聚乙二醇修饰的胸腺肽 1衍生物 技术领域 Polyethylene glycol modified thymosin 1 derivative TECHNICAL FIELD
本发明涉及长效胸腺肽 1衍生物的制备和应用。 具体涉及聚乙二 醇修饰的胸腺肽 1衍生物。 技术背景 The invention relates to the preparation and application of a long-acting thymosin 1 derivative. In particular, it relates to polyethylene glycol-modified thymosin 1 derivatives. technical background
胸腺是体内的重要免疫器官, 在淋巴系统发育和维持免疫系统的 正常功能中起重要作用。 尤其在抗感染, 抗肿瘤, 自身免疫性疾病和 器官移植等作用中具有特殊的意义。 随着机体年齢老化, 胸腺渐萎縮 退化, 肿瘤, 感染, 免疫性疾病等发病率逐渐增高。 近年国外学者将 胸腺肽提纯为几种单独成分, 发现其中胸腺肽 1 的生物学活性比胸腺 肽混合物活性高 1 0— 1 0 0 0倍。胸腺肽 1属于胸腺肽第五组分, 具 有免疫调节作用, 在体外可以进致敏细胞生成淋巴因子, 具有免疫调 节作用, 在体外可以促进致敏细胞生成淋巴因子, a_ I F N , γ— I N F , I L - 2; 增强细胞因子高亲和力 I L一 2的表达; 调控骨 髓前体细胞和脾细胞的末端脱氧核酸转移酶 (TdT)活性; 增强骨髓前 体细胞的 Thyl和 Lytl, 2, 3, 的表达; 加速 NK细胞的生成, 促进 NK 细胞的活性; 通过增强辅助 T细胞的活性可以促进混合淋巴细胞的反 应; 拮抗胸腺细胞成熟过程中的凋亡。 体内应用结果也表明其在 T细 胞发育和功能重中具有重要意义; 可促进淋巴细胞分泌 I L一 2和 I L一 2的表达; 增强宿主的抗感染能力; 促进病毒清除; 增强免疫功 能; 有抗氧化, 抑制癌细胞生长等重要作用; 临床上常应用于治疗肿 瘤, 抗病毒及免疫缺陷病人的辅助治疗, 并可以作为疫苗增强剂使用。 Chien等 (Chien, R-N et al, Hepatology, 1998 ; 27 : 1383-138) 用胸 腺肽 1治疗慢性乙型肝炎患者, 其 HBV DNA血清清除率和 HBeAg阴转
率高于未用胸腺肽 1治疗的对照组,且病理学检査也发现治疗组炎症 及纤维生成情况均显著底于对照组。在治疗肝纤维化方面胸腺肽 al也 有独到之处。 目前发现对肝纤维化只有 IFN有一定疗效, 但其存在低 反应性和剂量限制等缺点, Sherman等(Sherman, . E. 等, Hepatology, 1998 ; 27 : 1128-35)将 IFN和胸腺肽 1合用, 治疗慢性丙型肝炎, 随 机分组, 双盲对照, 观察到合用组各项检测指标均优于单用 IFN组和 未处理对照组, 其中 HCV RNA清除率为此 37. 1%, 明显高于单用组工 FN 组的确 16. 2%。 胸腺肽 1 对肝癌也有治疗作用, Stefanini 等 (Stefanini G. F.等, Hepato卜 Gastroenterology, 1998 ; 45 : 209-15 ) 用它治疗了不下 12例肝癌患者, 发现其存活期显著延长。 The thymus is an important immune organ in the body and plays an important role in the development of the lymphatic system and in maintaining the normal functioning of the immune system. Especially in anti-infection, anti-tumor, autoimmune disease and organ transplantation, it has special significance. As the body ages, the thymus gradually shrinks and degenerates, and the incidence of tumors, infections, and immune diseases gradually increases. In recent years, foreign scholars have purified thymosin into several separate components and found that the biological activity of thymosin 1 is 10-1000 times higher than that of thymosin mixtures. Thymosin 1 belongs to the fifth component of thymosin and has immunomodulatory effects. It can enter sensitized cells in vitro to generate lymphokines, has immunomodulatory effects, and can promote sensitized cells to produce lymphokines in vitro. A_ IFN, γ-INF, IL- 2; Enhance the expression of cytokine high affinity IL-2; Regulate terminal deoxynucleotidyl transferase (TdT) activity of bone marrow precursor cells and spleen cells; Enhance the expression of Thyl and Lytl, 2, 3, of bone marrow precursor cells; Accelerate The generation of NK cells promotes the activity of NK cells; it can promote the response of mixed lymphocytes by enhancing the activity of helper T cells; and it can antagonize apoptosis during thymocyte maturation. The in vivo application results also show that it has important significance in the development and function of T cells; it can promote the expression of IL-2 and IL-2 secreted by lymphocytes; enhance the host's anti-infective ability; promote virus elimination; enhance immune function; It plays an important role in oxidizing and inhibiting the growth of cancer cells; it is often used clinically as an adjuvant treatment for tumors, antiviral and immunodeficiency patients, and can be used as a vaccine enhancer. Chien et al. (Chien, RN et al, Hepatology, 1998; 27: 1383-138) treated with thymosin 1 in patients with chronic hepatitis B with HBV DNA serum clearance and HBeAg negative conversion The rate was higher than that of the control group not treated with thymosin 1, and pathological examination also found that the inflammation and fibrogenesis of the treatment group were significantly lower than those of the control group. Thymosin al is also unique in treating liver fibrosis. Currently, only IFN has certain effects on liver fibrosis, but it has the disadvantages of low reactivity and dose limitation. Sherman et al. (Sherman,. E. et al., Hepatology, 1998; 27: 1128-35) combined IFN with thymosin 1 For the treatment of chronic hepatitis C, randomized grouping, double-blind control, it was observed that the detection indicators of the combined group were better than those of the IFN group and the untreated control group alone, in which the HCV RNA clearance rate was 37.1%, which was significantly higher than The FN group alone was indeed 16. 2%. Thymosin 1 also has a therapeutic effect on liver cancer. Stefanini et al. (Stefanini GF et al., Hepato Gastroenterology, 1998; 45: 209-15) used it to treat no less than 12 patients with liver cancer and found that their survival period was significantly prolonged.
近年国内外已有胸腺肽 1 的产品, 商品名 "日达仙", 人工化学 合成, 不含其他血清产物如白蛋白等。 临床应用治疗乙型肝炎, 丙型 肝炎, 免疫缺陷病毒感染以及一些肿瘤, 如黑色素瘤, 肺癌, 白血病, 磷状上皮细胞癌, 结肠癌等, 效果较为显著。但是胸腺肽 1本身的药 物动力学性质较差, 在体内逗留时间较短而被排除, 其半衰期为 2小 时。 动物实验证明不能完全发挥其应有的药效。 这就需要频繁注射胸 腺肽 1, 因此导致治疗的不方便和治疗成本升高, 同时因为价格昂贵普 通病人难以承受, 治疗质量下降。研究表明, 体内胸腺肽 1是由体内 前胸腺肽 1衍生而来, 前胸腺肽 1和胸腺肽 1具有相同的生活活性, 其差别就在于它们分子量的大小不同。 前胸腺肽 1 其体内活性可能持 久些。 因为其分子量较大排除时间要更长些。 因此, 解决上述问题的 方法之一, 就是研究开发长效的胸腺肽 1衍生物, 用化学修饰的方法 来延长胸腺肽 1在体内的停留时间和稳定性, 将会达到减少治疗成本, 提高治疗质量的目的。 In recent years, domestic and foreign products of thymosin 1 have been traded under the trade name "Ridaxian". They are artificially synthesized and do not contain other serum products such as albumin. The clinical application is effective in treating hepatitis B, hepatitis C, immunodeficiency virus infection and some tumors, such as melanoma, lung cancer, leukemia, phosphorous epithelial cell carcinoma, colon cancer, etc. However, the pharmacokinetic properties of thymosin 1 itself is poor, and it is excluded because of its short residence time in the body, and its half-life is 2 hours. Animal experiments have proved that it cannot fully exert its due effect. This requires frequent injections of thymosin 1, which leads to inconvenience of treatment and increased treatment costs. At the same time, because of the high cost of ordinary patients, it is difficult to afford and the quality of treatment is reduced. Studies have shown that thymosin 1 in vivo is derived from prothymosin 1 in vivo, and prothymosin 1 and thymosin 1 have the same life activity, and the difference lies in their molecular weights. Prothymosin 1 may last longer in vivo. Because of its larger molecular weight, the elimination time is longer. Therefore, one of the methods to solve the above problems is to research and develop long-acting thymosin 1 derivatives, and use chemical modification to extend the residence time and stability of thymosin 1 in the body, which will reduce the cost of treatment and improve the quality of treatment. purpose.
借助于用聚乙二醇修饰蛋白成功的先例, 用同样的技术来修饰胸 腺肽 1衍生物将会改进其药代性质, 从而提高药效。 W0 03/037272公
开了有关用高聚物来修饰胸腺肽 1本身的方法及应用。 具体地说, 用 分子量为 20, 000左右的聚乙二醇来修饰胸腺肽 1本身所含五个氨基 之一或同时修饰其中多个氨基。正是因为胸腺肽 1有五个氨基(一个 N端氨基, 四个赖氨酸側链氨基), 再加上其化学修饰是建立这五个氨 基基础上的, 所以这种修饰方法的选择专一性只有通过复杂的化学合 成及保护机制才能实现, 因而, 对其进行大规模生产的成本高, 达到 商业化的可能性很小。 这需要寻找在工业上更易于制备的修饰性胸腺 肽 1衍生物和制备方法。 With the successful precedent of modifying proteins with polyethylene glycol, using the same technique to modify thymosin 1 derivatives will improve their pharmacokinetic properties and thus their efficacy. W0 03/037272 male Methods and applications for modifying thymosin 1 itself with polymers have been developed. Specifically, polyethylene glycol having a molecular weight of about 20,000 is used to modify one or five amino groups contained in thymosin 1 itself. It is precisely because thymosin 1 has five amino groups (one N-terminal amino group, four lysine side chain amino groups), and its chemical modification is based on these five amino groups, so the choice of this modification method is unique The property can only be achieved through complex chemical synthesis and protection mechanisms. Therefore, the cost of mass production is high and the possibility of commercialization is small. This requires finding a modified thymosin 1 derivative and a preparation method that are easier to prepare industrially.
鉴于上述观察和经过研究比较发现, 借助于反应专一的的化学修饰 方法,用聚乙二醇修饰胸腺肽 1的 C端来模拟体内前胸腺肽 1和胸腺肽 1的作用,这样会最小程度地减少或影响胸腺肽 1本身的生物活性的可 能性。 为此,只有用胸腺肽 1的衍生物而不是胸腺肽 1本身作为修饰的 前体。 就是说, 在胸腺肽 1的 c端加上修饰反应的连接点或把其序列 内的赖氨酸, 用精氨酸替换从而促成修饰反应的专一性。 这些修饰所 用前体的制备可以用本领域公知的任何技术进行完成, 优选用化学方 法或用重组 DNA方法。 In view of the above observations and comparisons, it is found that by using a reaction-specific chemical modification method, the C-terminus of thymosin 1 is modified with polyethylene glycol to simulate the effects of prothymosin 1 and thymosin 1 in vivo, which will minimize or reduce Possibility to affect the biological activity of thymosin 1 itself. For this reason, only derivatives of thymosin 1 were used as modified precursors, not thymosin 1 itself. That is, the junction of the modification reaction is added to the c-terminus of thymosin 1 or the lysine in the sequence is replaced with arginine to promote the specificity of the modification reaction. The preparation of the precursors for these modifications can be accomplished by any technique known in the art, preferably by chemical methods or by recombinant DNA methods.
本发明目的在于提供一类用聚乙二醇修饰胸腺肽 1衍生物 C端而产 生的一类新化合物, 它们的制备方法具有反应专一生产工艺简洁的特 点。 它们可以作为注射制剂或其它制剂, 用于免疫功能低下的疾病的 治疗及辅助治疗。 发明内容 The purpose of the present invention is to provide a new class of compounds produced by modifying the C-terminus of a thymosin 1 derivative with polyethylene glycol, and their preparation method has the characteristics of simple reaction-specific production process. They can be used as injection preparations or other preparations for the treatment and adjuvant treatment of immunocompromised diseases. Summary of the invention
为了克服现有技术的不足之处, 本发明涉及一种用聚乙二醇修饰的 胸腺肽 1衍生物及其可药用盐, 该聚乙二醇在胸腺肽 1衍生物的 C端 进行修饰。 In order to overcome the shortcomings of the prior art, the present invention relates to a thymosin 1 derivative modified with polyethylene glycol and a pharmaceutically acceptable salt thereof. The polyethylene glycol is modified at the C-terminus of the thymosin 1 derivative.
所述的聚乙二醇修饰的胸腺肽 1衍生物及其药用盐, 其特征在于
胸腺肽 1衍生物的 c端可以连接在聚乙二醇的一个末端或其两个末端。 另外胸腺肽 1衍生物的 c端可以连接在聚乙二醇的一个末端。聚乙二 醇的分子量范围为 5, 000-80, 000, 优选为 8, 000- 60, 000, 更优选为 10, 000-50, 000, 最优选的为 20, 000-40, 000。 The polyethylene glycol modified thymosin 1 derivative and a pharmaceutically acceptable salt thereof are characterized in that: The c-terminus of the thymosin 1 derivative may be attached to one or both ends of the polyethylene glycol. In addition, the c-terminus of the thymosin 1 derivative may be connected to one end of the polyethylene glycol. The molecular weight of polyethylene glycol ranges from 5,000 to 80,000, preferably from 8,000 to 60,000, more preferably from 10,000 to 50,000, and most preferably from 20,000 to 40,000.
本发明的聚乙二醇修饰的胸腺肽 1衍生物及其药用盐, 其特征在 于具有下式结构: The polyethylene glycol-modified thymosin 1 derivative and the pharmaceutically acceptable salt thereof are characterized in that they have the following structure:
A— S er— A sp— A la— A la— V al— A sp— T hr— S er— S er— Glu— He— Thr~Thr-B-Asp- Leu— C— Glu— D— E— Glu—Val -Val-Glu-Glu-Ala-Glu-Asn-X- Y-Z A— S er— A sp— A la— A la— V al— A sp— T hr— S er— S er— Glu— He— Thr ~ Thr-B-Asp- Leu— C— Glu— D— E — Glu—Val -Val-Glu-Glu-Ala-Glu-Asn-X- YZ
其中 A是 H或 Ac; Where A is H or Ac;
B, C, D, E是 Lys或 Arg, B, C, D, E are Lys or Arg,
X选自(Gly)n, (Gly-Ser)n, ( G ly- G ly- S er) n, (S er— Gly— Gly) n, X is selected from (Gly) n, (Gly-Ser) n, (G ly- G ly-S er) n, (S er— Gly— Gly) n,
n = 0 - 1 0 n = 0-1 0
Y是 C ys或高 Cys或 Lys或 Arg或 His, 并且 Y是聚乙二醇修饰 的残基; Y is Cys or high Cys or Lys or Arg or His, and Y is a polyethylene glycol modified residue;
Z是 OH或 NH2。 Z is OH or NH2.
所示结构为 SEQ ID NO: 1-12所述的序列。 The structure shown is the sequence described in SEQ ID NOs: 1-12.
所示的聚乙二醇修饰的胸腺肽 1衍生物及其药用盐, 其中 Polyethylene glycol modified thymosin 1 derivatives and pharmaceutically acceptable salts thereof,
A为 Ac; A is Ac;
B, C, D, E是 Lys; B, C, D, E are Lys;
n= 0; n = 0;
Y是 PEG40K修饰的 Cys。 Y is PEG40K modified Cys.
或者, Or,
A为 Ac; A is Ac;
B, C, D, E是 Arg;
n = 0; B, C, D, E are Arg; n = 0;
Y是 PEG40K修饰的 Lys。 Y is PEG40K modified Lys.
本发明还涉及聚乙二醇修饰的胸腺肽 1衍生物及其药用盐的制备 方法, 其中包括以下步骤: The invention also relates to a method for preparing a polyethylene glycol modified thymosin 1 derivative and a pharmaceutically acceptable salt thereof, which include the following steps:
用化学合成方法或基因工程法制备供修饰的前体胸腺肽 1衍生 物; 前体胸腺肽 1衍生物 c端与聚乙二醇进行衍生。 A chemical synthesis method or a genetic engineering method is used to prepare a precursor precursor thymosin 1 derivative; the precursor thymosin 1 derivative is c-terminally derivatized with polyethylene glycol.
该方法包括以下步骤: The method includes the following steps:
( 1 ) 制备或获得 ) 含麦克尔加成反应受体的聚乙二醇与含麦 克尔加成反应给体半胱氨酸的胸腺肽 1衍生物; 或 (b) 含麦克尔加 成反应给体的聚乙二醇与麦克尔加成反应受体的胸腺肽 1 衍生物; (2)进行麦克尔加成反应, 用其受体或给体衍生的聚乙二醇和胸腺肽 1衍生物进行反应, 形成聚乙二醇修饰的胸腺肽 1衍生物。 反应类型解释 (1) Preparation or acquisition) Polyethylene glycol containing a Michael's addition reaction receptor and a thymosin 1 derivative containing a Michael's addition donor cysteine; or (b) Michael's addition reaction containing The polyethylene glycol of the donor reacts with the thymosin 1 derivative of the Michael addition reaction receptor; (2) carrying out the Michael addition reaction, and reacting with its receptor or the donor-derived polyethylene glycol and the thymosin 1 derivative, A polyethylene glycol modified thymosin 1 derivative is formed. Response type explanation
在该方法中, 前体胸腺肽 1衍生物 c端与聚乙二醇进行衍生是通 过前体胸腺肽 1衍生物 c端的半胱氨酸、 高半胱氨酸、 赖氨酸、 精氨 酸或组氨酸作为聚乙二醇化学修饰点, 与聚乙二醇进行衍生。 In this method, the derivation of the c-terminus of the precursor thymosin 1 derivative with polyethylene glycol is through the cysteine, homocysteine, lysine, arginine or group of the c-terminal of the precursor thymosin 1 derivative. Amino acid is used as the modification point of polyethylene glycol, and is derived from polyethylene glycol.
通过反应专一的修饰反应而形成共价连接应选自以下的方法: The formation of covalent linkages through reaction-specific modification reactions should be selected from the following methods:
〈1>具有形成不对称双硫键能力的聚乙二醇或胸腺肽. 1衍生物反应; <2>具有羧基活化的聚乙二醇衍生物与含 C端赖氨酸或组氨酸的胸腺肽
1衍生物共价结合反应; <1> Polyethylene glycol or thymosin having the ability to form asymmetric disulfide bonds. 1 derivative reaction; <2> Polyethylene glycol derivatives having carboxyl activation and thymosin containing C-terminal lysine or histidine 1 Covalent binding reaction of derivatives;
〈3>具有醛基的聚乙二醇衍生物与含 C端赖氨酸的胸腺肽 1衍生物通过 还原胺化反应; <3> A polyethylene glycol derivative having an aldehyde group and a thymosin 1 derivative containing a C-terminal lysine undergo a reductive amination reaction;
〈4>具有异氰或异硫氰基团的聚乙二醇衍生物含 C 端赖氨酸的胸腺肽 <4> Polyethylene glycol derivative with isocyanide or isothiocyanate group Thymosin containing C-terminal lysine
1衍生物通过与氨基加成反应,形成脲或硫脲连接; 1 The derivative forms an urea or thiourea linkage through an addition reaction with an amino group;
〈5〉具有羰基活化的聚乙二醇与含 C端赖氨酸或组氨酸反应形成氨酯连 接。 <5> Polyethylene glycol with carbonyl activation reacts with C-terminal lysine or histidine to form a urethane linkage.
〈6>具有 2-酮基-苯乙醛的聚乙二醇衍生物与含 C 端精氨酸的胸腺肽 1衍生物反应; <6> A polyethylene glycol derivative having 2-keto-phenylacetaldehyde reacts with a C-terminal arginine-containing thymosin 1 derivative;
〈7>具有含巯基的胸腺肽 1衍生物与含易离去基因的聚乙二醇发生亲 核取代反应。 <7> A thiopeptide-containing thymosin 1 derivative undergoes a nucleophilic substitution reaction with polyethylene glycol containing an easily detachable gene.
行麦克尔加成反应为含活化的双硫键的聚乙二醇与含半胱氨酸的 胸腺肽 1衍生物反应。 The Michael reaction is performed by reacting polyethylene glycol containing an activated disulfide bond with a cysteine-containing thymosin 1 derivative.
所述的供聚乙二醇修饰的胸腺肽 1衍生物及其药用盐可用于治疗 与免疫系统调节物应答相关的疾病, 对免疫系统调节物应答相关的疾 病包括流行性感冒, 慢性肝炎, 免疫功能低下, 肿瘤病毒感染。 反应类型解释 The polyethylene glycol-modified thymosin 1 derivative and a pharmaceutically acceptable salt thereof can be used for treating diseases related to immune system regulators. The diseases related to immune system regulators include influenza, chronic hepatitis, and immunity. Low function, tumor virus infection. Response type explanation
换言之, 本发明的内容提供了一种用聚乙二醇修饰的胸腺肽 1 衍生物及其可药用盐, 该聚乙二醇在胸腺肽 1 衍生物的 C端进行修 饰, 优选具有下式结构: In other words, the content of the present invention provides a thymosin 1 derivative modified with a polyethylene glycol and a pharmaceutically acceptable salt thereof. The polyethylene glycol is modified at the C-terminus of the thymosin 1 derivative, and preferably has the following structure:
5 10 A— S er— Asp— Ala— Ala— Val— Asp— Thr— S er— S er 5 10 A— S er— Asp— Ala— Ala— Val— Asp— Thr— S er— S er
15 20 - Glu— 11 Θ— Thr— Thr-B-Asp - L eu— C - Glu- - E - Glu 15 20-Glu— 11 Θ— Thr— Thr-B-Asp-L eu— C-Glu--E-Glu
25 30 - Val-Val-Glu-Glu-Ala-Glu- Asn-X- Y-Z 其中 A是 H或 Ac; 25 30-Val-Val-Glu-Glu-Ala-Glu- Asn-X- Y-Z where A is H or Ac;
B, C, D, E是 Lys或 Arg, B, C, D, E are Lys or Arg,
X选自(Gly)n, (Gly-Ser)n, ( G ly- G ly- S er) n, ( S er— Gly— Gly) n, X is selected from (Gly) n, (Gly-Ser) n, (G ly- G ly- S er) n, (S er— Gly— Gly) n,
n = 0 _ 1 0 n = 0 _ 1 0
Y是 Cys或高 Cys或 Lys或 Arg或 His, 并且 Y是聚乙二醇修饰的 残基; Y is Cys or high Cys or Lys or Arg or His, and Y is a polyethylene glycol modified residue;
Z是 OH或 NH2, Z is OH or NH2,
如果 Y是 Cys或高 Cys则聚乙二醇是用硫醚键或双硫键而共价结合; 如果 Y是 Lys, 则聚乙二醇是用酰胺键或二级胺而共价结合; 如果 Y 是 His, 则聚乙二醇是与组氨酸的咪唑环山的氮形成酰咪唑而共价结 合; 如果 Y是 Arg, 则聚乙二醇是通过型成杂环而联接的。 聚乙二醇修饰的胸腺肽 1衍生物及其可药用盐优选为 SEQ ID NO: 1-12所述的序列, 最佳的选择性的氨基酸或基团具有以下定义- A为 Ac, B, C, D, E是 Lys, n= 0 , Y是 PEG40K修饰的 Cys;
或 If Y is Cys or high Cys, polyethylene glycol is covalently bonded using thioether or disulfide bonds; if Y is Lys, polyethylene glycol is covalently bonded using amide or secondary amines; if Y is His, then the polyethylene glycol is covalently bonded to the nitrogen of the imidazole ring of the histidine to form an acylimidazole; if Y is Arg, the polyethylene glycol is linked by forming a heterocycle. The polyethylene glycol modified thymosin 1 derivative and a pharmaceutically acceptable salt thereof are preferably the sequences described in SEQ ID NOs: 1-12, and the best selective amino acid or group has the following definitions-A is Ac, B, C, D, E are Lys, n = 0, Y is Cys modified by PEG40K; or
A为 Ac, B, C, D, E是 Arg, n = 0 , Y是 PEG40K修饰的 Lys。 本发明中所用的聚乙二醇的分子量范围为 5, 000-80, 000。其中以分 子量范围为 8, 000-60, 000 为佳, 以 10, 000- 50, 000 更好, 以 20, 000-40, 000最好。 A is Ac, B, C, D, E is Arg, n = 0, Y is PEG40K modified Lys. The molecular weight of the polyethylene glycol used in the present invention ranges from 5,000 to 80,000. Among them, the molecular weight range is preferably 8,000 to 60,000, more preferably 10,000 to 50,000, and most preferably 20,000 to 40,000.
本发明中胸腺肽 1衍生物的 c端可以连接在聚乙二醇的一个末端或 其两个末端, 以连接在一个末端的修饰方法为佳。 In the present invention, the c-terminus of the thymosin 1 derivative may be connected to one end or both ends of polyethylene glycol, and a modification method in which the c-terminus is connected to one end is preferred.
本发明提供的聚乙二醇修饰的胸腺肽 1衍生物是两性化合物, 所 属领域技术人员利用公知技术可使用本领域常用的酸性或碱性化合物 与之反应成盐。 The polyethylene glycol-modified thymosin 1 derivative provided by the present invention is an amphoteric compound, and those skilled in the art can use known techniques to react with the acidic or basic compounds commonly used in the art to form salts with them.
通常采用的形成酸加成盐的酸为: 盐酸, 氢溴酸, 氢碘酸, 碳酸, 磷酸, 对甲苯磺酸, 甲磺酸, 苯酸, 对溴苯基碳酸, 碳酸, 琥珀酸, 柠酸, 苯甲酸, 乙酸盐, 氟乙酸等。 这类盐的例子包括硫酸盐, 焦硫 酸盐, 硫酸氢盐, 亚硫酸盐, 亚硫酸氢盐, 磷酸盐, 磷酸氢盐, 磷酸 二氢盐, 偏磷酸盐, 焦磷酸盐, 盐酸盐, 溴化物, 碘化物, 乙酸盐, 三氟乙酸盐, 丙酸盐, 癸酸盐, 辛酸盐, 丙烯酸盐, 甲酸盐, 异丁酸 盐, 已酸盐, 庚酸盐, 丙炔酸盐, 苯酸盐, 丙二酸盐, 丁二酸盐, 辛 二酸盐,癸二酸盐,富马酸盐,马来酸盐,丁炔 -1, 4-二酸盐,乙炔 -1,6- 二酸盐, 苯甲酸盐, 氯苯甲酸盐, 甲基苯甲酸盐, 二硝基苯甲酸盐, 羟基苯甲酸盐, 甲氧基苯甲酸盐, 苯已酸盐, 苯丙酸盐, 苯丁酸盐, 柠檬酸盐, 乳酸盐, r-羟基丁酸盐, 甘醇酸盐, 酒石酸盐, 甲磺酸盐, 丙磺酸盐, 萘- 1-磺酸盐, 萘- 2-磺酸盐, 扁桃酸盐等, 优选的酸加成 盐是聚乙二醇修饰的胸腺肽 1 衍生物与盐酸盐, 氢溴酸, 乙酸, 三氟 乙酸, 尤其是乙酸形成的盐。 Acid addition salts commonly used are: hydrochloric acid, hydrobromic acid, hydroiodic acid, carbonic acid, phosphoric acid, p-toluenesulfonic acid, methanesulfonic acid, benzoic acid, p-bromophenylcarbonic acid, carbonic acid, succinic acid, lemon Acid, benzoic acid, acetate, fluoroacetic acid, etc. Examples of such salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, hydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, hydrochloride, Bromide, iodide, acetate, trifluoroacetate, propionate, caprate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propyne Acid salt, benzoate salt, malonate salt, succinate salt, suberate salt, sebacate salt, fumarate salt, maleate salt, butyne-1, 4-diacetate, acetylene- 1,6-Diacid, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, benzoate Acid salt, phenylpropionate, phenylbutyrate, citrate, lactate, r-hydroxybutyrate, glycolate, tartrate, mesylate, propanesulfonate, naphthalene-1- Sulfonate, naphthalene-2 sulfonate, mandelate, etc. The preferred acid addition salt is a polyethylene glycol modified thymosin 1 derivative with hydrochloride, hydrobromic acid, acetic acid, Trifluoroacetic acid, especially salts formed by acetic acid.
碱性物质也可以与聚乙二醇修饰的胸腺肽 1 反应形成盐, 这些 碱性物质包括铵, 碱金属或碱土金属的氢氧化物, 以及碳酸盐, 碳酸
氢盐等。 典型的有氢氧化钠, 氢氧化钾, 氢氧化铵, 碳酸钠, 碳酸钾 等。 Basic substances can also react with polyethylene glycol-modified thymosin 1 to form salts. These basic substances include ammonium, alkali metal or alkaline earth metal hydroxides, and carbonates, carbonates Hydrogen salt, etc. Typical are sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate and the like.
本发明所述的胸腺肽 1衍生物的制备方法可使用本领域公知的任 何方法进行制备, 此制备方法优选为固相和液相化学合成法。 固相方 法包括使用 Fmoc或 Boc保护的氨基酸, 用多肽合成法或手工合成法来 进行氨基酸序列的合成, 再经切除, 经高效液相 (HPLC) 分离纯化, 冻干。所得多肽为进行聚乙二醇修饰的中间体前体。本发明的胸腺肽 1 衍生物优选具有 SEQ ID NO : 1-24的氨基酸序列。 The method for preparing the thymosin 1 derivative according to the present invention can be prepared using any method known in the art, and the preparation method is preferably a solid-phase and liquid-phase chemical synthesis method. Solid-phase methods include the use of Fmoc or Boc-protected amino acids, synthesis of amino acid sequences by peptide synthesis or manual synthesis, and then excision, separation and purification by high-performance liquid phase (HPLC), and lyophilization. The obtained polypeptide is an intermediate precursor modified with polyethylene glycol. The thymosin 1 derivative of the present invention preferably has an amino acid sequence of SEQ ID NO: 1-24.
本发明所述的胸腺肽 1衍生物也可用基因工程法制备, 其步骤包 括; The thymosin 1 derivative according to the present invention can also be prepared by genetic engineering, and the steps include:
a. 按胸腺肽 1衍生物的氨基酸序列合成基因片段; a. Synthesis of gene fragments based on the amino acid sequence of a thymosin 1 derivative;
b . 基因片段经连接, 质粒构建, 受固体的培养, 转化, 克隆得到 菌株 5 b. Gene fragments are ligated, plasmid constructed, solid cultured, transformed and cloned to obtain strain 5
C . 菌株经发酵, 破壁, 抽提得到色涵体; C. Strains are fermented, broken, and extracted to obtain color culverts;
d. 色涵体裂解, 分离得粗品, 给高效液相仪 (HPLC) 分离纯化, 最后经冻干所得为进行聚乙二醇修饰的中间体前体。 d. The color culverts are lysed and separated into crude products, which are separated and purified by high performance liquid chromatography (HPLC), and finally lyophilized to obtain polyethylene glycol modified intermediate precursors.
本发明所述的用聚乙二醇修饰胸腺肽 1衍生物制备方法,其中包括: The method for preparing a thymosin 1 derivative modified with polyethylene glycol according to the present invention includes:
1、 制备或获得 (a) 含麦克尔加成反应受体的聚乙二醇与含麦克尔 加成反应给体半胱氨酸的胸腺肽 1衍生物; 或 (b) 含麦克尔加成反 应给体的聚乙二醇与麦克尔加成反应受体的胸腺肽 1衍生物; 1. Preparation or obtaining (a) a polyethylene glycol containing a Michael addition reaction receptor and a thymosin 1 derivative containing a Michael addition reaction donor cysteine; or (b) a Michael addition reaction A thymosin 1 derivative of a donor polyethylene glycol and a Michael addition reaction receptor;
2、进行麦克尔加成反应,用其受体或给体衍生的聚乙二醇和胸腺肽 1衍生物进行反应, 形成聚乙二醇修饰的胸腺肽 1衍生物, 如含马 来酰亚胺衍生的聚乙二醇与含半脱氨酸的胸腺肽 1 衍生物加成反应而 形成产品。 2. Carry out a Michael addition reaction, using its receptor or donor-derived polyethylene glycol and a thymosin 1 derivative to form a polyethylene glycol modified thymosin 1 derivative, such as a maleimide-containing derivative Polyethylene glycol reacts with a thymine-containing thymosin 1 derivative to form a product.
其中该方法优选通过以下步骤进行制备: The method is preferably prepared by the following steps:
( 1 ) 具有形成不对称双硫键能力的聚乙二醇或胸腺肽 1衍生物,
如含活化的双硫键的聚乙二醇与含半胱氨酸的胸腺肽 1 衍生物反应而 形成产品; (1) a polyethylene glycol or thymosin 1 derivative having the ability to form an asymmetric disulfide bond, For example, polyethylene glycol containing activated disulfide bonds reacts with cysteine-containing thymosin 1 derivatives to form products;
(2)具有羧基活化的聚乙二醇衍生物与含 C端赖氨酸或组氨酸的胸 腺肽 1 衍生物反应形成酰胺键而共价结合形成产品, 此时胸腺肽 1 内部所含的四个赖氨酸应用精氨酸替换; (2) A carboxyl group-activated polyethylene glycol derivative reacts with a C-terminal lysine or histidine-containing thymosin 1 derivative to form an amide bond and covalently bind to form a product. At this time, the four Replace lysine with arginine;
(3) 具有醛基的聚乙二醇衍生物与含 C端赖氨酸的胸腺肽 al衍生 物通过还原胺化反应形成产品, 此时胸腺肽 1 内部所含的四个赖氨酸 因为精氨酸替换。 (3) A polyethylene glycol derivative with an aldehyde group and a thymosin al derivative containing a C-terminal lysine are formed by a reductive amination reaction. At this time, the four lysines contained in thymosin 1 are arginine. replace.
(4)具有异氰或异硫氰基团的聚乙二醇衍生物含 C 端赖氨酸的胸腺 肽 1衍生物通过与氨基加成而形成产物,此时胸腺肽 1内部的四个赖 氨酸应为精氨所替换; (4) Polyethylene glycol derivative with isocyanide or isothiocyanate group. Thymosin 1 derivative containing C-terminal lysine is added to the amino group to form a product. At this time, the four lysines inside thymosin 1 Should be replaced by arginine;
(5)具有 2-酮基-苯乙醛的聚乙二醇衍生物与含 C端精氨酸的胸腺肽 (5) Polyethylene glycol derivative with 2-keto-phenylacetaldehyde and thymosin with C-terminal arginine
1衍生物反应形成的产品; 1 products formed by the reaction of derivatives;
(6) 具有含巯基的胸腺肽 1衍生物与易离去基因 (如 I, Br, CD 的聚乙二醇发生亲核取代反应形成的产品。 本发明的化合物是用聚乙二醇修饰的胸腺肽 1 衍生物, 亦包括为 进行聚乙二醇修饰而产生的新中间体, 是适用于治疗对免疫系统调节 物应答的各种疾病和指怔的免疫系统调节物。 本发明的增强免疫化合 物可用于丧失免疫和免疫抑制的患者重组免疫功能, 并能用于治疗免 疫缺陷疾病, 如流行性感冒, 慢性肝炎, 免疫功能低下, 肿瘤病毒感 染等疾病, 特别是对于治疗肿瘤, 病毒感染的病人可作辅助治疗。 (6) A product formed by a nucleophilic substitution reaction between a thiol-containing thymosin 1 derivative and a readily detachable gene (eg, I, Br, CD). The compound of the present invention is a thymosin modified with polyethylene glycol 1 Derivatives, which also include new intermediates produced for the modification of polyethylene glycol, are immune system modulators suitable for treating various diseases and fingertips that respond to immune system modulators. The immune-enhancing compounds of the present invention are useful It can be used to reconstitute the immune function in patients who have lost immunity and immunosuppression, and can be used to treat immunodeficiency diseases, such as influenza, chronic hepatitis, low immune function, tumor virus infection and other diseases, especially for the treatment of tumor, virus infection patients. For adjuvant therapy.
本发明提供了一类新的聚乙二醇修饰的胸腺肽 1衍生物, 利用聚乙 二醇修饰胸腺肽 1衍生物的 c端来模拟前胸腺肽 1。经药效药代实验证 明, 聚乙二醇修饰的胸腺肽 1衍生物完全具有胸腺肽 1的药效作用。 胸腺肽 1如果用皮下注射其体内半衰期只有近 2小时, 但是用本发明
中制备的聚乙二醇修饰的胸腺肽 1衍生物在体内半衰期延长至此 2-3 天。 其作免疫增强辅助治疗的用量可用常规的剂量滴定法测定, 其用 量可确定为 1- lOOmg/公斤体重 /周的范围内, 其中以 5-50mg/公斤体重 /周为佳, 以 20mg/公斤体重 /周为最优。 用聚乙二醇修饰的胸腺肽 1 衍生物可用合适药用液体如水制针剂而使用。 其药代性质得到了很大 的改善, 从而真正产生了长效胸腺肽 1衍生物。 . 与 N端聚乙二醇修饰的胸腺肽 1衍生物比较发现, 本发明所提供 的 C端聚乙二醇修饰的胸腺肽 1衍生物以及制备方法易于制备, 克服 了 N端聚乙二醇修饰的胸腺肽 1衍生物在工业生产中不易大规模产业 生产、 难于获得的问题, 且治疗活性更强。 附图说明 The present invention provides a new class of polyethylene glycol modified thymosin 1 derivatives. The c-terminus of thymosin 1 derivatives is modified by polyethylene glycol to simulate prothymosin 1. According to pharmacodynamic experiments, PEG-modified thymosin 1 derivatives have pharmacological effects of thymosin 1. Thymosin 1 has a half-life of only 2 hours in vivo if it is injected subcutaneously, but using the present invention The in vivo half-life of the polyethylene glycol modified thymosin 1 derivative prepared in the prolonged period was 2-3 days. The dosage for immune booster adjuvant therapy can be determined by conventional dose titration. The dosage can be determined within the range of 1-100 mg / kg body weight / week, of which 5-50 mg / kg body weight / week is preferred, and 20 mg / kg body weight / Week is optimal. The thymosin 1 derivative modified with polyethylene glycol can be used in a suitable pharmaceutical liquid such as an aqueous injection. Its pharmacokinetic properties have been greatly improved, thus truly producing long-acting thymosin 1 derivatives. Compared with the N-terminal polyethylene glycol modified thymosin 1 derivative, it was found that the C-terminal polyethylene glycol modified thymosin 1 derivative and the preparation method provided by the present invention are easy to prepare and overcome the N-terminal polyethylene glycol modified In industrial production, thymosin 1 derivatives are not easy to produce on a large scale, are difficult to obtain, and have stronger therapeutic activity. BRIEF DESCRIPTION OF THE DRAWINGS
图 1为受试品标准 ELISA校正曲线。 Figure 1 shows the standard ELISA calibration curve of the test product.
图 2为猕猴 sc PEG-TA1 (64 μ g. kg -1 ) 后血清抗原浓度一时间曲 线。 具体实施方式 Figure 2 is a curve of serum antigen concentration versus time for macaque sc PEG-TA1 (64 μg. Kg -1). detailed description
下面实施是对本发明的进一步阐述, 而不是对发明的限制。 The following implementation is a further explanation of the present invention, but not a limitation of the invention.
实施例一: Embodiment one:
Ac— S er— A sp— A la— A la— Val ~ As― Thr― Ser― Ser― Glu ― lie― Thr― Thr― Lys― Asp— L eu— Lys— G lu一 Lys一 Lys一 Glu— Val - Val - G lu - G lu - A la- G lu - A sn— Glu— Cys— PEG40K 的 制备 步骤 1 :固相化学合成法制备供聚乙二醇修饰的前体—— C端半胱氨 酸胸腺肽 1衍生物:
N2005/000452 Ac — Ser — A sp — A la — A la — Val ~ As-Thr-Ser-Ser-Glu-lie-Thr-Thr-Lys-Asp-Leu-Lys-G lu-Lys-Lys-Glu- Val-Val-G lu-G lu-A la- G lu-A sn— Glu— Cys— PEG40K Preparation Step 1: Preparation of polyethylene glycol modified precursor—C-terminal cysteine by solid-phase chemical synthesis Thymosin 1 derivative: N2005 / 000452
( 1 ) 所采用的氨基酸单 (1) the amino acid list used
Fmoc ~ Ala― OH Fmoc― Lys (Boc) 一 OH Fmoc ~ Ala― OH Fmoc― Lys (Boc)-OH
Fmoc— Asn (Trt)—OH Fmoc一 Ser (tBu)― OH Fmoc— Asn (Trt) —OH Fmoc- Ser (tBu) ― OH
Fmoc— Asp (OtBu)—OH Fmoc— Thr (tBu)—OH Fmoc— Asp (OtBu) —OH Fmoc— Thr (tBu) —OH
Fmoc— Cys (Trt)—OH Fmoc― Val― OH Fmoc— Cys (Trt) —OH Fmoc― Val― OH
Fmoc— Glu (OtBu)—OH Fmoc—— He—— OH Fmoc— Glu (OtBu) —OH Fmoc—— He—— OH
Fmoc—— Leu—— OH 上式中缩写表示: Fmoc—— Leu—— OH The abbreviation in the above formula means:
Fmoc: 9― 基甲氧幾基本 (9― f luorenylraethoxycarbonyl ) Boc- 叔丁氧幾基 (tert一 butyloxycarbonyl ) Fmoc: 9- f luorenylraethoxycarbonyl Boc- tert-butyloxycarbonyl
Trt: 三苯甲基 (trityl ) Trt: trityl
OtBu: 叔丁基 OtBu: tert-butyl
tBu: 叔丁基 (tert— butyl ) tBu: tert-butyl
(2) 合成所采用的设备及试剂 (2) Equipment and reagents used in synthesis
仪器: 多肽序列的合成釆用手工方法进行, 反应器具均为专制。 试剂: N, N—二甲基甲酰胺 (DMF), 二氯甲垸 (DCM), 六氢吡啶, 异丙醇,甲醇,二异丙基,碳二亚胺(N, N— Di i sopropy 1 carbodimi de ) , 1一羟基苯并三唑 (HOBt) Apparatus: The synthesis of the peptide sequence is carried out by manual methods, and the reaction apparatuses are all proprietary. Reagents: N, N-dimethylformamide (DMF), dichloroformamidine (DCM), hexahydropyridine, isopropanol, methanol, diisopropyl, carbodiimide (N, N-Di i sopropy 1 carbodimi de), 1-hydroxybenzotriazole (HOBt)
( 3) 操作 (3) Operation
于底部装有烧结玻璃过滤器和颈部装有机械搅拌器的肽合成烧瓶中 加入 10g接有 Fmoc— Cys (Trt)一 0H的王树脂(Fmoc— Cys (Trt )― OH— Wang resin) 0. 6mMol/g树脂, 6讓 ol )。 用 150ml , N—二甲基 甲酰胺(DMF)洗涤此树脂。 用 20%的六氢吡啶的 DMF溶液(300ml )分
二次每次各 10分钟来处理树脂, 以去除氨基保护基团 FmoC。 再用 DMF 450ml分三次洗涤树脂, 抽干。 以溶于 150ml DMF中的 15匪 ol Fmoc— Asn (Trt)—OH (8. 95g), 15讓 ol 1—羟基苯并三唑水和物 (2. 3g) 和 15醒 ol N, N—二异丙基碳化二亚胺(2. 3½1 )与去除了氨基保护基 因的树脂反应二小时形成 Fmoc— Asn (Trt )— Cys (Trt) 王树脂。 茚 三酮试验应显阴性。 然后, 按照下面的步骤继续进行固相合成。 其中, 依此将一种氨基酸连结于树脂上增长的肽链上 (另有说明)。 除外, 每 次洗涤用 20体积溶剂或试剂。 除另有说明外, 所有氨基酸的衍生物均 为 L构型。 Into a peptide synthesis flask equipped with a sintered glass filter at the bottom and a mechanical stirrer at the neck, 10 g of Fmoc-Cys (Trt)-0H king resin (Fmoc-Cys (Trt)-OH-Wang resin) was added. 0 6 mMol / g resin, 6 ng). This resin was washed with 150 ml of N-dimethylformamide (DMF). Use 20% hexahydropyridine in DMF solution (300ml) The resin was treated twice for 10 minutes each time to remove the amino protecting group Fmo C. The resin was washed three times with 450 ml of DMF and drained. 15 mol of Fmoc—Asn (Trt) —OH (8. 95 g), 15 mol of 1-hydroxybenzotriazole water compound (2.3 g) and 15 ol of N, N— Diisopropylcarbodiimide (2. 3½1) reacts with the resin from which the amino-protected gene has been removed for two hours to form Fmoc-Asn (Trt) -Cys (Trt) king resin. The ninhydrin test should be negative. Then, follow the steps below to proceed with solid phase synthesis. Among them, an amino acid is linked to the growing peptide chain on the resin (explained otherwise). Except, 20 volumes of solvent or reagents are used for each wash. Unless otherwise stated, all amino acid derivatives are in the L configuration.
1 ) 用溶于 DMF的 20%六氢吡啶溶液对所说的树脂进行洗涤。 1) The resin is washed with a 20% hexahydropyridine solution in DMF.
2) 用溶于 DMF的 20%六氢吡啶溶液中搅拌 30分钟; 2) Stir in 20% hexahydropyridine solution in DMF for 30 minutes;
3)用 DMF洗涤三次; 3 ) washing three times with DMF;
4) 与溶于 DMF中各为 15mmol的 Fmoc— Glu (OtBu)—OH, 1一羟基 苯并三唑水和物和 N, N—二异丙基碳化二亚胺搅拌 120分钟; 4) Stir with 15 mmoles of Fmoc-Glu (OtBu) -OH, 1-hydroxybenzotriazole and N, N-diisopropylcarbodiimide dissolved in DMF for 120 minutes;
5) 用异丙醇洗涤二次; 5) Wash twice with isopropanol;
6) 用 DMF洗涤三次; 6) Wash three times with DMF;
7) 试验茚三酮的显色反应, 如是阳性, 重复步骤 4一 6; 如阴性, 则进行下一个合成循环。 7) Test the color reaction of ninhydrin. If positive, repeat steps 4-6; if negative, proceed to the next synthesis cycle.
' 依此用下述 Fmoc—氨基酸且在每一循环步骤 7中用相应的氨基 酸重复合成循环; 'Repeat the synthesis cycle with the following Fmoc-amino acid and the corresponding amino acid in step 7 of each cycle;
Fmoc— Ala— OH, Fmoc— Glu (OtBu)—OH, Fmoc— Glu (OtBu)一 0rt, Fmoc— Ala— OH, Fmoc— Glu (OtBu) —OH, Fmoc— Glu (OtBu) — 0rt,
Fmoc— Val— OH, Fmoc— Val— 0H, Fmoc— Glu (OtBu),Fmoc— Val— OH, Fmoc— Val— 0H, Fmoc— Glu (OtBu),
Fmoc― Lys (Boc) ― OH, Fmoc一 Lys (Boc )― OH, Fmoc― Glu ( OtBu)一Fmoc-Lys (Boc)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Glu (OtBu)-
OH, OH,
Fmoc― Lys (Boc )一 0H, Fmoc一 Leu一 OH, Fmoc一 Asp (OtBu)― OH , Fmoc-Lys (Boc)-0H, Fmoc-Leu-OH, Fmoc-Asp (OtBu)-OH,
Fmoc— Lys (Boc)—OH, Fmoc— Thr (tBu)一 0H, Fmoc— Thr (tBu )—OH,
Fmoc— lie— OH, Fmoc— Glu (OtBu)—0H, Fmoc— Ser (tBu)—OH,Fmoc— Lys (Boc) —OH, Fmoc— Thr (tBu) —0H, Fmoc— Thr (tBu) —OH, Fmoc— lie— OH, Fmoc— Glu (OtBu) —0H, Fmoc— Ser (tBu) —OH,
Fmoc— Ser (tBu)—OH, Fmoc— Thr (tBu)—OH, Fmoc— Asp (OtBu)― OH, Fmoc— Ser (tBu) —OH, Fmoc— Thr (tBu) —OH, Fmoc— Asp (OtBu) ― OH,
Fmoc— Val— OH, Fmoc— Ala— OH, Fmoc— Ala— OH, Fmoc— Asp (O+Bu)—0H, Fmoc— Ser (+Bu)—OH, Fmoc— Val— OH, Fmoc— Ala— OH, Fmoc— Ala— OH, Fmoc— Asp (O + Bu) —0H, Fmoc— Ser (+ Bu) —OH,
最后用乙酸乙酰化后从而完成全部合成循环。 这样获得受保护的 c端半胱氨酸修改了的胸腺肽 1王树脂。 After the final acetylation with acetic acid, the entire synthesis cycle is completed. This obtained a protected c-terminal cysteine-modified thymosin 1 king resin.
Ac- Ser (tBu) 一 Asp (OtBu) - Ala- Ala- Val- Asp (OtBu) -Thr (tBu)一 Ser (tBu)—Ser (tBu)—Glu (OtBu)—lie— Thr (tBu)—Thr (tBu) — Lys (Boc)—Asp (OtBu)— Leu— Lys (Boc)—Glu (OtBu)— Lys (Boc)— Lys (Boc)—Glu (OtBu) —Val— Val— Glu (OtBu) —Glu (OtBu) — Ala— G lu (OtBu) — Asn (Trt)— Cys (Trt)—Wang resin重 40g。 Ac- Ser (tBu)-Asp (OtBu)-Ala- Ala- Val- Asp (OtBu) -Thr (tBu)-Ser (tBu) —Ser (tBu) —Glu (OtBu) —lie— Thr (tBu) — Thr (tBu) — Lys (Boc) — Asp (OtBu) — Leu — Lys (Boc) — Glu (OtBu) — Lys (Boc) — Lys (Boc) — Glu (OtBu) — Val — Val — Glu (OtBu) —Glu (OtBu) — Ala — G lu (OtBu) — Asn (Trt) — Cys (Trt) — Wang resin weighs 40g.
把获得的次肽树脂 (5.0g) 与含 2%苯甲硫醚, 2%甲醇, 4%三异 丙基硅垸, 2%二巯基乙垸 50ml三氟乙酸混合搅拌 2小时。 去除三氟 乙酸后, 用乙醚沉淀出产品粗品。 将粗品溶于 100ml浓度为 0.2M的醋 酸胺缓冲液中。 过滤后, 上清液待用制备 HPLC分离提纯。 The obtained hypopeptide resin (5.0 g) was mixed with 50 ml of trifluoroacetic acid containing 2% of anisole, 2% of methanol, 4% of triisopropylsiliconam, and 2% of dimercaptoacetamidine for 2 hours. After removal of trifluoroacetic acid, the crude product was precipitated with ether. The crude product was dissolved in 100 ml of a 0.2 M acetate buffer solution. After filtration, the supernatant was separated and purified by preparative HPLC.
以 10ml/分流速,在 234nm处检测,用 Vydac, C18拄 (2.2X25cm, 10u)。用线性梯度 0.5%乙蹿 /1分钟洗脱(缓冲液 Α·· 0.01%三氟乙酸水 溶液; 缓冲液 B: 0.01%三氟乙酸乙腈溶液)。 将含产品的组成合并, 冻 干得纯度为 99%的产品 0.4g。 用液质 (LC一 MS) 联用对分子量进行测 定表明具有正确的分子量 (M+2H) 2+=1606, (M+H) +=3210。 (计算的分 子量为 =3210, 参见图 2)。 对此肽酸水解后, 水解条件为: 用 6N HC1 在 110Ό水解 24小时。进行氨基酸分析表明此肽具有预计的氨基酸比 步骤 2··多肽前体与聚乙二醇反应,合成修饰的胸腺肽 1衍生物: 将 50mgC端半胱氨酸胸腺肽 1和 500mg用马来酰亚胺修改了的 甲氧基聚乙二醇(分子量为 40, 000 Dalton)溶于己于 5ml 0.1M磷酸
盐缓冲液中。 反应二小时后, 用 Superdex去盐, 然后再用反相制备高 效液相法分离出产品。 条件为; VydacC18柱 (2.2X25cm, 10μ); 线性 梯度 0.5%乙腈 /分钟, 乙腈和水都含 0.01%三氟乙酸; 检测 : 234mm; 流速: 10毫升 /分钟。 产品冻干后得 490mg。 实施例二: Detect at 234 nm at a flow rate of 10 ml / min using Vydac, C18 拄 (2.2X25cm, 10u). Elution was performed with a linear gradient of 0.5% acetamidine for 1 minute (buffer A ·· 0.01% trifluoroacetic acid aqueous solution; buffer B: 0.01% trifluoroacetic acid acetonitrile solution). The product-containing compositions were combined and lyophilized to obtain 0.4 g of a product having a purity of 99%. Liquid chromatography (LC-MS) was used to determine the molecular weight and the correct molecular weight (M + 2H) 2+ = 1606, (M + H) + = 3210. (Calculated molecular weight = 3210, see Figure 2). After hydrolysis of this peptide acid, the hydrolysis conditions are: Hydrolysis with 6N HC1 at 110 ° F for 24 hours. Amino acid analysis showed that this peptide has the expected amino acid ratio. Step 2 · The peptide precursor reacted with polyethylene glycol to synthesize a modified thymosin 1 derivative: 50 mg of C-terminal cysteine thymosin 1 and 500 mg were made with maleimide Modified methoxypolyethylene glycol (molecular weight 40,000 Dalton) dissolved in 5ml 0.1M phosphoric acid In salt buffer. After two hours of reaction, the product was desalted with Superdex, and then the product was separated by reversed-phase high performance liquid phase method. The conditions are: VydacC18 column (2.2X25cm, 10μ) ; linear gradient 0.5% acetonitrile / min, both acetonitrile and water contain 0.01% trifluoroacetic acid; detection: 234mm; flow rate: 10ml / min. The product was lyophilized to give 490 mg. Embodiment two:
Ac— S er— Asp— Ala— Ala— Val― Asp一 Thr一 Ser一 Ser― Glu— lie— Thr— Thr— Ar g— Asp - L eu-Arg- G lu-Arg— Arg— Glu— Val - Val - G lu— G lu-Ala— Glu-Asn— Lys— PEG40K Ac — Ser — Asp — Ala — Ala — Val — Asp — Thr — Ser — Ser — Glu — lie — Thr — Thr — Ar g — Asp-L eu-Arg- G lu-Arg — Arg — Glu — Val- Val-G lu— G lu-Ala— Glu-Asn— Lys— PEG40K
步骤 1:固相化学合成制备供聚乙二醇修饰的前体 C端赖氨酸 14, Step 1: Synthesis of PEGylated precursor C-terminal lysine 14 by solid-phase chemical synthesis,
17, 19, 20, 精氨酸取代的胸腺肽 1衍生物: 17, 19, 20, arginine substituted thymosin 1 derivatives:
固相化学合成法见实施例 1 固相化学制备 C端半胱氨酸胸腺肽 For solid-phase chemical synthesis, see Example 1. Preparation of C-terminal Cysteine Thymosin
1衍生物。合成用 10§接有?1110(—1^3(80( —(^的王树脂(0.6ramol/g 树脂, 6mmol)。 合成按照下述 Fmoc氨基酸且在每一循环步骤中用相应 的氨基酸重复合成循环: 1 derivative. 10 for § ? 1110 (—1 ^ 3 (80 (— (^ 's king resin (0.6ramol / g resin, 6mmol). Synthesis was performed according to the following Fmoc amino acid and the synthesis cycle was repeated with the corresponding amino acid in each cycle step:
Fmoc― Asn (Tre)一 OH, Fmoc— Ala—— OH, Fmoc— Glu (OtBu)—OH, Fmoc— Glu (OtBu)— 0rt, Fmoc― Asn (Tre)-OH, Fmoc-Ala-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OtBu)-0rt,
Fmoc一 Val一 OH, Fmoc― Val- ~ OH, Fmoc― Glu (OtBu), Fmoc— Arg (Pbf ) —OH, Fmoc— Arg (Pbf)—OH, Fmoc— Glu (OtBu)一 OH, Fmoc- Val- OH, Fmoc- Val- ~ OH, Fmoc- Glu (OtBu), Fmoc- Arg (Pbf) -OH, Fmoc- Arg (Pbf) -OH, Fmoc- Glu (OtBu)-OH,
Fmoc— Arg (Pbf)—OH, Fmoc一 Leu一 OH, Fmoc ~ Asp (OtBu)― OH, Fmoc— Arg (Pbf) —OH, Fmoc- Leu- OH, Fmoc ~ Asp (OtBu) ― OH,
Fmoc— Arg (Pbf)—OH, Fmoc— Thr (tBu)—OH, Fmoc— Thr (tBu)—OH, Fmoc― lie― OH, Fmoc— Glu (OtBu)—OH, Fmoc一 Ser (tBu)― OH, Fmoc— Ser (tBu)—OH, Fmoc— Thr (tBu)—OH, Fraoc一 Asp (OtBu)― OH, Fmoc— Arg (Pbf) —OH, Fmoc— Thr (tBu) —OH, Fmoc— Thr (tBu) —OH, Fmoc― lie― OH, Fmoc— Glu (OtBu) —OH, Fmoc—Ser (tBu) ― OH , Fmoc— Ser (tBu) —OH, Fmoc— Thr (tBu) —OH, Fraoc-Asp (OtBu) ― OH,
Fmoc— Val— OH, Fmoc— Ala— OH, Fmoc— Ala— OH, Fmoc— Asp (0+Bu)—OH Fraoc— Ser (+Bu)—— 01
最后用乙酸乙酰化后, 从而完成全部合成循环, 这样获得受保护 的多肽树脂, 重 40g。将粗品多肽从树脂中切割的方法与实施例一中的 方法相似。 所得的粗品多肽的分离提纯制备方法亦和实施例一中所给 的方法相似。 对此多肽进行水解, 水解条件为: 用 6NHCL在 110°C水 解 24小时。 进行氨基酸分析表明, 此肽具有预计的氨基酸比例。 Fmoc— Val— OH, Fmoc— Ala— OH, Fmoc— Ala— OH, Fmoc— Asp (0 + Bu) —OH Fraoc— Ser (+ Bu) —— 01 After final acetylation with acetic acid, the complete synthesis cycle is completed, so that a protected peptide resin is obtained, weighing 40 g. The method of cutting the crude polypeptide from the resin is similar to that in Example 1. The method for isolating and purifying the obtained crude polypeptide is also similar to the method given in Example 1. This peptide was hydrolyzed under the following conditions: 6NHCL at 110 ° C for 24 hours. Amino acid analysis showed that this peptide had the expected amino acid ratio.
多肽前体与聚乙二醇形成聚乙二醇修饰的胸腺肽 1 衍生物, 将 50mgC端赖氨酸, 14, 17, 19, 20精氨酸联代的胸腺肽 1衍生物溶于 5ml 0。 1M碳酸氢钠溶液中。 向此溶液加入 550mg用含羧基经 N—羟基 丁二酰亚胺活化的聚乙二醇 (分子量 40, 000Dalton)。 The peptide precursor and polyethylene glycol form a polyethylene glycol modified thymosin 1 derivative. 50 mg of C-terminal lysine, 14, 17, 19, 20 arginine-linked thymosin 1 derivative is dissolved in 5 ml of 0. 1M sodium bicarbonate solution. To this solution was added 550 mg of polyethylene glycol (molecular weight 40,000 Dalton) activated with N-hydroxysuccinimide containing a carboxyl group.
步骤 2: 多肽前体与聚乙二醇反应,合成修饰的胸腺肽 1衍生物: 反应 1¾应控制在 8— 9之间。 反应 4小时后用 Superdex去盐, 然 后再用反相制备高效液相法分离出产品。 条件为: Vydac C18 柱 (2. 2X25cm, 10μ); 线性梯度 0. 5%乙睛 /分钟, 乙睛和水均含 0. 01%三 氟乙酸; 检测; 234nm; 流速 10毫升 /分钟。 产品冻干后得出 490mg。 实施例三 药效实验 Step 2: The peptide precursor reacts with polyethylene glycol to synthesize the modified thymosin 1 derivative: The reaction 1¾ should be controlled between 8-9. After 4 hours of reaction, the product was desalted with Superdex, and then the product was separated by reversed-phase high performance liquid phase method. The conditions are: Vydac C18 column (2.2X25cm, 10μ) ; linear gradient 0.5% acetonitrile / min, both acetonitrile and water contain 0.01% trifluoroacetic acid; detection; 234nm; flow rate 10ml / min. The product was lyophilized to give 490 mg. Example 3 Pharmacodynamic Experiment
用日达仙为阳性对照, 检测样品对 T淋巴细胞产生细胞因子的影响 作用, 以确定样品对 T淋巴细胞的免疫活性是否有调节作用和作用的 强弱。 Using Ridaxian as a positive control, the effect of the sample on the cytokine production of T lymphocytes was tested to determine whether the sample has a regulatory effect on the immune activity of T lymphocytes and the strength of the effect.
[实验原理] [Experimental principle]
体外培养的淋巴细胞中 T 淋巴细胞在受到有丝分裂原刀豆素 A (ConA, Concanavalin A)刺激后, 可导致 T淋巴细胞活化, 产生细胞 因子合成、 细胞因子受体表达、 细胞分化及细胞增殖的细胞行为的变 化。 细胞因子的产生和细胞的增殖反应是免疫细胞的功能状态和细胞 活化的反映。 检测手段具有可靠性、 良好的重复性及简便性等特点, 故通过细胞因子产生和细胞增殖反应的测定来判断样品对 τ淋巴细胞
的功能影响作用一直得到广泛的应用。 T lymphocytes of lymphocytes cultured in vitro, stimulated by mitogen concanavalin A (ConA, Concanavalin A), can cause T lymphocytes to activate, produce cytokine synthesis, cytokine receptor expression, cell differentiation and cell proliferation. Changes in cell behavior. The production of cytokines and the proliferative response of cells are a reflection of the functional status of immune cells and cell activation. The detection method has the characteristics of reliability, good reproducibility, and simplicity. Therefore, the determination of the sample on τ lymphocytes is determined by the measurement of cytokine production and cell proliferation response. The effect of the function has been widely used.
[实验材料] - [Experimental Materials] -
1 . 样品: 日达仙、 PEG样品用 1640培养液稀释, 分别稀释成实验 所需浓度。 1. Samples: Zidaxian and PEG samples were diluted with 1640 medium and diluted to the concentration required for the experiment.
2.动物: 实验釆用 ICR小鼠, 雄性, 6- 8周龄, 购于中科院上海实 验动物中心, 动物合格证书号: SCXK (沪) 2002-0010。 动物购来后, 饲养于本所清洁级动物房, 一般饲养 3- 4天后, 用于实验。 2. Animals: ICR mice for experimental use, males, 6-8 weeks old, purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences, animal certificate number: SCXK (Shanghai) 2002-0010. After the animals were purchased, they were kept in the clean animal room of this institute, and generally used for experiments after 3-4 days.
2.试齐 [J : ConA (Concanaval in A) 购自 Si gma公司。 2. Trial [J: ConA (Concanaval in A) purchased from Si gma company.
RPMI 1640培养液为 GIBC0产品配制,含 10%灭活的胎牛血清, HEPES 缓冲液 10mM, 青霉素 100IU/mL, 链霉素 lOO g/mL, 谷氨酰胺 2mM, — 巯基乙醇 50μΜ, ΡΗ 7. 2ο RPMI 1640 culture medium is formulated with GIBC0 product, containing 10% inactivated fetal calf serum, 10 mM HEPES buffer, 100 IU / mL penicillin, 100 g / mL streptomycin, 2 mM glutamine, 50 μM mercaptoethanol, PF 7. 2ο
细胞因子 IFN- 、 IL-2等 ELISA试剂盒 (Becton Di ckinson co. 生产)。 ELISA kits such as cytokines IFN- and IL-2 (produced by Becton Di ckinson co.).
[实验方法] [experimental method]
脾细胞悬液的制备: 无菌取出脾脏, 用毛玻璃片将小鼠脾脏磨碎, 制成脾细胞悬液。 裂解红细胞后, 洗涤三次, 计数 (活细胞在 95 %以 上)。 用含 10%FBS (胎牛血清) RPMI 1640 培养液将脾细胞浓度调为 5 X 106 细胞 /ml。 Preparation of spleen cell suspension: The spleen was aseptically removed, and the spleen of the mouse was ground with ground glass to make a spleen cell suspension. After lysing red blood cells, wash three times and count (live cells above 95%). The spleen cell concentration was adjusted to 5 X 106 cells / ml with RPMI 1640 medium containing 10% FBS (fetal bovine serum).
一、 IL- 2和 IFN-γ含量的测定 (ELISA法) 1. Determination of IL-2 and IFN-γ (ELISA method)
获取培养上清液- 取小鼠脾细胞, 调至 5xl06/ml, 加入 24孔板中, 1ml/孔;各样品 每个浓度分别加于 24孔板, 0. 5ml/孔;加刺激物 ConA (20ug/ml) 0. 5ml/ 孔。 370C,C02培养箱中培养 24小时, 离心, 收培养上清液。 Obtain the culture supernatant-take mouse splenocytes, adjust to 5xl06 / ml, add to a 24-well plate, 1ml / well; add each concentration of each sample to a 24-well plate, 0.5ml / well; add stimulus ConA (20ug / ml) 0.5ml / well. Incubate in a 370C, CO2 incubator for 24 hours, centrifuge, and collect the culture supernatant.
二、 ELISA检测方法: Second, ELISA detection method:
抗体包被: 用包被稀释液将抗体稀释, 96孔酶标板加入稀释后的抗 体(Capture Ant ibody) , 50μ1/孔。 封板, 于 40C过夜。
封闭: 去抗体溶液, 用洗液(PBS/Tween溶液)洗 3次, 加入封闭 液 (PBS/10%FBS), lOOul/孔。 室温 1小时。 Antibody coating: Dilute the antibody with the coating dilution solution, and add the diluted antibody (Capture Ant ibody) to the 96-well microtiter plate, 50μ1 / well. Seal the plate and incubate at 40C overnight. Blocking: Remove antibody solution, wash 3 times with washing solution (PBS / Tween solution), add blocking solution (PBS / 10% FBS), 100ul / well. 1 hour at room temperature.
标准品和样品: 去封闭液, 用洗液(PBS/Tween溶液)洗 3次, 加 入标准品和样品, 50μΐ/孔。 室温 2小时。 Standards and samples: Remove blocking solution, wash 3 times with washing solution (PBS / Tween solution), add standards and samples, 50 μΐ / well. 2 hours at room temperature.
检测抗体和酶: 去标准品和样品, 用洗液 (PBS/Tween溶液)洗 3 次,加入稀释后的检测抗体(Detection Antibody)和酶(HRP), 50μ1/ 孔, 室温 1小时。 Detection of antibodies and enzymes: Remove standards and samples, wash 3 times with washing solution (PBS / Tween solution), add diluted detection antibody (Detection Antibody) and enzyme (HRP), 50μ1 / well, room temperature for 1 hour.
底物显色: 去检测抗体和酶, 用洗液(PBS/Tween溶液)洗 3次, 加入溶于柠檬酸, 双氧水的底物 (TMB) , 50μ1/孔。 室温,避光, 30 分钟。 显色后, 加终止液(2Ν H2S04), 25ul/孔。 Substrate coloration: Remove antibodies and enzymes, wash 3 times with washing solution (PBS / Tween solution), and add substrate (TMB) dissolved in citric acid and hydrogen peroxide, 50 μl / well. Room temperature, protected from light, 30 minutes. After color development, add stop solution (2N H2S04), 25ul / well.
0D值: 置酶标仪中, 于 450nm、 校正 570nm处, 测定 0D值。 0D value: Set the microplate reader at 450nm and calibrate at 570nm to measure the 0D value.
[实验结果] [Experimental results]
样品对 ConA诱导 T淋巴细胞细胞因子产生的影响作用检测:
Effects of samples on the production of T lymphocyte cytokines induced by ConA:
IFN-r (pg/ml) IL-2 (pg/ml) 样品 浓度 (ug/ml) Mean SD Mean SD 对照 4122 77 2920 24 日达仙 0. 001 4177 177 3231 30 IFN-r (pg / ml) IL-2 (pg / ml) Sample Concentration (ug / ml) Mean SD Mean SD Control 4122 77 2920 24 Dasin 0. 001 4177 177 3231 30
0. 01 4118 134 3582 25 0. 01 4118 134 3582 25
0. 1 4106 191 3434 1840. 1 4106 191 3434 184
1 4836 208 3692 191 4836 208 3692 19
10 4832 36 3614 30410 4832 36 3614 304
100 4095 427 3840 476100 4095 427 3840 476
PEG样品 0. 001 4809 151 3505 296 PEG sample 0.001 4809 151 3505 296
0. 01 4603 234 3438 117 0. 01 4603 234 3438 117
0. 1 4141 200 3339 1340. 1 4141 200 3339 134
1 4949 367 3214 551 4949 367 3214 55
10 3874 3 3348 29310 3874 3 3348 293
100 5467 73 3582 25 100 5467 73 3582 25
[实验结论] [Experimental results]
在 ConA激活诱导 T细胞产生细胞因子实验中,日达仙样品对 Τ细胞 的 IFN- 产生在 lug/ml和 10ug/m时有一定的促进作用,而 C-thymosin 2样品在多个浓度单位、 并与日达仙样品相比有较好的促进 T细胞的 IFN- 产生。 In the experiment of cytokine production by T cells induced by ConA, Zidaxian sample has a certain promotion effect on IFN- production of T cells at lug / ml and 10ug / m, while C-thymosin 2 sample has And compared with Zidaxian sample, it has better promotion of T cell IFN- production.
在 ConA激活诱导 T细胞产生细胞因子实验中,日达仙和 C- thymosin 2两样品对 T细胞的 IL-2产生的促进作用无明显的差异。
实施例四 药代实验 In the experiment of cytokine production by T cells induced by ConA, there was no significant difference in the effect of Zidaxian and C-thymosin 2 on IL-2 production of T cells. Example 4 Pharmacokinetic Experiment
1.实验目的 Experimental purpose
观察 PEG样品 (代号: PEG-TA1 ) 在猕猴体内的药代动力学特点. Observe the pharmacokinetics of PEG sample (code: PEG-TA1) in rhesus monkeys.
2. 试验材料和方法 2. Test materials and methods
2. 1受试品 2.1 Test article
受试品 PEG- TA1为无色澄清注射液, 4 °C保存。 The test product PEG-TA1 is a colorless and clear injection solution, stored at 4 ° C.
2. 2动物 2.2 animals
猕猴,军事医学科学院实验动物中心产(军医动字第 BDW95002号), 共 3只, 雌 1只, 雄 2只, 体重 3. 7土 0. 5 kg。 分笼用猴标准词料喂 养, 自由饮水, 每日给新鲜水果两次。 Macaque monkeys, produced by the Experimental Animal Center of the Academy of Military Medical Sciences (Medical Movement No. BDW95002), a total of 3, 1 female, 2 male, weighing 3. 7 soil 0.5 kg. Feed in separate cages with standard monkey food, drink freely, and give fresh fruit twice a day.
2. 3给药途径和剂量 2.3 routes of administration and dosage
皮下注射。 剂量根据临床人用剂量换算, 猕猴剂量为 64 g. kg -1。 2. 4取血时间及样品制备 Subcutaneous injection. The dose was converted according to the clinical human dose, and the macaque dose was 64 g. Kg -1. 2. 4 blood taking time and sample preparation
于药前、 药后 0. 5, 1, 2 , 4, 6, 8, 12, 24, 36, 48 h自后肢静脉 取血。 取血后 30分钟以内室温下离心 760 g X 15 min,。 避免溶血。 分离血清, -20 °C保存待测。 Blood was collected from the veins of the hind limbs before, 0.5, 1, 2, 4, 6, 8, 12, 12, 24, 36, 48 h after the medication. Centrifuge at room temperature for 760 g X 15 min within 30 minutes after blood collection. Avoid hemolysis. The serum was separated and stored at -20 ° C for testing.
2. 5检测方法 2.5 detection methods
ELISA法测定血清 PEG-TA1 浓度。 德国 Immundiagnostik 公司产 Thymosin a 1试剂盒。 Serum PEG-TA1 concentration was determined by ELISA. Thymosin a 1 kit from Immundiagnostik, Germany.
2. 6 药代动力学参数的估算及生物等效性的统计推断 2. 6 Estimation of pharmacokinetic parameters and statistical inference of bioequivalence
非房室 -统计矩法估算药代动力学参数。 Non-compartmental-statistical moment method to estimate pharmacokinetic parameters.
3. 结果 3. Results
3. 1 标准曲线 3.1 standard curve
药盒对于受试品在 1. 6- 16000 ng. mL-1范围内有良好的反应性, 呈 倒 "S "型曲线 (图 1 )。 实验中各板分别设标准曲线。 用同一板上的标 准曲线计算未知血清样品中 EP0归一浓度。
3. 2 血药浓度 The kit has good reactivity to the test product in the range of 1.6 to 16000 ng. ML-1, and has an inverted "S" curve (Figure 1). Standard curves were set for each plate in the experiment. Use the standard curve on the same plate to calculate the normalized EP0 concentration in the unknown serum sample. 3.2 blood concentration
表 1列出了 3只猕猴 sc 64 μ g. kg _1受试品用 ELISA法测得的去 本底血清抗原浓度。 图 2是平均血清抗原浓度 -时间曲线的比较。 从图 和表可以看到, 动物间个体差异值很大, 达峰时间从 4一 12小时不等, 平均值则于给药后 8 h达峰。 Table 1 lists the background serum antigen concentrations of three macaques sc 64 μ g. Kg _1 tested by ELISA. Figure 2 is a comparison of mean serum antigen concentration-time curves. It can be seen from the graph and table that the individual differences between animals are large, and the peak time varies from 4 to 12 hours, and the average value reaches a peak at 8 h after administration.
表 1、 猕猴 sc PEG-TA1 (64 g. kg -1 ) 后去本底血清抗原浓度一 时间变化 (ng. mL- 1 ) Table 1. Changes in serum antigen concentration over time (ng. ML- 1) after sc PEG-TA1 (64 g. Kg -1) in macaque monkeys.
Time/h 1# 2# 3# Mean 士 SD RSDTime / h 1 # 2 # 3 # Mean SD RSD
0. 5 169 135 一 152 土 24 150. 5 169 135 a 152 soil 24 15
1 一 237 1195 716 士 678 951 one 237 1195 716 taxi 678 95
2 722 565 1034 773 士 239 312 722 565 1034 773 taxis 239 31
4 636 880 1244 920 + 306 334 636 880 1244 920 + 306 33
6 一 2099 995 1547 ± 780 506 a 2099 995 1547 ± 780 50
8 936 2677 ― 1807 土 1231 688 936 2677 ― 1807 soil 1231 68
12 2163 2468 745 1792 + 919 5112 2163 2468 745 1792 + 919 51
24 1976 1716 965 1553 士 525 3424 1976 1716 965 1553 taxis 525 34
36 1091 663 985 913 土 223 2436 1091 663 985 913 dirt 223 24
48 926 463 ― 695 ± 328 47
48 926 463 ― 695 ± 328 47
3.3 PK参数 3.3 PK parameters
根据平均血药浓度一时间资料计算的药代动力学参数如表 2所示。 表 2、 猕猴 sc PEG-TA1 (64 μ g. kg -1) 后的平均 PK参数 参数 单位 mean ± SD RSD% Table 2 shows the pharmacokinetic parameters calculated based on the average blood concentration-time data. Table 2.Average PK parameters of macaque sc sc-PEG-TA1 (64 μ g. Kg -1) Parameter unit mean ± SD RSD%
AUC(0-48h) ng. h. mL - 1 60221 ± 16896 28AUC (0-48h) ng. H. ML-1 60221 ± 16896 28
AUC(O-oc) ng. h. mL-1 80742 ± 32324 40AUC (O-oc) ng. H. ML-1 80742 ± 32324 40
AUC(48h- ) ng. U. h. mL-1 20521 士 18277 89AUC (48h-) ng. U. h. ML-1 20521 ± 18277 89
AUC(48h-oc) % % 25.4 土 17.85 70AUC (48h-oc)%% 25.4 soil 17.85 70
MRT h 9.2 ± 0.7 8MRT h 9.2 ± 0.7 8
CL/F mL. h- 1. kg - 1 6.6 ± 2.8 43CL / F mL.h- 1. kg-1 6.6 ± 2.8 43
VSS mL. kg - 1 61 土 27 44 tl/2 h 20 ± 6 31VSS mL. Kg-1 61 soil 27 44 tl / 2 h 20 ± 6 31
Kel ― 0.03385 土 0.011 34 Kel ― 0.03385 soil 0.011 34
4. 结论 4 Conclusion
PEG化修饰后的 TAl总体上达峰时间延迟,末端消除相半衰期计算值也 显著比文献报道的未修饰 TA1原形药物为长。
The peak time of TAl modified by PEGylation is generally delayed, and the calculated half-life of the terminal elimination phase is also significantly longer than that of the unmodified TA1 prototype drug reported in the literature.