重组人表皮生长因子和本芴醇的聚乙二醇修饰研究
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摘要
随着药物研究的快速发展，通过聚乙二醇(polyethylene glycol，PEG)修饰来提高药物药效和顺应性已成为药学研究的一门重点。如蛋白质多肽类药物的稳定性差、半衰期短、免疫原性等缺陷和化学药物水溶差、毒性大等不足都限制了它们的给药途径和药效的发挥。
重组人表皮生长因子(rhEGF)为具有53个氨基酸残基、理论相对分子量为6045的单链多肽，现阶段主要用于消化道溃疡、口腔溃疡、烧伤创面、糖尿病足坏疽皮肤等的治疗。但其在胃液和创面的稳定性较差，常规制剂已不能满足治疗的需要。而复方蒿甲醚(一片片剂含蒿甲醚20mg和本芴醇120mg)作为WHO推崇的治疗疟疾一线药物在临床上已取得较好的疗效。但就本芴醇而言，其吸收缓慢、生物利用度低影响了它的药效发挥，故可以考虑结构改造来改善本芴醇的上述不足，以求达到本芴醇的单独给药目的。
通过聚乙二醇修饰可为上述问题的解决提供有益的借鉴和参考。本课题主要分为三部分：(1)PEG修饰剂的合成(2)重组人表皮生长因子(recombination human epidermal growth factor，rhEGF)的PEG修饰(3)本芴醇的PEG修饰及其生物利用度研究。
第一部分：课题涉及3种PEG修饰剂的合成：单甲氧基聚乙二醇胺(mPEG-NH_2)、单甲氧基聚乙二醇琥珀酸琥珀酰亚胺酯(mPEG_(5k)-SSA)、单甲氧基聚乙二醇丙醛(mPEG_(5k)-propionaldehyde)。首先用原料单甲氧基聚乙二醇(mPEG_(5k)-OH)通过酯化和还原得到mPEG-NH_2。再由mPEG-NH_2与琥珀酸酐及氮羟基琥珀酰亚胺(NHS)反应制得mPEG_(5k)-SSA。mPEG_(5k)-propionaldehyde是以mPEG_(5k)-OH为原料与3-氯丙醛二乙基缩醛反应先得到缩醛化合物，再在弱酸条件下分解生成醛类化合物。通过核磁共振(NMR)和红外(IR)检测证实所得产品为目标产物。
第二部分：rhEGF的PEG修饰，酯类修饰剂(mPEG_(5k)-SSA和mPEG_(20k)-NHS)在pH8.0的磷酸缓冲盐溶液中与rhEGF进行偶合反应，摩尔比为PEG∶rhEGF=9∶1；醛类修饰剂(mPEG_(5k)-propionaldehyde和mPEG_(5k)-ALD)在pH5.5的醋酸缓冲液中与rhEGF进行偶合反应，摩尔比为PEG∶rhEGF=3∶1，并在硼氢甲腈钠还原下生成稳定的腙类化合物。通过四种修饰剂的修饰可得到不同修饰率和修饰度(修饰度：蛋白上连接PEG的个数)的产物。mPEG_(5k)-SSA修饰生成含有两种不同修饰度的产品，mPEG_(20k)-NHS修饰生成一种修饰度的产品，mPEG_(5k)-propionaldehyde和mPEG_(5k)-ALD的修饰产品修饰度均为1。
第三部分：本芴醇的聚乙二醇修饰及其生物利用度研究，首先由本芴醇与琥珀酸酐反应得到含酯键的本芴醇琥珀酸，再与第一部分实验合成的mPEG-NH_2进行脱水反应生成可降解的水溶性PEG化本芴醇。通过质谱分析确证修饰产品为目标产物。并通过进一步的性质分析确证产物的纯度为86.71％，其中游离本芴醇占2.37％，其它为原料PEG。稳定性研究中发现其稳定性较差，在甲醇中三小时后降解53.83％。
在体内吸收研究中，取SD大鼠八只，随机分为木芴醇组(对照组)和PEG化本芴醇组(每组各含四只，雌雄各半)，肠溶胶囊给与药物(避免胃酸水解PEG化本芴醇)，一次性给与本芴醇15mg／kg和等摩尔量的PEG化本芴醇151mg／kg(PEG化本芴醇与本芴醇的分子量比值为10.1)。分别于给药后0.5、0.75、1、2、4、8、12、24、36、48、72h断尾取血。分析数据计算出k、K_a、t_(1／2)、AUC_(0-72)、T_(peak)、C_(max)，CL，V，MRT。可以看出PEG化本芴醇的相对生物利用度较本芴醇有所提高，达到317.95％。
Pegylation of drug is an important method to improve drug action and compliance. The shortcoming in stability, half life, toxicity and immunogenicity was a constraint for the drug delivery and effect.rhEGF is a polypeptide composed of 53 amino acid residues (Mr=6045). It has been used for the treatments of gastric and intestinal ulcers, burns and corneal wound, diabetic foot ulcers and so on. But its unstability in ulcers and wounds limits its effect by common preparation. Benflumetol is a new antimalarial drug developed in the Institute of Microbiology and Epidemiology. It was absorbed and eliminated slowly in vivo. While artemether was absorbed and eliminated quickly in vivo. So Co-artemether, which combinate of 20 mg artemether and 120 mg benflumetol in fixed tablet, was suggested as a first drug for the treatment of Plasmodium falciparum malaria by the WHO experts. In order to improve the absorption and bioavailability of benflumetol, it was PEGylated for the raise of oral absorption.
It could give some good suggests for these problems through Pegylation. There were there parts of this study: (1)synthesis of PEG derivatives (2)Pegylation of rhEGF and (3)Pegylation of benflumetol and bioavailability research of PEG-benflumetol.
In the part one: There PEG derivatives, which included mPEG-NH_2, mPEG_(5k)-SSA and mPEG_(5k)-propionaldehyde, were synthesized. mPEG-OH was esterized by p-toluene sulfonyl chloride and then got mPEG-NH2 through hydrogenising; mPEG-NH_2 reacted with succinic anhydride and N-hydroxy succinimide, at last got mPEG_(5k)-SSA; mPEG_(5k)-OH reacted with 3-chloropropionaldehyde diethyl acetal and got acetalational compound. mPEG_(5k)-propionaldehyde was gotten through this compound decomposed in weak acid. These compounds were identified by NMR and IR.
In the part two: the PEGlytion of rhEGF. The conjugation of rhEGF and Esters derivatives (mPEG_(5k)-SSA and mPEG20k-NHS, 9-fold molar excess) was carried out in phosphate buffer (50mM,pH 8.0) . The reaction between mPEG_(5k)-SSA and rhEGF produced a heterogeneous mixture of PEGylated rhEGF. The compounds in the conjugation of mPEG_(20k)-NHS and rhEGF conjugated same number of PEG derivatives. Aldehyde derivatives (mPEG_(5k)-propionaldehyde and mPEG_(5k)-ALD. 3-fold molar excess) and rhEGF were dissolved in sodium acetate buffer(50mM,pH 5.5) in the present of sodium cyanoborohydride as a reducing agent and forming a hydrazone linkage between rhEGF and PEG. In coincidence with expect, only mono-PEGylation rhEGF was gotten in the reaction between rhEGF and aldehyde derivatives. But the site of the conjugation has not been identified.
In the part there, benflumetol was PEGylated for the improvement of water solubility and the PEG-benflumetol was used for the absorption research in intestine. At first, benflumetol was esterized by succinic anhydride and we got a acid compound. PEG-benflumetol, which could be degrdned, was synthesized through a dehydrolysis reaction between the acid compound and mPEG- NH_2. The structure of PEG-benflumetol was identified by MADIL-TOF. And the purity of the product is 86.71% according to property analysis, which including 2.37% free benflumetol and raw material PEG for the rest. The stability of the product is not good from the 53.83% degradation after three hours in methanol.
Absorption of benflumetol and PEG-benflumetol was carried out in 8 SD rats in vivo. Each group had four rats (two female and two male) . A single dose of benflumetol (15mg/kg) and PEG-benflumetol (151mg/kg) was administered orally by enteric capsule. Blood samples were drawn at time points of 0.5h, 0.75h, 1h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, 72h after the administration.
After the calculation of k, K_a, t_(1/2), AUC0-72, T_(peak), C_(max), CL, V, MRT. we could decide that PEG-benflumetol get a good bioavailability. And the comparative bioavailability was 317.95%.
重组人表皮生长因子(rhEGF)为具有53个氨基酸残基、理论相对分子量为6045的单链多肽，现阶段主要用于消化道溃疡、口腔溃疡、烧伤创面、糖尿病足坏疽皮肤等的治疗。但其在胃液和创面的稳定性较差，常规制剂已不能满足治疗的需要。而复方蒿甲醚(一片片剂含蒿甲醚20mg和本芴醇120mg)作为WHO推崇的治疗疟疾一线药物在临床上已取得较好的疗效。但就本芴醇而言，其吸收缓慢、生物利用度低影响了它的药效发挥，故可以考虑结构改造来改善本芴醇的上述不足，以求达到本芴醇的单独给药目的。
通过聚乙二醇修饰可为上述问题的解决提供有益的借鉴和参考。本课题主要分为三部分：(1)PEG修饰剂的合成(2)重组人表皮生长因子(recombination human epidermal growth factor，rhEGF)的PEG修饰(3)本芴醇的PEG修饰及其生物利用度研究。
第一部分：课题涉及3种PEG修饰剂的合成：单甲氧基聚乙二醇胺(mPEG-NH_2)、单甲氧基聚乙二醇琥珀酸琥珀酰亚胺酯(mPEG_(5k)-SSA)、单甲氧基聚乙二醇丙醛(mPEG_(5k)-propionaldehyde)。首先用原料单甲氧基聚乙二醇(mPEG_(5k)-OH)通过酯化和还原得到mPEG-NH_2。再由mPEG-NH_2与琥珀酸酐及氮羟基琥珀酰亚胺(NHS)反应制得mPEG_(5k)-SSA。mPEG_(5k)-propionaldehyde是以mPEG_(5k)-OH为原料与3-氯丙醛二乙基缩醛反应先得到缩醛化合物，再在弱酸条件下分解生成醛类化合物。通过核磁共振(NMR)和红外(IR)检测证实所得产品为目标产物。
第二部分：rhEGF的PEG修饰，酯类修饰剂(mPEG_(5k)-SSA和mPEG_(20k)-NHS)在pH8.0的磷酸缓冲盐溶液中与rhEGF进行偶合反应，摩尔比为PEG∶rhEGF=9∶1；醛类修饰剂(mPEG_(5k)-propionaldehyde和mPEG_(5k)-ALD)在pH5.5的醋酸缓冲液中与rhEGF进行偶合反应，摩尔比为PEG∶rhEGF=3∶1，并在硼氢甲腈钠还原下生成稳定的腙类化合物。通过四种修饰剂的修饰可得到不同修饰率和修饰度(修饰度：蛋白上连接PEG的个数)的产物。mPEG_(5k)-SSA修饰生成含有两种不同修饰度的产品，mPEG_(20k)-NHS修饰生成一种修饰度的产品，mPEG_(5k)-propionaldehyde和mPEG_(5k)-ALD的修饰产品修饰度均为1。
第三部分：本芴醇的聚乙二醇修饰及其生物利用度研究，首先由本芴醇与琥珀酸酐反应得到含酯键的本芴醇琥珀酸，再与第一部分实验合成的mPEG-NH_2进行脱水反应生成可降解的水溶性PEG化本芴醇。通过质谱分析确证修饰产品为目标产物。并通过进一步的性质分析确证产物的纯度为86.71％，其中游离本芴醇占2.37％，其它为原料PEG。稳定性研究中发现其稳定性较差，在甲醇中三小时后降解53.83％。
在体内吸收研究中，取SD大鼠八只，随机分为木芴醇组(对照组)和PEG化本芴醇组(每组各含四只，雌雄各半)，肠溶胶囊给与药物(避免胃酸水解PEG化本芴醇)，一次性给与本芴醇15mg／kg和等摩尔量的PEG化本芴醇151mg／kg(PEG化本芴醇与本芴醇的分子量比值为10.1)。分别于给药后0.5、0.75、1、2、4、8、12、24、36、48、72h断尾取血。分析数据计算出k、K_a、t_(1／2)、AUC_(0-72)、T_(peak)、C_(max)，CL，V，MRT。可以看出PEG化本芴醇的相对生物利用度较本芴醇有所提高，达到317.95％。
Pegylation of drug is an important method to improve drug action and compliance. The shortcoming in stability, half life, toxicity and immunogenicity was a constraint for the drug delivery and effect.rhEGF is a polypeptide composed of 53 amino acid residues (Mr=6045). It has been used for the treatments of gastric and intestinal ulcers, burns and corneal wound, diabetic foot ulcers and so on. But its unstability in ulcers and wounds limits its effect by common preparation. Benflumetol is a new antimalarial drug developed in the Institute of Microbiology and Epidemiology. It was absorbed and eliminated slowly in vivo. While artemether was absorbed and eliminated quickly in vivo. So Co-artemether, which combinate of 20 mg artemether and 120 mg benflumetol in fixed tablet, was suggested as a first drug for the treatment of Plasmodium falciparum malaria by the WHO experts. In order to improve the absorption and bioavailability of benflumetol, it was PEGylated for the raise of oral absorption.
It could give some good suggests for these problems through Pegylation. There were there parts of this study: (1)synthesis of PEG derivatives (2)Pegylation of rhEGF and (3)Pegylation of benflumetol and bioavailability research of PEG-benflumetol.
In the part one: There PEG derivatives, which included mPEG-NH_2, mPEG_(5k)-SSA and mPEG_(5k)-propionaldehyde, were synthesized. mPEG-OH was esterized by p-toluene sulfonyl chloride and then got mPEG-NH2 through hydrogenising; mPEG-NH_2 reacted with succinic anhydride and N-hydroxy succinimide, at last got mPEG_(5k)-SSA; mPEG_(5k)-OH reacted with 3-chloropropionaldehyde diethyl acetal and got acetalational compound. mPEG_(5k)-propionaldehyde was gotten through this compound decomposed in weak acid. These compounds were identified by NMR and IR.
In the part two: the PEGlytion of rhEGF. The conjugation of rhEGF and Esters derivatives (mPEG_(5k)-SSA and mPEG20k-NHS, 9-fold molar excess) was carried out in phosphate buffer (50mM,pH 8.0) . The reaction between mPEG_(5k)-SSA and rhEGF produced a heterogeneous mixture of PEGylated rhEGF. The compounds in the conjugation of mPEG_(20k)-NHS and rhEGF conjugated same number of PEG derivatives. Aldehyde derivatives (mPEG_(5k)-propionaldehyde and mPEG_(5k)-ALD. 3-fold molar excess) and rhEGF were dissolved in sodium acetate buffer(50mM,pH 5.5) in the present of sodium cyanoborohydride as a reducing agent and forming a hydrazone linkage between rhEGF and PEG. In coincidence with expect, only mono-PEGylation rhEGF was gotten in the reaction between rhEGF and aldehyde derivatives. But the site of the conjugation has not been identified.
In the part there, benflumetol was PEGylated for the improvement of water solubility and the PEG-benflumetol was used for the absorption research in intestine. At first, benflumetol was esterized by succinic anhydride and we got a acid compound. PEG-benflumetol, which could be degrdned, was synthesized through a dehydrolysis reaction between the acid compound and mPEG- NH_2. The structure of PEG-benflumetol was identified by MADIL-TOF. And the purity of the product is 86.71% according to property analysis, which including 2.37% free benflumetol and raw material PEG for the rest. The stability of the product is not good from the 53.83% degradation after three hours in methanol.
Absorption of benflumetol and PEG-benflumetol was carried out in 8 SD rats in vivo. Each group had four rats (two female and two male) . A single dose of benflumetol (15mg/kg) and PEG-benflumetol (151mg/kg) was administered orally by enteric capsule. Blood samples were drawn at time points of 0.5h, 0.75h, 1h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, 72h after the administration.
After the calculation of k, K_a, t_(1/2), AUC0-72, T_(peak), C_(max), CL, V, MRT. we could decide that PEG-benflumetol get a good bioavailability. And the comparative bioavailability was 317.95%.
引文
1 Francesco MV, Gianfranco P, PEGylation, successful approach to drug delivery [J].DTT, 2005, 10(21): 1451-1458.
2 Roberts MJ, Bentley MD, Harris JM, et al. Chemistry for peptide and protein PEGylation[J]. Adv Drug Deliv Rev, 2002, 54(4):459-476.
3 宋丹 方秀斌．表皮生长因子的研究进展，国外医学免疫学分册．2002，8(4)：339-342．
4 Na DH, Lee KC, Dluca PP. PEGylation of Octreotide Ⅱ. Effect of N-terminal Mono-PEGylation on Biological Activity and Pharmacokinetics [J]. Pharm Res, 2005,22(5):743-749.
5 Walsh S, Shah A, Mond J. Improved pharmacokinetics and reduced antibody reactivity of lysostaphin conjugated to polyethylene glycol [J]. Antimicrob Agents Chemother2003, 47(2):554-558.
6 张修建 王清明 陈惠鹏等．药物的聚乙二醇修饰研究进展，解放军药学学报．2003，19(3)：213—216．
7 Pendri A ,Conover CD ,Greenwald RB. Antitumor activity of paclitaxel- 2'-glycinate conjugated to poly(ethylene glycol): a water soluble prodrug [J]. Anti-Cancer Drug Des, 1998, 13(5):387-395.
8 Conover CD, Greenwald RB ,Pendri A, et al. Camptothecin delivery systems:enhanced efficacy and tumor accumulation of camptothecin following its conjugation to polyethylene glycol via a glycine linker[J]. Cancer Chemother Pharmacol, 1998,42(5):407-414.
9 夏承建 王松 朱鹤孙．聚乙二醇二酸(2-羟甲基-3，5，6-三甲基吡嗪)酯的合成及其体外降解性能，精细化工．2004，21(9)：666-668．
10 孙卫华．抗疟疾复方制剂，中国药师．2006，9(10)：965—966．
11 刘克良 王良友．多肽和蛋白质的聚乙二醇化修饰方法，有机化学．2003，23(21)：1320-1323．
12 王良友 潘和平 柳川 高亚萍．人甲状旁腺素(1-34)的合成与聚乙二醇修饰，中国生化药物杂志．2005，26(2)：76-79．
13 姚文兵 吴梧桐 沈子龙等．聚乙二醇对干扰素α-2b的初步化学修饰研究，中国药科大学学报．2000，31(1)：74～77．
14 Benthey MD, Harris JM. Poly(ethylene glycol) aldehyde hydrates and related polymers and applications in modifying amines, US, 5990237 [P]. 1999-11-23.
15 Yamamoto Y, Tsutsumi Y, Yoshioka Y, et al. Site-specific PEGylation of a lysine-deficient TNF-alpha with full bioactivity [J]. Nat Biotechnol, 2003, 21(5): 546-552.
16 Lee H, Park TG. Preparation and characterization of mono-PEGylated epidermal growth factor: evaluation of in vitro biologic activity [J]. Pharm Res, 2002, 19(6): 845-851.
17 Lee H, Jang H , Ryu SH, et al. N-terminal site-specific mono-PEGylation of epidermal growth factor [J]. Pharm Res, 2003, 20(5): 818-825.
18 Mansor SM, Navaratnam V, Yahaya N, et al. Determination of a new antimalarial drug, benflumetol, in blood plasma by high-performance liquid chromatography [J]. J Chromatogr B Biomed Appl, 1996, 682(2): 321-325.
19 Zeng MY, Lu ZL, Yang SC, Min Zhang, et al. Determination of benflumetol in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection [J]. J Chromatogr B Biomed Appl, 1996, 681 (2): 299-306.
20 Hong L, Jong-Hwa S, Jeong-Sook P, Kun Han, et al. Polyethylene glycol-coated liposomes for oral delivery of recombinant human epidermal growth factor [J]. Int J Pharm 2003, 258 (1-2): 11-19.
2 Roberts MJ, Bentley MD, Harris JM, et al. Chemistry for peptide and protein PEGylation[J]. Adv Drug Deliv Rev, 2002, 54(4):459-476.
3 宋丹 方秀斌．表皮生长因子的研究进展，国外医学免疫学分册．2002，8(4)：339-342．
4 Na DH, Lee KC, Dluca PP. PEGylation of Octreotide Ⅱ. Effect of N-terminal Mono-PEGylation on Biological Activity and Pharmacokinetics [J]. Pharm Res, 2005,22(5):743-749.
5 Walsh S, Shah A, Mond J. Improved pharmacokinetics and reduced antibody reactivity of lysostaphin conjugated to polyethylene glycol [J]. Antimicrob Agents Chemother2003, 47(2):554-558.
6 张修建 王清明 陈惠鹏等．药物的聚乙二醇修饰研究进展，解放军药学学报．2003，19(3)：213—216．
7 Pendri A ,Conover CD ,Greenwald RB. Antitumor activity of paclitaxel- 2'-glycinate conjugated to poly(ethylene glycol): a water soluble prodrug [J]. Anti-Cancer Drug Des, 1998, 13(5):387-395.
8 Conover CD, Greenwald RB ,Pendri A, et al. Camptothecin delivery systems:enhanced efficacy and tumor accumulation of camptothecin following its conjugation to polyethylene glycol via a glycine linker[J]. Cancer Chemother Pharmacol, 1998,42(5):407-414.
9 夏承建 王松 朱鹤孙．聚乙二醇二酸(2-羟甲基-3，5，6-三甲基吡嗪)酯的合成及其体外降解性能，精细化工．2004，21(9)：666-668．
10 孙卫华．抗疟疾复方制剂，中国药师．2006，9(10)：965—966．
11 刘克良 王良友．多肽和蛋白质的聚乙二醇化修饰方法，有机化学．2003，23(21)：1320-1323．
12 王良友 潘和平 柳川 高亚萍．人甲状旁腺素(1-34)的合成与聚乙二醇修饰，中国生化药物杂志．2005，26(2)：76-79．
13 姚文兵 吴梧桐 沈子龙等．聚乙二醇对干扰素α-2b的初步化学修饰研究，中国药科大学学报．2000，31(1)：74～77．
14 Benthey MD, Harris JM. Poly(ethylene glycol) aldehyde hydrates and related polymers and applications in modifying amines, US, 5990237 [P]. 1999-11-23.
15 Yamamoto Y, Tsutsumi Y, Yoshioka Y, et al. Site-specific PEGylation of a lysine-deficient TNF-alpha with full bioactivity [J]. Nat Biotechnol, 2003, 21(5): 546-552.
16 Lee H, Park TG. Preparation and characterization of mono-PEGylated epidermal growth factor: evaluation of in vitro biologic activity [J]. Pharm Res, 2002, 19(6): 845-851.
17 Lee H, Jang H , Ryu SH, et al. N-terminal site-specific mono-PEGylation of epidermal growth factor [J]. Pharm Res, 2003, 20(5): 818-825.
18 Mansor SM, Navaratnam V, Yahaya N, et al. Determination of a new antimalarial drug, benflumetol, in blood plasma by high-performance liquid chromatography [J]. J Chromatogr B Biomed Appl, 1996, 682(2): 321-325.
19 Zeng MY, Lu ZL, Yang SC, Min Zhang, et al. Determination of benflumetol in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection [J]. J Chromatogr B Biomed Appl, 1996, 681 (2): 299-306.
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