TWI704926B - 醫藥組成物用於製備治療與缺氧誘導因子相關病理病症之藥物之用途 - Google Patents
醫藥組成物用於製備治療與缺氧誘導因子相關病理病症之藥物之用途 Download PDFInfo
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Abstract
本發明係關於治療與缺氧誘導因子(HIF)相關病症之方法，且詳言之係關於治療與HIF相關病症之方法，該方法包含投與包含運鐵蛋白之組成物。
Description
本發明係關於治療與缺氧誘導因子(HIF)相關病症之方法，且詳言之係關於治療與HIF相關病症之方法，該方法包含投與包含運鐵蛋白之組成物。
保護細胞，特別是神經元以避免由各種因子，包括中風、神經退化性疾病、外傷性損傷等造成之破壞對於細胞或神經元功能之長期恢復是重要的。藉由單一藥劑來治療性治療受傷細胞或神經元具有優勢，但是經常不足以調動有助於恢復完整功能所需要的複雜分子。
保護神經元或其他細胞避免缺氧或缺血事件或氧化之生理反應經常被認為藉由基因之表現來媒介，該等基因經由對於細胞損傷作出反應之關鍵調控途徑，缺氧誘導因子(HIF)信號傳遞途徑來上調。在腦中，咸信神經保護分子之上調係保護細胞避免無法補救的破壞之關鍵因素。然而，很少有藥物能夠充分用於預防、恢復或減少對於神經元及其他組織之破壞。另外其經常有毒、具有較短半衰期，或
兩者。舉例而言，國際專利申請案WO2006/20727提出使用去鐵胺(deferoxamine)作為抗禦再灌注之有害影響的神經保護劑；然而，投與去鐵胺由於其在血漿中之半衰期降低而造成問題。
運鐵蛋白係控制生物流體中之游離鐵之量的鐵結合血漿糖蛋白。運鐵蛋白充當鐵在循環中之主要轉運體，在循環中，其以無鐵去鐵運鐵蛋白(ApoTf)形式、單鐵運鐵蛋白、或二鐵全鐵運鐵蛋白(HoloTf)形式存在。通常，鐵結合至循環中之30%所有運鐵蛋白結合部位。ApoTf而非HoloTf之神經保護功能已經由Chen-Roetling等人(Chen-Roetling J.,Chen L.,and Regan R.F.Neuropharmacology,2011；60(2-3)：423-431.)揭示，其提出ApoTf可減輕大腦內出血之後的血紅蛋白之神經毒性。
本案發明人發現可增強在患者中投與之運鐵蛋白之神經保護性質，該增強係藉由將其與其他鐵螯合劑或與另一種鐵結合血漿蛋白質(諸如缺鐵乳鐵蛋白)組合來實現，該另一種鐵結合血漿蛋白質已被證明增加一些組織中之HIF-1α蛋白質量並且對於血漿EPO量具有作用(Zakharova E.T.等人Biometals(2012)25：1247-1259)。已經提出具有鐵螯合能力之分子藉由阻斷脯胺醯基羥化酶之活性而為HIF途徑活化劑。
因此，本發明係關於治療與缺氧誘導因子(HIF)相關病症之方法，該方法包括投與包含運鐵蛋白之組成物。本發
明亦係關於治療與缺氧誘導因子(HIF)相關病症之方法，其中所投與之組成物進一步包括運鐵蛋白與鐵螯合劑之組合。
如本文所使用，術語「運鐵蛋白」及其複數形式係指單獨的ApoTf或單獨的HoloTf或其混合物。
本發明進一步參考以下圖式來描述。
圖1顯示大部分ApoTf與大部分HoloTf之組成物在常氧(normoxic)及缺氧條件下、在治療後6小時誘導HIF-1α蛋白質。
圖2顯示大部分ApoTf與大部分HoloTf之組成物在常氧條件下、在治療後24小時誘導HIF-1α蛋白質。
圖3顯示ApoTf與HoloTf之混合物在治療後6小時誘導HIF-1α蛋白質。
圖4A顯示在HSA、去鐵運鐵蛋白或全鐵運鐵蛋白存在下、在常氧及缺氧條件下的Glut1之mRNA表現量。
圖4B顯示在HSA、去鐵運鐵蛋白或全鐵運鐵蛋白存在下、在常氧及缺氧條件下的VEGF之mRNA表現量。
圖5A顯示包括大部分HoloTf或大部分ApoTf之組成物的體外或體內毒性。
圖5B顯示使用包括大部分ApoTf或HoloTf之藥物來靜脈內治療之大鼠的修正貝德森(Bederson)及一般行為評分。
圖6A顯示在短暫大腦中動脈阻塞(MCAo)大鼠模型
中，ApoTf(385mg/kg，IV)或鹽水治療之梗塞體積之散佈圖。
圖6B顯示來自代表性對照及ApoTf治療大鼠之氯三苯四唑(TTC)染色冠狀切片。
圖7顯示藉由包括主要ApoTf及HoloTf之混合物來保護神經元細胞避免Aβ(1-42)之毒性影響。
圖8A顯示使用4mg/ml大部分ApoTf以及使用大部分ApoTf與DFO或IOX2之組合來治療SH-SY5Y神經元細胞。
圖8B顯示使用4mg/ml所指示的蛋白質以及使用所指示的蛋白質與10uM M30加之組合來治療SH-SY5Y神經元細胞。
圖8C顯示使用4mg/ml所指示的蛋白質以及使用所指示的蛋白質與200uM DFO之組合來治療SH-SY5Y神經元細胞。
圖9A顯示反應於大部分去鐵運鐵蛋白及DFO或IOX2組合的Glut1之mRNA表現量。
圖9B顯示反應於大部分去鐵運鐵蛋白及DFO或IOX2組合的VEGF之mRNA表現量。
圖10A顯示在正常氧量下使用4mg/mL大部分去鐵運鐵蛋白、大部分全鐵運鐵蛋白或每一種物質之各個混合物治療初級人類腎臟近端小管上皮(RPTEC)細胞6小時之後的HIF-1α量。
圖10B顯示在正常氧量下使用4mg/mL大部分去鐵運
鐵蛋白、大部分全鐵運鐵蛋白或每一種物質之各個混合物治療初級皮質上皮(HRCE)細胞6小時之後的HIF-1α量。
圖11A顯示初級人類腎臟近端小管上皮(RPTEC)或皮質上皮(HRCE)細胞在用大部分ApoTf或DFO治療48小時時的生存力。
圖11B顯示RPTEC或HRCE細胞在用4mg/mL大部分ApoTf、大部分HoloTf、運鐵蛋白之混合物治療72小時時的生存力。
圖12顯示在大部分ApoTf或DFO存在下、在人類初級腎細胞內的凋亡蛋白酶(caspase)3/7活化。
圖13A顯示在正常氧量下，肺細胞系NCI-H1650使用4mg/mL大部分去鐵運鐵蛋白、大部分全鐵運鐵蛋白或pd-轉鐵蛋白治療6小時之後的HIF-1α量。
圖13B顯示在正常氧量下，初級肝細胞使用4mg/mL大部分去鐵運鐵蛋白、大部分全鐵運鐵蛋白或pd-轉鐵蛋白治療6小時之後的HIF-1α量。
圖14A顯示人類肺細胞系NCI-H1650在使用4mg/mL大部分ApoTf、大部分HoloTf或pd-運鐵蛋白治療72小時時的生存力。
圖14B顯示初級人類肝細胞在使用4mg/mL大部分ApoTf、大部分HoloTf或pd-運鐵蛋白治療72小時時的生存力。
在一態樣中，本發明係關於治療與缺氧誘導因子(HIF)
相關病症之方法，包括投與包含運鐵蛋白之組成物。較佳地，運鐵蛋白係重組的來自血漿或化學合成之運鐵蛋白。
當運鐵蛋白係重組時，其可根據在蛋白質表現、產生及純化技術中已知之任何技術來獲得。舉例而言，運鐵蛋白之核酸序列可插入適合於表現於選定寄主細胞中之任何載體中，例如細菌(大腸桿菌、枯草芽孢桿菌(Bacilus subtilis)、鼠傷寒沙門菌(Salmonella typhimurium)、假單胞菌(Pseudomonas)、鏈黴菌屬(Streptomyces)及葡萄球菌屬(Staphylococcus))、酵母(酵母屬(Saccharomyces)、畢赤酵母(Pichia)或克魯維斯酵母屬(Kuyveromyces genus))、昆蟲細胞(中國桑蠶(Bombyx mori)、甘藍夜蛾(Mamestra brassicae)、草地貪夜蛾(Spodoptera frugiperda)、粉紋夜蛾(Trichoplusia ni)或黑腹果蠅(Drosophila melanogaster))或哺乳動物細胞(海拉細胞(HeLa cells)、幼倉鼠腎(BHK)細胞、猴腎細胞(COS-1)、人類肝細胞癌細胞(例如HepG2)、人類腺病毒轉化293細胞、小鼠L-929細胞、HaK倉鼠細胞系、來自Swiss、Balb-c或NIH小鼠之鼠科3T3細胞、CV-1細胞系、來自初級組織或初級外植體之活體外培養物之細胞品系)。設想來自血漿之運鐵蛋白自血漿之合適餾分分離。在一個較佳實施例中，運鐵蛋白自柯恩(Cohn)分餾過程之餾分IV，及最佳餾分IV-I或餾分IV-IV來分離。在另一較佳實施例中，運鐵蛋白來自純化α1-蛋白酶抑制劑(A1PI)之方法之廢物餾分。較佳地該純化方法如下：(a)藉由沉澱將污染性蛋白質之一部分自水溶液移
除以便獲得含有A1PI之純化溶液；(b)使純化溶液穿過陰離子交換樹脂以使得A1PI結合至陰離子交換樹脂；(c)洗提來自陰離子交換樹脂之A1PI以獲得含有A1PI之洗提溶液；(d)使洗提溶液穿過陽離子交換樹脂；(e)收集來自陽離子交換樹脂的含有A1PI之徑流；及(f)使步驟(c)之洗提溶液或步驟(e)之徑流與至少一種HIC介質之疏水性吸附劑接觸。
在最佳實施例中，用於以上提到之純化(A1PI)方法中的水溶液係血液、血漿或來自血漿餾分。
在另一實施例中，運鐵蛋白包括至少一種轉譯後修飾，較佳聚乙二醇化(pegylation)、糖基化(glycosylation)、多唾液酸化(polysialylation)或其組合。
在一個實施例中，用於本發明之治療方法中之運鐵蛋白係具有在SEQ ID NO：1中闡明的胺基酸序列之全長運鐵蛋白。另外實施例包括在本發明之治療方法中使用具有與SEQ ID NO：1至少50%、60%、70%、80%、90%、95%、96%、97%、98%或99%同源性或相似性之運鐵蛋白衍生物，只要野生型運鐵蛋白之70%、80%、90%、95%、96%、97%、98%、99%或100%鐵螯合活性保留。熟習此項技術者將容易認識到運鐵蛋白與胺基酸序列SEQ ID NO：1之間之同源性之差異可歸因於不影響鐵螯合功能之保守及/
或非保守胺基酸取代。
在另一實施例中，設想野生型運鐵蛋白(SEQ ID NO：1)之片段用於本發明之治療方法中。熟習此項技術者將容易選擇野生型運鐵蛋白之合適片段以使得其保持野生型運鐵蛋白之鐵螯合活性。
在另一實施例中，運鐵蛋白被修飾成增加其鐵結合親和力。熟習此項技術者將設想待修飾之殘基或區域可藉由在此項技術中已知之多種技術來判定，如例如定位誘變、丙胺酸篩選、晶體學或分析缺失及/或插入。
設想用於本發明之治療方法中之運鐵蛋白呈蛋白質偶聯物(protein conjugate)或融合蛋白質形式以便，例如，延長其在血液中之半衰期，其中運鐵蛋白偶聯或融合至任何其他蛋白質、蛋白質片段、蛋白質域或肽。在一個較佳實施例中，運鐵蛋白偶聯或融合至以下全長、片段域或肽：血清白蛋白(如例如，牛、兔或人)、匙孔血藍蛋白(keyhole limpet hemocyanin)、免疫球蛋白分子(包括免疫球蛋白之Fc域)、甲狀腺球蛋白、卵白蛋白、破傷風類毒素、或來自其他病源細菌如白喉、大腸桿菌、霍亂或幽門螺桿菌之類毒素，或減毒毒素衍生物、細胞因子、趨化因子(chemokines)、胰高血糖素樣肽-1(glucagon-like peptide-1)、毒蜥外泌肽-4(exendin-4)或XTEN。
用於本發明方法中之運鐵蛋白可為單獨Holo-Tf、單獨Apo-Tf或ApoTf與HoloTf之混合物。在一個較佳實施例中，用於本發明方法中之運鐵蛋白為ApoTf與HoloTf
之混合物並且，較佳，該混合物具有99：1與70：30之間之百分比(ApoTf：HoloTf)，甚至更佳，該混合物具有與自生物流體獲得或純化之餾分可比較的比例或百分比。在最佳實施例中，用於本發明方法中之ApoTf與HoloTf之混合物具有ApoTf與HoloTf之比例或百分比如存在於人類血液或血漿中之ApoTf與HoloTf之比例或百分比。
在本發明的方法中，組成物可進一步包括鐵螯合劑。在一個較佳實施例中，鐵螯合劑選自包括以下物質之群組：M30、去鐵胺(DFO)、地拉羅司(deferasirox)、去鐵酮(deferiprone)、地拉瑞汀(deferitrin)、L1NAl1、CP363、CP502、IOX2乙二胺四乙酸(EDTA)或其組合。在最佳實施例中，鐵螯合劑為去鐵胺(DFO)。
在本發明的方法中，治療缺氧誘導因子(HIF)相關病症包括用包含運鐵蛋白之組成物治療器官以準備移植至人體內。在一個較佳實施例中，器官選自包括腎臟、肝臟、肺及心臟之群組。
此外，在本發明的方法中，治療缺氧誘導因子(HIF)相關病症包括在移植之前或之後，將包含運鐵蛋白之組成物投與器官移植接受者。在一個較佳實施例中，本發明的方法包括在移植之前或之後，將包含運鐵蛋白之組成物投與器官移植接受者，並且其中移植器官先前用包含運鐵蛋白之組成物治療以準備移植至人體內。
在另一態樣中，本發明係關於治療缺氧誘導因子(HIF)相關病症之方法，其中該病症包括治療人之局部缺血，包
括向該人投與包含運鐵蛋白之組成物。在一個較佳實施例中，該局部缺血歸因於心臟驟停(cardiac arrest)、血栓性凝塊、外傷性損傷或中風。
在另一態樣中，本發明係關於治療缺氧誘導因子(HIF)相關病症之方法，其中該治療係在觀察到或預期組織/器官局部缺血或缺氧時術前投與人患者。
雖然本發明現在以下實例中結合某些較佳實施例來描述以使得其態樣可更完全理解及認識到，但是不意欲將本發明限制於此等具體實施例。相反地，意欲涵蓋所有替代方案、修改方案及等價方案，它們均可包括在如由附加申請專利範圍所界定的本發明的範疇之內。因此，包括較佳實施例之以下實例用來示出本發明之實踐，應理解所示詳細資料作為舉例並且目的僅為了例示性論述本發明之較佳實施例並且提供咸信本發明之配製程序以及原則及概念態樣的最適用及容易理解之描述。
以下實例中執行之實驗用包括去鐵形式或全鐵形式之運鐵蛋白來治療。測試廣泛多種運鐵蛋白混合物；包括大部分ApoTf、大部分HoloTf、pdTf及特別定義運鐵蛋白混合物之ApoTf與HoloTf之相對百分比在下表1中強調。
實例1. 包括大部分ApoTf及大部分HoloTf之組成物在常氧及缺氧條件下、在6小時治療之後誘導HIF-1α蛋白質。
在無血清培養基中培養之人類神經母細胞瘤SH-SY5Y細胞系細胞在常氧(21%氧)及缺氧條件(1%氧)下用來自血漿ApoTf及HoloTf(在兩種情況下以1mg/mL及4mg/mL之濃度)治療6小時。作為對照，使用未治療的細胞或用1mg/mL或4mg/mL濃度之人血清白蛋白(HSA)治療之細胞。在6小時之後，將細胞內蛋白質收穫並且針對HIF-1α蛋白質量藉由ELISA來測試。
如圖1所示，在常氧及缺氧條件下並且對於針對ApoTf所測試的兩種濃度而發生HIF-1α細胞蛋白質量之顯著增加。關於HoloTf，當細胞用1mg/mL濃度治療時，對於常氧及缺氧條件觀察到HIF-1α之細胞蛋白質量之顯著增加，並且當細胞用4mg/mL濃度治療時，對於常氧條件觀察到該顯著增加。當細胞用4mg/mL濃度HoloTf治療時，觀察到朝向HIF-1α之細胞蛋白質量增加之趨勢。
實例2. 包括大部分ApoTf及大部分HoloTf之組成物在常氧條件下、在24小時治療之後誘導HIF-1α蛋白質。
重複在實例1中執行之實驗但是執行治療24小時並且僅在常氧條件下。在24小時之後，將細胞內蛋白質收穫並且針對HIF-1α蛋白質量藉由ELISA來測試。圖2顯示對於此實驗所獲得的結果。如可以在該圖中看出，在所測試的兩種濃度下，ApoTf增加HIF-1α之細胞蛋白質量。對於
HoloTf，當治療使用4mg/mL濃度執行時，觀察到HIF-1α之細胞蛋白質量的顯著增加並且在使用1mg/mL濃度時，觀察到朝向該蛋白質增加之趨勢。
實例3. 在6小時治療之後，ApoTf與HoloTf之混合物誘導HIF-1α蛋白質。
在6小時之後，將細胞內蛋白質收穫並且針對HIF-1α蛋白質量藉由ELISA來測試。如圖3所示，在常氧條件下，用來自血漿之大部分ApoTf、大部分HoloTf或其混合物治療6小時之後觀察到HIF-1α細胞蛋白質量之增加。如亦可以在該圖中看出，ApoTf與HoloTf之混合物所有上調SH-SY5Y神經元細胞中之HIF-1α蛋白質。
實例4. 在HSA、去鐵運鐵蛋白或全鐵運鐵蛋白存在下、在常氧及缺氧條件下的Glut1及VEGF之mRNA表現量。
HIF-1α蛋白質之穩定性及增加通常導致上調HIF相關基因(由HIF靶向之基因轉錄增加)，即在其轉錄調控元件中具有HIF結合部位之基因。由HIF-1α蛋白質活化之兩種較好表徵基因係Glut1受體及VEGF。因此，為了分析此等HIF目標基因中之每一者的mRNA表現變化，將SH-SY5Y細胞系細胞培養並且在常氧(21%氧)或缺氧(1%氧)條件下用1mg/mL及4mg/mL濃度之大部分ApoTf或大部分HoloTf治療6小時。作為負性對照，細胞用HSA(1mg/mL或4mg/mL)治療或保持未治療。6小時之後，將細胞內mRNA收穫並且藉由qPCR針對Glut1及VEGF表現
量來測試。表現結果相對於在未治療細胞中觀察到之對應轉錄物之表現來計算。圖4A及4B顯示分別對於Glut1受體及VEGF所獲得的表現結果。圖中之值顯示為相對基因表現，並且目標基因(Glut1或VEGF)針對管家(β-肌動蛋白)表現來標準化。如可直接自該圖得到，在缺氧條件下，相對於HSA對照，在用去鐵運鐵蛋白治療時，Glut1(圖4A)及VEGF(圖4B)之表現顯著增加。引起關注地，在常氧條件下，全鐵運鐵蛋白，而非去鐵運鐵蛋白，僅增加Glut1之表現。
實例5. ApoTf與HoloTf之混合物在體外或體內不展示毒性。
由於已報告HoloTf在體內及體外對於細胞有毒，含有大部分ApoTf、大部分HoloTf或ApoTf+HoloTf混合物之各種組成物之毒性在SH-SY5Y細胞中加以測試。SH-SY5Y細胞用所指示濃度4mg/mL Tf(如圖5A指示)、M30或DFO治療72小時。72小時之後，細胞經受細胞滴定發光生存力檢定。對照細胞，即未治療的細胞，設定為100%生存力值。對於每個治療條件，顯示平均生存力及標準偏差。對於含有大部分ApoTf之任何組成物，未觀察到毒性影響，並且意外地，未觀察到大部分HoloTf之毒性或有害影響。
引起關注地，大部分ApoTf及大部分HoloTf之組成物都不顯示此等行為標準之顯著差異，表明在體內沒有HoloTf之有害影響。圖5B顯示使用包括大部分ApoTf或HoloTf之藥物來靜脈內治療之大鼠的修正貝德森及一般
行為評分。體內神經學功能藉由修正貝德森評分(貝德森等人，1986b；Crumrine等人，2011)使用以下定義來評估：評分0：無明顯神經學缺陷；評分1：存在身體扭轉；評分2：身體扭轉伴有右側虛弱；評分3：身體扭轉，右側虛弱伴有轉圈行為；及評分4：抽搐活動。
大鼠之一般行為評分由CALS人員為了監測手術程序之後動物恢復之目的而開發(標準CALS手術後護理)。將數值分配至預定行為觀察結果。
評分0：行為與正常雛期大鼠(即無同側缺陷)一致；評分1：生氣勃勃/活躍/有響應；大鼠自發移動並且在籠中探索，對於外部刺激作出響應，探索籠子頂部；評分2：安靜/警覺/有響應；行為保守但是對於外在刺激有響應，傾向於不豎起或探索籠子頂部；評分3：行為抑制：傾向於不移動除非受到刺激，快速地返回到嗜睡狀態，極少關注外部刺激；評分4：無響應：甚至在受刺激時，保持俯伏位置；及評分5：抽搐活動，需要安樂死。
實例6. 藉由運鐵蛋白之體內細胞保護
大腦中風之MCAo(大腦中動脈阻塞)大鼠模型用於評估藉由運鐵蛋白之細胞保護。藉由使用MCAo技術來手術誘導16隻大鼠之中風。8隻大鼠藉由在大腦中注射鹽水溶
液來治療並且其他8隻藉由在大腦中注射ApoTf來治療。圖6A及6B顯示在與對照大鼠(用鹽水溶液治療)相比時，在用包括大部分ApoTf之混合物治療之大鼠中觀察到梗死區域體積顯著降低。圖6A顯示在短暫MCAo中之ApoTf(385mg/kg，IV)或鹽水治療之梗塞體積之散佈圖；並且圖6B顯示來自代表性對照及ApoTf治療大鼠之TTC染色冠狀切片。
實例7. ApoTf及HoloTf保護SH-SY5Y避免Aβ 1-42毒性。
HIF途徑之上調已知在許多神經退化性疾病，包括導致破壞神經細胞及神經元之病狀中發揮保護作用。由於SH-SY5Y之治療使HIF上調，因此細胞用去鐵運鐵蛋白或全鐵運鐵蛋白治療應對於經受已知誘導神經退化性之物質之細胞提供保護效應。圖7突出了以下資料，該資料評估是否大部分去鐵運鐵蛋白及全鐵運鐵蛋白可保護SH-SY5Y細胞避免已知神經退化性毒素低聚Aβ1-42肽之毒性影響(圖7)。在生長培養基中培養之SH-SY5Y神經元細胞在正常氧量下用4mg/mL去鐵運鐵蛋白或全鐵運鐵蛋白治療24小時。24小時之後，細胞再用低聚Aβ1-42肽治療72小時。用低聚Aβ1-42治療之後，細胞經受ApoGlo凋亡蛋白酶3/7活化檢定。對照細胞，即未治療的細胞，設定為1標準化值。對於每個治療條件，顯示相對於對照細胞之平均凋亡蛋白酶誘導，及標準偏差。引起關注地，此等資料表明大部分ApoTf及HoloTf保護SH-SY5Y細胞
避免Aβ誘導毒性。此等資料亦進一步證實ApoTf或HoloTf缺乏固有毒性。
實例8. 與小分子HIF活化劑及ApoTf/HoloTf混合物之協同效應。
轉鐵蛋白可與其他HIF活化小分子，如其他鐵螯合劑或酶抑制劑協同起作用。此可允許投與較低量之此等小分子，誘導較少副作用但是保持較高治療量。為了判定是否去鐵運鐵蛋白增加鐵螯合劑、DFO及phd2抑制劑IOX2之效價；在無血清培養基中培養之SH-SY5Y神經元細胞在存在或不存在小分子藥物時、在正常氧量下用4mg/mL所指示的蛋白質治療。實驗結果顯示於圖8A、圖8B及圖8C。圖8A顯示之資料涉及用指示濃度之DFO或IOX2加上4mg/mL蛋白質之組合治療細胞。CoCl2用作實驗陽性對照。圖8B所示資料涉及用10uM M30加/減4mg/mL蛋白質之組合治療細胞。圖8C所示資料涉及用200uM DFO加/減4mg/mL蛋白質之組合治療細胞。在6小時之後，將細胞內蛋白質收穫並且針對HIF-1α蛋白質量藉由ELISA來測試。資料以具有標準偏差之pg/mL為單位來顯示。
實例9. 反應於大部分去鐵運鐵蛋白及DFO或IOX2組合的Glut1及VEGF之mRNA表現量。
另外，判定反應於大部分去鐵運鐵蛋白及DFO或IOX2組合之Glut1及VEGF之mRNA表現量。在無血清培養基中培養之SH-SY5Y神經元細胞在正常氧量下用4mg/mL人血清白蛋白或大部分去鐵運鐵蛋白治療。在指示時，
200uM DFO或1uM IOX2用HSA及大部分去鐵運鐵蛋白來共治療。6小時治療之後，將細胞內mRNA收穫並且藉由qPCR針對Glut1及VEGF表現量來測試。值顯示為相對基因表現，並且目標基因(Glut1或VEGF)針對管家(β-肌動蛋白)表現來標準化。顯示標準偏差。圖9A及圖9B表明隨著添加去鐵運鐵蛋白及HIF途徑之小分子活化劑，Glut1及VEGFmRNA量協同並且相加地增加。
實例10. 大部分ApoTf及大部分HoloTf之組成物在人類初級腎細胞中誘導HIF-1α蛋白質。
在此項技術中熟知用於治療與缺氧相關或引起之病症的許多小分子有毒並且具有許多副作用，例如DFO。該等小分子之最明顯副作用之一係腎毒性。因此，為了評估是否運鐵蛋白及/或混合物增加初級腎細胞中之HIF-1α量；獲得人類初級腎細胞，即初級人類腎臟近端小管上皮(RPTEC)或皮質上皮細胞(HRCE)。在無血清培養基中培養之初級人類腎臟近端小管上皮(RPTEC)或初級皮質上皮(HRCE)細胞在正常氧量下用4mg/mL大部分去鐵運鐵蛋白、大部分全鐵運鐵蛋白或每一種物質之各個混合物治療6小時。在6小時之後，將細胞內蛋白質收穫並且針對HIF-1α蛋白質量藉由ELISA來測試。圖10A及圖10B顯現HIF-1α量藉由分別主要由去RPTEC及HRCE中之鐵運鐵蛋白及全鐵運鐵蛋白的混合物組成之運鐵蛋白來誘導。
實例11. 人類初級腎細胞在運鐵蛋白或DFO存在下的生存力，包括在大部分ApoTf或DFO存在下、在人類
初級腎細胞內的凋亡蛋白酶3/7活化。
考慮到人類血漿蛋白質之預期安全概況，DFO及轉鐵蛋白(大部分去鐵、大部分全鐵及混合物)之毒性在初級人類腎細胞中評估。腎臟近端小管上皮(RPTEC)或皮質上皮(HRCE)細胞用指示濃度之大部分ApoTf或DFO治療48小時(圖11A)；並且RPTEC或HRCE細胞用4mg/mL大部分ApoTf、大部分HoloTf、運鐵蛋白之混合物治療72小時(圖11B)。48或72小時之後，細胞經受細胞滴定發光生存力檢定。對照細胞，即未治療的細胞，設定為100%生存力值。對於每個治療條件，顯示平均生存力及標準偏差。圖11A及圖11B顯示雖然DFO具有顯著毒性，運鐵蛋白分子沒有一種顯示對於此等初級腎細胞之任何有害影響。
為了評估在ApoTf或DFO存在下的人類初級腎細胞內之凋亡蛋白酶3/7活化；RPTE或HRC細胞用指示濃度之ApoTf或DFO治療48小時。48小時之後，細胞經受ApoGlo凋亡蛋白酶3/7活化檢定。對照細胞，即未治療的細胞，設定為1標準化值。對於每個治療條件，相對於對照細胞之平均凋亡蛋白酶活性，及標準偏差顯示於圖12中。
實例12. 在初級人類肝細胞或肺細胞系NCI-H1650中未觀察到HIF之上調。
如以上詳述，來自血漿之去鐵運鐵蛋白及全鐵運鐵蛋白增加人神經元細胞系SH-SY5Y中之HIF-1α之細胞量。除了神經元細胞以外，肝及肺器官移植物亦可受益於HIF
信號傳遞之誘導。因此，為了對其進行評估；判定轉鐵蛋白對於初級肝細胞及肺細胞系(NCI-H1650)中之HIF-1α量之效應。在無血清培養基中培養之肺細胞系NCI-H1650或初級肝細胞細胞在正常氧量下用4mg/mL大部分去鐵運鐵蛋白、大部分全鐵運鐵蛋白或pd-轉鐵蛋白治療6小時。在6小時之後，將細胞內蛋白質收穫並且針對HIF-1α蛋白質量藉由ELISA來測試。如圖13A及圖13B強調之資料顯示在NCIH1650或初級肝細胞中，HIF-1α量未藉由運鐵蛋白或混合物去鐵運鐵蛋白及全鐵運鐵蛋白來誘導。
實例13. 在運鐵蛋白存在下的NCI-H1650及人類初級肝細胞之生存力。
在給出人類血漿蛋白質之預期安全概況的情況下，評估NCI-H1650及初級人類肝細胞細胞中之轉鐵蛋白(大部分去鐵、大部分全鐵及pd-運鐵蛋白)之毒性。人類肺細胞系NCI-H1650及初級人類肝細胞用4mg/mL大部分ApoTf、大部分HoloTf或pd-運鐵蛋白治療72小時。72小時之後，細胞經受細胞滴定發光生存力檢定。對照細胞，即未治療的細胞，設定為100%生存力值。對於每個治療條件，平均生存力及標準偏差顯示於圖14A及圖14B中。資料顯示在肺細胞、NCI-H1650或初級肝細胞中，使用含有大部分HoloTf或大部分ApoTf之組成物，未觀察到毒性。
結論
在人類神經元細胞系SH-SY5Y中執行之實驗顯示來自血漿之去鐵運鐵蛋白及全鐵運鐵蛋白增加細胞量
HIF-1α。HIF-1α量之增加發生於常氧及缺氧條件下。在正常氧條件下將去鐵運鐵蛋白投與細胞將HIF-1α量提高至當細胞曝露於缺氧環境時觀察到之類似量。與較短時間相比，SH-SY5Y細胞在常氧條件下曝露於去鐵運鐵蛋白較長時間可在更大程度上增加HIF-1α之量。人類血清白蛋白負性對照對於HIF-1α量沒有效果。
ApoTf與HoloTf之各種混合物都在SH-SY5Y神經元細胞及初級腎細胞中使HIF-1α蛋白質上調。
在初級人類肝細胞或肺細胞系NCI-H1650中未觀察到HIF-1α之上調。
ApoTf與HoloTf之各種混合物都使SH-SY5Y神經元細胞中之HIF-1α目標基因上調。
在任何細胞類型(神經元、肺、腎或肝細胞)中或在體內，對於含有大部分HoloTf或大部分ApoTf之組成物，未觀察到毒性。
在局部缺血-再灌注之神經學壓力模型中在體內治療大鼠顯示運鐵蛋白(主要由ApoTf組成)保護大鼠細胞避免梗塞。
主要包括ApoTf或HoloTf之混合物保護神經元細胞避免Aβ(1-42)寡聚物之毒性影響。
僅由大部分ApoTf組成之混合物具有與M30或DFO之協同效應，並且此等協同活性僅發生於SH-SY5Y神經元細胞中。
Claims (15)
- 一種醫藥組成物用於製備治療與缺氧誘導因子相關病理病症之藥物之用途，該醫藥組成物包括治療有效量之運鐵蛋白，且其中該運鐵蛋白係去鐵運鐵蛋白與全鐵運鐵蛋白之混合物，該混合物呈99%去鐵-Tf：1%全鐵-Tf至30%去鐵-Tf：70%全鐵-Tf之比率。
- 如請求項1所記載之用途，其中該醫藥組成物進一步包含鐵螯合劑或PHD2酶抑制劑。
- 如請求項1所記載之用途，其中該與缺氧誘導因子相關病理病症與移植接受者之器官移植相關。
- 如請求項3所記載之用途，其中該器官已經用包含運鐵蛋白之醫藥組成物治療以準備移植至該接受者中。
- 如請求項4所記載之用途，其中該器官已經用包含運鐵蛋白及鐵螯合劑或PHD2酶抑制劑之醫藥組成物治療以準備移植至該接受者中。
- 如請求項1所記載之用途，其中該病症與手術之前的患者中之局部缺血或缺氧相關。
- 如請求項1所記載之用途，其中該病症係歸因於心臟驟停、血栓性凝塊或外傷性損傷之局部缺血。
- 如請求項1所記載之用途，其中在外科手術期間發生血液流動之中斷。
- 如請求項1所記載之用途，其中該病症歸因於選自由以下組成之群的神經退化性疾病：阿茲海默症、帕金森氏病、亨丁頓氏疾病及肌萎縮性側索硬化。
- 如請求項1所記載之用途，其中該運鐵蛋白係重組的運鐵蛋白。
- 如請求項1所記載之用途，其中該運鐵蛋白藉由聚乙二醇化、糖基化、多唾液酸化或其他物理修飾來修飾以延長該蛋白質之血漿半衰期，包括共價融合至延長血液中之半衰期的結構域，包括免疫球蛋白之Fc域、白蛋白、胰高血糖素樣肽-1、毒蜥外泌肽-4及XTEN之至少一者。
- 如請求項1所記載之用途，其中該運鐵蛋白被修飾成增加金屬結合親和力。
- 如請求項2所記載之用途，其中該鐵螯合劑係M30、去鐵胺(DFO)、地拉羅司(Deferasirox)、去鐵酮(deferiprone)、地拉瑞汀(deferitrin)、L1NAl1、CP363、CP502或乙二胺四乙酸(EDTA)。
- 如請求項2所記載之用途，其中該PHD2酶抑制劑係IOX2、IOX3、二甲基草醯甘胺酸或其他2-氧化戊二酸鹽(2-oxoglutarate)結合部位分子。
- 如請求項1所記載之用途，其中該醫藥組成物用於獲得至少25uM及直至250uM之去鐵胺(DFO)的血液濃度。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2013068504A1 (en) * | 2011-11-11 | 2013-05-16 | Fundació Institut D'investigació En Ciències De La Salut Germans Trias I Pujol | Apotransferrin for the treatment of brain stroke |
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| De Vries, B., Walter, S. J., von Bonsdorff, L., Wolfs, T. G., van Heurn, L. E., Parkkinen, J., & Buurman, W. A. (2004). Reduction of circulating redox-active iron by apotransferrin protects against renal ischemia-reperfusion injury1. Transplantation, 77(5), 669-675. |
| De Vries, B., Walter, S. J., von Bonsdorff, L., Wolfs, T. G., van Heurn, L. E., Parkkinen, J., & Buurman, W. A. (2004). Reduction of circulating redox-active iron by apotransferrin protects against renal ischemia-reperfusion injury1. Transplantation, 77(5), 669-675. Chen-Roetling, J., Chen, L., & Regan, R. F. (2011). Apotransferrin protects cortical neurons from hemoglobin toxicity. Neuropharmacology, 60(2-3), 423-431. Mole, D. R. (2010). Iron homeostasis and its interaction with prolyl hydroxylases. Antioxidants & redox signaling, 12(4), 445-458. * |
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