通用
本發明提供供與MRI一起使用之用於診斷涉及類澱粉蛋白斑之疾病的組合物及方法，該等疾病諸如阿兹海默症、癡呆、帕金森症(Parkinson's disease)及路易體症(Lewy body disease)。部分上，本發明係針對一種可穿過血腦屏障且與類澱粉蛋白斑及其他類似蛋白聚集體結合以使得其可藉由磁振造影(MRI)偵測到之新穎奈米顆粒。本文所述之組合物、技術及方法亦提供一種在早期診斷阿兹海默症之非侵入性手段。定義
如本文所使用，術語「超順磁」指磁性之形式，其呈現在例如小鐵磁性或次鐵磁性奈米顆粒中。在足夠小的奈米顆粒中，磁化可在溫度之影響下任意轉換方向。兩次轉換之間的典型時間稱作尼爾(Néel)弛緩時間。無外磁場存在下，當用於量測奈米顆粒之磁化之時間遠長於尼爾弛緩時間時，其磁化呈現為平均為零。如此，其據稱處於超順磁狀態。在此狀態下，外磁場能夠類似於順磁體而磁化奈米顆粒。然而，其磁化率比順磁體之磁化率大很多。 如本文所使用，術語「奈米顆粒」指具有小於約100奈米且大於約0.1奈米之實體尺寸之顆粒。 如本文所使用，術語「結合劑」指能夠結合或締合或錯合至類澱粉蛋白斑之試劑、組合物或化合物。 如本文所使用，片語「類澱粉蛋白斑」指共有特定結構特點之不可溶纖維蛋白聚集體。斑亦可稱作沈積物。此等摺疊異常結構更改其恰當組態，致使其錯誤地彼此相互作用或與其他細胞組分相互作用而形成不可溶原纖維。其已與多於20種嚴重人類疾病之病變相關。在器官中類澱粉原纖維之異常聚積可導致澱粉樣變性且可在各種神經退化病症中起一定作用。 如本文所使用，片語「蛋白聚集體」指蛋白聚集之產物，蛋白聚集係摺疊異常蛋白在胞內或胞外聚集、聚積及/或凝集在一起之生物現象。此等蛋白聚集體通常有毒且已涉及於已知為澱粉樣變性之各種疾病中，該等疾病包括阿兹海默症、帕金森症及朊病毒病。 如本文所使用，術語「指示」意謂具有一定疾病之特徵或表明呈現一定疾病之狀況。舉例而言，類澱粉蛋白斑指示阿兹海默症，因為其通常與此疾病相關。具有類澱粉蛋白斑之患者可能患有阿兹海默症，且因此此類斑可例如向醫生指示關於患者是否患有阿兹海默症之狀況。 如本文所使用，術語「親水性」指具有排斥非極性或不帶電化學基團(例如己烷)及吸引極性或帶電化學基團(例如水)之傾向之化學基團。「親水性」亦指傾向於溶解在水中、與水混合或由水潤濕之化學物質。 如本文所使用，術語「疏水性」指具有吸引非極性或不帶電化學基團(例如己烷)及排斥極性或帶電化學基團(例如水)之傾向之化學基團。「疏水性」亦指傾向於不溶解在水中、不與水混合或不由水潤濕之化學物質。 如本文所使用，片語「流體動力粒度」指可囊封有本發明之顆粒之假設三維球體的最小直徑。 如本文所使用，術語「可生物降解」指組合物藉由活有機體之作用分解，特定言之分解成無害產物之能力。 如本文所使用，術語「兩親媒性」用於將化學化合物描述為具有親水性及親脂疏水性特性。 如本文所使用，術語「共聚物」指相對於僅使用一種單體之均聚物，來源於兩種(或更多種)單體物種之聚合物。舉例而言，假定單體物種A及B，交替共聚物可具有-A-B-A-B-A-B-A-B-A-B形式。舉例而言，假定單體物種A及B，無規共聚物可具有-A-A-B-A-B-B-A-B-A-A-A-B-B-B-B-A形式。舉例而言，假定單體物種A及B，嵌段共聚物可具有-(A-A-A)-(B-B-B)-(A-A-A)-(B-B-B)-(A-A-A)-形式。組合物
在一些實施例中，本發明提供一種磁振造影(MRI)診斷劑組合物，其包括超順磁奈米顆粒、聚合物及類澱粉蛋白斑之至少一種結合劑。在此等實施例中之一些中，結合劑附著於奈米顆粒之表面。在此等實施例中之一些中，聚合物塗佈該具有結合劑附著於表面之奈米顆粒。 在一些實施例中，本文所描述之奈米顆粒具有包括黏附至奈米顆粒之表面且塗佈奈米顆粒之表面之聚合物之第一層。在一些實施例中，本文所描述之奈米顆粒具有包括類澱粉蛋白斑之至少一種結合劑之第二層。在一些實施例中，類澱粉蛋白斑之結合劑附著於奈米顆粒之表面。在一些實施例中，類澱粉蛋白斑之結合劑附著於尚未鍵結於聚合物之奈米顆粒之表面。在一些實施例中，附著於奈米顆粒之表面之類澱粉蛋白斑的結合劑能夠結合至類澱粉蛋白斑。在某些實施例中，奈米顆粒包括包含超順磁物質之核。在一些實施例中，聚合物及類澱粉蛋白斑之結合劑係黏附或鍵結於超順磁奈米顆粒之表面。在一些實施例中，聚合物及類澱粉蛋白斑之結合劑可在奈米顆粒之表面上緊密地締合。在一些實施例中，儘管聚合物及類澱粉蛋白斑之結合劑兩者皆附著於奈米顆粒之表面，但類澱粉蛋白斑之結合劑延伸出聚合物之表面。在又其他實施例中，聚合物及類澱粉蛋白斑之結合劑兩者皆鍵結於奈米顆粒之表面且類澱粉蛋白斑之結合劑充分暴露以能夠結合至類澱粉蛋白斑。 在一些實施例中，本發明提供磁振造影(MRI)診斷劑組合物，其包括：超順磁奈米顆粒、聚合物、及類澱粉蛋白斑之至少一種結合劑；其中結合劑附著至奈米顆粒之表面；且其中聚合物塗佈該具有結合劑附著於表面之奈米顆粒。在一些實施例中，聚合物及結合劑兩者皆附著於奈米顆粒之表面。在某些實施例中，聚合物及結合劑緊密地在奈米顆粒之表面上締合。在其他實施例中，聚合物塗佈結合劑及奈米顆粒之表面。在一些實施例中，聚合物塗佈奈米顆粒之表面但並不防止附著於奈米顆粒之表面之結合劑結合至類澱粉蛋白斑。 在一些實施例中，本發明提供結合劑係類澱粉蛋白斑的結合劑。在其他實施例中，本發明提供結合劑係類澱粉沈積物的結合劑。在某些實施例中，類澱粉蛋白斑係選自由蛋白聚集體、A-β聚集體及突觸核蛋白組成之群。在一些實施例中，類澱粉蛋白斑指示選自由以下組成之群的疾病：阿兹海默症、癡呆、帕金森症及路易體症。 在一些實施例中，本發明提供類澱粉蛋白斑能夠與薑黃素形成化學鍵。 在一些實施例中，本發明提供奈米顆粒包括鐵。在某些實施例中，本發明提供奈米顆粒包括磁鐵。在其他實施例中，本發明提供奈米顆粒包括選自由以下組成之群的金屬：鐵、金、鉑、銀及鈷。在又其他實施例中，本發明提供奈米顆粒包括選自由以下組成之群的半導體：硒化鎘、硫化鎘、硒化鉛、硫化鉛、硒化鋅、硫化鋅及其組合。在一些實施例中，奈米顆粒包括氧化鐵。 在一些實施例中，本發明提供奈米顆粒，其中奈米顆粒之尺寸小於30 nm。在某些實施例中，組合物之流體動力粒度小於300 nm。在一些實施例中，流體動力粒度係約10 nm至約300 nm。 在一些實施例中，本發明提供其中結合劑係生物類黃酮或染料之組合物。在一些此等實施例中，結合劑係選自由以下組成之群的染料：薑黃素、剛果紅、硫代黃素T/S、槲皮素、表兒茶素、橘皮苷、芸香苷及紅橘黃酮。在某些實施例中，染料係薑黃素。 在一些實施例中，本發明提供聚合物係親水性之組合物。在一些實施例中，親水性聚合物係選自由以下組成之群：聚乙二醇(PEG)、聚葡萄糖、聚乙烯吡咯啶酮(PVP)、脂肪酸、聚乙烯醇(PVA)、聚丙烯酸、多肽、磷酸膽鹼、聚(D,L-乳酸交酯)、聚(N-異丙基丙烯醯胺) (PolyNIPAAM)、幾丁聚糖、明膠、聚乳酸-共-乙醇酸(PLGA)、聚己內酯(PCL)及聚氰基丙烯酸丁酯(PBCA)。在一些此等實施例中，親水性聚合物係聚乙二醇(PEG)。 在一些實施例中，本發明提供進一步包括可生物降解兩親媒性共聚物之組合物。在一些實施例中，可生物降解兩親媒性共聚物係選自由以下組成之群：聚乳酸-共-乙醇酸(PLGA)、聚己內酯(PCL)、聚乳酸(PLA)、聚乙二醇(PEG)、甲氧基聚乙二醇(MePEG)、聚氧化乙烯(PEO)、聚丁二烯(PBD)、d-α-生育酚聚乙二醇1000丁二酸酯、PEG-PLA、PEG-PCL、PEG-PLGA、MePEG-PLA、MePEG-PCL、MePEG-PLGA、PEO-PBD及維生素E TPGS。 在一些實施例中，本發明提供進一步包括親水性聚合物穩定劑之組合物。在一些實施例中，親水性聚合物穩定劑係選自由以下組成之群：聚乙烯吡咯啶酮(PVP)、聚乙烯醇(PVA)、聚乙二醇(PEG)、羥丙基甲基纖維素(HPMC)，及泊洛沙姆(Poloxamer)、聚乳酸-共-乙醇酸(PLGA)、聚己內酯(PCL)、聚乳酸(PLA)、聚氰基丙烯酸丁酯(PBCA)及幾丁聚糖。 在一些實施例中，本發明提供進一步包括選自由苯紮氯銨(benzalkonium chloride)、苄索氯銨(benzethonium chloride)及西曲溴胺(cetrimide)組成之群的陽離子界面活性劑之組合物。 在一些實施例中，本發明提供進一步包括選自由多庫酯鈉(docusate sodium)及月桂基硫酸鈉組成之群的陰離子界面活性劑之組合物。 在一些實施例中，本發明提供進一步包括選自由以下組成之群的非離子界面活性劑之組合物：單油酸甘油酯、脫水山梨糖醇酯、聚氧乙烯脫水山梨糖醇脂肪酸酯及聚氧乙烯烷基醚。在一些實施例中，非離子界面活性劑係選自由以下組成之群的脫水山梨糖醇酯：脫水山梨糖醇單月桂酸酯、脫水山梨糖醇單油酸酯、脫水山梨糖醇單棕櫚酸酯、脫水山梨糖醇倍半油酸酯及脫水山梨糖醇三油酸酯。在一些實施例中，非離子界面活性劑係選自由以下組成之群的聚氧乙烯脫水山梨糖醇脂肪酸酯：聚山梨醇酯20、聚山梨醇酯40、聚山梨醇酯60、聚山梨醇酯65、聚山梨醇酯80及聚山梨醇酯85。在一些其他實施例中，非離子界面活性劑係選自由以下組成之群的聚氧乙烯烷基醚：聚乙二醇單十六基醚、聚乙二醇單月桂基醚、聚乙二醇單油基醚及聚乙二醇單十八基醚。在一些實施例中，泊洛沙姆係選自由以下組成之群：P124、P188、P237、P338及P407。 在一些實施例中，本發明提供奈米顆粒係氧化鐵且染料係薑黃素之組合物。在一些實施例中，本發明提供所計算最大負載係每公克氧化鐵75毫克薑黃素之組合物。在一些實施例中，本發明提供所計算最小負載係每公克氧化鐵5毫克薑黃素之組合物。醫藥組合物
本發明之醫藥組合物涵蓋藉由將本發明之化合物、奈米顆粒、或組合物與醫藥學上可接受之載劑及/或賦形劑或稀釋劑摻合而製備之組合物。此類組合物適合於在動物或人類中之醫藥用途。 本發明之醫藥組合物包括作為活性成分之本文所描述之化合物、奈米顆粒或組合物或其醫藥學上可接受之鹽，以及醫藥學上可接受之載劑及/或賦形劑或稀釋劑。醫藥組合物可視情況含有其他治療成分。 本發明化合物可作為均勻摻合物中之活性成分與適合的醫藥載劑及/或賦形劑根據習知醫藥混配技術組合。適合於投藥所期望的製備形式之任何載劑及/或賦形劑均可與本文所揭示之化合物一起使用。可藉由藥學技術所熟知之任何方法製備組合物。 組合物包括適合於局部、非經腸、經肺、經鼻、經直腸或經口投與之組合物。在任何給出案例中之最適合之投藥途徑將部分地視所診斷病況之性質及嚴重性而定。其他較佳組合物包括適合於全身(經腸或非經腸)投藥。全身投藥包括經口、經直腸、經舌下或經唇下投藥。組合物可藉由皮下、靜脈內、肌內或腹膜內注射(例如，經由注射器)投與。 用於經肺投與之組合物包括(但不限於)由本文所描述之化合物或其鹽之粉末及適合載劑及/或潤滑劑之粉末組成之乾燥粉末組合物。用於經肺投與之組合物可自熟習此項技術者已知之任何適合的乾燥粉末吸入器裝置吸入。 用於全身投與之組合物包括(但不限於)由如本文所述之組合物及適合載劑及/或賦形劑之粉末組成之乾燥粉末組合物。用於全身投與之組合物可由(但不限於)以下形式呈現：錠劑、膠囊、丸劑、糖漿、溶液及懸浮液。 在一些實施例中，本發明提供進一步包括醫藥界面活性劑之組合物。 在一些實施例中，本發明提供進一步包括低溫保護劑之組合物。在一些實施例中，低溫保護劑係選自由以下組成之群：葡萄糖、蔗糖(sucrose)、海藻糖、乳糖、麩胺酸鈉、PVP、HPβCD、CD、甘油、麥芽糖、甘露醇及蔗糖(saccharose)。 在一些實施例中，本發明提供包括如技術方案1之組合物及醫藥學上可接受之賦形劑的醫藥組合物。在此等實施例中之一些中，醫藥學上可接受之賦形劑包括鹽或稀釋劑。 在一些實施例中，本發明提供包括有效量之如技術方案1之組合物的組合物。在一些實施例中，組合物經調配用於經口投與或靜脈內投與且包括如技術方案1之組合物及選自由水溶液及緩衝溶液組成之群的至少一個成員。製備超順磁奈米顆粒之方法
本發明之奈米顆粒可藉由本文所述之方法來製備。舉例而言，結合薑黃素之氧化鐵奈米顆粒可藉由以下程序來製備。 在一些實施例中，氧化鐵奈米顆粒可藉由氧化-沈澱原理來製備，例如，Tada M, Hatanaka S, Sanbonsugi H, Matsushita N, Abe M. Method for synthesizing ferrite nanoparticles ~ 30 nm in diameter on neutral pH condition for biomedical applications, J. Appl. Phys. 2003;93(10):7566-7568；及Konwarh R, Saikia JP, Karak N, Konwar BK. 'Poly(ethylene glycol)-magnetic nanoparticles-curcumin' trio: Directed morphogenesis and synergistic free-radical scavenging. Colloids and Surfaces B: Biointerfaces 2010 12/1;81(2):578-586。在一些實施例中，將鹼溶液(例如KOH (1 mol/L))與2%聚乙二醇(PEG)之混合物逐滴添加至金屬鹵化物(例如FeCl2
)溶液中直至pH達約7.8。在持續攪拌下，添加過氧化氫產生暗黑沈澱。將產物純化，經去離子水洗滌且隨後經丙酮洗滌，且乾燥。 在一些實施例中，氧化鐵奈米顆粒可藉由描述於例如Aono H, Hirazawa H, Naohara T, Maehara T, Kikkawa H, Watanabe Y. Synthesis of fine magnetite powder using reverse coprecipitation method and its heating properties by applying AC magnetic field. Materials Research Bulletin 2005; 40(7): 1126-1135中之逆向共沈澱法來合成。在一些實施例中，將鹼溶液(例如，溶解於去氧純化水中之NaOH)逐滴添加至鐵溶液(例如，以2 mol Fe3 +
:1 mol Fe2 +
之比率溶解於去氧純化水中之FeCl3
·6H2
O及FeSO4
·7H2
O)中，同時攪拌。在添加鹼溶液期間形成暗黑沈澱。保持攪拌混合物隔夜，之後沈澱經水洗滌，在二甲基甲醯胺中再懸浮，且於4℃下儲存。 本文所述之方法適用於製備大小約10至30 nm之氧化鐵奈米顆粒。 本文所述之方法適用於製備以每公克氧化鐵約75 mg之最大負載而負載有薑黃素之奈米顆粒。在本文中最大負載定義為薑黃素在奈米顆粒上形成單層表面之負載。在一些實施例中，薑黃素之最大負載係每公克氧化鐵約1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74或75 mg。在其他實施例中，薑黃素之最大負載係每公克氧化鐵76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100 mg。在奈米顆粒上薑黃素之負載亦可根據薑黃素對氧化鐵之莫耳比來定義。在一些實施例中，奈米顆粒以在約0.05:1至約1:1範圍內之薑黃素對氧化鐵之莫耳比負載薑黃素。在一些實施例中，奈米顆粒以在約0.05:1至約0.5:1範圍內，在約0.3:1至約0.75:1範圍內，在約0.55:1至約1:1範圍內，在約0.05:1至約0.25:1範圍內，在約0.2:1至約0.4:1範圍內，約0.35:1至約0.55:1，約0.5:1至約0.7:1，約0.65:1至約0.85:1，或約0.8:1至約1:1之薑黃素對氧化鐵之莫耳比負載薑黃素。在一些實施例中，以約0.4:1之薑黃素對氧化鐵之莫耳比負載有薑黃素之奈米顆粒在後續經透析移除溶劑期間保持了其大小。 在一些實施例中，使用大功率音波處理製備奈米顆粒。在一些實施例中，奈米顆粒在超音波處理下分散於二甲基甲醯胺中一段設定之時間(例如1、2、3、4、5、6、7、8、9或10分鐘)。在一些實施例中，懸浮液進一步經真空濃縮器乾燥。 在一些實施例中，奈米顆粒囊封在聚合物微胞中。在一些實施例中，PEG-PLA共嵌段聚合物溶解於有機相中而奈米顆粒分散於水相中。在其他實施例中，PEG-PLA嵌段共聚物及結合薑黃素之奈米顆粒兩者均提供於有機相中同時亦提供水相。在某些實施例中，兩相均共注入多入口旋渦混合器中。在一些實施例中，在快速攪拌期間使奈米顆粒產生高能量。在某些實施例中，奈米顆粒經分離且防止聚集。在一些實施例中，向溶液中添加聚乙烯吡咯啶酮(PVP)或聚乙烯醇(PVA)。在一些實施例中，PVP或PVA溶解於與有機相共注入旋渦混合器中之水層中。 在一些實施例中，殘餘在奈米顆粒懸浮液中之溶劑經透析或離心過濾除去。在某些實施例中，藉由添加低溫保護劑，諸如蔗糖、甘露醇、β環糊精或葡萄糖，且隨後與奈米懸浮液共冷凍用於冷凍乾燥來實現乾燥。在一些實施例中，方法包括儲存經乾燥奈米顆粒。在一些實施例中，在儲存奈米顆粒之後，向奈米顆粒之溶液中添加聚山梨醇酯80或甘露醇以將其復原。 在一些實施例中，本發明提供一種標記類澱粉蛋白斑用於MRI偵測之方法，其包括使本文所描述之組合物與類澱粉蛋白斑接觸，由此標記類澱粉蛋白斑用於MRI偵測。在此等實施例中之一些中，類澱粉蛋白斑係選自由蛋白聚集體、A-β聚集體及突觸核蛋白組成之群。在某些實施例中，類澱粉蛋白斑指示選自由以下組成之群的疾病：阿兹海默症、癡呆、帕金森症及路易體症。 在一些實施例中，本發明提供一種診斷患者之疾病或病況之方法，其包括向患者投與本文所描述之組合物；用該組合物標記患者中之類澱粉蛋白斑；獲取經投與該組合物之患者之MRI圖像；分析該等影像以偵測患者中之類澱粉蛋白斑；及由此診斷患者之疾病或病況。 在一些實施例中，本發明提供一種方法，其中獲取MRI圖像包括用以下MRI參數操作MRI儀器：T2* - FLASH (2D)；TE = 25 ms，TR = 400 ms，FA = 200
；FOV=2×2，MTX = 400×400；平面內解析度=50 μm2
；切片厚度= 0.5 mm；及NEX = 80，掃描時間= 3.5小時。 在一些實施例中，本發明提供一種投與本文所描述之組合物之方法，其包括使該組合物與患者之血流接觸。 在一些實施例中，本發明提供一種製備本文所描述之組合物之方法，其包括製備鹼溶液、聚合物溶液及金屬鹵化物溶液；混合該鹼溶液、聚合物溶液及金屬鹵化物溶液以形成混合物；使該混合物與氧化劑接觸。 在此等實施例中之一些中，方法包括使用限制衝擊噴流混合器或多入口旋渦混合器以藉由快速奈米沈澱來製備組合物。 在一些實施例中，方法包括藉由冷凍乾燥、噴霧乾燥或真空濃縮來乾燥組合物。診斷方法
在一些實施例中，本發明提供輔助診斷存在類澱粉蛋白斑之疾病之方法，該疾病諸如阿兹海默症、癡呆、帕金森症及路易體症。在一些實施例中，方法包括向患者投與本發明之組合物。在一些實施例中，方法進一步包括使本發明之組合物與類澱粉蛋白斑結合或錯合。在一些實施例中，方法進一步包括使用MRI造影結合於本發明之組合物之類澱粉蛋白斑。在一些實施例中，方法進一步包括將患者診斷為患有或可能患有存在類澱粉蛋白斑之疾病，諸如阿兹海默症、癡呆、帕金森症及路易體症。在一些實施例中，診斷涉及針對指示存在類澱粉蛋白斑之對比來分析MRI圖像。在一些實施例中，藉由本發明之組合物在MRI圖像中誘導對比。在某些實施例中，診斷係針對本文所述之疾病之早期。患者群體
本文所描述之組合物及奈米顆粒適用於診斷各種疾病。在某些實施例中，本文所描述之組合物及奈米顆粒適用於診斷年齡為以下之個人：至少40歲；或至少45歲；或至少50歲；或至少55歲；或至少60歲；或至少65歲；或至少70歲；或至少75歲；或至少80歲；或至少85歲；或至少90歲；或至少95歲；或至少100歲。 習知診斷方法可用於補充本發明之方法。投藥
本文所描述之組合物、試劑及奈米顆粒亦可非經腸投與。此等活性化合物之溶液或懸浮液可在適當地與諸如羥基丙基纖維素之界面活性劑混合之水中製備。亦可在甘油、液體聚乙二醇及其於油中之混合物中製備分散液。在普通儲存及使用條件下，此等製劑含有防腐劑以防止微生物生長。 適合於可注射用途之醫藥形式包括無菌水溶液或分散液及用於臨時製備無菌可注射溶液或分散液之無菌粉末。在所有情況下，形式必須係無菌的且必須係達到可易於注射之程度之流體。其必須在製造及儲存條件下穩定，且必須保護其免受微生物(諸如細菌及真菌)之污染作用。載劑可為含有例如水、乙醇、多元醇(例如甘油、丙二醇及液體聚乙二醇)、其適合混合物及植物油之溶劑或分散液介質。 對於成年人，本發明之組合物單次投與之劑量在約0.5至約2 g範圍內。在一些針對成年人之實施例中，可使用較大或較小劑量，且可使用多次投與。確切劑量視投藥模式、涉及的組合物、所需診斷、投藥形式、待治療個體及待治療個體之體重、及主治醫師或獸醫之優先選擇及經驗而定。 通常，本發明之組合物可以每單位劑量包括較佳約1至500 mg活性成分及醫藥學上可接受之載劑之單位劑型分配。針對儲存及用途，此等製劑較佳含有防腐劑以防止微生物之生長。 提供一單位劑量之本文所述組合物的套組涵蓋於本發明內。提供許多單位劑量之本文所述組合物的套組涵蓋於本發明內。再進一步，提供若干單位劑量之本文所述組合物的套組涵蓋於本發明內。在一些實施例中，本發明之套組包括一單位劑量之本文所述之醫藥組合物。在某些實施例中，本發明之套組包括許多單位劑量之本文所述之醫藥組合物。在某些其他實施例中，本發明之套組包括一單位劑量之本文所述之醫藥組合物。 可藉由此項技術中已知之任何手段，諸如經口或經直腸、非經腸、腹膜內、靜脈內、皮下、真皮下、鼻內或肌內，投與適量候選化合物。 適合於經口投與之調配物可由以下組成：(a)液體溶液，諸如有效量之經封裝核酸懸浮於稀釋劑(諸如水、鹽水或PEG 400)中；(b)膠囊、藥包(sachets)或錠劑，各含有預定量之活性成分，呈液體、固體、顆粒或明膠；(c)在適當液體中之懸浮液；及(d)適合乳液。錠劑形式可包括以下中之一或多者：乳糖、蔗糖、甘露醇、山梨醇、磷酸鈣、玉米澱粉、馬鈴薯澱粉、微晶纖維素、明膠、膠態二氧化矽、滑石、硬脂酸鎂、硬脂酸及其他賦形劑、著色劑、填充劑、結合劑、稀釋劑、緩衝劑、濕潤劑、防腐劑、調味劑、染料、崩解劑及醫藥學上相容之載劑。 可自上述種類之無菌散劑、顆粒及錠劑製備注射溶液及懸浮液。適用於非經腸投與，諸如藉由關節內(關節中)、靜脈內、肌內、皮內、腹膜內及皮下途徑投與之調配物包括可含有抗氧化劑、緩衝劑、抑菌劑及使該調配物與預定接受者血液等張之溶質的水性及非水性等張無菌注射溶液，及可包括懸浮劑、增溶劑、增稠劑、穩定劑及防腐劑之水性及非水性無菌懸浮液。 本發明之組合物可與其他亦適用於治療、預防、抑制神經或心理病症之藥物組合使用。此類其他藥物可藉由其對應之常用途徑且以其對應之常用量來與本發明之化合物同時或相繼投與。當本發明之化合物與一或多種其他藥物同時使用時，含有此類其他藥物及該化合物之呈單位劑型之醫藥組合物係較佳的。當與一或多種其他活性成分組合使用時，可以比各自單獨使用時更低之劑量使用本發明之化合物及其他活性成分。 在用於經口、舌下、皮下、肌內、靜脈內、局部或經直腸投與之本發明之醫藥組合物中，活性成分本身，或與另一活性成分結合，可以混合有習知醫藥載劑之單位投藥形式投與動物及人類。適當之單位投藥形式包括經口形式，諸如，待經口獲取之錠劑、明膠、膠囊、散劑、顆粒及溶液或懸浮液；舌下及頰內投藥形式；氣霧劑、植入物、皮下、肌內、靜脈內、鼻內或眼內投藥形式；及經直腸投藥形式。實例 實例 1 - 結合薑黃素之氧化鐵奈米顆粒之合成及特徵化
藉由氧化-沈澱原理製造氧化鐵奈米顆粒。亦採用以下技術：Tada M, Hatanaka S, Sanbonsugi H, Matsushita N, Abe M. Method for synthesizing ferrite nanoparticles ~ 30 nm in diameter on neutral pH condition for biomedical applications, J. Appl. Phys. 2003;93(10):7566-7568；及Konwarh R, Saikia JP, Karak N, Konwar BK. 'Poly(ethylene glycol)-magnetic nanoparticles-curcumin' trio: Directed morphogenesis and synergistic free-radical scavenging. Colloids and Surfaces B: Biointerfaces 2010 12/1;81(2):578-586。 將KOH (1 mol/L)之鹼溶液與2%聚乙二醇(PEG)溶液之混合物逐滴添加至FeCl2
溶液中直至pH達到7.8。在持續攪拌下，添加過氧化氫以產生暗黑沈澱。將最終氧化鐵產物純化，用去離子水洗滌且隨後用丙酮洗滌，且乾燥。觀測到氧化鐵之粒度在10至30 nm範圍內。觀測到顆粒由PEG分離。圖1顯示PEG負載之氧化鐵之透射電子顯微鏡(TEM)圖像。 基於吸附等溫線研究，薑黃素以每公克氧化鐵75 mg薑黃素之所計算最大負載優先結合氧化鐵表面。圖2與圖3顯示吸附等溫線與朗謬模型，其指示在氧化鐵表面上薑黃素形成一個單層且結合在均質位點處。 在大功率音波處理下，部分地使用來自以上參考文獻及亦Zhang G, Guo B, Wu H, Tang T, Zhang BT, Zheng L, He Y, Yang Z, Pan X, Chow H, To K, Li Y, Li D, Wang X, Wang Y, Lee K, Hou Z, Dong N, Li G, Leung K, Hung L, He F, Zhang L, Qin L, A delivery system targeting bone formation surfaces to facilitate RNAi-based anabolic therapy, Nat Med 2012;18(2):307-314之技術來製備結合薑黃素之氧化鐵。在超音波處理下，在二甲基甲醯胺中將薑黃素溶液與預分散氧化鐵混合8分鐘。懸浮液經真空濃縮器進一步乾燥。圖4顯示結合薑黃素之PEG負載之氧化鐵奈米顆粒。 實例 2 - 藉由快速奈米沈澱 ( FNP ) 方法囊封結合 薑黃素之氧化鐵
結合薑黃素之氧化鐵囊封於聚合物微胞內部。PEG-PLA共嵌段聚合物溶解於有機相中而結合薑黃素之氧化鐵分散於水相中。兩相皆共注入多入口旋渦混合器中。PEG-PLA共嵌段聚合物之PLA尾端傾向於黏附於經薑黃素塗佈之表面且以此形成微胞結構。由於在快速混合期間產生高能量，因此將奈米顆粒分離且防止聚集。為增強儲存穩定性，向溶液添加聚乙烯吡咯啶酮(PVP)或聚乙烯醇(PVA)。溶劑移除及乾燥製程
藉由透析或離心過濾除去殘餘在奈米顆粒懸浮液中之溶劑。藉由添加低溫保護劑諸如蔗糖、甘露醇、β環糊精或葡萄糖且隨後與奈米懸浮液共冷凍用於冷凍乾燥來實現乾燥。經乾燥奈米顆粒可無限期儲存且隨後再懸浮於水溶液中。為增強奈米顆粒之血腦屏障滲透能力，在使用之前將聚山梨醇酯80或甘露醇添加至經復原溶液中。相關技術及材料可見於以下:Ren T, Xu N, Cao C, Yuan W, Yu X, Chen J等人 Preparation and Therapeutic Efficacy of Polysorbate-80-Coated Amphotericin B/PLA-b-PEG Nanoparticles. Journal of Biomaterials Science, Polymer Edition 2009;20(10):1369-1380；及Sun W, Xie C, Wang H, Hu Y. Specific role of polysorbate 80 coating on the targeting of nanoparticles to the brain. Biomaterials 2004 7;25(15):3065-3071。與現有商用技術之比較
當前藉由偵測類澱粉β蛋白斑診斷AD之方法包括藉由正電子發射斷層攝影法(PET)之放射性染料造影及藉由磁振造影(MRI)之超順磁奈米顆粒造影。本發明優於現有可商購之技術(例如Eli Lilly之PET產品(Amyvid™))，之一些優點呈現於表1中。
表1 用於診斷阿兹海默症之不同類澱粉蛋白斑造影方法之比較 本文描述結合薑黃素之磁性奈米顆粒之調配。鐵核之大小＜30 nm，且最終流體動力粒度＜300 nm。此等顆粒已針對類澱粉蛋白斑結合能力及血腦屏障滲透在活體外及活體內經測試，且針對MRI在活體內經測試。圖5顯示施用至APP(類澱粉前驅蛋白)轉殖基因小鼠大腦之連續切片之硫代黃素T、薑黃素及結合薑黃素之氧化鐵奈米顆粒的共焦螢光及相位對比圖像。全部三種反應劑結合類澱粉蛋白斑。 實例 3 - 藉由 結合薑黃素之磁性奈米顆粒之血腦屏障之滲透
已進行活體內測試以評估結合薑黃素之氧化鐵奈米顆粒滲透血腦屏障之能力。將奈米顆粒注入APP轉殖基因小鼠之靜脈中。在注射五小時之後，處死小鼠且收集大腦用於藉由普魯士藍反應進行鐵組織化學染色。用酸化之亞鐡氰化物處理大腦切片，且任何痕量之鐵(Fe+ 3
)變成亮藍色。圖6顯示在小鼠大腦切片中之氧化鐵顆粒。 由於薑黃素在UV光下呈現螢光，因此為確認薑黃素保持與氧化鐵結合，將在其中發現氧化鐵之切片藉由螢光顯微鏡法進一步檢測。圖7顯示在亮光及UV光下觀測之切片。可自螢光圖像鑑定出痕量之薑黃素。此等圖像指示結合薑黃素之氧化鐵可滲透轉殖基因小鼠之血腦屏障，其中薑黃素保持結合於氧化鐵表面。 實例 4 - 藉由結合薑黃素之磁性奈米顆粒靶向之類澱粉蛋白斑之磁振造影及免疫化學
亦對結合薑黃素之氧化鐵奈米顆粒進行針對MR造影及疾病特異性之活體內測試。對APP轉殖基因小鼠及非轉殖基因同窩對照給予相同劑量之奈米顆粒懸浮液之靜脈內注射。在注射5小時之後收集其大腦且固定在瓊脂糖凝膠中用於離體MRI。圖8顯示在APP小鼠大腦之MRI中存在許多黑點(上部圖像)但在對照大腦中並沒有(底部圖像)。因此鐵-薑黃素奈米顆粒僅特異性留存且經MRI顯現在AD小鼠之大腦而非對照小鼠中。 圖9顯示注射有結合薑黃素之磁性奈米顆粒之APP轉殖基因小鼠的典型大腦切片。紅色斑點指示經抗Aβ肽之4G8及6E10單株抗體之混合物染色的類澱粉蛋白斑，且藍色指示氧化鐵。放大視圖表明氧化鐵緊密貼近類澱粉蛋白斑。 圖10顯示匹配之MRI(左)及雙染色之APP轉殖基因小鼠大腦切片(右)。在MRI中發現之許多暗點與免疫標記之類澱粉蛋白斑(紅色)及氧化鐵(藍色)共定位。插圖係所選區域之40倍放大，顯示在亮視野圖像(左)中之類澱粉蛋白斑(紅色)及鐵(藍色)以及在螢光圖像(右)中之蛋白斑(橙色)。螢光係橙色的，因為薑黃素在520 nm下發射，而用於免疫標記蛋白斑之螢光標記在570 nm下發射。此等黃色及紅色之發射信號之組合產生亮橙色。圖像顯示結合薑黃素之磁性奈米顆粒能夠結合類澱粉蛋白斑且可經MRI或免疫組織化學顯現。 圖11顯示注射結合薑黃素之磁性奈米顆粒後相同年齡之非轉殖基因對照小鼠之切片。MRI未偵測到氧化鐵信號(左)。如所預期，在匹配之組織學切片上未偵測到類澱粉蛋白斑(紅色)或鐵(藍色)(右)。 針對薑黃素、氧化鐵及類澱粉蛋白斑之共定位，觀測注射有結合薑黃素之磁性奈米顆粒之APP轉殖基因小鼠的大腦切片(圖12)。在插圖中，亮視圖(左)顯示以藍色顯示之氧化鐵，且螢光視圖(右)顯示以紅色顯示之類澱粉蛋白斑及以黃色(或因類澱粉蛋白斑及薑黃素共定位而顯示之橙色)顯示之薑黃素。 實例 5 - 作為診斷劑之結合薑黃素之磁性奈米顆粒
為判定結合薑黃素之磁性奈米顆粒是否可用作AD診斷劑，需要關聯免疫組織化學與活體內MRI之結果。基於先前結果，活體內圖像可良好預測實際Aβ蛋白斑位置(示出於免疫組織化學中)。隨機選擇十個來自三個注射有結合薑黃素之磁性奈米顆粒之轉殖基因小鼠的活體內MRI大腦切片，且由不知情的操作員分析其匹配之免疫組織化學切片。圖13顯示MRI中之斑點密度對免疫組織化學中之Aβ密度之線性回歸。其有很強的相關性(皮爾森相關性(Pearson's correlation)，p=0.002)，且R2
值係0.72。結果顯示注射有結合薑黃素之磁性奈米顆粒之Tg小鼠的活體內MRI圖像係免疫組織化學中所發現之Aβ蛋白斑的良好預測子。因此，結合薑黃素之磁性奈米顆粒可在臨床上用於Aβ蛋白斑之診斷造影。 儘管出於清楚理解之目的已藉助於說明及實例相當詳細地描述了前述發明，但熟習此項技術者將瞭解，可在所附申請專利範圍之範疇內實踐某些改變及修改。此外，本文所引用之各參考文獻以全文引用之方式併入本文中，其併入程度如同與各參考文獻單獨地以引用之方式併入之程度相同。當本申請案與本文所提供之參考文獻之間存在衝突時，應以本申請案為準。 General purpose
The present invention provides compositions and methods for the diagnosis of diseases involving amyloid plaques for use with MRI, such as Alzheimer's disease, dementia, Parkinson's disease, and Lewy body disease (Lewy) Body disease). In part, the present invention is directed to a novel nanoparticle that can cross the blood-brain barrier and bind to amyloid plaques and other similar protein aggregates such that it can be detected by magnetic resonance imaging (MRI). The compositions, techniques, and methods described herein also provide a non-invasive means of early diagnosis of Alzheimer's disease.definition
As used herein, the term "superparamagnetic" refers to the form of magnetism that is present, for example, in small ferromagnetic or subferromagnetic nanoparticles. In sufficiently small nanoparticles, the magnetization can be arbitrarily switched in direction under the influence of temperature. The typical time between conversions is called the Néel relaxation time. In the absence of an external magnetic field, when the magnetization for measuring the nanoparticle is much longer than the Neil relaxation time, its magnetization appears to be zero on average. As such, it is said to be in a superparamagnetic state. In this state, the external magnetic field can magnetize the nanoparticle similar to the paramagnetic body. However, its magnetic susceptibility is much larger than that of a paramagnetic body. As used herein, the term "nanoparticle" refers to a particle having a physical size of less than about 100 nanometers and greater than about 0.1 nanometer. As used herein, the term "binding agent" refers to an agent, composition or compound that is capable of binding or associating or mismatching to an amyloid plaque. As used herein, the phrase "amyloid plaques" refers to insoluble fibrin aggregates that share specific structural characteristics. Spots can also be called sediments. These folded anomalous structures change their proper configuration such that they erroneously interact with each other or with other cellular components to form insoluble fibrils. It has been associated with more than 20 diseases of severe human disease. Abnormal accumulation of amylopectin-like fibers in organs can lead to amyloidosis and can play a role in various neurodegenerative disorders. As used herein, the phrase "protein aggregate" refers to a product of protein aggregation, a biological phenomenon in which a protein-aggregated abnormal protein aggregates, accumulates, and/or aggregates intracellularly or extracellularly. Such protein aggregates are generally toxic and have been implicated in a variety of diseases known to be amyloidosis, including Alzheimer's disease, Parkinson's disease, and prion diseases. As used herein, the term "indication" means a condition that is characteristic of a disease or that indicates a certain disease. For example, amyloid plaques indicate Alzheimer's disease as it is often associated with this disease. A patient with an amyloid plaque may have Alzheimer's disease, and thus such a plaque may, for example, indicate to a physician whether the patient has Alzheimer's condition. As used herein, the term "hydrophilic" refers to a chemical group that has the tendency to repel non-polar or non-electrochemical groups (eg, hexane) and to attract polar or electrochemical groups (eg, water). "Hydrophilic" also refers to chemicals that tend to dissolve in water, mix with water, or be wetted by water. As used herein, the term "hydrophobic" refers to a chemical group that has a tendency to attract non-polar or non-electrochemical groups (eg, hexane) and to repel polarity or to have electrochemical groups (eg, water). "Hydrophobic" also refers to chemicals that tend not to dissolve in water, do not mix with or are not wetted by water. As used herein, the phrase "hydrodynamic particle size" refers to the smallest diameter of a hypothetical three-dimensional sphere that can encapsulate the particles of the present invention. As used herein, the term "biodegradable" refers to the ability of a composition to decompose by the action of a living organism, specifically decomposing into a harmless product. As used herein, the term "amphiphilic" is used to describe a chemical compound as having hydrophilic and lipophilic hydrophobic properties. As used herein, the term "copolymer" refers to a polymer derived from two (or more) monomer species relative to a homopolymer using only one monomer. For example, assuming the monomer species A and B, the alternating copolymer may have the form -A-B-A-B-A-B-A-B-A-B. For example, assuming the monomer species A and B, the random copolymer may have the form -A-A-B-A-B-B-A-B-A-A-A-B-B-B-B-A. For example, assuming the monomer species A and B, the block copolymer may have the form -(A-A-A)-(B-B-B)-(A-A-A)-(B-B-B)-(A-A-A)-.combination
In some embodiments, the present invention provides a magnetic resonance imaging (MRI) diagnostic agent composition comprising at least one binding agent of superparamagnetic nanoparticle, a polymer, and an amyloid plaque. In some of these embodiments, the bonding agent is attached to the surface of the nanoparticle. In some of these embodiments, the polymer coats the nanoparticles having a binder attached to the surface. In some embodiments, the nanoparticles described herein have a first layer comprising a polymer adhered to the surface of the nanoparticle and coated with the surface of the nanoparticle. In some embodiments, the nanoparticles described herein have a second layer comprising at least one binding agent for amyloid plaques. In some embodiments, the amyloid plaque binding agent is attached to the surface of the nanoparticle. In some embodiments, the amyloid plaque binding agent is attached to the surface of the nanoparticle that has not been bonded to the polymer. In some embodiments, an amyloid plaque binding agent attached to the surface of the nanoparticle is capable of binding to an amyloid plaque. In certain embodiments, the nanoparticle comprises a core comprising a superparamagnetic material. In some embodiments, the binding agent of the polymer and the amyloid plaque is adhered or bonded to the surface of the superparamagnetic nanoparticle. In some embodiments, the binding agent of the polymer and the amyloid-like plaque can be closely associated on the surface of the nanoparticle. In some embodiments, although both the polymer and the amyloid plaque binding agent are attached to the surface of the nanoparticle, the amyloid plaque binding agent extends out of the surface of the polymer. In still other embodiments, both the polymer and the amyloid plaque binding agent are bonded to the surface of the nanoparticle and the amyloid plaque binding agent is sufficiently exposed to be capable of binding to the amyloid plaque. In some embodiments, the present invention provides a magnetic resonance imaging (MRI) diagnostic composition comprising: at least one binding agent of superparamagnetic nanoparticle, a polymer, and an amyloid-like plaque; wherein the binding agent is attached to the naphthalene a surface of the rice granule; and wherein the polymer coats the nanoparticle having the binding agent attached to the surface. In some embodiments, both the polymer and the binder are attached to the surface of the nanoparticle. In certain embodiments, the polymer and binder are closely associated on the surface of the nanoparticle. In other embodiments, the polymer coats the surface of the binder and the nanoparticles. In some embodiments, the polymer coats the surface of the nanoparticle but does not prevent binding of the binding agent attached to the surface of the nanoparticle to the amyloid plaque. In some embodiments, the invention provides a binding agent for a family of amyloid plaques. In other embodiments, the invention provides a binding agent for a binder-based starch deposit. In certain embodiments, the amyloid plaques are selected from the group consisting of protein aggregates, A-beta aggregates, and synuclein. In some embodiments, the amyloid plaques are indicative of a disease selected from the group consisting of Alzheimer's disease, dementia, Parkinson's disease, and Lewy body disease. In some embodiments, the invention provides amyloid plaques capable of forming chemical bonds with curcumin. In some embodiments, the invention provides that the nanoparticles comprise iron. In certain embodiments, the invention provides that the nanoparticles comprise a magnet. In other embodiments, the invention provides that the nanoparticles comprise a metal selected from the group consisting of iron, gold, platinum, silver, and cobalt. In still other embodiments, the present invention provides nanoparticle comprising a semiconductor selected from the group consisting of cadmium selenide, cadmium sulfide, lead selenide, lead sulfide, zinc selenide, zinc sulfide, and combinations thereof. In some embodiments, the nanoparticle comprises iron oxide. In some embodiments, the invention provides nanoparticles wherein the nanoparticles are less than 30 nm in size. In certain embodiments, the composition has a hydrodynamic particle size of less than 300 nm. In some embodiments, the hydrodynamic particle size is from about 10 nm to about 300 nm. In some embodiments, the invention provides compositions wherein the binding agent is a bioflavonoid or dye. In some such embodiments, the binding agent is selected from the group consisting of curcumin, Congo red, thioflavin T/S, quercetin, epicatechin, hesperidin, rutin, and Red tangerine flavones. In certain embodiments, the dye is curcumin. In some embodiments, the present invention provides a polymer-based hydrophilic composition. In some embodiments, the hydrophilic polymer is selected from the group consisting of polyethylene glycol (PEG), polydextrose, polyvinylpyrrolidone (PVP), fatty acids, polyvinyl alcohol (PVA), polyacrylic acid, Polypeptide, phosphorylcholine, poly(D,L-lactide), poly(N-isopropylacrylamide) (PolyNIPAAM), chitosan, gelatin, polylactic acid-co-glycolic acid (PLGA), Polycaprolactone (PCL) and polybutyl cyanoacrylate (PBCA). In some such embodiments, the hydrophilic polymer is polyethylene glycol (PEG). In some embodiments, the invention provides compositions further comprising a biodegradable amphiphilic copolymer. In some embodiments, the biodegradable amphiphilic copolymer is selected from the group consisting of polylactic acid-co-glycolic acid (PLGA), polycaprolactone (PCL), polylactic acid (PLA), polyethyl b. Glycol (PEG), methoxypolyethylene glycol (MePEG), polyethylene oxide (PEO), polybutadiene (PBD), d-α-tocopherol polyethylene glycol 1000 succinate, PEG- PLA, PEG-PCL, PEG-PLGA, MePEG-PLA, MePEG-PCL, MePEG-PLGA, PEO-PBD and Vitamin E TPGS. In some embodiments, the invention provides compositions further comprising a hydrophilic polymeric stabilizer. In some embodiments, the hydrophilic polymeric stabilizer is selected from the group consisting of polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyethylene glycol (PEG), hydroxypropyl methyl fiber (HPMC), and Poloxamer, polylactic acid-co-glycolic acid (PLGA), polycaprolactone (PCL), polylactic acid (PLA), polybutyl cyanoacrylate (PBCA) and several Butanose. In some embodiments, the invention provides a composition further comprising a cationic surfactant selected from the group consisting of benzalkonium chloride, benzethonium chloride, and cetrimide. In some embodiments, the invention provides a composition further comprising an anionic surfactant selected from the group consisting of docusate sodium and sodium lauryl sulfate. In some embodiments, the present invention provides a composition further comprising a nonionic surfactant selected from the group consisting of glycerol monooleate, sorbitan ester, polyoxyethylene sorbitan fatty acid ester, and Polyoxyethylene alkyl ether. In some embodiments, the nonionic surfactant is selected from the group consisting of sorbitan esters: sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate Ester, sorbitan sesquioleate and sorbitan trioleate. In some embodiments, the nonionic surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters: polysorbate 20, polysorbate 40, polysorbate 60, polysorbate Alcohol ester 65, polysorbate 80 and polysorbate 85. In some other embodiments, the nonionic surfactant is selected from the group consisting of polyoxyethylene alkyl ethers: polyethylene glycol monohexadecyl ether, polyethylene glycol monolauryl ether, polyethylene glycol Monooleyl ether and polyethylene glycol monostearyl ether. In some embodiments, the poloxamer is selected from the group consisting of P124, P188, P237, P338, and P407. In some embodiments, the invention provides a composition of nanoparticle iron oxide and a dye curcumin. In some embodiments, the present invention provides a composition having a calculated maximum load of 75 mg of curcumin per gram of iron oxide. In some embodiments, the present invention provides a composition having a calculated minimum load of 5 mg of curcumin per gram of iron oxide.Pharmaceutical composition
The pharmaceutical compositions of the present invention encompass compositions prepared by blending a compound, nanoparticle, or composition of the present invention with a pharmaceutically acceptable carrier and/or excipient or diluent. Such compositions are suitable for pharmaceutical use in animals or humans. The pharmaceutical composition of the present invention comprises, as an active ingredient, a compound, a nanoparticle or composition described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and/or excipient or dilution. Agent. The pharmaceutical composition may optionally contain other therapeutic ingredients. The compounds of the present invention can be combined as an active ingredient in a homogeneous blend with suitable pharmaceutical carriers and/or excipients according to conventional pharmaceutical compounding techniques. Any carrier and/or excipient suitable for the preparation form desired for administration may be employed with the compounds disclosed herein. The compositions can be prepared by any of the methods well known in the art of pharmacy. Compositions include compositions suitable for topical, parenteral, pulmonary, nasal, rectal or oral administration. The most appropriate route of administration in any given case will depend, in part, on the nature and severity of the condition being diagnosed. Other preferred compositions include those suitable for systemic (enteral or parenteral) administration. Systemic administration includes oral, transrectal, sublingual or transmucosal administration. The composition can be administered by subcutaneous, intravenous, intramuscular or intraperitoneal injection (e.g., via a syringe). Compositions for pulmonary administration include, but are not limited to, dry powder compositions consisting of a powder of a compound or a salt thereof described herein and a powder suitable for the carrier and/or lubricant. Compositions for pulmonary administration can be inhaled from any suitable dry powder inhaler device known to those skilled in the art. Compositions for systemic administration include, but are not limited to, dry powder compositions consisting of a composition as described herein and a powder suitable for carriers and/or excipients. Compositions for systemic administration can be presented by, but not limited to, the following forms: lozenges, capsules, pills, syrups, solutions, and suspensions. In some embodiments, the invention provides compositions further comprising a pharmaceutical surfactant. In some embodiments, the invention provides compositions further comprising a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of glucose, sucrose, trehalose, lactose, sodium glutamate, PVP, HPβCD, CD, glycerol, maltose, mannitol, and saccharose. ). In some embodiments, the invention provides a pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable excipient. In some of these embodiments, pharmaceutically acceptable excipients include salts or diluents. In some embodiments, the invention provides a composition comprising an effective amount of the composition of claim 1. In some embodiments, the composition is formulated for oral administration or intravenous administration and comprises a composition as in claim 1 and at least one member selected from the group consisting of aqueous solutions and buffer solutions.Method for preparing superparamagnetic nanoparticle
Nanoparticles of the invention can be prepared by the methods described herein. For example, iron oxide nanoparticle combined with curcumin can be prepared by the following procedure. In some embodiments, the iron oxide nanoparticles can be prepared by the oxidation-precipitation principle, for example, Tada M, Hatanaka S, Sanbonsugi H, Matsushita N, Abe M. Method for synthesizing ferrite nanoparticles ~ 30 nm in diameter on neutral pH condition for biomedical applications, J. Appl. Phys. 2003;93(10):7566-7568; and Konwarh R, Saikia JP, Karak N, Konwar BK. 'Poly(ethylene glycol)-magnetic nanoparticles-curcumin' trio: Directed morphogenesis and synergistic free-radical scavenging. Colloids and Surfaces B: Biointerfaces 2010 12/1; 81(2): 578-586. In some embodiments, a mixture of an alkali solution (eg, KOH (1 mol/L)) and 2% polyethylene glycol (PEG) is added dropwise to the metal halide (eg, FeCl)2
The solution is until the pH reaches about 7.8. Hydrogen peroxide was added to produce a dark precipitate with continuous stirring. The product was purified, washed with deionized water and then washed with acetone and dried. In some embodiments, the iron oxide nanoparticles can be described by, for example, Aono H, Hirazawa H, Naohara T, Maehara T, Kikkawa H, Watanabe Y. Synthesis of fine magnetite powder using reverse coprecipitation method and its heating properties by applying AC Magnetic field. Materials Research Bulletin 2005; 40(7): Reverse coprecipitation in 1126-1135 for synthesis. In some embodiments, an alkaline solution (eg, NaOH dissolved in deoxygenated water) is added dropwise to the iron solution (eg, at 2 mol Fe)3 +
:1 mol Fe2 +
Ratio of FeCl dissolved in deoxygenated water3
·6H2
O and FeSO4
·7H2
In O), stir at the same time. A dark precipitate formed during the addition of the alkaline solution. The mixture was kept stirring overnight, after which the precipitate was washed with water, resuspended in dimethylformamide and stored at 4 °C. The methods described herein are suitable for the preparation of iron oxide nanoparticles having a size of about 10 to 30 nm. The methods described herein are suitable for the preparation of nanoparticle loaded with curcumin at a maximum loading of about 75 mg per gram of iron oxide. The maximum load herein is defined as the loading of curcumin to form a single layer surface on the nanoparticles. In some embodiments, the maximum loading of curcumin is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 per gram of iron oxide. , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 , 68, 69, 70, 71, 72, 73, 74 or 75 mg. In other embodiments, the maximum loading of curcumin is 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 per gram of iron oxide. 93, 94, 95, 96, 97, 98, 99 or 100 mg. The loading of curcumin on the nanoparticles can also be defined by the molar ratio of curcumin to iron oxide. In some embodiments, the nanoparticle is loaded with curcumin at a molar ratio of curcumin to iron oxide in the range of from about 0.05:1 to about 1:1. In some embodiments, the nanoparticles are in the range of from about 0.05:1 to about 0.5:1, in the range of from about 0.3:1 to about 0.75:1, and in the range of from about 0.55:1 to about 1:1. In the range of from about 0.05:1 to about 0.25:1, in the range of from about 0.2:1 to about 0.4:1, from about 0.35:1 to about 0.55:1, from about 0.5:1 to about 0.7:1, from about 0.65:1 to About 0.85:1, or about 0.8:1 to about 1:1, the curcumin is loaded with curcumin on the molar ratio of iron oxide. In some embodiments, the curcumin-loaded nanoparticle of curcumin with about 0.4:1 of curcumin maintains its size during subsequent dialysis removal of the solvent. In some embodiments, nanoparticle is prepared using high power sonication. In some embodiments, the nanoparticles are dispersed in dimethylformamide under ultrasonic treatment for a set period of time (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes) . In some embodiments, the suspension is further dried by a vacuum concentrator. In some embodiments, the nanoparticles are encapsulated in a polymer micelle. In some embodiments, the PEG-PLA co-block polymer is dissolved in the organic phase and the nanoparticles are dispersed in the aqueous phase. In other embodiments, both the PEG-PLA block copolymer and the curcumin-bound nanoparticle are provided in the organic phase while also providing an aqueous phase. In certain embodiments, both phases are co-injected into a multi-entry vortex mixer. In some embodiments, the nanoparticles are produced with high energy during rapid agitation. In certain embodiments, the nanoparticles are separated and prevented from aggregating. In some embodiments, polyvinylpyrrolidone (PVP) or polyvinyl alcohol (PVA) is added to the solution. In some embodiments, the PVP or PVA is dissolved in an aqueous layer that is co-injected into the vortex mixer with the organic phase. In some embodiments, the solvent remaining in the nanoparticle suspension is removed by dialysis or centrifugal filtration. In certain embodiments, drying is achieved by the addition of a cryoprotectant, such as sucrose, mannitol, beta cyclodextrin or glucose, and then co-freezing with the nanosuspension for freeze drying. In some embodiments, the method includes storing the dried nanoparticle. In some embodiments, after storage of the nanoparticles, polysorbate 80 or mannitol is added to the solution of nanoparticles to restore it. In some embodiments, the invention provides a method of labeling amyloid plaques for MRI detection comprising contacting a composition described herein with an amyloid-like plaque, thereby labeling amyloid plaques for MRI detection Measurement. In some of these embodiments, the amyloid plaques are selected from the group consisting of protein aggregates, A-beta aggregates, and synuclein. In certain embodiments, the amyloid plaques are indicative of a disease selected from the group consisting of Alzheimer's disease, dementia, Parkinson's disease, and Lewy body disease. In some embodiments, the invention provides a method of diagnosing a disease or condition in a patient, comprising administering to the patient a composition described herein; labeling an amyloid plaque such as a patient with the composition; MRI images of the patient of the composition; analyzing the images to detect amyloid plaques in the patient; and thereby diagnosing the disease or condition of the patient. In some embodiments, the present invention provides a method wherein acquiring an MRI image comprises operating an MRI instrument with the following MRI parameters: T2* - FLASH (2D); TE = 25 ms, TR = 400 ms, FA = 200
;FOV=2×2, MTX = 400×400; in-plane resolution = 50 μm2
; slice thickness = 0.5 mm; and NEX = 80, scan time = 3.5 hours. In some embodiments, the invention provides a method of administering a composition described herein comprising contacting the composition with bloodstreams of a patient. In some embodiments, the present invention provides a method of preparing a composition described herein, comprising preparing an alkali solution, a polymer solution, and a metal halide solution; mixing the alkali solution, the polymer solution, and the metal halide solution to form a mixture; contacting the mixture with an oxidizing agent. In some of these embodiments, the method includes using a limited impact jet mixer or a multi-entry vortex mixer to prepare the composition by rapid nanoprecipitation. In some embodiments, the method comprises drying the composition by freeze drying, spray drying, or vacuum concentration.diagnosis method
In some embodiments, the invention provides methods of assisting in the diagnosis of a disease in the presence of amyloid plaques, such as Alzheimer's disease, dementia, Parkinson's disease, and Lewy body disease. In some embodiments, the method comprises administering to the patient a composition of the invention. In some embodiments, the method further comprises combining or mismatching the composition of the invention with an amyloid-like plaque. In some embodiments, the method further comprises binding to an amyloid plaque such as a composition of the invention using MRI contrast. In some embodiments, the method further comprises diagnosing the patient as having or likely having a disease in the presence of amyloid plaques, such as Alzheimer's disease, dementia, Parkinson's disease, and Lewy body disease. In some embodiments, the diagnosis involves analyzing the MRI image for a comparison indicating the presence of amyloid plaques. In some embodiments, the contrast is induced in an MRI image by the composition of the invention. In certain embodiments, the diagnosis is directed to the early stages of the disease described herein.Patient population
The compositions and nanoparticles described herein are useful for diagnosing various diseases. In certain embodiments, the compositions and nanoparticles described herein are suitable for use in diagnosing individuals under the age of at least 40 years old; or at least 45 years old; or at least 50 years old; or at least 55 years old; or at least 60 years old; Or at least 65 years old; or at least 70 years old; or at least 75 years old; or at least 80 years old; or at least 85 years old; or at least 90 years old; or at least 95 years old; or at least 100 years old. Conventional diagnostic methods can be used to supplement the methods of the present invention.Dosing
The compositions, agents, and nanoparticles described herein can also be administered parenterally. Solutions or suspensions of such active compounds can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. For adults, the compositions of the present invention are administered in a single dose in the range of from about 0.5 to about 2 g. In some embodiments directed to adults, larger or smaller doses may be used and multiple administrations may be used. The exact dosage will depend on the mode of administration, the composition involved, the desired diagnosis, the form of administration, the weight of the individual to be treated and the individual to be treated, and the preferences and experience of the attending physician or veterinarian. In general, the compositions of the present invention can be dispensed per unit dosage form, including preferably from about 1 to 500 mg of the active ingredient, and a pharmaceutically acceptable carrier. For storage and use, such formulations preferably contain a preservative to prevent the growth of microorganisms. Kits that provide a unit dose of a composition described herein are encompassed by the present invention. Kits that provide a number of unit doses of the compositions described herein are encompassed by the present invention. Still further, kits providing a number of unit doses of the compositions described herein are encompassed by the present invention. In some embodiments, a kit of the invention comprises a unit dose of a pharmaceutical composition described herein. In certain embodiments, the kit of the invention comprises a plurality of unit doses of the pharmaceutical compositions described herein. In certain other embodiments, the kit of the invention comprises a unit dose of a pharmaceutical composition described herein. Appropriate amounts of candidate compounds can be administered by any means known in the art, such as orally or rectally, parenterally, intraperitoneally, intravenously, subcutaneously, subdermally, intranasally or intramuscularly. Formulations suitable for oral administration may consist of (a) a liquid solution such as an effective amount of encapsulated nucleic acid suspended in a diluent such as water, saline or PEG 400; (b) capsules, sachets Or lozenges, each containing a predetermined amount of active ingredient in the form of a liquid, solid, granule or gelatin; (c) a suspension in a suitable liquid; and (d) a suitable emulsion. The tablet form may include one or more of the following: lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal cerium oxide, talc, stearic acid Magnesium, stearic acid and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Injection solutions and suspensions can be prepared from sterile powders, granules and lozenges of the above type. Formulations suitable for parenteral administration, such as by intra-articular (in-articular), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, may include antioxidants, buffers, bacteriostatic agents, and Aqueous and non-aqueous isotonic sterile injectable solutions for solubilizing the formulation with a predetermined recipient's blood, and aqueous and non-aqueous sterile suspensions which may include suspending, solubilizing, thickening, stabilizing and preserving agents . The compositions of the present invention can be used in combination with other drugs which are also suitable for the treatment, prevention, or inhibition of neurological or psychiatric conditions. Such other drugs may be administered simultaneously or sequentially with the compounds of the invention by their corresponding usual routes and in their corresponding usual amounts. When a compound of the present invention is used in combination with one or more other drugs, a pharmaceutical composition containing such other drug and the compound in unit dosage form is preferred. When used in combination with one or more other active ingredients, the compounds of the present invention and other active ingredients may be employed at lower doses than when used alone. In the pharmaceutical composition of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, topical or rectal administration, the active ingredient itself, or in combination with another active ingredient, may be mixed with a conventional medicine. The unit dosage form of the carrier is administered to animals and humans. Suitable unit dosage forms include oral forms such as lozenges, gelatin, capsules, powders, granules and solutions or suspensions to be obtained orally; sublingual and buccal administration forms; aerosols, implants, subcutaneous , intramuscular, intravenous, intranasal or intraocular administration; and transrectal administration.Instance Instance 1 - Synthesis and Characterization of Iron Oxide Nanoparticles Combined with Curcumin
Iron oxide nanoparticles are produced by the oxidation-precipitation principle. The following techniques are also employed: Tada M, Hatanaka S, Sanbonsugi H, Matsushita N, Abe M. Method for synthesizing ferrite nanoparticles ~ 30 nm in diameter on neutral pH condition for biomedical applications, J. Appl. Phys. 2003;93(10) :7566-7568; and Konwarh R, Saikia JP, Karak N, Konwar BK. 'Poly(ethylene glycol)-magnetic nanoparticles-curcumin' trio: Directed morphogenesis and synergistic free-radical scavenging. Colloids and Surfaces B: Biointerfaces 2010 12/ 1; 81 (2): 578-586. Add a mixture of KOH (1 mol/L) base solution and 2% polyethylene glycol (PEG) solution to FeCl dropwise2
In solution until the pH reached 7.8. Hydrogen peroxide was added to produce a dark precipitate with continuous stirring. The final iron oxide product was purified, washed with deionized water and subsequently washed with acetone and dried. The particle size of iron oxide was observed to be in the range of 10 to 30 nm. The particles were observed to be separated by PEG. Figure 1 shows a transmission electron microscope (TEM) image of PEG-loaded iron oxide. Based on the adsorption isotherm study, curcumin preferentially binds to the iron oxide surface with a maximum load calculated per gram of iron oxide 75 mg curcumin. Figures 2 and 3 show an adsorption isotherm and a recitation model indicating that curcumin forms a monolayer on the surface of the iron oxide and binds to the homogenous site. Under high-power sonication, some of the references from the above references are also used by Zhang G, Guo B, Wu H, Tang T, Zhang BT, Zheng L, He Y, Yang Z, Pan X, Chow H, To K, Li Y, Li D, Wang X, Wang Y, Lee K, Hou Z, Dong N, Li G, Leung K, Hung L, He F, Zhang L, Qin L, A delivery system targeting bone formation surfaces to facilitate RNAi-based Anabolic therapy, Nat Med 2012; 18(2): 307-314 to prepare iron oxide combined with curcumin. The curcumin solution was mixed with pre-dispersed iron oxide in dimethylformamide for 8 minutes under ultrasonic treatment. The suspension was further dried by a vacuum concentrator. Figure 4 shows PEG-loaded iron oxide nanoparticles coated with curcumin. Instance 2 - Rapid nanoprecipitation ( FNP ) Method of encapsulation Iron oxide of curcumin
The iron oxide combined with curcumin is encapsulated inside the polymer micelle. The PEG-PLA coblock polymer is dissolved in the organic phase and the iron oxide in combination with curcumin is dispersed in the aqueous phase. Both phases are injected into a multi-entry vortex mixer. The PLA tail of the PEG-PLA co-block polymer tends to adhere to the surface coated with curcumin and thereby form a microcell structure. Since high energy is generated during rapid mixing, the nanoparticles are separated and aggregation is prevented. To enhance storage stability, polyvinylpyrrolidone (PVP) or polyvinyl alcohol (PVA) is added to the solution.Solvent removal and drying process
The solvent remaining in the nanoparticle suspension is removed by dialysis or centrifugal filtration. Drying is achieved by the addition of a cryoprotectant such as sucrose, mannitol, beta cyclodextrin or glucose followed by co-freezing with the nanosuspension for freeze drying. The dried nanoparticle can be stored indefinitely and subsequently resuspended in an aqueous solution. To enhance the blood-brain barrier permeability of the nanoparticles, polysorbate 80 or mannitol is added to the reconstituted solution prior to use. Related technologies and materials can be found in the following: Ren T, Xu N, Cao C, Yuan W, Yu X, Chen J et al. Preparation and Therapeutic Efficacy of Polysorbate-80-Coated Amphotericin B/PLA-b-PEG Nanoparticles. Journal of Biomaterials Science, Polymer Edition 2009; 20(10): 1369-1380; and Sun W, Xie C, Wang H, Hu Y. Specific role of polysorbate 80 coating on the targeting of nanoparticles to the brain. Biomaterials 2004 7;25(15 ): 3065-3071.Comparison with existing commercial technologies
Current methods for diagnosing AD by detecting amyloid-like protein plaques include radioactive dye imaging by positron emission tomography (PET) and superparamagnetic nanoparticle imaging by magnetic resonance imaging (MRI). The present invention is superior to existing commercially available technologies (e.g., Eli Lilly's PET product (AmyvidTM)), some of which are presented in Table 1.
Table 1 Comparison of different types of amyloid plaque assays for the diagnosis of Alzheimer's disease This article describes the formulation of magnetic nanoparticles incorporating curcumin. The size of the iron core is <30 nm and the final hydrodynamic particle size is <300 nm. These particles have been tested in vitro and in vivo for amyloid plaque binding ability and blood-brain barrier penetration, and have been tested in vivo against MRI. Figure 5 shows confocal fluorescence and phase contrast images of thioflavin T, curcumin, and curcumin-bound iron oxide nanoparticles coated into serial sections of APP (plasmid-like precursor protein) transgenic mice. All three reactants bind to amyloid plaques. Instance 3 - By Infiltration of blood-brain barrier combined with curcumin magnetic nanoparticles
In vivo testing has been performed to assess the ability of the iron oxide nanoparticle bound to curcumin to penetrate the blood-brain barrier. Nanoparticles were injected into the veins of APP transgenic mice. Five hours after the injection, the mice were sacrificed and the brain was collected for iron histochemical staining by the Prussian blue reaction. Treatment of brain slices with acidified bismuth cyanide and any traces of iron (Fe+ 3
) turns bright blue. Figure 6 shows iron oxide particles in mouse brain sections. Since curcumin exhibits fluorescence under UV light, in order to confirm that curcumin remains bound to iron oxide, a section of iron oxide is found therein to be further detected by fluorescence microscopy. Figure 7 shows the sections observed under bright light and UV light. Trace amounts of curcumin can be identified from fluorescent images. These images indicate the blood-brain barrier of the iron oxide permeable transgenic mouse in combination with curcumin, wherein curcumin remains bound to the surface of the iron oxide. Instance 4 - Magnetic resonance imaging and immunochemistry of amyloid plaques by binding to curcumin magnetic nanoparticles
In vivo assays for MR imaging and disease specificity were also performed on iron oxide nanoparticles coated with curcumin. Intravenous injection of the same dose of the nanoparticle suspension was administered to the APP transgenic mouse and the non-transgenic gene littermate control. Brains were collected 5 hours after injection and fixed in an agarose gel for ex vivo MRI. Figure 8 shows that there are many black spots (upper image) in the MRI of the brain of APP mice but not in the control brain (bottom image). Therefore, the iron-curcumin nanoparticle was only specifically retained and visualized by MRI in the brain of AD mice but not in control mice. Figure 9 shows a typical brain section of an APP transgenic mouse injected with a magnetic nanoparticle incorporating curcumin. Red spots indicate amyloid plaques stained with a mixture of 4G8 and 6E10 monoclonal antibodies against Aβ peptide, and blue indicates iron oxide. The magnified view shows that the iron oxide is close to the amyloid plaque. Figure 10 shows matched MRI (left) and double-stained APP transgenic mouse brain sections (right). Many of the dark spots found in MRI are co-localized with amyloid plaques (red) and iron oxide (blue) such as immunolabels. The illustration is 40x magnification of the selected area, showing amyloid plaques (red) and iron (blue) in bright field images (left) and plaques (orange) in fluorescent images (right) . The fluorescence is orange because curcumin is emitted at 520 nm and the fluorescent label for immunolabeled protein spots is emitted at 570 nm. The combination of these yellow and red emission signals produces a bright orange color. The image shows that the magnetic nanoparticle combined with curcumin is capable of binding to amyloid plaques and can be visualized by MRI or immunohistochemistry. Figure 11 shows a section of non-transgenic control mice of the same age after injection of curcumin-loaded magnetic nanoparticles. No iron oxide signal was detected by MRI (left). As expected, no amyloid plaques (red) or iron (blue) (right) were detected on the matched histological sections. For the colocalization of curcumin, iron oxide and amyloid plaques, brain sections of APP transgenic mice injected with curcumin-loaded magnetic nanoparticles were observed (Fig. 12). In the illustration, the bright view (left) shows the iron oxide shown in blue, and the fluorescent view (right) shows the amyloid plaques in red and yellow (or colocalization due to amyloid plaques and curcumin) Displayed in orange) shows curcumin. Instance 5 - Curcumin-containing magnetic nanoparticle as a diagnostic agent
In order to determine whether the magnetic nanoparticle combined with curcumin can be used as an diagnostic agent for AD, it is necessary to correlate immunohistochemistry with MRI results in vivo. Based on previous results, in vivo images can well predict actual A[beta] plaque locations (shown in immunohistochemistry). Ten in vivo MRI brain sections from three transgenic mice injected with magnetic nanoparticles containing curcumin were randomly selected and analyzed by uninformed operators for their matched immunohistochemical sections. Figure 13 shows a linear regression of spot density in MRI versus A[beta] density in immunohistochemistry. It has a strong correlation (Pearson's correlation, p = 0.002), and R2
The value is 0.72. The results show that the in vivo MRI image of Tg mice injected with curcumin-loaded magnetic nanoparticles is a good predictor of A[beta] plaques found in immunohistochemistry. Therefore, the magnetic nanoparticle combined with curcumin can be clinically used for diagnostic imaging of Aβ plaques. Although the foregoing invention has been described with reference to the embodiments of the invention, In addition, each of the references cited herein is hereby incorporated by reference in its entirety in its entirety in the extent of the extent of the disclosure of the disclosure of each of each of the of In the event of a conflict between this application and the references provided herein, the present application shall prevail.