膜可通透的氮氧化物之合併使用俾以於活體内提供維續的抗氧化活性。較佳地，該氮氧化物藉由在該蛋白質之大部分胺基處進行標識，而被標識至一生物可相容的蛋白質或其片段。另外，在雙硫鍵處之標識增加該氮氧化物對該蛋白質之莫耳比例。藉由如此標識該蛋白質/片段，一會增進自由的氮氧化物與巨分子-結合的氮氧化物之間的相互反應之酸性微環境被生成之，這促進了因該等物種之有別的穩定性所致的電子旋轉轉移。就白蛋白而言，可以藉由活化TOPS將一氮氣化物共價地接合至白蛋白之主要膽红素結合位。藉由選擇位在該巨分子上之結合位置，氮氧化物之反應力被修飾，而此修飾可被用來改變該化合物之催化活性。這樣一個所想要的生物可相容的巨分子之實例是人類血清白蛋白（H S A)。血清白蛋白係為一種具有多個配位子-結合位置之血漿蛋白，且為有關位於血液内的許多配位子之輸送蛋白。氮氧化物可在許多的特殊配位子結合位置處被專一性地結合至人類血清白蛋白。氮氧化物-白蛋白可單獨地或加上一種低分子量氮氧化物化合物（例如TEMP0L)而被使用之。氮氣化物-標識的白蛋白在許多其中白蛋白現在被慣例地使用之應用（包括作為一腸道外膠體溶液、生物材料、生物可相容的表面塗層等等）上，亦可供作為具有實用性之一 a改良的〃販本的白蛋白（亦即藉由具有抗氧化活性而本紙張尺度適用中國國家標準（CNS ) Α4規格（210X297公董） ------辞I---,I--I (請先閲讀背面之注意事項再填寫本頁) 492866 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（）被改良）〇白蛋白可得自於血漿或可利用基因重組技術來製備之。HSA可以各種不同的形式，例如單體（正常的血發形式）、同型二聚物、寡聚物和聚集物（微球體），而被使用之。次又，白蛋白可用聚乙二醇予以處理俾以降低其免疫原性。可在數痼結合位置（包括膽紅素、FFA、蚓跺或Cii"結合位），藉由使用其已被活化俾以對位在蛋白質上的相關位置提供其結合專一性的氮氧化物化合物，來逹成白蛋白與氮氧化物之專一性標識。一較佳的實例是被共價地接合至 HSA的主要膽红素結合位之2,2,6,6-四甲基-卜羥氣基-4-哌啶叉琥珀酸（TOPS)。白蛋白之非-專一性標識可在大約 5 0種可接近的胺基基團處被達成之。白蛋白的溫度及化學處理容許氮氧化物對白蛋白的莫耳比例之增高。使用天然的白蛋白，可達到7以至60之莫耳比。使用經修飾的白蛋白，可達到高至95的氮氯化物對白蛋白的莫耳比。為了證實活體内有活性的氮氧化物之再生，配於磷酸緩衝的生理鹽液内之4-羥基-2, 2,6,6-四甲基六氫吡啶-N-羥氧基（TEMP0L)被注射至一被麻醉的小白鼠（體重40g)之尾部靜脈内以作為一對照組。該尾部之尖端被***至一電子旋轉共振（ESR)光譜計之樣品試管内。在注射之前，該小白鼠尾部顯示無esr信號。在0.5ml的6mM之注射後，偵測到一個e s r信號並觀察到其快速地衰減，這可從以3 0秒間隔來作記錄的光譜之3個連續掃描中看出（參照第18圖）。此結果顯示出該TEMP0L被還原成其esr-無活性的且無催本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） m· ΒιϋΒΙ nm iMI —i—ϋ I 1.1^1 am mu im§ \ V ^ US, (請先閱讀背面之注意事項再填寫本頁) 492866 經濟部中央標準局員工消費合作社印製 A7 B7 i、發明説明（）化活性的羥基胺U-NOH)衍生物（TEMPOL-H或T.PH)。為偵測 TEMP0L在小白鼠尾部内之血漿半衰期，連續地監測高視野波峰之強度歴時8分鐘（第19圖）。在8分鐘後，做一重複的 TEMP0L注射（參照第7B與7C圖）。在大約10秒内於小白鼠尾部得到其相應於TEMPOL (TPL)濃度之波峰強度的最大值，而後衰減至基線值，形成TEMP0L在活體内之半衰期約為50 秒（第19圔）。利用兩種方法來確認TEMPOL的半衰期：在間期下掃描整個光譜並連續的記錄高視野波峰之強度。源自該二方法之結果像為一致的。為了製備聚氮氧化物白蛋白（PNA)，令人類血清白蛋白（HSA，5¾溶液，Baxter Healthcare Corp.)與 6 莫耳當量的Br-TEMPO (Sigma)在60°C下反應S攪拌歷時10小時。對位於一 Vacutainer試管内之所形成的混合物（含有15ml 的HSA以及165mg的Β「-ΤΕΜΡ0)，使用一個0.22微米過濾器予以無菌過濾處理並轉移至一個配備有一痼lOkDa截留值超過據膜（Filtron Technology Corp.)之 150ml 之搜拌室 (St irredCel 1 Device)内。以林格氏液清洗該經過濾的反應混合物直至濾液，如藉由esr光譜學所偵測到的，含有低於ΙιηΜ自由的TEMPO。將亮橘色滯留液濃縮至25%HSA，並再次以一個0.22微米過濾器將之無菌過濾處理至10ml無菌管瓶（Abbott Laboratories)内，並儲存於4°C下直到使用時。巨分子聚氮氣化物之esr光譜示於第15C圖中。為增加氮氧化物在一聚氮氣化物白蛋白内之莫耳比，在尿素之存在下將雙硫鍵予以還原成競基乙酸鈉，並且予本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） m> ϋ-ϋ mmmimMt I —ΡΒϋ —Β_ιϋ m 1>- 1_ι^ϋ ϋϋ nail· m.— ^ V ·111 (請先閱讀背面之注意事項再填寫本頁) 492866 經濟部中央標準局員工消費合作社印製 A7 B7 __五、發明説明（）以加人一過量的Br-TEMPO以標識該斷開的雙硫鍵。藉由此方法，達到一平均約為10之莫耳比之增加，這類似於Chan et al., "Potential of Albumin Labeled with Nitroxi des as a COntrast Agent for Magnetic Resonance Imaging and Spectroscopy, TT B i c o nJ_u gate Chemistry, 1990，vol. U 32 -36中所描述之方法。一聚氮氧化物白蛋白溶液之濃度在一燒瓶内被調整至 17. 5mg/ml。此溶液以8M尿素予以稀釋並攪拌直至溶解。藉由加入2¾碳酸鈉將pH值調整至8.2。該pH值調整過的溶液予以完全地抽氣歴時1 5分鐘。接著，將其充塞以氬氣。在另一燒瓶中，製備一為3M的巯基乙酸鹽溶液，予以抽氣並充塞以氬氣。將該巯基乙酸鹽溶液加入至該聚氮氧化物白蛋白内直至最終濃度為0.3M。所形成的溶液予以抽氣並對混合物充塞以氬氣，並容許其在室溫及氬氣下於黑暗中靜置歴時20小時，而後藉由在室溫及氬氣下對3L的抽氣過的磷酸鹽-緩衝的生理鹽液 (pH 8.4，藉由2¾碩酸鈉來調整）進行透析歴時5小時。予以加入一過量的Br-TEMPO並在室溫及氬氣下再攪拌該混合物又24小時。令最終溶液對PBS (pH 7.4)進行透析歴時5小時以移除未反應的氮氧化物。可使用EPR光譜學來測定旋轉密度並可使用雙縮脲法來測定蛋白質。典型的結果敘述如下，其係有關3組之平均值： 1 .蛋白質濃度： (請先閲讀背面之注意事 #1 ，項再填· 裝 —--_--- 寫本頁) |線本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -71 _ 經濟部中央標準局員工消費合作社印製 492866 A7 B7 __ 五、發明説明（）胺基基團標識的聚氮氧化物白蛋白 53. Oing /πιΙ^Ο. 78πιΜ 胺基加上雙硫鍵Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs i. Description of the invention () The resonance spectrum of encapsulated heme is shown in Figure 10A. In this configuration, the nitrogen compound is inserted on the liposome membrane and can be found on the inner and outer surfaces of the bilayer water junction of the liposome. Substituting 16-DOXYL stearic acid for 3-D0XYL-cholin in the lipid composition shown in Fig. 10A will form an electron resonance spectrum shown in Fig. 10B 圔. The mobility of the nitrogen oxides reflected by the resonance spectrum is consistent with the explanation that the DOXYL- part of the stearic acid is mainly located inside the hydrophobicity of the lipid bilayer. 3-D0XYL cholestane and 16-D0XYL stearic acid were added to the lipid composition at the same molar ratio. The resonance spectrum of the double nitrogen oxide-labeled liposome-encapsulated heme is shown at 10C. In the figure. The resonance spectrum of Fig. 10C is a complex of Figs. 10A and 10B because the nitrogen oxide in this example is located at the membrane-water junction and inside the hydrophobic lipid bilayer. By placing the nitrogen oxide in these two positions, this embodiment provides an oxygen detoxification function at the junction between the hydrophobic interior of the lipid bilayer and the membrane-water interface, thus providing one of the oxygen detoxification capabilities of the encapsulated heme with additional retention. Additional benefits. Example 5-Preparation of a Nitrogen Oxide-labeled Conjugated Heme-A Physiologically Compatible Solution Consisted of Conjugated Heme Biocompatible macromolecular conjugates of agents. Plasma bulking agents, such as dextran (DX), polyoxyethylene (POE), and hydroxyethyl starch (HES), are used to increase the half-life of hemoglobin in the body. In this state, the heme molecule and the biocompatible macromolecule are collectively referred to as a heme conjugate. There are many convenient ways to incorporate a nitrogen oxide into a heme conjugate. For example, there is a method to simply use a nitrogen oxide-labeled blood paper size to apply the Chinese National Standard (CNS) A4 specification (210X297 mm) (please read the precautions on the back _ bee item before filling and loading- (Write this page) Line 492866 Printed by A7 B7 of the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention () Heme (such as TEMPO-PROXYL label DBBF-Hb) is used to replace the heme to be conjugated. This can be done by replacing the heme with 3-maleimidine-PR0XYréDBBF-Hb or 4- (2-bromoacetamido) -TEMP0-DBBF-Hb in the preparation of conjugated heme. Or pyridoxal can be heminized and completed. 4-Amino-TEMPO-labeled polysaccharide-conjugated heme images were prepared according to the procedures described in Tam et a 1., Proc. Natl. Head. Sci. · U: 2 1 28 (1 976)) of. Initially, an 8% heme solution in 0.15M NaCl and 5mM phosphate buffer (pH 7.4) was conjugated to periodic acid-gasified dextran to form a Schiff base (Sch iff- base) Intermediate. 20 mol-equivalent 4-amine-TEMPO was added to the heme to form a Schiff base between nitrogen oxides and the remaining reactive aldehyde groups on the polysaccharide. After incubation at 4 ° C for 30 minutes, a 50 mol equivalent dimethylamine borane in water was added. The solution was incubated at 4 ° C for another 2 hours. After that, the solution was diluted and reconstituted with lactated Ringer's buffer, and was filtered with Filtron Membrane Filtration Unit (Filtron Technology Co.) and sterile filtered. The 4-amino-TEMPO-labeled M-glycan- The electronic rotational resonance spectrum of the conjugated heme is a very asymmetrical three, which reflects that it has a high degree of freedom of movement (see Figure 11). The increased mobility of the TEMPO, which is covalently bonded to dextran, compared to the mobility of a tightly folded heme molecule, is consistent with being bonded to a flexible poly_ «glycan Nitride of the chain (see Figures 3 A and 3B). Therefore, the resonance spectrum of Figure 11 confirms that a novel nitrogen oxide-labeled glycan conjugated heme has been prepared. Example 6-Nitrogen oxide-labeled albumin (please read the precautions on the back before filling out this page) National Standard (CNS) A4 Specification (210X297 mm)-67--492866 A7 B7 printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of the Invention () One of the preferred embodiments of the present invention is the nitrogen oxide-label The combination of biocompatible macromolecules and low molecular weight membrane permeable nitrogen oxides is used in combination to provide sustained antioxidant activity in vivo. Preferably, the oxynitride is identified to a biocompatible protein or a fragment thereof by identifying at most of the amine groups of the protein. In addition, the identification at the disulfide bond increases the mole ratio of the nitrogen oxide to the protein. By identifying the protein / fragment in this way, an acidic microenvironment that promotes the interaction between free nitrogen oxides and macromolecule-bound nitrogen oxides is generated, which promotes differences due to these species. Electron rotation transfer due to stability. In the case of albumin, a nitride can be covalently attached to the main bilirubin binding site of albumin by activating TOPS. By selecting the binding position on the macromolecule, the reactive power of the nitrogen oxide is modified, and this modification can be used to change the catalytic activity of the compound. An example of such a desired biocompatible macromolecule is human serum albumin (HSA). Serum albumin is a plasma protein with multiple ligand-binding sites, and is a transporter for many ligands located in the blood. Nitrogen oxides can be specifically bound to human serum albumin at many specific ligand binding sites. Nitrogen oxide-albumin can be used alone or in combination with a low molecular weight nitrogen oxide compound (e.g., TEMP0L). Nitride-labeled albumin is also useful in many applications where albumin is now routinely used (including as a parenteral colloid solution, biomaterials, biocompatible surface coatings, etc.) One of the improved a albumin albumen (that is, by virtue of its anti-oxidant activity, the paper size applies the Chinese National Standard (CNS) A4 specification (210X297 public director) ------- I --- , I--I (Please read the notes on the back before filling this page) 492866 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention () Improved) 0 Albumin can be obtained from plasma or can be It is prepared using genetic recombination technology. HSA can be used in a variety of forms, such as monomers (normal blood forms), homodimers, oligomers, and aggregates (microspheres). Secondly, albumin can be treated with polyethylene glycol to reduce its immunogenicity. Provides specific nitrogen oxide compounds at several binding sites (including bilirubin, FFA, earthworm, or Cii " binding sites) by using them that have been activated to parasite relative positions on the protein , To form a unique identification of albumin and nitrogen oxides. A preferred example is 2,2,6,6-tetramethyl-hydroxyl-4-piperidineidenesuccinic acid (TOPS), which is covalently linked to the main bilirubin binding site of HSA. The non-specific identification of albumin can be achieved at about 50 accessible amine groups. The temperature and chemical treatment of albumin allow the molar ratio of nitrogen oxides to albumin to increase. Using natural albumin, a molar ratio of 7 to 60 can be achieved. With modified albumin, a molar ratio of nitrogen chloride to albumin up to 95 can be achieved. In order to confirm the regeneration of active nitrogen oxides in vivo, 4-hydroxy-2, 2,6,6-tetramethylhexahydropyridine-N-hydroxyoxyl (TEMP0L) was formulated in phosphate-buffered physiological saline. A control group was injected into the tail vein of an anesthetized mouse (weight 40 g). The tip of the tail was inserted into a sample test tube of an electronic rotation resonance (ESR) spectrometer. Prior to injection, the tail of the mouse showed no esr signal. After the injection of 0.5 ml of 6 mM, an esr signal was detected and its rapid decay was observed, which can be seen from three consecutive scans of the spectrum recorded at 30 second intervals (refer to Figure 18) . This result shows that the TEMP0L is reduced to its esr-inactive and non-reactive paper sizes. Applicable to China National Standard (CNS) A4 specifications (210X297 mm) m · ΒιϋΒΙ nm iMI —i—ϋ I 1.1 ^ 1 am mu im§ \ V ^ US, (Please read the notes on the back before filling out this page) 492866 A7 B7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs i. Description of the invention (Hydroxyamine U-NOH) derivatives (TEMPOL-H or T.PH). In order to detect the plasma half-life of TEMP0L in the tail of mice, the intensity of high-field peaks was continuously monitored for 8 minutes (Figure 19). After 8 minutes, repeat the TEMP0L injection (see Figures 7B and 7C). The maximum peak intensity corresponding to the concentration of TEMPOL (TPL) was obtained in the tail of the mouse in about 10 seconds, and then decayed to the baseline value to form a half-life of TEMP0L in vivo of about 50 seconds (19th). Two methods are used to confirm the half-life of TEMPOL: the entire spectrum is scanned at intervals and the intensity of high-field peaks is continuously recorded. The results from these two methods seem to be consistent. In order to prepare polynitrogen oxide albumin (PNA), human serum albumin (HSA, 5¾ solution, Baxter Healthcare Corp.) was reacted with 6 mol-equivalent Br-TEMPO (Sigma) at 60 ° C and stirred for 10 hours. hour. The resulting mixture (containing 15 ml of HSA and 165 mg of Beta-TEPPO) in a Vacutainer test tube was sterile-filtered using a 0.22 micron filter and transferred to a device equipped with a cut-off value of lOkDa exceeding the membrane (Filtron Technology Corp.) in a 150 ml search chamber (StirredCel 1 Device). The filtered reaction mixture was washed with Ringer's solution until the filtrate, as detected by esr spectroscopy, contained less than 1 μM free TEMPO. The bright orange retentate was concentrated to 25% HSA, and again sterile-filtered into a 10 ml sterile vial (Abbott Laboratories) with a 0.22 micron filter and stored at 4 ° C until use. The esr spectrum of the macromolecular polynitrogenate is shown in Figure 15C. In order to increase the molar ratio of nitrogen oxides in a polynitrogenate albumin, the disulfide bond was reduced to sodium sodium acetate in the presence of urea, And this paper size applies Chinese National Standard (CNS) A4 specifications (210X297 mm) m > ϋ-ϋ mmmimMt I —ΡΒϋ —Β_ιϋ m 1 >-1_ι ^ ϋ ϋϋ nail · m.— ^ V · 111 (Please read the notes on the back before filling this page) 492866 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 __V. Description of the invention () Add an excess of Br-TEMPO to identify the disconnected Disulfide bond. By this method, an average mole ratio increase of about 10 is achieved, which is similar to Chan et al., &Quot; Potential of Albumin Labeled with Nitroxi des as a COntrast Agent for Magnetic Resonance Imaging and Spectroscopy, The method described in TT B ico nJ_u gate Chemistry, 1990, vol. U 32-36. The concentration of a polynitrogen oxide albumin solution was adjusted to 17.5 mg / ml in a flask. This solution was administered with 8M urea. Dilute and stir until dissolved. The pH is adjusted to 8.2 by adding 2¾ sodium carbonate. The pH adjusted solution is completely aspirated for 15 minutes. Then, it is stuffed with argon. In another flask In the preparation, a 3M thioglycolate solution was prepared, evacuated and stuffed with argon. The thioglycolate solution was added to the polynitrogen oxide albumin until the final concentration was 0.3M. The resulting solution was Pump down and stuff the mixture with argon, and allow it to stand for 20 hours at room temperature and under argon in the dark, then by pumping 3L of phosphate- Buffered physiological saline (pH 8.4, adjusted by sodium bisulfate) for 5 hours. An excess of Br-TEMPO was added and the mixture was stirred at room temperature under argon for another 24 hours. The final solution was dialyzed against PBS (pH 7.4) for 5 hours to remove unreacted nitrogen oxides. Spin density can be measured using EPR spectroscopy and proteins can be measured using the biuret method. The typical results are described as follows, which are the average values of the three groups: 1. Protein concentration: (Please read the note # 1 on the back, and then fill in and fill out --- --- --- write this page) | Paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) -71 _ Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 492866 A7 B7 __ V. Description of the invention () Amino group-labeled polynitrogen oxide white Protein 53. Oing / πιΙ ^ Ο. 78πιΜ amino group plus a disulfide bond

13.9mg/inl=〇.20mM 2 ·旋轉密度：13.9mg / inl = 0.20mM 2 · Rotation density:

胺基基團標識的聚氮氧化物白蛋白 33mM 胺基加上雙硫鍵Polyamine oxide albumin labeled with amine group 33mM amine group plus disulfide bond

10 . 5mM 3 ·結合至蛋白質的氮氧化物之莫耳比的計算：胺基基團標識的聚氮氣化物白蛋白 33mM/0.78mM = 42氮氣化物/白蛋白胺基加上雙硫鍵 10.5mM/0.20niM = 52氮氧化物/白蛋白在此實施例中，該莫耳比增加1 0。為證實一聚氮氧化物-標識的巨分子增強該低分子量膜可通透的氮氧化物活體内活性，人類血清白蛋白被共價地標識以4-(2-溴乙醯胺基:)-TEMP0 (Br-TEMPO)，並在一劑量的TEMPO已被投藥後，將之輸入，並觀察到其被轉化成其還原狀態。如同上述之實施例，在TEMPO的注射後2小時，小白鼠尾部顯示出偵測不到esr信號。當0 . 2m 1的聚氮氯化物白蛋白經由尾部靜脈被注入後，於4分鐘内載出現TEMPO信號。該TEMPO信號強度持續超過2小時，具一約為40分鐘之半衰本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -72 - (請先閲讀背面之注意事項再填寫本頁) • - 裝---；---r丨訂 1 ----^線經濟部中央標準局員工消費合作社印製 492866 A7 B7 五、發明説明（）期（請亦參照第18圖）。藉由其等明顯有別的光譜圖形，該 TEMPO信號與聚氮氧化物白蛋白信號可相區別。在聚氮氣化物白蛋白之注射後偵測到的esr信號，全然是基於位在該巨分子上與該巨分子分離的氮氧化物之故此一可能性，利用下面實驗，而被排除了。如前述來進行此實驗，但使用P 5N] -TEMPO (參照第4C圖）以及白蛋白-[i 1] -TEMPO (參照第5C圖）。該等不同的氮物種提供了區別自由的與巨分子的氮氧化物之esr光譜之方法。該等結果（第18圖）確認了源自再生的氮氧化物之es「信號是衍生自1 5N同位素，而因此該低分子量膜可通透的P 5N] -TEMPO 之抗氧化活性在該巨分子聚氮氣化物之加入後被再生之。實施例7 —氮氧化物-標識的免疫球蛋白如上所述的，某些氮氣化物，當其被靜脈内注射時，已被顯示出具有非常短的血漿半衰期。由於想要具有一種具有一長血漿半衰期之抗氧化酵素模擬物，一氮氧化物化合物可被接合至一免疫球蛋白以提供長久的抗氧化酵素模擬物活性。免疫球蛋白係為一群由免疫条統的B細胞所製造的血漿蛋白，且其待徵在於具有兩個專一性配位子結合位（抗原-結合位）。氮氧化物在過去針對原發性及續發性免疫反應期間，免疫球蛋白的半抗原-結合專一性與親和力的研究中，已被用來作為探針之用。關於前面敘述氮氣化物-檫識的白蛋白之實施例，在前面氮氧化物-Η B 0 C之實施例中所證明的本案氛氧化物-標本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）辞l—丨丨丨丨—、可-----i---- (請先閲讀背面之注意事項再填寫本頁)10. 5mM 3 · Calculation of the molar ratio of nitrogen oxides bound to the protein: Amine-labeled polynitrogen albumin 33mM / 0.78mM = 42 nitrogen compounds / albuminamine plus disulfide bond 10.5mM /0.20niM = 52 nitrogen oxides / albumin In this example, the molar ratio is increased by 10. To confirm that a polynitrogen oxide-labeled macromolecule enhances the in vivo activity of nitrogen oxides permeable to this low molecular weight membrane, human serum albumin was covalently labeled with 4- (2-bromoacetamido :) -TEMP0 (Br-TEMPO), and after a dose of TEMPO has been administered, it is entered and observed to be converted to its reduced state. As in the above example, two hours after the injection of TEMPO, the tail of the mouse showed no detectable esr signal. When 0.2 m 1 of polynitrogen chloride albumin was injected through the tail vein, a TEMPO signal appeared within 4 minutes. The TEMPO signal strength lasts for more than 2 hours and has a half-decay of about 40 minutes. The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) -72-(Please read the precautions on the back before filling this page ) •-installed ---; --- r 丨 Order 1 ---- ^ Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 492866 A7 B7 V. Description of the invention () Period (please also refer to Figure 18). With their distinctly different spectral patterns, the TEMPO signal can be distinguished from the polynitrogen oxide albumin signal. The esr signal detected after the injection of polyazide albumin is entirely based on the nitrogen oxides that are separated from the macromolecule on the macromolecule. This possibility was eliminated using the following experiment. This experiment was performed as described above, but using P 5N] -TEMPO (see Figure 4C) and albumin- [i 1] -TEMPO (see Figure 5C). These different nitrogen species provide a way to distinguish free and esr spectra of macromolecular nitrogen oxides. These results (Figure 18) confirm that the es derived from regenerated nitrogen oxides "the signal is derived from the 1 5N isotope, and therefore the low molecular weight membrane is permeable to P 5N] -TEMPO. The molecular polynitride is regenerated after it is added. Example 7-Nitrogen oxide-labeled immunoglobulins As described above, certain nitrogen compounds, when injected intravenously, have been shown to have very short Plasma half-life. Since it is desired to have an antioxidant enzyme mimetic with a long plasma half-life, a nitrogen oxide compound can be conjugated to an immunoglobulin to provide long-term antioxidant enzyme mimetic activity. The immunoglobulin family is a group Plasma proteins produced by B-cells of the immune system and characterized by having two specific ligand binding sites (antigen-binding sites). Nitrogen oxides have been used in the past to target primary and secondary immune responses In the meantime, studies on hapten-binding specificity and affinity of immunoglobulins have been used as probes. Examples of nitrate-cognized albumin described above In the previous examples of nitrogen oxides-Η B 0 C, the atmospheric oxides-specimen paper size of this case applies the Chinese National Standard (CNS) A4 specification (210X297 mm). ---- i ---- (Please read the notes on the back before filling this page)

492866 五、發明説明（）識技術，可被容易地應用在氮氧化物-標識的免疫球蛋白之製造。免疫球蛋白具有專一性結合與長循環半衰期之優點，這使得本發明化合物之酵素模擬物活性可被標的至待殊的組織並且具有延長的活性。氮氧化物-標識的免疫球蛋白可被用於活體内以提供對抗由反應性氧物種所引起細胞性損害°氮氧化物—標識的免疫球蛋白可單獨地或與一低分子量氮氧化物組合而被使用之，俾以提供具一延長的血漿半衰期之抗氧化活性° 氮氧化物-標識的免疫球蛋白之製備可藉由該免疫球蛋白本身之專一性標識或藉由在一半抗原_結合位處之共價地標識。為避免氮氧化物-標識的免疫球蛋白之清除以為部分的身體之天然免疫反應，熟習此藝者可使用藉由習知技術來分解免疫球蛋白而生成的免疫球蛋白片段，例如 (Fab) 2，加上非-專一性-標識，一較佳的氮氧化物：免疫球蛋白之莫耳比可高達60 :1。對於供標的化一特定位置的酵素模擬物作用之用，氮氧化物標識的免疫球蛋白傺為一較佳的物種。例如，藉由選擇對一被認為與發炎或其他類似病狀有麗的抗原具有專一性之抗體，本發明之顯影增進效用及治療優點可被標的至一待定位置處。實施例8 —生物結構之顯影以及利用ESR的自由基反應在大多數情況下，自由基反應發生偽如此地快速以致於無法做EPR顯影（ERI)。但是，由於穩定的自由基之存在之故，氮氧化物可利用電子旋轉共振光譜學來作偵測。在 -74 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） ---------裝-- (請先閱讀背面之注意事項再填寫本頁) J— T - i 經濟部中央標準局員工消費合作社印製 492866 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（）現代的顯影儀器設備之發展下，依據一自由基濃度之一測定，可以進行整體的生物組織與器官之顯影。由於氮氧化物在投藥的數分鐘之内，於活體内被還原成無活性的衍生物，其實用性偽有限的。依據本發明，身體内有活性的氮氣化物位準可被維持一段延長的時間而容許較僅使用低分子量且膜可通透的氮氧化物時所看到的，要為改善的顯影對比以及較長的信號持續性。電子順磁性共振（EPR)光譜學係為一種藉由偵測不成對的電子在一磁場之存在下的能量狀態來觀察自由基之行為。此技術對於自由基而言偽為專一性的，因為僅有不成對的電子會被偵測到。使用其可測定電子順磁性共振的可用裝置，可獲得一巨觀的標的（包括活組織）之現時的顯影。EPR顯影（ERI)對於診斷或研究，提供了獲得多維度顯影 (包括光譜-空間顯影）之能力。使用氮氧化物對比劑的電子順磁性共振顯影（ER I)對於醫學顯影，大體上像一有價值的方法，特別是局部缺血的組織之顯影。但是，此技術之發展受限於氮氧化物在活體内會被快速地還原成非-順磁性物種。有關外源引入的氮氯化物之應用具有已證實的實用性以作為活體内之一相當低解析度的EPR顯影劑。參見L. J. Berliner, M Applications of In Vivo EPR,,T pp. 292-304 in EPR IMAGING AND IN VIVO EPR, G.R. Eaton, S.S· Eaton, K· Ohno, editors; CRC Press (1991)。重要地，Subrainanian及其同事建立了一種用以偵測自由基本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) -i§ iM9mt I ·ϋϋ m·^ mu-*J, Γ—ϋ ϋ«^— . - i 線 492866 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（）物種及活體内顯影之無線電頻傅立業（four ier)轉移EPR光譜計。參見 J· Bourg· et al., J. Mag. Res· B102，112 -115(1993)。一依據本發明製備的聚氮氧化物白蛋白（PNA)被投藥而分佈至血管及其他細胞外間隙。雖然本發明的組成物之濃度範圍可變化，一較佳的範圍是5-25g標識的PNA/dl以及0.卜200inM TEMP0L。利用電子順磁性共振（EPR)光譜學的抗氧化活性和可偵測性，對此目的而言是有用。另外，當被投藥以溫和劑量的小型低分子量膜可通透的氮氧化物時，該氮氧化物於身體内被維持在一有活性的自由基狀態歷時一段延長的時間。關於ER I顯影之一較佳的配方是被共價地標識以一高莫耳比的氮氧化物（7-95的氮氧化物對白蛋白莫耳比）之人類血清白蛋白（1.0-25.0 g/dl)。如本文各處所注意到的，該聚氮氧化物白蛋白（PNA)可從羥基胺形式的氮氧化物 (例如TPH)接受一個不成對的電子，再生出該氮氧化物至其自由基狀態（例如TPL)之活性。此多重組份条統之一基本優點是，雖然該巨分子或物種留在細胞外間隙内，該小型低分子量膜可通透的氮氧化物及其經還原的衍生物（羥基胺）自由地分佈在細胞内及細胞外間隙内。這産生一個循環，其中氮氧化物自由基在被還原前可於細胞内被偵測之，而後被該巨分子（細胞外）物種再生之。因此，一光譜學上可偵測的氮氣化物之一所欲濃度，可在活體内被雒持一段延長的時間。 I 今 (請先閱讀背面之注意事項再填寫本頁) -裝--- 訂*--- 線本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）經濟部中央標準局員工消費合作社印製 A7 B7 i、發明説明（）延長氮氧化物在活體内之短半衰期有助於克服在發展 ~以氮氣化物為主的顯影方法及醫學治療當中的一値主要困難。關於顯影，位在Johns Hopkins Uni vers ity的EPR 實驗室已發展出一種使用氮氧化物以作為對比劑之連續波 ER I儀器，俥供研究心臓局部缺血與再灌流之過程。但是，氮氧化物本身有限的半衰期已意謂著該再灌留相無法被研究之。在這些研究中，氮氧化物在大白鼠心臓内之三維光譜-空間EPR顯影，由於氮氧化物還原作用之故，遭受到自由基信號之快速的衰減。雖然已從一 3 - D光譜-空間實驗數據再建立出該大白鼠心臟之一橫截面的橫向2-D空間EPR 顯影，這是依據一個在12-分鐘獲得期間當中持續地衰減之EPR信號而得者。在心外膜、心肌中層以及心内膜内之不同的氮氧化物還原速率以為局部缺血期間之一函數，進一步降低了心臓的橫截面顯影之明確性。依據本發明，該ER I顯影可被改善之。一活體内氮氧化物之延長的有活性的半衰期容許了再灌流相之顯影，並對於組織及器官的局部缺血損害之進展提供額外的資料。本發明之組成物提供一穩定的氮氧化物信號適供用於在投藥後1 0分鐘内的顯影，並會持續達2 . 5小時。為了比較，源自一自由的氮氧化物本身之信號，在投藥後20分鐘内即有效地消失（第24圖）。對治療用途而言，能安全地維持有活性的氮氧化物位準歴時延長的時間之能力，表示了能夠提供一有助於局部缺血/再灌流損害、其中由氧衍生的毒性自由基傜為細胞損害之試劑的病理過程之預防的抗氧化本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） ---------ϋ-批衣II (請先閲讀背面之注意事項再填寫本頁) 訂 ;線 492866 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（）作用之能力。（參見第26圖與第27圖）。使用大體上如 Kuppusamy al·，Ρχ,,ο c ·_Η a 11 ·_A c a d 上 9 1 : 3388-3392 (1994)中所述的方法，使用在一濃度為4g白蛋白/ dl與2mM 15N-TEMP0L之聚氮氧化物白蛋白來作大白鼠心臓顯影。參照第24圖，15N-TEMP0L的信號強度，在聚氮氧化物白蛋白之存在下，可於一分離的大白鼠心臓内觀察到。該 EPR信號偽為穩定的，並且在強度上具一雙相的及漸次的降低。這與僅使用一低分子量膜可通透的氮氧化物（TP L) 之信號強度上的雙相快速降低成對比。在一 EPR顯影研究中，大白鼠心臓被灌注以一含有氮氧化物並加有或無聚氮氧化物白蛋白之溶液，而後停止灌注液流俾以形成局部缺血。第24圖顯示出，在有或無聚氮氧化物白蛋白之存在下，於局部缺血期間位在分離的大白鼠心臓内之I；1 5N] -TEMP0L的總信號強度。可看到該信號強度進行一雙相的衰減以及聚氮氯化物白蛋白之存在大大地減緩了該衰減。藉此穩定化該TEMP0L信號，聚氮氧化物白蛋白容許在一段延長的時間内可獲得高品質EPR顯影。參照第22圖，一個局部缺血的心臓之三維ERP顯影，在156分鐘的整體性局部缺血後，仍可被獲得之。該圖亦顯示出該顯影之截斷面圖。如第23圔中所例示的，該三維顯影亦可以橫截面來觀察之。這顯示出心臓内TEMP0L信號之差異性分佈；這於第28圖中被定量之。第24圖顯示出一条列的諸如第22圖中之顯影*這是在125分鐘的整體性局部缺血期本紙張尺度適用中國國家標準（CNS)A4規格（ 210X297公釐） -78- n.^ m -_-l = Βιϋ —I— i ! In m- (請先閱讀背面之注意事項再填寫本頁) ----線 492866 經濟部中央標準局員工消費合作社印製 A7 B7 i、發明説明（）間中，於一条列的連續時間處所獲得者。此例證了 PNA之存在，在解析度及信號持續性上，容許較僅使用TEMPOL時為佳的顯影。實施例9 —放射保護其被暴露於離子放射之活生物體蒙受有害的作用，這在高劑量的放射下係為致命性的。近來的證據暗示放射經由對DNA之損害造成細胞性傷害。在對DNA之總損害中，高達80¾偽源自於放射-誘發的從水衍生的自由基以及二级磺為主的基團。美國國防部已篩析過超過4,000種的化合物來尋找一活體内放射保護劑。雖然一試劑（WR-272 1 )顯示出選擇性放射保護作用，WR-2721於人類臨床試驗中無法展現出放射保護作用。一氮氣化物化合物（例如TPL)係為一非-硫醇放射保護劑，但如上所注意到的，（TPL)具有一非常短的活體内半衰期。其他的化合物偽為天然來源的巨分子（超氧化物歧化酶、IL-1以及GM-CSF)但是，由於其分子尺寸之故，其各個對於提供細胞内自由基的清除，具有有限的能力。美國國家癌症研究院嘗試使用氮氧化物化合物TEMPOL 以為一放射保護劑，俾以容許癌症病人之較高劑量位準的放射治療。研究人員很快地發現，該低分子量氮氣化物被快速地還原成一無活性形式，而且無法被投藥以安全的劑量。依據此處所掲露的本發明，被結合至巨分子化合物以罪為酵素模擬物之氮氧化物，可與低分子量氮氣化物一起本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）492866 V. Description of the invention () The recognition technology can be easily applied to the manufacture of nitrogen oxide-labeled immunoglobulins. The immunoglobulin has the advantages of specific binding and long circulating half-life, which enables the enzyme mimetic activity of the compounds of the present invention to be targeted to specific tissues and have prolonged activity. Nitrogen oxide-labeled immunoglobulins can be used in vivo to provide protection against cellular damage caused by reactive oxygen species. Nitrogen oxide-labeled immunoglobulins can be used alone or in combination with a low molecular weight nitrogen oxide And it is used to provide an antioxidant activity with an extended plasma half-life. Nitrogen oxide-labeled immunoglobulins can be prepared by the specific labeling of the immunoglobulin itself or by binding to Covalently identified location. In order to avoid the removal of nitrogen oxide-labeled immunoglobulin, which is part of the body's natural immune response, those skilled in the art can use immunoglobulin fragments generated by breaking down immunoglobulins by known techniques, such as (Fab) 2, plus non-specificity-labeling, a better NOx: immunoglobulin molar ratio can be as high as 60: 1. For the purpose of target enzyme simulants, a nitrogen oxide-labeled immunoglobulin 傺 is a better species. For example, by selecting an antibody specific for an antigen that is considered to be beautiful with inflammation or other similar conditions, the development-improving utility and therapeutic advantages of the present invention can be targeted to a pending location. Example 8-Development of Biological Structures and Free Radical Reactions Using ESR In most cases, the free radical reactions occur so quickly that EPR development (ERI) cannot be performed. However, due to the presence of stable free radicals, nitrogen oxides can be detected using electron resonance resonance spectroscopy. In -74-This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) --------- install-(Please read the precautions on the back before filling this page) J— T- i Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 492866 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the Invention () Under the development of modern developing equipment, it can be measured based on one of the free radical concentrations. Development of overall biological tissues and organs. Since the nitrogen oxides are reduced to inactive derivatives in vivo within minutes of administration, their usefulness is pseudo-limited. According to the present invention, the level of active nitrogen compounds in the body can be maintained for an extended period of time, allowing for better development contrast and better contrast to what is seen when only low molecular weight and membrane permeable nitrogen oxides are used Long signal persistence. Electron paramagnetic resonance (EPR) spectroscopy is an observation of the behavior of free radicals by detecting the energy state of unpaired electrons in the presence of a magnetic field. This technique is pseudo-specific for free radicals because only unpaired electrons are detected. With its available device for measuring the paramagnetic resonance of electrons, a great view of the current development of the target (including living tissue) can be obtained. EPR imaging (ERI) provides the ability to obtain multidimensional imaging (including spectral-spatial imaging) for diagnosis or research. Electronic paramagnetic resonance imaging (ER I) using nitrogen oxide contrast agents is generally a valuable method for medical imaging, especially for ischemic tissue. However, the development of this technology is limited by the rapid reduction of nitrogen oxides into non-paramagnetic species in vivo. The application of exogenously introduced nitrogen chlorides has proven utility as a relatively low-resolution EPR developer in vivo. See L. J. Berliner, M Applications of In Vivo EPR ,, T pp. 292-304 in EPR IMAGING AND IN VIVO EPR, G.R. Eaton, S.S. Eaton, K. Ohno, editors; CRC Press (1991). Importantly, Subrainanian and colleagues have established a method for detecting the basic paper size of free paper, which applies the Chinese National Standard (CNS) A4 specification (210X297 mm) (please read the precautions on the back before filling this page) -i§ iM9mt I · Ϋϋ m · ^ mu- * J, Γ—ϋ ϋ «^ —.-I-line 492866 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention () Radio frequency development of species and in vivo development (Four ier) transfer EPR spectrometer. See J. Bourg. Et al., J. Mag. Res. B102, 112-115 (1993). A polynitrogen oxide albumin (PNA) prepared according to the present invention is administered and distributed to blood vessels and other extracellular spaces. Although the concentration range of the composition of the present invention can be varied, a preferred range is 5-25 g of labeled PNA / dl and 0.1 to 200 inM TEMP0L. Utilizing the antioxidant activity and detectability of electron paramagnetic resonance (EPR) spectroscopy is useful for this purpose. In addition, when administered in a mild dose of small, low molecular weight membranes that are permeable to nitrogen oxides, the nitrogen oxides are maintained in the body in an active free radical state for an extended period of time. One of the preferred formulations for ER I development is human serum albumin (1.0-25.0 g) covalently labeled with a high mole ratio of nitrogen oxides (7-95 nitrogen oxide to albumin mole ratio). / dl). As noted throughout this article, the polynitrogen oxide albumin (PNA) can accept an unpaired electron from a nitrogen oxide in the form of a hydroxylamine (such as TPH), regenerating the nitrogen oxide to its free radical state ( Such as TPL). One of the basic advantages of this multiple recombinant system is that although the macromolecule or species remains in the extracellular space, the small, low molecular weight membrane is permeable to the nitrogen oxides and their reduced derivatives (hydroxyamines) freely. Distributed in the cell and extracellular space. This creates a cycle where nitrogen oxide free radicals can be detected inside the cell before being reduced, and then regenerated by the giant (extracellular) species. Therefore, a spectroscopically detectable concentration of one of the nitrogen compounds can be held in the body for an extended period of time. I today (please read the precautions on the back before filling this page) -Packing --- Order * --- The paper size of the thread is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) Employees of the Central Standards Bureau of the Ministry of Economic Affairs Cooperative printed A7 B7 i. Description of the invention () Prolonging the short half-life of nitrogen oxides in the living body helps to overcome one of the major difficulties in developing ~ nitrogenate-based imaging methods and medical treatment. Regarding imaging, the EPR laboratory at Johns Hopkins University has developed a continuous wave ER I instrument that uses nitrogen oxides as a contrast agent to study the process of cardiac ischemia and reperfusion. However, the limited half-life of nitrogen oxides has meant that the re-reserved phase cannot be studied. In these studies, the three-dimensional spectral-spatial EPR development of nitrogen oxides in the heart palpitations of rats suffered rapid attenuation of free radical signals due to the reduction of nitrogen oxides. Although a 3-D spectral-spatial experimental data has been used to establish a transverse 2-D spatial EPR development of a cross-section of the heart of the rat, this is based on an EPR signal that continuously decays during the 12-minute acquisition period. Winner. The different reduction rates of nitrogen oxides in the epicardium, the middle layer of the myocardium, and the endocardium are a function of the ischemic period, further reducing the clarity of the cross-sectional development of the palpitations. According to the present invention, the ERI development can be improved. The extended active half-life of nitrogen oxides in a living body allows development of the reperfusion phase and provides additional information on the progress of ischemic damage to tissues and organs. The composition of the present invention provides a stable nitrogen oxide signal suitable for development within 10 minutes after administration, and will last for 2.5 hours. For comparison, the signal from a free nitrogen oxide itself effectively disappeared within 20 minutes after administration (Figure 24). For therapeutic use, the ability to safely maintain active nitric oxide levels for extended periods of time indicates the ability to provide a toxic free radical derived from oxygen that contributes to ischemia / reperfusion damage傜 Antioxidant for the prevention of pathological process of cell damage reagents The paper size is applicable to Chinese National Standard (CNS) A4 specification (210X297mm) --------- ϋ-batch II (Please read the back first (Notes on this page, please fill out this page) Order; line 492866 Printed A7 B7 by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Ability to invent (). (See Figures 26 and 27). Using a method substantially as described in Kuppusamy al ·, Pχ ,, ο ·· Ηa 11 · _A cad on 9 1: 3388-3392 (1994), using a concentration of 4 g albumin / dl and 2 mM 15N- TEMP0L polynitrogen oxide albumin was used for the development of heart palpitations in rats. Referring to Fig. 24, the signal intensity of 15N-TEMP0L can be observed in the heart palpitations of an isolated rat in the presence of polynitrogen oxide albumin. The EPR signal is pseudo-stable and has a biphasic and a gradual decrease in intensity. This is in contrast to the rapid decrease in biphasic signal strength of nitrogen oxides (TP L), which is transparent with only a low molecular weight membrane. In an EPR imaging study, rat heart palpitations were perfused with a solution containing nitrogen oxides with or without polynitrogen oxide albumin, and then the perfusion fluid flow was stopped to form ischemia. Figure 24 shows the total signal intensity of I in the heart palpitations of isolated rats during ischemia in the presence or absence of polynitrogen oxide albumin; 15N] -TEMP0L. It can be seen that the signal intensity undergoes a biphasic attenuation and the presence of polynitrogen chloride albumin greatly slows the attenuation. This stabilizes the TEMP0L signal, and polynitrogen oxide albumin allows high-quality EPR development to be obtained for an extended period of time. Referring to Figure 22, a three-dimensional ERP visualization of an ischemic palpitation can still be obtained after 156 minutes of global ischemia. The figure also shows a cutaway view of the development. This three-dimensional development can also be viewed in cross section as exemplified in Section 23 (i). This shows the differential distribution of TEMP0L signals in the palpitations; this is quantified in Figure 28. Figure 24 shows a series of developments such as those in Figure 22 * This is a 125-minute total ischemic period. This paper is sized to the Chinese National Standard (CNS) A4 (210X297 mm) -78- n. ^ m -_- l = Βιϋ —I— i! In m- (Please read the notes on the back before filling out this page) ---- line 492866 Printed by the Consumers Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 i. Invention Description (), in a row of consecutive time places obtained. This exemplifies the existence of PNA, which allows better development in resolution and signal continuity than when only TEMPOL is used. Example 9-Radiation Protection Living organisms exposed to ionizing radiation suffer deleterious effects which are lethal at high doses of radiation. Recent evidence suggests that cellular damage caused by radiation damage to DNA. Of the total damage to DNA, up to 80 ¾ pseudo-derived from radiation-induced free radicals derived from water and secondary sulfo-based groups. The U.S. Department of Defense has screened more than 4,000 compounds to find an in vivo radioprotective agent. Although one agent (WR-272 1) showed selective radioprotective effect, WR-2721 failed to show radioprotective effect in human clinical trials. A nitride compound such as TPL is a non-thiol radioprotectant, but as noted above, (TPL) has a very short half-life in vivo. Other compounds are pseudo-naturally-derived macromolecules (superoxide dismutase, IL-1, and GM-CSF). However, due to their molecular size, each of them has a limited ability to provide intracellular free radical scavenging. The National Cancer Institute has tried to use the nitrogen oxide compound TEMPOL as a radioprotective agent, and to allow higher dose radiation treatment for cancer patients. Researchers quickly discovered that the low molecular weight nitrogen compound was quickly reduced to an inactive form and could not be administered at a safe dose. According to the invention disclosed here, nitrogen oxides that are bound to macromolecular compounds and are used as enzyme simulants can be used together with low molecular weight nitrogen compounds. This paper applies the Chinese National Standard (CNS) A4 specification (210X297 mm)

丨訂*-----9線 (請先閱讀背面之注意事項再填寫本頁) ^^66 A7 B7 ^___ 經濟部中央標準局員工消費合作社印製發明説明（），藉由與膜可通透的氮氧化物化合物之相互反應來解毒細胞内的自由基，而被用來解毒位於血管間隙內的氧基團。依據本發明，延長放射保護作用之期間容許氮氧化物化合物之使用，俾以保護對抗諸如癌症治療之醫療應用以及意外的暴露於放射源之有害作用。依據一由美國國家癌症研究院所發展的放射劑量標度，中國倉鼠細胞培養物在放射保護化學劑之存在下被暴露於離子放射。第16圖顯示出中國倉鼠V79細胞在12 Gray的幅射下之存活率。對照組，巨分子結合的氮氧化物（PNA) ，以及還原的氮氧化物（TPH)顯示出類似的存活率。但是，預混合以PNA的TPH造成至一放射保護性TPL的轉化（參見第17圖），這提高了該等V79細胞之存活（參見第16圖）。低分子量膜可通透的氮氧化物（例如TPL)已被證實會於C 3 Η小白鼠體内提供活體内放射保護。在這些研究中，被腹腔内投藥的TPL之最高耐受劑量，被發現是為275mg/kg ，這造成在注射後5-10分鐘，於全血内之最高的TPL位準 (約150/ig/ml)。在有或無TPL (275mg/kg)之存在下，於注射後5-10分鐘，將小白鼠暴露於全身放射。相對於對照組小白鼠之7.8 4G ray，在50¾的經TPL處理的小白鼠會於30天内死亡之放射劑量像為9.97Gray。由於TPL的放射保護作用偽衍生自該不成對的電子之反應力，當TPL藉由失去其不成對的電子而被還原成羥基胺時，其變成無活性的。依據本發明，有效的放射保護劑於活體内維持一治療濃度的呈其活性形式（自由基）狀態之 (請先閱讀背面之注意事項再填寫本頁) -裝- 訂線本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 492866 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（） TPL，並克服了此一問題：當僅有TPL被投藥時，要維持治療位準所需的劑量很高且有毒。 < 第19圖顯示出在腹腔内注射275mg/kg的TPLk後，TPL 的最高血清位準，約為在PNA之存在（0. 5ml/小白鼠，在20 g/dl的白蛋白濃度以及每一莫耳白蛋白有42莫耳TPL之下）下，腹腔内注射約l〇〇mg/kg的TEMP0L所觀察到的數值之1/5 。因此，TPL之血位準在PNA之存在下要增大10倍。此增進的TPL之血位準影響到TPL的細胞内位準，而後者則關傜到在細胞與胞核级之放射保護。此增進的保護，依據一於小白鼠進行的30-天存模型，於全身放射中被證實之。第20圔顯示出在一恆定的TPL 濃度（200mg/kg)下，藉由PNA之加入所得的放射保護之增進。該等結果顯示出TPL在PNA之存在下具有一明顯的放射保護作用。10隻小白鼠中有3隻（δΟί；)在lOGray致命放射下存活，相較於僅有TPL者，其於10隻小白鼠中僅有1隻存活。在一個對照組實驗（無TPL或僅有PNA)中’所有的小白鼠在第1 5天或該日附近死亡。因此，該膜不可通透的P N A顯示出不具放射保護作用，並且在細胞内位準下不會保護對抗放射損害。參照第21圖，其顯示出PNA會還原TPL的實驗數據，不達到類似的放射保護。在此實驗中’當僅使用PNA (每隻小白鼠為0.5 m 1)時，所有1 〇隻小白鼠在15天死亡。在第 20圖所用的TPL之1/4劑量下，該TPL濃度從200mg/ml下降本紙張尺度適用中國國家標準（CNS)A4規格（210X297公釐） -81 - (請先閱讀背面之注意事項再填寫本頁) 裝！--；---Γ、丨訂_------β線 492866 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（）至50mg/m卜PN A之存在能夠保護10隻小白鼠中有2隻免於致命的放射（lOGray)。這些結果證實了 PNA可以一為4之因子被用來降低TPL的劑量以逹到相同或更佳的放射保護。實施例10—活體内酵素模擬物如前所注意到的，氮氣化物（例如TEMP0L)已被顯示出具有模擬超氧化物歧化酶（S0D，其將超氧化物歧化成過氧化氫之含金屬酶）之催化活性。更甚者，在生物条統中，氮氧化物可與過氧化酶與假過氣化酶相互反應以達成一模擬觸酶（其將過氣化氫轉化成氧）的活性。此處所證實的是，使用氮氧化物以模擬一超氧化物氧化酶，俾以減輕與氧載體的代謝有關的氧化壓力。該衍生自含氮氣化物化合物之活性的生物作用包括藉由降低氣化壓力來幫助保護對抗反應性氧物種之細胞毒性。當依據本發明被投藥至活體内時，氮氧化物展現出額外複雜的抗氧化酵素模擬物活性。如前所注意到的，當被靜脈内注射時，TEMP0L已被顯示出具有非常短的血漿半衰期。由於其分子尺寸及電荷持徵之故，其容易地離開血管間隙。在許多醫應用中，希望能具有一種持續存在於血管間隙内之酵素模擬物。這依據本發明，藉由將一氮氧化物接至一生物上安全的且具有一所欲之血漿半衰期的巨分子（例如血紅素與白蛋白）。而被達成之。 t 一膜可通透的氮氧化物（例如TPL)，在其自由基狀態，已被顯示出在活體外及活體内具有酵素模擬物活性。但是，在活體内，主要是在細胞内間隙，其被諸如NADH之生 (請先閱讀背面之注意事 _ •項再填· 裝| 寫本頁) 丨訂”------A線本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）丨 Order * ----- 9 line (please read the notes on the back before filling in this page) ^^ 66 A7 B7 ^ ___ The invention description () is printed by the Consumer Consumption Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. Permeable nitrogen oxide compounds interact with each other to detoxify free radicals in cells, and are used to detoxify oxygen groups located in the vascular space. In accordance with the present invention, the use of nitrogen oxide compounds is allowed to prolong the period of radiation protection to protect against medical applications such as cancer treatment and the harmful effects of accidental exposure to radioactive sources. According to a radiation dose scale developed by the National Cancer Institute, Chinese hamster cell cultures are exposed to ionizing radiation in the presence of radioprotective chemicals. Figure 16 shows the survival rate of Chinese hamster V79 cells under 12 Gray radiation. In the control group, macromolecule-bound nitrogen oxides (PNA) and reduced nitrogen oxides (TPH) showed similar survival rates. However, premixing TPH with PNA caused transformation to a radioprotective TPL (see Figure 17), which improved the survival of these V79 cells (see Figure 16). Low molecular weight membranes that are permeable to nitrogen oxides (such as TPL) have been shown to provide in vivo radioprotection in C 3 Η mice. In these studies, the highest tolerated dose of TPL administered intraperitoneally was found to be 275 mg / kg, which resulted in the highest TPL level (about 150 / ig) in whole blood 5-10 minutes after injection. / ml). In the presence or absence of TPL (275 mg / kg), mice were exposed to systemic radiation 5-10 minutes after the injection. Compared with the 7.8 4G ray of the control group, the radiation dose of the 50% TPL-treated mice within 30 days was 9.97 Gray. Since the radioprotective effect of TPL is pseudo-derived from the reactive force of the unpaired electrons, when TPL is reduced to hydroxylamine by losing its unpaired electrons, it becomes inactive. According to the present invention, an effective radioprotective agent maintains a therapeutic concentration in its active form (free radicals) in the living body (please read the precautions on the back before filling out this page) National Standard (CNS) A4 specification (210X297 mm) 492866 A7 B7 printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention () TPL and overcome this problem: When only TPL is administered, it must be maintained The dose required for the treatment level is high and toxic. < Figure 19 shows that after intraperitoneal injection of 275 mg / kg of TPLk, the highest serum level of TPL is approximately in the presence of PNA (0.5 ml / mouse, albumin concentration at 20 g / dl and per One mole of albumin is under 42 moles of TPL). One-fifth the value observed by intraperitoneal injection of about 100 mg / kg of TEMP0L. Therefore, the blood level of TPL should increase 10-fold in the presence of PNA. This increased blood level of TPL affects the intracellular level of TPL, which is concerned with radioprotection at the cellular and nuclear levels. This enhanced protection was confirmed in whole body radiation based on a 30-day survival model in mice. The 20th stage shows the increase in radiation protection obtained by the addition of PNA at a constant TPL concentration (200 mg / kg). These results show that TPL has a significant radioprotective effect in the presence of PNA. Three of the 10 mice (δΟί;) survived the lethal radiation of 10Gray, compared with only TPL, only 1 of the 10 mice survived. In a control experiment (no TPL or PNA only), all of the mice died on or near the 15th day. Therefore, the membrane's impenetrable P N A appears to have no radioprotective effect and will not protect against radiation damage at the intracellular level. Referring to Figure 21, it is shown that PNA will reduce the experimental data of TPL without achieving similar radiation protection. In this experiment, when only PNA was used (0.5 m 1 per mouse), all 10 mice died on 15 days. At a 1/4 dose of TPL used in Figure 20, the TPL concentration drops from 200mg / ml. The paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) -81-(Please read the precautions on the back first Fill out this page again) -; --- Γ, 丨 order _------ β line 492866 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention () to 50mg / m PN A can protect 10 Two of the mice were protected from lethal radiation (lOGray). These results confirm that PNA can be used as a factor of 4 to reduce TPL doses to achieve the same or better radiation protection. Example 10-In vivo enzyme mimics As noted previously, nitrogen compounds (such as TEMP0L) have been shown to have mimetic superoxide dismutase (SOD), a metal-containing enzyme that disproportionates superoxide to hydrogen peroxide ) Catalytic activity. Furthermore, in biological systems, nitrogen oxides can interact with peroxidases and pseudo-pergassing enzymes to achieve a simulated catalase activity that converts hydrogen peroxide to oxygen. It is demonstrated here that the use of nitrogen oxides mimics a superoxide oxidase to reduce the oxidative stress associated with the metabolism of oxygen carriers. The biological effects derived from the activity of nitrogen-containing compounds include helping to protect against the cytotoxicity of reactive oxygen species by reducing the pressure of gasification. When administered according to the invention into a living body, nitrogen oxides exhibit additional complex antioxidant enzyme mimic activity. As noted previously, TEMP0L has been shown to have a very short plasma half-life when injected intravenously. Due to its molecular size and charge characteristics, it easily leaves the vascular space. In many medical applications, it is desirable to have an enzyme mimetic that persists in the vascular space. This is in accordance with the present invention by connecting a nitrogen oxide to a biologically safe macromolecule (e.g., heme and albumin) with a desired plasma half-life. And was achieved. t A membrane that is permeable to nitrogen oxides (such as TPL), in its free-radical state, has been shown to have enzyme mimetic activity in vitro and in vivo. However, in vivo, mainly in the intercellular space, it is born by, for example, NADH (please read the notes on the back _ • items and then fill and install | write this page) 丨 order "------ A line This paper size applies to China National Standard (CNS) A4 (210X297 mm)

二 )Α Η 經濟部中央標準局員工消費合作社印製 492866 Α7 Β7 五、發明説明（）物還原劑快速地還原成其無活性的經基胺衍生物（ΤΡΗ)。先前，依據一化學計量基礎，有活性的TPL還原成無活性的TP Η大體上偽無法回復的。因此，其作為一治療及診斷用工具即受到限制。依據本發明，一含多重組份組成物具有作為第一組份之膜可通透的氮氧化物，其展現出TPL (有活性）與ΤΡΗ (無活性）之間的一個動力平衡。2) Α Η Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 492866 Α7 B7 V. Description of the invention () The reducing agent is quickly reduced to its inactive amine derivative (TPY). Previously, the reduction of active TPL to inactive TP on a stoichiometric basis was generally pseudo-irreversible. Therefore, its use as a therapeutic and diagnostic tool is limited. According to the present invention, a multi-recombinant-containing composition has, as a first component, a membrane permeable nitrogen oxide, which exhibits a dynamic balance between TPL (active) and TPZ (inactive).

在活體内，無活性的TPH占優勢（>90幻。該二分子物種（TPL與TP Η :)容易地跨過細胞膜並分佈至細胞内與細胞外間隙。一種第二組份偽為一膜不可通透的巨分子聚氮氧化物，其分佈在細胞外間隙，主要是在血管區域内。該第一與第二組份展現出另一種先前於活體内未知的酵素模擬物功能。例如，此處所述的作為一多重組份条统之部分的聚氮氧化物白蛋白，藉經由一旋轉-轉移反應來氧化TP Η與TPL ，而發揮有如一被還原的-氮氧化物氧化酶之作用。因此 ’該巨分子聚氮氧化物白蛋白發揮有如一酵素模擬物之作用而將位於細胞内與細胞外間隙中的TPL/TPH平衡，移動本紙張尺度適^中國國家標準（CNS ) Α4規格（210X297公釐） -83 — (請先閱讀背面之注意事項再填寫本頁) -裝. 線 492866 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（）至約90¾。此酵素模擬物功能特別適合用於其中要産生必要的保護以對抗放射、局部缺血者等等之高劑量TPL，主要偽藉由其等之細胞性氧化還原機轉，而成為有毒的。例如，在一 7放射劑量之實例中，當該劑量被升高時，要提供有意義的放射保護作用所需的低分子量膜可通透的氮氯化物之數量可能變成大到細胞的氧化還原狀態被破壞，因而造成毒性。為了克服毒性障礙，本發明之多重組份条統從該被還原的TPH再生出TPL。此条統可被使用於其中一有活性的氮氣化物傺為所想要的任一種應用中。 TPL和一聚氮氧化物白蛋白之EPR物種分別被示於第3A 、3B與15C圖中。一還原的氮氣化物氧化酶之證實，藉由從TPH (EPR無活性的）至巨分子聚氮氧化物（第15C圖）的氧化還原而被顯示之，且其至TPL (EPR有活性的）之轉化（第 15A圔），傺如下予以進行：（1)將等莫耳比的TPH對一巨分子聚氮氧化物，於室溫下予以培育歷時30分鐘；（2)藉由一個10kD截留值膜離心該反應混合物；以及（3)該濾液的光譜被記錄並顯示於第15B圖中。TPH至TPL之定量轉化示於第17圖中。一合成的且經還原的氮氧化物（聚氮氧化物白蛋白）之製備偽藉由令人類血清白蛋白（HSA，25¾ Baxter Health-care Corp.)與40莫耳當量的4- (2-溴乙醯胺基）-TEMPO或 Br-TEMPO，在60 °C下反應並攪拌歴時10小時。對位於一個 Vaciitainer試管内之所形成的混合物（含有15ml的HSA以及本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） ........ - u In —II ϋϋ =__ am m I —li^rn ^ij ^ (請先閱讀背面之注意事項再填寫本頁) 492866 Α7 Β7 經濟部中央標準局員工消費合作社印製五、發明説明（） 165mg的Br-TEMPO)，使用一個0.22微米過濾器予以無菌過濾處理，並轉移至一個配備有一個l〇kDa截留值膜（Fi ltron Technology Corp.)之 150ml攪拌室（Stirred Cell Device) 内。以林格氏液清洗該經過濾的反應混合物直至濾液，如藉由ESR光譜學所偵測到的，含有低於1/ιΜ自由的TEMPO。將亮橘色滯留液濃縮至25%HSA，並再次以一個0.22微米過濾器將之無謹過濾處理至1〇πι1__管瓶（Abbott Laborato-r i es)内，並儲存於4 °C下直到使用時。為證實活體内TP Η 至TPL的酵素般的轉化，TPL之1 5 Ν穩定的同位素類似物被注射至一裝有套管的小白鼠之尾部靜脈内，並於該尾部直接監測EPR信號。一個0. 5ml的TPL溶液（40mM)至被麻醉的小白鼠之直接靜脈注射證實了 TPL的血漿内半衰期約為2分鐘。參照第18圖，使用一包含有一巨分子聚氮氧化物及一穩定的同位素1 5N TPL之混合物的後繼注射（約30分鐘之後），在1 5 N TPL的波峰強度内展現出呈一種雙相的變化。起初，1 5N TPL信號強度之一降低係有助於TPL擴散到血管間隙外，而後為其細胞内還原。此擴散速率，依據第18圖中的15N TPL信號強度之較慢的再出現，要快於TPH至TPL的再氧化。雖然TPH至TPL的再氧化要慢於初始擴散/還原速率，其快於TPL的恆定狀態細胞内還原。因此，第1δ圖中所示的TPL信號之再出現偵測了 ΤΡΗ至TPL的氧化還原，因而證實了在小白鼠活體内一合成製造的還原的氮氧化物氧化酶活性實施例11 一局部缺血與再灌流損害本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) •裝--- « fJMMmt ϋ_ι ι_·ϋ fammaf * 、可一線 492866 A7 B7 五、發明説明（）經濟部中央標準局員工消費合作社印製如前所注意到的，含氮氧化物化合物可被用於醫療顯影。一特別有用的應用是獲得心臟及他處内之局部缺血的組織之顯影，因為這可得到有關於氧代謝及再灌流損害之有價值的資料。但是，活體内自由基之快速的還原限制自由的氮氣化物在此方面之應用。但是，依據本發明，可以增進顯影能力空間地解析心臓内局部缺血的組織，俾以監測心肌的局部缺血之發展，研究心肌再灌流相之發展，以及觀察組織或器官的現時缺氣狀態。參照第23圖與第24圖，其等顯示出一被輸入以1 TE MPOL及一聚氮氧化物白蛋白之分離的大白鼠心臓之顯影。在第24圔中，EPR信號之強度被顯示出如同局部缺血期間之一函數。在下方曲線中，2mM的TEMPOL被輸入至一局部缺血的心臓。上方曲線追蹤2niM TEMPOL加上一個聚氮氧化物白蛋白（4g/dl的白蛋白，在每莫耳白蛋白有42莫耳的TEMPOL下）。該等實驗數據證實了當使用本發明之組成物時，該信號強度大體上要強得多，且被維續之。第25圖顯示出在局部缺血的組織處之顯影的活力。顯影之進展追蹤了在約125分鐘的局部缺血之進展。除了證明診斷用途外，該聚氮氣化物白蛋白與TEMPOL 之組合保護心臓免於局部缺血再灌流損害。第26圖顯示出僅有一氮氧化物或僅有聚氮氣化物白蛋白，不會影響冠狀動脈流之回收。但是，第27圖顯示出在30分鐘的整體性局部缺血，繼而45分鐘的血液流動後，僅在有該二者之存在下觀察到大體上改善的RPP回收（速度壓力産物）。更甚者 (請先閲讀背面之注意事_ -項再填- 裝— — :寫本頁) 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 492866 經濟部中央標準局員工消費合作社印製In vivo, inactive TPH predominates (> 90 phantoms). The two molecular species (TPL and TP Η :) easily cross the cell membrane and distribute to the intracellular and extracellular spaces. A second component is faked as a The impermeable membrane is a macromolecular polynitrogen oxide, which is distributed in the extracellular space, mainly in the vascular area. The first and second components exhibit the function of another enzyme mimic previously unknown in the living body. For example The polynitrogen oxide albumin described here as part of a multi-recombinant system, functions as a reduced nitrogen oxide oxidase by oxidizing TP Η and TPL through a spin-transfer reaction. Therefore, the macromolecular polynitrogen oxide albumin acts as an enzyme mimetic to balance the TPL / TPH in the intracellular and extracellular spaces, and the paper scale is suitable for China National Standards (CNS) Α4 Specifications (210X297 mm) -83 — (Please read the precautions on the back before filling out this page) -Installation. Line 492866 Printed by the Consumers Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of invention () to about 90¾ this The enzyme mimetic function is particularly suitable for high-dose TPL where the necessary protection is to be produced against radiation, ischemia, etc., which is mainly made toxic by their cellular redox machinery, for example, In an example of a 7 radiation dose, when the dose is increased, the amount of low molecular weight membrane permeable nitrogen chloride required to provide meaningful radiation protection may become so large that the redox state of the cell is destroyed Therefore, in order to overcome the obstacle of toxicity, the multi-recombination system of the present invention regenerates TPL from the reduced TPH. This system can be used for one of the active nitrogen compounds. Any desired one In application. TPL and an EPR species of polynitrogen oxide albumin are shown in Figures 3A, 3B, and 15C, respectively. A reduced nitrate oxidase was confirmed by the TPH (EPR inactive) to giant The redox of the molecular polynitrogen oxide (Figure 15C) is shown, and its conversion to TPL (EPR is active) (No. 15A 圔), 傺 is performed as follows: (1) the molar ratio of TPH pair The macromolecular polynitrogen oxide was incubated at room temperature for 30 minutes; (2) the reaction mixture was centrifuged through a 10 kD cut-off membrane; and (3) the spectrum of the filtrate was recorded and shown in Figure 15B. The quantitative conversion of TPH to TPL is shown in Figure 17. A synthetic and reduced nitrogen oxide (polynitrogen oxide albumin) was prepared pseudohuman serum albumin (HSA, 25¾ Baxter Health-care Corp .) Reaction with 40 mol equivalent of 4- (2-bromoacetamido) -TEMPO or Br-TEMPO at 60 ° C and stirring for 10 hours. For the resulting mixture in a Vacitainer tube (Contains 15ml of HSA and this paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) ..............-u In —II ϋϋ = __ am m I —li ^ rn ^ ij ^ ( Please read the precautions on the back before filling this page) 492866 Α7 Β7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (165mg of Br-TEMPO), use a 0.22 micron filter for aseptic filtration, Transfer to a membrane equipped with a 10 kDa cut-off membrane (Fi lt Ron Technology Corp.) in a 150 ml stirring cell (Stirred Cell Device). The filtered reaction mixture was washed with Ringer's solution until the filtrate, as detected by ESR spectroscopy, contained less than 1 / μM of free TEMPO. The bright orange retentate was concentrated to 25% HSA, and filtered again with a 0.22 micron filter into a 10 μm 1__ vial (Abbott Laborato-R es) and stored at 4 ° C Until when used. To confirm the enzymatic conversion of TPΗ to TPL in vivo, a 15 N stable isotope analog of TPL was injected into the tail vein of a cannulated mouse and the EPR signal was monitored directly at the tail. A direct intravenous injection of 0.5 ml of a TPL solution (40 mM) into anesthetized mice confirmed that the plasma half-life of TPL was about 2 minutes. Referring to FIG. 18, a subsequent injection (after about 30 minutes) using a mixture of a macromolecular polynitrogen oxide and a stable isotope of 15 N TPL showed a biphasic phase within the peak intensity of 15 N TPL The change. Initially, a reduction in one of the 15 N TPL signal strengths helped the TPL diffuse out of the interstitial space and then restore it intracellularly. This diffusion rate, based on the slower re-emergence of the 15N TPL signal strength in Figure 18, is faster than the reoxidation of TPH to TPL. Although the reoxidation of TPH to TPL is slower than the initial diffusion / reduction rate, it is faster than the constant state intracellular reduction of TPL. Therefore, the reappearance of the TPL signal shown in Fig. 1δ detected the redox of TPP to TPL, thus confirming the reduced NOx oxidase activity synthesized in vivo in a mouse. Example 11 A partial deficiency Blood and reperfusion damage The size of this paper applies the Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling out this page) • Packing --- «fJMMmt ϋ_ι ι_ · ϋ fammaf *, may First line 492866 A7 B7 V. Description of the invention () Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs As noted previously, nitrogen oxide compounds can be used for medical imaging. A particularly useful application is to obtain imaging of ischemic tissue in the heart and elsewhere, as this can provide valuable information on oxygen metabolism and reperfusion damage. However, the rapid reduction of free radicals in vivo limits the use of free nitrogen compounds in this regard. However, according to the present invention, it is possible to analyze the ischemic tissue in the cardiac parenchyma spatially to enhance the development ability, to monitor the development of myocardial ischemia, to study the development of myocardial reperfusion phase, and to observe the current state of lack of gas in the tissue or organ . Referring to Figures 23 and 24, they show the development of heart palpitations in a rat that was input with 1 TE MPOL and a polynitrogen oxide albumin. In Section 24, the intensity of the EPR signal is shown as a function of the ischemic period. In the lower curve, 2 mM TEMPOL is entered into an ischemic heart palpitation. The upper curve traces 2niM TEMPOL plus a polynitrogen oxide albumin (4g / dl albumin at 42 mol TEMPOL per mol albumin). These experimental data confirm that when using the composition of the present invention, the signal intensity is substantially much stronger and is maintained. Figure 25 shows the viability of development at the ischemic tissue. The development of imaging tracks the progress of ischemia at about 125 minutes. In addition to proven diagnostic use, this combination of polyazide albumin and TEMPOL protects palpitations from ischemia-reperfusion damage. Figure 26 shows that only nitrous oxide or only polynitride albumin does not affect the recovery of coronary flow. However, Figure 27 shows global ischemia at 30 minutes, followed by 45 minutes of blood flow, and a substantially improved RPP recovery (speed pressure product) was observed only in the presence of both. What's more (please read the notes on the back _-items and then fill--:: write this page) This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) 492866 Employees of the Central Standards Bureau of the Ministry of Economic Affairs Printed by a cooperative

AY B7 五、發明説明（），心臓的水腫以為局部缺血/再灌流之一結果，可被避免之。第2 8圖顯示出在超過1 0 0分鐘的局部缺血下，於局部缺血的心臓之各個解剖區域内的1 TPL濃度。TPL組織濃度之升高傺為PNA保護心臓之一可能的解釋。實施例1 2 --血管擴張以及以血紅素為主的红血球代用品 (H R C S))之血管中性配方第1 3圖顯示出本發明組成物對以血红素為主的氧載體 (HBOC)，待別是DBBF-Hb，之血管收縮作用的影響。此血管收縮作用於藉由於有意識的大白鼠模型中，本案溶液之一 10¾ v/v頂端承載被輸入時，測量平均動脈壓力（MAP)之增加而被證實之。參照第13圖，虛線表示在一 HBOC之輸入後，該平均動脈壓力為時間之一函數。依據本發明，被用於放射保護、 ER I以及局部缺血/再灌流損害保護之相同的PNA與TPU容液，被顯示出具有廣範圍的酵素模擬性以及基團解毒功能° PHS或TPL，當與HBOC—起被注射時，被發現不具有抗高血壓。更甚者，在有意識的大白鼠體内，PNA (5g/dl)或TPL (1 0 0 m Μ)本身作為1 0 % v / v頂端承載時，不生成顯著的血管擴張作用（第 29圖）。但是，PNA (5%//dl)/TPL (lOOmM) 作為10% v/v頂端承載時，觀察到其生成一顯著且維續的血管舒張作用。此血管舒張作用與這些大白鼠體内維續的血漿TPL位準相符（第14圖）。在無PNA下，這些大白鼠體内的TPL血漿半衰期為低於60分鐘。因此’藉由混合等量容AY B7 5. Description of the invention (), palpitation of palpitations as a result of ischemia / reperfusion can be avoided. Figure 28 shows the 1 TPL concentration in each anatomical region of ischemic palpitations under ischemia over 100 minutes. The increase in TPL tissue concentration is one of the possible explanations for PNA to protect the heart. Example 1 2-Vasodilator and vascular neutral formula of heme-based red blood cell substitute (HRCS) Figure 13 shows the composition of the present invention against heme-based oxygen carrier (HBOC), The difference is the effect of DBBF-Hb, the vasoconstrictor effect. This vasoconstriction effect was confirmed by measuring the increase in mean arterial pressure (MAP) when a 10¾ v / v top load of one of the solutions in this case was input in a conscious rat model. Referring to Figure 13, the dashed line indicates that the average pulse pressure is a function of time after an HBOC input. According to the present invention, the same PNA and TPU fluids used for radiation protection, ER I and ischemia / reperfusion damage protection have been shown to have a wide range of enzyme mimicry and group detoxification functions ° PHS or TPL, When injected with HBOC, it was found to have no antihypertensive. What's more, in conscious rats, PNA (5g / dl) or TPL (100m Μ) itself does not produce significant vasodilation when carried as 10% v / v apex (p. 29 Figure). However, when PNA (5% // dl) / TPL (100 mM) was carried as a 10% v / v apex, it was observed to generate a significant and sustained vasodilation effect. This vasodilation effect is consistent with the sustained plasma TPL level in these rats (Figure 14). In the absence of PNA, the TPL plasma half-life in these rats was less than 60 minutes. So ’by mixing equal volume

本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -ST .裝—I (請先閱讀背面之注意事項再填寫本頁) d 線 492866 A7 B7 五、發明説明（）積的 PNA (5g/dl)/100mM TPL 以及 DEEF-Hb 7.8g/(H，並於這些大白鼠體内頂端承載一為20% v/v，製造出一血管中性HRCS配方（第14圔）。於第29圖所觀察到的高血壓作用與血管平滑肌内TPL之維續的升高是相符的，這防止氯化氮 [亦即内皮衍生的鬆弛因子（EDRF)]之分佈，因而增進大白鼠體内的血管擴張以及降低MAP。在一血管中性的HRCS配方之例中，HBOC與PNA/TPL的血管收縮及血管擴張活性消除了彼此於活體内對氧化氮之作用。因此，依據自由基與氧化壓力之整體性保護，這個血管中性的HRCS配方是目前在臨床發展上是HBOC之一顯著的改良。 (請先閲讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消费合作社印製本紙張尺度適用中國國家標準（CNS ) A4規格（21〇X297公釐）This paper size applies Chinese National Standard (CNS) A4 specification (210X297mm) -ST.Packing -I (Please read the precautions on the back before filling this page) d line 492866 A7 B7 V. Description of invention () Product PNA (5g / dl) / 100mM TPL and DEEF-Hb 7.8g / (H, and carried a 20% v / v on the top of these rats to produce a vascular neutral HRCS formula (14th). The hypertensive effect observed in Figure 29 is consistent with the continued increase in TPL in vascular smooth muscle, which prevents the distribution of nitrogen chloride [ie, endothelial-derived relaxing factor (EDRF)], thereby enhancing the body of rats Vasodilation and MAP reduction in a blood vessel. In the case of a vascular neutral HRCS formulation, the vasoconstriction and vasodilation activities of HBOC and PNA / TPL eliminate each other's effect on nitric oxide in vivo. Therefore, according to free radicals and The overall protection of oxidative stress, this vascular-neutral HRCS formula is a significant improvement on the current clinical development of HBOC. (Please read the precautions on the back before filling this page) Paper size Chinese National Standard (CNS) A4 size (21〇X297 mm)

Claims

經濟部智总財/4/¾¾工消費合作社印製申請專利範圍第號專利再審查案中請專利範圍修正本修正日期：89年1〇月 1· -種供用於治療切斷局部缺血再灌流之損害的藥學組成物’其包含有：一被共價標識以一氮氧化物之人類血清白蛋白，其 :氮氧化物對白蛋白之莫耳比係為17至95,且其中該氮氧化物具有下述結構：〇其中R4R2各自係為一個具有H個碳原子的燒基，且 R3與R4各自係為氫或一個具有1-4個碳原子的烷基。 2.如申請專利範圍第w之藥學組成物，其中該人類血清白蛋白係重組型的。 3·如f請專利範圍第i項之㈣組成物，其巾該人類血清白蛋白係為單體的、二聚體的、聚合體的，或交聯形成一種微球體。 4.如申請專利範圍第W之藥學組成物，其中該氮氧化物係為2,2,6,6，-四甲基六氫吡啶_Ν·烴氧基（TEMp〇)。 5· —種供用於治療或診斷局部缺血再灌流之損害的藥學組成物，其包含有：一被共價標識以一氮氧化物之人類血清白蛋白，其中氮氧化物對白蛋白之莫耳比係為17至95,且其中該The Ministry of Economic Affairs, the Chief Financial Officer / 4 / ¾¾ Industrial and Consumer Cooperatives printed an application for the scope of patents and re-examination of the patent, requested the amendment of the scope of the patent. The perfusion-damaged pharmaceutical composition 'includes: a human serum albumin covalently labeled with a nitrogen oxide, wherein the molar ratio of nitrogen oxide to albumin is 17 to 95, and wherein the nitrogen oxidation The compound has the following structure: o wherein R4R2 is each an alkyl group having H carbon atoms, and R3 and R4 are each hydrogen or an alkyl group having 1-4 carbon atoms. 2. The pharmaceutical composition according to claim w, wherein the human serum albumin is of a recombinant type. 3. The composition of item (i) in the scope of patent, if the human serum albumin is monomeric, dimeric, polymeric, or crosslinked to form a microsphere. 4. The pharmaceutical composition according to claim W, wherein the nitrogen oxide system is 2,2,6,6, -tetramethylhexahydropyridine-N · alkoxy group (TEMpo). 5. · A pharmaceutical composition for treating or diagnosing the damage of ischemia-reperfusion, comprising: a human serum albumin covalently labeled with a nitrogen oxide, wherein nitrogen oxide is a mole of albumin The ratio is 17 to 95, and where

木紙張尺度適用中國國家襟準（CNS ) M規格（2丨0)<297公#Wood paper scale is applicable to China National Standard (CNS) M specification (2 丨 0) < 297 公 #

、11 (請先閲讀背面之注意事項再填寫本頁, 11 (Please read the notes on the back before filling in this page

申請專利範圍Patent application scope

氧化物具有下述結構：The oxide has the following structure:

其中1與以2各自係為一個具有1-4個妷原子的烷基，且 R3與R4各自係為氫或— 個具有1 _4個碳原子的烧基。 6. 如申請專利範圍第5項藥與貝之梁予組成物，其中該人類血清白蛋白係重組型的。 7. 如申請專㈣圍第5項之藥學組成物，其中該人類灰清蛋白係為單體的、二聚體的、聚合體的，或交聯形成一種微球體。 8. 如專利範圍第！至7項中任—項之藥學組成物，其中戎氮氧化物對白蛋白之莫耳比為3〇至95。 9·如：請專利範圍第⑴項中任—項之藥學組成物，其中該氮氧化物對白蛋白之莫耳比為42至62。 10·如申請專利範圍第5或6項之藥學組成物，其中該氮氧化物係為4，扣二甲基噚唑烷烴氧基（D0XYL)或2,2,5,5_ 四甲基咣咯烷-N-烴氧基（pr0XYL)。 11·種供用於治療或#斷局部缺血再灌流之損害的藥學組成物，其包含有：一種膜可通透的氮氧化物，以及種t氮氧化物-標識之生物上可相容的巨分子 (諸如糊精、羥乙基澱粉、白蛋白或血紅素），其中該氮 -2- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） (請先閱讀背面之注意事項再填寫本頁)Wherein 1 and 2 are each an alkyl group having 1 to 4 fluorene atoms, and R3 and R4 are each hydrogen or an alkyl group having 1 to 4 carbon atoms. 6. For example, the 5th medicine in the patent application scope and the composition of Bei Zhiliang, wherein the human serum albumin is a recombinant type. 7. For the pharmaceutical composition of claim 5, wherein the human ash albumin is monomeric, dimeric, polymeric, or cross-linked to form a microsphere. 8. Such as the scope of patents! The pharmaceutical composition of any one to seven items, wherein the molar ratio of nitrogen oxide to albumin is 30 to 95. 9. For example, the pharmaceutical composition according to any one of item (1) of the patent scope, wherein the molar ratio of the nitrogen oxide to albumin is 42 to 62. 10. The pharmaceutical composition as claimed in claim 5 or 6, wherein the nitrogen oxide is 4, dimethyl oxazolidinyloxy (DOXYL) or 2,2,5,5_tetramethylpyrrole Alkan-N-alkoxy (pr0XYL). 11. A pharmaceutical composition for treating or #damaging ischemia-reperfusion injury, comprising: a membrane permeable nitrogen oxide, and a nitrogen oxide-labeled biologically compatible Macromolecules (such as dextrin, hydroxyethyl starch, albumin, or heme), where the nitrogen-2- This paper size applies to China National Standard (CNS) A4 specifications (210 × 297 mm) (Please read the precautions on the back first (Fill in this page again)

經濟部智1財/ί^;"Η工消費合作社印製 492866 A8 B8 C8 D8Printed by the Ministry of Economic Affairs / 1 ^ &&; printed by the Ηconsumer cooperative 492866 A8 B8 C8 D8

經濟部智Λίτ/ί^ρ'工消費合作社印製申請專利範圍氧化物具有下述結構Printed by the Ministry of Economic Affairs of the Republic of China

其中R#R2各自係為-個具有1-4個碳原子的院基且 RAR4各自係為氫或-個具有丨_4個碳原子的烧基。 12·如申請專利範圍第"項之藥學組成物，其中該氮氧化物係為2,2,6,6，-四甲基六氫η比唆_N_烴氧基（TEMp〇)。 13.-種供用》治療或診斷局部缺血再灌流之損害的藥學組成物，其包含有·· 一種膜可通透的氮氧化物，以及一種聚氮氧化物-標識之生物上可相容的巨分子 (諸如糊精、羥乙基澱粉、白蛋白或血紅素），其中該氮氧化物具有下述結構：Wherein R # R2 is each a radical having 1 to 4 carbon atoms and RAR4 is each a hydrogen or an alkyl group having 4 carbon atoms. 12. The pharmaceutical composition according to item " of the scope of application for an application, wherein the nitrogen oxide system is 2,2,6,6, -tetramethylhexahydron η-N-alkoxy group (TEMp). 13.-Available for use "A pharmaceutical composition for the treatment or diagnosis of ischemia-reperfusion damage, comprising a membrane permeable nitrogen oxide and a polynitrogen oxide-labeled biologically compatible Macromolecules (such as dextrin, hydroxyethyl starch, albumin, or heme), where the nitrogen oxide has the following structure:

其中心與汉2各自係為一個具有1-4個碳原子的烷基，且 R3與R4各自係為氫或一個具有1-4個碳原子的烷基。 14 ·如申請專利範圍第13項之藥學組成物，其中該氮氧化物係為4，心二甲基哼唑烷-N-烴氧基（DOXYL)或2,2,5,5-四甲基°比咯烷烴氧基（pr〇xyl)。 (請先閱讀背面之注意事項再填寫本頁)Its center and Han 2 are each an alkyl group having 1-4 carbon atoms, and R3 and R4 are each hydrogen or an alkyl group having 1-4 carbon atoms. 14. The pharmaceutical composition according to item 13 of the application, wherein the nitrogen oxide system is 4, dimethyl dimethyl oxazolidine-N-alkoxy (DOXYL) or 2,2,5,5-tetramethyl Group ° is better than proxyl. (Please read the notes on the back before filling this page)

申μ專利範圍 15· ^申請專利範圍第⑴印項之藥學組成物，其中該聚氣虱化物-標識的之生物上可相容的巨分子係為在高於17 的莫耳比下被標識以氮氧化物的人類血清白蛋白。 16·如申請專利範圍第Μ 13項之藥學組成物化物對白蛋白之莫耳比為30至95。 17. 如申請專利範圍第^13項之藥學組化物對白蛋白之莫耳比為42至95。 ^乳乳 18. 如申請專利範圍第15項之藥學組成物，其中該生物上可相容的巨分子係為重組型人類白蛋白。 19·—種供用於治療或診斷局部缺血再灌流損害之穩定的生物可相容的氮氧化物的組成物，其包括：訂一種實質膜不可通透的且具有自由基電子之供給者氮氧化物，其自由基電子比一接受者氮氧化物之自由基電子要不穩定，一種膜可通透的接受者氮氧化物，其中該氮氧化物具有下列基礎結構：The scope of application for patent scope 15. The pharmaceutical composition of the first seal of the patent scope, wherein the polypeptide-labeled biocompatible macromolecule system is labeled at a mole ratio higher than 17 Human serum albumin with nitrogen oxides. 16. The molar ratio of the pharmaceutical composition to albumin according to item M 13 of the patent application range is 30 to 95. 17. For example, the molar ratio of the pharmaceutical composition to albumin No. ^ 13 is 42 to 95. ^ Milk 18. The pharmaceutical composition according to item 15 of the application, wherein the biocompatible macromolecule is recombinant human albumin. 19. · A stable biocompatible nitrogen oxide composition for the treatment or diagnosis of ischemia-reperfusion injury, comprising: Ordering a donor nitrogen that is impermeable to the substantial membrane and has free-radical electrons An oxide whose free radical electrons are less stable than the free radical electrons of a recipient nitrogen oxide. A recipient nitrogen oxide which is permeable to the membrane, wherein the nitrogen oxide has the following basic structure:

績濟部智慧时4¾¾工消費合作社印製其中R!與R2各自係為一具有W個碳原子的燒基，且R3 與R4各自係為氫或—具有14個碳原子的统基。 20.種供用於治療或診斷缺企再灌流損害之穩定的生物可相容的氮氧化物組成物，其包括： -4-:尸、度適用中國國家標導^ 492866 A8 B8 C8 D8 申請專利範圍一種實質膜不可通透的且具有自由基電子之供給者氮氧化物，其自由基電子比—接受者氮氧化物之自由基電子要不穩定，一種膜可通透的接受者氮氧化物，其中該氮氧化物具有下列基礎結構：Printed by the Ministry of Economic Affairs, Wisdom, Industrial and Consumer Cooperatives, where R! And R2 are each a radical with W carbon atoms, and R3 and R4 are each hydrogen or-a radical with 14 carbon atoms. 20. A stable biocompatible nitrogen oxide composition for treating or diagnosing reperfusion damage in a company, including: -4-: cadaver, degree applicable Chinese national standard ^ 492866 A8 B8 C8 D8 Patent application Scope A supplier of nitrogen oxides that is impermeable to a substantial membrane and has free radical electrons, whose free radical electrons are less stable than the free radical electrons of the recipient nitrogen oxides. , Where the nitrogen oxide has the following basic structure:

其中〜與尺2各自係為-具有1-4個碳原子的烷基，且r3 與R4各自係為氫或一具有μ個碳原子的烧基。 21· —種穩定的生物可相容的氮氧化物組成物，其包括：一種實質膜不可通透的且具有自由基電子之接受者氮氧化物，其自由基電子比一接受者氮氧化物之自由基電子要穩定，一種膜可通透的供給者氮氧化物，其中該氮氧化物具有下列基礎結構： (請先閲讀背面之注意事項再填寫本頁) 訂 f 經濟部智慧財.4AH(工消費合作社印製Wherein ~ and Chi 2 are each-an alkyl group having 1-4 carbon atoms, and r 3 and R 4 are each hydrogen or a sulphur group having μ carbon atoms. 21 · —A stable biocompatible nitrogen oxide composition comprising: a recipient nitrogen oxide having a substantially impermeable membrane and having free radical electrons, the free radical electrons being less than a recipient nitrogen oxide The free radical electrons must be stable. A kind of membrane-permeable supplier nitrogen oxides. The nitrogen oxides have the following basic structure: (Please read the precautions on the back before filling this page.) Order f Ministry of Economic Affairs. (Printed by Industrial and Consumer Cooperatives

其中Ri與尺2各自係為一具有^4個碳原子的烧基與〜各自係為氫或-具有1-4個碳原子的烧基。 22. —種生物可相容的氮氧化物組成物，其包括：衣纸張尺度適用中國國家縣 17^17^77^"297公釐） 4^2866Wherein Ri and Chi 2 are each an alkyl group having ^ 4 carbon atoms and ~ each are hydrogen or-an alkyl group having 1-4 carbon atoms. 22. A kind of biocompatible nitrogen oxide composition, including: paper size applicable to national counties of China 17 ^ 17 ^ 77 ^ " 297 mm) 4 ^ 2866

A8 B8 C8 D8 申請專利範圍一種實質膜不可通透的且具有自由基電子之接受者氮氧化物，其自由基電子比一接受者氮氧化物之自由基電子要穩定，一種膜可通透的供給者氮氧化物，其中該氮氧化物具有下列基礎結構：〇㊂:或其中1^與以2各自係為一具有1-4個碳原子的烷基，且與R4各自係為氫或一具有1-4個碳原子的烷基。 23· —種使用在防止活體組織被放射線損害的藥學組成物，其包含有：一種膜可通透的氮氧化物，以及一種生物上可相容之聚氮氧化物巨分子，其中該氮氧化物具有下述結構：A8 B8 C8 D8 Scope of patent application A substantially nitrogen-impermeable recipient nitrogen oxide with free radical electrons, whose free radical electrons are more stable than free radical electrons of a receiver nitrogen oxide, a membrane that is transparent A supplier of nitrogen oxides, wherein the nitrogen oxides have the following basic structure: 〇㊂: or 1 and 2 are each an alkyl group having 1-4 carbon atoms, and each of R 4 is hydrogen or a Alkyl groups having 1-4 carbon atoms. 23 · —A pharmaceutical composition for preventing living tissue from being damaged by radiation, comprising: a membrane permeable nitrogen oxide, and a biocompatible polynitrogen oxide macromolecule, wherein the nitrogen oxidation The object has the following structure:

其中心與!^各自係為一具有1-4個碳原子的烷基，且r3 與R4各自係為氫或一具有1-4個碳原子的烷基。 24· —種使用在防止活體組織被放射線損害的藥學組成物，其包含有： -6 - 木紙張尺度適用中國國家標準（CNS ) A4規格（21〇χ297公釐） (請先閱讀背面之注意事項再填寫本頁)Its center and! ^ Are each an alkyl group having 1-4 carbon atoms, and r3 and R4 are each hydrogen or an alkyl group having 1-4 carbon atoms. 24 · —A pharmaceutical composition used to prevent living tissues from being damaged by radiation, which includes: -6-Wood paper size is applicable to Chinese National Standard (CNS) A4 specification (21〇 × 297 mm) (Please read the note on the back first (Fill in this page again)

經濟部智慧財是A员工消費合作社印製 492866 8 8 8 8 ABCD 申請專利範圍一種膜可通透的氮氧化物，以及種生物上可相谷之聚氮氧化物巨分子，其中該氮氧化物具有下述結構：The smart money of the Ministry of Economic Affairs is printed by A employee consumer cooperative 492866 8 8 8 8 ABCD patent application scope a membrane permeable nitrogen oxides, and a kind of biologically similar poly nitrogen oxide macromolecules, where the nitrogen oxides Has the following structure:

其中心與尺2各自係為一具有1-4個碳原子的烷基，且R3 與R4各自係為氫或一具有1-4個碳原子的烷基。 25 ·如申请專利範圍第23或24項之藥學組成物，其中該生物上可相容之聚氮氧化物巨分子係為以在丨7至95之間的莫耳比例來標識之人類血清蛋白。 (請先閱讀背面之注意事項再填寫本頁) • 00IT t— 經濟部智S財是^;,h工消費合作社印製 -7. H浪尺度適用中_家標準（C M ) Μ規格（MW?公缝）Its center and foot 2 are each an alkyl group having 1-4 carbon atoms, and R3 and R4 are each hydrogen or an alkyl group having 1-4 carbon atoms. 25. The pharmaceutical composition of claim 23 or 24, wherein the biologically compatible polynitrogen oxide macromolecule is a human serum protein identified by a molar ratio between 7 and 95 . (Please read the notes on the back before filling this page) • 00IT t— printed by the Ministry of Economic Affairs and the Ministry of Economic Affairs ^; ? Sewing)