TW201006845A - Modified peptide of human acidic fibroblast growth factor - Google Patents
Modified peptide of human acidic fibroblast growth factor Download PDFInfo
- Publication number
- TW201006845A TW201006845A TW098119510A TW98119510A TW201006845A TW 201006845 A TW201006845 A TW 201006845A TW 098119510 A TW098119510 A TW 098119510A TW 98119510 A TW98119510 A TW 98119510A TW 201006845 A TW201006845 A TW 201006845A
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- Taiwan
- Prior art keywords
- afgf
- polypeptide
- human
- leu
- modified peptide
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- C—CHEMISTRY; METALLURGY
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
201006845 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種酸性纖維母細胞生長因子之改質肽，其 具有較佳的穩定性。 【先前技術】 酸性纖維母細胞生長因子(acidic fibroblast growth factor， aFGF) ’其係為最初由活體組織中，包含腦及海馬迴，所分離 之單鏈蛋白質，能夠在體外影響許多細胞種類之生長及分化。 aFGF係為肝素(heparin)_依賴性之細胞分裂劑㈣t〇gen)，且能 與目前已知的四種生長因子受體及其剪接型態牢固地結合。此 蛋白質可被定位在與運動及知覺功能相關之特定神經子集 合’且可自成人腦中分離。分離之3PGF係為一種神經母細胞 之細胞分裂劑，並促使神經炎自脊椎神經延長。 人類aFGF之天然肽係自人類大腦中分離，包含有154個 胺基酸。然而，天然之人類aFGF之肽胺基末端19個胺基酸 已被鑑定為與人類介白質-l(Interlukin-l, IL-1)同源。人類aFGF 與IL-1相似之多肽區位可能會造成自發性免疫反應，包括巨 嗟細胞的活化及造成細胞生長停滯(G. Venkataraman β α/.， iW.AS·，96:3658-63, 1999)。此外，促發炎細胞激素il-1及 FGF-l(aFGF)/FGF-2(bFGF)因具有相同的結構支架(scaff〇id)， 將會相互競爭酪胺酸激酶(tyrosine kinase)區位上相同之受體 結合區域(A· J. Minter et al.，J. Cell Physil.，167:229-37, 1996)。 【發明内容】 本發明係提供一種人類酸性纖維母細胞生長因子之改質 肽’命名為aFGF135 ’包含一天然之人類aFGF，其係為自該 天然之人類aFGF胺基末端刪除20個胺基酸，並增加一個丙 胺酸於縮短之人類aFGF之前。特定而言，aFGF多肽包含如 序列編號1之胺基酸序列，其具有相當高之穩定性且較已知 的aFGF多肽好。 3 201006845 本發明進提供—種醫藥組合物，其包含本發明之 aFGF夕肽以及醫藥上可接受之載體。 【實施方式】 本發明係提供一種人類酸性纖維母細胞生長因子之改質 肽缺命名為aFGFm ’包含-天然之人類aFGF，其係為自該 天w之人類aFGF胺基末酬除2〇健基酸，並增加—個丙 胺酸於縮蚊人類apGF之前。特定而言，aFGF多肽包含如 序列編號1之胺基酸序列。出乎預期地，改質之aFGF135多 肽在體溫下具有相當高地歡性，且具有於其它已知的a· 不同之立體結構。 根據本發明，具有如序列編號丨之aFGF135多肽有相當 高地穩定性。與天然之人類aFGF相比，aFGF135多肽自天然 之人類aFGF胺基末端刪除20個胺基酸（稱為「縮短^ aFGF」）’並在已刪除20個胺基酸之aFGF前增加一個丙胺酸。 根據本發明，因為人類aFGF與人類IL-1之前19個胺基酸已 被鑑定為同源’為了避免藉由共同的路徑產生類IL-1之反應， 該20個胺基酸係由天然之人類aFGF中刪除。令人意外的是， aFGF多肽在生理體溫下’較目前已知的aFGF，包括天然之 aFGF，有較佳的穩定度。如本發明之一具體實施例所示， aFGF135多肽在體溫下(例如約37°〇至少培養48個小時後， 仍保有正確的折疊而無任何降解的產生(如第二A圖所示）。在 本發明之另一具體實施例中’ aFGF135多肽在自我加速分解溫 度下(例如約54°C)培養一小時後(如第二B圖所示），仍保有其 完整的結構。由此可證，aFGF135多肽相較於已知的aFGF， 例如具有140個胺基酸及127個胺基酸之人類aFGF ,有較佳 的穩定度。 為了顯示aFGF135多肽與已知的aFGF之差異性，利用核 磁共振圖譜計算aFGF135多肽在三維空間中的正確結構，並 將其與已知的aFGF做比較。 在與 Bemett MJ ei a/. (Proiez>w，57(3):626-34, 2004)所公開 201006845 之具有140個胺基酸之人類aFGF(蛋白質資料庫(pDB)編碼為 1RG8)，及與 Lozano RM β a/. (Biochemistry，2;39(17):4982-93
2000)所公開之具有127個胺基酸之人類aFGF(蛋白質資料庫 〇ΌΒ)編碼為1DZD)相比下，多肽具有不同的結構特 徵1包括碳基末端及胺基末端的裸露以及左下角一個顯著的結 合=(loop)，如第五A圖所示。可推測因為aFGF135多肽具有 改質的序列（包含刪除20個胺基酸及增加一個丙胺酸），造成 與其它已知重組或天然aFGF相比有更穩定的結構。在本發明 的一個具體實施例中，如第三圖所示，針對穩定性進行與^解 有關之比較，商業上購得之重組aFGF(Pr〇mega公司)在'54。〇 培養一小時後在西方墨點試驗中產生出降解的條 aFGF135則相對地穩定。 本發明進一步提供一醫藥組合物，其包含本發明之aFGF 多肽以及醫藥上可接受之載體。 本發明之醫藥組合物可由傳統上習知之方法與一種或一 種以上之醫藥上可接受之載體製備。術語「醫藥上可接受之載 體」在本文中意指包含任何標準的醫藥上可接受之載體。此種 載體包含，但不限於：食鹽水、緩衝食鹽水、葡萄糖、水 油、乙醇或上述之組合物。
本發明之醫藥組合物可以選擇任何適合的形式投與 為直接塗覆在手術部位。 〃 。較佳 本發明進-步由下列實施例說明，但其目的僅作為例示而 非限制。 實施例1 : aFGF135多肽之克隆(ci〇ning) 人類全長aFGF多肽係為購自於ci〇ntech Laboratories Inc 3速克隆eDNA之產品。在建構前，設計兩解—性引子= 序列編號2: ATCA-3，
5 -ACTGAATTCtATGGCTGAAGGGGAA 5 201006845
序列編號 3: 5’-AAGAAGCTTCaAATC：AGAAC}A GACTGGCAGG-3， 在序列編號2中具有一個EcoRl限制酶切割位置(其標示 為▼)’而在序列編號3中具有一個Hind III限制酶切割位置(其 標示為▲)。將全長序列之產品利用上述引子進行聚合酶連鎖反 應(PCR)可得到485個鹼基對之PCR產物。將cDNA產物以 EcoRl及Hind III限制酶切割之後，其切割片段插入具有相同 切割位之P-UC18載體中。 〃 根據p-UC18-haFGF之模版，兩段專一,f·生引子設計如下：
序列編號 4: 5’-GGCATATGTGCTAATTAC：AAaAAGC CC-3， 序列編號 5: 5’-AAGAGATCTCTTT‘ AATC AGAAGAGACTGGC AGG-35 在序列編號4中具有一個Ned I限制酶切割位置(其標示 為）’而在序列編號5中具有一個Bglll限制酶切割位置(其 標示為▲)。由序列編號4及序列編號5放大之cDNA長度係為 較全長縮短57個核酸。aFGF135多肽僅有135個胺基酸，並 保留了 aFGF主要功能區域。此外，在胺基末端之第二個胺基 酸-甘胺酸(G)係被變更為丙胺酸(A)。如序列編號丨所示： △NYKKPKLLY。自 p-UC18 放大之 cDNA 片段與 Ned I 及 Bgl Π限制轉反應’其切割片段插入具有相同切割位之ρΕτ%載體 中。pET3c-haFGF因此而被建構。 實施例2 :表現並純化aFGF135多肽 在放大pET3ohaFGF之後，利用勝任細胞(c〇mpetent cell)BL21(DE3)(Novagen，德國)將載體轉化為DNA。在加入 隶終濃度為1 mMIPTG(異丙基β-D-l-硫代半乳糖普)培養 前，培養抗胺苄青黴素(ampicillin)之E. coli菌落並在lb培養 液中放大至OD6^到達0.3。在培養16個小時(土兩個小時)後， 蒐集細菌並以27,000xg離心以去除上清液。蒐集到的細菌以 201006845 PBS沖洗兩次’接著以高壓均質機(Niro s〇avi型號NS2〇〇6L，
Daken Stainless Products Ltd·，UK)打破。打破之樣本以 0.22 μιη 孔徑之篩過濾並用於蛋白質之純化。 人類aFGF135多肽以色層分析法分離，如以下之步驟： ⑴陽離子交換層析法(CMFF管柱，RM197，購自GE Healthcare Bio-Sciences USA Corp.); (2)親和層析法，其係對肝 素(heparin)專一(HeparinFF 管柱，RM244,購自 GEHealthcare Bio-Sciences USA Corp.); (3)大小排阻層析法(Superdex 75 pre-grade 管柱 ’ RM245 ’ 靖自 GE Healthcare Bio-Sciences USA Corp.)。上述管柱的緩衝液為磷酸鹽溶液= 51:49 ’ 0.1% EDTA-Na ’ pH6_8-7.2)。最後所得之產物即為本發 明之aFGF135多肽。aFGF135多肽的分子量由LC-MAMA方 法鑑定為15281 Da。 實施例3 : aFGF135多肽之穩定性試驗 (1)西方墨點法 純化之多肽加入4-20或10-20%之斜率凝膠(gradient gel) 中進行聚丙烯醯胺膠體電泳，接著以電泳轉印機(Polybl〇t Transfer System, Model SBD-1000; American Bionetics, Emeryville，CA)轉印至硝纖維膜上(0.05 Am; Schleicher & Sdiuell’Inc.，Keene，NH)。為了檢測aFGF多肽之穩定性，製備 Laemmli 緩衝液(2.4 ml 1 M Tris pH 6.8，0.8 g SDS-基液，4 ml 100%甘油’ 0.01%溴紛藍’ 0.02〇/〇 1 ml β_疏乙醇(電泳等級）， 以及2.8 ml水)含有或不含8 Μ尿素(用於打開潛在之聚結體） 作為焦集膠體(stacking gel)緩衝液，其中包含0.0625 Μ三羥甲 基氨基曱烷(Tris-base) ’ 1% SDS基液以及15 mM二硫蘇糖醇 (dithiothreitol)。樣本置於室溫1小時後，再置於4°c隔夜。進 行聚丙烯醯胺膠體電泳前將樣本煮沸。
在以含有3%奶粉之TBS轉移及阻斷非專一，I1 生蛋白質結 合位置後，硝纖維膜與以沖洗緩衝液(l〇mMTris-HCl，pH 7 201006845 8.0 ’ 〇·15 M NaCl，0.05°/。Tween-20)適當稀釋之不同抗體(aFGF 抗體以ΐ··5〇〇稀釋’購自R&DSystem,Inc.)在代下反應隔夜。 使用適當之二級抗體來與抗原_抗體組合物反應使其顯影，並 將其培養於 ProtoBlot Western Blot AP 系統中(Promega, Madison, WI) ° (2)降解測試 多肽分別培養於37〇c及54〇c下，其中54°c 為自我力。π速分解溫度。接著’將樣本進行西方墨點法分析。培 ^於37°C下之樣本之結果如第二a圖所示，其中道1至道3 為，養6小時、24小時及48小時後之結果，道4為生物 標誌且每個條帶上之數字為其所代表之分子量。aFGF1%多肽 在54 C下培養120(2小時)時間之結果如第二b圖所示;其中道 1至運3分別為培養10分鐘、60分鐘及120分鐘之結果，道 4為生物標痣且每個條帶上之數字為其所代表之分子量。 如第二A圖所示，aFGF135多肽在體溫下(約37。〇能保 結構至少48小時。這表示aFGF135多肽提供較長時 間之神經:保護效果，以及較佳之穩定性。 f ®所示，邮⑽多肽在5代下祕持完整之 J、時。當培養於54°C下2小時後，HFGF135多肽 不會轉化為其它結構，該結構可能造成具有不可 預。因此，aFGF多肽提供安全_存及運輸。 aFrFS>间的實驗條件下，同時使用一商業上講得之重組 ga/FGF”，購自 PlOmegaCGrP_輕行穩定性 所Ϊ之f 所不’ 條帶中具有黑色箭碩 ΐίίί %相反地，aFGF135多肽則維持原來結構而: 時’ #加人樣本於微盤中時，Pr_㈣卿產、 而aFGF135多狀則保持澄清。此結果證明 錢比起«上之aFGF有触之穩定性。 實施例4 : 核磁共振結構特徵 201006845 核磁共振光5普疋隶近用來確定生物上大分子，如蛋白質 或核酸，在原子解析度下之結構的常用工具。因此委由高磁場 核磁共振中心（中央研究院’台灣)針對aF GF丨3 5多肽鑑定其結 構特徵。 簡而言之’光譜係在298 K下以Bruker AVANCE 600紀 錄。碳基末端及胺基末端之化學位移係以TAL〇s軟體得到骨 架扭轉角(torsion angles)。骨架之循序共振判定
(sequence-specific resonance assignments)係藉由 HNC0、 HN(CA)CO、HACNCB、CBCA(CO)NH、HNCA 及 HN(CO)CA 試驗，側鏈判定(side-chain assignments)係藉由 15N-TOCSY_HSQC 及 I3c-noesy-hsqc 合日併 15n-noesy-hsqc 及 13c-noesy-hsqc 而完成。骨架圖如第 四圖所示。 為了結構計异，利用TALOS軟體計算出平面二面角的訊 息(dihedral angle) ’ N-NOESY-HSQC 及 13C-NOESY-HSQC 決 定出距離限制，配合D20交換的實驗結果列出26個可能的氫 鍵。aFGF135多肽的3D結構構築由CYANA軟體計算出如第 5A圖所示。aFGF135多肽的核磁共振數據及結構計算統計皆 詳列於表一。 _参一 aFGFl^l!.肽的核磁共振數攄及鈷椹玄十算統計
N0E距離數據 ' ---- - L 1639 869 206 538 26 總值（Total) 短距(Short range),丨 i-j 丨 中距(Medium range)，1 < 丨 i-j 丨 < 5 長距(Long range)，5 < | i-j | 固定氫鍵(Restrained hydrogen bonds) 扭轉角數據(Torsion angle constraints) 70 76 1_26±0.31 Φ Ψ CYANA 目標功能值(target fonction value；)
Ramachandranplot 統計值（％) 201006845 在主要區位之殘基(Residues in most favored regions) 65.9 在其它許可區域之殘基(Residues in additional allowed regions) 33 5 在通常許可區域之殘基(Residues in generously alio wed regions) 0.5 在不被允許區域之殘基(Residues in disallowed regions) 均方根離差(RMSD)來自於平均書(averaged coordinates)(A) 1.12士 0.36 1.56±0.28 〇.43±0.08 〇.93士0.11 0.54±0.07 1.05±0.06 全長之骨架RMSD 全長之重原子RMSD 次級結構之骨架RMSDb 次級結構之重原子RMSDb 殘基 7-12,16-30, 36-53, 58-85, 89-108, 111-131 之骨架RMSD c 殘基 7-12, 16-30, 36-53, 58-85, 89-108, 111-131 之骨架RMSDe _ a· 供之數據係根據CYANA檢測之超過平均20個代表溶解結構之 參 構造 b. 次級結構區域係根據MOLMOL選擇：7-11，16-21, 25-29, 39-43， 48-53, 59-62, 68-71, 80-85, 89-94, 127-131 c. 選擇之區域係根據文獻(Lozano RM.扣，施咖磁吵， 2:39(17):4982-93, 2000)所揭露之區域而決定 ’ ❹ aFGF 135多肽之二維立體結構及兩個已知的人類 多肽結構之比較如第五B圖及第五C圖所示。具有14〇個胺 基酸如序列編號6所示之aFGF多肽(pdb編碼irG8)，其結 構顯示於第五B圖；具有127個胺基酸如序列編號7所示之 aFGF多肽(PDB編碼1DZD)，其結構顯示於第五c圖。上述 兩者之結構皆不同於顯示於第五A圖之aFGF135多肽之結 構’因其<基末端及胺基末端藏於結構之巾且左下角缺少固 結合環(1—)。此絲鋼具奴㈣細序 aFGF135多肽，在結構上新穎且與已知^aFGF多;^構之 熟習技藝者將可明瞭’可對上述之具體實例進行變化而 不偏離其廣=發明概念。因此，翻瞭本發明並不限於所揭 不之特定具體貫例，其欲涵括由附呈之申續真 二 本發明精神及範圍中之修飾。之甲明專利耗圍所定義之 10 201006845 【圖式簡單說明】 將可幫助理解本敘述’結合附加之圖式 限於，狄狀細;，無論如何，本發明將不受 肽之分子量之圖譜，其係由 Φ 37。(^48^、^^^方墨點法之圖像，其展示31?(}17多肽在 時、料時及二別是f *6小 上·^數字為其所代表之分子k ^ U標▲，母個條帶 ，$4 綱票諸， ίϊΐτί夕為西方墨點法之圖像，其展示54。。下1小時間， 與商業上講得具有⑽個胺基酸之 i 標示為”p”)之降解比較;其中道"μ”為分子標 ί色構得人類aFGF有降解情形，其降解處以 儀二圖睹’1其if示由液態蛋白質核磁共振光谱分析 儀^測aFGF多肽之iH」5N_HSQC光譜。 之1H~HSQC光譜所預測之 碼為圖具有140個胺基酸之人類卿多肽(酬編 碼有m個胺基酸之人類卿多肽(腦編 【主要元件符號說明】 無 201006845 序列表 <110> 鄭宏志 <120>人類酸性纖维母細胞生長因子改質肽 <140> 098119510 <141> 2009-06-11 <150> US 61/060,262 <151> 2008-06-10 <160> 7 <170> Patentln 3.3 版 <210> 1 <211> 135
<212> PRT <213> 人工合成 <22〇> <223〉來自於人類酸性纖維母細胞生長因子（a-FGF)之多肽 <4〇0> 1
Ala Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His 15 10 15
Phe Leu Arg lie Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg 20 25 30
Ser Asp Gin His lie Gin Leu Gin Leu Ser Ala Glu Ser Val Gly Glu 35 40 45
Val Tyr lie Lys Ser Thr Glu Thr Gly Gin Tyr Leu Ala Met Asp Thr 50 55 60
Asp Gly Leu Leu Tyr Gly Ser Gin Thr Pro Asn Glu Glu Cys Leu Phe 65 70 75 80
Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr lie Ser Lys Lys 85 90 95
His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys 100 105 110
Lys Arg Gly Pro Arg Thr His Tyr Gly Gin Lys Ala lie Leu Phe Leu 115 120 125
Pro Leu Pro Val Ser Ser Asp 130 135 201006845 <210> 2 <211> 28 <212> DNA <213> 人工合成 <220> <223> 引子 <400> 2 actgaattca tggctgaagg ggaaatca <210> 3 <211> 30 <212> DNA 曹 <213> 人工合成 <220> <223> 引子 <400> 3 aagaagcttc aatcagaaga gactggcagg <210> 4 <211> 26 <212> DNA <213> <220> 人工合成 <223> 引子 <400> 4 ggcatatggc taattacaag aagccc <210> 5 <211> 33 <212> DNA <213> 人工合成 <220> <223>弓丨子 201006845 <40〇> 5 aagagatetc tttaatcaga agagactggc agg <210> 6 <211> 140 <212> PRT <213> 人工合成 <22〇> <223〉來自於人類酸性纖維母細胞生長因子（a-FGF)之多肽 <400> 6
Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 15 10 15
Ser Asn Gly Gly His Phe Leu Arg lie Leu Pro Asp Gly Thr Val Asp 20 25 30
Gly Thr Arg Asp Arg Ser Asp Gin His lie Gin Leu Gin Leu Ser Ala 35 40 45
Glu Ser Val Gly Glu Val Tyr lie Lys Ser Thr Glu Thr Gly Gin Tyr 50 55 60
Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gin Thr Pro Asn 65 70 75 80
Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95
Tyr lie Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys 100 105 110
Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gin Lys 115 120 125
Ala lie Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 <210> 7 <211> 127 <212> PRT <213〉人工合成 <220> <223>來自於人類酸性纖維母細胞生長因子（a_FGF)之多肽 1 <400> 201006845
Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg lie Leu Pro Asp Gly 15 10 15
Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gin His lie Gin Leu Gin 20 25 30
Leu Ser Ala Glu Ser Val Gly Glu Val Tyr lie Lys Ser Thr Glu Thr 35 40 45
Gly Gin Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gin 50 55 60
Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His 65 70 75 80
Tyr Asn Thr Tyr lie Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val 85 90 95
Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr 100 105 110
Gly Gin Lys Ala lie Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 115 120 125
Claims (1)
- 201006845 七、申請專利範圍： L —種人類酸性纖維母細胞生長因子(aFGF)之改質肽，包含一 天然之人類aFGF，其係為自該天然之人類aFGF胺基末端 刪除20個胺基酸，並增加一個丙胺酸於縮短該人類沾证 之前。 2. 3. 4. 5. 6. 7. 8. 如申請專利範圍第1項之改質肽，包含如序列標號1之胺 基酸序列。 如申請專利範圍第1項之改質肽，其具有如第五A圖之三 維立體結構。 如申請f利範圍第1項之改質肽，其具有相當高之穩定性。 —種醫藥組合物’其包含如申請專纖圍第丨項之經分離 之多肽以及一醫藥上可接受之載體。 —種醫藥組合物，其包含如申請專利範圍第 之多肽以及一醫藥上可接受之載體。 2項之經分離 3項之經分離 4項之經分離 —種醫藥組合物，其包含如申請專利範 之多肽以及一醫藥上可接受之載體。 藥利範圍第
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| WO2020173456A1 (en) * | 2019-02-28 | 2020-09-03 | Eusol Biotech Co., Ltd. | Use of recombinant protein for treating metabolic disorders |
| CN113727996A (zh) * | 2019-02-28 | 2021-11-30 | 雅祥生技医药股份有限公司 | 重组蛋白用于治疗代谢疾病 |
| CN113727996B (zh) * | 2019-02-28 | 2024-09-13 | 雅祥生技医药股份有限公司 | 重组蛋白用于治疗代谢疾病 |
Also Published As
| Publication number | Publication date |
|---|---|
| US20090305988A1 (en) | 2009-12-10 |
| TWI363761B (en) | 2012-05-11 |
| US7956033B2 (en) | 2011-06-07 |
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