TWI359019B

TWI359019B - Use of folates for the prevention and treatment of

Info

Publication number: TWI359019B
Application number: TW95129781A
Authority: TW
Inventors: Charalambos Antoniades; Cheerag Shirodaria; Keith M Channon; Rudolf Moser
Original assignee: Merck Eprova Ag
Priority date: 2006-08-14
Filing date: 2006-08-14
Publication date: 2012-03-01
Also published as: TW200808320A

Description

1359019 九、發明說明 . 【發明所屬之技術領域】 ^ 本發明有關葉酸化合物（folate )在預防及/或治療心血管疾病，諸如動脈粥狀硬化上之用途，特別是用於調節內皮型一氧化氮合成酶（eNOS )之用途。本發明另外有關由該等葉酸化合物與藥學可接受載體組成之藥學製劑，其係視需要與其他藥學活性劑合併使用，以及有關使用該 _ 等葉酸化合物或其藥學製劑之治療方法。【先前技術】一氧化氮（NO)係一種重要的訊息傳導分子。其鬆弛血管平滑肌細胞以使血管擴張。其亦抑制各種病理現象 ’諸如活化血小板與誘發炎性蛋白質。NO流失會導致高血壓與動脈粥狀硬化血管疾病。1359019 IX. The invention belongs to the technical field of the invention. The invention relates to the use of folic acid compound (folate) for preventing and/or treating cardiovascular diseases, such as atherosclerosis, in particular for regulating endothelial type oxidation. Use of nitrogen synthase (eNOS). The present invention further relates to a pharmaceutical preparation comprising the above-mentioned folic acid compound and a pharmaceutically acceptable carrier, which is used in combination with other pharmaceutically active agents, and a method of treatment using the folic acid compound or a pharmaceutical preparation thereof. [Prior Art] Nitric oxide (NO) is an important signaling molecule. It relaxes vascular smooth muscle cells to dilate blood vessels. It also inhibits various pathological phenomena such as activated platelets and induced inflammatory proteins. Loss of NO can lead to high blood pressure and atherosclerotic vascular disease.

該一氧化氮合成酶（NOS )係一群於02與菸鹼醯胺腺嘌呤二核苷酸磷酸鹽（NADPH)、黃素腺嘌呤二核苷酸（FAD )、黃素單核苷酸（FMN )、原血紅素、四氫生物喋呤（BH4)等輔因子存在之下，負責自L-精胺酸之末端氮原子合成NO之酶（EC 1.14.13.39)。內皮型NOS( eNOS)在血管中產生NO，並與調節血管功能有關（圖9 在其他心血管疾病與肺病當中，產生的反應性氧物質 (ROS )增加。產生過量R0S的重要後果係NO的氧化鈍化。例如’超氧化物迅速與NO反應形成過氧亞硝酸根（ -5- 135-9019 鹽），造成NO立即流失。ROS會將eNOS必要輔因子之 BH4氧化。此種反應導致eNOS產生超氧化物而非NO的狀況（圖10)。此種eNOS的「非偶聯」（uncoupling) 現象可能會延長氧化劑壓力。The nitric oxide synthase (NOS) is a group of 02 with nicotine indoleamine adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) An enzyme responsible for the synthesis of NO from the terminal nitrogen atom of L-arginine in the presence of cofactors such as protohemoglobin and tetrahydrobiopterin (BH4) (EC 1.14.13.39). Endothelial NOS (eNOS) produces NO in blood vessels and is involved in the regulation of vascular function (Fig. 9 In other cardiovascular diseases and lung diseases, reactive oxygen species (ROS) are increased. The important consequence of producing excessive ROS is NO. Oxidation passivation. For example, 'superoxide rapidly reacts with NO to form peroxynitrite (-5-135-9019 salt), causing immediate loss of NO. ROS will oxidize the BH4 of eNOS essential cofactor. This reaction leads to eNOS production. Superoxide rather than NO (Figure 10). This "uncoupling" of eNOS may prolong the oxidant pressure.

氧化應力經由ROS對於NO生物利用率之影響以及經由與無數對氧化還原敏感的訊息傳導途徑之交互作用而在諸如動脈粥狀硬化等心血管疾病上扮演重要角色。雖然已提出清除ROS作爲治療策略，將目標定爲心血管疾病（特別是動脈粥狀硬化）中之氧化應力，但使用「抗氧化劑」之臨床試驗結果令人失望（Griendling K.K.與 FitzGerald G.A.發表於 Circw/aiio”. 2003; 108:2034 )。葉酸化合物（folate)係一碳轉移反應中的必要輔因子，而且涉及人類、動物與植物細胞的關鍵合成作用，特別是DN A生物合成與甲基化循環。在藥物方面，目前葉酸化合物主要使用5-甲醯基-5,6,7,8-四氫葉酸（菊白葉酸 )或5-甲基-5,6,7,8-四氫葉酸（1^-甲基酸）之鈣鹽用於治療巨紅血球型貧血、用於加強葉酸化合物（folate )拮抗劑之適合性的解毒劑，特別是癌症治療中之胺基喋呤與甲胺喋玲（methotrexate) (「抗葉酸救援」）、用於增強氟化嘧啶之治療效果與用於治療諸如牛皮癣等自體免疫疾病、用於加強特定抗寄生物物質（例如三甲基苯嘧啶磺胺甲氧唑）之適合性，以及用於降低化學療法中之二去氮四氫葉酸化合物（dideazatetrahydrofolates)的毒性。此外，已進行葉酸（folic acid)對內皮功能的各種 1359019 硏究，但該等結果卻表示葉酸及其衍生物對於內皮功能的確切影響及機制仍未知之矛盾性質。例如，已意味總高半胱胺酸（tHcy ) ( Wald DS等人，發表於 2002; 325:1 202 )與葉酸化合物（folate) (Bunout D等人，發表於 2000; 1 6:434 )可能與心血管風險（WaldOxidative stress plays an important role in cardiovascular diseases such as atherosclerosis via the effects of ROS on NO bioavailability and through interaction with numerous redox-sensitive signaling pathways. Although ROS has been proposed as a treatment strategy, targeting oxidative stress in cardiovascular disease (especially atherosclerosis), the results of clinical trials using "antioxidants" have been disappointing (Griendling KK and FitzGerald GA) Circw/aiio". 2003; 108:2034 ) Folate is an essential cofactor in a carbon transfer reaction and involves key synthetic effects in human, animal and plant cells, particularly DN A biosynthesis and methylation. In terms of medicine, currently folic acid compounds mainly use 5-methylmercapto-5,6,7,8-tetrahydrofolate (Chrysanthemum folic acid) or 5-methyl-5,6,7,8-tetrahydrogen. A calcium salt of folic acid (1^-methyl acid) for the treatment of giant red blood cell anemia, an antidote for enhancing the suitability of folic acid antagonists, especially for the treatment of aminoguanidine and methylamine in cancer therapy Methrexate ("Anti-folate rescue"), used to enhance the therapeutic effect of fluorinated pyrimidines and for the treatment of autoimmune diseases such as psoriasis, for the enhancement of specific anti-parasitic substances (eg trimethyl sulfonamide) A Yl) The suitability and chemotherapy toxicity bis deaza-tetrahydro folate (dideazatetrahydrofolates) for reducing. In addition, various 1359019 studies on the endothelial function of folic acid have been carried out, but these results indicate that the exact influence and mechanism of folic acid and its derivatives on endothelial function are still unknown. For example, it has been suggested that total homocysteine (tHcy) (Wald DS et al., 2002; 325:1 202) and folate (funout D et al., published in 2000; 1 6:434) may And cardiovascular risk (Wald

DS等人，發表於2002; 325:1 202 )及內皮功能障礙有關（Doshi SN 等人，發表於 Ci'rcw/an'o/i. 2002; 105:22, Hyndman ME 等人，發表於 Am J Physiol Heart C ire Physiol. 2002; 282:H2167, Verhaar MC 等人，發表於 1 999; 1 00:3 3 5 )。不過，硏究指出以葉酸（ folic acid)降低tHcy可能延緩動脈粥狀硬化進展尙未獲得證實（Lange Η等人，發表於 W “g/ / Md. 2004; 3 5 0:267 3 )。事實上，對於患有中風（Toole JF等人，發表於 JAMA. 2004; 29 1 :565-575 )、心肌梗塞(Bonaa KH 等人，發表於#五《g/ «/Me 2006; 354:1578)或穩定冠狀動脈疾病（CAD) ( N Engl J Med. 2006; 3 54:1 567 )之病患的更大型試驗的晚近結果發現葉酸（folic acid)治療不會改善臨床結果。更新的硏究指出，葉酸經由其5-甲基四氫葉酸化合物（folate) (5-MTHF)的循環形式可在血管細胞中具有抗氧化性質並發揮生物效果，其與血漿 tHcy並無直接相關（Doshi SN等人，發表於jrieri〇ic/er Thromb Fwc 5ζ·ο/· 200 1 ; 2 1 : 1 1 96 ; Doshi SN 等人發表於 2002; 1 0 5:22 )。其他先前硏究已指出，葉酸化合物（folate )會影響NO-媒介之內皮功能，諸如藉由 1359019 該eNOS輔因子四氫生物喋呤（BH4)媒介改變eNOS調節作用（Stroes ES 等人，發表於 Cire 2000; 86:1129 :Verhaar MC 等人，發表於 C/rcw/fliiow. 1 998; 97:23 7; Hyndman ΜΈ 等人，發表於 A m J P hy s i o l H e ar t C i r cDS et al., 2002; 325:1 202) and endothelial dysfunction (Doshi SN et al., Ci'rcw/an'o/i. 2002; 105:22, Hyndman ME et al., published in Am J Physiol Heart C ire Physiol. 2002; 282: H2167, Verhaar MC et al., published at 1 999; 1 00:3 3 5 ). However, studies have pointed out that the reduction of tHcy with folic acid may delay the progression of atherosclerosis (Lange et al., published in W “g/ / Md. 2004; 3 50 0:267 3 ). On, for a stroke (Toole JF et al., published in JAMA. 2004; 29 1 : 565-575), myocardial infarction (Bonaa KH et al., published in #五"g/ «/Me 2006; 354:1578) Late results of larger trials in patients with stable coronary artery disease (CAD) (N Engl J Med. 2006; 3 54:1 567 ) found that folic acid treatment did not improve clinical outcomes. Folic acid, via its cyclic form of 5-methyltetrahydrofolate (folate) (5-MTHF), has antioxidant properties in vascular cells and exerts biological effects, which is not directly related to plasma tHcy (Doshi SN et al. , published in jrieri〇ic/er Thromb Fwc 5ζ·ο/· 200 1 ; 2 1 : 1 1 96 ; Doshi SN et al., 2002; 1 0 5:22). Other previous studies have indicated that folic acid compounds ( Folate ) affects the endothelial function of NO-mediated, such as by the 1390719 eNOS cofactor tetrahydrobiopterin ( BH4) Media alters eNOS regulation (Stroes ES et al., published in Cire 2000; 86:1129: Verhaar MC et al., published in C/rcw/fliiow. 1 998; 97:23 7; Hyndman ΜΈ et al., published in A m JP hy siol H e ar t C irc

Ρ/ιπίο/. 2000; 282:H2167)。此外，在營養領域的許多硏究指出含有葉酸（folic acid)合倂各種其他補充品（即 ’諸如維生素B6、B12、E與其他營養素）的各種維生素製劑的有益效果。此等製劑通常係硏發用於治療個別營養不足因而存在各種疾病風險（諸如，神經性精神病、血管腎與血液狀況）的病患（US 6,207,651)。很明確看出，雖然已廣泛使用葉酸（folic acid )，但其作用模式仍不清楚。而且雖然上述葉酸機制與效果的矛盾觀點，所有硏究均顯示葉酸化合物（folate )本身與內皮功能--諸如藉由eNOS在體內產生NO--並無直接影響 ’ （Verhaar MC 等人，發表於 1 99 8; 97:23 7 )’而且上述影響排除使用與其他活性劑合倂使用的葉酸化合物，此等其他活性即爲已知參與eNOS偶合之藥劑’諸如四氫生物喋呤（BH4 )或其衍生物，其實際上係NOS與該芳族胺基酸羥化酶的三種形式之天然輔因子，而且與各種生化反應有關；及/或該胺基酸精胺酸，其係內生 NO 的前驅體（US 6_544,994; US 6,995，158)。因此，目前對於礙有效率「抗氧化劑」治療的發展之血管壁中的氧化還原訊息傳導缺乏清楚瞭解。很清楚地，仍然極爲需要對於諸如動脈粥狀硬化等心血管疾病之有效 -8- 135*9019 率治療方法與預防及/或治療藥劑。Ρ/ιπίο/. 2000; 282:H2167). In addition, many studies in the field of nutrition point to the beneficial effects of various vitamin preparations containing various other supplements of folic acid (i.e., vitamins B6, B12, E and other nutrients). Such preparations are generally used to treat individual nutritional deficiencies and thus present a variety of disease risks (e.g., neuropsychiatric, vascular, renal and blood conditions) (US 6,207,651). It is clear that although folic acid has been widely used, its mode of action remains unclear. And despite the contradictory view of the above-mentioned folic acid mechanism and effect, all studies have shown that folate itself has no direct effect on endothelial function, such as the production of NO by eNOS in vivo (Verhaar MC et al., published in 1 99 8; 97:23 7 )' and the above effects exclude the use of folic acid compounds used in combination with other active agents, such as agents known to be involved in eNOS coupling, such as tetrahydrobiopterin (BH4) or a derivative thereof, which is actually a natural cofactor of three forms of NOS and the aromatic amino acid hydroxylase, and is associated with various biochemical reactions; and/or the amino acid arginine, which is endogenous NO Precursor (US 6_544,994; US 6,995,158). Therefore, there is currently a lack of clear understanding of redox signaling in the vessel wall that hinders the development of efficient "antioxidant" treatment. Clearly, there is still a great need for effective -8- 135*9019 treatments and prophylactic and/or therapeutic agents for cardiovascular diseases such as atherosclerosis.

本申請人目前意外地發現與先前技術陳述相左的是，葉酸化合物（folate )確實對於諸如動脈粥狀硬化之人類心血管疾病的特定氧化還原機制具有直接影響，表示該葉酸化合物發揮完全功效不一定需要存在任何其他活性劑（當然’可視需要包括一或多種另外的活性劑以求個別或協同效果）。特別是，已發現葉酸化合物即使在不存有任何其他活性劑或N0S輔因子（諸如BH4或精胺酸）並且在體內迅速達到的濃度範圍情況下，藉由避免過氧亞硝酸根 (鹽）媒介之四氫生物喋呤（BH4)氧化作用及逆轉 eNOS之非偶聯作用、增加血管BH4、BH4/總生物喋呤比與eNOS之二聚物：單體比、加強eNOS活性並另外使之具有在人類動脈粥狀硬化中一般「抗氧化劑」效果以外的特定效果，迅速改善NO-媒介之內皮功能，同時降低超氧化物產生。如此，葉酸化合物可作爲預防及/或治療心血管疾病，特別是動脈粥狀硬化之特定且有效細胞內化合物【發明內容】本發明第一方面中，提出葉酸化合物（folate )的新穎用途，其係用於製造包含葉酸化合物作爲預防及/或治療諸如動脈粥狀硬化之心血管疾病的活性劑之藥物。在不受到任何特定理論限制的情況下，藉由內皮型一氧化氮合成酶（eNOS )的調節，可能藉由清除反應性氧 135-9019 物質（ROS )、提高血管BH4及該BH4/總生物喋呤比，另外逆轉eNOS之非偶聯作用、提高eNOS二聚物：單體比以及直接增強eNOS活性，達到特定療效。因此，在體內迅速達到的濃度範圍情況下，5-甲基四氫葉酸（folateThe Applicant has unexpectedly discovered that contrary to the prior art statement, the folate compound does have a direct effect on the specific redox mechanism of human cardiovascular diseases such as atherosclerosis, indicating that the folate compound does not necessarily function fully. Any other active agent is required (of course, it may be desirable to include one or more additional active agents for individual or synergistic effects). In particular, folate compounds have been found to avoid peroxynitrite (salt) even in the absence of any other active agent or NOS cofactor (such as BH4 or arginine) and in a range of concentrations rapidly achieved in vivo. The tetrahydrogen biopterin (BH4) oxidizes and reverses the unconjugated effect of eNOS, increases the vascular BH4, BH4/total biotin ratio and eNOS dimer: monomer ratio, enhances eNOS activity and additionally It has a specific effect other than the general "antioxidant" effect in human atherosclerosis, rapidly improving the endothelial function of NO-media, while reducing superoxide production. Thus, a folic acid compound can be used as a specific and effective intracellular compound for preventing and/or treating cardiovascular diseases, particularly atherosclerosis. [Invention] In a first aspect of the present invention, a novel use of a folate compound is proposed. It is used to manufacture a drug comprising a folic acid compound as an active agent for preventing and/or treating cardiovascular diseases such as atherosclerosis. Without the limitation of any specific theory, by regulation of endothelial nitric oxide synthase (eNOS), it is possible to enhance the vascular BH4 and the BH4/total organism by scavenging reactive oxygen 135-9019 (ROS) In addition, the anti-coupling effect of eNOS is reversed, the eNOS dimer: monomer ratio is increased, and the eNOS activity is directly enhanced to achieve a specific therapeutic effect. Therefore, 5-methyltetrahydrofolate (folate) in the range of concentrations quickly reached within the body

)(5-MTHF)迅速改善NO-媒介之內皮依賴性之血管運動反應並降低血管體內與體外之超氧化物。此等變化並非由5-MTHF於體內之直接超氧化物清除作用或藉由體內之血漿中總高半胱胺酸之變化而解釋。此外，該等療效係由葉酸化合物（folate)對於人類動脈粥狀硬化的特定「抗氧化劑」效果所達成。在一特定具體實例中，該葉酸化合物（folate )包括喋酸單麩胺酸（葉酸（folic acid))、二氫葉酸、5-甲醯基四氫葉酸、5-甲基四氫葉酸、5,10-亞甲基四氫葉酸、 5, 10-次甲基四氫葉酸、10-甲醯基四氫葉酸或四氫葉酸、其多麩胺酸化合物、其光學異構物，特別是其光學純的天然異構物，以及光學異構物之混合物，特別是消旋混合物，以及其藥學可接受鹽與酯等等，視情況與一或多種其他藥學活性劑合倂使用。本發明另一方面中，乃提出一種藥學製劑用於治療及 /或預防心血管疾病，其特徵係該藥學製劑係由至少一種葉酸化合物（folate )或其藥學可接受鹽或酯以及至少— 種藥學可接受載體所組成。本發明另一方面，提出治療方法以預防及/或治療諸如動脈粥狀硬化之心血管疾病，特別是藉由內皮型一氧化 -10-(5-MTHF) rapidly improves the endothelium-dependent vascular motor response of NO-media and reduces superoxide in vivo and in vitro. These changes are not explained by direct superoxide scavenging of 5-MTHF in vivo or by changes in total homocysteine in plasma in vivo. In addition, these effects are achieved by the specific "antioxidant" effect of folate on human atherosclerosis. In a specific embodiment, the folic acid compound comprises frolic acid, folic acid, dihydrofolic acid, 5-methylmercaptotetrahydrofolate, 5-methyltetrahydrofolate, 5 , 10-methylenetetrahydrofolate, 5,10-methyltetrahydrofolate, 10-mercaptotetrahydrofolate or tetrahydrofolate, polyglutamic acid compounds thereof, optical isomers thereof, especially Optically pure natural isomers, as well as mixtures of optical isomers, especially racemic mixtures, as well as pharmaceutically acceptable salts and esters thereof, are optionally employed in combination with one or more other pharmaceutically active agents. In another aspect, the invention provides a pharmaceutical preparation for the treatment and/or prevention of cardiovascular diseases, characterized in that the pharmaceutical preparation is composed of at least one folic acid compound (folate) or a pharmaceutically acceptable salt or ester thereof and at least one species Composed of a pharmaceutically acceptable carrier. In another aspect of the invention, a method of treatment is provided for the prevention and/or treatment of cardiovascular diseases such as atherosclerosis, particularly by endothelial monooxy-10-

1359019 氮合成酶（eNOS)的調節作用、改善賴性之血管運動反應、提高血管BH4. 呤比、逆轉eNOS之非偶聯作用、提高體比、直接增強eNOS活性，可能藉由酸根（鹽）之反應性氧物質（ROS)等在一特定具體實例中，該等治療方對象施予葉酸化合物（folate)，諸如酸（folic acid))、二氫葉酸、5 -甲醒基四氫葉酸、5,10-亞甲基四氫葉酸、酸、10 -甲醯基四氫葉酸或四氫葉酸、、其光學異構物，特別是其光學純的天學異構物之混合物，特別是消旋混合物受鹽與酯，或由至少一種葉酸化合物與受載體組成之藥學製劑。其他具體實例涉及施予與一或多種倂使用之葉酸化合物（folate )。其他葉酸化合物或其藥學製劑的投藥途徑與他的具體實例可包括包含視情況需要與活性劑合倂使用之葉酸化合物或其藥學【實施方式】本發明有關葉酸化合物（folate )：血管疾病，諸如動脈粥狀硬化上之用途 NO-媒介之內皮依及該BH4/總生物喋 ;eNOS二聚物：單丨清除諸如過氧亞硝方法。法包括對於需要的喋酸單麩胺酸（葉 I基四氫葉酸、5-甲 5，10-次甲基四氫葉其多麩胺酸化合物然異構物，以及光，以及其藥學可接至少一種藥學可接其他藥學活性劑合的具體實例涉及該劑量形式。更再其一或多種其他藥學製劑的套組或容器 :預防及/或治療心，特別是用於調節 -11 -1359019 The regulation of nitrogen synthase (eNOS), the improvement of vasomotor response, the enhancement of vascular BH4. 呤 ratio, reverse the uncoupling of eNOS, increase the body ratio, directly enhance eNOS activity, possibly by acid (salt) Reactive oxygen species (ROS), etc. In a specific embodiment, the therapeutic subjects are administered folic acid, such as folic acid, dihydrofolic acid, 5-methyl ketone tetrahydrofolate, a mixture of 5,10-methylenetetrahydrofolate, acid, 10-methylmercaptotetrahydrofolate or tetrahydrofolate, its optical isomers, especially its optically pure astronomical isomers, especially The vortex mixture is subjected to a salt and an ester, or a pharmaceutical preparation comprising at least one folic acid compound and a carrier. Other specific examples relate to the administration of a folate compound (folate) for use with one or more hydrazines. The administration route of the other folic acid compound or a pharmaceutical preparation thereof and the specific examples thereof may include a folic acid compound containing the same as the case where it is required to be combined with the active agent or a pharmaceutical thereof. The present invention relates to a folic acid compound (folate): a vascular disease such as Use of atherosclerosis The endothelium of NO-media is dependent on the BH4/total biopterin; eNOS dimer: monoterpene removal such as peroxynitrite. The method includes the desired citrate monoglutamic acid (leaf I-based tetrahydrofolate, 5-methyl 5,10-methine tetrahydroleaf glutamic acid compound isomer, and light, and its pharmaceutically acceptable Specific examples of at least one pharmaceutically acceptable other pharmaceutically active agent are related to the dosage form. Further, a kit or container of one or more other pharmaceutical preparations: preventing and/or treating the heart, particularly for conditioning -11 -

1359019 內皮型一氧化氮合成酶（eNOS)之用途。本發明中，「葉酸化合物（folate)」或「葉 folate)化合物」等辭彙係與喋酸單麩胺酸（葉酸（ acid))與諸如二氫葉酸與四氫葉酸等還原形式二者，例如5 -甲醯基四氫葉酸、5 -甲基四氫葉酸、5, 1〇· 基四氫葉酸、5,10·次甲基四氫葉酸、10-甲醯基四氫與四氫葉酸、其多麩胺酸化合物、其光學異構物，特其光學純的天然異構物，以及光學異構物之混合物，是消旋混合物，亦包括其藥學可接受鹽與酯等等。較佳之葉酸化合物（folate )包括四氫葉酸，特四氫葉酸之天然非對映立體異構形式，諸如5 -甲醯； 6S)-四氣葉酸、5 -甲基-(6S)-四氨葉酸、5，1 0 -亞t (61〇-四氫葉酸、5，10-次甲基-(611)-四氫葉酸、] 醯基-(6R)-四氫葉酸、5-亞胺甲基-(6S)-四氫葉 (6S)-四氫葉酸或其藥學可接受鹽與酯類。更佳者^ 甲基-(6S)-四氫葉酸或5-甲基-(6R，S)-四氫葉酸其藥學可接受鹽與酯類。本文當中，與所使用之「藥學可接受」一辭有關與醋類應兼具藥理與藥學可接受兩種意義。諸如此類理與藥學可接受鹽類可爲鹼金屬或鹼土金屬鹽，較佳、鉀、鎂或銘鹽。諸如此類之藥理與藥學可接受酯類 C1-C4院醋、C5環烷酯或C6環烷酯、苯酯、C1_C4 本醋、卞醋或C1-C4烷基苄酯。此等酯類可爲單酯或。一醋可爲均相或多相。其中最佳者係均相二酯類，酸（ f ο 1 i c 有關亞甲葉酸別是特別別是 £ -( P基-〇-甲酸或 Ψ. 5-，或的鹽之藥係鈉可爲院基二酯諸如 -12- 13590191359019 Use of endothelial nitric oxide synthase (eNOS). In the present invention, the terms "folate" or "folate compound" are combined with citrate mono-glycolic acid (acid) and reduced forms such as dihydrofolate and tetrahydrofolate. For example, 5-methylmercaptotetrahydrofolate, 5-methyltetrahydrofolate, 5,1〇-tetrahydrofolate, 5,10-methine tetrahydrofolate, 10-methylmercaptotetrahydro and tetrahydrofolate The polyglutamic acid compound, its optical isomer, its optically pure natural isomer, and a mixture of optical isomers are racemic mixtures, including pharmaceutically acceptable salts and esters thereof and the like. Preferred folate compounds include tetrahydrofolate, a natural diastereomeric form of tetrahydrofolate, such as 5-methylhydrazine; 6S)-tetra-folate, 5-methyl-(6S)-tetraamine Folic acid, 5,10-t-t (61〇-tetrahydrofolate, 5,10-methine-(611)-tetrahydrofolate,] mercapto-(6R)-tetrahydrofolate, 5-imine -(6S)-tetrahydrofolate (6S)-tetrahydrofolate or a pharmaceutically acceptable salt thereof and an ester. More preferably ^methyl-(6S)-tetrahydrofolate or 5-methyl-(6R,S - tetrahydrofolate, its pharmaceutically acceptable salts and esters. In this context, the term "pharmaceutically acceptable" used in connection with vinegar should have both pharmacological and pharmaceutically acceptable meanings. The salts may be alkali metal or alkaline earth metal salts, preferably potassium, magnesium or salt. Such pharmacologically and pharmaceutically acceptable esters C1-C4 vinegar, C5 cycloalkyl or C6 cycloalkyl ester, phenyl ester, C1_C4 This vinegar, vinegar or C1-C4 alkyl benzyl ester. These esters may be monoesters or vinegar may be homogeneous or heterogeneous. The best ones are homogeneous diesters, acid (f ο 1 Ic related to methylene folic acid is not particularly £-( P-based-indole-formic acid or hydrazine. 5-, or the salt of the drug sodium can be a diester such as -12- 1359019

Cl-C4二烷酯，例如二甲酯或二乙酯。本文中，「心血管疾病j一辭較佳包括由氧化壓力所造成的徵候，更明確地說，係反應性氧物質造成之內皮輔因子四氫生物喋呤（BH4)的氧化作用，而且較佳包括狹心症、冠狀動脈疾病、高血壓、內皮功能障礙、動脈粥狀硬化等等，更佳係動脈粥狀硬化。Cl-C4 dialkyl ester, such as dimethyl or diethyl ester. In this article, "the term "cardiovascular disease" preferably includes signs caused by oxidative stress, more specifically, the oxidation of the endothelial cofactor tetrahydrobiopterin (BH4) caused by reactive oxygen species, and Good includes angina, coronary artery disease, hypertension, endothelial dysfunction, atherosclerosis, etc., and better atherosclerosis.

本文中，「調節內皮一氧化氮合成酶（eNOS)」一辭係有關藉由清除諸如過氧亞硝酸根（鹽）之反應性氧物質（ROS )、提高血管BH4、提高BH4/總生物喋呤比、逆轉eNOS之非偶聯作用、提高eNOS之二聚物：單體比以及直接加強eNOS活性，在體外與體內兩方面改善NO-媒介之血管內皮依賴性血管運動反應，並減少血管超氧化物本發明另外提出一種用於預防及/或治療心血管疾病之藥學製劑，特徵係其由至少一種葉酸化合物（f〇late) 或其藥學可接受鹽或酯以及至少一種藥學可接受載體所組成。本文中，「（本發明）藥學製劑」或「（本發明）製劑」一辭係有關經腸（例如口服、舌下或直腸）、非經腸或局部（例如經皮給藥）形式。不會與該活性成份反應的有機或無機物質可作爲載體，例如水、油、苄醇、聚乙二醇、三醋酸甘油酯或其他脂肪酸甘油酯、明膠、卵磷脂、環糊精、碳水化合物，諸如乳糖或澱粉、硬脂酸鎂、滑石或纖維素。錠劑、糖衣錠、膠囊、粉末、糖漿、濃縮液或 -13- 1359019 滴劑較佳係作爲口服給藥，栓劑較佳係用於直腸給藥，而水基或油基溶液或凍晶注射劑較佳係用於非經腸給藥。亦可使用懸浮液、乳液或植入物，而且可使用貼片或陶瓷作爲局部給藥。非經腸給藥之製劑包括該活性化合物的滅菌水性與非水性注射液，其較佳係與接受者的血液等滲壓》Herein, the term "regulating endothelial nitric oxide synthase (eNOS)" is related to the removal of reactive oxygen species (ROS) such as peroxynitrite (salt), increase of blood vessel BH4, and increase of BH4/total biopterin.呤 ratio, reverse the unconjugated effect of eNOS, improve the dimer of eNOS: monomer ratio and directly enhance eNOS activity, improve the vascular endothelium-dependent vasomotor response of NO-mediated in vitro and in vivo, and reduce vascular hyperactivity Oxide The invention further provides a pharmaceutical formulation for the prevention and/or treatment of cardiovascular diseases characterized by at least one folic acid compound or a pharmaceutically acceptable salt or ester thereof and at least one pharmaceutically acceptable carrier composition. Herein, the term "pharmaceutical preparation of the present invention" or "preparation (of the present invention)" relates to a form of enteral (e.g., oral, sublingual or rectal), parenteral or topical (e.g., transdermal administration). An organic or inorganic substance which does not react with the active ingredient can be used as a carrier, for example, water, oil, benzyl alcohol, polyethylene glycol, triacetin or other fatty acid glycerides, gelatin, lecithin, cyclodextrin, carbohydrates Such as lactose or starch, magnesium stearate, talc or cellulose. Tablets, dragees, capsules, powders, syrups, concentrates or-13-1359019 drops are preferably administered orally, suppositories are preferably used for rectal administration, and water-based or oil-based solutions or frozen-crystal injections are preferred. Good for parenteral administration. Suspensions, emulsions or implants can also be used, and patches or ceramics can be used for topical administration. Formulations for parenteral administration include sterile aqueous and non-aqueous injections of the active compound, preferably in isotonic pressure in the blood of the recipient.

此等製劑可另外包括安定劑、供該藥學活性化合物受控制釋放用之添加劑、抗氧化劑、緩衝劑、制菌劑與供獲得等滲壓溶液用之佐藥。水性與非水性滅菌懸浮液可包括懸浮添加劑與增稠劑。本發明製劑可以單劑量容器或多劑量容器存在，諸如熔接安瓿；其可以冷凍乾燥產物方式儲存，當時可藉由添加滅菌液體，例如水或鹽溶液而製備以供使用。同樣地，可使用滅菌粉末、顆粒或錠劑。雖然最佳的藥學製劑僅含有葉酸化合物（folate )或其藥學可接受鹽或酯作爲活性劑，但本發明所有製劑另外可含有一或多種獨立作用或與本發明製劑協同作用之其他藥學活性化合物。當包括葉酸化合物以外的其他藥學活性化合物時，則較佳製劑僅包括一種另外的藥學活性化合物。特別是，此等藥學活性化合物係直接包括在葉酸化合物循環裡或是影響該葉酸化合物循環的物質，或具有額外抗發炎效果之物質，諸如維生素、抗氧化劑、自由基清除劑、生物喋呤、脂質還原劑、免疫抑制劑、非類固醇抗發炎物質及/或其他活性成份。 -14- 1359019 較佳之維生素包括維生素B2、B6、BI2或抗壞血酸、麩胱甘肽、乙醯半胱胺酸、甜菜鹼。較佳之生物喋呤包括在所有氧化階段之生物喋呤以及生物喋呤之異構形式，尤其是L -紅生物喋呤、7,8 -二氫生物喋呤與 5,6,7,8-四氫生物喋呤，特別是 L-墨喋呤（ sepiapterin) 、D-新喋呤、黃喋呤與6-羥甲基-喋呤。較佳之抗氧化劑包括維生素£或0-胡蘿蔔素。Such formulations may additionally include a stabilizer, an additive for controlled release of the pharmaceutically active compound, an antioxidant, a buffer, a bacteriostat, and an adjuvant for obtaining an isotonic solution. Aqueous and non-aqueous sterile suspensions can include suspending additives and thickening agents. The formulations of the present invention may be presented in single-dose containers or in multi-dose containers, such as fusion ampoules; they may be stored as a lyophilized product, which may be prepared for use by the addition of a sterilizing liquid, such as water or a salt solution. Likewise, sterile powders, granules or lozenges can be used. While the most preferred pharmaceutical formulation contains only folate or a pharmaceutically acceptable salt or ester thereof as the active agent, all of the formulations of the present invention may additionally contain one or more other pharmaceutically active compounds which act independently or synergistically with the formulations of the present invention. . When a pharmaceutically active compound other than a folic acid compound is included, the preferred formulation comprises only one additional pharmaceutically active compound. In particular, such pharmaceutically active compounds are those which are included directly in the cycle of the folate compound or which affect the circulation of the folate compound, or substances which have an additional anti-inflammatory effect, such as vitamins, antioxidants, free radical scavengers, biopterin, Lipid reducing agents, immunosuppressive agents, non-steroidal anti-inflammatory substances and/or other active ingredients. -14- 1359019 Preferred vitamins include vitamin B2, B6, BI2 or ascorbic acid, glutathione, acetaminophen, and betaine. Preferred biopterin includes biopterin in all oxidation stages and isomeric forms of biopterin, especially L-red biopterin, 7,8-dihydrobiopterin and 5,6,7,8- Tetrahydrobiopterin, especially L-purine (septiapterin), D-neoquinone, xanthine and 6-hydroxymethyl-oxime. Preferred antioxidants include vitamins £ or 0-carotene.

較佳之脂質還原劑包括氯貝酸（clofibric acid)衍生物（貝特類（fibrates))，例如，氯貝特（clofibrate) 、苯扎貝特（bezafibrate )、艾托貝特（etofibrate )、非諾貝特（fenofibrate)、詹吉布羅齊（gemgibrozil)、離子交換樹脂，例如考來稀胺（colestyramine)或考來替伯 (Colestipol )、菸鹼酸（與其衍生物），例如阿西皮莫 (acipimox )、谷留醇（sitosterin) 、HMG-CoA-還原酶抑制劑，例如阿托伐他汀（atorvastatin )、洛伐他汀（ lovastatin)、普伐他汀（pravastatin)、辛伐他汀（ simvastatin )、氟伐他汀（fluvastatin )、羅蘇伐他汀（ rosuvastatin)或西立伐他汀（cerivastatin)與膽固醇吸收抑制劑，例如依澤提密（e z e t i m i b )。Preferred lipid reducing agents include clofibric acid derivatives (fibrates), for example, clofibrate, bezafibrate, etofibrate, non- Fenofibrate, gemgibrozil, ion exchange resins such as colestyramine or Colestipol, nicotinic acid (and its derivatives), such as asipimo (acipimox), sitosterin, HMG-CoA-reductase inhibitors, such as atorvastatin, lovastatin, pravastatin, simvastatin, Fluvastatin, rosuvastatin or cerivastatin and cholesterol absorption inhibitors, such as ezetimib.

較佳發炎抑制劑包括皮質類固醇、黴酚酸嗎啉乙酯（ mycophenolate mofetil)、雷帕黴素（rapamyein)、銘調磷酸酶（calcineurin )抑制劑、單株與多株抗體、以及生長因子，諸如紅血球生成素（erythropoetin)或GM-CSF -15- 1359019Preferred inflammatory inhibitors include corticosteroids, mycophenolate mofetil, rapamyein, calcineurin inhibitors, monoclonal and polyclonal antibodies, and growth factors, Such as erythropoetin or GM-CSF -15- 1359019

較佳非類固醇抗發炎物質包括潘托非林（pentoxyfyllin )、擴胺塞拉金（sulfasalazin)、金、阿斯匹靈、Ω-3 脂肪酸、血小板凝結抑制劑，諸如醣蛋白Ilb/IIIa受體抑制劑、荷爾蒙、類黃酮或其他非類固醇抗發炎羧酸，諸如阿斯匹靈、雙水楊酸酯（salsalate )、氟苯水楊酸（ diflunisal )或三水楊酸膽鹼鎂或其他非類固醇抗發炎丙酸，諸如布洛芬（ibuprofen)、萘普生（naproxen)、非諾洛芬（fenoprofen)、凱托洛芬（ketoprofen)、氟比洛芬（flurbiprofen)或奧沙普曉（oxaprozin)，或其他非類固醇抗發炎醋酸衍生物，諸如吲哚美洒辛（ indomethacin ) '托美汀（tolmetin)、舒林酸（sulindac )、雙氯芬酸（diclofenac)或依托度酸（etodolac)，或其他非類固醇抗發炎芬那酸鹽（fenamate )，諸如美洛芬那酸鹽（meclofenamate)或甲芬那酸（mefenamic acid) ，或其他非類固醇抗發炎烯醇酸衍生物，諸如吡羅昔康（ piroxicam)或苯丁 D比哩酮（phenylbutazone)，或其他非類固醇抗發炎萘康酮（naphthylkanone)，諸如萘丁美酮 (nabumetone )，以及COX-2抑制劑，諸如希樂考昔（ celecoxib)或羅非考昔（rofecoxib)。此類物質亦包括具有抗發炎效果之物質，諸如乙型阻斷劑、抗細細胞激素抗體，例如抗-TNF-甲型抗體，或保存器官用之灌流液，諸如 Eurollins、HTK 或 UW 溶液。除了葉酸化合物（folate )以外尙包括一或多種其他藥學活性化合物時之最佳製劑係由至少一種葉酸化合物或 -16- 1359019 其藥學可接受鹽或酯結合阿斯匹靈或抗壞血酸以及至少一種藥學可接受載體所組成的藥學製劑。本發明亦提供用於預防及/或治療心血管疾病之方法 ’其包括對於需要此種治療及/或預防的對象投予治療有效量之至少一種葉酸化合物或其藥學可接受鹽或酯，或由至少一種葉酸化合物（folate)或其藥學可接受鹽或酯與至少一種藥學可接受載體所組成之藥學製劑。Preferred non-steroidal anti-inflammatory substances include pentoxyfyllin, sulfasalazin, gold, aspirin, omega-3 fatty acids, platelet aggregation inhibitors, such as glycoprotein Ilb/IIIa receptors Inhibitors, hormones, flavonoids or other non-steroidal anti-inflammatory carboxylic acids such as aspirin, salsalate, diflunisal or choline trisalicylate or other non- Steroid anti-inflammatory propionic acid, such as ibuprofen, naproxen, fenoprofen, ketoprofen, flurbiprofen or oxaprozine Oxaprozin), or other non-steroidal anti-inflammatory acetic acid derivatives, such as indomethacin 'tolmetin, sulindac, diclofenac or etodolac, or Other non-steroidal anti-inflammatory fentanales such as meclofenamate or mefenamic acid, or other non-steroidal anti-inflammatory alkyd derivatives such as piroxicam Piroxicam) or phenylbutazone, or other non-steroidal anti-inflammatory naphthylkanone, such as nabumetone, and COX-2 inhibitors, such as celecoxib Or rofecoxib. Such substances also include substances having an anti-inflammatory effect, such as a type B blocker, an anti-cytokine antibody, such as an anti-TNF-type A antibody, or a perfusate for organ preservation, such as a Eurollins, HTK or UW solution. An optimal formulation in addition to a folic acid compound (folate) comprising one or more other pharmaceutically active compounds is a combination of at least one folic acid compound or -16- 1359019 pharmaceutically acceptable salt or ester thereof combined with aspirin or ascorbic acid and at least one pharmaceutically acceptable Pharmaceutical formulations comprising a carrier are acceptable. The present invention also provides a method for preventing and/or treating a cardiovascular disease comprising administering a therapeutically effective amount of at least one folic acid compound or a pharmaceutically acceptable salt or ester thereof to a subject in need of such treatment and/or prevention, or A pharmaceutical formulation consisting of at least one folate or a pharmaceutically acceptable salt or ester thereof and at least one pharmaceutically acceptable carrier.

該製劑每劑量包括介於O.OOlmg與l’OOOmg之活性成份。用於預防時，使用每劑量包含介於5μ§與l'OOOpg間之活性成份的製劑爲佳。用於治療時，使用每劑量中含有介於O.lmg與200mg該活性成份的製劑爲佳。該劑量係視治療形式、該製劑的應用形式、投藥途徑以及病患的年齡、體重、營養與狀況而定。可以低於最適量的較低劑量開始治療’而且可以提高該劑量以便達到最佳效果。用於預防的劑量較佳範圍介於每日5pg與5,000μβ間，特別是介於每日lOOpg與1’〇〇〇μ8間。治療的最適劑量範圍介於每日O.lmg與1 〇〇mg之間，特別是介於每曰〇.5mg與 5 mg之間。可以單次投藥或以重複劑量方式進行投藥。以上述描述爲基礎，熟悉本領域之人士可以立即推斷本發明的關鍵原理，而且在不違背本發明基本槪念與範圍情況下，可進行變化與添加，因而可以讓本發明適應不同需求與條件。本文中引用之所有專利申請案、專利與公告整體揭示係以提及的方式倂入本文中。以下列實施例中給定的產物 -17- 1359019 及/或處理條件取代本發明的一般或特別描述產物及/或處理條件，可以進行下列實施例並獲得相似結果。下列特定具體實例亦僅爲範例，不應視爲限制發明揭示的其餘部分實施例方法The preparation comprises an active ingredient in an amount of from 0.001 mg to 1 OO mg per dose. For prophylaxis, it is preferred to use a formulation comprising an active ingredient between 5 μ§ and 1'OOOpg per dose. For use in therapy, it is preferred to use a formulation containing between 0.1 mg and 200 mg of the active ingredient per dose. The dosage will depend on the form of treatment, the form of administration of the preparation, the route of administration, and the age, weight, nutrition, and condition of the patient. The treatment can be initiated at a lower dose than the optimal amount' and the dosage can be increased to achieve optimal results. The dosage for prevention is preferably in the range of between 5 pg and 5,000 μβ per day, especially between 100 pg and 1 〇〇〇μ8 per day. The optimal dose range for treatment is between 0.1 mg and 1 mg per day, especially between .5 mg and 5 mg per dose. It can be administered in a single dose or in repeated doses. Based on the above description, those skilled in the art can immediately infer the key principles of the present invention, and can make changes and additions without departing from the basic concepts and scope of the present invention, thereby adapting the present invention to different needs and conditions. . All patent applications, patents and publications cited herein are hereby incorporated by reference in their entirety. The following examples were carried out and similar results were obtained by substituting the products of the invention -17- 1359019 and/or treatment conditions given in the following examples for the general or specifically described products and/or treatment conditions of the invention. The following specific examples are merely examples and should not be construed as limiting the remainder of the disclosed embodiments.

病患：英國牛津的John Radcliffe醫院已對1 17位進行冠狀動脈繞道手術（CABG )的冠狀動脈病患進行硏究。排除標準係存在任何發炎、感染、肝病或腎病或惡性腫瘤。接受非類固醇抗發炎藥劑以及任何膳食補充品（諸如葉酸（folic acid)或抗氧化劑維生素）的病患亦排除在外。介於治療組別間的人口統計特徵或基線血漿tHcy並無明顯差異（未圖示）。此硏究協定係由地方硏究委員會核准，而且每個病患均簽下知情同意書。血管採集與體外硏究：如前述，在CABG期間對於總共 61位病患採集 SV ( n = 38 )與IMA ( n = 46 )樣本（Patient: John Radcliffe Hospital in Oxford, England, has studied 17 coronary artery patients undergoing coronary artery bypass surgery (CABG). Exclusion criteria are any inflammation, infection, liver disease or kidney disease or malignant tumor. Patients receiving non-steroidal anti-inflammatory agents and any dietary supplements such as folic acid or antioxidant vitamins are also excluded. There were no significant differences in demographic characteristics or baseline plasma tHcy between treatment groups (not shown). The study protocol was approved by the local research committee and each patient signed an informed consent form. Vascular collection and in vitro study: As described above, SV (n = 38) and IMA (n = 46) samples were collected for a total of 61 patients during CABG (

Channon KM, Trends Cardiovasc Med 2004; 14:323-327;Channon KM, Trends Cardiovasc Med 2004; 14:323-327;

Guzik TJ 等人，CircM/flizow 2002; 1 05:1 656- 1 662 ) » 在 30分鐘內將置於冰冷Krebs-Henseleit緩衝液的血管片段送至實驗室。於分析內皮功能、超氧化物/過氧亞硝酸根 (鹽）製造、放射性標定精胺酸/瓜胺酸轉換、西方點漬或組織5-MTHF與生物喋呤水準之前，在Krebs-Henseleit 緩衝液中以5-MTHF(0-100pM)培育SV與IMA片段45 -18- 1359019 分鐘。Guzik TJ et al., CircM/flizow 2002; 1 05:1 656- 1 662 ) » Transfer the vascular fragments placed in ice-cold Krebs-Henseleit buffer to the laboratory within 30 minutes. Klebs-Henseleit buffer before analysis of endothelial function, superoxide/peroxynitrite (salt) production, radiolabeled arginine/citrulline conversion, western blotting or tissue 5-MTHF and biopterin levels The SV and IMA fragments were incubated with 5-MTHF (0-100 pM) for 45 -18 - 1359019 minutes.

體內硏究：進行CABG的病患（n = 56)參與雙盲安慰劑控制硏究’其中這些病患接受靜脈輸注5-MTHF ( Merck Eprova，瑞士）或安慰劑’此係於CABG之前投藥。5-MTHF的投藥劑量係〇_i3mg/kg體重，在初步硏究中於投藥後丛即達到於血獎中濃度爲〜2-3μΜ，於輸注45 分鐘之後達到1-2μΜ。此濃度係以體外實驗中進行的劑量反應分析爲基礎而選定。SV與ΙΜΑ的樣本5係於輸注5-MTHF〜45分鐘後採集（採集之平均時間係輸注後45.7± 2.9分鐘）’並如下述分析NO -媒介之血管運動功能、血管超氧化物製造、組織5-MTHF與生物喋呤水準。血管運動硏究：如前述，使用等張力硏究評估內皮依賴性與非內皮依賴性擴張（Guzik TJ等人，C/rc 2000; 86·· e85)。使血管環平衡並被動地拉至 3g，於對 KC 1的收縮基線硏究中測得最適靜態張力。以苯腎上腺素 (3 X 1 0_6M )預收縮之後，在4個來自同一血管的相同大小片段中量化對於內皮媒介促效劑乙醯膽鹼（Ach，1(Γ9Μ 至1 0_5Μ )與緩激肽（ΒΚ，10_9Μ至 10_5Μ)的血管運動反應。在體外實驗當中，在將〇(對照組）、1、1〇或 ΙΟΟμΜ之5-MTHF添加至組織浴室中進行培育之後，血管舒張反應重複。最後，於存在NOS抑制劑NG-硝基L-精胺酸甲酯（L-NAME ; ΙΟΟμΜ )下評估對於NO施體硝普鹽（SNP，1(T1qM至10·6Μ)的舒張作用。超氧化物/過氧亞硝酸根（鹽）測量：如前述，使用 -19- 經顯光素（lucigenin) (5μΜ)強化之新鮮人類血管測量血管超氧化物的製造（Skatchkov ΜΡ等人，In vivo study: Patients with CABG (n = 56) participated in a double-blind placebo control study in which these patients received an intravenous infusion of 5-MTHF (Merck Eprova, Switzerland) or placebo, which was administered prior to CABG. The dose of 5-MTHF was 〇i3 mg/kg body weight, and in the initial study, the concentration reached ～2-3 μΜ in the blood prize after administration, and reached 1-2 μΜ after 45 minutes of infusion. This concentration was selected based on the dose response analysis performed in an in vitro experiment. Sample 5 of SV and sputum was collected after infusion of 5-MTHF for 45 minutes (the average time of collection was 45.7 ± 2.9 minutes after infusion) and analyzed for NO-media vasomotor function, vascular superoxide production, tissue as follows 5-MTHF and biopterin. Vascular motion studies: As described above, endothelium-dependent and non-endothelium-dependent expansion were assessed using isotonic studies (Guzik TJ et al., C/rc 2000; 86·e85). The vascular ring was equilibrated and passively pulled to 3 g, and the optimum static tension was measured in the contraction baseline of KC 1 . After pre-shrinking with phenylephrine (3×1 0_6M), the endothelium mediator acetylcholine (Ach, 1 (Γ9Μ to 1 0_5Μ) and bradykinin was quantified in 4 identically sized fragments from the same blood vessel. Vasomotor response (ΒΚ, 10_9Μ to 10_5Μ). In an in vitro experiment, the vasodilation reaction was repeated after adding sputum (control), 1, 1 〇 or ΙΟΟμΜ 5-MTHF to the tissue bath for incubation. The relaxation effect of NO-acting nitroprusside (SNP, 1 (T1qM to 10.6Μ)) was evaluated in the presence of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; ΙΟΟμΜ). /Peroxynitrite (salt) measurement: As described above, the production of vascular superoxide was measured using fresh human blood vessels reinforced with -19-lucigenin (5 μM) (Skatchkov et al.,

Biophys Res Commun. 1 9 9 9; 25 4:3 1 9 )。將來自同一病患的SV與IMA樣本縱切，使內皮表面露出，然後在37〇c 的充氧（95% 02/5% C02) Krebs-HEPES 緩衝劑（2mL, 口11値=7.4)中均衡20分鐘。藉由將[4八1^(10(^1\〇添加於均衡緩衝劑中，並與與基礎條件比較計算超氧化物訊息’測得N 0 S衍生之超氧化物。如前述，血管過氧亞硝酸根（鹽）係使用發光胺（ΙΟΟμΜ)代替顯光素（ lucigenin )，並扣除添加特定過氧亞硝酸根（鹽）清除劑尿酸（ImM)之後的剩餘訊息所測得（Laursen JB等人， Circulation, 2001; 1 03:1 2 82 )。氧化螢光顯微照片：以氧化螢光染料二氫溴化乙啶（ dihydroethidium) (DHE)測定血管冷凍超薄切片中在原位產生的超氧化物（Bendall JK等人，Ci.rcM/αίίοη，2005; 97:864) »在具有或不具L-NAME( ΙΟΟμΜ)的PBS中以 ϋΗΕ(2μ莫耳/ L)培育來自同一血管的成對冷凍超薄切片 (3 0 μΜ )。螢光影像（x40，Zeiss LSM 510 ΜΕΤΑ 雷射 6 掃描共焦顯微鏡）係自每個血管四分體獲得，結合該血管的發光側以量化內皮細胞螢光。在各實例中，以相同成像參數同時分析血管環片段（具有與不具L-NAME) "DHE 影像分析係以e試驗方式，並由兩個獨立硏究者，使用 Image-Pro Plus 軟體（Media Cybernetics)進行。超氧化物與過氧亞硝酸根（鹽）清除分析：使用黃嘌 -20- 1359019Biophys Res Commun. 1 9 9 9; 25 4:3 1 9 ). The SV and IMA samples from the same patient were slit longitudinally to expose the endothelium surface and then in an oxygenated (95% 02/5% C02) Krebs-HEPES buffer (2 mL, 11 値 = 7.4) at 37 °C. Balanced for 20 minutes. The N 0 S-derived superoxide was measured by adding [4 八 1 ^ (10 (^1 〇〇〇均衡均衡均衡均衡均衡均衡均衡均衡均衡均衡均衡均衡均衡均衡计算计算计算。。。。。。。。。。。。。。。。 Oxynitrite (salt) is replaced by luminescent amine (ΙΟΟμΜ) instead of luciferin (lucigenin) and subtracted from the remaining information after adding a specific peroxynitrite (salt) scavenger uric acid (ImM) (Laursen JB) Et al., Circulation, 2001; 1 03:1 2 82 ). Oxidized fluorescence micrographs: in situ generated in vascular frozen ultrathin sections using the oxidative fluorescent dye dihydroethidium (DHE) Superoxide (Bendall JK et al., Ci.rcM/αίίοη, 2005; 97:864) » Incubation of the same blood vessel with sputum (2 μmol/L) in PBS with or without L-NAME (ΙΟΟμΜ) Paired frozen ultrathin sections (30 μΜ). Fluorescence images (x40, Zeiss LSM 510 ΜΕΤΑ laser 6-scan confocal microscope) were obtained from each blood vessel quadrant, binding the luminescent side of the blood vessel to quantify endothelial cells Fluorescence. In each case, the vascular ring was analyzed simultaneously with the same imaging parameters. Fragments (with and without L-NAME) "DHE image analysis was conducted by e-test and by two independent researchers using Image-Pro Plus software (Media Cybernetics). Superoxide and peroxynitrite (Salt) Removal Analysis: Using Astragalus -20- 1359019

呤/黃嘌呤氧化酶系統評估5-MTHF的直接超氧化物清除效果。超氧化物係藉由在含有黃嘌呤（〇.133mM)與顯光素（lucigenin ) ( 5μΜ)，於存在 5 - Μ T H F ( 0 -1 Ο 0 μΜ ) 或相同濃度之抗壞血酸作爲正抑制之下，在37°C的Krebs HEPES緩衝劑（2mL，pH値=7.4 )中添加黃嘌呤氧化酶 (6.6 7MU/mL)誘發產生。過氧亞硝酸根（鹽）係藉由在如前述存有5-MTHF ( 0-100μΜ )或相同濃度之尿酸作爲正抑制之下，在37°C的Krebs HEPES緩衝劑（2mL，pH 値=7.4 ) 中添加 3-嗎啉斯德酮亞胺（3- morpholinosydnonimine )而誘發產生 ° 測定5-MTHF與tHcy:於投藥5-MTHF或安慰劑之前以及採集血管時收集血液樣本。藉由適用於IMx分析器（Abbot Diagnostics)的螢光偏振免疫分析法測量血漿總高半胱胺酸（tHcy )。如前述，藉由高效液相層析術（ HPLC )測定5-MTHF的血漿與組織水準（Leeming RJ等人，Metabolism. 1 9 9 0; 39:902 ) ° 測定生物喋呤水準：如前述，藉由HP LC測定人類血管中之生物喋哈水準（Alp NJ等人，《/ /«vei/. 2003; 1 1 2:725 )，並以pmol/g之組織表示。爲了檢驗5-MTHF 對於過氧亞硝酸根（鹽）誘發之BH4氧化作用的影響，在 37°C於存在或不存在5-MTHF(lpM)之下使用 SIN-1( 2μΜ )氧化 ΒΗ4 ( Ο.ΙμΜ) 14 分鐘（Milstein S 與 Katusic Z. Biochem Biophys Res Commun. 1 999; 263:68 1 )。評估完好血管中之eNOS活性：如前述，eNOS活性 -21 - 1359019 係藉由HPLC量化來自完好血管環之輻射定標精胺酸轉變成瓜胺酸的轉換率而評估（Bono J等人，iVWric Ox/de. 2006 ;(出版中））。The 呤/xanthine oxidase system evaluates the direct superoxide scavenging effect of 5-MTHF. Superoxide is obtained by containing astragalus (〇.133mM) and lucigenin (5μΜ) in the presence of 5 - THF THF ( 0 -1 Ο 0 μΜ ) or the same concentration of ascorbic acid as positive inhibition. The production was induced by the addition of xanthine oxidase (6.6 7 MU/mL) in Krebs HEPES buffer (2 mL, pH 7.4 = 7.4) at 37 °C. Peroxynitrite (salt) is Krebs HEPES buffer (2 mL, pH 値 = at 37 ° C) under positive inhibition of 5-MTHF (0-100 μΜ) or the same concentration of uric acid as described above. 7.4) was induced by the addition of 3-morpholinosydnonimine. Determination of 5-MTHF and tHcy: Blood samples were collected prior to administration of 5-MTHF or placebo and when blood vessels were collected. Plasma total homocysteine (tHcy) was measured by fluorescence polarization immunoassay for the IMx analyzer (Abbot Diagnostics). As described above, plasma and tissue levels of 5-MTHF were determined by high performance liquid chromatography (HPLC) (Leeming RJ et al., Metabolism. 1 9 9 0; 39: 902) ° Determination of biopterin levels: as described above, Biohatch levels in human blood vessels were determined by HP LC (Alp NJ et al., / / «vei/. 2003; 1 1 2: 725) and expressed in pmol/g tissue. To test the effect of 5-MTHF on peroxynitrite (salt)-induced BH4 oxidation, SIN-1 (2μΜ) yttrium oxide 4 was used at 37 ° C in the presence or absence of 5-MTHF (lpM). .ΙμΜ) 14 minutes (Milstein S and Katusic Z. Biochem Biophys Res Commun. 1 999; 263:68 1 ). Assessing eNOS activity in intact blood vessels: As previously described, eNOS activity-21 - 1359019 was assessed by HPLC quantification of the conversion rate of serotonin converted to citrulline from intact vascular rings (Bono J et al., iVWric Ox/de. 2006; (in press)).

西方點漬分析：如前述，藉由西方點漬法測量於存在或不存在ΙμΜ 5-MTHF之下培育45分鐘之成對IMA樣本中的eNOS之二聚物：單體比（Cai S等人，Car山·〇ναίίΓ hi. 2002; 55:83 8 )。使用化學發光劑顯現帶，並使用 NIH成像軟體量化。統計分析：使用供Windows用之SPSS 12.0統計套裝軟體（美國伊利諾州SPSS Inc)進行分析。正常分布數據係以平均値±SEM表示，而不正常分布變化（諸如血管超氧化物）係以中位數（第25至第75個百分位値）表示。以多重比較用之單向 ANOVA，然後藉由Bonferoni校正進行組別間之基線比較。重複測量係藉由雙向ANOVA評估每個血管環中5-MTHF對於血管舒張反應的影響。以 Mann-Whitney U試驗、Wilcoxon等級檢定或非成對或成對數據之t檢定評估5-MTHF培育對於超氧化物產生以及 5-MTHF或tHcy水準的影響。雙尾P<〇.〇5視爲具有統計意義。實施例1 : 5-MTHF在活體外對於內皮功能與人類血管超氧化物及過氧亞硝酸根（鹽）製造的影響硏究在基線與在組織室中以〇-1〇〇μΜ 5-MTHF培育 45分鐘之後的血管片段中，5-MTHF對於ACh與BK之血 -22- 1359019 管運動反應的影響。在基線處來自相同血管的4個環之間，對於乙醯膽鹼（Ach)或緩激肽（BK )的舒張反應相似。圖1顯示以ΙμΜ之5-MTHF培育45分鐘之後，對於 Ach (試驗小組Α)或ΒΚ (試驗小組Β )之最大舒張反應Western spotting analysis: As described above, the dimer of eNOS in a pair of IMA samples was measured by Western blotting in the presence or absence of ΙμΜ 5-MTHF for 45 minutes: monomer ratio (Cai S et al. ,Car Mountain·〇να ίίΓ hi. 2002; 55:83 8 ). Bands were visualized using chemiluminescent agents and quantified using NIH imaging software. Statistical Analysis: Analysis was performed using the SPSS 12.0 Statistical Suite for Windows (SPSS Inc, Illinois, USA). Normal distribution data is expressed as mean 値±SEM, and abnormal distribution changes (such as vascular superoxide) are expressed as median (25th to 75th percentile 値). One-way ANOVA for multiple comparisons, followed by Bonferoni correction for baseline comparisons between groups. Repeated measurements were performed by two-way ANOVA to assess the effect of 5-MTHF in each vascular ring on vasodilation. The effect of 5-MTHF incubation on superoxide production and 5-MTHF or tHcy levels was assessed by Mann-Whitney U test, Wilcoxon grade test or t-test of unpaired or paired data. Two-tailed P<〇.〇5 is considered statistically significant. Example 1: Effect of 5-MTHF on endothelial function and human vascular superoxide and peroxynitrite production in vitro. At baseline and in the tissue chamber, 〇-1〇〇μΜ 5-MTHF The effect of 5-MTHF on the movement response of ACh and BK blood-22-1359019 in the blood vessel fragments after 45 minutes of incubation. The diastolic response to acetylcholine (Ach) or bradykinin (BK) is similar between the 4 rings from the same blood vessel at baseline. Figure 1 shows the maximum diastolic response for Ach (test group Α) or ΒΚ (test group 之后) after incubation for 45 minutes with -μΜ in 5-MTHF.

大幅提高，但是對照組片段仍未改變。對於苯腎上腺素產生反應的收縮絕對値係7.9±0.8g，於培育之後保持不變（ *Ρ<0·05，**Ρ<0·01相對於基線）。更高濃度的5-MTHF (10或ΙΟΟμΜ)並沒有造成進一步提高對於Ach或ΒΚ 的最大舒張作用。同一病患中，對於Ach的最大舒張作用係與對於BK之舒張作用明顯相關（Γ = 0.746, P<0.001 )。已確認血管5-MTHF水準的劑量依賴性隨著5-MTHF培育濃度提高而使提高，自對照組中的0.40±0.6nmol/g組織至分別以1、10與ΙΟΟμΜ之5-MTHF培育25個SV之後的 3·40±0·7 1、21.3±8.10 與 57.2±18.9 nmol/g 組織（所有樣本均爲P<0.05與對照組）。在對照組血管片段與以5-MTHF培育之血管片段（數據未顯示）之間，對於內皮依賴型促效劑SNP的血管運動反應並無差異，表示5-MTHF 對於NO -媒介之內皮功能的特殊影響。在僅使用緩衝劑或與1、10與1〇〇μΜ之5-MTHF培育45分鐘之後，測定自SV與ΙΜΑ產生之血管超氧化物與過氧亞硝酸根（鹽）。圖2顯示以提高濃度之5-甲基四氫葉酸（folate) (5-MTHF)培育45分鐘之後，與來自相同病患的對照血管（以緩衝劑培育）相較，隱靜脈（ SV ’試驗小組A，n = 32與試驗小組β，n = 6 )與內乳動脈 -23- 1359019 (IMA ’試驗小組C，n = 23與試驗小組D，n = 6 )二者當中產生的超氧化物與過氧亞硝酸根（鹽）明顯減少（數値係以中位數（水平線）表示），該等數値係以第2 5至第 75個百分位（方塊）與範圍（鬚狀）的中位數（水平線 )表不。圖不*Ρ<0·01與對照組）。因此，以ΙμΜ之5-MTHF培育會大幅降低SV與ΙΜΑ二者中產生的超氧化物與過氧亞硝酸根（鹽），在更高5-MTHF濃度下不會進一Significantly improved, but the control fragment remained unchanged. The contraction absolute 値 7.9 ± 0.8 g for the phenylephrine-producing response remained unchanged after incubation (*Ρ < 0·05, ** Ρ < 0·01 vs. baseline). Higher concentrations of 5-MTHF (10 or ΙΟΟμΜ) did not cause further improvement in maximal relaxation of Ach or ΒΚ. In the same patient, the maximum diastolic effect on Ach was significantly associated with the diastolic effect on BK (Γ = 0.746, P<0.001). It has been confirmed that the dose-dependent dose of 5-MTHF in the blood vessels increases with the increase of the concentration of 5-MTHF, from 0.40±0.6 nmol/g tissue in the control group to 25-MTHF in 1,10 and ΙΟΟμΜ, respectively. 3·40±0·7 1 , 21.3±8.10 and 57.2±18.9 nmol/g tissues after SV (all samples were P<0.05 vs. control group). There was no difference in vasomotor response to the endothelium-dependent agonist SNP between the control vascular fragment and the vascular fragment cultured in 5-MTHF (data not shown), indicating 5-MTHF for NO-mediated endothelial function Special impact. Vascular superoxide and peroxynitrite (salt) produced from SV and sputum were measured after only 45 minutes of incubation with buffer or with 1, 10 and 1 μM of 5-MTHF. Figure 2 shows saphenous vein (SV 'test) after incubation with increasing concentrations of 5-methyltetrahydrofolate (5-MTHF) for 45 minutes compared to control vessels from the same patient (cultured with buffer) Group A, n = 32 vs. test group β, n = 6) and superoxide produced by the inner mammary artery -23- 1359019 (IMA 'test group C, n = 23 and test group D, n = 6) Significantly reduced with peroxynitrite (salt) (the median (horizontal line)), which is the 25th to 75th percentile (square) and range (sessor) The median (horizontal line) is not shown. The figure is not *Ρ<0·01 and the control group). Therefore, the cultivation of 5-MTHF with ΙμΜ significantly reduces the superoxide and peroxynitrite (salt) produced in both SV and strontium, and does not advance at higher concentrations of 5-MTHF.

實施例2: 5-MTHF在活體內對於內皮功能與人類血管超氧化物製造的影響在雙盲方式實驗中，病患係隨機接受5-MTHF或安慰劑。靜脈內接受5-MTHF(32.4±9.01nM)的病患與接受安慰劑（26.4±4.68nM，P = NS)的疾病相較，其 5-MTHF之基線血漿水準並無明顯差異。反之，於該經5-MTHF處理組別中採集的血漿時，5-MTHF水準明顯提高（2.28土 0.21μΜ ； Ρ<0.001相對於基線），但該經安慰劑處理組別保持不變（25.8±6.85nM，P = NS相對於基線，Ρ<0·001相對於經5-MTHF處理組別）。因此，經5-MTHF處理組別與安慰劑（SV 係 1 · 19 ± 0.25nmol/g，ΙΜΑ 係 1.54 士 0.38nmol/g，二者均爲Ρ<0·001)組別相較，SV中之組織 5-MTHF 水準（9.35 ± 1.72nmol/g )與 IMA 中之組織 5-MTHF水準（1 2.7±2.5 8nm〇l/g )明顯提高，確認靜脈內投藥5-MTHF實質上提高血管組織水準。於輸注45分鐘之 -24- 1359019Example 2: Effect of 5-MTHF on endothelial function and human vascular superoxide production in vivo In a double-blind mode experiment, patients were randomized to receive 5-MTHF or placebo. Baseline plasma levels of 5-MTHF were not significantly different between patients receiving intravenous 5-MTHF (32.4 ± 9.01 nM) and those receiving placebo (26.4 ± 4.68 nM, P = NS). Conversely, at the plasma collected in the 5-MTHF treatment group, the 5-MTHF level was significantly increased (2.28 ± 0.21 μΜ; Ρ < 0.001 vs. baseline), but the placebo-treated group remained unchanged (25.8 ±6.85 nM, P = NS vs. baseline, Ρ <0·001 vs. 5-MTHF treated group). Therefore, the 5-MTHF treatment group was compared with placebo (SV system 1 · 19 ± 0.25 nmol/g, lanthanide 1.54 ± 0.38 nmol/g, both Ρ < 0·001), SV The 5-MTHF level (9.35 ± 1.72nmol/g) and the tissue 5-MTHF level (1 2.7±2.5 8nm〇l/g) in IMA were significantly improved, confirming that intravenous administration of 5-MTHF substantially increased vascular tissue levels. . Infusion for 45 minutes -24-1359019

後，接受 5-MTHF (自 1 1 _0±0.62 至 9.42±0.46pmol/L， P<0.05)的病患之血漿tHcy適當降低，但安慰劑組則維持不變（自 12.0±1.23 至 11.6±1.26pmol/L，P=NS)。然後，藉由將得自接受安慰劑或5-MTHF之病患的血管片段對於ACh與BK之血管運動反應予以量化而評估體內投藥 5-MTHF對於內皮功能的影響。圖3表示與安慰劑組（分別爲 19.4±3.3%與 37.1±3.7%，二者均 P<〇.〇5 )相較，靜脈輸注5-MTHF對於ACh (試驗小組A 43.2±4.5%)以及 BK (試驗小組B 51.4±4.2%)之最大血管舒張作用具有驚人的影響，然而兩組之間對於硝普鹽（SNP )之非內皮依賴性血管運動反應則相同（圖3 )。此等血管中經5-MTHF處理組與經安慰劑處理組對於腎上腺素的預收縮絕對値分別爲 8.2 0±1.26g 和 7.72±1.31g(P = NS) (*P<0.05 相對於安慰劑組）。對於ACh或BK之最大舒張作用與血漿 tHcy ( ACh r = 0.272 > P = 0.1 1 4 ； BK r = - 0.0 9 0，P = 0 _ 6 0 5 )之間並無相關。在得自40位病患的SV與IMA成對片段中測量基礎超氧化物產生率（25位接受安慰劑與1 5位接受靜脈輸注5-MTHF)。與安慰劑組（n = 25,灰色方塊 ;二者均爲P<0.01，圖4)相較，靜脈輸注5-MTHF組中 ’ SV與IMA當中的血管超氧化物大幅降低（靜脈輸注5-MTHF約45分鐘之後，0.13mg/kg體重，n=15 ,白色方塊 )。使用5μΜ顯光素（lucigenin)強化化學發光，對於來自相同病患之隱靜脈（SV )與內乳動脈（IMA )成對樣本進行測量。數値係表示爲中位數（水平線）、第2 5至 -25- 1359019 第 75個百分位（方塊）與値域（range )(鬚狀）。 *Ρ<0·01相對於安慰劑組。實施例3 : 5-MTHF的直接超氧化物/過氧亞硝酸根（鹽）清除能力Afterwards, plasma tHcy was appropriately reduced in patients receiving 5-MTHF (from 1 1 _0 ± 0.62 to 9.42 ± 0.46 pmol/L, P < 0.05), but remained unchanged in the placebo group (from 12.0 ± 1.23 to 11.6 ± 1.26 pmol/L, P=NS). Then, the effect of 5-MTHF administration on endothelial function in vivo was evaluated by quantifying the vasomotor response of ACh and BK from a blood vessel fragment obtained from a placebo or 5-MTHF. Figure 3 shows that intravenous infusion of 5-MTHF for ACh (test group A 43.2 ± 4.5%) compared with placebo (19.4 ± 3.3% vs. 37.1 ± 3.7%, both P < 〇.〇5) The maximum vasodilation effect of BK (test group B 51.4 ± 4.2%) had a striking effect, whereas the non-endothelium-dependent vasomotor response to nitroprusside (SNP) was the same between the two groups (Fig. 3). The pre-contracted absolute enthalpy of epinephrine in the 5-MTHF-treated and placebo-treated groups in these vessels was 8.2 0 ± 1.26 g and 7.72 ± 1.31 g, respectively (P = NS) (*P < 0.05 vs. placebo) group). There was no correlation between the maximum relaxation effect of ACh or BK and plasma tHcy (ACh r = 0.272 > P = 0.1 1 4 ; BK r = - 0.0 9 0, P = 0 _ 6 0 5 ). Basal superoxide production rates were measured in SV and IMA paired sections from 40 patients (25 received placebo and 15 received intravenous infusion of 5-MTHF). Compared with the placebo group (n = 25, gray squares; both P < 0.01, Figure 4), the vascular superoxide in the SV and IMA was significantly reduced in the 5-MTHF group (intravenous infusion 5 - After about 45 minutes of MTHF, 0.13 mg/kg body weight, n=15, white squares). Chemiluminescence was enhanced using 5 μΜ Luciferin and the paired samples of saphenous vein (SV) and internal mammary artery (IMA) from the same patient were measured. The number system is expressed as the median (horizontal line), the 25th percentile (square) of the 25th to 25th, 1359019, and the range (saturated). *Ρ<0·01 vs. placebo group. Example 3: Direct superoxide/peroxynitrite (salt) removal ability of 5-MTHF

爲了硏究5-MTHF之直接清除作用算入其對於血管超氧化物產生與內皮功能的影響與否，評估5-MTHF清除超氧化物的能力並與習知超氧化物清除劑抗壞血酸（維生素 C)比較。添加於與血管組織中觀察到之相同水準黃嘌呤/ 黃嘌呤氧化酶系統產生的超氧化物中時，抗壞血酸具有有力清除效果，於ΙμΜ下減少50%可測量之超氧化物，然而低濃度5-MTHF ( l-ΙΟμΜ)對於可測量超氧化物則無可偵測效果（圖5)，僅於極高5-MTHF濃度（ΙΟΟμΜ)下觀察到適當超氧化物清除作用。與習知過氧亞硝酸根（鹽）清除劑尿酸比較，評估 5-MTHF之直接過氧亞硝酸根（鹽）清除能力。圖5顯示當添加於SIN-1系統時，5-MTHF具有與相同濃度尿酸之效果相當的有力清除效果，於ΙμΜ下可測量過氧亞硝酸根（鹽）減少75% (圖5 )，意味著5-MTHF在體外硏究中所使用的濃度下發揮明顯的過氧亞硝酸根（鹽）清除效果（數値係3個獨立實驗的平均値土SEM。*Ρ<0.01相對於維生素C ; tP<〇.〇5以及φΡ<0·01相對於ΟμΜ )。實施例4: 5-MTHF對於產生eNOS衍生之血管超氧化物 -26- 1359019 的影響與對eNOS活性的影響In order to investigate the direct clearance of 5-MTHF into its effects on vascular superoxide production and endothelial function, the ability of 5-MTHF to scavenge superoxide was evaluated and compared with the conventional superoxide scavenger ascorbic acid (vitamin C). When added to the superoxide produced by the same level of jaundice/xanthine oxidase system observed in vascular tissue, ascorbic acid has a powerful scavenging effect, reducing oxidizable superoxide by 50% under ΙμΜ, whereas low concentration 5 -MTHF (l-ΙΟμΜ) has no detectable effect on measurable superoxide (Fig. 5), and proper superoxide scavenging effect was observed only at very high 5-MTHF concentration (ΙΟΟμΜ). The direct peroxynitrite (salt) scavenging ability of 5-MTHF was evaluated in comparison with the conventional peroxynitrite (salt) scavenger uric acid. Figure 5 shows that when added to the SIN-1 system, 5-MTHF has a potent scavenging effect comparable to that of the same concentration of uric acid, and a reduction of 75% in peroxynitrite (salt) can be measured under ΙμΜ (Fig. 5), meaning 5-MTHF exerted a significant peroxynitrite (salt) scavenging effect at the concentrations used in in vitro studies (average alumina SEM of 3 independent experiments of several systems). *Ρ<0.01 vs. vitamin C; tP<〇.〇5 and φΡ<0·01 relative to ΟμΜ). Example 4: Effect of 5-MTHF on the production of eNOS-derived vascular superoxide -26- 1359019 and its effect on eNOS activity

藉由使用L-NAME量化NOS抑制作用，在SV與 IMA成對樣本中評估NO S衍生之超氧化物產生作用。在安慰劑組中的sv與IMA二者當中’使用L-NAME減少血管超氧化物產生，意味著對於NO S所致之血管超氧化物產生具有淨貢獻。反之’ L-NAME增加得自經5-MTHF處理之病患血管中的超氧化物產生作用，意味著淨NO產生 (圖6)。重要的是，SV或IMA當中可以L-NAME抑制的超氧化物產生作用與血漿 5-MTHF相關（分別係 r=0.511，ρ = 〇·〇〇6 及 r = 0.690，ρ = 0·0001 )，但與血槳 tHcy 水準無關（分別係 r = 0.40，ρ = 0·829 及 r = -0.286， p = 0.1 0 6 )。使用DHE之氧化螢光顯微照片係用以視察血管超氧化物產生作用，以及指明於eNOS媒介之內皮超氧化物產生作用的變化（圖6 ) 。L-NAME降低得自安慰劑組病患 _ 的血管中之內皮DHE螢光（表示eNOS非偶聯作用），然後而L-NAME增加得自經5-MTHF處理病患之血管中的內皮DHE螢光，此意味著此等血管中的eNOS偶合改善。重要的是，該血管壁中其他區域的DHE螢光不受5_ MTHF影響，提供部位內控制並證實NOS抑制之內皮特殊效果。使用HPLC測量來自12位病患之使用或使用5-MTHF培育45分鐘之IMA樣本當中經輻射標定精胺酸轉換成瓜胺酸的轉換率，進一步評估5-MTHF對於eNOS活性的影響。與來自相同病患之成對對照組血管（0. 1 4 土 -27- 1359019 0.02%/g，ρ<〇·〇5)比較，在 5-MTHF 培育的血管（0.20土 0.0 3 %/g組織）中觀察到瓜胺酸的產生明顯增加，此意味著5-MTHF直接提高人類血管中之eNOS活性。因此，5-MTHF減少血管產生超氧化物，並經由對於eNOS偶合作用的影響改善NO-媒介之內皮功能。The NO S-derived superoxide production was evaluated in SV and IMA paired samples by quantifying NOS inhibition using L-NAME. Using L-NAME to reduce vascular superoxide production in both sv and IMA in the placebo group meant a net contribution to vascular superoxide production by NO S . Conversely, 'L-NAME increased from superoxide production in the blood vessels of patients treated with 5-MTHF, meaning net NO production (Fig. 6). Importantly, the superoxide production that can be inhibited by L-NAME in SV or IMA is related to plasma 5-MTHF (r=0.511, ρ = 〇·〇〇6 and r = 0.690, ρ = 0·0001, respectively). However, it has nothing to do with the plasma tHcy level (r = 0.40, ρ = 0·829 and r = -0.286, p = 0.1 0 6 respectively). Oxidized fluorescence micrographs using DHE were used to inspect vascular superoxide production and to indicate changes in endothelial superoxide production in eNOS media (Fig. 6). L-NAME reduced endothelial DHE fluorescence in the blood vessels from the patients in the placebo group (indicating eNOS non-coupling), and then L-NAME increased from endothelial DHE in the blood vessels of patients treated with 5-MTHF Fluorescence, which means that the eNOS coupling in these blood vessels is improved. Importantly, DHE fluorescence in other areas of the vessel wall was not affected by 5_MTHF, providing intra-site control and confirming the endothelial specific effects of NOS inhibition. The effect of 5-MTHF on eNOS activity was further evaluated by HPLC using a conversion rate from radiolabeled arginine to citrulline in IMA samples from 12 patients or 45 min in 5-MTHF. Compared with the control group blood vessels (0. 14 soil -27- 1359019 0.02%/g, ρ<〇·〇5) from the same patient, the blood vessels cultured in 5-MTHF (0.20 soil 0.0 3 %/g) A significant increase in the production of citrulline was observed in the tissue, which means that 5-MTHF directly increases eNOS activity in human blood vessels. Thus, 5-MTHF reduces vascular production of superoxide and improves endothelial function of NO-mediated via effects on eNOS coupling.

實施例5: 5-MTHF對於人類血管中四氫生物喋呤水準與 eNOS二聚物：單體比之影響圖7顯示以ΙμΜ之5-MTHF體外培育SV 45分鐘會明顯增加血管ΒΗ4以及該BH4/tBio比。更顯著的是，與經安慰劑處理的病患相較，體內接受靜脈輸注5-MTHF的病患之BH4與BH4/tBio比的絕對値明顯升高（圖7 )。圖7亦表示5-MTHF亦會明顯減少曝於SIN-1所產生的過氧亞硝酸根（鹽）之後之BH4以及該BH4/tBio比二者的下降。此外，IMA環曝露於ΙμΜ之5-MTHF之後該二聚物：單體比亦會提高（圖7)。另外發現血管BH4/tBio比與中L-N ΑΜΕ所誘發之超氧化物產生的變化有重大相關。圖8顯示在來自相同病患之SV ( r = 0.495，Ρ = 0.002 )與 IMA ( r = 0.621 « Ρ = 0.001 )二者中 SV 與 IMA 之綜合相關 (紅色空心圓點：經安慰劑處理的IMA，紅色實心圓點：經5-MTHF處理之IMA ;黑色空心圓點：經安慰劑處理的 SV，黑色實心圓點：經5-MTHF處理之SV )。最後，SV 與IMA中的BH4/tBio比與血漿中5-MTHF具有明顯相關 (分別爲 r = 0.498，p = 0,001 與 r = 0.65 6 > p^O.OOO 1 ) ’但 -28- 1359019 與血漿中tHcy無明顯相關（分別爲r = 0.033，p = 〇.83 6與 r = -0_165，ρ = 0·374)，意味著5-MTHF本身才是避免體內人類血管之ΒΗ4氧化的關鍵參數，而非血漿中之tHcy。此等觀察意味著介於血管BH4有效性與人類血管中之 eNOS偶合作用之間的直接關係，其係NO-媒介之內皮功能與血管超氧化物產生二者的主要決定因素。Example 5: Effect of 5-MTHF on tetrahydrobiopterin and eNOS dimer: monomer ratio in human blood vessels Figure 7 shows that in vitro incubation of SV for 45 minutes with 5-MTHF in ΙμΜ significantly increased vasospasm 4 and the BH4 /tBio ratio. More significantly, the absolute sputum ratio of BH4 to BH4/tBio was significantly increased in patients receiving intravenous 5-MTHF in vivo compared with placebo-treated patients (Fig. 7). Figure 7 also shows that 5-MTHF also significantly reduced the decrease in BH4 and the BH4/tBio ratio after exposure to the peroxynitrite (salt) produced by SIN-1. In addition, the dimer:mer ratio was also increased after exposure of the IMA ring to 5-MTHF (Figure 7). It was also found that the vascular BH4/tBio ratio was significantly correlated with the change in superoxide production induced by L-N ΑΜΕ. Figure 8 shows a combination of SV and IMA in both SV ( r = 0.495, Ρ = 0.002 ) and IMA ( r = 0.621 « Ρ = 0.001 ) from the same patient (red hollow dots: placebo-treated IMA, red solid dots: IMA treated with 5-MTHF; black open dots: placebo treated SV, black solid dots: SV treated with 5-MTHF). Finally, the ratio of BH4/tBio in SV to IMA was significantly correlated with 5-MTHF in plasma (r = 0.498, p = 0,001 and r = 0.65 6 > p^O.OOO 1 ) 'but -28- 1359019 There was no significant correlation with tHcy in plasma (r = 0.033, p = 〇.83 6 and r = -0_165, ρ = 0·374), which means that 5-MTHF itself is the key to avoid the oxidation of human blood vessels in vivo. Parameters, not tHcy in plasma. These observations imply a direct relationship between the availability of vascular BH4 and eNOS coupling in human blood vessels, which is a major determinant of both NO-mediated endothelial function and vascular superoxide production.

【圖式簡單說明】圖1 ·對30名病患的隱靜脈片段以提高濃度之5-甲基四氫葉酸（folate) (5-MTHF)培育45分鐘進行等張力硏究。四組之間的基線舒張相同，而且以單一曲線表示。在對照血管中，血管內皮依賴性促效劑乙醯膽鹼（Ach ，試驗小組A )或緩激肽（BK，試驗小組B )造成之血管舒張並未改變，但以5-MTHF培育之後則會提高。於基線時，對於苯腎上腺素產生反應的收縮絕對値係7.9±0.8g，於培育之後保持不變。圖示*Ρ<0·05，**Ρ<〇.〇1相對於基線。圖2 ·以提高濃度之5-甲基四氫葉酸（folate) (5-MTHF )培育45分鐘之後，與相同病患的對照血管（以緩衝劑培育）相較，隱靜脈（SV，試驗小組A，n = 32與試驗小組B，n = 6 )與內乳動脈（IMA，試驗小組C，n = 23 與試驗小組D，n = 6 )二者當中產生的超氧化物與過氧亞硝酸根（鹽）明顯減少。該等數値係表示爲中位數（水平線）、第25至第75個百分位（方塊）與値域（鬚狀）。 -29- 1359019BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 - Isotonic force was studied by incubating a saphenous vein fragment of 30 patients with an increased concentration of 5-methyltetrahydrofolate (5-MTHF) for 45 minutes. Baseline diastolicity was the same between the four groups and was expressed as a single curve. In control blood vessels, vasodilation caused by the endothelium-dependent agonist acetylcholine (Ach, test group A) or bradykinin (BK, test group B) did not change, but after incubation with 5-MTHF Will improve. At baseline, the contractile absolute response to phenylephrine was 7.9 ± 0.8 g, which remained unchanged after incubation. The figure *Ρ<0·05, **Ρ<〇.〇1 is relative to the baseline. Figure 2 - Saphenous vein (SV, trial group) after incubation with increasing concentrations of 5-methyltetrahydrofolate (5-MTHF) for 45 minutes compared to control vessels of the same patient (cultured with buffer) Superoxide and peroxynitrite produced by A, n = 32 and test group B, n = 6) and internal mammary artery (IMA, test group C, n = 23 and test group D, n = 6) The root (salt) is significantly reduced. The numbers are expressed as the median (horizontal line), the 25th to 75th percentile (square), and the 値 field (saturated). -29- 1359019

圖3·相較於接受安慰劑（n= 17)的病患之隱靜脈片段，靜脈輸注 5-甲基四氫葉酸（folate ) 45分鐘（5-MTHF ’ 0.13mg/kg體重，n=15)之後獲得的隱靜脈片段對於乙醯膽鹼（Ach，試驗小組A)與緩激肽（BK，試驗小組B)二者的血管運動反應明顯較大。接受5-MTHF與接受安慰劑的病患之間對於硝普鹽（SNP )的血管運動反應無明顯差異。在5-MTHF與安慰劑處理的組別中，此等血管中對於苯腎上腺素的預收縮絕對値分別爲8.2 0± 1.26 g 與7.72±1.3 1g ( P=NS )。圖示*P<0.05相對於安慰劑組。圖4·靜脈輸注5-甲基四氫葉酸（folate) 45分鐘（ 5-MTHF，0.13mg/kg體重，n=15，白色方塊）之後的病患之完好血管片段所產生的超氧化物明顯低於接受安慰劑（ n = 25，灰色方塊）的病患。此等測量係使用5μΜ顯光素 (lucigenin )強化化學發光，對於來自相同病患之19組成對隱靜脈（SV)與內乳動脈（IMA )樣本進行。該等數値係表示爲中位數（水平線）、第25至第75個百分位（方塊）與値域（鬚狀）。圖示* Ρ<〇· 〇1相對於安慰劑組。圖5·使用黃嘌呤/黃嘌呤氧化酶系統（Α)評估5 -甲基四氫葉酸的超氧化物清除作用。5-MTHF僅在濃度 >10 μΜ下具有微弱超氧化物清除效果，然而維生素C在更低濃度下具有預期的清除效果。使用SIN-1 ( ΙμΜ )( Β )評估5-MTHF與尿酸之過氧亞硝酸根（鹽）清除作用。相較於相同濃度之尿酸的過氧亞硝酸根（鹽）清除效果，5-MTHF在低至ΙμΜ的濃度下具有強力直接過氧亞硝酸 -30- 1359019 根（鹽）清除效果。該等數値係爲3個獨立實驗的平均値士SEM。*P<0.0 1 相對於維生素 C ; tP<〇.〇5 以及 φΡ<0.01 相對於ΟμΜ。圖6·係靜脈輸注5-甲基四氫葉酸（5-MTHF)對於隱靜脈（SV)與內乳動脈（ΙΜΑ)中產生eNOS所衍生之Figure 3. Intravenous infusion of 5-methyltetrahydrofolate (folate) for 45 minutes compared to placebo in patients receiving placebo (n = 17) (5-MTHF ' 0.13 mg/kg body weight, n=15 The saphenous vein fragments obtained afterwards were significantly more responsive to vasomotor responses to both acetylcholine (Ach, test group A) and bradykinin (BK, test group B). There was no significant difference in vasomotor response to nitroprusside (SNP) between patients receiving 5-MTHF and placebo. In the 5-MTHF and placebo-treated groups, the pre-shrinking absolute enthalpies for phenylephrine in these vessels were 8.2 0 ± 1.26 g and 7.72 ± 1.3 1 g (P = NS ), respectively. The graph *P < 0.05 versus the placebo group. Figure 4. Intravenous infusion of 5-methyltetrahydrofolate (folate) After 45 minutes (5-MTHF, 0.13 mg/kg body weight, n=15, white squares), the superoxide produced by the intact blood vessel fragments of the patient is evident. Lower than patients receiving placebo (n = 25, gray squares). These measurements were enhanced by chemiluminescence using 5 μΜ lucigenin for 19 pairs of paired saphenous vein (SV) and internal mammary artery (IMA) samples from the same patient. These numbers are expressed as median (horizontal line), 25th to 75th percentile (square), and 値 (shall). Figure * Ρ <〇· 〇1 relative to the placebo group. Figure 5. Evaluation of the superoxide scavenging effect of 5-methyltetrahydrofolate using the xanthine/xanthine oxidase system (Α). 5-MTHF has a weak superoxide scavenging effect only at a concentration > 10 μΜ, whereas vitamin C has the expected scavenging effect at a lower concentration. The peroxynitrite (salt) clearance of 5-MTHF and uric acid was evaluated using SIN-1 ( ΙμΜ )( Β ). Compared with the peroxynitrite (salt) scavenging effect of uric acid of the same concentration, 5-MTHF has a strong direct peroxynitrite -30-1359019 root (salt) scavenging effect at concentrations as low as ΙμΜ. The numbers are the average SEM of 3 independent experiments. *P<0.0 1 relative to vitamin C; tP<〇.〇5 and φΡ<0.01 relative to ΟμΜ. Figure 6. Intravenous infusion of 5-methyltetrahydrofolate (5-MTHF) for the production of eNOS in the saphenous vein (SV) and internal mammary artery (ΙΜΑ)

超氧化物的影響。在安慰劑組病患（灰色方塊）中，SV 與IMA (試驗小組A)二者中以5μΜ顯光素（lucigenin )強化化學發光測量之超氧化物產生作用皆因NOS抑制劑L-NΑΜΕ而降低。不過，L-NΑΜΕ明顯提高靜脈輸注5-MTHF〜45分鐘（白色方塊）組別病患血管中所產生的超氧化物。使用二氫溴化乙陡（dihydroethidium)蛋光劑在原位偵測S V (試驗小組B與C，n= 1 0 )中產生之內皮衍生超氧化物，顯示在得自經5-MTHF處理的病患血管中，自血管內皮（箭頭）L-NAME誘發產生的超氧化物增加（白色方塊），而自經安慰劑處理的病患血管之血管內皮 L-NAME誘發產生的超氧化物減少（灰色方塊）。 *P<0.05，tP<0.01，ίΡ<0·005 5-MTHF 相對於安慰劑組。圖7 ·係隱靜脈（SV )與內乳動脈（IMA)中之組織生物喋呤水準。在SV(n = 20對）與IMA(n=12對）二者當中，以5-甲基四氫葉酸（5-MTHF; ΙμΜ)體外培育45 分鐘，相較於對照組乃提高了四氫生物喋呤（ΒΗ4 )水準 (試驗小組Α)與ΒΗ4/總生物喋呤（BH4/tBio)比（試驗小組B)。同樣地，與來自接受安慰劑病患的血管相較（ SV的n=19，IMA的n=19)，於5-MTHF浸漬45分鐘之 -31 - 1359019The effect of superoxide. In the placebo group (grey squares), the superoxide production measured by 5 μΜ lucigenin-enhanced chemiluminescence in both SV and IMA (test group A) was due to the NOS inhibitor L-NΑΜΕ. reduce. However, L-NΑΜΕ significantly increased the superoxide produced in the blood vessels of patients with intravenous infusion of 5-MTHF for ~45 minutes (white squares). Endothelial-derived superoxides produced in situ by SV (test groups B and C, n = 10) were detected in situ using dihydroethidium egg light, indicated in the treatment with 5-MTHF. In patients with blood vessels, the increase in superoxide induced by vascular endothelium (arrow) L-NAME (white squares), and the decrease in superoxide induced by vascular endothelial L-NAME from placebo-treated patients ( Gray squares). *P < 0.05, tP < 0.01, ί Ρ < 0·005 5-MTHF vs. placebo group. Figure 7 • Tissue biopsy in the saphenous vein (SV) and internal mammary artery (IMA). In the SV (n = 20 pairs) and IMA (n = 12 pairs), 5-methyltetrahydrofolate (5-MTHF; ΙμΜ) was incubated for 45 minutes in vitro, which improved tetrahydrogen compared to the control group. Biopterin (ΒΗ4) level (test group Α) vs. ΒΗ4/total biopterin (BH4/tBio) ratio (test group B). Similarly, compared to blood vessels from patients receiving placebo (n=19 for SV, n=19 for IMA), immersed in 5-MTHF for 45 minutes -31 - 1359019

後所獲得的20個血管樣本中的血管BH4(試驗小組C) 與BH4/tBio (試驗小組D )比較高。這兩組病患之間的人口統計特徵或用藥法並無明顯差異。灰色方塊：對照組或安慰劑組；白色方塊：5-MTHF; *Ρ<0·05，**Ρ<〇.〇1相對於對照組或安慰劑組。5-MTHF ( ΙμΜ )避免BH4 (試驗小組E )與BH4/tBio (試驗小組F )降低，此係因BH4 ( 0. ΙμΜ)曝於 SIN-1 (2μΜ) 14 分鐘所引發。tP<〇.〇5 以及本P<0.001相對於單獨使用BH4 ; *P<0.05，**P<0_01相對於BH4 + SIN-1。以5-MTHF體外培育IMA(n = 5對）大幅提高eNOS二聚物：單體比，此係藉由於量化eNOS帶強度（試驗小組Η ’ *Ρ<0·05相對於對照組）之後以免疫轉漬（試驗小組G)進行評估。圖8 . ΒΗ4/總生物喋呤（BH4/tBio )比係與體內接受安慰劑或 5-MTHF其中之一的病患的隱靜脈（SV， r = 0.495，P = 0.02 )與內乳動脈（IMA，r = 0.621，P = 0.001 )二者當中L-NΑΜΕ誘發之超氧化物製造改變相關。圖示的是SV與ΙΜΑ之結合相關。紅色空心圓點：經安慰劑處理的ΙΜΑ，紅色實心圓點：經5-MTHF處理之ΙΜΑ ;黑色空心圓點：經安慰劑處理的SV，黑色實心圓點：經5- MTHF處理之SV。圖9.內皮型一氧化氮合成酶（eNOS)將L-精胺酸催化成NO與L -瓜胺酸之催化反應。圖10·藉由eNOS製造NO，其係生理途徑（eNOS 偶合）與受損途徑（eNOS非偶聯作用）之比較。 -32-The blood vessel BH4 (test group C) in the 20 vascular samples obtained later was higher than BH4/tBio (test group D). There were no significant differences in demographic characteristics or medications between the two groups of patients. Gray squares: control group or placebo group; white squares: 5-MTHF; *Ρ<0·05, **Ρ<〇.〇1 relative to the control group or placebo group. 5-MTHF (ΙμΜ) avoided a decrease in BH4 (test group E) and BH4/tBio (test group F), which was triggered by exposure of BH4 (0. ΙμΜ) to SIN-1 (2 μΜ) for 14 minutes. tP < 〇. 〇 5 and this P < 0.001 vs. BH4 alone; *P < 0.05, **P < 0_01 relative to BH4 + SIN-1. In vitro incubation of IMA (n = 5 pairs) with 5-MTHF significantly increased the eNOS dimer: monomer ratio by quantifying the intensity of the eNOS band (test group Η '*Ρ<0·05 vs. control group) Immune transfer stains (test group G) were evaluated. Figure 8. The ΒΗ4/total sputum (BH4/tBio) ratio of the saphenous vein (SV, r = 0.495, P = 0.02) and the internal mammary artery (in patients with one of placebo or 5-MTHF) IMA, r = 0.621, P = 0.001) correlated with L-NΑΜΕ induced superoxide manufacturing changes. The illustration shows the combination of SV and sputum. Red open dots: placebo treated sputum, red solid dots: treated with 5-MTHF; black open dots: placebo treated SV, black solid dots: SV treated with 5-MTHF. Figure 9. Endothelial nitric oxide synthase (eNOS) catalyzes the catalytic reaction of L-arginine with NO and L-citrulline. Figure 10. Preparation of NO by eNOS, which is a comparison of the physiological pathway (eNOS coupling) with the damaged pathway (eNOS non-coupling). -32-

Claims

1359019 It,年又月α日修(ιό正本1359019 It, year and month, alpha day repair (ιό正本

附件3Α :第095129781號申請專利範圍修正本民國100年8月18日修正十、申請專利範圍 1·一種葉酸化合物（folate )之用途，其係用於製造基本上由至少一種葉酸化合物（folate)或其藥學可接受鹽或酯作爲活性劑所組成而供預防及/或治療心血管疾病之藥物，Annex 3: No. 09519781 Patent Application Amendment Amendment of August 18, 100 of the Republic of China, Application No. 1 1. Use of a folic acid compound (folate) for the manufacture of substantially at least one folic acid compound (folate) Or a pharmaceutically acceptable salt or ester thereof as an active agent for preventing and/or treating cardiovascular diseases,

其中該葉酸化合物係選自下列所組成之群：喋酸單麩胺酸 (葉酸（folic acid))、二氫葉酸、5-甲醯基四氫葉酸、 5 -甲基四氫葉酸、5, 10 -亞甲基四氫葉_、5,10-次甲基四氫葉酸、10 -甲醯基四氫葉酸或四氫葉酸、其多麩胺酸化合物、其光學異構物，以及光學異構物之混合物，以及其藥學可接受鹽與酯。 2.如申請專利範圍第1項之用途，其中該心血管疾病係動脈粥狀硬化。 3 ·如申請專利範圍第1或2項之用途，其中該光學異構物之混合物是消旋混合物。 4. 如申請專利範圍第1或2項之用途，其中該葉酸化合物係選自5-甲基- (6S)-四氫葉酸、5-甲基- (6R)-四氫葉酸、5-甲基- (6R，S) ·四氫葉酸、5-甲醯基- (6S) -四氫葉酸、5·甲醯基- (6R)-四氫葉酸或5-甲醯基-( 6R,S)-四氫葉酸，或其藥學可接受鹽或酯。 5. 如申請專利範圍第1或2項之用途，其中該葉酸化合物係選自5-甲基-(6S )-四氫葉酸或5-甲基-(6R，S )- 1359019 四氫葉酸，或其藥學可接受鹽或酯。 6. 如申請專利範圍第1項之用途，其中該葉酸化合物 (folate)具有改善NO·媒介之內皮依賴性血管運動反應以及減少血管超氧化物之效用。 7. 如申請專利範圍第1項之用途，其中該葉酸化合物具有清除反應性氧物質（R0S)、增加血管BH4、增加 BH4/總生物喋哈比、逆轉 eNOS之非偶聯作用、增加 eNOS二聚物：單體比以及直接加強eNOS活性之效用。 8. 如申請專利範圍第7項之用途，其中該反應性氧物質（ROS )係過氧亞硝酸根（鹽）。 9. 一種用於治療及/或預防心血管疾病之藥學製劑，其基本上由至少一種葉酸化合物（folate )或其藥學可接受鹽或酯與至少一種藥學可接受載體所組成，其中該葉酸化合物係選自下列所組成之群：喋酸單麩胺酸 (葉酸（folic acid))、二氫葉酸、5-甲醯基四氫葉酸、 5-甲基四氫葉酸、5,10-亞甲基四氫葉酸、5,10-次甲基四氫葉酸、10-甲醯基四氫葉酸或四氫葉酸、其多麩胺酸化合物、其光學異構物，以及光學異構物之混合物，以及其藥學可接受鹽與酯。 10. 如申請專利範圍第9項之藥學製劑，其中該光學異構物之混合物是消旋混合物。 11. 如申請專利範圍第9或10項之藥學製劑，其包含阿斯匹靈（aspirin )或抗壞血酸做爲另外之藥學可接受的活性成份及/或包含佐劑。Wherein the folic acid compound is selected from the group consisting of citric acid monoglutamic acid (folic acid), dihydrofolic acid, 5-methylmethyltetrahydrofolate, 5-methyltetrahydrofolate, 5, 10 - methylenetetrahydroleaf _, 5,10-methine tetrahydrofolate, 10-methylmercaptotetrahydrofolate or tetrahydrofolate, polyglutamic acid compounds, optical isomers thereof, and optical isoforms A mixture of constructs, as well as pharmaceutically acceptable salts and esters thereof. 2. The use of claim 1, wherein the cardiovascular disease is atherosclerosis. 3. The use of claim 1 or 2, wherein the mixture of optical isomers is a racemic mixture. 4. The use of claim 1 or 2, wherein the folic acid compound is selected from the group consisting of 5-methyl-(6S)-tetrahydrofolate, 5-methyl-(6R)-tetrahydrofolate, 5-A -(6R,S) ·Tetrahydrofolate, 5-methylindolyl-(6S)-tetrahydrofolate, 5·methylmercapto-(6R)-tetrahydrofolate or 5-methylindolyl-(6R,S - tetrahydrofolate, or a pharmaceutically acceptable salt or ester thereof. 5. The use of claim 1 or 2, wherein the folic acid compound is selected from the group consisting of 5-methyl-(6S)-tetrahydrofolate or 5-methyl-(6R,S)- 1359019 tetrahydrofolate, Or a pharmaceutically acceptable salt or ester thereof. 6. The use of the first aspect of the patent application, wherein the folate compound has an effect of improving an endothelium-dependent vasomotor response of the NO medium and reducing vascular superoxide. 7. For the use of the scope of claim 1, wherein the folic acid compound has a reactive oxygen species (ROS), increases blood vessel BH4, increases BH4/total biological hip-hop ratio, reverses the unconjugated effect of eNOS, and increases eNOS II. Polymer: monomer ratio and the effect of directly enhancing eNOS activity. 8. The use of claim 7 wherein the reactive oxygen species (ROS) is peroxynitrite (salt). A pharmaceutical preparation for treating and/or preventing cardiovascular diseases, which consists essentially of at least one folic acid compound (folate) or a pharmaceutically acceptable salt or ester thereof, and at least one pharmaceutically acceptable carrier, wherein the folic acid compound Is selected from the group consisting of citric acid monoglutamic acid (folic acid), dihydrofolate, 5-methylmethyltetrahydrofolate, 5-methyltetrahydrofolate, 5,10-methylene Tetrahydrofolate, 5,10-methine tetrahydrofolate, 10-mercaptotetrahydrofolate or tetrahydrofolate, a polyglutamic acid compound thereof, an optical isomer thereof, and a mixture of optical isomers, And pharmaceutically acceptable salts and esters thereof. 10. The pharmaceutical preparation according to claim 9, wherein the mixture of optical isomers is a racemic mixture. 11. The pharmaceutical preparation according to claim 9 or 10, which comprises aspirin or ascorbic acid as an additional pharmaceutically acceptable active ingredient and/or an adjuvant.