TWI472326B - Use of disodium norcantharidate for modulating the development of dendritic cells - Google Patents
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本揭示內容是有關於一單離自莞菁科昆蟲-大斑蝥(Mylabris Phalerata )的斑蝥素(cantharidin,CA)衍生物-去甲基斑蝥酸二鈉(disodium norcantharidate,DNCTD,在本文中稱「O6化合物」)的新穎用途,其可調控樹突細胞的發育及成熟,因此是潛在可用來製造抗排斥藥物的醫藥品或藥學組合物的小分子化合物。The present disclosure relates to a isolated from Wan phthalocyanine Coleoptera - Large cantharidin (Mylabris Phalerata) cantharidin (cantharidin, CA) derivative - demethylation cantharidic acid disodium (disodium norcantharidate, DNCTD, called "herein The novel use of the O6 compound"), which modulates the development and maturation of dendritic cells, is therefore a small molecule compound of a pharmaceutical or pharmaceutical composition potentially useful for the manufacture of anti-rejection drugs.
斑蝥(Mylabris )乃是指源自中國之橫紋大斑蝥(Mylabris Phalerata )或黃黑小斑蝥(Mylabris Cichorii )的乾燥蟲體,已被當作中藥材使用超過二千年之久,目前也仍是許多民俗療法的藥材之一。 Mylabris refers to the dry worms from China's Mylabris Phalerata or Mylabris Cichorii , which have been used as Chinese herbal medicines for more than two thousand years. It is one of the medicinal herbs of many folk treatments.
已知斑蝥具有抗腫瘤活性,可增加白血球細胞數目,但也會刺激泌尿系統造成不適。斑蝥中的活性成分為斑蝥素(cantharidin,CA),但斑蝥素合成不易,且本身具有高毒性,因此,長久以來,此領域中人士一直希望找到一種斑蝥素的衍生物,其仍具有有用的生物活性,但不會像CA這般具有強烈的毒性。據此,並開發出兩種斑蝥素的衍生物,分別為去甲基斑蝥素(norcantharidin,NCTD)和去甲基斑蝥酸二鈉。It is known that cantharidin has anti-tumor activity, which can increase the number of white blood cells, but it also stimulates the urinary system to cause discomfort. The active ingredient in cantharidin is cantharidin (CA), but cantharidin is not easy to synthesize and has high toxicity. Therefore, people in this field have long hoped to find a derivative of cantharidin, which is still useful. Biologically active, but not as strong as CA. Accordingly, two derivatives of cantharidin, norcantharidin (NCTD) and disodium methylphylloate, were developed.
本案發明人於之前的研究中發現,NCTD在活體外雖可使樹突細胞發育,但活性較差,而且還會促使 細胞凋亡;活體內,NCTD可使抗原呈現細胞及調控性T細胞聚集在器捐接受者脾臟,且不會引起強烈的免疫反應(參見Hsieh et al.,Transplantation2011: 92(8),848-857),因此適合做為免疫抑制劑使用。先前的研究雖然已確定NCTD較CA更不具細胞毒性,也較CA適合做為免疫抑制劑,但NCTD仍然具有一定程度的細胞毒性,會造成細胞凋亡,因此,此領域仍需要開發出一種毒性更低的斑蝥素衍生物,做為免疫抑制劑,用以製造抗排斥藥物。In the previous study, the inventors found that NCTD can develop dendritic cells in vitro, but its activity is poor, and it also promotes apoptosis. In vivo, NCTD can accumulate antigen-presenting cells and regulatory T cells. The donor donates the spleen and does not cause a strong immune response (see Hsieh et al., Transplantation 2011: 92(8), 848-857) and is therefore suitable for use as an immunosuppressive agent. Although previous studies have determined that NCTD is less cytotoxic than CA and is more suitable as an immunosuppressive agent than CA, NCTD still has a certain degree of cytotoxicity, which may cause apoptosis. Therefore, it is still necessary to develop a toxicity in this field. Lower cantharidin derivatives, used as immunosuppressive agents to make anti-rejection drugs.
本揭示內容至少部分係基於發現單離自莞菁科昆蟲-大斑蝥的斑蝥素(CA)衍生物,去甲基斑蝥酸二鈉,其不具細胞毒性、且具有可調控人類樹突細胞分化或成熟的特性而來。上述發現暗示本揭示內容中的去甲基斑蝥酸二鈉可作為能減少移植排斥之治療性藥劑的先導化合物。The present disclosure is based, at least in part, on the discovery of cantharidin (CA) derivatives, which are cytotoxic and have modulating dendritic cell differentiation in humans. Mature characteristics come. The above findings suggest that disodium desmethyl decanoate in the present disclosure can be used as a lead compound for a therapeutic agent that reduces graft rejection.
因此,本揭示內容之第一目的是關於以去甲基斑蝥酸二鈉來製造一種可抑制一樹突細胞之分化或成熟的醫藥品或藥學組合物的新穎用途。在本揭示內容中,該抑制是指減少該樹突細胞表面CD1a、CD40或CD83分子之表現量。Accordingly, a first object of the present disclosure is directed to the novel use of disodium desmethyl cinnamate to produce a pharmaceutical or pharmaceutical composition that inhibits differentiation or maturation of a dendritic cell. In the present disclosure, the inhibition refers to reducing the amount of expression of CD1a, CD40 or CD83 molecules on the surface of the dendritic cells.
因此,本發明目的之一實施方式是提供一種可抑制一樹突細胞之分化或成熟的醫藥品或藥學組合物。此醫藥品或藥學組合物包含一治療有效量之去甲斑蝥酸二鈉或其藥學上可接受的鹽類,以及一藥學上可接受的 佐劑。Accordingly, an embodiment of the object of the present invention is to provide a pharmaceutical or pharmaceutical composition which inhibits differentiation or maturation of a dendritic cell. The pharmaceutical or pharmaceutical composition comprises a therapeutically effective amount of disodium succinate or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable Adjuvant.
若以所述藥學組合物總重量為基準,本發明化合物-去甲基斑蝥酸二鈉,約占該藥學組合物總重量的0.1%至99%(重量%)。在某些實施方式中,本發明化合物的量至少約為該藥學組合物總重量的1%。在特定實施方式中,本發明化合物的量至少約為該藥學組合物總重量的5%。在其他實施方式中,本發明化合物的量至少約為該藥學組合物總重量的10%。在另外實施方式中,本發明化合物的量至少約為該藥學組合物總重量的25%。The compound of the present invention, disodium desmethyl decanoate, is present in an amount of from about 0.1% to about 99% by weight based on the total weight of the pharmaceutical composition, based on the total weight of the pharmaceutical composition. In certain embodiments, the amount of the compound of the invention is at least about 1% by weight based on the total weight of the pharmaceutical composition. In a particular embodiment, the amount of the compound of the invention is at least about 5% by weight based on the total weight of the pharmaceutical composition. In other embodiments, the amount of the compound of the invention is at least about 10% by weight based on the total weight of the pharmaceutical composition. In further embodiments, the amount of the compound of the invention is at least about 25% by weight of the total weight of the pharmaceutical composition.
本揭示內容之第二目的是關於在活體外,以去甲基斑蝥酸二鈉來抑制一樹突細胞分化或成熟的方法。所述方法包含在活體外使該樹突細胞接觸一足夠量之去甲基斑蝥素鈉或其藥學上可接受的鹽類。在一實例中,該足夠量之去甲斑蝥素鈉是介於約1 μM至約50 μM間;較佳是介於約5 μM至約45 μM間;更佳是介於約10 μM至約40 μM間;最佳是約20 μM。A second object of the present disclosure is directed to a method of inhibiting differentiation or maturation of a dendritic cell with disodium methyl succinate in vitro. The method comprises contacting the dendritic cells with a sufficient amount of sodium desmophyllin sodium or a pharmaceutically acceptable salt thereof in vitro. In one example, the sufficient amount of sodium norcantharidin is between about 1 μM and about 50 μM; preferably between about 5 μM and about 45 μM; more preferably between about 10 μM and about 40 μM; optimal is about 20 μM.
本揭示內容之第三目的是提供一種減輕移植排斥之方法。此方法包括對一個體施用一有效量之去甲基斑蝥酸二鈉或其藥學上可接受的鹽類,以便抑制該個體體內一人類樹突細胞分化或成熟成為T-淋巴細胞。該個體可以是一種哺乳動物,較佳是人類。A third object of the present disclosure is to provide a method of reducing transplant rejection. The method comprises administering to a subject an effective amount of disodium desmethyl decanoate or a pharmaceutically acceptable salt thereof to inhibit differentiation or maturation of a human dendritic cell into T-lymphocytes in the subject. The individual can be a mammal, preferably a human.
透過以下的詳細說明與附隨之申請專利範圍將可更了解本揭示內容的這些及其他特徵。These and other features of the present disclosure will be more apparent from the following detailed description and appended claims.
需知以上的概述及以下的詳細說明僅為例示,用來闡述本揭示內容,而非用以限制本揭示內容之範 疇。The above summary and the following detailed description are intended to be illustrative only, and are not intended to limit the scope of the disclosure Domain.
為讓本發明的上述與其他目的、特徵、優點與實施例能更明顯易懂,所附圖式之說明如下:第1圖繪示出依據本發明一實施方式,O6(即,DNCTD)對人類樹突細胞存活率之影響;第2圖繪示出依據本發明一實施方式,O6對人類樹突細胞之細胞形態的影響,其中(A)為控制組,且(B)為經20 μM之O6處理後之DC細胞;第3圖繪示出依據本發明一實施方式,O6對人類樹突細胞表面抗原分子表現的影響;第4圖繪示出依據本發明一實施方式,O6對異體培育DC細胞與T細胞的影響,其中(A)代表對增生之T細胞的影響,(B)則為所產生的干擾素-γ的影響;第5圖繪示出依據本發明一實施方式,O6對DC細胞所分泌之細胞素量的影響,其中(A)代表IL-10,(B)代表IL-12且(C)代表IL-23之分泌量;第6圖為依據本發明一實施方式,有或無經NCTD(0、10或20 μM)、CA(0、1.25或2.5 μM)或O6(0,20或40 μM)對DC細胞中PP2A之C單元蛋白表現的影響;第7圖為依據本發明一實施方式,有或無經NCTD(0、10或20 μM)、CA(0、1.25或2.5 μM)或O6(0,20或40 μM)對DC細胞中p70 S6激酶蛋白表現的影響;第8圖為依據本發明一實施方式,有或無經 NCTD(0、10或20 μM)、CA(0、1.25或2.5 μM)或O6(0,20或40 μM)處理之DC細胞的PP2B之A單元蛋白表現的影響;及第9圖為依據本發明一實施方式,有或無經NCTD(0、10或20 μM)、CA(0、1.25或2.5 μM)或O6(0,20或40 μM)處理之DC細胞的p-STAT3蛋白表現的影響。The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Effect of human dendritic cell survival rate; Figure 2 depicts the effect of O6 on cell morphology of human dendritic cells, in which (A) is the control group and (B) is 20 μM, in accordance with an embodiment of the present invention. The O6-treated DC cells; FIG. 3 illustrates the effect of O6 on the expression of human dendritic cell surface antigen molecules according to an embodiment of the present invention; and FIG. 4 illustrates the O6-transformed body according to an embodiment of the present invention. Incubating the effects of DC cells and T cells, wherein (A) represents the effect on the proliferating T cells, (B) is the effect of the interferon-γ produced; and FIG. 5 illustrates an embodiment according to the present invention, The effect of O6 on the amount of cytokines secreted by DC cells, wherein (A) represents IL-10, (B) represents IL-12 and (C) represents the secretion amount of IL-23; and FIG. 6 shows an embodiment according to the present invention. Mode, with or without NCTD (0, 10 or 20 μM), CA (0, 1.25 or 2.5 μM) or O6 (0, 20 or 40 μM) versus PP2A in DC cells Effect of unit protein expression; Figure 7 is an embodiment of the invention with or without NCTD (0, 10 or 20 μM), CA (0, 1.25 or 2.5 μM) or O6 (0, 20 or 40 μM) Effect on the expression of p70 S6 kinase protein in DC cells; Figure 8 is an embodiment of the present invention, with or without Effects of NCTD (0, 10 or 20 μM), CA (0, 1.25 or 2.5 μM) or O6 (0, 20 or 40 μM) DC cells on the performance of PP2B A-unit protein; and Figure 9 is based on this An embodiment of the invention, the effect of p-STAT3 protein expression in DC cells treated with or without NCTD (0, 10 or 20 μM), CA (0, 1.25 or 2.5 μM) or O6 (0, 20 or 40 μM) .
根據慣常的作業方式,圖中各種特徵與元件並未依比例繪製,其繪製方式是為了以最佳的方式呈現與本發明相關的具體特徵與元件。此外,在不同圖式間,以相同或相似的元件符號來指稱相似的元件/部件。The various features and elements in the figures are not drawn to scale, and are in the In addition, similar elements/components are referred to by the same or similar element symbols throughout the different drawings.
為了使本揭示內容的敘述更加詳盡與完備,下文針對了本發明的實施態樣與具體實施例提出了說明性的描述;但這並非實施或運用本發明具體實施例的唯一形式。實施方式中涵蓋了多個具體實施例的特徵以及用以建構與操作這些具體實施例的方法步驟與其順序。然而,亦可利用其他具體實施例來達成相同或均等的功能與步驟順序。The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.
雖然用以界定本發明較廣範圍的數值範圍與參數均是約略的數值,此處已盡可能精確地呈現具體實施例中的相關數值。然而,任何數值本質上不可避免地含有因個別測試方法所致的標準偏差。在此處,「約」通常係指實際數值在一特定數值或範圍的正負10%、5%、1%或0.5%之內。或者是,「約」一詞代表實際數值落在平均值的可接受標準誤差之內,視本發明所屬技術領域 中具有通常知識者的考量而定。除了實驗例之外,或除非另有明確的說明,當可理解此處所用的所有範圍、數量、數值與百分比(例如用以描述材料用量、時間長短、溫度、操作條件、數量比例及其他相似者)均經過「約」的修飾。因此,除非另有相反的說明,本說明書與附隨申請專利範圍所揭示的數值參數皆為約略的數值,且可視需求而更動。至少應將這些數值參數理解為所指出的有效位數與套用一般進位法所得到的數值。Although numerical ranges and parameters are used to define a broad range of values for the present invention, the relevant values in the specific embodiments have been presented as precisely as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Or, the word "about" means that the actual value falls within the acceptable standard error of the average value, depending on the technical field to which the present invention pertains. It depends on the consideration of the usual knowledge. Except for the experimental examples, or unless otherwise explicitly stated, all ranges, quantities, values, and percentages used herein are understood (eg, to describe the amount of material used, the length of time, the temperature, the operating conditions, the quantity ratio, and the like. Are all modified by "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters disclosed in the specification and the appended claims are intended to be At a minimum, these numerical parameters should be understood as the number of significant digits indicated and the values obtained by applying the general carry method.
除非本說明書另有定義,此處所用的科學與技術詞彙之含義與本發明所屬技術領域中具有通常知識者所理解與慣用的意義相同。此外,在不與上下文意衝突的情形下,本說明書所用的單數名詞涵蓋該名詞的複數型;而所用的複數名詞時亦涵蓋該名詞的單數型。The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein. In addition, the singular noun used in the specification encompasses the plural of the noun, and the plural noun of the noun is also included in the plural noun.
以下為本說明書中所用特定名詞的說明:在本文中,「抑制(inhibits or inhibiting)」一詞意指對樹突細胞或一個體施用所述化合物(即,去甲基斑蝥酸二鈉),以使至少45%、50%、55%、60%或65%的樹突細胞型態改變,包括培育物中每一樹突細胞上的樹突數目減少,樹突細胞表面抗原包括CD1a、CD40或CD83之表現量受到抑制,或刺激T-細胞增生能力下降,因此,「抑制」一詞在此亦表示抑制樹突細胞分化或成熟。In the following, the term "inhibits or inhibiting" means applying the compound to dendritic cells or a body (ie, disodium desmethyl decanoate), To alter at least 45%, 50%, 55%, 60%, or 65% of dendritic cell morphology, including a decrease in the number of dendrites on each dendritic cell in the culture, dendritic cell surface antigens including CD1a, CD40 or The amount of CD83 is inhibited, or the ability to stimulate T-cell proliferation is reduced. Therefore, the term "inhibition" is also used herein to inhibit dendritic cell differentiation or maturation.
「一有效量(an effective amount)」一詞意此一用量在經過適當的給藥期間後,能夠達到減少每一樹突細胞上之樹突數目、抑制樹突細胞表面抗原(包括CD1a、CD40或CD83)之表現量、或減少樹突細胞刺激T-細胞增 生能力,換言之,該有效量可達到所亟求之抑制樹突細胞分化或成熟的效果。The term "an effective amount" means that the amount of dendrites on each dendritic cell is reduced and the dendritic cell surface antigen (including CD1a, CD40 or CD83) performance, or decreased dendritic cells stimulate T-cell proliferation The ability to produce, in other words, the effective amount can achieve the desired effect of inhibiting dendritic cell differentiation or maturation.
「化合物(compound)」、「組合物(composition)」、「藥劑(agent)」或「醫藥品(medicine or medicament)」等詞在此可互相替換,且都是指當施用於一個體(人類或動物)時,能夠透過局部和/或全身性作用而誘發所亟求的藥學和/或生理反應的一種化合物或組合物。The words "compound", "composition", "agent" or "medicine or medicament" are used interchangeably herein and refer to when applied to a body (human Or an animal, a compound or composition capable of inducing a desired pharmaceutical and/or physiological response through local and/or systemic action.
「施用(administered、administering或、administration)」一詞在此係指直接施用所述的化合物或組合物,或施用活性化合物的前驅藥(prodrug)、衍生物(derivative)、或類似物(analog)等,而可於施用個體體內形成該活性化合物之一相當用量者。The term "administered, administered" or "administration" as used herein refers to the direct administration of the compound or composition, or the administration of a prodrug, derivative, or analog of the active compound. Alternatively, a substantial amount of the active compound can be formed in the subject to be administered.
本文中交替使用「個體(subject)」或「患者(patient)」等詞,其係指可接受所述化合物和/或方法治療的動物(包括人類)。「個體」或「患者」在此涵蓋了雄性與雌性兩種性別,除非另有具體說明。因此「個體」或「患者」包含任何哺乳類動物,較佳為人類,其可因利用所述化合物進行治療而獲益。The words "subject" or "patient" are used interchangeably herein to mean an animal (including a human) that is treatable by the compounds and/or methods. "Individual" or "patient" is used herein to encompass both male and female genders unless otherwise specified. Thus "individual" or "patient" encompasses any mammal, preferably a human, which may benefit from treatment with the compound.
本揭示內容至少有一部分是肇因於單離自莞菁科昆蟲-大斑蝥的斑蝥素(CA)之衍生物,去甲斑蝥酸二鈉,其本身不具細胞毒性、且具有可調控人類樹突細胞分化或成熟的特性而來。上述發現暗示本揭示內容中的去甲基斑蝥酸二鈉可作為能減少移植排斥之治療性藥劑的先導化合物。At least a part of the disclosure is a derivative of cantharidin (CA), which is a cytotoxic and has a modulating human dendrites, which is a derivative of cantharidin (CA), which is isolated from the insects of the genus Wanjing. The characteristics of cell differentiation or maturation. The above findings suggest that disodium desmethyl decanoate in the present disclosure can be used as a lead compound for a therapeutic agent that reduces graft rejection.
本揭示內容中該單離自莞菁科昆蟲-大斑蝥的斑蝥素(CA)衍生物,去甲基斑蝥酸二鈉(DNCTD,在本文實施例中稱O6化合物),具有以下的化學結構:
因此,本揭示內容之第一目的是關於以去甲基斑蝥酸二鈉來製造一種可抑制一樹突細胞之分化或成熟的醫藥品或藥學組合物的新穎用途。在本揭示內容中,抑制是指減少樹突細胞表面CD1a、CD40或CD83分子之表現量。Accordingly, a first object of the present disclosure is directed to the novel use of disodium desmethyl cinnamate to produce a pharmaceutical or pharmaceutical composition that inhibits differentiation or maturation of a dendritic cell. In the present disclosure, inhibition refers to reducing the amount of expression of CD1a, CD40 or CD83 molecules on the surface of dendritic cells.
依此發明目的所提供的一種實施方式是有關一種可抑制一樹突細胞之分化或成熟的醫藥品或藥學組合物。此醫藥品或藥學組合物包含一治療有效量之去甲斑蝥酸二鈉或其藥學上可接受的鹽類,以及一藥學上可接受的佐劑。One embodiment provided in accordance with the purpose of the present invention relates to a pharmaceutical or pharmaceutical composition which inhibits the differentiation or maturation of a dendritic cell. The pharmaceutical or pharmaceutical composition comprises a therapeutically effective amount of disodium succinic acid or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.
若以所述藥學組合物總重量為基準,則本發明化合物-去甲基斑蝥酸二鈉,約占該藥學組合物總重量的0.1%至99%(重量%)。在某些實施方式中,本發明化合物的量至少約為該藥學組合物總重量的1%。在特定實施方式中,本發明化合物的量至少約為該藥學組合物總重量的5%。在其他實施方式中,本發明化合物的量至少 約為該藥學組合物總重量的10%。在另外實施方式中,本發明化合物的量至少約為該藥學組合物總重量的25%。The compound of the present invention, disodium desmethyl decanoate, is present in an amount of from about 0.1% to about 99% by weight based on the total weight of the pharmaceutical composition, based on the total weight of the pharmaceutical composition. In certain embodiments, the amount of the compound of the invention is at least about 1% by weight based on the total weight of the pharmaceutical composition. In a particular embodiment, the amount of the compound of the invention is at least about 5% by weight based on the total weight of the pharmaceutical composition. In other embodiments, the amount of the compound of the invention is at least It is about 10% of the total weight of the pharmaceutical composition. In further embodiments, the amount of the compound of the invention is at least about 25% by weight of the total weight of the pharmaceutical composition.
本揭示內容之第二目的是關於在活體外以去甲斑蝥酸二鈉來抑制一樹突細胞分化或成熟的方法。所述方法包含在活體外使該樹突細胞接觸一足夠量之去甲基斑蝥素鈉或其藥學上可接受的鹽類。在一實例中,該足夠量之去甲基斑蝥素鈉介於約1 μM至約50 μM間;較佳是介於約5 μM至約45 μM間;更佳是介於約10 μM至約40 μM間;最佳是約20 μM。A second object of the present disclosure is directed to a method of inhibiting differentiation or maturation of a dendritic cell with disodium decanoate in vitro. The method comprises contacting the dendritic cells with a sufficient amount of sodium desmophyllin sodium or a pharmaceutically acceptable salt thereof in vitro. In one example, the sufficient amount of sodium desmethylcantharidin is between about 1 μM and about 50 μM; preferably between about 5 μM and about 45 μM; more preferably between about 10 μM and about 40 μM; optimal is about 20 μM.
本揭示內容之第三目的是提供一種減輕移植排斥之方法。此方法包括對一個體施用一有效量之去甲基斑蝥酸二鈉或其藥學上可接受的鹽類,以便抑制該個體體內一人類樹突細胞分化或成熟成為T-淋巴細胞。該個體可以是一種哺乳動物,較佳是人類。A third object of the present disclosure is to provide a method of reducing transplant rejection. The method comprises administering to a subject an effective amount of disodium desmethyl decanoate or a pharmaceutically acceptable salt thereof to inhibit differentiation or maturation of a human dendritic cell into T-lymphocytes in the subject. The individual can be a mammal, preferably a human.
在某些實施方式中,可經由口服、靜脈注射或肌肉注射方式施用去甲基斑蝥酸二鈉至一個體身上,所施用的有效用量約為1-100毫克/公斤個體體重間。每天可施用至該個體身上之去甲基斑蝥酸二鈉的有效用量約為10、20、30、40、50、60、70、80、90或100毫克/公斤體重;較佳是約30-70毫克/公斤體重,例如約30、40、50、60、70毫克/公斤體重;更佳是約50毫克/公斤體重。這些劑量可以單次施用或是分成多次在一天內施用。In certain embodiments, disodium desmethyl succinate can be administered to a subject via oral, intravenous or intramuscular injection at an effective level of between about 1 and 100 mg/kg of body weight. An effective amount of disodium methylphylloate applied to the individual per day is about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg body weight; preferably about 30- 70 mg/kg body weight, for example about 30, 40, 50, 60, 70 mg/kg body weight; more preferably about 50 mg/kg body weight. These doses can be administered in a single administration or divided into multiple administrations within one day.
在某些實施方式中,所述方法更包括在施用 去甲基斑蝥酸二鈉的同時、之前或之後,對該患者施用另一種已知可抑制樹突細胞分化或成熟的藥物,諸如地塞米松(dexamethasone,DEX)、環磷醯胺(Cyclophosphamide,CTX)、甲基强的松龍(Methylprednisolone,MP)之類的免疫抑制劑。In certain embodiments, the method further comprises administering Simultaneously, before or after demethylmethyl succinate is administered to the patient another drug known to inhibit dendritic cell differentiation or maturation, such as dexamethasone (DEX), cyclophosphamide, CTX), an immunosuppressive agent such as Methylprednisolone (MP).
因此,本揭示內容提供一種可用來抑制樹突細胞分化或成熟的藥品或藥學組合物。此藥品或藥學組合物包括一有效量之去甲基斑蝥酸二鈉;以及其藥學上可接受的佐劑。一般來說,去甲基斑蝥酸二鈉的含量佔整體藥學組合物重量約0.1%至99%(重量%)。在某些實施方式中,去甲基斑蝥酸二鈉的含量佔整體藥學組合物重量的至少1%(重量%)。在特定實施方式中,去甲基斑蝥酸二鈉的含量佔整體藥學組合物重量的至少5%(重量%)。在另一些實施方式中,去甲基斑蝥酸二鈉的含量佔整體藥學組合物重量的至少10%(重量%)。在又另一些實施方式中,去甲基斑蝥酸二鈉的含量佔整體藥學組合物重量的至少25%(重量%)。Accordingly, the present disclosure provides a pharmaceutical or pharmaceutical composition that can be used to inhibit dendritic cell differentiation or maturation. The pharmaceutical or pharmaceutical composition comprises an effective amount of disodium desmethyl decanoate; and a pharmaceutically acceptable adjuvant thereof. Generally, the amount of disodium methylphyllinate is from about 0.1% to about 99% by weight based on the total weight of the pharmaceutical composition. In certain embodiments, the amount of disodium methotrexate is at least 1% by weight of the total pharmaceutical composition. In a particular embodiment, the amount of disodium methotrexate is at least 5% by weight of the total pharmaceutical composition. In other embodiments, the amount of disodium methotrexate is at least 10% by weight based on the weight of the overall pharmaceutical composition. In still other embodiments, the amount of disodium methylbehenate is at least 25% by weight of the total pharmaceutical composition.
一般來說,本發明化合物(即,去甲斑蝥酸二鈉)可經由口服(如,口服用膠囊、藥包(sachets)、懸浮液或藥錠)、或腸胃外(parenterally)等適當方法施用。腸胃外(parenterally)施用方式包括例如肌肉內注射、靜脈注射、皮下注射或腹膜內注射等系統性方式施用。或是,也可透過穿皮膜方式施用,如局部皮膚塗抹或是吸入性(如支氣管內、鼻腔內、口腔內或鼻滴劑等);或是直腸內方式施用。施用時可單獨給藥或併同習知藥 學可接受輔劑一起給藥。在較佳實施方式中,可經由口服方式(如,透過食物)將本發明化合物投予個體。In general, the compounds of the invention (i.e., disodium canregestinate) can be administered by oral (e.g., orally, in capsules, sachets, suspensions or elixirs), or parenterally, and the like. . Parenterally administered means include systemic administration such as intramuscular injection, intravenous injection, subcutaneous injection or intraperitoneal injection. Alternatively, it may be applied by means of a film, such as topical skin application or inhalation (such as intrabronchial, intranasal, intraoral or nasal drops); or intrarectal administration. May be administered alone or in combination with conventional drugs The adjuvant can be administered together. In a preferred embodiment, the compounds of the invention can be administered to an individual via oral means (e.g., through food).
若以口服方式施用,可將本發明化合物(即,去甲基斑蝥酸二鈉)配方成為內含各種輔劑(如,微晶纖維素、碳酸鈣、磷酸二鈣及甘胺酸);各種崩解劑(如澱粉、藻酸及特定矽酸鹽);以及顆粒黏合劑(如,聚乙烯吡咯烷酮、蔗糖、明膠及相思樹膠(acacia))的藥錠。除此外,還可包含諸如硬脂酸鎂、十二烷基硫酸鈉及滑石等的潤滑劑。與此相關的較佳材料包括乳糖或牛奶中的糖以及高分子量聚乙二醇。上述的固態劑型也可非必要地包括塗層或殼,例如腸衣塗層,以及用來改善任一藥物活性成分釋放速率的塗層。這類塗層的實例已為此領域中人士所熟知。在一實例中,所述藥學組合物是被配方成為藥錠。在另一實例中,所述藥學組合物是被配方成為充填在軟或硬明膠膠囊內或是封裝在生物可分解之藥包內的顆粒。當使用方式是口服用懸浮液和/或特效藥液(elixirs)時,可組合活性成分與各種甜味劑或風味劑、著色劑或染料一起配方,需要時還可加入乳化劑和/或懸浮劑,以及諸如水、酒精、丙二醇、甘油等稀釋劑。If administered orally, the compound of the invention (ie, disodium desmethyl decanoate) can be formulated to contain various adjuvants (eg, microcrystalline cellulose, calcium carbonate, dicalcium phosphate, and glycine); Disintegrants (such as starch, alginic acid, and specific citrates); and granule binders (eg, polyvinylpyrrolidone, sucrose, gelatin, and acacia). In addition, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc may also be included. Preferred materials associated therewith include sugars in lactose or milk as well as high molecular weight polyethylene glycols. The above solid dosage forms may also optionally include a coating or shell, such as a casing coating, and a coating to improve the rate of release of any of the pharmaceutically active ingredients. Examples of such coatings are well known to those skilled in the art. In one example, the pharmaceutical composition is formulated as a tablet. In another example, the pharmaceutical composition is a granule formulated to be filled in a soft or hard gelatin capsule or encapsulated in a biodegradable kit. When used in the form of oral suspensions and / or special effects (elixirs), the active ingredients may be combined with various sweeteners or flavors, colorants or dyes, and emulsifiers and/or suspensions may be added if desired. And diluents such as water, alcohol, propylene glycol, glycerin.
在某些實施方式中,本發明藥學組合物是被配方成為適合口服的液體劑型。此類液體配方可更包括用來維持pH值的緩衝液。也可將此液體配方充填在軟膠囊內。此液體劑型可為一種溶液,懸浮液,乳化液,微乳化液,沉澱或是可攜帶本發明化合物、其之藥學上可接受的衍生物、異構物、代謝物、鹽類或溶合物之任 何液體介質。此液體可被設計成能改善本發明化合物其之藥學上可接受鹽類之溶解度,以便形成一種含有藥物的乳化液或分散液。當使用這種劑型時,是將活性化合物與至少一種藥學上可接受的輔藥(包括,但不限於,上述輔藥)一起混合後形成。In certain embodiments, the pharmaceutical compositions of the invention are formulated as liquid dosage forms suitable for oral administration. Such liquid formulations may further include a buffer to maintain the pH. This liquid formulation can also be filled in a soft capsule. The liquid dosage form can be a solution, suspension, emulsion, microemulsion, precipitate or can carry a compound of the invention, a pharmaceutically acceptable derivative, isomer, metabolite, salt or solvate thereof Ren What is the liquid medium. The liquid can be designed to improve the solubility of the pharmaceutically acceptable salts of the compounds of the invention to form a drug-containing emulsion or dispersion. When such a dosage form is employed, it is formed by mixing the active compound with at least one pharmaceutically acceptable adjuvant, including, but not limited to, the above-described adjuvants.
若以腸胃外(parenterally)方式施用,可將本發明化合物配方成為液態的藥學組合物,其可為能以靜脈內注射、肌肉內注射、皮下注射或腹膜內注射等方式施用的無菌溶液或懸浮液。可用來製造上述無菌注射溶液或懸浮液的稀釋劑包括,但不限於,1,3-丁二醇、甘露醇、水、林格氏溶液、等張性氯化鈉溶液。也可使用脂肪酸(如,油酸)及其之甘油酯衍生物,或是天然藥學可接受的油(如,橄欖油或菜籽油)來製造可供注射用的溶液或懸浮液。這類油性溶液或懸浮液中也可包含用來稀釋的醇類或羧甲基纖維素或類似的分散劑。也可使用其他常用的界面活性劑(如,Tweens或Spans系列)或乳化劑,或藥學領域製造配方時常用來增強生物可利用性的藥劑。If administered parenterally, the compound of the invention may be formulated as a liquid pharmaceutical composition which may be sterile solution or suspension which can be administered by intravenous, intramuscular, subcutaneous or intraperitoneal injection. liquid. Diluents which can be used in the manufacture of such sterile injectable solutions or suspensions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution, isotonic sodium chloride solution. Fatty acids (e.g., oleic acid) and their glyceride derivatives, or natural pharmaceutically acceptable oils (e.g., olive oil or rapeseed oil) can also be used to make solutions or suspensions for injectable use. Alcohols or carboxymethylcellulose or similar dispersing agents for dilution may also be included in such oily solutions or suspensions. Other commonly used surfactants (e.g., Tweens or Spans series) or emulsifiers, or agents commonly used in the pharmaceutical arts to enhance bioavailability can be used.
亦可將本發明之上述醫藥品或藥學組合物製成多種適用於黏膜給藥(mucosal application)的劑型,如經頰(buccal)和/或舌下(sublingual)藥物劑型單元,以遞送藥物穿過口腔黏膜。可使用多種生物可降解且藥學可接受的高分子輔劑,此種輔劑可使得藥學組合物具有可接受的吸附效果以及所欲的藥物釋放模式,且可和經頰和/或舌下藥物劑型單元中所含的欲施用活性成分 或其他成分相容。一般來說,上述的高分子輔劑包含親水性聚合物,其可黏附至口腔黏膜的濕潤表面。高分子輔劑的實施例包括但不限於丙烯酸聚合物與共聚物(acrylic acid polymers and copolymers);水解聚乙烯醇(hydrolyzed polyvinylalcohol);聚乙烯氧化物(polyethylene oxides);聚丙烯酸酯(polyacrylates);乙烯聚合物與共聚物(vinyl polymers and copolymers);聚乙烯吡咯啶;葡萄糖(dextran);瓜膠(guar gum);果膠(pectins);澱粉;及纖維素聚合物(cellulosic polymers)。The above pharmaceutical or pharmaceutical composition of the present invention may also be formulated into a variety of dosage forms suitable for mucosal application, such as buccal and/or sublingual pharmaceutical dosage units for delivery of a drug. Through the oral mucosa. A wide variety of biodegradable and pharmaceutically acceptable polymeric adjuvants can be used which provide the pharmaceutical compositions with acceptable adsorption and desired drug release patterns, as well as buccal and/or sublingual drugs. The active ingredient to be applied in the dosage unit Or other ingredients are compatible. In general, the above polymeric adjuvants comprise a hydrophilic polymer that adheres to the wetted surface of the oral mucosa. Examples of polymeric adjuvants include, but are not limited to, acrylic acid polymers and copolymers; hydrolyzed polyvinyl alcohol; polyethylene oxides; polyacrylates; Vinyl polymers and copolymers; polyvinylpyrrolidine; dextran; guar gum; pectins; starch; and cellulosic polymers.
當可理解,本發明化合物的劑量會因個體而異,這不僅是因為所用的特定化合物或組合物、給藥途徑、化合物(單獨或連同一或多種藥物)於患者體內所引發之所欲反應等因素之不同,還可能受到其他因素影響,例如:欲治療症狀的疾病狀態或嚴重程度;患者的年齡、性別或體重、患者的健康狀況;以及欲治療的病理狀態的嚴重程度、患者於同時進行的其他醫療或特殊飲食內容;以及本領域通常知識者可想到的其他因素;而負責照料的醫療人員最終可基於這些因素而判斷出適當的劑量。可調整給藥劑量與形式以提供較佳的治療反應。治療有效量同時也是指化合物或組合物所致的毒性或有害的效果不及於其所帶來的治療利益。在較佳的情形中,本發明之化合物或組合物於投藥時,應採用適當的劑量並持續一段時間,以減少出現症狀的次數和/或嚴重程度。It will be understood that the dosage of the compound of the invention will vary from individual to individual, not only because of the particular compound or composition employed, the route of administration, the compound (either alone or in combination with one or more drugs) Other factors may also be affected by other factors, such as the state or severity of the disease to be treated; the age, sex or weight of the patient, the health of the patient; the severity of the pathological condition to be treated, and the patient at the same time Other medical or special dietary content to be performed; and other factors conceivable by those of ordinary skill in the art; and the medical personnel responsible for care may ultimately determine the appropriate dosage based on these factors. The dosage and form of administration can be adjusted to provide a preferred therapeutic response. A therapeutically effective amount also refers to a toxic or detrimental effect of a compound or composition that is less than the therapeutic benefit it brings. In preferred instances, the compounds or compositions of the invention, when administered, should be administered in an appropriate dosage for a period of time to reduce the number and/or severity of symptoms.
以下實施例是用來闡明本揭示內容特定態樣,並幫助習知技藝者了解並實施本揭示內容。但本揭示內容範疇並不限於這些實施例中。The following examples are presented to illustrate certain aspects of the present disclosure and to assist those skilled in the art to understand and practice the present disclosure. However, the scope of the disclosure is not limited to these embodiments.
依以下流程I所述方式,製造去甲斑蝥酸二鈉(以下實施例中簡稱其為O6化合物)。Disodium decanoic acid disodium (abbreviated as O6 compound in the following examples) was produced in the manner described in Scheme I below.
在順丁烯二酸酐(maleic ahnydric)(11克,112.2毫莫耳)中加入300毫升之溶於甲苯的呋喃(furan)(30克,441.1毫莫耳)溶液,並於80℃下攪拌此混合溶液約12小時。之後,將反應混合物冷卻至室溫,並 過濾收集產物,接著以乙醚清洗所收集的產物,真空乾燥後可得去甲基去水斑蝥素(DHNCTD)之無色固體(14.7克,88.5毫莫耳,產率:78.9%),其熔點約為121-122℃,並以1 H-NMR及13 C-NMR鑑定其結構,光譜數據如下:1 H NMR(CDCl3 )δ 6.58(s,2H,H-5,6),5.47(s,2H,H-1,4),3.19(s,2H,H-2,3);13 C NMR(CDCl3 )δ 170.1(C=O),137.2(C-5,C-6),82.4(C-1,C-4),48.9(C-2,C-3)。In a maleic ahnydric (11 g, 112.2 mmol), 300 ml of a solution of furan (30 g, 441.1 mmol) dissolved in toluene was added and stirred at 80 ° C. The solution was mixed for about 12 hours. After that, the reaction mixture was cooled to room temperature, and the product was collected by filtration, and then the collected product was washed with diethyl ether, and dried in vacuo to give demethyl-s-hydrosporin (DHNCTD) as a colorless solid (14.7 g, 88.5 m. , yield: 78.9%), its melting point is about 121-122 ° C, and its structure is identified by 1 H-NMR and 13 C-NMR. The spectral data are as follows: 1 H NMR (CDCl 3 ) δ 6.58 (s, 2H, H-5,6), 5.47 (s, 2H, H-1, 4), 3.19 (s, 2H, H-2, 3); 13 C NMR (CDCl 3 ) δ 170.1 (C=O), 137.2 ( C-5, C-6), 82.4 (C-1, C-4), 48.9 (C-2, C-3).
在前述去甲基去水斑蝥素(4.7克,28.3毫莫耳)的THF(200毫升)溶液中加入10%之Pd/C(470毫克),在3大氣壓的氫氣、及室溫下攪拌反應混合物約8小時。以Celite 545® 過濾反應混合物,真空乾燥後可得去甲基斑蝥素(norcantharidin,NCTD)之無色固體(4.3克,產率:91.4%),其熔點約為115-116℃,並以1 H-NMR及13 C-NMR鑑定其結構,光譜數據如下:1 H NMR(CDCl3 )δ 5.05(dd,J =2.3,2.2 Hz,2H,H-1,4),3.18(s,2H,H-2,3),1.91(m,2H),1.64(m,2H);13 CNMR(CDCl3 )δ 171.1(C=O),80.2(C-1,C-4),50.6(C-2,C-3),28.1(C-5,C-6)。Add 10% Pd/C (470 mg) to a solution of the above-mentioned demethylhydrosporin (4.7 g, 28.3 mmol) in THF (200 ml), stir at 3 atmospheres of hydrogen, and at room temperature. The mixture was about 8 hours. The reaction mixture was filtered Celite 545 ®, available vacuum dried to methyl cantharidin (norcantharidin, NCTD) of a colorless solid (4.3 g, yield: 91.4%) having a melting point of about 115-116 deg.] C, and by 1 H The structure was identified by NMR and 13 C-NMR. The spectral data were as follows: 1 H NMR (CDCl 3 ) δ 5.05 (dd, J = 2.3, 2.2 Hz, 2H, H-1, 4), 3.18 (s, 2H, H) -2,3), 1.91 (m, 2H), 1.64 (m, 2H); 13 CNMR (CDCl 3 ) δ 171.1 (C=O), 80.2 (C-1, C-4), 50.6 (C-2) , C-3), 28.1 (C-5, C-6).
在前述去甲基斑蝥素(NCTD)(480毫克,2.9毫莫耳)的THF(50毫升)溶液中加入0.1N的碳酸氫鈉(5.7毫升,5.8毫莫耳)水溶液。在室溫下攪拌反應混合物約1小時,真空乾燥後可得O6化合物(466毫克,產率:69%) 的白色固體。分離出來的化合物利用1 H-NMR及13 C-NMR進行結構解析與鑑定。光譜數據如下:1 H NMR(D2 O)δ 4.73(dd,J =2.6,2.5 Hz,2H,H-1,4),2.93(s,2H,H-2,3),1.63(m,2H),1.51(m,2H);13 C NMR(D2 O)δ 178.1(C=O),79.1(C-1,C-4),54.0(C-2,C-3),28.3(C-5,C-6)。To a solution of the above-mentioned demethylcantharidin (NCTD) (480 mg, 2.9 mmol) in THF (50 ml) was added 0.1N aqueous sodium hydrogen carbonate (5.7 mL, 5.8 mmol). The reaction mixture was stirred at room temperature for about 1 hour, and dried in vacuo to give a white solid. The isolated compound was subjected to structural analysis and identification by 1 H-NMR and 13 C-NMR. The spectral data are as follows: 1 H NMR (D 2 O) δ 4.73 (dd, J = 2.6, 2.5 Hz, 2H, H-1, 4), 2.93 (s, 2H, H-2, 3), 1.63 (m, 2H), 1.51 (m, 2H); 13 C NMR (D 2 O) δ 178.1 (C=O), 79.1 (C-1, C-4), 54.0 (C-2, C-3), 28.3 ( C-5, C-6).
已知將人類單核細胞或小鼠骨髓細胞培育在粒細胞-巨噬細胞群落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)下,會誘發產生粒細胞、巨噬細胞以及衍生自單核細胞的樹突細胞(參閱Liu and Nussenzweig,Immuno Rev.,2010 234:45-54)。依此,在本實驗中先收集來自健康人週邊血液之單核細胞,接著利用市售的miniMACS系統分離並純化出CD14+細胞。將所分離出的CD14+細胞培育在RPMI 1640培養基中,此培養基中添加有10%胎牛血清(fetal calf serum,FCS)、100 ng/ml之GM-CSF、50 ng/ml之IL-4,並將細胞維持在37℃的潮溼環境下(其氣體組成為5% CO2 及95%空氣)。It is known that the cultivation of human monocytes or mouse bone marrow cells under the granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the production of granulocytes, macrophages and derived from Dendritic cells of monocytes (see Liu and Nussenzweig, Immuno Rev., 2010 234:45-54). Accordingly, in the present experiment, monocytes derived from the blood surrounding the healthy person were collected first, and then CD14+ cells were isolated and purified using a commercially available miniMACS system. The isolated CD14+ cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 100 ng/ml GM-CSF, and 50 ng/ml IL-4. The cells were maintained in a humidified environment at 37 ° C (the gas composition was 5% CO 2 and 95% air).
為了誘使DCs往分化或成熟路徑上發育,將不成熟的DCs培育在含有多種細胞素組合的培養基中,所使用的細胞素組合包括有5 ng/ml之TNF-α、5 ng/ml之IL-1、15 ng/ml之IL-6、與1μg/ml之前列腺素E2。在部分實驗中,培養基中額外添加有不同濃度的試驗化合物(即,0、1.25、2.5、5、10或20 μM之O6);之後,再 於特定期間收集所培育的細胞進行細胞存活、細胞型態、細胞表面抗原分子表現、細胞免疫功能分析,及進一步確認O6之生物活性途徑。In order to induce DCs to develop on the differentiation or maturation pathway, immature DCs were grown in a medium containing a combination of cytokines using a combination of 5 ng/ml TNF-α, 5 ng/ml. IL-1, 15 ng/ml of IL-6, and 1 μg/ml of prostaglandin E2. In some experiments, different concentrations of test compound (ie, 0, 1.25, 2.5, 5, 10, or 20 μM of O6) were added to the medium; The cultured cells are collected for a specific period of time for cell survival, cell type, cell surface antigen molecule expression, cellular immune function analysis, and further confirmation of the biological activity pathway of O6.
在本實例中,在第8天收集所培育的DCs,並以苯酚藍(trypan blue)染料對細胞進行染色,並計算細胞數目,其中死亡細胞將呈現藍色。結果示於第1圖。由圖上可觀察到,即使培養基內所添加的O6濃度高達20 μM,細胞存活率仍高達80%,表示O6本身並不具有細胞毒性,這點與其類似物,即,已知具有細胞毒性的CA或NCTD,並不相同,也突顯相較於CA或NCTD,O6做為調控樹突細胞發育或抗排斥治療藥劑的優勢。In this example, the incubated DCs were collected on day 8 and the cells were stained with trypan blue dye and the number of cells was counted, with the dead cells appearing blue. The results are shown in Figure 1. It can be observed from the figure that even if the concentration of O6 added in the medium is as high as 20 μM, the cell viability is as high as 80%, indicating that O6 itself is not cytotoxic, which is similar to its analog, ie, known to be cytotoxic. CA or NCTD, which is not the same, also highlights the advantages of O6 as a therapeutic agent for dendritic cell development or anti-rejection compared to CA or NCTD.
在本實例中,細胞型態分析係透過劉氏染色法,先對DCs進行染色後,再以顯微鏡觀察細胞型態。In this example, the cell type analysis was performed by staining the DCs by Liu's staining method, and then observing the cell type by a microscope.
劉氏染色 將待染色細胞轉移到載玻片上並以溶液A(0.5克的甲基藍(methylene blue)以及1.7克的伊紅黃(Eosin yellow),皆溶於1,000 ml的甲醇中)覆蓋。45秒後,加入溶液B(1.3克的天藍染料(azure)、1.4克的甲基藍、23.38克的磷酸氫二鈉(Na2 HPO4 )與6.5克的磷酸二氫鉀(KH2 PO4 ),皆溶於1,000 ml的蒸餾水中),其中溶液B與溶液A的添加量為2:1。輕吹溶液表面,以使得兩種溶液混合均勻。將玻片靜置約90秒,而後以流動的水迅速地洗去染料。之後利用顯微鏡來觀察染色細胞的外觀。 Liu staining The cells to be stained were transferred to a glass slide and covered with solution A (0.5 g of methylene blue and 1.7 g of Eosin yellow, both dissolved in 1,000 ml of methanol). After 45 seconds, solution B (1.3 g of azure, 1.4 g of methyl blue, 23.38 g of disodium hydrogen phosphate (Na 2 HPO 4 ) and 6.5 g of potassium dihydrogen phosphate (KH 2 PO 4 ) were added. ), all dissolved in 1,000 ml of distilled water), wherein the addition amount of solution B and solution A is 2:1. The surface of the solution was lightly blown to make the two solutions homogeneously mixed. The slide was allowed to stand for about 90 seconds and then the dye was quickly washed away with running water. The microscope was then used to observe the appearance of the stained cells.
結果 將過劉氏染色法後所觀察到的細胞型態示於第2圖。可發現相對於控制組,經過20 μM之O6處理後的DC細胞,其中會形成貼附的細胞數目變少,同時其數突數目也變少或變短,表示DC細胞已往成熟路徑上發育。 Results The cell type observed after the Liu staining method is shown in Fig. 2. It can be found that DC cells treated with 20 μM of O6 relative to the control group have less number of cells to be attached, and the number of the number of cells is also reduced or shortened, indicating that the DC cells have developed on the mature pathway.
細胞表面抗原分子表現模式係透過細胞流式分析進行。The cell surface antigen molecule expression pattern is performed by flow cytometry analysis.
細胞流式分析 分別透過異硫氰酸酯螢光素(Fluorescein isothiocyanate,FITC)及藻紅蛋白(phycoerythrin,PE)-共軛之單株抗體來對細胞表面抗原分子(包括CD14、CD1a、CD83、CD40、CD80、CD86等)進行雙色免疫標定。所使用抗體如下:抗-CD14(IgG-FITC)、抗-CD1a-PE、抗-CD80-PE、抗-CD83-PE、抗-CD40-PE、抗-CD86-PE、抗-人類白血球抗原(human leukocyte antigen,HLA)-DR-PE、及抗-DC-SIGN-PE。操作時,每次將1 x 106 個細胞注入至FACS caliber流式細胞儀中進行分析,並使用儀器配備的CellQuest軟體分析實驗結果。 Cell flow analysis was performed on cell surface antigen molecules (including CD14, CD1a, CD83, respectively) by fluorescein isothiocyanate (FITC) and phycoerythrin (PE)-conjugated monoclonal antibodies. Two-color immunolabeling was performed on CD40, CD80, CD86, and the like. The antibodies used were as follows: anti-CD14 (IgG-FITC), anti-CD1a-PE, anti-CD80-PE, anti-CD83-PE, anti-CD40-PE, anti-CD86-PE, anti-human leukocyte antigen ( Human leukocyte antigen, HLA)-DR-PE, and anti-DC-SIGN-PE. During the operation, 1 x 10 6 cells were injected into the FACS caliber flow cytometer for analysis and the results were analyzed using the CellQuest software equipped with the instrument.
結果 可發現相對於控制組,O6並不會明顯抑制CD14、DC-SIGN、CD80、CD86或是抗-人類白血球抗原(HLA)-DR之表現;相反的,隨著O6濃度增加,經過O6處理的DC細胞表面抗原分子CD1a、CD40、或CD83之表現模式會出現明顯降低的變化(第3圖)。 The results showed that O6 did not significantly inhibit the expression of CD14, DC-SIGN, CD80, CD86 or anti-human leukocyte antigen (HLA)-DR relative to the control group; conversely, with the increase of O6 concentration, O6 treatment Significantly reduced changes in the expression pattern of DC cell surface antigen molecules CD1a, CD40, or CD83 (Fig. 3).
在此實驗中,透過異體培育DC細胞與T細胞, 來刺激T細胞增生,並測量細胞培育物上清液內培育細胞之分泌物量(如,細胞素或干擾素-γ的量),來確認細胞功能。相關操作方式及結果如下述。In this experiment, DC cells and T cells were cultured through allogeneic cells. To stimulate T cell proliferation, and measure the amount of secretions (eg, the amount of cytokine or interferon-γ) in the cells cultured in the cell culture supernatant to confirm cell function. The relevant operation methods and results are as follows.
異體刺激T細胞增生 首先,依本實施例所述方式,收集單核細胞培育物中未貼附的細胞,並由其中純化出T細胞,接著利用市售miniMACS系統套組來擴增所純化出來的T細胞。之後,收集自單核細胞衍生而來且經實驗化合物O6(或DNCTD)處理過的DC細胞,並施以6 MeV的X光(約30 Gy)照射(劑量為每分鐘4.0 Gy),再將此經過輻射照射的DC細胞與上述擴增之T細胞,以1:10或1:30的比例共同培育約5天。接著,在細胞培育物中加入5 μM螢光素二醋酸鹽琥珀醯亞胺脂(carboxy fluorescein succinimidyl ester)並將之留置於細胞培育物中約18小時。接著,收集所培育的細胞並以流式細胞儀進行分析,並以抗-CD4-PE及抗-CD25-FITC單株抗體來標定T細胞。 Allogeneic Stimulation of T Cell Proliferation First, unattached cells in monocyte cultures were harvested as described in the Examples, and T cells were purified therefrom, and then purified by amplification using a commercially available miniMACS system kit. T cells. Thereafter, DC cells derived from monocytes and treated with the test compound O6 (or DDNCT) were collected and irradiated with 6 MeV X-rays (about 30 Gy) (dose of 4.0 Gy per minute), and then The irradiated DC cells were incubated with the above expanded T cells for about 5 days at a ratio of 1:10 or 1:30. Next, 5 μM carboxy fluorescein succinimidyl ester was added to the cell culture and left in the cell culture for about 18 hours. Next, the cultured cells were collected and analyzed by flow cytometry, and T cells were labeled with anti-CD4-PE and anti-CD25-FITC monoclonal antibodies.
細胞素含量測定 DC細胞與上述透過異體刺激而增生的T細胞,在培育過程中會分別分泌出細胞素(如,IL-10、IL-12)與干擾素-γ,因此,可透過酵素免疫分析來測量細胞培育物上清液中的細胞素與干擾素-γ的含量。 Cytokine content determination DC cells and the above-mentioned T cells proliferated by allogeneic stimulation will secrete cytokines (such as IL-10, IL-12) and interferon-γ, respectively, during the incubation process. Therefore, they can be immunized by enzymes. Analysis was performed to measure the content of cytokines and interferon-γ in the supernatant of the cell culture.
結果 如上述,將經過不同濃度之O6(0、1.25、2.5、5、10或20 μM)處理後的DC細胞與T細胞(naïve T cells)分別以1:10或1:30之比例,共同培育約5天,結果示於第4圖。可發現將DC細胞與T細胞共同培 育,可刺激T細胞增生;經過O6處理後,其刺激T細胞增生的能力會受到調控(第4A圖);干擾素-γ的分泌量模式亦然(第4B圖)。 Results As described above, DC cells treated with different concentrations of O6 (0, 1.25, 2.5, 5, 10 or 20 μM) and T cells (naïve T cells) were combined at a ratio of 1:10 or 1:30, respectively. The cultivation was carried out for about 5 days, and the results are shown in Fig. 4. It can be found that DC cells and T cells can be stimulated to stimulate T cell proliferation; after O6 treatment, their ability to stimulate T cell proliferation will be regulated (Fig. 4A); interferon-γ secretion mode is also the same ( 4B picture).
至於DC所分泌之細胞素量,其中IL-10的分泌量會現隨著O6濃度增加而受到抑制;IL-23的分泌量會現隨著O6濃度增加而受到增加;至於IL-12的分泌量,則沒有明顯變化(第5圖)。As for the amount of cytokines secreted by DC, the amount of IL-10 secreted will be inhibited as the concentration of O6 increases; the amount of IL-23 secreted will increase as the concentration of O6 increases; as for the secretion of IL-12 There is no significant change in the amount (Figure 5).
為確認O6在細胞內的生物機轉,分別利用免疫墨點分析來測定有或無O6(0、20或40 μM)處理時,不同訊遞路徑上相關蛋白質的表現量,包括細胞中普遍存在之絲氨酸/蘇胺酸磷酸酶或鈣調蛋白(clacuneurin)路徑上的第IIA或IIB型磷酸酶(phosphotase 2A or 2B,PP2A or PP2B)之單元蛋白質及哺乳動物拉巴黴素路徑上的p70S6K蛋白質。To confirm the biotransformation of O6 in cells, immunoblotting analysis was used to determine the expression of related proteins on different signaling pathways, including ubiquitous cells, in the presence or absence of O6 (0, 20 or 40 μM). a unit protein of phosphonate/threonine phosphatase or calmodulinin pathway IIA or IIB phosphatase (phosphotase 2A or 2B, PP2A or PP2B) and p70S6K protein on the mammalian rapamycin pathway.
免疫墨點分析 利用已知的溶解緩衝液使培育後的細胞溶解,並利用布拉德福(Bradford)法來測量蛋白質總量。將萃取出的粗單白(about 50-100 μg)加入至0.15%的十二基硫酸鈉聚丙醯胺凝膠(sodium dodecyl sulfate polyacrylamide gel)中,再利用電泳法分離其中的蛋白質。其後將分離後的蛋白質轉印至硝化纖維膜上,並利用含有4%脫脂牛奶的TBST緩衝液(0.8%氯化鈉、0.02%氯化鉀、25 mM Tris-HCl/pH 8.0以及0.1%的Tween-20)來移除非專一結合。將各種抗體(包括p-stat3、stat3、β-actin、PP2B A單元、PP2A C單元、p-p70 S6K、 p70 S6K(購自Transduction Laboratories,Lexington,KY,USA))分別溶解於含有2%脫脂牛奶的TRST緩衝液中,並分別和分離後的蛋白帶(protein band)於4℃下培育至少一夜。清洗後,以和辣根過氧化酶形成共軛的二次抗體(horseradish conjugated secondary antibodies)在室溫下培育約30分鐘,而後利用ECL化學冷光試劑(Amersham Pharmacia Biotech,US)偵測蛋白帶並利用X-光將底片曝光,便於以肉眼觀察表現的蛋白帶。 Immunoblot analysis The incubated cells were lysed using a known lysis buffer and the total amount of protein was measured using the Bradford method. The extracted crude white (about 50-100 μg) was added to 0.15% of sodium dodecyl sulfate polyacrylamide gel, and the protein was separated by electrophoresis. Thereafter, the separated protein was transferred to a nitrocellulose membrane, and TBST buffer (0.8% sodium chloride, 0.02% potassium chloride, 25 mM Tris-HCl/pH 8.0, and 0.1%) containing 4% skim milk was used. Tween-20) to remove non-specific combinations. Various antibodies (including p-stat3, stat3, β-actin, PP2B A unit, PP2A C unit, p-p70 S6K, p70 S6K (purchased from Transduction Laboratories, Lexington, KY, USA)) were separately dissolved in 2% degreased The milk was in TRST buffer and incubated with the separated protein band at 4 ° C for at least one night. After washing, the cells were incubated with horseradish conjugated secondary antibodies for about 30 minutes at room temperature, and then the protein bands were detected using ECL chemical luminescence reagent (Amersham Pharmacia Biotech, US). The film was exposed by X-ray to facilitate visual observation of the expressed protein band.
結果請參閱第6-9圖;可發現,經過20或40μM之O6處理的細胞,PP2A之C單元蛋白以及p70S6K的表現量並未受到抑制(第6、7圖),相反的,PP2B之A單元蛋白表現量則會受到抑制(第8圖),顯示O6的生物活性是透過調控鈣調蛋白路徑上PP2A之C單元蛋白以及PP2B蛋白質的A單元來達成。The results are shown in Figures 6-9; it can be seen that the amount of C2 protein of PP2A and p70S6K was not inhibited by cells treated with 20 or 40 μM of O6 (Figures 6 and 7). Conversely, A of PP2B The amount of expression of the unit protein was inhibited (Fig. 8), indicating that the biological activity of O6 was achieved by regulating the C-unit protein of PP2A on the calmodulin pathway and the A unit of the PP2B protein.
此外,發明人更進一步藉由測量諸如stat3及磷酸化之stat3等調控蛋白的表現量,來確認O6的作用。實驗發現,相較於控制組,經過20或40μM之O6處理的DC細胞中,磷酸化之stat3的表現量明顯下降(參見9圖),此一觀察也確認stat3蛋白質會參與O6促使人類DC細胞成熟並分化的過程。In addition, the inventors further confirmed the effect of O6 by measuring the amount of expression of regulatory proteins such as stat3 and phosphorylated stat3. The experiment found that the phosphorylated stat3 was significantly decreased in DC cells treated with 20 or 40 μM of O6 compared to the control group (see Figure 9). This observation also confirmed that stat3 protein is involved in O6 promoting human DC cells. The process of maturity and differentiation.
當可理解上述實施方式與實施例僅為例示,且熟習此技藝者可對齊進行各種修飾。上文提出之說明書、實施例與資料的目的在於使本說明書的結構完備,並作為實作本發明之例示。雖然本揭示內容已以實施方式揭露如上,然其並非用以限定本揭示內容,任何熟習 此技藝者,在不脫離本揭示內容之精神和範圍內,當可作各種之更動與潤飾,因此本揭示內容之保護範圍當視後附之申請專利範圍所界定者為準。It will be understood that the above-described embodiments and examples are merely illustrative, and that those skilled in the art can align various modifications. The description, examples, and materials set forth above are intended to be illustrative of the present invention and are illustrative of the invention. Although the disclosure has been disclosed above in the embodiments, it is not intended to limit the disclosure, any familiarity The scope of the present disclosure is defined by the scope of the appended claims, and the scope of the disclosure is subject to the scope of the appended claims.
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Hsieh Chen-Hsi et al.;"Norcantharidin Modulates Development of Dendritic Cells and Prolongs Skin Allograft Survival";Transplantation,2011,vol.92,no.8,pages 848-857. 连慕兰等;"去甲斑螯酸鈉對CHO、HeLa和小兒包皮細胞的殺傷效應";解剖學報,1991,第22卷,第3期,第279-285頁 * |
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