TWI344964B - Novel peptides that bind to the erythropoietin receptor - Google Patents

Description

九、發明說明： 【韻^明所屬之_45"領域】 相關申請案之交互參照 對美國臨時專利申請案第60/469,993及60/470,244號請 求優先權’二案申請曰皆為2003年5月12曰。此等前案内容 全文以引用方式併入此處。 發明領域 本發明係有關胜肽化合物，其為紅血球生成受|| (EPO-R)激動劑。本發明亦係關於使用此種胜肽化合物治療 紅血球製造不足或缺陷關聯病症之治療方法。也提供包含 本發明之胜肽化合物之藥學組成物。 【先前技術3 發明背景 紅血球生成素（EPO)是一種含165胺基酸之糖蛋白激 素，分子量約為34千道爾呑(kD)，及較佳糖化位置係位於 胺基酸位置24、38、83及126。最初製造為前驅蛋白帶有— 個含23胺基酸之信號胜肽。EPO可以三種形式出現：邙、 及asialo。α形式及yS形式之碳水化合物成分略有差異， 但具有相同強度、生物活性及分子量。Asialo形式為〇；或万 形式之端末碳水化合物（唾液酸)被去除。也曾經報告蝙碼 EP0之DNA序列[Lin之美國專利第4,703,008號]。 EP0刺激紅血球前驅細胞之有絲***及分化’如此可 保證紅血球的製造。當發生組織缺氧情況時’紅血球生成 素於腎臟製造。於Ep〇誘生紅血球前驅細胞分化時，誘生 求蛋白的合成，刺激血質複合物的合成以及增加鐵蛋白 文益的數目。此等變化允許細胞攝取更大量鐵，及合成功 能血色素，於成熟的紅血球與氧氣結合。如此紅血球及其 血色素於身體的氧供應上扮演關鍵要角。此等變化係經由 EPO與紅血球前驅細胞表面之適當受器交互作用所引發 [例如參考 Graber 及 Krantz (1978) Arm. Rev· Med. 29.51-66] » 虽身體處於健康狀態，組織由現有紅血球數目可接受 足夠氧合作用時，EPO係以極低濃度存在於血漿。此種正 常低濃度即足夠刺激補充正常經由老化而損失的紅血球。 於組織缺氧條件下，當血循環中血球轉運的氧氣減少 時，血循環中之EPO含量增高。組織缺氧例如可能係經由 下列因素所引起，經由出血而實質上血液流失、經由過度 暴露於輻射而紅血球受破壞、因高海拔或長期昏迷造成氧 ^取篁降低、或各種形式的貧血。回應於此種組織缺氧的 壓力，EPO濃度升咼，經由刺激紅色袓先細胞增生，來增 加紅血球的製造。當血循環的紅血球數目大於正常組織氧 需求所需時，血循環之EPO濃度下降。 因EPO為紅血球生成過程所必需，此種Epo激素可能可 用於以紅血球製造量低或缺陷為特徵之血液病的診斷與治 療。晚近研究提供預測EPO用於多種疾病狀態、病症及血 液不規則狀態之治療功效的基礎，該等疾病包括：万地中 海型貧血[參考 Vedovato 荨人（1984) Acta. Haematol. 71:211_213];囊性纖維變性[參考vichinsky等人（1984)】 1344964Nine, invention description: [Yu ^ Ming belongs to the _45" field] The cross-references of the relevant applications for the United States Provisional Patent Application Nos. 60/469,993 and 60/470,244 request priority 'two cases are all in 2003 5 12 months. The contents of these prior matters are hereby incorporated by reference in their entirety. FIELD OF THE INVENTION The present invention relates to peptide compounds which are erythropoiesis-derived || (EPO-R) agonists. The invention is also directed to a method of treating such a deficiency or defect-related disorder in the treatment of red blood cells using such a peptide compound. A pharmaceutical composition comprising the peptide compound of the present invention is also provided. [Prior Art 3 Background] Erythropoietin (EPO) is a glycoprotein hormone containing 165 amino acids having a molecular weight of about 34 kDa, and a preferred saccharification position at the amino acid position 24, 38 , 83 and 126. Originally produced as a precursor protein with a signal peptide containing 23 amino acids. EPO can appear in three forms: 邙, and asialo. The carbohydrate components of the alpha form and the yS form are slightly different, but have the same strength, biological activity and molecular weight. The Asialo form is 〇; or the 10,000-type terminal carbohydrate (sialic acid) is removed. The DNA sequence of the bat code EP0 has also been reported [U.S. Patent No. 4,703,008 to Lin]. EP0 stimulates mitosis and differentiation of red blood cell precursor cells' to ensure the production of red blood cells. When a tissue hypoxia occurs, 'erythropoietin is produced in the kidney. When Ep〇 induces red blood cell precursor cell differentiation, it induces protein synthesis, stimulates the synthesis of blood complexes, and increases the number of ferritin. These changes allow the cells to take up a larger amount of iron, and the successful hemoglobin, which combines mature red blood cells with oxygen. Such red blood cells and their hemoglobin play a key role in the body's oxygen supply. These changes are triggered by the interaction of EPO with appropriate receptors on the surface of the red blood cell precursor cells [eg eg Graber and Krantz (1978) Arm. Rev. Med. 29.51-66] » Although the body is in a healthy state, the number of tissues is from the number of existing red blood cells When adequate oxygenation is acceptable, EPO is present in the plasma at very low concentrations. This normal low concentration is sufficient to stimulate the red blood cells that are normally lost through aging. Under hypoxic conditions, when the oxygen transported by the blood cells in the blood circulation is reduced, the EPO content in the blood circulation is increased. Tissue hypoxia may be caused, for example, by substantial blood loss through bleeding, red blood cells destroyed by excessive exposure to radiation, decreased oxygen levels due to high altitude or prolonged coma, or various forms of anemia. In response to this tissue hypoxia stress, the EPO concentration rises and increases the production of red blood cells by stimulating red sputum cell proliferation. When the number of red blood cells circulating in the blood is greater than that required for normal tissue oxygen demand, the EPO concentration of the blood circulation decreases. Because EPO is required for erythropoiesis, this Epo hormone may be used for the diagnosis and treatment of blood diseases characterized by low red blood cell production or defects. Recent studies provide the basis for predicting the therapeutic efficacy of EPO for a variety of disease states, disorders, and blood irregularities, including: 10,000 Mediterranean anemia [Ref. Vedovato 荨人 (1984) Acta. Haematol. 71:211_213]; Fibrosis [Ref. vichinsky et al. (1984)] 1344964

Pediatric 105:15-21];妊娠病症及月經病症[參考Cotes等人 (193) Brit. J. Ostet. Gyneacol. 90:304-311];早產兒早期貧血 [參考Haga等人（1983) Acta Pediatr. Scnad. 72; 827-831];脊 索受傷[參考 Claus-walker 等人（1984) Arch Phys. Med. 5 Rehabil. 65:370-374];太空飛行[參考Dunn等人（1984) Eur. J. Appl. Physiol. 52:Π8-182];急性失血[參考Miller等人（1982) Brit. J. Haematol. 52:545-590];老化[參考Udupa等人（1984) J. Lab. Clin. Med. 103:574-580及581-588及Lipschitz等人 (1983)血液63:502-509];多種伴隨有紅血球生成異常之腫瘤 10 疾病狀態[參考Dainiak等人（1983) ACNcer 5:1101-1106及 Schwartz等人（1983) Otolaryngol. 109:269-272];以及腎功能 不全[參考Eschblach等人（1987) N. Eng. J. Med. 316:73-78]。 對純化後之均質EPO已經決定其特徵[Hewick之美國 專利第4,677,195號]。編碼EPO之DNA序列經純化、選殖、 15 及表現來製造有相同生化性質及免疫性質之重組多肽及天 然EPO。也已經製造具有與天然EP0相同寡醣之重組EPO分 子[參考Sasaki等人（1987) J. Biol. Chem. 262:12059-12076]。 Ε Ρ Ο之生物功效顯然部分係經由與細胞膜結合受器交 互作用媒介。初步研究使用分離自小鼠脾臟之未成熟紅色 20 細胞，初步研究提示EPO結合細胞表面蛋白質包含二多 肽，其分別具有約略分子量85,000道爾吞及1〇〇,〇〇〇道爾吞 [Sawyer 等人（1987) Proc. Natl. Acad. Sci. USA 84:3690-3694]。EPO結合位置數目計算得每個細胞表面平 均800至1000。此等結合位置中，約有300個結合EP0具有 7 1344964 Κ3^90ρΜ(微微莫耳濃度），而其餘結合EP〇具有約57〇pM 之較低親和力[Sawyer 等（1987) J. Biol. Chem. 262:5554-5562]»獨力研究提示由小鼠注射佛蘭白血病病毒 (Friend leukemia virus)之貧血種系（FVA)，製備得EPO反應 ， 5 性脾臟紅母細胞，共有約400個高EPO結合位置及低親和力Pediatric 105:15-21]; gestational disorders and menstrual disorders [Ref. Cotes et al. (193) Brit. J. Ostet. Gyneacol. 90:304-311]; early anemia in preterm infants [Ref. Haga et al. (1983) Acta Pediatr Scnad. 72; 827-831]; spinal cord injury [cf. Claus-walker et al. (1984) Arch Phys. Med. 5 Rehabil. 65: 370-374]; space flight [cf. Dunn et al. (1984) Eur. J Appl. Physiol. 52: Π 8-182]; acute blood loss [Ref. Miller et al. (1982) Brit. J. Haematol. 52: 545-590]; aging [cf. Udupa et al. (1984) J. Lab. Clin. Med. 103:574-580 and 581-588 and Lipschitz et al. (1983) Blood 63:502-509]; a variety of tumors associated with abnormal erythropoiesis 10 disease states [Reference Dainiak et al. (1983) ACNcer 5:1101- 1106 and Schwartz et al. (1983) Otolaryngol. 109:269-272]; and renal insufficiency [cf. Eschblach et al. (1987) N. Eng. J. Med. 316: 73-78]. The homogenized EPO after purification has been characterized (Hewick's U.S. Patent No. 4,677,195). The DNA sequence encoding EPO is purified, cloned, and expressed to produce recombinant polypeptides and natural EPO having the same biochemical and immunological properties. Recombinant EPO molecules having the same oligosaccharides as native EP0 have also been produced [see Sasaki et al. (1987) J. Biol. Chem. 262: 12059-12076]. The biological effects of Ε Ρ 显然 are clearly partly through interaction with the cell membrane receptor. Preliminary studies using immature red 20 cells isolated from mouse spleens, preliminary studies suggest that EPO-binding cell surface proteins contain two polypeptides, each with an approximate molecular weight of 85,000 Torr and 1 〇〇, aw 尔 吞 [Sawyer Et al. (1987) Proc. Natl. Acad. Sci. USA 84:3690-3694]. The number of EPO binding sites was calculated to average 800 to 1000 per cell surface. Of these binding positions, approximately 300 bound EP0 have 7 1344964 Κ3^90ρΜ (pico molar concentration), while the remaining bound EP〇 have a lower affinity of about 57〇pM [Sawyer et al. (1987) J. Biol. Chem 262:5554-5562]»Independent research suggests that the mouse is injected with the affluent germline (FVA) of Friend leukemia virus to prepare an EPO response, 5 spleen red mother cells, and a total of about 400 high EPOs. Combined position and low affinity

^ EPO結合位置分別具有Kd值約100 pM至800 pM^ EPO binding sites have Kd values of approximately 100 pM to 800 pM, respectively

[Landschulz等人（1989)血液73:1476-1486]。 p 隨後研究工作指出兩種形式EPO受器（EPO-R)係由單 一基因所編碼。此基因已經經過選殖[例如參考j〇nes等人 10 (1990)血液 76，31-35 ; Noguchi 等人（1991)血液 78:2548-2556 ; Maouche等人（1991)血液78:2557-2563]。例[Landschulz et al. (1989) Blood 73: 1476-1486]. p Subsequent research indicates that the two forms of EPO receptor (EPO-R) are encoded by a single gene. This gene has been selected for breeding [eg reference to J〇nes et al. 10 (1990) Blood 76, 31-35; Noguchi et al. (1991) Blood 78: 2548-2556; Maouche et al. (1991) Blood 78: 2557-2563 ]. example

如鼠EPO-R蛋白質及人EPO-R蛋白質之DNA序列及編碼胜 肽序列述於D’Andrea等人之PCT公告案第WO 90/18822 號。本研究模型提示EPO結合至EPO-R，結果導致兩個 15 EPO-R分子的二聚合及活化，結果導致隨後信號轉導步驟 _ [例如參考Watowich等人（1992) Proc. Natl. Acad. Sci. USA 89:2140-2144]。 可取得EPO-R選殖基因，有助於此種重要受器之激動 劑與拮抗劑的研究。可取得重組受器蛋白質，允許研究於 ^ 2〇 多種隨機及半隨機胜肽分集產生系統的受器-配位子交互 作用的研究，此等系統包括「胜肽於殖體」系統[述於美國 專利第6,270，170號];「胜肽於噬菌體」系統[述於美國專利 第 5,432,018號及Cwirla等人（1990) Proc. Natl. Acad. Sci. USA 87:6378-6382] ;「編碼合成存庫」（ESL)系統[述於美國 8 1344964 專利申請案第946,239號’申請日1992年9月16日；及「極 大規模制動聚合物合成系統[述於美國專利第5,143,854 號；PCT公告案第90/15070號；Fodor等人（1991)科學 251:767-773 ; Dower及Fodor (1991) Ann. Ep. Med. Chem· 5 26:271-180 ;及美國專利第5,424,186號]。 與EPO-R交互作用之至少某種程度之胜肽已經被識別 出，例如述於Wrighton等人之美國專利第5,773,569 ; 5,830,851及5,986,047號；\¥1^匕〇11等人之？(：1'公告案第*0 96/40749號；Johnson及Zivin之美國專利第5,767,078號及 10 PCT公告案第96/40772號；Balu之PCT公告案第WO 01/38342號；以及Smith-Swintosky等人之WO 01/91780號。 特別一組含有胜肽部分之胜肽經過識別，其成員結合至 EPO-R，且刺激EPO依賴型細胞增生。但至今為止識別之含 胜肽部分之胜肽，可於試管試驗刺激EPO依賴型細胞增 15 生，具有EC50值約20奈莫耳濃度(nM)至約250nM。如此要 求20nM至250nM胜肽濃度來刺激藉EPO刺激之最大細胞增 生的50%。 已知E P Ο - R激動劑有廣大潛力用於由此受器媒介之重 要生物活動之研究，以及用於治療疾病，仍然需要識別具 20 有增強能力及活性之胜肽EPO-R激動劑。本發明提供此種 化合物。 本節及全文說明書所引用及/或討論之參考文獻僅未 為澄清本發明之說明，絕非承認該參考文獻為本發明之「先 前技術」。 9 1344964 【明内3 發明概要 本發明提供新穎胜肽化合物，其為強度與活性劇增之 EPO-R激動劑。此等胜肽化合物為具有胺基酸序列 * 5 (AcG)GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1)之 , 胜肽單體之同質二元體，或具有胺基酸序列 (AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO： φ 2)之胜肽單體之同質二元體 '具有胺基酸序列 (AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG) (SEQ ID NO: 10 3)之胜肽單體之同質二元體；此處各個胺基酸係以標準單 字母縮寫表示。「（AcG)」為N-乙醯基甘胺酸，「（i-nai)」為 1-萘基丙胺酸及「（MeG)」為N-曱基甘胺酸也稱作肌胺酸。 胜肽一元體之各個胜肽單體含有一個分子内雙硫鍵介於單 體之半胱胺酸殘基間。 15 胜肽單體可藉共價附接至分支第三醯胺鍵聯基而二聚 • 合。第三醯胺鍵聯基可表示為： -c1o-ch2-x-ch2-c2o- ★ 此處x為nc〇-(CH2)2_n1h-;鍵聯基之c1與第一胜肽單體c 端離胺酸殘基之ε胺基形成醯胺鍵結；鍵聯基之〔2與第二 ^ 20胜肽單體c端離胺酸殘基之ε胺基形成醯胺鍵結 :以及X之 Ν係透過胺基曱酸酯鍵聯或醯胺鍵聯而附接至活化聚乙二 醇(PEG)部分’此處PEG具有分子量約20 000至約4〇〇〇〇道 爾吞（「約」一詞指示於準備PEG時，有些分子比所述分子 量更重或更輕）。 10 1344964 當同質二元體之各個單體具有胺基酸序列 (AcG)GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1)及 鍵聯基之N1係透過胺基曱酸酯鍵聯附接至活化聚乙二醇 (PEG)部分時，本發明之新穎胜肽化合物表示如後：The DNA sequences and coding sequences of the mouse EPO-R protein and the human EPO-R protein are described in PCT Publication No. WO 90/18822 to D'Andrea et al. This model suggests that EPO binds to EPO-R, resulting in dimerization and activation of two 15 EPO-R molecules, resulting in subsequent signal transduction steps [eg, see, for example, Watowich et al. (1992) Proc. Natl. Acad. Sci USA 89:2140-2144]. The EPO-R selection gene can be obtained to facilitate the study of agonists and antagonists of such important receptors. Recombinant receptor proteins can be obtained, allowing for the study of receptor-coordination interactions in a variety of random and semi-random peptide diversity production systems, including "peptides in colony" systems U.S. Patent No. 6,270,170; "Peptide in Phage" System [described in U.S. Patent No. 5,432,018 and Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382]; Synthetic Repository (ESL) system [described in U.S. Patent No. 8, 1 344,964, U.S. Patent Application Serial No. 946, 239, filed on Sep. 16, 1992; and "Extremely Large Scale Brake Polymer Synthesis System [described in U.S. Patent No. 5,143,854; PCT Notice Case No. 90/15070; Fodor et al. (1991) Science 251: 767-773; Dower and Fodor (1991) Ann. Ep. Med. Chem. 5 26:271-180; and U.S. Patent No. 5,424,186]. At least some degree of peptides of the EPO-R interaction have been identified, for example, in U.S. Patent Nos. 5,773,569, 5,830,851 and 5,986,047 to Wrighton et al.;\¥1^匕〇11, etc. (:1' Bulletin No. *0 96/40749; US Patent Nos. 5,767,078 and 10 PCT by Johnson and Zivin Prosecution No. 96/40772; PCT Bulletin No. WO 01/38342 to Balu; and WO 01/91780 by Smith-Swintosky et al. A special set of peptides containing a peptide moiety is identified and its members are bound to EPO-R, and stimulates EPO-dependent cell proliferation. However, the peptides that have been identified to date with the peptides can be stimulated by EPO-dependent cells in a test tube with an EC50 value of about 20 nanomolar (nM). Up to about 250 nM. This requires a peptide concentration of 20 nM to 250 nM to stimulate 50% of the maximum cell proliferation stimulated by EPO. It is known that EP Ο-R agonists have broad potential for the study of important biological activities of this receptor media, As well as for the treatment of diseases, it is still necessary to identify a peptide PEO ER-R agonist having enhanced ability and activity. The present invention provides such a compound. The references cited and/or discussed in this section and the entire specification are not clarified. The description of the invention is by no means an admission that the reference is a "prior art" of the present invention. 9 1344964 [Inventive 3 Summary of the Invention The present invention provides a novel peptide compound which is an EPO-R agonist with a sharp increase in strength and activity. The peptide compound is a homodimer having a amino acid sequence * 5 (AcG) GLYACHMGPIT (l-nal) VCQPLRK (SEQ ID NO: 1), a peptide monomer, or an amino acid sequence (AcG) GLYACHMGPIT (l-nal) VCQPLR(MeG)K (SEQ ID NO: φ 2) The homodimer of the peptide monomer' has an amino acid sequence (AcG) GLYACHMGPIT (l-nal) VCQPLR (MeG) (SEQ ID NO: 10 3) The homodimer of the peptide monomer; here each amino acid is represented by a standard one-letter abbreviation. "(AcG)" is N-ethinylglycine, "(i-nai)" is 1-naphthylalanine and "(MeG)" is N-mercaptoglycine also known as sarcosine. Each peptide monomer of the peptide monolayer contains an intramolecular disulfide bond between the cysteine residues of the monomer. 15 peptide monomers can be dimerized by covalent attachment to the branched third amine linkage. The third indole linkage can be expressed as: -c1o-ch2-x-ch2-c2o- ★ where x is nc〇-(CH2)2_n1h-; the c1 of the linkage group and the c-terminus of the first peptide monomer The ε-amine group of the lysine residue forms a guanamine bond; the bond group [2] forms a guanamine bond with the ε amine group of the amino acid residue at the c-terminus of the second -20 peptide monomer: and X The lanthanide is attached to the activated polyethylene glycol (PEG) moiety via an amine phthalate linkage or a guanamine linkage. Here PEG has a molecular weight of from about 20 000 to about 4 Torr. The term indicates that some molecules are heavier or lighter than the molecular weight when preparing PEG. 10 1344964 When each monomer of a homomeric binary body has an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1) and a N1 line of a linkage group is attached via an amine phthalate linkage By the activation of the polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention are represented as follows:

當同質二元體之各個單體具有胺基酸序列 (AcG)GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1)及 鍵聯基之N1係透過醯胺鍵聯附接至活化聚乙二醇(PEG)部 分時，本發明新穎胜肽化合物表示如後：When the individual monomers of the homogeneous binary have an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1) and the N1 system of the linkage is attached to the activated polyethylene via a guanamine linkage In the case of an alcohol (PEG) moiety, the novel peptide compounds of the invention are represented as follows:

(AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 11 1344964(AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 11 1344964

2)及鍵聯基之N1係透過胺基甲酸醋鍵聯附接至活化聚乙二 醇(PEG)部分時，本發明新穎胜肽化合物表示如後：2) When the N1 line of the bonding group is attached to the activated polyethylene glycol (PEG) moiety through the urethane linkage, the novel peptide compound of the present invention is expressed as follows:

-PEGjo^ok 當同質二元體之各個單體具有胺基酸序列 5 (AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 2)及鍵聯基之N1係透過醯胺鍵聯附接至活化聚乙二醇(peg) 部分時，本發明新穎胜肽化合物表示如後：-PEGjo^ok When the individual monomers of the homomorphic entity have the amino acid sequence 5 (AcG) GLYACHMGPIT (l-nal) VCQPLR(MeG)K (SEQ ID NO: 2) and the N1 line of the linkage group through the guanamine When the linkage is attached to the activated polyethylene glycol (peg) moiety, the novel peptide compounds of the invention are expressed as follows:

胜肽單體可藉共價附接至分支第三醯胺鍵聯基而二聚 10 合。第三醯胺鍵聯基可表示為： -c】o-ch2-x-ch2-c2o- 此處X為nco-(ch2)2-nh-c3o;鍵聯基之c1與第一胜肽單體 12 1344964 C端離胺酸殘基之ε胺基形成醯胺鍵結；鍵聯基之C2與第二 胜肽單體C端離胺酸殘基之ε胺基形成疏胺鍵結。本發明之 胜肽二元體進一步包含如下結構式之間隔基部分： -βΗ-ΚΗΑ-θΗ-Ν2!!- 5 此處C4為共價鍵結至X之C3之間隔基；間隔基之Ν1透過胺基 甲酸酯鍵聯或醢胺鍵聯共價附接至活化聚乙二醇(PEG)部 分；以及間隔基之N2係透過胺基曱酸酯鍵聯或醯胺鍵聯共 價附接至活化PEG部分，此處PEG具有分子量約10,〇〇〇至約 50,000道爾吞（「約」一詞表示於準備PEG時，某些分子可 10 能比所述分子量更重或更輕）。各個PEG部分個別為10,000 道爾呑（10kD)、20kD、30kD、40kD或 50kD。 當同質二元體之各個單體具有胺基酸序列 (AcG)GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1)及 間隔基之N1及N2皆係透過胺基曱酸酯鍵聯附接至活化聚乙 15 二醇(PEG)部分時，本發明新穎胜肽化合物表示如後：The peptide monomer can be dimerized by covalent attachment to the branched third guanamine linkage. The third indole linkage can be expressed as: -c]o-ch2-x-ch2-c2o- where X is nco-(ch2)2-nh-c3o; the linkage of c1 with the first peptide The C-terminus of the body 12 1344964 forms a guanamine bond with the epsilon amine group of the amine acid residue; the C2 of the bond group forms a serotonin bond with the ε amine group of the amine acid residue at the C-terminus of the second peptide monomer. The peptide peptide of the present invention further comprises a spacer moiety of the formula: -βΗ-ΚΗΑ-θΗ-Ν2!!- 5 where C4 is a spacer covalently bonded to C3 of X; Covalently attached to the activated polyethylene glycol (PEG) moiety via a urethane linkage or a guanamine linkage; and the N2 linkage of the spacer is covalently attached via an amine phthalate linkage or a guanamine linkage Connected to the activated PEG moiety, where PEG has a molecular weight of about 10, 〇〇〇 to about 50,000 Torr (the term "about" means that certain molecules can be heavier or lighter than the molecular weight when preparing PEG. ). Each PEG moiety is individually 10,000 Doha (10 kD), 20 kD, 30 kD, 40 kD or 50 kD. When each monomer of the homodimer has an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1) and the spacers N1 and N2 are attached via an amine phthalate linkage By the activation of the polyethylene glycol diol (PEG) moiety, the novel peptide compounds of the invention are represented as follows:

較佳具體例中，二胜肽單體之C端離胺酸為胺酸。 熟諳技蟄人士由前述化學結構式，瞭解兩個線性15£；(3部分 13 1344964 係藉離胺酸接合（例如呈mPEGrLys-NHS或呈 mPEG2-Lysinol-NPC)，也較佳為L離胺酸，獲得如下立體化 學。In a preferred embodiment, the C-terminal amino acid of the dipeptide monomer is an amine acid. Those skilled in the art are aware of two linear 15 £s from the aforementioned chemical structural formula; (3 part 13 1344964 is linked by an aminic acid (for example, mPEGrLys-NHS or mPEG2-Lysinol-NPC), also preferably L-amine For the acid, the following stereochemistry was obtained.

(AcGJGLYA^HMGPnXl-NaOviQPLR-NH^L.^ A(AcGJGLYA^HMGPnXl-NaOviQPLR-NH^L.^ A

5 另外’一或多個離胺酸殘基可為D離胺酸獲得其它熟諳 技藝人士瞭解之立體化合物。 當同質二元體之各個單體具有胺基酸序列 (AcG)GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1)及 間隔基之N1及N2皆係透過酿胺鍵聯附接至活化聚乙二醇 ® 10 (PEG)部分時，本發明新穎胜肽化合物表示如後： •’5 Further, one or more of the amino acid residues may be a stereo compound which is known to those skilled in the art from D to the amine acid. When each monomer of the homomorphic entity has an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1) and the spacers N1 and N2 are attached to the activated poly In the case of the ethylene glycol® 10 (PEG) moiety, the novel peptide compounds of the present invention are expressed as follows: • '

V x^-PEGk^cok ο PEGi〇^〇k 14 1344964 再度’本化合物之離胺酸分子較佳皆為L離胺酸，獲得 如下立體化學。V x^-PEGk^cok ο PEGi〇^〇k 14 1344964 Again, the lysine molecules of the present compound are preferably L-amino acids, and the following stereochemistry is obtained.

另外’一或多個離胺酸殘基可為D離胺酸獲得其它熟諳 5 技藝人士瞭解之立體化合物。當同質二元體之各個單體具有 胺基酸序列（AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 2)及間隔基之N]及N2皆係透過胺基甲酸酯鍵 聯附接至活化聚乙二醇(PEG)部分時，本發明新賴胜肤化合 物表示如後：Further, the one or more lysine residues may be a stereo compound known to those skilled in the art from D to the amine acid. When each monomer of the homodimer has an amino acid sequence (AcG) GLYACHMGPIT (l-nal) VCQPLR(MeG)K (SEQ ID NO: 2) and a spacer N] and N2 are all permeable to the uric acid When the ester linkage is attached to the activated polyethylene glycol (PEG) moiety, the novel Lai Sheng skin compound of the present invention is expressed as follows:

較佳於本分子接合胜肽單體及線性PEG部分之離胺酸 殘基皆為L離胺酸’獲得如下立體化學： 15 1344964Preferably, the molecules of the conjugated peptide monomer and the linear PEG moiety are all from the amine acid residue of the L-amino acid. The following stereochemistry is obtained: 15 1344964

P£G10_5dkP£G10_5dk

10-50K 另外’一或多個離胺酸殘基可為D離胺酸獲得其它熟諳 技藝人士瞭解之立體化合物。 當同質二元體之各個單體具有胺基酸序列 5 (AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 2)及間隔基之N1及N2皆係透過醯胺鍵聯附接至活化聚乙二 醇(PEG)部分時’本發明新穎胜肽化合物表示如後： 0 (AcGXH-YACHMGPrr(l-ittl)VCQPLR(MeiFurther, one or more of the amino acid residues may be a stereo compound which is known to those skilled in the art from D to the amine acid. When each monomer of the homodimer has an amino acid sequence 5 (AcG) GLYACHMGPIT (l-nal) VCQPLR(MeG)K (SEQ ID NO: 2) and the spacers N1 and N2 are linked through a guanamine linkage When attached to an activated polyethylene glycol (PEG) moiety, the novel peptide compound of the invention is represented as follows: 0 (AcGXH-YACHMGPrr(l-ittl)VCQPLR(Mei

〇 Ο V NH〇 Ο V NH

Ε^ιο^οκΕ^ιο^οκ

NH ·· ΗΝ xj^^PEGi tte〇K Ο (AcOjOLYAiptMGHTO^naDV^QPLRtMeGy^H)/ 較佳於本分子接合胜肽單體及線性PEG部分之離胺酸 殘基皆為L離胺酸，獲得如下立體化學。 16 1344964NH ·· ΗΝ xj^^PEGi tte〇K Ο (AcOjOLYAiptMGHTO^naDV^QPLRtMeGy^H)/ Preferably, the amino acid residues of the conjugated peptide monomer and the linear PEG moiety are L-amino acid. The following stereochemistry. 16 1344964

其它具體例中，一或多個離胺酸殘基可為D離胺酸獲得 其它熟諳技藝人士瞭解之立體化合物。 胜肽單體也可藉附接至離胺酸鍵聯基而二聚合，藉此 5 一個胜肽單體之C端附接至離胺酸之ε胺基，而第二胜肽單 體之C端附接至離胺酸之α胺基。 本發明之胜肽二元體進一步包含如下結構式之間隔基 部分： -N1H-(CH2)2-0-(CH2)2-N2H- 10 於一端，間隔基之N1係透過醯胺鍵聯附接至離胺酸鍵聯基 之羰基碳。於相反端，間隔基之N2透過胺基曱酸酯鍵聯或 酿胺鍵聯附接至活化聚乙二醇(PEG)部分，此處PEG具有分 子1約20,〇〇〇至約40,000道爾呑（「約」一詞表示於準備pEG 時，有些分子可能比所述分子量更重或更輕）。 15 當間隔基係透過胺基曱酸酯鍵聯附接至活化聚乙二醇 (PEG)部分時’本發明新穎胜肽化合物表示為： 17 1344964In other embodiments, one or more of the lyophilic acid residues may be D-amino acid obtained from other stereochemical compounds known to those skilled in the art. The peptide monomer can also be dimerized by attachment to an amide linkage, whereby the C-terminus of the 5 peptide monomers is attached to the epsilon amine group of the amine acid, and the second peptide monomer The C-terminus is attached to an alpha amine group of an amine acid. The peptide peptide of the present invention further comprises a spacer moiety of the formula: -N1H-(CH2)2-0-(CH2)2-N2H- 10 at one end, and the spacer N1 is attached via a guanamine bond Attached to the carbonyl carbon of the amine linkage. At the opposite end, the N2 of the spacer is attached to the activated polyethylene glycol (PEG) moiety via an amine phthalate linkage or a urethane linkage, where PEG has a molecule of about 20, 〇〇〇 to about 40,000 channels. Er (the term "about" means that some molecules may be heavier or lighter than the molecular weight when preparing pEG). 15 When the spacer is attached to the activated polyethylene glycol (PEG) moiety via an amino phthalate linkage, the novel peptide compound of the invention is represented by: 17 1344964

〇-peg2IM0K〇-peg2IM0K

mwt 當間隔基係透過酿胺鍵聯附接至活化聚乙二醇(peg) 部分時，本發明新穎胜肽化合物表示為：Mwt When the spacer is attached to the activated polyethylene glycol (peg) moiety via a chiral linkage, the novel peptide compound of the invention is represented by:

本發明進一步提供包含此等胜肽化合物之藥學組成 物，以及使用此等胜肽化合物治療各種醫療情況之方法。 I：實施方式】 較佳實施例之詳細說明 定義： 於胜肽之胺基酸殘基縮寫如後：苯基丙胺酸為Phe或 F;白胺酸為Leu或L;異白胺酸為I〗e或I;蛋胺酸為Met4M; 網胺酸為Val或V ;絲胺酸為Ser或S ;脯胺酸為pro或p ;蘇 胺酸為Thr或T ;丙胺酸為Ala或A ;酪胺酸為Tyr或Y ;組胺 酸為His或Η ;麩胺為Gln或Q ;天冬醯胺為Asn或N ;離胺酸 為Lys或K ;天冬酸為Arg或D ;麩胺酸為Glu或E ;半胱胺酸 為Cys或C ;色胺酸為Trp或w ;精胺酸為Arg或R ;及甘胺酸 為Gly或G。胜肽之非習常胺基酸縮寫如後：萘基丙胺酸 為Ι-nal或Np ; N-甲基甘胺酸（也稱作為肌胺酸）為]yieG或 18 15 1344964The invention further provides pharmaceutical compositions comprising such peptide compounds, and methods of using the peptide compounds to treat various medical conditions. I: Embodiments The detailed description of the preferred embodiment is defined as follows: The amino acid residue of the peptide is abbreviated as follows: phenylalanine is Phe or F; leucine is Leu or L; isoleucine is I 〖e or I; methionine is Met4M; retic acid is Val or V; serine is Ser or S; proline is pro or p; threonine is Thr or T; alanine is Ala or A; Tyrosine is Tyr or Y; histidine is His or Η; glutamine is Gln or Q; asparagine is Asn or N; lysine is Lys or K; aspartic acid is Arg or D; glutamine The acid is Glu or E; the cysteine is Cys or C; the tryptophan is Trp or w; the arginine is Arg or R; and the glycine is Gly or G. The non- conventional amino acid abbreviation of the peptide is as follows: naphthylalanine is Ι-nal or Np; N-methylglycine (also known as sarcosine) is]yieG or 18 15 1344964

Sc ;及乙醯化甘胺酸(N-乙醯基甘胺酸）為AcG。 如此處使用，「多肽」或「蛋白質」等詞表示α胺基酸 經由醯胺鍵結接合在一起之胺基酸單體之聚合物。因此多 肽長度至少兩個胺基酸殘基，通常更長。通常「胜肽」一 5 詞表示長度只有數個胺基酸殘基之多肽》本發明之新穎 EPO-R激動劑胜肽較佳長度不超過50個胺基酸殘基。更佳 長度約17至約40胺基酸殘基。與胜肽相反，多肽可包含任 何數目之胺基酸殘基。因此多肽一詞包括胜肽及更長之胺 基酸序列。 10 如此處使用，「藥學上可接受」一詞表示「通常被視為 安全」之分子實體及組成物’例如為生理可财受，且典型 不會產生過敏反應等非期望之反應，例如投予人體時不會 造成胃部不適、頭昏眼花等。較佳用於此處，「藥學上可接 受：」一詞表示由聯邦法規或州政府核准、或列舉於美國藥 15典或其它公認藥典可供動物使用，特別人類使用。「載劑」 一詞表示藉其來投予化合物之稀釋劑、佐劑、賦形劑或媒 劑。此卓樂學載劑可為無函液體，例如水及油類，包括石 油、動植物或合來源之油類，例如花生油、大豆油、礦油、 芝麻油等。水或水溶液、食鹽水溶液及水性葡萄糖溶液及 20甘油溶液較佳用作為載劑，特別用於注射溶液劑。適當藥 學載劑述於「雷明頓藥學科學」，作者E.w Martin。 如此處使用「激動劑」一詞表示生物活性配位子，其 結合至其互補生物活性受器，且活化受器，引發受器之生 物反應，或增強既存之受器生物活性。 19 1344964 屬於ΕΡΟ-R激動劑之新Μ胜肽 本發明提供新穎胜肽化合物，其為強度與活性劇增之 EPO-R激動劑。此等胜肽化合物為具有胺基酸序列 (AcG)GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1)之 * 5 胜肽單體之同質二元體，或具有胺基酸序列 (AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 2)之胜肽單體之同質二元體；此處各個胺基酸係以標準單 | 字母縮寫表示。「（AcG)」為N·乙醯基甘胺酸，「（Ι-nal)」為 1-萘基丙胺酸及「（MeG)」為N-甲基甘胺酸也稱作肌胺酸。 10 胜肽二元體之各個胜肽單體含有一個分子内雙硫鍵介於單 體之半胱胺酸殘基間。此等單體可示意表示如後： (AcG)GLYA<!：HMGPrr(l*Ml)V(!；〇PLRK (AcG)GLYACHMGPIT(l-nal)VCQPLRK ^ _ 或 - ；及Sc; and acetylated glycine (N-ethinylglycine) are AcG. As used herein, the terms "polypeptide" or "protein" refer to a polymer of an amino acid group in which an alpha amino acid is bonded via a guanamine linkage. Thus the polypeptide is at least two amino acid residues in length, usually longer. Generally, the word "peptide" is a polypeptide having a length of only a few amino acid residues. The novel EPO-R agonist peptide of the present invention preferably has a length of no more than 50 amino acid residues. More preferably, the length is from about 17 to about 40 amino acid residues. In contrast to the peptide, the polypeptide may comprise any number of amino acid residues. Thus the term polypeptide includes both the peptide and the longer amino acid sequence. 10 As used herein, the term "pharmaceutically acceptable" means that the molecular entity and composition "usually considered safe" is, for example, physiologically acceptable, and typically does not produce an undesired reaction, such as an allergic reaction. It does not cause stomach upset, dizziness, etc. when given to the human body. Preferably used herein, the term "pharmaceutically acceptable:" means approved by the federal or state government, or listed in the US Pharmacopoeia or other recognized pharmacopoeia for use by animals, particularly humans. The term "carrier" means a diluent, adjuvant, excipient or vehicle with which the compound is administered. The Zhuo Le learning carrier can be a non-functional liquid such as water and oil, including oils, animals, plants or oils of various origins, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or an aqueous solution, an aqueous salt solution and an aqueous dextrose solution and a 20 glycerin solution are preferably used as a carrier, particularly for injectable solutions. The appropriate pharmaceutical carrier is described in "Remington Pharmaceutical Sciences" by E.w Martin. The term "agonist" as used herein refers to a biologically active ligand that binds to its complementary biologically active receptor and activates the receptor, initiates a biological response of the receptor, or enhances the existing receptor biological activity. 19 1344964 Novel oxime peptides belonging to ΕΡΟ-R agonists The present invention provides novel peptide compounds which are EPO-R agonists with a dramatic increase in strength and activity. These peptide compounds are homodimers of a *5 peptide monomer having an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1), or have an amino acid sequence (AcG) GLYACHMGPIT (l-nal) VCQPLR(MeG)K (SEQ ID NO: 2) is a homodimer of the peptide monomer; here each amino acid is represented by the standard single | letter abbreviation. "(AcG)" is N. acetylglycolic acid, "(Ι-nal)" is 1-naphthylalanine and "(MeG)" is N-methylglycine, also called sarcosine. Each peptide monomer of the 10 peptide peptide contains an intramolecular disulfide bond between the cysteine residues of the monomer. These monomers can be schematically represented as follows: (AcG)GLYA<!:HMGPrr(l*Ml)V(!;〇PLRK(AcG)GLYACHMGPIT(l-nal)VCQPLRK^ _ or - ;

(AcG)GLYA(lHMGPm(lHial)viQPLRiMeaiK (AcG)GLYACHMGPIT(l-n#l)VCQPLK<MeG)K 或 此等胜肽單體二聚合來提供具有增強之EP〇_R激動劑 丨活性之胜肽二元體。鍵聯基(LK)部分為分支第三醯胺，其 15經由同時附接至各個單體之C端離胺酸殘基而橋接二胜肽 單體C端。第三醯胺鍵聯基可表示為： m -C,0-CH2-X-CH2-C20- /此處X為ΝΟΟ-βΗ2)2-：^!!-;鍵聯基之c1與第一胜肽單體c 端離胺酸殘基之ε胺基形成醯胺鍵結；鍵聯基之c2與第二 20胜肽單體匚端離胺酸殘基之ε胺基形成醯胺鍵結；以及X之 Ν1係透過胺基甲酸酯鍵聯或醯胺鍵聯而附接至活化聚乙二 醇(PEG)部分’此處PEG具有分子量約20,000至約40,000道 20 1344964 爾吞（「約」一詞指示於準備PEG時，有些分子比所述分子 量更重或更輕）。 苐二酿胺鍵聯基可表示為： -C^O-CHyX-CHa-C2。- 5 此處X為NC0-(CH2)2-NH-C30;鍵聯基之C1與第一胜肽單體 C端離胺酸殘基之£胺基形成醯胺鍵結；鍵聯基之C2與第二 胜肽單體C端離胺酸殘基之ε胺基形成醯胺鍵結。本發明之 胜肽二元體進一步包含如下結構式之間隔部分： -βίΗα^νθΗ-Ν2^ 10 此處間隔基之c4係共價鍵結至X之C3;間隔基之Ν1係透過胺 基曱酸酯鍵聯或醯胺鍵聯附接至活化PEG部分，此處PEG 具有分子量約20,000至約40,000道爾吞（「約」一詞表示於 準備PEG時，有些分子可能比所述分子量更重或更輕）。 如此本發明之新穎胜肽也含有一個PEG部分’其係透 15過胺基甲酸酯鍵聯或醯胺鍵聯共價附接至胜肽二元體之第 三醯胺鍵聯基。PEG為藥學上可接受之水溶性聚合物。用 於本發明之PEG可為具有分子量為約20千道爾吞(20K)至約 60Κ之線性非分支pEG(「約」一詞表示於PEG製備中，有些 分子比所述分子量更重，有些比所述分子量更輕）。最佳PEG 20 具有分子量約30K至約40K。熟諳技藝人士可基於下列考量 選擇所需聚合物大小：例如期望劑量；循環時間；對蛋白 質分解之抗;!生；對生物活性之影響（若有）；處理上容易；抗 原性程度或缺抗原性；以及其它已知對治療性胜肽之影響。 本發明之胜肽、胜肽二元體及其它基於胜肽之分子可 21 1344964 使用多種化學之任一種，鍵聯水溶性聚合物至分子(例如胜 肽+間隔基）之受器結合部分而附接至水溶性聚合物（例如 PEG)。典型具體例採用單一附接接合讓水溶性聚合物共價 附接至受器結合部分，但於其它具體例中，可使用多重附 * 5接接合，包括各種變化，其中不同種水溶性聚合物附接至 爻器接合部分之分開附接接合，可包括共價附接接合至間 隔基及/或接合至一或二胜肽鏈。若干具體例中，二元體或 • 更尚級多元體將包含多種不同胜狀鏈（亦即非同質二元體 或其它非同質多元體）。舉例言之但非限制性，二元體可包 1〇 含一第一胜肽鏈其具有PEG附接接合，以及一第二胜肽鏈 缺乏PEG附接接合，或利用與第一胜肽鏈不同的鍵聯化 學’且於某些變化例，間隔基可含有或缺乏pEG附接接合， 且該間隔基於PEG化時可利用與第一胜肽鏈及/或第二胜肽 鏈不同的鍵聯化學》另一具體例採用PEG附接至受器結合 15 部分之間隔基部分，不同水溶性聚合物（例如碳水化合物） | 軛合至分子胜肽部分之胺基酸之一的支鏈。 多種聚乙二醇(PEG)可用於受器結合部分（胜肽+間隔 基)之PEG化。實質上可使用任何適當反應性PEG試劑。較 佳具體例中，反應性PEG試劑當軛合至受器結合部分時， 〆 20 將導致生成胺基甲酸酯鍵或醯胺鍵。適當反應性PEG包括 (但非限制性）NOF公司（Yebisu Garden Place Tower，20-3 Ebisu 4-chome, Shibuya-ku ’ 東京 150-6019)之藥物輸送系統 型錄（2003年）購得，以及由Nektar治療公司（阿拉巴馬州 35806漢茲維爾發現大道490號)之分子工程型錄(2003年）購 22 1344964(AcG)GLYA(lHMGPm(lHial)viQPLRiMeaiK(AcG)GLYACHMGPIT(ln#l)VCQPLK<MeG)K or these peptide monomers are dimerized to provide a peptide II with enhanced EP〇_R agonist activity Yuan body. The linker (LK) moiety is a branched third guanamine which bridges the C-terminus of the dipeptide monomer via simultaneous attachment to the C-terminal amine acid residue of each monomer. The third indole linkage can be expressed as: m -C,0-CH2-X-CH2-C20- / where X is ΝΟΟ-βΗ2)2-:^!!-; the linkage group c1 and the first The c-terminus of the peptide monomer forms a guanamine bond from the epsilon amine group of the amine acid residue; the c2 of the bond group forms a guanamine bond with the ε amine group of the second 20-peptide monomer terminal and the amino acid residue And X Ν 1 is attached to the activated polyethylene glycol (PEG) moiety via a urethane linkage or a guanamine linkage. Here PEG has a molecular weight of from about 20,000 to about 40,000 channels 20 1344964 ernt (" The term "about" indicates that some molecules are heavier or lighter than the molecular weight when preparing PEG). The hydrazine amine linkage can be expressed as: -C^O-CHyX-CHa-C2. - 5 where X is NC0-(CH2)2-NH-C30; the C1 of the bond group forms a guanamine bond with the amine group of the C-terminus of the first peptide monomer from the amine acid residue; C2 forms a guanamine linkage with the epsilon amine group of the amino acid residue at the C-terminus of the second peptide monomer. The peptide peptide of the present invention further comprises a spacer moiety of the following structural formula: -βίΗα^νθΗ-Ν2^ 10 where the c4 group of the spacer is covalently bonded to C3 of X; the Ν1 of the spacer is permeable to the amine group An acid ester linkage or a guanamine linkage is attached to the activated PEG moiety, where PEG has a molecular weight of from about 20,000 to about 40,000 Dolphant (the term "about" means that some molecules may be heavier than the molecular weight when preparing the PEG. Or lighter). Thus, the novel peptide of the present invention also contains a PEG moiety which is covalently attached to the third guanamine linkage of the peptide binary via a urethane linkage or a guanamine linkage. PEG is a pharmaceutically acceptable water soluble polymer. The PEG used in the present invention may be a linear non-branched pEG having a molecular weight of about 20 kD (20K) to about 60 F (the term "about" is used in the preparation of PEG, and some molecules are heavier than the molecular weight, some Lighter than the molecular weight). The preferred PEG 20 has a molecular weight of from about 30K to about 40K. A skilled artisan can select the desired polymer size based on the following considerations: for example, the desired dose; cycle time; resistance to protein breakdown; growth; effects on biological activity (if any); ease of treatment; degree of antigenicity or lack of antigen And other effects known to be therapeutic peptides. The peptide of the present invention, the peptide binary and other peptide-based molecules can be used in any of a variety of chemistries to bond a water-soluble polymer to a receptor binding moiety of a molecule (eg, a peptide + spacer). Attached to a water soluble polymer (eg PEG). A typical embodiment uses a single attachment joint to covalently attach a water soluble polymer to the receptor binding portion, but in other embodiments, multiple attachments can be used, including various variations, among which different types of water soluble polymers A separate attachment joint attached to the jaw engagement portion can include covalent attachment to the spacer and/or to the one or two peptide chain. In a number of specific examples, a binary or a more versatile polymorph will contain a plurality of different winning chains (i.e., non-homologous or other non-homogeneous plurals). By way of example and not limitation, the binary may comprise a first peptide chain having a PEG attachment junction, and a second peptide chain lacking a PEG attachment linkage, or utilizing a first peptide chain Different linkage chemistry 'and in some variations, the spacer may contain or lack a pEG attachment junction, and the spacer may utilize a different bond than the first peptide chain and/or the second peptide chain based on PEGylation Another specific example uses a PEG attached to the spacer portion of the acceptor binding 15 moiety, a different water soluble polymer (e.g., carbohydrate) | a branch that is conjugated to one of the amino acids of the molecular peptide moiety. A variety of polyethylene glycols (PEG) can be used for PEGylation of the receptor binding moiety (peptide + spacer). Essentially any suitable reactive PEG reagent can be used. In a preferred embodiment, when the reactive PEG reagent is conjugated to the acceptor binding moiety, hydrazine 20 will result in the formation of a urethane linkage or a guanamine linkage. Suitable reactive PEGs include, but are not limited to, the NOF Corporation (Yebisu Garden Place Tower, 20-3 Ebisu 4-chome, Shibuya-ku 'Tokyo 150-6019) drug delivery system catalogue (2003), and Molecular Engineering Catalogue (2003) by Nektar Therapeutics Inc. (490 Discovery Avenue, Huntsville, Alabama) purchased 22 1344964

得。例如（但非限制性)下列PEG試劑於多個具體例經常為較 佳：mPEG2-NHS、mPEG2-ALD、多臂 PEG、mPEG(MAL)2、 mPEG2(MAL)、mPEG-NH2、mPEG-SPA、mPEG-SBA、 mPEG-硫醋、mPEG-複醋、mPEG-BTC、mPEG-ButyrALD、 5 mPEG-ACET、雜官能基 PEGs (NH2-PEG-COOH， Boc-PEG-NHS ，Fmoc-PEG-NHS ，NHS-PEG-VS ， NHS-PEG-MAL)、PEG丙烯酸酯（ACRL-PEG-NHS)、PEG-磷脂質（例如mPEG-DSPE)、山布來特(SUNBRITE)系列多臂 PEGs包括藉熟諳技藝人士選用之化學活化之以甘油為主之 10 PEGs之GL系列、任一種山布來特活化PEGs(包括但非限於 羧基-PEGs ' p-NP-PEGs、Tresyl-PEGs、醛 PEGs、乙醛 -PEGs、胺基-PEGs、毓基-PEGs、順丁烯二醯亞胺基-PEGs、 羥基-PEG-胺、胺基-PEG-COOH、羥基-PEG-醛、羧酸酐型 -PEG、官能化PEG-磷脂質及其它類似及/或適當之如熟諳技 15 藝人士選用於特殊應用及用途之反應性PEG)。 本發明之新穎胜肽也含有二PEG部分，其透過胺基甲 着 酸酯鍵聯或醯胺鍵聯而共價附接至間隔基部分，其中間隔 接部分係共價鍵結至胜肽二元體之第三醯胺鍵聯基。用於 一 本發明之此一具體例之兩個PEG部分各別為線性，且可於 20 單一附接點鍵聯。各個PEG部分較佳具有分子量約1〇千道 爾吞（10K)至約60K(「約」一詞表示於準備peg時，有些分 子可能比所述分子量更重或更輕）❶以線性PEG部分為特 佳。更佳’兩個PEG部分各自具有分子量約20K至約40K， 又更佳約20K至約40K。又更佳兩個PEG部分各自具有分子 23 1344964 量約20K。熟諳技藝人士可基於下列考量選用所需聚合物大 小：例如期望劑量、循環時間；對蛋白質分解之抗性；對 生物活性之影響（若有）；操作容易；抗原性程度或缺抗原 性；其它已知PEG對治療性胜肽之影響。 5 本發明也包含胜肽激動劑其為具有胺基酸序列 (AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG) (SEQ ID NO: 3)之胜肽單體之同質二元體，此處各個胺基酸係以標準單 子母縮寫表示，「（ACG)」為N-乙醯基甘胺酸，「（1 _nai)」為 1-萘基丙胺酸及「（MeG)」為N-甲基甘胺酸，也稱作為肌胺 10 酸。胜肽二元體之各個胜肽單體含有分子内雙硫鍵於單體 之半胱胺酸殘基間。此種單體可示意表示如後： (AcG)OLYA<!HMGPIT(l-Ml)viQl>LR(MeG)或(Ac〇)GLYACHMGFrr(l-P«l)VCQPm(MeO) 此等單體胜肽經過二聚合來提供EPO-R激動劑活性增 強之胜肽二元體。鍵聯基(LK)部分為離胺酸殘基，鍵聯基 15 部分藉同時附接至各個單體之C端胺基酸來橋接兩個胜肽 單體之C端個胜肽單體之C端係附接至離胺酸之ε -胺 基，以及第二胜肽單體之C端係附接離胺酸之α-胺基。例 如，二元體可以結構式顯示於式I且摘要顯示如式II : ΜGot it. For example, but not by way of limitation, the following PEG reagents are often preferred in a number of specific examples: mPEG2-NHS, mPEG2-ALD, multi-arm PEG, mPEG (MAL) 2, mPEG2 (MAL), mPEG-NH2, mPEG-SPA , mPEG-SBA, mPEG-sulfuric acid, mPEG-complex vinegar, mPEG-BTC, mPEG-ButyrALD, 5 mPEG-ACET, heterofunctional PEGs (NH2-PEG-COOH, Boc-PEG-NHS, Fmoc-PEG-NHS , NHS-PEG-VS, NHS-PEG-MAL), PEG acrylate (ACRL-PEG-NHS), PEG-phospholipid (eg mPEG-DSPE), SUNBRITE series of multi-arm PEGs including cooked 谙Chemically activated glycerol-based GL series of 10 PEGs, glycerol-activated PEGs (including but not limited to carboxyl-PEGs 'p-NP-PEGs, Tresyl-PEGs, aldehyde PEGs, acetaldehyde) -PEGs, Amino-PEGs, Mercapto-PEGs, Maleimide-PEG-PEGs, Hydroxy-PEG-amines, Amino-PEG-COOH, Hydroxy-PEG-aldehydes, Carboxylic anhydrides-PEG, Functional PEG-phospholipids and other similar and/or suitable reactive PEGs selected by those skilled in the art for specific applications and uses. The novel peptide of the present invention also contains a diPEG moiety which is covalently attached to the spacer moiety via an aminoester linkage or a guanamine linkage, wherein the spacer moiety is covalently bonded to the peptide II The third indoleamine linkage of the moiety. The two PEG moieties used in this particular embodiment of the invention are each linear and can be bonded at 20 single attachment points. Each PEG moiety preferably has a molecular weight of from about 1 kilogram to 10 kilograms (about 10K) to about 60K (the term "about" means that some molecules may be heavier or lighter than the molecular weight when preparing peg) and the linear PEG moiety It is especially good. More preferably, the two PEG moieties each have a molecular weight of from about 20K to about 40K, and more preferably from about 20K to about 40K. Still more preferably, the two PEG moieties each have a molecular weight of about 20K. A skilled artisan can select the desired polymer size based on considerations such as desired dosage, cycle time; resistance to protein breakdown; effects on biological activity (if any); ease of handling; degree of antigenicity or lack of antigenicity; The effect of PEG on therapeutic peptides is known. 5 The present invention also encompasses a peptide agonist which is a homodimeric monomer having a peptide monomer having an amino acid sequence (AcG) GLYACHMGPIT (l-nal) VCQPLR (MeG) (SEQ ID NO: 3), each of which is The amino acid is represented by a standard abbreviation, "(ACG)" is N-acetylglycine, "(1 _nai)" is 1-naphthylalanine and "(MeG)" is N-methyl. Glycine, also known as myosin 10 acid. Each peptide monomer of the peptide binary contains an intramolecular disulfide bond between the cysteine residues of the monomer. Such a monomer can be schematically represented as follows: (AcG)OLYA<!HMGPIT(l-Ml)viQl>LR(MeG) or (Ac〇)GLYACHMGFrr(lP«l)VCQPm(MeO) Dimerization provides a peptide peptide with enhanced EPO-R agonist activity. The linking group (LK) moiety is an amino acid residue, and the linking group 15 is partially attached to the C-terminal amino acid of each monomer to bridge the C-terminal peptide monomer of the two peptide monomers. The C-terminus is attached to the epsilon-amine group of the amine acid, and the C-terminus of the second peptide monomer is attached to the a-amine group of the amine acid. For example, a binary body can be shown in formula I and the summary is shown in formula II:

式IIFormula II

24 20 1344964 式I及式II中，N2表示離胺酸之ε胺基之氮原子，以及 Ν1表示離胺酸之α-胺基之氮原子。 本發明之胜肽二元體進一步包含如下結構式之間隔基 部分： 5 ^^-(^2)2-0-(^2)2^¾ 於一端，間隔基之N1係透過醯胺鍵聯附接至離胺酸鍵 聯基之羰基碳。於相反端，間隔基之N2透過胺基曱酸酯或 醯胺鍵聯附接至活化聚乙二醇(PEG)部分，此處PEG具有分 子量約10,000至約60,000道爾吞（「約」一詞表示於準備peg 10 時，有些分子可能比所述分子量更重或更輕）。更佳PEG具 有分子量約20,000至40,000道爾吞。 如此本發明之新穎胜肽也含有一個PEG部分，其係共 價附接至胜肽二元體。PEG為藥學上可接受之水溶性聚合 物。用於本發明之PEG可為具有分子量可為約20千道爾吞 15 (20K)至約60K之線性非分支PEG(「約」一詞表示於pEG製 備中’有些分子比所述分子量更重，有些比所述分子量更 輕）。最佳PEG具有分子量約20K至約40K，又更佳具有分子 量約30K至約40K。熟諳技藝人士可基於下列考量選擇所需 聚合物大小：例如期望劑量；循環時間；對蛋白質分解之 20抗性；對生物活性之影響（若有）；處理上容易；抗原性程度 或缺抗原性；以及其它已知對治療性胜肽之影響。 當同質二元體之各個單體具有胺基酸序列 (AcG)GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1)及 鍵聯基之N1係透過胺基甲酸酯鍵聯附接至活化聚乙二醇 25 1344964 (PEG)部分時，本發明之新穎胜肽化合物表示如後：24 20 1344964 In the formulae I and II, N2 represents a nitrogen atom of the epsilon amine group of the amine acid, and Ν1 represents a nitrogen atom of the α-amino group of the amine acid. The peptide peptide of the present invention further comprises a spacer moiety of the formula: 5 ^^-(^2)2-0-(^2)2^3⁄4 at one end, the spacer N1 is linked through a guanamine linkage Attached to the carbonyl carbon of the amine linkage. At the opposite end, the spacer N2 is attached to the activated polyethylene glycol (PEG) moiety via an amine phthalate or guanamine linkage, where PEG has a molecular weight of from about 10,000 to about 60,000 dolphine ("about" one The words indicate that some molecules may be heavier or lighter than the molecular weight when preparing peg 10.) More preferred PEGs have a molecular weight of from about 20,000 to 40,000 dolphins. Thus, the novel peptide of the present invention also contains a PEG moiety which is covalently attached to the peptide binary. PEG is a pharmaceutically acceptable water soluble polymer. The PEG used in the present invention may be a linear non-branched PEG having a molecular weight of from about 20 kilo Torr 15 (20K) to about 60K (the term "about" is used in the preparation of pEG" and some molecules are heavier than the molecular weight. Some are lighter than the molecular weight). The preferred PEG has a molecular weight of from about 20K to about 40K, and more preferably has a molecular weight of from about 30K to about 40K. A skilled artisan can select the desired polymer size based on the following considerations: for example, the desired dose; cycle time; resistance to protein breakdown 20; effect on biological activity (if any); ease of handling; degree of antigenicity or lack of antigenicity And other effects known to be therapeutic peptides. When the individual monomers of the homogeneous binary have an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1) and the N1 line of the linkage is attached to the activation via a urethane linkage In the case of the polyethylene glycol 25 1344964 (PEG) moiety, the novel peptide compounds of the invention are represented as follows:

當同質二元體之各個單體具有胺基酸序列 (AcG)GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1)及 鍵聯基之N1係透過醯胺鍵聯附接至活化聚乙二醇(PEg)部 分時，本發明新穎胜肽化合物表示如後： (AcO)OLYACHMGPIT(l-naJ)VCQPLS：When the individual monomers of the homogeneous binary have an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1) and the N1 system of the linkage is attached to the activated polyethylene via a guanamine linkage In the case of the alcohol (PEg) moiety, the novel peptide compounds of the present invention are expressed as follows: (AcO)OLYACHMGPIT(l-naJ)VCQPLS:

NH t^EG2〇-4〇kNH t^EG2〇-4〇k

(AcG)GLYA<&iMGPIT(l *nel)viQPI 當同質二元體之各個單體具有胺基酸序列 (AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 10 2)及鍵聯基之N1係透過胺基甲酸酯鍵聯附接至活化聚乙二 醇(PEG)部分時，本發明新穎胜肽化合物表示如後·· 26 1344964(AcG)GLYA<&iMGPIT(l*nel)viQPI When the individual monomers of the homomorphic entity have an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 10 2) And the N1 line of the linkage group is attached to the activated polyethylene glycol (PEG) moiety through a urethane linkage, and the novel peptide compound of the present invention is represented by the latter 26 2634464

-PEG2O40K 當同質二元體之各個單體具有胺基酸序列 (AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 2)及鍵聯基之N1係透過醯胺鍵聯附接至活化聚乙二醇(PEG) 5 部分時，本發明新穎胜肽化合物表示如後： Ο (AeG)OLYA<iHMGPrT(I -nal)vlQPLR(MeG). ο Ο 〇20-ΜΚ-PEG2O40K When the individual monomers of the homoduplex have an amino acid sequence (AcG) GLYACHMGPIT (l-nal) VCQPLR(MeG)K (SEQ ID NO: 2) and the N1 system of the linkage group is attached via a guanamine bond Upon addition to the activated polyethylene glycol (PEG) 5 moiety, the novel peptide compounds of the present invention are represented as follows: Ο (AeG)OLYA<iHMGPrT(I-nal)vlQPLR(MeG). ο Ο 〇20-ΜΚ

Ο (AcO)GLYACHMGPm^n^)VCQPLR(MeG) 本發明之較佳胜肽二元體包括（但非限制性）： 27 1344964Ο (AcO) GLYACHMGPm^n^) VCQPLR (MeG) The preferred peptide binary of the present invention includes, but is not limited to: 27 1344964

(AcG)GLYACHMGPrT(l^iaI)VCQPI Ο I 〇(AcG)GLYACHMGPrT(l^iaI)VCQPI Ο I 〇

V NH -FEGjokV NH -FEGjok

(AcG)GL YACHMOPITY1 -naQVCQPI(AcG)GL YACHMOPITY1 -naQVCQPI

(AcOGLYAdHMGPrTil-nalJVCQPI(AcOGLYAdHMGPrTil-nalJVCQPI

OO

°y NH 0 〇 -PEGsok (AcG)GLYACHMGPIT( l -nal) VCQPLR-°y NH 0 〇 -PEGsok (AcG)GLYACHMGPIT( l -nal) VCQPLR-

OO

(AcG)GLYA<!hMGPIK1 -naJ)viQPLR-NH JL 〇 〇(AcG)GLYA<!hMGPIK1 -naJ)viQPLR-NH JL 〇 〇

(AcG)GLYACHMGPIT(t-nal)VCQPLR-HN ^-NH2 28 1344964 (AcG)GLYACHMGPrr(l -nal)V6QPLR- 0(AcG)GLYACHMGPIT(t-nal)VCQPLR-HN ^-NH2 28 1344964 (AcG)GLYACHMGPrr(l -nal)V6QPLR- 0

NH (AcG)OLYACHMGPIT(l-Dal)VajPLR-HN ^NH (AcG)OLYACHMGPIT(l-Dal)VajPLR-HN ^

V NHV NH

Ni£GNi£G

20K (AcG)GLYA(iHMGPIT(l-nal)VCQ]20K (AcG)GLYA(iHMGPIT(l-nal)VCQ]

0 PLR-NH0 PLR-NH

NHNH

°v NH 〇 0°v NH 〇 0

EGEG

30K (AcG)GLYACHMOPIT(l-nal)VCQPLR-30K (AcG)GLYACHMOPIT(l-nal)VCQPLR-

OO

(AcG)GLYA(iHMGPriXl-nel)viQPLR-NH JL(AcG)GLYA(iHMGPriXl-nel)viQPLR-NH JL

NH2NH2

NH (AcG)GLYACHMGPIT(l-nal)VCQPLR-NH (AcG)GLYACHMGPIT(l-nal)VCQPLR-

OO

V NH 〇 0V NH 〇 0

4QK 29 13449644QK 29 1344964

30 1344964 (AcG)GLYAiHMOPnr(卜mW：QPLR(McGVnh Ο Ν °ν ,ΝΗ Ο30 1344964 (AcG)GLYAiHMOPnr(卜mW:QPLR(McGVnh Ο Ν °ν ,ΝΗ Ο

ε〇2〇κ (AcG)GLYACHMqPlT(l-nal)VCQPLR(MeG)-〇〇2〇κ (AcG)GLYACHMqPlT(l-nal)VCQPLR(MeG)-

ΟΟ

(AcG)GLYACHMGPIT(l-nBl)viQPLR(MeG)-NH Ο (AcG)GLYACHMGPIT(l-na〇VCQPLR(MeG) ~ΗΝ(AcG)GLYACHMGPIT(l-nBl)viQPLR(MeG)-NH Ο (AcG)GLYACHMGPIT(l-na〇VCQPLR(MeG) ~ΗΝ

Ο V ,ΝΗ U ° EGsqkΟ V , ΝΗ U ° EGsqk

(AcG}GLYA(£M(3Tr(I-Ml)viQI»LR(MeG)-NH JL(AcG}GLYA(£M(3Tr(I-Ml)viQI»LR(MeG)-NH JL

V ΝΗ π ° (AcG)OLYACHMGPrr(l-na〇VfQPU^MeG)~HN_V ΝΗ π ° (AcG)OLYACHMGPrr(l-na〇VfQPU^MeG)~HN_

Ο 當同質二元體之各個單體具有胺基酸序列 (AcG)GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1)及 31 1344964 間隔基之β及N2皆係透過胺基甲酸酯鍵聯附接至活化聚乙 二醇(PEG)部分時，本發明新穎胜肽化合物表示如後：Ο When each monomer of the homodimer has an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1) and 31 1344964, the β and N2 of the spacer are all permeable to the urethane bond. When attached to an activated polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention are expressed as follows:

0 —PEG10h5〇K Ο V ,ΝΗ0 —PEG10h5〇K Ο V ,ΝΗ

,O-PEG1<W0K 當同質二元體之各個單體具有胺基酸序列 5 (AcG)GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID ΝΟ_· 1)及 間隔基之Ν1及Ν2皆係透過醯胺鍵聯附接至活化聚乙二醇 (PEG)部分時’本發明新賴胜肽化合物表示如後：, O-PEG1 < W0K When each monomer of the homomorphic entity has an amino acid sequence 5 (AcG) GLYACHMGPIT (l-nal) VCQPLRK (SEQ ID ΝΟ _ 1) and the spacers Ν 1 and Ν 2 are all passed through the guanamine When the linkage is attached to the activated polyethylene glycol (PEG) moiety, the novel lysin compound of the present invention is expressed as follows:

(AcG)GLYA<lHMGPniCl-iial)V<!：QPLR-NH IL PEGi〇^s〇k \ HNG而(AcG)GLYA<lHMGPniCl-iial)V<!:QPLR-NH IL PEGi〇^s〇k \ HNG

阳 T (Ac〇)GLYACHMGPIT(l-n«l)VCQPLR--HN/ 當同質二元體之各個單體具有胺基酸序列 10 (AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 2)及間隔基之N1及N2皆係透過醯胺鍵聯附接至活化聚乙二 醇(PEG)部分時，本發明新穎胜肽化合物表示如後： 32 1344964阳T(Ac〇)GLYACHMGPIT(ln«l)VCQPLR--HN/ When the individual monomers of the homomorphic entity have the amino acid sequence 10 (AcG) GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO 2) and when the spacers N1 and N2 are attached to the activated polyethylene glycol (PEG) moiety through a guanamine linkage, the novel peptide compounds of the present invention are expressed as follows: 32 1344964

(AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 5 2)及間隔基之N1及N2皆係透過酿胺鍵聯附接至活化聚乙二 醇(PEG)部分時，本發明新穎胜肽化合物表示如後：(AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 5 2) and the spacers N1 and N2 are attached to the activated polyethylene glycol (PEG) moiety via a pull amine linkage. The novel peptide compounds of the present invention are expressed as follows:

本發明之較佳胜肽二元體包括（但非限制性）：Preferred peptide peptides of the invention include, but are not limited to:

33 134496433 1344964

34 134496434 1344964

(AcO)GLYA(iHMGPIT(l-Da])V(!：QPL Ο 〇 〇 Υ PEG50K ΝΗ O'/O—EGsok ^ΝΗ 〆 ΗΝ、一n_f(AcO)GLYA(iHMGPIT(l-Da))V(!:QPL Ο 〇 〇 PEG PEG50K ΝΗ O'/O—EGsok ^ΝΗ 〆 ΗΝ, one n_f

(AcG)GLYACHMGPIT(l-na])VCQPLR-HN(AcG)GLYACHMGPIT(l-na])VCQPLR-HN

Ο τ (AcG)GLYA« dHMGPIT(l-nal)viQPL]Ο τ (AcG)GLYA« dHMGPIT(l-nal)viQPL]

R-NH Ο ΝΗ ΝV ΝΗ ΟR-NH Ο ΝΗ ΝV ΝΗ Ο

(AcG)GLYACHMGPrr(l-iial)VCQPLR-HN ^(AcG)GLYACHMGPrr(l-iial)VCQPLR-HN ^

ο °γhn^,peg10K Οο °γhn^,peg10K Ο

PEG10K (AcG)GLYA<fHMGPm(i-iial)viQPLR-h ΟPEG10K (AcG)GLYA<fHMGPm(i-iial)viQPLR-h Ο

ΝΗ、.〇 9 0 〇yPEG- 、Ν ,Λ\、八 /ΝΗ (AcG)GLYACHMGPIT(1-dsI)VCQ； »PLR—ΗΝΝΗ,.〇 9 0 〇yPEG-, Ν, Λ\, 八/ΝΗ (AcG) GLYACHMGPIT(1-dsI)VCQ; »PLR—ΗΝ

Ο V ΝΗΟ V ΝΗ

ΗΝ^ΡΕ〇2〇κ 35 1344964 (AcG)GLYA^HMGPITXl-nal)V<!x?PLR- 0 Q 0 (AcG)GLYACHMGPIT(i-nal)VCQPLR-HN )‘ΗΝ^ΡΕ〇2〇κ 35 1344964 (AcG)GLYA^HMGPITXl-nal)V<!x?PLR- 0 Q 0 (AcG)GLYACHMGPIT(i-nal)VCQPLR-HN )‘

V々 Y HN 丫 PEGV々 Y HN 丫 PEG

V /NHV / NH

SQKSQK

(AcG)GLYA(!HMOPIT(l-iial)V(i：Q!»LR-NH^ JL °v m (AcG)GLYACHMGPIT(l-na1)VCQPLRr(AcG)GLYA(!HMOPIT(l-iial)V(i:Q!»LR-NH^ JL °v m (AcG)GLYACHMGPIT(l-na1)VCQPLRr

o (AcO)GLYA(iHMGPIT(l^al)VCQ]o (AcO)GLYA(iHMGPIT(l^al)VCQ]

•PLRHNH• PLRHNH

OO

NHNH

(AcG)GLYACTMGPrr(l-nal)VCQPLR—HN(AcG)GLYACTMGPrr(l-nal)VCQPLR—HN

peg40Kpeg40K

V Ο o PEG40kV Ο o PEG40k

X 〇 0X 〇 0

V PEG.V PEG.

NV NHNV NH

OO

,NH, NH

HN^PEG50K O 36 1344964HN^PEG50K O 36 1344964

37 1344964 (AcC)GLYAc!HMGPrr(l-nal)VCQPLR(MeG)—NH、37 1344964 (AcC)GLYAc!HMGPrr(l-nal)VCQPLR(MeG)—NH,

NH 9 0 V* •peg4 Ϊ HN^o—PEG40K 0NH 9 0 V* • peg4 Ϊ HN^o—PEG40K 0

(AcO)GLYACHMGPIT(l-nal)VCQPLR(MeG>-HN (AcG)GLYAcWGPIT(l-naI)VCQPLR(McG)-(AcO)GLYACHMGPIT(l-nal)VCQPLR(MeG>-HN (AcG)GLYAcWGPIT(l-naI)VCQPLR(McG)-

〇 O O-PEGsok〇 O O-PEGsok

0Y HNx^o-peG5〇K0Y HNx^o-peG5〇K

(AcG)GLYACHM〇Prr(l-f»al)VCQPLR(McG)—HN (Ai^^YACHMGPIT(l-naDV([QPLR(McG)-(AcG)GLYACHM〇Prr(l-f»al)VCQPLR(McG)—HN (Ai^^YACHMGPIT(l-naDV([QPLR(McG)-

O V°〇 O VY hn^peg10K o (AcG)GLYACHMGPIT(l -naJ: ^VCQPLR(MeG)-HNy 38 1344964O V°〇 O VY hn^peg10K o (AcG)GLYACHMGPIT(l -naJ: ^VCQPLR(MeG)-HNy 38 1344964

39 134496439 1344964

當間隔基係透過胺基甲酸酯鍵聯附接至活化聚乙二醇 (PEG)部分時，本發明新穎胜肽化合物表示為： (AcG)GLYACHMGPIT(l -Ml)V(!：QPLR(MeG). (AcG)GLYA<piMGPrr〇~Ml>V〒QPLR(MeG)，When the spacer is attached to the activated polyethylene glycol (PEG) moiety through a urethane linkage, the novel peptide compound of the invention is represented by: (AcG)GLYACHMGPIT(l -Ml)V(!:QPLR( MeG). (AcG)GLYA<piMGPrr〇~Ml>V〒QPLR(MeG),

ο -ο Ο Λ ο -PEG2M0kο -ο Ο Λ ο -PEG2M0k

當間隔基係透過酿胺鍵聯附接至活化聚乙二醇(PEG) 部分時，本發明新穎胜肽化合物表示為： (AcG)GLYA(?HMGPnXl-nal)viQPLR(MeG)-When the spacer is attached to the activated polyethylene glycol (PEG) moiety via a chiral linkage, the novel peptide compound of the invention is represented by: (AcG)GLYA(?HMGPnXl-nal)viQPLR(MeG)-

(AcG)GLYACHMGPIT(l-nal)VCQPLIKMeG)-HN(AcG)GLYACHMGPIT(l-nal)VCQPLIKMeG)-HN

κκ\Κκ\

PEG20"40K -Ο 此種二元體結構式可寫成[Ac-胜肽，雙硫]2Lys-間隔基 -PEG2(M()K來表示N端乙醯化胜肽鍵結至離胺酸之α胺基及 10 ε胺基二者，各個胜肽含有一個分子内雙硫回路以及一個 間隔基分子形成離胺酸C端與PEG部分間之共價鍵聯，此處 該PEG具有分子量約20,〇〇〇至約40,000道爾吞。 40 1344964 較佳本發明之胜肽二元體包括(但非限制性）： (AcO)GLYACHMGPIT(PEG20"40K-Ο This binary structure can be written as [Ac-peptide, disulfide] 2Lys-spacer-PEG2 (M()K to indicate N-terminal acetylated peptide bonding to lysine Both the alpha amine group and the 10 ε amine group, each peptide contains an intramolecular disulfide loop and a spacer molecule forms a covalent linkage between the C-terminus of the amine acid and the PEG moiety, where the PEG has a molecular weight of about 20 , 〇〇〇 to about 40,000 dolphine. 40 1344964 Preferably, the peptide peptide of the present invention includes, but is not limited to: (AcO) GLYACHMGPIT (

Unal)V(!：QPLR(MeG)-NH Ο人Unal)V(!:QPLR(MeG)-NH Ο人

O-PEGO-PEG

20K Ο Ο20K Ο Ο

(AcG)GLYACHMGPnxl-naI)VCQPUt(MeG) -HN -ο (AcG)GLYACHNTCPIT(l.iial)V0QPLRCMeG)- (AcG)GLYACHMGPIT(i-nal)va3PLR(MeG) •ΗΝ ΝΗ(AcG)GLYACHMGPnxl-naI)VCQPUt(MeG) -HN -ο (AcG)GLYACHNTCPIT(l.iial)V0QPLRCMeG)- (AcG)GLYACHMGPIT(i-nal)va3PLR(MeG) •ΗΝ ΝΗ

Ο -ο Λ-，Ο -ο Λ-,

EG30K ο (Αςσ)ΟΙΎΑ<ίΗΜΟΡ1Τ(1-ω1>νί(3ΡΙΛ(ΜβΟ)· 又 〇-peg4〇k οEG30K ο (Αςσ)ΟΙΎΑ<ίΗΜΟΡ1Τ(1-ω1>νί(3ΡΙΛ(ΜβΟ)·又 〇-peg4〇k ο

(AcG)GLYACHMGPrr(l -nal)VCQPLR(McG)-HN -Ο (AcG)GLYAi(AcG)GLYACHMGPrr(l -nal)VCQPLR(McG)-HN -Ο (AcG)GLYAi

c!HMGPlT(l-nal)viQPLR(MeG)H (AcG)GLYACHMGPIT(l-iud)VCQPLR(MeG) -ίc!HMGPlT(l-nal)viQPLR(MeG)H (AcG)GLYACHMGPIT(l-iud)VCQPLR(MeG) -ί

·〇 Λρε, ©20Κ Ο -ο (AcQ)GLYACHMGPm(l-nal) VCQPLR(MeG)- Ο人 (AcG)GLYACHOVlGPnXl-nal)VCQPLR(McG) ·ί·〇 ερε, ©20Κ Ο -ο (AcQ)GLYACHMGPm(l-nal) VCQPLR(MeG)- Ο人 (AcG)GLYACHOVlGPnXl-nal)VCQPLR(McG) ·ί

Ο ο O^EG2〇k •ο (AcG)GLYACHMGPrT(l-nal)VCQPLR(McO)- 〇Λρο ο O^EG2〇k •ο (AcG)GLYACHMGPrT(l-nal)VCQPLR(McO)- 〇Λρ

EG40K Ο Ο (AcO)GLYACHMGPrT(l-naJ)VCQPLR(McG) · •Ο 41 20種習知胺基酸之立體異構物（例如D胺基酸）、非天然 胺基酸如a,a-二取代胺基酸、N-烷基胺基酸、乳酸、及其它 非習知胺基酸也可作為本發明化合物之適當成分。非習知 胺基酸例如包括（但非限制性）：冷-丙胺酸、3-吡啶基丙胺 酸、4-羥基脯胺酸、〇-磷基絲胺酸、N_甲基甘胺酸、N-乙 酿基絲胺酸' N-甲醯基蛋胺酸、3-甲基組胺酸、5-羥基離胺 酸、新白胺酸及其它類似之胺基酸及亞胺基酸。其它修改 亦屬可能，包括胺基端的修改、羧基端的修改、一或多個 天然基因編碼胺基酸以非習知胺基酸置換、一或多個胺基 酸殘基支鏈的修改、胜肽磷酸化等。 本發明之胜肽序列可單獨存在或結合胜肽N端延長及/ 或C端延長存在。此種延長可為天然編碼胜肽序列選擇性含 有非天然序列或實質上不含非天然序列；延長可包括如熟 諳技藝人士期望之任何添加、刪失、點突變或其它序列修 改或組合。例如但非限制性，天然序列可為全長序列或部 分長度序列，可包括胺基酸取代來提供透過支鏈軛合而附 接碳水化合物、PEG、其它聚合物等之位置。變化例中， 胺基酸取代結果導致序列經過人化而變成與人類免疫系統 可相容。可提供全部各類融合蛋白質，包括鄰近或接近本 發明之EPO-R活化序列含或不含非免疫球蛋白間隔基序列 之免疫球蛋白序列。一類具體例為免疫球蛋白鏈含有 EPO-R活化序列來替代重鏈及/或輕鏈之可變區區）。 本發明胜肽化合物之事備_ 胜肽之合成 1344964 本發明胜肽可藉業界已知之傳統方法製備。標準方法 包括排它固相合成法、部分固相合成法、片段縮合法、傳 統溶液合成法及重組DNA技術[例如參考j. Am. Chem. Soc. 1963 85:2149]。 於一具體例，胜肽二元體之胜肽單體可個別合成，及 於合成後二聚合。 另一具體例中，二元體之胜肽單體透過其C端藉一分支 第三酿胺鍵聯基。部分鍵聯，該鍵聯基有二官能基可作為 胜肽合成之引發位置，以及有一第三官能基(例如缓基或胺 10基)其可結合至另—分子部分(例如可存在於固體支持體表 )此種If况下’於固相合成技術之變化技術，兩個胜狀 單體可直接於鍵聯基LK部分之兩個反應性氮基合成。此種 合成可為循序進行或同時進行。 15 20 〃體例中，於固相合成技術之變化技術，兩個胜 肽早體可直接於鍵聯SLk部分之兩個反應性氮基合成。此 種合成可為循序進行或同時進行。本具體财，使用-種 離胺S夂鍵聯基(LK)部分有兩個胺基可料胜肽合成之引發 位置’以及有—個第三官能基(例如離胺酸之減或離胺酿 胺基離⑯酸殘基其中缓基已經被轉成醯胺部分 -C〇NH2)其允許結合至另—分子部分(例如可存在於固體支 持體表面）。 ““人進订循序合成二元體之胜肽鏈至一鍵聯基時，鍵 聯基分子之兩個胺基官能基可以兩種不同之可正交去除之 胺基保護紐護。經鎌之鍵聯基透_縣的第三個官 43 1344964 能基而偶合至一固體支持體。第一胺基保護基被去除，二 元體之第一胜肽於第一胺基保護基被去除，二元體之第一 胜肽於第一脫保護之胺部分合成。然後第二胺保護基被去 除’二元體之第二胜肽於第二脫保護之胺部分合成。例如， , 5 鍵聯基之第一胺基部分可以Alloc保護，第二胺基部分以EG40K Ο Ο (AcO) GLYACHMGPrT (l-naJ) VCQPLR (McG) · • Ο 41 20 stereoisomers of conventional amino acids (such as D amino acids), unnatural amino acids such as a, a- Disubstituted amino acids, N-alkylamino acids, lactic acid, and other non-conventional amino acids are also suitable components of the compounds of the present invention. Non-proprietary amino acids include, for example, but are not limited to, cold-alanine, 3-pyridyl alanine, 4-hydroxyproline, 〇-phosphosine, N-methylglycine, N-ethyl basal acid 'N-methyl methionine, 3-methylhistamine, 5-hydroxy lysine, neoleucine and other similar amino acids and imino acids. Other modifications are also possible, including modification of the amino terminus, modification of the carboxy terminus, substitution of one or more of the native gene encoding amino acids with a non-conventional amino acid, modification of one or more amino acid residue branches, Peptide phosphorylation and the like. The peptide sequence of the present invention may be present alone or in combination with the N-terminal elongation and/or C-terminal elongation of the peptide. Such extension may be such that the naturally-encoded peptide sequence optionally contains a non-native sequence or is substantially free of non-native sequences; elongation may include any addition, censoring, point mutation or other sequence modification or combination as desired by those skilled in the art. For example and without limitation, the native sequence can be a full length sequence or a partial length sequence, and can include amino acid substitutions to provide attachment of carbohydrates, PEG, other polymers, and the like through branching conjugates. In a variant, the amino acid substitution results in a sequence that is humanized to become compatible with the human immune system. All types of fusion proteins can be provided, including immunoglobulin sequences that are adjacent to or near the EPO-R activation sequence of the invention with or without non-immunoglobulin spacer sequences. A specific example is the immunoglobulin chain containing an EPO-R activation sequence in place of the variable region of the heavy and/or light chain). The preparation of the peptide compound of the present invention - Synthesis of the peptide 1344964 The peptide of the present invention can be prepared by a conventional method known in the art. Standard methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, conventional solution synthesis, and recombinant DNA techniques [see, for example, j. Am. Chem. Soc. 1963 85:2149]. In one embodiment, the peptide monomers of the peptide binary can be synthesized separately and polymerized after synthesis. In another embodiment, the peptide monomer of the binary form a branched third amine linkage through its C-terminus. Partial linkage, the linkage has a difunctional group as the initiation site for peptide synthesis, and a third functional group (eg, a slow or amine 10 group) that can bind to another molecule moiety (eg, can be present in a solid Supporting the surface of the body. Under the condition of the solid phase synthesis technique, the two victoric monomers can be directly synthesized from the two reactive nitrogen groups of the LK moiety of the linking group. This synthesis can be carried out sequentially or simultaneously. In the 15 20 〃 system, in the variation technique of the solid phase synthesis technique, the two peptide precursors can be directly synthesized from the two reactive nitrogen groups of the linked SLk moiety. Such synthesis can be carried out sequentially or simultaneously. This specific use, the use of a kind of amine-free S夂 linkage (LK) moiety has two amine-based peptides for the initiation of the synthesis of the peptide 'and a third functional group (such as the reduction of amines or amines) The amine group is separated from the 16 acid residue in which the slow group has been converted to the indole moiety -C〇NH2) which allows for binding to another molecular moiety (for example, may be present on the surface of the solid support). "When a human is ordered to synthesize a peptide chain of a binary body to a one-linking group, the two amine functional groups of the linking molecule can be protected by two different orthogonally removable amine groups. The third official of the county, the first official of the county, 43 1344964, is coupled to a solid support. The first amino protecting group is removed, the first peptide of the dimer is removed from the first amine protecting group, and the first peptide of the binary is synthesized in the first deprotected amine moiety. The second amine protecting group is then removed. The second peptide of the binary is synthesized in the second deprotected amine moiety. For example, the first amine moiety of the 5-bonding group can be Alloc protected, and the second amine moiety can be

Fmoc保護。此種情況下，Fmoc基（但非Alloc基）可以弱鹼[例 β 如20%哌啶於二曱基甲醯胺(DMF)]處理去除，合成第一胜 &gt; 肽鏈。隨後，Alio基可以適當反應劑[例如Pd(PPh3)/4-甲基 嗎啉及氯仿]去除而合成第二胜肽鏈。注意當使用不同硫醇 10 保護基來保護半胱胺酸用來控制雙硫鍵之生成（容後詳述) 時’此項技術甚至必須用於二元體胜肽鏈之最終胺基酸序 列為相同時。 若欲進行二元體之二胜肽鏈同時與鍵聯基合成，則鍵 聯基之兩個胺官能基以相同可去除之胺保護基保護。經保 15 護之鍵聯基透過鍵聯基之第三官能基而偶合至固體支持 丨體。此種情況下，鍵聯基分子之兩個經保護官能基同時被 脫去保護’兩個胜肽鏈同時於脫保護之胺合成。注意使用 本技術’二元體胜肽鏈序列係相同，半胱胺酸殘基之硫醇 保護基皆須相同。 * 20 較佳胜肽合成方法為固相合成。固相胜肽合成程序為 業界眾所周知[例如參考Stewart固相胜肽合成（Freeman公 司’舊金山）1969;諾瓦拜耳康（N〇 vabi〇chem)公司2002/2003 通用型錄’美國聖地牙哥；Goodman胜肽及擬胜肽之公点 (Houben-Weyl，Stuttgart) 2002]。於固相合成，合成典型係 44 1344964 使用〇：胺基經保護之樹脂始於胜肽c端。適當起始物料例如 可經由將所需α胺基酸附接至氣甲基化樹脂、羥基甲基樹 脂、聚苯乙烯樹脂、二苯甲胺樹脂等而製備。此種氣甲基 化樹脂係以商品名生物珠(BIO-BEADS) SX-1由拜耳雷(Bi〇 5 Rad)實驗室（加州李奇蒙）出售。羥基甲基化樹脂之製備也已 經有說明[Bodonszky等人（1966) Chem. Ind.倫敦38:1597]。 二苯甲基胺(BHA)樹脂也經過描述[Piett^Marshall (1970) Chem. Commun.650]’鹽酸鹽形式可得自貝克曼（Beckman) 儀器公司（加州保羅奥圖）。例如α胺基經保護之胺基酸可根 10 據Gisin (1973) Helv. Chim. Acta 56:1467所述方法，借助於 碳酸氫铯催化劑而偶合至氯甲基化樹脂。 初步偶合後’ α胺基保護基例如使用三氟乙酸(TFA) 或鹽酸(HC1)溶液於有機溶劑於室溫去除。隨後，α胺基經 保護之胺基酸循序偶合至成長中之結合於支持體之胜肽 15鏈。α胺基保護基為已知可用於逐步合成胜肽業界之保護 基，包括：醯基類保護基(例如甲醯基、三氟乙醯基、乙醯 基）、芳香族胺基甲酸酯類保護基[例如苄氧羰基((：1)幻及經 取代之Cbz] ’脂肪族胺基曱酸酯保護基[例如第三丁氧羰基 (Boc)、異丙氧幾基、環己氧羰基]、及烷基類保護基(例如 20节基、三苯甲基）、芴基曱氧羰基(Fmoc)、丙烯基氧羰基 (Alloc)及1-(4,4-二曱基-2,6-二酮基環己-1-亞基）乙基(Dde)。 支鏈保護基（典型為醚類、酯類、三苯曱基、PMC等） 於偶合過程維持完好，於胺基端保護基脫保護期間或偶合 期間不會裂解去除。支鏈保護基必須於胜肽成品合成完成 45 時，於不會變更目標胜肽之條件下可被去除。Tyr之支鏈保 護基包括四氫吡喃基、第三丁基、三苯曱基、苄基、Cbz、 Z-Br-Cbz及2,5-二氣苄基。Asp之支鏈保護基包括苄基、2 6_ 二氣苄基、曱基、乙基及環己基。Thr及Ser之支鏈保護基 5 包括乙酿基、苯曱酿基、三苯甲基、四氫0比喃基、苄基、 2,6-二氣苄基及cbz。Arg之支鏈保護基包括硝基、甲苯磺 酿基(Tos)、Cbz金剛烷基氧羰基、三甲苯甲醯基磺醯基 (Mts)、2,2,4,6,7-五甲基二氫笨并呋喃-5-磺醯基(Pbf)、4-曱 氧基-2,3,6-三甲基-苯靖酿基(Mtr)或B〇(^ Lys之支鏈保護基 10包括Cbz、2-氣苄氧羰基（2-Cl-Cbz)、2-溴苄氧羰基 (2-Br-Cbz)、Tos或Boc。 於去除α胺基保護基後，其餘經保護之胺基酸以期望 順序逐步偶合。各個經保護之胺基酸通常係以約3倍過量使 用適當羧基活化劑反應，羧基活化劑例如為2-(lH-苯并*** 15 基广1，1，3,3·四曱基服鏘六氟磷酸鹽（HBTU)或二環己基 甲二酿亞胺(DCC)於溶液，例如於二氯甲烷(CH2C12)、Ν-甲 基°比&quot;各啶酮、二曱基甲醯胺(DMF)或其混合物反應。 於完成預定胺基酸序列後，所需胜肽由樹脂支持體解 偶合，解偶合之方式係使用試劑例如三氟乙酸(TFA)或氟化 2〇氮(HF)處理’該等試劑不只由樹脂裂解胜肽，同時也裂解 全部剩餘之支鏈保護基。當使用氯甲基化樹脂時，氟化氫 處理結果導致生成自由態胜肽酸。當使用二苯甲基胺樹脂 時’氟化氫處理結果直接獲得自由態胜肽醯胺。另外，當 使用氣曱基化樹脂時，支鏈經保護之胜肽可以氨處理胜肽 46 1344964 樹脂解偶合’獲得所需支鏈經保護之醯胺，或使用烷基胺 解偶合獲得支鏈經保護之烷基醯胺或二烷基醯胺。隨後以 尋常方式使用氟化氫處理去除支鏈保護 ，獲得自由態醯胺 類、院基醯胺類、或二烷基醯胺類。 5 製備本發明之酯類時，採用用於製備胜肽酸之樹脂， 支鍵經保護之胜肽係使用鹼及適當醇(例如甲醇）裂解。然後 支鍵保護基以尋常方式經由使用氟化氫處理去除而獲得所 需酯。 此等程序也可用來合成胜肽，其中20個天然基因編碼 10胺基酸以外之胺基酸於本發明之任一種化合物之一、二或 多個位置取代。可取代於本發明胜肽之合成胺基酸包括（但 非限制性)N-甲基、L-經基丙基、L-3、4-二經基苯基丙胺醯 基、5胺基酸類如L-5-羥基離胺醯基及D-(5-曱基丙胺醯 基、L-α-曱基丙胺醯基、召胺基酸類及異喳啉基。D_胺基 15 酸及非天然合成胺基酸也可結合於本發明胜肽。 胜肽改性 也可改性本發明胜肽化合物之胺基端及/或羧基端，來 製造其它本發明化合物。例如胺基端可以乙酸或其齒化衍 生物如α -氣乙酸、α -溴乙酸或α -蛾乙酸乙醯化。 20 可以其它支鏈置換20種天然基因編碼胺基酸（或立體 異構D胺基酸）之天然支鏈，例如使用下列基團置換：例如 烷基、低碳烷基、環狀4-、5- ' 6-至7員烷基、醯胺、醯胺 低碳烧基、醯胺二(低碳烷基）、低碳烷氧基、羥基、羧基及 其低碳酯衍生物；以及使用4-、5-、6-至7員雜環置換。特 47 1344964 別可採用脯胺酸類似物，其中脯胺酸殘基之環大小由5員改 支成為4、6或7員。環狀基團可為飽和或不飽和，若屬不飽 和，則可為芳香族或非芳香族。雜環基較佳含有一或多個 氮、乳、及/或疏雜原子。雜環基例如包括d夫η贄基、咬η南夷、 • 5咪唑啶基、咪唑基、咪唑啉基、異噻唑基、異噚唑基、嗎 . 琳基(例如嗎啉環）、噚唑基、哌畊基(例如1-哌《4基）、„底咬 基(例如1-哌啶基、哌啶環）、吡喃基、吡井基、Β比唑咬基、 | 吡唑啉基、吡唑基、嗒畊基、吡啶基、嘧啶基、咣咯啶基(例 如1-吡咯啶基）、吡咯啉基、吡咯基、噻二唑基、嘴唾基、 10 吩基、硫嗎嚇·基(例如硫嗎啦環)及三σ坐基。此等雜環美可 經取代或未經取代。當一個基團經取代時，取代基可為院 基、烷氧基、自原子、氧或經取代或未經取代之苯基。 可藉磷酸化及其它方法方便改性胜肽[例如述於H ru b y 等人（1990) Biochem J. 268:249-262]。 15 本發明胜肽化合物也可用作為具有類似生物活性之非 • 胜肽化合物的結構模型。熟諳技藝人士瞭解多項技術可用 於組成具有先導胜肽化合物之相同或類似的預定生物活性 之化合物’但就溶解度' 安定性、及對水解及蛋白質分解 •Ψ 的敏感度而言比先導化合物有更有利的活性[參考M〇rgan -20 及Gainor (1989) Ann. Rep. Med. Chem. 22:243-252]。此等技 術包括以膦酸酯類、醯胺酸酯類、胺基甲酸酯類、磺醯胺 類、第二胺類及N-曱基胺基酸組成之主鏈置換胜肽主鏈。 雙硫鍵之生成 本發明化合物含有兩個分子内雙硫鍵。此種雙硫鍵可 48 經由各個胜肽單體之半脱胺酸殘基氧化生成。 :具體例中，半胱胺酸鍵結生成之控制係經由選擇可 有效取佳生成就異構物之氡化賴別及^執行。例 如，當氧化劑為DMSO或峨(l2)時，（比較分子内雙琉鍵之生 成)優先達纽肽二元體氧化而生成兩個分子内雙硫鍵(各 個胜肽鏈上各一個雙硫鍵）。 其它具體例中，半胱胺酸鍵結之生成係經由於胜狀合 成期間適當選用硫醇保護基而生成。彳物，當需要有兩個 分子内雙硫鍵之二元體時，第—單體胜肽鏈係以核心序列 使用第-硫醇保護基[例如三笨f基、 及HM-二甲基-认二彻己小亞基 等]保護之兩個半胱胺酸殘基合成，然後’第二單體胜肽係 使用核心序列以與第-硫醇保護基不同的第二硫醇保護基 [例如乙酿胺基曱基(Acm)、第三丁基(tBu)等]保護之兩個半 胱胺酸殘基合成。隨後’第—硫醇保護基被去除執行第一 單體之雙硫環化，然後第二硫醇保護基被去除，執行第二 單體之雙硫環化。 其它本發明具體例提供雙硫衍生物類似物，其中一個 硫已經由CH2基或其它硫iS0tere置換。此等類似物可由本發 明化合物製備，其中各個胜肽單體含有至少一個c殘基或高 半脱殘基，以及含有α-胺基丁酸來替代第二匸殘 基，替代係使用業界已知方法透過分子内或分子間異位進 行[例如參考Barker等人 1992] J. Med. Chem. 35:2040-2048 及Or等人（1991)J. Org. Chem. 56:3146-3149]。熟諳技藝人士 1344964 瞭解此種替代也可使用α-胺基-r-丁酸及高半胱胺酸之其 它同系物進行。 μ除了别述環化策略之外，也可採用其它非雙硫胜肽環 化策略其匕環化策略例如包括醯胺環化策略及涉及硫-醚 Α 5鍵生成之％化策略。如此，本發明化合物可以具有分子内 . 酿賴或分子㈣·觀之環化形式存在 。例如可合成胜 肽其中核w序列之一個半胱胺酸以離胺酸置換，第二個 • +脱胺酸以麵胺酸置換。隨後，環狀單體可經由二殘基之 支鏈間的醯胺鍵結生成。另外，可合成胜月太，其中核心序 10歹J之辦耽胺酸以離胺酸（或絲胺酸）置換。然後經由離胺 文（或4 fe馱）殘基之支鏈與核心序列之第二個半胱胺酸殘 基間之硫-醚鍵結形成環狀單體。如此，除了雙硫環化策略 之外’醯胺環化策略及硫-醚環化策略皆方便用來環化本發 月化0物另外，胜肽之胺基可以α取代乙酸加端基，其 I5中α取代基為離去基例如α鹵乙酸如〇氯乙酸、α溴乙酸 • 或α -碘乙酸。 分支第三醯胺鍵聯基之添加 胜肽單體可藉分支第三醯胺鍵聯基部分二聚合。一個 具體例中’鍵聯基於胜肽合成過程結合入胜肽。例如，當 ^ 20鍵聯基Lk部分含有兩個官能基，其可用作為胜肽合成之引 發位置’以及一個或多個其它官能基(例如羧基或胺基）其可 結合至一或多個其它分子部分時，鍵聯基可軛合至固體支 持體。隨後，兩個胜肽單體可以多種固相合成技術，直接 合成至鍵聯基LK部分之兩個反應性氮基。 50 1344964 另—具體例中’鍵聯基可於胜肽合成後軛合至胜肽二 兀體之兩個胜肽單體。此種軛合可藉業界確立之方法達 成。一具體例中，鍵聯基含有兩個官能基其適合附接至合 成胜肽單體之目標官能基。例如含有兩個缓基之鍵聯基可 5預先活化或於適當偶合劑存在下，與二胜肽單體各別之目 標離胺酸支鏈胺基反應。 例如胜肽單體可化學偶合至第三醯胺鍵聯基， A*-C10-CH2-X-CH2-C20-B* 此處.X為NCO-(CH2)2-NH-Y及γ為適當保護基’例如第三 10 丁氧羰基(Boc)保護基；A*為適當官能基例如N-氧基丁二醯 亞胺，A*用來軛合鍵聯基之之第一胜肽單體之c端離胺 酸殘基之卜胺基；以及為適當官能基例如N_氧基丁二醯 亞胺，用來軛合鍵聯基之c2之第二胜肽單體之c端離胺 酸殘基之ε -胺基。 15 此外，例如胜肽單體可化學偶合至第三醯胺鍵聯基， A^-C^-C^-X-C^-C'O-B* 此處：X為NCO-(CH2)2-NH-C3〇- ; A*為適當官能基例如N_ 氧基丁二醯亞胺，A*用來輛合鍵聯基之c〗之第一胜肽單體 之C端離胺酸殘基之£_胺基；以及B*為適當官能基例如&amp; 20氧基丁二醯亞胺，用來軛合鍵聯基之c2之第二胜肽單體 C端離胺酸殘基之^_胺基；以及第三醯胺鍵聯基係化學鍵 結至間隔基部分，Fmoc protection. In this case, the Fmoc group (but not the Alloc group) can be removed by treatment with a weak base such as 20% piperidine in dimercaptocaramine (DMF) to synthesize the first &gt; peptide chain. Subsequently, the Alio group can be removed by a suitable reactant [e.g., Pd(PPh3)/4-methylmorpholine and chloroform] to synthesize a second peptide chain. Note that when different thiol 10 protecting groups are used to protect the cysteine from the formation of disulfide bonds (described in detail later), this technique must even be used for the final amino acid sequence of the binary peptide chain. For the same time. If the dipeptide chain of the binary is to be synthesized simultaneously with the linkage, the two amine functional groups of the linkage are protected with the same removable amine protecting group. The protected linkage is coupled to the solid support steroid via a third functional group of the linkage. In this case, the two protected functional groups of the linking molecule are simultaneously deprotected from the synthesis of the two peptide chains simultaneously with the deprotected amine. Note that the use of the present technology 'the binary peptide sequence is the same, and the thiol protecting groups of the cysteine residues must be the same. * 20 The preferred peptide synthesis method is solid phase synthesis. Solid phase peptide synthesis procedures are well known in the industry [eg reference to Stewart solid phase peptide synthesis (Freeman's 'San Francisco) 1969; Nyva Bi〇chem's 2002/2003 general catalogue 'San Diego, USA; Goodman peptide and the peptide of the peptide (Houben-Weyl, Stuttgart) 2002]. For solid phase synthesis, a typical system is synthesized. 44 1344964 The use of hydrazine: an amine-protected resin begins at the c-terminus of the peptide. A suitable starting material can be prepared, for example, by attaching a desired α-amino acid to a gas methylated resin, a hydroxymethyl resin, a polystyrene resin, a benzhydryl resin, or the like. Such a gas methylated resin is sold under the trade name Biobeads (BIO-BEADS) SX-1 from the Bi〇 5 Rad laboratory (Liqimen, Calif.). The preparation of hydroxymethylated resins has also been described [Bodonszky et al. (1966) Chem. Ind. London 38: 1597]. The benzhydrylamine (BHA) resin is also described [Piett^Marshall (1970) Chem. Commun. 650]' hydrochloride form available from Beckman Instruments Inc. (Paul Otto, Calif.). For example, the alpha amine-protected amino acid root 10 is coupled to the chloromethylated resin by means of a cesium hydrogencarbonate catalyst according to the procedure described by Gisin (1973) Helv. Chim. Acta 56:1467. After the preliminary coupling, the [alpha]amino protecting group is removed, for example, using a solution of trifluoroacetic acid (TFA) or hydrochloric acid (HC1) in an organic solvent at room temperature. Subsequently, the alpha amino-protected amino acid is sequentially coupled to the growing peptide 15 chain which binds to the support. The alpha-amino protecting group is a protecting group known to be useful in the stepwise synthesis of the peptide industry, including: fluorenyl protecting groups (eg, formazan, trifluoroethenyl, ethenyl), aromatic urethanes. Protecting group [eg benzyloxycarbonyl ((: 1) phantom and substituted Cbz] 'aliphatic amino phthalate protecting group [eg third butoxycarbonyl (Boc), isopropoxy, cyclohexyloxycarbonyl) And alkyl protecting groups (eg 20-membered, trityl), fluorenyl oxycarbonyl (Fmoc), acryloxycarbonyl (Alloc) and 1-(4,4-dimercapto-2, 6-diketocyclohex-1-ylidene)ethyl (Dde). Branched protecting groups (typically ethers, esters, triphenylsulfonyl, PMC, etc.) remain intact during the coupling process at the amine end The protecting group is not cleaved or removed during the deprotection or during the coupling. The branched protecting group must be removed when the peptide synthesis is completed 45, and the target peptide is not changed. The branch protecting group of Tyr includes tetrahydrogen. Pyranyl, tert-butyl, triphenylsulfonyl, benzyl, Cbz, Z-Br-Cbz and 2,5-digasbenzyl. The branched protecting group of Asp includes benzyl, 2 6 -diqibenzyl , 曱基,乙The base and cyclohexyl group. The branch protecting group 5 of Thr and Ser includes an ethyl, benzoyl, trityl, tetrahydro 0, benzyl, 2,6-dibenzyl and cbz. The branched chain protecting group of Arg includes nitro, tosyl sulphonate (Tos), Cbz adamantyl oxycarbonyl, toluene sulfonyl sulfonyl (Mts), 2,2,4,6,7-pentamethyl Dihydro benzofuran-5-sulfonyl (Pbf), 4-decyloxy-2,3,6-trimethyl-benzoinyl (Mtr) or B〇(^ Lys branched protective group 10 Including Cbz, 2-benzyl benzyloxycarbonyl (2-Cl-Cbz), 2-bromobenzyloxycarbonyl (2-Br-Cbz), Tos or Boc. After removal of the α-amino protecting group, the remaining protected amine group The acid is coupled stepwise in the desired order. Each protected amino acid is typically reacted in about a 3-fold excess using a suitable carboxyl activator such as 2-(lH-benzotriazole 15 based broad 1,1,3 , 3 · tetradecyl hexafluorophosphate (HBTU) or dicyclohexyl propylene diamine (DCC) in solution, for example, in dichloromethane (CH2C12), Ν-methyl ° ratio Reaction with dimercaptocaramine (DMF) or a mixture thereof. After completion of the predetermined amino acid sequence, the desired peptide is made of resin. Holder decoupling, decoupling is carried out using reagents such as trifluoroacetic acid (TFA) or fluorinated diazoxide (HF). These reagents not only cleave the peptide by the resin, but also cleave all remaining branch protecting groups. When a chloromethylated resin is used, the hydrogen fluoride treatment results in the formation of a free-form peptide. When a benzhydrylamine resin is used, the result of hydrogen fluoride treatment directly obtains the free-form peptide guanamine. In addition, when a gas-based group is used. In the case of a resin, the branched protected peptide can be ammonia treated with a peptide 46 1344964 resin decoupled to obtain the desired branched chain protected guanamine, or alkylamine decoupled to obtain a branched protected alkyl guanamine Or dialkyl decylamine. The branching protection is then removed in a conventional manner using hydrogen fluoride treatment to obtain free-form guanamines, deuterated guanamines, or dialkyl decylamines. 5 When preparing the ester of the present invention, a resin for preparing a peptide acid is used, and the peptide-protected peptide is cleaved using a base and a suitable alcohol such as methanol. The bond protecting group is then removed in a conventional manner by treatment with hydrogen fluoride to obtain the desired ester. These procedures can also be used to synthesize peptides in which 20 natural genes encoding amino acids other than 10 amino acids are substituted at one, two or more positions of any of the compounds of the present invention. Synthetic amino acids which may be substituted for the peptides of the present invention include, but are not limited to, N-methyl, L-propylpropyl, L-3,4-di-phenylphenylalaninium, 5-amino acids Such as L-5-hydroxyl-aminopurine and D-(5-mercaptopropylamine sulfhydryl, L-α-mercaptopropylamine sulfhydryl, arylamino acid and isoindolyl. D_amino 15 acid and non Natural synthetic amino acids can also be incorporated into the peptides of the present invention. The peptide modification can also modify the amine end and/or carboxyl end of the peptide compound of the present invention to produce other compounds of the invention. For example, the amine end can be acetic acid. Or a dentate derivative thereof such as α-gas acetic acid, α-bromoacetic acid or α-mothranacetate. 20 Other natural branches may be substituted for the amino acid encoding the amino acid (or stereoisomeric D-amino acid). Naturally branched, for example, substituted with: for example, alkyl, lower alkyl, cyclic 4-, 5- to 6- to 7-membered alkyl, decylamine, decylamine, lower alkyl, decyl Lower alkyl), lower alkoxy, hydroxy, carboxy, and lower aliphatic ester derivatives thereof; and 4-, 5-, 6- to 7-membered heterocyclic substitutions. Special 47 1344964 may be similar to lysine Object, of which The ring size of the acid residue is changed from 5 to 4, 6 or 7. The cyclic group may be saturated or unsaturated, and if it is unsaturated, it may be aromatic or non-aromatic. Containing one or more nitrogen, milk, and/or heteroatoms. Heterocyclic groups include, for example, d-n-n-decyl, n-n-Nan, • 5 imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, iso Carbazolyl, aryl. (eg morpholine ring), carbazolyl, piperage (eg 1-pipe "4 base", "bottom bite (eg 1-piperidinyl, piperidine)), pyridyl Meryl, pyridyl, pyridazole, benzoxyl, pyrazolyl, pyridyl, pyridyl, pyrimidinyl, pyridinyl (eg 1-pyrrolidyl), pyrrolinyl, Pyrrolyl, thiadiazolyl, succinyl, 10 phenyl, thiophene (for example, thiourapine) and tris(s). These heterocyclic rings may be substituted or unsubstituted. When substituted, the substituent may be a pendant group, an alkoxy group, an atom, an oxygen or a substituted or unsubstituted phenyl group. Phosphorylation and other methods may be used to facilitate the modification of the peptide [for example, in H ru by Et al. (1990) Bioche m J. 268:249-262]. 15 The peptide compounds of the present invention can also be used as structural models of non-peptide compounds having similar biological activities. Those skilled in the art understand that a number of techniques can be used to form the same or with a peptide compound. A similar bioactive compound is intended to have a more favorable activity than the lead compound in terms of solubility 'stability' and sensitivity to hydrolysis and proteolysis. [Ref. M〇rgan -20 and Gainor (1989) Ann. Rep. Med. Chem. 22: 243-252]. These techniques include phosphonates, phthalates, urethanes, sulfonamides, secondary amines, and N-decylamines. The backbone of the base acid composition replaces the peptide backbone. Formation of Disulfide Bonds The compounds of the invention contain two intramolecular disulfide bonds. Such a disulfide bond can be formed by oxidation of a semi-deaminating acid residue of each peptide monomer. In a specific example, the control of the formation of cysteine bond formation can be carried out by selecting and purifying the isomerization of the isomer. For example, when the oxidizing agent is DMSO or hydrazine (12), (comparing the formation of intramolecular bismuth bonds) preferentially oxidizes the dacombinide to form two intramolecular disulfide bonds (one disulfide in each peptide chain) key). In another specific example, the formation of a cysteine bond is formed by appropriately selecting a thiol protecting group during the synthesis of the winning form. For the sputum, when a binary body having two intramolecular disulfide bonds is required, the first monomeric peptide chain system uses a thiol protecting group as a core sequence [eg, a tris-f-group, and an HM-dimethyl group). - Recognition of two small subunits, etc.] The two cysteine residues are protected, and then the 'second monomer peptide is a second thiol protecting group that uses a core sequence to differ from the first thiol protecting group. [For example, two cysteine residues protected by anthranyl sulfhydryl (Acm), tert-butyl (tBu), etc.] are synthesized. Subsequent removal of the &lt;RTI ID=0.0&gt;&gt;&gt; </RTI> thiol protecting group is carried out to carry out the disulfide cyclization of the first monomer, and then the second thiol protecting group is removed, and the disulfide cyclization of the second monomer is carried out. Other specific examples of the invention provide disulfide derivative analogs in which one sulfur has been replaced by a CH2 group or other sulfur iStere. Such analogs can be prepared from the compounds of the invention wherein each peptide monomer contains at least one c residue or a high half-depleted residue, and alpha-aminobutyric acid is substituted for the second anthracene residue, which is used in the industry. The method is known to be carried out by intramolecular or intermolecular ectopic [see, for example, Barker et al. 1992] J. Med. Chem. 35:2040-2048 and Or et al. (1991) J. Org. Chem. 56:3146-3149]. Skilled person 1344964 It is understood that this alternative can also be carried out using alpha-amino-r-butyric acid and other homologues of homocysteine. In addition to other cyclization strategies, other non-disulfide peptide cyclization strategies may be employed, such as a guanidine cyclization strategy and a %ification strategy involving thio-ether Α 5 bond generation. Thus, the compounds of the present invention may exist in the form of intramolecular or cyclized forms of the molecule (tetra). For example, a peptide can be synthesized in which one cysteine of the nuclear w sequence is replaced with an amine acid, and the second + deaminating acid is replaced with a face acid. Subsequently, the cyclic monomer can be formed via a guanamine linkage between the branches of the two residues. In addition, it is possible to synthesize Shengyuetai, in which the core sequence is substituted with aminic acid (or seric acid). The cyclic monomer is then formed via a sulphur-ether bond between the branch of the amine (or 4 fe) residue and the second cysteine residue of the core sequence. Thus, in addition to the disulfide cyclization strategy, the indole cyclization strategy and the thio-ether cyclization strategy are conveniently used to cyclize the fluorinated product. In addition, the amino group of the peptide can be substituted with an acetic acid plus a terminal group. The alpha substituent in I5 is a leaving group such as alpha haloacetic acid such as hydrazine chloroacetic acid, alpha bromoacetic acid or alpha-iodoacetic acid. Addition of a branched third indole linkage The peptide monomer can be dimerized by a branched third indole linkage moiety. In a specific example, the linkage is incorporated into the peptide based on the peptide synthesis process. For example, when the moiety of the L20 linkage group contains two functional groups, which can be used as the initiation site for the synthesis of the peptide, and one or more other functional groups (such as a carboxyl group or an amine group), which can be bonded to one or more other In the molecular moiety, the linking group can be conjugated to a solid support. Subsequently, the two peptide monomers can be directly synthesized into two reactive nitrogen groups of the LK moiety of the linking group by various solid phase synthesis techniques. 50 1344964 In another embodiment, the 'bonding group' can be conjugated to the two peptide monomers of the peptide dimer after synthesis of the peptide. Such conjugates can be achieved by methods established by the industry. In one embodiment, the linking group contains two functional groups which are suitable for attachment to the target functional group of the synthetic peptide monomer. For example, a linking group containing two suspending groups can be preactivated or reacted with the respective target of the dipeptide monomer alone from the branched amine amine group in the presence of a suitable coupling agent. For example, the peptide monomer can be chemically coupled to the third guanamine linkage, A*-C10-CH2-X-CH2-C20-B* where X is NCO-(CH2)2-NH-Y and γ is A suitable protecting group such as a third 10 butyloxycarbonyl (Boc) protecting group; A* is a suitable functional group such as N-oxybutylenimide, and A* is used to conjugate a first peptide monomer of a bonding group a c-terminal amide group of a second peptide monomer for c2 conjugated to a bonding group, and a suitable functional group such as N-oxybutaneimine Ε-amino group of the residue. In addition, for example, a peptide monomer can be chemically coupled to a third guanamine linkage, A^-C^-C^-XC^-C'OB* where: X is NCO-(CH2)2-NH- C3〇- ; A* is a suitable functional group such as N-oxybutaneimine, and A* is used to bond the C-terminal amino acid residue of the first peptide monomer of the bond group. And B* is a suitable functional group such as &amp; 20 oxybutaneimine, which is used to conjugate the C-terminal of the second peptide monomer of the bond C2 to the amine group of the amine acid residue; The third indole linkage is chemically bonded to the spacer moiety,

Y-NH-(CH2)4-C4H-NH-Y 此處：X之C3係共價鍵結至間隔基之C4;以及γ為適當保護 51 1344964 基例如第三丁氧羰基(Boc)保護基》 離胺酸鍵聯基之添加 胜月太單體可藉離胺酸鍵聯基LK部分二聚合。一具體例 中，離胺酸鍵聯基係於胜肽合成過程結合入胜肽。例如當 ，5離紐鍵縣^部分含有兩個官能基其可作為⑽合成引 發位置，以及一個第三官能基(例如羧基或胺基）其可結合至 另一個分子部分時，鍵聯基可輕合至固體樓體。隨後，二 • 胜肽單體可以固相合成技術之變化方法，直接合成至離胺 酸鍵聯基lk部分之二反應性氮基。Y-NH-(CH2)4-C4H-NH-Y where: C3 of X is covalently bonded to the C4 of the spacer; and γ is suitably protected 51 1344964, such as a third butoxycarbonyl (Boc) protecting group The addition of a sulphate linkage can be carried out by partial condensation of the amino acid linkage group LK. In one embodiment, the lysine linkage is incorporated into the peptide during the peptide synthesis process. For example, when the 5 moiety of the bond contains two functional groups which can serve as (10) a synthetic initiation site, and a third functional group (such as a carboxyl group or an amine group) which can be bonded to another molecular moiety, the linkage can be Lightly fit to a solid building. Subsequently, the dipeptide monomer can be directly synthesized to the di-reactive nitrogen group of the lk moiety of the amine linkage by a solid phase synthesis technique.

10 另一具體例中，此處胜肽二元體係藉離胺酸鍵聯基LK 部分二聚合’該鍵聯基可於胜肽合成後軛合至胜肽二元體 之二胜肽單體。此種軛合可藉業界確立之方法達成。一個 具體例中’鍵聯基含有至少兩個適合附接至合成胜肽單體 之目標官能基之官能基。例如離胺酸的兩個自由胺基可與 15 二胜肽單體各別之C端羧基反應。 I 間隔基之添加 本發明胜肽化合物進一步包含一間隔基部分。一具體 例中’間隔基可於胜肽合成過程結合入胜肽。例如當間隔 基含有一自由胺基以及第二官能基(例如羧基或胺基）其可 一 20 結合另一分子部分時，間隔基可軛合至固體撐體。 一具體例中，含有二官能基之間隔基首先透過第一官 能基偶合至固體撐體。其次，離胺酸鍵聯基LK部分具有二 官能基可作為胜肽合成引發位置，以及一第三官能基(例如 羧基或胺基)其可結合另一分子部分，該離胺酸鍵聯基透過 52 間隔基之第二官能基其鍵聯基之第三官能基軛合至間隔 基。隨後，二胜肽單體可以固相合成技術之變化法直接合 成於鍵聯基LK部分之兩個反應性氮基。例如固體撐體偶合 有自由胺基之間隔基，可透過鍵聯基之自由缓基而與離胺 5醆鰱聯基反應。 另一具體例中’間隔基可於胜肽合成後軛合至胜肽二 疋體。此種軛合可藉業界確立之方法達成。一具體例中， 鍵聯基含有至少一個適合附接至合成胜肽之目標官能基之 g能基《例如具有自由胺基之間隔基可與胜肽C端羧基反 1〇 應。另—例中’具有自由羧基之鍵聯基可與離胺酸醯胺之 自由胺基反應。 聚乙二醇(PEG)之附接 近年來，水溶性聚合物如聚乙二醇(PEG)用於共價修飾 具有治療及診斷重要性之胜肽。附接此種聚合物相信可提 15 高生物活性、延長血循環時間、降低免疫原性、增加水中 溶解度及增加對蛋白酶消化之抗性。例如，共價附接PEG 至治療性多肽例如介白質類[Knauf等人（1988) J. Biol. Chem. 263;15064; Tsutsumi 等人（1995)控制釋放期刊 33:447]、干擾素(Kita等人（1990)藥物指定輸送6:157)、催化 20 酶(Abuchowski等人（1977) J. Biol. Chem· 252:582)，超氧化 物歧化酶（Beauchamp等人（1983) Anal. Biochem. 131:25)及 腺苷去胺酶（Chen 等人（1981) Biochim. Biophy. Acta 660:293)已報告可延長活體内半生期，及/或降低免疫原性 及抗原性。 53 1344964 本發明胜肽化合物可包含聚乙二醇(PEG)部分’其係透 過胺基甲酸醋鍵聯或酸胺鍵聯共價附接至胜肽二元體之分 支第三醯胺鍵聯基或間隔基。用於本發明之PEG例如為線 性未分支PEG ’分量約20千道爾吞(20K)至約40K(「約」一 Λ 5詞表示PEG製備中，有些分子比所述分子量更高，有些分 子比所述分子量更低）。較佳PEG具有分子量約30K至約 * 40K。In another specific example, the peptide binary system herein is partially dimerized by an amine acid linkage LK. The linkage can be conjugated to the peptide peptide of the peptide binary after synthesis of the peptide. . Such conjugates can be achieved by methods established by the industry. In one embodiment, the 'bonding group' contains at least two functional groups suitable for attachment to the target functional group of the synthetic peptide monomer. For example, two free amine groups from the amine acid can react with the respective C-terminal carboxyl groups of the 15 dipeptide monomer. I. Addition of Spacer The peptide compound of the present invention further comprises a spacer moiety. In a specific example, the spacer can be incorporated into the peptide during the peptide synthesis process. For example, when the spacer contains a free amine group and a second functional group (e.g., a carboxyl group or an amine group) which can bind to another molecular moiety, the spacer can be conjugated to the solid support. In one embodiment, the spacer containing a difunctional group is first coupled to the solid support through the first functional group. Secondly, the difunctional group of the amine acid linkage LK has a difunctional group as a peptide synthesis initiation site, and a third functional group (such as a carboxyl group or an amine group) which can bind to another molecular moiety, the isoleucine linkage group. The third functional group of the bonding group is conjugated to the spacer through the second functional group of the 52 spacer. Subsequently, the dipeptide monomer can be directly synthesized in the solid phase synthesis technique by two reactive nitrogen groups in the LK moiety of the linkage group. For example, a solid support couples a free amine group spacer which is reactive with an amine 5 linkage through a free moieving group of the linkage. In another embodiment, the spacer can be conjugated to the peptide dimer after synthesis of the peptide. Such conjugates can be achieved by methods established by the industry. In one embodiment, the linker contains at least one g-energy group suitable for attachment to a target functional group of the synthetic peptide. For example, a spacer having a free amine group can be reacted with a C-terminal carboxyl group of the peptide. In another example, a bond having a free carboxyl group can be reacted with a free amine group of a guanamine amine. Attachment of polyethylene glycol (PEG) In recent years, water-soluble polymers such as polyethylene glycol (PEG) have been used to covalently modify peptides of therapeutic and diagnostic importance. Attachment of this polymer is believed to provide high biological activity, prolonged blood circulation time, reduced immunogenicity, increased solubility in water, and increased resistance to protease digestion. For example, covalent attachment of PEG to therapeutic polypeptides such as the white matter class [Knauf et al. (1988) J. Biol. Chem. 263; 15064; Tsutsumi et al. (1995) Controlled Release Journal 33:447], Interferon (Kita Et al. (1990) Drug Delivery 6: 157), Catalytic 20 Enzyme (Abuchowski et al. (1977) J. Biol. Chem. 252: 582), Superoxide Dismutase (Beauchamp et al. (1983) Anal. Biochem. 131:25) and adenosine deaminase (Chen et al. (1981) Biochim. Biophy. Acta 660:293) have been reported to prolong in vivo half-life and/or reduce immunogenicity and antigenicity. 53 1344964 The peptide compound of the present invention may comprise a polyethylene glycol (PEG) moiety which is covalently attached to the branch of the peptide binary by a urethane linkage or an acid amine linkage to a third amine linkage. Base or spacer. The PEG used in the present invention is, for example, a linear unbranched PEG 'component of about 20 kilo Torr (20K) to about 40K ("about" Λ 5 words means that in PEG preparation, some molecules are higher than the molecular weight, some molecules Lower than the molecular weight). Preferably, the PEG has a molecular weight of from about 30K to about *40K.

&amp; 本發明使用之PEG之另一例為分子量約ιοκ至約60K 之線性PEG(「約」一詞表示peg製備中，有些分子比所述 10分子量更更’有些分子比所述分子量更低）。較佳PEG之分 子量約20K至約40K。更佳PEG之分子量約20K。 共價附接PEG之方法(PEG化）之範例說明如後。範例說 明並非意圖為限制性。熟諸技藝人士瞭解於業界已經確立 多種共價附接寬廣範圍之PEG之方法。如此業界已知已經 15 藉多種附接方法附接PEG之胜肽化合物皆涵蓋於本發明之 | 範圍。 例如PEG可透過反應性基團共價鍵結至鍵聯基，於該 反應性基團可結合活化PEG分子（例如自由胺基或缓基）。 &quot; PEG分子可使用有不同反應性部分之甲氧化PEG(「mPEG _j) 一 20 附接至胺基酸。此種聚合物包括mPEG-丁二醯亞胺基丁二 酸醋、mPEG-丁二酿亞胺基碳酸醋、mPEG-醯亞胺酸醋、 mPEG-4-硝基苯基碳酸S旨及mPEG-氰尿醯氯。同理，peg分 子可使用有自由胺基之甲氧化PEG (mPEG-NH2)附接至叛 基0 54 1344964 若干具體例中，鍵聯基或間隔基含有端末胺基（亦即位 於間隔基末端）。此端末胺基可與適當活化PEG分子例如 mPEG-對硝基苯基碳酸酯（mPEG-NPC)反應來製造穩定共 價胺基曱酸酯鍵。另外，此端末胺基可與適當活化PEG分 5 子例如含有反應性N-羥基-丁二醯亞胺(NHS)基團之mPEG-丁二醯亞胺基丁酸酯（mPEG-SB A)或mPEG- 丁二醯亞胺基 丙酸醋（mPEG-SPA)反應，來形成穩定共價胺基曱酸酯鍵。 其它具體例中，鍵聯基反應基含有羧基，其可被活化而於 適當反應條件下與含胺PEG分子生成共價鍵。適當PEG分子 10 包括mPEG-NH2,以及適當反應條件包括曱二醯亞胺媒介之 醯胺生成反應條件等。 E_PO-R激動劑活性檢定分析： 試管内功能檢定分析 試管内競爭結合檢定分析可定量試驗胜肽與EPO競爭 15 結合HPO-R之能力。例如（例如參考美國專利5,773,569所 述）’人EPO-R之胞外領域(EPO結合蛋白質EBP)可於大腸桿 菌以重組方式製造，重組蛋白質偶合至固體支持體例如微 力價皿或合成珠[例如沙風林克（Sulfolink)珠得自皮爾斯 (Pierce)化學公司（伊利諾州洛克福)]^然後經過制動之EBP 20 與加標記之重組EPO共同培養’或與加標記之重組EPO及試 驗胜肽共同培養。試驗胜肽之一系列稀釋液用於此種實 驗。未添加試驗胜肽之檢定分析點定義EPO總結合至EBP。 用於含試驗胜肽反應，EP0結合量經過定量，且以占對照（總 量= 100%)結合之百分比表示。此等值對胜肽濃度作圖。IC50 55 1344964 值定義為可降低EPO結合至EBP達50%(亦即50%抑制EPO 結合)之試驗胜肽濃度。 不同的试管s式驗競爭結合檢定分析測定以二珠粒： EP0輛合珠粒及EP〇-R|fe合珠粒之接近程度之函數變化產 參5生的光信號做測定。珠粒接近程度可經由EPO結合至EPO-R 而產生。與EPO競爭結合至EPO-R之試驗胜肽可防止此項結 « 合’造成發光的下降。可獲得發光減低5〇%之試驗胜肽濃 &gt; 度定義為IC50值。 本發明胜肽可極為有效與EP0競爭結合至EP0_R。此種 1〇增強功能可由本發明化合物於實質較低濃度胜肽（亦即具 有極低IC50值)可抑制EPO的結合來表示。 本發明之單體胜肽及二元體胜肽EPO_R激動劑之生物 活性及強度’特別結合EPO受器之生物活性及強度，可使 用試管試驗基於細胞之功能檢定分析測定。 15 一種檢定分析係基於可表現人EP0-R之鼠前B細胞 .系’進一步使用fOS啟動基因驅動之蟲螢光素酶通報子基因 組成體轉移感染。此種細胞當暴露於EP〇或其它EP0-R激動 劑時’此種細胞係藉合成蟲螢光素酶來回應。當添加蟲螢 光素姆時造成發光。如此此種細胞之EP0-R活化程度可透 2〇 * 過測定蟲螢光素酶活性來量化。試驗胜狀活性之測量方 式’係經由添加試驗胜肽之系列稀釋液至細胞，然後培養4 卜時。培養後’蟲螢光素酶基質添加至細胞，測定發光。 可導致半最大發光之試驗胜肽濃度記錄作為EC50。 本發明胜肽於本檢定分析顯示促進EP0-R發訊依賴型 56 1344964 蟲螢光素酶表現的能力大增。此種功能增強可以其於實質 上較低胜肽濃度獲得半最大蟲螢光素酶活性表示（亦即具 有極低EC50值）。本檢定分析為估計本發明之EPO-R激動劑 胜肽強度及活性之較佳方法》 5 可使用FDC-P1/ER細胞[Dexter等人(1980) J. Exp. Med. 152:1036-1047]進行另一項檢定分析，該細胞為已經穩定轉 移感染EPO-R之明確特徵化之未轉形鼠骨髓衍生細胞系。 此等細胞具有EPO依賴型增生。 一項檢定分析中，細胞於所需生長因子存在下生長至 10 半靜態密度(例如參考美國專利5，773，569)。然後細胞於PBS 洗滌，於不含生長因子之全培養基饑餓沁-24小時，及測定 細胞存活能力（例如藉萃龐藍(trypan blue)染色測定)後，製 備備用溶液(於不含生長因子之全培養基）獲得每5〇微升含 約105細胞》接受試驗之胜肽EpO_R激動劑化合物之一系列 15稀釋液(典型為自由態液相胜肽，而非與噬菌體結合或其它 結合胜肽或制動胜肽）於96孔組織培養孔板内製作成每孔 終容積50微升。細胞(5〇微升）添加至各孔，細胞培養24-48 小時’此時陰性對照將死亡或靜止。然後藉業界已知技術 例如MTT檢定分析測定細胞增生，MTT檢定分析測定H3-20胸腺嘧。定之結合量作為細胞增生的指標[參考Mosmann (1983)免疫方法期刊65:55 63p胜肽係對砂〇_尺表現性細胞 系及親代非表現性細胞系二者評比。獲得半最大值細胞增 生之試驗胜肽濃度記錄作為EC50。 本發明胜肽於本檢定分析顯示促進EPO依賴型細胞生 57 長能力大增。此種功能增強可以其於實質低濃度胜肽獲得 半最大細胞增生刺激活性表示（亦即其具有極低E c 5 〇值）。 本檢定分析為估計本發明之]£1&gt;〇_11激動劑胜肽強度及活性 之較佳方法。 於另一檢定分析’細胞於補充EP〇之培養基生長至穩 定期，細胞經收集然後又於不含EP〇之培養基培養18小 時。細胞分成細胞密度相等的三組：一組未添加因子（陰性 對照）、一組含EPO(陽性對照）、及一實驗組含試驗胜肽。 然後培養後之細胞於各個時間點收集、固定、使用DNA結 合螢光染料（例如propidium碘或赫斯特染料（Hoechst dye)，皆係得自西格瑪（sigma)公司）染色。然後例如使用 FACS掃描流量細胞計測定螢光。細胞週期各期之細胞百分 比隨後例如使用CellFIT軟體（貝通迪金森(Becton Dickinson) 公司）之SOBR模型測定。使用EPO或活性胜肽處理的細胞比 較陰性對照組，顯示有較大百分比細胞於S期（藉螢光增加 作為DNA含量增高之指標測定）。 類似檢定分析可使用FDCP-1[例如參考Dexter等人 (1980) J. Exp. Med. 152:1036-1047]或TF-1 [Kitamura等人 (1989)血液73:375-380]進行。FDCP-1為生長因子依賴型鼠 多潛力原始造血祖先細胞系，當補充以經過WEHI-3調理之 培養基(含IL-3之培養基’ ATCC編號TIB-68)培養時可增生 但不會分化。用於此實驗，FDCP-1細胞系以人EPO-R或鼠 EP0-R轉移感染來產生FDCP-1-hEPO-R細胞系或 FDCP-1-mEPO-R細胞系，其於EPO存在下可增生但不會分 1344964 化。TF-1為epo依賴型細胞系。TF-1也可用來測定胜肽 EPO-R激動劑對細胞增生的影響。 於又另一檢定分析，可採用Krystal (1983) Exp. Hematol 11:649-660敘述之將Η3-胸腺嘧啶結合於脾細胞之 5 微檢定分析之程序來確定本發明化合物用作為Ε Ρ Ο激動劑 的能力。簡言之，B6C3FN、鼠每日注射苯基肼(60毫克/千 克）共2日。於第三日，取出脾細胞，使用ΜΤΤ檢定分析確 定其於24小時時間增生之能力。 ΕΡΟ結合至紅血球生成素反應性細胞系之EPO-R，誘生 10 酪胺酸磷酸化受器及多種胞内蛋白質，包括She，vav及 JAK2激酶。因此，另一項試管試驗檢定分析測定本發明胜 肽誘生EPO-R及下游胞内信號轉導子蛋白質之酪胺酸磷酸 化的能力。藉前述結合及增生檢定分析辨識之活性胜肽， 提引出磷酸化樣式接近類似EPO於紅血球生成素反應性細 15 胞之磷酸化樣式。用於本檢定分析，FDC-P1/ER細胞[Dexter 等人（1980) J. Exp Med 152:1036-47]於補充EPO培養基維持 及生長至穩定期。然後細胞於不含EPO之培養基培養24小 時。然後特定數目之細胞與試驗胜肽於37°C培養約1〇分 鐘。含EP0之細胞對照試樣也於各個檢定分析同時進行。 2〇 然後經處理之細胞藉離心收集，再懸浮於SDS溶解緩衝 液’接受SDS聚丙烯醯胺凝膠電泳。凝膠内經過凝膠電泳 的蛋白質轉移至硝基纖維素◊於墨點上含磷酸路胺酸之蛋 白質藉標準免疫技術觀察。例如墨點可使用抗碟酸赂胺酸 抗體(例如小鼠抗鱗酸酷胺酸IgG ’得自亞普史鐵特(upstate) 59 生技A司)¼測，洗滌，然後使用第二抗體[例如過氧化酶標 ^之^羊抗小鼠1gG，得自科高及沛力（Kirkegaard &amp; Perry) 貫所至A司（華盛頓特區探測。隨後，含填酸絡胺酸之蛋 白貝可藉才示準技術包括比色檢定分析 '化學發光檢定分析 5或螢光檢疋分析觀察。例如，化學發光檢定分析可使用得 自阿莫山（Amersham)公司之虹西方墨點系統進行。 另種可用來評比本發明胜肽之活性之基於細胞之試 s内檢D析為群落檢定分析，該檢定分析係使用 鼠骨髓 、細胞或周邊血球。鼠骨耀可得自鼠股骨，人周邊血球樣本 1〇可付自健康捐血人。以周邊血液為例，首先由血液中分離 單核細胞，例如經飛可海帕克(Ficoll-Hypaque)梯度[幹細胞 技術公司（加拿大溫哥華離心分離。用於本檢定分析，進 行含有核之細胞數目計算來確定原先血樣中之核化細胞濃 度。特定數目之細胞遵照製造商指示[幹細胞技術公司（加拿 15大溫哥華接種於甲基纖維素。實驗組使用試驗胜肽處理， 1%性對照組使用EP0處理，陰性對照組未接受處理。然後 各組之生長群落量於特定培養時間後，通常為1〇曰至18曰 評分。活性胜肽可促進群落的生成。 其匕可用於驗證本發明化合物活性之試管試驗生物檢 20 定分析揭示於Greenberger等人（1983) Proc. Natl. Acad. Sci. USA 80:2931-2935 (EPO依賴型造血祖先細胞系）；Queue及&amp; Another example of PEG used in the present invention is a linear PEG having a molecular weight of from about ιοκ to about 60K (the term "about" means that in the preparation of peg, some molecules are more molecular than the molecular weight of 10, some molecules are lower than the molecular weight) . Preferably, the PEG has a molecular weight of from about 20K to about 40K. More preferably, the molecular weight of PEG is about 20K. An example of a method of covalently attaching PEG (PEGylation) is as follows. The examples are not intended to be limiting. Those skilled in the art understand that the industry has established a variety of methods for covalently attaching a wide range of PEGs. Thus, it is known in the art that peptides which have been attached to PEG by a variety of attachment methods are encompassed by the scope of the present invention. For example, PEG can be covalently bonded to a linking group through a reactive group, and the reactive group can bind to an activated PEG molecule (e.g., a free amine group or a slow group). &quot; PEG molecules can be attached to the amino acid using methacrylic PEG ("mPEG _j)-20 with different reactive moieties. Such polymers include mPEG-butyl succinimide succinate, mPEG-butyl Di-i-imine carbonate, mPEG-hydrazide vinegar, mPEG-4-nitrophenyl carbonate S and mPEG-cyanuridine chloride. Similarly, peg molecules can be used with free amine-based methacrylic PEG (mPEG-NH2) Attached to Rebel 0 54 1344964 In several embodiments, the linker or spacer contains a terminal amine group (ie, located at the end of the spacer). This terminal amine group can be suitably activated with a PEG molecule such as mPEG- The p-nitrophenyl carbonate (mPEG-NPC) is reacted to produce a stable covalent amine phthalate linkage. In addition, the terminal amine group can be reacted with a suitable activated PEG, for example, containing a reactive N-hydroxy-butane An imine (NHS) group of mPEG-butanediamine-based butyrate (mPEG-SB A) or mPEG-butanediamine-based propionic acid vinegar (mPEG-SPA) to form a stable covalent amine Further oxime bond. In other embodiments, the linker reactive group contains a carboxyl group which can be activated to form a covalent bond with the amine-containing PEG molecule under appropriate reaction conditions. The appropriate PEG molecule 10 includes mPEG-NH2, and the appropriate reaction conditions include the indoleamine production reaction conditions of the indole diimide medium, etc. E_PO-R agonist activity assay: In-tube functional assay analysis In-tube competition binding assay can be quantified The test peptide competes with EPO for the ability to bind to HPO-R. For example (see, for example, U.S. Patent No. 5,773,569), the extracellular domain of human EPO-R (EPO binding protein EBP) can be produced recombinantly in E. coli, recombinant protein. Coupling to a solid support such as a micro-valve or synthetic bead [eg, Sulfolink beads from Pierce Chemical Company (Rockford, Ill.)] ^ then braked EBP 20 and labeled recombination EPO co-cultured or co-cultured with labeled recombinant EPO and test peptides. A series of dilutions of the test peptide was used for this experiment. The assay point without the addition of the test peptide defines the total binding of EPO to EBP. Containing the test peptide reaction, the EP0 binding amount is quantified and expressed as a percentage of the control (total = 100%) binding. This value is plotted against the peptide concentration. IC50 55 1344 The 964 value is defined as the concentration of the test peptide that reduces EPO binding to EBP up to 50% (ie, 50% inhibition of EPO binding). Different test tubes are tested for competitive binding assays to determine two beads: EP0 beads and The change in the proximity of EP〇-R|fe beads is measured by the light signal of the ginseng. The proximity of the beads can be produced by EPO binding to EPO-R. The test for competing with EPO to EPO-R The peptide prevents the junction from causing a decrease in luminescence. The test peptide concentration obtained by reducing the luminescence by 5% is defined as the IC50 value. The peptide of the present invention is extremely effective in competing with EP0 for binding to EP0_R. Such a 〇 enhancing function can be represented by a compound of the present invention which inhibits the binding of EPO at a substantially lower concentration of a peptide (i.e., having a very low IC50 value). The biological activity and strength of the monomeric peptides and the binary peptide EPO_R agonists of the present invention, in particular in combination with the biological activity and strength of the EPO receptor, can be determined by assays based on cell-based functional assays in vitro. 15 One assay was based on a pre-B cell that expresses human EP0-R. The system further utilizes the fOS promoter-driven luciferase reporter gene constitutive transfer infection. Such cells are responded to by synthetic luciferase when exposed to EP 〇 or other EP0-R agonists. It causes luminescence when worm fluorescein is added. The degree of EP0-R activation of such cells can be quantified by measuring luciferase activity. The measurement of the activity of the test is carried out by adding a serial dilution of the test peptide to the cells, and then culturing for 4 times. After the culture, the luciferase substrate was added to the cells, and luminescence was measured. The test peptide concentration that results in half-maximal luminescence is recorded as the EC50. The peptides of the present invention have been shown to have an increased ability to promote the expression of EP0-R signaling-dependent 56 1344964 luciferase in this assay. Such enhanced function may result in a semi-maximal luciferase activity at a substantially lower peptide concentration (i.e., having a very low EC50 value). This assay is a preferred method for estimating the strength and activity of the EPO-R agonist peptide of the present invention. 5 FDC-P1/ER cells can be used [Dexter et al. (1980) J. Exp. Med. 152: 1036-1047 Another assay was performed, which was a well-characterized untransformed murine bone marrow-derived cell line that had been stably metastatically infected with EPO-R. These cells have EPO-dependent proliferation. In one assay, cells were grown to a 10 semi-static density in the presence of the desired growth factor (see, e.g., U.S. Patent 5,773,569). The cells were then washed in PBS, hungry for -24 h in growth medium without growth factors, and assayed for cell viability (eg, by trypan blue staining) to prepare a stock solution (without growth factor) Full medium) obtained a series of 15 dilutions of each of the peptides of the peptide EpO_R agonist (containing about 105 cells per 5 microliters) (typically a free-form liquid phase peptide, rather than a phage-binding or other binding peptide or The brake peptide was prepared in a 96-well tissue culture well plate to a final volume of 50 μL per well. Cells (5 μL) were added to each well and the cells were cultured for 24-48 hours. At this point the negative control will die or quiescent. The cell proliferation is then determined by techniques known in the art, such as MTT assay, and the MTT assay is assayed for H3-20 thymidine. The binding amount is used as an indicator of cell proliferation [Ref. Mosmann (1983) Immunol. Journal of 65:55 63p peptide system for the comparison of the sand 〇 尺 表现 表现 表现 亲 亲 亲 亲 亲 亲 。 。 。 。 。 。 。 。 。 。 。 。 。 The test peptide concentration at which the half maximal cell growth was obtained was recorded as the EC50. In the present assay, the peptide of the present invention showed an increase in the ability to promote EPO-dependent cells. Such enhanced function may be obtained by obtaining a semi-maximal cell proliferative stimulating activity at a substantially low concentration peptide (i.e., having a very low Ec5 〇 value). This assay is a preferred method for estimating the strength and activity of the peptide of the present invention. In another assay, the cells were grown in a medium supplemented with EP 至 to a steady state, and the cells were collected and cultured for 18 hours in an EP-free medium. The cells were divided into three groups of equal cell density: one group of unadded factors (negative control), one group containing EPO (positive control), and one experimental group containing test peptides. The cultured cells are then collected, fixed, and stained with DNA-binding fluorescent dyes (e.g., propidium iodine or Hoechst dye, all from sigma) at various time points. Fluorescence is then measured, for example, using a FACS scan flow cytometer. The percentage of cells in each phase of the cell cycle is then determined, for example, using the SOBR model of CellFIT software (Becton Dickinson). Cells treated with EPO or active peptides compared to the negative control group showed a larger percentage of cells in the S phase (as measured by an increase in fluorescence as an indicator of increased DNA content). Similar assays can be performed using FDCP-1 [for example, see Dexter et al. (1980) J. Exp. Med. 152: 1036-1047] or TF-1 [Kitamura et al. (1989) Blood 73: 375-380]. FDCP-1 is a growth factor-dependent murine multi-potential primitive hematopoietic progenitor cell line that proliferates but does not differentiate when cultured in WEHI-3 conditioned medium (IL-3 containing medium ATCC number TIB-68). For this experiment, the FDCP-1 cell line was infected with human EPO-R or murine EP0-R to produce FDCP-1-hEPO-R cell line or FDCP-1-mEPO-R cell line, which can be used in the presence of EPO. Proliferation but not divided into 1344496. TF-1 is an epo-dependent cell line. TF-1 can also be used to determine the effect of peptide EPO-R agonists on cell proliferation. For yet another assay, the compound of the invention can be used as a Ε Ρ Ο 可采用 可采用 5 ( ( 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 胸 胸 胸The ability of the agent. Briefly, B6C3FN and rats were injected daily with phenylhydrazine (60 mg/kg) for 2 days. On the third day, spleen cells were removed and assayed for their ability to proliferate in 24 hours using a sputum assay. The EPO-R, which binds to the erythropoietin-reactive cell line, induces 10 tyrosine phosphorylation receptors and a variety of intracellular proteins, including She, vav and JAK2 kinases. Thus, another tube assay assay analyzes the ability of the peptides of the invention to induce tyrosine phosphorylation of EPO-R and downstream intracellular signal transducer proteins. By the above-mentioned binding and proliferative assay analysis, the active peptide was extracted, and the phosphorylation pattern was similar to that of EPO-like erythropoietin-reactive phosphorylation. For the assay, FDC-P1/ER cells [Dexter et al. (1980) J. Exp Med 152:1036-47] were maintained and grown to a stationary phase in supplemented EPO medium. The cells were then cultured in an EPO-free medium for 24 hours. A specific number of cells are then incubated with the test peptide at 37 ° C for about 1 minute. Cell control samples containing EP0 were also performed simultaneously in each assay. 2〇 The treated cells were then collected by centrifugation and resuspended in SDS Lysis Buffer' subjected to SDS polypropylene gel electrophoresis. The protein subjected to gel electrophoresis in the gel was transferred to nitrocellulose, and the protein containing phospholamic acid on the ink dot was observed by standard immunological techniques. For example, the ink dot can be tested with an anti-sphingic acid antibody (for example, mouse anti-squaric acid IgG 'from Upstate 59 Biotech A), washed, and then used a secondary antibody. [For example, the peroxidase standard ^^ goat anti-mouse 1gG, obtained from Kirkegaard &amp; Perry to A Division (Washington, DC detection. Subsequently, protein containing lysine Borrowing techniques include colorimetric assays for chemiluminescence assays 5 or fluorescence assays. For example, chemiluminescence assays can be performed using the Western Western blot system from Amersham. To evaluate the activity of the peptide of the present invention, the cell-based test s internal assay D is a community assay, which uses murine bone marrow, cells or peripheral blood cells. The rat bone can be obtained from the mouse femur, human peripheral blood cell sample 1 〇 can be paid from healthy donors. Taking peripheral blood as an example, mononuclear cells are first isolated from the blood, for example, by a Ficoll-Hypaque gradient [Stem Cell Technology Corporation (Vancouver, Vancouver, Canada). In The number of cells containing the nucleus is calculated to determine the concentration of nucleated cells in the original blood sample. A specific number of cells are in accordance with the manufacturer's instructions [Stem Cell Technology Inc. (Canada 15 Vancouver is inoculated with methylcellulose. The experimental group is treated with the test peptide) The 1% control group was treated with EP0, and the negative control group was not treated. Then the growth community amount of each group was usually scored from 1〇曰 to 18曰 after the specific culture time. The active peptide can promote the formation of the community. A test tube bioassay that can be used to verify the activity of a compound of the invention is disclosed in Greenberger et al. (1983) Proc. Natl. Acad. Sci. USA 80: 2931-2935 (EPO-dependent hematopoietic progenitor cell line); Queue and

Wojchowski (1991) J. Β〇1· Chem. 266:609-614 (B6SUt.EP細 胞之蛋白質酷·胺酸麟酸化）；Dusanter-Fourt等人（1992) J. Biol. Chem. 287:10670-10678(人EPO反應性細胞之EPO受 60 1344964 器之酪胺酸磷酸化）；Quelle等人（1992) J. Biol. Chem. 267:17055-17060 (FDC-ER細胞之溶胞蛋白質pp 100之酪胺 酸填酸化）；Worthington 等人（1987) Exp. Hernatol. 15:85-92(血色素之比色檢定分析）；Kaiho及Miuno (1985) 5 Anal. Biochem. 149:117-120(血色素使用 2,7-二胺基芴之檢 測）；Patel 等人（1992) J. Biol. Chem. 267:21300·21302 (c-myb之表現）；Withuhn等人（1933)細胞74:227-236 (JAK2 之結合與酪胺酸磷酸化）；Leonard等人（1993)血液 82:1071-1079 (GATA轉錄因子之表現）；及Ando等人（1993) 10 Proc. Natl· Acad. Sci. USA 90:9571-9575(經由循環D2及D3 調節G]之變遷）。 分子裝置公司設計之一種稱作為微生理儀之儀器，據 報告可成功地用於測定激動劑及拮抗劑對各種受器的影 響。本裝置之基礎係測定胞外介質回應於受器活化之酸化 15 速率改變。 活體内功能檢定分析 可用於評比試驗胜肽強度之一種活體内功能檢定分析 為紅血球增多性外血氧不足小鼠生物檢定分析。用於此項 檢定分析，小鼠接受交替調理週期數日。此週期中，小鼠 20 介於低於大氣壓條件與周圍大氣壓條件間交替。隨後小鼠 於投予試驗樣本前2-3日維持於周圍壓力。於陽性對照小鼠 之試驗胜肽樣本或陽性對照小鼠之Ε Ρ Ο標準經皮下注射入 經過調理後的小鼠體内。放射性標記鐵（例如&amp;59)於2日後 投予’投予放射性標記鐵後2日採血樣。然後藉標準技術則 61 1344964 定各血樣之血容及放射性測量值。注射活性試驗胜肽之小 鼠血樣（由於紅血球血色素結合Fe59)顯示比未接受試驗胜 月太或接受EPO之小鼠更高的放射性。 ίο 另一項可用於評比試驗胜肽活性之活體内功能檢定分 析為網狀細胞檢定分析❶用於本檢定分析，正常未經處理 小鼠連續3曰皮下注射EPO或試驗胜肽。於第三曰，小鼠也 於腹内注射鐵葡萄聚糖。於第5日，由小鼠採集血樣。血液 中之網狀細胞百分比（％)係藉噻唑橙染色及流動細胞計分 析（格狀計數計畫）測定。此外以人工測定血容。修正後之網 狀細胞百分比係使用下式測定： % RETIC校正=% RETIC觀察x(血容_/血容正常） 活性試驗化合物顯示％ RETICftΛ濃度比未試驗胜肽或Ερ〇 之小鼠高。 本發明之ΕΡΟ激動劑之用途 15 20 本發明胜肽化合物可用於試管内作為瞭解ΕΡΟ生物角 色的工具’包括評比多項因素，該等因素被視為可能影響 ΕΡΟ的產量以及ΕΡΟ結合至EPO-R(例如EPO/EPO-R信號轉 導/受器活化機轉）’或受ΕΡΟ產量以及ΕΡΟ結合至EPO-R的 影響。本胜肽由於提供重要結構-活性-關係資訊，有助於開 發其它化合物’故本發明胜肽也可用於開發其它可結合至 EPO-R之化合物。 此外，基於本發明胜肽結合至EPO-R的能力，本發明 胜肽可用作為於活細胞、固定後的細胞、生物體液、組織 均化物、純化後之天然生物物質等檢測EPO-R之試劑。例 62 1344964 如經由標記此等胜肽，可識別表面具有ΕΡΟ-R之細胞。此 外，基於其結合EPO-R之能力，本發明胜肽可用於原位染 色' FACS(螢光活化細胞篩選）分析、西方墨點分析、 ELISA(酶聯結免疫吸附檢定分析）等。此外，基於本發明化 5 合物結合至EPO-R的能力，本發明胜肽可用於受器的純 化，或用於純化細胞表面（或滲透細胞内側）表現EPO-R之細 胞。 本發明胜肽也可用作為商業試劑用於各項藥學研究與 診斷目的。此等用途包括（但非限制性）：（1)用作為校準標 10 準來定量於多種功能檢定分析，候選EPO-R激動劑之活 性；（2)用作為阻斷劑用於隨機胜肽篩選，亦即用於尋找新 EPO-R胜肽配位子族群，該胜肽可用來阻斷本發明EPO胜肽 的回收；（3)用於與EPO-R共同結晶，亦即可生成本發明胜 肽結合至EPO-R之晶體，因而藉X光結晶攝影術可測定受器 15 /胜肽結構；（4)用於測定紅血球前驅細胞經由引發分化，而 誘生球蛋白合成及血質複合物合成以及增加鐵蛋白受器數 目之能力；（5)用來維持EPO依賴型細胞系如 FDCP-1-mEPO-R及TF-1細胞系之增生及生長；⑹於使用放 射性產色基團標記本發明胜肽相關之用途；以及其它研 2〇究及診斷應用，其中EP〇-R較佳經活化，或此種活化方便 對已知量之EPO-R激動劑等校準。 於本發明之又另一方面，提供治療方法及藥物之製 造。本發明胜肽化合物可投予溫血動物包括人類來模擬 EP〇於活體内結合至EPO-R。如此，本發明涵蓋Ep〇缺乏相 63 1344964 關病症之治療性處理方法’該方法包含以足夠刺激EP〇省 因而改善活體内EPO缺乏相關症狀之數量，投予本發明化 合物。例如，本發明胜肽可用於治療腎機能不全及/或末期 腎农竭/透析，愛滋病相關貧血；慢性發炎疾病（例如類風濕 * 5性關節炎及慢性腸發炎）及自體免疫病引起的的貧血；以及 用於手術前增加病人紅血球數目。其它可藉投予本發明胜 肽治療處理之疾病狀態、病症及血液不規則狀態包括：沒 I 型地中海貧血；囊性纖維變性；妊娠及月經病症；未成熟 之早期貧血；脊索受傷；太空飛行；急性血液流失；老化； 10中風、缺血（包括中樞神經系統及心臟）；及多種伴隨有紅血 球生成異常之腫瘤疾病狀態。 其它具體例中，本發明胜肽化合物可用於治療非以紅 金球過低或缺乏為特徵的病症，例如用於作為輸血前的前 處理。此外，投予本發明化合物可能導致出血時間縮短， 15如此可於手術前投予病人，或用於預期可能發生出血的適 丨應症。此外，本發明化合物可用於活化巨核細胞的活化。 因EPO顯示對血管内皮細胞具有有絲***原性及趨化 效果’以及對中樞膽鹼激性神經元有作用[例如參考 Amagnostou 專人（1990) Proc· Natl. Acad. Sci. USA .20 87:5978-5982及Konishi等人（1993)腦研究609:29-35]，本發 明化合物也可用於治療多種血管病症：例如促進傷口癒 合、促進側支冠狀血管生長(例如心肌梗塞後可能發生）；瘡 傷的處理；及血管移植後處理。本發明化合物也可用於治 療多種神經病症’通常係以乙醯膽鹼絕對濃度比其它神經 64 1344964 活性物質（例如神經傳遞物質）濃度低，或乙酿膽驗相對浪度 比其它神經活性物質相對濃度低為特徵。 藥學組成物 於本發明之又另一方面，提供前述Ep〇R激動劑脉月太 5化合物之藥學組成物。可藉投予此等組成物改善或調節之 病情包括前文列舉之病情。此等藥學組成物例如可藉經 口、腸道外（肌肉、腹内、靜脈(IV)或皮下注射）、經皮（被 動滲透，或使用離子電泳或電泳）、經黏膜（經鼻、經***、 經直腸或經舌下）途徑投藥，或使用可生物分解之植入物， 10其可以適合各種投藥途徑之劑型調配。通常本發明含拍·藥 學組成物包含有效量之本發明之EPO-R激動劑胜肽或衍生 物產物，連同藥學上可接受之稀釋劑、保藏劑、增溶劑、 乳化劑、輔劑及/或載劑。此等組成物包括含有不同緩衡劑 (例如Tris-HCl、乙酸鹽、磷酸鹽）、不同pH及離子強度之豨 15釋劑；添加劑例如清潔劑及增溶劑（例如吞恩(Tween) 20、 吞恩80、玻力索貝（Polysorbate) 80)、抗氧化劑（例如抗壞血 酸、偏亞硫酸氫納）、保藏劑（例如柳硫汞、苄醇)及增量物 質（例如乳糖、甘露糖醇）；攙混材料於聚合物化合物顆粒製 劑例如聚乳酸、聚乙醇酸等之顆粒製劑或攙混於微脂粒。 20 也可使用玻尿酸。此種組成物影響本蛋白質及衍生物之物 理狀態、安定性 '活體内釋放速率及活體内廓清率。例如 參考雷明頓製藥科學，第18版（1990年默克出版公司，賓州 伊斯頓18042) 1435-1712頁，以引用方式併入此處。組成物 也可製備成液體形式，或乾粉(例如凍乾)形式。 65 1344964 經口輸送 口服固體劑型預期供此處使用，口服固體劑型概略述 於雷明頓製藥科學第18版（1990年默克出版公司，賓州伊斯 頓18042)第89章，以引用方式併入此處。固體劑型包括錠 ^ 5 劑、膠囊劑、丸劑、***錠或菱型錠、豆狀膠囊、片劑、 散劑或粒劑。此外，微脂粒或類蛋白質包封也可用於調配 » 本組成物(例如類蛋白質微球，報告於美國專利第4,925,673 | 號）。微脂粒包封也可使用，微脂粒可使用各種聚合物衍生 (例如美國專利第5,013,556號）。可能之固體治療用劑型之說 10 明述於Marshall，K.:近代藥物G.s. Banker及C.T. Rhodes第 10章，1979年，以引用方式併入此處。通常配方包括Ep0_R 激動劑胜肽（或其化學改性形式）以及惰性成分，惰性成分允 許保護對抗胃部環境，而於腸道釋放出生物活性物質。 此處預期也涵蓋口服投藥用液體劑型，包括藥學上可 15 接受之乳液劑、溶液劑、懸浮液劑及糖漿劑，其可含有其 &amp; 它成分’包括惰性稀釋劑；輔劑例如濕潤劑、乳化劑及懸 浮液劑；及甜味劑、矮味劑及香味劑。 胜肽可經化學改性’讓衍生物之口服輸送為有效。通 常，預期化學改性係附接至少一部分至成分分子本身，此 -20處該部分允許(a)抑制蛋白質分解；以及(b)由胃或腸吸收入 血流。也希望增高各成分整體安定性’且延長於體内擔環 時間。如前文討論PEG化為較佳供製藥用之化學改性。其 它可用於改性之部分包括：丙二醇、乙二醇與丙二醇之共 聚物、羧甲基纖維素、葡萄聚糖、聚乙烯醇、聚乙烯吡咯 66 1344964 。定酮、聚脯胺酸、聚-1,3-二氧戊環及聚-1,3,6-三氧辛環[例 如參考Abuchowski及Davis (1981)「水溶性聚合物-酶加合 物」’酶作為藥物。Hocenberg及Roberts編輯（威利科技公 司.紐約州紐約）367-383頁；及Newmark等人（1982) J. Appl. 5 Biochem. 4:185-189]。 用於口服配方，釋放位置可為胃、小腸（十二指腸、空 腸或迴腸）或大腸。熟諳技藝人士已經可取得不會溶解於 胃’但可於十二指腸或腸道其它部位釋放出材料之配方。 較佳釋放藥物係經由保護胜肽（或衍生物）、或經由釋放胜肽 10 (或衍生物)超出胃部環境例如於腸道，來避免對胃部環境造 成有害影響。 為了確保全胃抗性，需要有至少對pH 5.0不透性之包 衣。可用作為腸衣之更常見惰性成分為乙酸偏苯三酸纖維 素(CAT)、苯二甲酸羥基丙基曱基纖維素(HPmcP)、HPMCP 15 5〇、HPMCP 55、聚乙酸苯二曱酸乙烯酯（PVAP)、優拉吉 (Eudragit) L30D、亞奎堤瑞（Aquateric)、乙酸苯二甲酸纖維 素(CAP)、優拉吉l、優拉吉S及蟲膠。此等包衣可呈混合膜 使用。 包衣或包衣混合物也可用於非意圖保護不接觸胃部之 20鍵劑。包括糖衣或讓錠劑更容易吞嚥包衣。膠囊可由硬殼 (例如明膠）組成，供輸送乾治療劑（亦即粉末），用於液體治 療藥物’可使用軟明膠殼。豆狀膠囊之殼體材料可為厚澱 粉紙或其它食用紙。用於丸劑、菱型錠、模製錠或錠濕磨 劑，可使用濕質塊技術。 67 1344964 胜肽（或衍生物）可以細小多顆粒，呈大小約1毫米之粒 劑或顆粒片含於配方。供膠囊投藥用之材料配方可呈粉 末、微壓縮柱塞或甚至錠形式。此等治療劑可藉壓縮製備。 也可含括著色劑及/或矯味劑。例如胜肽（或衍生物）可 ^ 5調配(例如藉微脂粒或微球包囊調配），然後進一步含於食用 產品例如含著色劑及矯味劑之冰凍飲料。 可使用惰性材料稀釋或增加胜肽（或衍生物）體積。此等 φ 稀釋劑包括碳水合物特別為甘露糖醇、α乳糖、無水乳糖 纖維素、蔗糖、改性葡萄聚糖及澱粉。某些無機鹽也可用 1〇作為填充劑’包括三破酸妈、碳酸鎮及氯化納。若干市售 稀釋劑為法斯特福羅Fast_F1〇)、伊米德斯(Emdex) 、STA-Rx 1500、伊米肯皮斯(Emc〇mpress)及亞微希爾（Avicell)。 朋散劑ΊΓ 3括於調配成固體劑型的治療劑配方。用作 為崩散θ丨之材料包括(但非限制性)殿粉，包括市售以殿粉為 μ主之崩散劑、伊斯普羅塔(Expl〇tab)、乙醇酸澱粉鈉、安伯 • 萊特(Ambei*lite) ϋ纖維錢、超戊糖果膠、褐藻酸 納、明膠、撥皮、酸性缓曱基纖維素鈉、天然海綿及矣土 〜 纟部皆可使用。崩散劑也可為不溶性陽離子交換樹脂。粉 狀樹膠可用作為崩散劑及黏結劑，也包括粉狀樹勝例如壤 20月曰卡拉揚膠或西黃箸膠。褐藤酸及褐藻酸納鹽也可 為崩散劑。 黏結劑可用來將胜肽（或衍生物)結合在-起形成硬質 錠d包括彳于自天然之產物例如***膠、西黃蓍膠礙粉 及明膠。其匕錢劑包括甲基纖維素(MC)、乙基纖維素吨 68 1344964 及緩甲基纖維素(CMC)。聚乙烯基吡咯啶酮(PVP)及羥基丙 基甲基纖維素(HPMC)也可用於醇溶液來造粒胜肽（或衍生 物）〇 抗摩擦劑也可含括於胜肽（或衍生物）調配物，來防止調 5 配過程中的沾黏。潤滑劑可用作為胜肽（或衍生物）與壓模壁 間之該層，潤滑劑包括（但非限制性硬脂酸包括其鎂鹽及 約鹽、聚四氟乙烯(PTFE)、液體石蠟、植物油及蠟。也可 使用可溶性潤滑劑例如硫酸月桂酯鈉、硫酸月桂酯鎂、各 種分子量之聚乙二醇卡玻瓦司（Carbowax) 4000及卡玻瓦司 10 6000 。 可添加滑動劑，滑動劑可改良調配過程之藥物流動性 質’且可輔助壓縮過程的重新排列。滑動劑包括澱粉、滑 石、熱原矽氧及水合矽鋁酸鹽。 . 為了輔助胜肽（或衍生物）溶解於水性環境，可添加界面 15 活性劑作為濕潤劑。界面活性劑包括陰離子清潔劑如硫酸 月桂酯鈉 '二辛基續基丁二酸鈉及二辛基續酸納。可使用 陽離子清潔劑，包括氯化苄烷鑕或氯化苯乙鏘。可含括於 調配物中作為界面活性劑之可能之非離子清潔劑表單包括 勞拉馬克喬(lauromacrogol) 400、玻利奥克斯(p〇ly〇xyi) 40 2〇 硬脂酸酯、聚氧伸乙基氫化蓖麻油10、50及60、一硬脂酸 甘油酷、玻麗索貝20 ' 40、60 ' 65及80 '荒糖脂肪酸醋、 甲基纖維素及羧曱基纖維素。此等界面活性劑也可單獨或 呈不同比例之混合物存在於蛋白質或衍生物調配物。 可促進胜肽（或衍生物）吸收之添加劑為例如脂肪酸 69 類’例如油酸、亞油酸及亞麻酸。 也希望使用控制釋放口服調配物《胜肽（或衍生物）可攙 混於惰性基體，惰性基體允許藉擴散或滲濾機轉例如樹膠 釋放。緩慢變性之基體也可攙混於調配物。若干腸衣也具 有延長釋放效果。另一種控制釋放形式係基於歐若斯(0r0s) 治療系統(歐哲(Alza)公司）之方法，換言之，藥物包於半透 膜内，半透膜由於滲透作用允許水進入而經由單一小開口 將藥物推送出。 其它包衣也可用於配方。此等包衣包括多種糖，可於 包衣盤施用。胜肽（或衍生物）也可被製作成膜衣錠，本例使 用之材料分成兩組。第一組為非腸溶材料，包括曱基纖維 素、乙基纖維素、羥乙基纖維素、甲基羥-乙基纖維素、羥 基丙基纖維素、羥基丙基-甲基纖維素、致基-曱基纖維素 鈉、普維隆(providone)及聚乙二醇類。第二組包括腸溶材 料，通常為苯二曱酸之酯類。 可使用混合材料來提供最佳膜衣。包膜衣可於盤式包 衣機或於流化床進行或藉壓縮包衣而進行。 腸道外輸送 根據本發明供腸道外投藥用之製劑包括無菌水性或非 水性溶液劑、懸浮液劑或乳液劑。非水性溶劑或媒劑例如 為丙一醇、I乙一醇、植物油如撖獲油及玉米油、明踢及 注射用有機醋類如油酸乙酯。此等劑型也可含有輔劑例如 保藏劑、濕潤劑、乳化劑及分散劑。其滅菌方式例如可利 用通過滯留細菌之過濾膜過濾，經由攙混滅菌劑於組成物 i344_ 滅菌，經由以光妝射組成物滅菌，或經由加熱組成物滅菌β 此等劑型也可使用無菌水或若干其它無菌注射用介質而恰 於使用前調配。 敏直腸或經***輸送 5 供直腸或***投藥用之組成物較佳為栓劑，其除了活 採物質之外’含有賦形劑如可可脂或栓劑堪。供經鼻或舌 卞投藥用之組成物也可使用業界眾所周知之標準賦形劑製 權。 經肺輸送 1〇 本發明也涵蓋經肺輸送EPO-R激動劑胜肽（或其衍生 物）。胜肽（或衍生物)於哺乳類吸入時輸送至哺乳類肺臟， 通過肺上皮襯層進入血流[例如參考Adjei等人（1990)製藥 研究7:565_569 ; Adjei等人（1990)國際製藥期刊 63:135-144(柳普賴德(leuprolide)乙酸鹽）；Braquet 等人（1989) 15 心血管藥理學期刊13(sup5):143-146(内皮素-1) ; Hubbard等 人（1989)内科年報，VOL. III, 206-212 (a 1-抗胰蛋白酶）； Smith等人（1989) J. Clin. Invest 84:1145-1146 (α-l-蛋白 酶）；Oswein等人（1990)「蛋白質之氣溶膠化作用」，第二屆 呼吸藥物輸送研討會議事錄科羅拉多州奇思同（重組人生 長激素）；Debs等人（1988) J. Immunol. 140:3482-3488(干擾 素-r及腫瘤壞死因子a);及Platz等人之美國專利第 5,284,656號(粒性細胞群落刺激因子）。經肺部輸送藥物達成 系統性效果之方法及組成物說明於Wong等人之美國專利 第 5,451，569號]。 71 1344964 涵蓋於本發明之實務為寬廣多種設計經肺臟輸送治療 產品之機械裝置，包括(但非限制性）霧化器、定量吸入器、 及乾粉吸入器’全部皆為熟諳技藝人士眾所周知。若干市 售適合供本發明實務之裝置特例為亞哲文特⑽ravem)霧 • 5化器（莫林克特(Mallindaodt)公司，蒙大拿州聖路易）；亞可 • 恩(Acorn)I1霧化器（馬克斯特(Marquest)醫學產品公司，科羅 拉多州英吾市），文托林(Ventolin)定量吸入器（葛蘭素(Glax〇) φ 公司，北卡羅萊那州研究三角公園市）；及史賓海勒 (Spinhaler)乾粉吸入器（費遜斯(Fis〇ns)公司，麻省貝德福）。 10 全部此等裝置皆要求使用適合配送胜肽（或衍生物）之 調配物。典型地此等調配物係依據使用之裝置類型而定， 除了尋常稀釋劑、輔劑及/或治療用載劑之外，涉及使用適 當推進劑材料。也涵蓋使用微脂粒、微囊或微球，包括複 合物或其它類型之載劑。依據化學改性類型或使用之裝置 15類型而定，化學改性胜肽也製備於不同調配劑。 φ 適合用於霧化器包括噴射霧化或超音波霧化之調配物 典型包含胜肽（或衍生物）以約〇1至25毫克生物活性蛋白質 /毫升溶液溶解於水。調配物也包括緩衝液及單糖(例如供蛋 白質穩定與滲透壓調節之用）。霧化器調配物也含有界面活 - 20性劑’來減少或防止形成氣溶膠時，由於溶液霧化造成胜 肽（或衍生物）的凝集。 用於定量吸入器裝置之調配物通常包含細分粉末，含 有借助於界面活性劑懸浮於推進劑胜肽（或衍生物）。推進劑 可為任一種用於此項目的之材料，例如氣氟化碳、氫氯氟 72 1344964 化碳'氫氟化碳或烴’包括三氯氟甲炫、一氯二氟甲烧、 二氯四氟乙烷、及U，l，2-四氟乙烷或其組合°適當界面活 性劑包括三油酸山梨糖醇酯及大豆卵磷脂。油酸也可用作 為界面活性劑。 5 由乾粉吸入器裝置配送之調配物包含含胜狀（或衍生 物)之細分乾粉，也包括增量劑如乳糖、山梨糖醇、蔗糖戋 甘露糖醇，其含量可輔助乾粉由裝置分散，例如占調配物 之50至90%重量比。胜肽（或衍生物）最佳係製備成顆粒形 式，平均粒徑小於10毫米（或微米），最佳為0 5毫米至5毫米 10 供最有效輸送至遠端肺臟。 經鼻輸送 本發明也涵蓋經鼻輸送EPO-R激動劑胜肽（或衍生 物）。經鼻輸送允許治療產品投予鼻後，胜肽直接進入血 流’而產品無須沉積於肺臟。經鼻輸送之調配物包括使用 15 葡萄聚糖或環糊精調配之調配物。 劑量 對全部胜肽化合物而言，進行進一步研究將瞭解有關 於各種病人治療各種病情之適當劑量，熟諳技藝人士考慮 治療内容 '接受者年齡及一般健康情況將可決定適當劑 20 量。劑量之選擇係依據期望療效、投藥途徑、及預定治療 時間決定。通常0.001至10毫克/千克體重每曰劑量投予哺乳 類。通常供靜脈注射或輸注用，劑量可降低。此等投藥計 晝可依據循環半生期及使用之調配物而改變。 本發明胜肽（或其衍生物）可結合一或多種其它活性成 73 1344964 分或藥學組成物投藥。 其次將藉以下貫施例說明本發明。但於說明書中任何 為位置使用此等及其它實施例僅供舉例說明之用，而非限 ♦ 5制本發明之範圍及定義或限於任何舉例說明之形式。同 • 理，本發明非僅限於此處所述任何特佳具體例。確實本發 明之多項修改及變化於熟諳技藝人士研讀本說明書將顯然 .自明，且可未悖離本發明之精髓及範圍而達成。因此本發 明僅受隨後之申請專利範圍各項及申請專利範圍之相當例 10 之完整範圍所限。 貫施例1 :藉固相合成合成EPO-R激動劑胜肽二元體 步驟1 -Cbz-TAP之合成：含有市售二胺（rTAp」得自 亞力胥(Aldrich)化學公司）(1〇克，67·47毫莫耳)於無水DCM (1〇〇毫升）之溶液冷卻至〇°C。氣曱酸苄酯（4.82毫升，33.7 15毫莫耳）於無水DCM(50毫升）之溶液經添加漏斗以6_7小時 _ 時間緩慢添加’整個過程維持反應混合物溫度於，然後 讓其冷卻至室溫（約25。〇。又經16小時後，DCM經真空去 . 除’殘餘物分溶於3N鹽酸與醚。水層經收集，以50%水性 氫氧化鈉中和至pH 8-9及以乙酸乙酯萃取。乙酸乙酯層於 2〇無水硫酸鈉脫水’然後真空濃縮’獲得粗產物 mono-Cbz-TAP(5克，約50%產率）。此化合物未經進一步純 化即用於次一反應。 NHCbzWojchowski (1991) J. Β〇1· Chem. 266: 609-614 (protein cool acid nucleotification of B6SUt.EP cells); Dusanter-Fourt et al. (1992) J. Biol. Chem. 287:10670- 10678 (EPO of human EPO-reactive cells is phosphorylated by tyrosine of 60 1344964); Quelle et al. (1992) J. Biol. Chem. 267:17055-17060 (FDC-ER cell lysate protein pp 100 Tyrosine acid acidification); Worthington et al. (1987) Exp. Hernatol. 15:85-92 (colorimetric assay for hemoglobin); Kaiho and Miuno (1985) 5 Anal. Biochem. 149:117-120 (hemoglobin use) Detection of 2,7-diaminopurine); Patel et al. (1992) J. Biol. Chem. 267:21300·21302 (performance of c-myb); Withuhn et al. (1933) cells 74:227-236 ( Combination of JAK2 and phosphorylation of tyrosine); Leonard et al. (1993) Blood 82: 1071-1079 (performance of GATA transcription factors); and Ando et al. (1993) 10 Proc. Natl· Acad. Sci. USA 90: 9571-9575 (adjustment of G through loop D2 and D3). An instrument designed by Molecular Devices to be used as a microphysiometer has been reported to be successfully used to determine the effects of agonists and antagonists on various receptors. The basis of the device is the determination of the rate of acidification of the extracellular medium in response to activation of the receptor. In vivo functional assay analysis An in vivo functional assay that can be used to assess the strength of a peptide in a test. Bioassay for mice with erythrocytosis. For this assay analysis, mice received alternating conditioning cycles for several days. During this cycle, mouse 20 alternates between subatmospheric conditions and ambient atmospheric conditions. The mice were then maintained at ambient pressure 2-3 days prior to the administration of the test sample. The test peptide samples of the positive control mice or the positive control mice were injected subcutaneously into the conditioned mice. Radiolabeled iron (e.g., &amp; 59) was administered 2 days after the administration of radiolabeled iron. The blood volume and radioactivity measurements of each blood sample are then determined by standard techniques 61 1344964. The blood sample of the mouse injected with the activity test peptide (due to erythrocyte hemoglobin binding to Fe59) showed higher radioactivity than the mice that did not receive the test Shengtai or EPO. Ίο Another in vivo functional assay that can be used to assess peptide activity is a reticulocyte assay used in this assay. Normal untreated mice were injected subcutaneously with EPO or test peptides for 3 consecutive days. In the third episode, mice were also injected intraperitoneally with iron glucomannan. On day 5, blood samples were taken from mice. The percentage (%) of reticulocytes in the blood was determined by thiazole orange staining and flow cytometry analysis (lattice counting). In addition, the blood volume is measured manually. The corrected percentage of reticulocytes was determined using the following formula: % RETIC correction = % RETIC observation x (blood volume _ / normal blood volume) The activity test compound showed a % RETICft concentration higher than that of the untested peptide or Ερ〇 mice. Use of the agonist of the present invention 15 20 The peptide compound of the present invention can be used in a test tube as a tool for understanding the role of sputum organisms' including a number of factors that are considered to affect the yield of sputum and the binding of hydrazine to EPO-R. (eg EPO/EPO-R signal transduction/receiver activation) 'either by the yield and the effect of hydrazine binding to EPO-R. The peptide is useful for the development of other compounds by providing important structure-activity-relationship information. Thus, the peptide of the present invention can also be used to develop other compounds that can bind to EPO-R. In addition, based on the ability of the peptide of the present invention to bind to EPO-R, the peptide of the present invention can be used as a reagent for detecting EPO-R in living cells, cells after fixation, biological fluids, tissue homogenates, purified natural biological substances, and the like. . Example 62 1344964 By labeling such peptides, cells having a ΕΡΟ-R surface can be identified. Further, based on its ability to bind EPO-R, the peptide of the present invention can be used for in situ coloring 'FACS (fluorescence activated cell screening) analysis, Western blot analysis, ELISA (enzyme-linked immunosorbent assay), and the like. Furthermore, based on the ability of the compound of the present invention to bind to EPO-R, the peptide of the present invention can be used for purification of the receptor, or for purifying cells expressing EPO-R on the cell surface (or inside the osmotic cell). The peptide of the present invention can also be used as a commercial reagent for various pharmaceutical research and diagnostic purposes. Such uses include, but are not limited to, (1) quantification as a calibration target for multiple functional assays, activity of candidate EPO-R agonists, and (2) use as a blocker for random peptides Screening, that is, for finding a new EPO-R peptide peptide ligand group, the peptide can be used to block the recovery of the EPO peptide of the present invention; (3) for co-crystallization with EPO-R, Inventive peptides bind to the crystal of EPO-R, so X-ray crystallography can be used to determine the receptor 15 / peptide structure; (4) used to determine the red blood cell precursor cells through the initiation of differentiation, and induced globulin synthesis and blood quality Complex synthesis and the ability to increase the number of ferritin receptors; (5) to maintain proliferation and growth of EPO-dependent cell lines such as FDCP-1-mEPO-R and TF-1 cell lines; (6) use of radioactive chromophores The group is labeled for use with the peptide of the present invention; and other research and diagnostic applications, wherein EP〇-R is preferably activated, or such activation is conveniently calibrated to a known amount of EPO-R agonist. In yet another aspect of the invention, methods of treatment and manufacture of medicaments are provided. The peptide compounds of the present invention can be administered to warm-blooded animals including humans to mimic the binding of EP to EPO-R in vivo. Thus, the present invention encompasses a therapeutic treatment of Ep〇 deficiency phase 63 1344964's' which comprises administering a compound of the invention in an amount sufficient to stimulate the EP to improve the symptoms associated with EPO deficiency in vivo. For example, the peptide of the present invention can be used for the treatment of renal insufficiency and/or terminal renal abortion/dialysis, AIDS-related anemia, chronic inflammatory diseases (such as rheumatoid arthritis and chronic intestinal inflammation), and autoimmune diseases. Anemia; and increase the number of red blood cells in the patient before surgery. Other disease states, conditions, and blood irregularities that can be treated by the peptide treatment of the present invention include: no type I thalassemia; cystic fibrosis; pregnancy and menstrual conditions; immature early anemia; spinal cord injury; space flight Acute blood loss; aging; 10 stroke, ischemia (including the central nervous system and heart); and a variety of tumor diseases accompanied by abnormal red blood cell production. In other embodiments, the peptide compounds of the present invention are useful for treating conditions that are not characterized by low or low red gold balls, e.g., for pre-treatment prior to transfusion. In addition, administration of a compound of the invention may result in a shortened bleeding time, 15 so that it can be administered to a patient prior to surgery, or for an appropriate condition for which bleeding may be expected. Furthermore, the compounds of the invention are useful for activating the activation of megakaryocytes. Because EPO shows mitogenic and chemotactic effects on vascular endothelial cells' and effects on central choline-induced neurons [eg reference to Amagnostou (1990) Proc. Natl. Acad. Sci. USA. 20 87:5978- 5982 and Konishi et al. (1993) Brain Research 609: 29-35], the compounds of the invention may also be used to treat a variety of vascular disorders, such as promoting wound healing, promoting collateral vessel growth (eg, may occur after myocardial infarction); sores Treatment; and post-vascular graft treatment. The compounds of the invention are also useful in the treatment of a variety of neurological disorders 'generally at a lower concentration of acetylcholine than other nerves 64 1344964 active substances (eg, neurotransmitters), or the relative volatility of the bile test is relative to other neuroactive substances. Low concentration is characteristic. Pharmaceutical Compositions In yet another aspect of the present invention, there is provided a pharmaceutical composition of the aforementioned Ep〇R agonist. Conditions that can be improved or adjusted by administering such compositions include the conditions listed above. Such pharmaceutical compositions can be, for example, by oral, parenteral (muscle, intra-abdominal, intravenous (IV) or subcutaneous injection), transdermal (passive infiltration, or using ion electrophoresis or electrophoresis), transmucosal (transnasal, transvaginal) , transrectal or sublingual route of administration, or the use of biodegradable implants, 10 which can be formulated for a variety of administration routes. Generally, the pharmaceutical compositions of the present invention comprise an effective amount of an EPO-R agonist peptide or derivative product of the present invention, together with a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and/or Or carrier. Such compositions include 豨15 release agents containing various buffering agents (eg, Tris-HCl, acetate, phosphate), different pH and ionic strength; additives such as detergents and solubilizers (eg, Tween 20, Thon 80, Polysorbate 80), antioxidants (such as ascorbic acid, sodium metabisulfite), preservatives (such as mercapto, benzyl alcohol) and bulk substances (eg lactose, mannitol) The mash compound is mixed with the granule preparation of the polymer compound granule preparation such as polylactic acid, polyglycolic acid or the like or mixed with the vesicle. 20 Hyaluronic acid can also be used. Such a composition affects the physical state, stability of the protein and its derivatives, the rate of release in vivo, and the rate of clearance in vivo. See, for example, Remington's Pharmaceutical Sciences, 18th Edition (Murck Publishing Company, 1990, Easton, TX 18042), pages 1435-1712, incorporated herein by reference. The composition can also be prepared in liquid form, or in the form of a dry powder (e.g., lyophilized). 65 1344964 Oral delivery of oral solid dosage forms is intended for use herein, and oral solid dosage forms are outlined in Remington's Pharmaceutical Sciences, 18th Edition (1990 Merck Publishing Company, Easton, Pennsylvania 18042), Chapter 89, by reference Enter here. Solid dosage forms include ingots, capsules, pills, buccal or diamond ingots, lenticular capsules, tablets, powders or granules. In addition, liposome or protein-like encapsulation can also be used to formulate » this composition (eg, protein-like microspheres, as reported in U.S. Patent No. 4,925,673). Liposomes can also be used, and the vesicles can be derivatized using various polymers (e.g., U.S. Patent No. 5,013,556). A description of possible solid therapeutic dosage forms is described in Marshall, K.: Modern Medicine G.s. Banker and C.T. Rhodes Chapter 10, 1979, incorporated herein by reference. Typical formulations include the Ep0_R agonist peptide (or a chemically modified form thereof) and an inert ingredient which allows protection against the stomach environment and release of the biologically active substance in the intestinal tract. It is contemplated herein to also encompass oral pharmaceutical dosage forms, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain &amp; the ingredients 'including inert diluents; adjuvants such as wetting agents , emulsifiers and suspensions; and sweeteners, dwarfs and fragrances. The peptide can be chemically modified to allow oral delivery of the derivative to be effective. Generally, it is contemplated that the chemical modification will attach at least a portion to the component molecule itself, where this moiety allows (a) inhibition of protein breakdown; and (b) absorption into the bloodstream by the stomach or intestine. It is also desirable to increase the overall stability of each component' and extend the time in the body. As previously discussed, PEGylation is preferred for chemical modification for pharmaceutical use. Other modifiable parts include: propylene glycol, a copolymer of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrole 66 1344964. Ketone, polylysine, poly-1,3-dioxolane, and poly-1,3,6-trioxocyclo [see, for example, Abuchowski and Davis (1981) "Water Soluble Polymer-Enzyme Adducts "Enzymes as drugs. Edited by Hocenberg and Roberts (Willie Technology, Inc., New York, NY), pp. 367-383; and Newmark et al. (1982) J. Appl. 5 Biochem. 4:185-189]. For oral formulation, the release site can be the stomach, small intestine (duodenum, jejunum or ileum) or large intestine. Those skilled in the art can already obtain a formulation that does not dissolve in the stomach but can release material in the duodenum or other parts of the intestine. The preferred release of the drug avoids deleterious effects on the stomach environment by protecting the peptide (or derivative) or by releasing the peptide 10 (or derivative) beyond the stomach environment, such as the intestinal tract. In order to ensure total gastric resistance, a coating that is at least impervious to pH 5.0 is required. The more common inert ingredients that can be used as casings are cellulose acetate trimellitate (CAT), hydroxypropyl decyl cellulose (HPmcP), HPMCP 15 5 , HPMCP 55, polyvinyl acetate phthalate. (PVAP), Eudragit L30D, Aquateric, cellulose acetate (CAP), yuraji, yuraji S and shellac. These coatings can be used as a hybrid film. The coating or coating mixture can also be used to protect the 20-key agent from contact with the stomach. It includes sugar coatings or makes it easier to swallow the coating. Capsules may be composed of a hard shell (e.g., gelatin) for delivery of a dry therapeutic agent (i.e., a powder) for use in a liquid therapeutic drug. A soft gelatin shell may be used. The shell material of the bean-shaped capsule may be thick paper or other edible paper. For pellets, diamond ingots, molded ingots or ingot wet abrasives, wet block technology can be used. 67 1344964 The peptide (or derivative) may be finely multiparticulate, in the form of granules or granules of about 1 mm in size. The formulation of the material for capsule administration can be in the form of a powder, a micro-compressed plunger or even an ingot. These therapeutic agents can be prepared by compression. Coloring agents and/or flavoring agents may also be included. For example, the peptide (or derivative) can be formulated (e.g., by liposome or microsphere encapsulation) and then further included in an edible product such as a frozen beverage containing a coloring agent and a flavoring agent. The inert peptide can be used to dilute or increase the volume of the peptide (or derivative). These φ diluents include carbon hydrates, particularly mannitol, alpha lactose, anhydrous lactose cellulose, sucrose, modified dextran, and starch. Some inorganic salts can also be used as a filler, including tri-dead acid, carbonic acid and sodium chloride. Several commercially available thinners are FastFo F. (Fast_F1), Emdex, STA-Rx 1500, Emc〇mpress and Avicell. The powder ΊΓ 3 is included in the formulation of the therapeutic agent formulated into a solid dosage form. Materials used as disintegration θ丨 include, but are not limited to, temple powders, including commercially available disintegrating agents with a powder of the main powder, Expl〇tab, sodium starch glycolate, Amber Wright (Ambei*lite) ϋ fiber money, ultra-pentose candy gel, sodium alginate, gelatin, scalp, acid buffered sodium cellulose, natural sponge and bauxite ~ 纟 can be used. The disintegrating agent can also be an insoluble cation exchange resin. Powdered gum can be used as a disintegrating agent and a binder, as well as a powdery tree, such as the soil, carrageenan or scutellaria. The brown acid and the sodium alginate salt can also be disintegrating agents. The binder can be used to bind the peptide (or derivative) to form a hard ingot d, including products derived from nature such as acacia, scutellaria, and gelatin. Its money-reducing agents include methyl cellulose (MC), ethyl cellulose tons 68 1344964 and slow methyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) can also be used in alcoholic solutions to granulate peptides (or derivatives). Anti-friction agents can also be included in peptides (or derivatives). ) Formulations to prevent sticking during the dispensing process. Lubricants can be used as the layer between the peptide (or derivative) and the wall of the stamp. Lubricants include (but without limitation, stearic acid including its magnesium and about salts, polytetrafluoroethylene (PTFE), liquid paraffin, Vegetable oils and waxes. Soluble lubricants such as sodium lauryl sulfate, magnesium lauryl sulfate, Carbowax 4000 of various molecular weights and Carbows 10 6000 can also be used. The agent can improve the drug flow properties of the formulation process and can assist in the rearrangement of the compression process. The slip agent includes starch, talc, pyrogen, and hydrated yttrium aluminate. To assist the peptide (or derivative) to dissolve in water. Environment, interface 15 active agent can be added as a wetting agent. Surfactant includes anionic detergents such as sodium lauryl sulfate 'dioctyl sulfonate sodium succinate and dioctyl benzoate. Can use cationic detergents, including chlorine Benzalkanal or chlorinated phenethyl hydrazine. Possible nonionic detergent forms that can be included as a surfactant in the formulation include lauromacol 400, Boliox (p〇ly) Xyi) 40 2 〇 stearate, polyoxyethylidene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, borissol 20 '40, 60 '65 and 80 'salt fatty vinegar, Methylcellulose and carboxymethylcellulose. These surfactants may also be present in a protein or derivative formulation, either alone or in a mixture of different ratios. Additives that promote absorption of the peptide (or derivative) are, for example, fatty acids 69. Classes such as oleic acid, linoleic acid and linolenic acid. It is also desirable to use controlled release oral formulations "peptides (or derivatives) which can be mixed with an inert matrix which allows for the release of, for example, gum by diffusion or percolator. The slowly denatured matrix can also be mixed with the formulation. Several casings also have an extended release effect. Another controlled release form is based on the method of the Oros (0r0s) treatment system (Alza), in other words, the drug package. Within the semipermeable membrane, the semipermeable membrane allows water to enter due to osmosis and pushes the drug out through a single small opening. Other coatings can also be used in the formulation. These coatings include a variety of sugars that can be applied to the coating tray. ( The derivative can also be made into a film-coated tablet. The materials used in this example are divided into two groups. The first group is a non-enteric material, including thiol cellulose, ethyl cellulose, hydroxyethyl cellulose, methyl hydroxy group. - ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium thioglycolate, providone and polyethylene glycol. The second group includes enteric materials , usually an ester of phthalic acid. A mixed material can be used to provide an optimum film coat. The coat can be carried out in a pan coater or in a fluidized bed or by a compression coating. The preparation for parenteral administration of the present invention comprises a sterile aqueous or non-aqueous solution, suspension or emulsion. The non-aqueous solvent or vehicle is, for example, propanol, I-ethyl alcohol, vegetable oil such as sein oil and corn oil, Organic vinegar such as ethyl oleate for bright kicking and injection. These dosage forms may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. The sterilization method can be carried out, for example, by filtering through a filtration membrane that retains bacteria, sterilizing the composition i344_ via a mash-mixing sterilizing agent, sterilizing by using a glazing composition, or sterilizing β by heating the composition. Several other sterile injectable media are formulated just prior to use. Transrectal or transvaginal delivery 5 The composition for rectal or vaginal administration is preferably a suppository which contains excipients such as cocoa butter or a suppository in addition to the active substance. Compositions for nasal or lingual administration can also be prepared using standard excipients well known in the art. Transpulmonary delivery 1〇 The invention also encompasses delivery of an EPO-R agonist peptide (or a derivative thereof) via the lung. The peptide (or derivative) is delivered to the mammalian lung when inhaled by the mammal and enters the bloodstream through the lung epithelial lining [see, for example, Adjei et al. (1990) Pharmaceutical Research 7:565_569; Adjei et al. (1990) International Pharmaceutical Journal 63: 135-144 (leuprolide acetate); Braquet et al. (1989) 15 Journal of Cardiovascular Pharmacology 13 (sup5): 143-146 (endothelin-1); Hubbard et al. (1989) Annual Report of Internal Medicine , VOL. III, 206-212 (a 1-antitrypsin); Smith et al. (1989) J. Clin. Invest 84: 1145-1146 (α-l-protease); Oswein et al. (1990) "Protein Aerosolization, the second session of the Respiratory Drug Delivery Symposium, Colorado Chiss (Recombinant Human Growth Hormone); Debs et al. (1988) J. Immunol. 140:3482-3488 (interferon-r and tumor) Necrosis factor a); and U.S. Patent No. 5,284,656 to Platz et al. (granulocyte community stimulating factor). The method and composition of the system for the delivery of a drug through the lungs is described in U.S. Patent No. 5,451,569 to Wong et al. 71 1344964 The teachings encompassed by the present invention are a wide variety of mechanical devices for the delivery of therapeutic products via the lung, including, but not limited to, nebulizers, metered dose inhalers, and dry powder inhalers&apos;, all of which are well known to those skilled in the art. A number of commercially available special devices suitable for the practice of the present invention are Yazhevent (10) ravem) 5 • (Mallindaodt, St. Louis, Montana); Acorn I1 atomization (Marquest Medical Products, Inc., Yingwu, CO), Ventolin metered dose inhaler (Glax〇 φ, Research Triangle Park, North Carolina); and Spencer Spinhaler dry powder inhaler (Fis〇ns, Bedford, MA). 10 All such devices require the use of a formulation suitable for dispensing peptides (or derivatives). Typically, such formulations will depend on the type of device used, and in addition to the usual diluents, adjuvants, and/or therapeutic carriers, the use of suitable propellant materials. The use of vesicles, microcapsules or microspheres, including complexes or other types of carriers, is also contemplated. Depending on the type of chemical modification or the type of device used, the chemically modified peptides are also prepared in different formulations. φ Formulations suitable for nebulizers including spray atomization or ultrasonic atomization typically comprise a peptide (or derivative) dissolved in water at a level of from about 1 to 25 mg of bioactive protein per ml of solution. Formulations also include buffers and monosaccharides (e.g., for protein stability and osmotic pressure regulation). The nebulizer formulation also contains an interfacial agent to reduce or prevent agglomeration of the peptide (or derivative) due to solution atomization when the aerosol is formed. Formulations for metered dose inhaler devices typically comprise a finely divided powder comprising a propellant peptide (or derivative) suspended by means of a surfactant. The propellant can be any material used in this project, such as carbon fluorinated carbon, hydrochlorofluorocarbon 72 1344964 carbonized carbon hydrofluorocarbon or hydrocarbon 'including chlorotrifluoromethane, chlorodifluoromethane, two Chlorotetrafluoroethane, and U,l,2-tetrafluoroethane or combinations thereof. Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid can also be used as a surfactant. 5 The formulation delivered by the dry powder inhaler device comprises a finely divided dry powder containing a win (or derivative), and also includes a bulking agent such as lactose, sorbitol, sucrose and mannitol, in an amount to assist in dispersing the dry powder by the device. For example, 50 to 90% by weight of the formulation. The peptide (or derivative) is preferably prepared in a granular form having an average particle size of less than 10 mm (or micrometers), preferably from 0.5 mm to 5 mm 10 for most efficient delivery to the distal lung. Nasal delivery The present invention also encompasses transnasal delivery of EPO-R agonist peptides (or derivatives). Nasal delivery allows the therapeutic product to be administered to the nose, and the peptide enters the bloodstream directly, and the product does not need to be deposited in the lungs. Nasal delivery formulations include formulations formulated with 15 dextran or cyclodextrin. Dosage For all peptide compounds, further studies will be conducted to understand the appropriate doses for various patients to treat various conditions, and those skilled in the art will consider the treatment content 'recipient age and general health status will determine the appropriate dose of 20 doses. The choice of dosage is based on the desired therapeutic effect, the route of administration, and the time of the scheduled treatment. Usually, 0.001 to 10 mg/kg of body weight per dose is administered to the mammal. Usually for intravenous or infusion, the dose can be reduced. These dosing schedules may vary depending on the cycle half-life and the formulation used. The peptide of the present invention (or a derivative thereof) can be administered in combination with one or more other activities into a fraction of 73 1344964 or a pharmaceutical composition. Next, the present invention will be described by way of the following examples. However, the use of these and other embodiments in the specification is for illustrative purposes only and is not intended to limit the scope and the scope of the invention. In the meantime, the invention is not limited to any particularly specific examples described herein. It is to be understood that many modifications and variations of the present invention will be apparent to those skilled in the art. Therefore, the present invention is limited only by the scope of the following claims and the full scope of the equivalent of the patent application. Example 1: Synthesis of EPO-R agonist peptide peptide by solid phase synthesis Step 1 - Synthesis of Cbz-TAP: Contains commercially available diamine (rTAp from Aldrich Chemical Company) (1 The solution of gram, 67.47 mmoles in anhydrous DCM (1 mL) was cooled to 〇 °C. A solution of benzyl plutonate (4.82 ml, 33.7 15 mmol) in anhydrous DCM (50 mL) was added to a funnel over 6-7 s _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ (About 25. 〇. After another 16 hours, DCM was removed by vacuum. The residue was dissolved in 3N hydrochloric acid and ether. The aqueous layer was collected and neutralized to pH 8-9 with 50% aqueous sodium hydroxide and Extracted with ethyl acetate. The ethyl acetate layer was dried <RTI ID=0.0></RTI> to <RTI ID=0.0> One reaction. NHCbz

DCMDCM

74 1344964 步驟2-Cbz-TAP-Boc之合成：於Cbz-TAP(5克，17.7毫 莫）於己烷(25毫升）之激烈攪拌之懸浮液内加入二丁醚(3.86 克，17.7毫莫耳），於室溫持續攪拌隔夜。反應混合物以DCM (25毫升）稀釋，以丨〇%水性檸檬酸(2次）、水(2次)及食鹽水 5 洗滌。有機層以無水硫酸鈉脫水及真空濃縮。粗產物(5克) 直接用於次一反應。74 1344964 Step 2 - Synthesis of Cbz-TAP-Boc: Dibutyl ether (3.86 g, 17.7 mmol) was added to a vigorously stirred suspension of Cbz-TAP (5 g, 17.7 mmol) in hexanes (25 mL) Ear), stirring continuously at room temperature overnight. The reaction mixture was diluted with DCM (25 mL) and washed with EtOAc EtOAc (EtOAc) The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product (5 g) was used directly in the next reaction.

步驟3 - Boc-TAP之合成：得自前一反應之粗產物溶解 於曱醇(25毫升），於5%鈀/碳(5% w/w)存在下於氣球壓力下 10 氫化16小時。混合物經過濾，以甲醇洗滌，濾液經真空濃 縮獲得粗產物H-TAP-Boc產物（產率3.7克）。步驟1-3後 Boc-TAP之總估計產率為44%(基於Cbz-Cl用量計算）。Step 3 - Synthesis of Boc-TAP: The crude product from the previous reaction was dissolved in methanol (25 mL) and hydrogenated at balloon pressure for 16 hours in the presence of 5% palladium/carbon (5% w/w). The mixture was filtered, washed with EtOAc (EtOAc)EtOAc. The total estimated yield of Boc-TAP after steps 1-3 was 44% (calculated based on the amount of Cbz-Cl).

步驟4 -天塔膠(TentaGel)鍵聯基之合成：溴化天塔膠 15 (2.5克’ 〇.48毫莫耳/克’得自德國雷普(Rapp)聚合物公司）， 酚系键聯基5當量)及碳酸鉀(5當量)於2〇毫升DMF加熱至70 °C經歷14小時❶冷卻至室溫後，樹脂經洗滌(0· 1 N Ha、水、 ACN、DMF、MeOH)及乾燥獲得琥珀色樹脂。Step 4 - Synthesis of TentaGel Bonding Group: Brominated Tianta Gum 15 (2.5 g '〇.48 mmol/g' from Rapp Polymers, phenolic bond) 5 equivalents of the base and potassium carbonate (5 equivalents) were heated to 70 ° C in 2 mL of DMF for 14 hours. After cooling to room temperature, the resin was washed (0·1 N Ha, water, ACN, DMF, MeOH). And drying to obtain an amber resin.

75 1344964 步驟5 -天塔膠鍵聯基-TAP(Boc)之合成：2.5克如上所 得樹脂及H-TAP-Boc(1.5克，5當量）及冰醋酸AcOH(34微 升，5當量)攝取於1:1 MeOH-THF混合物及振搖隔夜。1M氰 基硼氫化鈉（5當量）於THF之溶液添加至其中又振搖7小 5 時。樹脂以過濾洗滌(DMF，THF，0.1NHC1 ’水，MeOH) 及乾燥。小量樹脂使用Bz-Cl及DIEA於DCM苯甲醯化，於 70%TFA-DCM裂解，及藉LCMS及HPLC檢驗。75 1344964 Step 5 - Synthesis of Tianta Glue-TAP (Boc): 2.5 g of the above-obtained resin and H-TAP-Boc (1.5 g, 5 equivalents) and glacial acetic acid AcOH (34 μL, 5 equivalents) Mix in 1:1 MeOH-THF and shake overnight. A solution of 1 M sodium cyanoborohydride (5 eq.) in THF was added thereto and shaken again for 7 hours. The resin was washed with filtration (DMF, THF, 0.1NH.sub.1 water, MeOH) and dried. A small amount of resin was deuterated in DCM using Bz-Cl and DIEA, cleavage in 70% TFA-DCM, and by LCMS and HPLC.

步驟6-天塔膠鍵聯基-TAP-Lys之合成：如上所得樹脂 10 使用Fmoc-Lys(Fmoc)-OH活性溶液（由5當量胺基酸及5當量 HATU以0.5 Μ溶解於DMF製備，接著加入10當量DIEA處 理），讓其溫和振搖14小時。樹脂經洗滌(DMF、THF、DCM、 MeOH)，及乾燥獲得經保護之樹脂。殘餘胺基經由使用1〇% 乙酐’ 20%吡啶於DCM溶液處理樹脂20分鐘，接著如前述 15 洗滌。Fmoc基之去除方式，係經由於30%哌啶於DMF溫和 振搖樹脂20分鐘，接著洗滌(DMF、THF、DCM、MeOH) 及乾燥。Step 6 - Synthesis of Tianta Glue-TAP-Lys: Resin 10 obtained above was prepared by dissolving Fmoc-Lys(Fmoc)-OH active solution (5 equivalents of amino acid and 5 equivalents of HATU in 0.5 Torr in DMF). Then 10 equivalents of DIEA treatment) was added and allowed to shake gently for 14 hours. The resin was washed (DMF, THF, DCM, MeOH) and dried to give a purified resin. The residual amine group was treated with a 1% by weight acetic anhydride &lt; 20% pyridine in DCM solution for 20 minutes, followed by washing as before. The Fmoc group was removed by gently shaking the resin with 30% piperidine in DMF for 20 minutes, followed by washing (DMF, THF, DCM, MeOH) and dried.

步驟7 -天塔膠鍵聯基-TAP-Lys(胜肽)2之合成：如上所 76 1344964 得樹脂接受Fmoc-胺基酸偶合，使用HBTU/HOBt活化，以 及以哌啶去除Fmoc之重複循環處理，來同時營建二胜肽 鏈。可方便地於得自應用生物系統公司之ABI 433自動化胜 肽合成儀進行。於最終Fmoc去除後，端末胺基以乙酐（1〇 當量）及DIEA (20當量)於DMF 20分鐘醯化，接著如前文說 明洗滌。Step 7 - Synthesis of tyrosin-linked TAP-Lys (peptide) 2: as above 76 1344964 Resin accepts Fmoc-amino acid coupling, activates with HBTU/HOBt, and repeats the cycle of removal of Fmoc with piperidine Processing, to build a second peptide chain at the same time. It is conveniently carried out from Applied Biosystems' ABI 433 automated peptide synthesizer. After the final Fmoc removal, the terminal amine groups were deuterated in acetic acid (1 当量 equivalent) and DIEA (20 eq.) in DMF for 20 minutes, followed by washing as previously described.

ίο 步驟8 -由樹脂裂解：如上所得樹脂於室溫懸浮於TFA (82.5%)、齡(5%)、乙二硫醇(2.5%)、水(5%)及硫菌香醚(5%) 之溶液3小時。也可使用另一種裂解混合液，例如TFA (95%)、7]c(2.5%)及三異丙基矽烷基(2 5%)。TFA溶液冷卻至5 °C，倒入***内來沉澱胜肽。於減壓下過濾及脫水，獲得所 需胜肽。使用C19管柱經製備性HPLC純化，獲得純質胜肽。Ίο Step 8 - Cleavage by resin: The resin obtained above was suspended at room temperature in TFA (82.5%), aged (5%), ethanedithiol (2.5%), water (5%) and thiocyanate (5%). ) The solution was 3 hours. Another cleavage mixture can also be used, such as TFA (95%), 7]c (2.5%), and triisopropyldecyl (2 5%). The TFA solution was cooled to 5 ° C and poured into diethyl ether to precipitate the peptide. Filtration and dehydration under reduced pressure gave the desired peptide. Purification by preparative HPLC using a C19 column gave a pure peptide.

步驟9-胜狀氧化而生成分子内雙硫鍵：胜肽二元體溶 解於20% DMSO/水（1毫克乾重胜肽/毫升），讓其於室溫靜置 36小時。胜肽之純化方式係經由將反應混合物載猗炱C18 HPLC管柱（瓦特氏(waters)公司碟塔派免(Delta-Pak) C18，15 77 1344964 微米粒徑，300埃孔徑，40毫米x 200毫米長度），接著為線 性ACN水/0.01% TFA梯度，由5%至95% ACN經歷40分鐘時 間。凍乾含所需胜肽之洗提分，獲得產物，呈絮狀白色固體。Step 9 - Oxidation of the oxime to form an intramolecular disulfide bond: The peptide peptide was dissolved in 20% DMSO/water (1 mg dry weight peptide/ml) and allowed to stand at room temperature for 36 hours. The peptide was purified by loading the reaction mixture on a C18 HPLC column (Waters Corporation Delta-Pak C18, 15 77 1344964 micron particle size, 300 angstrom aperture, 40 mm x 200 Millimeter length) followed by a linear ACN water/0.01% TFA gradient from 5% to 95% ACN for 40 minutes. The elution fraction containing the desired peptide was lyophilized to give the product as a flocculent white solid.

含還原雙硫鍵之 二元逋胜肽（XYZ)Binary peptides (XYZ) containing reduced disulfide bonds

DMSO/H2ODMSO/H2O

S—SS-S

含氧化雙琉鍵之 二元體胜肽（XYZ) 5 步驟10 -端末NH2基之PEG化： 透過胺基曱酸酯鍵之PEG化：胜肽二元體混合1.5當量 (莫耳基準）活性PEG物種(mPEG-NPC，得自曰本NOF公司） 於無水DMF獲得澄清溶液。5分鐘後，4當量DIEA添加至前 述溶液。混合物於周圍溫度攪拌14小時，接著以C18反相 10 HPLC純化。藉MALDI質譜證實PEG化胜肽之結構式。純化 後之胜肽也經由陽離子交換層析術純化，摘述如後。 (AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)ί Ο ?ΝΗ- (AcGjGLYACHMGPrrCI-naQVCQPLRtMeO^HN^Binary peptides containing oxidized biguanide bonds (XYZ) 5 Step 10 - PEGylation of terminal NH2 groups: PEGylation via amino phthalate linkages: peptide equivalents mixed 1.5 equivalents (mole basis) activity PEG species (mPEG-NPC, available from Sakamoto NOF) A clear solution was obtained in anhydrous DMF. After 5 minutes, 4 equivalents of DIEA were added to the above solution. The mixture was stirred at ambient temperature for 14 hours then purified by C18 reverse phase 10 HPLC. The structural formula of the PEGylated peptide was confirmed by MALDI mass spectrometry. The purified peptide was also purified by cation exchange chromatography, as described later. (AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)ί Ο ?ΝΗ- (AcGjGLYACHMGPrrCI-naQVCQPLRtMeO^HN^

mPEG-NPC DIEA, DMF (AcG)GLYACHMGPIT(l.na1)viQPLR(MeG)—). NH/ O A. O-PEGjok 〇 (AcQGLYACHMGPnXI-aaQVCQPLR^MeGHIN^NH-VQ^ 78 0 1344964 經由醯胺鍵結之PEG化：胜肽二元體混合L5當量（莫耳 基準）活性PEG物種（PEG-SPA-NHS ’得自美國席爾瓦特 (Shearwater)公司）於無水DMF獲得澄清溶液。5分鐘後，4 當量DIEA添加至前述溶液。混合物於周圍溫度搜拌2小 5 時，接著以C18反相HPLC純化。藉MALDI質譜證實PEG化 胜月大之結構式。純化後之胜狀也經由陽離子交換層析術純 化，摘述如後。mPEG-NPC DIEA, DMF (AcG)GLYACHMGPIT(l.na1)viQPLR(MeG)—). NH/ O A. O-PEGjok 〇(AcQGLYACHMGPnXI-aaQVCQPLR^MeGHIN^NH-VQ^ 78 0 1344964 via guanamine bond PEGylation: peptide peptide mixed L5 equivalent (mole reference) active PEG species (PEG-SPA-NHS 'from Shearwater, USA) to obtain a clear solution in anhydrous DMF. After 5 minutes, 4 Equivalent DIEA was added to the above solution. The mixture was mixed at ambient temperature for 2 hours, and then purified by C18 reverse phase HPLC. The structure of PEGylation was confirmed by MALDI mass spectrometry. The purified form was also subjected to cation exchange chromatography. Purification, as described below.

PEG.SPA.NHSPEG.SPA.NHS

D1EA.DMFD1EA.DMF

步驟11 -離子交換純化：除了保有起始二元體胜肽之 10能力之外，若干交換支持體也研究其由未反應（或水解)pEG 分離前述胜肽-PEG軛合物之能力。離子交換樹脂(2_3克）載 於1厘米管柱，接著轉成鈉形式(〇2 N氫氧化鈉載於管柱， 直至洗提分為pH 14,約5倍管柱容積），而非轉成氫形式(使 用0.1 N鹽酸或〇.1 M乙酸洗提直至洗提分匹配負載阳約$ 15倍管柱容積）’接著以2 5 % A C Ν/水洗滌至ρ Η 6。軛合前之胜 肽或胜肽-PEG輕合物溶解於25% ACN/水（1〇毫克/毫升）， pH以THA調整至小於3，職載荷至管柱。以2_3管桂容積 79 1344964 25% ACN/水洗滌且收集5毫升洗提分後，以0.1 Μ乙酸銨於 25% ACN/水洗提讓胜肽由管柱釋放出，再度收集5毫升洗 提分。藉HPLC分析顯示該洗提分含有所需胜肽。以氣化光 散射檢測器（ELSD)分析，指示當胜肽保留於管柱，且使用 5 乙酸銨溶液(通常於洗提分4及洗提分10間洗提出）洗提時， 未觀察得污染物未經輛合之PEG。當胜肽於初洗提緩衝液 ' (通常為前二洗提分）洗提時，未觀察得所需PEG-軛合物的 | 分離及過量PEG。 下列管柱可成功地保有胜肽及胜肽-PEG輛合物，成功 10 地由非輕合物胜肽純化胜狀-PEG輥合物： 表1 :離子交換樹脂 支持體 來源 Mono S HR 5/5強陽離子交換預載荷管柱 阿莫山(AmershairO 生科公司 SE53纖維素，微粒強陽離子交換支持體 瓦特曼公司 SP西法羅斯(Sepharose)快速流動強陽離子交換支持體 阿莫山生科公司 ’ 實施例2 :藉片段縮合合成EPO-R激動劑胜肽二元體 步驟1 - (Cbz)2-Lys之合成：離胺酸於標準條件下與氯 、15 甲酸苄酯溶液反應，獲得於兩個胺基使用Cbz基團保護之離 胺酸。Step 11 - Ion exchange purification: In addition to the ability to retain the initial binary peptide, several exchange supports were also investigated for their ability to separate the aforementioned peptide-PEG conjugates from unreacted (or hydrolyzed) pEG. The ion exchange resin (2_3 g) was loaded on a 1 cm column and then converted to sodium form (〇2 N sodium hydroxide was placed on the column until elution was divided into pH 14, about 5 times the column volume) instead of Hydrogen form (extracted with 0.1 N hydrochloric acid or 〇.1 M acetic acid until the elution fraction matches the loading cation of about 15 15 column volumes)' then washed to ρ Η 6 with 25 % AC Ν/water. Pre-Conjugation The peptide or peptide-PEG light compound is dissolved in 25% ACN/water (1 mg/ml) and the pH is adjusted to less than 3 with THA and the load is applied to the column. After washing with 2_3 tube volume 79 1344964 25% ACN/water and collecting 5 ml of elution fraction, the peptide was released from the column by 0.1 Μ ammonium acetate in 25% ACN/water, and 5 ml of the elution fraction was collected again. . Analysis by HPLC showed that the elution fraction contained the desired peptide. Analysis by gasification light scattering detector (ELSD), indicating that when the peptide remains in the column and is eluted with 5 ammonium acetate solution (usually eluted in elution 4 and elution 10), it is not observed. Contaminants are not PEG. When the peptide was eluted in the initial elution buffer ' (usually the first two washes), no separation of the desired PEG-conjugate and excess PEG were observed. The following columns successfully retained the peptide and peptide-PEG complexes and were successfully purified from the non-light compound peptides. PEG roll compounds: Table 1: Ion exchange resin support source Mono S HR 5 /5 strong cation exchange preloaded column Amoshan (AmershairO Biotech SE53 cellulose, fine particle cation exchange support body Wattman SP Sepharose fast flow strong cation exchange support Amoshan Biotech company' 2: Synthesis of EPO-R agonist peptide peptide by fragment condensation Step 1 - Synthesis of (Cbz)2-Lys: Amino acid is reacted with chlorine and 15 benzyl acetate solution under standard conditions to obtain two amines. The base is protected with an amine acid using a Cbz group.

步驟2 - Boc-TAP之合成：Boc-TAP係如實施例1步驟1 至3所述合成。 80 1344964 步驟 3 - (Cbz)-Lys 與 Boc-TAP 偶合：（cbz)2-Lys 與 Boc-TAP於標準偶合條件下偶合獲得(Cbz)2-Lys-TAP-Boc。Step 2 - Synthesis of Boc-TAP: Boc-TAP was synthesized as described in steps 1 to 3 of Example 1. 80 1344964 Step 3 - Coupling of (Cbz)-Lys with Boc-TAP: (cbz)2-Lys coupled with Boc-TAP under standard coupling conditions to obtain (Cbz)2-Lys-TAP-Boc.

步驟4 _ Lys-TAP-Boc :前一反應所得粗產物溶解於甲 5醇(25毫升），於5%鈀/碳(5% w/w)於氣球壓力下氮化16小 時。混合物經過濾，以甲醇洗滌，濾液經真空濃縮，猂得 粗產物Lys-TAP-Boc產物。Step 4 _ Lys-TAP-Boc: The crude product obtained from the previous reaction was dissolved in methyl alcohol (25 ml) and nitrided at 5% palladium on carbon (5% w/w) under balloon pressure for 16 hours. The mixture was filtered, washed with EtOAc (EtOAc)EtOAc.

10 步驟5 -藉片段縮合合成胜肽單體：四個胜肤單體序列 之胜肽片段藉標準技術合成。鎌部分經保護之胜狀片尸 進行二分開偶合反應回合。第一回合中，單體之叫該半‘ 偶合其中二胜肽片段形成，單體之c端該半藉偶合另二胜: =形成，第二偶合回合，N端半與c端半偶合生成 遵單體。紐賴藉標準彳績㈣⑽峨去解。’、 81 134496410 Step 5 - Synthesis of peptide monomer by fragment condensation: The peptide fragments of the four peptide monomers are synthesized by standard techniques. Part of the protected smear piece is subjected to two separate coupling reaction rounds. In the first round, the monomer is called the half-coupled with the formation of the two peptide fragments, and the c-terminal of the monomer is coupled with the other two: = formation, the second coupling round, and the N-terminal half and the c-terminal semi-coupling Follow the monomer. Nilai relied on standard performance (4) (10) to solve the problem. ’, 81 1344964

OtBu Tit 偶合OtBu Tit coupling

FhiocU t-f-d-na^-V-όγΡ-Οί OlBu TrtFhiocU t-f-d-na^-V-όγΡ-Οί OlBu Trt

偶合 S(Aan) CAeC)-G-L-Y-A-C-H-M Offiu TitCoupling S(Aan) CAeC)-G-L-Y-A-C-H-M Offiu Tit

Offlu Trt PbfOfflu Trt Pbf

偶合Coupling

备脫保護 (AcG)-G-Standby protection (AcG)-G-

S(Acm) -Tj-( l-nal)-V&gt;C-Q-P-L-R-(MeG)-OH OtBu Tit Pbf 步驟6-胜肽單體氧化而生成分子内雙硫鍵：然後〇Bn 脫去保護之縮合胜肽單體使用碘化物氧化而生成單體之經 Acm保護之二半胱胺酸殘基間的分子内雙硫鍵。 S(Acm)S(Acm) -Tj-( l-nal)-V&gt;CQPLR-(MeG)-OH OtBu Tit Pbf Step 6 - Oxidation of the peptide monomer to form an intramolecular disulfide bond: then 〇Bn removes the protective condensation The peptide monomer is oxidized with iodide to form an intramolecular disulfide bond between the Acm-protected cysteine residues of the monomer. S(Acm)

Trt PbfTrt Pbf

OlBu ΤΠOlBu ΤΠ

OffluOfflu

t-P-I-T-(l-oaI)-V-C-Q-P-L.R^MeG)-OH 1 II 5 (AcG&gt;G.L-yA-C-H-M-0-l OtBu Titt-P-I-T-(l-oaI)-V-C-Q-P-L.R^MeG)-OH 1 II 5 (AcG&gt;G.L-yA-C-H-M-0-l OtBu Tit

Ofiu Tit Pbf 步驟7 - Lys-TAP-Boc偶合至經氧化之〇Bn脫去保護之 單體而生成胜肽二元體：Lys-TAP-Boc於標準條件下偶合至 兩倍莫耳過量之經氧化之OBn脫去保護之單體，形成胜肽 二元體。然後胜肽二元體於標準條件下脫去保護。 82 1344964Ofiu Tit Pbf Step 7 - Lys-TAP-Boc coupling to the oxidized oxime Bn deprotected monomer to form a peptide binary: Lys-TAP-Boc coupled to twice the molar excess under standard conditions The oxidized OBn removes the protected monomer to form a peptide binary. The peptide binary is then deprotected under standard conditions. 82 1344964

(Ai^)-G-L.yA&lt;：.^M.G.P-I.T&lt;l^).vAQ.p.L-R^MeG)-OH(Ai^)-G-L.yA&lt;:.^M.G.P-I.T&lt;l^).vAQ.p.L-R^MeG)-OH

OtBu Trt W&gt;£OtBu Trt W&gt;£

OiBu Tr I i,. 氧化 ：\ NHBocOiBu Tr I i,. Oxidation :\ NHBoc

HjH NH •0 inOBoc oHjH NH • 0 inOBoc o

NH· _0 °®u ^ C®« in kt (AcG&gt;04^yAX-H-M-G-P-I-T^i -naJ&gt;V-&lt;i-Q-p^L*R^McG； 咖u Tn Ofiu Tff Pbf 脫保護 (Αϋ〇ΗΗ^Υ-Α·€-Η-Μ«&lt;3·Ρ·Ι·Τ&lt;1-ι»1)ΛΛ&lt;^&lt;}·ρ·ΐ^(Μβ〇: (AcG)-G*L-Y-ΑΌ-Η-Μ-G-P-l-T-{I ^al)^*(L&lt;^P-L4lr&lt;MeG)-—HN'NH· _0 °®u ^ C®« in kt (AcG&gt;04^yAX-HMGPIT^i -naJ&gt;V-&lt;iQp^L*R^McG; 咖u Tn Ofiu Tff Pbf Deprotection (Αϋ〇ΗΗ^ Υ-Α·€-Η-Μ«&lt;3·Ρ·Ι·Τ&lt;1-ι»1)ΛΛ&lt;^&lt;}·ρ·ΐ^(Μβ〇: (AcG)-G*LY-ΑΌ -Η-Μ-GPlT-{I ^al)^*(L&lt;^P-L4lr&lt;MeG)--HN'

NH- 0 0 步驟8 -脫保護二元體之peg化：然後脫去保護之胜肽 二元體如實施例1步驟10所述PEG化。 步驟9 ·離子交換純化：PEG化胜肽二元體然後如實施 5 例1步驟11所述純化。 實施例3 :試管内活性檢定分析 本實施例說明多個試管内檢定分析，其可用於評比本 發明EP0-R激動劑胜肽之活性及強度。檢定分析結果證實 本發明新穎胜肽結合至EPO-R且活化EPO-R發訊。此外，檢 10 定分析結果顯示新穎胜肽組成物比較前述EPO模擬胜肽， EP0-R結合親和力及生物活性出乎意外地增高。 83 1344964 Ε Ρ Ο - R激動劑胜肽二元體係根據實施例丨或實施例2提 供之方法製備。此等胜狀二元體之強度係使用一系列試管 活性檢定分析評比，包括：通報子檢定分析、增生檢定分 析、重複結合檢定分析及C/BFU-e檢定分析❶四項檢定分析 β 5 進一步說明其細節如後。 此等試管内活性檢定分析結果摘述於表2。 • 1.通報子檢定分析 • 本檢定分析係基於鼠前Β細胞系衍生之通報子細胞 Baf3/EpoR/GCSFR fos/lux。此通報子細胞系可表現嵌合體 10嗳器，包含人EP0受器至胞外部分之人GCSF受器之胞内部 分。此細胞系進一步以f0S起動基因驅動子蟲螢光素酶通報 子基因組成體轉移感染。此嵌合體受器經由加入紅血球生 成劑活化’結果導致蟲榮光素酶通報子基因表現，因此當 加入蟲螢光素酶酶基質蟲螢光素時發光。如此此種細胞之 15 EPO-R活化程度可藉蟲螢光素酶活性之量測而定量。 灸 Baf3/EpoR/GCSFR fos/lux細胞於DMEM/F12培養基（吉 伯可(Gibco)公司培養，培養基内補充10%胎牛血清(fbs ; 海克隆(Hyclone))，10% WEHI-3上清液（由 WEHI-3細胞， • ATCC # TIB-68)培養所得上清液，青黴素/鏈黴素。檢定分 -20 析前約18小時，細胞藉轉移至DMEM/F12培養基補充10% FBS及0.1% WEHI-3上清液而ft乏。檢定分析當天，細胞以 補充10% FBS(不含WEHI-3上清液）之DMEM/F12培養基洗 滌，然後lxlO6細胞/毫升於已知濃度之試驗胜肽存在下培 養，或使用EP0(R&amp;D系統公司，明尼蘇達州明那玻利）作 84 1344964 為陽性對照，於補充10% FBS(不含WEHI-3上清液）之 DMEM/F12培養。試驗胜肽之一系列稀釋液於本檢定分析 同時測試。檢定分析孔板於37。(：於5%二氧化碳氣氛下培養 4小時。隨後添加蟲螢光素（史得力葛羅(Steady_G1〇);普羅 5 米加(Promega)公司，威斯康辛州馬里森)至各孔。培養5分 鐘後，於派克(Packard)公司桌面光度計（派克儀器公司，伊 利諾州道納葛羅吾）測定發光。光強度值相對於試驗胜肽濃 度作圖’使用Graph Pad軟體分析。結果獲得半最大發光之 試驗胜肤浪度記錄作為EC50[參考表2 :通報子EC50]。 10 2.增生檢定分析 本檢定分析係基於鼠前B細胞系Baf3轉移感染來表現 人EPO-R。所得細胞系Baf3/Gal4/Elk/EPOR之增生係與 EPO-R活化相關。細胞增生程度使用MTT量化，此處MTT 檢定分析之信號係與活存細胞數目成正比。 15 BaF3/Gal4/Elk/EPOR細胞於旋轉瓶内於補充1〇% FBS(海克隆公司）及2% WEHI-3上清液(ATCC # TIB-68)之 DMEM/F12培養基（吉伯可公司）培養。培養後細胞於旋轉瓶 内以細胞密度lxlO6細胞/毫升，於補充10% FBS及0.1% WEHI-3上清液之DMEM/F12培養基匱乏隔夜《然後匱乏細 20 胞使用杜別克PBS(吉伯可公司）洗兩次，再懸浮於補充10% FBS(不含WEHI-3上清液）至細胞密度lxl〇6細胞/毫升。50 微升液分（約5 0，000細胞）細胞懸浮液重複三次接種於9 6孔， 檢定分析孔板。整份50微升試驗EPO模擬胜肽之一系列稀 釋液或50微升EPO(R&amp;D系統公司，明尼蘇達州明尼玻利 85 市）或阿月&amp;尼司（Aranesp)(島碧生成素a： (darbepoeitin α )， 購自安京（Amgen)公司之EPO-R激動劑）於補充10% fbs(不 含WEHI-3上清液I)之DMEM/F12培養基添加至96孔檢定分 析孔板（最終各孔容積100微升）。例如試驗12種不同稀釋 液’試驗胜狀（或對照EPO胜肽）終濃度為8l〇pM至0.0045ΡΠ1 之範圍。然後接種後之孔板細胞於37。(：培養48小時。其次 10微升MTT(羅斯診斷公司）添加至各培養皿之各孔，然後讓 其培養4小時。藉加入1〇% SDS + 0.01N HC1停止反應。孔 板於37°C隔夜。然後藉分光光譜術測定各孔於595奈米波長 之吸光比。吸光比讀數對試驗胜肽濃度作圖，使用Graph Pad軟體算出EC50。可獲得半最大吸光比之試驗胜肽濃度記 錄作為EC50[參考表2 :增生EC50]。 3.競爭結合檢定分析 使用檢定分析從事競爭結合計算，檢定分析中光信號 係依據二珠粒接近程度之函數變化而產生：鏈絲菌抗生物 素施體珠粒載有生物素化EP〇_r結合胜肽追蹤劑，以及結 合有EPO-R之受體珠粒。藉非輻射能量移轉發光，此時當 發光時由第一珠粒放出孤氧，第二珠粒接觸孤氧造成發 光。此種珠粒集合可於商業上獲得（派克公司^珠粒接近係 由於EPO-R結合胜肽追蹤劑結合至Ep〇 R所產生。試驗胜肽 與EP0-R結合胜肽追蹤劑競爭結合至Ep〇_R，將防止此種結 合，造成發光的減少。 該方法之進一步細節如後：添加4微升試驗EPO-R激動 劑胜肽之一系列稀釋液或陽性對照或陰性對照至384孔板 1344964 各孔。隨後，加入每孔2微升/受體珠粒混合液。受體珠粒 混合液之組成為：15微升5毫克/毫升鏈絲菌抗生物素受體 珠粒（派克公司），15微升5亳克/毫升單株抗體abl79(此抗體 辨識重組EPO-R所含人胎盤鹼性磷酸酶蛋白質部分），蛋白 5 質A塗覆受器珠粒(蛋白質A結合至abl79抗體；派克公司）， 112.5微升1:6.6重組EPO-R稀釋液（中國倉鼠卵巢細胞製 造’呈結合至人胎盤鹼性磷酸酶蛋白質部分（其含有abl79 目標抗原決定部位）之融合蛋白質，及607.5微升阿爾發奎斯 特（Alphaquest)緩衝液4 mM HEPES, pH 7.4; 1 Mm MgCl2; 10 0.1% BSA，0.05%吞恩20)。輕敲桌面混合。加入2微升/孔生 物素化EPO-R結合胜肤追縱劑，AF33068 (30 nM終濃度）。 AF33068為一EPO-R結合胜肽（參考表3「通報子EC50 (pM)」) 係根據實施例1所述方法製備。 AF33068 15NH- 0 0 Step 8 - Pegification of the deprotected binary: then deprotected peptide The PEGylation was carried out as described in Step 10 of Example 1. Step 9 - Ion exchange purification: PEGylated peptide peptides were then purified as described in Example 1, Step 1, Step 11. Example 3: In-tube activity assay analysis This example illustrates a plurality of in-vitro assay assays that can be used to assess the activity and strength of the EP0-R agonist peptide of the present invention. The results of the assay confirmed that the novel peptide of the present invention binds to EPO-R and activates EPO-R signaling. In addition, the results of the assay showed that the novel peptide composition compared to the aforementioned EPO mimetic peptide, the EP0-R binding affinity and biological activity unexpectedly increased. 83 1344964 Ε Ο R - R agonist peptide binary system was prepared according to the method provided in Example 丨 or Example 2. The strength of these winning binary bodies was evaluated using a series of test tube activity assays, including: notification subtest analysis, hyperplastic assay analysis, repeated binding assay analysis, and C/BFU-e assay analysis. Four assays analyzed β 5 further Explain its details as follows. The results of these in vitro assays are summarized in Table 2. • 1. Notification of sub-assay analysis • This assay is based on the reporter cell Baf3/EpoR/GCSFR fos/lux derived from the rat sputum cell line. This reporter cell line can represent a chimera, including the internal portion of the human GC0 receptor from the human EP0 receptor to the extracellular portion. This cell line was further transfected with the f0S promoter gene-driven luciferase reporter gene assembly. This chimera receptor is activated by the addition of a red blood cell generator&apos; resulting in the expression of the luciferase reporter gene, thus emitting light when the luciferase enzyme matrix luciferin is added. The extent of 15 EPO-R activation of such cells can be quantified by measurement of luciferase activity. Moxibustion Baf3/EpoR/GCSFR fos/lux cells were cultured in DMEM/F12 medium (Gibco) supplemented with 10% fetal bovine serum (fbs; Hyclone), 10% WEHI-3 supernatant The supernatant (by WEHI-3 cells, • ATCC # TIB-68) was cultured to obtain penicillin/streptomycin. The assay was divided into -20 minutes before the precipitation, and the cells were transferred to DMEM/F12 medium to supplement 10% FBS and 0.1% WEHI-3 supernatant and ft. On the day of assay analysis, the cells were washed with DMEM/F12 medium supplemented with 10% FBS (without WEHI-3 supernatant), and then tested at a known concentration of lxlO6 cells/ml. Culture in the presence of peptides, or use EP0 (R&amp;D Systems, Minneapolis, Minnesota) for 84 1344964 as a positive control, supplemented with DMEM/F12 supplemented with 10% FBS (without WEHI-3 supernatant) A series of dilutions of the test peptide was tested simultaneously in this assay. The assay plate was assayed at 37. (: incubated for 4 hours in a 5% carbon dioxide atmosphere. Subsequent addition of luciferin (Steady_G1〇) Promega 5 (Marison, Wisconsin) to each hole. After 5 minutes of incubation, at Parker (Packard) company's desktop photometer (Parker Instruments, Donna Gloria, Ill.) measures luminescence. Light intensity values are plotted against test peptide concentration's using Graph Pad software analysis. The result is a semi-maximum luminescence test. The skin wave was recorded as EC50 [Ref. Table 2: Bulletin EC50]. 10 2. Proliferation assay This assay is based on the Bf3 metastasis of the pre-B cell line to express human EPO-R. The resulting cell line Baf3/Gal4/Elk The proliferation of /EPOR is associated with EPO-R activation. The degree of cell proliferation is quantified using MTT, where the signalling of the MTT assay is directly proportional to the number of viable cells. 15 BaF3/Gal4/Elk/EPOR cells are replenished in a rotating vial 1〇% FBS (Hylon) and 2% WEHI-3 supernatant (ATCC # TIB-68) in DMEM/F12 medium (Jibo Co.) culture. After culture, the cells were centrifuged to a cell density of lxlO6 cells. /ml, DMEM/F12 medium supplemented with 10% FBS and 0.1% WEHI-3 supernatant was lacking overnight. Then the depleted fine cells were washed twice with Dubec PBS (Gibb) and resuspended in supplement 10%. FBS (without WEHI-3 supernatant) to cell density lxl〇6 Cells / ml was dispensed .50 l (about 5 0,000 cells) of the cell suspension was inoculated in triplicate in 96 hole plate assay. A full 50 microliter test EPO mimetic peptide series dilution or 50 microliter EPO (R&amp;D Systems, Minneapolis, Minnesota 85) or Arny & Aranesp A: (darbepoeitin α), purchased from Amgen's EPO-R agonist) added to DMEM/F12 medium supplemented with 10% fbs (without WEHI-3 supernatant I) to 96-well assay Orifice plate (final volume of each hole is 100 microliters). For example, test 12 different dilutions' test traits (or control EPO peptides) to a final concentration ranging from 8 l 〇pM to 0.0045 ΡΠ1. The plate cells after inoculation were then at 37. (: culture for 48 hours. Next, 10 μl of MTT (Rose Diagnostics Inc.) was added to each well of each culture dish, and then allowed to culture for 4 hours. The reaction was stopped by adding 1% SDS + 0.01 N HCl. The plate was at 37°. C overnight. Then the absorbance ratio of each well at 595 nm was measured by spectroscopic spectroscopy. The absorbance ratio reading was plotted against the test peptide concentration, and the EC50 was calculated using Graph Pad software. The test score of the semi-maximum absorbance ratio was obtained. As EC50 [Reference Table 2: Proliferation EC50] 3. Competitive binding assay analysis uses competitive assays to perform competitive binding calculations. The optical signal in the assay is based on a change in the proximity of the two beads: Streptavidin-resistant biotin donor beads The pellet carries a biotinylated EP〇_r binding peptide tracer and an acceptor bead that incorporates EPO-R. The non-radiative energy shifts the luminescence, at which time the solitary oxygen is released from the first bead when illuminated. The second bead contacts the orphaned oxygen to cause luminescence. Such a bead collection can be obtained commercially (Pike's Co., Ltd. bead-proximate system is produced by EPO-R binding peptide tracer binding to Ep〇R. Test peptide and EP0 -R combined with peptide Competition for binding to Ep〇_R will prevent this binding, resulting in reduced luminescence. Further details of this method are as follows: Add 4 μl of test EPO-R agonist peptide one dilution or positive control or negative Control to each well of 384-well plate 1344. 4. Subsequently, add 2 μl/receptor bead mixture per well. The composition of the acceptor bead mixture is: 15 μl of 5 mg/ml Streptavidin Receptor Beads (Pike), 15 μl of 5 g/ml monoclonal antibody abl79 (this antibody recognizes the human placental alkaline phosphatase protein fraction contained in recombinant EPO-R), protein 5 A coated cap beads ( Protein A binds to abl79 antibody; Parker), 112.5 μl of 1:6.6 recombinant EPO-R dilution (Chinese hamster ovary cell manufacturing 'binding to human placental alkaline phosphatase protein fraction (which contains abl79 target epitope) Fusion protein, and 607.5 μl Alphaquest buffer 4 mM HEPES, pH 7.4; 1 Mm MgCl2; 10 0.1% BSA, 0.05% ton 20). Tap the tabletop mix. Add 2 μl /hole biotinylated EPO-R combined with skin refining agent, AF330 68 (30 nM final concentration) AF33068 is an EPO-R binding peptide (refer to Table 3 "Notifier EC50 (pM)") prepared according to the method described in Example 1. AF33068 15

Biotin*GGLYAi CHMGPITWVijQPLRG^ K-NH,Biotin*GGLYAi CHMGPITWVijQPLRG^ K-NH,

Bioda^3GLYACHMGPrTWVCQPLRG 〆 離心1分鐘混合。板使用派克頂封密封且包裹於金屬箔 内。於至溫培養隔仪。18小時後使用阿爾發查斯特讀取哭 (派克公司）讀取發光。發光對胜肽濃度作圖，使用Graph Pad 或Excel分析。 比較不含試驗胜肽之觀察值，可獲得發光減少5〇%之 2〇 試驗胜肽濃度記錄為IC50[參考表2 : AQ IC50]。 4. C/BFU-e檢定分析 EPO-R發訊模擬骨髓幹細胞分化成為增生中的紅血球 87 前驅細胞。本檢定分析測定試驗胜肽模擬得自一次人骨髓 多重作用幹細胞之紅血球前驅細胞之增生與分化。 用於本檢定分析，於補充10% FBS(海克隆公司）之 IMDM培養基（吉伯可公司）製作試驗胜肽之一系列稀釋 5 液。然後系列稀釋液或陽性對照EPO胜肽添加至曱基纖維 素來獲得終容積1.5毫升。然後甲基纖維素與胜肽混合物徹 底翻轉。整份（100,000細胞/毫升）人骨髓衍生得自CD34+細 胞(保伊堤克斯(Poietics)/肯布拉斯(Cambrex))解凍。然後解 床後之細胞溫和添加至50毫升試管内之〇.1毫升1毫克/毫升 10 DNAse(幹細胞h其次40-50毫升IMDM培養基溫和添加至細 胞：前10毫升培養基係順著50毫升試管旁逐滴添加，然後 其餘培養基則沿試管旁緩慢配送。然後細胞於9〇〇rpm離心 20分鐘，藉溫和吸取小心去除培養基。細胞再懸浮於1毫升 IMDM培養基，於血球計玻片計算每毫升細胞密度（玻片上 15為一整份10微升細胞懸浮液，細胞密度為平均數χΐ〇,0〇〇細 胞/毫升）。然後細胞於IMDM培養基稀釋至15,〇〇〇細胞/毫升 細胞密度。然後1〇〇微升稀釋後之細胞添加至各15毫升甲 基纖維素加胜肽試樣(檢定分析中之終細胞濃度為丨〇 〇 〇細 胞/毫升）’混合物經翻轉。讓混合物内之氣泡消失然後使 20用鈍端針頭吸取1毫升。將得自各樣本之0.25毫升吸取之混 合物添加至24孔孔板（飛爾空（Falcon)品牌）四孔之各孔。接 種混合物於37°C於5%二氧化碳於潮濕培育器培養14日。使 用相位顯微鏡(5倍-10倍物鏡，最終放大倍率1〇〇倍)計算紅 血球群落。比較使用EP0陽性對照組觀察，生成之群=數 88 1344964 目為最大值90%之試驗胜肽濃度記錄作為EC9〇[參考表2 : C/BFU-e EC90]。 5.放射性配位子競爭結合檢定分析 其它放射性配位子競爭結合檢定分析也可用於測定本 5 發明胜肽之1Cso值。此檢定分析係測定I-EPO與EPOR之結 合。檢定分析較佳根據如下範例方案進行：Bioda^3GLYACHMGPrTWVCQPLRG 混合 Mix by centrifugation for 1 minute. The plate is sealed with a Parker top seal and wrapped in a metal foil. Gently incubate the instrument. After 18 hours, use Alpha Chastain to read the cry (Pike) to read the luminescence. Luminescence is plotted against peptide concentration and analyzed using Graph Pad or Excel. Comparing the observations without the test peptide, the luminescence was reduced by 5%. 2 The test peptide concentration was recorded as IC50 [Ref. Table 2: AQ IC50]. 4. C/BFU-e assay analysis EPO-R signaling simulates the differentiation of bone marrow stem cells into proliferating red blood cells 87 precursor cells. This assay analyzes the test peptide to mimic the proliferation and differentiation of red blood cell precursor cells derived from a single human bone marrow multiplexed stem cell. For the purpose of this assay, a series of dilutions of one of the test peptides were prepared by supplementing 10% FBS (Hai Keng) IMDM medium (Jibo Co.). A serial dilution or positive control EPO peptide was then added to the thiol cellulose to obtain a final volume of 1.5 ml. The methylcellulose and peptide combination are then thoroughly inverted. Whole (100,000 cells/ml) human bone marrow derived from CD34+ cells (Poietics/Cambrex) was thawed. Then the cells after the bed was gently added to a 50 ml test tube. 1 ml of 1 mg/ml 10 DNAse (stem cells h followed by 40-50 ml of IMDM medium gently added to the cells: the first 10 ml of the medium was passed alongside the 50 ml tube Add dropwise, then the rest of the medium is slowly distributed along the tube. Then the cells are centrifuged at 9 rpm for 20 minutes, and the medium is carefully removed by gentle aspiration. The cells are resuspended in 1 ml of IMDM medium and counted per ml of cells on a hemocytometer slide. Density (15 is a whole 10 microliters of cell suspension on a slide with an average cell density of 0 〇〇 cells/ml). The cells are then diluted to 15 in IMDM medium at a cell density of 〇〇〇 cells/ml. One microliter of diluted cells were then added to each 15 ml sample of methylcellulose conjugated peptide (the final cell concentration in the assay was 丨〇〇〇 cells/ml). The mixture was inverted. The bubbles disappeared and then 20 ml was pipetted with a blunt end needle. 0.25 ml of the extracted mixture from each sample was added to each of the four wells of a 24-well plate (Falcon brand). The inoculation mixture was cultured for 14 days at 37 ° C in 5% incubator in a humidifier. The red blood cell population was calculated using a phase microscope (5 times -10 times objective lens, final magnification 1 〇〇). Comparison using the EP0 positive control group, Generated group = number 88 1344964 The test peptide concentration of 90% of the maximum value was recorded as EC9 〇 [Reference Table 2: C/BFU-e EC90] 5. Radioactive ligand competition binding assay analysis of other radioactive ligands The competitive binding assay can also be used to determine the 1 Cso value of the peptide of the present invention. This assay analyzes the binding of I-EPO to EPOR. The assay is preferably performed according to the following exemplary protocol:

A.材料 重組人EPOR/Fc 嵌合體 •識別：重組人EPOR/Fc嵌合體 :靈参寧9：6?盟系統公司(美國明尼蘇達州明那玻利〕 •批邊：EOK033071 •儲存：4°C 蛾化重組人紅血球 .識別：（3[125!]碘酪胺醯基)紅血球生成素，人重袓， 高特異性，370 kBq，10 μα •供應商：阿莫山生科公司(美國紐澤西州匹兹卡威） •型號：Μ219-10μα •批故： •儲存：4°C 蛋白質-G西法羅斯 •識別：蛋白質-G西法羅斯4快速流動 •供應商：阿莫山生科公司(真國紐拿西州匹茲卡咸、 •型號：17-0618-01 •批號： •儲存：4°C 檢定分析緩衝液 •磷酸化緩衝食鹽水(PBS)，pH 7.4，含有0.1%牛血:杳白 蛋白及0.1%疊氣化納 丁孤有曰 •儲存：4eCA. Material Recombinant Human EPOR/Fc Chimera • Recognition: Recombinant Human EPOR/Fc Chimera: Lingshen Ning 9:6 Alliance System Corporation (Minneapolis, Minnesota, USA) • Boundary: EOK033071 • Storage: 4° C moth recombination human red blood cells. Identification: (3[125!] iodotyrosamine) erythropoietin, human weight, high specificity, 370 kBq, 10 μα • Supplier: Amoshan Biotech (USA) Pitzkawi, Jersey) • Model: Μ219-10μα • Accusation: • Storage: 4°C Protein-G Sifaros • Identification: Protein-G Sifaros 4 Fast Flow • Supplier: Amoshan Biotech ( Pittsburgh, New Jersey, USA • Model: 17-0618-01 • Batch number: • Storage: 4°C assay buffer • Phosphorylation buffered saline (PBS), pH 7.4, containing 0.1% bovine blood : 杳 albumin and 0.1% stacked gas Nadine orphaned • Storage: 4eC

B·適當受器濃度之測定 10 於1毫升檢定分析緩衝液重新調配一小瓶5 0微克;東乾 重組EPOr胞外領域融合至人igG1 Fc部分。為了測定檢定分 析之受器正確用量，1〇〇微升一系列稀釋之此受器製劑組合 12x75毫米聚丙烯試管内約2〇,〇〇〇 cpm於200微升碘化重組 人紅血球生成素（Ιι'ΕΡ0)。試管加蓋，於雷伯奎克 15 (LabQuake)旋轉振搖機於4°C溫和混合隔夜。 次日，50微升50%蛋白質-G西法羅斯漿液添加至各試 89 1344964 管。然後試管於4°C培養2小時，溫和混合。然後試管於4000 RPM (3297XG)離心15分鐘，將蛋白質-G西法羅斯造粒。小 心去除上清液拋棄。以1毫升4。(：檢定分析缓衝液洗三次 後，小片粒於瓦拉威惹(Wallac Wizard)珈瑪計數器計算數 k 5目。然後結果經分析，求出達到50 %最大結合值所需稀釋 程度。 C.胜肽IC5Q之測定 .為了測定AF37702之IC5〇 ’ 1〇〇微升胜肽之一系列稀釋 液組合100微升重組紅血球生成素受器（1〇〇微微克/管）於 10 12x75毫米聚丙烯試管。然後100微升碘化重組人紅血球生 成素（125I-EPO)添加至各管’試管加蓋且於4°c溫和混合隔 夜。 次日，結合之125i-epo如前文說明定量。結果經分析， 使用得自GraphPad軟體公司（加州聖地牙哥）之Graphpad 15 Prism 4.0版計算IQ。值。對各試驗胜肽重複檢定分析兩次或 兩次以上，共測得3個或3個以上重複lc5〇測定值。 90 1344964 ^&lt;^^^±i-^M^^wll^M^^:fs&lt; C/BFU-e EC90 (nM) &lt;N (N 卜 &lt;N IC50 (pM) I_ Ο r—^ 增生 EC50 (nM) II 通報子 EC50 (pM) II 150 胜肽二元體 o (AcQ)OLYA(!HMGPrr( l-n*〇V&lt;!x3PLR(MeG)_mi NH^-PEQsok 〇° (AcG)aLYACTMGPIT(l-nal)VCQPLR(MeG)*HN/^VNH--y_^/r^ UJ 〇=^ Ό 1 i 1 I ri I ! i ! LI I ^ r | έ 1 l 化合物命名 AF35645 AF35527B. Determination of the appropriate receptor concentration 10 Reconstitute a vial of 50 μg in 1 ml assay buffer; Concans recombinant EPOr extracellular domain was fused to the human igG1 Fc fraction. In order to determine the correct amount of the assay for the assay, 1 〇〇 microliter of a series of diluted preparations of this receptor was combined with a 12x75 mm polypropylene tube for about 2 〇, 〇〇〇cpm at 200 μl of iodinated recombinant human erythropoietin ( Ιι'ΕΡ0). The tubes were capped and gently mixed overnight at 4 °C on a LabQuake rotary shaker. The next day, 50 μl of 50% protein-G sifaros slurry was added to each test 89 1344964 tube. The tubes were then incubated at 4 ° C for 2 hours with gentle mixing. The tube was then centrifuged at 4000 RPM (3297XG) for 15 minutes to pellet the protein-G sifaros. Carefully remove the supernatant and discard it. Take 1 ml of 4. (: After the assay analysis buffer was washed three times, the small pieces were counted in the Wallac Wizard gamma counter to calculate the number k 5 mesh. Then the results were analyzed to determine the degree of dilution required to reach 50% of the maximum binding value. Determination of peptide IC5Q. For the determination of AF37702's IC5〇' 1〇〇 microliter peptide series of dilutions 100 μl recombinant erythropoietin receptor (1 〇〇 picogram / tube) in 10 12x75 mm polypropylene Tubes. Then 100 microliters of iodinated recombinant human erythropoietin (125I-EPO) was added to each tube's tube-capped and gently mixed overnight at 4 ° C. The next day, the combined 125i-epo was quantified as described above. For analysis, IQ was calculated using Graphpad 15 Prism version 4.0 from GraphPad Software, Inc. (San Diego, Calif.). Repeated assay analysis for each test peptide was repeated two or more times for a total of 3 or more replicates. Lc5 〇 measured value. 90 1344964 ^&lt;^^^±i-^M^^wll^M^^:fs&lt;C/BFU-e EC90 (nM) &lt;N (N 卜&lt;N IC50 (pM) I_ Ο r—^ hyperplasia EC50 (nM) II reporter EC50 (pM) II 150 peptide binary o (AcQ)OLYA (!HMGPrr( ln*〇 V&lt;!x3PLR(MeG)_mi NH^-PEQsok 〇° (AcG)aLYACTMGPIT(l-nal)VCQPLR(MeG)*HN/^VNH--y_^/r^ UJ 〇=^ Ό 1 i 1 I ri I ! i ! LI I ^ r | έ 1 l Compound nomenclature AF35645 AF35527

91 1344964 實施例4 :活體内活性檢定分析 本例驗證可用於評比本發明EPO-R激動劑胜肽之活性 及強度之活體内檢定分析。Ep〇 R激動劑胜肽二元體係根 據實施例1或實施例2提供之方法製備。胜肽單體及二元體 ‘ 5 d體内活㈣使用—系列檢定分析包括多重血球胞外缺 • 氧小鼠生物檢定分析及網狀細胞生物檢定分析評比。兩種 檢定分析之進一步細節說明如後。 • 丨·多重血球胞外缺氧小鼠生物檢定分析 試驗胜肤係於多重血球胞外缺氧小鼠生物檢定分析測 1〇定活體内活性，該檢定分析係由Ctoes及Bangham (1961)， 自然191:1065-1067所述方法修正。本檢定分析檢驗試驗胜 肽用作為EPO模擬物：亦即活化Ep〇_R且誘生新紅血球合成 之能力。紅血球之合成係基於放射性標記鐵結合於合成紅 血球之血色素定量。 15 讓BDF1小鼠對周圍條件馴化7-10日。測定全部小鼠體 # 重，不使用體重太輕的小鼠（小於15克）。小鼠接受於低氣壓 艙連續調理週期共計14日^ 24小時週期係有18小時於〇4〇土 • 0.02%大氣壓及6小時於周圍壓力組成。小鼠調理後，於給 藥前又維持於周圍壓力72小時。 -20 試驗胜肽或重組人EP〇標準品於PBS + 〇.1% BSA媒劑 PBS/BSA)稀釋。胜肤早體備用溶液首先於二甲亞鑛(dms〇) 增溶。陰性對照組包括一組小鼠單獨注射PBS/BSA，一組 小鼠單獨注射1%DMS0。各劑量組各有1〇頭小鼠。小鼠皮 下注射(頸背）0.5毫升適當試樣。 92 於注射試樣後48小時，小鼠腹内注射〇2毫升Fe、枉邦 2司NEN)，劑量約0.75微居禮/小鼠。投予&amp;59後24小時稱 量小鼠體重，投予Μ後48小時犧牲小鼠。由各動物藉心臟 穿刺採集血樣’測定也容(使用肝素作為抗凝血劑）。各血樣 5 =2毫升使用派克伽瑪計數器分析…結合量。無反應之小 乳（亦即放射性結合低於陰性對照組之小鼠）由適當資料集 合中剔除。血容值低於陰性對照組53%之小鼠也被剔除。 · 結果係衍生自各實驗劑量10頭小鼠的集合。求出各組 、〜合於血樣之放射性平均量[每分鐘計數值(CpM)]。 · 10 2♦網狀細胞檢定分析 正常BDF1小鼠連續3曰給藥（〇 5毫升’皮下注射)Ερ〇 對妝或試驗胜肽。於第三日，給予小鼠(〇1毫升，腹内注射） 鐵葡萄聚糖（1〇〇毫克/毫升）。於第五日，小鼠以二氧化碳麻 和’藉心臟穿刺採血。各血樣之網狀細胞百分比（％)係藉嘍 15坐橙染色及流動細胞計分析（格子計數計畫）以人工方式測 疋血谷。校正後之網狀細胞數目係使用下式測定： % RETIC校正後==% RETICfewX(血容㈣/血容μ) — 3.血液學檢定分析 正常CD1小鼠每週四次大劑量靜脈注射Epo陽性對 ， 2〇照、试驗胜狀或媒劑。一定範圍之陽性對照及試驗胜肽劑 -%· 里（以毫克/千克表示）係經由改變調配物之活性化合物濃度 &quot;式驗。左射量為5毫升/千克。媒劑對照組包含12頭動物， 其餘各給藥組每組8頭。每日記錄存活情況，以及每週記錄 體重》 93 1344964 給藥小鼠為空腹小鼠’隨後吸入異氟烷(is〇flurane)麻 醉，於第1曰（媒劑對照小鼠）及第15日及第29曰（4小鼠/組/ 曰）經心臟穿刺或腹腔主動脈穿刺採集血樣。血液轉移至瓦 秋天那（Vacutainer)品牌試管内。較佳抗凝血劑為伸乙基二 5 胺四乙酸(EDTA)。 參 使用業界眾所周知之自動化臨床分析儀（例如固特 • (Coulter)公司製造）評比血樣之紅血球合成終點及生理例如 如容(1^1;)、血色素(Hgb)及總紅血球數目（rbc)。 ί 實施例5 :具有胺基酸序列（AcG)GLYACHMGPnXl-nal) VCQPLRK (SEQ ID NO: 1)之胜肽單體之Ep〇_j^動劑胜 肽同質二元體之合成 步驟1 -胜肽單體之合成：胜肽單體係使用標準Fm〇c 化學於八61431八胜肽合成儀，使用丁〇七八14樹脂（0.18毫莫 耳/克雷普聚合物，德國）合成。用於合成有醯胺化羧基端之 15 胜肽單體，完全組裝後之胜肽由樹脂使用82.5% TFA、5% &gt; 水、6.25%茴香醚、6.25%乙二硫醇裂解。脫保護產物由樹 脂過濾、’使用乙謎沉澱。徹底乾燥後，產物藉C18反相高效 液相層析術，以乙腈/水於0.1%三氟乙酸之梯度純化。胜肽 ’ 結構藉電噴灑質譜術證實。胜肽以1毫克/毫升濃度溶解於 -20 DMS0 :水1:1溶液，來影響雙硫鍵之生成。產物藉C18反相 高效液相層析術’以乙腈/水於0.1%三氟乙酸之梯度純化。 胜肽單體顯示如後。 (AcG)GLYACHMGPrT(l-nal)VCXJPLRK.NH2 94 1344964 15 步驟2 -三官能鍵聯基之合成：於亞胺基⑽二㈣ ⑽.〇克，52.8毫莫耳及Bg略丙胺酸(削克，52 8毫莫耳） 於刚毫升顧之溶液内於室溫以1〇分鐘時間加入二異丙 基曱二酿亞胺(8.0毫升，511毫莫耳）。添加期間反應混合物 溫熱至約HTC ’然後㈣分鐘時間冷卻回室溫。讓反應混 合物攪拌隔夜，過渡出咖之二異丙基脈。於減壓下蒸發 去除溶劑獲得膠狀物，殘餘物溶解於乙酸乙_，再度過渡 去除額外沉澱脲。有機相置於分液漏斗，洗蘇(飽和複酸氣 鈉、食鹽水、0.5 N鹽酸、食鹽水），脫水(硫酸鎮)過渡及於 減壓下蒸發，獲得二S旨產物呈無色油。二賴取於甲醇： THF 1:1混合物（100毫升），於其中加水(25毫升），然後加入 氫氧化鈉(5克，125毫莫耳）。測得pH大於1〇。反應混合物 於室溫攪拌2小時，然後u6N鹽酸酸化至pH〗。水相以氯化 鈉飽和，及以乙酸乙酯萃取4次。組合有機相經洗滌（食鹽 水）’脫水(硫酸鎂）及於減壓下濃縮獲得白色半固體。固體 溶解於50毫升DCM，於其中加入3〇〇毫升己烷形成白色漿 液。於減壓下去除溶劑，獲得二酸，呈白色固體（14 7克， 91.5%產率共二步驟）。於二酸（1克，3 29毫莫耳)於2〇毫升 20 DMF之溶液内加入N-羥基丁二醯亞胺（77〇毫克，6 69毫莫 耳）及二異丙基曱二醯亞胺（1〇〇毫升，6 38毫莫耳）及4-二曱 基胺基吡啶(3毫克’ 0.02毫莫耳）。反應混合物攪拌隔夜， 於減壓下去除溶劑。殘餘物攝取於乙酸乙酯，及過濾去除 沉殿脲。有機相置於分液漏斗，洗務(飽和碳酸氫鈉、食鹽 水、0.5 N鹽酸、食鹽水），脫水(硫酸鎂），過濾及於減壓下 95 1344964 濃縮，獲得二-NHS酯產物，呈白色固體（1.12克，68%產率）。91 1344964 Example 4: In vivo activity assay analysis This example validates an in vivo assay for assessing the activity and intensity of the EPO-R agonist peptide of the present invention. The Ep〇R agonist peptide binary system was prepared according to the method provided in Example 1 or Example 2. Peptide monomer and binary ‘5 d in vivo live (IV) use—series assay analysis includes multiple blood cell extracellular deficiency • oxygen mouse bioassay analysis and reticular cell bioassay analysis. Further details of the two assays are described below. • 丨·Multiple Hematocrit Extracellular Hypoxia Mice Bioassay Test The succulent test is based on the bioassay of multiple blood cells in extracellular hypoxic mice. The assay is determined by Ctoes and Bangham (1961). Natural method 191: 1065-1067 method correction. This assay analyzes the test peptide as an EPO mimetic: that is, the ability to activate Ep〇_R and induce new red blood cells to synthesize. The synthesis of red blood cells is based on the quantification of radiolabeled iron bound to synthetic hemoglobin. 15 BDF1 mice were domesticated for 7-10 days. All mouse body weights were determined, and mice that were too light (less than 15 grams) were not used. The mice received a continuous conditioning cycle in a low pressure chamber for a total of 14 days ^ 24 hours period with 18 hours in 〇 4 bauxite • 0.02% atmospheric pressure and 6 hours in ambient pressure. After conditioning, the mice were maintained at ambient pressure for 72 hours prior to administration. -20 Test peptide or recombinant human EP standard was diluted in PBS + 〇.1% BSA vehicle PBS/BSA). The Shengfu early body backup solution was first solubilized in the dimethyl submine (dms〇). The negative control group included a group of mice injected with PBS/BSA alone, and a group of mice injected with 1% DMS0 alone. Each dose group had 1 taro mouse. Mice were injected subcutaneously (neck back) with 0.5 ml of the appropriate sample. 92 48 hours after the injection of the sample, the mice were intraperitoneally injected with 毫升2 ml of Fe, 枉邦 2 Division NEN) at a dose of about 0.75 microcures per mouse. Mice were weighed 24 hours after administration &amp; 59, and sacrificed 48 hours after administration of sputum. Blood samples were taken from each animal by cardiac puncture, and the measurement was also performed (heparin was used as an anticoagulant). Each blood sample 5 = 2 ml was analyzed using a Parker gamma counter... binding amount. Unresponsive small milk (i.e., mice with less radioactive binding than the negative control group) were excluded from the appropriate data collection. Mice with a blood volume lower than 53% of the negative control group were also excluded. The results were derived from a collection of 10 mice at each experimental dose. The average amount of radioactivity (counting per minute (CpM)) of each group and the blood sample was determined. · 10 2♦ Reticulocyte assay analysis Normal BDF1 mice were given 3 consecutive doses (〇 5 ml 'subcutaneous injection) Ερ〇 for makeup or test peptides. On the third day, mice (〇1 ml, intraperitoneal injection) of iron glucomannan (1 mg/ml) were administered. On the fifth day, the mice were bled with carbon dioxide and by heart puncture. The percentage of reticular cells (%) of each blood sample was measured by artificial sputum 15 by orange staining and flow cytometry analysis (grid counting program). The corrected number of reticulocytes was determined using the following formula: % RETIC corrected ==% RETICfewX (blood volume (four) / blood volume μ) — 3. Hematology assay analysis of normal CD1 mice four times a week high dose intravenous Epo Positive pair, 2 photos, test traits or vehicle. A range of positive controls and test peptides -%· expressed in mg/kg are modified by varying the concentration of active compound in the formulation. The left shot is 5 ml / kg. The vehicle control group contained 12 animals, and each of the remaining drug-administered groups contained 8 animals each. Survival was recorded daily, and body weight was recorded weekly. 93 1344964 The mice were vaccinated with fasting mice followed by inhalation of isoflurane, on the first sputum (media control mice) and on the 15th day. And 29th (4 mice/group/曰) blood samples were taken by cardiac puncture or abdominal aortic puncture. The blood is transferred to the Vacutainer brand test tube. A preferred anticoagulant is exoethyldiaminetetraacetic acid (EDTA). Refer to the well-known automated clinical analyzers (such as those manufactured by Coulter Inc.) to evaluate the erythrocyte synthesis endpoints and physiology of the blood samples such as Rong (1^1;), hemoglobin (Hgb), and total red blood cell count (rbc).实施 Example 5: Amino acid sequence (AcG) GLYACHMGPnXl-nal) VCQPLRK (SEQ ID NO: 1) peptide peptide monomer Ep〇_j^activator peptide homodimer synthesis step 1 - win Synthesis of Peptide Monomers: The peptide single system was synthesized using standard Fm〇c chemistry on an eight-61431 eight-peptide synthesizer using Dingqi 7:8 14 resin (0.18 mmol/Krepp polymer, Germany). For the synthesis of 15 peptide monomers with a guanidine carboxyl end, the fully assembled peptide was cleaved from the resin using 82.5% TFA, 5% &gt; water, 6.25% anisole, 6.25% ethanedithiol. The deprotected product was filtered by resin and precipitated using a puzzle. After thorough drying, the product was purified by C18 reverse phase high performance liquid chromatography eluting with acetonitrile/water eluting with 0.1% trifluoroacetic acid. The peptide structure was confirmed by electrospray ionization mass spectrometry. The peptide was dissolved in -20 DMS0: water 1:1 solution at a concentration of 1 mg/ml to affect the formation of disulfide bonds. The product was purified by C18 reverse phase high performance liquid chromatography eluting with EtOAc/water eluting The peptide monomer is shown as follows. (AcG)GLYACHMGPrT(l-nal)VCXJPLRK.NH2 94 1344964 15 Step 2 - Synthesis of trifunctional linkages: imino group (10) di(tetra) (10). gram, 52.8 millimolar and Bg adenine (sharp , 52 8 mM) Diisopropyl hydrazine iodide (8.0 ml, 511 mmol) was added to the solution of MgSO. The reaction mixture was warmed to about HTC' during the addition and then cooled back to room temperature over a period of (four) minutes. The reaction mixture was allowed to stir overnight and transitioned to the diisopropyl vein of the coffee. The solvent was removed by evaporation under reduced pressure to give a gum, which was dissolved in ethyl acetate. The organic phase was placed in a separatory funnel, washed with sodium (saturated sodium sulphate, brine, 0.5 N hydrochloric acid, brine), dehydrated (sulphuric acid) and evaporated under reduced pressure to give the product as a colorless oil. The mixture was taken up in methanol: THF 1:1 mixture (100 ml), water (25 ml) was added and then sodium hydroxide (5 g, 125 mM) was added. The pH was measured to be greater than 1 Torr. The reaction mixture was stirred at room temperature for 2 hours and then acidified to pH with EtOAc. The aqueous phase was saturated with sodium chloride and extracted 4 times with ethyl acetate. The combined organic phases were washed (salt water) &apos; dehydrated (MgSO4) and concentrated under reduced pressure to afford white semi solid. The solid was dissolved in 50 ml of DCM, and 3 ml of hexane was added thereto to form a white slurry. The solvent was removed under reduced pressure to give the diacid as a white solid (14 g, 91. Add N-hydroxybutaneimine (77 mg, 6 69 mmol) and diisopropyl hydrazine to a solution of 2 mM 20 DMF of diacid (1 g, 3 29 mM) Imine (1 ml, 6 38 mmol) and 4-didecylaminopyridine (3 mg '0.02 mmol). The reaction mixture was stirred overnight and the solvent was evaporated under reduced pressure. The residue was taken up in ethyl acetate and filtered to remove the precipitated urea. The organic phase was placed in a separatory funnel, washed (saturated sodium hydrogen carbonate, brine, 0.5 N hydrochloric acid, brine), dried (MgSO4), filtered, and concentrated under reduced pressure 95 1344964 to give the bis-NHS ester product. White solid (1.12 g, 68% yield).

步驟3-三官能鍵聯基偶合至胜肽單體：為了偶合至鍵 聯基，2當量胜肽混合1當量三官能鍵聯基於無水DMF，獲 得澄清溶液，2分鐘後加入5當量DIEA。混合物於周圍溫度 攪拌14小時。於減壓下去除溶劑，粗產物溶解於80% TFA 於DCM 30分鐘來去除Boc基，接著以C18反相HPLC純化。 二元體結構式係藉電喷灑質譜術證實。此偶合反應將鍵聯 基附接至各個單體之離胺酸殘基之ε胺基之氮原子。 96 1344964 (AoG)GLYACHMGPrT(l-naI)VCQPLRK-NH2 ΟStep 3 - Trifunctional linkages are coupled to the peptide monomer: for coupling to the linkage, 2 equivalents of the peptide are mixed with 1 equivalent of a trifunctional linkage based on anhydrous DMF to give a clear solution, and 2 equivalents of DIEA are added after 2 minutes. The mixture was stirred at ambient temperature for 14 hours. The solvent was removed under reduced pressure and the crude material was dissolved in &lt;RTI ID=0.0&gt;&gt; The binary structure was confirmed by electrospray ionization mass spectrometry. This coupling reaction attaches a bond to the nitrogen atom of the epsilon amine group of the amine acid residue of each monomer. 96 1344964 (AoG)GLYACHMGPrT(l-naI)VCQPLRK-NH2 Ο

ΟΟ

Ο V ΝΗΟ V ΝΗ

ΝΗ2ΝΗ2

(AcO)GLYACHMGPIT(l-nBl)VCQPLR-HN(AcO)GLYACHMGPIT(l-nBl)VCQPLR-HN

Ο 97 1344964 步驟4 _胜肽二元體之peg化Ο 97 1344964 Step 4 _Pepsylation of peptide peptides

透過胺基甲酸酯鍵之PEG化：胜肽二元體混合等量（莫 耳基準）活性PEG物種(mPEG-NPC，得自曰本NOF公司）於無 水DMF獲得澄清溶液5分鐘後’ 4當量DIEA添加至前述溶 ♦ 5 液。混合物於周圍溫度攪拌14小時，接著以C18反相HPLC 純化。藉MALDI質譜證實PEG化胜肽之結構式。純化後之 ^ 胜肽也經由陽離子交換層析術純化，摘述如後。PEGylation through urethane linkage: peptide peptide mixed equal amount (mole reference) active PEG species (mPEG-NPC, obtained from Sakamoto NOF) after obtaining clear solution in anhydrous DMF for 5 minutes ' 4 Equivalent DIEA is added to the aforementioned solvent. The mixture was stirred at ambient temperature for 14 hours then purified by C18 reverse phase HPLC. The structural formula of the PEGylated peptide was confirmed by MALDI mass spectrometry. The purified peptide was also purified by cation exchange chromatography and summarized as follows.

(AcG)GLYACHMGPIT(l-iBl)VCQPLR-i(AcG)GLYACHMGPIT(l-iBl)VCQPLR-i

NH 0NH 0

OO

N V (AcO)GLYACHMGPir(l.nal)VCQPLR-N V (AcO) GLYACHMGPir (l.nal) VCQPLR-

inPEQ-NPCinPEQ-NPC

DOEA,DMPDOEA, DMP

(AcG)GLYACHMGPrT(l-nal)V(!QPLR-NH NH c(AcG)GLYACHMGPrT(l-nal)V(!QPLR-NH NH c

〇 O〇 O

、NH 〇-pEGa〇K, NH 〇-pEGa〇K

N V NHN V NH

O 98 1344964 經由醯胺鍵結之PEG化：胜肽二元體混合等量（莫耳基 準）活性PEG物種（PEG-SPA-NHS，得自美國席爾瓦特 (Shearwater)公司）於無水DMF獲得澄清溶液。5分鐘後，4 當量DIEA添加至前述溶液。混合物於周圍溫度授拌2小 5 時，接著以C18反相HPLC純化。藉MALDI質譜證實pEG化 胜肽之結構式。純化後之胜肽也經由陽離子交換層析術純 化，摘述如後。O 98 1344964 PEGylation via guanamine linkage: peptide peptide mixed equal (mole reference) active PEG species (PEG-SPA-NHS, available from Shearwater, USA) in anhydrous DMF Clarify the solution. After 5 minutes, 4 equivalents of DIEA was added to the aforementioned solution. The mixture was stirred at ambient temperature for 2 hours 5 and then purified by C18 reverse phase HPLC. The structural formula of the pEG-derived peptide was confirmed by MALDI mass spectrometry. The purified peptide was also purified by cation exchange chromatography and summarized as follows.

NH2 O (AoG)QLYACHMC3&gt;rr(l-naI)VCQPLR- NH,NH2 O (AoG)QLYACHMC3&gt;rr(l-naI)VCQPLR- NH,

I I (AcO)OLYACHMGPrT(l-oaI)VCQPLR- ΡΒΟθ： DIEA,I I (AcO)OLYACHMGPrT(l-oaI)VCQPLR- ΡΒΟθ: DIEA,

ίΡΑ-NHSΡΑ-NHS

DMFDMF

NHNH

(AcG)GLYA0aMGHT(l-naI)V(lQPLR-NH JL(AcG)GLYA0aMGHT(l-naI)V(lQPLR-NH JL

(AcG)GLYACHMGPrr(l-na])VCQPLR-HN °v m(AcG)GLYACHMGPrr(l-na])VCQPLR-HN °v m

NH 99 0 1344964 步驟5_胜肽之離子交換純化：除了保有起始二元體胜 肽之能力之外，若干交換支持體也研究其由未反應（或水 解)PEG分離前述胜肽-PEG軛合物之能力。離子交換樹脂 (2-3克)載於1厘米管柱，接著轉成鈉形式(〇2 N氫氧化鈉載 • 5於管柱，直至洗提分為PH 14，約5倍管检容積），而非轉成 , 氫形式（使用0.1 N鹽酸或0.1 Μ乙酸洗提直至洗提分匹配負 載pH，約5倍管柱容積）’接著以25% ACN/水洗蘇至pH 6。 . 桄合前之胜肽或胜肽-PEG輛合物溶解於25% ACN/水（10毫 克/毫升）’ pH以THA調整至小於3，然後載荷至管柱。以2-3 10管柱容積25%ACN/水洗滌且收集5毫升洗提分後，以0.1 μ 乙酸銨於25% ACN/水洗提讓胜肽由管柱釋放出，再度收集 5毫升洗提分。藉HPLC分析顯示該洗提分含有所需胜肽》 以氣化光散射檢測器（ELSD)分析，指示當胜肤保留於管 柱’且使用乙酸銨溶液(通常於洗提分4及洗提分1〇間洗提 15出）洗提時，未觀察得污染物未經扼合之PEG。當胜狀於初 | 洗提緩衝液（通常為前二洗提分）洗提時，未觀察得所需 PEG-軛合物的分離及過量PEG。 下列管柱可成功地保有胜肽及胜肽-PEG軛合物，成功 * · 地由非軛合物胜肽純化胜肽_PEG軛合物： -2〇 表3:離子交換樹脂 支持體 來源 Mono S HR 5/5強陽離子交換預載荷管柱 阿莫山(Amersham) 生科公司 SE53纖維素’微粒強陽離子交換支持體 瓦特曼公司 SP西法羅斯(Sepharose)快速流動強陽離子交換支持體 阿莫山生科公司 100 1344964 實施例6 :具有胺基酸序列(AcG)GLYACHMGPIT(l-nal) VCQPLRK (SEQ ID NO: 1)之胜肽單體之EPO-R激動劑胜 肽同質二元體之合成 具有胺基酸序列（AcG)GLYACHMGPITU -nal) VCQPLR (MeG)K (SEQ ID NO: 2)之胜肽單體之EPO-R激動劑胜肽同 質二元體係如實施例1所述合成，但於步驟1 ’合成之胜肽 單體為： 10 (AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K 此處PEG係透過胺基甲酸酯鍵聯附接至鍵聯基，本合成終 產物之結構式顯示如後：NH 99 0 1344964 Step 5 - Ion exchange purification of peptides: In addition to the ability to retain the initial binary peptide, several exchange supports were also investigated for their separation of the aforementioned peptide-PEG yoke from unreacted (or hydrolyzed) PEG. The ability of the compound. The ion exchange resin (2-3 g) was loaded on a 1 cm column and then converted to sodium form (〇2 N sodium hydroxide • 5 on the column until the elution was divided into PH 14, about 5 times the tube volume) Instead of converting, the hydrogen form (extracted with 0.1 N hydrochloric acid or 0.1 hydrazine acetic acid until the elution fraction matches the loading pH, about 5 times the column volume)' is then washed with 25% ACN/water to pH 6. The peptide or peptide-PEG complex before compounding is dissolved in 25% ACN/water (10 mg/ml) pH adjusted to less than 3 with THA and then loaded onto the column. After washing with 2-3 10 column volume 25% ACN/water and collecting 5 ml of elution fraction, the peptide was released from the column by 0.1 μ ammonium acetate in 25% ACN/water, and 5 ml of the elution was collected again. Minute. By HPLC analysis, the elution fraction contains the desired peptide. Analysis by gasification light scattering detector (ELSD), indicating that when the skin is retained on the column 'and using ammonium acetate solution (usually in elution 4 and elution) When eluted in 1 minute, the PEG without contaminant was not observed. The separation of the desired PEG-conjugate and excess PEG were not observed when the precursor was eluted from the initial elution buffer (usually the first two washes). The following columns successfully retained the peptide and peptide-PEG conjugates, successfully purified from the non-conjugate peptides. PEG conjugates: -2 〇 Table 3: Sources of ion exchange resin supports Mono S HR 5/5 strong cation exchange preloaded column Amorshan (Amersham) Bios company SE53 cellulose 'fine particles strong cation exchange support body Wattman company SP West Faros (Sepharose) fast flow strong cation exchange support Amo Shansheng 100 1344964 Example 6: Synthesis of EPO-R agonist peptide homologous binary with amino acid sequence (AcG) GLYACHMGPIT (l-nal) VCQPLRK (SEQ ID NO: 1) Amino acid sequence (AcG) GLYACHMGPITU-nal) EPO-R agonist peptide of the peptide peptide monomer of VCQPLR (MeG)K (SEQ ID NO: 2) is synthesized as described in Example 1, but Step 1 'The synthesized peptide monomer is: 10 (AcG) GLYACHMGPIT (l-nal) VCQPLR(MeG)K Here, the PEG is attached to the bonding group through a urethane linkage, and the final product of the synthesis is The structural display is as follows:

若PEG係透過醯胺鍵聯附接至鍵聯基，則本合成終產 物結構式顯示如後：If the PEG is attached to the linkage via a guanamine linkage, the structural formula of the synthetic end product is shown as follows:

101 1344964 實施例7 :試管内活性檢定分析 本實施例說明多個試管内檢定分析，其可用於評比本 發明EPO-R激動劑胜肽之活性及強度。檢定分析結果證實 本發明新穎胜肽結合至EPO-R且活化EPO-R發訊。此外，檢 , 5 定分析結果顯示新穎胜肽組成物比較前述EPO模擬胜狀， EPO-R結合親和力及生物活性出乎意外地增高。 EPO-R激動劑胜肽單體及二元體係根據實施例1或實 _ 施例2提供之方法製備。此等胜肽二元體之強度係使用一系 列試管活性檢定分析評比，包括：通報子檢定分析、增生 1〇 檢定分析、重複結合檢定分析及C/BFU-e檢定分析。四項 檢定分析進一步說明其細節如後。 此等試管内活性檢定分析結果摘述於表4。 1.通報子檢定分析 本檢定分析係基於鼠前B細胞系衍生之通報子細胞 15 Baf3/EpoR/GCSFR fos/lux。此通報子細胞系可表現嵌合體 | 受器，包含人EPO受器至胞外部分之人GCSF受器之胞内部 分。此細胞系進一步以fos起動基因驅動子蟲螢光素酶通報 子基因組成體轉移感染。此嵌合體受器經由加入紅血球生 成劑活化，結果導致蟲螢光素酶通報子基因表現，因此當 • 20 加入蟲螢光素酶酶基質蟲營光素時發光。如此此種細胞之 EP0-R活化程度可藉蟲螢光素酶活性之量測而定量。101 1344964 Example 7: Intra-tube activity assay analysis This example illustrates a plurality of in-vitro assay assays that can be used to assess the activity and strength of the EPO-R agonist peptide of the present invention. The results of the assay confirmed that the novel peptide of the present invention binds to EPO-R and activates EPO-R signaling. In addition, the results of the analysis showed that the novel peptide composition was compared with the aforementioned EPO simulation, and the EPO-R binding affinity and biological activity unexpectedly increased. The EPO-R agonist peptide monomer and binary system were prepared according to the method provided in Example 1 or Example 2. The strength of these peptide peptides was assessed using a series of tube activity assays, including: reporter assay, hyperplasia assay, repeated binding assay, and C/BFU-e assay. The four verification analyses further illustrate the details as follows. The results of these in vitro assays are summarized in Table 4. 1. Bullet assay analysis This assay is based on the pre-murine B cell line-derived reporter cells 15 Baf3/EpoR/GCSFR fos/lux. This reporter cell line can represent a chimera receptor, which contains the internal portion of the human GCSF receptor from the human EPO receptor to the extracellular portion. This cell line was further infected with a fos-inducing gene-driven luciferase reporter gene assembly. This chimera receptor is activated by the addition of a red blood cell-forming agent, resulting in the expression of the luciferase reporter gene, so that it emits light when the luciferase enzyme matrix is added. The degree of EP0-R activation of such cells can be quantified by measurement of luciferase activity.

Baf3/EpoR/GCSFR fosMux細胞於DMEM/F12培養基（吉 伯可(Gibco)公司培養，培養基内補充10%胎牛血清(FBS ; 海克隆（Hyclone))，10% WEHI-3上清液（由 WEHI-3細胞， 102 1344964 ATCC # TIB-68)培養所得上清液，青黴素/鏈黴素。檢定分 析前約18小時’細胞藉轉移至DMEM/F12培養基補充10% FBS及0.1%WEHI-3上清液而匱乏。檢定分析當天，細胞以 補充10% FBS(不含WEHI-3上清液）之DMEM/F12培養基洗 5滌，然後1x106細胞/毫升於已知濃度之試驗胜肽存在下培 養，或使用epo(r&amp;d系統公司，明尼蘇達州明那玻利）作為 陽性對照，於補充10%FBS(不含WEHI-3上清液)之DMEM/ F12培養。試驗胜狀之一系列稀釋液於本檢定分析同時測 試。檢定分析孔板於37°C於5%二氧化碳氣氛下培養4小 10時。隨後添加蟲螢光素（史得力葛羅(Steady-Glo);普羅米加 (Promega)公司’威斯康辛州馬里森)至各孔。培養5分鐘後， 於派克(Packard)公司桌面光度計（派克儀器公司，伊利諾州 道納葛羅吾）測定發光。光強度值相對於試驗胜肽濃度作 圖，使用Graph Pad軟體分析。結果獲得半最大發光之試驗 15 胜肽濃度記錄作為EC50。 2.增生檢定分析 本檢定分析係基於鼠前B細胞系Baf3轉移感染來表現 人EP0-R。所得細胞系Baf3/Gal4/Elk/EPOR之增生係與 EPO-R活化相關。細胞增生程度使用MTT量化，此處MTT 20 檢定分析之信號係與活存細胞數目成正比。Baf3/EpoR/GCSFR fosMux cells were cultured in DMEM/F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 10% WEHI-3 supernatant (by WEHI-3 cells, 102 1344964 ATCC # TIB-68) The resulting supernatant was cultured for penicillin/streptomycin. Approximately 18 hours prior to assay analysis, cells were transferred to DMEM/F12 medium supplemented with 10% FBS and 0.1% WEHI-3. The supernatant was scarce. On the day of the assay, the cells were washed with DMEM/F12 medium supplemented with 10% FBS (with no WEHI-3 supernatant), and then 1x106 cells/ml in the presence of a known concentration of the test peptide. Culture, or use epo (r&amp;d Systems, Minneapolis, Minnesota) as a positive control, supplemented with DMEM/F12 supplemented with 10% FBS (without WEHI-3 supernatant). The dilutions were tested simultaneously in this assay. The assay plates were incubated at 37 ° C in a 5% carbon dioxide atmosphere for 4 hours and 10 hours. Then add luciferin (Steady-Glo; Promega ( Promega) company "Marison, Wisconsin" to each hole. After 5 minutes of incubation, at the Packard table Surface luminometer (Pike Instruments, Donna Gloria, Ill.) measured luminescence. Light intensity values were plotted against test peptide concentration and analyzed using Graph Pad software. Results were obtained for semi-maximal luminescence test 15 peptide concentration record As EC50. 2. Proliferation assay This assay is based on the Bf3 metastasis of the murine pre-B cell line to express human EP0-R. The resulting cell line Baf3/Gal4/Elk/EPOR is associated with EPO-R activation. The degree is quantified using MTT, where the signalling of the MTT 20 assay is directly proportional to the number of viable cells.

BaF3/Gal4/Elk/EP〇R細胞於旋轉瓶内於補充10% FBS(海克隆公司）及2% WEHI-3上清液(ATCC # TIB-68)之 DMEM/F12培養基（吉伯可公司）培養。培養後細胞於旋轉瓶 内以細胞密度lxlO6細胞/毫升，於補充10% FBS及0.1% 103 1344964 WEHI-3上清液之DMEM/F12培養基匱乏隔夜。然後匱乏細 胞使用杜別克PBS(吉伯可公司）洗兩次，再懸浮於補充1〇% FBS(不含WEHI-3上清液)至細胞密度1χΐ〇6細胞/毫升。50微 升液分（約50,000細胞）細胞懸浮液重複三次接種於96孔檢 • 5 定分析孔板。整份50微升試驗EPO模擬胜肽之一系列稀釋 液或50微升EPO (R &amp; D系統公司，明尼蘇達州明尼玻利市） 或阿能尼司（Aranesp)(島碧生成素〇： (darbepoeitin α )，購自 _ 安京（Amgen)公司之EPO-R激動劑）於補充1〇% FBS(不含 WEHI-3上清液I)之DMEM/F12培養基添加至％孔檢定分析 10 孔板（最終各孔容積1〇〇微升）。例如試驗12種不同稀釋液， 試驗胜肽（或對照EPO胜肽）終漠度為81 〇pM至〇.〇〇45Pm之 範圍。然後接種後之孔板細胞於37。(：培養48小時。其次10 微升MTT(羅斯診斷公司）添加至各培養孤之各孔，然後讓其 培養4小時。藉加入1〇% SDS + 0.01N HC1停止反應。孔板 15於37 C隔夜。然後藉分光光t普術測定各孔於595奈米波長之 吸光比。吸光比讀數對試驗胜狀濃度作圖，使用GraphPad 軟體算出EC50。可獲得半最大吸光比之試驗胜肽濃度記錄 作為EC50。 3·競爭結合檢定分析 20 使用檢疋分析從事競爭結合計算，檢定分析中光信號 係依據二珠粒接近程度之函數變化而產生：鏈絲菌抗生物 素施體珠粒載有生物素化ΕΡΟ-R結合胜肽追蹤劑，以及結 合有EPO-R之受體珠粒。藉非輻射能量移轉發光，此時當 發光時由第一珠粒放出孤氧，第二珠粒接觸孤氧造成發 104 1344964 光。此種珠粒集合可於商業上獲得（派克公司）。珠粒接近係 由於ΕΡΟ-R結合胜肽追蹤劑結合至EP〇_R所產生。試驗胜肽 與EPO-R結合胜肽追蹤劑競爭結合至EP〇_R，將防止此種結 合，造成發光的減少。 5 該方法之進一步細節如後：添加4微升試驗ΕΡΟ-R激動 劑胜肽之一系列稀釋液或陽性對照或陰性對照至384孔板 各孔。隨後，加入每孔2微升/受體珠粒混合液。受體珠粒 混合液之組成為：15微升5毫克/毫升鏈絲菌抗生物素受體 珠粒（派克公司）’ 15微升5毫克/毫升單株抗體abl79(此抗體 10 辨識重組ΕΡΟ-R所含人胎盤鹼性磷酸酶蛋白質部分），蛋白 質A塗覆受器珠粒(蛋白質A結合至abl79抗體；派克公司）， 112.5微升1:6.6重組ΕΡΟ-R稀釋液（中國倉鼠卵巢細胞製 造，呈結合至人胎盤驗性破酸峰蛋白質部分（其含有沾179 目標抗原決定部位)之融合蛋白質，及607.5微升阿爾發奎斯 15 特（Alphaquest)緩衝液4mM HEPES，pH 7.4; IMm MgCl2; 0.1% BSA，0.05%吞恩20)。輕敲桌面混合。加入2微升/孔生 物素化ΕΡΟ-R結合胜肽追蹤劑，AF33068 (30nM終漠度）。 AF33068為一ΕΡΟ-R結合胜肽（參考表3「通報子EC50 (pM)」) 係根據實施例1所述方法製備。 AF33068BaF3/Gal4/Elk/EP〇R cells were supplemented with DMEM/F12 medium supplemented with 10% FBS (Haylon) and 2% WEHI-3 supernatant (ATCC # TIB-68) in a rotating flask (Jibo Co., Ltd.) )to cultivate. After the culture, the cells were depleted in a rotating flask at a cell density of lxlO6 cells/ml in DMEM/F12 medium supplemented with 10% FBS and 0.1% 103 1344964 WEHI-3 supernatant overnight. The depleted cells were then washed twice with Dubec PBS (Giboco) and resuspended in 1% FBS (without WEHI-3 supernatant) to a cell density of 1χΐ〇6 cells/ml. 50 microliters of liquid fraction (approximately 50,000 cells) of the cell suspension was inoculated three times in 96 wells. A full 50 microliter test EPO mimetic peptide series dilution or 50 microliters EPO (R &amp; D Systems, Minneapolis, Minnesota) or Aranesp (Island Bismuth) : (darbepoeitin α ), purchased from _ Angen (Amgen) EPO-R agonist) added to 1%% FBS (without WEHI-3 supernatant I) in DMEM/F12 medium added to % well assay 10 well plates (final volume of each hole is 1 μL). For example, testing 12 different dilutions, the test peptide (or control EPO peptide) has a final indifference ranging from 81 〇pM to 〇.〇〇45Pm. The plate cells after inoculation were then at 37. (: culture for 48 hours. Next, 10 μl of MTT (Rose Diagnostics Inc.) was added to each well of each culture, and then allowed to culture for 4 hours. The reaction was stopped by adding 1% SDS + 0.01 N HC1. Orifice plate 15 at 37 C overnight. Then the absorbance ratio of each well at 595 nm is measured by spectrophotometry. The absorbance ratio reading is plotted against the test winner concentration, and the EC50 is calculated using GraphPad software. The test peptide concentration of the half maximum absorbance ratio can be obtained. Record as EC50. 3. Competing binding assay analysis 20 Using the profiling analysis to perform competitive binding calculations, the optical signal in the assay is based on a change in the proximity of the two beads: Streptococcus avidin donor beads contain biotin The phlegm-R-binding peptide tracer and the acceptor beads combined with EPO-R. The non-radiative energy shifts the luminescence, when the luminescence releases the solitary oxygen from the first bead, and the second bead contacts the lone Oxygen causes 104 1344964 light. This bead collection is commercially available (Pike). The bead is close to the ΕΡΟ-R-binding peptide tracer binding to EP〇_R. Test peptide and EPO- R-binding peptide Competition for binding to EP〇_R will prevent this binding and result in reduced luminescence. 5 Further details of this method are as follows: Add 4 μl of test ΕΡΟ-R agonist peptide to a series of dilutions or positive control Or a negative control to each well of a 384-well plate. Subsequently, a 2 μl/receptor bead mixture per well is added. The composition of the acceptor bead mixture is: 15 μl of 5 mg/ml Streptavidin Body Beads (Parker) '15 μl of 5 mg/ml monoclonal antibody abl79 (this antibody 10 recognizes the human placental alkaline phosphatase protein fraction contained in recombinant ΕΡΟ-R), protein A coated receptor beads (protein A binds to abl79 antibody; Parker), 112.5 microliters of 1:6.6 recombinant ΕΡΟ-R dilution (manufactured by Chinese hamster ovary cells, which binds to the human placenta-tested acid-breaking peak protein fraction (which contains the 179 target epitope) Fusion protein, and 607.5 μl Alphaquest buffer (4 mM HEPES, pH 7.4; IMm MgCl2; 0.1% BSA, 0.05% ton 20). Tap the tabletop mix. Add 2 μl/ Pore biotinylated ΕΡΟ-R binding peptide tracking , AF33068 (30nM final degree desert). AF33068 is a ΕΡΟ-R binding peptide (see Table 3 "communications sub EC50 (pM)") is prepared according to the method described in Example 1. AF33068

Biotin-OGLYACHMGPrrWVCQPLRG^ Biotiii-GGLYACHMGPnWVCQPLRG ^ 離心1分鐘混合。板使用派克頂封密封且包裹於金屬箱 内。於室溫培養隔夜。18小時後使用阿爾發奎斯特讀取器 105 20 (派克公司）讀取發光。發光對胜肽濃度作圖，使用Graph Pad 或Excel分析。 比較不含試驗胜肽之觀察值，可獲得發光減少50%之 試驗胜肽濃度記錄為IC50。 4. C/BFU-e檢定分析 EPO-R發訊模擬骨髓幹細胞分化成為增生中的紅血球 前驅細胞。本檢定分析測定試驗胜肽模擬得自一次人骨髓 多重作用幹細胞之紅血球前驅細胞之增生與分化。 用於本檢定分析，於補充10% FBS(海克隆公司）之 IMDM培養基（吉伯可公司）製作試驗胜肽之一系列稀釋 液。然後系列稀釋液或陽性對照EPO胜肽添加至甲基纖維 素來獲得終容積1.5毫升。然後甲基纖維素與胜肽混合物徹 底翻轉。整份（100,000細胞/毫升）人骨髓衍生得自CD34+細 胞(保伊堤克斯/肯布拉斯）解凍。然後解凍後之細胞溫和添 加至50毫升試管内之0.1毫升1毫克/毫升DNAse(幹細胞）。其 -人40-50毫升IMDM培養基溫和添加至細胞：前1〇毫升培養 基係順著50毫升試管旁逐滴添加，然後其餘培養基則沿試 管旁緩慢配送。然後細胞於900rpm離心20分鐘，藉溫和吸 取小心去除培養基。細胞再懸浮於丨毫升IMDM培養基，於 血球s十玻片計算母毫升細胞密度（玻片上為一整份1〇微升 細胞懸浮液，細胞岔度為平均數X10 000細胞/毫升）^然後 細胞於IMDM培養基稀釋至15,〇〇〇細胞/毫升細胞密度。然 後100微升稀釋後之細胞添加至各15毫升甲基纖維素加胜 肽試樣(檢定分析中之終細胞濃度為1〇〇〇細胞/毫升），混合 1344964 物經翻轉。讓混合物内之氣泡消失，然後使用鈍端針頭及 取1毫升。將得自各樣本之0.25毫升吸取之混合物添加至24 孔孔板（飛爾空（Falcon)品牌）四孔之各孔。接種混合物於^ C於5%·一氣化峡於潮濕培育器培養14日。使用相位顯微鏡 5 (5倍-10倍物鏡，最終放大倍率100倍）計算紅血球群落。比 較使用EPO陽性對照組觀察，生成之群落數目為最大值9〇% 之試驗胜肽濃度記錄作為EC90。Biotin-OGLYACHMGPrrWVCQPLRG^ Biotiii-GGLYACHMGPnWVCQPLRG ^ Centrifuge for 1 minute to mix. The panels are sealed with a Parker top seal and wrapped in a metal box. Incubate overnight at room temperature. After 18 hours, the Alpha Quest reader 105 20 (Pike) was used to read the luminescence. Luminescence is plotted against peptide concentration and analyzed using Graph Pad or Excel. Comparing the observations without the test peptide, the test peptide concentration at which the luminescence was reduced by 50% was recorded as IC50. 4. C/BFU-e assay analysis EPO-R signaling simulates the differentiation of bone marrow stem cells into proliferating red blood cell precursor cells. This assay analyzes the test peptide to mimic the proliferation and differentiation of red blood cell precursor cells derived from a single human bone marrow multiplexed stem cell. For the purpose of this assay, a series of dilutions of the test peptide was prepared by supplementing 10% FBS (Hai Keng) IMDM medium (Jibo Co.). Serial dilutions or positive control EPO peptides were then added to methylcellulose to obtain a final volume of 1.5 ml. The methylcellulose and peptide combination are then thoroughly inverted. A whole (100,000 cells/ml) of human bone marrow derived from CD34+ cells (Paulie/Kenbras) was thawed. The thawed cells were then gently added to 0.1 ml of 1 mg/ml DNAse (stem cells) in a 50 ml test tube. - 40-50 ml of IMDM medium was gently added to the cells: the first 1 ml of the culture system was added dropwise alongside the 50 ml tube, and the remaining medium was slowly dispensed alongside the test tube. The cells were then centrifuged at 900 rpm for 20 minutes and the medium was carefully removed by gentle aspiration. The cells were resuspended in 丨ml of IMDM medium, and the cell density of the mother cells was calculated on the hematocrit of the blood cells (one whole microliter of cell suspension on the slide, the cell length was an average of X10 000 cells/ml) ^ then the cells Dilute to 1.5 cells in IMDM medium at a cell density of 〇〇〇 cells/ml. Then, 100 μl of the diluted cells were added to each 15 ml of methylcellulose vasopeptide sample (the final cell concentration in the assay was 1 〇〇〇 cells/ml), and 1344964 was mixed and inverted. Let the bubbles in the mixture disappear, then use a blunt end needle and take 1 ml. The 0.25 ml of the extracted mixture from each sample was added to each of the four wells of a 24-well plate (Falcon brand). The inoculation mixture was cultured in a humidified incubator for 14 days at 5%. Red blood cell populations were calculated using a phase microscope 5 (5x-10x objective, final magnification 100x). When compared with the EPO-positive control group, the test peptide concentration at which the number of colonies generated was 9% by maximum was recorded as EC90.

107 1344964107 1344964

C/BFU-e EC90 (nM) &lt;N 1 AQ 1 IC50 i (PM) 增生 EC50 (nM) 1 1 1 ff 〇 ^0¾ ^ w ^ 胜肽二元體 _ _ … ______ ________1 )0 ^NH^O-PEGaoK I 1 rM |l ! I I L| r ^ 3 X» u ^ 1- 化合物命名 AF36205 108 1344964 實施例8 :活體内活性檢定分析 本例驗證可用於評比本發明EP〇 - R激動劑胜肽之活性 及強度之活體内檢定分析。EPCMU^動劑胜肽單體及二元 體係根據實施例1提供之方法製備。胜肽單體及二元體之活 5體内活性係使用一系列檢定分析包括多重血球胞外缺氧小 鼠生物檢定分析及網狀細胞生物檢定分析評比。兩種檢定 分析之進一步細節說明如後。 1.多重血球胞外缺氧小鼠生物檢定分析 試驗胜肽係於多重血球胞外缺氧小鼠生物檢定分析測 10 疋活體内活性’該檢定分析係由Ctoes及Bangham (1961)， 自然191:1065-1067所述方法修正。本檢定分析檢驗試驗胜 肽用作為EPO模擬物：亦即活化EP0-R且誘生新紅血球合成 之能力。紅血球之合成係基於放射性標記鐵結合於合成紅 血球之血色素定量。 15 讓BDF1小鼠對周圍條件馴化7-10日。測定全部小鼠體 重’不使用體重太輕的小鼠（小於15克）。小鼠接受於低氣壓 艙連續調理週期共計14曰。24小時週期係有18小時於0.40士 0.02%大氣壓及6小時於周圍壓力組成。小鼠調理後，於給 藥前又維持於周圍壓力72小時。 20 試驗胜肽或重組人EPO標準品於PBS + 0.1% BSA媒劑 PBS/BSA)稀釋。胜肽單體備用溶液首先於二曱亞鑛(DMS0) 增溶。陰性對照組包括一組小鼠單獨注射PBS/BSA，一組 小鼠單獨注射1% DMS0。各劑量組各有1〇頭小鼠。小鼠皮 下注射（頸背）〇·5毫升適當試樣。 109 1344964 於注射試樣後48小時，小鼠腹内注射〇2毫升^(枉邦 公司NEN)，劑量約〇.75微居禮/小鼠。投予&amp;59後％小時稱 量小鼠體重，投予W後48小時犧牲小鼠。由各動物藉心臟 穿刺採集血樣，測定血容(使用肝素作為抗凝血劑）。各灰樣 .5 (0.2亳升使用派克㈣計數器分析Fe59結合^。無反應之小 • 鼠（亦即放射性結合低於陰性斜照組之小鼠）由適當資料集 • 合中剔除。金容值低於陰性對照組53%之小鼠也被剔除。 Φ 肖果係衍生自各實驗劑量10頭小鼠的集合。求出各組 結合於血樣之放射性平均量[每分鐘計數值((:1&gt;]^)]。 10 2.網狀細胞檢定分析 正常BDFH、鼠連續3日給藥（〇5毫升，皮下注射)Ep〇 對照或試驗胜肽。於第三日，給予小鼠(〇1毫升，腹内注射) 鐵葡萄聚糖(100毫克/毫升）。於第五日，小鼠以二氧化碳麻 Sr，藉心臟穿刺採血。各血樣之網狀細胞百分比係藉嘍 15唑橙染色及流動細胞計分析（格子計數計晝）以人工方式測 • 定血容。校正後之網狀細胞數目係使用下式測定： % RETIC校正後=% RETICft察值x(血容個別/血容正常） 3.血液學檢定分析 正常CD1小鼠每週四次大劑量靜脈注射Ep〇陽性對 • 2〇照、試驗胜肽或媒劑》—定範圍之陽性對照及試驗胜肽劑 里（以毫克/千克表示）係經由改變調配物之活性化合物濃度 試驗。注射量為5毫升/千克。媒劑對照組包含12頭動物， 其餘各給藥組每組8頭。每日記錄存活情況，以及每週記錄 體重。 110 1344964 給藥小鼠為空腹小鼠，隨後吸入異氟烷(isoflurane)麻 醉，於第1日（媒劑對照小鼠）及第15曰及第29曰（4小鼠/組/ 曰）經心臟穿刺或腹腔主動脈穿刺採集血樣。血液轉移至瓦 秋天那（Vacutainer)品牌試管内。較佳抗凝血劑為伸乙基二 5 胺四乙酸(EDTA)。 使用業界眾所周知之自動化臨床分析儀（例如固特 (Coulter)公司製造）評比血樣之紅血球合成終點及生理例如 血容(Hct)、血色素(Hgb)及總紅血球數目（RBC)。 實施例9 :具有胺基酸序列(AcG)GLYACHMGPIT( 1-nal) 10 VCQPLRK (SEQ ID NO: 1)之胜肽單體之EP0-R激動劑胜 肽同質二元體之合成 步驟1 -胜肽單體之合成：胜肽單體係使用標準Fmoc 化學於八81431八胜肽合成儀，使用丁〇-11八厘樹脂(0.18毫莫 耳/克雷普聚合物’德國）合成。用於合成有醯胺化羧基端之 15 胜肽單體’完全組裝後之胜肽由樹脂使用82.5% TFA、5% 水' 6.25%茴香醚、6.25%乙二硫醇裂解。脫保護產物由樹 脂過濾’使用***沉澱。徹底乾燥後，產物藉C18反相高效 液相層析術，以乙腈/水於0.1%三氟乙酸之梯度純化。胜肽 結構藉電喷灑質譜術證實。胜肽以1毫克/毫升濃度溶解於 20 DMSO:水1:1溶液，來影響雙硫鍵之生成》產物藉C18反相 高效液相層析術，以乙腈/水於0.1%三氟乙酸之梯度純化。 胜肽單體顯示如後。 (AcG)GLYACHMGPIT(l-nal)VCQPLRK-NH2 步驟2 -三官能鍵聯基之合成：於亞胺基乙酸二乙酯 111 1344964 (10.0克’ 52.8毫莫耳及Boc-沒-丙胺酸(1〇〇克，52 8毫莫耳） 於100毫升DCM之溶液内於室溫以1〇分鐘時間加入二異丙 基曱二醯亞胺(8.0毫升’5U毫莫耳）。添加期間反應混合物 溫熱至約贼，然後以20分鐘時間冷卻回室溫。讓反應潘 4 5纟物隔夜’過濾出沉殿之二異丙基脲。於減壓下蒸發 去除溶劑獲得膠狀物，殘餘物溶解於乙酸乙酯，再度過濾 ' 去除額外沉澱脲。有機相置於分液漏斗，洗條(飽和碳酸1 φ 納、食鹽水、〇·5 N鹽醆、食鹽水卜脫水(硫酸鎮}過濾、及於 減壓下蒸發，獲得二酿產物呈無色油。二醋攝取於甲醇： H) THF 1:1混合物（100毫升）’於其中加水(25毫升），然後加入 氮氧化鈉(5克，125毫莫耳）。測得pH大於1〇。反應混合物 於室溫授拌2小時，然後以6 N鹽酸酸化至pH i。水相以氣 化鈉飽和，及以乙酸乙酿萃取心欠。組合有機相經洗條（食 鹽水），脫水(硫酸鎂）及於減壓下濃縮獲得白色半固體。固 15體溶解於50毫升DCM，於其中加入3〇〇毫升己烧形成白色聚 _ 液。於減壓下去除溶劑，獲得二酸，呈白色固體(147克， 91.5%產率共二步驟）。於二酸(1克，3 29毫莫耳)於2〇毫升 ·· DMF之〉谷液内加入Ν'羥基丁二醯亞胺(770毫克，6.69毫莫 耳)及一異丙基曱二酿亞胺（1 〇〇毫升，毫莫耳)及心二甲 -20基胺基η比咬(3毫克’ 0〇2毫莫耳卜反應混合物撥拌隔夜， 於減壓下去除溶劑。殘餘物攝取於乙酸乙醋，及過溏去除 &quot;L瓜服。有機相置於分液漏斗，洗制飽和碳酸氫納、食鹽 水、〇·5 Ν鹽酸、食鹽水），脫水(硫酸鎮），過渡及於減壓下 濃縮’獲得二-NHS酷產物，呈白色固體（1 12克，68%產率）。 112 丄〕44%4C/BFU-e EC90 (nM) &lt;N 1 AQ 1 IC50 i (PM) hyperplasia EC50 (nM) 1 1 1 ff 〇^03⁄4 ^ w ^ peptide binary _ _ ... ______ ________1 ) 0 ^NH^ O-PEGaoK I 1 rM |l ! IIL| r ^ 3 X» u ^ 1- Compound nomenclature AF36205 108 1344964 Example 8: In vivo activity assay analysis This example validation can be used to evaluate the EP 〇-R agonist peptide of the present invention In vivo assay analysis of activity and intensity. The EPCMU kinetic peptide monomer and binary system were prepared according to the method provided in Example 1. The activity of the peptide monomer and the binary body 5 in vivo activity using a series of assays including multiple blood cell extracellular hypoxic mouse bioassay analysis and reticulocyte bioassay analysis. Further details of the two assays are described below. 1. Multiple blood cell extracellular hypoxia mice bioassay analysis test peptides in multiple blood cells extracellular hypoxic mice bioassay analysis 10 疋 in vivo activity 'The assay was analyzed by Ctoes and Bangham (1961), Nature 191 : 1065-1067 The method is modified. This assay analyzes the test peptide as an EPO mimetic: that is, the ability to activate EP0-R and induce new red blood cells to synthesize. The synthesis of red blood cells is based on the quantification of radiolabeled iron bound to synthetic hemoglobin. 15 BDF1 mice were domesticated for 7-10 days. All mice were measured for body weight&apos; mice that were too light (less than 15 grams) were not used. Mice received a continuous conditioning cycle of 14 Torr in a low pressure chamber. The 24-hour cycle consisted of 18 hours at 0.40 ± 0.02% atmosphere and 6 hours at ambient pressure. After conditioning, the mice were maintained at ambient pressure for 72 hours prior to administration. 20 Test peptides or recombinant human EPO standards were diluted in PBS + 0.1% BSA vehicle PBS/BSA). The peptide monomer monomer solution was first solubilized in the Dioxin (DMS0). The negative control group included a group of mice injected with PBS/BSA alone, and a group of mice injected with 1% DMS0 alone. Each dose group had 1 taro mouse. The mice were injected subcutaneously (neck back) with 5 ml of the appropriate sample. 109 1344964 48 hours after the injection of the sample, the mice were intraperitoneally injected with 2 ml of ^ (NEN), at a dose of about 75.75 microcures per mouse. The body weight of the mice was weighed by the administration of &amp; 59, and the mice were sacrificed 48 hours after the administration of W. Blood samples were taken from each animal by cardiac puncture and blood volume was measured (heparin was used as an anticoagulant). Each ash sample .5 (0.2 liters using the Parker (4) counter to analyze Fe59 binding ^. Non-reactive small mice (ie, mice with radioactive binding below the negative slant group) were excluded from the appropriate data set. Mice with a value lower than 53% of the negative control group were also excluded. Φ Xiaoguo was derived from a collection of 10 mice at each experimental dose. The average amount of radioactivity bound to the blood sample was determined [counts per minute ((: 1&gt] ;]^)]. 10. 2. Reticulocyte assay analysis Normal BDFH, rats were administered for 3 consecutive days (〇5 ml, subcutaneous injection) Ep〇 control or test peptide. On the third day, mice were given (〇1 ml) , intraperitoneal injection) iron glucomannan (100 mg / ml). On the fifth day, the mice were treated with carbon dioxide hemp Sr, blood sampling by heart puncture. The percentage of reticulocytes in each blood sample was stained with 15 oxazolium orange and flow cells. The analysis (grid counting) is done manually to determine the blood volume. The corrected number of reticulocytes is determined using the following formula: % RETIC corrected = % RETICft value x (blood volume / normal blood volume) 3 Hematological examination analysis of normal CD1 mice four times a week high dose intravenous E P〇 positive pair • 2 test, test peptide or vehicle” - a range of positive control and test peptide agent (expressed in mg / kg) is tested by changing the active compound concentration of the formulation. The injection volume is 5 ML/kg. The vehicle control group contained 12 animals, and the remaining drug-administered groups received 8 animals each. Survival was recorded daily, and body weight was recorded every week. 110 1344964 The mice were administered as fasting mice, followed by inhalation of isoflurane. Isoflurane anesthesia, blood samples were taken by cardiac puncture or abdominal aortic puncture on day 1 (media control mice) and 15th and 29th (4 mice/group/曰). Blood transfer to tile fall In the Vacutainer brand test tube, the preferred anticoagulant is exoethyldiaminetetraacetic acid (EDTA). Use the well-known automated clinical analyzer (such as Coulter) to evaluate the red blood cell synthesis of blood samples. End points and physiology such as blood volume (Hct), hemoglobin (Hgb) and total red blood cell count (RBC). Example 9: having an amino acid sequence (AcG) GLYACHMGPIT (1-nal) 10 VCQPLRK (SEQ ID NO: 1) EP0-R agonist peptide of peptide monomer Synthesis of Qualitative Binary Steps 1 - Synthesis of peptide monomer: The peptide single system uses standard Fmoc chemistry on the eight 81431 eight-peptide synthesizer, using Ding 〇-11 octadecyl resin (0.18 mmol/Kray Polymeric polymer [Germany] synthesis. Used to synthesize 15 peptide monomers with amidated carboxyl end. 'Completely assembled peptides are made from resin. 82.5% TFA, 5% water' 6.25% anisole, 6.25% Ethylene Thiol cleavage. The deprotected product was filtered from the resin 'precipitated with diethyl ether. After thorough drying, the product was purified by C18 reverse phase high performance liquid chromatography eluting with acetonitrile/water eluting with 0.1% trifluoroacetic acid. The peptide structure was confirmed by electrospray ionization mass spectrometry. The peptide was dissolved in 20 DMSO:water 1:1 solution at a concentration of 1 mg/ml to affect the formation of disulfide bonds. The product was subjected to C18 reversed-phase high performance liquid chromatography with acetonitrile/water in 0.1% trifluoroacetic acid. Gradient purification. The peptide monomer is shown as follows. (AcG)GLYACHMGPIT(l-nal)VCQPLRK-NH2 Step 2 - Synthesis of trifunctional linkages: diethyl iminoacetate 111 1344964 (10.0 g '52.8 mmoles and Boc-no-alanine (1 〇〇克,52 8 mmoles) Diisopropyl phthalimide (8.0 ml '5 U mmol) was added to a solution of 100 ml of DCM at room temperature over 1 Torr. The temperature of the reaction mixture during the addition. Heat to about thief, then cool back to room temperature in 20 minutes. Let the reaction pan 4 5 纟 overnight 'filter out the diisopropyl urea of the sinking. Evaporate under reduced pressure to remove the solvent to obtain a gel, the residue is dissolved In ethyl acetate, re-filtered 'to remove additional precipitated urea. The organic phase was placed in a separatory funnel, and the strip was washed (saturated carbonate 1 φ sodium, brine, 〇·5 N salt 醆, brine dehydrated (sulfate sulphate), And evaporating under reduced pressure, the obtained product was obtained as a colorless oil. The diacetic acid was taken up in methanol: H) THF 1:1 mixture (100 ml) was added water (25 ml), then sodium oxynitride (5 g, 125 mmol.) The pH was measured to be greater than 1 Torr. The reaction mixture was stirred at room temperature for 2 hours and then acidified with 6 N hydrochloric acid. pH i. The aqueous phase is saturated with gasified sodium, and extracted with acetic acid. The combined organic phase is washed with water (salt brine), dehydrated (magnesium sulfate) and concentrated under reduced pressure to obtain a white semi-solid. Dissolve in 50 ml of DCM, add 3 ml of hexane to give a white poly-form. The solvent is removed under reduced pressure to give the diacid as a white solid (147 g, 91.5% yield a total of two steps). Acid (1 g, 3 29 mM) was added to Ν'hydroxybutaneimine (770 mg, 6.69 mmol) and isopropyl hydrazine in 2 ml of DMF. Amine (1 〇〇 ml, millimolar) and dimethyl-20-amino-amine η than bite (3 mg '0 〇 2 mmol) reaction mixture was mixed overnight, the solvent was removed under reduced pressure. In the acetic acid ethyl acetate, and the sputum removal &quot;L melon. The organic phase is placed in a separatory funnel, washed with saturated sodium bicarbonate, brine, 〇·5 Ν hydrochloric acid, brine), dehydrated (sulfate town), transition Concentrate under reduced pressure to give the bis-NHS product as a white solid (1 12 g, 68% yield). 112 丄] 44% 4

步驟3-三官能鍵聯基偶合至胜肽單體：為了偶合至鍵 聯基’ 2當量胜肽混合丨當量三官能鍵聯基於無水1)1^1?，獲 侍澄清溶液’ 2分鐘後加入5當量DIEA。混合物於周圍溫度 授掉14小時。於減壓下去除溶劑，粗產物溶解於80% TFA 於DCM 30分鐘來去除B〇c基，接著以C18反相HPLC純化。 二元體結構式係藉電噴灑質譜術證實。此偶合反應將鍵聯 基附接至各個單體之離胺酸殘基之ε胺基之氮原子。 卜 MVOQPLRK-NH:Step 3 - Trifunctional linkages are coupled to the peptide monomer: for coupling to the linkage '2 equivalents of peptides, mixed oxime equivalents, trifunctional linkages based on anhydrous 1) 1^1?, obtained a clear solution' 2 minutes later Add 5 equivalents of DIEA. The mixture was allowed to stand at ambient temperature for 14 hours. The solvent was removed under reduced pressure and the crude material was dissolved in &lt;RTI ID=0.0&gt;&gt; The binary structure was confirmed by electrospray ionization mass spectrometry. This coupling reaction attaches a bond to the nitrogen atom of the epsilon amine group of the amine acid residue of each monomer. Bu MVOQPLRK-NH:

ΝΗχ 113 1344964 步驟4 -包含藉離胺酸mPEG2-離胺醇-NPC鍵聯之二 線性PEGS鏈之PEG部分之合成 離胺醇可由市面上購得，離胺醇使用過量mPEG2-NPC 處理獲得mPEG2-離胺醇，隨後使用NPC處理來獲得 , 5 mPEG2-離胺醇-NPC。ΝΗχ 113 1344964 Step 4 - Synthesis of a PEG moiety comprising a bilinear PEGS chain linked by an aminic acid mPEG2-isamino alcohol-NPC linkage. The oleyl alcohol is commercially available and is treated with an excess of mPEG2-NPC to obtain mPEG2 from the amine alcohol. - 5 m PEG 2-isoamine-NPC, obtained from an amine alcohol followed by NPC treatment.

mPEG2-Lys-NHS 本產物係由市面上購得，例如得自分子工程型錄(2003 | 年），内塔(Nektar)治療公司（阿拉巴馬州35806漢茲維爾發現 大道490號），項目編號2Z3X0T01。 1〇 步驟5 -胜肽二元體之PEG化： 經由胺基甲酸酯鍵之PEG化： 胜肽二元體與PEG (mPEG2·離胺醇-NPC)以1:2莫耳比 於無水DMF混合獲得澄清溶液。5分鐘後，4當量DIEA添加 至前述溶液。混合物於周圍溫度攪拌14小時，接著以Cl8 15 反相HPLC純化。藉MALDI質譜證實PEG化胜肽之結構式。 • 純化後之胜肽也經由陽離子交換層析術純化，摘述如後。 114 1344964 0mPEG2-Lys-NHS This product is commercially available, for example, from Molecular Engineering Catalogue (2003 | year), Nektar Treatment Company (490, Discovery Avenue, Huntsville, Alabama 35806), project No. 2Z3X0T01. 1〇Step 5 - PEGylation of peptide peptides: PEGylation via urethane linkages: peptide peptides and PEG (mPEG2·isoamine alcohol-NPC) at 1:2 molar ratio to anhydrous DMF was mixed to obtain a clear solution. After 5 minutes, 4 equivalents of DIEA were added to the aforementioned solution. The mixture was stirred at ambient temperature for 14 hours then purified by EtOAc EtOAc. The structural formula of the PEGylated peptide was confirmed by MALDI mass spectrometry. • The purified peptide is also purified by cation exchange chromatography, as described below. 114 1344964 0

(AcG)GLYAOEIMGPIT(l-naI)V&lt;iQPLR-(AcG)GLYAOEIMGPIT(l-naI)V&lt;iQPLR-

I nPEG^七ysinol-NPC D1EA,DMFI nPEG^seven ysinol-NPC D1EA, DMF

經由醯胺鍵之PEG化： 胜肽二元體與PEG (mPEGr離胺醇-NHS得自美國席爾 瓦特公司）以1:2莫耳比於無水DMF獲得澄清溶夜。5分鐘 5 後，4當量DIEA添加至前述溶液。混合物於周圍溫度攪拌 14小時，接著以C18反相HPLC純化。藉MALDI質譜證實peg 化胜肽之結構式。純化後之胜肽也經由陽離子交換層析術 純化，摘述如後。 115 1344964PEGylation via a guanamine bond: Peptide Binary and PEG (mPEGr-aminol-NHS available from Schwart, USA) gave a clear nights with a 1:2 molar ratio to anhydrous DMF. After 5 minutes 5, 4 equivalents of DIEA were added to the aforementioned solution. The mixture was stirred at ambient temperature for 14 hours then purified by C18 reverse phase HPLC. The structural formula of the peg-winning peptide was confirmed by MALDI mass spectrometry. The purified peptide was also purified by cation exchange chromatography and summarized as follows. 115 1344964

EG20KEG20K

Ο 步驟6-胜肽之離子交換純化：除了保有起始二元體胜 肽之能力之外’若干交換支持體也研究其由未反應（或水 解)PEG分離前述胜肽，PEG輕合物之能力。離子交換樹脂 5 (2-3克)載於1厘米管柱，接著轉成鈉形式(〇2 n氣氧化納載 於管柱，直至洗提分為pH Η，約5倍管柱容積），而非轉成 氫形式(使用0·1 Ν鹽酸或0.1 Μ乙酸洗提直至洗提分匹配負 载ΡΗ ’約5倍管桎容積），接著以25%ACN/水洗滌至ρΗ6。 輕合如之胜肽或胜肽_PEG輥合物溶解於25% ACN/水（10毫 116 1344964 克/毫升），pH以THA調整至小於3，然後載荷至管柱。以2-3 管柱容積25% ACN/水洗滌且收集5毫升洗提分後，以(U M 乙酸銨於25% ACN/水洗提讓胜肽由管柱釋放出，再度收集 5毫升洗提分。藉HPLC分析顯示該洗提分含有所需胜狀。 5 以氣化光散射檢測器（ELSD)分析，指示當胜肽保留於管 柱，且使用乙酸銨溶液（通常於洗提分4及洗提分1〇間洗提 出）洗提時，未觀察得污染物未經軛合之PEG 〇當胜肽於初 洗提緩衝液（通常為前二洗提分）洗提時，未觀察得所需 PEG-軛合物的分離及過量PEG。 10 下列管柱可成功地保有胜肽及胜肽-PEG軛合物，成功 地由非軛合物胜肽純化胜肽-PEG軛合物： 表5 :離子交換樹脂 支持體 來源 Mono S HR 5/5強陽離子交換預載荷管柱 阿莫山(Amersham) 生科公司 SE53纖維素，微粒強陽離子交換支持體 瓦特曼公司 SP西法羅斯(Sepharose)快速流動強陽離子交換支持體 阿莫山生科公司 實施例6 :具有胺基酸序列(AcG)GLYACHMGmXl-nal) 15 VCQPLRK (SEQ ID NO: 1)之胜肽單體之EPO-R激動劑胜 肽同質二元體之合成 具有胺基酸序列（AcG)GLYACHMGPIT( 1 -nal) VCQPLR (MeG)K (SEQ ID NO: 2)之胜肽單體之EPO-R激動劑胜肽同 質二元體係如實施例1所述合成，但於步驟1，合成之胜肽 20 單體為：Ο Step 6-Peptide ion exchange purification: In addition to the ability to retain the starting binary peptide, 'several exchange supports also studied to separate the above peptides from unreacted (or hydrolyzed) PEG, PEG light compounds ability. Ion exchange resin 5 (2-3 g) was loaded on a 1 cm column and then converted to sodium form (〇2 n gas oxide was supported on the column until elution was divided into pH Η, approximately 5 times the column volume). Instead of converting to hydrogen form (extracted with 0.1 Ν hydrochloric acid or 0.1 Μ acetic acid until the elution fraction matches the load ΡΗ 'about 5 times the volume of the tube), it is washed with 25% ACN/water to ρΗ6. The light-weight peptide or peptide PEG coating was dissolved in 25% ACN/water (10 ng 116 1344964 g/ml), the pH was adjusted to less than 3 with THA, and then loaded onto the column. After washing with 2-3 column volume 25% ACN/water and collecting 5 ml of elution fraction, the peptide was released from the column by UM ammonium acetate in 25% ACN/water, and 5 ml of the elution fraction was collected again. HPLC analysis showed that the elution fraction contained the desired triumph. 5 Analyzed by gasification light scattering detector (ELSD), indicating that the peptide remained in the column and used ammonium acetate solution (usually in elution fraction 4 and When the elution is washed, the PEG is not observed. The unconjugated PEG is not observed when the peptide is eluted in the initial elution buffer (usually the first two washes). Separation of the desired PEG-conjugate and excess PEG. 10 The following column successfully retained the peptide and peptide-PEG conjugate, successfully purifying the peptide-PEG conjugate from the non-conjugate peptide: Table 5: Ion exchange resin support source Mono S HR 5/5 strong cation exchange preloaded column Amor Hill (Amersham) Biosystems SE53 cellulose, particle strong cation exchange support Wattman SP Sefarose (Sepharose) fast Flowing Strong Cation Exchange Support Amoshan Biotech Company Example 6: Having an Amino Acid Sequence (Ac G) GLYACHMGmXl-nal) 15 EPO-R agonist of the peptide monomer of VCQPLRK (SEQ ID NO: 1) The synthesis of the peptide homogenous binary has an amino acid sequence (AcG) GLYACHMGPIT( 1 -nal) VCQPLR ( The EPO-R agonist peptide of MeG)K (SEQ ID NO: 2) is synthesized as described in Example 1, but in Step 1, the synthesized peptide 20 monomer is:

(AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K 117 1344964(AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K 117 1344964

此處PEG係透過胺基甲酸醋鍵聯附接至鍵聯基，本合 成終產物之結構式顯示如後：Here, the PEG is attached to the bonding group through the urethane linkage, and the structural formula of the final product of the synthesis is as follows:

(AcG)GLYACHMGPIT(l-nal)VCQPLR(Mt&lt;?&gt;-NH(AcG)GLYACHMGPIT(l-nal)VCQPLR(Mt&lt;?&gt;-NH

若PEG係透過酿胺鍵聯附接至鰱聯基，則本合成終產 物結構式顯示如後：If the PEG is attached to the hydrazine via a stilbene linkage, the structural formula of the final product is shown as follows:

實施例11 :試管内活性檢定分析 本實施例說明多個試管内檢定分析，其可用於評比本 發明EPO-R激動劑胜肽之活性及強度。檢定分析結果證實 本發明新穎胜肽結合至EPO-R且活化EPO-R發訊。此外，檢 定分析結果顯示新穎胜肽組成物比較前述EPO模擬胜狀， EPO-R結合親和力及生物活性出乎意外地增高。 118 1344964 ΕΡΟ-R激動劑胜肽單體及二元體係根據實施例丄或實 施例2提供之方法製備。此等胜肽二元體之強度係使用一系 列試管活性檢定分析評比，包括：通報子檢定分析、增生 檢定分析、重複結合檢定分析及C/BFU_e檢定分析。四項 5檢定分析進一步說明其細節如後。 此等試管内活性檢定分析結果摘述於表4。 1-通報子檢定分析 本檢定分析係基於鼠前B細胞系衍生之通報子細胞 Baf3/EpoR/GCSFR fGS/iup此通報子細胞系可表現嵌合體 ίο受器，包含人EP0受器至胞外部分之人GCSF受器之胞内部 分。此細胞系進一步以fos起動基因驅動子蟲螢光素酶通報 子基因組成體轉移感染。此嵌合體受器經由加入紅血球生 成劑活化，結果導致蟲螢光素酶通報子基因表現，因此當 加入蟲螢光素酶酶基質蟲螢光素時發光。如此此種細胞之 15 EPO-R活化程度可藉蟲螢光素酶活性之量測而定量。Example 11: Intra-tube activity assay analysis This example illustrates a plurality of in-vitro assay assays that can be used to assess the activity and strength of the EPO-R agonist peptide of the present invention. The results of the assay confirmed that the novel peptide of the present invention binds to EPO-R and activates EPO-R signaling. In addition, the results of the assay showed that the novel peptide composition compared to the aforementioned EPO mimetic, the EPO-R binding affinity and biological activity unexpectedly increased. 118 1344964 ΕΡΟ-R agonist peptide monomer and binary system were prepared according to the methods provided in Example 实 or Example 2. The strength of these peptide peptides was assessed using a series of tube activity assays, including: reporter assays, proliferative assays, repeated binding assays, and C/BFU_e assays. The four 5 test analyses further illustrate the details as follows. The results of these in vitro assays are summarized in Table 4. 1-Reporter assay This assay is based on the murine pre-B cell line-derived reporter cell Baf3/EpoR/GCSFR fGS/iup. This reporter cell line can represent chimeric IgG receptors, including human EP0 receptors to extracellular Part of the person's intracellular part of the GCSF receptor. This cell line was further infected with a fos-inducing gene-driven luciferase reporter gene assembly. This chimera receptor is activated by the addition of a red blood cell-forming agent, resulting in the expression of the luciferase reporter gene, and thus emits light when the luciferase enzyme matrix luciferin is added. The extent of 15 EPO-R activation of such cells can be quantified by measurement of luciferase activity.

Baf3/EpoR/GCSFR f〇s/lux細胞於DMEM/F12培養基（吉 伯可(Gibco)公司培養’培養基内補充1〇%胎牛血清(FBS ; 海克隆(Hyclone))，1〇% WEHI_3 上清液（由 WEHI_3 細胞， ATCC # TIB-68)培養所得上清液，青黴素/鏈黴素。檢定分 2〇析前約18小時，細胞藉轉移至DMEM/F12培養基補充10% FBS及0.1% WEHI-3上清液而匱乏。檢定分析當天，細胞以 補充10% FBS(不含WEHI-3上清液）之DMEM/F12培養基洗 滌，然後1 X 1〇ό細胞/毫升於已知濃度之試驗胜肽存在下培 養’或使用EPO (R &amp; D系統公司，明尼蘇達州明那玻利） 119 1344964 作為陽性對照，於補充10% FBS(不含WEHI-3上清液）之 DMEM/F12培養。試驗胜肽之一系列稀釋液於本檢定分析 同時測試。檢定分析孔板於37°C於5%二氧化碳氣氛下培養 4小時。隨後添力σ蟲螢光素（史得力葛羅(steady-Glo);普羅 ^ 5米加(Promega)公司’威斯康辛州馬里森)至各孔。培養5分 鐘後，於派克(Packard)公司桌面光度計（派克儀器公司，伊 利諾州道納葛羅吾）測定發光。光強度值相對於試驗胜肽濃 | 度作圖，使用Graph Pad軟體分析。結果獲得半最大發光之 試驗胜肽濃度記錄作為EC50。 10 2.增生檢定分析 本檢定分析係基於鼠前B細胞系Baf3轉移感染來表現 人EPO-R。所得細胞系Baf3/Gal4/Elk/EPOR之增生係與 EP0-R活化相關。細胞增生程度使用MTT量化，此處MTT 檢定分析之信號係與活存細胞數目成正比。 15 BaF3/Gal4/Elk/EPOR細胞於旋轉瓶内於補充1〇% &gt; FBS(海克隆公司）及2% WEHI-3上清液(ATCC # TIB-68)之 DMEM/F12培養基（吉伯可公司）培養。培養後細胞於旋轉瓶 内以細胞密度lxlO6細胞/毫升，於補充10% FBS及0.1% * WEHI-3上清液之DMEM/F12培養基匱乏隔夜。然後匱乏細 * 20 胞使用杜別克PBS(吉伯可公司）洗兩次，再懸浮於補充1〇% FBS(不含WEHI-3上清液）至細胞密度Ιχίο6細胞/毫升。50 微升液分（約50,000細胞）細胞懸浮液重複三次接種於96孔 檢定分析孔板。整份50微升試驗EPO模擬胜肽之一系列稀 釋液或50微升EPO(R&amp;D系統公司，明尼蘇達州明尼玻利 120 1344964 市）或阿犯尼司（Aranesp)(島碧生成素〇； (darbepoeitin α )，購 自安京（Amgen)公司之EPO-R激動劑）於補充i〇% FBS(不含 WEHI-3上清液I)之DMEM/F12培養基添加至96孔檢定分析 孔板(最終各孔容積1〇〇微升）。例如試驗12種不同稀釋液，試 5驗胜狀（或對照EPO胜肽）終濃度為81 ΟρΜ至0.0045Pm之範 圍。然後接種後之孔板細胞於37°C培養48小時。其次10微升 MTT(羅斯診斷公司）添加至各培養孤之各孔，然後讓其培養4 小時。藉加入10% SDS + 0.01N HC1停止反應。孔板於37。(：隔 夜。然後藉分光光譜術測定各孔於595奈米波長之吸光比。 10 吸光比s賣數對試驗胜狀漢度作圖，使用Graph Pad軟體算出 EC50。可獲得半最大吸光比之試驗胜肽濃度記錄作為EC5〇。 3.競爭結合檢定分析 使用檢定分析從事競爭結合計算，檢定分析中光信號 係依據二珠粒接近程度之函數變化而產生：鏈絲菌抗生物 15 素施體珠粒載有生物素化EPO-R結合胜肽追蹤劑，以及結 合有EPO-R之受體珠粒。藉非輻射能量移轉發光，此時當 發光時由第一珠粒放出孤氧，第二珠粒接觸孤氧造成發 光。此種珠粒集合可於商業上獲得（派克公司）。珠粒接近係 由於EPO-R结合胜肽追蹤劑結合至EPO-R所產生。試驗胜肽 20 與EPO-R結合胜肽追蹤劑競爭結合至EPO-R，將防止此種結 合，造成發光的減少。 該方法之進一步細節如後：添加4微升試驗EPO-R激動 劑胜肽之一系列稀釋液或陽性對照或陰性對照至3 84孔板 各孔。隨後，加入每孔2微升/受體珠粒混合液。受體珠粒 121 1J44964Baf3/EpoR/GCSFR f〇s/lux cells were supplemented with 1% fetal bovine serum (FBS; Hyclone) in DMEM/F12 medium (Gibco's culture medium) on 1%% WEHI_3 The supernatant (by WEHI_3 cells, ATCC # TIB-68) was cultured to obtain penicillin/streptomycin. The assay was divided into 2 minutes before decantation, and the cells were transferred to DMEM/F12 medium supplemented with 10% FBS and 0.1%. WEHI-3 supernatant was scarce. On the day of assay analysis, cells were washed with DMEM/F12 medium supplemented with 10% FBS (without WEHI-3 supernatant), then 1 X 1 〇ό cells/ml at known concentrations Culture in the presence of test peptides or use EPO (R & D Systems, Minneapolis, Minnesota) 119 1344964 as a positive control, supplemented with DMEM/F12 supplemented with 10% FBS (without WEHI-3 supernatant) Culture. A series of dilutions of the test peptide was tested at the same time in this assay. The assay plate was incubated at 37 ° C for 4 hours in a 5% carbon dioxide atmosphere, followed by the addition of σ luciferin (steady gyro -Glo); Pro + 5 meters (Promega) 'Marison, Wisconsin' to each hole. After 5 minutes of incubation, The luminescence was measured by a Packard desktop photometer (Parker Instruments, Inc., Douglas, Ill.). The light intensity values were plotted against the test peptide concentration and analyzed using Graph Pad software. The test peptide concentration was recorded as EC50. 10 2. Proliferation assay This assay is based on the mouse pre-B cell line Baf3 metastasis to express human EPO-R. The resulting cell line Baf3/Gal4/Elk/EPOR proliferation line and EP0 -R activation-related. The degree of cell proliferation was quantified using MTT, where the signalling of the MTT assay was directly proportional to the number of viable cells. 15 BaF3/Gal4/Elk/EPOR cells were supplemented with 1%% &gt; FBS in a rotating vial ( Hypoclosure) and 2% WEHI-3 supernatant (ATCC # TIB-68) in DMEM/F12 medium (Jibo Co.) culture. After culture, the cells were centrifuged at a cell density of lxlO6 cells/ml. 10% FBS and 0.1% * WEHI-3 supernatant DMEM/F12 medium was lacking overnight. Then the depleted fine * 20 cells were washed twice with Dubec PBS (Gibob) and resuspended in 1% FBS ( Contains no WEHI-3 supernatant) to cell density Ιχίο6 cells /ml. 50 μl of the aliquot (approximately 50,000 cells) of the cell suspension was re-inoculated three times in 96 well assay assay plates. A full 50 microliter test EPO mimetic peptide series dilution or 50 microliters EPO (R&amp;D Systems, Minneapolis, Minnesota 120 1344964) or Aranesp (Islandiamycin) (darbepoeitin α), purchased from Amgen's EPO-R agonist) in DMEM/F12 medium supplemented with i〇% FBS (without WEHI-3 supernatant I) to 96-well assay Orifice plate (final volume of each hole is 1 〇〇 microliter). For example, test 12 different dilutions, try 5 test (or control EPO peptide) final concentration of 81 ΟρΜ to 0.0045Pm range. The plated cells after inoculation were then cultured at 37 ° C for 48 hours. Next, 10 μl of MTT (Rose Diagnostics Inc.) was added to each well of each culture and allowed to incubate for 4 hours. The reaction was stopped by the addition of 10% SDS + 0.01 N HC1. The orifice plate is at 37. (: Overnight. Then absorbance spectroscopy is used to determine the absorbance ratio of each well at 595 nm. 10 Absorbance ratio s sells the plot to the test win, and calculate the EC50 using Graph Pad software. The half maximum absorbance ratio can be obtained. The test peptide concentration was recorded as EC5〇. 3. Competitive binding assay analysis uses competitive assays to perform competitive binding calculations. The optical signal in the assay is based on a change in the proximity of the two beads: Streptomyces antibiotic 15 donor beads Contains a biotinylated EPO-R-binding peptide tracer and an acceptor bead that incorporates EPO-R. The non-radiative energy shifts the luminescence, at which point the solitary oxygen is released by the first bead when illuminated, and the second The beads are contacted with orphaned oxygen to cause luminescence. This bead collection is commercially available (Pike). The bead proximity is due to the binding of EPO-R to the peptide tracer to EPO-R. Test peptide 20 and EPO The -R-binding peptide tracker competes for binding to EPO-R, which will prevent this binding, resulting in a decrease in luminescence. Further details of this method are as follows: Add 4 μl of test EPO-R agonist peptide to a series of dilutions Positive pair Or a negative control to each well of 384 well plate. Subsequently, 2 microliters / acceptor bead mixture to each well. Acceptor beads 121 1J44964

&gt;見合液之組成為：15微升5毫克/毫升鏈絲菌抗生物素受體 珠粒(派克公司），15微升5毫克/毫升單株抗體abl79(此抗體 辨識重組EPO-R所含人胎盤鹼性磷酸酶蛋白質部分），蛋白 質A塗覆受器珠粒(蛋白質a結合至abl79抗體；派克公司）， U2.5微升1:6.6重組EPO-R稀釋液（中國倉鼠卵巢細胞製 造，呈結合至人胎盤驗性填酸酶蛋白質部分（其含有abl79 目標抗原決定部位)之融合蛋白質，及607.5微升阿爾發奎斯 特（Alphaquest)緩衝液 4mM HEPES, pH 7.4; IMm MgClsO.l% BSA，0.05%呑恩20)。輕敲桌面混合。加入2微 升/孔生物素化EPO-R結合胜肽追蹤劑，AF33068 (30nM終 濃度）。AF33068為一 EPO-R結合胜肽（參考表3「通報子EC50 (PM)」）係根據實施例1所述方法製備。&gt; See the composition of the mixture is: 15 μl of 5 mg / ml Streptavidin antibiotic label (Pike), 15 μl of 5 mg / ml monoclonal antibody abl79 (this antibody identifies recombinant EPO-R Human placenta alkaline phosphatase protein fraction), protein A coated receptor beads (protein a binds to abl79 antibody; Parker), U2.5 microliter 1:6.6 recombinant EPO-R dilution (Chinese hamster ovary cells) Manufactured as a fusion protein that binds to the human placental virulence enzyme protein portion (which contains the abl79 target epitope), and 607.5 microliters of Alphaquest buffer 4 mM HEPES, pH 7.4; IMm MgClsO. L% BSA, 0.05% 呑 20). Tap the desktop mix. Add 2 μl/well biotinylated EPO-R binding peptide tracer, AF33068 (30 nM final concentration). AF33068 is an EPO-R binding peptide (refer to Table 3 "Notifier EC50 (PM)") prepared according to the method described in Example 1.

離心1分鐘混合。板使用派克頂封密封且包裹於金屬落 内。於至培養隔夜。18小時後使用阿爾發奎斯特讀取器 (派克公司）讀取發光。發光對胜肽濃度作圖，使用Graph pad 或Excel分析。 比較不含試驗胜肽之觀察值，可獲得發光減少5〇%之 試驗胜肽濃度記錄為IC50 » 4. C/BFU-e檢定分析 EPO-R發訊模擬骨髓幹細胞分化成為增生中的紅血球 前驅細胞。本檢定分析測定試驗胜肽模擬得自—次人骨髓 122 1344964 多重作用幹細胞之紅血球前驅細胞之增生與分化。 用於本檢定分析’於補充10% FBS(海克隆公司）之 IMDM培養基（吉伯可公司）製作試驗胜肽之一系列稀釋 液。然後系列稀釋液或陽性對照EPO胜肽添加至甲基纖維 5素來獲得終容積L5毫升。然後甲基纖維素與胜肽混合物徹 底翻轉。整份（100,000細胞/毫升）人骨髓衍生得自CD34+細 胞(保伊堤克斯/肯布拉斯)解凍。然後解凍後之細胞溫和添 加至50毫升試管内之0.1毫升1毫克/毫升DNAse(幹細胞）。其 次40-50毫升IMDM培養基溫和添加至細胞：前1〇毫升培養 10基係順著50毫升試管旁逐滴添加，然後其餘培養基則沿試 管旁緩慢配送。然後細胞於900rpm離心20分鐘，藉溫和吸 取小心去除培養基。細胞再懸浮於1毫升IMDM培養基，於 血球計玻片計算每毫升細胞密度（玻片上為一整份1〇微升 細胞懸浮液，細胞密度為平均數XI〇,〇〇〇細胞/毫升）。然後 15細胞於IMDM培養基稀釋至15,000細胞/毫升細胞密度。然 後100微升稀釋後之細胞添加至各1.5毫升甲基纖維素加胜 肽試樣(檢定分析中之終細胞濃度為1〇〇〇細胞/毫升），混合 物經翻轉。讓混合物内之氣泡消失，然後使用鈍端針頭吸 取1毫升。將得自各樣本之0·25毫升吸取之混合物添加至24 20孔孔板（飛爾空（Falcon)品牌）四孔之各孔。接種混合物於37 C於5%二氧化故於潮濕培育器培養14日。使用相位顯微鏡 (5倍-10倍物鏡，最終放大倍率卿倍)計算紅血球群落。比 較使用EPO陽性對照組觀察，生成之群落數目為最大值9〇% 之試驗胜狀濃度記錄作為EC90。 123 1344964Mix by centrifugation for 1 minute. The plate is sealed with a Parker top seal and wrapped in a metal drop. So that it is cultivated overnight. The luminescence was read after 18 hours using an Alpha Quest reader (Pike). Luminescence is plotted against peptide concentration and analyzed using Graph pad or Excel. Comparing the observations without the test peptide, the concentration of the test peptide obtained by reducing the luminescence by 5〇% was recorded as IC50 » 4. C/BFU-e assay analysis EPO-R signaling to simulate the differentiation of bone marrow stem cells into proliferative red blood cell precursors cell. This assay analyzes the test peptides obtained from the proliferation and differentiation of red blood cell precursor cells from the human bone marrow 122 1344964 multiplexed stem cells. For this assay analysis, a series of dilutions of the test peptide was prepared by supplementing 10% FBS (Hai Keng) IMDM medium (Jibo Co.). A serial dilution or positive control EPO peptide was then added to the methylcellulose to obtain a final volume of L5 ml. The methylcellulose and peptide combination are then thoroughly inverted. A whole (100,000 cells/ml) of human bone marrow derived from CD34+ cells (Paulie/Kenbras) was thawed. The thawed cells were then gently added to 0.1 ml of 1 mg/ml DNAse (stem cells) in a 50 ml test tube. The next 40-50 ml of IMDM medium was gently added to the cells: the first 1 ml of the culture was added 10 bases down the 50 ml tube, and the remaining medium was slowly dispensed alongside the test tube. The cells were then centrifuged at 900 rpm for 20 minutes and the medium was carefully removed by gentle aspiration. The cells were resuspended in 1 ml of IMDM medium and the cell density per ml was calculated on a hemocytometer slide (one whole microliter of cell suspension on the slide, cell density XI〇, 〇〇〇 cells/ml). The 15 cells were then diluted in IMDM medium to a cell density of 15,000 cells/ml. Then, 100 μl of the diluted cells were added to each 1.5 ml of methylcellulose vasopeptide sample (the final cell concentration in the assay was 1 〇〇〇 cells/ml), and the mixture was inverted. Let the bubbles in the mixture disappear and then use a blunt end needle to absorb 1 ml. The 0. 25 ml of the extracted mixture from each sample was added to each of the four wells of a 24 20-well plate (Falcon brand). The inoculated mixture was incubated at 37 C for 5% in a humidified incubator for 14 days. Red blood cell populations were calculated using a phase microscope (5x-10x objective, final magnification). When compared with the EPO-positive control group, the test scent concentration with a maximum number of colonies of 9〇% was recorded as EC90. 123 1344964

茬令^逛^昶^:伽^)額^-^^荽：9&lt; C/BFU-e EC90 (pM) m Si ^ 〇 /-^s 增生 EC50 (pM) iTi VD 通報子 EC50 (PM) m 胜肽二元體 1 I 〇 (AeO)GLYACHMGFnxl mOVOQMJ^MeOKNH^^ 舰 0 〇 ο^〇-ρε6.κ 丫 —PEGjok 广 Γ &lt;AoO)GLYAjMMG?rr(l«nal)Vp}PUl(^e〇HD/^-NH2 化合物命名 AF37702 124 實施例12:活體内活性檢定分析 本例驗證可用於評比本發明EPO-R激動劑胜肽之活性 及強度之活體内檢定分析。EPO-R激動劑胜肽單體及二元 體係根據實施例1提供之方法製備。胜肽單體及二元體之活 體内活性係使用一系列檢定分析包括多重血球胞外缺氧小 鼠生物檢定分析及網狀細胞生物檢定分析評比。兩種檢定 分析之進一步細節說明如後。 1.多重血球胞外缺氧小鼠生物檢定分析 試驗胜肽係於多重血球胞外缺氧小鼠生物檢定分析測 疋活體内活性’該檢定分析係由Ctoes及Bangham (1961)， 自然191:1065-1067所述方法修正。本檢定分析檢驗試驗胜 肽用作為EP0模擬物：亦即活化EPO-R且誘生新紅血球合成 之能力。紅血球之合成係基於放射性標記鐵結合於合成紅 血球之血色素定量。 讓BDF1小鼠對周圍條件馴化7_10日。測定全部小鼠體 重’不使用體重太輕的小鼠（小於15克）。小鼠接受於低氣壓 艙連續調理週期共計14日。24小時週期係有18小時於〇.4〇土 〇·〇2%大氣壓及6小時於周圍壓力組成。小鼠調理後，於給 藥前又維持於周圍壓力72小時。 試驗胜肽或重組人EP0標準品於pbs + 0.1% BSA媒劑 PBS/BSA)稀釋。胜肽單體備用溶液首先於二曱亞鑛(DMS〇) 增溶。陰性對照組包括一組小鼠單獨注射pBS/BSA，一組 小鼠單獨注射1%DMSC^各劑量組各有10頭小鼠。小鼠皮 下注射（頸背）0_5毫升適當試樣。 1344964 於注射試樣後48小時，小鼠腹内注射〇2毫升杜邦 公司NEN) ’劑量約0.75微居禮/小鼠。投予^後24小時稱 量小鼠體重，投予W後48小時犧牲小鼠。由各動物藉心臟 穿刺採集血樣，測定血容(使用肝素作為抗凝血劑）。各血樣 5 5 (〇_2毫升使用派克珈瑪計數器分析Fe59結合量。無反應之小 . I亦即放射性結合低於陰性對照組之小鼠）由適當資料集 合中剔除。血容值低於陰性對照組53%之小鼠也被剔除。 齡 縣係衍生自各實_量_小鼠雜^求出各組 結合於血樣之放射性平均量[每分鐘計數值((：1&gt;1^)]。 10 2.網狀細胞檢定分析 正常BDF1小鼠連續3日給藥（〇 5毫升，皮下注射)Ep〇 對照或試驗胜肽。於第三日，給予小鼠(〇1毫升，腹内注射） 鐵葡萄聚糖（100毫克/毫升）。於第五日，小鼠以二氧化碳麻 醉，藉心臟穿刺採血。各血樣之網狀細胞百分比（％)係藉噻 15唑橙染色及流動細胞計分析（格子計數計晝）以人工方式測 丨 定血容。校正後之網狀細胞數目係使用下式測定： % RETIC校正後=% RETICWx(血容個別/血容正常） 3.血液學檢定分析茬令^逛^昶^: 伽^) Amount ^-^^荽:9&lt; C/BFU-e EC90 (pM) m Si ^ 〇/-^s Proliferation EC50 (pM) iTi VD regenerant EC50 (PM) m peptide peptide 1 I 〇(AeO)GLYACHMGFnxl mOVOQMJ^MeOKNH^^ Ship 0 〇ο^〇-ρε6.κ 丫—PEGjok 广Γ &lt;AoO)GLYAjMMG?rr(l«nal)Vp}PUl(^ e〇HD/^-NH2 Compound Nomenclature AF37702 124 Example 12: In vivo Activity Assay Analysis This example demonstrates an in vivo assay for assessing the activity and intensity of the EPO-R agonist peptide of the present invention. EPO-R agonist The peptide monomer and binary system were prepared according to the method provided in Example 1. The in vivo activity of the peptide monomer and the binary was analyzed using a series of assays including multiple blood cell extracellular hypoxic mice bioassay analysis and reticular Cell bioassay analysis and evaluation. Further details of the two assays are described as follows. 1. Multiple blood cell extracellular hypoxic mice bioassay analysis test peptides in multiple blood cells extracellular hypoxic mice bioassay analysis test in vivo Activity 'This assay was corrected by the method described by Ctoes and Bangham (1961), Nature 191: 1065-1067. This assay analyzes The test peptide is used as an EP0 mimetic: that is, the ability to activate EPO-R and induce the synthesis of new red blood cells. The synthesis of red blood cells is based on the quantification of radiolabeled iron bound to synthetic red blood cells. Let BDF1 mice acclimate to surrounding conditions 7_10 Days. All mice were weighed 'not using mice that are too light (less than 15 grams). The mice received a continuous conditioning cycle of 14 hours in a low pressure chamber. The 24-hour cycle was 18 hours in 〇.4〇土〇 〇 2% atmospheric pressure and 6 hours at ambient pressure. After conditioning, the mice were maintained at ambient pressure for 72 hours before administration. Test peptide or recombinant human EP0 standard in pbs + 0.1% BSA vehicle PBS/BSA )dilution. The peptide monomer monomer solution was first solubilized in the Dioxin (DMS®). The negative control group consisted of a group of mice injected with pBS/BSA alone, and one group of mice injected with 1% DMSC alone. Each group had 10 mice in each dose group. The mouse was injected subcutaneously (neck back) with 0_5 ml of the appropriate sample. 1344964 At 48 hours after the injection of the sample, the mice were intraperitoneally injected with 2 ml of DuPont NEN) dose of about 0.75 microcures per mouse. The body weight of the mice was weighed 24 hours after administration, and the mice were sacrificed 48 hours after the administration of W. Blood samples were taken from each animal by cardiac puncture and blood volume was measured (heparin was used as an anticoagulant). Each blood sample 5 5 (〇_2 ml was analyzed for the amount of Fe59 binding using a Parker's counter. The small amount of no reaction. I, that is, the mouse with radioactive binding lower than the negative control group) was excluded from the appropriate data collection. Mice with a blood volume lower than 53% of the negative control group were also excluded. The age of the county is derived from the actual amount of _ mice mixed to determine the average amount of radioactivity bound to each blood sample [counts per minute ((: 1 &gt; 1 ^)]. 10 2. Reticulocyte assay analysis of normal BDF1 small The rats were administered for 3 consecutive days (〇5 ml, subcutaneous injection) of Ep〇 control or test peptide. On the third day, mice (〇1 ml, intraperitoneal injection) of iron glucomannan (100 mg/ml) were administered. On the fifth day, the mice were anesthetized with carbon dioxide and collected by blood puncture. The percentage of reticular cells (%) of each blood sample was determined by artificial sputum determination by thioloxyl orange staining and flow cytometry analysis (grid counting). The corrected number of reticulocytes was determined using the following formula: % RETIC corrected = % RETICWx (blood volume / normal blood volume) 3. Hematology analysis

X 正常CD1小鼠每週四次大劑量靜脈注射Ep〇陽性對 2〇照、試驗胜肽或媒劑。一定範圍之陽性對照及試驗胜肽劑 重（以毫克/千克表示）係經由改變調配物之活性化合物濃度 试驗。注射量為5毫升/千克。媒劑對雜包含12頭動物， 其餘各給藥組每組8頭。每日記錄存活情況，以及每遇記錄 126 1344964 給藥小鼠為空·腹小鼠，隨後吸入異氟烷(iS〇flurane)麻 醉，於第1曰（媒劑對照小鼠）及第15曰及第29曰（4小鼠/組/ 曰）經心臟穿刺或腹腔主動脈穿刺採集血樣。血液轉移至瓦 秋天那（Vacutainer)品牌試管内。較佳抗凝血劑為伸乙基二 5 胺四乙酸(EDTA) ° 使用業界幕所周知之自動化臨床分析儀（例如固特 (Coulter)公司製造）評比血樣之紅血球合成終點及生理例如 血容(Hct)、血色素(Hgb)及總紅血球數目（RBC)。 * * * φ) 10 本發明之範圍並非由此處所述特定具體例所限。確實 除此處所述之外，本發明之各項修改為熟諳技藝人士由前 文說明及附圖顯然自明。此等修改意圖皆落入隨附之請專 利範圍之範圍。 進-步須瞭解，全部數值皆為近似值，僅供舉例說明 15 之用。 各參考文獻包括專利案、專利申請案及各公開文獻皆 引用且討論於本發明之說明。此等參考文獻之引用及/或討 ^ 論僅為澄清本發明之說明，而絕非承認任何一個參考文獻 為本發明之「先前技術」。本說明書引用及討論之全部參考 2〇文獻全文以引用方式併入此處，彷彿各參考文獻各卿入 ' 本文以供參考般。 ‘ 【圖式簡單說明】 (無） 【主要元件符號說明】 * (無） 127X Normal CD1 mice were given a large dose of Ep〇 positive for 4 times a week, test peptide or vehicle. A range of positive controls and test peptide doses (expressed in mg/kg) are tested by varying the concentration of active compound in the formulation. The injection volume is 5 ml / kg. The vehicle mixture contained 12 animals, and the remaining drug administration groups contained 8 animals each. Survival was recorded daily, and 126 1344964 mice were recorded as empty abdomen mice, followed by inhalation of isoflurane (iS〇flurane) anesthesia, in the first sputum (media control mice) and 15th 曰And 29th (4 mice/group/曰) blood samples were taken by cardiac puncture or abdominal aortic puncture. The blood is transferred to the Vacutainer brand test tube. The preferred anticoagulant is exoethyldiaminetetraacetic acid (EDTA) °. The erythrocyte synthesis endpoint and physiology, such as blood volume, of the blood sample are evaluated using an automated clinical analyzer (such as that manufactured by Coulter). (Hct), hemoglobin (Hgb) and total red blood cell count (RBC). * * * φ) 10 The scope of the present invention is not limited by the specific examples described herein. Indeed, various modifications of the invention are apparent to those skilled in the art and These modifications are intended to fall within the scope of the accompanying patents. It should be understood that all values are approximate and are for illustrative purposes only. Each of the references, including patents, patent applications, and publications are hereby incorporated by reference. The citation and/or discussion of these references is merely illustrative of the invention and is not an admission that any reference is a prior art of the invention. The entire disclosure of the present specification is hereby incorporated by reference. ‘ [Simple description of the diagram] (none) [Description of main component symbols] * (none) 127

Claims (1)

1344964 1第93134267號專利申請案申請專利 範圍修正本 修正曰 期：99年12月 公告本 十、申請專利範圍： 1. 一種結合且活化紅血5 f)年IL月/〈日則東)正本 托生成素受器（EPO-R)之1 匕合物， 該化合物包含胜肽二元體，具有下式：1344964 1 Patent Application No. 93134267 Patent Application Amendment This Amendment Period: December 1999 Announcement Ten, Patent Application Range: 1. A combination of activated red blood 5 f) annual IL month / <日则东) original A compound of Etomycin receptor (EPO-R), which comprises a peptide binary having the following formula: (AcG)GLYA(?HNiGPIT(l&lt;ial)VCQPLR-&gt;NH(AcG)GLYA(?HNiGPIT(l&lt;ial)VCQPLR-&gt;NH 5 其中 (i) 於胜肽二元體之各個胜肽單體，各個胺基酸係 以標準單字母縮寫表示，AcG為N-乙醯基甘胺酸，以及 Ι-nal為1-萘基丙胺酸； (ii) 胜肽二元體之各個胜肋·單體介於各單體之兩 10 個半胱胺酸(C)殘基間含有一個分子内雙硫鍵； (iii) [「PET」]包含至少一個線性聚乙二醇(PEG)部 分’，各個PEG部分具有分子量約20,〇〇〇至約4〇,〇〇〇道爾 吞。 2. 如申請專利範圍第1項之化合物，其中各個pEG具有分 15 子量約30,000道爾吞。 3. —種如申請專利範圍第1項之化合物用於製造一藥物的 用途’該藥物係用於治療以紅血球生成素缺乏或紅血球 族群低或缺陷為特徵之病症β 成’其中該病症係選自由下 病或惡性病相關貧血二透析;愛滋病、自體免疫 性.單本&amp;曰 ，冷型地中海貧血；囊性纖維變 备性血；r ★期貧血慢性發炎病相關貧血；脊索受傷； =;及伴隨有紅血球生成異常之腫瘤 疾病狀態。 如申請專·_3項之用途，其中各個觸具有分子 置約30,〇〇〇道爾吞。 -種藥學組成物，包含如中請專利範㈣丨項之化合物 及藥學上可接受之載劑。 如申吻專利範圍第6項之藥學組成物，其中各個pEG具 有分子量約30,000道爾吞。 一種結合且活化紅血球生成素受器（Ep〇_R)之化合物， 該化合物包含胜肽二元體，具有下式：5 wherein (i) each peptide monomer of the peptide peptide, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, and Ι-nal is 1-naphthyl Amino acid; (ii) each of the peptides of the peptide is composed of an intramolecular disulfide bond between two of the ten cysteine (C) residues of each monomer; (iii) [" PET"] comprises at least one linear polyethylene glycol (PEG) moiety, each having a molecular weight of from about 20, 〇〇〇 to about 4 〇, 〇〇〇Dowagen. 2. A compound as claimed in claim 1 wherein each pEG has a sub-quantity of about 30,000 dolphins. 3. The use of a compound as claimed in claim 1 for the manufacture of a medicament for the treatment of a condition characterized by a deficiency or deficiency of erythropoietin or a red blood cell population, wherein the condition is selected Free dialysis or malignant disease-related anemia; AIDS, autoimmune. Single &amp; 曰, cold thalassemia; cystic fibrosis; blood; =; and tumor disease status accompanied by abnormal red blood cell production. For example, if the application is for the purpose of the _3 item, each of the touches has a molecular weight of about 30, which is 〇〇〇道尔吞. A pharmaceutical composition comprising a compound of the formula (IV), and a pharmaceutically acceptable carrier. For example, the pharmaceutical composition of claim 6 wherein each pEG has a molecular weight of about 30,000 doxantane. A compound that binds to and activates a erythropoietin receptor (Ep〇_R), the compound comprising a peptide binary having the formula: (i) 於胜肽一元體之各個胜肽單體，各個胺基酸係 以標準單字母縮寫表示，AcG為N-乙醯基甘胺酸，i_naj 為〗-萘基丙胺酸，及MeG為N-甲基甘胺酸； (ii) 胜肽二元體之各個胜肽單體介於各單體之兩 個半胱胺酸(C)殘基間含有一個分子内雙硫鍵； (ill) PEG包含具有分子量約2〇,〇〇〇至約40 000道爾 吞之線性未分支聚乙二醇分子。 9·如申印專利範圍第8項之化合物其中各個PRO具有分 子量約30,000道爾吞。 1〇.—種如申請專利範圍第8項之化合物用於製造一藥物的 用途’該藥物係用於治療以紅血球生成素缺t或紅血球 族群低或缺陷為特徵之病症。 U·如申請專㈣圍第_之用途’其中該病症係選自由下 列組成之群組：末期腎衰竭或透析；愛滋病、自體免疫 =或惡性病相關貧血；6型地中海貧血；囊性纖維變 ♦糾早產兒早期貧灰；慢性發炎病相關貧血；脊索受傷； 血n老化；及伴隨有紅血球域之 疾病狀態。 12.2請專利範圍第_之用途，其中各 量約3〇，_道爾吞。 13·:，成物’包含如申請專利範圍第8項之化合物 14及樂學上可接受之載劑。 有八二專圍第13項之藥學組成物，其中各個PEG具 有刀子量約3〇，_道爾吞。 15. —種結合且活化紅血球生成素受器（EPO-R)之化合物， 該化合物包含胜肽二元體，具有下式：(i) each peptide monomer of the peptide monopeptide, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, i_naj is 〖-naphthylalanine, and MeG is N-methylglycine; (ii) each peptide monomer of the peptide complex contains an intramolecular disulfide bond between the two cysteine (C) residues of each monomer; PEG comprises linear unbranched polyethylene glycol molecules having a molecular weight of from about 2 Å to about 40,000 Torr. 9. A compound of the eighth aspect of the patent application wherein each PRO has a molecular weight of about 30,000 dolphins. 1) A use of a compound of claim 8 for the manufacture of a medicament for treating a condition characterized by erythropoietin deficiency or a low red blood cell population or defect. U. If the application is for (4), the use of the disease is selected from the group consisting of: end stage renal failure or dialysis; AIDS, autoimmune = or malignant disease-related anemia; type 6 thalassemia; cystic fiber Change ♦ correct early premature infants with poor ash; chronic inflammatory disease-related anemia; spinal cord injury; blood n aging; and accompanied by the red blood cell disease state. 12.2 Please use the patent scope _, which is about 3 〇, _Doltun. 13:: The product 'includes the compound 14 as set forth in claim 8 and the learned carrier. There are eighty-two pharmacy compositions of the 13th item, in which each PEG has a knife amount of about 3 〇, _Doltun. 15. A compound that binds to and activates a erythropoietin receptor (EPO-R), the compound comprising a peptide binary having the formula: 〇 (i) 於胜肽二元體之各個胜肽單體，各個胺基酸係 以標準單字母縮寫表示’ AcG為N-乙醯基甘胺酸，以及 Ι-nal為1-蔡基丙胺酸； (ii) 胜肽二元體之各個胜肽單體介於各單體之兩 個半胱胺酸(C)殘基間含有一個分子内雙硫鍵； (iii) PEG包含具有分子量約2〇,〇〇0至約4〇 〇〇〇道爾 吞之線性未分支聚乙二醇分子。 16. 如申請專利範圍第15項之化合物，其中各個ρΕ(}具有分 子量約30,000道爾吞。 17. -種如f請專利範圍第15項之化合物用於製造一藥物 的用途，該藥物剌於治療以紅血球生成素缺乏或紅血 球族群低或缺陷為特徵之病症。 18. 如申請專利範圍第17項之用途，其中該病症係選自由下 列組成之群組：末㈣衰蝎或透析；愛滋病、自體免疫 病或惡性病相關貧血；点型地中海貧血；囊性纖維變 性，早產兒早期貧血；慢性發炎病相關貧血；脊索受傷； 急性血液流失；老化；及伴隨有紅血球生料常之腫瘤 疾病狀態。 19. 如申咕專利範圍第17項之用途，其中各個pEG具有分子 里約30,〇〇〇道爾吞。 20. —種藥學組成物，包含如申請專利範圍第15項之化合物 及藥學上可接受之載劑。 21. 如申請專利範圍第20項之藥學組成物，其中各個pEG具 有分子量約30,000道爾吞。 22. —種結合且活化紅血球生成素受器（Ep〇R)之化合物， 該化合物包含胜肽二元體，具有下式： (AcGJGLYAOiMGPnXl -nal)viQPLR(MeG)〇(i) for each peptide monomer of the peptide binary, each amino acid is represented by a standard one-letter abbreviation 'AcG is N-ethinylglycine, and Ι-nal is 1-caffeyl propylamine (ii) each peptide monomer of the peptide binary contains an intramolecular disulfide bond between the two cysteine (C) residues of each monomer; (iii) the PEG comprises a molecular weight of about 2〇, 〇〇0 to about 4 〇〇〇〇Dolphine linear unbranched polyethylene glycol molecules. 16. A compound according to claim 15 wherein each ρΕ(} has a molecular weight of about 30,000 dolphine. 17. a compound such as f, wherein the compound of claim 15 is used for the manufacture of a medicament, For the treatment of conditions characterized by erythropoietin deficiency or low red blood cell population or defects. 18. The use of claim 17 wherein the condition is selected from the group consisting of: (4) dysfunction or dialysis; AIDS Autoimmune disease or malignant disease-related anemia; point-type thalassemia; cystic fibrosis, early anemia in premature infants; anemia associated with chronic inflammatory disease; spinal cord injury; acute blood loss; aging; and tumors often associated with red blood cells The state of the disease 19. The use of the scope of claim 17 wherein each pEG has about 30 molecules in the molecule, and is a pharmaceutical composition comprising a compound as claimed in claim 15 And a pharmaceutically acceptable carrier. 21. The pharmaceutical composition of claim 20, wherein each pEG has a molecular weight of about 30,000 dolphins. And activation in combination of erythropoietin receptors (Ep〇R) of the compound, the compound comprising a peptide dyad, having the formula: (AcGJGLYAOiMGPnXl -nal) viQPLR (MeG) (AcG)GLYACHMGPIT(I-nal)VCQPLR(MeG)(AcG)GLYACHMGPIT(I-nal)VCQPLR(MeG) 其中 (i)於胜肽二元體之各個胜狀單體，各個胺基酸係 以標準單字母縮寫表示’ AcG為N-乙醯基甘胺酸，i_nal 為1-萘基丙胺酸，及MeG為N-甲基甘胺酸； (11)胜肽二元體之各個胜肽單體介於各單體之兩 個半胱胺酸(C)殘基間含有一個分子内雙硫鍵； (iii) PEG包含具有分子量約2〇,〇〇〇至約4〇 〇〇〇道爾 吞之線性未分支聚己二醇分子。 如申專利圍第22項之化合物，其中各個ρΕ〇具有分 子量約30,000道爾呑。 24.—種如申請專利範圍第22項之化合物用於製造—藥物 的用途„玄藥物係用於治療以紅血球生成素缺乏或紅血 球族群低或缺陷為特徵之病症。 25·如申請專利範圍第24項之用途，其中該病症係選自由下 列組成之群組：末期腎衰竭或透析；愛滋病、自體免疫 病或惡性病相關貧血；沒型地中海貧金；囊性纖維變 眭，早產兒早期貧血；慢性發炎病相關貧血；脊索受傷； 〜ί·生血液流失；老化；及伴隨有紅血球生成異常之腫瘤 疾病狀態。 如申叫專利範圍第24項之用途，其中各個pEG具有分子 量約30,〇〇〇道爾吞。 .種藥學組成物，包含如申請專利範圍第22項之化合物 及藥學上可接受之載劑。 申μ專利範圍第27項之藥學組成物，其中各個pEG具 有分子量約30,000道爾吞。 種結合且活化紅血球生成素受器（ep〇r)之化合物， 該化合物包含胜肽二元體，具有下式： (AcG)GLYA&lt;!HMOPrT(l-naI)V&lt;!：QPLR(MeG).Wherein (i) each of the pheno-monomers of the peptide complex, each of the amino acids is represented by a standard one-letter abbreviation 'AcG is N-ethinylglycine, i_nal is 1-naphthylalanine, and MeG is N-methylglycine; (11) each peptide monomer of the peptide binary contains an intramolecular disulfide bond between the two cysteine (C) residues of each monomer; (iii) PEG comprises a linear unbranched polyhexylene glycol molecule having a molecular weight of from about 2 Å to about 4 Torr. The compound of claim 22, wherein each ρ Ε〇 has a molecular weight of about 30,000 Dao. 24. The use of a compound as claimed in claim 22 for the manufacture of a drug - a drug for the treatment of a condition characterized by a deficiency of erythropoietin or a low red blood cell population or a defect. The use of 24 items, wherein the condition is selected from the group consisting of: end stage renal failure or dialysis; AIDS, autoimmune disease or malignant disease-related anemia; no type of thalassemia; cystic fibrosis, early premature infants Anemia; chronic inflammatory disease-related anemia; spinal cord injury; ~ ί · raw blood loss; aging; and tumor disease state accompanied by abnormal erythropoiesis. For example, the application of patent scope range 24, wherein each pEG has a molecular weight of about 30, A pharmaceutical composition comprising a compound of claim 22 and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 27, wherein each pEG has a molecular weight of about 30,000 dolphins. A compound that binds to and activates a erythropoietin receptor (ep〇r), the compound comprising a peptide binary having Formula: (AcG) GLYA &lt; HMOPrT (l-naI) V &lt;:!! QPLR (MeG). g (AcG)GLYACHMCTIT(l-iia〇VCQPLR(MeG)-HN NHg (AcG)GLYACHMCTIT(l-iia〇VCQPLR(MeG)-HN NH (i)於胜肽二元體之各個胜肽單體，各個胺基酸係 以標準單字母縮寫表示’ AcG為N-乙醯基甘胺酸，i_nai 為1-萘基丙胺酸，及MeG為N-甲基甘胺酸； (η)胜肽二元體之各個胜肽單體介於各單體之兩 個半胱胺酸(C)殘基間含有一個分子内雙硫鍵； (m) PEG包含具有分子量約2〇 〇〇〇至約4〇 〇〇〇道爾 吞之線性未分支聚乙二醇分子。 3〇.如申請專利個第29項之化合物，其#個咖具有分 子量約30,000道爾吞。 種如_請專利範圍第29項之化合物用於製造一藥物 的用途’該藥物係用於治療以紅血球生成素缺乏或紅血 球族群低或缺陷為特徵之病症。 32·=Γ利範圍第31項之用途，其中該病症係選自由下 病二末期腎衰竭或透析;愛滋病、自體免疫 性::產兒早目：貧也；万型地中海貧金；囊性纖維變 ;慢性發炎病相關貧^脊索受傷； 1 病狀=失；老化；及伴隨有紅血球生成異常之腫瘤 ’其中各個PEG具有分子 33.如申請專利範圍第31項之用途 量約30,000道爾吞。 34. —種藥學組成物，包含如申請專利範圍第29項之化合物 及藥學上可接受之載劑。 35. 如申請專利範圍第34項之藥學組成物，其中各個PEG具 有分子量約30,000道爾吞。 36. —種結合且活化紅血球生成素受器（ep0_r)之化合物， 該化合物包含胜肽二元體，具有了式： (AcG)GLYAC&amp;MGPIT(l-iial)viQPLR(MeG)H (AcG)GLYACHMGPIT(l.iial)VCQPLR(MeG) -l(i) for each peptide monomer of the peptide binary, each amino acid is represented by a standard one-letter abbreviation 'AcG is N-ethinylglycine, i_nai is 1-naphthylalanine, and MeG Is N-methylglycine; each peptide monomer of the (η) peptide complex contains an intramolecular disulfide bond between the two cysteine (C) residues of each monomer; m) PEG comprises a linear unbranched polyethylene glycol molecule having a molecular weight of from about 2 Torr to about 4 Torr. 3. For example, if the compound of claim 29 is applied, the #咖 has a molecular weight of about 30,000 dolphins. The use of a compound such as the scope of claim 29 for the manufacture of a medicament for treating a condition characterized by a deficiency in erythropoietin or a low or a red blood group. 32·=The use of the 31st item of the profit range, wherein the condition is selected from the second stage of renal failure or dialysis; AIDS, autoimmune:: early childhood: poor also; 10,000 types of thalassemia; cystic Fibrosis; Chronic inflammatory disease associated with poor spinal cord injury; 1 Symptoms = loss; aging; and tumors with abnormal erythropoiesis. Each PEG has a molecule 33. The application amount is about 30,000 Dao as claimed in claim 31. swallow. 34. A pharmaceutical composition comprising a compound as claimed in claim 29 and a pharmaceutically acceptable carrier. 35. The pharmaceutical composition of claim 34, wherein each PEG has a molecular weight of about 30,000 dolphins. 36. A compound that binds to and activates a erythropoietin receptor (ep0_r), the compound comprising a peptide binary having the formula: (AcG)GLYAC&amp;MGPIT(l-iial)viQPLR(MeG)H (AcG) GLYACHMGPIT(l.iial)VCQPLR(MeG) -l NH N=EG s 0 -O (i) 於胜肽二元體之各個胜肽單體，各個胺基酸係 以標準單字母縮寫表示，AcG為N-乙醯基甘胺酸，i_nai 為1-萘基丙胺酸，及MeG為N-曱基甘胺酸； (ii) 胜肽二元體之各個胜肽單體介於各單體之兩 個半胱胺酸(C)殘基間含有一個分子内雙硫鍵； (iii) PEG包含具有分子量約2〇 〇〇〇至約4〇 〇〇〇道爾 吞之線性未分支聚乙二醇(PEG)部分。 37‘如申請糊範圍第36項之化合物，其巾各個PEG具有分 子量約20,〇〇〇道爾吞。 38· —種如申請專利範圍第％項之化合物用於製造—藥物 的用途’該藥物係用於域以紅血球生成素缺乏或紅血 球族群低或缺陷為特徵之病症。 39. 如申請專利範圍第38項之用途，其中該病症係選自由下 列組成之群組：末期腎衰蝎或透析；愛滋病、自體免疫 病或惡性病相關貧血；冷型地中海貧血；囊性纖維變 性；早產兒早期貧也；慢性發炎病相關貧血；脊索受傷； 急性血液流失；老化；及伴隨有紅血球生成異常之腫瘤 疾病狀態。 40. 如申請專利範圍第38項之用途，其中各個pEG具有分子 量約30,〇〇〇道爾吞。 41. 一種藥學組成物，包含如申請專利範圍第36項之化合物 及藥學上可接受之載劑。 42. 如申請專利範圍第41項之藥學組成物，其中各個pEG具 有分子量約20,000道爾吞。 43. —種結合且活化紅血球生成素受器（EPO-R)之化合物， 該化合物包含胜肽二元體，具有下式：NH N=EG s 0 -O (i) For each peptide monomer of the peptide binary, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, and i_nai is 1 -naphthylalanine, and MeG is N-mercaptoglycine; (ii) each peptide monomer of the peptide binary is contained between two cysteine (C) residues of each monomer An intramolecular disulfide bond; (iii) PEG comprises a linear unbranched polyethylene glycol (PEG) moiety having a molecular weight of from about 2 Torr to about 4 Torr. 37 'As applied for the compound of the range 36 of the paste, each PEG of the towel has a molecular weight of about 20, 〇〇〇dol. 38. Use of a compound as claimed in claim % for the manufacture of a drug. The drug is used in a disorder characterized by a deficiency in erythropoietin or a low red blood cell population. 39. The use of claim 38, wherein the condition is selected from the group consisting of: end stage renal failure or dialysis; AIDS, autoimmune disease or malignant disease-related anemia; cold thalassemia; cystic Fibrosis; early premature infants are also poor; chronic inflammatory disease-related anemia; spinal cord injury; acute blood loss; aging; and tumor disease state accompanied by abnormal red blood cell production. 40. For the purposes of claim 38, each of the pEGs has a molecular weight of about 30, doldorf. 41. A pharmaceutical composition comprising a compound as claimed in claim 36 and a pharmaceutically acceptable carrier. 42. The pharmaceutical composition of claim 41, wherein each pEG has a molecular weight of about 20,000 dolphins. 43. A compound that binds to and activates a erythropoietin receptor (EPO-R), the compound comprising a peptide binary having the formula: (AcGJGLYACHMGPrrOHBalJVCQPLR-NH(AcGJGLYACHMGPrrOHBalJVCQPLR-NH (AcO)GLYACHMGPrT(l-oa〇VCQPLR-HN 1-1 Ο(AcO)GLYACHMGPrT(l-oa〇VCQPLR-HN 1-1 Ο 其中 (i)於胜肽二元體之各個胜肽單體，各個胺基酸係 15 鲁 20 以標準單字母縮寫表示，AeG為乙酿基甘胺酸，以及 Ι-nal為1-萘基丙胺酸； ⑻胜狀二讀之各個胜肽單體介於各單體之兩 個半耽胺酸(C)殘基間含有一個分子内雙硫鍵； (⑴)[「PEG」]包含至少二線性聚乙 鍵聯於單一附接 ； _〇道竭吞。 合分子量約_〇至約 利範圍第43項之化合物，其中各個咖具有分 子ϊ約20,〇〇〇道爾吞。 申請專利範圍第43項之化合物用於製造一藥物 ΙΙί’·物仙於治細紅血球生成素缺乏或紅血 球族群低或缺陷為特徵之 46.2請專利範圍第45項之用途，其中該病症係選自由下 病戈2群組末期腎衰蝎或透析；愛滋病、自體免疫 病相關貧血1型地中海貧血；囊性纖維變 急-^炎病相關貧血;脊索受傷; 疾病狀態。 ’及伴隨有紅血球生成異常之腫瘤 47.t申請專利範㈣45項之用途，其中各刪G具有分子 置約20,〇〇〇道爾吞。 種藥學㈣物’包含如申請專利範圍第43項之化合物 及樂學上可接受之载劑。 申請專利範圍第48項之藥學組成物 ’其中各個PEG具 有分子量約20,000道爾吞。 10 50. —種結合且活化紅血球生成素受器（EPO-R)之化合 物，該化合物包含胜肽二元體，具有下式：Wherein (i) each peptide monomer of the peptide peptide, each amino acid system 15 Lu 20 is represented by a standard one-letter abbreviation, AeG is an ethyl glycosidic acid, and Ι-nal is a 1-naphthyl group. Alanine; (8) Each peptide monomer of the second reading of the winning monomer contains an intramolecular disulfide bond between the two half-proline (C) residues of each monomer; ((1)) ["PEG"] contains at least The two-linear polyethylation bond is attached to a single attachment; A compound having a molecular weight of about 〇 〇 to about 43 of the approximate range, wherein each coffee has a molecular weight of about 20, 〇〇〇dolp. The compound of claim 43 is used for the manufacture of a drug ΙΙ ' 物 于 于 于 于 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Lower disease group 2 end stage renal failure or dialysis; AIDS, autoimmune disease related anemia type 1 thalassemia; cystic fibrosis acute - ^ inflammatory disease associated anemia; spinal cord injury; disease state. 'and tumors associated with abnormal erythropoiesis 47.t application for patent (4) 45 applications, each of which has a molecular weight of about 20, 〇〇〇道尔吞. The pharmaceutically acceptable (tetra) article comprises a compound as described in claim 43 and a grammatically acceptable carrier. The pharmaceutically acceptable composition of claim 48 wherein each PEG has a molecular weight of about 20,000 dolphins. 10 50. A compound that binds to and activates a erythropoietin receptor (EPO-R), the compound comprising a peptide binary having the formula: 其中 (i)於胜肽二元體之各個胜肽單體，各個胺基酸係 以標準單字母縮寫表示，AcG為N-乙醢基甘胺酸，以及 1_nal為1_萘基丙胺酸，及MeG為N-甲基甘胺酸； (11)胜肽二元體之各個胜肽單體介於各單體之兩 個半耽胺酸(C)殘基間含有一個分子内雙硫鍵； (U1) PEG包含二線性聚乙二醇(PEG)部分，具有組 合分子量約1 〇,〇〇〇至約30,000道爾吞。 如申凊專利範圍第5〇項之化合物，其中各個PEG具有分 子量約20,000道爾吞。 種如申請專利範圍第50項之化合物用於製造一藥物 “藥物係用於治療以紅血球生成素缺乏或紅血 53 :族:低或缺陷為特徵之病症。 9会月專和範圍第52項之用途，其中該病症係選自由下 群、卫·末期腎衰竭或透析；愛滋病、自體免疫 1344964 =生病相_貧血1型地中海貧血;囊性纖維變 q兒早期貧血；慢性發炎病相關貧血；脊索受傷； 二血液机失，老化；及伴隨有紅血球生成異常之腫瘤 疾病狀態。 54‘如申請專利範圍第52項之用途，其中各個舰具有分子 量約20,〇〇〇道爾吞。 %-種藥學組成物’包含如巾請專利範圍㈣項之化合物 及藥學上可接受之載劑。 10 如申明專利範圍第55項之藥學組成物，其中各個pEG具 有分子量約20,〇〇〇道爾呑。 57.—種結合且活化紅血球生成素受器（Ep〇幻之化合物， 該化合物包含胜肽二元體，具有下式·Wherein (i) each peptide monomer of the peptide peptide, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, and 1_nal is 1-naphthylalanine, And MeG is N-methylglycine; (11) each peptide monomer of the peptide complex contains an intramolecular disulfide bond between the two semi-proline (C) residues of each monomer (U1) PEG comprises a dilinear polyethylene glycol (PEG) moiety having a combined molecular weight of about 1 〇, 〇〇〇 to about 30,000 Torr. A compound according to claim 5, wherein each PEG has a molecular weight of about 20,000 doxane. A compound such as the patent application 50 is used to manufacture a drug "drugs are used to treat conditions characterized by erythropoietin deficiency or red blood 53: family: low or defective. 9 month and scope 52 Use, wherein the condition is selected from the group consisting of the lower group, Weiwei end stage renal failure or dialysis; AIDS, autoimmune 1344496 = ill phase _ anemia type 1 thalassemia; cystic fibrosis q early anemia; chronic inflammatory disease related anemia ; spinal cord injury; 2 blood loss, aging; and tumor disease state accompanied by abnormal red blood cell formation. 54 'As claimed in the scope of application of the 52nd, each ship has a molecular weight of about 20, 〇〇〇道尔吞. - Pharmaceutical composition 'comprising a compound of the scope of claim (4) and a pharmaceutically acceptable carrier. 10 The pharmaceutical composition of claim 55, wherein each pEG has a molecular weight of about 20, a sputum 57. A combination and activation of erythropoietin receptor (Ep phantom compound, this compound contains a peptide binary, with the following formula (i) 於胜狀二元體之各個胜狀單體，各個胺基酸係 以標準單字母縮寫表示，AcG為N-乙醯基甘胺酸，以及 Ι-nal為1-蔡基丙胺酸； (ii) 胜肽二元體之各個胜肽單體介於各單體之兩 12 5 個半胱胺酸(C)殘基間含有一個分子内雙硫鍵； 於單：:包乙二_)部— 爾吞。 、有&amp;刀子1約10,0⑻至約30,000道 1::專利範圍第57項之化合物，其中 子1約20,000道爾吞。 頁刀 ^如申⑺專利㈣第57項之化合物用於製造 10 15(i) for each singular monomer of the winning binary, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, and Ι-nal is 1-caffeyl alanine (ii) each peptide monomer of the peptide peptide contains an intramolecular disulfide bond between two 12 5 cysteine (C) residues of each monomer; _) Department - Ertun. There are &amp; knives 1 about 10,0 (8) to about 30,000. 1:: Compound of the 57th patent range, which contains about 20,000 dolphins. Page knife ^The compound of the 57th item of Shen (7) patent (4) is used for manufacturing 10 15 ^用途’該__於治療以紅㈣生成素缺乏或Μ 球族群低或缺陷為特徵之病症。 如U利範ϋ第59項之料，其中該病症係選自由下 歹L且成之群ia .末期腎衰竭或透析；愛滋病、自體免疫 病或惡性病相關貧血1型地中海貧血；囊性纖維變 (生，早產兒早期貧血；慢性發炎病相關貧血’·脊索受傷； 急性血液流失；老化；及伴隨有紅血球生成異常之腫瘤 疾病狀態。^ Use 'This __ treats a condition characterized by a red (four) pheromone deficiency or a low or defective spheroid population. Such as U Lifan ϋ Item 59, wherein the condition is selected from the group consisting of 歹L and ia. End stage renal failure or dialysis; AIDS, autoimmune disease or malignant disease-related anemia type 1 thalassemia; cystic fiber Change (birth, early anemia in premature infants; anemia associated with chronic inflammatory disease) · spinal cord injury; acute blood loss; aging; and tumor disease state accompanied by abnormal red blood cell production. 61.如申凊專利範圍第59項之用途，其中各個PEG具有分子 量約20,〇〇〇道爾吞。 62. 一種藥學組成物’包含如中請專利範圍第57項之化合物 及藥學上可接受之載劑。 2〇 63·如申請專利範圍第62項之藥學組成物，其中各個PEG具 有分子量約20,〇〇〇道爾吞。 64. 一種結合且活化紅血球生成素受器（EPO-R)之化合物， 該化合物包含胜肽二元體，具有下式： 13 134496461. The use of claim 59, wherein each PEG has a molecular weight of about 20, dolphal. 62. A pharmaceutical composition comprising a compound of claim 57 and a pharmaceutically acceptable carrier. 2 〇 63. The pharmaceutical composition of claim 62, wherein each PEG has a molecular weight of about 20, doldorf. 64. A compound that binds to and activates a erythropoietin receptor (EPO-R), the compound comprising a peptide binary having the formula: 13 1344964 5 (i) 於胜肽二元體之各個胜肽單體，各個胺基酸係 以標準單字母縮寫表示，AcG為N-乙醯基甘胺酸，i_nal 為1-萘基丙胺酸’及MeG為N-甲基甘胺酸； (ii) 胜肽二元體之各個胜肽單體介於各單體之兩 個半胱胺酸(C)殘基間含有一個分子内雙硫鍵； (m) PEG包含二線性聚乙二醇(pEG)部分，具有組 。为子里約1〇,〇〇〇至約3〇 〇〇〇道爾呑。 •如申印專利範圍第64項之化合物，其中各個PEG具有分 子量約20,〇〇〇道爾吞。 •種如中4專利範圍第64項之化合物用於製造一藥物 的用途，該单物在i m 萊物係用於治療以紅血球生成素缺乏或紅血5 (i) for each peptide monomer of the peptide peptide, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, i_nal is 1-naphthylalanine' and MeG is N-methylglycine; (ii) each peptide monomer of the peptide binary contains an intramolecular disulfide bond between the two cysteine (C) residues of each monomer; (m) PEG comprises a dilinear polyethylene glycol (pEG) moiety with a group. For the child, about 1 〇, 〇〇〇 to about 3 〇〇〇 〇〇〇 呑 呑. • A compound of claim 64, wherein each PEG has a molecular weight of about 20, doldorf. • The use of a compound according to item 64 of the Chinese Patent No. 4 for the manufacture of a medicament for the treatment of erythropoietin deficiency or red blood in the i m system ，其中該病症係選自由下 列組成之群組： 病或惡性病相關貧血； 性；早產兒早期貧 末期腎衰竭或透析；愛滋病、自體免疫 曼血；/5型地中海貧血；囊性纖維變 灰；慢性發炎病相關貧血；脊索受傷； 14 1^44^4 急性血液流失；老化； 及伴隨有紅jk球生成異常之腫瘤 疾病狀態。 5 10 队如申請專利範圍第66項之用途，其中各個舰具有分子 量約20,〇〇〇道爾吞。 種藥子、.且成物，包含如申請專利範圍第料項之化合物 及藥學上可接受之栽劑。 如申請專㈣圍⑽奴藥學喊物，其巾各個舰具 有分子量約20,000道爾吞。 71. -種結合且活化紅血球生成素受器（Ep〇-R)之化合物， 該化合物包含胜狀二元體，具有下式：Wherein the condition is selected from the group consisting of: anemia associated with a disease or malignant disease; sex; early stage renal failure or dialysis in preterm infants; AIDS, autoimmune human blood; type 5 thalassemia; cystic fibrosis Gray; chronic inflammatory disease-related anemia; spinal cord injury; 14 1^44^4 acute blood loss; aging; and tumor disease state accompanied by abnormal red jk ball formation. 5 10 The team used the application of the 66th scope of the patent, in which each ship has a molecular weight of about 20, 〇〇〇道尔吞. A medicine, a composition, and a compound as claimed in the scope of the patent application and a pharmaceutically acceptable carrier. For example, if you apply for special (4) Wai (10) Pharmacy shouts, the hulls of each towel have a molecular weight of about 20,000 dolphins. 71. A compound that binds to and activates a erythropoietin receptor (Ep〇-R), the compound comprising a triumphant binary having the formula: (AcG)GLYA(}HMGPn&lt;l-niU)vii 〇(AcG)GLYA(}HMGPn&lt;l-niU)vii 〇 V jtm PEG (AcO)GLYA(piMGPlT(i-Ml)VCQPLRWINV jtm PEG (AcO)GLYA(piMGPlT(i-Ml)VCQPLRWIN ΟΟ 其中 (i) 於胜肽二元體之各個胜肽單體，各個胺基酸係 以標準單字母縮寫表示，AcG為N-乙醯基甘胺酸，以及 15 Ι-nal為1-萘基丙胺酸； (ii) 胜肽二元體之各個胜肽單體介於各單體之兩 個半胱胺酸(C)殘基間含有一個分子内雙硫鍵； 15 1344964 (iii) PEG包含具有分子量約20,000至約40,000遒爾 吞之線性未分支聚乙二醇(PEG)部分。 72. —種結合且活化紅血球生成素受器（EPO-R)之化合物’ 該化合物包含胜肽二元體，具有下式：Wherein (i) each peptide monomer of the peptide peptide, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, and 15 Ι-nal is 1-naphthyl Alanine; (ii) each peptide monomer of the peptide complex contains an intramolecular disulfide bond between the two cysteine (C) residues of each monomer; 15 1344964 (iii) PEG contains A linear unbranched polyethylene glycol (PEG) moiety having a molecular weight of from about 20,000 to about 40,000 mil. 72. A compound that binds and activates a erythropoietin receptor (EPO-R). The compound comprises a peptide binary having the following formula: (i)於胜肽二元體之各個胜肽單體，各個胺基酸係 以標準單字母縮寫表示，AcG為N-乙醯基甘胺酸，以及 Ι-nal為1-萘基丙胺酸； (η)胜肽二元體之各個胜肽單體介於各單體之兩 個半胱胺酸(C)殘基間含有一個分子内雙硫鍵； (Hi) PEG包含具有分子量約1 〇,〇〇〇至約6〇 〇〇〇道爾 吞之線性未分支聚乙二醇(PEG)部分。 16(i) each peptide monomer of the peptide peptide, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, and Ι-nal is 1-naphthyl alanine (η) each peptide monomer of the peptide peptide contains an intramolecular disulfide bond between the two cysteine (C) residues of each monomer; (Hi) PEG contains a molecular weight of about 1 〇, 〇〇〇 to a linear unbranched polyethylene glycol (PEG) moiety of about 6 〇〇〇〇. 16